[Emerging Infectious Diseases | Volume 4 No. 2 / April - June 1998]


International EditorsUpdate

   * Emerging Infectious DiseasesSoutheast Asia Sai Kit Lam

Perspectives

   * Wild Primate Populations in Emerging Infectious Disease Research: The
     Missing Link? N.A. Wolfe, A.A. Escalante, W.B. Karesh, A. Kilbourn, A.
     Spielman, and A.A. Lal
   * A Method Accommodating Analysis of Error Facilitates Comparison and
     Clustering of Molecular Fingerprints. H. Salamon, M.R. Segal, A. Ponce
     de Leon, and P.M. Small
   * Legal Issues Associated with Antimicrobial Drug Resistance. D.P.
     Fidler
   * Interactions of Rickettsial Pathogens with Arthropod Vectors. A.F.
     Azad and C.B. Beard
   * Could Myocarditis, Insulin-Dependent Diabetes Mellitus and Guillain
     Barr Syndrome be caused by One or More Infectious Agents Carried by
     Rodents? B. Niklasson, B. Hrnfeldt, and B. Lundman
   * Multidrug-Resistant Mycobacterium tuberculosis: Molecular
     Perspectives. A. Rattan, A. Kalia, and N. Ahmad

Synopses

   * Hospitalization for Unexplained Illnesses Among U.S. Persian Gulf War
     Veterans. J.D. Knoke and C. Gray
   * Could the Agricultural Use of Burkholderia (Pseudomonas) cepacia Pose
     a Threat to Human Health? A. Holmes, J. Govan, and R. Goldstein
   * The Epidemiology of Haemophilus influenzae Invasive Diseases in the
     United States, 1994-1995: Near Disappearance of a Child Vaccine
     Preventable Disease. K.M. Bisgard, A. Kao, J. Leake, P.M. Strebel,
     B.A. Perkins, M. Wharton
   * Multiply Resistant Enterococci: The Nature of the Problem and an
     Agenda for the Future. M.M. Huycke, D.F. Sahm, and M.S. Gilmore
   * Enteroaggregative Escherichia coli (EAEC) in Diarrhea and
     Malnutrition. J.P. Nataro, T. Steiner, and R.L. Guerrant
   * C3 Binding Among Campylobacter jejuni Strains from Patients with
     Guillain-Barr Syndrome or with Uncomplicated Enteritis. B.M. Allos,
     F.T. Lippy, A. Carlsen, R.G. Washburn, and M.J. Blaser

Dispatches

   * An Apparently New Virus (Family Paramyxoviridae) Infectious for Pigs,
     Humans, and Fruit Bats. A.W. Philbey, P.D. Kirkland, A.D. Ross, R.J.
     Davis, A.B. Gleeson, R.J. Love, A.R. Gould, P.W. Daniels, and A.D.
     Hyatt
   * Probable Human Infection with a Newly Described Virus in the Family
     Paramyxoviridae. K. Chant, R.Chan, M.Smith, D.E Dwyer, P. Kirkland,
     and the NSW Expert Group
   * Emergence of the M Phenotype of Erythromycin-Resistant Pneumococci in
     South Africa. C.D. Widdowson and K.P. Klugman
   * Reemergence of Plasmodium vivax Malaria in the Republic of Korea. B.H.
     Feighner, P. Son Il, W.L. Novakoski, L.L. Kelsey, and D. Strickman
   * Molecular and Epidemiologic Analysis of Dengue Virus Isolates from
     Somalia, N. Kanesa-thasan, G.J. Chang, B.L. Smoak, A. Magill, M.J.
     Burrous, and C.H. Hoke, Jr.
   * Molecular Characterization of a Novel Spotted Fever Group Rickettsia
     species from Ixodes scapularis in Texas. A.N. Billings, G.J. Teltow,
     and D.H. Walker
   * Dual Infection with Erlichia chaffeensis and a Spotted Fever Group
     Rickettsia: A Case Report. D.J. Sexton, G.R. Corey, C. Carpenter, L.Q.
     Kong, T. Gandhi, E. Breitschwerdt, B. Hegarty, S.-M. Cheng, H.-M.
     Feng, X.-J. Yu, J. Olano, D.H. Walker, and S.J. Dumler
   * New Probable Vectors of Rift Valley Fever in West and Central Africa.
     D. Fontenille, M. Traore-Lamizana, M. Diallo, J. Thonnon, J.P.
     Digoutte, and H.G. Zeller
   * Mycobacterium tuberculosisInfection as a Zoonotic Disease: Evidence of
     Possible Transmission between Humans and Elephants. K. Michalak, C.
     Austin, S. Diesel, J.M. Bacon, P. Zimmerman, and J.N. Maslow
   * Molecular Fingerprinting of Multiresistant Salmonella typhi. M.D.
     Hampton, L.R. Ward, B. Rowe, and E.J. Threlfall
   * Using Nurse Hot Line Calls for Disease Surveillance. J.S. Rodman, F.
     Frost, and W. Jakubowski
   * Clostridium septicum Infection and Hemolytic Uremic Syndrome. M.
     Barnham and N. Weightman
   * Persistent Infection of Pets within a Household with Three Bartonella
     Species. D.L. Kordick and E.B. Breitschwerdt

Commentary

   * Vector-borne Disease Surveillance and Natural Disasters Roger S. Nasci
     and Chester G. Moore

Letters

   * Similarity of Chemokines Charge and the V3 Domain of HIV-1 env
     Protein Ben Berkhout and Atze T. Das
   * Detection of Glycoprotein of Burkholderia pseudomallei Natalja P.
     Khrapova, Nikolay G. Tikhonov, Yelena V. Prokhvatilova
   * Forging New Perspectives on Disease Surveillance Harley Feldbaum
   * Provide a Context for Disease Emergence Toby D. St. George
   * Resistance to Dryness of Escherichia coli O157:H7 Strains from
     Outbreak in Sakai City, Japan, 1996 Yoshio Iijima, Mayumi Matsumoto,
     Kumiko Higuchi, Taro Furuta, and Takeshi Honda
   * Irradiation Pasteurization of Solid Foods Stephen Moses and Robert C.
     Brunham
   * Emerging Infectious Diseases in Brazil Luiz Jacintho da Silva
   * Reply to L.J. da Silva Hooman Momen
   * A Brief Update on Rabbit Hemorrhagic Disease Virus Lorenzo Capucci and
     Antonio Lavazza
   * Rabbit Hemorrhagic Disease C. Mead
   * Reply to Drs. Capucci, Lavazza, and Mead Alvin W. Smith, Neil J.
     Cherry, and David O. Matson

News and Notes

   * The National Food Safety Initiative
   * Conference on Global Disease Elimination and Eradication as Public
     Health Strategies,
     February 26, 1998
   * New and Reemerging Infectious Diseases: A Clinical Course
   * Erratum



Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

--------------------------------------------------------------------------------------

Update

International Editors Update

--------------------------------------------------

In this new section, we welcome commentary and updates from our newly
formed Board of International Editors.

Emerging Infectious DiseasesSoutheast Asia

Sai Kit Lam
University of Malaya, Kuala Lumpur, Malaysia

Dr. Lam is professor and head, Department of Medical Microbiology, Faculty of 
Medicine, University of Malaya. He serves on the editorial board of several 
medical journals and is the director of the WHO National Influenza Center, 
the WHO National Center for Rapid Viral Diagnosis, and the WHO Collaborating 
Center for Arbovirus Reference and Research for Dengue and Dengue Hemorrhagic Fever.

The recent emergence of a new strain (H5N1) of influenza A virus, the
so-called avian or bird flu, in Hong Kong underlines the importance of the
Southeast Asia region as an epicenter not only for influenza A viruses but
also for other microbial agents. In late 1992, Vibrio cholerae O139
appeared on the Indian subcontinent; and within a few months, it had spread
to China, Nepal, Pakistan, Malaysia, and as far as Russia.

The World Health Organization (WHO) has reported that infectious diseases
account for more than 17 million deaths per year worldwide and that at
least 30 new infectious diseases have emerged within the last 2 decades. Up
to half of the 5.8 billion people on earth are at risk for many endemic
diseases, with the most overpopulated and economically depressed countries
in Southeast Asia at highest risk. Although vaccines and antibiotics are
available for many diseases, in 1995 alone, respiratory infections such as
pneumonia killed 4.4 million people, 4 million of them children. Diarrheal
diseases, including cholera, typhoid, and dysentery killed 3.1 million,
most of them children. Tuberculosis (TB) killed almost 3.1 million, malaria
2.1 million, hepatitis B more than 1.1 million, and measles more than 1
million.

Faced with the reality of infectious diseases on a daily basis, Southeast
Asian nations have not been able to give emerging disease surveillance the
priority status it deserves. Much of the surveillance in the region is
centered in a few well-established laboratories where there is adequate
expertise, sufficient funding, and personal interest. Because of influenza
A, it is widely believed that Asia is the epicenter of new influenza
strains; the emergence of the avian influenza strain in Hong Kong supports
this contention. As a result of the activities of the Pacific Basin
Respiratory Virus Research Group, an excellent surveillance program for
respiratory viral infections is in place. If this small cluster of avian
flu had occurred elsewhere, it would most likely have been missed. All the
110 WHO Influenza Centers, particularly those in Southeast Asia, have been
alerted and are frequently updated about the Hong Kong outbreak. Diagnostic
reagents for the identification of the H5N1 strain will be made available
to these centers as part of the surveillance program.

Enteroviruses

Enteroviruses are frequently associated with the hot and humid climate of
tropical countries in Southeast Asia. The viruses are responsible mainly
for inapparent and mild infection, but occasionally they can give rise to
infection of the central nervous system, resulting in aseptic meningitis
and (rarely) paralysis; polioviruses are a good example. Hand, foot, and
mouth disease, caused by enteroviruses, such as coxsackie A16 and
enterovirus 71, is common in many countries in the region. In a recent
outbreak of hand, foot, and mouth disease in Malaysia, a new clinical
entity of encephalomyelitis emerged, which resulted in several deaths among
children under 5 years of age. Four such deaths were investigated
thoroughly in the University Hospital in Kuala Lumpur. All four children
were seen in the Emergency Department in a state of respiratory and
cardiovascular instability with a history of fever (3 to 5 days) associated
with reduced oral intake. One child had flaccid paralysis with hyporeflexia
of the lower limbs. A constant feature in these patients was the very rapid
vascular changes, followed rapidly by cardiac decompensation. Pulmonary
edema followed, suggesting a concomitant increase in pulmonary vascular
permeability. Consent for postmortem was obtained with some difficulty
because of religious and cultural reasons. The midbrain, pons, medulla, and
spinal cord were extensively damaged, with the worst damage at the level of
the medulla. The cerebrum and myocardium were relatively normal.
Enterovirus 71 was isolated from the medulla and spinal cord of three
children and from the cerebrum of the other child, where no brain stem
material was available. Other tissue sites including the cerebrospinal
fluid did not yield virus. The need for postmortem examinations in
investigating fatal emerging diseases must be addressed in areas where such
examinations are not possible for cultural reasons.

Arboviruses

Arthropod-borne viruses or arboviruses are yet another group of viruses
synonymous with countries in the region and cause considerable sickness and
death. Some of these viruses, e.g., Murray Valley encephalitis virus in
Australia, have restricted geographic distribution but may have the ability
to transcend geographical barriers, as in the emergence of Japanese
encephalitis virus in the Torres Strait of northern Australia and in Papua
New Guinea.

Dengue

Dengue is by far the most important arbovirus infection in Southeast Asia.
According to WHO, dengue has been reported in over 100 countries worldwide
and poses a threat to approximately 2 billion people. At the 46th World
Health Assembly held in Geneva in May 1993, a resolution was passed to make
the prevention and control of dengue a priority.

Dengue is an acute viral infection characterized by abrupt onset of fever,
severe headache, pain behind the eyes, muscle and joint pains, and rash.
The hemorrhagic form of dengue fever, dengue hemorrhagic fever (DHF), was
recognized as a new disease in the Philippines in 1953 and has been seen in
India, Malaysia, Singapore, Indonesia, Vietnam, Cambodia, and Sri Lanka.
During 1956 to 1992, 1,335,049 cases of DHF (13,723 deaths) were recorded
in Vietnam alone. The rise of dengue in tropical and subtropical areas of
the world is explained by factors such as rapid population growth,
expanding urbanization, inadequate municipal water supplies, and
difficulties in refuse disposal. These lead to an abundance of new breeding
sites for the mosquito vectors, while human migration patterns disperse
vectors and viruses into new areas.

The resurgence of DHF in several countries where only dengue fever had been
reported has become worrisome. Sri Lanka reported its first outbreak in
1989, and India and China also faced the same dilemma. DHF/dengue shock
syndrome has also been reported for the first time in New Caledonia and
Tahiti. The large outbreak in Cuba in 1981 showed how the disease has
spread to the Americas and other countries such as Venezuela and Brazil. In
Malaysia, where the disease is endemic, encephalitis cases associated with
dengue viruses have been documented, and vertical transmission has also
been reported in the last two years. Other unusual manifestations observed
rarely include acute renal failure and hemolytic uremic syndrome. The
emergence of such unusual clinical manifestations should also be monitored
and documented in other dengue-endemic countries. Despite intensive efforts
by the countries in the region to control the vectors, the disease is still
on the rise. A tetravalent live attenuated vaccine developed at Mahidol
University, Thailand, is being field-tested, and the preliminary results
are promising.

Disease Spread

Rapid transportation has been blamed for the spread of diseases, and this
is especially so in countries in the region. Tourism is an important
industry in Southeast Asia, and a series of conferences on travel medicine
has highlighted the problems, particularly in infectious diseases. Another
vast movement of people between countries in the region is that of migrant
workers. In Malaysia, an estimated 1.7 million workers are migrant workers,
of which only 1.2 million came in legally. Most of the workers are from
Indonesia, Myanmar, Philippines, and Pakistan, and they work in the
agricultural sector as well as in construction and domestic services. Those
who come in legally undergo a medical examination that includes screening
for some infectious diseases. Diseases associated with migrant workers are
chloramphenicol-resistant typhoid, multidrug-resistant TB, leprosy,
malaria, HIV/AIDS, and filariasis. In 1993, the first case of kala-azar
caused by Leishmania donovani was reported in a Bangladeshi migrant worker
in Malaysia, and since then, four other cases have been identified, all of
them in Bangladeshi workers. This disease had not been present in Malaysia.

In 1997, the theme "Emerging Infectious Diseases: Global AlertGlobal
Response" was chosen by WHO to celebrate World Health Day. This year, as
WHO celebrates its 50th anniversary, the region is still plagued with major
public health problems threatening the lives of millions of people in the
region. These problems underline the need to strengthen epidemiologic
surveillance and concentrate public health activities in Southeast Asia,
the epicenter of many emerging diseases.

Address for correspondence: S. K. Lam, Department of Medical Microbiology,
Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; fax:
603-758-2801; e-mail: lamsk@medicine.med.um.edu.my.


Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/update.htm

Please note that figures and equations are not available in ASCII format; 
their placement within the text is noted by [fig] and [eq], respectively. 
Greek symbols are spelled out. The following codes are used: 
(ft) for footnote; (sup) for superscript; (sub) for subscript; 
/= for greater than or equal to. 

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[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Perspectives

Wild Primate Populations in Emerging Infectious Disease Research:  The
Missing Link?

Nathan D. Wolfe,* Ananias A. Escalante, William B. Karesh, Annelisa
Kilbourn, Andrew Spielman,* and Altaf A. Lal
*Harvard School of Public Health, Boston, Massachusetts, USA; Centers for
Disease Control and Prevention, U.S. Public Health Service, Chamblee,
Georgia, USA; Wildlife Health Sciences, Wildlife Conservation Society,
Bronx, New York, USA.
-----------------------------------------------------------------------
           Wild primate populations, an unexplored source of
           information regarding emerging infectious disease,
           may hold valuable clues to the origins and evolution
           of some important pathogens. Primates can act as
           reservoirs for human pathogens. As members of
           biologically diverse habitats, they serve as
           sentinels for surveillance of emerging pathogens and
           provide models for basic research on natural
           transmission dynamics. Since emerging infectious
           diseases also pose serious threats to endangered and
           threatened primate species, studies of these diseases
           in primate populations can benefit conservation
           efforts and may provide the missing link between
           laboratory studies and the well-recognized needs of
           early disease detection, identification, and
           surveillance.

Infectious diseases respect no species or geographic boundaries. For a
parasite, closely related hosts offer new environments in which infection,
maintenance, replication, and transmission remain possible. The anthropoid
primates (which include humans) and to a lesser degree simian primates
share broadly similar physiologic and genetic characteristics and thus
susceptibility to many viruses, bacteria, fungi, protozoa, helminths, and
ectoparasites (1) that have the potential to cross primate-species
boundaries (2).

Similarities in pathogen susceptibility have made nonhuman primates ideal
laboratory models. During the 20th century, laboratory research on captive
primates has elucidated the life cycle and pathogenesis of many infectious
agents and facilitated drug and vaccine development. Nevertheless, the
ecology of infectious agents found in wild populations of primates has only
recently been addressed. Just as captive primates have proved invaluable
for research at the level of the organism, wild populations can provide the
opportunity to study infectious disease phenomena at the population and
ecosystem levels. Research at these levels addresses such pressing
questions as the origin(s) of pathogens, determinants of pathogen
emergence, and factors influencing maintenance of pathogens in animal
reservoirs.

During the past two decades unknown human diseases, including AIDS, Ebola
fever, hantavirus infection, and dengue hemorrhagic fever, have emerged
from enzootic foci. The emergence of these and other diseases has been
linked to the interface of tropical forest communities with high levels of
biodiversity and agricultural communities with relative genetic homogeneity
and high population densities of humans, domestic animals, and crops. This
interface poses a high risk for the emergence of novel disease (3-5).

Since most nonhuman primates live in tropical forest habitats, most
interactions between humans and wild nonhuman primates occur in this
high-risk interface, which has recently increased because of expanded
ecotourism and forest encroachment. These interactions can lead to pathogen
exchange through various routes of transmission (Table). Arthropod vectors,
shared water, and hunting of wild animals have facilitated pathogen
exchange and may have played an important role in pathogen transfers since
ancient times. In the recent past, laboratory research has led to
accidental human exposure to such agents as primate malaria parasites (12)
and a simian immunodeficiency virus (SIV) (16). The potential for exchange
through xenotransplantation has been discussed (17), and infection from
vaccine contaminated with SV40, a primate papovavirus, led to the exposure
of millions of persons in the 1950s (15). Conversely, pathogen transmission
from humans to nonhuman primates places both captive and wild animals at
serious risk for diseases such as measles and tuberculosis (TB), which are
deadly in many nonhuman primate species. In general, as levels of
interaction increase, so does pathogen exchange, resulting in further risks
to both humans and nonhuman primates.

The need for improved surveillance and basic research on emerging
infectious disease is well documented (3,18-20). Wild primates can serve as
sentinels by signaling which pathogens pose a risk for humans in the
immediate area (21) as well as in distant countries (5). Here, we describe
potential benefits of incorporating wild populations into emerging
infectious disease research.

Pathogen Origins

Nonhuman primates are infected with the closest relatives of important
human pathogens. The construction of molecular phylogenies has played an
important role in studying the evolution and classification of many
pathogens. While trees that result from these analyses must be interpreted
with care, they can provide valuable information on the history of
pathogens. Furthermore, the selection of genes that are evolving at the
appropriate rate should allow phylogenetic analyses to assess both ancient
and more recent epidemic origins (22).

Human herpes simplex virus infection is common in nonhuman primates and may
reflect contact with humans. A study of viral infection in nonhuman
primates found that chimpanzees and gorillas were seropositive to human
herpes simplex virus-1 and -2 strains, but orangutans and gibbons were not
(23).

Research on SIV phylogeny (24,25) has shown that HIV-1 and HIV-2 are each
more closely related to primate pathogens than they are to one another.
HIV-1 is in a group with SIV(sub CPZ) (26), a chimpanzee (Pan troglodytes) virus,
while HIV-2 falls within a clade consisting of West African primate viruses
(Figure 1; 24). In fact, Mindell (27) has argued that HIVs and SIVs should
be referred to as `primate immunodeficiency viruses' to more accurately
reflect their heritage. Preliminary evidence suggests that the origins of
global T-cell lymphotropic virus-1 subtype diversity may be analogous,
leading Liu et al. (28) to propose that HTLV-I subtypes emerged from three
separate nonhuman primate reservoirs.

Table. Routes of pathogen exchange between human and nonhuman primates
--------------------------------------------------------------------------
						   Direction of 
Route of exchange  	Pathogen       exchange  Evidence(sup a)  Reference
--------------------------------------------------------------------------
                                        Nonhuman
                                        primate to
Animal bite        	Herpes B        human           E        	 6(sup b)
                                        Nonhuman
                                        primate to
                        Monkeypox       human           E            7
                                        Human to
                                        nonhuman
Fecal-oral         	Poliovirus      primate         L           2(sup b)
                                        Chimpanzee
                                        to
                        Poliovirus      chimpanzee      E            8
                                        Nonhuman
Hunting, food prep                 	    primate to
& eating           	Ebola           human           E            9
                        Mycobacterium   Among
Nasal secretions   	leprae          primates       P, L     	10(sup b)
                                        Human to
                                        nonhuman
Respiratory droplet	Tuberculosis    primate         L       	11(sup b)
                                        Both
Vector-borne       	Malaria         directions     L,E       	12(sup b)
                                        Both
                        Filaria         directions     L,E       	 8(sup b)
                                        Human to
                                        nonhuman
Water-mediated     	Dracunculiasis  primate         L        	 13
                                        Nonhuman
                                        primate to
                        Schistosomiasis human           E        	 14
                                        Nonhuman
                                        primate to
Xenotransplantation	SV40            human        E(sup c)     15(sup b)  
---------------------------------------------------------------------------
(sup a)L = laboratory; E = epidemiologic; P = evidence that parasites
live naturally in multiple primate hosts.
(sup b)Evidence reviewed.
(sup c)The only current evidence for xenotransplantation includes SV40
spread through vaccine production.

This pattern is not unique to viruses, as demonstrated through decades of
research on primate malarias. More than 26 species of Plasmodia infect
primates (12,29). Both morphologic and molecular analyses show human and
nonhuman primate malarias interdigitating on phylogenetic trees (Figure 2;
30,33). Preliminary evidence suggests that this group of parasites has a
range of coevolutionary scenarios, including the speciation of P. vivax and
related parasites in Asian primates, the recent exchange of parasites
between humans and New World monkeys (30), and perhaps an ancient exchange
of a falciparum-like parasite from a bird or lizard to an African hominoid
(31,34,35). Further analyses are likely to lead to surprises. For example,
recent research has shown that the diversity of human P. vivax (previously
considered to be a single species) also includes a P. vivax-like parasite,
a widespread pathogen, which is most closely related to P. simiovale, a
primate malaria of Asian macaques (Macaca sp.) (30,36).

					[fig 1]
            Figure 1. Relationships among primate and human
            lentiviruses: Phylogeny of primate lentiviruses
            based on the gag gene obtained by (25). The names
            of the strains are indicated in parentheses. Hosts
            are indicated on trees. The description of the
            lentivirus strains is provided in (25).

A major limitation of these studies is that they rely on very small numbers
of nonhuman primate isolates only two SIV(sub cpz) isolates for the HIV-1 clade,
a single P. simiovale isolate for malaria phylogeny. In addition, very few
data are available on the distribution of these pathogens in wild
populations; more data on the distribution and extent of shared pathogens
should provide clues to origins (37), thereby pointing to general
conditions that may contribute to disease emergence.

Molecular phylogenies can also play a direct role in the control of
pathogens, for example, in charting their antigenic diversity, a task
necessary for vaccine design (38). Because nonhuman primate pathogens are
often evolutionary outgroups, vaccines that are targeted at antigens shared
by human and nonhuman pathogens should provide more universal coverage.
Molecular phylogenies can also assist in determining the rate of evolution
of vaccine candidate antigens. Because low rates of mutation often indicate
selective constraint, this technique may point to candidate antigens that
cannot easily mutate to evade vaccine-induced antibodies. Similar analyses
identifying highly constrained gene products or metabolic pathways as
targets for drug development may contribute to slowing the emergence of
drug resistance.

Reservoirs

The study of pathogen transmission has been encumbered by the use of
inappropriate terminology. Such terms as "primary hosts," "reservoir
hosts," "carriers," "arthroponoses," "xenonoses," and "zoonoses" pose
premature assumptions and belie aspects of the origins and natural
transmission cycles of pathogens. The factors that determine pathogen
viability within a vertebrate host may correspond only roughly, or not at
all, to species boundaries. Primate pathogens do not adhere to the
faithfully maintained sanctity of the distinction between humans and
nonhumans.

                         [fig 2]
          Figure 2. Relationships between primate and human
          parasites: Malaria phylogeny based on the
          circumsporozoite protein gene. The alignment does not
          include the central repeat region. P. falciparum (Pfa),
          P. vivax (Pvi), and P. malariae (Pma) are from humans;
          P. cynomolgi (Pcy), P. simiovale (Pso), and P. knowlesi
          (Pkn) are from macaques; P. simium (Psi) and P.
          brasilianum (Pbr) are from New World monkeys; P.
          reichenowi (Pre) is from chimpanzees; P. gallinacium
          (Pga) is from birds; and P. berghei (Pbe) and P. yoelii
          (Pyo) are from rodents. The numbers in the names
          indicate different isolates as described in (30). The
          sequence of P. gallinacium was reported by (31). The
          numbers on the branches are bootstrap % based on 500
          pseudoreplications. The tree was estimated by the
          neighbor-joining method with the Tajima and Nei
          distance (32).

The misconception of an evolutionary trend toward increasing host
specificity (39) has contributed to the belief that pathogen exchange
should be rare. An apparently restricted host range, however, may be the
product of specific ecologic conditions and not of an intrinsic
characteristic of the pathogen. In many cases, ecologic changes can broaden
the host range of a pathogen. For example, the filarid worm Loa loa remains
outside human populations, primarily because of vector behavior (40);
changes in ecology, such as the availability of a novel host, can change
vector behavior and expand the pathogen's host range. Similar phenomena may
have played a role in the emergence of a range of flaviviruses (e.g.,
yellow fever, dengue, and Japanese encephalitis) from primarily
forest-dwelling nonhuman primate cycles. For these flaviviruses, primate
populations may also continue to play a role by introducing novel genetic
variants, which, at least in the case of dengue, may be involved in
pathogenesis.

Another misconception is that primate populations are too sparse to
maintain human pathogens. A number of variables influence whether or not a
pathogen is maintained in a given population. The capacity for latency, for
example, may decrease the host population size necessary to maintain
pathogens. This characteristic has evolved independently in pathogens as
diverse as herpesviruses and Plasmodium and may help explain sustained
transmission in hosts with low population density. Orangutans (Pongo
pygmaeus), for example, are thought to act as hosts to two distinct
Plasmodium species (41), despite an estimated population size of two per
km2. While molecular phylogenies have not yet been used to verify that
these parasites are unique, the presence of a dormant hypnozoite stage
might allow for sustained transmission even at this extreme. Where human
and nonhuman hosts overlap, both must be factored into epidemiologic
models. Small populations of nonimmune humans alone may not be capable of
maintaining a pathogen, but when nearby nonhuman hosts are considered, a
critical population size may be reached.

Surveys to assess pathogen prevalence among nonhumans can play an important
role in control strategies. Eradication programs must consider animal
reservoirs; even if complete human coverage is achieved, long-term
reemergence from animal reservoirs can undo the best eradication efforts.
For example, it is not known if poliovirus infections can be maintained in
nonhuman primate populations. Seroprevalence surveys conducted before and
after eradication may prove invaluable.

Accidental exposure to infected laboratory workers has led to poliovirus
infections of chimpanzees and gorillas since the 1940s (1). Poliovirus can
infect not only our closest living relatives, chimpanzees, gorillas
(Gorilla gorilla), and orangutans (Pongo pygmaeus) (2), but also more
distantly related anthropoids like the colobus monkeys (e.g., Colobus
abyssinicus kikuyuensis [=guereza]) (42). Antibodies and shed virus have
also been found in recently imported animals (8), and some chimpanzees may
act as symptomless carriers (2). Long-term research by Jane Goodall on wild
Tanzanian chimpanzees documented the potential for transmission of
poliovirus (or a similar virus) in free-ranging chimpanzee populations
(43). Since no samples were collected, it is impossible to determine if the
epidemic described by Goodall was part of a natural chimpanzee cycle or the
result of introduction from local human populations or researchers. As
poliovirus eradication efforts intensify, it may be useful to monitor virus
prevalence in humans living near primate habitats.

Control efforts that rely on antimicrobial drugs must also take into
account the potential for nonhuman primate reservoirs. Despite
demonstrations that mass administration of diethylcarbamazine citrate
successfully controlled Brugia malayi (a filarid worm), Mak et al. found a
high prevalence of B. malayi after a large-scale administration of
chemotherapy (44). Research showed that even though periodic prevalence of
B. malayi decreased, subperiodic prevalence remained high. The maintenance
of subperiodic B. malayi was eventually attributed to mosquitoes infected
by leaf monkeys (Presbytis obscura). In this particular free-ranging
primate host, approximately 83% of the monkeys were infected (44).

Nonhuman "reservoirs" may also confer potential benefits. It is at least
theoretically possible that nonhuman primate populations may provide a
barrier to the spread of drug-resistant pathogens; while these pathogens
benefit from resistance in environments where drug pressure exists, drug
resistance can be costly, and resistant pathogens may not compete
effectively against susceptible `wild-type' pathogens in the absence of
drug pressure. As drug-free populations, reservoirs may provide havens for
susceptible pathogens, thereby decreasing the rate at which drug-resistant
genes spread and increasing the rate at which susceptibility may return
after drug pressure ends. This hypothesis may help explain why the rates of
drug-resistant gram-negative enteric bacteria of wild baboons (Papio
cynocephalus) living with limited human contact are significantly lower
than those of baboons living with human contact (45).

Sentinel Surveillance

In tropical lowland forests, which contain the greatest biodiversity of
terrestrial habitats (46), exist rarely seen or unknown pathogens with the
potential to enter human populations. These pathogens may affect residents
of and visitors to forested regions (21) and act as the source of
introduction of infectious agents to distant susceptible populations (47).
Increasing human contact with forested systems almost certainly leads to a
corresponding increase in the emergence of infections in the human
population. Nevertheless, predicting which pathogens humans may encounter
and be susceptible to remains a methodologic challenge.

Surveillance methods for predicting emerging pathogens include surveillance
of vectors or forest-dwelling human populations and wildlife epidemiology
(epidemiologic study of infections in wild populations) (48). These
approaches have limitations. While vector sampling may prove the easiest
method for widespread surveillance, the pathogens identified from vectors
may be difficult or impossible to culture. Even when successful, vector
sampling is likely to identify a range of pathogens, only some of which may
infect humans. Studies of human populations, while providing valuable
information, are limited to regions in which forest-dwelling human
populations exist.

Epidemiologic research among free-ranging primate populations has the
potential to predict which pathogens might enter human populations as
contact with forested regions increases. In addition to their physiologic
similarities to humans, primates have other characteristics that contribute
to their accumulation of infectious agents. Primates live primarily in
forested environments (49); in general, they have large bodies and live in
large groups-characteristics that may attract vectors (50). Furthermore,
dependence on fruit, a characteristic of most primates, requires mobility
(both terrestrially and arboreally), a trait that may increase exposure to
pathogens (51,52).

Despite the lack of organized attempts to document the distribution of
pathogens in wild populations, recognized "die-offs" in wild primate
populations have played an important role in identifying novel pathogens.
In 1956, for example, a novel flavivirus was identified through the
investigation of large-scale deaths of bonnet macaques (Macaca radiata) and
hanuman langurs (Presbytis entellus) in the Kyanasur Forest of India
(Seymour, 1981, cited in [2]) caused by Kyanasur Forest virus. More
recently, in 1995, deaths in a chimpanzee population studied by Christoph
Boesch in the Ti Forest, Cte d'Ivoire, and a single human case following
a necropsy led to the identification of a novel strain of Ebola virus
(9,53). The single human case in the Swiss researcher foreshadowed the
localized mini-outbreak of Ebola hemorrhagic fever in Mayibout, a village
in the northeast of Gabon in January 1996. The Gabon epidemic was linked to
the handling, preparation, and consumption of a chimpanzee that had been
found dead; 29 of 37 identified cases involved exposure to the dead
chimpanzee (54). Close monitoring of such populations, as is being
conducted in the Ti Forest, has the potential to identify emergence-linked
behavior, such as the consumption of specific plants or insects, which may
lead to the still elusive reservoir of Ebola virus. Considering the
exceptionally small percentage of wild primate populations under long-term
study, these examples represent only the tip of the iceberg. More
systematic monitoring of wild primate populations will likely provide a
substantial payoff in our understanding, identification, and possible
control of novel pathogens, both for humans and endangered primates.

Surveillance for certain types of human-nonhuman primate contact may be
particularly useful. Hunting, which involves tracking, capturing, handling,
transporting, preparing, and consuming meat, may play a particularly
important role in pathogen exchange. In addition to the recent evidence of
hunting-mediated Ebola transmission, the hunting of a red colobus (Colobus
pennanti oustaleti [=badius]) has been implicated in a localized epidemic
of monkeypox, an orthopoxvirus similar to smallpox, which continued for
four generations of human-to-human contact (55). Another example is the
increased risk for feline plague among cats that hunt rodents (7).
Necropsies share many characteristics with hunting and are appropriately
considered a high-risk activity.

Bites from wild primates may also play a role in the transmission of
certain pathogens. For example, chimpanzee-to-human transmission of
monkeypox occurred when a wild chimpanzee bit a 2-year-old girl (6).
Further sampling demonstrated high prevalence in forest squirrel
populations (up to 49% among Funisciurus lemniscatus) (56), underlining the
need for comprehensive studies before determining the ultimate source and
reservoir of pathogens. Bites and scratches have transmitted pathogens to
laboratory workers. The transmission of B-virus, a herpesvirus infecting
rhesus macaques (Macaca mulatta), has caused death in 18 of 24 known human
cases (57). Surveillance and education in human populations that hunt
nonhuman primates, as well as follow-up of reported primate bites in
nonlaboratory settings, may be indicated.

Ecologic and Social Factors Involved in Natural Transmission

The potential for disease emergence and reemergence depends on the
interaction of complex social, ecologic, and genetic factors at the host,
vector, and pathogen levels (58). Because wild populations of primates
display diverse social behavior and live in a range of ecologic
environments, they exemplify natural transmission. Furthermore, monitoring
pathogens in wild primate populations does not involve the treatment or
behavior change interventions that monitoring pathogens in humans requires.
Exceptions include the increasingly common need for wildlife medicine to
maintain the health of endangered species and decrease the impact of
pathogens from humans and domestic animals on free-ranging animals (59).
Nevertheless, for many pathogens, basic epidemiologic research, and not
treatment, remains the primary goal of wildlife medicine (59).

Studies of wild populations can highlight factors associated with the
pathogen exchange across species boundaries. Some parasites, for example,
the chewing licepocket gopher system, complete their entire life cycle on
a single host. This high level of host specificity may contribute to the
close cospeciation between pocket gophers and their respective lice; the
phylogenetic trees of host and parasite are nearly mirror images (60).
Nevertheless, such host-specific parasites may be the exception rather than
the rule. Many intestinal parasites, for example, seem to be generalists.
One epidemiologic study of wild primates in the Kibale Forest National
Park, Uganda, evaluated the role of primate distribution on the
distribution of intestinal amoebas (61); the park contains a number of
spatially separate primate groups, each of which consists of multiple
primate species. Most variation in amoeba prevalence was explained by group
membership, and little was explained by species, which suggests that these
parasites treated the primate groups as biologic islands (62), spreading
easily among diverse members of the same island but rarely spreading to new
islands.

Long-term behavioral research combined with occasional pathogen sampling
may provide valuable data. Long-term research on the SIVs of East African
primates, for example, has provided evidence for a recent cross-species
transmission of SIV between baboons and African grivet monkeys
(Cercopithecus aethiops aethiops) (62). This research has documented the
incidence of SIV(sub agm) among Ethiopian grivet monkeys for more than 20 years
(63). By examining prevalence in age groups over time, this research
demonstrated the minimal impact of SIV(sub agm) on the survival of grivets, the
predominance of sexual transmission, and the lack of maternal transmission.

An ecologic approach to pathogen transmission can benefit our understanding
of emerging diseases. By determining how humans become a part of the life
cycle of pathogens rather than how pathogens enter human populations, we
can better understand the factors associated with emergence and improve the
quality of public health responses. How humans become part of the life
cycle of pathogens depends on human migrations, environmental changes, and
cultural and social factors, in the context of evolutionary history of the
pathogens, vectors, and hosts, which make up an infectious system. Ecologic
and evolutionary studies of wild animals in general, and primate
populations in particular, can address questions arising from these complex
interactions.

The Future

Traditionally, the study of wild populations of primates has been the
domain of primatologists and wildlife veterinarians, who have worked to
overcome logistic difficulties in the field, develop methods, and address
ethical issues. Opportunities exist for collaborative work on infectious
disease; the benefits of such efforts are considerable. The translocation
of animals from vulnerable forest fragments to forest reserves, an
increasingly common conservation effort, is an opportunity for pathogen
sampling. Other possibilities are provided by collaboration with long-term
behavioral research on free-ranging animals. While behavioral research
sites may only provide fecal and urine samples, the study of intestinal
parasites is often possible; recent advances in urinalysis demonstrate that
urine may be a source of both antigen and antibody from systemic
infections, such as malaria (64). The distribution of appropriate necropsy
protocols and sample collection kits would improve data collection and
decrease risks associated with necropsies. Urgently needed are strategies
for noninvasive remote diagnosis. For example, combined with sensitive
molecular diagnostics, a remote tissue-biopsy dart system (65) may have
potential for obtaining epidemiologic samples.

Efforts to preserve endangered primates and monitor disease emergence have
some common objectives. Pathogen exchange is a two-way street, and exposure
to human pathogens poses a serious threat to endangered animals (11). While
the main focus of this article has been risk to humans, nonhuman primates
are frequently more threatened by microorganisms indigenous to humans than
vice versa (2). TB, which is often fatal to nonhuman primates, represents a
serious threat to laboratory primate communities, which commonly are
infected by humans and can occasionally reinfect laboratory workers (66).
TB is prevalent in wild populations, as demonstrated by the presence in
wild olive baboons (Papio cynocephalus) of Mycobacterium bovis infection,
an infection that most likely originated from cattle (67).

Both hunting and forest encroachment threaten endangered primates and
increase the possibility of human and nonhuman infectious disease
emergence. Research on the pathogens of primates and humans on forest
boundaries helps assess risks to wild primates and to humans. In addition
to their value in the study of infectious disease and human evolution, many
primates are valuable natural resources in their home countries. The
veterinary expertise and wildlife management skills of conservation
organizations can both supplement the basic pathogen research and control
work of the public health community and benefit from it.

The examination of pathogen exchange in regions of host overlap may
identify social factors that influence pathogen emergence. Data on forest
use by human communities surrounding forest reserves and levels of
crop-raiding by nonhuman primates have been collected as part of ongoing
conservation projects. A comparison of these data with human and nonhuman
primate pathogen prevalence may provide a measure of forest-oriented
behavioral risks. Further research comparing infection rates among hunters
and nonhunters could confirm the findings and determine the role of
behavioral control measures to decrease risk.

Much remains to be done. Recent evidence has suggested that some wild
primates self-medicate with plants in their environment (68). An
understanding of the underlying wildlife epidemiology, therefore, combined
with long-term dietary data and analysis of plant chemistry, may lead to
new chemotherapeutic drugs (69). In addition, research on the dynamics of
primate pathogens in their natural hosts may elucidate novel host
resistance mechanisms. In particular, evidence of impaired host survival
and reproduction, as well as long-term host-parasite association, may point
to hosts that are likely to have evolved genetically mediated resistance.
Such resistance mechanisms in humans (70) have begun to play an
increasingly important role in vaccine and drug development (71,72). While
the findings from captive primate studies have played an important role in
medicine in the 20th century, this period has also been marked by a notable
absence of research on the basic ecology of disease systems. Perhaps by
learning from primates in their natural environments we may better prepare
ourselves for the disease threats to humans and wildlife populations in the
coming century.

Acknowledgments

We thank Adria Tassy Prosser and Daniel G. Colley for valuable suggestions
and Mary Bartlett for editorial assistance. Nathan Wolfe is supported by a
Taplin Graduate Fellowship and a National Science Foundation-Predoctoral
Fellowship. Ananias A. Escalante is supported by the ASM/NCID Postdoctoral
Research Associates Program.

Nathan Wolfe is a doctoral candidate in the Department of Immunology and
Infectious Disease at the Harvard School of Public Health. Mr. Wolfe has
conducted research on self-medicating behavior among chimpanzees and
mountain gorillas in Uganda and is collaborating with Drs. Altaf Lal and
Ananias Escalante on malaria evolution research.

Address for correspondence: Altaf A. Lal, Centers for Disease Control and
Prevention, Mail Stop F12, 4770 Buford Hwy, Chamblee, Georgia 30341, USA;
fax: 770-488-4454; e-mail: aal1@cdc.gov.

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Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/wolfe.htm

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[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Perspectives

Accommodating Error Analysis in Comparison and Clustering of Molecular
Fingerprints

Hugh Salamon,* Mark R. Segal,* Alfredo Ponce de Leon, and Peter M. Small
*University of California, San Francisco, California, USA; Instituto
Nacional Nutricin, Zubriran, Mexico City, Mexico; and Stanford University
Medical Center, Stanford, California, USA

  -------------------------------------------------------------------------
      Molecular epidemiologic studies of infectious diseases rely on
      pathogen genotype comparisons, which usually yield patterns
      comprising sets of DNA fragments (DNA fingerprints). We use a
      highly developed genotyping system, IS6110-based restriction
      fragment length polymorphism analysis of Mycobacterium
      tuberculosis, to develop a computational method that automates
      comparison of large numbers of fingerprints. Because error in
      fragment length measurements is proportional to fragment length
      and is positively correlated for fragments within a lane, an
      align-and-count method that compensates for relative scaling of
      lanes reliably counts matching fragments between lanes. Results
      of a two-step method we developed to cluster identical
      fingerprints agree closely with 5 years of computer-assisted
      visual matching among 1,335 M. tuberculosis fingerprints. Fully
      documented and validated methods of automated comparison and
      clustering will greatly expand the scope of molecular
      epidemiology.

The combination of conventional epidemiologic investigations with molecular
techniques for genotyping pathogens has elucidated the epidemiology of many
infectious diseases. The most frequently used genotyping techniques (e.g.,
pulsed-field gel electrophoresis, restriction fragment length polymorphism
[RFLP], and randomly amplified polymorphic DNA) yield fragment-based data.
Fewer than 100 patterns can be compared visually. For larger numbers,
commercially available computer programs can be used to identify a
manageable subset of potentially matching patterns, which are then compared
visually. This approach is accurate but cumbersome and excessively
labor-intensive as the number of isolates exceeds several hundred.
Furthermore, the results of computer-assisted matching are not as
reproducible as systematic computational methods. These limitations
significantly constrain the size, scope, and standardization of molecular
epidemiologic investigations. We present an approach by which identical
patterns can be identified from large collections of DNA fingerprints.

The number of IS6110 fingerprints continues to increase, with many studies
across the globe producing IS6110 data to characterize Mycobacterium
tuberculosis isolates. Such molecular epidemiologic studies provide
information about tuberculosis (TB) transmission patterns. Studies in
Ethiopia, Tunisia, and The Netherlands (1), South Africa (2), India (3),
Denmark and Greenland (4,5), the United States (6), and Tanzania (7), among
many others, exploit IS6110-based RFLP genetic fingerprints.

We developed an automated computational system, in which a statistical
analysis of the error in measuring fragment sizes provides a conceptual
framework for comparing sets of fragment lengths. The computational
approach to lane comparisonalign-and-count method (ACM)permits
calculation of the number of fragments that match between two IS6110-based
RFLP fingerprints. The parameters of the computational ACM are adjusted to
provide the same high sensitivity as the labor-intensive visual inspection
used over the last 5 years.

We developed an approach to identifying a set of identical fingerprints
when the identity of the fingerprints is nontransitive. We also explored
improving the specificity among matched fingerprints to reveal additional
information in RFLP lanes.

Data Acquisition

Genotyping M. tuberculosis isolates with IS6100-based RFLP fingerprinting
was performed as described in van Embden et al., 1993 (8).
Computer-assisted comparison and clustering were performed on the RFLP
lanes as described in Woelffer et al., 1996 (9).

Internal standards were used to quantitate fragment sizes visualized with a
DNA probe to IS6110. Two films' exposures were scanned into Whole Band
Analyzer software (BioImage, Ann Arbor, MI, USA); one was obtained when
probing for the internal standard, the other when probing for IS6110. The
resulting images were aligned with three registration marks that gave
reference to the original nylon membrane. The Whole Band Analyzer software
quantitated fragment lengths for the IS6110-visualized bands, which were
inspected and edited manually in the software package. The resulting
collections of fragment lengths for each lane (bacterial isolate or
laboratory strain H37Rv) were exported to our ACM software and compared
with other lanes.

Mathematical Methods

The following is a descriptive summary of the principles underlying the
analysis (Appendix).

Analysis of Error in Data

The magnitude and characteristics of experimental error were empirically
assessed by analyzing variation in the results obtained from a reference
strain included in each experiment (gel). The absolute and proportional
differences in the measured fragment lengths of biologically identical
samples of this strain were calculated; results showed that the error in
measurement was proportional to fragment length and greater between than
within gels.

Align-and-Count Matching Algorithm

A method for matching was developed to accommodate the empirically defined
error. The fragment length data from two lanes were scaled through a range
of values, and the maximum number of mutually closest bands falling within
a threshold tolerance was reported (Figure 1). The acceptable tolerance was
smaller when lanes from the same gel were compared than when lanes from
different gels were compared. An animated demonstration of this method is
accessible on the Internet (URL for use with a graphics-capable Web
browser: http://molepi.stanford.edu/hugh/acm/counting).

A Graph-Theoretic Approach to Identical Fingerprints

We considered pairs of patterns identical if all fragments matched.
However, because the results might be nontransitive (A might be identical
to B, B identical to C, but A not identical to C), the identification of
groups of matched patterns is more complicated. A graph-theoretic approach
was used to assemble clusters of matching fingerprints.

Alignment and Analysis of Residual Error

We further aligned collections of lanes determined to match according to the 
above algorithms. The optimal alignment was defined as that which minimized 
the proportional error between putatively identical fragments. This analytic 
step, which is comparable to the experimental approach of rerunning clustered 
strains in the same gel, improved the ability to distinguish similar, but 
nonidentical fingerprints.

 			[fig 1]
Figure 1. The align-and-count method finds the 
maximum number of mutually closest bands within 
a threshold deviation value[Delta], for a search 
across a ranges of scaling values. The two lanes are 
scaled incrementally, thus searching for the best 
alignment.                                

Error in Analysis of H37Rv Data

Investigating the absolute error for pairs of 116 H37Rv lanes (Figure 2), 
we found that the error was consistently higher in gel-to-gel comparisons 
than in comparisons of lanes from the same gel. Error was proportional 
to fragment length for the range of fragment lengths from 0.9 kbp up to at 
least 5 kbp (Figure 2), which included 90% of the bands in the fingerprint 
data from San Francisco. The latter empirical result was consistent with the 
fact that the distance migrated by a DNA fragment on an electrophoresis gel 
is typically proportional to the logarithm of the fragment length. 
Furthermore, the error found when we compared one band of a lane to that 
band in another lane was positively correlated to the error
found for the other bands; in other words, if the first band in lane A was
larger than the average measurement for that band, it was likely that the
other bands in lane A would be larger than the average measurements. This
positive correlation was intuitively evident in comparing lane maps (i.e.,
when comparing graphic representations of the fragment lengths). In Figure
3, the set of lane maps on the left are measurements of a genotype found in
San Francisco. With the exception of the fourth lane from the right, they
represent a set of identical patterns. Note that the error is mostly a
scaling error and that if one fragment is larger than average for that
lane, the others are very likely to be larger also. These two
observationsthat error is proportional to fragment length and positively
correlated for bands within a given lanesuggests two classes of error: one
is a property of each band; the second is a property of each lane. This
analysis motivated us to develop a computational algorithm that scales
fingerprints and measures the number of mutually closest bands within
threshold sizes of each other for the best alignment, i.e., the scaling
that maximizes the number of matching fragments.

 	[fig 2]          				[fig 2]           
 Figure 2. Means and two            Figure 3. Additional alignment
 standard errors of the mean        of very similar patterns can
 error bars for pairwise            identify clearly distinct
 comparisons among 116 12-banded    patterns. Measurement noise
 H37Rv lanes show that error is     obscures the detailed
 consistently larger when           relationships between 26
 comparing lanes between gels       patterns that were identified
 than when comparing lanes from     from 1,335 as being very
 the same gel. The x-axis           similar. However, after
 corresponds to w(b), and the       alignment to a consensus
 y-axis to d(b), as presented in    pattern, a clearly distinct
 the text. It is evident that       pattern (an outlier from the
 error is proportional to           other members of this
 fragment length in the range of    autocluster) can be readily
 fragment lengths found in          identified. Fragment lengths are
 H37Rv. The data exhibit 2% to      given in kilobasepairs (kb).
 3% error for between gel
 comparisons, but only
 approximately 1% error on
 average for within gel
 comparisons.

Alignment and Residual Error for H37Rv Lanes

By optimally aligning (i.e., minimizing the sum of proportional errors)
pairs of H37Rv lanes, we find a distribution of scaling factors and of
residual error. Table 1 shows 6,641 pairwise comparisons of 91 replicate
lanes (members of each pair are taken from different gels). The mean value
of s, the scaling factor, for these 6,641 alignments is 0.0212, and the
standard deviation of s is 0.0189. The reduction in error due to alignment
is approximately twofold, to approximately 1% with a standard deviation
also of approximately 1% (Table 1); therefore, a search range, S, of 0.10
will allow for virtually every incidence of scaling error in the data.
(Assuming normally distributed scaling and noting that 0.10 lies more than
four standard deviations about the mean scaling error, we conclude that
scaling error will not be compensated in fewer than 1 out of 10,000
independent pairwise comparisons.) Employing a deviation tolerance,
[Delta], of 0.045 should ensure a sensitivity very close to
100% for matching individual bands, after alignment. (Assuming normally
distributed differences between replicate band measurements and a deviation
tolerance of 4.5%, which is approximately 3.5 standard deviations above the
mean fragment length error after alignment, one should falsely conclude two
identical bands do not match with an approximate probability of 0.0002.)
These parameter values, together with the number of incremental searches,
I, set to 100, empirically gave results agreeing closely with visual
inspection by experienced researchers who matched entire lanes. Similarly,
one may use analysis of within gel lane error to determine the parameter
values for the ACM to match lanes from the same gel.

Table 1. Pairwise comparisons (n=6,641) of lanes
across gels characterize unaligned proportional error
and residual error

-----------------------------------------------------

                    Unaligned            Aligned
                     pairwise            pairwise
                   proportional        proportional  
          Mean        error               error
H37Rv    kilo      ------------     ----------------
band     bases    mean      s.d.     mean      s.d.
b(sup *)w(sub b)  r(b)            r(sup a)(b)
----------------------------------------------------
1        5.029   0.0253   0.0196    0.0099    0.0079

2        4.853   0.0249   0.0202    0.0084    0.0075

3        3.533   0.0253   0.0303    0.0109    0.0203

4        2.814   0.0237   0.0283    0.0105    0.0182

5        2.153   0.0202   0.0186    0.0086    0.0125

6        1.892   0.0210   0.0204    0.0100    0.0085

7        1.800   0.0226   0.0220    0.0092    0.0082

8        1.684   0.0227   0.0193    0.0083    0.0083

9        1.541   0.0221   0.0198    0.0079    0.0081

10       1.397   0.0225   0.0200    0.0093    0.0078

11       1.314   0.0231   0.0200    0.0111    0.0090

12      0.0936   0.0281   0.0249    0.0142    0.0157

mean             0.0235   0.0219    0.0099    0.0110

  --------------------------------------------------
(sup *)Symbols as in Appendix.

Both the range of scaling factors and the threshold deviations are derived
from empirical investigation of the San Francisco data. An adjustment for
larger error in measurement is included for the more rare larger fragment
length bands. In applying the ACM to San Francisco bacterial fingerprints,
[Delta] is allowed to increase at a rate of 0.005/kbp above
a value of 7 kbp.

San Francisco Bacterial Genetic Fingerprint Comparisons

To evaluate the performance of the ACM, we analyzed (by computer-assisted
visual inspection and by ACM) 125 lanes from bacterial isolates obtained in
the first half of 1996. We evaluated ACM's performance by first determining
whether a 1996 lane matched all bands to lanes in visually defined clusters
(from previous years), matched other 1996 lanes, or did not find any
identical matches at all. The automated matching of the 1996 lanes agreed
nearly perfectly with the visual analysis; the few conflicting results were
due to inconsistencies in the existing data (e.g., two bands of nearly
identical size being edited sometimes as one band and sometimes as two).
Humans often compensate for such inconsistencies, whereas a computational
method would have to have such capabilities explicitly built in.

Using ACM, we analyzed all 890,445 pairwise comparisons of isolates from
1,335 TB cases in San Francisco from 1991 to mid-1996. The autoclusters
defined from the pairwise comparisons agreed closely with the clusters
defined by computer-assisted visual inspection (Table 2). An example of one
autocluster is shown in Figure 3. Without additional alignment, noise in
fragment length measurements makes it difficult to determine if these
putative clusters include individual patterns which, although similar, are
distinct (outliers) or if the cluster contains identifiable subgroups of
patterns (subclusters).

Table 2. Preliminary autoclustering agrees closely with
results obtained by computer-assisted visual inspection
(CAVI)

  -----------------------------------------------------
                        Clustered  Not clustered
                        by CAVI      by CAVI      Total
  -----------------------------------------------------

Autoclustered              540           27       567
Not autoclustered           49          719       768
Total                      589          746     1,335
-------------------------------------------------------

We then further analyzed autoclusters and identified more precisely
nonidentical genotypes. Refinement of clusters begins with defining a
consensus pattern for the cluster (consisting of the collection of mean
fragment lengths for each band). Then the fragment lengths for each
putative member of the autocluster are aligned to the consensus pattern.
After alignment, a pattern can be easily identified as an outlier (Figure
3).

If the analysis of a refined, aligned cluster shows multiple outliers,
realignment of subsets of lanes can be used to reveal subclusters of
identical patterns. In Figure 4 the distributions of fragment lengths
before (a,b) and after (c,d) an initial alignment are presented for an
autocluster of 84 2-banders from San Francisco. Given that the aligned
bands are clearly split into two distributions, we split the autocluster
into two subclusters. A set of 26 fingerprints (group 1) is aligned to its
assembled mean-value lane (Figure 4 e,f), as is a set of 58 fingerprints
(group 2, Figure 4 g,h). The contrast between the original fragment length
data and the two well-aligned groups of fingerprints shows that the higher
fragment length band is shifted between the two groups and no clear
outliers exist after alignment. Figure 5 shows that alignment greatly
improves a difficult-to-resolve clustering issue among four lanes.

Preliminary investigation with a polymorphic GC-rich sequence (PGRS)
fingerprinting method shows that the two subclusters exhibit distinct
fingerprints, which further validates the increased specificity in IS6110
fingerprints. Of 81 PGRS genotyped autoclustered 2-banders, 63 fall into
eight visually defined clusters, the remaining being unique PGRS patterns
among the members of the IS6110 2-banded autocluster. Each cluster consists
of isolates that all fall into one or the other IS6110-refined subcluster.

Conclusions

We have developed and validated a systematic approach to pairwise
comparison and clustering of identical patterns in a large data set of DNA
fragment-based genotypes. Incorporating a control pattern in each
experiment allows the nature and magnitude of error in DNA fragment length
measurements to be determined. An analysis of measurement error provides
parameter values to use with algorithms that accommodate these errors.
Relative scaling of entire lanes, an important characteristic of the error
generated in quantitating fragment lengths from RFLP patterns used to type
M. tuberculosis isolates, arises in part from aligning two images of a gel,
one for internal lane size standards and one for data fragments; the image
of internal standards and the image of data fragments are registered by
three marks. Error in registration of the two images occurs, leading to the
scaling effect in the fragment lengths reported. We strongly suggest that
software for analyzing internal lane size standards and data fragments from
separate images permit (and encourage) use of more than three registration
marks. While internal standards compensate for idiosyncrasies in
lane-specific fragment mobility, the limitations imposed by poor
registration methods can result in increased scaling error. We have
demonstrated that allowing for scaling, as in ACM, greatly assists in
automating matching; incorporating alignment of pairs of lanes into the
method has provided fully automated lane matching that agrees closely with
results of the well-established method of computer-assisted visual
comparison. We successfully address the nontransitivity of pairwise
identity and once again use scaling of lanes to ensure the reliability of
automated clustering of identical patterns.

Alignment of DNA fragment patterns removes noise in clusters of
fingerprints, showing further specificity in genotyping. This is analogous
to the experimental approach of rerunning similar patterns on a single gel
to reduce intergel noise. Mathematical transformation of the fragment
length data yields similar information for far less cost in labor and
materials. Further automated clustering within sets of aligned patterns
could exploit the fact that we assume putative homology of fragments. As
the aligned patterns have the same number of fragments, the residual error
presents a multidimensional clustering problem; each pattern may deviate
from the mean-value pattern in any of the fragment lengths. This clustering
may prove more straightforward than the more general problem of clustering
among patterns differing in numbers of fragments.

		[fig 4]
Figure 4. Histograms of the fragment lengths for 84 two-banded
patterns connected by identity (autoclustered with in-house 
software) exhibit enough spread in values to make detecting 
outliers and band shifts difficult (a,b). Aligning the 84 lanes 
to the mean-value lane for this collection reveals that the 
lanes do not align well, but instead shows bimodal distributions 
for the fragment lengths (c,d). Dividing the 84 fingerprints
into two sets and separating the distinct distributions detected 
when aligning all 84 fingerprints show that 26 fingerprints align 
well to their mean-value lane (e,f), and the remaining 58 also 
align well to their respective mean value lane (g,h). The smaller
fragment length fragment does not appear shifted between the two 
sets of 2-banders(comparing e to g), but the larger fragment is 
clearly shifted (comparing f to h). 


Numerous commercial packages of computer software are available to compare 
and match DNA fragment patterns. The availability of these systems fosters
unquestioning application of turnkey pattern matching and clustering methods,
which are not always fully documented or validated (for fragment-based genotypes)
in the scientific literature. While possibly acceptable for small studies 
when visually validated, this approach to data analysis is risky for large 
studies. The methods for matching DNA fragment patterns presented in this
paper should be an adjunct to software packages that quantitate fragments. 
We provide a systematic approach to analysis of fragment length estimates, 
appropriate whether the numerical data are generated by hand using a ruler 
and arithmetic or are output by a multithousand dollar gel analyzer. This
approach can thus be used by molecular epidemiologists working with both
large and small budgets around the world. We anticipate that our methods 
can be incorporated into existing commercial software packages with broad
distribution and encourage similar documentation in peer-reviewed journals
of other methods provided in software packages.

In addition, our focus on the use of fragment length data, as opposed to the
comparison of actual images, will fostercomparison of data generated in 
different laboratories that use different proprietary software. We are 
working on a World-Wide Webbased system to facilitate IS6110 genotype 
data sharing.

The availability of a precise and validated method to count the number of
matching fragments in a pairwise fashion among very large numbers of
patterns now permits an assessment of the importance and usefulness of
approaches that exploit fingerprint similarity. In conjunction with the
growing understanding of the underlying biology of IS6110 instability and
the relevant statistical issues, this may greatly expand the scope of TB
molecular epidemiology.

A general question arises when comparing molecular fingerprints: how many
bands need to match to indicate a close biologic relationship? Aside from
the issues of defining biologic "closeness"too often ignored in
epidemiologic studiestechnical issues are relevant to ACM. The usefulness
of the matched band information output by ACM depends on at least several
factors: the underlying band size distribution from which fingerprint bands
are sampled, the independence of sampled bands, measurement error (both
scaling and independent band error), the length of the gel, and the number
of fragments. At one extreme, where error is large and few bands are
observed, even in the case of a perfect match, statistical analysis may
fail to reject coincidental band matches. By using computer simulation and
sets of assumptions regarding band size distributions, one may learn about
the role of coincidental band matching. We are actively researching these
issues for IS6110 fingerprint comparisons. Furthermore, we intend to
provide a general computational framework in which one may assess error in
laboratory measurement, the appropriateness of ACM for analyzing data (and
appropriate parameter values to use with the method), and the role of
coincidence in band matching. Information regarding computer programs for
various tasks, including ACM matching itself, will be made available on the
Internet at http://molepi.stanford.edu/hugh/acm.

		fig 5]
Figure 5. Prior to alignment of two sets of 2-banders, 
lanes are difficult to cluster(lanes a-d are from the 
distributions in Figures 4a and 4b). Subsequent to 
alignment, lanes are much easier to cluster (lanes a' 
and b' are specific examples from the distributions 
in Figures 4e and 4f; lanes c' and d' likewise correspond 
to Figures 4g and 4h). Fragment lengths are given in 
kilobasepairs (kb).

A study of M. tuberculosis isolates from northern Tanzania demonstrates the 
utility of partial matching (3). The study brings into focus difficulties 
inherent in employing one-parameter tolerance for DNA fragment-based genotype
matching, a technical issue effectively addressed by ACM. Gillespie et al. 
(7) also call attention to the poor specificity of low copy number IS6110-
based fingerprints, exacerbated by the use of the Dice coefficient. We are 
pursuing alternative similarity measures that use the numbers of matching
fragments identified by ACM and are tailored to the needs of epidemiologic
investigations. Dendogram clustering methods often provided in software 
targeted at DNA-fragment genotype management and analysis could in some 
instances fail to reconstruct the correct relationships among infectious 
organism isolates, even when presented perfect clock-like genetic
distances. This may result in part from the fact that fast-evolving markers
are characterized for isolates sampled over a period; samples are not
contemporaneous. In conjunction with our efforts to define similarity
measures, we are also working to modify phylogenetic inference tools used
in clinical and molecular epidemiologic settings to better handle the data
typically analyzed in molecular fingerprint management and analysis
software.

Appendix

Analysis of Error in Data

To characterize error in fragment length measurement, pairs of 12 band
7H37Rv lanes are compared; the difference between fragment lengths of each
band is calculated as follows. Let w(sub i,b) be the measured fragment length of
band b of lane i. In general, let B be the number of bands. To compare
lanes i and j, we can calculate the absolute difference between the
measured lengths,

d(sub i,j)(b) = |w(sub i,b) - w(sub j,b)|.

Let lower case DELTA(sub i,j) be an indicator that equals 1 when lanes i and j are
from the same electrophoresis gel and 0 otherwise. Let n be the total number of 
replicate lanes. The mean absolute difference for a fragment over all pairwise
comparisons of lanes from different gels is found by,

(eq) d(b)(overscore)=1(over)t(sup i=n-1)EPSILON(sub i=1)(sup j=n)EPSILON(sub j>i)
d(sub i,j)times (1-lower case delta(sup i,j)(sup 3)

where (eq) t= EPSILON(sup i=n-1)(sub i=t) EPSILON(sup i=n-1)
(sub i=t)(1-lower case delta(sup i,j) is the number of pairs of lanes from
different gels.

Similarly, we calculate the proportional difference between measurements
for a fragment,
(eq)r(sub i,j)(b)=|w(sub i,b)- w(sub j,b)| over (w(sub i,b)+ 
w(sub j,b))/2(sup 2) 
and its mean for comparisons between lanes from different gels,

(eq) r(b)(overscore)=1(over)t(sup i=n-1)EPSILON(sub i=1)(sup j=n)EPSILON(sub j>i)
r(sub i,j)times (1-lower case delta(sup i,j)(sub -)

Calculating fragment measurement errors for the 
(eq) u= EPSILON(sup i=n-1)(sub i=1) EPSILON(sup j=n-1)(sub j>i)times
lower case delta(sup i,j) pairwise comparisons of lanes from the same gel 
are also performed.

Align-and-Count Matching Algorithm

The Align-and-Count Method (ACM) for counting the matching bands between
two lanes is defined as follows. Consider two lanes, lane A with m bands,

w(sub A,x) , x = 1,...,m,

and lane B with n bands,

wB,y , y = 1,...,n.

We count the mutually closest measured fragment lengths within a
proportional deviation factor, [delta] , over a range of alignments (Figure 1).
Alignments are searched by scaling the fragment lengths. Multiplying the
fragment lengths in a lane by a scaling factor reflects the phenomenon that
error in fragment lengths is proportional to fragment length and is
positively correlated for fragments in a lane.

Define

(eq)Match [w(sub A,x),w(sub B,y)]={1 over 0 if |w (sub B,y)- w (sub A,x)|</=[delta] and
					      x and y  are mutually closest bands otherwise
By mutually closest we mean that there is no band in lane B closer to band
x of lane A than band y, and there is no band in lane A closer to band y of
lane B than band x.

Lanes are incrementally scaled (I increments), and the maximum number of
matching bands is reported. Specifically, the Jth increment in the search
scales the lanes as follows:

(eq)Omega(sub A,x)(J)= w(sub A,x)*[1+(J-I/2)*S], x=1,...,m,

and

(eq)Omega(sub B,y)(J)= w(sub B,y)*[1+(J-I/2)*S], y=1,...,n.

Here S defines the range of scaling factors (Figure 1). In this way the
lanes slide past each other as J goes from 1 to I, always scaling the bands
proportionally. At one extreme, lane A is scaled (S/2 * 100)% larger while
B is scaled (S/2 * 100)% smaller. At the other extreme the scaling is
reversed.

The number of matching bands reported, k, is the maximum of K(J) over J =
1,...,I:

(eq)K(J)x=m over under x=1 EPSILON  y=n over under y=1 EPSILON Match(Omega(sub A,x)
(J,(Omega(sub B,y)(J).


We have described a method to find the number of matching bands k, when
comparing a lane with m bands and a lane with n bands. This algorithm has
three parameters: S, the range of scaling factors to align the lanes, [delta],
the cutoff proportional difference for mutually closest bands to be
considered matches, and I, the number of increments used to search the
range of scaling. Note that [delta], in general, may be a function of the
fragment lengths of mutually closest bands under inspection.

A Graph-Theoretic Approach to Identical Fingerprints

If all bands in lane j match their aligned counterparts in lane i, the two
lanes are defined as identical. A common feature of ACM and all
band-sharing approaches is that identity may not be transitive. This
situation may arise when accumulated errors exceed tolerance or
(proportionally) small changes in fragment size occur in biologic samples.

To analyze identical fingerprints, we define sets of fingerprints connected
by identity. Let each of T fingerprints exhibiting the same number of bands
be a node in a graph; let an edge joining two nodes indicate that two
fingerprints are identical. By an algorithm attributable to Dijkstra (10),
we determine the number of steps, P, of identity between two fingerprints
when such a pathway exists. The algorithm finds the shortest path between
two nodes. We use a code modified from that presented in Tenenbaum et al.
(10). From this analysis we assemble collections of fingerprints connected
by identity, which serve as tentative clusters of identical fingerprints.
We refer to these tentative clusters as autoclusters. The distributions of
fragment lengths for these collections of lanes are subsequently plotted,
allowing outliers and band-shifts to be identified.

Alignment and Analysis of Residual Error

We aligned two replicate fingerprint patterns by using the following least
squares regression formula. We minimize

(eq)Q(s)=B over b=1 under EPSILON[log(w(sub i,b)]-log[s.w(sub j,b)](sup 2)

with respect to the scaling factor s by which lane j is aligned to lane i.
Log-transformed fragment lengths are used to reflect the fact that
measurement error is proportional to fragment length. The minimization has
the closed form solution

(eq)s=exp[1/B, B over, b=1 under EPSILON log w[(sub i,b)over w(sub j,b))] 

Residual error in band size measurement after lane alignment is evaluated
in a fashion analogous to that used for unaligned lanes. For example, the
proportional difference between band b of lane j aligned to lane i and band
b of lane i is

(eq)r(sub i,j)(b)=|w(sub i,b)- w(sub j,b)| over (w(sub i,b)+ 
w(sub j,b))/2(sup 2)

For alignment of sets of lanes, the mean fragment length is calculated for
each fragment. A mean-value lane is constructed from these mean lengths.
Each lane is then aligned to the mean-value lane. The alignment is plotted
and analyzed for outliers, providing refined clusters of identical
fingerprints.

Acknowledgments

We thank Philip Hopewell, for his interest in and support for continued
development of M. tuberculosis genetic fingerprinting in TB research; Mike
Cummings, for his participation in numerous meetings regarding analysis of
the fingerprint data, his input and discussions in the evolution of ideas
leading to the work presented herein, and his comments on manuscripts;
Melvin Javonillo, for help in accessing fingerprint data; Daniel Ruderman,
for help with the issue of logarithmic alignment; and Marcel Behr and Sam
Warren, for comments on manuscripts.

This research was supported by NIH grants AI35969, AI34238.

Hugh Salamon is a postdoctoral researcher at Stanford University Medical
Center; he is focusing on molecular epidemiology and Mycobacterium
tuberculosis genomics. In his work as computer programmer in the Department
of Medicine at the University of California, San Francisco, he writes
custom software for many analyses and maintains a web site
(http://molepi.stanford.edu) at Stanford University.

Address for correspondence: Hugh Salamon, Division of Infectious Diseases
and Geographic Medicine, Stanford University Medical Center, Rm S 156,
Stanford, CA 94305-5107, USA; fax: 650-498-7011; e-mail:
hugh@molepi.stanford.edu.

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     al. Transmission of tuberculosis among the urban homeless. JAMA
     1996;275:305-7.
  7. Gillespie SH, Kennedy N, Ngowi FI, Fomukong NG, al-Maamary S, Dale JW.
     Restriction fragment length polymorphism analysis of Mycobacterium
     tuberculosis isolated from patients with pulmonary tuberculosis in
     northern Tanzania. Trans R Soc Trop Med Hyg 1995;89:335-8.
  8. van Embden JD, Cave MD, Crawford JT, Dale JW, Eisenach KD, Gicquel B,
     et al. Strain identification of Mycobacterium tuberculosis by DNA
     fingerprinting: recommendations for a standardized methodology. J Clin
     Microbiol 1993;31:406-9.
  9. Woelffer GB, Bradford WZ, Paz A, Small PM. A computer-assisted
     molecular epidemiologic approach to confronting the reemergence of
     tuberculosis. Am J Med Sci 1996;311:17-22.
 10. Tenenbaum AM, Langsam Y, Augenstein MJ. Data structures using C.
     Englewood Cliffs (NJ): Prentice-Hall; 1990. p. 514-6.


Emerging Infectious Diseases
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Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/salamon.htm

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[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Perspectives

Legal Issues Associated with Antimicrobial Drug Resistance

David P. Fidler
Indiana University School of Law, Bloomington, Indiana, USA

  --------------------------------------------------
      An effective public health strategy against the development of
      antimicrobial drug resistance needs to be informed by legal as
      well as scientific analysis. This article describes some legal
      issues arising from current efforts against antimicrobial
      resistance and underscores the interdependence between law and
      public health in these efforts.

The development of antimicrobial resistance in many pathogenic microbes
poses one of the most serious problems in the control of infectious
diseases (1-3). Antimicrobial resistance results from the ability of
microbes to adapt to anthropogenic pressures; therefore, it is not a
passing trend but likely a permanent feature in the fight against
infectious diseases (4). This article highlights the legal issues involved
in addressing the problem of antimicrobial resistance.

Law and Global Public Health

Globalization interferes with infectious disease control at the national
level (1,5-7). While microbes move freely around the world, unhindered by
borders, human responses to infectious diseases are conditioned by
jurisdictional boundaries. Therefore, public health responses to infectious
diseases must constantly navigate the mazes created by international and
national law. Although national law now dominates legal approaches to
infectious diseases, the global nature of the emerging infectious disease
problem points towards a larger future role for international law.

Especially in federal systems, countries often divide authority for public
health among various levels of government. In the United States, for
example, states have primary power for public health because the
Constitution did not grant the federal government explicit public health
powers (8). While the federal government has authority to act in the public
health context (9), its statutes and regulations derive from other federal
powers. Most U.S. public health law is at the state level. The emerging
infectious disease threat points to a larger role not only for
international but also for federal law. Federal agencies create networks
that allow the state and national governments to cooperate on issues such
as antimicrobial resistance, but public health law remains primarily a
state domain; therefore, state laws on public health may need to be
reevaluated in the context of emerging infectious disease control (10).

Public Health Strategies to Address Antimicrobial Resistance

The dominant public health strategy against antimicrobial resistance
contains improved surveillance of resistant pathogens, as well as rational
use and increased research and development of new antimicrobial drugs (1).
These elements fit within the larger strategy to address emerging
infectious diseases, which stresses surveillance, applied research,
prevention and control, and infrastructure development (1).

Surveillance

Surveillance is critical to the control and prevention of infectious
diseases (1). Since no national or global surveillance system exists for
monitoring antibiotic resistance, improved surveillance is a top priority
(11). Surveillance is also useful in addressing the threat of biological
weapons and protecting the community from highly contagious infectious
diseases. Law is critical to surveillance not only because reporting
information must be a legal duty, but also because law is needed to deal
with the tensions sometimes arising between individual privacy rights and
the community's interest in being protected from infectious diseases.

  Reporting

International and domestic surveillance systems are based on the legal duty
to report certain public health information. The International Health
Regulations mandate, for example, that member states of the World Health
Organization (WHO) report outbreaks of plague, cholera, and yellow fever to
WHO (12). Similarly, state public health departments in the United States
legally mandate the reporting of cases of certain diseases from health-care
providers to public health agencies (13).

Existing laws at the international and national level require reporting of
a limited number of diseases, do not require systematic reporting of
antimicrobial resistance, and receive inadequate compliance or
noncompliance. WHO, however, has proposed including surveillance of
antimicrobial resistance in the revisions of the International Health
Regulations (14-16) and requiring drug resistance reporting.

In the United States, surveillance of antimicrobial resistance has been
described as woeful (17). In 1992 less than $55,000 was spent on antibiotic
resistance surveillance for human pathogens in the United States at the
local, state, and federal levels combined (11). While the need for improved
surveillance of antimicrobial resistance is recognized (1), state reporting
laws have not changed much. For example, although the Council of State and
Territorial Epidemiologists recommended in 1995 that states make
drug-resistant Streptococcus pneumoniae reportable (18), not all states
have (D. Bell, pers. comm.).

Several factors explain the lag of state responses to federal
recommendations on resistance surveillance. First, state laws on infectious
diseases often do not keep pace with new scientific findings (10). In
addition, many states do not have within their legal systems the
flexibility needed to respond to new threatsas was the case, for example,
when the Council of State and Territorial Epidemiologists recommended that
Escherichia coli O157:H7 be made reportable (13). Second, adding new
surveillance responsibilities is difficult because of inadequate resources
(13). These two factors indicate inadequate emphasis on public health
within many state governments. The structure of public health law in the
United States can, therefore, impede antimicrobial resistance strategies.
As at the international level, if reporting is not required, surveillance
for antimicrobial resistance is compromised.

Changes in diagnostic testing in the United States also raise public health
and legal concerns about surveillance. Privatization of laboratory services
by state legislatures may compromise national surveillance of emerging
infectious diseases and investigation of outbreaks because many
surveillance systems rely on data from state laboratories (13). In
addition, because of economic pressures, hospitals increasingly rely on
testing done in areas outside their jurisdiction; therefore, accurate
information on infectious diseases in their areas may not be available.
Diagnostic testing done by private laboratories may require closer federal
regulation to protect the quality of needed surveillance data (D. Bell,
pers. comm.).

Creation of a legal duty does not ensure the success of a policy. WHO
member states have routinely ignored required outbreak reporting of plague,
cholera, and yellow fever (4,19,20). Reporting of diseases within U.S.
states is sometimes poor, haphazard, and unhelpful (21). Fulfillment of
legal duties often hinges on sufficient resources. In many developing
countries public health systems may be inadequate (22). Thus, financial and
technical leadership is needed from national governments towards local
authorities and from international organizations towards developing
countries. In addition to legal requirements, national regulatory barriers
may also hinder global surveillance. Such surveillance will require many
countries to import and use equipment, software, and reagents to detect and
report on antimicrobial resistance. Eliminating barriers could improve
prospects for global surveillance. A precedent can be found in the proposed
Convention on the Provision of Telecommunication Resources for Disaster
Mitigation and Relief Operations, which obligates the parties, where
possible, to lower or remove regulatory barriers for using
telecommunication resources during disasters (23).

  Privacy Issues

Surveillance systems sometimes have to balance the privacy of the patient
with the need for useful scientific and medical information and the need of
the community to be protected from the spread of infectious diseases (9).
As the HIV/AIDS epidemic has demonstrated, the privacy issue is
particularly acute in sexually transmitted diseases. Because in some
sexually transmitted diseases, e.g., gonorrhea, the infectious organisms
are developing resistance to antibiotics, the inherent privacy concerns are
compounded by surveillance-related privacy concerns. Increased surveillance
for antimicrobial resistance may heighten privacy concerns with respect to
other diseases, such as multidrug-resistant tuberculosis (MDRTB).

Different systems of law deal with privacy concerns differently, which
could create legal problems for global surveillance of antimicrobial
resistance. The differences between U.S. and European Union policies
illustrate this difficulty. In the United States, the privacy of
health-related information is of concern. Health information gathered by
public health agencies is regulated largely by the Constitution and by
state statutes. While the Constitution "requires reasonable levels of
privacy and security when the government collects personally identifiable
data through . . . disease reporting" (9), a recent survey of U.S. state
legislation on public health information privacy concluded that many
states' safeguards of public health privacy are insufficient (24). Laws
protecting private sector records are even weaker (9). Some states regulate
private dissemination of health-related information through
physician-licensing systems, common-law tort rules on invasion of privacy,
or statutes (25). Nevertheless, such legal regulations may not adequately
protect privacy, as health-related information is increasingly manipulated
electronically by health-care providers, health maintenance organizations,
and insurance companies (9). Lack of laws adequately protecting the privacy
of health-care information has led to calls for federal regulation
(9,25,26), and some legislative proposals have been introduced in Congress
(27).

The European Union has a strict law forbidding the processing of health
information data without the written permission of the patient (28). This
law places other strict conditions on the use of health data directly
affecting European Union surveillance efforts. The contrast between
American and European legal protection for health-related information may
affect global surveillance efforts because European law permits states to
withhold personal data from those who cannot adequately protect these data
(26).

A particularly important development is the growth of privately owned
infectious disease information. Private initiatives are building global
information-sharing networks on various disease issues through the Internet
and other information technologies (4,29,30); private companies are
starting to monitor and test bacterial resistance globally (31); and some
for-profit companies gather and sell epidemiologically useful information.
These private efforts raise legal questions: privacy issues arise with the
dissemination of epidemiologic data by private companies; this
dissemination is treated differently in different countries; jurisdictional
problems arise regarding legal regulation of information sharing in
cyberspace (32) (the quality of health information on the Internet, for
example, is being questioned [33-35]); and legal (and ethical) concerns
arise with the practice of selling epidemiologic data, especially with data
gathering, and whether governments can compel disclosure of privately
gathered information in the interest of public health.

  Biological Weapons

While most of the pathogenic agents considered to be the most likely
candidates for use as biological weapons do not exhibit resistance (36),
the potential use of resistant pathogens as weapons is of concern because
resistance blunts one of the few lines of defense against a biological
weapons attack. For the U.S. Department of Defense, antibiotic resistance
is one of the criteria for characterizing suspicious outbreaks of
infectious disease that might point to a possible biological weapons event
(37).

The main source of international law on biological weapons, the 1972
Biological Weapons Convention, prohibits the development, production, and
stockpiling of biological and toxin weapons (38). Negotiations are under
way to strengthen the Biological Weapons Convention through a protocol that
both establishes compliance procedures and commits states to improving
domestic and global surveillance of infectious diseases (37). Calls for
reform are also being made in domestic legal systems (37). The
comprehensive statutory and regulatory system in the United States that
governs the acquisition, use, and transfer of biological agents that pose a
threat to public health (39) might serve as a model for legislation in
other countries.

  Personal Control Measures

The authority of public health officials to detain or isolate persons
infected with highly contagious and resistant pathogens in order to protect
the community is another important legal issue. MDRTB, for example, is a
threat to public health because it is highly infectious (10). To deal with
its TB epidemic, New York City has issued dozens of orders to detain MDRTB
patients for isolation and compulsory treatment (40). Controversy exists
about the proper scope of detaining patients for infectious disease control
purposes (13). While U.S. courts have upheld detaining infected patients to
protect public health (41), governments today face heightened judicial
scrutiny of personal control measures in the public health context (10,42).
In one case in New Jersey, the court held that public health authorities,
in applying a 1912 TB control statute, had to comply with contemporary
notions of due process and the Americans with Disabilities Act in order to
detain and isolate a patient with MDRTB (43). At a time when antimicrobial
resistance may have created a greater need for personal control measures
for public health (e.g., with MDRTB), the status of U.S. law on the scope
and nature of the government's power to undertake such measures seems
unsettled (13). U.S. public health law may need to be modified to allow
public health officials to control demonstrated threats of risk through
flexible policy options that minimize infringements on individual rights
(10).

The proper scope of personal control measures may appear pertinent only to
industrialized countries, given that only those countries can identify
resistant pathogens and their human hosts. In addition, the notion of
personal control measures against drug-resistant malaria patients in Africa
seems far-fetched, given the scale of the problem. Nevertheless, the
importance of international human rights law to effective public health
policiesas seen in the context of HIV/AIDS (44)demonstrates that
complacency towards individual rights in any public health policy is
dangerous legally and medically.

Rational Use of Antimicrobial Drugs

Antimicrobial drug misuse in both industrialized and developing countries
is a problem in connection not only with human treatment, but also with
food production (11,45,46). More rational use of antimicrobial drugs in
every countryfor disease treatment and food productionmust be at the core
of the response to antimicrobial resistance.

Education has been suggested as the primary tactic for improving
antimicrobial drug use (1,16). WHO has recommended "a code of practice for
prudent use of antimicrobials in food animal production" (47).

However, the global scope of antimicrobial resistance indicates that an
integrated strategy operating at both the national and international legal
levels is needed. Such an integrated strategy faces political and legal
challenges. Building effective national legal regimes regarding prudent
antimicrobial use in many developing countries is unrealistic absent
financial, political, and legal support from the international community
(46). International legal harmonization of principles for prudent
antimicrobial drug use will have to include monitoring and enforcement, as
well as financial, technical, and legal assistance by industrialized
countries to developing countries.

The international legal strategy needed will be difficult to create. WHO's
limited powers to adopt regulations (48) do not seem to extend to creating
regulations regarding the use of drugs. WHO has not, for example, proposed
revising the International Health Regulations to rationalize the use of
antimicrobial drugs. WHO has authority to adopt a convention on the use of
antimicrobial drugs (49), but it has not done so (4). As illustrated by its
proposal for a code of practice, rather than regulations, for antimicrobial
use in food animal production, WHO historically has preferred not to use
international legal powers to advance global health. Lessons from
international environmental efforts suggest that international law must
play a major role in setting international standards for implementation
domestically and creating the political, technical, and financial
conditions necessary to integrate international and national law (19).

The misuse of antibiotics in food production also raises concerns under
international trade law. If antimicrobial drugs routinely used by food
producers lose their effectiveness against animal-borne or plant-borne
diseases, exports of contaminated food may be restricted by countries
applying sanitary and phytosanitary measures (SPS measures) under the World
Trade Organization (WTO) and other international trade agreements to keep
such food out of their territories as threats to human, animal, or plant
health (50). In addition, exports might be disrupted by countries applying
SPS measures under international trade agreements against products
suspected of containing harmful residues of antimicrobial drugs. The Codex
Alimentarius Commission already sets standards for residues from veterinary
drugs in food through its Committee on Residues of Veterinary Drugs in
Foods, and WHO has recommended that this Codex committee discuss
antimicrobial resistance (47). Codex standards have become important in
international trade law through the WTO Agreement on the Application of
Sanitary and Phytosanitary Measures (SPS Agreement) (50), which uses these
standards as a basis for international harmonization of SPS measures. The
importance of Codex food safety standards to international trade law was
seen in the Beef Hormones Case, in which the WTO held that the European
Union violated the SPS Agreement for not providing scientific justification
for a beef hormone regulation stricter than the relevant Codex standards
(51-53).

The inability of many governments to regulate antimicrobial drug use and
the consequent misuse of these drugs raise the possibility that countries
might use trade restrictions on food produced with improper use of
antimicrobial drugs. In this situation, the food products might have
residue levels below those established as maximums by Codex, but the trade
restriction is intended to pressure the exporting country to improve
regulation of antimicrobial drug use and the process by which the product
is made. In the context of environmental protection, trade restrictions
seeking to change a production process in another country, rather than to
protect against health dangers from a particular product, have been ruled
incompatible with international trade law (54-56). Although "relevant
processes and production methods" (SPS Agreement, art. 5.2) form part of a
risk assessment under the SPS Agreement, the risk must be a specific health
risk from the product (e.g., highly inconsistent residue levels created by
inadequate antimicrobial regulation) rather than fear of the health
consequences of antimicrobial misuse. To avoid losing trade restrictions as
part of a general strategy to combat antimicrobial misuse, legitimate trade
restrictions against countries that systematically neglect recognized
principles and practices for antimicrobial use might be considered; such a
move would elevate the status of Codex's Code of Practice for Control of
the Use of Veterinary Drugs and Guidelines for the Establishment of a
Regulatory Programme for Control of Veterinary Drug Residues in Foods, as
the SPS Agreement has elevated the importance of Codex's Maximum Residue
Levels for Veterinary Drugs in Foods.

The International Conference on Harmonization, a multilateral effort
between the United States, Japan, and the European Union to harmonize
pharmaceutical regulatory systems, is another forum for discussing
antimicrobial drug resistance. If national regulatory systems begin to
grapple with the overuse of antimicrobial drugs, the International
Conference on Harmonization might provide a forum to discuss a harmonized
approach to more rational drug use in the United States, Japan, and the
European Union. As structured, the Conference does not include other
countries where overuse of antimicrobial drugs is a serious problem.

National laws for improving antimicrobial drug use face difficulties in
industrialized countries; realistic national strategies confront legal and
political hurdles. In the United States, state legislatures probably have
the power to regulate how physicians prescribe antimicrobial drugs, but any
attempt to legislate more rational use of drugs might evoke negative
reactions from physicians and their medical associations, who might oppose
the government's efforts to interfere with their professional judgment
(57). If formal legislative regulation would not prove feasible, an
alternative would be self-regulation by the medical and veterinary
professions through practice guidelines, for example (57). A peer review
process to monitor antimicrobial drug use has also been recommended (1).
Managed care organizations may be included in the effort to control misuse
of antimicrobial drugs, given their power and economic incentives to curb
such misuse by physicians.

Formal regulation of antimicrobial use may be needed, which would involve
monitoring and enforcement. In the United States, Congress could regulate
use of antimicrobial drugs by monitoring interstate commerce in these
products. Congress probably does not have the authority to regulate
antimicrobial prescription practices directly; such authority rests with
the states. The U.S. Food and Drug Administration (FDA) has authority to
restrict the postapproval marketing of new drugs designed for treating
serious or life-threatening illnesses and has indicated that these
regulations can be used specifically in cases of new antimicrobial drugs
(17,58). Restricted distribution is, however, a disincentive to the
development of new drugs, and the regulations do not address misuse of
existing products. Another regulatory strategy available to the FDA for
dealing with resistance in existing products is to modify labeling
requirements; modifying labels is, however, somewhat cumbersome (17).
Effective regulatory changes in the United States will be jeopardized by
the lack of similar changes in other countries.

Perhaps the most powerful U.S. federal strategy would be to make
implementation of state policies to curb the misuse of antimicrobial drugs
mandatory before states receive federal funds earmarked for public health.
Although states might argue that this would encroach on their traditional
public health rights and powers, such a federal enactment would be
constitutional. In addition, using federal funds to improve antimicrobial
use policies nationwide fits with the need for federal political leadership
as well as financial and technical assistance. State governments might,
therefore, welcome federal money conditioned on implementing policies the
money supports. Pharmaceutical companies, worried about the federalization
of policies affecting their economic relationships with local and state
health-care providers, might oppose these mandates. In addition, federal
leadership in this way would also run counter to trends in other areas,
such as welfare reform, which are moving responsibilities from the federal
to the state level.

In countries where governments subsidize the purchase of antimicrobial
drugs, legislative or regulatory changes in these subsidies could lead to a
decline in the use of the drugs. When Iceland ended government subsidies of
antimicrobial drugs, their use in Iceland declined, while sales kept
increasing in other Nordic countries (59). Although Iceland's legislative
change was made for political, not medical reasons (59), it illustrates the
possible impact of legislative and regulatory controls on the use of
antimicrobial drugs.

Improving physicians' awareness of antimicrobial resistance does not
address, however, misuse of these drugs by patients (60). MDRTB has
developed largely because of improper adherence by patients to TB therapy
(10); directly observed therapy was initiated as a result. Because patient
misuse of antimicrobial drugs also contributes to the public health crisis,
proper completion of antimicrobial therapy must also be addressed.

New Antimicrobial Drug Research and Development (R&D)

As the antimicrobial arsenal shrinks, new drug R&D becomes critical (1).
Legislation at the domestic level would ensure adequate funding for public
sector involvement. For diseases that pose an especially large or complex
problem (e.g., malaria), international law can play a role by structuring
international cooperation through such institutions as WHO or the World
Bank. The recently proposed Multilateral Initiative on Malaria, an
international multiagency malaria control program, involves, for example,
plans to coordinate R&D on antimalarial products supported by WHO and the
World Bank (61-63).

While public involvement and funds are important, the real engine of
pharmaceutical development is the private sector. However, pharmaceutical
companies have only recently brought new antimicrobial drugs forward for
regulatory approval. Critical disincentives continue to constrain private
R&D investment in new drugs: intellectual property protection, regulatory
approval procedures, and perceived antitrust law limitations on
collaborative R&D.

  Intellectual Property Protection

Pharmaceutical companies fear that their R&D efforts can be undermined by
loss of intellectual property rights. Lack of secure patents deters
pharmaceutical companies from some R&D activities on new drugs. While the
WTO Agreement on Trade-Related Aspects of Intellectual Property Rights
offers pharmaceutical companies better international legal rules on patent
protection (64), the loss of patented agents remains a concern. Many WTO
member states, especially in developing countries, have to upgrade their
national laws to fulfill the agreement obligations, a process that could
take years. International law on intellectual property protection is, thus,
a critical piece of the overall strategy against antimicrobial resistance.

Other patent issues are relevant to the problem of antimicrobial
resistance. Pharmaceutical companies that developed antibiotics years ago
but never commercially exploited them might pursue more antimicrobial R&D
if their earlier antibiotics (now without patent protection) were given
extra legal protection, either under patent law or a legal regime like the
Orphan Drug Act (17). The efficacy of such legal strategies depends, of
course, on the number of promising antibiotics potentially in the R&D
pipeline.

  Regulatory Approval Procedures

Another legal deterrent to antimicrobial development is the varied,
complex, and costly regulatory approval procedures pharmaceutical companies
face in the United States and other countries. Unilateral efforts are being
made in the United States and the European Union to streamline drug
approval regimes, and the International Conference on Harmonization
represents an international effort at harmonization.

Another approach involves creating "expedited approval of new
antimicrobials" (1). FDA, which has already created accelerated approval
rules for drugs that treat serious or life-threatening illnesses, has
indicated that these rules could be used to review new antibiotic
treatments (17).

  Antitrust Law Limitations on Collaborative R&D

In some circumstances, collaborative R&D efforts by a number of
pharmaceutical companies might be indicated. Pharmaceutical companies have,
however, noted the difficulties that antitrust laws create for
collaborative R&D efforts. For example, in response to calls for
collaborative R&D on antimalarial drugs, pharmaceutical companies are
concerned about sharing intellectual property and about laws against
cartels (65). Both U.S. antitrust law and European Union competition law,
however, permit collaboration in certain areas (66,67). The Inter-Company
Collaboration for AIDS Drug Development and the developing Multilateral
Initiative on Malaria set precedents and might be replicated in the area of
antimicrobial resistance.

Conclusion

Strategies to address antimicrobial resistance as a public health and legal
challenge must consider three levels of interdependence: among the
antimicrobial drug surveillance, use, and R&D components of the public
health strategy; among the levels of lawnational and international; and
between the public health and legal aspects of dealing with antimicrobial
resistance.

Each element of the public health strategy against antimicrobial resistance
affects and depends on the other elements. More rational use of
antimicrobial drugs and increased R&D depend on accurate surveillance.
Limitations on the use of new drugs negatively affect the incentives
pharmaceutical companies have to develop new drugs. Continued misuse of
antimicrobial drugs places more pressure on both surveillance and new R&D.
The effectiveness of new drugs will have to be measured by accurate
surveillance and will continue to be undercut by their misuse. The lack of
new R&D will force continued use of existing products, reducing their
effective life-span. Thus, any public health strategy addressing
antimicrobial resistance must consider all three components.

Because antimicrobial resistance is a global problem, national legal
reforms taken in one or a few countries would suffer if other countries did
not take similar actions. For example, since drug-resistant pathogens
travel easily in today's world, national legal reforms to rationalize
antimicrobial use in a few countries might be subverted if such misuse is
not curtailed in many other countries. The creation of new international
legal duties would likewise be undermined if such duties were not
translated into national law. Thus, any legal strategy against
antimicrobial resistance must be pursued at both the national and
international levels.

Achieving the public health objectives of an antimicrobial resistance
strategy involves, at each stage, legal considerations and often calls for
legal decisions. National and international law are integral to the public
health mission in every country; the interdependence of public health and
law forms part of the large multidisciplinary challenge created by the
global problem of emerging infectious diseases.

Confronting antimicrobial resistance requires not only a scientific and
public health strategy but also a legal strategy. Including law in the
developing discourse will broaden and strengthen the strategy for combating
antimicrobial resistance.

Acknowledgments

I thank Dr. Polly F. Harrison, Terry P. O'Brien, Dr. David Bell, Fred Cate,
Beth Cate, Roger Dworkin, Ken Dau-Schmidt, Larry Gostin, Mary Ann Torres,
Cynthia Baraban, and members of the Institute of Medicine's Forum on
Emerging Infections for their help and encouragement during the preparation
of this article; I also thank the anonymous reviewers of Emerging
Infectious Diseases for their comments and suggestions.

An earlier draft of this article was originally presented as a working
paper to the Institute of Medicine's Forum on Emerging Infections in July
1997. Research for this article was supported by a research grant from the
Indiana University School of Law-Bloomington.

Address for correspondence: David P. Fidler, Indiana University School of
Law, 211 South Indiana Avenue, Bloomington, IN 47405-1001, USA; fax:
812-855-0555; e-mail: davidfidler@law.indiana.edu.

Mr. Fidler is an associate professor of law at Indiana University School of
Law-Bloomington, where he teaches public and private international law. Mr.
Fidler is at work on a book entitled International Law and Infectious
Diseases, to be published by Oxford University Press.

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Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/fidler.htm

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[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Perspectives

Rickettsial Pathogens and Their Arthropod Vectors

Abdu F. Azad* and Charles B. Beard
*University of Maryland School of Medicine, Baltimore, Maryland, USA; and
Centers for Disease Control and Prevention, Atlanta, Georgia, USA

---------------------------------------------------------------------------
      Rickettsial diseases, important causes of illness and death
      worldwide, exist primarily in endemic and enzootic foci that
      occasionally give rise to sporadic or seasonal outbreaks.
      Rickettsial pathogens are highly specialized for obligate
      intracellular survival in both the vertebrate host and the
      invertebrate vector. While studies often focus primarily on the
      vertebrate host, the arthropod vector is often more important
      in the natural maintenance of the pathogen. Consequently,
      coevolution of rickettsiae with arthropods is responsible for
      many features of the host-pathogen relationship that are unique
      among arthropod-borne diseases, including efficient pathogen
      replication, long-term maintenance of infection, and
      transstadial and transovarial transmission. This article
      examines the common features of the host-pathogen relationship
      and of the arthropod vectors of the typhus and spotted fever
      group rickettsiae.

Rickettsial diseases, widely distributed throughout the world in endemic
foci with sporadic and often seasonal outbreaks, from time to time have
reemerged in epidemic form in human populations. Throughout history,
epidemics of louse-borne typhus have caused more deaths than all the wars
combined (1). The ongoing outbreak of this disease in refugee camps in
Burundi involving more than 30,000 human cases is a reminder that
rickettsial diseases can reemerge in epidemic form as a result of the
catastrophic breakdown of social conditions (2). In addition to explosive
epidemics, sporadic but limited outbreaks of louse-borne typhus and other
rickettsial diseases have been reported throughout the world. In the United
States, a drastic increase of murine typhus in the 1940s, Rocky Mountain
spotted fever (RMSF) in the late 1970s, and the human ehrlichioses in the
1990s attests to the potential emergence of these infections in populations
at risk (3).

The rickettsiae's obligate intracellular existence in both mammalian and
arthropod hosts serves as an excellent model for the study of complex
host-parasite interactions. Rickettsial associations with obligate
blood-sucking arthropods represent the highly adapted end-product of eons
of biologic evolution. The ecologic separation and reduced selective
pressure due to these associations may explain rickettsial genetic
conservation. Their intimate relationships with vector hosts (Table 1) are
characterized by efficient multiplication, long-term maintenance,
transstadial and transovarial transmission, and extensive geographic and
ecologic distribution. Although rickettsiae have a symbiotic relationship
with their arthropod hosts, in some instances, they act as true
parasites-for example, members of the Wolbachia and Orientia tsutsugamushi
alter reproduction and manipulate cellular processes in their arthropod
hosts (4), and the agent of epidemic typhus, Rickettsia prowazekii, kills
its vector, the human body louse (5).

Although rickettsiae are maintained in nature through arthropod vectors,
they frequently infect vertebrates, which in turn allow new lines of
vectors to acquire infection from the rickettsemic hosts. The involvement
of vertebrates, including humans, in the rickettsial cycle is variable and
in some cases complicated (Figure). With the exception of epidemic typhus,
humans are not essential in the rickettsial cycle. Humans acquire
rickettsial infection from the infected vectors. While tick-borne
rickettsiae are transmitted to humans by tick salivary secretions, flea-
and louse-borne rickettsiae are transmitted to humans through contamination
of broken skin and mucosal surfaces by infected vector feces.

Table 1. Epidemiologic features of the pathogenic rickettsiae(sup a)
--------------------------------------------------------------------------------

Rickettsia                        Natural cycle(sup b)     Geographic
species             Disease       Vectors      Hosts      distribution
--------------------------------------------------------------------------------
Typhus group:
Rickettsia    Epidemic typhus     Human     Humans       Worldwide
prowazekii                        body lice
              Recrudescent typhus None      Humans       Worldwide
                                  Lice,     Flying       Eastern USA
                                  fleas     squirrels
R. typhi      Murine typhus       Fleas     Rodents      Worldwide
                                  Fleas     Opossums     USA
R. felis      Murine typhuslike   Fleas     Opossums     USA
Spotted Fever group:
R. rickettsii Rocky Mountain      Ticks     Small        North & South
              spotted fever                 mammals,     America
                                            dogs,
                                            rabbits,
                                            birds
R. conorii    Boutonneuse fever   Ticks     Rodents,     Africa,
                                            dogs         Southern
                                                         Europe, India
R. sibirica   North Asia tick     Ticks     Rodents      Eurasia, Asia
              typhus
R. japonica   Japanese spotted    Ticks     Rodents,     Japan
              fever                         dogs
R. australis  Queensland tick     Ticks     Rodents      Australia
              typhus
R. akari      Rickettsialpox      Mites     House mice,  Worldwide
                                            rats
Ehrlichioses group:
Ehrlichia     Human monocytic     Ticks     Humans, deer USA, Europe
chaffeensis   ehrlichiosis
Ehrlichia Sp  Human granulocytic  Ticks     Humans,      USA, Europe
              ehrlichiosis                  deer,
                                            rodents
Others:
Coxiella      Q fever             Ticks     Small        Worldwide
burnetii                                    mammals,
                                            sheep,
                                            goats,
                                            cattle, dogs
Orientia      Scrub typhus        Mites     Rodents      Asia, Indian
tsutsugamushi                                            subcontinent,
                                                         Australia
--------------------------------------------------------------------------------
(sup a)Not listed are R. helvetica, R. honei, and R. slovaca (6).
*sup b)Evidence for arthropod serving as a vector or vertebrate serving as a host is based on the rickettsial isolation from field-collected specimens,
experimental studies, and indirect evidence for rickettsial presence or
exposure to rickettsiae (hemolymph test and serosurveys).

Although rickettsiae have common      
features with their vertebrate and    
invertebrate hosts, they differ       [fig]
considerably in terms of arthropod    Figure. Composite diagram of the
vectors, geographic distribution, and life cycle of Rocky Mountain spotted
virulence (Table 1). In this article, fever, rickettsialpox, and murine
we focus on the members of the typhus typhus. A. Life cycle of Rickettsia
group (TG) and spotted fever group    rickettsii in its tick and mammalian
(SFG) rickettsiae to construct a      hosts (7); B. Rickettsia akari life
conceptual framework of the natural   cycle; and C. Rickettsia typhi life
history of human rickettsioses and    cycle.
evaluate feeding behavior of the
vectors with regard to rickettsial
maintenance and transmission.

Tick-borne Rickettsial Pathogens

The development and extensive use of the hemolymph test (which has been
fundamental to tick and rickettsial surveys), improved isolation methods,
and the application of molecular techniques have helped identify 14 valid,
relatively distinct SFG rickettsiae (Tables 1, 2). Except for mite-borne R.
akari, all SFG rickettsiae are transmitted by ixodid ticks (Tables 1, 2).
In addition to R. rickettsii, the etiologic agent of RMSF, eight other
tick-borne rickettsial species are human pathogens (Table 1; 6). The
remaining SFG rickettsiae are isolated only from ticks and have low or no
pathogenicity to humans or certain laboratory animals (7). However, some of
these rickettsiae could be etiologic agents of as-yet-undiscovered, less
severe rickettsioses.

Distribution of SFG rickettsiae is limited to that of their tick vectors.
In the United States, a high prevalence of SFG species in ticks cannot be
explained without the extensive contributions of transovarial transmission.
The transovarial and transstadial passage of SFG rickettsiae within tick
vectors in nature ensures rickettsial survival without requiring the
complexity inherent in an obligate multihost reservoir system. Although
many genera and species of ixodid ticks are naturally infected with
rickettsiae, Dermacentor andersoni and D. variabilis are the major vectors
of R. rickettsii. SFG infection rates vary considerably by state. For
example, the infection rate for adult D. variabilis collected from
vegetation and mammalian hosts was 2% to 9% in Connecticut, 5% in New York,
6% in Kentucky-Tennessee and Maryland, 8.8% in Arkansas, and 10% in Alabama
(5,8,9). Rickettsia and tick surveys indicate that R. rickettsii is less
prevalent in vector ticks than some other SFG rickettsiae. In most cases,
the SFG-positive ticks, including D. andersoni and D. variabilis, are
infected with nonpathogenic rickettsiae rather than with R. rickettsii
(Table 2). The low prevalence of R. rickettsii in SFG-positive ticks is
intriguing. Interspecific competition among ticks may result in stable
maintenance of SFG rickettsiae through transovarial transmission and may
cause the gradual replacement of R. rickettsii in the tick population. Very
little is known about the process of interspecific competition between
prokaryotic microorganisms in ticks. Burgdorfer et al. (10) reported that
D. andersoni from the east side of Bitterroot Valley in western Montana
contained a nonpathogenic SFG-rickettsia, which they named East Side agent.
East Side agent has recently been described as a new species, R. peacockii
(11). This rickettsia is rarely present in tick hemolymph and is readily
missed by the standard hemolymph test. The rickettsiae are confined
primarily to the tick posterior diverticulae of the midgut, the small
intestine, and most importantly, the ovaries. R. peacockii is maintained in
the tick population through transovarial transmission, and the infected
ticks were shown to be refractory to ovarian infection with R. rickettsii.
However, these ticks acquired experimental infection with R. rickettsii and
transmitted rickettsiae horizontally (10,11). Thus, ticks constitutively
infected with R. peacockii and infected experimentally with R. rickettsii
were unable to transmit R. rickettsii to their progeny. In effect,
infection of D. andersoni with R. peacockii blocked the subsequent ability
of the ticks to transmit R. rickettsii transovarially (10,11). The
phenomenon of transovarial interference provided an explanation for the
curious long-standing disease focus in Bitterroot Valley. Most cases of
RMSF have occurred among residents on the west side of the valley where D.
andersoni were abundant; on the east side, D. andersoni were also abundant
and were reported to feed on residents, yet few (if any) locally acquired
cases of RMSF occurred there (8,10). In the presence of R. peacockii, R.
rickettsii could not be maintained transovarially-it could only be
transmitted horizontally, and thus long-term maintenance could not be
sustained. Transovarial interference by tick-associated symbionts such as
R. peacockii is unlikely to be confined only to D. andersoni ticks on the
east side of Bitterroot Valley, Montana. Burgdorfer et al. (10) stated that
transovarial interference of R. rickettsii in D. andersoni ticks may be
mediated by other nonpathogenic SFG rickettsiae-R. montana and R.
rhiphicephali. Tick surveys for SFG rickettsiae generally report finding R.
rickettsii only in a minority of ticks with rickettsiae (Table 3). Most
infected ticks harbor nonpathogenic species. Thus, transovarial
interference may have epidemiologic significance: it may explain why ticks
collected from various geographic regions are not infected with two or more
species of SFG rickettsiae.

Table 2. Epidemiologic characteristics of the North American tick-borne
rickettsia(sup a)
---------------------------------------------------------------------------
                                                Natural cycle
Rickettsia                         ----------------------------------------
species           Disease          Vectors                   Hosts
---------------------------------------------------------------------------
Rickettsia        Rocky Mountain   Dermacentor, Amblyomma,   Small mammals,
rickettsii        spotted fever    Rhipicephalus,            dogs, rabbits,
                                   Haemaphysalis             birds
R. akari          Rickettsialpox   Liponyssoides             House mice,
                                                             rats
R. amblyommii                      R. amblyommii A.          Small mammals
                                   americanum Small mammals
R. bellii                          D. andersoni, D.          Rodents, dogs
                                   variabilis,
                                   D. occidentalis, D.
                                   albopictus,
                                   H. leporispalustris
R. canada                          Haemaphysalis             Rabbits,
                                                             hares, birds
R. montana                         D. andersoni, D.          Rodents, dogs
                                   variabilis
R. parkeri                         A. americanum, A.         Domestic
                                   maculatum                 animals,
                                                             birds, rodents
R. peacockii                       D. andersoni              Rodents, deer
R. rhipicephali                    R. sanguineus, D.         Small mammals
                                   andersoni,
                                   D. variabilis, D.
                                   occidentalis
---------------------------------------------------------------------------
(sup a)Excluding four as yet undescribed species of SFG rickettsiae (WB-8-2,
364-D, Tillamook, and the D. parumapertus agent).



Table 3. Species composition of tick-borne rickettsiae isolated from
hemolymph-positive Dermacentor ticks(sup a)

---------------------------------------------------------------------------


            California     Montana       Ohio      Long Island   Maryland
         D.occidentalis D.andersoni  D.variabilis D.variabilis D.variabilis 
Rickettsial (No.            (No.        (No.      (No.            (No.
 sp         isolates)      isolates)    isolates) isolates)       isolates) 
---------------------------------------------------------------------------
R. rickettsii 0 (0)         9 (10)     18 (4)      0 (0)         8 (2)
R.
rhipicephali  96 (79)       44 (47)    0 (0)       0 (0)         0 (0)
R. montana    -             7 (8)      59 (13)     100 (100)     0 (0)
Other 
SFG(sup b)    -             -          5 (1)(sup c) -           88 (23)(sup d)
R. bellii     4 (3)         39 (41)    18 (4)      0 (0)         4 (1)
Total number
isolates      82            106        22          100          26
---------------------------------------------------------------------------
(sup a)Shows a compilation of various statewide surveys, comparing the species
composition of SFG rickettsiae in Dermacentor spp. ticks that tested
positive by immunofluorescence assay.
(sup b)SFG, spotted fever group.
(sup c)R. amblyommii.
(sup d)Mouse anti-sera made against Maryland isolates reacted with WB-8-2
(unnamed SFG rickettsiae).

The tick-rickettsia interrelationships are complex, and the mechanisms used
by rickettsiae to survive in unfed overwintering ticks or during molting
are poorly understood. How changes (e.g., before and after blood meal) in
tick gut physiology influence rickettsial growth, cell division, and
differential expression of rickettsial surface protein is not well
understood. Although experiments (8,10) have deciphered the phenomenon of
reactivation of rickettsial virulence after infected ticks take a blood
meal, the underlying molecular events have yet to be elucidated. Other
aspects of rickettsia-tick interactions need to be studied. For example,
after feeding, a tick larva enters a quiescent period before emerging as a
questing nymph the following year. How rickettsiae survive within the tick
during this quiescent period and regain infectivity during the nymphal
feeding is poorly understood. Although the precise mechanisms of
rickettsial reactivation are not known, temperature shift and blood intake
are believed to reactivate rickettsiae. As in the Borrelia
burgdorferi-Ixodes scapularis model, transmission of rickettsiae probably
cannot occur until after 24 hours of tick attachment, which allows time for
rickettsial growth.

Insect-Borne Rickettsial Pathogens

Unlike SFG, TG rickettsiae are associated with insects (Table 1). Their
association with blood-sucking insects such as human body lice (Pediculus
humanus) and fleas (Xenopsylla cheopis and other rodent fleas [13-15])
provides rickettsiae the potential to spread rapidly among susceptible
populations. Both fleas and human body lice, intermittent feeders, are
capable of multiple feeding and thus of transmitting rickettsiae to several
hosts. Outbreaks of epidemic typhus thereby can result from rapid
transmission of R. prowazekii from human to human by infected lice. Unlike
ticks that transmit SFG rickettsiae, lice infected with R. prowazekii die
within 2 weeks after imbibing infected blood. R. typhi, the etiologic agent
of murine typhus, does not shorten the life span of fleas (15). Although R.
typhi and R. felis are maintained transovarially in fleas (15-18), there is
no evidence that R. prowazekii is maintained in human body lice vertically.
Since lice die of R. prowazekii infection, the role of the reservoir in
maintaining the rickettsiae in nature becomes essential. R. prowazekii
sequesters in its human host; persistence of rickettsiae occurs despite the
strong, long-lasting immunity after infection with R. prowazekii. Patients
with recrudescent typhus (Brill-Zinsser disease) serve as potential
reservoirs capable of infecting lice. Although a search for a zoonotic
cycle of R. prowazekii in areas with louse-borne typhus epidemics (e.g.,
Ethiopia and Burundi) proved to be unsuccessful, flying squirrels
(Glaucomys volans) in the eastern United States are naturally infected with
this organism. Flying squirrel ectoparasites (lice and fleas) were
implicated in the transmission of R. prowazekii between the squirrels and
from squirrels to humans (5). Although the distribution of G. volans
extends to the entire eastern United States as well as to isolated areas of
Mexico and Guatemala, the search for an extrareservoir of R. prowazekii was
not pursued further. Consequently, the importance of the R. prowazekii and
squirrel system remains unclear. In the absence of a zoonotic cycle,
conditions such as widespread lice infestations, active human infection,
reactivation of latent infection in patients with recrudescent typhus could
easily ignite a resurgence of louse-borne typhus. Louse-borne typhus
continues to occur in epidemics following the breakdown of social,
economic, or political systems, as exemplified by recent outbreaks in
Burundi and remote parts of South America. Therefore, active surveillance
to monitor louse-borne typhus and prevent its spread is indicated.

In contrast to louse-borne typhus, murine typhus is prevalent throughout
the world and accounts for widespread illness in areas infested with many
rats and fleas. Murine typhus occurs in epidemics or has a high prevalence,
is often unrecognized and substantially underreported, and although it may
be clinically mild, can cause severe and even fatal cases (19). Thousands
of human cases used to occur annually in the United States (13,14).
Outbreaks have been reported in Australia and recently in China, Kuwait,
and Thailand (13,15). The classic cycle of R. typhi involves rats (Rattus
rattus and R. norvegicus) and primarily the rat flea, X. cheopis (13,14).
X. cheopis is the main vector, and transmission is affected by contact with
rickettsia-containing flea feces or tissue during or after blood feeding.
Reported cases of murine typhus in the United States are from south and
central Texas and the Los Angeles and Orange County area of California
(21-25). However, most of the cases are attributed to opossum-cat flea
cycles. Both opossums and domestic cats collected from the case areas were
seropositive for R. typhi antibodies. The cat flea, Ctencephalides felis,
which is a competent vector of murine typhus, is the most prevalent flea
species (97%) collected from opossums, cats, and dogs in southern Texas; no
fleas were recovered from rats in this area. In addition to R. typhi, R.
felis was also found in opossums and their fleas (15). This finding,
consistent with surveys in other areas of the country (14,20), further
documents the reduced role of rat and X. cheopis in the maintenance of
murine typhus within endemic-disease areas of the United States. The
maintenance of R. typhi in the cat flea and opossum cycle is therefore of
potential public health importance since C. felis is a prevalent and
widespread pest that avidly bites humans (12,15).

In many parts of the world, murine typhus infection is intimately
associated with introduced commensal rodents (R. rattus, R. norvegicus, and
Mus musculus) and their ectoparasites, particularly fleas. Although R.
typhi have been isolated from other commensal rodents and even shrews, they
do not seem to play a role in the transmission of murine typhus to humans
(13,14). In Rangoon (Myanmar), of four species of murines and one shrew
commonly found in buildings, 7% (M. musculus) to 30% (R. rattus) and 38%
(Bandicota bengalensis) of those tested were seropositive to R. typhi. In
contrast, 62% of R. rattus collected from buildings in Addis Ababa
(Ethiopia) and 49% of those collected in Sarawak (Nepal) were seropositive
for murine typhus (Azad et al., unpub. data). Infection rates in X. cheopis
fleas collected from rats were 7% to 18%. While infection rates vary
considerably among indoor rats and their fleas, murine typhus infection
seems clearly associated with indoor rat populations throughout the world.
However, in the absence of indoor rats, murine typhus infection is
maintained in suburban and rural cycles when native animals seek shelter in
human habitations where food and hospitable environments are plentiful.

Pathogen-Arthropod Interaction

The process of displacement of pathogenic rickettsiae with nonpathogenic
endosymbionts in ticks through transovarial interference is of potential
epidemiologic importance. The displacement might occur only if transovarial
maintenance of pathogenic rickettsiae harms the host or the maintenance of
nonpathogenic organisms confers important advantages to the tick progeny.
Burgdorfer et al. (10), in experimental studies with the D. andersoni-R.
rickettsii model, observed that maintenance of this pathogen in ticks over
several generations resulted in unusually high death rates among engorged
females and reduced numbers and fertility of deposited eggs. If tick
infection with pathogenic rickettsiae in nature adversely affected egg
maturation, oviposition, and embryogenesis, the balance would favor a tick
population infected with the nonpathogenic rickettsiae, and over time such
tick populations would displace those infected with R. rickettsii. Also,
ticks infected experimentally with R. montana and R. rhipicephali could not
maintain R. rickettsii through transovarial transmission, which suggests an
interference phenomenon. Such a precedent exists-several tick species carry
nonpathogenic rickettsiae (e.g., D. andersoni in east side Bitterroot
Valley of western Montana, A. americanum in Maryland) frequently
encountered in tick samples from different parts of the United States
(8,9).

Recent work in our laboratory with TG rickettsiae suggests that
interspecific competition between closely related rickettsiae may control
rickettsial establishment in arthropods. Studies have identified both R.
typhi and R. felis in opossums and in their cat fleas in endemic-disease
regions in Texas and California (21-25). However, infection with both
rickettsiae has not been observed in individual fleas (20-24). The
intermittent feeding behavior of cat fleas associates them with various
hosts, which increases the likelihood of infection with more than one
pathogenic organism. Cat fleas constitutively infected with R. felis and
experimentally infected with R. typhi contained both rickettsial species.
While the R. felis infection rate in the infected flea population was 86%
to 94%, prevalence of dually infected fleas was 13% and 26% (25). While no
other relationships involving multiple bacterial infections in arthropods
have been studied as thoroughly, the results from the few available studies
show that dual infections in arthropod vectors are rare (e.g., human
granulocytic ehrlichia was recently identified in 2.2% and 4% of I.
scapularis ticks coinfected with B. burgdorferi) (26).

Whether infection with nonpathogenic rickettsiae presents an advantage to
the tick population is difficult to ascertain because of lack of
experimental data. There is no experimental evidence for
rickettsial-induced postzygotic reproductive incompatibility,
parthenogenesis, and feminization of genetic males as observed for members
of the genus Wolbachia (4,27). Although a rickettsial relative is
associated with male killing in the ladybird beetle (27), we do not know
whether any nonpathogenic members of the SFG rickettsiae can induce
reproductive incompatibility or sex distortion in ticks. A compilation of
data from various laboratories that maintain cat flea colonies indicate
that after several generations R. felis infection in fleas approaches 100%
(28). Whether the high infection rate is the result of selection through
reproductive incompatibility remains to be elucidated. Flea samples from
opossums, cats, and dogs in different parts of the United States were
infected with R. felis. Since it is maintained transovarially, R. felis
could be used as a marker to follow changes in the infection rates over
time.

Several questions remain: 1) why does infection with nonpathogenic
rickettsiae prevent establishment of virulent species in the ovaries of
infected ticks; 2) what are the molecular bases for transovarial
interference; 3) are nonpathogenic rickettsiae selected favorably by their
arthropod hosts through reproductive incompatibility; and 4) why are
nonpathogenic rickettsiae found more often in ticks than a virulent
species.

Summary and Conclusions

Ixodid ticks, fleas, and lice are temporary obligate ectoparasites often
found on the same vertebrate hosts. They differ substantially with respect
to feeding behavior and digestion of blood meals (30), which can affect
rickettsiae transmission from vector to vertebrate hosts. While all stages
of ticks and lice are blood feeders, only adult fleas take blood from the
host. Fleas and lice feed intermittently and digest blood meals rapidly,
while ixodid ticks feed for several days and digest meals very slowly
(29,30). SFG rickettsiae are associated with ixodid ticks, while TG
rickettsiae are transmitted by fleas and lice. Efficient transovarial and
transstadial transmission of rickettsiae in ticks ensures rickettsial
survival while maintaining the genetic integrity of the SFG rickettsiae.
This mechanism also allows ticks to serve as reservoir host for SFG
rickettsiae. The SFG rickettsiae are transmitted by tick bites, whereas TG
rickettsiae are deposited with insect feces at the bite site. At each life
stage (larva, nymph, and adult), ticks feed only once; if undisturbed, they
transmit rickettsiae to a single host, whereas repeated feedings allow
fleas and lice to infect several hosts. Thus, vector feeding behavior
accounts for the observed differences in disease epidemiology and natural
history between tick- and insect-borne rickettsioses. Despite their strong
similarities, SFG and TG rickettsiae have major differences in terms of
growth, entry, and exit from host cells. Rickettsial characteristics
related to vector compatibility were discussed in a recent publication by
Hackstadt (31).

The molecular aspects of rickettsia-vector interactions are not well
understood largely because only a few laboratories have addressed the
mechanisms by which arthropods and rickettsiae interact. These
intracellular organisms have long been associated with arthropods, and yet
we know very little about their relationship with arthropod vectors.

This work was supported by grants R37 AI 17828 and R01 AI 43006 from the
National Institutes of Health.

Dr. Abdu Azad is professor of microbiology and immunology, University of
Maryland School of Medicine, Baltimore. His laboratory research focuses on
host parasite interactions with particular interest in the molecular
aspects of rickettsial pathogenesis.

Dr. Charles Ben Beard is research entomologist and chief, Vector Biology
Activity, Entomology Branch, Division of Parasitic Diseases, NCID, CDC.
Research in his laboratory focuses on the molecular biology of insect
disease vectors and the molecular epidemiology of opportunistic infections
in persons with AIDS.

Address for correspondence: Abdu F. Azad, Department of Microbiology and
Immunology, University of Maryland School of Medicine, 655 W. Baltimore
Street, Baltimore, MD 21201 USA; fax: 410-706-0282; e-mail:
aazad@umaryland.edu.

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     et al. Ixodes dammini as a potential vector of human granulocytic
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     identification of rickettsia-like microorganisms associated with
     colonized cat fleas (Ctenocephalides felis). Insect Molecular Biology
     1994;3:27-33.
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     fleas. Ann N Y Acad Sci 1990;590:70-5.
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Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/azad.htm

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[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Perspectives

Could Myocarditis, Insulin-Dependent Diabetes Mellitus, and Guillain-Barr
Syndrome Be Caused by One or More Infectious Agents Carried by Rodents?

Bo Niklasson,*, Birger Hrnfeldt, Berit Lundman,
*Swedish Institute for Infectious Disease Control, Stockholm,
Sweden;National Defense Research Establishment, Ume, Sweden; Department
of Animal Ecology, Ume University, Ume, Sweden; Department of Advanced
Nursing, University of Ume, Ume, Sweden; Research and Development Unit,
Sundsvalls Hospital, Sundsvall, Sweden

  --------------------------------------------------
      The numbers of small rodents in northern Sweden fluctuate
      heavily, peaking every 3 or 4 years. We found that the
      incidence of Guillain-Barr syndrome and insulin-dependent
      diabetes mellitus, as well as the number of deaths caused by
      myocarditis, followed the fluctuations in numbers of bank
      voles, although with different time lags. An environmental
      factor, such as an infectious agent, has been suggested for all
      three diseases. We hypothesize that Guillain-Barr syndrome,
      myocarditis, and insulin-dependent diabetes mellitus in humans
      in Sweden are caused by one or more infectious agents carried
      by small rodents. Also, a group of novel picornaviruses
      recently isolated from these small rodents is being
      investigated as the possible etiologic agent(s).

Nephropathia epidemica (NE) is a disease caused by Puumala virus (genus
Hantavirus, family Bunyaviridae). The vector and natural reservoir of
Puumala virus is a small rodent, the bank vole (Clethrionomys glareolus)
(1). In most parts of southern Sweden, bank vole populations are noncyclic,
whereas in the north, populations fluctuate on a 3- or 4-year cycle of
abundance (2-4). NE is endemic only in the northern part of the country,
and the number of human cases cycles with the bank vole population (5-6).

We found the incidence of myocarditis, Guillain-Barr syndrome (GBS), and
insulin-dependent-diabetes mellitus (IDDM) to lag behind the population
density fluctuations of bank voles, although with different time delays. We
hypothesize that in Sweden the bank vole is a vector for one or more
infectious agents, which are pathogenic and cause these diseases in humans.

The Study

Density of Bank Voles

Long-term records (>10 years) on small rodent abundance are scarce.
However, in Sweden such data are available from the area with cyclic rodent
populations (2, 4-8). We used data from Grims (5940' N, 1525' E), where
small rodents have been collected every autumn since 1973 (6-8).
Snap-trapping was performed where possible in six adjacent 5x5-km areas,
each with four 1-ha plots. Of 24 permanent 1-ha plots, 20 were trapped. At
each trapping, approximately 940 traps were set during three consecutive
days, amounting to approximately 2,800 "trap-nights."

Species, date, and location of trapped animals were recorded (4, 6-8). As
an index of bank vole abundance, we calculated the number of animals
trapped per 100 trap nights (i.e., density). Bank vole abundance fluctuated
in cycles with 3- or 4-year intervals between density peaks (Figure 1).

[fig 1]
Figure 1. Bank vole abundance in Grims, 1973-150;1994. Untransformed data.


Incidence of Disease

We used the national census to calculate (according to counties and
age groups) annual disease incidences per 100,000 population. Because of
the lack of long-term records on noncyclic small rodent populations,
we analyzed data from the counties with cyclic rodent populations: 
Norrbotten, Vsterbotten, Vsternorrland, Jmtland, Gvleborg, Kopparberg, 
Vrmland, rebro, Vstmanland, Uppsala, Stockholm, Sdermanland, Gteborg, 
-lvsborg, Jnkping, and Kronoberg (2-3). We excluded Stockholm and Uppsala
counties as they are predominantly urban and on the border between counties 
with cyclic and noncyclic rodents.

Myocarditis

Myocarditis is a clinical condition in which the heart is infiltrated by
inflammatory cells. Diagnosis is very difficult during the acute stage and
is often made by postmortem microscopic investigation of the myocardium.
All causes of death in Sweden are registered by the Swedish Cause-of-Death
Statistics, Statistics Sweden, S-115 81 Stockholm, Sweden. Our study
included all deaths between 1970 and 1986, in the age group 11 to 46 years,
caused by myocarditis (ICD 8 code 422), if the diagnosis was given as
either the direct (primary) cause of death or as the first out of six
recorded contributing causes of death. The age group 11 to 46 years was
chosen because congenital or arteriosclerotic cardiovascular disease is
less common in this age group. In 201 (92%) of 218 cases, the diagnoses
were based on clinical or forensic autopsies. The remaining diagnoses were
based on clinical examination before death. We did not use data collected
after 1986, as the disease classification changed from ICD 8 to ICD 9 in
1987, making comparison with the period after 1986 difficult.

Guillain-Barr Syndrome

In different parts of Europe (9), reported incidence of GBS varies
considerably depending on study method. Our data, from the hospital
discharge registry, Swedish National Board of Health and Welfare, S-106 30
Stockholm, Sweden, are considered adequate for epidemiologic surveillance
of GBS (9). We selected patients hospitalized with the diagnosis GBS (ICD 8
code 354) and gathered information on age, sex, county of residence, county
of hospitalization, diagnosis, date of admission and date of discharge. We
only analyzed data of GBS patients 46 years of age and younger, as the
diagnoses in older patients frequently are obscured by nonspecific
illnesses and other problems (10).

Insulin-Dependent Diabetes Mellitus

IDDM was studied in Medelpad (part of Vternorrland County), an area with
cyclic rodent populations. The patients were identified through registries
of patients with diabetes at the only hospital and at 16 out of the 17
health-care centers, as well as through common registries of diagnosis at
one health-care center. The World Health Organization (WHO) diagnostic
criteria were applied. We analyzed case data in all age groups because
precision in the diagnosis of IDDM is not age related.

Statistical Analysis

A cross-correlation function (CCF) (11) can indicate any direct or delayed
dependence between two different time series. A CCF graph is a plot of
positive and negative correlation coefficients between pair-wise values
from the two series. The correlations have been calculated between the two
series for different time shifts (lags), with lag 0 meaning no time shift,
lag -1 meaning the first series has been shifted backwards one time unit,
and lag 1 meaning the first series has been shifted forward by one time
unit.

We used CCFs, calculated in Statistical Package for the Social Sciences
(SPSS) (12), to indicate any temporal association between the incidence in
cyclic versus noncyclic areas of GBS and of deaths from myocarditis,
respectively. We also used CCFs to indicate any direct (at lag 0) or
delayed dependence (at negative lags) of the different disease incidences
on vole density. At negative lags, the vole time series is the leading
indicator that is shifted "backwards" along the time scale, relative to the
disease series. Thus, the correlation coefficients at negative lags are in
focus, as they indicate any disease incidence dependence on past vole
densities, i.e., on densities 1, 2, or 3 years ago. At positive lags, the
disease time series is shifted "backwards" relative to the vole time
series. The correlation coefficients at positive lags may be large because
of the similarities between the two series, but we see no causal
relationship between vole density and past disease incidence.

Log transformation of the time series was used because of the variable
amplitudes of the fluctuations in the disease and vole series. In the case
of IDDM, we also ran one CCF with the disease and vole time series
difference-transformed by one year.

Findings

Myocarditis

A total of 218 patients, ages 11 to 46 years, died of acute myocarditis in
1970 to 1986; 148 in counties with cyclic rodent density and 70 in counties
with noncyclic rodent density. Sex ratio was 2:1 (146 male, 72 female
patients). Myocarditis death incidences in cyclic and noncyclic areas
(Figure 2) were not correlated as revealed by cross-correlation of the
disease series (CCF not shown; n = 17 computable 0-order correlations).
Cross-correlation of the annual incidence of myocarditis deaths in 1970 to
1986 in the cyclic area with bank vole abundance (Figure 3) showed that the
incidence of myocarditis was most highly correlated with vole abundance in
the previous year, according to the high positive and significant
correlation at lag -1 in the CCF (Figures 4, 5) (r = 0.635, p < 0.05, n =
13).

 [fig 2]                              [fig3]
 Figure 2. Incidence of death from    Figure 3. Myocarditis deaths,
 myocarditis, 19701986.              19741986 relative to bank vole
 Untransformed data.                  abundance 1 year previously (vole
                                      data from 1973-1985). Untransformed
                                      data.

 [fig 4]                              [fig 5]
 Figure 4. Cross-correlation          Figure 5. Myocarditis deaths,
 function of incidence of death from  19741986, relative to bank vole
 myocarditis with bank vole           abundance 1 year previously (vole
 abundance, 19731986. Time series    data from 1973-1985). Log
 are log transformed; n = 14          transformed data. r = 0.635, n = 13.
 computable 0-order correlations.
 Lines represent + 2 SE. The
 standard error is based on the
 assumption that the series are not
 cross-correlated and one of the
 series is white noise.

Guillain-Barr Syndrome

In five cyclic counties and two noncyclic counties, where complete
data were available, 258 GBS patients (</= 46 years of age) were hospitalized
in 1973 to 1982; the sex ratio was 1.1:1 (135 male, 123 female
patients), 104 from the cyclic and 154 from the noncyclic area.

No correlation was found between GBS in the cyclic and noncyclic areas by
cross-correlation of the disease series (CCF not shown; n = 10 computable
0-order correlations). Cross-correlation of the annual incidence of GBS in
cyclic areas in 1973 to 1982 with bank vole abundance (Figure 6) suggested
that the incidence of GBS co-varied with current vole abundance, as shown
by the high positive and significant correlation at lag 0 in the CCF
(Figures 7, 8) (r = 0.757, p < 0.05, n = 10).

 [fig 6]
Figure 6. Time series of Guillain-Barr syndrome incidence, 1973&#150;1982, 
relative to bank vole abundance in the same years. Untransformed data.


 [fig 7]                              [fig 8]
 Figure 7. Cross-correlation          Figure 8. Guillain-Barr syndrome
 function of Guillain-Barr syndrome  incidence, 19731982, relative to
 incidence with bank vole abundance,  bank vole abundance in the same
 19731982. Time series are log       years. Log transformed data. r =
 transformed; n = 10 computable       0.757, n = 10.
 0-order correlations. Lines
 represent + 2 SE. The stan-dard
 error is based on the assumption
 that the series are not
 cross-correlated and one of the
 series is white noise.

Insulin-Dependent Diabetes Mellitus

A total of 318 cases of IDDM were recorded in 1971 to 1991,
representing a sex ratio of 1.3:1 179 male, 139 female patients). No
cross-correlation was found between the annual incidence of IDDM in the
cyclic area and bank vole abundance(Figure 9)(CCF not shown; n = 19
computable 0-order correlations), as was found for myocarditis and GBS
with vole abundance. On the other hand, after the alternative
transformation (to log transformation) by differencing the IDDM and vole
time series by 1 year, changes of the IDDM incidence from one year to the
next covaried with the changes of vole abundance 2 years previously. This
was suggested by the high positive and significant correlation at lag -2 in
the CCF (Figures 10,11) (r = 0.595, p < 0.05, n = 16).

 [fig 9]
 Figure 9. Time series of insulin-dependent diabetes mellitus incidence in 
 1975-1991 relative to bank vole abundance 2 years previously (vole data 
 from 1973-1989). Untransformed data.

 [fig 10]                             [fig 11]
 Figure 10. Cross-correlation         Figure 11. Change of
 function of insulin-dependent        insulin-dependent diabetes mellitus
 diabetes mellitus incidence with     incidence, 19751991, relative to
 bank vole abundance, 19731991.      change of bank vole abundance 2
 Time series are differenced (1); n   years previously, after
 = 18 computable 0-order              transformation of time series by
 correlations. Lines represent +/- 2  differencing (1) (vole data from
 SE. The standard error is based on   1973-1989). r = 0.595, n = 16.
 the assumption that the series are
 not cross-correlated and one of the
 series is white noise.

Calculation of Confidence Limits

The confidence limits (+/-2 SE) for the most important cross correlations, at
lag 0 for GBS (Figure 7), lag -1 for myocarditis (Figure 4), and at lag -2
for IDDM (Figure 10), were calculated in an alternative way to the default
calculation in SPSS (12) according to formula 11.1.11, p. 377 (11). The
alternative calculations were made according to formula 11.1.7, p. 376
(11). These calculations were made assuming that 1) the three disease time
series represented white noise (i.e. contain no autocorrelation); 2) the
vole time series contained autocorrelation, but it was zero after lag 5; 3)
for the CCF of GBS with the vole time series, correlation existed at lag 0
and +1 but was zero at other lags; 4) for the CCF of myocarditis with the
vole series, correlation existed at lag -1 but was zero at other lags; and
5) for the CCF of the differenced IDDM with the differenced vole time
series, correlation existed at lag 0 to lag -4 but was zero at other lags
and the differenced vole time series contained autocorrelation up to, but
not after, lag 7. The calculations of the alternative confidence limits
showed that the most important cross-correlations were significant at p <
0.05. The confidence limits for GBS at lag 0 were 0.757 +/- 0.542; for
myocarditis at lag -1, 0.635 +/- 0.450; and for the differenced IDDM at lag
-2, 0.595 +/- 0.478.

Conclusions

Zoonotic disease reservoir or vector population density data can indicate a
link between the etiologic agent and its reservoir/vector and between the
agent and the disease. This type of data linked rodents to NE in Sweden in
the 1930s and to Korean hemorrhagic fever in the 1950s (13-14). In a
previous study, we found that the Puumala virus infection rate in bank
voles in the spring tracked the vole density in the previous autumn and
that infection rate peaked as vole population declined (5). We believe that
this explains the approximately 9-month time lag in the incidence of NE in
the spring relative to vole abundance in the previous autumn, although the
incubation period in humans is only 2-6 weeks.

Our present data show significant temporal correlations of bank vole
population density with incidence of death from myocarditis (Figures 3-5)
and incidence of GBS (Figures 6-8). Our data also show a positive and
significant correlation between changes in IDDM incidence and changes in
bank vole density (Figures 9-11). We hypothesize that these three diseases
are caused or triggered by one or more infectious agents carried by bank
voles in Sweden. In this context, several sudden deaths among Swedish
orienteers were recognized during 1989 to 1992. Six of approximately 200
elite orienteers died of acute myocarditis (15). Orienteering involves
finding the fastest/shortest way between several checkpoints, often in
forested areas. Because of extensive exposure to nature, it has been
speculated that the sudden deaths of the orienteers were caused by a
vector-borne (rodent or arthropod) infectious agent.

If our hypothesis is correct, the different time lags of different diseases
relative to vole abundance, as seen in the present study, may depend in
part on the specific infection rate dynamics of any agent in the bank vole
population as seen in the case of NE (5). The time elapsed from infection
to disease onset or death may also be important in IDDM and myocarditis.

The disease time series presented here were not very long, and the results
should therefore be treated with caution. The present findings do not prove
any direct link between the diseases investigated and the bank vole. Many
infectious diseases and other biological phenomena have a cyclic variation,
and the relationships presented here may be spurious or may involve another
reservoir or vector population (e.g., small rodents and small game species
that fluctuate synchronously with the bank vole at these latitudes)
(4-5,7-8,16-17).

The identification of the etiologic agent or agents is critical to "prove"
our hypothesis. However, as seen in several zoonotic diseases, humans are
often dead-end hosts, and it may be easier to isolate the etiologic agent
from the reservoir or vector than from the patient. Also, because of the
time elapsed between primary infection and onset of disease, the infectious
agent may be present only in very small amounts or absent. It is also
possible that several human diseases with no etiologic agent identified are
caused by agents that are difficult to cultivate, at least by standard
techniques.

Thus, because we need to identify the etiologic agent(s) and it is
difficult to isolate any virus from humans, we have initially attempted to
isolate new viruses from small rodents, the primary suspects. The bank vole
is the most common wild-living Swedish mammal. Its abundance varying by
>300 times (4), this rodent is likely to have a high and highly variable
potential over time to transmit any etiologic agent to humans. In addition,
bank voles are known to enter buildings, thus transporting disease risk
closer to humans.

Three novel virus isolates from bank voles, resembling picornaviruses in
size and morphology, have been found. The first isolate was named Ljungan
after the Ljungan river in Medelpad County, Sweden, where the animals were
trapped. The second and third isolate originated from animals trapped in
Vsterbotten County. The amino acid sequences of predicted Ljungan virus
capsid proteins were closely related (approximately 70% similarity) to the
human pathogen echovirus 22. Partial 5' noncoding region sequence of
Ljungan virus was most closely related to cardioviruses. Two additional
isolates were serologically and molecularly related to the prototype
(Niklasson et al., unpub. observation). Echovirus 22 is a known human
pathogen, and other known cardioviruses can induce not only myocarditis but
also neurologic diseases and IDDM in several species of animals.

We hope to elucidate the role of Ljungan virus as a human pathogen by
serologic assays for Ljungan virus, diagnostic polymerase chain reaction
based on generated sequence data, and specific antisera for viral antigen
detection. Studies are also under way to identify and determine the role of
other viruses of these small rodents in inducing myocarditis, GBS, and IDDM
in humans.

Acknowledgments

We thank Sara Sjstedt for statistical help and Stiftelsen Olle Engkvist,
Byggmstare and by the Swedish Environment Protection Agency (via the
National Environmental Monitoring Programme) for financial support for the
vole trapping.

Address for correspondence: Bo Niklasson, Swedish Institute for Infectious
Disease Control, S-105 21 Stockholm, Sweden; fax: 46-8-735-66-15; e-mail
BO.NIKLASSON@SMI.KI.SE

References

  1. Niklasson B, Le Duc J. Epidemiology of nephropathia epidemica in
     Sweden. J Infect Dis 1987;155:269-76.
  2. Hansson L, Henttonen H. Gradients in density variations of small
     rodents: the importance of latitude and snow cover. Oecologia 1985;
     67:394-402.
  3. Hansson L, Henttonen H. Rodent dynamics as community processes. Trends
     in Ecology & Evolution 1988;3:195-200.
  4. Hrnfeldt B. Delayed density dependence as a determinant of vole
     cycles. Ecology 1994;75:791-806.
  5. Niklasson B, Hrnfeldt B, Lundkvist , Bjrsten S, LeDuc J. Temporal
     dynamics of Puumala virus antibody prevalence in voles and of
     nephropathia epidemica incidence in humans. Am J Trop Med Hyg
     1995;53:134-40.
  6. Hrnfeldt B. Smsdggdjursinventering i PMK:s referensomrden - rapport
     frn verksamheten 1992. Solna, Sweden: Swedish Environmental
     Protection Agency; 1994. Report 4294.
  7. Lindstrm ER, Andrn H, Angelstam P, Cederlund G, Hrnfeldt B,
     Jderberg L, Lemnell P-A, Martinsson B, Skld K, Swenson JE. Disease
     reveals the predator: sarcoptic mange, red fox predation and prey
     populations. Ecology 1994;75:1042-9.
  8. Lindstrm ER, Hrnfeldt B. Vole cycles, snow depth and fox predation.
     Oikos 1994;70:156-60.
  9. Jiang GX, de Pedro-Cuesta J, Fredriksson G. Guillain-Barr syndrome in
     South-West Stockholm, 1973-1991. Quality of registered hospital
     diagnosis and incidence. Acta Neurol Scand 1995;91:109-17.
 10. Winner SJ, Evans JG. Age-specific incidence of Guillain-BarrT syndrome
     in Oxfordshire. Quarterly Journal of Medicine, New Series
     1990;77:1297-1304.
 11. Box GEP, Jenkins GM. Time series analysis, forecasting and control.
     San Francisco: Holden Day; 1970.
 12. SPSS Base System Syntax Reference Guide, Release 6.0. Carey (NC): SPSS
     Inc.; 1993.
 13. Myhrman G. En njursjukdom med egenartad symptombild. (In Swedish).
     Nord Med Tidskr 1934;7:793-94.
 14. Gajdusek DC. Virus haemorrhagic fevers-special reference to
     haemorrhagic fevers with renal syndrome (epidemic haemorrhagic fever).
     J Pediatr 1962;60:841-57.
 15. WesslTn L, Pshlson C, Friman G, Fohlman J, Lindquist O, Johansson C.
     Myocarditis caused by Chlamydia pneumonie (TWAR) and sudden unexpected
     death in a Swedish elite orienteer. Lancet 1992;340:427-8.
 16. Hrnfeldt B. Synchronous population fluctuations in voles, small game,
     owls and tularemia in northern Sweden. Oecologia 1978; 32:141-152.
 17. Hrnfeldt B, Lfgren O, Carlsson B-G. Cycles in voles and small game
     in relation to variations in plant production indices in northern
     Sweden. Oecologia 1986;68:496-502.


Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/niklason.htm

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[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Perspectives

Multidrug-Resistant Mycobacterium tuberculosis: Molecular Perspectives

Ashok Rattan, Awdhesh Kalia, and Nishat Ahmad
All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India

  --------------------------------------------------
      Multidrug-resistant strains of Mycobacterium tuberculosis
      seriously threaten tuberculosis (TB) control and prevention
      efforts. Molecular studies of the mechanism of action of
      antitubercular drugs have elucidated the genetic basis of drug
      resistance in M. tuberculosis. Drug resistance in M.
      tuberculosis is attributed primarily to the accumulation of
      mutations in the drug target genes; these mutations lead either
      to an altered target (e.g., RNA polymerase and
      catalase-peroxidase in rifampicin and isoniazid resistance,
      respectively) or to a change in titration of the drug (e.g.,
      InhA in isoniazid resistance). Development of specific
      mechanismbased inhibitors and techniques to rapidly detect
      multidrug resistance will require further studies addressing
      the drug and drug-target interaction.

In the last decade, tuberculosis (TB) has reemerged as one of the leading
causes of death (nearly 3 million deaths annually) (1). The estimated 8.8
million new cases every year correspond to 52,000 deaths per week or more
than 7,000 each day, which translates into more than 1,000 new cases every
hour, every day (2,3). These death rates, however, only partially depict
the global TB threat; more than 80% of TB patients are in the economically
productive age of 15 to 49 years. The emergence of AIDS and decline of
socioeconomic standards contribute to the disease's resurgence in
industrialized countries (4). In most developing countries, although the
disease has always been endemic, its severity has increased because of the
global HIV pandemic and extensive social restructuring due to rapid
industrialization and conflicts. A major public health problem worldwide,
TB is now a global emergency (Figure 1).

                           [fig 1]
Figure 1. Global incidence of tuberculosis. Of the estimated 8.8 million 
cases worldwide, more than 40% of the cases are in Southeast Asia; India 
has approximately 53.3% of those cases. A, Americas; Afr, Africa; WP, 
Western Pacific; E, Europe; M, Eastern Mediterranean; and SEA, Southeast 
Asia; Ind, Indonesia; B, Bangladesh; Thai, Thailand; My, Myanmar. 
*Others include Bhutan, 0.05%; Nepal, 1.2%; Maldives, 0.001%; Sri Lanka, 
1%; DPR Korea, 1.2%. (Data from reference 2). 


Short-course chemotherapy forms the backbone of antitubercular
chemotherapy (5). Proper prescriptions and patient compliance
almost always cure. In fact, TB incidence was steadily declining in
most industrialized countries, until the trend was reversed (6). Further
contributing to the increased death rate is the emergence of new strains
of M. tuberculosis resistant to some or all current antitubercular drugs.
The resistance is attributed primarily to improper prescriptions
or patient noncompliance and is often a corollary to HIV infection (7-9).
Multidrug-resistant TB (MDRTB), associated with high death rates of
50% to 80%, spans a relatively short time (4 to 16 weeks) from diagnosis
to death (10). Delayed recognition of drug resistance, which results in
delayed initiation of effective therapy, is one of the major factors
contributing to MDRTB outbreaks, especially in health-care facilities
(11,12). In most countries, MDRTB has increased in incidence and interferes
with TB control programs, particularly in developing countries, where
prevalence rates are as high as 48% (13,14). The high infection and death
rates pose an urgent challenge to rapidly detect cases.

In the past few years, genetic and molecular insights have unraveled the
mechanisms involved in the acquisition of drug resistance by Mycobacterium
tuberculosis (MTB), concomitant with the development of various molecular
strategies to rapidly detect MDRTB. In this review, we examine the status
of the mechanisms of resistance to antitubercular drugs.

MDRTB and the Mechanisms of Resistance

Currently TB is treated with an initial intensive 2-month regime comprising
multiple antibioticsrifampicin (RIF), isoniazid (INH), pyrazinamide (PZA),
and ethambutol (EMB) or streptomycin (SM)to ensure that mutants resistant
to a single drug do not emerge (15). The next 4 months, only RIF and INH
are administered to eliminate any persisting tubercle bacilli. INH and RIF,
the two most potent antituberculous drugs, kill more than 99% of tubercule
bacilli within 2 months of initiation of therapy (16,17). Along with these
two drugs, PZA, with a high sterilizing effect, appears to act on
semidormant bacilli not affected by any other antitubercular drugs (18).
Using these drugs in conjunction with each other reduces antitubercular
therapy from 18 months to 6 months. Therefore, the emergence of strains
resistant to either of these drugs causes major concern, as it leaves only
drugs that are far less effective, have more toxic side effects, and result
in higher death rates, especially among HIV-infected persons.

The phrase "MDR state" in mycobacteriology refers to simultaneous
resistance to at least RIF and INH (19) (with or without resistance to
other drugs). Genetic and molecular analysis of drug resistance in MTB
suggests that resistance is usually acquired by the bacilli either by
alteration of the drug target through mutation (20) or by titration of the
drug through overproduction of the target (21). MDRTB results primarily
from accumulation of mutations in individual drug target genes (Table). The
probability of resistance is very high for less effective antitubercular
drugs such as thiacetazone, ethionamide, capreomycin, cycloserine, and
viomycin [10(sub -3)]; intermediate for drugs such as INH, SM, EMB, kanamycin,
and p-amino salicylic acid [10(sub -6)]; and lowest for RIF [10(sub -8)] (22,23).
Consequently, the probability of a mutation is directly proportional to the
bacterial load. A bacillary load of 109 will contain several mutants
resistant to any one antitubercular drug (24). Because the mutations
conferring drug resistance are chromosomal, the likelihood of a mutant
being simultaneously resistant to two or more drugs is the product of
individual probabilities; thus the probability of MDR is multiplicative.
Resistance to a drug does not confer any selective advantage to the
bacterium unless it is exposed to that drug (19). Under such circumstances,
the sensitive strains are killed and the drug-resistant mutants flourish.
When the patient is exposed to a second course of drug therapy with yet
another drug, mutants resistant to the new drug are selected, and the
patient may eventually have bacilli resistant to two or more drugs. Serial
selection of drug resistance, thus, is the predominant mechanism for the
development of MDR strains; the patients with MDR strains constitute a pool
of chronic infections, which propagate primary MDR resistance. In addition
to accumulation of mutations in the individual drug target genes, the
permeability barrier imposed by the MTB cell wall can also contribute to
the development of low-level drug resistance. Studies addressing resistance
to SM have found evidence of such a two-step mechanism for the development
of drug resistance (119,120).

Table. Gene loci involved in conferring drug-resistance in Mycobacterium
tuberculosis.
--------------------------------------------------------------------------------
                                                   Reported
                                                   frequency
                                                      in
                                                   resistant
                                                 strains(sup a)
Drug             Gene       Product                   (%)         Reference
--------------------------------------------------------------------------------
Rifampicin       rpoB       B-subunit of RNA        >95           45-48,68-71
                            polymerase
Isoniazid        katG       Catalase-peroxidase      60-70        39-48
                 oxyR-ahpC  Alky hydro-reductase    ~20           36
INH-Ethionamide  inhA       Enoyl-ACP reductase     <10           46-48
Streptomycin     rpsL       Ribosomal protein S12    60           46-48
                 rrs        16s rRNA                <10           113-117
Fluoroquinolone  gyrA       DNA gyrase              >90           107
Pyrazinamide     pncA       Amidase                  70-100       92-94
Ethambutol       embCAB     EmbCAB                   69           88
--------------------------------------------------------------------------------
(sup a)Mutation frequencies are as determined by sequencing and polymerase chain
reaction-single strand conformational polymorphism (PCR-SSCP) analysis.

Resistance to INH

INH (isonicotinic acid hydrazide, 4-pyridinecarboxylic acid hydrazide),
highly active against the MTB complex (M. tuberculosis, M. bovis, M.
africanum, and M. microti), has very low MICs (0.02 g/ml to 0.06 g/ml)
(25). The mechanism of action of INH, as well as mechanisms conferring INH
resistance, are complex and not completely understood (Figure 2). However,
evidence suggests that INH inhibits the biosynthesis of cell wall mycolic
acids (long-chain alpha-branched -hydroxylated fatty
acids), thereby making the mycobacteria susceptible to reactive oxygen
radicals and other environmental factors. Activation of INH to an unstable
electrophilic intermediate requires the enzyme catalase-peroxidase (KatG,
coded by katG) and an electron sink (hydrogen peroxide) (26), although
hydrazine formed after INH spontaneously decomposes may also mediate
activation of INH (27). Nevertheless, KatG is the only enzyme capable of
activating INH, and consequently, KatG mutant MTB strains are invariably
INH resistant.

                          [fig 2]
Figure 2. Mechanism of action of isoniazid (INH); acquisition of resistance
and combating oxidative stress. DPR, divergent promoter region.

Early studies by Middlebrook demonstrated that INH resistance was
associated with loss of catalase activity (28). Genetic studies
demonstrated that transformation of INH-resistant M. smegmatis and MTB
strains with a functional KatG restored INH susceptibility and put
forth the hypothesis that katG deletion may cause INH resistance in
MTB (29,30). However, in the absence of a peroxide-inducible genetic
response, mediated in most bacteria by the transcription factor OxyR (31),
KatG is the only peroxide-inducible MTB protein (32). Consequently, MTB
resistance to INH is paradoxical; it has to sacrifice KatG function. MTB's
ability to adapt to the loss of KatG function and combat organic peroxides
is remarkable. Studies conducted by Sherman et al. demonstrated that all
KatG mutant MTB strains overexpressed a 22-kD protein at levels
significantly higher than INH-sensitive strains (33). Sequence analysis
confirmed that this protein was similar to the earlier reported MTB AhpC
protein. AhpC can detoxify organic peroxides and is homologous to other
bacterial and eukaryotic proteins with alkyl hydroperoxidase and
thioredoxin-dependent peroxidase activities (34,35). The 5' regions (39 to
81 bp upstream from the ahpC start codon) of each AhpC-upregulated (and
katG mutant) isolate contained mutations that could increase promoter
activity; it was proposed that compensatory mutations in the ahpC promoters
were selected in katG mutant strains to combat oxidative stress (33).
Subsequent studies using immunoblotting experiments demonstrated the
consistency of AhpC upregulation among clinical isolates with complete
deletion of katG (36,37). katG mutant isolates with variable residual KatG
activity did not have this strict linear relationship. Characterization of
the oxyR-ahpC region further demonstrated that mutations responsible for
AhpC upregulation occurred at low frequencies and were primarily G>C to A>T
transitions localized in the oxyR-ahpC intervening region (36). Although
the sequence alterations in the oxyR-ahpC region were predominantly
restricted to INH-resistant isolates, not all alterations detectably
increased the AhpC levels. The apparent rarity of AhpC upregulation among
INH-resistant and katG mutant isolates could be attributed partially to the
rare occurrence of MTB strains with complete katG deletion (38-41,44).
Alternatively, among katG mutant isolates, selection of AhpC upregulatory
mutations may be subject to the selective pressure exerted by residual
catalase-peroxidase activity (36). However, AhpC upregulation was not
observed among MTB isolates with katG315 codon mutations, which reportedly
lead to more than a 20-fold decrease in KatG activity and confer high MICs
against INH (>90 g/ml) (42, 43). This inconsistency and rarity of AhpC
upregulation among katG mutant INH-resistant isolates indicates a more
complex relationship between the two and underlines the need for in-depth
studies to determine precisely the conditions regulating AhpC expression.

Clinical studies to validate the paradigm of katG deletions and INH
resistance showed that complete deletion rarely occurred (38-41). We
constructed a 35-mer oligonucleotide probe specific for katG gene. Southern
hybridization demonstrated the presence of katG in all INH-resistant
isolates, precluding complete deletion of katG gene as a dominant mechanism
for INH resistance (44). Previous studies using polymerase chain reaction
(PCR) amplification had also established these findings; sequence analysis
of katG from INH-resistant strains showed randomly distributed mutations,
including point mutations and deletions and insertions of up to 1 to 3
bases (38-41). These mutations could disrupt the katG gene, leading to the
production of an inactive gene product or a gene product with compromised
peroxidative activity. PCR amplification of the katG gene followed by
single strand conformational polymorphism (SSCP) detected mobility shifts
supporting the presence of these mutations and thereby INH resistance.

Our analysis of the katG gene by PCR-SSCP resulted in the amplification of
the 237 bp fragment of the katG gene and demonstrated a 67.3% (n = 19)
correlation between mutations in the katG gene and INH resistance (45). The
results were consistent with those from earlier studies indicating that
katG gene mutations had a correlation rate of less than 60% to 70% with INH
resistance (46-48). Sequence analysis of INH-resistant strains
demonstrating altered SSCP patterns showed that the most common mutation
was G>T transversion in codon 463 (42). In this G>T change, Leu is
substituted for Arg, and the restriction site for NciI and MspI is lost
(40). Polymorphism in the katG locus can then be easily detected by
restriction digestion. Recent kinetic and spectroscopic studies have
demonstrated striking similarities between KatG from wild-type strains and
the R463L mutant isolates (49). Both enzymes had similar visible and
electron-paramagnetic-resonance spectra and similar ability to oxidize INH
and inactivate InhA. Further, when the INH-resistant katG-defective strains
of M. smegmatis with wild-type katG or the R463L katG were transformed, INH
susceptibility was restored to about the same extent (50). These
similarities do not support the contention that the R463L mutation of katG
allows discrimination against INH as a substrate and thereby confers
resistance to INH. Although the exact role of the R463L mutation of katG
requires further scrutiny, this mutation may be a frequent polymorphism and
may not affect INH susceptibility.

Other common mutations resulting in an attenuated KatG have been identified
primarily as missense mutations that result in single amino acid
substitutions (46-48). While the data point towards mutations in the katG
gene as the dominant mechanism for INH resistance, they also point to other
factors that could mediate MTB acquisition of resistance to INH.

Mutations in the oxyR regulon, from which AhpC is divergently transcribed,
could explain the acquisition of INH resistance in the remaining
INH-resistant isolates (33,51). OxyR confers high-level intrinsic
resistance to INH in Escherichia coli and Salmonella Typhimurium; mutations
in the oxyR or AhpC restore INH susceptibility in these species (51). The
MTB oxyR regulon is much smaller than in M. leprae and other
mycobacteriabecause of two important deletions of 29 bp and 372 bp
(32,52). In addition to these deletions, the oxyR regulon carries many
frame shift mutations, which result in low expression of this regulon and
eventually lead to low-level expression of AhpC (consistent with the
finding of low-level expression of AhpC in INH-sensitive strains vs.
INH-resistant strains) (33). A related member of the genus resistant to
INH, M. leprae, however, has a complete oxyR-ahpC region that is
transcriptionally fully active and may play a role in the detoxification of
active INH intermediates (52). By analogy, therefore, the loss of the OxyR
function, in conjunction with its putative effects on ahpC expression,
could explain the exquisite specificity of INH for the MTB complex.
However, evidence from recent studies does not indicate a direct role for
oxyR or the ahpC genes in determining susceptibility to INH (36,37).
Polymorphisms in oxyR do not have any preferential predisposition and exist
among both INH-resistant and -susceptible isolates with about the same
frequency (36). The relationship of AhpC overexpression to INH resistance
is more complex. Earlier observations based on transformation of M.
smegmatis strains suggested a possible involvement of AhpC overexpression
in acquiring INH resistance (53). Transformation of M. smegmatis isolates
with multicopy constructs of ahpC led to almost a fivefold increase in the
MIC for INH. However, an increasing body of evidence precludes any direct
role of AhpC in determining INH susceptibility among MTB isolates. MTB
transformants bearing multicopy constructs of ahpC did not demonstrate
significant increase in the MIC for INH, thus any direct role for AhpC in
acquisition of INH resistance was ruled out (37).

Efforts to determine the factors involved in resistance to INH led to the
discovery of the inhA locus, which was proposed as the primary target for
coresistance to INH and ethionamide (54). This locus is composed of two
open reading frames (ORFs), designated orf1 and inhA, separated by a 21-bp
noncoding region. InhA, an enoyl-ACP reductase (55), more than 40%
homologous to the EnvM protein, catalyzes an early step in fatty acid
synthesis among enterobacteria. Like EnvM, InhA activity is also thought to
use NAD(H) as cofactor. INH susceptibility could result from incorporation
of iso-NAD, which is formed as a consequence of the action of KatG on INH,
and thus hinders the enzymatic activity of InhA and blocking fatty acid
synthesis (56). A T>G transversion, observed in few of the resistant
strains, at position 280 in the inhA gene, results in the ser94 to ala94
replacement (54). This replacement, thought to alter the binding affinity
of InhA to NAD(H), ultimately results in INH resistance (57).
Alternatively, because of mutations in the putative promoter region,
hyperexpression of InhA could result in INH resistance.

Studies conducted in clinical settings to provide corroborating evidence of
mutations in the inhA locus and INH resistance have shown approximately 10%
correlation (46-48). Analysis of 37 INH-resistant isolates by Kapur et al.
demonstrated no ser94-ala94 substitution in the resistant isolates. Only
one isolate had a missense mutation: ATC>ACC at position 47, resulting in
substitution of Ile16 by Thr16. Morris et al. also demonstrated the lack of
mutations in the inhA gene among 42 INH-resistant MTB isolates. However,
five of the INH-resistant isolates showed single nucleotide mutations in
the putative inhA regulatory region upstream of orf1.

Subsequent biochemical characterization of InhA function demonstrated that
it catalyzed the reduction of 2-trans-octenoyl-acyl carrier protein and
also that protein of enoyl CoA esters (58-59), thereby acting at the final
step in chain elongation in fatty acid synthesis (58). This observation
contradicted earlier biochemical evidence suggesting that an enzyme
involved in the synthesis of an unsaturated 24-carbon fatty acid was the
target for activated INH (60,61). Thus, the targets identified
biochemically and by complementation of M. smegmatis are different. Lipid
pulse labeling experiments demonstrated that the lipid biosynthetic
response of M. smegmatis and MTB after exposure with INH were different
(62), indicating a different mechanism of action for the INH intermediate
in the two species. Transformation of M. smegmatis with single-copy alleles
of mutant inhA loci did not result in significant resistance to INH,
indicating the presence of a different promoter in M. smegmatis. Further,
the inability of multicopy vector constructs bearing the inhA gene to
significantly increase the MIC for INH provided substantiating evidence for
the limited involvement of this locus in mediating INH resistance among MTB
isolates. These data, along with clinical evidence, preclude the likelihood
that inhA is the primary target for the activated form of INH.

Functional characterization of inhA mutations, occurring with katG
mutations (as observed in isolates with very high MICs) (46) in relation to
lipid metabolism of INH-resistant isolates, could perhaps resolve this
discrepancy and delineate the roles of the respective loci in the mechanism
of action of INH and subsequent acquisition of drug resistance.

In summary, mutations in the katG and the inhA genes are associated with
approximately 70% to 80% of INH-resistant MTB isolates; molecular
mechanisms operating in the remaining isolates are still unknown. The role
of the MTB cell wall as an important permeability barrier needs to be
explored in greater detail, particularly with reference to INH resistance
(56).

Resistance to RIF

RIF, first introduced in 1972 as an antitubercular drug, is extremely
effective against MTB. It has MICs of 0.1 g to 0.2 g (16,63). Because of
its high bactericidal action, RIF, along with INH, forms the backbone of
short-course chemotherapy (5). Although rare, resistance to RIF is
increasing because of widespread application and results in selection of
mutants resistant to other components of short-course chemotherapy. In this
context, resistance to RIF can be assumed to be a surrogate marker for
MDRTB (19). RIF had long been believed to target the mycobacterial RNA
polymerase and thereby kill the organism by interfering in the
transcription process (64). Using purified RNA polymerase from M.
smegmatis, strain mc(sup 2)155, Levin and Hatfull demonstrated that RIF
specifically inhibited the elongation of full-length transcripts and had
virtually no effect on the initiation of transcription (65).

RNA polymerase, a complex oligomer composed of four different subunits 
(alpha,,'and sigma, encoded by rpoA, rpoB, rpoC, and rpoD, respectively), 
is highly conserved among bacterial species (66). Characterization of the 
rpoB gene in E. coli demonstrated that RIF specifically interacted with 
the  subunit of RNA polymerase, thereby hindering transcription, and that
mutations in the rpoB locus conferred conformational changes leading to
defective binding of the drug and consequently resistance (67).
Subsequently, the rpoB locus from MTB was characterized and mutations
conferring the resistant trait were identified (Figure 3; 68-71). Most
mutations were determined to be restricted to an 81-bp core region and are
dominated by single nucleotide changes, resulting in single amino acid
substitutions, although inframe deletions and insertions also occur at
lower frequencies. Changes in the codons Ser531 and His526 have been
documented in more than 70% of the RIF-resistant isolates. A very small
number of mutations in RIF-resistant isolates do not map in this 81-bp core
region; it is speculated that additional mechanisms, including RIF
permeability and mutations in alternate subunits of RNA polymerase, may
also be involved in conferring the resistance phenotype.

                          [fig 3]
Figure 3. Single amino acid substitutions in the 81 bp core-region of
the rpoB gene responsible for conferring rifampicin (RIF) resistance 
(Insertions and deletions that confer the RIF-resistance phenotype are 
not depicted). Amino acids are represented with single letter abbreviations. 
Changes in codon Ser531 and His526 account for more than 70% of the 
mutations with RIF resistance (depicted in shaded ellipses).


The consistency of mutations in the rpoB locus and the RIF- resistant
phenotype (>95%) has marked clinical implications. Because it may act as a
surrogate marker for MDRTB, RIF resistance has prompted development of
various diagnostic tests to improve the sensitivity of mutation detection.
Although automated sequencing has been unambiguously applied to
characterize mutations associated with RIF resistance, a number of other
techniques such as PCR-SSCP (41,45-48,121), dideoxy fingerprinting (72),
heminested PCR (73), PCR heteroduplex analysis (70), and line probe
hybridization (74,75) have been successfully applied to detecting these
mutations. Such novel strategies to detect drug-resistant MTB isolates have
been described elsewhere (76). PCR-SSCP analysis for detection of mutations
responsible for conferring drug-resistance is increasingly useful. In
particular, the development of nonisotopic PCR-SSCP analysis has simplified
the procedure, enhancing its utility in routine laboratories (41,45).
However, results obtained with SSCP analysis should be interpreted with
caution as the technique only detects mutations and gives no information on
the nature of associated mutation. For example, silent mutations in the
rpoB gene have been identified that give altered mobility patterns on SSCP
analysis but have no association with RIF resistance, which underlines the
need for caution in interpreting results and phenotypic or genotypic
correlation (77).

Resistance to EMB

EMB [dextro-2,2'-(ethyldiimino)-di-1onol], synthetic compound with profound
antimycobacterial effects (78), is a first-line anti-MTB drug with a broad
spectrum of activity, unlike INH. EMB is also advocated in disseminated M.
avium complex infections, particularly in HIV-infected persons (79). Until
recently, EMB's mechanism of action and the genetic basis for resistance to
it were largely obscure. Specificity of EMB for mycobacterial species,
however, indicated that its target may have been involved in the
construction of the outer cell wall. Synergy resulting from
coadministration of EMB and other drugs gave further evidence for the
involvement of EMB in obstructing the formation of cell wall. The
synergistic effect was explained as a consequence of increased permeability
of the mycobacterial cell wall leading to increased drug uptake (80,81).
Indeed, earlier studies of Takayama and colleagues demonstrated that
administration of EMB led to rapid cessation of mycolic acid transfer to
the cell wall and equally rapid accumulation of trehalose mono- and
di-mycolates (82,83). Mycolic acids attach to the 5'-hydroxyl groups of
D-arabinose residues of arabinogalactan and form
mycolyl-arabinogalactan-peptidoglycan complex in the cell wall. Disruption
of the arabinogalactan synthesis inhibits the formation of this complex and
may lead to increased permeability of the cell wall. Subsequently, it was
demonstrated that EMB specifically inhibited arabinogalactan synthesis
(84).

A breakthrough was achieved in defining the precise cellular target for EMB
with the isolation and identification of
-D-arabinofuronosyl-1-monophosphoryl decaprenol (DPA), which accumulates
rapidly (less than 2 minutes) on exposure of EMB- sensitive cells to EMB
(86). DPA is an arabinosyl donor; cell-free assay systems developed for DPA
established that it was one of the major intermediates of arabinan
synthesis. It was later shown that EMB specifically inhibited arabinosyl
transfer, suggesting that arabinosyl transferase was the primary cellular
target for EMB (Figure 4).

                        [fig 4]
Figure 4. Mechanism of action of ethambutol (adapted from 84-88).
EMB interacts with the EmbCAB proteins encoded by the embC, embA, and embB
genes, leading to inactivation of arabinogalactan synthesis. Mutations in 
the embB locus cause alterations in EmbB, possibly leading to an altered 
target for EMB. Alternatively, hyperexpression of the EmbCAB proteins could 
lead to EMB resistance. Inlet box: Organization of the emb operon in 
Mycobacterium tuberculosis(MTB).

Identification of arabinosyl transferase as the primary target for
EMB helped unravel the genetic basis for EMB resistance. Using target
overexpression by a plasmid vector, Belanger et al. cloned the emb locus
from an EMB-resistant strain of M. avium (86). Transformation of this
emb locus conferred resistance to M. smegmatis mc(sup 2)155 strain and also
demonstrated that the level of resistance conferred depended on the
copy number of the gene, which was consistent with the notion of drug
resistance due to target overexpression. Site-directed mutagenesis and 
overlapping clone analysis localized a 9.8-kb EMB resistance locus, subsequently
shown to be ubiquitous among mycobacteria. Sequence analysis of this locus
revealed three complete ORFsdesignated embR, embA, and embB. The embR ORF
is separated by a 178 bp divergent promoter region from the embA and embB
ORFs. Characterization of the embR ORF showed that the region was strongly
homologous with a family of transcriptional activators of Streptomyces and
thus could play a role in modulating the expression of embA and embB.
Importantly, the embB ORF lacks a potential ribosome binding site and is
thus translationally coupled to embA, which suggests that a heterodimeric
enzyme complex may be the target for EMB. Mapping studies further
demonstrated that both embA and embB, along with the divergent promoter
region, were essential to EMB resistance.

In contrast to the organization of the emb locus in M. avium, molecular
genetic approaches applied to MTB revealed a highly conserved 14-kb region
comprising three homologous ORFs designated embC, embA, and embB preceded
by a predicted coding region and by orfX (which encodes a putative protein
belonging to the short chain alcohol dehydrogenase family) (87). Primer
extension analysis of the emb region supported the notion of its
organization as an operon and further indicated the polycistronic nature of
its transcripts. The emb genes are translationally coupled the absence of
any untranslated intercistronic region between the emb genes so indicated).
However, the presence of a secondary stem loop structure between the embA
and the embB genes indicates that the embB gene in MTB could be
differentially regulated. The embCAB proteins are believed to be integral
membrane proteins, consistent with their role in the synthesis of various
arabinan-linkage motifs of the arabinogalactan and lipoarabinomannan
(86,87).

Identification of the embCAB genes prompted a detailed analysis of the
molecular mechanisms responsible for conferring resistance to EMB in MTB
isolates. Preliminary studies documented among EMB-resistant isolates
missense substitutions in the conserved embB codon 306 that coded for
methionine; their role in conferring resistance to EMB was confirmed by
gene tranfer assays (87). Recent analysis of the embCAB region has
confirmed the predominance of embB Met306 substitutions among EMB-resistant
clinical isolates of MTB (approximately 89% among EMB-resistant isolates
with single amino acid substitutions) (88). Sequence analysis of 118
clinical isolates of MTB showed five mutants of the embB codon 306, all
leading to substitution of Met with Val, Leu, or Ile. MTB strains with
Met306Leu and Met306Val substitutions demonstrated a higher MIC for EMB (40
g/ml) than those for organisms with Met306Ile substitutions (20 g/ml).
The embB codon 306 may contain important structure-function information;
structural alterations in this codon may have a detrimental effect on the
interaction of EMB and EmbB, thereby resulting in a EMB-resistant
phenotype.

Sequence alterations in the embCAB region correlate with approximately 70%
of EMB-resistant strains. Overexpression of the EmbB protein has been
documented to mediate resistance in M. smegmatis (87), and a homologous
mechanism may operate in MTB, perhaps accounting for the remaining 30% of
the EMB-resistant isolates. A full understanding of the mechanisms for
acquisition of EMB resistance among these isolates requires further
studies.

Resistance to PZA

PZA, a structural analog of nicotinamide, was shown to have considerable
anti-MTB activity in 1952, but it became an important component of
short-course chemotherapy only in the mid- 1980s. PZA, active against
semidormant bacilli not affected by any other drug, has strong synergy with
INH and RIF and shortens the chemotherapeutic schedule for antitubercular
treatment from 9 to 12 months to 6 months (15). Depending on the assay
system and conditions applied, MICs of PZA vary from 8 g/ml to 60 g/ml.
However, even at very high MICs, PZA has no significant bactericidal effect
and is primarily considered a "sterilizing drug" (18). Activity of PZA is
highly specific for MTB; PZA has scant or no effect on other mycobacteria,
including M. bovis, which demonstrate high-level intrinsic resistance to
PZA (89). Naturally resistant strains of M. bovis lack the enzyme Pzase,
which hydrolyzes PZA to pyrizinoic acid, the presumed active form of PZA
(90,91). PZA in this context is similar to INH; it is transported as a
neutral species into the cell, where it is converted into its active form.
This notion was strengthened by evidence provided by in vitro studies that
demonstrated the susceptibility of PZA-resistant MTB and M. bovis to
pyrizinoic acid. MTB Pzase has both pyrazinamidase and nicotinamidase
activities (90). Using sequence information of E. coli nicotinamidase,
Scorpio and Zhang isolated the mycobacterial pncA gene, which codes for the
amidase (92). Characterization of the pncA gene from M. bovis isolates
identified a single point mutation that results in the substitution of His
to Asp at position 57. This substitution results in the production of an
ineffective Pzase in M. bovis strains. Point mutations in the pncA gene of
PZA-resistant MTB strains were also identified. Substitution of Cys138 with
Ser, Gln141 with Pro, and Asp63 with His and deletion G nucleotide at
positions 162 and 288 resulted in a defective Pzase. Transformation of
Pzase-resistant strains with functional construct of MTB pncA gene restored
susceptibility to PZA, providing further evidence that mutations in the
pncA gene were responsible in conferring the resistant phenotype.
Subsequent characterization of the pncA gene from clinical isolates of MTB
confirmed these findings (93,94). Mutations including missense alterations,
nucleotide insertions or deletions, and termination mutations have been
found in the pncA gene from PZA-resistant MTB isolates. These sequence
alterations are interspersed along the entire length of the pncA gene,
demonstrate limited degree of clustering, and vary in frequency from 70% to
100% (93,94). The absence of correlating mutations in the pncA gene from
PZA- resistant MTB isolates indicates that perhaps at least one additional
mechanism mediates resistance to PZA.

The cellular target for PZA, however, has not been identified, although the
apparent similarity of PZA to nicotinamide suggests that enzymes involved
in pyridine nucleotide biosynthesis are probable targets. Implication of
the pncA gene in conferring PZA-resistant phenotype has profound clinical
applications. Application of PCR-SSCP for detection of mutations in the
pncA gene could help circumvent the difficulties in determining PZA
susceptibilities (96) and rapidly discriminate between MTB and M. bovis
(96).

Resistance to Fluoroquinolones (FQ)

FQs as antimycobacterial agents were first described in 1984 and have
primarily been used as therapeutic alternatives in MDRTB cases (97). DNA
gyrase (Gyr), a member of the type II DNA topoisomerases (98), is the
primary target for FQ action. Gyr introduces negative supercoils in closed
circular DNA molecules and is a heterotetramer (A2B2), coded by gyrA and
gyrB respectively (99,100). Quinolone sensitivity is determined by the GyrA
protein, which contains the cleavage/religation activity (100), while GyrB
contains the intrinsic coumarin-sensitive ATPase activity (101).

FQs, synthetic derivatives of nalidixic acid, act by inhibiting DNA
supercoiling and relaxation activity of Gyr without affecting the ATPase
activity (102) and enhance the rate of DNA cleavage by Gyr.
Quinolone-mediated cleavage of double-stranded DNA results in a 4 bp 5'
overhangs on either strand, to which GyrA subunits become attached
covalently by O4 phosphotyrosine bond (103). Gyr catalyzes the cutting of
DNA, denaturation of the overhang, and strand separation. The exact
mechanism of inhibition of Gyr activity with respect to quinolones remains
unknown. However, quinolone drugs bind with a greater affinity to
single-stranded DNA than double-stranded DNA and possibly do not bind to
Gyr at all (104). Consequently, by binding to the single-stranded DNA, the
quinolones may inhibit religation, thereby imposing an effective
transcriptional block (105), culminating in cellular death. However,
questions about the specific interaction of quinolones and the Gyr/DNA
complex remain unsolved (106).

Cloning and expression of the MTB gyrA and gyrB genes allowed mapping
of mutations that confer resistance to FQs (107). Mutations were found to
be clustered in a small region in GyrA that is close, approximately 40
residues amino-terminal, in the linear amino acid sequence to the
active site tyrosine, Tyr122 (E. coli numbering) (108). Other single amino
substitutions, for residues 88 to 94, were also identified in
ciprofloxacin-resistant MTB isolates (Figure 5). Because polymorphism
encountered at codon 95 (Ser95>Thr95) occurred in both resistant and
susceptible isolates, it may not be involved in acquiring the FQ-resistant
phenotype. Alternative mechanisms to gyrA mutations, including changes in
cell wall permeability and active quinolone efflux pumping, have also been
proposed and could account for the low-level resistance among MTB isolates.

                            fig 5]
Figure 5. Single-amino acid substitutions responsible for conferring 
resistance to fluoroquinolones (FQ). Mutation in the Ser95 codon (shown 
in stippled box), observed in both FQ-sensitive and FQ-resistant isolates, 
rules out its role in acquisition of resistance.

Newer FQ derivatives such as sparfloxacin have shown greater anti-MTB
potency (MIC = 0.2 g/ml) than ciprofloxacin and ofloxacin, giving hope for
better therapeutic alternatives for MDRTB. However, FQ susceptibility in
the treated patient population must be continuously monitored to prevent
low-level FQ-resistant strains from acquiring additional mutations that
lead to high-level resistance (109).

Resistance to Streptomycin and Other Inhibitors Of Protein Synthesis

Various drugs exert their antibacterial effects by inhibiting the protein
transitional machinery. Among these, aminoglycosides, macrolides,
tetracyclines, and basic peptides like viomycin and capreomycin are active
against mycobacteria (110). SM, one of the oldest drugs known to be active
against MTB, disrupts the decoding of aminoacyl-tRNA and thus inhibits mRNA
translation or causes inefficient translation (111). One of the most common
mechanisms for acquisition of resistance to SM is acetylation of the drug
by aminoglycoside-modifying enzymes (111,112). However, this mechanism is
not found in MTB. Instead, resistance to SM is attributed, at least
partially, to two distinct classes of mutations including point mutations
in S12 ribosomal protein, encoded by rpsL gene (113), and mutations in the
rrs operon encoding the 16S rRNA (114).

Point mutations in the rpsL gene result in single amino acid substitutions
(114-117) that affect higher order structures of 16S rRNA and thereby
confer SM resistance. Mapping of the mutations in the rpsL gene
demonstrated that they primarily affected one of the two critical lysine
residues at positions 43 and 88 and led to the substitution with either
arginine at 88 or arginine and threonine at position 43 (115). An
SM-resistant isolate (>60 g/ml) showed an A>G transversion at position 904
in the 16S rRNA with an additional single A>C transversion in the rpsL
gene, which resulted in the substitution of Lys-Gln at position 88 (115).
Because each of the corresponding mutations in the small subunit rRNA or
the ribosomal protein S12 confer the resistant phenotype in E. coli, these
mutations mediated ribosomal drug resistance and were responsible for
conferring high-level SM resistance. Mutations in the rpsL gene accounted
for more than two thirds of SM-resistant cases.

The genesis of SM resistance in some of the SM-resistant isolates is due to
point mutations in the 16S rRNA. Mutations in the rrs locus have been
mapped to two regions, the 530 loop and the 915 region. Within the 530
loop, C>T transitions at 491, 512, and 516, in addition to the A>C
transversion at position 513, are consistent with the SM-resistant
phenotype (114) pseudoknot formation within the MTB 16S rRNA. Base pairing
between residue 524-526 (of the 530 region of the hairpin loop) and residue
504-507 (of the adjacent 510 region bulge loop) (118) results in SM
resistance in clinical isolates of MTB (114). Further, G-U wobble base
pairing between residues 522-501 stabilizes the pseudoknot formation and
thereby confers resistance to SM. It can thus be concluded that SM
resistance in MTB stems from alterations of the drug target and not by drug
modification.

However, no mutations in the rpsL and the rrs genes are detected in a
significant number of SM-resistant isolates (46,48). Curiously,
intrinsically SM-resistant strains of M. gordonae, M. szulgae, and M. avium
do not show any alterations in the rpsL or the rrs genes, suggesting a
probable third factor in conferring SM resistance. Earlier studies have
documented the inhibitory effect of SM on protein synthesis in vitro to the
same extent as observed in wild-type MTB strains. The same inhibitory
effect was not observed on whole cells, suggesting the probable role of
cell wall permeability barrier in conferring SM resistance (119). More
recently, it has been demonstrated that membrane-active substances
augmented the MIC for SM in strains with alterations in the rrs genes, thus
providing further evidence for a probable role of the MTB-permeability
barrier in mediating resistance to SM (120).

Resistance to Other Drugs

Related aminoglycosides such as kanamycin, amikacin, and paromomycin
demonstrate no obvious cross-resistance to SM and thus are alternatives in
cases of SM resistance. Viomycin and capreomycins are bacteriostatic agents
that act by binding to the 50S ribosomal subunit and inhibit the
translocation reaction (111). Although cross-resistance between viomycin
and capreomycin does occur, the exact mechanism for acquisition of drug
resistance is not known.

Conclusions

Molecular insights suggest that accumulation of mutations in the individual
drug target genes is the primary mechanism of MDRTB. Morris and colleagues'
investigation of the molecular mechanisms of drug resistance in MDR strains
found that 25 of 44 SM-resistant strains had mutations in the rpsL gene,
while five others had rrs gene perturbations (48). The rpoB gene had
mutations in 28 of 29 RIF-resistant strains. Mutation in the katG gene was
seen in 20 of the 42 INH-resistant stains, while five had inhA gene
mutations. Of the 20 MDRTB strains, 11 had mutations in genetic markers
associated with resistance to each of these three drugs.

Similarly, Heym et al. reported that resistance to antitubercular agents in
their collection of strains resulted from alterations to chromosomal genes
encoding the drug targets; they excluded the possibility that MDRTB stemmed
from acquisition of genes for novel resistance determinants (46). MDR
appeared to result from the stepwise acquisition of new mutations in the
genes for different drug targets. In all cases exactly the same mutations
or combination of mutations were observed, regardless of the patient's HIV
status.

Thus, the origin of MDRTB is due more to treatment difficulties, including
noncompliance and administration of inadequate treatment regime, and not to
the emergence of novel resistance mechanisms; this is reassuring for the
future of short-course chemotherapy. Administration of directly observed
combination chemotherapy (or Directly Observed Treatment Short-Course
[DOTS]) appears to be the most effective way to ensure a decrease in
primary resistance, acquired resistance, and relapses (3). DOTS has been
successfully implemented in diverse geographic areas including Tanzania,
Guinea, China, Bangladesh, New York City, and Peru, which reported more
than a 90% cure rate (3). Nearly 70 countries have adapted DOTS as a part
of their national TB control programs and achieve good cure rates.
Successful implementation of DOTS in the coming decades requires not only a
concerted effort from various funding agencies but also a strong social and
political commitment. Apart from strategic interventions based on strong
political will, grass-roots action will have to be strengthened mainly at
the primary health-care level to check the unlimited upsurge of this
preventable fatal disease. Basic research will have to be continually
updated to prevent the drug-resistant strains from becoming an unmanageable
clinical paradigm. DOTS currently is our only option to reverse the global
TB epidemic and prevent MDRTB.

The inability to detect resistance early, however, is one of the major
factors involved in the genesis and control of MDRTB; this invariably
results in prolonged exposure to drugs that are virtually ineffective. One
of the major consequences of unraveling the genetic basis of drug
resistance in MTB is the development of various molecular strategies to
rapidly detect MDRTB (76). However, the sheer multiplicity of gene loci to
be investigated for diagnosis of MDRTB renders most of the approaches
mentioned above as tedious and resource-intensive for a routine laboratory
service program, particularly in developing countries like India, with
limited resources and high disease incidence. Resistance to most anti-MTB
drugs, with the exception of RIF, cannot be attributed to a single locus in
substantial percentage (>90%), which is perhaps the greatest deterrent in
the development of single amplificationbased methods for rapid detection
of resistance.

Working out the exact biochemical details of drug-drug target interaction
acquires considerable attention in the era of MDRTB, because only then will
more rational structure- and mechanism-based approaches to inhibitor design
be possible. Clearly, a concerted global effort is required to defeat TB
resurgence.

Acknowledgments

We are indebted to Dr. Ellen Jo Baron and Prof. Eric C. Bottger for their
suggestions and encouragement; Dr. James M. Musser for providing recent
data on ethambutol resistance; and Swathi Arur, Nadeem Hasan, and Koninika
Ray for help in preparing this manuscript.

Work in our laboratory is supported by financial grants from the Department
of Biotechnology, Government of India.

Dr. Ashok Rattan is an additional professor, Department of Microbiology,
All India Institute of Medical Sciences, New Delhi, India. His research
focuses on drug resistance and application of cost-effective methods for
surveillance of MDRTB strains.

Address for Correspondence: Ashok Rattan or Awdhesh Kalia, TB and STD
Section, Department of Microbiology, All India Institute of Medical
Sciences, Ansari Nagar, New Delhi 110 029, India; fax: 91-11-686-2663;
e-mail: rattan@medinst.ernet.in or awdhesh@aiims.ernet.in.

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Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/rattan.htm

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[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Synopses

Hospitalizations for Unexplained Illnesses among U.S. Veterans of the
Persian Gulf War

James D. Knoke and Gregory C. Gray
Naval Health Research Center, San Diego, California, USA

  --------------------------------------------------
      Persian Gulf War veterans have reported a variety of symptoms,
      many of which have not led to conventional diagnoses. We
      ascertained all active-duty U.S. military personnel deployed to
      the Persian Gulf War (552,111) and all Gulf War era military
      personnel not deployed (1,479,751) and compared their postwar
      hospitalization records (until 1 April 1996) for one or more of
      77 diagnoses under the International Classification of Diseases
      (ICD-9) system. The diagnoses were assembled by the Emerging
      Infections Program, Centers for Disease Control and Prevention,
      and are here termed "unexplained illnesses." Deployed veterans
      were found to have a slightly higher risk of hospitalization
      for unexplained illness than the nondeployed. Most of the
      excess hospitalizations for the deployed were due to the
      diagnosis "illness of unknown cause" (ICD-9 code 799.9), and
      most occurred in participants of the Comprehensive Clinical
      Evaluation Program who were admitted for evaluation only. When
      the effect of participation in this program was removed, the
      deployed had a slightly lower risk than the nondeployed. These
      findings suggest that active-duty Gulf War veterans did not
      have excess unexplained illnesses resulting in hospitalization
      in the 4.67-year period following deployment.

The Persian Gulf War was one of the briefest full-scale conflicts in U.S.
history. For a 2-month period of fighting ending in March 1991, nearly
700,000 U.S. service members were deployed to the Persian Gulf region.
Since returning from the war, many veterans have reported unexplained
symptoms (1-5), prompting allegations of a new disease or diseases
(2,3,5,6). Numerous expert panels and research projects have examined
illness and death among Gulf War veterans (4,7,8). However, with the
exception of self-reported symptoms (8-14), no consistent pattern of
increased illness or death has been reported (10,14-18).

The U.S. Department of Defense (DoD) conducted an epidemiologic comparison
of the postwar DoD hospitalizations of service members deployed to the Gulf
War and service members of the same era not deployed (15). In the 2-year
period after the war, no consistent increase in the overall risk for
hospitalization (or specific risk for hospitalization for various broad
diagnostic categories) was found for those deployed. This study relied upon
diagnoses based on the International Classification of Diseases, 9th
Revision, Clinical Modification (ICD-9) system (19). Medical providers may
not consistently classify a new or poorly recognized syndrome, and
consequently a true difference in hospitalization risk could be spread
across numerous diagnostic categories and remain undetected.

The present study compares the postwar DoD hospitalizations for diagnoses
consistent with an unexplained illness of Persian Gulf War veterans and
their nondeployed peers.

Analytic Approach

Study Population

The study population consisted of active-duty service members (Army, Air
Force, Navy, Marine Corps, and Coast Guard) who were either deployed to the
Persian Gulf War for 1 or more days during the Gulf War deployment period
(8 August 1990 through 31 July 1991) or were not deployed but were on
active duty for at least part of the Gulf War deployment period. All
deployed (n = 552,111) and nondeployed (n = 1,479,751) service members who
remained on active duty at the end of the period were included in the study
population.

The study was restricted to active-duty personnel because they are rarely
hospitalized outside DoD facilities. Members of the U.S. Reserve and
National Guard forces were not studied because only a fraction of their
postdeployment hospitalizations were in DoD facilities.

Study Outcome

The study outcome was determined by 77 ICD-9 diagnoses assembled by the
Emerging Infections Program, Centers for Disease Control and Prevention, to
monitor death certificates for unexplained deaths (20). These diagnoses
span several of the 17 major categories delineated in ICD-9 and include
selected diagnoses from diseases of the blood, nervous system, circulatory
system, respiratory system, and digestive system; infectious and parasitic
diseases; and symptoms, signs, and ill-defined conditions. These diagnoses
primarily relate to nonspecific infections and other ill-defined
conditions, and for convenience they are termed "unexplained illnesses" in
this study.

All study population admissions to U.S. military hospitals worldwide
(reported to the DoD computerized hospitalizations database by 1 October
1996), subsequent to the Gulf War deployment period and before 1 April
1996, were evaluated for unexplained illnesses among as many as eight ICD-9
diagnoses coded for each admission. For another analysis, only the first
coded diagnosis (the principal diagnosis) was ascertained. The DoD ICD-9
coding guidelines define the principal diagnosis as "the condition
established, after study, to be responsible for the admission."
Hospitalizations prior to the conclusion of the deployment period were not
evaluated because access to care for the deployed differed markedly from
that for the nondeployed during this time. Outpatient visits were not
studied because they are not computerized centrally by DoD.

Data

Demographic variables available for use as covariates included age,
race/ethnicity, occupation, rank, salary, branch of service, length of
service, marital status, and gender. Time-dependent variables were
evaluated as of 31 July 1990. A previous study (15) found hospitalization
for any reason in the 12 months preceding the Gulf War deployment period to
be an important predictor of postwar hospitalization. This indicator
variable may be a surrogate for baseline health status and was also used as
a covariate. Demographic data, including deployment status, were obtained
from the Defense Manpower Data Center, Seaside, California. Hospitalization
information was obtained from the Data Processing Center, Fort Detrick,
Frederick, Maryland. Comprehensive Clinical Evaluation Program data were
obtained from the Deployment Surveillance Team, Falls Church, Virginia.

Statistical Analysis

Frequencies of selected diagnoses and causes of death were calculated. The
Cox proportional hazards survival analysis model (21) was used to obtain
the risk ratio (RR) and 95% confidence interval (CI) of deployment status
(deployed relative to nondeployed) for an event consisting of
hospitalization with an unexplained illness, adjusting for the covariates.
Follow-up time was computed from 1 August 1991 until hospitalization in any
DoD hospital worldwide with at least one unexplained illness, separation
from the service, or until 31 March 1996, whichever occurred first. All
data management and statistical calculations were performed with the
Statistical Analysis System (22).

Findings

Frequent Unexplained Illnesses

Our study population consisted of 25,495 first hospitalizations (with at
least one unexplained illness among the eight possible diagnoses), 6,672 in
the deployed and 18,823 in the nondeployed. For these hospitalizations, the
10 most frequent first unexplained illnesses among all eight possible
diagnoses were tabulated (Table 1). Eight of these diagnoses occurred with
similar proportional distributions between the two groups. The diagnosis
"nonspecific abnormal findings in the amniotic fluid" accounted for a
higher proportion of hospitalizations among the nondeployed than among the
deployed. This is consistent with a higher proportion of women among the
nondeployed (12.7%) than among the deployed (6.1%). The tenth most frequent
diagnosis, "illness of unknown cause," accounted for a considerably greater
proportion of hospitalizations among the deployed (8.3%) than among the
nondeployed (1.8%).

Table 1. Frequencies of the 10 most common diagnoses for first hospitalizations
with an unexplained illness, considering all eight coded diagnosesa

------------------------------------------------------------------------------
                                          Frequency              Percentage 
                                                              of all diagnoses
                                   ------------------------ --------------------
ICD-9                                               Non-                Non-
Code      Description            Total  Deployed  deployed  Deployed  deployed
-----------------------------------------------------------------------------------
008.8     Unspecified             2286    581      1705        8.7       9.1
          intestinal infections
047.9     Unspecified viral        946    236       710        3.5       3.8
          meningitis
079.9     Unspecified viral       3540    896      2644       13.4      14.1
          infections
465.9     Acute URIb,             1999    527      1472        7.9       7.8
          unspecified site
486       Pneumonia, organism     2840    719      2121       10.8      11.3
          unspecified
780.6     Pyrexia of unknown       961    233       728        3.5       3.9
          origin
785.6     Enlarged lymph          1587    423      1164        6.3       6.2
          nodes
786.0     Dyspnea                 2736    703      2033       10.5      10.8
792.3     Nonspecific
          abnormal amniotic       1317    188      1129        2.8       6.0
          fluid
799.9     Illness of unknown       892    555       337        8.3       1.8
          cause
Subtotal                        19,104   5061    14,043       75.9      74.6
Other                             6391   1611      4780       24.1      25.4
Total                           25,495   6672    18,823      100.0     100.0
-------------------------------------------------------------------------------------
(sup a)Only the first unexplained illness coded for each hospitalization was
tabulated.
(sup b)URI, upper respiratory infection.

An unexplained illness was the principal diagnosis in 13,490 first
hospitalizations, 3,525 from the deployed and 9,965 from the nondeployed.
The proportional distributions of the 10 diagnoses reported in Table 1
showed some rearrangements when only the principal diagnosis was considered
(Table 2). The percentages of "acute upper respiratory infection,
unspecified site"; "nonspecific abnormal amniotic fluid"; and "illness of
unknown cause" were dramatically lower as the principal diagnosis than they
were as any diagnosis. However, "illness of unknown cause" remained the
only diagnosis that accounted for an appreciably larger proportion of
admissions among the deployed (2.0%) than among the nondeployed (0.3%).

Screening of Covariates

The demographic characteristics of the deployed and nondeployed groups have
been reported (15). All available covariates were included in a preliminary
Cox proportional hazards model to assess their effect on the risk for
hospitalization for an unexplained illness (Table 3). All available
covariates (one variable at a time) were also included in a series of
preliminary models for this same purpose. These two approaches to screening
covariates gave somewhat different results, probably because of
multicolinearity among the covariates. The second approach was less useful
than the first, so it is not further reported here.

Given the other covariates, age, marital status, and length of service were
only minimally related to risk (possibly because of colinearity) and were
not included in subsequent model analyses. The covariates retained included
race (coded as white; black; or other, including unknown); rank (coded as
enlisted or warrant or commissioned officer, including unknown); salary
(coded as less than $1,000; $1,000 to $1,399; or at least $1,400 a month,
including unknown); and branch of service (coded as Army or other). The
military system has a large number of occupational categories; however,
only "health-care worker" appeared to have an appreciably higher risk for
hospitalization for an unexplained illness than other occupational
categories. Consequently, occupation was simply coded as health-care worker
or other (including unknown). Prewar hospitalization status was coded as
"yes" if one was hospitalized for any reason during the 12 months before 1
August 1990 and as "no" otherwise. All of these other covariates, as well
as gender (with unknowns included with men), had a highly statistically
significant effect on the risk for hospitalization for an unexplained
illness and were included in all subsequent model analyses. Other possible
covariates, whose effect on risk was either less highly significant or
nonsignificant, were not included so as to minimize unnecessary computation
and variance inflation.

Survival Analysis, Using All Eight Diagnoses

A model analysis including deployment status and the selected covariates
showed that deployment status was significantly associated with
hospitalization for an unexplained illness when all eight possible
diagnoses were used (Table 4). Separate model analyses for the two
deployment status groups indicated that the parameter estimates were
similar, and the effect of the covariates on the probability of
hospitalization was essentially independent of deployment status.

Table 2. Frequencies of the 10 most common diagnoses for first hospitalization
with an unexplained illness, considering only the principal diagnosis
--------------------------------------------------------------------------------------
                                                                   Percentage of all
                                              Frequency               diagnoses
                                       ------------------------   --------------------
ICD-9                                                     Non-                Non-
Code      Description                Total    Deployed  deployed  Deployed  deployed
--------------------------------------------------------------------------------------

008.8     Unspecified intestinal      1807       467      1340      13.2      13.5
          infections
047.9     Unspecified viral            912       228       684       6.5       6.9
          meningitis
079.9     Unspecified viral           2620       661      1959      18.7      19.8
          infections
465.9     Acute URI, unspecified       487       151       336       4.3       3.4
          site
486       Pneumonia, organism         2350       615      1735      17.5      17.4
          unspecified
780.6     Pyrexia of unknown           382        93       289       2.6       2.9
          origin
785.6     Enlarged lymph nodes        1000       274       726       7.8       7.3
786.0     Dyspnea                     1176       302       874       8.6       8.8
792.3     Nonspecific abnormal           5         1         4       0.0       0.0
          amniotic fluid
799.9     Illness of unknown cause     101        72        29       2.0       0.3

Subtotal                            10,840      2864      7976      81.2      80.0
Other                                 2650       661      1989      18.8      20.0
Total                               13,490      3525      9965     100.0     100.0
---------------------------------------------------------------------------------------

The probabilities under the separate models of hospitalization with an
unexplained illness, at the mean values of the included covariates, are
presented as a function of follow-up time in Figure 1. These probabilities
for the two groups were virtually coincidental and linear over time, until
late 1994 when the deployed group's probability increased. This shift in
the historical track for the deployed group suggests that the hazards for
the two groups were not proportional over time and that additional analyses
may be indicated.

The Comprehensive Clinical Evaluation Program

In May 1994, DoD announced a Comprehensive Clinical Evaluation Program
(CCEP), offering thorough clinical examinations and evaluations to Gulf War
veterans who sought them. This program was implemented in June 1994. The
divergence in probability curves (Figure 1) in the last quarter of 1994
prompted us to investigate whether the introduction of CCEP may have
affected the probability of hospitalization.

Referring to any hospitalization for unexplained illness after 1 June 1994
of a CCEP participant as a CCEP hospitalization, we found that 837 first
unexplained illness hospitalizations in the deployed were CCEP
hospitalizations, as were 55 in the nondeployed. (For the purpose of CCEP
participation, deployment status was self-determined.) Many of the
nondeployed (according to Defense Manpower Data Center data) CCEP
participants may have been in the Persian Gulf region after 1 August 1991.
Of these 892 hospitalizations, 59% of the first diagnoses for unexplained
illnesses were "illness of unknown cause," the most nonspecific diagnosis
in ICD-9. Furthermore, with 128 DoD hospitals operating worldwide in 1995,
60% of these hospitalizations were in six facilities-three Army hospitals
and three Air Force hospitals-and fewer than 1% were in any Navy facility.

Table 3. Frequencies and risk ratios under the Cox proportional hazards model
for all hospitalizations with at least one unexplained illness, all covariates,
1 August 1991 to 1 April 1996

------------------------------------------------------------------------------------
                                                                            95%        
                                                      Non-     Risk      Confidence  
Variable                                 Deployed   deployed ratio(sup a) interval
-------------------------------------------------------------------------------------
Not deployed                                        1,479,751
Deployed                                  552,111               1.08     1.05-1.11
Not hospitalized before war(sup b)        513,486   1,375,169
Hospitalized before war                    38,625     104,582   1.73     1.66-1.79
Not married                               258,821     602,553
Married                                   291,009     875,309   1.05     1.02-1.08
Unknown marital status                       2282        1889   0.64     0.41-1.00
Male                                      517,223   1,291,323
Female                                     33,690     188,273   2.13     2.06-2.20
Unknown gender                               1198         155   0.60     0.20-1.75
White                                     383,704   1,110,949
Black                                     130,249     286,427   1.05     1.02-1.08
Hispanic                                   12,900      26,696   0.91     0.83-1.00
Other                                      23,592      54,128   0.91     0.85-0.97
Unknown race/ethnicity                       1666        1551   1.08     0.69-1.68
Age in years:         17-21               142,547     343,535
                      22-25               146,652     306,272   0.94     0.90-0.97
                      26-31               138,173     373,698   0.95     0.90-1.00
                      >/=32               123,353     455,302   1.05     0.99-1.12
                      Unknown                1386         944   1.04     0.57-1.88
Infantry, gun crews, seamanship           140,742     273,079
Electronic equipment repair                45,263     159,195   0.99     0.93-1.04
Communications/intelligence                55,665     120,108   1.00     0.95-1.06
Health care                                28,398     107,591   1.46     1.39-1.53
Other technical                            11,633      33,246   1.02     0.93-1.11
Administration                             62,194     255,576   0.96     0.93-1.02
Construction/related trades                19,424      47,800   1.11     1.06-1.17
Trainees, undesignated                       6421      64,625   1.02     0.95-1.10
Unknown job category                       18,147      80,911   1.18     1.11-1.25
Enlisted                                  489,034   1,238,790
Warrant Officer                              7760      13,555   0.79     0.69-0.89
Commissioned Officer                       53,121     227,381   0.70     0.65-0.75
Unknown status                               2196          25   0.43     0.20-0.93
Salary/month:             <$1000          122,161     328,187
                     $1000-$1399          269,500     573,995   0.81     0.77-0.85
                     $1400-$2099           75,523     252,923   0.68     0.63-0.72
                     $2100-$3199           60,528     200,295   0.73     0.68-0.79
                        >/=$3200           22,203     124,326   0.74     0.66-0.83
                         Unknown             2196          25   -

Army                                      258,858     459,644
Navy                                      141,604     423,138   0.61     0.59-0.64
Marine Corps                               83,377     113,467   0.66     0.63-0.70
Air Force                                  67,942     447,125   0.76     0.73-0.78
Coast Guard                                   330      36,377   0.42     0.37-0.48
Service in months:          < 22          124,629     262,260
                           22-54          162,123     269,727   1.05     1.00-1.11
                          55-126          133,608     363,138   0.97     0.92-1.02
                          >/=127          131,751     584,626   1.12     1.07-1.18
-----------------------------------------------------------------------------------------
(sup a)Risk ratios are relative to the first group in each variable category. When the
algorithm failed to converge, because of lack of cases in the category, a dash
(-) is given.
(sup b)In the 12 months preceding 1 August 1990.



Table 4. Frequencies and risk ratios under the Cox proportional hazards model
for all hospitalizations with at least one unexplained illness, selected
demographic variables, 1 August 1991 to 1 April 1996

--------------------------------------------------------------------------------------------
                                                                                   95%                                           
                                                            Non-      Risk      Confidence 
Variable                                   Deployed       deployed  ratio(sup a) interval
--------------------------------------------------------------------------------------------
Not deployed                                  -          1,479,751      
Deployed                                   552,111         -           1.06      1.03-1.09
Not hospitalized before war(sup b,c)       513,486       1,375,169
Hospitalized before war                     38,625         104,582     1.71      1.65-1.78
Male(sup c)                                518,421       1,291,478
Female                                      33,690         188,273     2.11      2.04-2.18
White                                      383,704       1,110,949
Black                                      130,249         286,427     1.04      1.01-1.08
Other race/ethnicity(sup c)                 38,158          82,375     0.91      0.86-0.96
All other occupations(sup c)               523,713       1,372,160
Health-care worker                          28,398         107,591     1.44      1.38-1.50
Enlisted                                   489,034       1,238,790
Officer(sup c)                              63,077         240,961     0.66      0.62-0.70
Salary/month:            <$1,000           122,161         328,187
                   $1,000-$1,399           269,500         573,995     0.77      0.74-0.79
                 >/=$1,400(sup c)          160,450         577,569     0.82      0.78-0.87
Army                                       258,858         459,644
Other branches                             293,253       1,202,107     0.60      0.58-0.62 
----------------------------------------------------------------------------------------------
(sup a)Risk ratios are relative to the first category for each variable.
(sup b)In the 12 months preceding 1 August 1990.
(sup c)Includes unknown.



These unusual results prompted us to make inquiries at some of the DoD
hospitals reporting large numbers of hospitalizations for "illness of
unknown cause." We learned that several of the larger Army and Air Force
hospitals established special wards for CCEP participants reaching phase 2
of the evaluation process, admitted them for several days, and performed
extensive evaluations (including invasive procedures and sleep studies)
during hospitalization. These facilities also gave at least some of these
participants a diagnosis of "illness of unknown cause." This coding
practice for evaluation admissions may have resulted from a memo dated 25
August 1994 from the Headquarters, U.S. Army Medical Command at Fort Sam
Houston, San Antonio, Texas, which directed Army facilities to code CCEP
participants with "unexplained complaints with no confirmed diagnosis" as
799.9. The practice of admitting CCEP participants was discontinued by
mid-1995. The probability of hospitalization curves tended to become
parallel again about mid-1995 (Figure 1). Thus, we inferred that a
substantial majority of the CCEP hospitalizations were primarily for
evaluation.

Independent evaluation supported this inference. Since DoD computerized
hospital records do not include cause of hospitalization or any other
indication of whether a patient may have been hospitalized primarily for
evaluation, we randomly selected for chart review 50 CCEP hospitalizations
from each of the Army and Air Force facilities with the greatest numbers of
CCEP hospitalizations. Both of these facilities were located in the same
metropolitan area, San Antonio, Texas. The selected CCEP hospitalizations
were independently evaluated by two clinicians, one from each facility.
Seventy-nine charts were reviewed, 44 from the Air Force facility and 35
from the Army facility. The other 21 records were located in satellite
facilities or otherwise not readily accessible. The two clinicians agreed
that 77 of these hospitalizations were for evaluation only and would not
have been considered hospitalizations had the CCEP not been in effect. One
of the clinicians thought that two of the hospitalizations were for
clinical management and would have occurred regardless of CCEP.

 [fig 1]
 Figure 1. Probability of hospitalization for unexplained illness, deployed
 and nondeployed veterans.



Survival Analysis, Using All Eight Diagnoses, Censoring CCEP Participants

We repeated the survival analyses (reported in Table 4; Figure 1),
censoring all CCEP participants on 1 June 1994. This change resulted in
5,835 first hospitalizations for an unexplained illness in the deployed
group and 18,768 in the nondeployed. No important differences were observed
in the effects of the covariates between the models censoring CCEP
participants on 1 June 1994 and those reported in Table 4. However, when
CCEP participants were censored, the risk for hospitalization for an
unexplained illness was lower in the deployed than in the nondeployed (RR =
0.93, CI = 0.91 to 0.96), and the probability of hospitalization for an
unexplained illness was generally lower over time for the deployed than for
the nondeployed (Figure 2). Also, the proportional hazards assumption now
appeared reasonable.

CCEP participants may truly have been at increased risk for hospitalization
for an unexplained illness, in spite of many CCEP hospitalizations having
been for evaluation only. Consequently, the true risk for the deployed is
likely to be inter- mediate to that depicted in Figures 1 and 2. However,
our record review, which concluded that the preponderance of CCEP
hospitalizations were for evaluation only, suggested that the results are
more closely depicted in Figure 2 than Figure 1.

[fig 2]
Figure 2. Probability of hospitalization for unexplained illness, deployed
and nondeployed veterans, censoring comprehensive clinical evaluation
program participants on 1 June 1994.



Survival Analyses, Using Principal Diagnosis Only

Model analysis of hospitalizations for which the principal diagnosis was an
unexplained illness showed that deployment status was not a statistically
significant predictor (RR = 0.99, CI = 0.95 to 1.03). When CCEP
participants were censored on 1 June 1994, model analysis results were
similar and showed that the deployed were slightly less likely than the
nondeployed to be hospitalized (RR = 0.93, CI = 0.89 to 0.97). Model
analysis results for hospitalization for an unexplained illness using only
the principal diagnosis thus were similar to the results when all eight
diagnoses were employed, except that there were many fewer admissions with
"illness of unknown cause" as a principal diagnosis than as a secondary
diagnosis.

Deaths

During hospitalization with an unexplained illness 348 veterans died. Of
those who died, 86 had been deployed, and 262 had not. Of the 348 deaths,
only 62 had an unexplained illness indicated as the underlying cause. Of
these 62, 17 had been deployed and 45 had not. Deployed veterans with an
unexplained illness as the underlying cause of death included four with
"unspecified septicemia"; six with "pneumonia, organism unspecified"; three
with "other diseases of the lung"; and one each with "other primary
cardiomyopathies," "thrombotic microangiopathy," "unexplained death," and
"respiratory failure."

Conclusions

Because they live in close quarters and are required to travel, military
personnel commonly acquire diseases endemic to the regions they visit and
may serve as reservoirs and vectors for disease transmission when they
return home. A number of military deployment-related epidemics have
occurred in recent years. Persian Gulf War veterans have been diagnosed
with a new manifestation of leishmaniasis (23). Veterans of the
peace-keeping effort in Somalia have had increased hospitalization rates
for malaria (24), deployed Navy personnel have brought nonendemic strains
of HIV virus into the United States (25), and Russian soldiers have been
implicated in the mass epidemics of diphtheria in Eastern Europe (26).
These and other observations make surveillance for unexplained illnesses
among U.S. military personnel an important federal public health issue.

We designed this study to screen for unexplained illnesses using a
collection of ICD-9 diagnoses (20). Since unexplained illnesses generally
have no specific ICD-9 diagnoses, a nonspecific diagnosis from this
collection is likely to be used by hospital coders when an unexplained
illness is encountered. The DoD worldwide hospitalization data subsequent
to the Persian Gulf War were analyzed using the Cox proportional hazards
model for evidence of unexplained illnesses. Analyses were performed both
with all eight coded diagnoses and with only the principal diagnosis.

Model analysis showed that Gulf War veterans were more likely than their
peers to be hospitalized for unexplained illness. After 4.67 years of
follow-up, the cumulative probability of hospitalization for unexplained
illness was 0.0185 in the deployed and 0.0166 in the nondeployed (Figure
1). This increased hospitalization risk of 11% for the deployed was a
consequence of the recruiting for free clinical evaluations beginning in
June 1994, with most of the resulting CCEP hospitalizations being for
medical evaluation and not for clinical management. When CCEP participants
were censored on 1 June 1994, deployed Gulf War veterans were not at
greater risk than those not deployed. The slightly lower hospitalization
risk for the deployed than for the nondeployed (Figure 2) is consistent
with a healthy service member effect; that is, those selected for
deployment are, on average, slightly healthier than those not selected.

This study had a number of limitations. Some miscoding of Gulf War
deployment status was suggested by the finding that some nondeployed
veterans were evaluated under the CCEP as being Gulf War veterans. However,
veterans who did not serve in the Gulf region until after 31 July 1991 were
eligible for the CCEP, and this apparent discrepancy was less than 6%.
Also, many personnel separated from the service during the period of
follow-up; 59.8% of the deployed and 54.3% of the nondeployed had separated
by 1 April 1996. However, the fact that separating veterans receive
thorough medical screening and have the potential of receiving lifelong
disability benefits motivates them to be thorough in their reporting of
illness before separation. The broad categorization of deployment status
may have masked illness due to time- and geography-specific exposures, and
illnesses not serious enough to require hospitalization have not been
captured by our analyses. Finally, the collection of diagnoses used as the
outcome measure was not designed specifically for our purposes, but it does
have the advantage of prior development by other researchers (20).

This study also had its strengths. The large group sizes, along with the
availability of numerous important covariates (demographic variables and
prewar hospitalization), offer unusually high statistical power for the
detection of differences in the hospitalization risk between groups.
Additionally, discharge diagnoses are thoroughly recorded and edited by DoD
hospitals, and active-duty personnel have few opportunities to be
hospitalized outside the DoD system, which ensures high-quality data with
few missing values.

In summary, these analyses show a slightly greater hospitalization risk for
unexplained illness among deployed Gulf War veterans than among those not
deployed. However, the excess hospitalizations for the deployed are
attributable to participation in CCEP; most of these hospitalizations were
for medical evaluation, not clinical management. Consequently, before
initiation of CCEP, active-duty Gulf War veterans were not at increased
risk for hospitalization for unexplained illness.

Acknowledgments

We thank Drs. Edwin C. Matthews and Gregg T. Anders for completing the
record review of the 79 randomly selected CCEP hospitalizations. We also
thank Michael A. Dove and Wayne F. Woo for their assistance in obtaining
the deployment status and covariate data.

This is Naval Health Research Center report no. 96-35, supported by the
Department of Defense (Health Affairs) and the Naval Medical Research and
Development Command, Department of the Navy, Bethesda, Maryland, under work
unit no. 63738DP4464.001-6423.

James D. Knoke is statistician, Emerging Illness Division, Health Sciences
and Epidemiology Department, Naval Health Research Center, San Diego. His
work concentrates on methodology and applications in clinical trials and
epidemiology. He has special interests in the areas of survival analysis,
longitudinal analysis, multivariate analysis, and nonparametric methods. He
serves as an investigator in a number of epidemiologic studies among
Persian Gulf War veterans.

Captain Gregory C. Gray is head, Emerging Illness Division, Health Sciences
and Epidemiology Department, Naval Health Research Center, San Diego. He is
a medical epidemiologist, specializing in general preventive medicine and
public health. He has experience conducting epidemiologic investigations
among military populations, particularly those involving respiratory
pathogens. He serves as principal investigator for a number of large
epidemiologic studies among Persian Gulf War veterans.

Address for correspondence: James D. Knoke, Naval Health Research Center,
P.O. Box 85122, San Diego, CA 92186, USA; fax: 619-553-7601; e-mail:
knoke@nhrc.navy.mil.

References

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  2. Why are we sick? Army Times 1994 Apr 25:12-23.
  3. Cowley G, Hager M, Liu M. Tracking the second storm. Newsweek 1994 May
     16:56-7. Persian Gulf Veterans Coordinating
  4. Board. Unexplained illnesses among Desert Storm veterans: a search for
     causes, treatment, and cooperation. Arch Intern Med 1995;155:262-8.
  5. Brown D. Diagnosis unknown: Gulf War syndrome. The Washington Post
     1994 Jul 24;Sect. A:1 (col. 2-4).
  6. Nicolson GL, Rosenberg-Nicolson NL. Doxycycline treatment and Desert
     Storm. JAMA 1995;273:618-9.
  7. Research Working Group of the Persian Gulf Veterans Coordinating
     Board. Federally Sponsored Research on Persian Gulf Veterans'
     Illnesses for 1995. Washington: Department of Veterans Affairs; 1996.
     Annual Report to Congress.
  8. Institute of Medicine. Health consequences of service during the
     Persian Gulf War: recommendations for research and information
     systems. Washington: National Academy Press; 1996.
  9. Unexplained illness among Persian Gulf veterans in an Air National
     Guard unit: Preliminary report-August 1990-March 1995. MMWR Morb Mortal
     Wkly Rep 1995;44:443-7.
 10. DeFraites RF, Wanat ER, Norwood AE, Williams S, Cowan D, Callahan D.
     Investigation of a suspected outbreak of an unknown disease among
     veterans of Operation Desert Shield/Storm, 123rd Army Reserve Command,
     Fort Benjamin Harrison, Indiana, April 1992. Washington: Epidemiology
     Consultant Service, Division of Preventive Medicine, Walter Reed Army
     Institute of Research; 1992.
 11. Presidential Advisory Committee on Gulf War Veterans' Illnesses. Final
     Report, Dec 1996. Washington: U.S. Government Printing Office; 1996.
 12. Defense Science Board. Report of the Defense Science Board Task Force
     on Persian Gulf War Health Effects. Washington: Office of the Under
     Secretary of Defense for Acquisition and Technology; 1994.
 13. National Institutes of Health Technology Assessment Workshop Panel.
     The Persian Gulf experience and health. JAMA 1994;272:391-6.
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     symptoms among active-duty Seabees: Gulf War veterans versus era
     controls. In: Proceedings of the 123rd Annual Meeting of the American
     Public Health Association; 1995 Oct 31; San Diego, California.
     Washington: American Public Health Association; 1995.
 15. Gray GC, Coate BD, Anderson CM, Kang HK, Berg SW, Wignall FS, et al.
     The postwar hospitalization experience of U.S. Persian Gulf War
     veterans compared with other veterans of the same era. N Engl J Med
     1996;335:1505-13.
 16. Kang HK, Bullman TA. Mortality among U.S. veterans of the Persian Gulf
     War. N Engl J Med 1996;335:1498-504.
 17. Writer JV, DeFraites RF, Brundage JF. Comparative mortality among U.S.
     military personnel in the Persian Gulf region and worldwide during
     Operations Desert Shield and Desert Storm. JAMA 1996;275:118-21.
 18. Cowan DN, DeFraites RF, Wishik SM, Goldenbaum MB, Wishik SM. The risk
     of birth defects among children of Gulf War veterans. N Engl J Med
     1997;336:1650-6.
 19. The International Classification of Diseases, 9th Revision, Clinical
     Modification. 3rd ed. Washington: U.S. Department of Health and Human
     Services; 1991.
 20. Perkins BA, Flood JM, Danila R, Holman RC, Reingold AL, Klug LA, et
     al. Unexplained deaths due to possibly infectious causes in the United
     States: defining the problem and designing surveillance and laboratory
     approaches. Emerg Infect Dis 1996;2:47-53.
 21. Kalbfleisch JD, Prentice RL. The Statistical Analysis of Failure Time
     Data. New York: John Wiley & Sons; 1980.
 22. SAS Institute Inc. SAS Language Reference. Version 6. 1st ed. Cary
     (NC): SAS Institute Inc.; 1990.
 23. Magill AJ, Grogl M, Gasser RA Jr, Sun W, Oster CN. Visceral infection
     caused by Leishmania tropica in veterans of Operation Desert Storm. N
     Engl J Med 1993;328:1383-7.
 24. Wallace MR, Sharp TW, Romajzl PJ, Batchelor RA, Thornton SA, Longer
     CF, et al. Malaria in Mogadishu, Somalia. Clin Infect Dis
     1993;17:510-1.
 25. Brodine SK, Mascola JR, Weiss PJ, Ito SI, Porter KR, Artenstein AW, et
     al. Detection of diverse HIV-1 genetic subtypes in the USA. Lancet
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 26. Maurice J. Russian chaos breeds diphtheria outbreak. Science
     1995;267:1416-7.

Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/knoke.htm

Please note that figures and equations are not available in ASCII format; 
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-------------------------------------------------------------------------------------------
-------------------------------------------------------------------------------------------


[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Synopses

Agricultural Use of Burkholderia (Pseudomonas) cepacia: A Threat to Human
Health?

Alison Holmes,* John Govan, and Richard Goldstein
*Imperial College School of Medicine, Hammersmith Hospital, London, United
Kingdom; University of Edinburgh Medical School, Edinburgh, United
Kingdom; and Maxwell Finland Laboratory for Infectious Diseases,
BostonUniversity School of Medicine, Boston, Massachusetts, USA

  --------------------------------------------------
      In the past 2 decades, Burkholderia cepacia has emerged as a
      human pathogen causing numerous outbreaks, particularly among
      cystic fibrosis (CF) patients. One highly transmissible strain
      has spread across North America and Britain, and another
      between hospitalized CF and non-CF patients. Meanwhile, the
      organism has been developed as a biopesticide for protecting
      crops against fungal diseases and has potential as a
      bioremediation agent for breaking down recalcitrant herbicides
      and pesticides. However, B. cepacia is inherently resistant to
      multiple antibiotics; selection of strains "safe" for
      environmental application is not at present possible
      phenotypically or genotypically; molecular epidemiology and
      phylogenetic studies demonstrate that highly transmissible
      strains emerge randomly; and the organism has a capacity for
      rapid mutation and adaptation (facilitated by numerous
      insertion sequences), and a large, complex genome divided into
      separate chromosomes. Therefore, the widespread agricultural
      use of B. cepacia should be approached with caution.

Burkholderia (previously known as Pseudomonas) cepacia, a nutritionally
versatile, gram-negative organism, was first described in 1949 by Walter
Burkholder of Cornell University, as the phytopathogen responsible for a
bacterial rot of onions (1) (Figure 1). Ironically, B. cepacia is now being
considered by agricultural microbiologists as an agent to promote crop
growth.

[fig]    
Figure 1. B. cepacia causes an onion rot known as slippery skin (1). The onions
shown were inoculated with three strains of B. cepacia. Rot occurred in onion1
left), which was inoculated with strain originally isolated from onions. Rot did not
occur with environmental isolates tested or with strains from CF lung. 

B. cepacia is inherently resistant to
multiple antibiotics, can metabolize
diverse substrates, and is found in
soil and in moist environments. The
organism has a particular
predilection for the lung in patients
with cystic fibrosis (CF) and has
emerged as an important opportunistic
human pathogen in hospitalized and
immunocompromised patients (2,3).

B. cepacia as a Human Pathogen

In CF Patients

CF is a recessively transmitted genetic disease that occurs in
approximately 1 in 2,500 Caucasians (carrier frequency of 1 in 25). The
condition is characterized by a generalized dysfunction of the exocrine
glands, giving rise to a broad spectrum of clinical syndromes, especially
malabsorption due to pancreatic insufficiency and chronic progressive lung
disease giving rise to bronchiectasis. B. cepacia is associated with
increased illness and death among CF patients. In the early 1980s, the
organism emerged as a major threat, causing superinfection in as many as
40% of patients in some CF centers (4-6). While in some patients indolent
pulmonary infection occurs with only gradual deterioration in lung function
similar to that associated with Pseudomonas aeruginosa, approximately 35%
of B. cepaciainfected patients contract accelerated pulmonary
deterioration or fulminant, necrotizing pneumonia with rapidly fatal
bacteremia (3,7-10), sometimes referred to as "cepacia syndrome" (7).
Unlike infection with P. aeruginosa, B. cepacia infection significantly
increases death rates among CF patients (11) at all levels of lung
function.

Over the last 20 years, CF survival rates have increased as a result of
improved treatment, the median length of survival increasing from early
childhood to more than 29 years. As a result, approximately one third of CF
patients are adults. Pulmonary infection causes the most illness and
ultimately more than 90% of CF-related deaths. B. cepacia's emergence as a
pathogen coincided with social and medical grouping of CF patients in
specialized units, clinics, and social groups. Studies subsequently
demonstrated that social contact between CF patients was an important
factor in transmission of B. cepacia (12).

The threat of B. cepacia infection led to severe control measures,
affecting not only the treatment but also the social and family lives of CF
patients. CF centers adopted stringent infection control policies, assuming
that all B. cepacia isolates were highly transmissible (13,14). CF summer
camps in North America were closed, and many lung transplant centers ceased
to accept B. cepaciainfected CF patients as transplant candidates. Newly
formed national and international associations for CF adults and CF
families (providing conferences, holidays, and support groups) addressed
the issue of B. cepacia transmission by abandoning many activities and
adopting exclusion and segregation measures (13).

Numerous CF-associated B. cepacia epidemics have now been described, and
the epidemic strains have been characterized (15-18). One particular highly
transmissible strain, epidemically spread within and between CF centers on
both sides of the Atlantic, carries the cblA gene (18). This gene encodes
for the major structural subunit of unique mucin binding cable pili (4).
These enormously long pili (radiating 2 to 4 microns) are peritrichously
arranged and are intertwined to form cablelike structures that adhere to CF
mucin (4,18) (Figure 2). This cblA+ strain has spread across Canada and has
now been isolated in 50% of CF centers in the United Kingdom (19). Another
strain of B. cepacia has spread among CF centers in four regions of France
(20).

[fig]
Figure 2. Transmission electron micrograph of Toronto/Edinburgh epidemic clone of B.
cepacia expressing CF mucous-binding Cbi adhesin pili. High resolution was achieved by
using a JEOL 100CX electron microscope and negative staining. Reprinted with permission
from Richard Goldstein and Journal of Bacteriology (J Bacteriol 1995;177:1039-52).
    
However, it has become clear that
transmissibility varies markedly from
strain to strain, and that most
strains are not involved in epidemics
but appear to be independently
acquired and show no evidence of
transmission. For example, in 8 years
no cases of transmission were
detected at the University of North
Carolina CF center, despite clinical
and social contact between patients
and the absence of stringent
infection control measures (21).
Independent acquisition of B. cepacia
with no evidence of transmission
between CF patients was also reported
from Denmark (22). Lack of transmission of some strains has also been
observed between siblings with CF (23).

In Patients Without CF

B. cepacia was first reported as a human pathogen causing endocarditis in
the 1950s. Since then the organism has caused numerous catheter-associated
urinary tract infections, wound infections, and intravenous
catheterassociated bacteremias. In 1971, it was reported as the causative
organism of "foot rot" in U.S. troops on swamp training exercises in
northern Florida; it was also isolated from troops serving in the Mekong
Delta, Vietnam (24).

In the 1980s the number of nosocomial infections with B. cepacia increased
markedly, with deaths particularly associated with lung infections (25).
Numerous small focal hospital outbreaks involving non-CF patients have
usually been due to a contaminated common source, such as disinfectant
preparations or intravenous solutions (2,26,27). The organism's unusually
broad metabolic capabilities, which enable it to survive and proliferate in
water-based environments, probably account for its propensity to cause
nosocomial outbreaks (2,27-29). Because of its resistance to multiple
antibiotics, once acquired, the organism can be difficult to treat.

B. cepacia is rarely found in the non-CF patient (30); however, when it is
found the organism can spread to and from CF patients. Transmission between
CF and non-CF patients has been associated with serious illness and death
(31;Holmes et al., unpub. data) and presents a greater nosocomial threat
than previously recognized. In addition, although B. cepacia is not a
common commensal organism, hospitalized patients without CF may harbor it
and pose an infection threat to vulnerable CF patients. This possible
source of infection may account for the known association between
hospitalization and of B. cepacia infection (32). B. cepacia may be
underreported because selective media for B. cepacia, which greatly
increase the yield (33,34), are rarely used except in specimens from CF
patients.

B. cepacia as an Agricultural Agent

While emerging as a human pathogen, B. cepacia has attracted intense
interest from the agricultural industry as a possible biologic control
agent. The organism, which has been shown to have remarkable potential as
an agent for both biodegradation and biocontrol, is being considered as a
plant-growth-promoting rhizobacterium (23,35-43).

B. cepacia has extraordinary metabolic versatility and can degrade
chlorinated aromatic substrates (toxic compounds found in complex
pesticides and herbicides, some with carcinogenic potential) for use as
carbon energy sources. One important toxic compound degraded by B. cepacia
is 2,4,5 chlorophenoxy acetic acid (2,4,5-T), a potent herbicide that is
not easily biodegradable and persists for long periods in the environment
(37).

B. cepacia can also antagonize and repress many soilborne plant pathogens.
It can prevent leaf and stem blight caused by the fungus Alternaria by
inhibiting spore germination. Economically important crop diseases such as
blight due to A. solani and the blight caused by A. brassicae and A.
brassicola, which affects the oil-producing plants rape and canola, can be
controlled by B. cepacia. The organism is also being used to prevent the
blight of ginseng plants due to A. panax (41) and is effective against the
fungus Aphanomyces euteiches, which causes root rot in peas, alfalfa, and
snap beans (39,40). It can prevent Pythium diseases of cucumber and peas,
and Rhizoctonia solani stem rot of poinsettia (42). To prevent these plant
diseases, B. cepacia provides a seemingly environmentally friendly
alternative to potent and toxic fungicides, which cannot be broken down in
the environment.

The forestry industry also sustains large economic losses from the
pathogenic effects of fungi such as Fusarium, Pythium, Rhizoctonia,
Cylindrocarpum, and Botrytis. These widespread fungal pathogens cause
seedling loss in nurseries and may kill or stunt transplanted seedlings. A
strain of B. cepacia has been developed as a successful seed and root
inoculant, which can suppress these fungi on a variety of conifers (43).

Numerous patents are being sought for specific agricultural applications of
different strains of B. cepacia. The ecologic and economic benefits could
be enormous if the organism's antifungal activity is used to enhance crop
yields and reduce the need for pesticides and its ability to degrade
complex herbicides and pesticides is harnessed for bioremediation.

Molecular Epidemiology of B. cepacia

Evolution of Highly Transmissible Strains

B. cepacia isolates are closely related (a panmictic population structure)
(18,31,44) and epidemic isolates are scattered throughout (18,31), as
demonstrated by rrn-based phylogenic trees, which include large numbers of
environmental and clinical isolates. Such a phylogeny indicates that in B.
cepacia strains, genetic changes conferring high transmissibility are
occurring at random, or, given the right epidemiologic circumstances,
random environmental strains are innately highly transmissible. In
addition, the widespread geographic distribution of different epidemic
strains of B. cepacia would also suggest that highly transmissible strains
are emerging independently and randomly.

Taxonomy

Isolates presumptively identified as B. cepacia belong to at least five
genomovars (23,45,46). This group of phenotypically similar subpopulations
is referred to as the B. cepacia complex (6,46). After multiphasic
taxonomic analysis, the species names B. multivorans and B. vietnamiensis
have been proposed for genomovars II and V, respectively (45). The
pathogenic and epidemic potential of each of these subpopulations of the B.
cepacia complex requires further examination. Although it appears that
strains associated with cepacia syndrome belong to genomovar III (46),
isolates belonging to each of these five genomovars have been cultured from
CF patients.

The Unusual Genome

B. cepacia's capacity to propagate as an environmental microbe and as an
opportunistic pathogen may be due to its possession of an unusually large
(more than four times that of H. influenzae, two times that of E. coli, and
half again as large as P. aeruginosa), complex, and variable genome. The
genome contains numerous insertion sequences and is divided into one to
four circular replicons (47,48). A few other bacterial species of
agricultural and medical importance also have multiple chromosomes:
Brucella melitensis, B. abortus, B. suis, B. ovis, Rhodobacter sphaeroides,
and Agrobacterium tumefaciens (49-51).

This unusual genomic arrangement may account for B. cepacia's nutritional
versatility and adaptability. Such a division of genomic content would
allow for high levels of homologous and illegitimate recombination. The
resultant chromosomal rearrangements and associated mutations could provide
a basis for spontaneous "pulsed" evolutionary spurts, such as that seen
from soil to the CF lung, suited for rapid adaptation to radical changes in
environmental growth conditions.

Selection of Strains for Agricultural Use

Because evolution of highly transmissible strains occurs randomly and
transmission of B. cepacia between CF and non-CF populations can cause
severe illness and death, the deliberate widespread environmental
application of strains of this organism should be considered carefully.
Although B. cepacia does not appear to survive on dry surfaces for more
than 1 week, it can survive for many months in water. The agricultural
application of B. cepacia will lead to environmental and water
contamination and increased human exposure. In addition to medical,
metabolic, and taxonomic issues, increasing knowledge of B. cepacia raises
important ecological issues, including the evolution of pathogenicity and
multiresistant environmental bacteria through horizontal gene transfer. The
agricultural use of B. cepacia risks this hazard of horizontal gene
transfer between the strains applied and existing soil organisms and the
subsequent emergence of pathogenic, highly resistant organisms.

Recently, in an attempt to assess the human risk associated with the use of
rhizophere isolates as field inoculants, two clinical isolates of different
strain types from two CF patients and two agricultural isolates from the
rhizopheres of rice and maize were examined (38). The clinical isolates had
identical 16S ribosomal rDNA sequences, but differences were seen between
the soil isolates and clinical isolates. The results were presented as
evidence of evolutionary divergence of the rhizophere isolates from their
clinical counterparts. Alternatively, in the light of the multiple replicon
model, this difference in 16S rrn is most likely due to the multiple
replicons carrying varying sequences of the 16s gene. Furthermore, evidence
based on four isolates alone is inadequate to support the safe application
of rhizophere isolates. We have seen diversity among 16s rrn that is
unrelated to source in a large collection of B. cepacia isolates (Holmes,
Truong, Geisselsoder, Goldstein, unpub. data).

The possibility of highly virulent strains of B. cepacia with the potential
to survive intracellularly exists if the organism acquires virulence genes
from B. pseudomallei, a very closely related pseudomonad. B. pseudomallei
is a soil saprophyte that gives rise to melioidosis, a life-threatening
tropical disease seen mostly in Southeast Asia. The disease has a broad
clinical spectrum and can remain dormant for years before giving rise to
sepsis and death. The pattern of disease produced by B. pseudomallei may
warn of a spectrum of clinical consequences from B. cepacia acquisition.
The transfer of genetic material between these two closely related
organisms in the environment is highly probable, with the subsequent
emergence of a hybrid pathogen. This speculation is supported by recent
detection of insertion sequences within B. pseudomallei that have been
identified in B. cepacia, including an isolate belonging to the highly
transmissible transatlantic epidemic lineage (18,52).

With the potential emergence of diseases related to new and developing
practices in the food and agricultural world, it seems prudent that
communication and information sharing between medical microbiologists,
agricultural microbiologists, and public health professionals be optimized
and promoted. Meanwhile, it is impossible to identify, phenotypically or
genotypically, strains of B. cepacia that could safely be used in
agriculture. Even if environmental strains incapable of human infection
could be identified, their potential to evolve into human pathogens
remains. Current molecular genetic evidence indicates that the deliberate
environmental distribution of the organism might pose a hazard to human
health, regardless of which particular strains are selected. Until more is
known about the organism and the risks from environmental application, a
moratorium should be called on the widespread use of B. cepacia in
agriculture.

This work was supported in part by a research grant award to RG from the
National Institutes of Health (NIDDK RO1 DK50838).

Address for correspondence: Alison Holmes, Department of Infectious
Diseases, Hammersmith Hospital, Du Cane Rd, London W12 0NN, United Kingdom;
fax: 44-181-383-3394; e-mail: aholmes@rpms.ac.uk.

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Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/holmes.htm

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[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Synopses

Haemophilus influenzae Invasive Disease in the United States, 1994-1995:
Near Disappearance of a Vaccine-Preventable Childhood Disease

Kristine M. Bisgard, Annie Kao, John Leake, Peter M. Strebel,  Bradley A.
Perkins, and Melinda Wharton
Centers for Disease Control and Prevention, Atlanta, Georgia, USA

---------------------------------------------------------------------------
         We analyzed national Haemophilus influenzae (Hi)
         surveillance data from 1994 and 1995 to describe the
         epidemiology of Hi invasive disease among persons of all
         ages. Serotype data were available for 376 (56%) of 669
         reported Hi cases among children aged 4 years or
         younger; 184 (49%) were H. influenzae type b (Hib).
         Among children aged 4 or younger, incidence (per
         100,000) of all Hi invasive disease was 1.8 in 1994 and
         1.6 (p < 0.05) in 1995. Children aged 5 months or
         younger had the highest average annual incidence rate of
         Hib invasive disease (2.2 per 100,000); children aged 6
         to 11 months had the next highest rate (1.2 per 100,000)
         (p < 0.05). Of 181 children with Hib invasive disease
         whose age in months was known, 85 (47%) were too young
         (aged 5 months or younger) to have completed a primary
         series with an Hib-containing vaccine. Of the 83
         children with known vaccination status who were eligible
         to receive a primary series (aged 6 months or older), 52
         (63%) were undervaccinated, and the remaining 31 (37%)
         had completed a primary series in which vaccine failed.
         Among persons aged 5 years or older with Hi invasive
         disease, the lowest average annual incidence was among
         those 20 to 39 years of age (0.15 per 100,000), and the
         highest was among those aged 80 years or older (2.26 per
         100,000). Among persons aged 5 years or older, serotype
         data were available for 1,372 (71%) of the 1,940 Hi
         invasive disease cases; 159 (28%) of the 568 Hi cases
         with known serotype were due to Hib.

Before the first Haemophilus influenzae type b (Hib) polysaccharide vaccine
was introduced in 1985, Hib was the most common cause of bacterial
meningitis in children under 5 years of age (approximately 12,000 cases per
year, most in children younger than 18 months [1]); approximately 5% of
affected children died. Neurologic sequelae developed in 15% to 30% of the
surviving children (1). An additional estimated 7,500 cases of other
invasive Hib infections also occurred annually in young children (1). The
cumulative risk for Hib invasive disease before the age of 5 was one in 200
children, similar to the risk for poliomyelitis during the 1950s (1,2).

The incidence of Hib invasive disease among children aged 4 years or
younger has declined by 98% since the introduction of Hib conjugate
vaccines, initially licensed in 1989 for use in children aged 15 months or
older, then for infants beginning at 2 months of age in 1990 (3-6). One
goal of the Childhood Immunization Initiative (7) was to eliminate invasive
Hib disease among children aged 4 years or younger by 1996; however,
approximately 300 cases of H. influenzae (Hi) invasive disease per year
continue to be reported (3).

Moreover, Hi invasive disease is an important cause of illness and death in
immunocompromised persons and adults with certain underlying medical
conditions (8-11). The average incidence of Hi invasive disease from 1988
to 1990 was estimated at 1.7 cases per 100,000; 50% of cases examined in
this study were due to Hib (8). Prior studies of Hi invasive disease among
adults found case-fatality rates of 26% to 36% (8-11).

In this article, we summarize the epidemiology of Hi and Hib invasive
disease among children aged 4 years or younger and persons aged 5 years or
older in the United States during 1994 and 1995.

Data Collection

We used three sources of surveillance data to document the number of
reported Hi invasive disease cases with onset in 1994 and 1995. Cases were
reported to the Centers for Disease Control and Prevention (CDC). Two
sources were passive surveillance systems: the National Notifiable Diseases
Surveillance System (NNDSS) and the National Bacterial Meningitis and
Bacteremia Reporting System (NBMBRS); the third source was the active,
laboratory-based surveillance sites.

The NNDSS case reports of Hi invasive disease are transmitted
electronically from state health departments to CDC as part of the weekly
notifiable disease reporting system. These reports contain only demographic
information. Supplemental information, including Hi serotype, infection
site, outcome, and Hib vaccination status and specific vaccine received
among children aged 4 years or younger is transmitted electronically or
mailed to CDC on NBMBRS reporting forms. During 1994, 10.5 million persons
were under active, laboratory-based surveillance for Hi invasive disease:
these included residents of the state of Oklahoma; three counties in the
San Francisco Bay area of California; eight counties in the metropolitan
area of Atlanta, Georgia; and four counties in Tennessee (3). In 1995, 12.8
million persons were under surveillance; an additional county in Tennessee
was added, and Maryland was included in place of Oklahoma. Each site was
contacted every 2 weeks to identify Hi invasive disease cases; surveillance
personnel then collected detailed data on these cases. Data on cases from
the active surveillance sites were also mailed to CDC on a form similar to
the NBMBRS report form.

Information from the three data sources was combined, and duplicates (cases
with identical date of birth, onset, county of residence, and demographic
data) were eliminated. After the end of each calendar year, each state
health department was contacted by telephone to send information on Hi
invasive disease cases among children aged 4 years or younger not
previously reported to CDC and to provide missing critical supplementary
information, such as vaccinations received.

Our analysis included confirmed and probable cases. A confirmed case of Hi
invasive disease was defined as an illness clinically compatible with
invasive disease (such as meningitis, pneumonia, cellulitis, epiglottitis,
peritonitis, pericarditis, septic arthritis, empyema, and abscesses) with
isolation of Hi from a normally sterile site (e.g., cerebrospinal fluid or
blood). A probable case was defined as an illness clinically compatible
with detection of Hib antigen in cerebrospinal fluid (12). Up to three
infection sites could be recorded from a list including primary bacteremia,
meningitis, pneumonia, cellulitis, epiglottitis, peritonitis, pericarditis,
septic arthritis, or another specified site.

Completion of a primary vaccination series was defined according to
recommendations from the Advisory Committee on Immunization Practices (4).
Hib vaccine doses administered fewer than 14 days before the onset of
disease were excluded.

Data Analysis

U.S. census population estimates for 1994 and 1995 were used to calculate
age- and race-specific average annual incidence rates (calculated by
[events/2]  [population/2]) for all Hi invasive disease and for documented
Hib disease (13). The difference between two rates was regarded
statistically significant at the 0.05 level if it exceeded the following: 2
x [R1(sup 2)/N1 + R2(sup 2)/N2](sup 1/2) , where R1 is the rate corresponding 
to N1 events and R2 is the rate corresponding to N2 events (14). County average 
annual incidence rates of Hi invasive disease were calculated by using 1990 
county population estimates and were mapped with Atlas Mapmaker (15). For Hi
invasive disease cases among children aged 4 years or younger, we looked at
the proportion due to Hib, the Hib vaccination history, and the
age-specific and geographic distribution of residual Hib cases. A
chi-square test of independence was used to test the association between
age category and outcome.

Findings

In 1994 and 1995, 1,277 and 1,332 cases of invasive Hi disease were
reported, respectively. Although the overall incidence of invasive Hi
disease remained constant, the incidence of invasive Hi disease among
children aged 4 years or younger was lower in 1995 (305 cases; 1.56 per
100,000) than in 1994 (364 cases; 1.84 per 100,000) (p < 0.05). During 1994
and 1995, children 5 months or younger had the highest reported disease
incidence of Hi invasive disease (8.02 per 100,000 children), Hib invasive
disease (2.20 per 100,000), and Hi invasive disease of unknown serotype
(3.57 per 100,000) (Table 1). The incidence of Hi invasive disease, Hib
invasive disease, and Hi invasive disease of unknown serotype was
significantly greater (p < 0.05) among children aged 5 months or younger
than among children 6 to 11 months of age, who had the next highest rates;
a high rate of Hi invasive disease was also observed among adults aged 80
years or older. The lowest disease rates were found in persons 5 to 19 and
20 to 39 years of age.

Hi isolates were serotyped for only 36% of 2,609 reported cases. A higher
percentage of isolates were serotyped among children aged 4 years or
younger (56%) than among older persons (29%) (Table 2). Among Hi cases in
children aged 4 years or younger with reported serotype information, Hib
was the most common single serotype (184 type b of 376 serotyped isolates);
Hib represented 49% of invasive disease and 65% of meningitis. The average
annual incidence for Hib invasive disease was 0.47 per 100,000 children
aged 4 years or younger. Among persons aged 5 years or older, Hib accounted
for 28% of serotyped isolates.

Among the 433 Hi case-patients younger than 1 year of age for whom age in
months was reported during the study period, most (72%) were aged 5 months
or younger; 29% were younger than 1 month of age (Figure 1). Similarly, of
184 children aged 4 years or younger with Hib invasive disease, most (133,
72%) cases were in children younger than 1 year of age.

Table 1. Number of cases and average annual incidence rates of Hi and Hib
invasive disease and Hi invasive disease of unknown serotype, by age(sup a),
United States, 1994 - 1995    

			    Hi                  Hib          Hi serotype unknown
             ------------------- -------------------- --------------------------
    Age
    group   No.(sup b)Incid.(sup c)No.(sup b)Incid.(sup c)No.(sup b)Incid.(sup c
    ----------------------------------------------------------------------------
     0-5 mo   310      8.02         85          2.20        138         3.57
     6-11 mo  123      3.18         45          1.16         45         0.16
     1-4 yr   219      0.69         51          0.16         99         0.31
     5-19 yr  179      0.16         23          0.02        108         0.10
     20-39 yr 249      0.15         25          0.02        166         0.10
     40-59 yr 405      0.33         42          0.03        280         0.23
     60-79 yr 715      1.01         51          0.07        539         0.76
     80 + yr  361      2.26         18          0.11        152         0.95
     ---------------------------------------------------------------------------
     (sup a)Does not include 31 cases missing age and 17 patients aged <1
     year but missing age in months.
     (sup b)Number of cases over the 2-year period.
     (sup c)Incidence per 100,000 population.

The highest average annual incidence rates (5.1 per 100,000) of Hi invasive
disease among case-patients aged 4 years or younger were in American
Indians; comparisons with other race or ethnic groups were statistically
significant (p /= 0.05) (Table 3). Similar race and ethnic differences were
noted for Hib invasive disease incidence but not for Hi invasive disease of
unknown serotypes. Among case-patients aged 5 years or older, differences
between race or ethnic groups were less marked. The incidence rates among
male and female case-patients aged 5 years or older and among case-patients
aged 4 years or younger were equivalent.

Infection site data were available for 1,556 (60%) case-patients (Table 4).
Bacteremia was the most frequently reported site of Hi invasive disease for
both children aged 4 years or younger (62%) and persons aged 5 years or
older (51%). The second most frequently reported site of invasive disease
among children aged 4 years or younger was meningitis (44%), and among
persons aged 5 years or older it was pneumonia (36%).

Table 2. Serotype information for reported Hi invasive disease (n=2609) and
meningitis cases (n=380), stratified by age, United States, 1994 - 1995

          --------------------------------------------------------
                    No. (%) of children	 No. (%) of persons
			    aged 0-4 years  	   aged >/=5 years                     
			     Invasive            Invasive
          Serotype     disease Meningitis  disease   Meningitis
          --------------------------------------------------------
          Serotype b   184 (27)  101 (47)   159 (8)     41 (25)
          Other(sup a) 114 (17)   39 (18)   171 (9)     26 (15)
          Non-typeable  78 (12)    16 (8)   238 (12)    25 (15)
          Unknown      293 (44)   57 (27)  1,372(71)    75 (45)
          Total        669 (100) 213 (100) 1,940(100)  167 (100)
		--------------------------------------------------------
          (sup a)Includes serotypes a, c, d, e, f.


Outcome data were available for 1,380 (53%) of 2,609 case-patients with Hi
invasive disease. Among case-patients with known outcome, the overall
case-fatality ratio (CFR) was 14%. The CFR differed by age; the CFRs among
case-patients aged 0 to 1 year, 1 to 29 years, 30 to 59 years, and 60 years
of age or older were 10%, 6%, 13%, and 24%, respectively (p < 0.001).

During 1994 and 1995, a moderate seasonal pattern of Hi and Hib invasive
disease was observed, with more cases occurring during the winter months
(Figure 2); this pattern was similar for children 4 years of age or younger
(data not shown).

              [fig 1]
              Figure 1. Reported Hi invasive disease cases by
              age and serotype, children aged <1 year,a United
              States, 1994-1995.
              (sup a)N=433; excluding 17 children aged <1 year
              missing age in months.



Table 3. Number of cases and average annual incidence rates of Hi and Hib
invasive disease and Hi invasive disease of unknown serotype, by race and
ethnicity,a United States, 1994 - 1995

                                                           Persons aged >/=5
                               Children aged 0-4 year           years
                          -------------------------------------------------
                                                              Hi: unknown
                      All Hi         	Hib           serotypes        All Hi
               ------------------- ------------------ -------------- ---------------
Race/ethnicity No.(sup b)Incid.(sup c)No.(sup b)Incid.(sup c)No.(sup b)Incid.(sup c)
-------------------------------------------------------------------------------------
Non-Hispanic
   White            338    1.34        102       0.40       150    0.59   1,269 0.35
   Black            122    2.08         32       0.55        52    0.89     258 0.45
   American Indian   18    5.10         10       2.83         3    0.84      12 0.37
   Asian or Pacific
   Islander          13    0.86          3       0.20         6    0.40      21 0.13
Hispanic             94    1.49          7       0.11        31    0.49      70 0.15
-------------------------------------------------------------------------------------
(sup a)Does not include 84 (13%) of children aged /= 4 years, and 310 (16%) of
persons aged >/= 5 years for whom race and ethnicity data were missing.
(sup b)Number of cases over the 2-year period.
(sup c)Per 100,000 population.

To determine the remaining cases of Hib invasive disease, data on the
number of Hib vaccine doses received were reviewed for the 184 reported
cases among children aged 4 years or younger by the age (in months) of the
case-patient (Table 5). Eighty-five (47%) of the 181 children aged 4 years
or younger with known age in months were too young (5 months or younger) to
have completed a primary series with the most commonly used Hib vaccines
(three-dose primary series). Of the 149 children aged 4 years or younger
with known vaccination status, 83 were eligible (aged 6 months or older) to
receive a primary series; of these, 52 (63%) were undervaccinated.
Thirty-one (37%) of the 83 children had completed a primary series with
either three doses of HbOC (Wyeth-Lederle Laboratories, Pearl River, NY) or
PRP-T (Pasteur Mrieux Connaught, Lyon, France), or with two doses of
PRP-OMP (Merck, Inc., West Point, PA), or one dose with any Hib-containing
vaccine on or after 15 months of age. Manufacturer and vaccine lot
information was available for 22 of the 31 children who had received a
primary series. No single lot predominated, and no child received vaccine
from the same lot for all doses.

 Table 4. Reported infection site(sup a) among Hi invasive disease cases,
(sup b)United States, 1994 - 1995

                                         Number (%)
                              ----------------------------------
                               Children aged       Persons aged          
		Site            0 - 4 years(sup c) >/= 5 years(sup d)	
		----------------------------------------------------
            Bacteremia(sup e)		299 (62)  		551 (51)
            Meningitis			213 (44) 		167 (16)
            Pneumonia 			 42  (9)  		390 (36)
            Cellulitis		 	 19  (4)		  7  (1)
            Epiglottitis 		  8  (2)		 19  (2)
            ----------------------------------------------------
            (sup a)Includes case-patients with multiple infection
            sites, such as meningitis and bacteremia.
		(sup b)Case-patients with known infections site(n=1,556)
            (sup c)N=485.
            (sup d)N=1,071.
            (sup e)Bacteremia is reported following positive blood
            culture results, with or without foci.



              [fig 2]
              Figure 2. Reported Hi invasive disease cases by
              serotype and month of onset,a United States,
              1994-1995.
              (sup a)N=2,609; excluding 10 cases with unknown month
              of onset.



Table 5. Children aged </= 4 years with invasive Hib disease,(sup a) 
by number of Hib vaccine doses received and age group, United States, 
1994 - 1995

                          No. of children
                         receiving vaccine(sup b)
                     -------------------------
                                               Vaccination
           Age                                  status
          (mos.) Rec.(sup c)0   1   2   3  4   unknown   Total
            ----------------------------------------------------

            0-1        0    15                     2        17

            2-3        1    17  12                 9        38

            4-5        2     7  13   2             8        30

            6-11       3    13   8  11   6         7        45

            12-18      4     5   1   2   7  2      0        17

            19-59      4    11   5   1   7  4      6        34

            Total      4    68  39  16  20  6      32      181
            ----------------------------------------------------
            (sup a)Excludes three children aged <1 year but missing
            age in months; vaccination status was unknown  for
            two of the children, and one child was
            unvaccinated.
            (sup b)Five children completed a primary series with 2
            doses of PedvaxHIB (at ages 7, 11, 13, 14, and 43
            months); two children completed a primary series
            with 1 dose at >/=15 months of age.
            (sup c)Cumulative recommended doses for PRP-T and HbOC
            vaccines.



Overall, 49 states and 673 (21%) of the 3,137 U.S. counties reported at
least one Hi invasive disease case with disease onset during the 2-year
period; of these, 327 (49%) counties (from 47 states) reported two or more
Hi cases (Figure 3A). Forty-seven states and 339 (11%) of counties reported
Hi invasive disease cases among children aged 4 years or younger, and 42
states and 137 (4%) counties reported Hib cases during the 2-year period;
25 (18%) of these counties reported two or more Hib cases (range 2 to 8
cases). In these 25 counties, the incidence for Hib invasive disease was
0.41 to 12.19 per 100,000 children aged 4 years or younger (Figure 3B).
Among the 14 counties with two Hib cases, the median time between cases was
139 days (range 14 to 334 days). In three of the 11 counties with three or
more Hib invasive disease cases, children were too young (aged 5 months or
younger) to have completed a primary series with an Hib vaccine; in the
remaining eight counties, 20 (67%) of 30 children had been eligible for
vaccination (aged 6 months or older) and only one child had completed a
primary series. In these 11 counties, the incidence for Hib invasive
disease was 0.41 to 4.31 per 100,000 children aged 4 years or younger.

                        [fig 3]
            Figure 3. A. Average annual incidence of Hi
            invasive disease among persons of all ages, by
            county, United States, 1994 - 1995. B. Reported
            number of Hib invasive disease cases among children
            aged </= 4 years, by county, United States, 1994 -
            1995.



Conclusions

Invasive Hib disease among children aged 4 years or younger in the United
States has become rare because of the introduction and widespread use of
conjugate Hib vaccines. Although national vaccination coverage of children
19 to 35 months of age with three or more doses of Hib conjugate vaccines
only reached 90% in 1995 and some metropolitan areas had lower coverage
rates (16,17), all Hi invasive disease incidence in 1994 and 1995 was 98%
lower than during the prevaccine era. During that era, Hib invasive disease
was the cause of more than 95% of Hi invasive disease (18-22). While our
results were not directly comparable to prevaccine Hib invasive disease
estimates because of reliance on passive reporting and lack of serotype
data (23), in the active surveillance sites, the race-adjusted incidence of
Hib invasive disease declined 99% from 1989 to 1995 (3). Before Hib
conjugate vaccine availability in 1988, the highest Hib invasive disease
incidence had been in children 6 to 17 months of age (estimated annual
incidence of 275 and 223 per 100,000 for children 6 to 11 and 12 to 17
months of age, respectively), compared with children 5 months of age or
younger (estimated annual incidence of 148 per 100,000) (18-22). However,
during 1994 and 1995, children aged 5 months or younger had the highest
incidence of Hib invasive disease of all age groups (2.2 per 100,000)
(Table 1); these children were too young to have completed a primary
vaccination series.

Consistent with previous studies of Hib invasive disease in children aged 4
years or younger in the prevaccine era, we found higher Hi and Hib disease
incidence among American Indian (the highest incidence rates), black, and
Hispanic children than among Asian or Pacific islander and white children
(18,20). In the prevaccine era, Hib invasive disease rates among American
Indian children were approximately five times higher than among
non-American Indian children in Alaska (annual incidence of 601 per 100,000
children) (20). Although important progress has been made in reducing Hib
invasive disease in this population, American Indian children continue to
have higher disease incidence than other racial or ethnic groups. The
reasons for the higher Hib invasive disease incidence among some ethnic or
racial groups or some regions are unknown but could be associated with one
or more of the following: low Hib vaccine coverage levels in an area (e.g.,
an inner-city "pocket of need"), low socioeconomic status, cofactors, host
susceptibility, and high levels of carriage in the population. In 1996, a
study of Alaskan native children aged 6 years or younger with high
vaccination coverage (>90%) found a carriage level of 8%, a rate similar to
that in the prevaccine era (24). However, a 1992 study in the Atlanta,
Georgia, metropolitan area documented a low (0.2%) carriage level among
children with a 75% Hib vaccination coverage (25). In the United States,
the reduction in the number of reported cases has been greater than
expected when the efficacy of Hib conjugate vaccines and the coverage of a
primary series alone are taken into account; this is likely due to the
reduction of pharyngeal carriage in vaccinated children, which protects
nonvaccinated persons in the community (i.e., herd immunity) (18,26). High
carriage levels are unlikely in most areas of the United States; the
difference in the results of the two studies (24,25) may be due to the use
of different Hib conjugate vaccines, as well as to different host and
environmental factors. In European countries that use Hib conjugate
vaccines, increasing vaccine use has been associated with diminishing rates
of both Hib pharyngeal carriage and invasive Hib disease (26).

With the marked decline among children, the occurrence of Hib invasive
disease among older persons (aged 5 years or older) has achieved greater
prominence. Although serotype data were missing from most cases, reported
cases among both children (n = 184) and adults (n = 159) indicate
persistent circulation of Hib. Reported Hib invasive disease cases and
nasopharyngeal carriage among children have decreased; however, because
these data were from passive reporting sources it is not clear if a similar
decrease in invasive Hib disease in older children and adults has occurred
nationally. Because of underreporting of adult Hib invasive disease cases
nationally, 1994 to 1995 incidence rates cannot be directly compared with
rates found in prior studies (8-11). In our study, 28% of serotyped
isolates were Hib; however, among adults in the Atlanta, Georgia,
metropolitan active surveillance site, Hi invasive disease cases due to Hib
declined in 1990 to 1991 from 50% to 0% in 1995 to 1996 (8,27).
Nevertheless, because previous studies have found that most Hi and Hib
invasive disease occurred among immunocompromised patients or patients with
underlying conditions (e.g., chronic lung disease, splenectomy, leukemia,
HIV infection, sickle-cell disease), health-care providers should consider
Hib vaccination for patients at high risk (6). Because of continued
occurrence of Hib invasive disease among older persons and infants too
young to be vaccinated, additional strategies may be needed to eliminate
Hib invasive disease among children aged 4 years or younger. In addition,
ongoing surveillance for Hi invasive disease is needed to monitor incidence
among older persons; case investigations may include assessments for
underlying immunosuppressive conditions or ascertainment of other cofactors
such as prior or concomitant viral infections.

Continued decline in incidence of Hib invasive disease in the United States
requires that children aged 4 years or younger be appropriately vaccinated
to protect them and other children and infants in the community. Intensive
efforts to increase vaccination levels among children in areas with
coverage rates lower than 90% are needed to reach the elimination goal.
Children should receive a primary Hib vaccination series at ages 2, 4, and
6 months with either HbOC or PRP-T, or at ages 2 and 4 months with PRP-OMP
(6). A booster dose with any of the four licensed Hib conjugate vaccines
should be given at 12 to 15 months of age (6). Among children who were
eligible for vaccination, timely completion of a primary series might have
prevented 63% of reported Hib invasive disease cases in 1994 and 1995. Few
(28) Hib cases among children 4 years or younger were reported in those who
had completed a primary series with Hib vaccine, which suggests that
vaccine failure was uncommon. However, because of passive reporting,
vaccination status and vaccine failure may be underestimated. Hib invasive
disease in children too young to have completed a primary series will
continue to be a barrier for elimination unless Hib nasopharyngeal carriage
rates in the population (including adults) approach zero.

Because the highest Hib invasive disease incidence occurs among infants,
all infants should be appropriately vaccinated, beginning at 2 months of
age. In a case-control study for undervaccination among Hib invasive
disease cases among children aged 2 to 18 months in the United States, risk
factors for undervaccination included low maternal education and having a
single mother (29); special outreach efforts may be needed to vaccinate
children at risk. Other programmatic strategies to increase vaccination
rates include linkage of the Special Supplemental Nutrition Program for
Women, Infants and Children (WIC) to vaccination assessment and referral
(30), implementation of immunization registries and reminder/recall systems
to encourage parents to vaccinate their children in a timely fashion
(28,31,32), and monitoring clinic coverage levels and providing feedback to
clinic staff (33). In addition to high vaccination coverage of other
children in the community to help reduce carriage, breast-feeding of
infants should be promoted because it is protective against invasive Hi
disease in this age group (21). For children aged 4 years or younger with
Hib invasive disease who have completed a primary Hib vaccination series,
case investigations may include examining reasons for vaccine failure, such
as measuring antibodies to the polyribosylribitol phosphate (PRP) capsule
of Hib; undetectable levels suggest lack of immune response (18).

To monitor the changing epidemiology of Hib invasive disease and look for
an increase or decline in incidence among children and adults, serotype
information for all Hi invasive disease cases is essential and will
generate data needed to examine new strategies for elimination.
Surveillance is necessary for detecting pockets of continuing transmission
and identifying areas in need of improved vaccine coverage (3). Each case
of Hib invasive disease is a sentinel for circulation of Hib in a
community. In addition, Hib invasive disease cases among children aged 4
years or younger may be a marker for low community coverage. Therefore,
community immunization policies, practices, and coverage levels should be
reviewed, and recommendations should be made, if needed, to improve Hib
vaccination coverage levels.

Acknowledgments

The authors thank Wendy Wattigney for her review of the manuscript and
assistance with statistical analysis and Shalani Desai for her meticulous
assistance in data cleaning.

Dr. Bisgard is a medical epidemiologist in CDC's National Immunization
Program. Her research interests include vaccine effectiveness, uses of new
vaccines, and outbreak control of vaccine-preventable diseases; her areas
of expertise include vaccine preventable diseases (Hib, pertussis, and
diphtheria) and infectious disease epidemiology.

Address for correspondence: Kristine M. Bisgard, Centers for Disease
Control and Prevention, 1600 Clifton Road, Mail Stop E61, Atlanta, GA
30333, USA; fax: 404-639-8616; e-mail: kmb6@cdc.gov.

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 30. Campbell JR, Szilagyi PG, Rodewald LE, Doane C, Roghmann KJ.
     Patient-specific reminder letters and pediatric well-child-care show
     rates. Clin Pediatr (Phila) 1994;May:268-72.
 31. Abramson JS, O'Shea MO, Ratledge DL, Lawless MR, Givner LB.
     Development of a vaccine tracking system to improve the rate of
     age-appropriate primary immunization in children of lower
     socioeconomic status. J Pediatr 1995;126:583-6.
 32. Dini EF, Linkins RW, Chaney M. Effectiveness of computer-generated
     telephone messages in increasing clinic visits. Arch Pediatr Adolesc
     Med 1995;149:902-5.
 33. Centers for Disease Control and Prevention. Recommendations of the
     Advisory Committee on Immunization Practices: programmatic strategies
     to increase vaccination rates assessment and feedback of
     provider-based vaccination coverage information. MMWR Morb Mortal Wkly
     Rep 1996;45:219-20.


Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/bisgard.htm

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[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Synopses

Multiple-Drug Resistant Enterococci:  The Nature of the Problem and an
Agenda for the Future

Mark M. Huycke,* Daniel F. Sahm, and Michael S. Gilmore
*University of Oklahoma Health Sciences Center, Oklahoma, USA; Department
of Veterans Affairs Medical Center, Oklahoma City, Oklahoma, USA; and MLR
Pharmaceutical Services, Inc., Reston, Virginia, USA

  --------------------------------------------------
      Enterococci, leading causes of nosocomial bacteremia, surgical
      wound infection, and urinary tract infection, are becoming
      resistant to many and sometimes all standard therapies. New
      rapid surveillance methods are highlighting the importance of
      examining enterococcal isolates at the species level. Most
      enterococcal infections are caused by Enterococcus faecalis,
      which are more likely to express traits related to overt
      virulence butfor the momentalso more likely to retain
      sensitivity to at least one effective antibiotic. The remaining
      infections are mostly caused by E. faecium, a species virtually
      devoid of known overt pathogenic traits but more likely to be
      resistant to even antibiotics of last resort. Effective control
      of multiple-drug resistant enterococci will require 1) better
      understanding of the interaction between enterococci, the
      hospital environment, and humans, 2) prudent antibiotic use, 3)
      better contact isolation in hospitals and other patient care
      environments, and 4) improved surveillance. Equally important
      is renewed vigor in the search for additional drugs,
      accompanied by the evolution of new therapeutic paradigms less
      vulnerable to the cycle of drug introduction and drug
      resistance.

The past few years have witnessed increasing interest in enterococci. Until
recently, these ordinary bowel commensals languished as misclassified
streptococci, commonly perceived "with the exception of endocarditis and
rare cases of meningitis  [as] not  a major cause of serious infection"
(1). In the last decade, however, enterococci have become recognized as
leading causes of nosocomial bacteremia, surgical wound infection, and
urinary tract infection (2,3). Two types of enterococci cause infections:
1) those originating from patients' native flora, which are unlikely to
possess resistance beyond that intrinsic to the genus and are unlikely to
be spread from bed to bed, and 2) isolates that possess multiple antibiotic
resistance traits and are capable of nosocomial transmission. The
therapeutic challenge of multiple-drug resistant (MDR) enterococcithose
strains with significant resistance to two or more antibiotics, often
including, but not limited to, vancomycinhas brought their role as
important nosocomial pathogens into sharper focus.

The accretion and spread of antibiotic resistance determinants among
enterococci, to the point where some clinical isolates are resistant to all
standard therapies, highlight both the vulnerability of our present
armament as well as the looming prospect of a "postantibiotic era" (4).
This review focuses on the magnitude and nature of the problem posed by
enterococci in general, and MDR enterococci in particular. For many points,
only representative citations are provided.

Habitat and Microbiology

Enterococci normally inhabit the bowel. They are found in the intestine of
nearly all animals, from cockroaches to humans. Enterococci are readily
recovered outdoors from vegetation and surface water, probably because of
contamination by animal excrement or untreated sewage (5). In humans,
typical concentrations of enterococci in stool are up to 10(sup 8) CFU per gram
(6). Although the oral cavity and vaginal tract can become colonized,
enterococci are recovered from these sites in fewer than 20% of cases. The
predominant species inhabiting the intestine varies. In Europe, the United
States, and the Far East, Enterococcus faecalis predominates in some
instances and E. faecium in others (6). Ecologic or microbial factors
promoting intestinal colonization are obscure. Of 14 or more enterococcal
species (7), only E. faecalis and E. faecium commonly colonize and infect
humans in detectable numbers. E. faecalis is isolated from approximately
80% of human infections, and E. faecium from most of the rest. Infections
to other enterococcal species are rare.

Enterococci are exceedingly hardy. They tolerate a wide variety of growth
conditions, including temperatures of 10C to 45C, and hypotonic,
hypertonic, acidic, or alkaline environments. Sodium azide and concentrated
bile salts, which inhibit or kill most microorganisms, are tolerated by
enterococci and used as selective agents in agar-based media. As
facultative organisms, enterococci grow under reduced or oxygenated
conditions. Enterococci are usually considered strict fermenters because
they lack a Kreb's cycle and respiratory chain (8). E. faecalis is an
exception since exogenous hemin can be used to produce d, b, and o type
cytochromes (9,10). In a survey of 134 enterococci and related
streptococci, only E. faecalis and Lactococcus lactis expressed
cytochrome-like respiration (11). Cytochromes provide a growth advantage to
E. faecalis during aerobic growth (9). E. faecalis cytochromes are only
expressed under aerobic conditions in the presence of exogenous hemin
(9,10,12) and, therefore, may promote the colonization of inappropriate
sites.

Enterococci are intrinsically resistant to many antibiotics. Unlike
acquired resistance and virulence traits, which are usually transposon or
plasmid encoded, intrinsic resistance is based in chromosomal genes, which
typically are nontransferrable. Penicillin, ampicillin, piperacillin,
imipenem, and vancomycin are among the few antibiotics that show consistent
inhibitory, but not bactericidal, activity against E. faecalis. E. faecium
are less susceptible to -lactam antibiotics than E. faecalis because the
penicillin-binding proteins of the former have markedly lower affinities
for the antibiotics (13). The first reports of strains highly resistant to
penicillin began to appear in the 1980s (14,15).

Enterococci often acquire antibiotic resistance through exchange of
resistance-encoding genes carried on conjugative transposons,
pheromone-responsive plasmids, and other broad-host-range plasmids (6). The
past two decades have witnessed the rapid emergence of MDR enterococci.
High-level gentamicin resistance occurred in 1979 (16) and was quickly
followed by numerous reports of nosocomial infection in the 1980s (17).
Simultaneously, sporadic outbreaks of nosocomial E. faecalis and E. faecium
infection appeared with penicillin resistance due to -lactamase production
(18); however, such isolates remain rare. Finally, MDR enterococci that had
lost susceptibility to vancomycin were reported in Europe (19,20) and the
United States (21).

Among several phenotypes for vancomycin-resistant enterococci, VanA
(resistance to vancomycin and teicoplanin) and VanB (resistance to
vancomycin alone) are most common (22). In the United States, VanA and VanB
account for approximately 60% and 40% of vancomycin-resistant enterococci
(VRE) isolates, respectively (23). Inducible genes encoding these
phenotypes alter cell wall synthesis and render strains resistant to
glycopeptides (22).

VanA and VanB types of resistance are primarily found among enterococci
isolated from clinical, veterinary, and food specimens (24), but not other
common intestinal or environmental bacteria. In the laboratory, however,
these genes can be transferred from enterococci to other bacteria (22). For
example, Staphylococcus aureus has been rendered vancomycin-resistant
through apparent transfer of resistance from E. faecalis on the surface of
membrane filters and on the skin of hairless obese mice (25), which
indicates that there is no biologic barrier to the emergence of
vancomycin-resistant S. aureus. Clinical isolates of highly
vancomycin-resistant S. aureus have yet to be identified, although strains
with reduced susceptibility to vancomycin have appeared (26). The mechanism
of resistance for these strains remains undetermined but does not appear to
involve genes associated with VanA or VanB phenotypes.

Epidemiology

Enterococci account for approximately 110,000 urinary tract infections,
25,000 cases of bacteremia, 40,000 wound infections, and 1,100 cases of
endocarditis annually in the United States (2,27,28). Most infections occur
in hospitals. Although several studies have suggested an increase in
nosocomial infection rates for enterococci in recent years, National
Nosocomial Infections Surveillance system data show little change in the
percentage of enterococcal bloodstream (12% vs. 7%), surgical site (15% vs.
11%), and urinary tract (14% vs. 14%) infections over the past 2 decades
(3,29). Adequate surveillance data prior to 1980 are not available.
Enterococcal infection deaths have also been difficult to ascertain because
severe comorbid illnesses are common; however, enterococcal sepsis is
implicated in 7% to 50% of fatal cases (6). Several case-control and
historical cohort studies show that death risk associated with
antibiotic-resistant enterococcal bacteremia is severalfold higher than
death risk associated with susceptible enterococcal bacteremia (30). This
trend will likely increase as MDR isolates become more prevalent.

Colonization and infection with MDR enterococci occur worldwide. Early
reports showed that in the United States, the percentage of nosocomial
infections caused by VRE increased more than 20-fold (from 0.3% to 7.9%)
between 1989 and 1993, indicating rapid dissemination. New database
technologies, such as The Surveillance Network (TSN) Database-USA, now
permit the assessment of resistance profiles according to species. TSN
Database electronically collects and compiles data daily from more than 100
U.S. clinical laboratories, identifies potential laboratory testing errors,
and detects emergence of resistance profiles and mechanisms that pose a
public health threat (e.g., vancomycin-resistant staphylococci).

Data collected by the TSN Database between 1995 and September 1, 1997
were analyzed to determine whether the earlier increase in vancomycin
resistance was unique to vancomycin, whether it represented a continuing
trend, and whether speciation is quantifiably important in analyzing
this trend. E. faecalis resistance to ampicillin and vancomycin is uncommon 
Figure 1); little change in resistance prevalence occurred from 1995 to 1997.
In contrast, E. faecium vancomycin and ampicillin resistance increased alarmingly. 
In 1997, 771 (52%) of 1,482 of E. faecium isolates exhibited vancomycin resistance,
and 1,220 (83%) of 1,474 isolates exhibited ampicillin resistance (Figure 1).
E. faecium resistance notwithstanding, E. faecalis remained by far the most 
commonly encountered of the two enterococcal species in TSN Database.
E. faecalis to E. faecium total isolates were approximately 4:1 (Figure 1),
blood isolates 3:1, and urine isolates 5:1. This observation underscores important
differences in the survival strategies and likelihood of therapeutic success, critical
factors usually obscured by lumping the organisms together as Enterococcus
species or enterococci. Widespread emergence and dissemination of
ampicillin and vancomycin resistance in E. faecalis would significantly
confound the current therapeutic dilemma. There is little reason to suspect
that vancomycin and ampicillin resistances only provide selective advantage
for the species faecium and not faecalis. The relative absence of these
resistances in E. faecalis may simply reflect a momentary lack of
penetrance and equilibration of the traits. Because of these important
differences between the two species, meaningful surveillance of
enterococcal resistance must include species identification.

[fig 1]
Figure 1. Epidemiology of enterococcal infection based on 15,203 susceptibility
results obtained by The Surveillance Network (TSN) Database-USA, 1995 to Sep 1, 1997. The 
increase in total numbers between 1995 and 1996 represents additional reporting centers
coming on line. Numbers for 1997 represent total collected for the partial year to Sep 1,
1997. 

Although exact modes of nosocomial transmission for MDR enterococci are
difficult to prove, molecular microbiologic and epidemiologic evidence
strongly suggest spread between patients, probably on the hands of
health-care providers or medical devices, and between hospitals by patients
with prolonged intestinal colonization. At least 16 outbreaks of MDR
enterococci have been reported since 1989 (31); all but two were due to E.
faecium. This disparity, particularly in view of the higher numbers of
clinical E. faecalis isolates, may reflect a reporting bias due to the
novelty of the combinations of resistance that occur in E. faecium. When
isolates from outbreaks of MDR enterococci have been analyzed by genetic
fingerprints, more than half involve clonally related isolates (18,32).

Prior treatment with antibiotics is common in nearly all patients colonized
or infected with MDR enterococci (33-35). Clindamycin, cephalosporin,
aztreonam, ciprofloxacin, aminoglycoside, and metronidazole use is equally
or more often associated with colonization or infection with MDR
enterococci than vancomycin use. Other risk factors include prolonged
hospitalization; high severity of illness score; intraabdominal surgery;
renal insufficiency; enteral tube feedings; and exposure to specific
hospital units, nurses, or contaminated objects and surfaces within
patient-care areas.

Infection Control

Controlling the spread of MDR enterococci among inpatients is difficult. We
know relatively little about the biology of enterococcal transmission or
the specific microbial factors favoring colonization by exogenous
enterococcal strains. Nevertheless, VRE infection control policies, which
could apply to MDR enterococci, were recently published by the Hospital
Infection Control Practices Advisory Committee (36). Control methods
include routine screening for vancomycin resistance among clinical
isolates, active surveillance for VRE in intensive care units, contact
isolation to minimize person-to-person transmission, and vancomycin
restriction.

These measures to limit VRE spread, however, have failed on occasion (35).
Not all hospitals can or are willing to perform active surveillance.
Because more patients are typically colonized with VRE (3% to 47%) than are
infected (35,37,38), and because intestinal colonization can be prolonged,
passive surveillance by routine cultures allows colonized inpatients to go
unidentified and serve as point sources for continued spread of VRE. Even
if all colonized inpatients are successfully identified, VRE may be spread
by health-care workers through either inadequate hand washing (39) or
through contact with items such as bedrails, sinks, faucets, and doorknobs
(enterococci can persist for weeks on environmental surfaces) (40).
Decontamination efforts must be rigorous.

The Hospital Infection Control Practices Advisory Committee strongly
recommended restricting oral and parenteral vancomycin to control VRE (36).
However, limiting use of vancomycin while ignoring widespread use of other
broad spectrum antibiotics likely will not lead to maximal control of VRE
or of MDR enterococci.

Antibiotics may promote colonization and infection with MDR enterococci by
at least two mechanisms. First, many broad spectrum antibiotics have little
or no anti-enterococcal activity, and administration commonly leads to
overgrowth of susceptible (or resistant) enterococci at sites at risk for
infection. Second, most antibiotics substantially reduce the normal
resistance of the intestinal tract to colonization by exogenous organisms
(41). Colonization resistance results primarily from the "limiting action"
of the normal anaerobic flora, and to a lesser extent from an intact
mucosa, gastric acid secretion, intestinal motility, and
intestinal-associated immunity (41). Antibiotic-induced alterations in the
protective flora of the intestine provide large footholds for colonization
with exogenous pathogens such as MDR enterococci (41). Antibiotic
restriction programs would be more effective if they included prudent
prescribing of all antibiotics, not just single agents such as vancomycin.
This approach substantially decreased intestinal colonization with VRE in
one hospital pharmacy that restricted vancomycin, cefotaxime, and
clindamycin (42).

At a minimum, a successful program for control of MDR enterococci requires
effective passive and active surveillance to identify colonized and
infected patients, absolute adherence to contact isolation by health-care
workers, rigorous decontamination of patient-contact areas and judicious
use or restriction of vancomycin and other broad spectrum antibiotics.

Therapeutic Approaches

Suitable antibiotics often are not available to treat MDR enterococcal
infections, e.g., endocarditis or bacteremia, in the presence of
neutropenia. Combinations of penicillin with vancomycin, ciprofloxacin with
ampicillin, or novobiocin with doxycycline, among others, have been used
(43) but can be unpredictable and remain clinically unproven. In one report
chloramphenicol successfully treated chloramphenicol-susceptible infections
(44), but these findings await confirmation in controlled trials.

Promising new antibiotics for MDR enterococcal infection under
investigation include fluoroquinolones, streptogramins, oxazolidinones,
semisynthetic glycopeptides, and glycylcyclines. Clinafloxacin, a
fluoroquinolone with improved potency against enterococci compared with
ciprofloxacin, has excellent activity against VRE, appears bactericidal in
vitro, and has been effective in treatment of enterococcal infections in a
murine model (45). Although single-step resistance to clinafloxacin could
not be detected in vitro, multistep resistance is readily achieved. Should
this agent gain approval for treatment of enterococcal infections,
selection for resistance may limit its effectiveness.

Quinupristin/dalfopristin (Synercid) is a combination of streptogramins A
and B that inhibits protein synthesis and has a narrower spectrum of
activity against enterococci than clinafloxacin (46). Many, but not all, E.
faecium isolates with VanA and VanB phenotypes are susceptible (47);
however, E. faecalis is uniformly resistant, and superinfection has been
reported during therapy (48). In addition, quinupristin/dalfopristin is
bacteriostatic only, potentially allowing emergence of resistance (49). For
these reasons the drug may have only a limited role in treating MDR
enterococcal infections. Novel oxazolidinones and glycylcyclines have also
shown potent activity against enterococci, including MDR enterococci
(50,51), but await further testing.

The substantial drawback of the broad spectrum approach is that the more
organisms affected (both protective commensals as well as pathogens), the
more opportunities for resistance to evolve. Broad spectrum antibiotics
permit empiric therapy in the absence of a specific diagnosis and generate
a more substantial return on investment in the short term. However, broad
spectrum antibiotics affect not only disease-causing organisms but also
commensals present in numbers large enough to generate resistance by
otherwise rare mutations or genetic exchange events. As long as market
forces favor development of broad spectrum therapeutics, a cycle of drug
introduction followed by emergence of resistance undoubtedly will continue.

Targeted Therapeutics

In contrast to the historical reliance on broad spectrum antibiotic
therapy, the continuing development and introduction of rapid diagnostic
techniques (52) may allow a more focused approach to infectious disease
therapy. Any of the myriad microbial-host interactions that subvert the
host response or damage tissues during an infection represent potential
therapeutic targets. However, many key interactions in disease pathogenesis
are specific to the organism involveda characteristic that is both a
strength and a weakness. Because of the specificity of these interactions,
rapid and accurate diagnosis is required. However, therapeutics aimed only
at interaction between host and a specific pathogen should leave the
diverse commensal flora essentially unaffected. As a result, the targeted
population would be restricted to the relatively small numbers of
disease-producing bacteria and would not likely reach the numbers or
diversity required to make development of resistance a statistical
probability.

The current spectrum of approaches to identify new antiinfective compounds
has two extremes: 1) screening vast libraries of compounds to identify
substances that by chance inhibit a microbial property and 2) detailed
study of interactions between host and parasite to identify critical events
leading to host tissue damage or compromise (53).

With a long-term view toward new therapeutic approaches as well as optimal
use of existing therapies, we and others have begun examining in detail the
interactions between enterococci and host (6). A major obstacle is that
enterococci also form part of the commensal or autochthonous flora; as
such, they are nearly devoid of traits traditionally associated with overt
pathogens and have subtle interactions with the host. Using inocula with as
few as 10 organisms, we have developed sensitive biologic systems for
examining the host-parasite interactions (54).

Although E. faecium strains are
resistant to vancomycin and
ampicillin more often than E.
faecalis strains, the relative
proportion of infections caused by
these species has not dramatically
changed in recent years (Figure 1).
Since both organisms are frequently
isolated from the commensal flora, this bias suggests that E. faecalis
traits confer a greater degree of intrinsic virulence, for example,
cytolysin production, pheromone-responsive plasmid transfer (and
accompanying production of aggregation substance), extracellular superoxide
production, and a newly identified surface protein tentatively termed Esp
(5,56,57) (Figure 2). These properties provide logical points of departure
for developing new targeted therapeutic approaches to enterococcal disease;
examination of more subtle interactions between E. faecium and host will
follow as an understanding of enterococcal biology evolves.

[fig 2]
Figure 2. Virulence traits and their association with enterococcal species.

Targeting the E. faecalis Cytolysin

Cytolysin is disproportionately expressed by E. faecalis strains associated
with disease (5,55,56). This cytolysin causes rupture of a variety of
target membranes, including bacterial cells, erythrocytes, and other
mammalian cells, with activity observed as a hemolytic zone on some types
of blood agar. Cytolysin contributes to the toxicity or lethality of
infection in several infection models and is associated with a fivefold
increased risk of sudden death from nosocomial bacteremia (54,56-59).
Cytolysin also contributes to the appearance of enterococci in a murine
bacteremia model (Figure 3; 45,60), an observation consistent with the
disproportionate representation of cytolytic strains among human blood
isolates (56,62).

[fig 3]
Figure 3. Cytolysin favors the appearance of circulating enterococci. In this
experiment, 10(sup)7 CFU of E. faecalis, either cytolytic FA2-2(pAM714) (60)
or noncytolytic FA2-2(pAM771) (64), were intraperitoneally injected (45) into groups of
five BalbC mice. Viable bacteria in liver, spleen, and the bloodstream were enumerated 48
hrs following injection, and significance assessed by Student's t-test. (P. Coburn, L.E.
Hancock, and M.S. Gilmore, in preparation).

Beginning with E.W. Todd in 1934 (63) and
culminating in a recent study (64), the
E. faecalis cytolysin has now been
characterized as a unique, extensively
modified bacterial toxin (Figure 4). The
cytolysin maturation pathway is ideally
designed for therapeutic targeting
because the two structural subunits are
activated by an extracellular protease,
an event that is accessible and
potentially inhibitable by a novel
therapeutic. Moreover, the activator
protease, CylA, belongs to the subtilisin
class of serine proteases (64), whose
structure-function relationships and
inhibitor design we are beginning to
understand. Investigations are in
progress to design and test inhibitors of
extracellular cytolysin activation to
determine whether a reduction by several
logs in the levels of circulating
enterococci can be attained as would be predicted
by the observed behavior
of cytolysin mutants (Figure 3).

[fig 4]
Figure 4. Cytolysin is expressed and processed through a complex maturation pathway
(64). The cytolysin precursors, CylL(sub L) and CylL(sub S), are ribosomally
synthesized. The putative modification protein, CylM, is required for the expression of
CylL(sub L) and CylL(sub S) in an activatable form, and the secreted forms, CylL(sub L)
and CylL(sub S) were recently shown to possess the amino acid lanthionine as the
result of posttranslational modification (64). CylL(sub L) and CylL(sub S) both
are secreted by CylB (65), which is accompanied by an initial proteolytic trimming event
(64) converting each to CylL(sub L)' and CylL(sub S)', respectively. Once
secreted, CylL(sub L)' and CylL(sub S)' are both functionally inactive until six
amino acids are removed from each amino terminus. This final step in maturation is
catalyzed by CylA (64), a subtilisin-type serine protease. Since this final catalytic
event is essential, occurs extracellularly, and is catalyzed by a class of enzyme for
which a substantial body of structural information exists, it represents an ideal
therapeutic target. As shown in Figure 3, inhibition of cytolysin by
mutation (or potentially by therapeutic intervention) results in a reduction by several
orders of magnitude in the number of circulating organisms. 

An inhibitor of cytolysin activation,
accompanied by appropriate rapid
diagnostics, would be of potential
value in treating bacteremia caused
by cytolytic strains of E. faecalis
without affecting commensal flora.
Development of resistance should be
exceedingly improbable because of the
small number of bacteria targeted and
because unlike antibiotics, cytolysin
inhibitors would not act directly on
the organism itself.

Other Enterococcal Targets

Several laboratories are using
information on the E. faecalis genome
and genomes of other pathogens to
identify therapeutic targets (66) and
facilitate studies on pathogenesis
for the remaining 60% of noncytolytic
enterococcal infections. The genome
of an E. faecalis strain that caused
multiple hospital infections (56) was
sampled at high frequency by sequence
analysis. Several sequences appeared
to have a role in host-parasite
interaction. The gene specifying Esp
encodes an apparent surface protein
of unusual repeating structure (67).
Although a role for this protein in
enterococcal infection has yet to be
determined, its distribution among
clinical and commensal strains is
tantalizing: 29 of 30 strains with
this gene were recovered from
patients with bacteremia or
endocarditis; one of 34 isolates
obtained from healthy volunteers
possessed Esp. The core of this large
protein (inferred mass of 202 kDa)
consists of a series of 82 amino acid
repeats encoded by highly conserved
tandem 246 base pair repeats. Lack of
divergence in repeat sequences suggests that recombination occurs at high
frequency, perhaps during infection. Moreover, the number of repeats
observed in homologous genes from different E. faecalis isolates is 3 to 9
(67). This gene is flanked by a sequence similar to the transposase of
IS905. None of 24 clinical or laboratory E. faecium isolates had this gene
(67; V. Shankar, G. Lindahl, and M. Gilmore, unpub. data).

A second promising lead involves a series of genes encoding products highly
related to enzymes involved in O-antigen synthesis in gram-negative
bacteria (68). Preliminary evidence suggests that in E. faecalis these
genes contribute to cell wall carbohydrate synthesis and that this
carbohydrate relates to persistence in vivo. A knockout in one of these
genes results in a strain with normal in vitro growth, but after
subcutaneous injection, the mutant was more readily cleared than the wild
type parental strain (68). One of the genes studied was present in all E.
faecalis strains examined, whereas another occurs only in E. faecalis
strains that share a periodate-susceptible epitope (68). Collectively,
these data indicate that enzymes for synthesis of E. faecalis surface
carbohydrates are important for persistence in vivo and may represent a
useful therapeutic target. Taking a different approach, Arduino et al.
(69,70) identified a protease-resistant, periodate susceptible substance
associated with some strains of E. faecium, but not E. faecalis, which
conferred resistane to phagocytosis in vitro. The relationship between the
putative carbohydrate of E. faecalis under study above and the inhibitory
substance of E. faecium remains to be determined. It may be found that many
enterococci produce such carbohydrates at biologically significant levels
in vivo, but only some strains of E. faecium do so in vitro.

Finally, recent observations indicate that nearly all E. faecalis strains,
and only a few E. faecium strains, generate substantial extracellular
superoxide. When E. faecalis isolates from patients with endocarditis and
bacteremia were compared with isolates from healthy volunteers (71), on
average, extracellular superoxide production was 60% higher among blood
isolates than commensal strains. These data raised several questions: Do E.
faecalis that produce larger amounts of extracellular superoxide possess
greater metabolic flexibility, facilitating adaptation to nonintestinal
infection sites? Does free radical production lead to host cell damage,
allowing release of normally sequestered nutrients (e.g., hemin) that might
promote enhanced E. faecalis growth through cytochrome formation? Might
antioxidants modulate colonization or invasive infection? Answers to these
questions may provide new insights into the transition from intestinal
colonization to infection and may suggest new preventive strategies.

Obstacles to Further Development

Although important insights into enterococcal biology and pathogenesis are
being gleaned from a reverse genetic approach, a paucity of information
still exists on how enterococci colonize the intestinal tract and cause
infection. For example, do E. faecalis or E. faecium colonize the colon
through specific interactions with ligands on human epithelial cells or
intestinal mucin? Do MDR enterococci possess alternate binding activities
that enable them to colonize the intestinal tract at new sites without
competing with the indigenous enterococci? Do probiotics have a role in
restoring colonization resistance to an intestinal ecology altered by broad
spectrum antibiotics?

Is enough being done to combat the emergence of highly resistant nosocomial
pathogens? To effectively compete, industry remains highly responsive to
market opportunities. Research in the public sector has been slow to
respond, and as a result, our understanding of the biology of enterococcal
infection is inadequate. Reasons for the modest public sector response
include the following. 1) The emergence of resistant enterococci coincided
with a reduction of public support for non-AIDS related infectious disease
research. 2) The pathogenesis of nosocomial infection deviates from
paradigms established for obligate pathogens. 3) The research
infrastructure is relatively small because of the low importance
traditionally attached to enterococci as etiologic agents of human disease
and the deemphasis on antibiotic resistance research in the 1980s.

Conclusions

Historically, substantial resources have been invested in developing an
in-depth understanding of the molecular biology of model organisms. During
the 1960s and 1970s, when gram-negative organisms were leading causes of
hospital- and community-acquired infections and gram-positive organisms
were typically sensitive to existing antibiotics (72), a sizable investment
in gram-negative model organisms was appropriate. However, with the
emergence of gram-positive organisms as leading causes of both hospital-
and community-acquired infection in the 1990s, a reevaluation of public
research priorities is warranted.

Since antibiotic use became widespread 50 years ago, bacteria have steadily
and routinely developed resistance. Control of the emergence of resistance
will depend on new approaches to prudent antibiotic use in hospitals and
clinics, based in part on improved surveillance for MDR enterococci and on
better systems to encourage staff adherence to contact isolation
procedures. Equally important will be development of new drugs with
narrower spectra of activity aimed at known and potentially new targets and
the evolution of market conditions that favor their use.

Portions of the work described were supported by Veterans Administration
Merit Review Program, grants from the Public Health Service (EY08289 and
AI41108), and an unrestricted award from Research to Prevent Blindness,
Inc.

Dr. Huycke is an associate professor in the Infectious Diseases Section,
Department of Medicine, Oklahoma University Health Sciences Center. He is
interested in enterococcal pathogenesis as it relates to extracellular
superoxide production by E. faecalis.

Address for correspondence: Michael Gilmore, Department of Microbiology and
Immunology, University of Oklahoma Health Sciences Center, PO Box 26901,
Oklahoma City, OK 73190, USA; fax: 405-271-8128; e-mail:
mgilmore@aardvark.ouknor.edu.

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Emerging Infectious Diseases
National Center for Infectious Diseases

Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/huycke.htm

Please note that figures and equations are not available in ASCII format; 
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[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Synopses

Enteroaggregative Escherichia coli

James P. Nataro,* Theodore Steiner, and Richard L. Guerrant
*University of Maryland School of Medicine, Baltimore, Maryland, USA; and
University of Virginia School of Medicine, Charlottesville, Virginia, USA

  --------------------------------------------------
      Enteroaggregative Escherichia coli (EAEC), an increasingly
      recognized cause of diarrhea in children in developing
      countries, has been particularly associated with persistent
      diarrhea (more than 14 days), a major cause of illness and
      death. Recent outbreaks implicate EAEC as a cause of foodborne
      illness in industrialized countries. The pathogenesis of EAEC
      infection is not well understood, but a model can be proposed
      in which EAEC adhere to the intestinal mucosa and elaborate
      enterotoxins and cytotoxins, which result in secretory diarrhea
      and mucosal damage. EAEC's ability to stimulate the release of
      inflammatory mediators may also play a role in intestinal
      illness.

Since first described in 1987, enteroaggregative Escherichia coli (EAEC)
have been recognized increasingly as agents of diarrhea in developing
countries and (more recently) in industrialized countries. We review the
data supporting the pathogenicity of EAEC, discuss clinical and
epidemiologic features, and summarize the current status of EAEC
pathogenesis research.

History

E. coli have been implicated as agents of diarrheal disease since the 1920s
(1). In 1979, Cravioto et al. reported that most enteropathogenic E. coli
(EPEC) adhered to HEp-2 cells in culture and that the adherence phenotype
could be used to differentiate EPEC. Subsequently, many E. coli not of EPEC
serogroups were also found to adhere to semiconfluent HEp-2 cells, but the
adherence phenotype was clearly different from that induced by EPEC (2,3).
Whereas the adherence pattern of EPEC was localized, the non-EPEC pattern
was diffuse. The diffuse adherent strains were later subdivided into two
further phenotypic subcategories: aggregative and true "diffuse" (4). Only
the aggregative were associated with pediatric diarrhea in Santiago, Chile
(4), prompting the investigators to propose two independent categories:
EAEC and diffusely adherent E. coli. At the same time, non-EPEC, HEp-2
adherent strains (termed enteroadherent E. coli) were associated with
pediatric and traveler's diarrhea in Mexico (5,6). These enteroadherent
strains were later found to belong to the EAEC or diffusely adherent E.
coli categories (7).

Thus, EAEC are now defined as E. coli that do not secrete heat-labile or
heat-stable enterotoxins and adhere to HEp-2 cells in an aggregative (AA)
pattern (Figure 1). This definition may encompass both pathogenic and
nonpathogenic clones that share a factor(s) conferring a common phenotype.


[fig 1]
Figure 1. Aggregative pattern of adherence in the HEp-2 assay after 3 hours
incubation of bacteria with HEp-2 cells. Note bacteria on the surface of the
HEp-2 cells as well as on the glass substratum.

Pathogenesis

Histopathology

EAEC strains adhere to the mucosal epithelium of gnotobiotic piglets in a
thick mucus-containing blanket, irrespective of whether or not the strain
induces diarrhea in this animal (8; Nataro, unpub. obs.; Figure 2). In
these studies, the intestinal epithelium displayed pitting of goblet cells,
suggesting stimulation of mucus secretion. EAEC strains adhered to sections
of pediatric small bowel mucosa in an in vitro organ culture (IVOC) model
(9); the bacteria were also embedded within a mucus-containing biofilm. Two
further observations support a role of mucus in EAEC pathogenesis: EAEC
bound rabbit mucus avidly in an in vitro model (10) and volunteers fed EAEC
strains excrete mucoid stools (11). The formation of a heavy mucus biofilm
may contribute to EAEC diarrheagenicity and, perhaps, to its ability to
cause persistent colonization and diarrhea.

[fig 2]
Figure 2. Biofilm (arrow), containing aggregating bacteria and mucus, adhering 
to the mucosa of a gnotobiotic piglet fed enteroaggregative E. coli strain 042
and sacrificed after 3 days. This piglet did not contract diarrhea. 
(Reprinted with permission of James Nataro and Clinical Microbiology
Reviews. Clin Microbiol Rev 1998;11:142-201.)



In addition to forming the characteristic mucus biofilm, many EAEC strains
induce cytotoxic effects on the intestinal mucosa. Infection with EAEC
strains in rabbit and rat ileal loop models resulted in a destructive
lesion demonstrable by light microscopy (7). EAEC induced shortening of the
villi, hemorrhagic necrosis of the villous tips, and a mild inflammatory
response with edema and mononuclear infiltration of the submucosa.
Transmission electron microscopy showed normal microvillar architecture and
no invasion of enterocytes; both light and electron microscopy showed
adherent bacteria without the attaching and effacing lesion, which is
characteristic of EPEC.

Mucosal damage has also been demonstrated in ileum specimens taken after
patients died of EAEC persistent diarrhea during an outbreak in the
malnutrition ward of Mexico City Hospital (12). This effect was reproduced
in the IVOC model; EAEC induced exfoliation of enterocytes from the mucosal
surface of pediatric intestinal biopsies (9). Indeed, EAEC strains elicit
cytotoxic effects on T84 cells (human intestinal carcinoma) in vitro (13;
Figure 3). In the in vitro model, EAEC induced the microvillar membrane to
vesiculate and the cells from the monolayer to exfoliate. This effect was
accompanied by increased vacuole formation and separation of the nucleus
from the surrounding cytoplasm. Damage was most prominent in areas where
EAEC were adhering to the cells. In both the T84 and IVOC systems, the
toxic effects required genes encoding the adherence fimbriae (14), as well
as other genes on the 65MDa plasmid.

Adherence

Adherence of EAEC to the intestinal mucosa has been well studied. A
flexible, bundle-forming EAEC fimbrial structure 2 nm to 3 nm in diameter,
designated aggregative adherence fimbriae I (AAF/I) (15), mediates HEp-2
adherence and human erythrocyte hemagglutination in EAEC strain 17-2. The
genes for AAF/I are organized as two separate gene clusters on the 60 MDa
plasmid separated by 9 kb of intervening DNA (16-18). Region 1 genes encode
the fimbrial structure itself; nucleotide sequence analysis of the Region 1
cluster suggests that AAF/I is related to the Dr family of adhesins (18).
Region 2 genes encode a transcriptional activator of AAF/I expression
(AggR), which is a member of the AraC family of DNA binding proteins (17).
The AAF/I fimbriae are bundle-forming fimbriae but do not show homology
with members of the so-called type 4 class of fimbriae (19).

[fig 3]
Figure 3. Effects of enteroaggregative E. coli strain
042 on T84 cells in culture. Polarized T84 monolayers were
washed and inoculated with 10(sup 6) CFU of 042 and allowed to
coincubate at 37C for 6 hrs. Transmission electron microscopy
reveals adherence of bacteria to the apical surface of the T84
cells without internalization. The apical brush border is
effaced; cells are ballooning and will eventually be extruded from
the monolayer. (J.P. Nataro and S. Sears, unpub. data). Bar, 1 mm.


A second fimbrial structure, designated AAF/II, has been identified (14);
it is distinct from but related to AAF/I. Insertional mutants in AAF/II
genes no longer adhered to colonic mucosa in IVOC. DNA probe analysis
suggested that AAF/I and AAF/II were each present in only a minority of
EAEC strains and thus, as is the case with enterotoxigenic E. coli,
intestinal colonization of EAEC appears to be mediated by one or more of
several different fimbrial antigens.

In some EAEC strains, AA may be due to factors other than the AAF fimbriae
(20,21). An afimbrial outer membrane protein or a 38 kDa outer membrane
protein may be responsible for AA in some strains (20,21).

EAEC ST-Like Toxin

Savarino et al. have identified an open reading frame encoding a 4,100 Da
homologue of the heat-stable enterotoxin designated EAST1 (22,23), which is
a 38-amino acid protein featuring four cysteine residues, instead of six
(which are characteristic of E. coli ST). A role for EAST1 in human disease
has not been demonstrated, although EAST1 clones yield net increases in
short circuit current in the rabbit mucosal Ussing chamber model (22). Of
the four strains given to volunteers, one that induced diarrhea, as well as
one that did not, secreted EAST1 (11).

EAST1 has been found in approximately 40% of EAEC strains and in a similar
proportion of nonpathogenic E. coli strains (24). Other E. coli categories,
most notably EHEC, elaborate EAST1 with a higher frequency than EAEC (24).

Invasiveness

Some EAEC strains may invade intestinal epithelial cells in vitro (25).
However, human intestinal explants do not internalize EAEC, and clinical
evidence for or against a role for invasiveness is lacking (9).

Other EAEC Toxins

In an outbreak of EAEC diarrhea in Mexico, Eslava et al. identified (in
serum from patients in the outbreak) an approximately 108 kDa protein that
induces exfoliation of enterocytes in the rat ileal loop model. DNA
sequence analysis of the plasmid-borne gene (C. Eslava, J. Czeczulin, F.
Navarro-Garcia, I. Henderson, A. Cravioto, J.P. Nataro, unpub. obs.)
encoding this protein suggests that the toxin is a member of the family of
proteins (26) called autotransporters because their secretion through the
bacterial outer membrane is mediated by the carboxy terminus of the
molecule. The 108 kDa toxin (termed Pet for plasmid-encoded toxin) also
induces enterotoxic activity from rat intestinal tissue mounted in the
Ussing chamber (F. Navarro-Garcia, C. Eslava, J.P. Nataro, A. Cravioto,
unpub. obs.). A 120 kDa EAEC supernatant protein had been described that
elicited rises in intracellular calcium in HEp-2 cells (27). Whether or not
this protein is related to the protein described by Eslava et al. remains
to be determined.

Intestinal Inflammation

An ongoing study of infant diarrhea in Fortaleza, Brazil (28-30), has shown
that children with EAEC infection have intestinal inflammation, as measured
by the presence of inflammatory markers in the stool (31). These markers
include lactoferrin, a stable neutrophil product and sensitive marker for
fecal neutrophils; interleukin-8 (IL-8), a neutrophil chemokine; and
interleukin-1 (IL-1), a polyfunctional proinflammatory cytokine. Fecal
lactoferrin, IL-8, and IL-1 were elevated in children with persistent
diarrhea due to EAEC but not in controls (children in the same cohort
without stool pathogens and free of diarrhea for 3 weeks before and after
the time of the stool sample in prospective surveillance). Fecal
lactoferrin and IL-1b were also elevated in children with EAEC but no
diarrhea for 3 weeks. The origin of these inflammatory factors remains
unknown, although they may be the result of direct stimulation of cytokine
release from the epithelium.

While studying cytokine release in vitro, Steiner et al. observed IL-8
release from Caco-2 intestinal epithelial cells exposed to cell-free
filtrates of EAEC strains 042 or 17-2. This release was apparently mediated
by a heat-stable, high molecular weight, chromosomally encoded protein
(31). Lipopolysaccharide does not appear responsible for this effect as it
is not blocked by polymyxin B and is not elicited by normal E. coli flora.

The relationship between IL-8 release and the symptoms of EAEC infection is
not known, especially since severe infiltration by neutrophils has not yet
been reported in EAEC histopathologic specimens. However, a hypothetical
role for IL-8 release in EAEC diarrhea can be envisioned. IL-8 released
from epithelial cells in response to EAEC could act as the first step in a
secretory cascade by recruiting neutrophils, since neutrophils in the
intestinal epithelium release 5'-adenosine monophosphate, which is
converted by a 5'-nucleotidase on the apical surface of enterocytes to
adenosine, an agonist for chloride secretion (32). A similar mechanism of
neutrophil chemotaxis and cytokine release may also be involved in the
diarrhea produced by other microbial products, such as Clostridium
difficile toxin A (33,34).

Strain Heterogeneity

EAEC strains exhibit considerable heterogeneity. Studies at the University
of Maryland found that at a dose of 10(sup 10) CFU with bicarbonate, strain 042
(which produces AAF/II fimbriae, EAST1, and the 108 kDa Pet toxin) elicited
loose stools in 4 of the 5 adult volunteers (11). Three other strains, all
of which expressed AAF/I and one of which secreted EAST1, did not produce
any intestinal symptoms. These data suggest that virulence is likely to be
heterogeneous and that Pet may play an important role.

A Model of EAEC Pathogenesis

Extrapolating from the observations described above, we propose a
three-stage model of EAEC pathogenesis. Stage I involves initial adherence
to the intestinal mucosa and the mucus layer; this initial adherence is
apparently mediated by the AAF fimbriae. Stage II comprises enhanced mucus
production, apparently leading to the deposit of a thick mucus-containing
biofilm encrusted with EAEC. The blanket may promote persistent
colonization and nutrient malabsorption. Stage III, suggested from
histopathologic and molecular evidence, includes the elaboration of toxins
or inflammation, which result in damage to the mucosa and intestinal
secretion. Malnourished hosts may be unable to repair the mucosal damage
and may thus become prone to the persistent diarrhea syndrome.

The site of EAEC infection in the human intestine has yet to be clearly
demonstrated. IVOC experiments have shown that EAEC strains can adhere to
both small and large bowel mucosa (9). Tissue specimens in these studies
were derived from pediatric patients and were not formalin-fixed before
they were incubated with EAEC strains. These features may explain the
discrepant results obtained by other investigators (35,36). The short
incubation period observed in some humans challenged with strain 042 (as
short as 8 hours) is also consistent with involvement of the small bowel in
diarrheagenicity (11).

Clinical Features

The clinical features of EAEC diarrhea are increasingly well defined in
outbreaks, sporadic cases, and the volunteer model. Typical illness is
manifested by a watery, mucoid, secretory diarrhea with low grade fever and
little to no vomiting (37,38). However, up to one-third of patients with
EAEC diarrhea have had grossly bloody stools (39); this outcome may be
strain dependent. In volunteers infected with EAEC strain 042, the diarrhea
was mucoid, of low volume, and without occult blood or fecal leukocytes;
all patients remained afebrile. In such volunteers, the incubation period
of the illness was 8 to 18 hours (11).

The duration of EAEC diarrhea is its most striking feature. In a community
surveillance study in Anapur-Palla in northern India, the mean duration of
EAEC-associated diarrhea cases in children under 3 years of age was 17
days, longer than that associated with any other pathogen (37). In this
study, of the 41 cases of diarrhea, fever was found in 12%, gross blood in
12%, vomiting in 7%, and persistence of the episode more than 14 days in
44%.

A large percentage of patients excreting EAEC have detectable fecal
lactoferrin and supranormal levels of IL-8 in the stool (31). Although this
observation suggests that EAEC infection may be accompanied by mucosal
inflammation, most patients lack overt clinical evidence of inflammation.

Diagnosis

EAEC colonization is detected by isolating E. coli from stool samples and
demonstrating the AA pattern in the HEp-2 assay. Analysis of small bowel
aspirates has not increased yield (40). Implication of EAEC as the cause of
the patient's disease must be done cautiously, given the high rate of
asymptomatic colonization in many populations (4,30,41-43). If no other
organism is implicated in the patient's illness and EAEC is isolated
repeatedly, it may be considered to be a probable cause of the patient's
illness.

Despite various methods of performing the HEp-2 assay (4,44,45),
comparative studies suggest that the technique first described by Cravioto
et al. (45,46) (i.e., a single 3-hour incubation of bacteria with cells,
without a change in medium during the assay) best discriminates among the
three adherence patterns. Because AAF adhesins are expressed maximally in
static L-broth cultures at 37C (15), we incubate all HEp-2 assay inocula
in this manner.

A DNA fragment probe has proven highly specific in detecting EAEC strains.
This probe was developed by Baudry et al. (47), who tested fragments
derived empirically from the plasmids of strains 17-2 and 042. A 1.0 kb
plasmid-derived Sau3a fragment hybridized with 56 (89%) of 63 EAEC strains
(defined by HEp-2 assay); of 376 strains representing normal flora and
other diarrheagenic categories, only 2 hybridized with the probe. The
correlation of the EAEC probe-positivity with AA varies by location. In
some studies, the correlation achieves the 89% sensitivity reported by
Baudry et al. (47,48), while in other studies, the sensitivity may be
substantially lower (40). The epidemiologic significance of probe-positive
versus probe-negative strains has not been determined. The nucleotide
sequence of the AA probe represents a cryptic open reading frame adjacent
to the plasmid replicon (J.P. Nataro, unpub. obs.). A polymerase chain
reaction assay using primers derived from the AA probe sequence shows
similar sensitivity and specificity (49).

Epidemiology

Volunteer studies and outbreak investigations have shown that at least some
EAEC strains are human pathogens. A growing number of studies have
supported the association of EAEC with diarrhea in developing countries;
the increasing number of such reports and the rising proportion of
diarrheal cases in which EAEC are implicated suggest that EAEC are
important emerging agents of pediatric diarrhea.

EAEC in Sporadic Diarrhea

The earliest epidemiologic reports showed that EAEC were most prominently
associated with persistent (lasting >/=14 days) cases of pediatric diarrhea
(Table 1). In some of these studies, EAEC cultured from the stool during
the first few days of diarrhea were predictive of a longer illness (37,43).

The importance of EAEC in diarrheal disease appears to vary geographically.
On the Indian subcontinent, six studies (which included hospitalized
patients with persistent diarrhea [50], outpatients visiting health clinics
[51], and cases of sporadic diarrhea detected on household surveillance
[37]) have demonstrated the importance of EAEC in pediatric diarrhea
(37,38,48,50,51,53).

EAEC and persistent diarrhea syndrome have been consistently associated
(28,29,30,40). In Brazil, EAEC have been implicated in up to 68% of
persistent diarrhea cases (40), which represent a disproportionate share of
deaths due to diarrhea. EAEC have also been implicated as causes of
sporadic diarrhea in Mexico, Chile, Bangladesh, and Iran (4,39,42,43).

Table 1. Epidemiologic studies associating enteroaggregative Escherichia coli
(EAEC) with diarrhea ( p < 0.05)

----------------------------------------------------------------------------------

Source or                                  EAEC in cases
reference   Syndrome        Site           vs controls           Comments
-----------------------------------------------------------------------------------
4         All             Santiago, Chile  84(33%)/253 vs        Community and
          diarrhea                         20(15%)/134(sup a)    hospital-based. First
                                                                 description of EAEC
37        Persistent      Anapur-Palla,    18(30%)/61 vs.        Household surveillance of
          diarrhea        India            20(10%)/201(sup b)    ruralchildren </=3 yrs;
                                                                 first association of EAEC
                                                                 with PD
50        Persistent      New Delhi,       18(20%)/92 vs         Case-control study of non-
          diarrhea        India            6(7%)/92(sup b)       bloody PD in children </= 2
                                                                 yrs
28        Persistent      Fortaleza,       8(20%)/40 vs.         Household surveillance of
          diarrhea        Brazil           2(5%)/38(sup b)       children </= 5 yr

39        All diarrhea;   Morales, Mexico  78(12%)/636(sup a)    Household surveillance of
          persistent                       and 29(51%)/57(sup b) children </= 2 yrs
          diarrhea                         vs 5(5%)/100

43        Persistent      Mirzapoor,       17(27%)/62 vs         Household surveillance of
          diarrhea        Bangladesh       5(18%)/28(sup c)      children </= 6 yrs. EAEC
                                                                 isolated in first 2 days
                                                                 of episode predicts
                                                                 persistence
29        Persistent      Fortaleza,       8(20%)/40 vs          Outpatients </= 29 months
          diarrhea        Brazil           2(5%)/38(sup b)       with PD
51        All             New Delhi,       9(21%%)/42 vs         Children </= 4 yrs referred
          diarrhea        India            4(4%)/107(sup b)      to hospital
38        Acute           Calcutta,        17(11%)/159 vs        EAEC associated with acute
          diarrhea        India            3(2%)/174(sup d)      secretory diarrhea in
                                                                 hospitalized pediatric
                                                                 patients
42        All             Tehran, Iran     99(32%)/309 vs        Pediatric outpatients.
          diarrhea                         17(17%)/100(sup a)    High rate of antibiotic
                                                                 resistance among EAEC
39        Persistent      Fortaleza,       38(68%)/56 vs         Outpatients </= 3 yrs; EAEC
          diarrhea        Brazil           13(31%)/42(sup b)     associated with more
                                                                 cases  of  PD than all
                                                                 other agents combined
48        Acute diarrhea  Vellore, India   60(8%)/794 vs         Outpatients </= 3 yrs with
                                           2(4%)/566(sup a)      diarrhea </= 3days
52        All diarrhea    Wurbuerg,        16(2%)/79 vs          Hospitalized children < 16
                          Germany          0/580(sup a)          yrs. First description of
                                                                 EAEC disease burden in
                                                                 industrialized countries
41        Acute diarrhea  Caracas,         138(27%)/513 vs       Inpatient and outpatient
                          Venezuela        36(15%)/241(sup a)    children </= 2 yrs with
                                                                 diarrhea </= 3 days. EAEC
                                                                 only associated with
                                                                 diarrhea in infants </= 2
                                                                 mos of age
Jalla-     All diarrhea   Lwiro, Zaire     29(25%)/115 vs        Outpatients </= 5 yrs. First
luddin et                                  3(8%)/34(sup a)       description of EAEC in
al., 1997,                                                       Africa
pers.
comm.
---------------------------------------------------------------------------------------
(sup a)All diarrhea vs. asymptomatic.
(sup b)Persistent diarrhea (PD) vs. asymptomatic.
(sup c)Persistent diarrhea vs. acute diarrhea.
(sup d)Secretory vs. invasive diarrhea.

The epidemiologic characteristics of EAEC (e.g., likely sources, reservoirs
of infection, routes of transmission, seasonality, and age-distribution)
are largely unknown. Most studies in which EAEC are implicated as causes of
diarrhea have isolated the organism from infants and small children, yet
volunteer studies and outbreak investigations suggest that school-age
children and adults are also susceptible (54-56). Regarding sources of
infection, EAEC strains have been isolated from the lacteal contents of
infant feeding bottles in Brazil (57).

Recent studies have suggested that EAEC are found frequently in stool
samples of patients in industrialized countries and may be important agents
of diarrhea. EAEC were isolated from 2% of pediatric patients with diarrhea
in Germany; the pathogen was associated with diarrheal illness (58). The
patients from whom EAEC were isolated had characteristically prolonged
illness, often followed by apparent abdominal pain mimicking infant colic.

EAEC Outbreaks

Several outbreaks of EAEC diarrhea have now been reported (Table 2). The
first detailed description of an outbreak from which EAEC were clearly
implicated involved 19 infants in a hospital nursery in Nis, Serbia, during
9 days in 1995 (52). Twelve of the 19 infants had the same
multidrug-resistant EAEC strain of serogroup O4, while none of five well
neonates had this organism (p = 0.02). The illness lasted 3 to 9 days (mean
5.2 days) in 16 babies, but in three infants, persistent diarrhea
developed, lasting 18 to 20 days. Infants with diarrhea typically had
liquid green stools. In three infants, mucus was visibly apparent; no
infant had bloody stools. All but three infants required intravenous
hydration but all survived. The source of infection was unclear.

In two outbreaks of severe lethal diarrhea in malnutrition wards of Mexico
City hospitals (12), persistent diarrhea developed in affected infants, and
five patients died despite aggressive support. Autopsy findings from
infants who died in these outbreaks showed severe necrotic lesions of the
ileal mucosa.

During a diarrhea epidemic in a village in southern India in January 1996,
EAEC were identified in the stools of 11 of 20 persons who were thought to
have outbreak-related diarrhea; 1 of 11 stools from asymptomatic controls
from the same village (p < 0.02) contained EAEC (59). Intake of water from
open wells was associated with diarrheal illness; however, EAEC were not
identified from this source, and contaminated water was not definitively
implicated as the source of the epidemic.

EAEC outbreaks also occur in industrialized countries. In a massive
outbreak of EAEC diarrhea in Gifu Prefecture, Japan, in 1993, 2,697
children at 16 schools became ill after consuming contaminated school
lunches (54). The illness was characterized by abdominal pain, nausea, and
severe diarrhea, which was protracted in at least 30 cases. The incubation
period for this illness averaged 40 to 50 hours. A single EAEC strain of
serotype O:H10 was implicated, but the organism was not found in any of the
foods served in the implicated lunch.

Four outbreaks of EAEC-related diarrhea occurred in the United Kingdom in
1994 (56), which involved 19, 10, 51, and 53 persons, respectively, most of
them adults. Only in the last two outbreaks was a single EAEC strain
implicated. Although each of these outbreaks was linked to consumption of a
meal, no single vehicle has been implicated. The illnesses were
characterized by vomiting and diarrhea, occasionally with fever. The mean
duration of illness in one outbreak was 68 hours. Strains of E. coli
O44:H18 implicated in outbreaks in the United Kingdom (and previously
presumed to be EPEC) are in fact EAEC (55). This report mentions three EAEC
outbreaks, two among children and one among adult patients.

Table 2. Outbreaks of enteroaggregative Escherichia coli diarrhea
---------------------------------------------------------------------------------
Reference        Setting           Patients        Illness
---------------------------------------------------------------------------------

52          Nursery; Nis,          19 infants      Watery diarrhea
            Serbia, 1995      
     
56          Conference Center,     51 adults       Watery diarrhea for 3-7 days
            UK, 1994               

56          Hotel, UK, 1994        53 adults       Watery diarrhea, mean duration
                                                   68 hr

59          Village in southern    20 children     Watery diarrhea, mean duration
            India, 1996            and adults      11 days

12          Nursery, Mexico        Not reported    Severe diarrhea; 5 deaths
            City                 

54          16 schools, Gifu,      2,697 school    Diarrhea; some cases
            Japan                  children        protracted

---------------------------------------------------------------------------------

EAEC and Malnutrition

Perhaps even more significant than the association of EAEC with diarrhea
may be the recent data from Fortaleza, Brazil, that link EAEC with growth
retardation in infants (31). In this study, EAEC isolated from the stools
of infants was associated with a low z-score for height or weight,
irrespective of the presence of diarrheal symptoms. Such an association has
been suggested for Cryptosporidium (60). A study among an Australian
aborigine population supports the association of EAEC with growth
retardation in the absence of diarrhea (S. Elliott, J.P. Nataro, unpub.
data). Given the high prevalence of asymptomatic EAEC excretion in many
areas (4,30,41-43), if this observation holds up in further studies, the
contribution of EAEC to human disease might be much greater than is
currently thought.

Conclusions

EAEC is an emerging diarrheal pathogen linked to acute and persistent
diarrhea in both developing and industrialized countries. Both sporadic
diarrhea and outbreaks (possibly foodborne) have been described. The
mechanisms of pathogenesis are being elucidated; likely not all EAEC
strains are equally pathogenic. The mechanism of the association between
EAEC and malnutrition is not entirely clear, although plausible
pathogenetic models can be proposed. Malnutrition predisposes to persistent
diarrhea (61,62); whether this is due to impaired host immunity (preventing
normal killing of pathogens) or to altered intestinal physiology
(predisposing to more severe infections) is not known. However, it seems
also plausible that asymptomatic colonization with EAEC may lead to
malnutrition caused by increased metabolic demand secondary to intestinal
inflammation, persistent mucosal damage due to cytotoxins, and/or the
presence of a barrier to the absorption of nutrients imposed by the
mucus/bacteria biofilm. A vicious cycle may thus operate wherein
malnutrition and infection perpetuate and enable each other (Figure 4). The
pathophysiologic machinery that propels children through this circle is
probably multifactorial and opens several possible avenues for intervention
against this important global problem.

[fig 4]
Figure 4. Relationship between diarrhea and malnutrition (31, 6062).

Supported by Public Health Service grant AI 33096 to JPN.

Dr. Nataro is associate professor of pediatrics, medicine, and microbiology
and immunology at the University of Maryland School of Medicine, Baltimore.
His research focuses on the pathogenesis and epidemiology of the various
pathotypes of diarrheagenic Escherichia coli, with particular interest in
their role in persistent diarrhea among children in developing countries.

Address for correspondence: James P. Nataro, Center for Vaccine
Development, University of Maryland School of Medicine, 685 W. Baltimore
St., Baltimore, MD 21201, USA; fax: 410-706-6205; e-mail:
jnataro@umppa1.ab.umd.edu.

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 52. Cobeljic M, Miljkovic-Selimovic B, Paunovic-Todosijevic D, Velickovic
     Z, Lepsanovic Z, Zec N, et al. Enteroaggregative Escherichia coli
     associated with an outbreak of diarrhoea in a neonatal nursery ward.
     Epidemiol Infect 1996;117:11-6.
 53. Bhan MK, Bhandari N, Sazawal S, Clemens J, Raj P, Levine MM, Kaper JB.
     Descriptive epidemiology of persistent diarrhea among young children
     in rural northern India. Bull World Health Organ 1989;67:281-8.
 54. Itoh Y, Nagano I, Kunishima M, Ezaki T. Laboratory investigation of
     enteroaggregative Escherichia coli O untypeable:H10 associated with a
     massive outbreak of gastrointestinal illness. J Clin Microbiol
     1997;35:2546-50.
 55. Smith HR, Scotland SM, Willshaw GA, Rowe B, Cravioto A, Eslava C.
     Isolates of Escherichia coli O44:H18 of diverse origin are
     enteroaggregative. J Infect Dis 1994;170:1610-3.
 56. Smith HR, Cheasty T, Rowe B. Enteroaggregative Escherichia coli and
     outbreaks of gastroenteritis in UK. Lancet 1997;350:814-5.
 57. Morais TB, Gomes TAT, Sigulem DM. Enteroaggregative Escherichia coli
     in infant feeding bottles. Lancet 1997;349:1448-9.
 58. Huppertz HI, Rutkowski S, Aleksic S, Karch H. Acute and chronic
     diarrhoea and abdominal colic associated with enteroaggregative
     Escherichia coli in young children living in western Europe. Lancet
     1997;349:1660-2.
 59. Pai M, Kang G, Ramakrishna BS, Venkataraman A, Muliyil J. An epidemic
     of diarrhoea in south India caused by enteroaggregative Escherichia
     coli. Indian J Med Res 1997;106:7-12.
 60. Checkley W, Gilman RH, Epstein LD. Asymptomatic and symptomatic
     cryptosporidiosis: their acute effect of weight gain in Peruvian
     children. Am J Epidemiol 1997;145:156-63.
 61. Guerrant R, Schorling J, McAuliffe J, Souza MD. Diarrhea as a cause
     and effect of malnutrition: diarrhea prevents catch-up growth and
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Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/nataro.htm

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[[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Perspectives

Campylobacter jejuni Strains from Patients with Guillain-Barr Syndrome

Ban Mishu Allos,* Frank T. Lippy,* Andrea Carlsen,* Ronald G. Washburn,
and Martin J. Blaser*
*Vanderbilt University School of Medicine, The Department of Veterans
Affairs Medical Center, Nashville, Tennessee, USA; and The Bowman Gray
School of Medicine, Winston-Salem, North Carolina, USA

---------------------------------------------------------------------------
      Guillain-Barr syndrome (GBS), an acute demyelinating
      peripheral neuropathy, may be triggered by an acute infectious
      illness; infection with Campylobacter jejuni is the most
      frequently reported antecedent event. In Japan, O:19 is the
      most common serotype among GBS-associated C. jejuni strains. To
      determine whether serotype O:19 occurs among GBS-associated
      strains in the United States and Europe, we serotyped seven
      such strains and found that two (29%) of seven GBS-associated
      strains from patients in the United States and Germany were
      serotype O:19. To determine whether GBS-associated strains may
      be resistant to killing by normal human serum (NHS), we studied
      the serum susceptibility of 17 GBS- and 27 enteritis-associated
      strains (including many O:19 and non-O:19 strains) using C.
      jejuni antibody positive (pool 1) or negative (pool 2) human
      serum. Using pool 1 serum we found that one (6%) of 18 serotype
      O:19 strains compared with 11 (42%) of 26 non-O:19 strains were
      killed; results using pool 2 serum were nearly identical.
      Finally, 8 O:19 and 8 non-O:19 strains were not significantly
      different in their ability to bind complement component C3.
      Serotype O:19 C. jejuni strains were overrepresented among
      GBS-associated strains in the United States and Germany and
      were significantly more serum-resistant than non-O:19 strains.
      The mechanism of this resistance appears unrelated to C3
      binding.

Guillain-Barr syndrome (GBS) is an acute demyelinating polyneuropathy
characterized by an immunologic attack upon peripheral nerve myelin (1,2).
The trigger for this immune attack is unknown; however, GBS is frequently
preceded by an acute infectious illness (3). In recent years, infection
with Campylobacter jejuni has emerged as one of the most common antecedent
events associated with GBS. Up to 40% of patients with GBS have culture or
serologic evidence of C. jejuni infection when neurologic symptoms begin
(4-6). The many variants of GBSincluding acute motor axonal neuropathy,
acute inflammatory demyelinating neuropathy, and Miller Fisher
syndromehave also been associated with preceding C. jejuni infection
(7-9).

C. jejuni infections are common in the United States, affecting
approximately 1% of the population each year (10). Typically, they cause a
self-limited gastrointestinal illness characterized by diarrhea, abdominal
pain, and fever. However, approximately 1 in 2,000 C. jejuni infections may
be complicated by GBS (11). Because C. jejuni infections occur far more
commonly than GBS, either host (12) or strain (5,13) characteristics may
determine which infected persons contract GBS. Several reports from Japan
showed that a particular serotype, O:19, was overrepresented among C.
jejuni strains isolated from GBS patients (5,13). Among 12 Japanese
patients with GBS and culture-confirmed C. jejuni infection, 10 (83%) were
infected with strains of O:19, a serotype that accounts for only 2% of
randomly selected C. jejuni isolates in Japan (5). However, in England,
none of four C. jejuni strains isolated from patients with GBS were
serotype O:19 (6). Other Campylobacter serotypes predominate among GBS
patients in other parts of the world. All nine C. jejuni strains isolated
from GBS patients in South Africa were serotype O:41 (14); this serotype
accounts for fewer than 2% of isolates from South African children with
uncomplicated enteritis. With Lior typing (a serotyping system based upon
heat-labile antigens), all four GBS-associated C. jejuni strains in Germany
were serotype O:11 (15).

Whether the predominance of O:19 strains among GBS patients is limited to
Japan is not clear. Furthermore, the reason for the O:19 overrepresentation
among Japanese GBS strains is unknown, but their resistance to host
bacterial clearance mechanisms is one possibility. C. jejuni strains vary
in their susceptibility to the bactericidal activity in normal human serum
(NHS) (16). Bacteria that are more resistant to nonspecific serum killing
may elicit more extensive specific responses to eliminate the organism;
such elicited immune responses may have the potential of greater tissue
injury and myelin damage.

To determine whether C. jejuni serotype O:19 is an important cause of GBS
outside Japan, we serotyped GBS-associated strains from the United States
and Germany. To investigate the immunologic response to GBS-associated C.
jejuni strains, we performed two additional sets of experiments. First, we
examined the susceptibility of C. jejuni strains to NHS. Our hypothesis was
that GBS-associated Campylobacter strains, more specifically O:19 strains,
are more serum-resistant than those from patients with uncomplicated
enteritis. To further characterize the mechanism of resistance, we
determined whether C. jejuni strains isolated from GBS patients differ in
their capacity to bind complement component C3.

Statistical Analysis

We determined mean values, standard deviations, odds ratios, p-values,
paired T-tests, 95% confidence limits, and interquartile ranges. Software
included Lotus 123 and EpiInfo (a statistical software package designed by
the Centers for Disease Control and Prevention [CDC]).

Serotyping of Strains

Seven GBS-associated strains were serotyped with the O (formerly
heat-stable) serotyping scheme of Penner and Hennessy (17) and the
heat-labile serotyping scheme of Lior (18). Serotyping was conducted (19)
at the CDC Campylobacter Reference Laboratory. A GBS-associated strain was
defined as one isolated from a patient with GBS (n = 6) or from a patient
infected during an outbreak in which at least one infected person
contracted GBS (n = 1). Four strains were from patients in the United
States, and three were from patients in Germany; four of these were
isolated from women.

Serotype O:19 was present in one of four U.S. GBS-associated C. jejuni
isolates and one of three German isolates. Thus, 2 (29%) of 7
GBS-associated C. jejuni isolates from patients in the United States and
Germany were O:19. The O and heat-labile serotypes of the seven
GBS-associated strains are shown in Table 1.

Table 1. Serotypes of Guillain-Barr syndromeassociated Campylobacter jejuni
strains from the United States and Germany

----------------------------------------------------------------------
                          
   Strain designation   (O)(sup a)(HL)(sup b)
-----------------------    sero-    sero-                      Age/
CDC(sup c)Vanderbilt(sup d)type     type    Location           sex
----------------------------------------------------------------------
D459          93-002        19       77     Florida            79F
D2769         93-006         2        4     Maine              F
D4262         84-158        19       84     Germany            59F
D4266         84-196         2        4     Germany            24M
D4267         84-197         8,17    40     Germany            71F
D4271         86-381         2        4     Wisconsin          30M
D4808         93-001         1        4     Washington         57M
----------------------------------------------------------------------
(sup a)O = heat-stable.
(sup b)HL = heat-labile.
(sup c)Designated strain number assigned by the Centers for Disease Control and
Prevention Campylobacter Reference Laboratory.
(sup d)Designated strain number in the Vanderbilt Campylobacter/Helicobacter
strain collection.

Serum Bactericidal Assays

We studied 44 GBS- and enteritis-associated strains from all over the
world. We divided C. jejuni isolates into two major groups. Group 1 (17
GBS-associated strains) was divided into two subgroups. Subgroup 1A
(GBS-associated/type O:19) consisted of seven C. jejuni O:19 isolates:
three from the United States, three from Japan, and one from Germany.
Subgroup 1B (GBS-associated/non-O:19 serotype) consisted of 10 C. jejuni
isolates with serotypes other than O:19: three from the United States, two
from Germany, and five from England. Group 2 (27 enteritis-associated
strains from patients with uncomplicated enteritis but no known GBS
association) was also divided into two subgroups. Subgroup 2A (not
GBS-associated/type O:19) consisted of 11 C. jejuni O:19 isolates: 10 from
the United States and one from Japan. Subgroup 2B (not
GBS-associated/non-O:19 serotype) consisted of 16 C. jejuni isolates from
U.S. patients with serotypes other than O:19. Serum-resistant C. fetus
strain 23D and its spontaneous serum-susceptible mutant, 23B, were used as
controls (20).

Strains were grown on trypticase soy blood agar plates (BBL Cockeysville,
MD, USA) at 37C for 48 hours in an incubator containing 10% CO2, 5% O2,
and 85% N2. The bacteria were harvested in 0.15M saline and centrifuged at
8,000 g for 15 minutes. The supernatant was discarded, the pellet was
resuspended in 300 l of saline, and 10-fold serial dilutions were
performed in saline. From the 10(sup -4), 10(sup -5), and 10-6 dilutions, 
400 l aliquots of cells were added to 1.2 ml of Hanks balanced salt solution
(HBSS).

Two sources of NHS were used in these assays. Pool 1 consisted of serum
from five healthy adults that was pooled, aliquotted, and frozen at -70C.
The level of C. jejuni antibodies in pool 1 serum was determined by
enzyme-linked immunosorbent assay (ELISA) (4). The optical density ratio
(ODR) for immunoglobulin G (IgG) was 5.14; the ODR for IgM was 0.66.
Documentation of previous C. jejuni infection was not required to be
included in this pool. In contrast, pool 2 consisted of serum from two
healthy adults with only low-titer antibody. The ODR for pool 2 serum in
the IgG assay was 0.37; and the ODR in the IgM assay was 0.30. As a
control, the NHS was heated to 56oC for 30 minutes (heat inactivated NHS
[HINHS]) to ablate all complement activity.

The assay to determine susceptibility to NHS was performed in sterile,
disposable 96-well microtiter U-bottom plates (Falcon MICROTEST III, Becton
Dickinson & Co., Franklin Lakes, NJ, USA). From each of the bacterial
suspensions, a 150 l aliquot was added to duplicate wells. In addition, 50
l of NHS or HINHS (diluted to 40% with HBSS) was added; the final serum
concentration in the suspension was 10%. After the assay plate was
incubated at 37C for 1 hour, 50 l of the suspension from each well was
poured onto blood agar plates and incubated for 48 hours; the number of
CFUs was then calculated. The difference between the counts for cells
incubated with NHS and HINHS was expressed as median log(sub 10) kill for each
strain; greater than 1 log10 kill was considered a serum-susceptible strain
(21). If less than 1 log10 kill occurred, the strain was considered
serum-resistant. The identical procedure was performed using pool 1 and
pool 2 serum.

The resistance of the 44 C. jejuni strains studied to killing by NHS is
shown in Table 2. Of these strains, 12 (27%) were resistant to killing by
C. jejuni antibody-positive pool 1 serum and 10 (23%) were resistant to
killing by C. jejuni antibody-negative pool 2 serum; thus, as described
previously (22), antibodies to C. jejuni had little impact on
serum-killing. The GBS-associated strains were no more likely to be
resistant to serum-killing than were the strains from patients with
uncomplicated enteritis. However, O:19 strains were significantly more
likely than other C. jejuni serotypes to resist serum-killing, regardless
of GBS-association and serum pool used (Table 2). In serum pool 1, only 1
of 18 O:19 strains was serum-susceptible compared with 11 (42%) of 26
non-O:19 strains (odds ratio = 12.5, p = 0.008). Similarly, in serum pool
2, no O:19 strain was serum-susceptible compared with 10 (38%) of the
non-O:19 isolates.

Table 2. Susceptibility of Campylobacter jejuni strains to killing by normal
human serum (NHS)

---------------------------------------------------------------------------------                           
                   	        No. (%) with                Median log kill
                    No. of    >1 log kill(sup a,b)     (interquartile range) 
		  O    strains  ------------------------- -----------------------
Illness   serotype studied Pool 1(sup c) Pool 2(sup c)  Pool 1     Pool 2
---------------------------------------------------------------------------------

GBS(sup d)   19      7     1 (14)           0 (0)       0.53        0.39 
                                                    (0.18-0.63) (0.08-0.52)
Enteritis    19     11      0 (0)           0 (0)       0.37        0.48 
                                                    (0.09-0.47) (0.29-0.50)
GBS          non-19 10     3 (30)           3 (30)      0.70        0.43 
                                                    (0.44-1.18) (0.03-1.32)
Enteritis    non-19 16     8 (50)           7 (44)      0.90        0.59 
                                                    (0.24-2.00) (0.35-2.53)
All          19     18     1 (6)            0 (0)       0.38        0.45 
                                                    (0.17-0.58) (0.23-0.52)
All          non-19 26    11 (42)          10 (38)      0.70        0.54 
                                                    (0.26-1.86) (0.17-2.27)
---------------------------------------------------------------------------------
(sup a)Bacterial suspensions were incubated in either NHS or heat inactivated NHS
at 37C for 1 hr as described in text, and net CFU (NHS minus HINHS)
determined. Values greater than 1 log(sub 10) kill were considered to denote a
serum-susceptible strain.
(sup b)Comparison of O:19 strains with non-O:19 strains: Pool 1, odds ratio = 12.5,
p = 0.008; Pool 2, odds ratio = undefined, p = 0.003.
(sup c)Pool 1 consists of C. jejuni-antibody positive serum from five healthy
adults; Pool 2 consists of C. jejuni-antibody negative serum from two healthy
adults.
(sup d)GBS = Guillain-Barr syndrome.

(sup 125)I-C3 Binding Assays

Eight strains from Group 1 (four randomly selected from each subgroup) and
eight from Group 2 (four randomly selected from each subgroup) were grown
on blood agar plates as described above. C. fetus strains 23D and 23B again
served as controls and the assays were conducted (21). In brief, bacteria
from each plate were harvested in 1.5 ml of HBSS and were centrifuged at
8,000 g for 15 minutes; the pellet was resuspended in 0.5 ml HBSS and
adjusted to OD(sub 450)0 = 3.0. For each strain studied, a suspension of 4 l
(sup 125)I-C3 (with 20,000 cpm to 65,000 cpm) and 2.5 l of either NHS or HINHS
from pool 2 and 100 l of the bacteria-HBSS suspension were incubated at
37oC for 15 minutes. (sup 125)I-C3 was prepared in the laboratory of one of the
authors (RGW) (21). The suspensions were then centrifuged twice at 175xg
for 5 minutes, the pellet was resuspended in HBSS, and the supernatant was
discarded. The bottom 5 mm (containing the pellet) of each tube was
clipped, and emissions were determined in a gamma counter. The counts in
the negative control mixtures containing HINHS were subtracted from the NHS
counts. An assay was considered valid only if the net pellet counts (NHS
minus HINHS) for control strain 23B were at least four times higher than
for 23D. To control for nonspecific binding, the counts for each strain
studied were expressed as the ratio of net counts relative to the serum
susceptible control (23B). Each strain was assayed two to four times.

Mean binding of serum-susceptible control strain 23B was 497 cpm, whereas
mean binding of serum-resistant control strain 23D was 54. The mean ratio
of C3-binding for strain 23D to 23B of 0.114 was as expected (21). In
contrast, the mean ratio for C3 binding to C. jejuni strains in comparison
with strain 23B was 0.022 to 0.464 (mean 0.216). The eight O:19 strains
were not significantly different from the eight non-O:19 strains in their
ability to bind (sup 125)I-C3 (Table 3).

Table 3. Comparison of (sup 125)I-C3 binding to Campylobacter
jejuni strains
--------------------------------------------------------------
Measure                   Serotype of      C. jejuni strain
--------------------------------------------------------------
                             O:19              non-O:19
                            (n = 8)             (n = 8)
Counts per minute(sup a)   85 +/-51            98 +/- 48
Mean binding ratio(sup b)  0.187 +/-           0.245 +/-
                            0.116               0.130
--------------------------------------------------------------
(sup a)Bacterial cells were incubated with pool 2 normal human
serum or heat inactivated normal human serum and (sup 125)I-C3
(mean 40,416 [range 20,234-63,503]. cpm) at 37C for 15
minutes. The cells were centrifuged and washed; net (NHS
minus HINHS) 125I binding was measured as counts per minute.
p-value = 0.20 (paired T-test, 1-tailed).
(sup b)The mean binding ratio is an average of the ratio of the net
counts for each study strain relative to the net counts of
the serum-susceptible control (23B). p-value = 0.31 (paired
T-test, 1-tailed).

Conclusion

This study of GBS patients in the United States and Germany confirms the
observation made in Japan that serotype O:19 is overrepresented among
patients with C. jejuni-induced GBS. Of 298 randomly collected
Campylobacter isolates from patients with enteritis in the United States,
only 3% were serotype O:19 (19). A similarly low prevalence of O:19 strains
is found in all parts of the world, including North and South America,
Asia, and Europe (5,6,19,23). Although specific serotyping surveys have not
been done in Germany, it is unlikely that serotype O:19 is more frequent
among German C. jejuni isolates. Thus, the GBS-associated strains in our
study were more than 11 times as likely to belong to this serotype (p =
0.03). Although the association among GBS and C. jejuni serotype O:19 was
not as marked as in Japan (where more than 80% of GBS-associated isolates
are O:19), this serotype clearly is overrepresented among GBS-associated
strains in other countries. We conclude that the association of O:19
strains with GBS is not just a local phenomenon in Japan but likely
reflects a fundamental characteristic of O:19 strains. O-serotype 2 and
heat-labile serotype 4, which were common among the GBS strains, are
commonly represented among infected persons in the United States (23).

Despite the frequency of Campylobacter infections in GBS patients, such
strains are difficult to obtain for several reasons. First, in most C.
jejuni-infected patients, stools are clear before neurologic symptoms
begin. Second, most neurologists do not culture stool samples when GBS is
first diagnosed. And finally, even if a stool culture is ordered and
Campylobacter is present, few microbiology laboratories save their
isolates; by the time the case is reported, the strain has been discarded.
Thus, these seven strains represent one of the largest collections of
GBS-associated strains described. Additionally, these are likely to be
representative of the population of GBS-associated C. jejuni strains.
Unless serotype O:19 strains persist in stools longer, are more easily
cultured, or are less likely to be discarded by microbiology laboratories
(and no data support any of these possibilities), these strains probably
are not different in any systematic way from other GBS-associated isolates.

Most C. jejuni strains are susceptible to killing by human serum (22), but
because we studied highly selected strains (most either serotype O:19 or
from GBS patients), a high proportion of strains in this investigation were
resistant. In this context, the finding that C. jejuni strains from GBS
patients were no more likely than the strains from patients with
uncomplicated enteritis to be serum resistant is not surprising. Even when
the analysis was limited only to serotype O:19 strains, no differences were
found between GBS- and enteritis-associated strains. However, serotype O:19
strains were substantially less serum-susceptible than strains of other
serotypes and were 12 times as likely to be serum-resistant. Although
serotype O:19 represents only a small percentage of C. jejuni strains from
patients with uncomplicated enteritis in the United States or Japan, it is
the most common serotype identified in GBS patients in both locales. The
relatively small variation in serum susceptibility of the O:19 strains is
consistent with the close genetic relationship observed among these strains
(24).

To better understand the basis for the relative serum-resistance among the
O:19 strains, we compared their ability to bind C3 in relation to non-O:19
strains. Because C3 binding occurs after activation by either the
alternative or classical complement pathways, it is a screen for
differences in these early steps. The serum-resistance of C. fetus is
explained by the inability of C3 to bind to the cell surface (25). In
contrast, C3 binding is normal in serum-resistant Salmonella, but the C5-9
membrane attack complex does not insert properly (26). In the present
study, the lack of substantial C3 binding differences among O:19 and
non-O:19 C. jejuni strains suggests that the early steps in complement
activation are similar among strains. However, clear differences in
serum-susceptibility bespeak either rapid inactivation of C3 or reduced
assembly or function of the membrane attack complex in the more resistant
strains. Future studies should address this point.

The increasing awareness of the importance of C. jejuni infection in
triggering GBS is another example of how previously well-described diseases
have emerged as sequelae of acute infectious illnesses. This study attempts
to begin to characterize the nature of this association; however, there is
much to learn about how an acute gastrointestinal infection results in
ascending paralysis. One fact is quite clear: many more people are infected
with C. jejuni than contract GBS subsequently. Perhaps some persons are
predisposed to contracting GBS after infection with campylobacters that
might cause only uncomplicated enteritis in another patient. Conversely, as
we have suggested in this paper, some strains may be more likely than
others to trigger GBS. No associations between human leukocyte antigen
(HLA) types and GBS have been found (27,28). However, in Great Britain and
Japan, an association between HLA type and C. jejuni-associated GBS has
been suggested (9,29). Perhaps some combination of familial susceptibility,
HLA type, strain serotype, or other host or strain characteristics together
play a role in the pathogenesis of C. jejuni-induced GBS.

The relative serum-resistance of O:19 strains correlates with mechanism.
Furthermore, the relevance of these in vitro assays to the susceptibility
of organisms in vivo cannot be known with certainty. We speculate that the
relative insensitivity of these strains to the lytic effects of complement
allows them to trigger a heightened specific immunologic response. We
further speculate that this heightened immunologic response leads to injury
of peripheral nerve structures. Since only a small fraction of infections
caused by C. jejuni O:19 lead to GBS (estimated incidence 1 in 158) (30),
additional factors also must be involved in vivo.

Acknowledgments

We thank Charlotte Patton for serotyping the strains and Manfred Kist for
supplying the strains from Germany.

This work was supported by grant NIH-1-K08-NS01709 of the NINDS, NIH (BMA);
NIH grant KO4-AI01036 (RGW); and the Medical Research Service of the
Department of Veterans Affairs (MJB).

Ban Mishu Allos is an assistant professor of medicine in the Division of
Infectious Diseases, Department of Medicine and the Department of
Preventive Medicine at Vanderbilt University School of Medicine in
Nashville, Tennessee. Her clinical work relates to HIV and other infectious
diseases; her research has focused on foodborne illness in general and
Campylobacter jejuni as a cause of Guillain-Barr syndrome in particular.

Address for correspondence: Ban Mishu Allos, A-3310 MCN, Division of
Infectious Diseases, Vanderbilt University School of Medicine, Nashville,
TN 37232, USA; fax: 615-343-6160; e-mail:
Ban.mishu.allos@mcmail.vanderbilt.edu.

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     Africa associated with Campylobacter jejuni O:41 strains. J Infect Dis
     1997;(Suppl 2):S139-S43.
 15. Enders U, Karch H, Toyka KV, Heesemann J, Hartung HP. Campylobacter
     jejuni and Guillain-Barr syndrome. Ann Neurol 1994;35:249.
 16. Blaser MJ, Perez-Perez G, Smith PF, Patton CM, Tenover FC, Lastovica
     AJ, et al. Extraintestinal Campylobacter jejuni and Campylobacter coli
     infections: host factors and strain characteristics. J Infect Dis
     1986;153:552-9.
 17. Penner JL, Hennessy JN. Passive hemagglutination technique for
     serotyping Campylobacter fetus subsp. jejuni on the basis of soluble
     heat-stable antigens. J Clin Microbiol 1980;2:378-83.
 18. Lior H, Woodward DL, Edgar JA, Laroche LJ, Gill P. Serotyping of
     Campylobacter jejuni by slide agglutination based on heat-labile
     antigenic factors. J Clin Microbiol 1982;15:761-8.
 19. Patton CM, Nicholson, MA, Ostroff SM, Ries AA, Wachsmuth IK, Tauxe RV.
     Common somatic O and heat-labile serotypes among Campylobacter strains
     from sporadic infections in the United States. J Clin Microbiol
     1993;31:1525-30.
 20. Blaser MJ, Smith PF, Hopkins JA, Heinzer I, Bryner JH, Wang WLL.
     Pathogenesis of Campylobacter fetus infections. Serum resistance
     associated with high molecular weight surface proteins. J Infect Dis
     1987;155:696-709.
 21. Gonzales-Valencia G, Perez-Perez GI, Washburn RG, Blaser MJ.
     Susceptibility of Helicobacter pylori to the bactericidal activity of
     human serum. Helicobacter 1996;1:28-33.
 22. Blaser MJ, Smith PF, Kohler PF. Susceptibility of Campylobacter
     isolates to the bactericidal activity of human serum. J Infect Dis
     1985;151:227-35.
 23. Patton CM, Wachsmuth IK. Typing schemes-are current methods useful? In:
     Nachamkin I, Blaser MJ, Tompkins LS, editor. Campylobacter
     jejuni-current strategy and future trends. Washington: American Society
     for Microbiology; 1992. p. 110-28.
 24. Fujimoto S, Allos BM, Patton CM, Blaser MJB. Restriction fragment
     length polymorphism analysis and random amplified polymorphic DNA
     analysis of Campylobacter jejuni strains isolated from patients with
     Guillain-Barr syndrome. J Infect Dis 1997;176:1105-8.
 25. Blaser MJ, Smith PF, Repine JE, Joiner KA. Pathogenesis of
     Campylobacter fetus infection. J Clin Invest 1988;81:1434-44.
 26. Joiner KA, Hammer CH, Brown EJ, Cole RJ, Frank MM. Studies on the
     mechanism of bacterial resistance to complement-mediated killing. I.
     Terminal complement components are deposited and released from
     Salmonella minnesota S218 without causing bacterial death. J Exp Med
     1982;155:809-19.
 27. Hillert J, Osterman PO, Olerup O. No association with HLA-DR, -DQ, or
     -DP alleles in Guillain-Barr syndrome. J Neuroimmunol 1991;31:67-72.
 28. Winer JB, Briggs D, Welsh K, Hughes RAC. HLA antigens in the
     Guillain-Barr syndrome. J Neuroimmunol 1988;18:13-6.
 29. Yuki N, Fujimoto S, Yamada S, Tsujino Y, Kinoshito A, Itoh T. Serotype
     of Campylobacter jejuni, HLA, and the Guillain-Barr syndrome. Muscle
     Nerve 1992;968-9.
 30. Allos BM, Blaser MJ. Campylobacter jejuni infection and the
     Guillain-Barr syndrome: mechanisms and implications. International
     Journal of Medical Microbiology, Virology, Parasitology, and
     Infectious Diseases 1994;281:544-8.


Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/allos.htm

Please note that figures and equations are not available in ASCII format; 
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-------------------------------------------------------------------------------------------

[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Dispatches

An Apparently New Virus (Family Paramyxoviridae) Infectious for Pigs,
Humans, and Fruit Bats

Adrian W. Philbey,* Peter D. Kirkland,* Anthony D. Ross,* Rodney J. Davis,*
Anne B. Gleeson,* Robert J. Love, Peter W. Daniels, Allan R. Gould, and
Alex D. Hyatt
*Elizabeth Macarthur Agricultural Institute, Camden, New South Wales,
Australia; University of Sydney, Camden, New South Wales, Australia; and
CSIRO Australian Animal Health Laboratory, Geelong, Victoria, Australia

  --------------------------------------------------
      We isolated an apparently new virus in the family
      Paramyxoviridae from stillborn piglets with deformities at a
      piggery in New South Wales, Australia. In 1997, the pregnancy
      rate and litter size at the piggery decreased markedly, while
      the proportion of mummified fetuses increased. We found
      serologic evidence of infection in pigs at the affected piggery
      and two associated piggeries, in humans exposed to infected
      pigs, and in fruit bats. Menangle virus is proposed as a common
      name for this agent, should further studies confirm that it is
      a newly recognized virus.

Viruses in the family Paramyxoviridae have been associated with new
diseases in a variety of species, including humans, throughout the world.
Morbilliviruses related to canine distemper virus have caused disease
outbreaks in seals, dolphins, porpoises, and lions (1-6). Equine
morbillivirus (EMV) (also called Hendra virus or bat paramyxovirus),
responsible for the deaths of horses and humans with respiratory or
neurologic disease in Australia (7,8), is thought to have originated from
fruit bats (Pteropus sp.) (9-11). La Piedad Michoacan paramyxovirus (blue
eye paramyxovirus) causes encephalitis and death in piglets and
reproductive disease in adult pigs in Mexico (12). We have isolated an
apparently new virus in the family Paramyxoviridae from stillborn piglets
with abnormalities of the brain, spinal cord, and skeleton at a commercial
piggery with 2,600 sows in New South Wales, Australia. Serologic studies
indicate that at least two humans exposed to affected pigs have been
infected with the virus, possibly with resultant illness, and that fruit
bats are a potential source of infection.

From mid-April to early September 1997 at the affected piggery, the
farrowing rate decreased from an expected 82% to 60%; the number of live
piglets declined in 27% of litters born; the proportion of mummified and
stillborn piglets, some with deformities, increased; and occasional
abortions occurred. The disease occurred sequentially in all four breeding
units at the piggery, affecting the progeny of sows of all parities and
gilts. No disease was observed in postnatal animals of any age. Affected
stillborn piglets frequently had severe degeneration of the brain and
spinal cord (which were almost absent in some piglets), arthrogryposis,
brachygnathia, and occasionally fibrinous body cavity effusions and
pulmonary hypoplasia. Histologic examination of the brain and spinal cord
showed extensive degeneration and necrosis of gray and white matter
associated with infiltrations of macrophages and other inflammatory cells.
Neurones contained intranuclear and intracytoplasmic inclusion bodies. Some
piglets had nonsuppurative myocarditis.

A virus producing cytopathic effects in cell cultures, including
vacuolation of cells and formation of syncytia, was isolated at the
Elizabeth Macarthur Agricultural Institute, Menangle, in baby hamster
kidney cells (BHK21) from lung (n = 9 isolates), brain (n = 8), and heart
(n = 5) of affected piglets. The isolates have been shown by electron
microscopy to be morphologically consistent with viruses in the family
Paramyxoviridae, which comprise spherical to pleomorphic particles 30 nm to
more than 100 nm long, contain "herringbone" nucleocapsids with a diameter
of 19  4 nm and a pitch of 5.8  0.4 nm, and are surrounded by an envelope
with a single fringe of surface projections 17  4 nm long (Figure). The
virus grows in a wide range of cell types from many species, including
porcine and human cells, and is nonhemadsorbing and nonhemagglutinating,
using erythrocytes from several species; in this respect, the virus differs
from La Piedad Michoacan paramyxovirus, which is hemagglutinating (13).
Studies at the Australian Animal Health Laboratory, Geelong, indicate that
the virus is probably a new member of the family Paramyxoviridae. EMV and
La Piedad Michoacan paramyxovirus have been excluded on the basis of
absence of specific polymerase chain reaction products and limited
sequencing, and EMV has been further excluded on the basis of lack of
serologic cross-reactivity and differing appearance by electron microscopy.
Serologic tests at the Elizabeth Macarthur Agricultural Institute and the
Australian Animal Health Laboratory on sera from sows have also excluded
other porcine reproductive pathogens, such as ruminant and porcine
pestiviruses (including classic swine fever virus), porcine reproductive
and respiratory syndrome (Lelystad) virus, porcine parvovirus,
encephalomyocarditis virus, and Aujeszky's disease virus. Pestivirus
infection was also excluded by negative antigen capture enzyme-linked
immunosorbent assay results on tissues from affected piglets.

A high proportion (> 90%) of serum collected from pigs of all age
categories at the affected piggery (n = 88) from May to September 1997
contained high titers (>/= 256) of neutralizing antibodies against the virus.
In contrast, serum and plasma samples collected from pigs at the affected
piggery before May 1997 (n = 120) tested negative. Porcine sera (n = 50)
from two piggeries that receive only weaned pigs from the affected piggery
neutralized the virus at dilutions of 16 to 4,096. Virus-neutralizing
antibodies were also detected in body cavity fluids from some stillborn
piglets. Testing of porcine sera (n = 1,114) from other piggeries
throughout Australia, including piggeries with reproductive problems,
indicates that infection is confined to the affected piggery and the two
associated piggeries.

Serum from two humansone working at the affected piggery and one working
at one of the associated piggerieshad neutralizing antibody titers of 128
and 512. These workers had unexplained febrile illnesses in the weeks after
exposure to potentially infective material. Further details are provided in
the article by Chant et al. in this issue.

 [fig]
 Figure. Transmission electron micrographs of Menangle virus negatively stained
 with 2% phosphotungstic acid. A. Depicts the pleomorphic nature of the virion;
 bar=100nm. B. Shows a disrupted virion, the virus envelope with surface
 projections (hollow arrow) and nucleocapsids (solid arrow); bar=100nm.

A large breeding colony of gray-headed fruit bats (Pteropus poliocephalus),
as well as little red fruit bats (P. scapulatus), roosts within 200 m of
the affected piggery from October to April; therefore, fruit bats were
investigated as a potential source of infection. In a preliminary study, 42
of 125 serum samples collected from fruit bats in New South Wales and
Queensland were positive in the virus neutralization test, with titers of
16 to 256. Positive samples were from 26 of 79 gray-headed fruit bats, 11
of 20 black fruit bats (P. alecto), 4 of 10 spectacled fruit bats (P.
conspicillatus), 0 of 15 little red fruit bats, and one unidentified
species. This panel included positive samples collected in 1996 before the
pigs were infected, as well as positive samples collected in November 1997
from a colony of gray-headed fruit bats 33 km from the piggery, supporting
the hypothesis that fruit bats were the primary source of the virus. Other
species in the vicinity of the affected piggery, including rodents (n =
19), birds (n = 13), cattle (n = 60), sheep (n = 70), cats (n = 25), and a
dog, were seronegative.

Along with EMV (7-11) and a lyssavirus causing encephalitis (14,15), this
is the third virus causing disease in humans or domesticated animals that
appears to have emerged from fruit bats in Australia in recent years.
Menangle virus is proposed as a common name for this agent, should further
studies confirm that it is a newly recognized virus.

Acknowledgments

We thank Bryan Eaton, Peter Hooper, Jacquie Kattenbelt, Brenda
King, Chris Morrissey, Paul Selleck, and Harvey Westbury from the CSIRO
Australian Animal Health Laboratory, Geelong, for their invaluable
contributions with serology, histopathology, and PCR and Keith Hart and
Andrew Glover, Moss Vale Rural Lands Protection Board, Camden, for
collection of serum samples from rodents, birds, cattle, sheep, and cats at
the affected piggery. Sera from humans were provided by the Public Health
Unit, South Western Sydney Area Health Service, Liverpool, under the
directorship of Kerry Chant. Peter Young and Hume Field from the Animal
Research Institute, Department of Primary Industries, Yeerongpilly,
Queensland, kindly provided serum samples from fruit bats. We appreciate
contributions by technical staff in the virology section and regional
veterinary laboratory at the Elizabeth Macarthur Agricultural Institute,
Menangle, and at the University of Sydney, Camden, throughout this
investigation.

Adrian Philbey is a veterinary research officer at Elizabeth Macarthur
Agricultural Institute, New South Wales. His research interests include
veterinary pathology with a special interest in virology, use of molecular
techniques for pathogen identification in domestic animals, vaccine
development for infectious diseases in domestic animals, and rabbit
calicivirus.

Address for correspondence: Adrian W. Philbey, Elizabeth Macarthur
Agricultural Institute, PMB 8, Camden, New South Wales 2570, Australia;
fax: 61-2-46-40-6429; e-mail: Adrian.Philbey@agric.nsw.gov.au.

References

  1. Osterhaus ADME, Vedder EJ. Identification of virus causing recent seal
     deaths. Nature 1988;335:20.
  2. Osterhaus ADME, Groen J, De Vries P, UytdeHaag FGCM. Canine distemper
     virus in seals. Nature 1988;335:403-4.
  3. Kennedy S, Smyth JA, McCullough SJ, Allan GM, McNeilly F, McQuaid S.
     Confirmation of cause of recent seal deaths. Nature 1988;335:404.
  4. Kennedy S, Smyth JA, Cush PF, McCullough SJ, Allan GM, McQuaid S.
     Viral distemper now found in porpoises. Nature 1988;336:21.
  5. Domingo M, Ferrer L, Pumarola M, Marco A, Plana J, Kennedy S, et al.
     Morbillivirus in dolphins. Nature 1990;348:21.
  6. Roelke-Parker ME, Munson L, Packer C, Kock R, Cleaveland S, Carpenter
     M, et al. A canine distemper virus epidemic in Serengeti lions
     (Panthera leo). Nature 1996;379:441-5.
  7. Murray K, Selleck P, Hooper P, Hyatt A, Gould A, Gleeson L, et al. A
     morbillivirus that caused fatal disease in horses and humans. Science
     1995;268:94-7.
  8. Allworth T, O'Sullivan J, Selvey L, Sheridan J. Equine morbillivirus
     in Queensland. Communicable Diseases Intelligence 1995;19:575.
  9. Young P. Possible reservoir host of equine morbillivirus identified.
     Communicable Diseases Intelligence 1996;20:262.
 10. Young PL, Halpin K, Selleck PW, Field H, Gravel JL, Kelly MA, et al.
     Serologic evidence for the presence in Pteropus bats of a
     paramyxovirus related to equine morbillivirus. Emerg Infect Dis
     1996;2:239-40.
 11. Halpin K, Young P, Field H. Identification of likely natural hosts for
     equine morbillivirus. Communicable Diseases Intelligence 1996;20:476.
 12. Stephan HA, Gay GM, Ramirez TC. Encephalomyelitis, reproductive
     failure and corneal opacity (blue eye) in pigs, associated with a
     paramyxovirus infection. Vet Rec 1988;122:6-10.
 13. Moreno-Lpez J, Correa-Girn P, Martnez A, Ericsson A.
     Characterization of a paramyxovirus isolated from the brain of a
     piglet in Mexico. Arch Virol 1986; 91:221-31.
 14. Fraser GC, Hooper PT, Lunt RA, Gould AR, Gleeson LJ, Hyatt AD, et al.
     Encephalitis caused by a lyssavirus in fruit bats in Australia. Emerg
     Infect Dis 1996;2:327-31.
 15. Allworth A, Murray K, Morgan J. A human case of encephalitis due to a
     lyssavirus recently identified in fruit bats. Communicable Diseases
     Intelligence 1996;20:504.


Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/philbey.htm

Please note that figures and equations are not available in ASCII format; 
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-------------------------------------------------------------------------------------------
-------------------------------------------------------------------------------------------

[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Dispatches

Probable Human Infection with a Newly Described Virus in the Family
Paramyxoviridae

Kerry Chant,* Raymond Chan, Mitchell Smith,* Dominic E. Dwyer, Peter
Kirkland, and the NSW Expert Group 
*Public Health Unit, Sydney,
Australia; South Western Area Pathology Service, Sydney, Australia;
Institute of Clinical Pathology and Medical Research (ICPMR), Westmead,
Australia; and Elizabeth Macarthur Agricultural Institute, Menangle, New
South Wales, Australia
Stephen Conaty, NSW Department of Health; Yvonne Cossart, University of
Sydney; Gwendolyn Gilbert, ICPMR; Richard Jane, NSW Department of
Agriculture; Robert Love, University of Sydney; Jeremy McAnulty, NSW
Department of Health; William Rawlinson, University of New South Wales and
SEALS Department, Prince of Wales Hospital, Sydney; and Evan Sergeant, NSW
Department of Agriculture.

  --------------------------------------------------
      After an apparently new virus in the family Paramyxoviridae was
      isolated from pigs in August 1997, an investigation was carried
      out to assess its risk for humans. More than 250 persons with
      potential exposure to infected pigs were tested serologically.
      Two piggery workers with intense occupational exposure had high
      convalescent-phase antibody titers to this new virus. In early
      June 1997, both workers had an influenzalike illness with rash;
      serologic testing showed no alternative cause. Strong evidence
      indicates that the two men became ill from this new virus, but
      the mode of transmission from pigs to humans remains unknown.

Zoonotic illnesses due to viruses in the family Paramyxoviridae (i.e.,
Newcastle disease virus [1] and equine morbillivirus [2]) have been
described. An apparently new virus in the family Paramyxoviridae, isolated
from pigs in a piggery near Sydney, Australia, is described by Philbey et
al. in this issue. We describe epidemiologic investigations to assess the
risk the virus poses for humans and detail two probable cases of human
disease.

Piggery A, from which the virus was isolated, is a large commercial piggery
with animals housed in four discrete production units. At approximately 6
weeks of age, some pigs are transported to piggeries B and C for growing to
slaughter weight. Animals from piggery A are also supplied to a university
and a hospital for research.

After the new virus was isolated, an assay for neutralizing antibodies that
allowed testing of sera from animals and humans was developed at the
Elizabeth Macarthur Agricultural Institute. All 33 workers at piggery A
were tested with this assay for antibodies to the new agent. Other workers
who had come into contact with potentially infectious pigs from piggery A
were also tested: abattoir workers (n = 142), workers at the grower
piggeries B and C (n = 5), researchers and animal handlers (n = 41),
veterinarians and pathology laboratory workers (n = 24), and others (n =
6). Sixty delinked sera from women receiving routine prenatal screening
were tested as controls and were seronegative for the agent. Two workers
were seropositive, with virus neutralizing antibody titers of 128 and 512.
These results were confirmed by repeat collection and testing. Both workers
received a detailed clinical review and extensive serologic testing, the
results of which are described below.

In early June 1997, Patient 1 had sudden onset of malaise and chills
followed by drenching sweats and fever. He was confined to bed with severe
headaches and myalgia for the next 10 days. He had no cough, vomiting, or
diarrhea. After the third day, he went to a locum physician, who prescribed
amoxicillin. A day later, he noted a spotty red rash. He went to his usual
physician, who noted tenderness in both hypochondria, lymphadenopathy, and
a rubelliform rash and diagnosed acute Epstein-Barr virus infection.
However, subsequent review of the patient's medical notes showed a positive
result for Epstein-Barr virus immunoglobulin G (IgG) in 1991. He returned
to work after 14 days' absence but tired easily. He reported a 10-kg weight
loss during his illness.

Results of a clinical examination 2 months after this illness were normal
except for mild tenderness in the right hypochondrium. Results of
urinalysis, full blood count, erythrocyte sedimentation rate, C-reactive
protein concentration, and blood chemistries were normal. An upper
abdominal ultrasound indicated that liver size was at the upper limits of
normal, and the spleen was enlarged (15 cm long).

The worker had frequent prolonged contact with birthing pigs. He reported
that splashes of amniotic fluid and blood to the face were not uncommon and
that he often received minor wounds to his hands and forearms. His partner
tested negative for neutralizing antibodies to this agent.

Six other workers at the piggery reported an influenzalike illness during
the winter months, yet records showed that only two had more than 1 day off
work, the most being 4 days.

Patient 2 worked at piggery B. He also had onset of illness in early June
1997 characterized by fever, chills, rigors, drenching sweats, marked
malaise, back pain, severe frontal headache, and photophobia. He had no
cough, vomiting, or diarrhea. The headache resolved after 4 to 5 days. Four
days after onset of the illness, he noted on the torso a spotty, red,
nonpruritic rash, which lasted 7 days. He had largely recovered after 10
days, noting a 3-kg weight loss. No investigations were performed.

Results of a clinical examination 2 months after his illness were
essentially normal. Results of urinalysis, full blood count, erythrocyte
sedimentation rate, and C-reactive protein were within normal limits. Blood
chemistry results were normal except for mildly elevated liver function. He
was hepatitis Cantibody positive and IgA-deficient. An upper abdominal
ultrasound showed mild hepatomegaly with normal texture. The spleen was at
the upper limit of normal size.

This worker did not have contact with birthing pigs; however, he performed
autopsies on pigs without wearing gloves or protective eye wear. As with
Patient 1, exposure to pig secretions (e.g., feces, urine) was common. The
worker had received a delivery of young pigs from piggery A the week before
his illness.

Both workers had a similar illness in early June 1997. Both had high
convalescent-phase neutralizing antibody titers to this new virus.
Serologic testing of all 33 workers at piggery A for other human
paramyxoviruses excluded cross-reactivity as a cause for this finding.
Extensive serologic testing of both patients in September 1997 (Table) did
not identify an alternative cause for the illnesses.

The unexplained splenomegaly in the first worker may be an incidental
finding. The second worker had evidence of hepatitis C virus infection,
abnormal liver function tests, hepatomegaly, and IgA deficiency. None of
these findings need be implicated in the patient's presumed infection with
this paramyxovirus.

While single high antibody titers must be interpreted with caution, the
timing of the described illnesses in relation to the disease in pigs, the
similarity of the two cases, the exclusion of cross-reactivity due to
preexisting antibody to other paramyxoviruses, and the absence of serologic
results suggesting another cause constitute strong evidence that the
illness in these two men was caused by this new virus.

Although respiratory transmission is proposed as a mode of spread in pigs,
the mode of transmission from pigs to humans is unknown. If spread to
humans is by the respiratory route, infectivity appears much lower than in
pig-to-pig transmission. Alternatively, a different transmission mode such
as parenteral or permucosal exposure may be involved. Philbey et al. in
this issue present evidence suggesting that bats may be involved in the
ecology of this new virus. Neither of the two patients had contact with
bats.

   Table. Results of case investigations

  ------------------------------------------------------------------
                                                     Titer
                                         ---------------------------
   Serologic test(sup a)                 Patient 1       Patient 2
  ------------------------------------------------------------------
   Neutralizing antibody to new agent   128             512
   Antibody to equine morbillivirus      negative        negative
   CMV IgG                               positive        positive
   CMV IgM                               negative        negative
   EBV-VCA IgG                           positive        positive
   EBV-VCA IgM                           negative        negative
   EBV-EBNA IgG                          negative        positive
   Rubella IgG                           positive        positive
   Rubella IgM                           negative        negative
   Mycoplasma (CFT)                      < 4(sup b)      4(sup b)      
   Mycoplasma IgM                        negative        negative
   Adenovirus (CFT)                      8(sup b)	   8(sup b)
   Enterovirus (CFT)                     8(sup b)	   8(sup b)
   Influenza A (CFT)                     32(sup b)       16(sup b)
   Influenza B (CFT)                     8(sup b)        <4 (negative)
   Leptospiral agglutinins               negative        negative
   Brucella antibodies                   negative        negative
   Toxoplasma IgG                        positive        positive
   Toxoplasma IgM                        negative        negative
   Q Fever (phase 2 - CFT)               negative        negative
   Measles IgG                           positive        positive
   Measles IgM                           negative        negative
   Mumps IgG                             positive        positive
   Mumps IgM                             negative        negative

   RSV (CFT)                             < 8(sup b)      anticomple-
                                                         mentary
   Parainfluenza 1 (CFT)                 32(sup b)       < 16(sup b)
   Parainfluenza 2 (CFT)                 16(sup b)       < 16(sup b)
   Parainfluenza 3 (CFT)                 < 8(sup b)      < 16(sup b)
   HBsAg                                 negative        negative
   anti-HBc                              negative        negative
   anti-HCV                              negative        positive(sup c)
   anti-HIV (Western Blot)               negative        negative
   anti-HIV (EIA)                        negative        negative
  -------------------------------------------------------------------
   (sup a)CMV, cytomegalovirus; IgG, immunoglobulin G; EBV, Epstein-Barr
   virus; VCA, viral capsid antigen; EBNA, Epstein-Barr nuclear antigen;
   CFT, complement fixation test; RSV, respiratory syncytial virus;
   HBsAg, hepatitis B surface antigen; HBc, hepatitis B core; HCV,
   hepatitis C virus; EIA, enzyme immunoassay.
   (sup b)Not significant.
   (sup c)By two screening enzyme immunoassays.

Human infection with this new virus seems confined to those with intense
occupational exposure to recently infected pigs. Sentinel pig surveillance
indicates that the virus continues to circulate at piggery A. Ongoing
surveillance for influenzalike illnesses and a serologic testing program
have been instituted for the workers. In January 1998, 19 of 21 previously
seronegative workers had repeat testing; all remained negative. The
occupational health and safety body of the state, in conjunction with
agricultural and health authorities, has issued appropriate safety
guidelines for the industry.

Acknowledgments

We thank the Microbiology Department, Prince of Wales Hospital; CIDMLS,
ICPMR, Westmead Hospital; Dan Russell, Mid Western NSW Public Health Unit;
Paul Van Buynder, Southern NSW Public Health Unit; and staff of South
Western Sydney Public Health Unit.

Dr. Kerry Chant is director and medical officer of health, South Western
Sydney Public Health Unit, and conjoint lecturer, University of New South
Wales. Dr. Chant is responsible for infectious disease surveillance,
prevention and control, health-related environmental health issues, and
food safety in southwestern Sydney. Her research interests include
infectious disease and infection control issues in particular related to
blood-borne viruses and childhood immunization.

Address for correspondence: Peter Kirkland, Virology Laboratory, Elizabeth
Macarthur Agricultural Institute, Menangle, NSW Australia; fax:
61-2-4640-6429; e-mail: kirklap@agric.nsw.gov.au.

References

  1. Trott DG, Pilsworth R. Outbreaks of conjunctivitis due to the
     Newcastle disease virus among workers in chicken-broiler factories.
     BMJ 1965;5477:1514-7.
  2. O'Sullivan JB, Allworth AM, Paterson DL, Snow TM, Boots R, Gleeson LJ,
     et al. Fatal encephalitis due to novel paramyxovirus transmitted from
     horses. Lancet 1997;349:93-5.


Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/chant.htm

Please note that figures and equations are not available in ASCII format; 
their placement within the text is noted by [fig] and [eq], respectively. 
Greek symbols are spelled out. The following codes are used: 
(ft) for footnote; (sup) for superscript; (sub) for subscript; 
/= for greater than or equal to.

-------------------------------------------------------------------------------------------
-------------------------------------------------------------------------------------------

[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Dispatches

Emergence of the M Phenotype of Erythromycin-Resistant Pneumococci in South
Africa

Carol A. Widdowson and Keith P. Klugman
South African Institute for Medical Research, Johannesburg, South Africa
-------------------------------------------------------------------------
      Erythromycin-resistant pneumococci have been isolated in South
      Africa since 1978; however, from 1987 to 1996, resistance to
      macrolides was only detected in 270 (2.7%) of 9,868 blood or
      cerebrospinal fluid (CSF) pneumococcal isolates, most of which
      were obtained from the public sector. In South Africa,
      macrolide use in the public sector is estimated at 56% of that
      in the private sector. Most erythromycin-resistant strains
      (89%) exhibited resistance to erythromycin and clindamycin
      (macrolide-lincosamide-streptogramin B phenotype). In the
      United States, most erythromycin-resistant pneumococci exhibit
      the newly described M phenotype (resistance to erythromycin
      alone), associated with the mefE gene. The M phenotype in South
      Africa increased significantly in the last 10 years, from 1 of
      5,115 to 28 of 4,735 of blood and CSF isolates received from
      1987 to 1991 compared with 1992 to 1996 [p = 5x10(sup -7)]. These
      data suggest that, although macrolide resistance in pneumococci
      remains low in the public sector, the mefE gene is rapidly
      emerging in South Africa.

Resistance to erythromycin in pneumococci has been observed since 1967 (1)
and was first reported in South African multiresistant pneumococcal strains
in 1978 (2). Until recently, the only mechanism described for resistance to
erythromycin in the pneumococcus was the N(sup 6)-methylation of a specific
adenine residue (A2058) in 23S rRNA, which resulted in reduced affinity
between the antibiotic and the ribosome (3,4). This resistance is
associated with the gene ermAM (5), first described in Streptococcus
sanguis (6). Since then, other mechanisms of erythromycin resistance in the
pneumococcus have been reported. In fact, most resistance in the United
States appears to be due to efflux of the antibiotic from the cell,
associated with the gene mefE (7,8). While ermAM confers coresistance to
most macrolides, lincosamides, and streptogramin B antibiotics (resulting
in the so-called MLS phenotype) (3,9), mefE confers resistance only to the
14- and 15-membered macrolides (resulting in the M phenotype) (7,8). We
report the emergence of M-phenotype erythromycin resistance in South
African blood and cerebrospinal fluid (CSF) pneumococcal isolates from 1987
to 1996.

The South African Institute for Medical Research (SAIMR), Johannesburg,
South Africa, regularly receives all pneumococcal isolates from
participating laboratories in eight of the nine provinces of South Africa.
We examined all erythromycin-resistant blood and CSF isolates received by
SAIMR from 1987 to 1996.

Erythromycin-, clindamycin-, and penicillin-resistance phenotypes were
determined by using disk diffusion assays (erythromycin, 15g/disk,
clindamycin, 2 g/disk, oxacillin, 1 g/disk) on 5% horse blood agar plates
(Mueller-Hinton base) after overnight growth at 37C under aerobic
conditions. Strains showing resistance (zone diameters </= 20mm for
erythromycin, </= 18mm for clindamycin, and </= 20mm for oxacillin) on the disk
diffusion plates were tested by the agar dilution method to obtain MICs
according to the National Committee for Clinical Laboratory Standards
guidelines (10). We evaluated (by the chi-square test) increases in the
prevalence of erythromycin resistance and the incidence of resistance to
erythromycin and susceptibility to clindamycin, which represents the M
phenotype.

The M phenotype increased significantly from 1987 to 1991 compared with
1992 to 1996 in blood and CSF isolates-from 1 of 5,115 isolates to 28 of
4,753 isolates (p = 5x10(sup -7), odds ratio [OR] 30.13, 95% confidence interval
[CI] 4.1-221) (Table 1; Figure).

Data on oral macrolides in the public sector (from the Division of Medical
Schemes Supplies and Pharmaceutical Services of the Department of Health)
show that 16.4 million defined daily doses (ddd) of macrolides were
purchased for the estimated 30.3 million persons who obtain health care
from the public sector (0.54 ddd per capita). Private sector use for the
year ending December 1997 (Intercontinental Medical Statistics South
Africa, Pty, Ltd., unpub. data) show that 7.3 million ddd of macrolide were
purchased in an estimated population of 7.57 million (0.96 ddd per capita).

Table 1. Prevalence of South African erythromycin-resistant pneumococcal
isolates, 19871996(sup a)
------------------------------------------------------------------
                   Total        No. of E-R      No. of M strains
Years       isolates(sup b) strains (%)(sup c) (% of E-R strains)
------------------------------------------------------------------
1987-1991      5,115          128 (2.5)            1 (0.8)
1992-1996      4,753         142 (3.0)            28 (19.7)
Total          9,868         270 (2.7)            29 (10.7)
  ----------------------------------------------------------------
(sup a)Of 9,868 blood and cerebrospinal fluid (CSF) isolates received by the
SAIMR from 1987 to 1996, 270 were fully resistant to erythromycin. While
the number of erythromycin-resistant blood and CSF isolates received
increased from 1987 to 1991 compared with 1992 to 1996 (2.5% to 3.0%), the
increase was not significant. There was no significant relationship
between erythromycin resistance and the M phenotype within any given
province throughout the 10 years.
(sup b)Blood and cerebrospinal fluid isolates.
(sup b)E-R= erythromycin-resistant.



                 [fig]
  Figure. Number of erythromycin-resistant blood and cerebro-
  spinal fluid isolates of pneumococci.

  --------------------------------------------------

All 78 MLS isolates hybridized with the ermAM probe or produced a
616-bp-amplification product during polymerase chain reaction (PCR)
amplification using the ermAM-specific primers. The 12 M isolates tested
contained the mefE gene as shown by a 348bp-amplification product when
amplified using primers specific for mefE. There were no
erythromycin-resistant isolates that contained neither the ermAM nor the
mefE gene.

(ft)
DNA was extracted from pneumococcal isolates by using a lysis solution
consisting of 0.1% sodium deoxycholate as described in (11), except that we
used plate rather than broth cultures.
        Seventy-eight MLS strains were probed for the ermAM gene by using
dot blots. The probe (supplied by P. Courvalin, Pasteur Institute, Paris,
France) (Escherichia coli JM83/pUC19 560bp Ssp1 intragenic fragment of
ermB) was labeled with digoxygenin by using random primed labeling (DIG DNA
Labeling and Detection Kit; Boeringer, Mannheim, Germany). Hybridization
and detection were performed following manufacturer's instructions (DIG DNA
Labeling and Detection Kit; Boeringer, Mannheim, Germany). PCR was also
used to detect ermAM in 30 strains according to standard conditions, with
an annealing temperature of 58C. We used the following primers: forward
primer, 5'-CGAGTGAAAAAGTACTCAACC, reverse primer,
5'-GGCGTGTTTCATTGCTTGATG).
        Published primers for the mefE gene (5'-AGTATCATTAATCACTAGTGC, and
5'-TTCTTCTGGTACTAAAAGTGG) (12) were used to detect mefE through PCR
amplification in 13 M strains. Amplification was performed in a Perkin
Elmer Cetus DNA Thermal Cycler under standard reaction conditions, with an
annealing temperature of 56oC.


Erythromycin-resistant strains were serotyped by using the quellung
reaction and antisera from the Staten Seruminstitut, Copenhagen, Denmark.
Over the 10 years, the five most common serotypes and groups among the
erythromycin-resistant isolates in decreasing order of frequency were
serotype 14, serogroups 6, 19, 23, and serotype 1 (Table 2). Serotype 1
erythromycin-resistant pneumococci appeared only after 1992; serotype 14
was the most common in MLS isolates; serogroup 23 was the most common
serogroup in M isolates (Table 2). Serotypes 14 and serogroups 6, 23, and
19 are the most common serotypes and groups isolated from children with
serious infections (13,14). Of the 157 isolates from patients whose age was
supplied, 98 (62%) were obtained from children (</= 12 years). There was a
trend that was not significant toward more macrolide resistance in children
than adults (OR 1.17 [95% CI 0.98-1.39]). This trend may have been
significant if age data had been supplied with all the isolates received.
The proportion of pediatric isolates did not change significantly over the
10-year period (63% during 1987 to 1991 and 62% the 1992 to 1996 period).

Table 2. Serotype distribution among erythromycin-resistant pneumococci
-----------------------------------------------------------------------
Serotype/           No. (%) of       No. (%) of          Total no.
group           MLS(sup a)isolates   M isolates       (%) of isolates
  ---------------------------------------------------------------------
14                  96 (39.8)         9 (31.0)          105 (38.9)
 6                  71 (29.5)         3 (10.3)           74 (27.4)
19                  36 (14.9)         3 (10.3)           39 (14.4)
23                  26 (10.8)        10 (34.5)           36 (13.3)
 1                   7 (2.9)          1 (3.5)             8 (3.0)
Other                5 (2.1)          3 (10.3)            8 (3.0)
Total              241               29                 270
-----------------------------------------------------------------------
(sup a)MLS, macrolides-lincosamides-streptogramin B.

Approximately half of all the macrolide-resistant isolates were also either
intermediate or fully resistant to penicillin (60 of the 128 isolates from
the 1987 to 1991 period, and 72 of the 142 isolates from the 1992 to 1996
period). There was a trend (not significant) toward greater resistance to
penicillin in MLS strains. Of the 78 strains with MICs available, 38 (49%)
were fully resistant (MIC(sup 3) 2g/ml) to penicillin, while the rest showed
intermediate resistance (1 g </= MIC </= 0.12 g). Previous data have
indicated that penicillin-intermediate resistance is far more common in
South Africa than full resistance to penicillin (15). In 1992, Friedland
and Klugman (15) reported that only 3 of 35 penicillin-resistant strains
showed high-level resistance.

Coresistance to penicillin limits the use of most macrolides as treatment
of penicillin-resistant pneumococcal infections. New semisynthetic
macrolides such as the ketolide RU 64004 (16) are being developed, however,
that do not show cross-resistance to penicillin or to erythromycin.

Compared with total erythromycin resistance (middle ear fluid, blood, and
CSF) in Europe (17-19), the United Kingdom (18), and the United States
(19,21,22), the overall prevalence of macrolide resistance is low. In
Europe erythromycin resistance varies by country. In Slovakia, almost all
pneumococcal isolates are resistant (17), whereas in Portugal only 0.6% of
isolates are resistant and the proportion appears to be declining (23). In
the United Kingdom, erythromycin resistance increased from 3.3% to 8.6%
between 1989 and 1992 in England and Wales (20). In the United States, 10%
of pneumococcal isolates appear to be erythromycin resistant (21). Most
isolates received by SAIMR are from the public sector, where macrolides are
not normally prescribed for pneumococcal infections. Only 4 of the 128
erythromycin-resistant isolates from 1987 to 1991 and 14 of the 142
erythromycin-resistant isolates from 1992 to 1996 were from the private
sector. Resistance data from the private sector may show much higher levels
of macrolide resistance, a contention supported by previous South African
resistance data (1986), where the carriage rates of multiresistant
pneumococci were 17.7% in children from more affluent communities and 0% in
children from less affluent areas (24). MIC data were available for 15 of
the 20 multiresistant isolates, and all 15 were fully resistant to both
erythromycin and clindamycin (24).

Before the M phenotype was observed, erythromycin resistance was assumed to
indicate cross-resistance to lincosamides and streptogramin B antibiotics
in the pneumococcus. The increase in the incidence of M phenotype may
warrant investigation into the use of these antibiotics for the treatment
of pneumococcal infections. Sutcliffe et al. (7) suggested that clindamycin
be considered for the treatment of bacteremia and middle ear and sinus
infections caused by Streptococcus pneumoniae. Treatment with clindamycin
is feasible only if infection with gram-negative pathogens has been
excluded and if the S. pneumoniae phenotype is known because the strain may
show MLS resistance and studies indicate that many penicillin-resistant
strains are also clindamycin-resistant (16,25). Visalli and colleagues (25)
found that clindamycin concentrations of only 0.06 g/ml were required to
inhibit 90% of penicillin-susceptible strains when grown in air, while
clindamycin concentrations of > 64 g/ml were required to inhibit 90% of
penicillinintermediate and resistant strains.

Studies of streptogramin use against pneumococci show some promise. The
streptogramin RP 59500, a mixture of type A streptogramin, dalfopristin,
and type B streptogramin quinupristin, is active against pneumococci
regardless of their susceptibilities to penicillin or erythromycin (26,27).
In contrast to erythromycin, RP 59500 is rapidly bactericidal (26).
Clinical and bacteriologic failure has, however, already been reported
using pristinamycin (28), an oral streptogramin combination from which RP
59500 was derived.

The M phenotype is thus relatively new in South African pneumococci but is
emerging as an important factor in erythromycin-resistant pneumococci.
Although the low overall rate of resistance makes the use of streptogramins
and lincosamides potentially more feasible for the treatment of
pneumococcal infections, coresistance to penicillin and the present high
rate of MLS resistance necessitate antibiotic susceptibility testing before
these antibiotics are administered.

Acknowledgments

The authors thank Patrice Courvalin for the probe used in this study, Thora
Capper for assistance with MICs, Avril Wasas for performing serotyping, and
Robyn Hubner for assistance with data analysis.

This work was supported by the Medical Research Council, the South African
Institute for Medical Research, and the University of the Witwatersrand.

Carol Widdowson is completing her Ph.D. at the South African Institute for
Medical Research, through the University of the Witwatersrand. Her research
focuses mainly on resistance to the nonbetalactam antibiotics such as
erythromycin, tetracycline, chloramphenicol, and streptomycin, in the
pneumococcus.

Keith Klugman is the director of the South African Institute for Medical
Research. He also heads the Pneumococcal Research Unit of the Medical
Research Council, the South African Institute for Medical Research, and the
University of the Witwatersrand. He has an interest in all aspects of
pneumococcal research.

Address for correspondence: Carol A. Widdowson, Department of Clinical
Microbiology and Infectious Diseases, SAIMR, P.O. Box 1038, Johannesburg
2000, South Africa; fax: 27-11-489-9332; e-mail:
174carol@chiron.wits.ac.za.

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     Chemother 1996;40:2562-6.
 13. Robbins JB, Austrian R, Lee C-J, Rastogi SC, Schiffman G, Henrichsen
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 15. Friedland IR, Klugman KP. Failure of chloramphenicol therapy in
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     meningitis in England and Wales 1982 to 1992. Commun Dis Rep CDR Rev
     1995;5:R45-R50.
 21. Doern GV, Brueggemann AB, Holley Jr HP, Rauch AM. Antimicrobial
     resistance of Streptococcus pneumoniae recovered from outpatients in
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     pneumoniae in the United States: an update from the Centers for
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     Infect Dis 1996;174:986-93.
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     isolates of unusual multiply resistant pneumococci. The American
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 25. Visalli MA, Jacobs MR, Appelbaum PC. Susceptibility of
     penicillin-susceptible and -resistant pneumococci to dirithromycin
     compared with susceptibilities to erythromycin, azithromycin,
     clarithromycin, roxithromycin, and clindamycin. Antimicrob Agents
     Chemother 1997;41:1867-70.
 26. Pankuch GA, Lichtenberger C, Jacobs MR, Appelbaum PC. Antipneumococcal
     activities of RP 59500 (quinupristin-dalfopristin), penicillin G,
     erythromycin, and sparfloxacin determined by MIC and rapid time-kill
     methodologies. Antimicrob Agents Chemother 1996;40:1653-6.
 27. Barry AL, Fuchs PC. In vitro activities of a streptogramin (RP59500),
     three macrolides, and an azalide against four respiratory tract
     pathogens. Antimicrob Agents Chemother 1995;39:238-40.
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     Clin Microbiol Infect Dis 1995;14:341-2.


Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/widdowsn.htm

Please note that figures and equations are not available in ASCII format; 
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[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Dispatches

Mycobacterium tuberculosis infection as a Zoonotic Disease: Transmission
between Humans and Elephants

Kathleen Michalak,* Connie Austin, Sandy Diesel,* J. Maichle Bacon,* Phil
Zimmerman, and Joel N. Maslow
*McHenry County Department of Health, Woodstock, Illinois, USA; Illinois
Department of Public Health, Springfield, Illinois, USA; University of
Illinois, College of Medicine at Rockford, Rockford, Illinois, USA; and
Boston University School of Medicine and the VA Medical Center, Boston,
Massachusetts, USA

  --------------------------------------------------
      Between 1994 and 1996, three elephants from an exotic animal
      farm in Illinois died of pulmonary disease due to Mycobacterium
      tuberculosis. In October 1996, a fourth living elephant was
      culture-positive for M. tuberculosis. Twenty-two handlers at
      the farm were screened for tuberculosis (TB); eleven had
      positive reactions to intradermal injection with purified
      protein derivative. One had smear-negative, culture-positive
      active TB. DNA fingerprint comparison by IS6110 and TBN12
      typing showed that the isolates from the four elephants and the
      handler with active TB were the same strain. This investigation
      indicates transmission of M. tuberculosis between humans and
      elephants.

Mycobacterium tuberculosis and M. bovis, related organisms of the M.
tuberculosis complex, infect a wide variety of mammalian species. M. bovis
is pathogenic for many animal species, especially bovidae, cervidae, and
occasionally carnivores. Human disease with M. bovis is well described and
historically was a common cause of tuberculosis (TB) transmitted by
infected dairy products. As a result of milk pasteurization and TB
eradication programs in most industrialized countries, zoonotic
transmission of M. bovis through domestic livestock is now rare. In
contrast, a similar eradication program has not been conducted for wild or
exotic animals, which therefore remain an uncommon source for M. bovis
exposure. Zoonotic transmission of M. bovis has been reported from seals,
rhinoceros, and elk (1-4).

M. tuberculosis, the most common species to cause TB, classically causes
disease in humans. Animal infection with M. tuberculosis, while uncommon,
has been described among species (e.g., birds, elephants, and other
mammals) with prolonged and close contact with humans (5-10). Transmission
of M. tuberculosis between animals and humans has not been reported. This
paper describes M. tuberculosis transmission from elephants to humans.

The Outbreak

In March 1996, five elephants from an exotic animal farm in Illinois were
in California as part of a circus act. One elephant (with chronic,
unexplained weight loss since October 1995) died under anesthesia on August
3, 1996, during a diagnostic dental work-up. Necropsy showed widespread
consolidation of lung tissue with caseous necrosis of the lungs and
mediastinal lymph nodes. Short, fat, relatively scant numbers of acid-fast
bacilli were observed in necropsy tissues. A presumptive diagnosis of M.
tuberculosis was made. The remaining four elephants were recalled to the
farm in Illinois. A second elephant died en route on August 6, 1996.
Necropsy revealed copious respiratory and trunk exudates and caseous
necrosis of the lung.

To determine the risk for and possibility of infection among the animal
trainers and caretakers, an epidemiologic investigation was initiated. The
remaining elephants in the herd and the elephant handlers and trainers who
were still traveling were recalled to the farm and examined for evidence of
M. tuberculosis infection. All elephants were empirically begun on
antituberculous therapy in early December 1996.

Epidemiologic Investigation

The exotic animal farm was visited on numerous occasions to evaluate the
type and degree of contact between elephants and employees. The farm,
located in a rural area and surrounded by barbed wire and trees, originally
housed 18 Asian and 2 African elephants. Thirteen elephants were tethered
on a chain in one large barn, four were housed in a separate large room
(two in a common stall), and a baby elephant was in a third room with 5-6
tigers. A separate barn housed approximately 80 tigers.

TB Screening of Employees

The animal handlers (trainers and caretakers) who had direct contact with
the elephants were administered purified protein derivative (PPD) skin
tests. Initial screening was performed in August 1996, with subsequent
screenings in December 1996 and March, June, and September of 1997. Testing
was performed by the McHenry County Department of Health, except in two
handlers who had subsequent skin tests performed elsewhere. As part of the
screening process, handlers were questioned about their risk factors for
TB, including previous bacillus Calmette-Gurin (BCG) vaccination.

Handlers were tested by the two-step method using 5 tuberculin units of PPD
(0.1 ml) by intradermal injection in the flexor surface of the forearm. A
positive result was defined as an induration of >5 mm. Handlers with
positive skin tests were evaluated by a TB health-care worker and had chest
radiographs taken. Sputum samples from any handler with a chest radiograph
consistent with TB were submitted to the Illinois Department of Public
Health Laboratory. Samples were examined by direct microscopy for acid-fast
organisms, stained with fluorochrome, and processed for culture by standard
methods.

Examination of Isolates

The human isolate and the four elephant isolates were sent to the National
Tuberculosis Genotyping and Surveillance Network at the Michigan Community
Public Health Agency for restriction fragment length polymorphism (RFLP)
analysis. Southern blots of Pvu II restricted whole chromosomal DNA,
resolved in 1% agarose gels, were probed with a DNA fragment corresponding
to the right side of IS6110 and detected by chemiluminescence (11). The
number and size of the hybridizing fragments for each isolate were compared
in the same gel. Isolates with identical RFLP patterns or with </= 2 band
differences were considered to represent the same strain. Additionally, Pvu
II digested DNA was similarly typed after probing with the repetitive
element TBN12.

Epidemiologic Findings

Elephant handlers worked in very close proximity with the elephants around
the clock, whereas tiger handlers had little direct contact with the
elephants. Most of the elephant handlers lived on the farm in a separate
section of the barn; four lived in trailers on the grounds. The handlers'
living quarters had a separate ventilation system from the elephants';
however, the doors between the two quarters were open for unknown periods.
Handlers indicated that they held social events in a building connected to
the elephant barn.

Necropsies of elephants were performed on the farm and were attended by a
number of elephant and tiger handlers (including the handler with the
active case). The necropsy of the elephant that died in 1994, also
performed on the farm, showed caseous necrosis of the lungs and pleural
exudates whose culture yielded M. tuberculosis.

In addition to the three elephants that died of M. tuberculosis infection,
a fourth living elephant was also infected with the mycobacterium; this
infection was diagnosed in late December 1996 from a trunk culture obtained
in October 1996. Subsequent cultures from this and the other animals have
been negative for mycobacteria. Another elephant from this farm died of M.
tuberculosis infection in 1981 (5), but contact between this elephant and
the present herd or any of the handlers could not be established.

Twenty-two handlers at the exotic animal farm had moderate to frequent
contact with the infected animals; 12 were elephant handlers and 10 were
tiger handlers. Initial PPD testing was performed for 14 handlers in August
1996, 2 in October 1996, and 5 in December 1996. One who was PPD-positive
in November 1995 reported receiving BCG more than 10 years before.

Eleven (50%) of 22 handlers were found PPD-positive as part of this
investigation. Eight of the 11 had positive PPD skin test results upon
initial testing, with a median induration of 12 mm (range, 10 to 19 mm).
Four of the eight were elephant handlers and four were tiger handlers. The
skin test reaction of three handlers converted from negative to positive
with a median induration of 12 mm (range, 8 to 15 mm). The three PPD
converters were initially tested in August 1996; one was positive upon
retesting in January 1997, and two tested positive in April 1997 (Table).

Eight of the 11 handlers reported that they had negative skin tests in the
past and had not received BCG. The other three reported some type of
reaction from a previous skin test in the past but did not know the
results. All three also reported receiving BCG more than 10 years before.
Eight of the 10 handlers with negative PPD skin tests had at least one
negative follow-up test at 3 months; two left the farm and did not receive
follow-up testing.

The attack rates were approximately equal for the elephant and tiger
handlers. Of the 12 elephant handlers tested, 6 (50%) were PPD-positive
with two conversions documented in April 1997; of the 9 tiger handlers, 5
(56%) were PPD-positive, with one conversion documented in January 1997.
Overall, a very high rate (52%) of handlers tested positive.

All 12 handlers with positive PPDs (including the one with the known
positive PPD) received an evaluation and chest radiograph; one had
irregular nodules and interstitial changes in the right apex without
retraction of the lungs, consistent with active TB, but no cough, chest
pain, fever, night sweats, weight loss, or fatigue.

Three sputum samples were smear-negative for acid-fast bacilli, although
one yielded M. tuberculosis upon culture. Isoniazid (INH), rifampin,
pyrazinamide, and ethambutol treatment was initiated in September 1996, and
after 2 months, was reduced to INH and rifampin when the isolate showed no
resistance to antituberculous medications. Subsequent chest radiographs
revealed improvement or clearing of the initial lesions. Nine of the
remaining 11 PPD-positive handlers were prescribed INH prophylaxis; two
declined because of the risk for adverse reactions.

Table. TB PPDa skin test results of animal handlers, Aug
1996  Sep 1997
--------------------------------------------------
                         Positive Negative
--------------------------------------------------
Previously positive      1
Elephant handlers        4        6
Tiger handlers           4        4
Elephant handlers
(converted)              2
Tiger handlers
(converted)              1
Total                    12       10
--------------------------------------------------
(sup a)Tuberculin purified protein derivative.

Molecular Analysis of Elephant and Human Isolates

The sputum isolate from the handler with active TB was compared with the
isolates from the three animals that died and the living elephant whose
infection was diagnosed during the investigation. The isolates had
identical IS6110 RFLP pattern, differing by </= 2 bands (Figure 1).
Additionally, all isolates had the identical TBN12 RFLP pattern, except the
isolate from the elephant that died in August 1996, which demonstrated a
shift of one band (Figure 2).

Conclusions

Infection with M. tuberculosis or M. bovis has not been reported in
nondomesticated Asian or African elephants. M. tuberculosis infection in
domesticated elephants was first reported in 1875 by Garrod and has been
recognized in the ancient Ayurvedic literature (10); humans have been
considered the source of infection. A trainer with cavitary TB was
suspected as the source of infection (8) for one Asian elephant that died
of M. tuberculosis, although subsequent analysis showed the animal and
human isolates to be of two different phage types.

This report describes the first case of zoonotic M. tuberculosis
transmission. The epidemiologic investigation strongly suggests M.
tuberculosis transmission between humans and elephants, as evidenced by DNA
fingerprinting. RFLP analysis comparing Southern blots of chromosomal DNA
probed with IS6110 and TBN12 indicated that four elephant isolates had
identical patterns with the human isolate, differing by </= 2 bands. The
addition or loss of a single band has been demonstrated in other outbreak
settings, and the repetitive element that generates patterns has
characteristics of a mobile genetic element (11).

Eleven (50%) of 22 employees screened were skin-test positive, with no
difference between tiger and elephant handlers. This is a higher rate of
positives than documented in animal handlers exposed to M. bovis-infected
animals (3,4). Since the handlers had no accurate history of tuberculin
skin testing, it was not possible to determine when conversions took place.
The original source of infection for both elephants and humans is unknown.

The possible mechanisms of transmission include close contact while
handling and training elephants, cleaning the barn, participating in
elephant necropsies, and living in close proximity to the elephant barn.

Human-to-human transmission of TB is unlikely because the only handler with
active disease did not have cough. Of the three sputum samples initially
collected, two were smear- and culture-negative; the third had low numbers
of acid-fast bacilli manifested by a negative sputum smear, thus posing a
low infectivity risk to others. In contrast, the three elephants that died
had evidence of widespread pulmonary disease and, therefore, represented a
greater risk for dissemination.

Three handlers converted from negative to positive during the course of the
investigation; their relevant exposure is unknown. The source may have been
one elephant found antemortem to be culture-positive for M. tuberculosis,
although this animal did not return to the farm until November. Contact
with this animal is unlikely for handlers whose PPD tests converted in
December and unknown for the two handlers whose test results were positive
in April (the latter two had not been retested since August).

TB is transmitted through close prolonged contact with a person (or animal)
with active TB. The risk for TB transmission from an animal with a case of
active TB is higher for daily handlers than for persons with only brief
contact, e.g., members of the public viewing a performance or receiving
elephant rides. In this outbreak, screening of all persons who had (or
thought they had) contact with an elephant that died of M. tuberculosis
identified three PPD-positive cases but no cases of active TB (8). Because
the real risk for transmission to the general public was poorly understood,
this case received considerable media attention as well as mention in the
medical literature (7,12).

Veterinary practices should be initiated to reduce the risks for exposure
to animals infected with M. tuberculosis. No data are available on TB
incidence among domesticated elephants in the United States. An estimate
can be derived from a retrospective study of 379 zoo elephants of which
eight (2.3%) had M. tuberculosis infection (10).

Reliable diagnosis and prevention of TB in all domesticated and exhibited
animals is ideal. Short of this, possible ways to prevent and decrease
zoonotic spread of any mycobacterial infection (M. tuberculosis or M.
bovis) include 1) regular skin testing of handlers or keepers; 2) a high
index of suspicion of TB in elephants with unexplained weight loss, cough,
or rhinorrhea; 3) public health measures of contact tracing and
notification; and 4) active and effective treatment of infected personnel
and animals (13).

[fig 1]                              [fig 2]
Figure 1. IS6110 restriction         Figure 2. TBN12 restriction
fragment length polymorphism         fragment length polymorphism
results. Lane 1, elephant            results. Lane 1, elephant
isolate (died August 6, 1996);       isolate (died August 6, 1996);
Lane 2, elephant isolate (died       Lane 2, elephant isolate (died
1994); Lane 3, living elephant       1994); Lane 3, living elephant
trunk culture (October 1996);        trunk culture (October 1996);
Lane 4, elephant lung tissue         Lane 4, elephant lung tissue
isolate (died August 3, 1996);       isolate (died August 3, 1996);
Lane 5, elephant lymph node          Lane 5, elephant lymph node
tissue isolate (died August 3,       tissue isolate (died August 3,
1996); Lane 6, human sputum          1996); Lane 6, human sputum
isolate (September 1996).            isolate (September 1996).
Provided by State of Michigan        Provided by State of Michigan
Community Public Health Agency.      Community Public Health Agency.

Acknowledgments

We thank Stephen Dietrich and Laura Mosher, North Central Area TB-RFLP
Laboratory, Michigan Department of Public Health; George J. Dizikes, Chief
of Molecular Diagnostics, Illinois Department of Public Health, Division of
Laboratories; and Janet Payeur, National Veterinary Services Laboratory,
U.S. Department of Agriculture, MB Section. This research was supported in
part by the Centers for Disease Control and Prevention, National
Tuberculosis Genotyping and Surveillance Network cooperative agreement.

Kathleen Michalak is the Director of Nursing for McHenry County Department
of Health. She has worked to improve the use of community resources, and
improved service to client, providers, and the community as a whole.

Address for correspondence: Kathleen Michalak, McHenry County Department of
Health, 2200 N. Seminary Avenue, Woodstock, IL 60098, USA; fax:
815-338-7661.

References

  1. Thoen CO. Tuberculosis (Revised 1995). Zoonosis updates. Journal of
     the American Veterinary Medical Association 1995;2:155-8.
  2. Thompson PJ, Cousins DV, Gow BL, Collins DM, Willamson BH, Dagnia HT.
     Seals, seal trainers, and mycobacterial infection. American Review of
     Respiratory Diseases 1993;147:164-7.
  3. Dalovisio JR, Stetter M, Mikota-Wells S. Rhinoceros' rhinorrhea: cause
     of an outbreak of infection due to airborne Mycobacterium bovis in
     zookeepers. Clin Infect Dis 1992;15:598-600.
  4. Fanning A, Edwards S. Mycobacterium bovis infection in human beings in
     contact with elk (Cervus elaphus) in Alberta, Canada. Lancet
     1991;338:1253-5.
  5. Saunders G. Pulmonary Mycobacterium tuberculosis infection in a circus
     elephant. J Am Vet Med Assoc 1983;183:1311-2.
  6. Pinto MRM, Jainudeen MR, Panabokke RG. Tuberculosis in a domesticated
     Asiatic Elaphas maximus. Vet Rec 1973;93:662-4.
  7. Frankel D. Elephants pack their trunks and leave the circus. Lancet
     1997;349:1675.
  8. Greenberg AS, Jung RC, Gutter AK. Hazel Elephant is dead (of
     Tuberculosis). American Review of Respiratory Diseases 1981;124:341.
  9. Jones WD, Good RC. Hazel elephant redux. American Review of
     Respiratory Diseases 1982;125:270.
 10. Mikota S, Sargent EL, Ranglack GS. Medical management of the elephant.
     Tuberculosis and Tuberculin Testing 1994;33-9.
 11. Small P, Moss A. Molecular epidemiology and the new tuberculosis.
     Infect Agents Dis 1993;2:132-8.
 12. Furley CW. Tuberculosis in elephants. Lancet 1997;350:224.
 13. Centers for Disease Control and Prevention. Guidelines for preventing
     the transmission of Mycobacterium tuberculosis in health-care
     facilities. MMWR Morb Mortal Wkly Rep 1994;43:63-4.


Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/michalak.htm

Please note that figures and equations are not available in ASCII format; 
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-------------------------------------------------------------------------------------------
-------------------------------------------------------------------------------------------

[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Dispatches

New Vectors of Rift Valley Fever in West Africa

D. Fontenille,* M. Traore-Lamizana,* M. Diallo,* J. Thonnon, J.P.
Digoutte, and H.G. Zeller
*Laboratoire de zoologie mdicale, Institut Francais de recherche
scientifique pour le dveloppement en coopration (ORSTOM), Institut
Pasteur, BP 1386, Dakar, Sngal; Institut Pasteur, BP 220, Dakar,
Sngal; Institut Pasteur, BP 1274, Antananarivo, Madagascar

  --------------------------------------------------
      After an outbreak of Rift Valley fever in Southern Mauritania
      in 1987, entomologic studies were conducted in a bordering
      region in Sngal from 1991 to 1996 to identify the sylvatic
      vectors of Rift Valley fever virus. The virus was isolated from
      the floodwater mosquitoes Aedes vexans and Ae. ochraceus. In
      1974 and 1983, the virus had been isolated from Ae. dalzieli.
      Although these vectors differ from the main vectors in East and
      South Africa, they use the same type of breeding sites and also
      feed on cattle and sheep. Although enzootic vectors have now
      been identified in West Africa, the factors causing outbreaks
      remain unclear.

Rift Valley fever (RVF) virus belongs to the genus Phlebovirus, family
Bunyaviridae. RVF virus epizootics, which cause abortions and deaths in
young ungulates and epidemics with hemorrhagic fever and other symptoms,
have occurred throughout sub-Saharan Africa and Egypt (1). The virus was
first isolated in 1930 in Kenya. The most recent outbreaks occurred in
Egypt in 1977-1978 and 1993, South Mauritania in 1987, Madagascar in
1990-1991 (2), and Northern Kenya and Somalia in 1997 (3). The virus is
transmitted by mosquitoes and by aerosols of viremic blood during
hemorrhage.

After the virus was isolated from livestock, humans, and mosquitoes,
transmission cycles were proposed for East and South Africa (1). In East
and South Africa, the virus is transmitted from floodwater Aedes (subgenera
Aedimorphus and Neomelaniconion) to vertebrates or from mosquito to
mosquito by vertical transmission. Vectors (Ae. cumminsii, Ae.
circumluteolus, and Ae. mcintoshi) breed in temporary flood ponds, which
flood extensively during heavy rainfall. After such rainfall, the number of
mosquitoes increases dramatically, and epizootic and epidemic cycles can
occur. During epidemics, other mosquito species can serve as vectors;
transmission also occurs by aerosol from the blood of viremic vertebrates.
Data obtained in the last few years, however, have shown that the pattern
of RVF transmission is different in West Africa (4).

Table 1. Rift Valley fever virus isolates in West and Central Africa

----------------------------------------------------
                    No. of
Host               isolates   Location       Year(s)
----------------------------------------------------
Aedes dalzieli        3    Kdougou, Sngal 1974
Ae. dalzieli          1    Kdougou, Sngal 1983
Ae. ochraceus         3    Barkedji, Sngal 1993
Ae. vexans           10    Barkedji, Sngal 1993
Ae. cumminsii         1    Burkina-Faso      1983
Ae. furcifer          1    Burkina-Faso      1983
Culex antennatus      1    Nigeria           1967-70
Culicoides sp.        2    Nigeria 1967      1967

Ae. palpalis          1    Central African   1969
                                  Republic

Mansonia africana     1    Central African   1969
                                  Republic
Amblyomma
 variegatum 
(on cattle           1    Central African    1983
 in a slaughterhouse)             Republic
Humans               2    Sngal            1975
                     1    Sngal            1980
                   201    Mauritania         1987
                    12    Central African
                                  Republic   1971-90
Bats                 2    Guinea             1981-83
Sheep                1    Barkedji, Sngal  1993
Bovine               1    Kolda, Sngal     1993
  --------------------------------------------------

Before 1991, RVF virus had been isolated from mosquitoes, humans, and bats
in different West African countries (Table 1). Serologic data have
demonstrated active transmission of the virus throughout West Africa (5).

In Sngal, RVF virus was isolated from Aedes dalzieli in 1974 and 1983.
RVF virus had never been isolated from this species in East and South
Africa. The only known RVF outbreak, resulting in more than 200 human
deaths, occurred in Southern Mauritania near the village of Rosso, on the
Sngal River (6). Serologic surveys of cattle after the outbreak showed
that the epizootic was widespread. Animals with positive immuglobulin (IgM)
were recorded in The Gambia 340 km south of the epidemic (7). However,
surveys conducted 1 to 2 years after the outbreak showed a decrease in RVF
seroprevalence, which suggested that the transmission had ceased (8). No
human cases were observed in Sngal after the outbreak. In 1995 and 1996,
IgM-positive sheep and cattle were observed again along the Sngal River,
demonstrating that the virus remains present in the region (Thonnon, unpub.
data). Because the virus was not isolated from approximately the 500,000
mosquitoes captured in the Rosso area the year after the outbreak, we
decided to identify the mosquitoes or other arthropod species involved in
the RVF-endemic cycles. We wanted to determine where the virus was when
there were no visible manifestations in humans or cattle and whether the
mosquito vectors were the same there as in East Africa.

On the basis of the epidemiology of the RVF virus in East and South Africa
and the few isolations from West Africa, we selected for a study of the
sylvatic vectors of RVF two sites in different bioclimatic areas in
Sngal: Kdougou, where the virus had been isolated before, and Barkedji,
where temporary ground pools occur. We focused on mosquitoes and sand flies
because RVF is a phlebovirus and research had shown that Phlebotomus
duboscqi can transmit the virus (9). This research also first identified in
the sahelian region the sylvatic vectors of RVF (10), which are different
from those in East and South Africa.

Entomologic surveys were conducted from 1991 to 1996. The Kdougou area
(12ll'N, 1233'W), in southeastern Sngal in the Sudano-Guinean
bioclimatic zone, has a rainy season (May through October) and an average
rainfall of 1,100 mm. The Barkedji area (1517'N, 1417'W), in northern
Sngal in the sahelian Ferlo region, has a short rainy season (July to
September) with an average rainfall of 250-350 mm. Temporary ground pools
fill soon after the first rains and remain the only source of water during
the dry season until January.

Hematophagous arthropods were collected each year in July, October, and
November in Kdougou, and monthly in Barkedji. Insects were captured by
four methods: when they landed to bite human volunteers from 17:30 to
22:30; with Centers for Disease Control and Prevention (CDC)dry ice light
traps; with CDC light traps located in sheepfolds, and with animal-baited
(one sheep or three chickens) intermittent light traps. In Kdougou,
arthropods were collected in villages and in a forest, and in Barkedji, at
the edge of three temporary ground pools.

Arthropods were sorted and pooled by species, sex, location, and date in
the field. Pools of arthropods (fewer than 100) were put in liquid nitrogen
and stored at -70C. Viruses were isolated on AP61 (Ae. pseudoscutellaris)
and Vero cell cultures. Some mosquito pools were injected into suckling
mice. Viruses were detected by immunofluorescence assay that used specific
mouse immune ascitic fluids (11). Viruses were identified by complement
fixation and neutralization tests. Blood-fed mosquitoes collected in the
traps were preserved so that the blood meal source could be identified by
enzyme-linked immunosorbent assay (ELISA) (12).

More than 228,000 mosquitoes from 52 species in Barkedji and 250,000
mosquitoes from 102 species in Kdougou were collected and tested for virus
isolation. Additionally, 233,000 sand flies from 11 species and 35,000 sand
flies from 25 species were caught in Barkedji and Kdougou, respectively
(Table 2). In Barkedji, Aedes species represented 28.8% of the mosquitoes
collected. Ae.(Aedimorphus) vexans was the most abundant Aedes species
collected, followed by Ae.(Adm) ochraceus; Ae. (Neomelaniconion) mcintoshi
and Ae. (Adm) dalzieli were rare. Sand flies were abundant during the dry
season (December through May). In Kdougou, Aedes species represented 50.6%
of the mosquitoes collected. Ae. dalzieli was the predominant Aedes
species; Ae. vexans, Ae. mcintoshi, and Ae. ochraceus were much less
abundant.

Table 2. Arthropod collections in Kdougou and Barkedji, 1991 to 1996
------------------------------------------------------------------------------------
                           Mosquitoes           Aedes spp.          Sand flies
Location      Year         No.    (Pools)       No.   (Pools)       No.      (Pools)
------------------------------------------------------------------------------------
Barkedji      1991      34,327    (1,042)    11,23      (338)     3,370         (56)
              1992      42,804    (1,534)      5,78     (352)    30,547        (269)
              1993      64,810    (2,023)     6,056     (300)    72,104        (651)
              1994      22,470      (927)     8,701     (365)    42,730        (416)
              1995      40,952    (1,413)    16,124     (561)    66,300        (667)
              1996      23,041      (764)    16,243     (453)    18,934        (192)
Total                  228,404    (7,703)    64,139   (2,369)   233,895      (2,251)
Kdougou      1991      48,377    (1,264)    28,579     (730)       200          (2)
              1992      37,685    (1,364)    26,889     (804)     1,032          (9)
              1993      71,992    (2,493)    31,224   (1,136)
              1994      48,751    (1,877)    23,839     (956)
              1995      17,756    (1,014)    12,590     (616)    23,122        (238)
              1996      25,768    (1,284)    14,869     (679)    11,215        (115)
Total                  250,329    (9,296)   126,659   (4,914)    35,569        (364)
Total        (1991-96) 478,733   (16,999)   190,798   (7,283)   269,464      (2,615)
------------------------------------------------------------------------------------
In Barkedji, 10 RVF virus isolates came from Ae. vexans and 3 from Ae.
ochraceus collected around three temporary ground pools and near cattle
droves in CO2-CDC light traps in October and November 1993. In November, an
RFV virus isolate was obtained from one of the sheep. West Nile, Ngari, and
Wesselsbron viruses were also isolated from vectors or potential RVF
vectors. Five viruses were isolated from sand flies (Table 3).

In Kdougou, no RVF virus was isolated from any vector during the study
period, although the virus had been found four times in earlier isolates
obtained in 1974 and 1982 from Ae.dalzieli. This study found other viruses
isolated from Ae. dalzieli including 42 viral strains belonging to seven
different viruses, mostly alphavirus and flavivirus, one strain of
Wesselsbron virus was isolated from Ae. ochraceus, and five different
viruses were isolated from sand flies (Table 3).

Even if the vectorial competence of Ae.vexans, Ae. ochraceus, and Ae.
dalzieli had not been experimentally confirmed, they would be likely
enzootic vectors of RVF virus in Sngal. These three species are different
from the main known East African vectorsAe.cumminsii, Ae. circumluteolus,
and Ae. mcintoshiwhich are also in West Africa, but whose role in RVF
virus transmission has not been demonstrated. A much lower number of Ae.
mcintoshi than Ae.vexans or Ae. ochraceus were captured each year: when RVF
virus was isolated in 1993 in Barkedji, 6,958 Ae. vexans, 1,069 Ae.
ochraceus, but only 58 Ae. mcintoshi were captured and tested.

Other Sngalese floodwater zoophilic mosquitoes such as Ae.(Adm) minutus,
Ae. (Adm) fowleri, and Ae. (Adm) argenteopuntatus should be suspected as
potential sylvatic RVF vectors because they belong to the same Aedes
subgenera of known vectors and have almost the same breeding sites and
trophic behavior (10). Moreover, females from a colony of Sngalese Ae.
fowleri were able to transmit RVF virus experimentally (13). Mosquito
species belonging to other genera (e.g., Culex, Mansonia) should be
implicated during an outbreak only after the virus is amplified, as in East
Africa (3).

The taxonomic status of Ae. vexans, the
chief enzootic vector of RVF virus, has to
be clarified. Ae. vexans has a worldwide
distribution. In 1975, G.B. White wrote
that the African specimens of Ae. vexans
belong to the same subspecies Ae. vexans               [fig]
arabiensis, which was recorded in            Distribution of Aedes vexans 
Mauritania, Sngal, The Gambia, Ghana,      and Culex poicilipes captured
Nigeria, Sudan, Ethiopia, Somalia, and       by monthly rainfall, Barkedji,
South Africa (14). In Barkedji, in the dry   Sngal, 1991-1996.
sahelian region of Sngal, female Ae.
vexans lay eggs on the soil of temporary
ground pools. The adult Ae. vexans appear only 4 days after the first rain,
a very short larval development period. Ae. vexans can be abundant (more
than 4,000 per light trap). However, their density decreases quickly about
2 months after the ground pools flood, while the density of other species,
such as Culex poicilipes, Mimomyia splendens, or Mansonia africana,
increases at the end of the rainy season (Figure).

Evaluating the trophic preferences of mosquitoes is difficult and highly
dependent on the sampling methods. Some species are highly host-specific,
but most species feed on a range of different host animals. Despite
numerous human bites by Ae. vexans and Ae. ochraceus around the pools,
these species seemed not very host-specific. Females that fed on cattle,
sheep, goats, horses, and even chickens were identified. Other sylvatic
vectors or potential vectors of RVF virus also showed equally low host
specificity (Table 4).

 Table 3. Potential Rift Valley fever sylvatic vectors and
 viral isolates, Sngal, 1991 to 1996
 ------------------------------------------------------------
                                    No.  Virus isolates(sup a)
Vector species               No.   pools   (No. strains)
-------------------------------------------------------------

 Barkedji

     Aedes vexans         42,055  1,428  WN-NRI (1), RVF
                                         (10), WN (2)

     Ae. mcintoshi          758      88  NRI (1)

     Ae. ochraceus        3,672     228  WSL (1), RVF (3)

     Ae. dalzieli           105      34  (0 virus)

 Phlebotominae spp.     233,895   2,251  SAB (63), CHP (7), GF
                                         (5), Ar D 88909 (1),
                                         Ar D 95737 (12)

 Kdougou

     Ae. vexans           1,194      81  (0 virus)

     Ae. mcintoshi          536     107  (0) virus)

     Ae. ochraceus          915     110  WSL (1)

     Ae. dalzieli(sup b) 31,809     821  CHIK (8), BBK (1),
                                         WSL (2), KED (6), BOU
                                         (1), PGA (2), ZIKA
                                         (22)

 Phlebotominae spp.      35,569     364  SAB (11), CHP (4),
                                         TETE (1), Ar D 111740
                                         (1), Ar D 95737 (2)
--------------------------------------------------------------------
(sup a)WN: West-Nile virus, NRI: Ngari virus, RVF: Rift Valley fever
 virus, WSL: Wesselsbron virus, SAB: Saboya virus, CHP:
 Chandipura virus, GF: Gabek Forest virus, CHIK: Chikungunya
 virus, BBK: Babanki virus, KED: Kdougou virus, BOU: Bouboui
 virus, PGA: Pongola virus, ZIKA: Zika virus, TETE: Tete virus.
 Ar D 88909, Ar D 95737, and Ar D 111740: not yet identified
 viruses.
 (sup b)4 RVF strains were isolated in 1974 and 1982.

Because of low host specificity, many different viruses, including RVF, can
be transmitted to many vertebrate species. More than 30 viruses belonging
to different groups have been isolated from Ae. vexans throughout the world
(15)the three from West Africa include West Nile virus (a flavivirus from
birds) and Ngari virus (isolated from ill humans). Fifteen viruses, also
from different arbovirus groups, have been isolated from Ae. dalzieli in
West Africa (16). Few viruses were isolated from Ae. ochraceus and Ae.
mcintoshi, probably because of the lower number of pools tested.

Table 4. Host choices of Rift Valley fever virus vectors
captured with light traps or animal-bait traps in Sngal,
1991-1996
------------------------------------------------------------------
                          No. of host-specific blood feedings
                     ---------------------------------------------    
Vector                Cow     Sheep    Horse   Chicken Human(sup a)
------------------------------------------------------------------
Aedes vexans           35      33       38        7       + + +
Ae. ochraceus          8       18        5        1        + +
Ae. mcintoshi          7       20        2        2        + +
Ae. dalzieli           52      114       1        21      + + +
  ----------------------------------------------------------------
(sup a)++: numerous bites; +++: very numerous bites.

The risk for a new outbreak of RVF in Sngal is highly speculative. An
increase in IgG prevalence in livestock in different West African countries
the year before the 1987 Mauritanian outbreak demonstrated that the virus
was present in endemic cycles in this area (17). This epizootic was caused
by three factors: a dam was built near Rosso on the Sngal River, mosquito
density increased probably because of the flooding of the river bank in
1987, and the livestock density increased. In 1993, when RVF virus was
isolated in Barkedji from floodwater Aedes and from one sheep, the
situation was different. No environmental or climatic changes were
identified; in particular, the rainfall was not higher than in previous
years. However, a 15% IgM prevalence was observed in 1994 and 1995 among
herds studied each year since 1990 along the Sngal River, demonstrating
RVF virus circulation. Enzootic vectors of RVF virus are now identified in
West Africa, but factors causing outbreaks need to be further studied.

Acknowledgments

We thank Leonard Munstermann, who organized the Emerging Vectors of
Emergent Diseases symposium, sponsored by the American Commitee of Medical
Entomology, held in Orlando during the XX meeting of the American Society
of Tropical Medicine and Hygiene (7-11 December 1997), where these data
were presented and Scott McKeown for help with the English translation.

This work was supported by the Institut Francais de Recherche Scientifique
pour le Dveloppement en coopration (ORSTOM) and by the Dakar Pasteur
Institute.

Address for correspondence: Didier Fontenille, Laboratoire ORSTOM de
zoologie mdicale, Institut Pasteur, BP 1386, Dakar, Sngal; fax: 221 839
92 10; e-mail: fontenil@dakar.orstom.sn.

References

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     arboviruses: epidemiology and ecology. Boca Raton (FL): CRC Press;
     1988. p. 51-76.
  2. Peters CJ. Emergence of Rift Valley fever. In: Saluzzo JF, Dodet B,
     editors. Factors in the emergence of arbovirus diseases. Paris:
     Elsevier; 1997. p.253-64.
  3. World Health Organization. Emerging and other communicable diseases.
     Disease outbreaks reported, 28 January 1998: Rift Valley fever and
     haemorrhagic disease in Kenya and Somalia. [cited 1998 Mar 17]:[1
     screen]. Available from URL: http://www.who.ch/emc/oldnews.htm.
  4. Zeller HG, Fontenille D, Traore-Lamizana M, Thiongane Y, Digoutte JP.
     Enzootic activity of Rift Valley fever virus in Sngal. Am J Trop Med
     Hyg 1997;56:265-72.
  5. Zeller HG, Bessin R, Thiongane Y, Bapetel I, Teou K, Gbaguidi Ala M,
     Nde Atse A, Sylla R, Digoutte JP, Akakpo JA. Rift Valley fever
     antibody prevalence in domestic ungulates in several West-African
     countries (1989-1992) following the 1987 Mauritanian outbreak. Res
     Virol 1995;146:81-5.
  6. Jouan A, Le Guenno B, Digoutte JP, Philippe B, Riou O, Adam F. A Rift
     Valley fever epidemic in Southern Mauritania. Ann Virol
     1988;139:307-8.
  7. Ksiazek TG, Jouan A, Meegan JM, Le Guenno B, Wilson ML, Peters CJ,
     Digoutte JP, Guillaud M, Merzoug NO, Touray EM. Rift Valley fever
     among domestic animals in the recent West African outbreak. Res Virol
     1989;140:67-7.
  8. Thiongane Y, Gonzalez JP, Fati A, Akakpo JA. Changes in Rift Valley
     fever neutralizing antibody prevalence among small domestic ruminants
     following the 1987 outbreak in the Sngal river basin. Res Virol
     1991;142:67-70.
  9. Turell MJ, Perkins PV. Transmission of Rift Valley fever virus by the
     sand fly, Phlebotomus duboscqi (Diptera: Psychodidae). Am J Trop Med
     Hyg 1990;42:185-8.
 10. Fontenille D, Traore-Lamizana M, Zeller H, Mondo M, Diallo M, Digoutte
     JP. Rift Valley fever in Western Africa: isolations from Aedes
     mosquitoes during an interepizootic period. Am J Trop Med Hyg
     1995;52:403-4.
 11. Digoutte JP, Calvo-Wilson MA, Mondo M, Traore-Lamizana M, Adam F.
     Continuous cell lines and immune ascitic fluid pools in arbovirus
     detection. Res Virol 1992;143:417-22.
 12. Beier JC, Perkins PV, Wirtz RA, Koros J, Diggs D, Gargam TPII, Koech
     DK. Bloodmeal identification by direct enzyme-linked immunosorbent
     assay (ELISA) tested on Anopheles (Diptera: Culicidae) in Kenya. J Med
     Entomol 1988;25:9-16.
 13. Turrel MJ, Faran ME, Cornet M, Bailey CL. Vector competence of
     Sngalese Aedes fowleri (Diptera: Culicidae) for Rift Valley fever
     virus. J Med Entomol 1988;25:262-6.
 14. White GB. Note on a catalogue of Culicidae of the Ethiopian region.
     Mosquito Systematics 1975;7:303-44.
 15. Horsfall WR, Novak RJ. The inland floodwater mosquito, Aedes vexans:
     an annotated bibliography to world literature. Pt I. J Am Mosq Control
     Assoc; 1991: 1-43.
 16. Thonnon J. Rapport annuel 1995 du Centre Collaborateur OMS de
     Reference et de Recherche pour les Arbovirus et les virus de fievres
     hemorragiques (CRORA). Sngal: Institut Pasteur de Dakar; 1996.
 17. Saluzzo JF, Digoutte JP, Chartier C, Martinez D, Bada R. Focus of Rift
     Valley fever transmission in southern


Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/fontnile..htm

Please note that figures and equations are not available in ASCII format; 
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-------------------------------------------------------------------------------------------

[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Dispatches

Reemergence of Plasmodium vivax Malaria in the Republic of Korea

Brian H. Feighner, Son Il Pak, William L. Novakoski, Lori L. Kelsey, and
Daniel Strickman
U.S. Army, Republic of Korea

  --------------------------------------------------
      Plasmodium vivax malaria reemerged in the Republic of Korea in
      1993. The number of cases has tripled each year since, with
      more than 1,600 cases reported in 1997. All 27 cases in U.S.
      troops resolved uneventfully with chloroquine/primaquine
      therapy. Disease is localized along the western Demilitarized
      Zone and presents minimal risk to tourists.

Plasmodium vivax malaria has been endemic on the Korean peninsula for
centuries (1). The primary vector is believed to be Anopheles sinensis, a
zoophilic and facultatively anthropophilic mosquito, which breeds in fresh,
sun-exposed water such as that found in rice fields (2). During the Korean
War (19501953), U.S. troops were exposed to malaria (3) and received
weekly chloroquine chemoprophylaxis. This regimen generally succeeded in
preventing malarial attacks while U.S. troops were in the Republic of
Korea. As the troops returned home, however, U.S. military hospitals were
inundated with cases of malariaat one point, as many as 629 cases per week
(3). More than 3,000 cases of malaria were documented in U.S. troops that
served during the war. Subsequent U.S. military research led to the current
use of primaquine therapy (4). The outbreak of malaria continued on the
peninsula after the armistice. In the 1960s and 1970s, the Republic of
Korea government combined active and passive case detection with extensive
use of pesticides in an ambitious eradication project (1). Indigenous
transmission of malaria appeared to cease in the mid-1970s, and in 1979 the
Republic of Korea was declared malaria-free by the World Health
Organization (5).

In 1993, P. vivax malaria reemerged in the Republic of Korea along the
western edge of the Demilitarized Zone (DMZ) (6). Since the reemergence,
the annual number of cases has increased geometrically, tripling (1995,
1996) or quadrupling (1997) in each of the past 4 years (Table 1). In 1997,
the Republic of Korea Army administered antimalarial chemoprophylaxis to
approximately 8,000 of its troops at greatest risk (U.S. Army/Republic of
Korea Army/Republic of Korea Malaria Symposium, pers. comm.). U.S. troops
in Korea were not placed on antimalarial chemoprophylaxis in 1997 after
local and U.S. Army experts concluded that personal protective measures and
community mosquito control would adequately limit transmission (7).

Table 1. Annual number of Plasmodium vivax malaria cases
in U.S. Forces-Korea (USFK), Republic of Korea (ROK) Army,
and ROK Civilians, 19931997

  --------------------------------------------------
                   ROK        ROK
Year    USFK      Army     Civilians          Total
  --------------------------------------------------

1993	  0          1           1                2

1994	  1         18          20               39

1995	 19          8          19              118

1996	 11        285          71              367

1997	 27      1,154         461            1,642
  --------------------------------------------------

Cases in 1997 were distributed as expected from June to October, with a
peak of 518 cases in August (Table 2). As in past years, more than 80% of
the cases were associated with military service along the western DMZ.
During 1997, 34 cases of P. vivax malaria in U.S. troops were attributed to
exposure in the Republic of Korea. This number includes 27 cases in U.S.
Forces-Korea troops, one case in a soldier stationed in Okinawa, Japan, who
was temporarily assigned to the Republic of Korea in August 1997, and six
cases in U.S. troops exposed in the summer of 1996. The U.S. Forces-Korea
incidence of P. vivax in all malarious areas between July 1, 1997, and
September 30, 1997, was estimated to be 0.5 cases per 1,000 per week. The
incidence northwest of the Imjin River was approximately one case per 1,000
per week. Site-specific rates, as well as rates for the Republic of Korea
Army, cannot be released because of security concerns; however, the
Republic of Korea Army has 50 times more exposed soldiers than does U.S.
Forces-Korea.

Table 2. Monthly distribution of Plasmodium vivax
malaria cases in the Republic of Korea (ROK), by
onset of disease, 1997

  --------------------------------------------------
                     ROK       ROK
Month   USFK(sup a) Army     Civilian      Total
  --------------------------------------------------
Jan          0        0         0            0
Feb          0        0         0            0
Mar          0        5         2            7
Apr          1       10         3           14
May          0       31         4           35
Jun          0      145        38          183
Jul          2      260       102          364
Aug         11      317       190          518
Sep         10      320        75          405
Oct          3       55        38           96
Nov          0        9         9           18
Dec          0        2       NA(sup b)      2
  --------------------------------------------------
(sup a)USFK, U.S. Forces-Korea.
(sup b)NA, Not available.

The epidemic remains focused along the western DMZ. Before 1997, almost all
civilian cases were attributed to exposure in areas within 10 km of the
DMZ. In 1997, however, dozens of cases occurred among civilians farther
than 10 km from the DMZ. The area where U.S. troops are exposed is strictly
controlled; tourists are not allowed in this area after dark. Other areas
under the Republic of Korea Army control, e.g., Yonchon and Chorwon, are
accessible to tourists but are seldom visited. The risk to foreign
tourists, therefore, appears to be negligible at this time.

Of the 27 U.S. Forces-Korea cases, 18 were in U.S. soldiers and 9 were in
Korean soldier augmentees to the U.S. Army. All cases were in men 19 to 40
years of age (median 21.5 years). With one exception, the cases occurred in
junior enlisted soldiers. As in the past 3 years, no cases occurred in
African American soldiers, who account for almost 30% of the exposed U.S.
troops. This is consistent with the known natural immunity against P. vivax
in African Americans, most of whom do not have the Duffy antigen on their
red blood cells (8). This relative immunity of African Americans was also
seen during the Korean War (3). The time from onset of symptoms to
diagnosis was 1 to 25 days (median 5 days). Three patients with mild
illness were treated as outpatients. All patients quickly recovered after
standard chloroquine therapy. All patients were also treated with
primaquine.

The Korean strain of P. vivax malaria has been well characterized by both
Korean and U.S. medical personnel (1,3). This type of malaria is termed
"temperate climate" P. vivax malaria (3). Transmission is restricted to
only a few summer months, when the Korean peninsula is warm enough to
support the parasite's reproductive cycle in the mosquito (1,3). This
Korean P. vivax malaria is remarkable in that 40% to 50% of cases have no
symptoms until 6 to 9 months after infection (3,9). The long incubation
period interferes with determining the start of the transmission season.
Malaria cases begin to occur in the late spring or early summer, before the
weather is warm enough to support malaria transmission. These early season
cases, arising from infections acquired the previous fall, blend into and
fuel infections transmitted each summer. Another result of this long
incubation period, given the extraordinary (>90%) annual turnover in U.S.
troops, is the return of infected soldiers to the United States before any
symptoms appear.

Malaria in the Republic of Korea is also characterized as epidemic or
unstable because conditions usually do not support widespread disease (10).
Factors contributing to this instability include the extremely short
transmission season and the zoophily of the major vector. Areas with
unstable malaria usually have low to very low rates of disease but can
occasionally have high rates in response to climatic, livestock, or
sociopolitical aberrations (10). The current epidemic may fit this pattern.
Heavy rains from 1994 to 1996 led to widespread crop failures in the
Democratic Peoples Republic of Korea. As a result, domestic farm animal
populations, the preferred source of blood for A. sinensis, fell sharply.
These factors may have contributed to the geometric increase of disease,
but the lack of data makes this explanation speculative.

Most Korean officials perceive the current epidemic to be a Democratic
Peoples Republic of Korea public health problem spilling over the DMZ and
discount the importance of indigenous transmission in the Republic of
Korea. Broad-scale epidemiologic investigation is necessary to confirm
indigenous transmission. Although it is not known if the epidemic will
continue its geometric increase and geographic expansion in 1998, a
decrease in disease without extensive intervention seems unlikely.

Acknowledgments

The authors thank Colonel Daniel F. Perugini, U.S. Forces-Korea Surgeon,
and Colonel Kenneth G. Torrington, Deputy Commander, 18th Medical Command,
for their support and critical review of this manuscript.

All equipment, supplies, and author salaries were received from the U.S.
Army.

Address for correspondence: Brian H. Feighner, HHC, 18th MEDCOM, Unit
#15281, Box 763, APO AP 96205-0054 USA; 822-7916-3028; e-mail:
ltc_brian_feighner@smtplink.seoul. amedd.army.mil.

References

  1. Paik YH, Ree HI, Shim JC. Malaria in Korea. Japanese Journal of
     Experimental Medicine 1988;58:55-66.
  2. Ree HI, Hong HK, Paik YH. Study on natural infection of P. vivax in
     Anopheles sinensis in Korea. Korean J Parasitol 1967;5:3-4.
  3. Hankey DD, Jones R, Coatney GR, Alving AS, Coker WG, Garrison PL, et
     al. Korean vivax malaria. I. Natural history and response to
     chloroquine. Am J Trop Med Hyg 1953;2:958-69.
  4. Jones R, Jackson LS, DiLorenzo A, Marx RL, Levy BL, Kenny EC, et al.
     Korean vivax malaria. III. Curative effect and toxicity of primaquine
     in doses from 10 to 30 mg daily. Am J Trop Med Hyg 1953;2:977-82.
  5. World Health Organization. Synopsis of the world malaria situation,
     1979. Wkly Epidemiol Rec 1981;56:145-9.
  6. Cho SY, Kong Y, Park SM, Lee JS, Lim YA, Chae SL, et al. Two vivax
     malaria cases detected in Korea. Korean J Parasitol 1994;4:281-4.
  7. Craig S, Ockenhouse C, Evans E, Keep L, Hewitson W, Bell C. EPICON
     final report: malaria in U.S. troops, Republic of Korea. Aberdeen
     Proving Grounds (MD): U.S. Army Center for Health Promotion and
     Preventive Medicine; 1996.
  8. Miller LH, Mason SJ, Clyde DF, McGinness MH. The resistance factor to
     Plasmodium vivax in blacks. The Duffy-blood-group genotype, FyFy. N
     Engl J Med 1976;295:302-4.
  9. Shute PG, Lupasco G, Branzei P, Maryon M, Constantinescu P,
     Bruce-Chwatt LJ, et al. A strain of Plasmodium vivax characterized by
     prolonged incubation: the effect of numbers of sporozoites on the
     length of the prepatent period. Trans R Soc Trop Med Hyg
     1977;70:474-81.
 10. Gilles HM, Warrell DA. Bruce-Chwatt's essential malariology. 3rd ed.
     Boston: Edward Arnold; 1993. p. 127-31.


Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/feighner.htm

Please note that figures and equations are not available in ASCII format; 
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/= for greater than or equal to. 

-------------------------------------------------------------------------------------------
-------------------------------------------------------------------------------------------

[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Dispatches

Molecular and Epidemiologic Analysis of Dengue Virus Isolates from Somalia

Niranjan Kanesa-thasan,* Gwong-Jen J. Chang, Bonnie L. Smoak,* Alan
Magill,* M. Jeanne Burrous,* and Charles H. Hoke, Jr.*
*Walter Reed Army Institute of Research, Washington, DC, USA; and Centers
for Disease Control and Prevention, Ft. Collins, Colorado, USA

  --------------------------------------------------
      Nucleotide sequence analysis was performed on 14 dengue virus
      isolates (13 dengue-2 viruses and 1 dengue-3 virus) recovered
      from febrile soldiers in Somalia in 1993. The dengue-2 viruses
      were most closely related to dengue-2 virus recovered in
      Somalia in 1984. However, differences in nucleotide sequence
      (0.35% to 1.35%) were evident among the 1993 isolates. These
      differences were closely associated with the geographic
      location of the infection as well as with different times of
      infection at the same location. Genetic difference between
      strains was not associated with differences in clinical
      features. Molecular analysis of dengue viruses is a useful
      adjunct to epidemiologic investigation of their distribution
      over distance and time.

Dengue fever was a major cause of illness among U.S. troops in Somalia
during 1993 (1). We identified 14 virus isolates from soldiers with dengue
fever: 12 dengue-2, 1 dengue-3, and 1 mixed dengue-2 and -3 (2). Nucleotide
sequence followed by phylogenetic analysis was performed to further
characterize these isolates. The genotype of these virus isolates was
subsequently combined with information collected during epidemiologic
investigation of the febrile patients. The molecular epidemiologic analysis
showed that endemic strains of dengue-2 virus were segregated in specific
geographic locations (Figure 1).

[fig 1]
Figure 1. Map of Somalia showing towns from which dengue viruses were recovered.

Analytic Approach

Ninety patients with temperatures of at least 38.1C who were admitted to the
86th Evacuation Army Hospital in Somalia from 27 February to 3 April 1993
underwent a standardized clinical evaluation (history and physical
examination) and were asked to fill out an epidemiologic questionnaire at
admission. Clinical evaluations were repeated daily until discharge. Serum
samples were obtained from patients at admission and discharge and frozen
at -70C for later testing. For this report, the geographic site of
infection for each patient was defined as the site of principal activity 2
weeks before onset of illness and was assigned without knowledge of the
dendrogram.

Virus isolations were performed on 81 available admission sera by injecting
them into Toxorhynchites splendens mosquitoes and C6/36 cells (Aedes
albopictus mosquito cell line) (3). Dengue virus isolates were identified
by indirect immunofluorescence assay (IFA), by nested reverse
transcriptase-polymerase chain reaction (RT-PCR) for dengue virus, and by
virus neutralization (3). Virus seeds were obtained from culture fluids
collected 14 days after incubation on C6/36 cells (single amplification in
C6/36 cells) and stored at -70C for genotypic analysis.

Nucleotide sequencing of the envelope (E) gene from position 639 to 1,233
was obtained for all 14 Somalia isolates. This genomic region was amplified
directly by RT-PCR from extracted viral RNA and sequenced by the
dye-terminator method of ABI (Applied Biosystems, Foster City, CA) (5). The
nucleotide sequence was resolved by a 377 automated DNA sequencer (Applied
Biosystems). The sequences, combined with the E-gene database of other
viruses (5,6), were phylogenetically analyzed by genetic distance or
discrete character-state methods (7). Viruses were classified into a
specific serotype and genotype according to phylogenetic clustering with
other dengue viruses.

Findings

The patients were men ages 19 to 39 years (median age 22); 12 were
Caucasian and 2 (patients 11 and 12) were African American; all had signs
and symptoms consistent with dengue fever (Table 1). The sequence analysis
identified 13 dengue-2 viruses and one dengue-3 virus among the 14
isolates. These results supported previous identification by IFA and
RT-PCR, except that no mixed infection with dengue-2 and -3 was found in
the isolate from patient 12 after amplification in C6/36 cell culture.

Table 1. Patients from whom dengue-2 virus isolates were recovered

-------------------------------------------------------------------------------------------
                                            Dates of    Max.
Patient                                     illness     temp  Head-  Body-  Eye
No(sup a)    Location         Unit          (1993)      (F)  ache   ache   pain     Rash
------------------------------------------------------------------------------------------

23      Mogadishu/Airport  711 Postal      5-9 Mar      102.0  No    Yes    Yes      Yes

        Kismayu/ICRC
20      Hospital           2/87th          4-10 Mar     102.0  Yes    No    No        No

        Kismayu/ICRC
21      Hospital           2/87th          4-8 Mar      102.6  Yes    No    Yes       Yes

02      Kismayu/Airport    710 MSB         27 Feb-5 Mar 103.0  Yes    Yes   Yes       Yes

11      Kismayu/Airport    3/14th          27 Feb-2 Mar 102.0  Yes    Yes   NA(sup b) Yes
                           
12      Kismayu/Airport    129th Postal    28 Feb-4 Mar 104.2  Yes    Yes   Yes       No

13      Kismayu/Airport    3/14th 27       Feb-3 Mar    101.8  Yes    Yes   NA        Yes

56      Mogadishu/Airport  LSB             1-4 Apr      103.0  Yes    Yes   No        No
                           
57      Mogadishu/Airport  70th Transport  2-5 Apr      102.4  Yes    Yes   Yes       No

58      Mogadishu/Stadium  1/7th           3-6 Apr      102.8  Yes    Yes   Yes       No

30      Afgoi              984 MP          11-15 Mar    103.4  No     Yes   Yes       No

39      Mog/Checkpoint 57  1/7th           21-25 Mar    103.8  Yes    Yes   Yes       No

46      Mog/Checkpoint 57  1/7th           26-28 Mar    103.2  Yes    Yes   Yes       Yes

------------------------------------------------------------------------------------------
(sup a)Patients are grouped according to the degree of homology of isolate sequences.
Each group represents a single branch in the phylogenetic dendrogram.
(sup b)NA = not available

The single dengue-3 isolate was recovered from a soldier stationed at the
port in Mogadishu. The sequence was dengue-3 genotype III, which closely
resembles Mozambique 85 strain (6) and strains from Sri Lanka, India, and
Samoa.

The 13 dengue-2 isolates were all genotype IV and most closely related
genetically to the 1984 isolate from Somalia (Figure 2). Phylogenetic
clustering of genotype IV viruses was supported by 98% bootstrap confidence
level, which also included viruses from Burkina Faso, Indonesia, Sri
Lanka, and the Seychelles (5). An apparent genetic drift of 1.18% to
1.85% nucleotide sequence difference within dengue-2 viruses was observed
between 1984 and 1993 (data not shown). A similar level of genetic
difference (0.34% to 1.35%) was also observed among the 13 dengue-2 virus
isolates collected in 1993.

[fig 2]
Figure 2. Phylogenetic tree of the dengue-2 viruses, including Somalia 1993 virus
isolates, using nucleotide sequence from position 639 to 1,233 of the envelope protein
gene. This strict consensus tree was constructed by the following programs of the PHYLIP
3.5 package: SEQBOOT, DNAPARS, CONSENSUS, and DRAWGRAM. Strain ID is listed first,
followed by abbreviation for country of isolation, and the last two digits of the year of
isolation. 


Nucleotide sequence alignment from E gene position 638 to 1,233 showed 24 nucleotide 
changes with four amino acid alterations at the E-291, 300, 352, and 409 between 
1984 and 1993 isolates (Table 2). Transition mutations were the predominant changes with
a transition to transversion ratio of six to one. All 1993 isolates shared
six unique nucleotide changes at position 792, 871, 885, 1,074, 1,137, and
1,161. However, only position 871, C to A change, altered amino acid
sequence of Gln in the Somalia 1984 isolate to Lys in the 1993 dengue-2
virus isolates.

Viruses with certain nucleotide sequence differences appeared to be
segregated by location. Virus isolates from Kismayu had a unique U change
at position 1,167 that the Somalia 1984 dengue-2 virus and the Mogadishu
and Afgoi isolates did not have (Table 2). Identical strains were isolated
from soldiers infected in the same location at the same time.
Distinguishable strains were collected from patients apparently infected at
different locations within the same city, e.g., during a 1-week period at
the ICRC hospital and the airport in Kismayu (Tables 1, 2). Yet, greater
differences were found between dengue-2 viruses recovered from cases at
different times from the same location within the city of Mogadishu.
Sequences from viruses recovered in March (patient 23) or in April
(patients 56, 57) were as dissimilar as sequences from viruses collected at
the same time from different locations.

Conclusions

The data show distinct clustering of dengue-2 viruses within specific
locales in Somalia. Identical dengue-2 viruses were acquired at the same
time in the same locale, as shown by the four isolates from persons
presumably infected at the airport in Kismayu. Moreover, virus isolates
recovered from persons infected in three widely dispersed towns (Mogadishu,
Kismayu, and Afgoi) were clearly distinguishable by nucleotide sequence
analysis. The number of virus isolates studied in this analysis is small,
and there is some imprecision in assigning the geographic location of
infection. However, these data suggest a selective preference for a single
circulating dengue-2 virus in a particular geographic location.

The distances between towns exceeded the flight range of mosquito vectors,
unless they were transported by vehicles or by air. Movement of vectors is
refuted by the consistency of isolates recovered at different locations.
Additionally, no large-scale movements of troops occurred during this
study. The local population was stable, and no large refugee movements
occurred. Hence, the isolates reflect viruses circulating among the
indigenous populations, a consequence of moving populations unexposed to
dengue into endemic-disease locations.

The analysis also shows that dengue-2 viruses with unique genetic sequences
may circulate at different times within the same location. The six isolates
recovered from soldiers infected in Mogadishu show consistent differences
over time during the short course of this analysis (2 months). These
findings suggest spatial and temporal clustering of dengue viruses causing
infections within Somalia. These differences are not readily explained by
immigration of infective vector mosquitoes. The distances between locations
within towns (e.g., 8 km) exceeded the flight range of Aedes aegypti.
However, surveillance for vector mosquitoes was not routinely performed
during the time of this analysis, and mosquitoes may have been carried by
transport vehicles within the town.

Table 2. Nucleotide sequences at the E-639 to 1233 region of Somalia dengue-2
isolates

--------------------------------------------------------------------------------------

E gene       SOM                                        Patient(sup c)
position     1984(sup b)
nt/aa(sup a) nt/aa                   11  13  02  12 21  20 23  30 39 46  56  57   58

--------------------------------------------------------------------------------------

654/218      uuA/Leu                                              G  G

661/221      Cug/Leu                                                     U   U

720/240      uuC/Phe                                       U

723/241      aaA.Lys                                              G   G

729/243      ccU/Pro                                           C

783/261      caU/His                                C   C

792/264      cuC/Leu                  U  U   U   U  U   U  U   U  U   U  U   U    U

801/267      gcC/Ala                                              U   U

870/290      gaC/Asp                                           U
             
871/291      Caa/Gln --->arrow>Lys    A  A   A   A  A   A  A   A  A   A  A   A    A
             
885/295      aaA/Lys                  G  G   G   G  G   G  G   G  G   G  G   G    G

900/300      UCu/Ser                                           A         A   A    A

972/324      guA/Val                                G   G

1,050/350    cgC/Arg                                           U

1,056/352    auU/Ile                                C   C      A         A   A    A

1,074/358    guU/Val                  C  C   C   C  C   C  C   C  C   C  C   C    C
             
1,079/360    gAa/Glu --->arrow>Gly                                G   G 
             
1,082/361    aAa/Lys --->arrow>Arg                                                G
             
1,104/368    gaA/Glu                                G   G

1,137/379    auC/Ili                  U  U   U   U  U   U  U   U  U   U  U   U    U

1,161/387    cuG/Leu                  A  A   A   A  A   A  A   A  A   A  A   A    A

1,167/389    cuC/Leu                  U  U   U   U  U   U  U

1,209/403    gaG/Glu                                       A
             
1,225/409    Gcg/Ala --->arrow>Pro                         C
             
--------------------------------------------------------------------------------------
(sup a)nt/aa = nucleotide/amino acid.
(sup b)Somalia 1984 dengue-2 virus.
(sup c)Capital letter (A, U, G, C) = different nucleotide sequence; empty cell indicates
identical nucleotide sequence.

No apparent differences in clinical features of the illness caused by these
different dengue-2 strains were observed. This may reflect the small sample
size, intrinsic variability in host response, and restricted time of
analysis. Reports from Australia suggest that clinical features may be
modified over the course of a dengue epidemic, perhaps reflecting a change
in virulence of circulating virus (8). Only sporadic cases of dengue fever
were detected, reflecting endemic transmission during this investigation;
hence we cannot directly comment on transmission during epidemics.

Isolation of dengue viruses in C6/36 insect cells from viremic serum was
required to increase virus titer for genetic analysis. Amplification of the
virus isolates on insect cells may have induced or selected specific
genetic changes. A comparison of the RNA T1 fingerprints of the New Guinea
C isolate and C6/36 cell passage showed that under the conditions of
laboratory passage in experimental hosts, during a single infection in
humans, dengue-2 was subject to very little selective pressure (10). In
this study, the identical isolation procedure was followed, and four
identical gene sequences representing four sets of independent isolates
were obtained for all isolates (Table 2); these results indicate that, if
present, the same selection process was exerted on all isolation attempts.

Hence, nucleotide sequences obtained by direct sequencing of RT-PCR
amplified product from serum samples of viremic patients were certainly
representative of original virus genotype. Dengue-2 was the dominant virus
in the mixed dengue-2, and -3 infected serum from patient 12. Failure to
recover dengue-3 virus from this patient may be due to a direct growth
competition of viruses in C6/36 cells. Titer of dengue-3 virus after C6/36
cell passage was insufficient to be amplified by RT-PCR directly.

The stability or relatedness of strains over time can only be surmised from
these limited data. The dengue-2 viruses recovered in 1993 from this
investigation are genetically distinct from the Somalia 1984 dengue-2
isolate. The extent of homology preserved for 10 years suggests that only 6
(1%) of 596 positions (Table 2) of nucleotides changed because of genetic
drift. Remarkable genetic stability has been proposed for dengue viruses
(10). The ability of genotyping methods to reliably resolve small (<1%)
differences in sequence and the expanding access to these methods are
powerful epidemiologic and clinical tools. These methods may further refine
the ability to track the circulation or spread of dengue viruses within a
city or country (9), as has been done with other viruses (11). These
techniques may also provide insight into the evolution of dengue viruses at
specific locations and perhaps detect the frequency of dual infections or
the presence of recombination within dengue viruses (12).

Dr. Kanesa-thasan focuses on tropical medicine, specifically tropical
virology and immunology, at the Department of Virus Diseases, Walter Reed
Army Institute of Research in Washington, D.C.

Address for correspondence: Niranjan Kanesa-thasan, Department of Virus
Diseases, DCD&I, Walter Reed Army Institute of Research, 14th and Dahlia
Streets, N.W., Washington, DC 20307-5100, USA; fax: 202-782-0442; e-mail:
maj.niranjan.kanesa@wrsmtp-ccmail-army.mil.

References

  1. Sharp TW, Wallace MR, Hayes CG, Sanchez JL, DeFraites RF, Arthur RR,
     et al. Dengue fever in U.S. troops during Operation Restore Hope,
     19921993. Am J Trop Med Hyg 1995;53:89-94.
  2. Kanesa-thasan N, Iacono-Connors L, Magill A, Smoak B, Vaughn D, Dubois
     D, et al. Dengue serotypes 2 and 3 in U.S. forces in Somalia. Lancet
     1994;343:678.
  3. Gubler DJ, Kuno G, Sather GE, Velez M, Oliver A. Mosquito cell
     cultures and specific monoclonal antibodies in surveillance for dengue
     viruses. Am J Trop Med Hyg 1984;33:158-65.
  4. Tanaka M. Rapid identification of flavivirus using the polymerase
     chain reaction. J Virol Methods 1993;41:311-22.
  5. Lewis JA, Chang G-J, Lanciotti RS, Kinney RM, Mayer LW, Trent DW.
     Phylogenetic relationships of dengue-2 viruses. Virology
     1993;197:216-24.
  6. Lanciotti RS, Lewis JG, Gubler DJ, Trent TW. Molecular evolution and
     epidemiology of dengue-3 viruses. J Gen Virol 1994;75:65-75.
  7. Felsenstein J. PHYLIP (Phylogeny Inference Package) [computer program]
     Version 3.5c. Seattle (WA): Department of Genetics, University of
     Washington; 1993.
  8. Harley D, Murray-Smith S, Weinstein P. Comparative severity of dengue
     symptoms in outbreaks in tropical Queensland. Communicable Disease
     Intelligence 1995;19:442-6.
  9. Trent DW, Grant JA, Rosen L, Monath TP. Genetic variation among dengue
     2 viruses of different geographic origin. Virology 1983;18:271-84.
 10. Monath TP. Dengue: the risk to developed and developing countries.
     Proc Natl Acad Sci U S A 1994;91:2395-400.
 11. McDade JE, Anderson BE. Molecular epidemiology: applications of
     nucleic acid amplification and sequence analysis. Epidemiol Rev
     1996;18:90-7.
 12. Burke DS. Recombination in HIV: an important viral evolutionary
     strategy. Emerg Infect Dis 1997;3:153-9.


Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/kanesa.htm

Please note that figures and equations are not available in ASCII format; 
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[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Dispatches

Molecular Characterization of a Novel Rickettsia Species from Ixodes
scapularis in Texas

Adrian N. Billings,* Glenna J. Teltow, Scott C. Weaver,* and David H.
Walker*
*University of Texas Medical Branch, Galveston, Texas, USA; Bureau of
Laboratories, Texas Department of Health, Austin, Texas, USA

---------------------------------------------------------------------------
      A novel Rickettsia species of undetermined pathogenicity was
      detected in Ixodes scapularis. DNA sequencing showed the
      highest nucleotide sequence similarities with R. australis for
      the 17 kDa gene, R. helvetica for gltA, and R. montana for
      rompA. The new organism, provisionally designated as genotype
      Cooleyi, is highly divergent in three conserved genes from
      recognized Rickettsia species.

Rickettsia spp., obligately intracellular, gram-negative,
vector-transmitted bacteria, are divided antigenically into the typhus
group (TG) and the spotted fever group (SFG). In nature, TG rickettsiae
inhabit either flea (Rickettsia typhi and R. felis), louse (R. prowazekii),
or tick (R. canada) arthropod hosts. Except for R. akari, whose
invertebrate host is a mite, all SFG rickettsiae reside in tick hosts. SFG
rickettsiae, excluding R. akari, are maintained in nature by a cycle
involving both transovarial and transstadial passage of the microorganism
in the tick and, for some species, amplification in small mammal hosts.
Ticks infected with pathogenic rickettsiae (i.e., R. rickettsii, R.
conorii, R. sibirica, R. australis, R. japonica, R. africae, and R. honei)
that feed on humans, who are dead-end hosts, may transmit an SFG
rickettsiosis (i.e., Rocky Mountain spotted fever [RMSF], boutonneuse
fever, North Asian tick typhus, Queensland tick typhus, Japanese spotted
fever, African tick bite fever, or Flinders Island spotted fever,
respectively).

In the United States, Dermacentor andersoni and D. variabilis are the
classic vectors of R. rickettsii to humans; other recognized vectors
include Rhipicephalus sanguineus for R. rickettsii in Mexico and for R.
conorii in Eurasia and Africa, Amblyomma cajennense for R. rickettsii in
Brazil, and Ixodes holocyclus for R. australis in Australia. Other
rickettsiae, such as R. parkeri and R. amblyommii, have been isolated from
human-biting ticks; however, these rickettsiae have not been proven
pathogenic for humans. The presence of apparently nonpathogenic rickettsiae
whose vector host and geographic distribution overlap with pathogenic
rickettsiae may interfere with the transmission of virulent rickettsiae to
humans, as may be the case with R. peacockii (nonpathogenic) (1,2) and R.
rickettsii in D. andersoni in the Bitterroot Valley of Montana.

The black-legged tick, I. scapularis, is of major public health importance
because it feeds indiscriminately on numerous vertebrate hosts, including
humans, and transmits Borrelia burgdorferi, Babesia microti, and human
granulocytotropic ehrlichia to humans. Rickettsiae have been detected in I.
scapularis (3-8); however, the ability of black-legged ticks to serve as
vectors of rickettsiae to humans or to other small mammals important in
maintaining and spreading rickettsiae in nature has not been examined.
Because rickettsiae are regularly detected by immunofluorescence staining
in I. scapularis specimens sent to the Texas Department of Health (5,7) and
because the range of I. scapularis and RMSF cases overlaps in Texas (9), we
characterized the immunofluorescence-detected microbial agent in this tick
collected from Anderson County in eastern Texas.

We examined the field-collected I. scapularis, preserved in alcohol, to
determine species, sex, and stage of development. Midgut tissues were
removed and smeared on two glass slides. One tissue smear was screened for
rickettsiae by the Gimenez staining technique. The smear was further
analyzed by using a fluorescein isothiocyanate-labeled rabbit antibody
against R. rickettsii that cross-reacts with many other SFG rickettsiae
(10).

DNA from the tick was extracted with phenol:chloroform (1:1) by the
standard method (11) after proteinase K and sodium dodecyl sulfate
digestion of homogenized tick tissue. The DNA was resuspended in 50 l of
sterile water.

A volume of 10 l of the polymerase chain reaction (PCR) product was
separated by electrophoresis on 6% polyacrylamide gels. After
electrophoresis, gels were stained with ethidium bromide, and amplicons
were visualized with UV transillumination. Each PCR product was sequenced
after DNA purification by a QIAquick Spin PCR Purification Kit (Qiagen,
Santa Clarita, CA). Perkin Elmer Ready Reactions (Foster City, CA) and
AmpliTaq FS with a Perkin Elmer ABI Prism 377 Automated DNA Sequencer
(Foster City, CA) were used for sequencing.

Nucleotide and deduced amino acid sequences were aligned by using the
PILEUP program (12). Phylogenetic analyses were conducted by using the PAUP
parsimony algorithm (13), and the NEIGHBOR and FITCH distance matrix
programs were implemented in the PHYLIP package (14). Parsimony analysis
was implemented by using the heuristic algorithm and both ordered
(transition:transversion = 3:1) and unordered characters. Because
nucleotide sequences generally differed by less than 20%, distance methods
used the Jukes-Cantor formula (15). Sequences were added at random with 10
replications. Bootstrap resampling with 200 replicates was used to place
confidence values on groupings within trees.

Rickettsiae were visualized in a Gimenez-stained tick smear. A duplicate
slide contained rickettsiae stained by an SFG-specific fluorescein
isothiocyanate-labeled antibody (Figure 1).

PCR amplification was performed on the fluorescent antibody-positive tick,
and the DNA sequences of the Rickettsia-specific 17 kDa gene, gltA, and
rompA were determined. Comparison of the highly conserved 17 kDa gene
sequences showed the highest level of nucleotide sequence similarity (94.0%
identical) between the I. scapularis rickettsia and R. australis (Figure
2A). Comparison of the I. scapularis rickettsia gltA partial nucleotide
sequence showed the highest nucleotide sequence similarity (95.6%
identical) with R. helvetica (Figure 2B). Comparison of the amplified 532
bp-product of rompA over a length of 441 nucleotides with all GenBank's
rompA sequences showed the highest nucleotide sequence similarity (87.8%
identical) with the established species R. montana (Figure 2C). A portion
of rompB was not amplified for the rickettsia in this tick. The expected
PCR product of 650 bp was obtained from the positive control, R. rickettsii
DNA.

                             [fig.1]
     Figure 1. Photomicrograph of crushed midgut tissues of the
     fluorescent antibody-positive Ixodes scapularis specimen. Slides
     were incubated with fluorescein isothiocyanate-conjugated rabbit
     antibodies to Rickettsia rickettsii and examined by fluorescent
     microscopy. Magnification 400X.

All three genes (17 kDa, gltA, and rompA) analyzed from this rickettsia had
distinct sequences. Indeed, comparison of 17 kDa, gltA, and rompA partial
nucleotide sequences showed at least 7.5%, 4.4%, and 12% differences,
respectively, with the closest rickettsial species for each of the three
genes analyzed. Both parsimony and distance phylogenetic methods indicated
that the most closely related rickettsia was different at each of the three
genes. However, bootstrap values for grouping the I. scapularis rickettsia
with other rickettsiae were low, with the exception of its pairing with R.
montana for the rompA gene (bootstrap value 99%, Figure 2C), perhaps
because only relatively short partial sequences of the three genes were
used for analysis. Otherwise, the three phylogenetic methods generally
yielded trees with similar branching patterns; minor differences were
observed within the SFG and in the position of the R. akari, R. australis,
and R. felis groups for the gltA gene (Figure 2B). Figures 2A and B
generally support the monophyletic nature of SFG. The few exceptions were
the grouping of R. australis and R. akari with R. felis at the gltA gene
(Figure 2B). This grouping of R. australis and R. akari with TG rickettsiae
was supported by a bootstrap value of only 47%. In contrast, TG sequences
never made up a monophyletic group when either gltA or 17 kDa sequences
were analyzed by any of the phylogenetic methods. However, groupings
placing TG sequences with sequences for other organisms such as R. bellii,
AB Bacterium, or I. scapularis rickettsia were only weakly supported by
bootstrap analyses.

                         [fig.2]
    Figure 2. Unrooted phylogenetic trees showing relationship of
    Cooleyi genotype to other rickettsiae. Scale bar represents
    genetic distance of 5% by using the Jukes-Cantor formula.
    Bootstrap values were derived by using the Fitch algorithm.
    Circled bootstrap values are for nodes indicated by the dashed
    line. Dotted lines show the actual position of some closely
    related spotted fever group species. A. 17 kDa. B. gltA. C. rompA.

    A 10-l aliquot of phenol:chloroform-extracted DNA was used as a
    template to amplify portions of four rickettsial genes: 17 kDa
    protein gene, gltA (citrate synthase), rickettsial outer membrane
    protein A (rompA), and rickettsial outer membrane protein B
    (rompB). In all amplification reactions, R. rickettsii DNA was
    used as a positive control. A 434 bp-portion of the Rickettsia
    genus-specific 17 kDa protein was amplified (16). A 381 bp-portion
    of the rickettsial gltA gene was amplified from extracted DNA by
    using primer pairs Rp CS.877p and Rp CS.1258n (17). A 532
    bp-portion of the rickettsial rompA gene was amplified from
    extracted tick DNA by using primer pairs Rr190.70n and Rr190.602p
    (17). Primer pair BG1-21 and BG2-20 (BG-12) was used in a
    polymerase chain reaction (PCR) for the amplification of a 650
    bp-portion of the rompB gene (18).

    GenBank accession numbers for the portions of the I. scapularis
    rickettsia 17 kDa, rompA, and gltA gene sequences are AFO31534,
    AFO31535, and AFO31536, respectively. GenBank accession numbers
    for the available 17 kDa gene sequences included in phylogenetic
    analyses are R. canada, M82879; R. felis, M82878; R. prowazekii,
    M28482; R. typhi, M28481; AB Bacterium, U04162; R. amblyommii,
    U11013; R. japonica, D16515; R. honei, M99391; R. rickettsii,
    M16486; R. conorii, M28480; R. montana, U11017; R. rhipicephali,
    U11020; R. parkeri, U17008; and R. australis, M74042. GenBank
    accession numbers for the available gltA sequences included in
    phylogenetic analyses are AB Bacterium, U20242; R. bellii, U59716;
    R. typhi, U20245; R. prowazekii, U20244; R. montana, U74756; R.
    slovaca, U59725; R. conorii, U20243; R. sibirica, U59734; R.
    africae, U59733; Thai tick typhus rickettsia, U59726; R.
    rickettsii, U59729; R. honei, AFO22817; R. parkeri, U59732; R.
    japonica, U59724; R. massiliae, U59719; R. rhipicephali, U59721;
    R. aeschlimanni, U74757; R. felis, U33922; R. australis, U59718;
    R. akari, U59717; R. canada, U20241; and R. helvetica, U59723.
    GenBank accession numbers for the rompA sequences included in
    phylogenetic analyses are R. massiliae, U43793; R. rhipicephali,
    U43803; R. aeschlimanni, U43800; R. conorii, U43794; Israeli tick
    typhus rickettsia, U43797; R. peacockii, U55821; R. slovaca,
    U43808; R. parkeri, U43802; R. africae, U43790; R. sibirica,
    U43807; Thai tick typhus rickettsia, U43809; R. rickettsii,
    U55822; R. japonica, U43795; and R. montana, U43801.

When R. helvetica sequence information becomes available and is analyzed,
it will be interesting to view the placement of the I. scapularis
rickettsia and R. helvetica within the 17 kDa and rompA phylogeny. The
closest rickettsial species at the 17 kDa, R. australis, and at the gltA
gene, R. helvetica, are both Ixodes-associated rickettsiae. That a segment
of the rompB was not amplified when the BG-12 primer pair was used is not
surprising; the rompB segments of R. akari, R. bellii, R. helvetica, R.
massiliae, and the I. scapularis rickettsia identified recently in
Minnesota were also not amplified when this primer pair was used (8,18).
Indeed, the highest sequence similarity of the gltA gene was with R.
helvetica, which occurs in Swiss I. ricinus ticks (19).

Because I. scapularis readily feeds upon humans in the northeastern United
States and occasionally in Texas, this rickettsia, genetically distinct
from R. rickettsii, may cause an unrecognized rickettsiosis manifesting as
a clinical disease different from the severe illness caused by R.
rickettsii. In fact, I. scapularis has been associated epidemiologically
with a fatal illness in Texas considered to be RMSF (5). Elliott and
colleagues reported a fatal case of SFG rickettsiosis in a patient who
jogged with a friend at a state park. Ticks collected at that site were
only I. scapularis, some infected with an SFG rickettsia. An I. scapularis
tick removed from the scalp of the patient's friend during an interview was
later found to contain a rickettsia. In addition, rickettsiae of lower
virulence may exist in Texas. A serologic study by Taylor and others
suggested that 9% to 21% of sixth-grade students in Burleson and Mansfield
Counties of Texas had prior immune stimulation by a rickettsial agent (7).
None of the 32 children with serologic evidence of prior rickettsial
infection had ever been treated for or diagnosed with RMSF. This study
implies that these children were infected with a rickettsia of low
virulence, or contrarily, that these children experienced asymptomatic
immune stimulation with rickettsial antigens.

On the other hand, if not pathogenic for humans, the I. scapularis
rickettsia could interfere with the transmission of R. rickettsii by other
ticks to animals by inducing cross-protective immunity or by an
interference mechanism such as that of R. peacockii (1,2). Noda et al. have
proposed that a rickettsia found in laboratory-reared I. scapularis from
Minnesota is an endosymbiont of the tick because the microorganism appears
to be localized only to the ovaries (8). The potential pathogenicity of a
rickettsia confined to the ovaries raises the question of whether or not it
could escape from this intracellular niche to become infectious. Also,
naturally occurring dual infections of rickettsiae in a tick have never
been established. If this rickettsia is restricted to the ovaries, this
tick may be precluded from serving as a vector of rickettsiosis under
natural conditions. However, because the black-legged tick is a three-host
tick, it may potentially serve as a vector of pathogenic rickettsiae to a
wide range of susceptible hosts, including mammals, birds, and reptiles,
from which other tick vectors could become infected after a rickettsemic
blood meal. SFG rickettsiae have been detected in 10.2% to 16.7% of I.
scapularis ticks in Texas (5,7), 5.6% in Connecticut (4), and 11.1% to
66.7% in South Carolina (3). While the numbers of ticks in these
collections have been low, the prevalence of SFG rickettsiae in these ticks
is among the highest recorded. Because rickettsiae have been regularly
detected in unengorged black-legged ticks from different geographic
locations (Texas, South Carolina, and Connecticut), we believe that I.
scapularis is infected with SFG rickettsiae and that infection in these
ticks likely represents more than ingestion in blood meals from
rickettsemic animals.

Because for each of the three genes analyzed phylogenetically a robust
relationship with other known Rickettsia species does not appear to exist,
we believe that this rickettsia is genetically unique, and therefore, we
provisionally designate this agent genotype Cooleyi after Dr. Robert A.
Cooley, who served the Montana State Board of Entomology from 1899 to 1944.
Isolation of this new rickettsial agent, characterization of its
pathogenicity as the potential etiologic agent of an emerging infectious
disease, and investigation of its role in its ecologic niche are subjects
of interest for future study.

Address for correspondence: David H. Walker, University of Texas Medical
Branch, 301 University Blvd., 1.116 Keiller Bldg., Galveston, Texas
77555-0609, USA; fax: 409-772-2500; e-mail: dwalker@utmb.edu.

Acknowledgments

The authors thank Josie Ramirez and Kay Kantowski for their expert
secretarial assistance. This research was supported by grants from the
National Institutes of Health (AI 21242 and AI 31431-05A1).

Adrian Billings is a Ph.D. candidate in the experimental pathology graduate
program, University of Texas Medical Branch. His research interests include
the epizootiology and pathogenesis of tick-borne zoonoses.

References

  1. Neibylski ML, Schrumpf ME, Burgdorfer W, Fischer ER, Gage KL, Schwan
     TG. Rickettsia peacockii sp. nov., a new species infecting wood ticks,
     Dermacentor andersoni, in Western Montana. Int J Syst Bacteriol
     1997;47:446-52.
  2. Burgdorfer W, Hayes SF, Marvos AJ. Nonpathogenic rickettsiae in
     Dermacentor andersoni: a limiting factor for the distribution of
     Rickettsia rickettsii. In: Burgdorfer W, Anacker RL, editors.
     Rickettsiae and rickettsial diseases. New York: Academic Press, Inc.;
     1981; p. 585.
  3. Loving SM, Smith AB, DiSalvo AF, Burgdorfer WB. Distribution and
     prevalence of spotted fever group rickettsiae in ticks from South
     Carolina, with an epidemiological survey of persons bitten by infected
     ticks. Am J Trop Med Hyg 1978;27:1255-60.
  4. Magnarelli LA, Anderson JF, Philip RN, Burgdorfer W. Antibodies to
     spotted fever-group rickettsiae in dogs and prevalence of infected
     ticks in southern Connecticut. Am J Vet Res 1982;43:656-9.
  5. Elliott LB, Fournier PV, Teltow GJ. Rickettsia in Texas. Ann N Y Acad
     Sci 1990;590:221-6.
  6. Magnarelli LA, Andreadis TG, Stafford KC, Holland CJ. Rickettsiae and
     Borrelia burgdorferi in ixodid ticks. J Clin Microbiol
     1991;29:2798-804.
  7. Taylor JB, Tanner WB, Rawlings JA, Buck J, Elliott LB, Dewlett HJ, et
     al. Serological evidence of subclinical Rocky Mountain spotted fever
     in Texas. J Infect Dis 1985;151:367-8.
  8. Noda H, Munderloh UG, Kurtii TJ. Endosymbionts of ticks and their
     relationship to Wolbachia spp. and tick-borne pathogens of human and
     animals. Appl Environ Microbiol 1997;63:3926-32.
  9. Billings AN, Walker DH. Tick-borne diseases in Texas. Texas Medicine.
     In press 1998.
 10. Hebert AG, Tzianabos T, Gamble WC, Chappell WA. Development and
     characterization of high-titered, group-specific fluorescent-antibody
     reagents for direct identification of rickettsiae in clinical
     specimens. J Clin Microbiol 1980;11:503-7.
 11. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning. A Laboratory
     Manual. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press;
     1989.
 12. Devereux J, Haeberli P, Smithies O. A comprehensive set of sequence
     analysis programs for the VAX. Nucleic Acids Res 1984;12:387-395.
 13. Swofford DL. PAUP: phylogenetic analysis using parsimony [computer
     program]. Version 3.0. Champaign (IL): Illinois Natural History
     Survey; 1991.
 14. Felsenstein J. PHYLIP (Phylogeny Inference Package [computer
     program]). Version 3.5p. Seattle: Department of Genetics, University
     of Washington; 1993.
 15. Jukes TH, Cantor CR. Evolution of protein molecules. In: Munro HN,
     editor. Mammalian protein metabolism. New York: Academic Press; 1969;
     p. 21-132.
 16. Webb L, Carl M, Malloy DC, Dasch GA, Azad AF. Detection of murine
     typhus infection in fleas by using the polymerase chain reaction. J
     Clin Microbiol 1990;28:530-4.
 17. Regnery RL, Spruill CL, Plikaytis BD. Genotypic identification of
     rickettsiae and estimation of intraspecies sequence divergence for
     portions of two rickettsial genes. J Bacteriol 1991;173:1576-89.
 18. Eremeeva M, Yu X, Raoult D. Differentiation among spotted fever group
     rickettsiae species by analysis of restriction fragment length
     polymorphism of PCR-amplified DNA. J Clin Microbiol 1994;32:803-10.
 19. Beati L, Peter O, Burgdorfer W, Aeschlimann A, Raoult D. Confirmation
     that Rickettsia helvetica sp. nov. is a distinct species of the
     spotted fever group of rickettsiae. Int J Syst Bacteriol
     1993;43:521-6.


Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/billings.htm
Please note that figures and equations are not available in ASCII format; 
their placement within the text is noted by [fig] and [eq], respectively. 
Greek symbols are spelled out. The following codes are used: 
(ft) for footnote; (sup) for superscript; (sub) for subscript; 
/= for greater than or equal to.

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[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Dispatches

Dual Infection with Ehrlichia chaffeensis and a Spotted Fever Group
Rickettsia:  A Case Report

Daniel J. Sexton,* G. Ralph Corey,* Christopher Carpenter,* Li Quo Kong,*
Tejel Gandhi,* Edward Breitschwerdt, Barbara Hegarty, Sheng-Ming Chen,
Hui-Min Feng, Xue-Jie Yu, Juan Olano, David H. Walker, and Stephen J.
Dumler
*Duke University Medical Center, Durham, North Carolina, USA; North
Carolina State University, Raleigh, North Carolina, USA; University of
Texas Medical Branch at Galveston, Galveston, Texas, USA; and Johns
Hopkins Medical Center, Baltimore, Maryland, USA
---------------------------------------------------------------------------
      Well-documented cases of simultaneous human infection with more
      than one tick-borne pathogen are rare. To our knowledge only
      two dual infections have been reported: simultaneous human
      infection with the agent of human granulocytic ehrlichiosis and
      Borrelia burgdorferi and simultaneous human infection with B.
      burgdorferi and Babesia microti (1-2). Rocky Mountain spotted
      fever has long been known to be endemic in North Carolina;
      cases of human ehrlichial infection were recognized there soon
      after Ehrlichia chaffeensis was recognized as an important
      cause of tick-borne disease in the southeastern United States.
      Because both Rocky Mountain spotted fever and ehrlichiosis are
      prevalent in North Carolina, occasional cases of simultaneous
      human infection by rickettsial and ehrlichial agents would not
      be surprising; however, no such cases seem to have been
      reported.

Case Report

A 44-year-old man with a history of hepatitis C infection and regular use
of cocaine was asymptomatic until June 6, 1995, when he noted the acute
onset of myalgia, headache, arthralgia, fever (40C), nonproductive cough,
nausea, and vomiting. The following day he sought medical care and received
oral trimethoprim/sulfamethoxazole for a presumed respiratory tract
infection. Over the next 3 days, his symptoms worsened; on the fourth day
he noticed a maculopapular rash and came to our medical center. Initial
examination showed tachycardia (pulse 125 beats per minute), fever
(38.6C), and a macular red rash localized to his right arm. The patient
reported a tick bite 1 day before his symptoms began. In addition, he
reported removing an engorged tick from his pet dog and crushing it between
his fingers 4 days before he became ill. Laboratory studies showed
leukopenia (white blood cell count 2,700/mm(sup 3)), thrombocytopenia (platelet
count 69,000/mm(sup 3)), hyponatremia (serum sodium 130 meq/dL), and mildly
elevated serum concentrations of hepatic enzymes (serum aspartate
aminotransferase level of 160 IU/L [normal range 10 to 60 IU]). A screening
test for HIV infection was negative. Therapy with intravenous doxycycline
was begun (100 mg twice daily). A skin biopsy showed a perivascular
lymphocytic infiltrate in the superficial and deep dermis. Direct
immunofluorescent antibody staining of the fresh frozen skin biopsy with
antiserum against Rickettsia rickettsii demonstrated organisms typical of
spotted fever group rickettsiae within endothelial cells. Sections were
also stained with antiserum against human albumen as a negative control.
Parallel staining of a section of positive control tissue was also
performed (3). Three days after admission, the patient was afebrile and
asymptomatic. He was discharged and instructed to continue therapy with
oral doxycycline for an additional 7 days. At follow-up 12 weeks later, he
felt well. His white blood count was 7,600/mm(sup 3, and his platelet count was
166,000/mm(sup 3.

Serologic Tests

Serum samples obtained 5, 24, and 55 days after onset of illness were
tested for antibodies to Ehrlichia chaffeensis and R. rickettsii antigens
by immunofluorescence assay (IFA) except that E. chaffeensis (Arkansas
strain) was used instead of E. canis (4,5).

A fourfold IFA antibody rise (1:32 to 1:128) occurred against E.
chaffeensis antigen, but antibodies against R. rickettsii antigen were not
detected in either the acute- or first convalescent-phase serum sample.
However, a serum sample collected 55 days after onset contained an antibody
titer of 1:40 to R. rickettsii antigen. Significant in vitro T-cell
proliferation was seen in response to the antigens of both E. chaffeensis
and R. rickettsii (Table 1).

Cell Culture Isolation and In Vitro Lymphocyte Stimulation

A sample of blood taken before the patient was treated with doxycycline was
centrifuged in two steps to remove red blood cells and to pellet buffy coat
cells. A portion of cellular material was then resuspended in plasma and
injected into Vero cells and 030 cells (North Carolina State University
canine monocytoid cell line). Vero cell cultures were then maintained with
medium 199 and 030 cells with RPMI 1640 containing 10% fetal bovine serum,
2 mM L-glutamine, and 0.075% sodium bicarbonate. Cultures were examined
weekly for rickettsia-like organisms (5) or morulaelike structures (6) by
light microscopy. Cell suspensions were stained by Gimenez, Wright, and
direct immunofluorescent antibody (DFA) for R. rickettsii (rabbit anti-R.
rickettsii, Centers for Disease Control and Prevention [CDC], Atlanta, GA)
and E. chaffeensis (rabbit anti-ehrlichia fluorescein
isothiocyanate-labeled conjugates, J.E. Dawson, CDC, Atlanta, GA) (6,7).

          Table 1. Patient's T-lymphocyte proliferation when
          stimulated with antigens of Rickettsia rickettsii or
          Ehrlichia chaffeensis
         ------------------------------------------------------
          Mitogenic            Quantity of        Stimulation
          stimulus             antigen (g)          index
         ------------------------------------------------------
          R. rickettsii            2.0               17.9
                                   1.0               26.7
                                   0.2               14.8
          E. chaffeensis           4.0               65.3
                                   2.0               19.1
                                   0.4               48.7
          Phytohemagglutinin       5.0              271.2
                                   1.0                8.2
         ------------------------------------------------------

Approximately 12 weeks after onset of illness, venous blood was obtained
and placed in tubes with EDTA anticoagulant. The specimen was shipped
overnight on wet ice and then processed as follows. An equal volume of
medium was added to a 10-ml aliquot of the blood sample, loaded onto 6 ml
of Ficoll-paque (Pharmacia, Uppsala, Sweden), and centrifuged at 770 x g
for 20 min at room temperature. The upper layer of plasma was removed. Then
the interface (containing most of the lymphocytes) and the Ficoll-paque
(containing most of the monocytes) were harvested and washed once with
medium. The pellet was suspended in medium (Dulbecco modified Eagle medium
[DMEM]) containing 10% fetal bovine serum, 2 mM glutamine, 5 x 10(sup -5) M
2-mercaptoethanol, 10 M HEPES buffer, and 50 g/ml of gentamicin. The
concentration of cells was adjusted to 1 x 10(sup 6)/ml in DMEM. A 0.1-ml portion
of these cells was then aliquoted into each well of a 96-well plate. Serial
dilutions of inactivated intact rickettsial or ehrlichial antigen (0.2g to
4 g/well) were added to individual wells in the same plate in triplicate.
The plate was then incubated at 37C in a 5% CO2 atmosphere for 6 days.
Afterwards, 3H-thymidine was added to each well (1 microcurie/well), and
the plates were again incubated at 37C overnight. On the next day the
cells were harvested and dried; -fluor cocktail scintillation fluid was
then added, and the mixture was counted in a -counter. Count per minute
rates greater than threefold of the unstimulated control were considered
positive.

After 8 days of incubation in Vero cells, a spotted fever group rickettsia
was detected by direct immunofluorescent antibody from a sample of blood
obtained immediately before doxycycline therapy. However, the rickettsial
organism did not thrive with subsequent in vitro passage, and further
attempts to recover the organism in vitro or by injecting into guinea pigs
were unsuccessful. Subsequent attempts to amplify rickettsial 17kDa protein
gene and the 16SrRNA gene by PCR with stored cell culture materials from
passages 1 and 2 were negative. In addition, morulae consistent with
ehrlichial organisms were detected by light and direct fluorescence in
blood taken from the patient before antimicrobial therapy. Subsequent
attempts to isolate ehrlichiae with further passage in 030 cells were
unsuccessful.

Polymerase Chain Reaction (PCR) Amplification/Sequencing

DNA was extracted from acute-phase blood with a commercially available kit
(IsoQuick; Orca Research Inc., Bothell, WA) for preparation of DNA from
whole blood, according to manufacturer's instructions. The dried pellet was
then resuspended in DNAase-free water in preparation for PCR analysis.

A pair of primers that recognize sequences of the nadA gene of E.
chaffeensis was used to analyze the patient's sample (Table 2). The PCR
products were then separated by electrophoresis in a 1% agarose gel,
stained with ethidium bromide, and visualized under UV light (Figure A).

The amplification conditions for the 120 kDA protein gene were essentially
the same as the ones used for the nadA gene, except that nested PCR was
performed (9-10). The outside primers were pXCF3 and pXAR4, and the nested
primers were pXCF3b and pXAR5 (Table 2). The nested primers obtained a
product similar to the 1.1 kb product amplified from the Arkansas strain
and one repeat unit larger than the Sapulpa strain (Figure B).

Nested PCR amplification of the 120 kDa protein gene of E. chaffeensis
yielded a PCR product from the patient's blood similar to the repeat region
of the E. chaffeensis Arkansas strain. The nadA gene of E. chaffeensis was
amplified by a single pair of primers; the sequence of the nadA segment was
determined to be similar in size to that of E. chaffeensis (Figure).

Case Findings

This patient had clinical features of infection with both R. rickettsii and
E. chaffeensis. His rash, although limited and purely macular, was
compatible with R. rickettsii infection (11). Compelling data support the
conclusion that the patient was infected with a spotted fever group
rickettsia: 1) IFA staining of a skin specimen showed organisms typical of
spotted fever group rickettsia, 2) a spotted fever group rickettsia was
initially recovered in tissue culture from a blood sample taken before
therapy, and 3) T-cell lymphocytes harvested from a convalescent-phase
blood sample contained specific memory cells reactive to R. rickettsii from
which a T-lymphocyte cell line was subsequently established.

     Table 2. Sequences of primers used in amplification of the NadA
     gene and the 120 kDa protein gene of Ehrlichia chaffeensis
    -----------------------------------------------------------------
 	Gene        		Primers 		Sequence
    -----------------------------------------------------------------
     NadA gene                ECHNADA1  5' TCATTTCGTGCTTTCTTATTG 3'
                              pXCR6     5' TGCCCACATATGCGTTTG 3'
     120 kDa protein gene     pXCF3     5' CAGAATTGATTGTGGAGTTGG 3'
                              pXAR4     5' ACATAACATTCCACTTTCAAA 3'
     120 kDa protein gene     pXCF3b    5' CAGCAACAGCAAGAAGATGAC 3'
     nested                   pXAR5     5' ATCTTTCTCTACAACAACCGG 3'
  -------------------------------------------------------------------

We do not know why a typical vigorous convalescent-antibody response to R.
rickettsii did not develop in this patient, even though antibody response
to E. chaffeensis was typical. It is possible, but highly unlikely, that
the spotted fever group rickettsia isolated from the patient was a species
other than R. rickettsii. We cannot state with certainty that the isolate
detected in vitro in Vero cells and then lost in subsequent passage was
indeed R. rickettsii and not a closely related species such as R.
amblyommii (12). It is also possible that coinfection with E. chaffeensis
suppressed a normal antibody response to R. rickettsii antigens.
Alternately, if infection with E. chaffeensis was transmitted by a tick
bite several days before the second bite by a tick infected with a spotted
fever group rickettsia, antimicrobial therapy may have blunted a typical
antibody response to rickettsialbut not to previously present
ehrlichialantigens (early antimicrobial therapy can impair or blunt the
antibody response in patients with R. rickettsii infection [13]). In
addition, a previous report described a patient with documented
rickettsemia who was treated within 12 hours of onset of symptoms and
failed to develop a convalescent-phase antibody response (14).

                        	[fig]
     Figure. Polymerase chain reaction (PCR) products. A. With primers
     for the NadA gene. Lane 1: Patient's sample with an approximately
     1.02-kb product; Lane 2: Negative control; Lane 3: Positive
     control. Ehrlichia chaffeensis (Arkansas strain)infected DH82
     cells; Lane 4: Molecular size markers: (Phi)(Chi)174 phage DNA cleaved
     with HaeIII. B. With nested primers for the 120 kDa protein gene.
     Lane 1: Patient's sample with an approximately 1.1-kb product;
     Lane 2: Positive control. E. chaffeensis (Sapulpa strain)DH82
     infected cells; Lane 3: Positive control. E. chaffeensis
     (Arkansas strain)infected DH82 cells; Lane 4: Molecular size
     markers: (Phi)(Chi)174 phage DNA cleaved with HaeIII.

     Amplification conditions. Ten microliters of the patient's DNA
     was added to a 90-l PCR reaction tube containing 10 l of 10X
     PCR buffer (Boehringer Mannheim, Indianapolis, IN), 1 l of PCR
     primers ECHNADA1 and pXCR6, final concentration 1 M each; 2 l
     deoxynucleotide triphosphates (final concentration, 200 M); and
     1 l of Taq polymerase (Boehringer Mannheim, Indianapolis, IN).
     Water was added to bring the volume to 100 l. The mixture was
     placed in a Progene FPROGO2Y thermocycler (Techne, Princeton,
     NJ). A DNA lysate prepared from E. chaffeensis-infected DH82
     cells was used as a positive control. The cycling program
     consisted of 3 min at 94C followed by 30 cycles, each of 30 sec
     at 94C, 1 min at 55C, and 2 min at 72C, and an additional
     cycle with an extension step of 3 min at 72C.

     To confirm the identity of the PCR product, we sequenced the 590
     bp of the nadA gene product. After amplification, the PCR
     products were purified by QIAquick, a PCR purification kit
     (QIAgen, Santa Clarita, CA). The nucleotide sequence was then
     determined by the dideoxynucleotide method of cycle sequencing
     with Taq polymerase (ABI Prism 377 DNA sequencer, Perkin-Elmer
     Corp., Foster City, CA). The sequencing reaction was carried out
     for each strand of DNA. The sequencing data were analyzed by
     Genetics Computer Group, Wisconsin Package software; 99.8%
     identity of the 590-bp overlap with nadA nucleotide sequence of
     Arkansas strain (Gen Bank accession number U90900) was obtained
     (8).

Had we not performed a skin biopsy or attempted to isolate rickettsiae from
the blood, we might have logically (but erroneously) concluded that the
illness was due solely to E. chaffeensis infection.

Concurrent infection with E. chaffeensis is supported by three different
lines of evidence: 1) a greater than fourfold rise in IFA titer against E.
chaffeensis antigens, 2) in vitro stimulation of T cells harvested from a
convalescent-phase blood sample with the antigen of E. chaffeensis causing
a strong lymphoproliferative response, and 3) PCR products similar in size
to the repeat region of the 120 kDA surface protein gene of E. chaffeensis
Arkansas strain and specific for the E. chaffeensis nadA gene recovered
from a sample of blood obtained before therapy. Although we did not
sequence amplicon generated from the 120 kDa protein gene, the
oligonucleotide primers that were used in PCR amplification are derived
from regions of the 120 kDa protein gene that are unique to E. chaffeensis.
These regions are not present in other bacteria, including closely related
Ehrlichia species.

We are unable to determine if the patient was infected by two different
ticks or one tick simultaneously infected with E. chaffeensis and R.
rickettsii or another spotted fever group rickettsia. Coinfection of Ixodes
scapularis ticks with the agent of human granulocytic ehrlichiosis or a
closely related organism and B. burgdorferi has been described (15-17);
however, to our knowledge, simultaneous infection of a tick with R.
rickettsii and E. chaffeensis has not. Furthermore, the major vector of E.
chaffeensis in North Carolina is thought to be Amblyomma americanum (18),
and the primary vector for R. rickettsii in this area is Dermacentor
variabilis. Although E. chaffeensis has been detected on one occasion in D.
variabilis (19), the evidence that A. americanum is a vector for Rocky
Mountain spotted fever elsewhere is circumstantial; to our knowledge, R.
rickettsii has never been isolated from A. americanum (W. Burgdorfer, pers.
comm.).

Most likely E. chaffeensis was transmitted to the patient from A.
americanum, and the spotted fever rickettsia was transmitted from D.
variabilis. Indeed, a walk in the fields of the Piedmont region of North
Carolina in the spring frequently results in attachment of numerous ticks
of both species. Since the patient had direct contact or bites with two
different ticks before his illness, we suspect that he was infected by
separate ticks rather than by one tick infected with both pathogens.

Finally, this case is remarkable in an additional way. Despite coinfection
with two pathogens, the patient had a relatively mild illness. He did not
have shock, major organ dysfunction, or central nervous system involvement;
and with doxycycline therapy, recovery was prompt and uneventful.

On the basis of this case report, we cannot predict that future cases will
have a benign course or an impaired or absent convalescent-phase antibody
response to one or more of the infecting agents. However, the patient's
illness illustrates that coinfection with a spotted fever group rickettsia
and E. chaffeensis may occur in areas where both diseases are endemic. The
frequency of human coinfection with ehrlichial and rickettsial agents is
unknown. Serologic studies have demonstrated that approximately one third
of patients with antibodies to E. chaffeensis also have antibodies to one
or more rickettsiae (20). Whether this phenomenon is due to the presence of
cross-reactive antigens or actual immune stimulation by simultaneous or
sequential multiple tick-borne pathogens is unknown.

Dr. Sexton is a professor in the Department of Medicine, Division of
Infectious Diseases, Duke University Medical Center, Durham, North
Carolina. His research interests include the clinical and epidemiologic
features of Rocky Mountain spotted fever and other tick-borne diseases.

Address for correspondence: Daniel J. Sexton, Box 3605, Duke University
Medical Center, Durham, NC 27710, USA; fax: 919-681-7945; e-mail:
Sexto002@mc.duke.edu.

References

  1. Nadelman RB, Horowitz HW, Hsieh T-C, Wu JM, Aguero-Rosenfeld ME,
     Schwartz I, et al. Simultaneous human granulocytic ehrlichiosis and
     Lyme borreliosis. N Engl J Med 1997;337:27-30.
  2. Krause PJ, Telford SR III, Spielman A, Sikand V, Ryan R, Christianson
     D, et al. Concurrent Lyme disease and babesiosis: evidence for
     increased severity and duration of illness. JAMA 1996;275:1657-60.
  3. Procop GW, Burchette JL, Howell DN, Sexton DJ. Immunoperoxidase and
     immunofluorescent staining of Rickettsia rickettsii in skin biopsy. A
     comparative study. Arch Pathol Lab Med 1997;121:894-9.
  4. Philip RN, Casper EA, Ormsbee RA, Peacock MG, Burgdorfer W.
     Microimmunofluorescence test for the serological study of Rocky
     Mountain spotted fever and typhus. J Clin Microbiol 1976;3:51-61.
  5. Dawson JE, Rikihisa Y, Ewing SA, Fishbein DB. Serologic diagnosis of
     human ehrlichiosis using two Ehrlichia canis isolates. J Infect Dis
     1991;163:564-7.
  6. Lange JV, Walker DH, Wester TB. Documented Rocky Mountain spotted
     fever in wintertime. JAMA 1982;247:2403-4.
  7. Iqbal Z, Rikihisa Y. Re-isolation of Ehrlichia canis from blood and
     tissue of dogs after doxycyline treatment. J Clin Microbiol
     1994;32:1644-9.
  8. Xu X-J, Walker DH. Sequence and characterization of an Ehrlichia
     chaffeensis gene encoding 314 amino acids highly homologous to the NAD
     A enzyme. FEMS Microbiol Lett 1997;154:53-8.
  9. Xu X-J, Crocquet-Valdes PA, Walker DH. Cloning and sequencing of the
     gene for 120-kDA immunodominant surface protein of Ehrlichia
     chaffeensis. Gene 1997;184:149-54.
 10. Xu X-J, Piesman JF, Olson JG, Walker DH. Geographic distribution of
     different genetic types of Ehrlichia chaffeensis. Am J Trop Med Hyg
     1997;56:679-80.
 11. Sexton DJ, Corey GR. Rocky Mountain spotless and almost spotless
     fever: a wolf in sheep's clothing. Clin Infect Dis 1992;15:439-48.
 12. Anderson BE, Sims KG, Olson JG, Childs JE, Piesman JF, Happ CM, et al.
     Amblyomma americanum: a potential vector of human ehrlichiosis. Am J
     Trop Med Hyg 1993;49:239-44.
 13. Schubert JH. Serologic titers in rickettsial infection as affected by
     antibiotic treatment. Pub Health Lab 1952;10:38-41.
 14. Davis JP, Wilfert CM, Sexton DJ, Burgdorfer W, Casper EA, Philip RN.
     Serologic comparison of R. rickettsii isolated from patients in
     Montana and North Carolina. In: Rickettsiae and rickettsial diseases.
     New York: Academic Press, Inc.; 1981 p. 139-47.
 15. Pancholi P, Kolbert CP, Mitchell PD, Reed KD Jr, Dumler JS, Bakken JS,
     et al. Ixodes dammini as a potential vector of human granulocytic
     ehrlichiosis. J Infect Dis 1995;172:1007-12.
 16. Telford SR III, Dawson JE, Katavolos P, Warner CK, Kolbert CP, Persing
     DH. Perpetuation of the agent of human granulocytic ehrlichiosis in a
     deer tick-rodent cycle. Proc Natl Acad Sci U S A 1996;93:6209-14.
 17. Schwartz I, Fish D, Daniels TJ. Prevalence of the rickettsial agent of
     human granulocytic ehrlichiosis in ticks from a hyperendemic focus of
     Lyme disease. N Engl J Med 1997;336:49-50.
 18. Ewing SA, Dawson JE, Kocan AA, Barker RW, Warner CK, Panciera RJ, et
     al. Experimental transmission of Ehrlichia chaffeensis (Rickettsiales:
     Ehrlichieae) among white-tailed deer by Amblyomma americanum (Acarei:
     Ixodidae). J Med Entomol 1995;32:368-74.
 19. Anderson BE, Sumner JW, Dawson JE, Tzianabos T, Greene CR, Olson JC,
     et al. Detection of the etiologic agent of human ehrlichiosis by
     polymerase chain reaction. J Clin Microbiol 1992;30:775-80.
 20. Dawson JE, Fishbein DB, Eng TR, Redus MA, Greene NR. Diagnosis of
     human ehrlichiosis with the indirect fluorescent antibody test:
     kinetics and specificity. J Infect Dis 1990;162:91-5.


Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/sexton.htm

Please note that figures and equations are not available in ASCII format; 
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-------------------------------------------------------------------------------------------
-------------------------------------------------------------------------------------------

[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Dispatches

Molecular Fingerprinting of Multidrug-Resistant Salmonella enterica
Serotype Typhi

M. D. Hampton, L. R. Ward, B. Rowe, and E. J. Threlfall
Central Public Health Laboratory, London, United Kingdom

  --------------------------------------------------
      For epidemiologic investigations, the primary subdivision of
      Salmonella Typhi is vi-phage typing; 106 Vi-phage types are
      defined. For multidrug-resistant strains the most common types
      have been M1 (Pakistan) and E1 (India, Pakistan, Bangladesh,
      and the Arabian Gulf); a strain untypable with the Vi phages
      has been responsible for a major epidemic in Tajikistan. Most
      often, isolates from the Indian subcontinent have been
      resistant to ampicillin, chloramphenicol, streptomycin,
      sulfonamides, tetracyclines, and trimethoprim; but in the 1997
      Tajikistan outbreak, the epidemic strain was also resistant to
      ciprofloxacin. For multidrug-resistant strains, subdivision
      within phage type can be achieved by plasmid profile typing and
      pulsed-field gel electrophoresis.

In the United Kingdom, the incidence of multidrug-resistant (MDR)
Salmonella Typhi isolates has increased from 1.5% in 1989 to 34% in 1995
(1). The most common MDR Vi-phage types have been M1 and E1, isolated from
patients who recently returned from Southeast Asia or the Indian
subcontinent (1). Although the increasing incidence of MDR isolates of
Vi-phage types El and M1 has been particularly noteworthy, outbreaks caused
by drug-sensitive strains of these Vi-phage types have continued to be
identified. For example, all four patients in a July 1997 outbreak had
recently traveled to the Dominican Republic; the causative strain was drug
sensitive and belonged to Vi-phage type E1 (2).

Discrimination within phage type is desirable for studying the epidemiology
of MDR strains of S. Typhi and for determining their phylogenetic
relationships with established drug-sensitive types. Insertion sequence 200
profiling can differentiate between drug-sensitive and drug-resistant
strains of Vi-phage types M1 and E1 but is unsuitable for subdivision
within these phage types (3).

We used plasmid profile typing and pulsed-field gel electrophoresis (PFGE)
for the molecular characterization and subdivision of isolates of S. Typhi
of Vi-phage types E1, M1, and untypable Vi strain (UVS) from outbreaks and
sporadic cases. We selected a panel of 18 strains comprising the type
strains of S. Typhi Vi-phage types E1 and M1, as well as drug-sensitive and
drug-resistant isolates of these phage types from patients infected in
several countries of the Indian subcontinent. We also included UVS and
Vi-phage type E1 isolates from the 1997 epidemic in Tajikistan and an
isolate of Vi-phage type E1 from an infection acquired in the Dominican
Republic in 1997 (Table). The type strains of Vi-phage types E1 and M1 were
isolated in Russia and Canada in 1918 (E1) and 1939 (M1), which
demonstrates the subsequent global dissemination of these phage types over
a prolonged period.

Table. Drug-resistant and drug-sensitive isolates of Salmonella Typhi phage types
E1, M1, and UVS
---------------------------------------------------------------------------------
Laboratory
accession   Phage Country of   Year of                    Mol wt of    Pulsed-field
number      type  origin     isolation Antibiogram(sup a) plasmid DNA  profile
---------------------------------------------------------------------------------
E1          E1    Russia         1918     $             -(sup b)      X1
P3047990    E1    India          1993     $               -           X2
P2967750    E1    India          1992     ACSSuTTm        120         X2a
P3639160    E1    Pakistan       1994     ACSSuTTm        120         X2a
P3640980    E1    Pakistan       1994     ACSSuTTm        120         X2a
P4632360    E1    Bangladesh     1997     ACSSuTTm        120         X2a
P4663970    E1    Bangladesh     1997     ACSSuTTm        120         X2a
P4405140    UVS   Tajikistan     1997     ACSSuTTmCp      120    80   X2a
P4405200    UVS   Tajikistan     1997     ACSSuTTmCp      120    80   X2a
P4466680    E1    India          1997     ACSSuTTm        120         X3
P2951290    E1    Bangladesh     1992     ACSSuTTm        120         X4
P3166750    E1    Bangladesh     1993     ACSSuTTm        120         X4
P4219910    E1    Pakistan       1997     ACSSuTTmCp      120         X4
P4405160    E1    Tajikistan     1997     ACSSuTTm        120         X5

P4543210    E1    Dominican      1997     $               -           X6
                  Republic
M1          M1    Canada         1939     $               -           X7
P3112100    M1    Pakistan       1992     $               -           X8
P3044890    M1    Pakistan       1992     ACSSuTTm        120         X8a
  --------------------------------------------------
(sup a)A, ampicillin; C, chloramphenicol; S, streptomycin; Su, sulphonamides; 
T, tetracyclines; Tm, trimethoprim; Cp, ciprofloxacin; 
$, drug-sensitive.
(sup b)Plasmid DNA not detected.

All drug-sensitive isolates of Vi-phage types E1 and M1 were plasmid-free,
whereas MDR isolates were characterized by a plasmid of approximately 120
megadaltons (MDa). The two UVS isolates also had a 120-MDa plasmid and an
additional plasmid of 80 MDa. Conjugation and incompatibility testing
showed that the 120-MDa plasmids encoded resistance to ampicillin,
chloramphenicol, streptomycin, sulphonamides, tetracyclines, and
trimethoprim and belonged to incompatibility group H(sub 1). In contrast, the
80-MDa plasmids in the two UVS isolates from Tajikistan did not code for
drug resistance and were not further characterized by incompatibility
grouping. In isolates of Vi-phage type E1 and UVS with additional
resistance to ciprofloxacin, resistance to this antimicrobial was
chromosomally encoded.

PFGE following digestion of genomic DNA with Xba I showed 19 to 23
fragments per strain, with 13 fragments common to each and 1 to 12 band
differences between patterns. Several differences were identified, both
between and within phage types (Figure 1). For Vi-phage type E1, six
pulsed-field profiles (PFPs) (S. Typhi PFP Stp X1 through PFP Stp X6) and
one subtype (PFP Stp X2a) were identified and defined. For PFPs Stp X1, X2,
X5, and X6, subdivision was based on the numbers of fragments (from 150 kb
to >500 kb) and for PFPs X3 and X4, subdivision was based on the number and
size of fragments in the 20-kb to 50-kb range, with PFP X3 also lacking a
300-kp band present in PFP X4. The PFPs of the two UVS isolates from
Tajikistan were indistinguishable from PFP X2a. For Vi-phage type M1, two
PFP types (PFPs X7 to X8) and one subtype (PFP X8a) were identified. PFPs
X7 and X8 were designated as separate types on the basis of the numbers of
fragments in the 250-kb to 500-kb range. PFP X2a could be distinguished
from PFP X2, and PFP X8a could be distinguished from PFP X8 by the presence
of three additional fragments of approximately 50 kb, 30 kb, and 25 kb. As
these three fragments were observed in isolates of PFPs X2a, X3, X4, X5,
and X8a, all of which had incompatibility group H(sub 1) plasmids but were not
identified in isolates of PFPs X1, X2, X6, X7, and X8 (none of which
possessed H1 plasmids), it is likely that these three fragments are
plasmid-derived. These plasmid-containing derivatives of PFPs X2 and X8
have been designated PFPs X2a and X8a, respectively.

A dendogram (Figure 2) analyzed by pairwise fragment comparison using the
Dice coefficient and by data clustering using the unweighted pair group
arithmetic averaging method (UPGMA) clearly illustrated the relationships
described above. The Dice coefficient values, excluding plasmid DNA, were
from 66.7% to 97.6%. The PFPs of Vi-phage type E1 and UVS and Vi-phage type
M1 clustered as two distinct groups with the branch-point for the E1 and
UVS PFPs X1-X6 cluster at approximately 80.5% and the M1 PFPs X7-X8 cluster
at approximately 86%. Within Vi-phage type E1, PFPs X1 and X6 (isolates
from Russia and the Dominican Republic) are distinct from the closely
related PFP X2-X5 cluster (the Indian subcontinent and Tajikastan), which
suggests a recent evolutionary divergence in Asia within this universally
common phage type.

      [fig 1]                    				[fig 2]                    

Figure 1. Pulsed-field gel                      Figure 2. Dendogram of
electrophoresis profiles of                     Salmonella Typhi
Xba I-digested genomic DNA                      pulsed-field profiles Stp
from drug-resistant and                         X1 -X8a. Genetic similarity
drug-sensitive isolates of                      was calculated by the Dice
Salmonella Typhi Vi-phage                       coefficient (S) and
types E1, M1, and UVS.                          clustered by unweighted
Tracks 1-20 contained 1 and                     pair group arithmetic
20, lambda 48.5-kb ladder                       averaging method.
(Sigma); 2, M1 type strain
(S. Typhi PFP Stp X7); 3,
P3044890 (PFP X8a); 4,
P3112100 (PFP X8); 5, E1
type strain (PFP X1); 6,
P3640980 (PFP X2a); 7,
P3639160 (PFP X2a); 8,
P2967750 (PFP X2a); 9,
P4405140 (PFP X2a); 10,
P4405200 (PFP X2a); 11,
P4632360 (PFP X2a); 12,
P4663970 (PFP X2a); 13,
P3047990 (PFP X2); 14,
P4543210 (PFP X6); 15,
P4466680 (PFPX3); 16,
3166750 (PFP X4); 17,
P2951290 (PFP X4); 18,
P4219910 (PFP X4); and 19,
P4405160 (PFP X5). PFGE
conditions: 180V (5.4 V/cm);
45h; Ramp, 0.5 - 45s.

These results demonstrate that PFGE, coupled with plasmid profile analysis,
is a useful method for discriminating MDR isolates of S. Typhi Vi-phage
type E1. The results also suggest that isolates of Vi-phage type E1 with
the X2a pulsed-field profile have been derived within the Indian
subcontinent from a drug-sensitive strain of Vi-phage type E1 already
endemic in this area. This drug-sensitive progenitor strain is
genotypically distinct from the type strain of Vi-phage type E1, isolated
in Russia in 1918 and more recently in the Dominican Republic. In turn,
isolates of Vi-phage type E1 of PFPs X3-X5 are closely related to PFP X2
and could have been derived from PFP X2 in the course of its recent
epidemic history. The two UVS isolates from Tajikistan had a PFP
indistinguishable from PFP X2a but differed by plasmid profile and
antibiogram and by at least three bands from that of the MDR Vi-phage type
E1 isolate from Tajikistan (PFP X5). This suggests that despite the recent
observation that MDR strains of Vi-phage type E1 and UVS are in circulation
in Tajikistan (4), the epidemic ciprofloxacin-resistant UVS strain is more
likely to have originated in the Indian subcontinent than in Tajikistan.
Likewise, the drug-sensitive and drug-resistant isolates of Vi-phage type
M1 from Pakistan are genotypically distinct from the type strain of
Vi-phage type M1 isolated in Canada in 1939. Again, this suggests that MDR
strains of Vi-phage type M1 have been derived in Pakistan from a
drug-sensitive progenitor strain of Vi-phage type M1 already endemic in
that country.

A previous PFGE-based study of S. Typhi from Southeast Asia demonstrated
that multiple genetic variants associated with sporadic cases and
occasional outbreaks are simultaneously present in that area (5). The study
also demonstrated considerable genetic diversity between strains,
suggesting considerable movement of these strains within endemic-disease
regions. However, the study did not use Vi-phage typing for the preliminary
identification and subdivision of isolates. In our study, we combined
Vi-phage typing with plasmid analysis and PFGE to provide a method of
subdividing MDR isolates of Vi-phage types El and M1 endemic in India,
Pakistan, and Bangladesh; we also compared the resulting patterns with
those from historic isolates and isolates from recent outbreaks of MDR S.
Typhi in Tajikistan. Although further work is needed to establish the
applicability of combining phage typing with plasmid typing and PFGE for
studying the epidemiology of MDR S. Typhi, our studies demonstrate that
subdivision within the most common phage types can be achieved and may be
useful both for investigating outbreaks and for providing information about
the evolutionary history of MDR epidemic strains.

Acknowledgments

We thank Drs. D. Murdoch and N. Banatvala of MERLIN for providing the
isolates of S. Typhi from the 1997 epidemic in Tajikistan.

M.D. Hampton is a biomedical scientist.

Address for correspondence: E. J. Threlfall, WHO Collaborating Centre for
Phage Typing and Resistance in Enterobacteria, Laboratory of Enteric
Pathogens, Central Public Health Laboratory, 61 Colindale Avenue, London
NW9 5HT, United Kingdom; fax: 44-181-905-9929; e-mail:
jthrelfall@phls.co.uk.

References

  1. Rowe B, Ward LR, Threlfall EJ. Multiresistant Salmonella typhi a
     world-wide epidemic. Clin Infect Dis 1997;24:S106-9.
  2. Typhoid fever associated with travel to the Dominican Republic. Commun
     Dis Rep CDR Weekly 1997;7:299-302.
  3. Threlfall EJ, Torre E, Ward LR, Rowe B, Gibert I. Insertion sequence
     IS200 can differentiate drug-resistant and drug-sensitive Salmonella
     typhi of Vi-phage types E1 and M1. J Med Microbiol 1993;39:454-8.
  4. Murdoch DA, Banatvala N, Bone A, Shoismatulloev BI, Ward LR, Threlfall
     EJ. Epidemic ciprofloxacin-resistant Salmonella typhi in Tajikistan.
     Lancet 1998;351:339.
  5. Thong K-L, Puthucheary S, Yassim RM, Sudarmono P, Padmidewi M,
     Soewandojo E, et al. Analysis of Salmonella typhi isolates from
     southeast Asia by pulsed-field gel electrophoresis. J Clin Microbiol
     1995;33:1938-41.


Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/hampton.htm

Please note that figures and equations are not available in ASCII format; 
their placement within the text is noted by [fig] and [eq], respectively. 
Greek symbols are spelled out. The following codes are used: 
(ft) for footnote; (sup) for superscript; (sub) for subscript; 
/= for greater than or equal to. 

-------------------------------------------------------------------------------------------
-------------------------------------------------------------------------------------------

[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Dispatches

Clostridium septicum Infection and Hemolytic Uremic Syndrome

M. Barnham* and N. Weightman
*Harrogate General Hospital, Harrogate, United Kingdom; and Northallerton
Friarage Hospital, North Yorkshire, United Kingdom

---------------------------------------------------------------------------
      Five cases of Clostridium septicum infection secondary to
      Escherichia coli O157induced hemolytic uremic syndrome have
      been reported. We report on three cases (one of which is
      included in the above five) of dual Cl. septicum and E. coli
      infection; all three patients were exposed to farm animals. A
      common zoonotic source for Cl. septicum and E. coli O157
      infections should be considered. Patients with hemolytic uremic
      syndrome should be treated aggressively and monitored closely
      for Cl. septicum superinfection.

Severe infection with Clostridium septicum in humans is relatively rare. In
recent years, the annual incidence of reported bacteremic infection in
England and Wales has been 0.4 to 1.0 cases per million population,
although compared with other clostridial bacteremias, Cl. septicum
infection has been second only to Cl. perfringens (Public Health Laboratory
Service, Communicable Disease Surveillance Centre, Colindale: pers. comm.).

In 1877, Cl. septicum, then known as the "vibrion septique," was the first
pathogenic anaerobe cultured by Pasteur and Joubert (1). The organism
reportedly caused much of the gas gangrene in wounded soldiers during the
First and Second World Wars (2), although the bacteriologic techniques of
the time were primitive, and findings varied. In addition to gas gangrene,
Cl. septicum can cause other severe focal or disseminated infections by
spontaneous invasion from the gut of compromised patients (3,4). Strong
associations have been established between these spontaneous forms of
infection and colonic malignancy (especially in the cecum) (5), acute
leukemia or cyclical neutropenia (with the development of neutropenic
enterocolitis) (6,7), and diabetes. The organism may be more likely than
other clostridia to establish infection in viable tissues by virtue of its
aerotolerance and ability to establish infection (at least in animal
models) from a small inoculum (4).

We have diagnosed serious Cl. septicum infection (three bacteremic) in four
English patients in a period of 13 years. One of these infections developed
as a complication of Escherichia coli O157-induced hemolytic uremic
syndrome (HUS); with four similar case reports (8-11), this condition
appears to be emerging as one with a high risk of Cl. septicum
superinfection. Details of five published reports of HUS-associated
invasive Cl. septicum infection are shown in the Table. Mucosal damage and
thrombocytopenia from, respectively, Shiga toxin-producing E. coli
enteritis and HUS appear to provide highly suitable conditions for invasion
by Cl. septicum in infected patients (5). Caya et al. (7) speculate that
thrombocytopenia may impair endothelial resistance to clostridial invasion,
and there is gathering evidence for the importance of platelets in host
defense against infection (12). When antibiotics are not given, as in the
management of E. coli-associated HUS, clostridia have more opportunity to
thrive unhindered. The five patients in this case sequence did not show
granulocytopenia, a feature thought to potentiate invasive clostridial
infection in those with neutropenic enterocolitis (6) and in leukemic
patients (7); in the latter group, the thrombocytopenia that commonly
coexists with neutropenia might be relevant in the evolution of infection.
Four of the five patients died, and the three examined at autopsy had
colonic ulceration and hemorrhagic necrosis. Unusual clinical features
included intracranial infection with Cl. septicum in four and clostridial
cellulitis in the abdominal wall in two.

Table. Hemolytic uremic syndrome-associated Clostridium septicum infection

---------------------------------------------------------------------------



    Sex,  E.                  Cytopenias(sup c)
Country, (sup a)coli                 ---------------
year       age (sup b)   Cl. septicum                   Clinical
(ref)     (yrs) in feces isolated from granulo-thrombo- features-outcome
---------------------------------------------------------------------------
USA,      M,1 NS(sup d) Rash aspirate, No     Yes     Abdominal rash,
1988 (8)                PM(sup e)blood                sepsis, convulsions,
                                                      cerebritis, necrosis
                                                      colon-died
England,  M,4 O157      Blood, PM      No     Yes     Convulsions,
1993 (9)                brain                         cerebritis, necrosis
                                                      colon-died
USA,      F,2 E.        Blood,         No     Yes     Sepsis, cerebritis,
1993 (10)     coli      cerebrospinal                 meningitis, necrosis
                        fluid                         colon-died

USA,      M,2 O157      Abscess pus    No     Yes     Brain abscess-
1995 (11)                                             recovered

England,  M,2 O157      Blood          No     Yes     Septic shock-died
1997
(this
study)
---------------------------------------------------------------------------
(sup a) M, male; F, female.
(sup b)Escherichia coli.
(sup c)At HUS = at the time that hemolytic uremic syndrome was evident.
(sup d)NS, not stated.
(sup e)PM, postmortem.

Both Cl. septicum and E. coli O157 have herbivore animal reservoirs. We
propose as a possible sequence of events for our three cases a) acquisition
of the organisms from animals, b) colonization of the colon of the patient,
leading to c) invasion by Cl. septicum as described above. Two of three
cases of serious Cl. septicum infection also were linked to sheep; the
remaining case, in an elderly man from a suburban setting, who had
carcinoma of the cecum and Cl. septicum septicemia, resembles the typical
case descriptions recorded by others (3,5).

In May 1997, a 2-year-old boy was hospitalized with a 7-day history of
vomiting and blood-flecked watery diarrhea; E. coli O157 phage-type 1,
VT-type 1+2 had been isolated from fecal specimens. On admission, the
patient was pale and uncomfortable with a petechial rash on the trunk and
an oral temperature of 38.2C; the abdomen was soft with no tenderness, and
blood pressure was normal. Investigations showed hemoglobin 9.5 g/dL with
some burr cells and microspherocytes present, elevated total white cell
count, and thrombocytopenia. Renal function was impaired, with blood urea
elevated; serum creatinine was within the reference range. Blood cultures
were taken on admission. After diagnosis of HUS, the patient was given
intravenous fluid replacement and was transferred to the local teaching
hospital where blood cultures were repeated and antibiotics were initially
withheld.

Peritoneal dialysis initially produced clinical improvement, but 32 hours
after admission, the patient suddenly became irritable and confused and had
a large hematemesis; his blood pressure fell to 100/40 mm Hg, and he
exhibited clinical shock. After transfusion with fresh frozen plasma and
albumen, cefuroxime, and metronidazole, he was transferred to the intensive
care unit, intubated, and ventilated. The pulse rate rose to 163/min and
the circulating white blood cell count to 34.0 x 10(sup 9)/L. The abdomen became
distended, and at the peritoneal dialysis catheter entry site, an area of
superficial skin ischemia resembling fasciitis developed, which later
progressed over the right side of the abdomen. A plain radiograph showed
dilated loops of bowel with thickened walls but no free gas. A diagnosis of
ischemic bowel was made, but 8 hours after his sudden deterioration and
before an exploratory laparotomy could be performed, the patient went into
cardiorespiratory arrest and died. Both sets of blood cultures yielded Cl.
septicum, the first becoming positive after 48 hours of incubation. An
autopsy was not performed.

The boy had played on a dairy and sheep farm, where E. coli O157 with
identical banding on digested DNA pulsed-field gel electrophoresis analysis
to that from the boy was isolated from five cows, but no search was made
for Cl. septicum.

A 64-year-old hiker in August 1984 slipped against a rock on a sheep trail
and cut her leg a few centimeters above the ankle. Emergency treatment
consisted of wound suturing and a tetanus toxoid booster. Three days later,
the patient returned to the Emergency Department flushed and sweating with
a grossly swollen, discolored, blistered, and foul-smelling leg. The limb
was clearly gangrenous to the lower third of the thigh with patchy erythema
and edema in the buttock and lower abdominal wall; radiographs showed large
quantities of gas in the soft tissues of the leg. Findings included an oral
temperature of 38.8C, blood pressure 100/60 mm Hg, hemoglobin 14.1 g/dL,
white blood cell count 5.3 x 10(sup 9)/L (including lymphopenia of 0.58 x 10(sup 9)/L),
and thrombocytopenia (platelets 101 x 10(sup 9)/L). Cl. septicum was isolated in
pure culture from blood as well as from swabs of blisters on the leg, where
it was found together with E. coli and Bacillus cereus.

The patient's leg was amputated mid thigh, and she received gas gangrene
antiserum, blood transfusion, and intravenous benzyl penicillin and
metronidazole. Postoperative complications included hypotension, atrial
fibrillation, and acute renal failure, but she responded to treatment and
recovered.

A 71-year-old male, sheep-farm laborer was cleaning out sheep dipping pits
in September 1985 when he caught his arm in the slurry machinery, causing a
traumatic amputation above his elbow joint with dislocation of the head of
the humerus. Emergency hospital treatment included general resuscitation,
blood transfusion, and surgical excision of damaged bone and soft tissues
together with reduction of the dislocated shoulder.

Despite postoperative treatment with cefuroxime and sodium fusidate, Cl.
septicum, E. coli, and enterococci were repeatedly isolated from the wound;
by the sixth postoperative day, a copious and very offensive watery
discharge exuded from the wound, with crepitus under the edges. At this
time the patient's general condition remained good except for an oral
temperature of 38.5C and circulating white cell count of 16.5 x 10(sup 9)/L;
platelet counts remained within the normal range. Surgical exploration
revealed local necrosis of soft tissues in the wound down to the bone;
devitalized tissues were excised, and drainage was promoted. Intravenous
benzyl penicillin, ampicillin, and metronidazole led to rapid resolution of
infection and a good recovery.

Opinions differ as to whether Cl. septicum is part of the normal human
fecal flora (5,13). Studies have reported the presence of the organism in
fecal specimens of 2% to 3% of healthy persons consuming relatively large
quantities of beef (14), but it is uncertain whether the organism was
resident or transient. Earlier studies (14) involving very small numbers of
healthy persons are unconvincing. Human infection with Cl. septicum is
assumed to be essentially autogenous, and the possibilities of
environmental or foodborne exposure originating from animals have not been
adequately discussed. Cl. septicum is found in the feces of herbivores, and
it is readily cultivated from soil in areas where they graze; the dominant
causative clostridium in malignant edema (posttraumatic gangrene) in
domestic animals, it causes a form of necrotizing enteritis (braxy) in
young sheep grazing on frozen vegetation during the autumn and winter
months (15).

The three cases described above suggest an infection of animal origin. In
the first case, E. coli O157 was almost certainly acquired by the young boy
from a farm contaminated by sheep and cattle, suggesting coexposure to
clostridia. The hiker's wound was probably contaminated with spores from an
area of intensive sheep grazing; Northern England is considered an area of
high risk for Cl. septicum infection in sheep (16). In the third case,
organisms from the slurry or sheep dip were probably injected directly into
the wound; sheep dips can become highly contaminated with Cl. septicum,
causing high incidence of infection (15).

We need to learn more about the role of animals in the epidemiology of
colonization and disease with Cl. septicum in humans. E. coli O157 and
associated HUS appear to be increasing in many parts of the industrialized
world; severe Cl. septicum infection can occur as a secondary disease. The
occurrence of at least five such dual infections suggests important
elements linking the evolution and pathogenesis of the two infections;
furthermore, acquisition of both organisms from a common zoonotic source
remains a possibility. A high level of suspicion for Cl. septicum
superinfection in patients with HUS, rapid diagnosis, prompt antimicrobial
therapy, and urgent surgical attention, as required, can improve the
survival rate of patients with this life-threatening infection.

Address for correspondence: M. Barnham, Department of Microbiology,
Harrogate General Hospital, Knaresborough Road, Harrogate, United Kingdom
HG2 7ND; fax: 44-1423-883-391.

References

  1. Sebald M, Hauser D. Pasteur, oxygen and the anaerobes revisited.
     Anaerobe 1995;1:11-6.
  2. MacLennan JD. Histotoxic clostridial infections of man. Bacteriology
     Reviews 1962;26:176-276.
  3. Koransky JR, Stargel MD, Dowell VR. Clostridium septicum
     bacteremia-its clinical significance. Am J Med 1979;66:63-6.
  4. Stevens DL, Musher DM, Watson DA, Eddy H, Hamill RJ, Gyorkey F, et al.
     Spontaneous, nontraumatic gangrene due to Clostridium septicum.
     Reviews of Infectious Diseases 1990;12:286-96.
  5. Kornbluth AA, Danzig JB, Bernstein LH. Clostridium septicum infection
     and associated malignancy. Medicine 1989;68:30-7.
  6. Clostridium septicum and neutropenic enterocolitis. Lancet 1987;2:608.
  7. Caya JG, Farmer SG, Ritch PS, Wollenberg NJ, Tieu TM, Oechler HW, et
     al. Clostridial septicemia complicating the course of leukemia. Cancer
     1986;57:2045-8.
  8. Riccio JA, Oberkircher OR. Clostridium septicum sepsis and cerebritis:
     a rare complication of the hemolytic-uremic syndrome. Pediatr Infect
     Dis J 1988;7:342-5.
  9. Randall JM, Hall K, Coulthard MG. Diffuse pneumocephalus due to
     Clostridium septicum cerebritis in hemolytic uremic syndrome.
     Neuroradiology 1993;35:218-20.
 10. Broughton RA, Lee EY. Clostridium septicum sepsis and meningitis as a
     complication of the hemolytic-uremic syndrome. Clin Pediatr (Phila)
     1993;32:750-2.
 11. Chiang V, Adelson PD, Poussaint TY, Hand M, Churchwell KB. Brain
     abscess caused by Clostridium septicum as a complication of
     hemolytic-uremic syndrome. Pediatr Infect Dis J 1995;14:72-4.
 12. Yeaman MR. The role of platelets in antimicrobial host defense. Clin
     Infect Dis 1997;25:951-70.
 13. Lorber B. Gas gangrene and other clostridium-associated diseases. In:
     Mandell GL, Bennett JE, Dolin R, editors. Principles and practice of
     infectious diseases. 4th ed. New York: Churchill Livingstone; 1995. p.
     2182-95.
 14. George WL, Finegold SM. Clostridia in the human gastrointestinal
     flora. In: Borriello SP, editor. Clostridia in gastrointestinal
     disease. Boca Raton (FL): CRC Press; 1985. p. 1-37.
 15. Radostits OM, Blood DC, Gay CC. Veterinary medicine: a textbook of the
     diseases of cattle, sheep, pigs, goats and horses. 8th ed. London:
     Bailliere Tindall; 1994. p. 686-9.
 16. Martin WB, Aitken ID. Diseases of sheep. 2nd ed. Oxford: Blackwell
     Scientific Publications; 1991. p. 109-13.


Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/barnham.htm

Please note that figures and equations are not available in ASCII format; 
their placement within the text is noted by [fig] and [eq], respectively. 
Greek symbols are spelled out. The following codes are used: 
(ft) for footnote; (sup) for superscript; (sub) for subscript; 
/= for greater than or equal to.

-------------------------------------------------------------------------------------------
-------------------------------------------------------------------------------------------

[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Dispatches

Persistent Infection of Pets within a Household with Three Bartonella
Species

Dorsey L. Kordick and Edward B. Breitschwerdt
North Carolina State University, Raleigh, North Carolina, USA

  --------------------------------------------------
      We monitored by blood culture and immunofluorescence assay
      (IFA) bartonella infection in one dog and eight cats in a
      household to determine the prevalence and persistence of the
      infection as well as its transmissibility to humans.
      Ectoparasite control was rigorously exercised. During a 3-year
      period, Bartonella clarridgeiae was recovered from one cat on
      two occasions, and B. henselae was isolated from another cat on
      four occasions. During a 16-month period, B. vinsonii subsp.
      berkhoffii was isolated from the dog on 8 of 10 culture
      attempts. Despite extensive household contact, the pet owner
      was seronegative to all three species by IFA for
      Bartonella-specific immunoglobulin G.

Bartonella species are hematotropic bacteria that have been isolated from
humans, cats, dogs, wildlife species, rodents, and arthropods. The
mammalian reservoir for B. clarridgeiae and B. henselae is the domestic
cat, in which asymptomatic infection can be maintained for long periods
(13). Feline infection with B. henselae is widespread throughout the
world, and B. clarridgeiae has been isolated from cats in Europe and
various regions of the United States (46). Chomel et al. have demonstrated
flea transmission of B. henselae between cats (7). Prolonged bacteremia has
been demonstrated in cats, but to our knowledge persistent asymptomatic B.
vinsonii subsp. berkhoffii infection in dogs, as described in this report,
has not been previously documented. Human exposure to B. henselae can
result in cat-scratch disease (CSD) or other medical conditions (8), and B.
clarridgeiae was recently implicated as the cause of CSD in a veterinarian
(6).

B. vinsonii was originally isolated by Baker in 1946; splenic homogenates
were cultured from voles that inhabited a former immigrant quarantine
station on Grosse Isle, Canada (9). No other isolates of B. vinsonii were
described until 1995 when we recovered a similar organism from a dog with
valvular endocarditis (10). Subsequent genotypic evaluation of the vole and
canine isolates resulted in taxonomic division of the species into
subspecies. The vole and canine isolates were designated B. vinsonii subsp.
vinsonii and B. vinsonii subsp. berkhoffii, respectively (11). Although the
type strain of B. vinsonii subsp. berkhoffii was isolated from a dog with
cardiac disease, the organism can also cause subclinical infection. The
reservoir for B. vinsonii subsp. berkhoffii has not been established, but
seroepidemiologic evidence supports the potential for tick transmission
(12).

We describe infection of three pets within the same household with three
different Bartonella species and the subclinical persistence of B. vinsonii
subsp. berkhoffii infection in a dog for 14 months. This report is a
component of other ongoing studies of Bartonella infection in animals. In
conjunction with an earlier study, a veterinary student offered her cats as
controls for an investigation into the prevalence of B. henselae associated
with cases of CSD (1). Three cats and the dog were subsequently monitored
for bacteremia and seroreactivity during a bartonella antimicrobial
efficacy study, in which the cats received 22.7 mg enrofloxacin q12h PO for
28 days and the dog received 25 mg doxycycline q12h PO for 28 days (13).

Blood samples were collected from the dog and additional cats acquired
later by the student. At each collection, all animals were assessed for
general health. For the dog, packed cell volume and total plasma protein
were determined from EDTA blood samples.

Sera were analyzed by immunofluorescence antibody assay (IFA) for
reactivity against B. clarridgeiae, B. henselae, and B. vinsonii subsp.
berkhoffii (1). All positive serum samples (dog, cat, and human) were
reassayed at least three separate times.

Blood samples from the dog and cats were placed into 1.5-ml pediatric
isolator tubes (Wampole Laboratories, Cranbury, NJ) for Bartonella culture
on rabbit blood agar (1). Isolates were identified by polymerase chain
reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of
the 16S rRNA gene and 16S-23S intergenic spacer (ITS) region using Dde I,
Mnl I, Hae III, and Alu I restriction endonuclease (6). DNA from the type
strains of B. clarridgeiae (ATCC 51734), B. henselae (ATCC 49882), and B.
vinsonii subsp. berkhoffii (ATCC 51672), in addition to B. vinsonii subsp.
berkhoffii strains cultured from two healthy dogs, was analyzed for
comparison.

The dog, cats, and pet owner remained healthy throughout the study. The
packed cell volume and total plasma protein values of the dog remained
within reference ranges. These values were not routinely assessed in the
cats. The source, age, and order of introduction of animals into the
household are depicted in Table 1. Bartonella-specific immunoglobulin G
(IgG) was detected at various titers in the animals (Table 2).

During the 3-year period (December 1993 to January 1997), B. clarridgeiae
was recovered from one cat on two occasions, and B. henselae was isolated
from another cat on four occasions. B. vinsonii subsp. berkhoffii was
isolated from the dog on 8 of 10 culture attempts during 16 months of
observation (Table 2). Only one species of Bartonella was identified from
any individual animal. In all three animals, blood cultures performed after
completion of antimicrobial therapy were negative.

Table 1. Animals included in the study

---------------------------------------------------------------------------------
                                                    Date
Animal      Breed(sup a) Sex(sup b)  Birthdate      acquired      Source/state
---------------------------------------------------------------------------------
Cats
A           DSH        	  M/C         10/91        12/91      Pet store/CA
B           DSH      	  F/S         10/90        12/90        Stray/DE
C           DLH      	  F/S          6/92         6/92        Stray/NC
D           DSH      	  M/C          4/93         5/93        Stray/NC
E           DSH      	  F/S          5/93         7/93        Stray/NC
F           DLH      	  M/C          4/94         6/94        Stray/NC
G           DLH      	  F/S          6/95        10/95        Stray/NC
H           MC       	  M/C          6/96         9/96        Family/NC
Dog
I           PM       	  F/S          4/95         6/95        Family/NC
--------------------------------------------------------------------------------
(sup a)DSH, Domestic shorthair; DLH, Domestic longhair; MC, Maine coon; PM,
Pekingese mix.
(sup b)M/C, Male/castrated; F/S, Female/spayed.

When compared with type strains, PCR-RFLP analysis of the 16S rRNA gene and
the 16S-23S ITS region identified the feline isolates as B. clarridgeiae or
B. henselae. Endonuclease digestion of the 16S gene of the isolates from
the dog as well as two isolates from other subclinically bacteremic dogs
produced restriction patterns identical to the type strain of B. vinsonii
subsp. berkhoffii; however, digestion of the ITS region resulted in a
different restriction profile for the subclinically bacteremic dogs
(Figure).

[fig]
Figure. A. Polymerase chain reaction-restriction fragment length
polymorphism (PCR-RFLP) analysis of the 16S rRNA gene of B. vinsonii
subsp. berkhoffii isolates using Dde I demonstrating differentiation
between subspecies of B. vinsonii. B. PCR-RFLP analysis of the
16S-23S intergenic spacer region of B. vinsonii subsp. berkhoffii
isolates using Hae III. Strain differences were detected between B.
vinsonii subsp. berkhoffii (T), which was the type strain cultured
from a dog with endocarditis, and isolates A (described in this
report), B, and C from subclinically infected dogs.


Table 2. Blood culture and immunofluorescence antibody assay
results

------------------------------------------------------------------------
                     Culture      Colony          Serologic results
Date           ID results(sup a) counts/ml  Bh         Bc           Bvb
------------------------------------------------------------------------
12/4/93         A   + (Bh)         97      256   	 32           512
                D                         ND(sup b)   ND            ND
1/15/94         C   + (Bc)         67      <16   	<16           <16
5/25/94         A   + (Bh)         13      128    	 16           256
                C   + (Bc)        113      128   	<16           <16
                D                         256       1024           512
8/19/94         A   cont                   128   	<16           128
                C   + (Bc)        240       64   	<16           <16
9/18/94         F   nd                     <16   	<16           <16
12/9/94(sup c)  A                          32  	<16           128
12/13/94        C                          32   	<16            64
                D                         128       1024           512
5/6/95(sup d)   A                          64   	<16            64
                C   + (Bc)         9        32   	<16            32
                F                         <16   	<16           <16
5/27/95         A                          64   	 16            64
                C                         <16   	<16           <16
                F                         <16   	<16           <16
6/10/95         A                          32   	<16            64
                C                          32   	<16           <16
                F                         <16   	<16           <16
6/22/95         A                          32   	<16           128
                C                         <16   	<16            32
                F                         <16   	<16           <16
7/30/95         A                          32   	<16           256
                C                          32   	<16           <16
                F                         <16   	<16           <16
11/19/95        A                          16   	<16           256
                B                         <16   	<16            32
                C                          32   	<16            32
                D                          64   	<16            64
                E                         <16   	<16           256
                F                         <16   	<16            32
                G                          32   	<16           128
                I   + (Bvb)      >1000     128   	<16           512
2/25/96         I   + (Bvb)       369       64   	<16           256
3/24/96         I   + (Bvb)       164       32   	<16           256
4/21/96         I   + (Bvb)       154       16   	<16           128
6/22/96         I   + (Bvb)       357       16   	<16           128
8/24/96         I   + (Bvb)       12       <16   	<16            64
10/21/96        I                         <16   	<16            64
12/18/96        I   + (Bvb)        6        16   	<16            64
1/22/97         student            nd       32   	 16            32
1/14/97         A                          16   	<16           128
                B                         <16   	<16           <16
                C                         <16   	<16            16
                D                          64   	<16            64
                G                         <16   	<16            32
                H                          64   	<16           256
1/30/97(sup e)  I   + (Bvb)        1        16   	<16            64
3/21/97         I                          16   	<16            32
--------------------------------------------------------------------------
(sup a)Bh, B. henselae; Bc, B. clarridgeiae; Bvb, B. vinsonii
subsp. berkhoffii; cont, contaminated; nd, not done.
(sup b)ND, no data.
(sup c)Four weeks before sample was drawn, the cat received
amoxicillin clavulanate (62.5 mg q12h, for 10 days) for the
treatment of an oral abscess.
(sup d)After collection of blood samples, these animals received
enrofloxacin at 22.7 mg q12h for 28 days.
(sup e)After collection of blood samples, the dog received 28 days
of doxycycline hyclate at 25 mg q12h for 28 days.

We did not detect bacteremia temporally associated with exposure to known
bacteremic animals in any pet newly introduced to the household. Chomel et
al. (7) reported that as few as five infected fleas could transmit B.
henselae infection between cats, but normal daily contact between infected
and uninfected cats in ectoparasite-free surroundings was insufficient to
produce bacteremia in the uninfected cats. Despite their immunologic status
(seronegative) and the occasional presence of fleas, some cats in this
household did not seroconvert or become bacteremic after continued exposure
to infected resident animals. Although we have observed that infection with
B. henselae is not cross-protective against B. clarridgeiae (Kordick and
Breitschwerdt, unpub. data) and that dual infection with B. clarridgeiae
and B. henselae can occur in cats (2,6), similar data are not available
with regard to B. vinsonii subsp. berkhoffii. B. vinsonii subsp. berkhoffii
bacteremia has not been reported in cats. Intradermal injection of B.
henselae into dogs has induced seroconversion but not bacteremia (14). It
is therefore not surprising that B. vinsonii subsp. berkhoffii was not
recovered from any of the cats in the study or, conversely, that B.
henselae or B. clarridgeiae were not recovered from the dog.

Interpretation of Bartonella IFA serologic results for species
identification has been compromised because of presumed cross-reactivity.
The dog in this study never seroconverted to B. clarridgeiae or B.
henselae; however, the cat infected with B. henselae had a consistently
elevated level of antibodies reactive against B. vinsonii subsp. berkhoffii
antigen. The issue of serologic cross-reactivity among Bartonella species
deserves additional study.

Subtyping of bacteria after endonuclease digestion of the 16S-23S rRNA ITS
region has been performed by other investigators because the spacer region,
not appearing to encode functional proteins, demonstrates sequence
variability in an operon that has been useful for taxonomic classification
(15,16). In this study, different restriction profiles were obtained from
isolates derived from asymptomatic dogs compared with the type strain
obtained from a dog with endocarditis. Since our sample size was small,
further examination of these isolates by pulsed-field gel electrophoresis
or bacteriophage analysis may determine whether PCR-RFLP analysis is
epidemiologically useful in predicting the pathogenicity of different
strains.

The pet owner (student) remained healthy throughout the study; however, she
reported that 2 years before the study she had debilitating fatigue of 1
month duration without fever or lymphadenopathy. At the time, she lived in
California and had recently acquired Cat A. A mononucleosis test was
negative; her physician suspected a viral infection. Whether the fatigue
was related to Bartonella exposure (CSD) or the cat was bacteremic is not
possible to determine retrospectively; however, the cat was bacteremic with
B. henselae when cultured 2 years later as part of another study (1).
Bartonella transmission from dogs and cats to humans appears to be
relatively inefficient. It is not surprising, therefore, that despite
continuous exposure to three Bartonella species, the student's serum sample
was minimally reactive to Bartonella antigens.

Acknowledgments

The authors thank Jennifer Goetz for her cooperation in obtaining samples
for this study and Craig E. Greene and David A. Bemis for providing their
canine isolates of B. vinsonii subsp. berkhoffii for analysis. Financial
support was provided by Pfizer Animal Health (Exton, PA), Bayer AG
(Leverkusen, Germany), and the State of North Carolina.

Address for correspondence: Edward B. Breitschwerdt, North Carolina State
University, College of Veterinary Medicine, Department of Companion Animal
and Special Species Medicine, 4700 Hillsborough Street, Raleigh, NC 27606,
USA; fax: 919-829-4336; e-mail: ed_breitschwerdt@ncsu.edu.

References

  1. Kordick DL, Wilson KH, Sexton DJ, Hadfield TL, Berkhoff MA,
     Breitschwerdt EB. Prolonged Bartonella bacteremia in cats associated
     with cat-scratch disease patients. J Clin Microbiol 1995;33:324-551.
  2. Bergmans AMC. Cat scratch disease: studies on diagnosis and
     identification of reservoirs and vectors. [dissertation]. Utrecht, The
     Netherlands: Utrecht Univ.; 1996.
  3. Abbott RC, Chomel BB, Kasten RW, Floyd-Hawkins KA, Kikuchi Y, Koehler
     JE, Pedersen NC. Experimental and natural infection with Bartonella
     henselae in domestic cats. Comp Immunol Microbiol Infect Dis
     1997;20:41-51.
  4. Jameson P, Greene C, Regnery R, Dryden M, Marks A, Brown J, et al.
     Prevalence of Bartonella henselae antibodies in pet cats throughout
     regions of North America. J Infect Dis 1995;172:1145-9.
  5. Heller R, Artois M, Xemar V, DeBriel D, Gehin H, Jaulhac B, et al.
     Prevalence of Bartonella henselae and Bartonella clarridgeiae in stray
     cats. J Clin Microbiol 1997;35:1327-31.
  6. Kordick DL, Hilyard EJ, Hadfield TL, Wilson KH, Steigerwalt AG,
     Brenner DJ, Breitschwerdt EB. Bartonella clarridgeiae, a newly
     recognized zoonotic pathogen causing inoculation papule, fever and
     lymphadenopathy (cat scratch disease). J Clin Microbiol 1997;35:181-38.
  7. Chomel BB, Kasten RW, Floyd-Hawkins K, Chi B, Yamamoto K,
     Roberts-Wilson J, et al. Experimental transmission of Bartonella
     henselae by the cat flea. J Clin Microbiol 1996;34:1952-6.
  8. Anderson BE, Neuman MA. Bartonella spp. as emerging human pathogens.
     Clin Microbiol Rev 1997;10:203-19.
  9. Baker JA. A rickettsial infection in Canadian voles. J Exp Med
     1946;84:37-50.
 10. Breitschwerdt EB, Kordick DL, Malarkey DE, Keene B, Hadfield TL,
     Wilson K. Endocarditis in a dog due to infection with a novel
     Bartonella subspecies. J Clin Microbiol 1995;33:154-60.
 11. Kordick DL, Swaminathan B, Greene CE, Wilson KH, Whitney AM, O'Connor
     SP, et al. Bartonella vinsonii subsp. berkhoffii subsp. nov., isolated
     from dogs; Bartonella vinsonii subsp. vinsonii and emended description
     of Bartonella vinsonii. Int J Syst Bacteriol 1996;46:704-9.
 12. Pappalardo BL, Correa MT, York CC, Peat CY, Breitschwerdt EB.
     Epidemiologic evaluation of the risk factors associated with exposure
     and seroreactivity to Bartonella vinsonii in dogs. Am J Vet Res
     1997;58:467-71.
 13. Kordick DL, Papich MG, Breitschwerdt EB. Efficacy of enrofloxacin or
     doxycycline for treatment of Bartonella henselae or Bartonella
     clarridgeiae infection in cats. Antimicrob Agents Chemother
     1997;41:2448-55.
 14. Chomel BB. Cat-scratch disease and bacillary angiomatosis. Rev Sci
     Tech 1996;15:106173. 
 15. Matar GM, Swaminathan B, Hunter SB, Slater LN,
     Welch DF. Polymerase chain reaction-based restriction fragment length
     polymorphism analysis of a fragment of the ribosomal operon from
     Rochalimaea species for subtyping. J Clin Microbiol 1993;31:1730-4.
 16. Frothingham R, Wilson KH. Sequence-based differentiation of strains in
     the Mycobacterium avium complex. J Bacteriol 1993;175:2818-25.


Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/kordick.htm

Please note that figures and equations are not available in ASCII format; 
their placement within the text is noted by [fig] and [eq], respectively. 
Greek symbols are spelled out. The following codes are used: 
(ft) for footnote; (sup) for superscript; (sub) for subscript; 
/= for greater than or equal to. 

-------------------------------------------------------------------------------------------
-------------------------------------------------------------------------------------------

[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Dispatches

Using Nurse Hot Line Calls for Disease Surveillance

Jane Somsel Rodman,* Floyd Frost,* and Walter Jakubowski
*The Southwest Center for Managed Care Research, Albuquerque, New Mexico,
USA; and U.S. Environmental Protection Agency, Cincinnati, Ohio, USA

  --------------------------------------------------
      Nurse hot line calls are a potential source of public health
      surveillance data and may help identify epidemics of emerging
      infectious diseases. In this study, nurse hot line data from
      Milwaukee, Wisconsin, showed more than a 17-fold increase in
      calls for diarrhea during the 1993 Milwaukee cryptosporidiosis
      outbreak. Moreover, consistent patterns of seasonal variation
      in diarrhea- and vomiting-related calls were detected from the
      Baltimore, Maryland, and Albuquerque, New Mexico, hot lines.
      Analysis of nurse hot line calls may provide an inexpensive and
      timely method for improving disease surveillance.

Most disease outbreaks are identified by an alert health care provider or
citizen (1-3); some are identified by analyses of laboratory-confirmed
reportable diseases (4,5)-in these outbreaks, because of delays in specimen
collection and analysis, laboratory confirmation is often received by
public health agencies days to weeks after the onset of illness, and
consequently, outbreak investigations are delayed. Some outbreaks may not
be detected at all because of limitations of the disease surveillance
system (6). Several new surveillance methods for more timely detection of
outbreaks have been proposed (6,7). Among these is the analysis of calls to
nurse hot lines, also known as nurse call lines.

During the past 5 years, computerized hot line systems for sorting patient
calls have been developed and marketed. Patient calls concerning illness or
injury are received by trained nurses, who categorize symptoms and provide
advice on the basis of approved protocols. The computerized hot line
systems integrate structured interview, record keeping, and treatment
protocol. These systems have been expanding-one nurse hot line vendor alone
has installed more than 240 systems throughout the United States and
Canada. These systems have been highly successful not only because they
provide patient services, but also because they save physicians' time
(8-10).

During the structured interview, nurses follow symptom-based guidelines
developed by the software vendors and modified and approved by local
physicians. As part of the interview process, information about the illness
and the patient is computerized and can usually be retrieved with minimal
cost. For example, two hot line systems in Albuquerque, New Mexico, receive
more than 50,000 calls each per year; the systems' software can provide
summary reports of calls. A major advantage of nurse hot line systems for
disease surveillance is, therefore, their ability to provide timely
information from a large number of callers. Disadvantages are that hot
lines provide symptom-based rather than pathogen-based reports and that
persons who call several times can be counted as separate callers. For hot
lines connected with health maintenance organizations, the data may be
expressed in terms of the population, whereas call systems that serve an
unknown number of clients cannot.

Our study, which provides preliminary data on the potential value of nurse
hot line data for disease surveillance, had two specific goals: to evaluate
whether nurse hot line calls for diarrhea-related illness during the 1993
Milwaukee cryptosporidiosis outbreak increased and could have identified
the outbreak earlier than other methods and to evaluate whether nurse hot
line calls for diarrheal diseases reflected an expected seasonal variation.

We telephoned directors of three nurse hot lines: Covenant Healthcare
System (CHS), Milwaukee, Wisconsin; Lovelace Health Hot Line (LHH),
Albuquerque, New Mexico; and Helix Telehealth Center (THC), Baltimore,
Maryland; they agreed to provide hot line call data at no cost. CHS and THC
used the Ask-A-Nurse system, which defined diarrhea as a sudden increase in
number and volume of bowel movements causing rapid evacuation of water and
electrolytes. When the caller described multiple symptoms, detailed
guidelines were used to choose the major symptom, and the call was
documented in only one category. LHH used the Sharp Focus System and
defined diarrhea as five or more watery stools every 6 hours. Records of
daily CHS adult and pediatric (age < 13 years) diarrhea-related calls for
two Milwaukee hospitals were examined for the period March 4 to May 10,
1993, to determine whether the number of calls increased during the
cryptosporidiosis outbreak (11). Hot line calls to LHH and THC from July
1994 to July 1996 for pediatric (ages < 13 years) diarrhea and adult (ages
> 18 years) vomiting were plotted for each month. Temporal patterns of
calls for respiratory conditions and skin rashes were also examined.

From March 1 to April 1, 1993, and from April 25 to May 10, 1993, the CHS
hot line in Milwaukee received an average of 0.66 adult diarrhea calls per
day. An increase in diarrhea calls more than four standard deviations
higher than the background rate (p < 0.001) was noted on April 2 (Figure
1), 4 days before a television news story about dramatic increases in cases
of diarrhea (7). The number of calls remained high through April 16, with a
peak in adult calls on April 10, 3 days after the Milwaukee Health
Department reported the outbreak. Adult diarrhea calls peaked at more than
17 times the average number of calls received per day before and after the
outbreak and returned to this number on April 25. Pediatric
diarrhea-related calls increased from 18 in March to 38 in April and
declined to 17 in May and 11 to 14 per month from June through August (not
shown).

[fig 1]
Figure 1. Calls to Covenant Healthcare System hot line, Milwaukee,
Wisconsin, March 1May 10, 1993.



Analysis of the calls by zip code indicated that most callers lived near
the two hospitals (located in the north and northwest parts of the city),
which were least affected by the outbreak. These data may not represent
illness rates in different parts of the city.

To determine whether hot line calls reflect seasonal disease patterns,
adult vomiting- and pediatric diarrhea-related calls to the THC and LHH hot
lines were plotted for 24 consecutive months (Figure 2). Each condition
showed a strong seasonal pattern in both health-care systems. Adult
vomiting-related calls to LHH (Figure 2) and to THC (Figure 2) showed
increases during November and December. Pediatric diarrhea calls to LHH
showed a major increase from October to December each year (Figure 2),
whereas calls to THC peaked from February to May (Figure 2). Respiratory
conditions peaked in the winter, and skin rashes peaked in the summer (not
shown).

[fig 2]
Figure 2. Seasonal variation of calls to Lovelace Health Hot Line (LHH) and
Helix Telehealth (THC), 1994-1996.



The diarrheal disease outbreak in Milwaukee (first noticed by a pharmacist
on April 1, 1993 [7], and reported by the Milwaukee Health Department on
April 7 [11]), was the largest reported waterborne outbreak in U.S.
history, affecting almost half the city's population. Nurse hot line data
showed an increase in diarrhea-related calls on April 2, a day after the
pharmacist noted an increase in antidiarrhea drug sales and 5 days before
the health department reported the outbreak. Media exposure on April 4 did
not affect the initial increase detected by hot line data but may have
affected the peak on April 10. The hot lines detected an increase in
diarrhea even though almost all the callers lived in neighborhoods least
affected by the outbreak. Since hot lines serve the areas in which they are
located, no citywide analysis of disease patterns was possible.

The collection of hot line data was passive, but because reports came
regularly, no data were missed. Unlike systems monitoring school
absenteeism or nursing home illnesses, nurse hot lines provide
symptom-based illness reports. Although they do not identify specific
pathogens, hot line calls can provide a sentinel of increased illnesses for
various conditions, including diarrhea, vomiting, and respiratory illness.
Information on symptoms can be obtained rapidly.

The dramatic increase in the number of hot line calls during the Milwaukee
cryptosporidiosis outbreak indicates that call volume may prospectively
identify an outbreak. If hot lines serve a large area, they could also
provide geographic information on the populations affected. As use of hot
line data becomes more established, better estimates of expected daily and
monthly variation in call volume should emerge, making detection of excess
illness easier. Analysis of another outbreak may confirm the results of our
study. However, a survey of state public health epidemiology programs found
that no state uses or plans to use nurse hot line information for disease
surveillance (12). With the decline in resources for state disease
surveillance programs (13), nurse hot lines can inexpensively enhance
existing surveillance programs.

Acknowledgments

The research described in this article has been supported by the U.S.
Environmental Protection Agency through assistance agreement CR 824227 to
The Lovelace Institutes.

Jane S. Rodman, postdoctoral research associate, Southwest Center for
Managed Care Research, Albuquerque, New Mexico, has conducted research on
cell biology structure and function and serologic surveillance of
infectious diseases. Her areas of expertise include cell biology methods,
Cryptosporidium, and viral serology tools.

Address for correspondence: Floyd Frost, Southwest Center for Managed Care
Research, 2425 Ridgecrest Drive SE, Albuquerque, NM 87108, USA; fax:
505-262-7043; e-mail; ffrost@ed.lrri.org.

References

1. Kirner JC, Littler JD, Angelo LA. A waterborne outbreak of giardiasis in
   Camas, Wash. Journal of the American Water Works Association 1978;70:35-40.
2. Foster LR. Surveillance for waterborne illness and disease reporting:
   state and local responsibilities. In: Craun GF, editor. Methods for the
   investigation and prevention of waterborne disease outbreaks. Washington:
   U.S. EPA Office of Research and Development; 1990. Report No.:
   EPA/600/1-90/005a.
3. Karalekas PC, Taylor FB. Regulations and surveillance. In: Craun GF,
   editor. Waterborne diseases in the United States. Boca Raton (FL): CRC
   Press; 1986.
4. Goldstein ST, Juranek DD, Ravenholt O, Hightower AW, Martin DG, Mesnik
   JL, et al. Cryptosporidiosis: an outbreak associated with drinking water
   despite state-of-the-art water treatment. Ann Intern Med 1996;124:459-68.
5. Leland D, McAnulty J, Keene W, Sterens G. A cryptosporidiosis outbreak
   in a filtered-water supply. Journal of the American Water Works Association
   1993;85:34-42.
6. Frost FJ, Craun CF, Calderon RL. Waterborne disease surveillance.
   Journal of the American Water Works Association 1996;88:66-78.
7. Rodman JS, Frost F, Davis-Burchat L, Fraser D, Langer J, Jakubowski W.
   Pharmaceutical sales-a method of disease surveillance. Journal of
   Environmental Health 1997;60:8-14.
8. Papzian R. Hold the phone. Harvard Health Letter 1992;18:1-3.
9. Glasoper A, McGrath K. Telephone triage: extending practice. Nursing
   Standard 1993;7:34-6.
10. Curran CR. An interview with Sandra C. Matherly and Shannon Hodges.
    Nursing Economics 1995;3:125-8.
11. MacKenzie WR, Hoxie JN, Proctor ME, Gradus MS, Blair KA, Peterson DE,
    et al. A massive outbreak in Milwaukee of cryptosporidium infection
    transmitted through the public water supply. N Engl J Med 1994;331:161-7.
12. Frost FJ, Calderon RL, Craun GL. Waterborne disease surveillance:
    findings of a survey of state and territorial epidemiology programs.
    Journal of Environmental Health 1995;58:6-11.
13. Berkelman RL, Bryan RT, Osterholm MT, LeDuc JW, Hughes JM. Infectious
    disease surveillance: a crumbling foundation. Science 1994;264:368-70.


Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/rodman.htm

Please note that figures and equations are not available in ASCII format; 
their placement within the text is noted by [fig] and [eq], respectively. 
Greek symbols are spelled out. The following codes are used: 
(ft) for footnote; (sup) for superscript; (sub) for subscript; 
/= for greater than or equal to. 


-------------------------------------------------------------------------------------------
-------------------------------------------------------------------------------------------

[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Commentary

Vector-borne Disease Surveillance and Natural Disasters

Roger S. Nasci and Chester G. Moore
Centers for Disease Control and Prevention, Fort Collins, Colorado 80522,
USA

Natural disasters, such as hurricanes and floods, are frequently followed
by a proliferation of mosquitoes and requests from residents and government
agencies for widespread application of insecticides. However, nuisance
mosquito species, which often require emergency control measures, rarely
present a threat to public health (1). Natural disasters in the continental
United States have rarely been accompanied by epidemics of
mosquito-transmitted disease (2). Nevertheless, public health disaster
response policies should include a provision for monitoring increases in
the prevalence of potentially infectious mosquitoes and the risk for
arboviral disease in the affected areas.

Four major arboviruses (viruses transmitted by mosquitoes or other
arthropods) are of human and veterinary public health importance in the
United States: eastern equine encephalomyelitis (EEE), western equine
encephalomyelitis (WEE), St. Louis encephalitis (SLE), and LaCrosse (LAC)
encephalitis. Because of its unique ecology, LAC virus does not occur in an
epidemic form and would not increase as a result of floods or hurricanes.
The three viruses of primary concern (EEE, SLE, WEE) overlap in their
distribution, but each has a distinct ecology, involving different mosquito
species and avian amplifier hosts (3)(sub 1). Despite these differences,
populations of primary or secondary vector species of each virus may
increase significantly in response to heavy rainfall or flooding.
Therefore, under certain circumstances, disasters might produce increases
in disease risk.

Several major floods and hurricanes have been evaluated since 1975 (Table).
The major geographic and ecologic regions of the United States, some of
which contain each of the common epidemic arboviruses (EEE, SLE, WEE), are
included. With the exception of the Red River flood of 1975, disasters have
not increased transmission of arboviruses to humans or domestic animals.
The single case of EEE associated with Hurricane Fran in 1996 may have been
acquired before the hurricane. Similarly, cases of EEE and WEE in domestic
animals, especially horses and emus, do not appear to increase after floods
or hurricanes (SLE does not cause disease in most domestic animals).

Despite the fact that epidemics of arboviral encephalitis have rarely
followed hurricane- or flood-related disasters in the United States, these
events can increase the risk for human arboviral disease in certain
circumstances. In nine of the ten events in which surveillance has been
conducted (Table), arbovirus activity was detected in surveillance programs
initiated after the event, which indicates the existence of an enzootic
transmission cycle in the area. Under these circumstances, increases in
vector mosquito population density can enhance transmission and increase
the prevalence of potentially infectious mosquitoes, which in turn
increases the risk for human disease, particularly when coupled with
expanded human exposure to mosquitoes after a disaster (e.g., residents and
recovery workers removing debris, restoring housing, and living in
substandard conditions).

Public health policies dealing with flood-related disasters must include
provisions addressing mosquito-transmitted disease. Preferably, data from
an existing arbovirus surveillance program can be evaluated to determine
the status of transmission cycles before the event and determine the risk
for disease. For example, California's long-term surveillance has
established a baseline to which post-disaster virus activity can be
compared (4-7). After the 1994-95 winter flood in California, Moore (unpub.
data) used those data to determine that WEE seroconversion rates in
sentinel chickens did not differ from rates in previous years. However, in
most hurricane- and flood-prone areas, routine surveillance is not
conducted.

 Table. Natural disasters in the continental United States since 1975(sub a)

 ---------------------------------------------------------------------------------------------
       State/                    Surveillance Activity       Human         Veterinary
 Year  region     Event             done?     detected?      cases         cases
 ---------------------------------------------------------------------------------------------
 1975  N.D.,      Red River          Yes      WEE(sub b)in   55 WEE,       281 WEE(estimated)
       Minn.      flood                       mosquitoes     12 SLE(sub c)                                           
 
 1989  S.E.       Hurricane          Yes      EEE(sub d)in   None          (No data)
       United     Hugo                        mosquitoes
       States  
 1992  Fla., La.  Hurricane          Yes      None           None           None
                  Andrew
 1993  Ariz.      Gila River         Yes      SLE, WEE       None           None
                  flood                        in
                                               mosquitoes
 1993  Mid-       Mississippi,       Yes      WEE - S.D.,    None           None
       western,   Missouri        (7 states)  SLE - Il.
       United     river
       States,    flooding
 1994  Ala.,      Tropical           Yes      EEE-Al, Fl     None           EEE in horses,
       Fla.,      storm Alberto                                               emus -
       Ga                                                                     Al, Fl
 1995  Calif.     Winter &           Yes      WEE, SLE       None           WEE
                  spring floods               in sentinel
                                              flocks
 1996  Calif.     Winter flood       Yes      WEE, SLE       None           None
                                              in chickens,
                                              WEE in
                                              mosquitoes
 1996  Oregon,    Winter flood        No      No            None            None
       Wash.
 1996  N.C.       Hurricane          Yes      EEE in        1 EEE           EEE in horses
                  Fran                        mosquitoes
 1997  Colo.      Summer floods      Yes      WEE in        None            None
                                              chickens
 1997  N.D.,      Red River        Sporadic   None          None            None
       Minn.      flood                       reported
 -----------------------------------------------------------------------------------------
 (sub a)Surveillance data collected by the Division of Vector-Borne Infectious
 Diseases, Centers for Disease Control and Prevention (CDC). State and local
 health departments assisted during emergency response. Federal Emergency
 Management Agency and Emergency Response Coordination Group, National
 Centers for Environmental Health, CDC, provided field support.
 (sub b) Western equine encephalitis
 (sub c) St. Louis encephalitis
 (sub d) Eastern equine encephalitis

In the absence of an existing program, arbovirus surveillance should be
initiated as part of the disaster response plan. At the very least, these
programs should determine population indexes of key vector species, virus
infection rates in those species, and seroprevalence in sentinel or wild
animals. Additional useful information includes prevailing weather
conditions, time of year, human exposure, human population at risk,
historical patterns of arboviral disease, and the potential for imported
diseases (3).

(sub 1)Additional information may be obtained at
http://www.cdc.gov/ncidod/dvbid/arbor/arboinfo.htm

References

  1. Centers for Disease Control and Prevention. Emergency mosquito control
     associated with Hurricane Andrew-Florida and Louisiana, 1992. MMWR
     1993;42:240-2.
  2. Centers for Disease Control and Prevention. Rapid assessment of
     vectorborne diseases during the Midwest flood-United States, 1993.
     MMWR 1994;43:481-3.
  3. Moore, C.G., R.G. McLean, C.J. Mitchell, Nasci RS, Tsai TF, Calisher,
     CH, et al. Guidelines for arbovirus surveillance programs in the
     United States. Fort Collins, CO: U.S. Dept. of Health and Human
     Services, Centers for Disease Control and Prevention; 1993.
  4. Emmons RW, Ascher MS, Dondero DV, Enge B, Milby MM, Hui LT, et al.
     Surveillance for arthropod-borne viral activity and disease in
     California during 1991. In: Proceedings of the California Mosquito and
     Vector Control Association; 1992.
  5. Emmons RW, Ascher MS, Dondero DV, Enge B, Reisen WK, Milby MM, et al.
     Surveillance for arthropod-borne viral activity and disease in
     California during 1992. Proceedings of the California Mosquito and
     Vector Control Association; 1993.
  6. Emmons RW, Ascher MS, Dondero DV, Enge B, Reisen WK, Milby MM, et al.
     Surveillance for arthropod-borne viral activity and disease in
     California during 1993. Proceedings of the California Mosquito and
     Vector Control Association; 1994.
  7. Reeves WC. Epidemiology and control of mosquito-borne arboviruses in
     California, 1943-1987. Sacramento, CA: California Mosquito and Vector
     Control Association; 1990.

Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/nasci.htm

Please note that figures and equations are not available in ASCII format; 
their placement within the text is noted by [fig] and [eq], respectively. 
Greek symbols are spelled out. The following codes are used: 
(ft) for footnote; (sup) for superscript; (sub) for subscript; 
/= for greater than or equal to. 

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[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

 Letters

  --------------------------------------------------

Similarity of Chemokines Charge and the V3 Domain of HIV-1 env Protein

To the Editor: Most clinical HIV-1 isolates can infect CD4+ peripheral
blood T lymphocytes, monocytes, and cultured macrophages (macrophage or
M-tropic) but not transformed T-cell lines. In contrast, HIV-1 strains
adapted for growth in transformed T-cell lines (T-cell line or T-tropic) do
not infect primary monocytes or macrophages. This difference in tropism
appears to be a consequence of specific amino acid changes in the env
protein. Changes in env responsible for an M- to T-tropism shift often
involve the acquisition of multiple positively charged residues in the
hypervariable V3 loop domain (1). However, some non-V3 determinants are
also important for viral tropism. Although both types of viruses use CD4 as
receptor, the CXCR4 chemokine receptor (previously designated LESTR/fusin)
is the unique cofactor for entry of T-tropic HIV-1 strains (2). The CCR5
chemokine receptor was subsequently demonstrated to be the cofactor for
M-tropic HIV-1 isolates (3). Although some direct evidence for cell surface
association of the CD4-env complex and the CXCR4 coreceptor was obtained
(4), little detail is available on the molecular forces responsible for
these protein-protein interactions. In particular, there is no direct
evidence to indicate that the V3 loop binds to the chemokine receptor.

Jiang (5) reported that the extracellular domain of the CXCR4 coreceptor
for T-tropic HIV-1 isolates is more negatively charged than the CCR5
coreceptor. Because T-tropic isolates have evolved a positively charged V3
domain, it was suggested that coreceptor-env binding involves the
interaction between oppositely charged residues. We now expand this
analysis by showing that the chemokines corresponding to the different
receptors have a similarly unbalanced composition of charged amino acids.
So far, the CXCR4 receptor has been demonstrated to bind only the SDF-1
chemokine (6). The CCR5 receptor binds more than one chemokine, including
RANTES, MIP-1[alpha.gif (53 bytes)] , and MIP-1 (7). The amino acid
sequence of these four chemokines is presented in the Figure. We listed the
number of positive residues (arginine [R] and lysine [K]) and negative
residues (aspartic acid [D] and glutamic acid [E]), and calculated the net
charge. SDF-1 appears to have the highest number of positive residues and
the lowest number of negative residues, resulting in a net charge of +11.
All other chemokines have much less positively charged amino acids,
resulting in a net charge for MIP-1[alpha.gif (53 bytes)] and MIP-1 of -2
and -1, respectively. The RANTES chemokine has an intermediate charge of
+7, which may correlate with the unique receptor use of this chemokine
(e.g., RANTES, but not MIP-1[alpha.gif (53 bytes)] and MIP-1, binds the
CCR3 receptor [7]). These results are consistent with the idea that
positive charges in SDF-1 interact with negative charges in the CXCR4
receptor, and this binding may thus resemble the HIV-1 env-CXCR4
interaction.

 [fig]
 Figure. Amino acid alignment of the four chemokines was performed with
 the PC/Gene program. An overall identity and similarity of 12.4% and
 40.2% was calculated, respectively. The initiator methionine contained
 within each sequence is removed in the processing of the chemokine. The
 SDF-1 form shown is the -form; the alpha-form lacks the
 C-terminal amino acids RFKM, thus reducing the net charge to +9.
 * = a perfectly conserved residue.
 . = a conservative change.

Early evidence that both the chemokines and HIV-1 bind to the same domain
of the chemokine receptor comes from virus inhibition studies. Several
-chemokines suppress infection with M-tropic but not T-tropic HIV isolates
(8), and SDF-1 specifically blocks entry of T-tropic isolates (6). Although
direct blocking of the receptor may explain part of this chemokine-mediated
inhibition, it has also been proposed that internalization of the receptor
contributes to the antiviral effect (9). Irrespective of the precise
antiviral mechanism, the combined results of this analysis and the one
presented by Jiang (5) suggest that both the SDF-1 chemokine and the V3
loop of T-tropic HIV-1 viruses use positively charged amino acids for an
electrostatic interaction with the negatively charged CXCR4 receptor. To
examine whether the similarity between the chemokine and env V3 domain is
also apparent at the primary sequence level, we performed an amino acid
alignment; however, we found no conserved motifs (data not shown). A
detailed mutational analysis is required to further our understanding of
the env-coreceptor interaction.

Ben Berkhout and Atze T. Das
Department of Human Retrovirology, Academic Medical Center, University of
Amsterdam, Amsterdam, The Netherlands (b.berkhout@amc.uva.nl)

References

  1. De Jong J-J, Ronde de A, Keulen W, Tersmette M, Goudsmit J. Minimal
     requirements for the human immunodeficiency virus type 1 V3 domain to
     support the syncytium-inducing (SI) phenotype: analysis by single
     amino acid substitution. J Virol 1992;66:6777-80.
  2. Feng Y, Broder CC, Kennedy PE, Berger EA. HIV-1 entry cofactor:
     functional cDNA cloning of a seven-transmembrane, G protein coupled
     receptor. Science 1996;272:872-7.
  3. Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, et al.
     Identification of a major co-receptor for primary isolates of HIV-1.
     Nature 1996;381:661-6.
  4. Lapham CK, Ouyang J, Chandrasekar B, Nguyen NY, Dimitrov DS, and
     Golding H. Evidence for cell-surface association between fusin and the
     CD4-gp120 complex in human cell lines. Science 1996;274:602-5.
  5. Jiang S. HIV-1 co-receptors binding. Nat Med 1997;3:367-8.
  6. Oberlin E, Amara A, Bachelerie F, Bessia C, Virelizier J-L,
     Arenzana-Seisdedos F, et al. The CXC chemokine SDF-1 is the ligand for
     LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1.
     Nature 1996;382:833-5.
  7. Premack BA, Schall TJ. Chemokine receptors: gateways to inflammation
     and infection. Nat Med 1996;2:1174-8.
  8. Cocchi F, DeVico AL, Garzino-Demo A, Arya SK, Gallo RC, Lusso P.
     Identification of RANTES, MIP-1 alpha and MIP-1 beta as the major
     HIV-suppressive factors produced by CD8+ T cells. Science
     1995;270:1811-5.
  9. Amara A, Le Gall S, Schwartz O, Salamero J, Montes M, Loetscher P, et
     al. HIV coreceptor downregulation as antiviral principle:
     SDF-1a-dependent internalization of the chemokine receptor CXCR4
     contributes to inhibition of HIV replication. J Exp Med
     1997;186:139-46.

  --------------------------------------------------

Detection of Glycoprotein of Burkholderia pseudomallei

To the Editor: Melioidosis, a potentially fatal disease that is difficult
to diagnose and treat, is common in areas with subtropical climate (e.g.,
Singapore, the southern provinces of China) and is hyperendemic in
Thailand. The etiologic agent, Burkholderia pseudomallei (Pseudomonas
pseudomallei), is widely distributed in Southeast Asia and northern
Australia. The agent has the potential to become established in regions
with similar climate conditions, particularly if animals infected with B.
pseudomallei are imported from endemic-disease zones (1-3).

Rapid and reliable detection of B. pseudomallei and its antigens has many
potential applications. Recently, we developed a monoclonal antibody
immunoenzyme test system for the detection of minimal concentrations of a
B. pseudomallei glycoprotein, which is considered one of the pathogenicity
factors for this microorganism. This glycoprotein, called Ag8 by N.N. Piven
and V.I. Ilyukhin (4), is present in different strains of B. pseudomallei
and B. mallei but not in other Burkholderia spp. (B. aeruginosa, B. putida,
B. cepacia, B. malthophilia, B. fluorescens, B. pseudoalcaligenes). Ag8 is
composed of 10% protein and 90% carbohydrate, has molecular mass 800 kDa,
and is localized in an extracellular capsulelike substance surrounding B.
pseudomallei cells (5).

We developed an immunoenzyme test system with three monoclonal antibodies
(Mab) to different epitopes of Ag8 (Mab 2A6-IgG3, Mab 2H7-IgG1, Mab
1G2-IgG2b) and one antibody to epitopes common for Ag8 and LPS of B.
pseudomallei (mab 1ES-IgG2b). A sandwich enzyme-linked immunosorbent assay
(ELISA) was used for the detection of Ag8 in different test samples (6).
The sensitivity of the immunoenzyme test system was determined with a
standard antigen sample. Minimal sensitivity (37 ng/ml of carbohydrate) was
observed when polyclonal immunoglobulins were used as "catching"
antibodies. Maximal sensitivity (0.37 ng/ml of carbohydrate) was noted when
either Mabs 2A6 or mixtures of Mabs were used as catching antibodies.

The test system was further evaluated with samples of extracellular
antigens (extracts of cultural media, fractions after gel chromatography of
extracellular antigens) and bacterial suspensions of B. pseudomallei and B.
mallei strains isolated in different regions of the world. Levels of Ag8 in
cultural media varied considerably depending on periods of cultivation of
bacteria. Additionally, the level of Ag8 varied among strains of B.
pseudomallei and B. mallei. Among 61 strains of B. pseudomallei from the
museum collection (most of which were isolated in Southeast Asia and
northern Australia), three had increased ability to produce Ag8. These
strains had been isolated from clinical specimens (blood, abscesses of
hospitalized melioidosis patients) in Vietnam. The strains gave results
typical of B. pseudomallei species in all routine serologic tests
(agglutination test, immunofluorescence assay, immunodiffusion test). In
contrast, the B. pseudomallei glanders agent (16 strains from the museum
collection) had reduced ability for Ag8 production; ELISA titers of Ag8
were a thousandfold less in culture fluids in these strains.

The ELISA technique not only facilitates diagnosis of disease but also
provides a rational basis for selecting strains for vaccine production. It
also has considerable utility for studying the pathogenicity of B.
pseudomallei.

Natalja P. Khrapova, Nikolay G. Tikhonov, Yelena V. Prokhvatilova
Volgograd Antiplague Research Institute, Volgograd, Russia

References

  1. Dodin A. La mlioidose: un probleme mondial. Recherche
     1976;7(68):574-7.
  2. Galimand M, Dodin A. Le point sur la melioidose dans le monde. Bull
     Soc Pathol Exot 1982;75,4:375-83.
  3. Larionov GM, Pogasiy NI. A methodology for determination of reservoirs
     of the causative agents of sapronoses. Med Parazitol (Mosk)
     1995;4:45-9.
  4. Piven NN, Ilyukhin VI. Antigenic structure of Pseudomonas
     pseudomallei. Zh Microbiol Epidemiol Immunobiol 1981;4:78-82.
  5. Kapliev VI, Piven NN, Khrapova NP. Determination of antigens on
     melioidosis agents with the use of monoclonal antibodies at the
     ultrastructural level. Mikrobiol Z 1992;54,1:85-9.
  6. Voller A, Bidwell DE, Bartlett A. Enzyme immunoassays in diagnostic
     medicine. Bull World Health Organ 1976;53,1:55-65.

  --------------------------------------------------

Forging New Perspectives on Disease Surveillance

To the Editor: Recognizing disease emergence as a paradigm uniquely
influenced by human activity demands a reevaluation of traditional disease
surveillance systems. Part of a surveillance program should be focused on
the areas of human activity where disease emergence is most likely to
occur. A system that monitors areas known to be involved in disease
emergence, such as development projects, agriculture, climate, and refugee
movements, may greatly increase our ability to detect and prevent
outbreaks.

Large development projects entail ecologic upheavals that can facilitate
disease emergence. Construction of a dam in 1987 in Mauritania resulted in
increased mosquito breeding sites and in an explosion in the mosquito
population; epidemics of Rift Valley fever quickly followed (1). The
Southeastern Anatolia Irrigation Project on the Euphrates and Tigris Rivers
in Turkey, which will provide irrigation for 1.7 million hectares, has
already increased malaria and leishmaniasis cases in the local population
(2). The massive Three Gorges Dam Project on the Yangtze River in China,
which will create a reservoir 760 km long, must be evaluated for its impact
on local disease. With knowledge of endemic diseases and their reservoirs
and vectors in these areas of ecologic change, public health workers can
anticipate disease epidemics and implement prevention measures.

Incorporating climate predictions into a disease surveillance system would
supplement resources in an area known to affect disease emergence. The U.S.
Agency for International Development's (USAID) Famine Early Warning System
monitors the African continent for two major factors implicated in
emergence: temperature and precipitation. Focused on countries at high risk
for food shortages and famine, the early warning system is an example of a
predictive and preventative surveillance system. Precipitation,
temperature, and plant health data from satellites are evaluated as
indicators of crop failure. These data are supplemented by information from
field representatives who directly observe agricultural production. USAID's
system and other global monitoring systems can provide a base level of
surveillance that can add to our knowledge of climatologic influence on
disease emergence.

The beginning of the Zairian refugee crisis in 1994 illustrates the need
for surveillance among refugee populations. In July 1994, 500,000 to
800,000 Rwandan Hutus fled into the North Kivu region of Zaire. In the
month between July 14 and August 14, 48,347 of these refugees died,
predominantly of infectious diseases (3). Similar infectious disease
epidemics occurred among Kurdish refugees in 1991 (4), Somali refugees in
1992 (5), and Burundian refugees in Rwanda in 1993 (6). In the wake of
intense political, social, or physical disruption, the movement of large
numbers of people creates ideal conditions for disease outbreaks. When
moving into new areas, refugees may not be equipped immunologically against
endemic diseases. Most refugee camps are overcrowded, with inadequate
sanitation or medical care (7). Refugees often have severe shock or stress,
which in combination with poor nutrition, weakens immune defenses.
Therefore, refugee populations are extremely vulnerable and should be
closely monitored for infectious disease outbreaks. The United Nations High
Commissioner for Refugees, the International Rescue Committee, and the
Human Rights Watch can provide rapid notification about disease emergence
in refugee populations.

Recognizing human involvement as a common critical factor in emergence
creates the possibility of refining international disease surveillance. The
Centers for Disease Control and Prevention, the World Health Organization,
and national governments should foster relationships with organizations
already placed to provide disease emergence information in populations and
locations implicated in disease emergence. These relationships will
increase the scope and efficiency of our efforts to prevent human disease.

Harley Feldbaum,
Wesleyan University, Middletown, Connecticut, USA

References

  1. Monath T. Arthropod-borne viruses. In: Morse S, editor. Emerging
     viruses. New York: Oxford University Press; 1993. p. 144.
  2. Aksoy S, Ariturk S, Armstrong MYK, Chang KP, Dortbudak Z, Gottlieb M,
     et al. The GAP Project in Southeastern Turkey: the potential for
     emergence of diseases. Emerg Infect Dis 1995;1:62-3.
  3. Goma Epidemiology Group. Public health impact of Rwandan refugee
     crisis: what happened in Goma, Zaire, in July, 1994? Lancet
     1995;345:339-44.
  4. Yip R, Sharp TW. Acute malnutrition and high childhood mortality
     related to diarrhea. JAMA 1993;270:587-90.
  5. Moore PS, Marfin AA, Quenemoen LE, Gessner BD, Ayub YS, Miller DS, et
     al. Mortality rates in displaced and resident populations of central
     Somalia during 1992 famine disaster. Lancet 1993;42:913-7.
  6. Centers for Disease Control and Prevention. Health status of displaced
     persons following civil warBurundi, December 1993-January 1994. MMWR
     Morb Mortal Wkly Rep 1994;43:701-3.
  7. Wilson ME. Travel and the emergence of infectious diseases. Emerg
     Infect Dis 1995;1:39-46.

  --------------------------------------------------

Provide a Context for Disease Emergence

To the Editor: When a disease emerges, the trend is to assume that another
important and spreading infection is about to devastate humans or animals.
Some qualification of the term "emergence" is needed to put emerging
diseases into a context for each target species. There may be a cause for
alarm and further action or, alternatively, no real change except in
knowledge. In Australia, for example, an old disease "emerged" in a new
area, while in another, a disease new to the continent emerged. The two
disease agents were Ross River virus (which causes fever and polyarthritis
in humans) and bluetongue virus (which often causes fatal disease in
sheep). Both causative viruses are insect-borne.

Ross River virus is probably of very ancient lineage as an infection
transmitted between marsupials and indigenous mosquitoes and was on the
Australian continent long before humans first entered (some tens of
thousands of years ago). In 1975, the infection was not known to occur in
Tasmania, the state separated from the Australian mainland by a wide
stretch of sea. In that year, my group detected a clear-cut seroconversion
to Ross River virus in sentinel cows in northern Tasmania (1). Cooperative
investigations found antibody-positive sera first in marsupials and then in
persons who had never left Tasmania. The existing clinical condition of
polyarthritis was linked to Ross River virus only after the causative virus
was recognized indirectly (2). The marsupial populations of mainland
Australia and Tasmania were continuous until the seas rose at the end of
the last ice age. Ross River disease had emerged in Tasmania, but only from
obscurity.

Bluetongue viruses occur widely in southern and eastern Asia (3). This
general picture has been established only since the discovery of bluetongue
virus in Australia. Overt disease occurs in sheep on the fringes of the
endemic-disease region and in susceptible sheep imported into various
countries within the region (3). In contrast, at least eight members of the
bluetongue group of viruses have entered Australia and some of these the
Pacific countries; they must have arrived after ruminant populations were
introduced in New Guinea, Australia, and the Pacific islands. They spread
through the ruminant-dependent Culicoides species vectors, also introduced
(4). Emergence of bluetongue in Australia has so far meant one sheep dead
of bluetongue disease in 1989 (in a population of 100 million sheep) since
the discovery of the presence of bluetongue virus in Australia in 1977 (4).
However, the potential for a major epidemic remains and the discovery
caused major trade difficulties. In both these situations the disease was
emergent but its potential was very different.

Since the latter part of 1994, two newly recognized zoonotic viruses have
been reported from Australia: paramyxovirus (equine morbillivirus), which
caused deaths in horses and humans (5), and lyssavirus (closely related to
rabies virus), which has also caused a human death in Australia (6). In
both instances, strong evidence indicates that bats are the maintenance
hosts. Bats are probably the oldest form of placental mammal in Australia,
with fossil evidence from the Middle Miocene era, circa 15 million years
ago (7). Some species of bats migrate between various countries of southern
Asia and the Pacific; probably they migrated more in the ice ages when sea
distances were shorter. This past continuity of the bat populations and the
inadequate study of the Microchiroptera in Asia for rabies viruses led me
to forecast in 1989 that rabies (or a rabieslike virus) was established in
bats in Australia (8). These recently recognized disease agents are both
emergent but have not become important for humans or animals.

As a hypothetical example, Ebola virus has been shown by Swanepoel and
others to multiply well in bats of three species (9). Two of the species
used experimentally belong to the genus Tadarida, which is well represented
in Africa, Asia, and Australia (10). Evidence that bats are the reservoir
hosts of Ebola virus (Reston), which is known to infect monkeys in the
Philippines should be sought in the Philippines and for its silent presence
in bats in Australia. If such evidence were found, Ebola would be labeled
as emerging in Australia and other countries between Australia and the
Philippines, although no cases of disease might ever occur east of the line
where monkeys are indigenous.

The scientist who looks for evidence of a disease agent in a country or
region and the regulator who has to deal with the public health or economic
consequences of a new or newly recognized disease have conflicting
interests. The national and international reaction to the discovery of many
agents in countries where they had not been found does not take into
account the measure of risk for the disease. The term "emergent" disease
needs some qualifiers to diminish fear and overreaction.

One way would be to rate the risk for the disease on a scale of 1 to 5;
another would be to provide a context that notes the capacity of the agent
to spread and cause illness and death.

Toby D. St. George
Virus Consultants International, Queensland, Australia

References

  1. St. George TD. The use of sentinel herds for monitoring the
     distribution of endemic diseases of livestock in Australia. In:
     Veterinary Update. Brisbane, Australia: University of Queensland;
     1992. p. 393-414.
  2. McManus TJ, Marshall ID. The epidemiology of Ross River virus in
     Tasmania. In: St George TD, Kay BH, Blok J, editors. Arbovirus
     research in Australia. Proceedings of the 4th Symposium of the
     Commonwealth Scientific and Industrial Research
     Organization/Queensland Institute of Medical Research (CSIRO/QIMR);
     1986; Brisbane, Australia: Brisbane, Australia: CSIRO/QIMR; 1986. p.
     127-31.
  3. Bluetongue disease in southeast Asia and the Pacific. In: St George
     TD, Peng KG, editors. Australian Centre for International Research
     (ACIAR) Proceedings No. 66. Canberra, Australia: ACIAR; 1995. p.
     15-41.
  4. St George TD. Arboviruses infecting livestock in the Australian
     region. In: St George TD, Kay BH, Blok J, editors. Arbovirus research
     in Australia. Proceedings of the 4th Symposium of the Commonwealth
     Scientific and Industrial Research Organization/Queensland Institute
     of Medical Research (CSIRO/QIMR); 1986; Brisbane, Australia: Brisbane,
     Australia: CSIR/QIMR; 1986. p. 23-5.
  5. Young PL, Halpin K, Selleck PW, Field H, Gravel JL, Kelly MA, et al.
     Serological evidence for the presence in Pteropus bats of a
     paramyxovirus related to equine morbillivirus Emerg Infect Dis
     1996;2:239-40.
  6. Fraser GC, Hooper PT, Lunt RA, Gould AR, Gleeson LJ, Hyatt AD, et al.
     Encephalitis caused by a lyssavirus in fruit bats in Australia. Emerg
     Infect Dis 1996;2:327-31.
  7. Archer M. A review of the origins and radiation of Australian mammals.
     In: Keast A, editor. Ecological biogeography of Australia. The Hague:
     W. Junk; 1981. p. 1473-82.
  8. St George TD. Are there common features in lyssavirus diseases?
     Arbovirus Research in Australia. In: Uren MF, Blok J, Manderson LH,
     editors. Proceedings of the 5th symposium of the Commonwealth
     Scientific and Industrial Research Organization/Queensland Institute
     of Medical Research (CSIRO/QIMR); 1989; Brisbane, Australia. Brisbane,
     Australia: CSIRO/QIMR; 1989; p. 264-7.
  9. Swanepoel R, Leman PA, Burt FJ, Zachariades NA, Braack TG, Rollin PE,
     et al. Experimental inoculation of plants and animals with Ebola
     virus. Emerg Infect Dis 1996;2:321-5.
 10. Hill JE, Smith JD. Bats, a natural history. Austin (TX): University of
     Texas Press; 1984.

  --------------------------------------------------

Resistance to Dryness of Escherichia coli O157:H7 Strains from Outbreak in
Sakai City, Japan, 1996

To the Editor: A large outbreak of Escherichia coli O157:H7 with more than
6,000 cases occurred in Sakai City, Osaka Prefecture, Japan, in July 1996
(1); after the outbreak, more than 1,000 secondary infections occurred in
the families of the patients (2). We studied the resistance of E. coli
O157:H7 to dryness because the survival on surfaces of inert materials
under dry conditions may be related to the transmissibility of the strains.

E. coli O157:H7 strains grown on 3.0% nutrient broth with 1.5% agar for 20
to 22 hours at 37C were suspended at a concentration of approximately 5 x
108 cfu/ml in a 10% skim milk, 0.5% NaCl solution. Aliquots (10 l) of
bacterial suspensions were spread to approximately 10 cm2 on plastic petri
plates for bacterial culture and dried under the air flow of a clean bench
until no aliquots were evident. After storage in the dark at room
temperature, bacteria were harvested with saline and gauze and the viable
number was counted (n = 3).

The log reductions 12 hours after drying were employed to show the
resistance levels of E. coli O157:H7 to dry stress. The log reductions of
the three strains from the Sakai City outbreak (RIMD0509950, RIMD0509894,
and RIMD0509951) were 0.04  0.34, 0.14  0.06, and 0.20  0.60,
respectively (mean = 0.13), whereas those of the E. coli O157:H7 strains
from the other cases varied from 0.71  0.18 to 4.57  1.02. (RIMD0509861,
0.71  0.18; ATCC35150, 1.15  0.35; RIMD0509826, 1.23  0.60; RIMD0509742,
1.28  0.18; ATCC43890, 1.31  0.17; RIMD0509933, 1.48  0.19; RIMD0509932,
1.48  0.22; RIMD0509765, 3.60  0.24; RIMD0509764, 4.57  1.02; mean =
1.87). Although the strain from the Sakai City outbreak (RIMD0509950)
survived for at least 35 days under the dry conditions, the strains from
the other cases had no viable cells after 7 days. The log reductions of E.
coli JM109 and DH10B strains were more than 10, and no viable cells were
detected in 12 hours.

The strains from the Sakai City outbreak also showed marked acid
resistance. Acid resistance of E. coli O157:H7 has been reported to depend
on rpoS, which is induced in a stationary phase (3). Since the E. coli
O157:H7 strains in a stationary phase were more resistant to dry and acid
stresses than those in a log phase, rpoS may also be associated with
resistance to dry stress. However, no deletions of rpoS were detected by
polymerase chain reaction analysis in the E. coli O157:H7 strains used in
this experiment. Further study on the mechanism of resistance will be
needed to establish new strategies for eradicating the bacteria.

A case-control study by the Ministry of Health and Welfare of Japan showed
that uncooked radish sprouts were the vehicle of the largest outbreak in
Sakai City (4). In two small outbreaks of E. coli O157:H7 in March 1997,
the vehicle of infection might have been radish sprouts; therefore, the
possible contamination of white radish seeds with E. coli O157:H7 has been
discussed (5). If such contamination was present, dry resistance might be
involved in the survival on or in white radish seeds because the bacteria
were exposed to dry conditions for a long period before sprouting. We
propose that dry resistance be considered an important factor in infection.

Yoshio Iijima,* Mayumi Matsumoto,* Kumiko Higuchi,* Taro Furuta,* and
Takeshi Honda
*Saraya Biochemical Laboratory, Kashiwara, Japan; Institute for Microbial
Diseases, Osaka University, Suita, Japan

References

  1. National Institute of Health and Infectious Diseases Control Division,
     Ministry of Health and Welfare of Japan. Verocytotoxin-producing
     Escherichia coli (Enterohemorrhagic E. coli) infections, Japan,
     1996-June, 1997. Infectious Agent Surveillance Report 1997;18:153-4.
  2. Takatorige T. Study on the patients of the diarrheal disease outbreak
     among schoolchildren in Sakai City; oc-currence of infections among
     the families of the school-children. In: Report of Osaka Prefecture
     Research Society for Enterohemorrhagic E. coli Infectious Disease,
     Osaka; 1997. p. 85-96. (in Japanese)
  3. Lin J, Smith MP, Chapin KC, Baik HS, Bennett GN, Foster JW. Mechanisms
     of acid resistance in enterohemorrhagic Escherichia coli. Appl Environ
     Microbiol 1996;62:3094-100.
  4. Michino H, Araki K, Minami S, Takaya S, Sakai N. Investigation of
     large-scale outbreak of Escherichia coli O157:H7 infection among
     schoolchildren in Sakai City, 1996. In: Proceedings of the 32nd Joint
     Conference on Cholera and Related Diarrheal Diseases, U.S.-Japan
     Cooperative Medical Science Program(USJCMSP); 14-16 Nov 1996; Nagasaki
     University, Nagasaki, Japan. USJCMSP: 1996. p. 84-9.
  5. Nathan R. American seeds suspected in Japanese food poisoning
     epidemic. Nat Med 1997;3:705-6.

  --------------------------------------------------

Irradiation Pasteurization of Solid Foods

To the Editor: Osterholm and Potter have made a strong case for irradiation
pasteurization of solid foods that enter kitchens as raw agricultural
commodities, such as meat, poultry, and seafood (1). Irradiation
pasteurization was advocated to protect against foodborne diseases caused
by common pathogens such as Campylobacter, Cryptosporidium, Escherichia
coli, Listeria, Salmonella, and Toxoplasma (2). An additional rationale for
irradiation pasteurization is bacterial resistance to antimicrobial drugs,
a major health concern, which will undoubtedly increase in magnitude unless
new approaches become available (3). The widespread use of antibiotics in
animal husbandry may be the cause of some of this resistance, for example,
in vancomycin-resistant enterococci associated with the agricultural use of
glycopeptide antibiotics (4,5). Furthermore, resistance to glycopeptide
antibitiotics can be transferred from enterococci to other gram-positive
organisms, at least in the laboratory (6). Thus, resistant bacterial
strains from animal sources may enter the human population through
contaminated food without necessarily causing immediate disease but
resulting in expanded human reservoirs of antimicrobial resistance through
horizontal gene transfer (7). When such bacterial strains are subsequently
transmitted to a susceptible person, serious disease could result, which
would be exceedingly difficult to treat (8). Irradiation pasteurization of
solid foods could reduce the magnitude of transfer of resistance genes
through contaminated foods.

Stephen Moses and Robert C. Brunham
University of Manitoba, Winnipeg, Canada

References

  1. Osterholm MT, Potter ME. Irradiation pasteurization of solid foods:
     taking food safety to the next level. Emerg Infect Dis 1997;3:575-6.
  2. Monk JD, Beuchat LR, Doyle MP. Irradiation inactivation of food-borne
     microorganisms. Journal of Food Protection 1995;58:197-208.
  3. Gold HS, Moellering RC. Antimicrobial-drug resistance. N Engl J Med
     1996;335:1445-53.
  4. Bates J, Jordens JZ, Griffiths DT. Farm animals as a putative
     reservoir for vancomycin-resistant enterococcal infection in man. J
     Antimicrob Chemother 1994;34:507-14.
  5. Van de Bogaard AE, Jensen LB, Stobberingh EE. Vancomycin-resistant
     enterococci in turkeys and farmers [letter]. N Engl J Med
     1997;337:1558-9.
  6. Leclerq R, Derlot E, Weber M, Duval J, Courvalin P. Transferable
     vancomycin and teicoplanin resistance in Enterococcus faecium.
     Antimicrob Agents Chemother 1989;33:10-5.
  7. Davies J. Inactivation of antibiotics and the dissemination of
     resistance genes. Science 1994;264:375-82.
  8. Swartz MN. Hospital-acquired infections: diseases with increasingly
     limited therapies. Proc Natl Acad Sci U S A 1994;91:2420-7.

  --------------------------------------------------

Emerging Infectious Diseases in Brazil

To the Editor: Hooman Momen's update on emerging infectious diseases in
Brazil (1) appears to be based solely on notifiable disease data, which
cannot adequately describe the current situation. Additional data in
several areas may be useful.

Parasitic diseases: Dr. Momen's update restricts itself to protozoal
diseases and does not distinguish between mucocutaneous and visceral
leishmaniasis. Visceral leishmaniasis is in fact expanding in many suburban
and urban areas in the northeast. Mucocutaneous leishmaniasis, after a
small retreat following extensive deforestation, has made a comeback; and
in many suburban areas in Rio de Janeiro and So Paulo, in the southeast,
transmission is occurring, probably because of changes in sandfly ecology
(1).

A helminthic disease of interest is mansoni schistosomiasis, which has been
expanding its area of transmission, reaching over to Santa Catarina, in the
south, to Par in the north, expanding also westward, to Mato Grosso and
Mato Grosso do Sul. The number of cases, as well as the associated illness,
has possibly been reduced, but there is no doubt that the disease can be
found in a much larger area than 20 years ago. Other emerging helminthiases
of interest, albeit not of public health concern, are onchocerciasis, still
restricted to the Yanomami group in Roraima, bordering Venezuela;
Angiostrongylus costaricensis infection (2), found in the south, Rio Grande
do Sul; and some cases of lagochilascariasis, reported from Par.

Viral diseases: As Dr. Momen pointed out, dengue is by far the most serious
emerging viral disease in Brazil, and the area occupied by Aedes aegypti is
expanding. Dengue hemorrhagic fever has occurred occasionally, but no
outbreaks have been recorded. However, measles is no longer a problem; the
outbreaks have been controlled.

There is no evidence to support that hepatitis B is declining because of
vaccination. Vaccination is still restricted to areas of high prevalence.
Other states are beginning vaccination programs in newborns, but it will be
some time before these programs have any effect on prevalence. As to
hepatitis C, because diagnostic testing is only recently becoming
widespread, we are probably experiencing an increase in detection rather
than in incidence.

Other notable agents are Mayaro and Oropouche viruses, which are
arthropod-borne and among the most common causes of febrile illness in the
Amazon region. Aedes albopictus, found all over the country, could be a
potential vector (3). Apart from HIV, other retroviruses are cause for
concern: HTLV-I and HTLV-II screening is recommended for blood banks, and
enough data exist to conclude that the infection is widespread in the
country but with a low prevalence (0.4% and 0.1%, respectively). Clusters
of disease have not been identified, but adult T-cell leukemia/lymphoma is
far from a curiosity (4).

Bacterial diseases: Brazilian purpuric fever, caused by Haemophilus
influenzae biogroup aegyptius, was first reported in outbreaks in the
central-south part of the country (western So Paulo, eastern Mato Grosso
do Sul, and northwestern Paran) about 10 years ago, causing a syndrome
much like meningococcemia (5). For enteric infections, the limited data
available present interesting trends. Salmonella Enteritidis is rising and
S. Typhimurium is declining in So Paulo and the southern states. These
trends may reflect improved sanitation and increased use of industrialized
foods and contaminated animal feeds (6).

Fungal diseases are not reportable, but many epidemiologic studies have
been conducted. Paracoccidioidomycosis (South American blastomycosis) was
unheard of in the Amazon region, never being found in native habitants;
however, because of environmental and socioeconomic changes, the infection
is now being identified (7).

Antimicrobial resistance is a serious problem, not only within hospitals,
but also in the community. Penicillin-resistant pneumococcus is not yet a
widespread problem, but it has been detected (8); the same situation exists
with regard to Mycobacterium tuberculosis (9).

The problem of emerging infectious disease is gaining increasing attention
in Brazil, and published reports together with notifiable disease data
underline the main points of concern.

Luiz Jacintho da Silva
Clinica Medica, FCM, Unicamp, Campinas, Brazil

References

  1. Gomes AM. Sandfly ecology in the State of So Paulo. Mem Inst Oswaldo
     Cruz 1994;89:457-60.
  2. Rambo PR, Agostini AA, Graeff-Teixeira C. Abdominal angiostrongylosis
     in southern Brazilprevalence and parasitic burden in mollusk
     intermediate hosts from eighteen endemic foci. Mem Inst Oswaldo Cruz
     1997;92:9-14.
  3. Smith GC, Francy DB. Laboratory studies of a Brazilian strain of Aedes
     albopictus as a potential vector of Mayaro and Oropouche viruses. J Am
     Mosq Control Assoc 1991;7:89-93.
  4. Farias de Carvalho SM, Pombo de Oliveira MS, Thuler LC, Rios M, Coelho
     RC, Rubim LC, et al. HTLV-I and HTLV-II infections in hematologic
     disorder patients, cancer patients, and healthy individuals from Rio
     de Janeiro, Brazil. J Aquir Immune Defic Syndr Hum Retrovirol
     1997;15:238-42.
  5. The Brazilian Purpuric Fever Study Group. Brazilian purpuric fever
     identified in a new region of Brazil. J Infect Dis 1992;165:S16-9.
  6. Tavechio AT, Fernandes SA, Neves BC, Dias AM, Irino K. Changing
     patterns of Salmonella serovars: increase of Salmonella Enteritidis in
     So Paulo, Brazil. Rev Inst Med Trop So Paulo 1996;38:315-22.
  7. Coimbra Jr CE, Wanke B, Santos RV, do Valle AC, Costa RL,
     Zancope-Oliveira RM. Paracoccidiodin and histoplasmin sensitivity in
     Tupi-Monde Amerindian populations from Brazilian Amazonia. Ann Trop
     Med Parasitol 1994;88:197-207.
  8. Brandileone MC, Vieira VS, Casagrande ST, Zanella RC, Guerra ML,
     Bokermann S, et al. Prevalence of serotypes and antimicrobial
     resistance of Streptococcus pneumoniae strains isolated from Brazilian
     children with invasive infections Pneumococcal Study Group in Brazil
     for the SIREVA project. Regional System for Vaccines in Latin America.
     Microb Drug Resist 1997;3:141-6.
  9. Pinto WP, Hadad DJ, Palhares MC, Ferrazoli L, Telles MA, Ueki SY, et
     al. Drug resistance of M.tuberculosis isolated from patients with HIV
     infection seen at an AIDS Reference Center in So Paulo, Brazil. Rev
     Inst Med Trop So Paulo 1996;38:15-21.

  --------------------------------------------------

Reply to L.J. da Silva

To the Editor: Dr. da Silva's letter raises several important points. My
article, however, was never intended to be comprehensive. The choice of
which emerging infectious diseases to include was difficult, especially in
a country where many endemic infections continue at a high prevalence and
others, thought to be controlled, are reemerging.

As Dr. da Silva states, many reports (in Portuguese and English) discuss
infectious diseases in Brazil; however, this information is rarely current.
The information about measles in my article is a case in point. At the time
of my article, an outbreak causing national concern was occurring in
Brazil; it has since been controlled. A further problem is that the most
detailed and reliable studies are generally of only a regional or local
nature, for example, the recent excellent report by Merchan-Hamann (1) on
the situation of endemic diseases in north and northeastern Brazil and
other references cited by Dr. da Silva. To obtain current information at
the national level and provide numerical data rather than merely discuss
current trends, I focused on notifiable diseases.

As Dr. da Silva states, schistosomiasis has continued to decrease both in
the number of cases and associated illness. Onchocerciasis has been
restricted to a small focus in northern Brazil for many years, and a recent
report of a new focus in the state of Goias has yet to be confirmed. In my
opinion, neither infection could be considered emerging. An important
helminthiasis that perhaps should be mentioned is Bancroftian filariasis
with a main focus in Recife and minor foci in Belem and Alagoas. Because of
traditional and novel control strategies, the number of cases is declining
in all foci.

The information I used about hepatitis is confirmed by the National
Reference Center on Viral Hepatitis of the Ministry of Health. Febrile
illnesses in the Amazon are the great enigma and probably provide the cover
for many new diseases that may still emerge. For example, only
approximately 20% of blood slides taken from suspected malaria patients in
the Amazon are confirmed as positive, which leaves at least one million
cases of febrile illness per year undiagnosed. I am unaware of any data
that show Mayaro and Oropouche viruses as the most common cause of these
illnesses. Dr. da Silva's letter provides useful additional information on
bacterial diseases, antimicrobial resistance, and a number of
low-prevalence diseases that may in time prove to be important emerging
infections.

Hooman Momen
Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil

Reference:

  1. Merchan-Hamann E. Diagnostico macroregional da situacao das endemias
     das regioes norte e nordeste. Informe Epidemiologico do SUS
     1997;4:43-114.

  --------------------------------------------------

A Brief Update on Rabbit Hemorrhagic Disease Virus

To the Editor: We read with interest the paper by A. Smith et al. (Emerg
Infect Dis 1998; 1:13-20) on calicivirus emergence from ocean reservoirs.
Our attention was drawn particularly to the data and comments regarding
rabbit hemorrhagic disease (RHD), a recently emerged and devastating
disease of just one rabbit species, Oryctolagus cuniculus. We have been
involved in RHD research and diagnosis since 1989. Like D. Gregg's
laboratory at the Foreign Animal Diseases, U.S. Department of Agriculture,
Greenport, USA, our laboratory at the Istituto Zooprofilatico Sperimentale
della Lombardia e dell'Emilia, Brescia, Italy, was in 1991 designated a
Reference Laboratory for RHD by the International Office of Epizootics
(OIE), Paris, France. Although other aspects of the article by Dr. Smith
and colleagues appear unclear (e.g., the fact that feline calicivirus is
classified among human pathogens like Norwalk virus), we will confine our
comments to a few main points concerning RHD virus (RHDV).

Is RHDV a calicivirus or a parvovirus? RHD is caused by a calicivirus
(1-3). The articles cited by Dr. Smith date back to 1991 and are part of a
book review promoted and edited by OIE (4). This landmark review includes
papers from China and the United States supporting the parvovirus
hypothesis and papers from Europe concluding that RHDV is a calicivirus. A
retrospective reading of those articles may explain the reasons for the
misinterpretation of some results. However, this occurred in 1991 and,
after 7 years, more than 50 published articles consider RHDV a calicivirus.
Actually, RHDV is one of the best characterized caliciviruses, and the
publication of its full genome sequence in 1991 was the first of a
Caliciviridae member (5).

Diagnostic tools have been developed by our and other laboratories (3,4,6).
Thanks also to specific monoclonal antibodies produced towards RHDV and
European brown hare syndrome virus (EBHSV) by our colleague E. Brocchi, we
standardized different enzyme-linked immunosorbent assays (ELISAs) for the
diagnosis of related diseases (4,6-8). In particular, we developed five
different ELISAs for serology that allow the detection of antibodies
specific for RHDV or EBHSV or that are cross-reactive. In addition, we can
define the antibody response in rabbits and hares in terms of
isotype-involved immunoglobulin M (IgM), IgA, and IgG (9). Today the main
difficulty is the qualitative distinction between RHDV and rabbit
calicivirus (RCV, a recently identified nonpathogenic calicivirus)
antibodies because of the close antigenic profiles of these viruses (6).
Finally, RHDV- and EBHSV-specific polymerase chain reaction has been
developed in at least five laboratories besides ours. We have sent these
reagents and/or diagnostic methods to at least 19 laboratories outside
Italy, including Australia, New Zealand, and the United States.

Does RHDV infect humans? This question has arisen together with the
prospect of using RHDV as a biologic control agent in countries like
Australia and New Zealand, when they were free of RHDV. In Europe, where
the disease naturally occurred and quickly spread, no particular control on
human health was planned. In Italy only, between 1987 and 1990, hundreds of
millions of rabbits died of RHD in regions where the average density of
humans is very high. As a consequence of the use of the vaccine since 1991,
the incidence of RHD among breeding rabbits decreased drastically and
quickly. Nevertheless, the disease is still endemic, mainly in small farms
and among wild rabbits. EBHS also is endemic in wild hares, and hunters are
highly exposed to the virus since hares are their main target. However,
neither in humans nor in animal species other than rabbits and hares have
any diseases similar to RHD ever been reported. In relation to the
likelihood of mild or inapparent infections, we used 100 human sera
randomly selected from blood donors to carry out a preliminary
standardization of an RHD-ELISA that has been periodically used to control
the sera of the RHD laboratory staff. Very recently, we tested nine sera
from laboratory personnel exposed to RHDV; again no positive result was
noted by RHD-ELISA. These findings have limited epidemiologic value, but
considering the high level of exposure of part of the sample, it is evident
that RHDV infection in humans is unlikely to be the rule.

Lorenzo Capucci and Antonio Lavazza
Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia,
Brescia, Italy

References

  1. Murphy FA. Virus taxonomy. In: Fields BN, Knipe DM, Howley PM,
     editors. Virology. 3rd ed. New York: Lippincott-Raven Publishers;
     1996. p. 15-57.
  2. Ohlinger VF, Hass B, Meyers G, Weiland F, Thiel HJ. Identification and
     characterization of the virus causing rabbit hemorrhagic disease. J
     Virol 1990;64:3331-6.
  3. Rodk L, md B, Valcek L, Vesel T, Stepnek J, Hampl J, et al.
     Enzyme-linked immunosorbent assay of antibodies to rabbit haemorrhagic
     disease virus and determination of its major structural proteins. J
     Gen Virol 1990;71:1075-80.
  4. Capucci L, Scicluna MT, Lavazza A. Diagnosis of viral hemorrhagic
     disease of rabbits and European brown hare syndrome. Rev Sci Tech
     1991;10:347-70.
  5. Meyers G, Wirblich C, Thiel HJ. Rabbit hemorrhagic disease
     virus-molecular cloning and nucleotide sequencing of a calicivirus
     genome. Virol 1991;184:664-76.
  6. Lavazza A, Capucci L. Viral haemorrhagic disease of rabbits. In:
     Office International des Epizooties, Paris, France, Manual of
     standards for diagnostic test and vaccine. Paris: the Office; 1996. p.
     589-98.
  7. Capucci L, Fusi P, Lavazza A, Pacciarini ML, Rossi C. Detection and
     preliminary characterization of a new rabbit calicivirus related to
     hemorrhagic disease virus but nonpathogenic. J Virol 1996;70;8614-23.
  8. Capucci L, Frigoli G, Ronsholt L, Lavazza A, Brocchi E, Rossi C.
     Antigenicity of the rabbit hemorrhagic disease virus studied by its
     reactivity with monoclonal antibodies. Virus Res 1995;37:221-38.
  9. Capucci L, Nardin A, Lavazza A. Seroconversion in an industrial unit
     of rabbits infected with a non-pathogenic rabbit haemorrhagic
     disease-like virus. Vet Rec 1997;140:647-50.

  --------------------------------------------------

Rabbit Hemorrhagic Disease

To the Editor: The recent article on calicivirus by Smith et al. (1) is
misleading in its use of the study concerning human health aspects of
rabbit hemorrhagic disease (RHD) by Mead et al. (2).

The RHD exposure categories of "low" and "high" used by Mead et al. and
mentioned in the first column of page 18 (1) are not related to the
categories of "low" and "high" given in the same paragraph at the top of
the second column. The reader might easily assume that it was Mead et al.
who considered that JulDec 1995 was "a low exposure period." This is not
sosuch a classification is made by Smith et al.

Further, the reader might assume that it was the study by Mead et al. that
concluded "that exposure to RHD virus remains a plausible explanation for
increased disease incidence." Again this is an inference drawn by Smith et
al. and is the opposite of the conclusion of Mead et al.

The basis of exposure in the study by Mead et al. is at an individual
levelthe respondents were chosen either because they had been handling
rabbits or as controls in determining the level of disease. In contrast,
Smith et al. consider exposure at a broad environmental level and disregard
whether the respondents had been handling infected rabbits or not.
Actually, more contact with rabbits occurred during the first half of the
study than during the second.

Smith et al. do not mention the conclusions of Mead et al.: These neither
showed any significant difference between levels or types of illness in
those exposed and those not exposed to RHD virus nor demonstrated any
association between the exposure to RHD and number of episodes of illness
in the subsequent 1 to 2 months.

The results of the study by Mead et al. may be summarized by noting that
the average number of episodes of illness over the 13-month reporting
period was 2.6 for respondents who had not been exposed to RHD virus, 2.2
for those classified as having a low level of exposure, and 2.3 for those
classified as having a high level.

The study by Mead et al. concluded that, on the basis of the health survey
and the lack of any serologic reaction of the respondents, there was
considerable support to the view that RHD virus is not associated with
infection or disease in humans. The results of the study have been
submitted for publication in a scientific journal.

Reference 31 should refer to the Bureau of Resource Sciences (not Studies).

C. Mead
Convenor, Rabbit Calicivirus Human Health Study Group, Department of Health
and Family Services, Canberra, Australia

References

  1. Smith AW, Skilling DE, Cherry N, Mead JH, Matson DO. Calicivirus
     emergence from ocean reservoirs: zoonotic and interspecies movements.
     Emerg Infect Dis 1998;4:13-20.
  2. Mead C, Kaldor J, Canton M, Gamer G, Crerar S, Thomas S. Rabbit
     calicivirus and human health. Canberra, Australia: Department of
     Primary Industries and Energy, Australian Government (Released under
     the Official Information Act). Report of the Rabbit Calicivirus Human
     Health Study Group; 1996.

  --------------------------------------------------

Reply to Drs. Capucci, Lavazza, and Mead

To the Editor: We are aware of Capucci and Lavazza's excellent work.
Indeed, one of the best characterized calicivirus genomes is that detected
in rabbit hemorrhagic disease (RHD); however, the virus' infectivity,
pathogenesis, modes of transmission, reservoirs, survival in nature, host
of origin, virulence factors, number of neutralization serotypes, and
multispecies infectivity are poorly characterized. Propagating this virus
in vitro could provide insight for addressing questions relevant to
caliciviruses that cannot be propagated in vitro.

We are unclear about the confusion regarding Norwalk virus and feline
calicivirus (FCV). Both are caliciviruses. Norwalk virus is a human
pathogen. FCV is in a different genus (1) that includes strains infecting
humans (2). We know of no documented FCV infections in humans nor of
detailed studies to search for such occurrences, although some evidence
suggests the possibility (3).

Capucci and Lavazza's remaining questions address the etiology of RHD,
diagnostic reagents, and possible human infection. They report nine
laboratory workers as antibody negative but do not report test results on
persons at high risk, such as rabbit farm workers, nor do they mention
having positive control human or primate sera. Koch's postulates have been
fulfilled for RHD: a parvovirus was isolated in vitro and was cell-passaged
15 times; at a second laboratory, the parvovirus was identified in
materials causing RHD (4,5). In Europe the parvovirus etiology for RHD was
deemed hypothetical but has not been refuted on a scientific basis. The
calicivirus consistently identified in European materials has not been
isolated in vitro, and Koch's postulates have not been fulfilled. Are the
parvovirus-associated outbreaks of RHD in Mexico and China (4,5) and the
calicivirus-associated RHD outbreaks in Europe identical disease
manifestations of two different viruses? Is RHD multifactorial requiring
two or more agents? Is RHD caused by only a calicivirus or only a
parvovirus? A calicivirus and a parvovirus can be isolated in vitro from
the same fecal sample of a sick rabbit (N. Keefer, D.E. Skilling, A.W.
Smith, unpub. data).

Our comments on RHD diagnostic assays referred to those used in Australia
(6,7) to screen humans and experimentally infected animals to support
legalizing the spread of RHD in Australia and New Zealand.

Public health protection requires prudent avoidance of pathogens associated
with risk of adverse outcome, not necessarily proof of causation (8). In
this context, human health risk for RHD goes largely unaddressed. The
deliberate introduction of a new disease agent (RHD) known to cause death
in mammals requires prudence rather than proof of human illness, especially
when the scientific literature includes reports that the agent has induced
antibody reactions in a wide range of mammalian and avian species (6).

Mead et al. (9) conclude, "No significant association between exposure to
RCV and subsequent bouts of sickness could be demonstrated." Their recorded
data do not support a statistically significant risk of illness because
sample sizes in the monthly groups were too small for any meaningful
interpretation. Mead et al. (9) state a "lack of any serologic reaction of
the respondents," but a 50% cut-point was used for the competitive ELISA
test, and some individual sera were repeated up to six times with percent
inhibition reactions ranging from approximately 1% to 44% in one instance
and 12% to 100% in another. Results were selected from these laboratory
data and reported "lack of serologic reaction."

We derived our findings from data obtained under a freedom of information
request. Mead et al. used the same data to support an opposite conclusion.
Opposing conclusions "red flag" the quality of the study. In summary, the
reporting of negative results of such a study cannot be used to support the
important biologic, health, and political conclusion that humans are not at
risk from infection with RHD.

We encourage a well-designed longitudinal study of persons at high risk of
RHD exposure to answer conclusively whether RHD has infected humans. If
"the rule" means that most humans exposed to RHD would become infected, we
agree with Dr. Capucci "that infection is unlikely to be the rule," but
transmission of equine morbillivirus, Rift Valley fever, and H5N1 influenza
to humans is also "unlikely to be the rule" (10).

Alvin W. Smith,* Neil J. Cherry, and David O. Matson
*Oregon State University, Corvallis, Oregon, USA; Lincoln University,
Christchurch, New Zealand; Center for Pediatric Research, Norfolk,
Virginia, USA

References

  1. Berke T, Golding B, Jiang X, Cubitt WD, Wolfaart M, Smith AW, et al. A
     phylogenetic analysis of the caliciviruses. J Med Virol
     1997;52:419-24.
  2. Smith AW, Berry ES, Skilling DE, Barlough JE, Poet SD, Berke T, et al.
     In vitro isolation and characterization of a calicivirus causing a
     vesicular disease of the hands and feet. Clin Infect Dis
     1998;26:434-9.
  3. Cubitt WD. Proceedings of the European Society of Veterinary Virology.
     Reading, United Kingdom: Reading University, Oct 1996.
  4. Xu WY. Viral hemorrhagic disease in rabbits in the People's Republic
     of China: epidemiology and virus characterization. Rev Sci Tech,
     1991;10:2393-408.
  5. Gregg DA, House C, Meyer R, Berninger M. Viral haemorrhagic disease of
     rabbits in Mexico: epidemiology and viral characterization. Rev Sci
     Tech, 1991;10:2434-51.
  6. Bureau of Resource Sciences, Australia. Rabbit calicivirus disease.
     Canberra, Australia: Australian Government Printing Office; 1996.
  7. Collins BJ, White JR, Lenhaus C, Boyd V, Westbury HA. A competition
     ELISA for the detection of antibodies to Rabbit Hemorrhagic Disease
     Virus. Vet Microbiol 1995;43:85-96.
  8. Brad-Hill A. The environment and disease: association or causation?
     Proceedings of the Royal Society of Medicine, 1965;58:295-300.
  9. Mead C, Kaldor J, Canton M, Gamer G, Crerar S, Thomas S. Rabbit
     calicivirus and human health. Canberra, Australia: Department of
     Primary Industries and Energy, Australian Government (Released under
     the Official Information Act). Report of the Rabbit Calicivirus Human
     Health Study Group; 1996.
 10. Vogel G. Sequence offers clues to deadly flu. Science News. Science
     1998;279:324.


Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/letters.htm

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[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

News and Notes

The National Food Safety Initiative

For many years, the Centers for Disease Control and Prevention (CDC) and
state and local health departments have investigated and controlled
outbreaks of foodborne diseases and conducted limited passive surveillance
for specific foodborne pathogens. However, recent changes in the food
supply and in the consuming public have changed the type and range of
infections caused by food and have made traditional approaches to disease
surveillance increasingly inadequate. In particular, new technology which
facilitates the national and international distribution of food to a
population of consumers increasingly vulnerable to infection by new
microorganisms has changed the character of foodborne disease in the United
States in ways that demand more and better information for decision making.

Foodborne Disease Surveillance

In 1997, President Clinton announced an administration initiative to create
a nationwide early warning system for foodborne diseases, enhanced food
safety inspection, and expanded food safety research, training, and
education. The National Food Safety Initiative will meet the challenges
presented by the changing nature of our food safety concerns by
strengthening and broadening surveillance and response systems at CDC and
the state and local health departments. The following areas of national
surveillance for foodborne diseases are addressed by the initiative: rapid
identification of and effective response to foodborne hazards;
identification of large, diffuse outbreaks that have a low attack rate
within one health jurisdiction but affect the health of many people within
a larger geographic area; and evaluation of the nature and scope of food
safety problems. In addition, the National Food Safety Initiative will 1)
link CDC, state and local health departments, and other federal agencies
within a powerful electronic network developed by CDC for rapid sharing of
microbial subtyping information, including the digitalized images of
subtyping patterns needed for molecular epidemiology, and 2) will enhance
and expand the activities of FoodNet, the interagency foodborne disease
component of CDC's emerging infectious disease active surveillance program
to improve the quantitative base for foodborne disease.

Strengthening and Broadening the Base of Surveillance

Under the National Food Safety Initiative, CDC is building epidemiologic
and laboratory capacity in state and local health departments so they can
quickly and effectively identify foodborne hazards, investigate and control
outbreaks, and develop effective programs to prevent additional illnesses
from the same source. Routine surveillance and outbreak response, critical
to the proper functioning of health departments, have been neglected at the
state and local levels because of limited resources and competing
priorities. Therefore, CDC is providing training and equipment needed by
the health departments to update surveillance systems and diagnostic
capability for bacterial, parasitic, and viral foodborne diseases. In
addition, CDC, state epidemiologists, and state public health laboratory
directors are working to define the elements of core capacity for effective
response to food safety problems. The results of this evaluation will
provide guideposts for efficient allocation of initiative resources in 1999
and beyond to construct a truly effective national early warning system for
foodborne diseases.

CDC's response to foodborne hepatitis A virus infections, supported in 1998
by the National Food Safety Initiative, illustrates the value of better
surveillance. Existing hepatitis A surveillance data document food as a
vehicle of transmission in only 2% to 5% of cases. On the basis of these
data, economic analysis suggests that routine immunization of food workers
to prevent foodborne transmission of hepatitis A virus (with a vaccine
already available) is not a cost-effective nationwide public health
response. However, existing surveillance data document a source of exposure
(food or otherwise) for only half of the reported cases of hepatitis A.
Therefore, CDC is collaborating with health departments in three states to
more fully describe the epidemiology of hepatitis A infections for which
routine investigation fails to determine a source. In addition, CDC will
evaluate the effectiveness of the algorithm used to determine which patrons
of food service establishments with infected food workers will receive
immune globulin for postexposure prophylaxis and will establish a genetic
sequence-based library of hepatitis A virus strains circulating in the
United States and other countries to permit the effective use of molecular
epidemiology in investigations of hepatitis A outbreaks and sporadic cases.

Diarrheal disease results in large numbers of physician visits and
hospitalizations each year in the United States. However, the infectious
cause of the illness is frequently not determined, which prevents good
surveillance and impedes development of effective prevention strategies.
Therefore, CDC is embarking on studies of mild and severe diarrhea to
determine to the greatest extent possible the bacterial, parasitic, and
viral agents involved. Similarly, applied research at CDC that is improving
diagnostic and subtyping methods for parasitic foodborne diseases and
caliciviruses will support future surveillance and outbreak response for
these foodborne pathogens, as well as help define the extent of their
contribution as causes of sporadic foodborne disease.

Establishing Electronic Networks and Generating Quantitative Data

Another aspect of CDC's response to foodborne disease under the National
Food Safety Initiative is the development of an electronic network that
links public health laboratories for rapid data sharing, including the
digitalized images of pulsed-field gel electrophoresis (PFGE) patterns.
These patterns can be used to distinguish related cases that may be part of
an outbreak from background disease so that risk factors can be identified
and control measures can be established. By the end of 1997, CDC had
laboratories in 16 state health departments trained and equipped to perform
PFGE subtyping of Escherichia coli serotype O157:H7 with standardized
techniques so the results could be compared among laboratories.
Microbiologists in federal regulatory laboratories were also trained in
these standardized techniques so E. coli O157:H7 isolated from food and
animal samples could be directly compared with clinical isolates from human
cases. Laboratories in four states were established as regional resources
to make PFGE subtyping available to surrounding states, and one was
directly linked to CDC's PFGE library of strains so it could query CDC's
database in addition to being able to submit electronic data for analysis.
The other three regional laboratories and laboratories in USDA and FDA will
be directly linked to CDC's database early in 1998; the remaining six
FoodNet sites will be linked later in the year. In addition, in 1998 six
more states will be trained and equipped to perform PFGE subtyping of E.
coli O157:H7, training will be provided to health department laboratories
in two foreign countries, PFGE subtyping of Salmonella Typhimurium will
begin, and methods for subtyping Salmonella Enteritidis and other foodborne
pathogens will be developed.

Changes in food safety regulations (e.g., regulatory use of Hazard Analysis
and Critical Control Points systems and international requirements for
quantitative risk assessment) and increasing emphasis on the
cost-effectiveness of prevention programs require epidemiologic data on
foodborne disease that cannot be provided by routine passive surveillance
systems based on laboratory and physician diagnoses. In response, CDC
collaborated with U.S. Department of Agriculgure, Federal Drug
Administration, and the five state health departments enrolled in CDC's
emerging infections active surveillance program in 1995 to create FoodNet.
FoodNet expands the quantitative database on foodborne diseases, expedites
response to certain food safety concerns, and provides a test bed for
evaluating new prevention strategies. FoodNet activities include 1)
comprehensive documentation of foodborne infections through active
laboratory-based surveillance, 2) determination of health and food-handling
practices of the population under surveillance, 3) determination of
health-care practices by surveys of the physicians and diagnostic
laboratories in the surveillance areas, and 4) implication of specific
foods as vehicles for various foodborne pathogens by conducting
case-control studies of sporadic infections.

These efforts have shown that Campylobacter is the most commonly diagnosed
cause of diarrheal disease, that great geographic variations in the
frequency of E. coli O157:H7 infections and other foodborne pathogens
suggest a need for targeted control measures, and that approximately 1.4
diarrheal illness episodes occur per person per year in the United States.

FoodNet began with active surveillance of sporadic infections with seven
bacterial pathogens in the five sites. In 1997, two new sites were added,
and in 1998 an eighth active surveillance site will be added; active
surveillance for Cryptosporidium, Cyclospora, and Calicivirus infections
will be included, and intensive investigation of foodborne disease
outbreaks will be initiated. Many bacterial pathogens, including those that
are foodborne, are becoming resistant to antibiotics. To measure this
change in resistance, CDC, in collaboration with FDA, USDA, and the states,
began monitoring resistance levels of several foodborne bacteria. This
information is being used to promote the prudent use of antibiotics in
human health and in agriculture.

Other Food Safety Initiative Projects

In 1999, in addition to continuing and extending surveillance activities,
CDC plans to support the following projects through the National Food
Safety Initiative: school-based health education to teach foodborne disease
prevention to a new generation of consumers; a foodborne disease diagnosis
and surveillance component to CDC's Field Epidemiology Training Program,
which helps Ministries of Health in foreign countries (many of which are
major food exporters to the United States) improve their surveillance and
diagnostic capabilities; and new methods of determining the risk for
exposure to pesticide residues in food. The National Food Safety Initiative
is building the public health framework for an effective and efficient
response when novel foods from new areas that contain unfamiliar foodborne
hazards are introduced into the marketplace.

Sue Binder, Rima Khabbaz, Bala Swaminathan, Robert Tauxe, and Morris Potter

Centers for Disease Control and Prevention, Atlanta, Georgia, USA

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Meeting Summary

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Conference on Global Disease Elimination and Eradication as Public Health
Strategies, February 26, 1998

The goals of the conference were to assess the role of elimination and
eradication in decreasing global disease and in using health resources more
effectively. Two hundred invited representatives from 81 organizations and
34 countries participated in the multidisciplinary conference; the
proceedings will be published in late 1998 in a supplement to the Bulletin
of the World Health Organization.

The working definitions during the conference were those developed at the
Dahlem Workshop on the Eradication of Infectious Diseases, March 1997.
Disease control: reduction of disease incidence, prevalence, illness, or
death to a locally acceptable level as a result of deliberate efforts;
continued intervention measures are required to maintain the reduction.
Disease elimination: reduction to zero of the incidence of a specific
disease in a defined geographic area as a result of deliberate efforts;
continued intervention measures are required. Disease eradication:
permanent reduction to zero of the worldwide incidence of infection caused
by a specific agent as a result of deliberate efforts; intervention
measures are no longer needed.

The successful smallpox program and the ongoing poliomyelitis and
dracunculiasis (Guinea worm disease) programs served as models in the
discussions on eradication. The malaria, yellow fever, and yaws programs of
earlier years were recognized as unsuccessful but to have contributed to
better understanding of the biologic, social, political, and financial
complexities and responsibilities of disease eradication. The conference
addressed five major areas: sustainable development, noncommunicable
diseases, bacterial diseases, parasitic diseases, and viral diseases. The
following is a summary of the conclusions.

Sustainable Development

Eradication programs should have two objectives: 1) eradication of the
disease and 2) strengthening and further development of health systems,
especially functions such as monitoring and surveillance, supervision, and
program management. Potential risks of eradication to the health system and
health development include the diversion of resources from basic services
and other priorities in countries where the disease being eradicated is
perceived to be of lower priority. An additional concern is the failure to
accurately estimate the human and financial needs of the eradication
efforts. Potential benefits for health development should be identified and
delineated at the start of any eradication initiative. Measurable targets
should be set, and the program should be held accountable for achieving
them. Resources for eradication activities should be supplementary to those
available for basic health-care services.

Successful eradication programs are powerful examples of effective
management and can build management capacities that can be carried to other
health programs. Therefore, eradication programs should incorporate efforts
to design program activities that enhance leadership development and
managerial skills among health personnel. These programs also should
actively aid in the development and implementation of surveillance systems
that can be readily adapted to meet the needs of other national priority
programs after eradication goals have been achieved.

Noncommunicable Diseases

The conference concluded that better control was achievable for
micronutrient deficiencies (iodine, vitamin A, iron, and folic acid) and
lead intoxication, even though none of these conditions meet the
requirements for eradication. Recommendations were made for reducing
protein/energy malnutrition and lead intoxication and for accelerating
attainment of global goals for control of micronutrient deficiencies.
Micronutrient supplementation should be enhanced by food fortification and
the opportunities presented by existing health infrastructure and
immunization programs.

Bacterial Diseases

No bacterial diseases were judged to be candidates for eradication within
the next 10 to 15 years. Haemophilus influenzae type b (Hib) and congenital
syphilis are candidates for regional elimination, and trachoma is a
candidate for regional elimination over a somewhat longer term. By the
strict definition, neonatal tetanus elimination cannot be guaranteed,
although the World Health Organization "elimination goal" of <1 case/1,000
live births in every district is attainable.

Eradication was deemed to be a long-term vision for tuberculosis and Hib.
Each of the bacterial diseases considered at the conference represents a
major disease, and each has substantial research needs before long-term
goals can be achieved. Aggressive action was strongly recommended to
improve global control of these conditions.

Parasitic Diseases

The availability of potent, long-acting drugs makes possible the control of
onchocerciasis (river blindness) and lymphatic filariasis on a scale
heretofore unconsidered. Even though no parasitic diseases were candidates
for eradication within the next 10 to 15 years, the group recommended
onchocerciasis as a candidate for elimination. Lymphatic filariasis caused
by Wucheraria bancrofti also could be eliminated and possibly eradicated at
some time in the future.

Filariasis caused by Brugei malayi could be eliminated from many areas, as
could American trypanosomiasis (Chagas disease).

Viral Diseases

The group urged stronger international efforts to control rabies, yellow
fever, and Japanese encephalitis by using existing tools. None of the three
was considered a candidate for eradication. Hepatitis A eradication was
deemed biologically feasible, but further demonstration of sustainable
elimination is a prerequisite.

Rubella and measles were considered possible candidates for eradication
within the next 10 to 15 years. The eradication of rubella as an add-on to
measles eradication was thought biologically plausible, but several issues
first needed to be addressed: the human and financial cost of rubella
disease, the human and marginal costs of adding rubella to a measles
eradication effort, and demonstration that elimination is programmatically
feasible and sustainable in a large geographic area.

Measles eradication was concluded to be biologically plausible with the
present vaccine. In the Americas, measles transmission appears to have been
interrupted for variable time intervals in many countries. Elimination has
yet to be demonstrated in other regional settings. The group recommended
that industrialized countries proceed with elimination of measles as a step
toward eradication. In other countries, accelerating measles control should
be the priority, especially in areas with high death rates. Developing
countries should proceed cautiously to more costly measles elimination
programs to avoid undermining the polio eradication effort. Experience
gained from regional and country interventions should be used to refine the
strategies for eventual eradication.

General Comments

The short list of conditions for elimination and eradication within the
next 10 to 15 years was concluded to be a reflection of current limitations
in our knowledge. Additional basic and operational research was strongly
urged. Some participants expressed concern over the use of the term
"elimination," on the grounds that the distinctions between elimination and
control and elimination and eradication were unclear. Further discussion
and possible revision of these terms were recommended.

In summary, the conference provided a multidisciplinary forum for
addressing issues around elimination and eradication and their relationship
to sustainable health development. There was widespread agreement that an
eradication program could have many positive effects on systems development
and that explicit efforts should be made to maximize these positive effects
as well as to minimize any negative effects. Community mobilization and
organization should be seen as a component of sustainable health
development, with the additional potential for disease control and
eradication. The conclusions and recommendations of the conference should
be brought to other forums to expand international health goals and
strengthen the mutual ties between sustainable health development and
disease control, elimination, and eradication.

For further information, contact Dr. Walter R. Dowdle, 404-371-0466,
e-mail: wdowdle@taskforce.org; or Dr. Richard Goodman, 404-639-7400,
e-mail: rag4@cdc.gov.

Dick Conlon,
Task Force for Child Survival Carter Center, Atlanta, Georgia, USA

Conference sponsors included Burroughs Wellcome Fund; CARE; The Carter
Center; Centers for Disease Control and Prevention (CDC); CDC Foundation;
Children's Vaccine Initiative; The Edna McConnell Clark Foundation; The
Fogarty International Center; Glaxo Wellcome; International Life Sciences
Institute (ILSI); International Union of Microbiological Societies (IUMS);
Merck & Co., Inc. Vaccine Division; National Countil for International
Health (NCIH); National Institute of Allergy and Infectious Diseases
(NIAID); Pan American Health Organization (PAHO); Pasteur Merieux Connaught
USA; The Rockefeller Foundation; Rollins School of Public Health of Emory
University; The Task Force for Child Survival and Development; United
National Children's Fund; United National Development Programme (UNDP); The
World Bank; World Federation of PUblic Health Associations (WFPHA); World
Health Organization; and Wyeth-Lederle Vaccines and Pediatrics.

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New and Reemerging Infectious Diseases: A Clinical Course

Atlanta, Georgia, USA, June 13-15, 1998

Jointly sponsored by the Emory University School of Medicine, Centers for
Disease Control and Prevention, and National Foundation for Infectious
Diseases, New and Reemerging Infectious Diseases: A Clinical Course focuses
on the epidemiology, recognition, treatment, and management of new and
reemerging infectious diseases. The course will bring together the foremost
infectious disease clinicians and epidemiologists to present pertinent
information on emerging infections and prospective therapeutic agents.

For more information, contact Kip Kantelo at NFID, 4733 Bethesda Avenue,
Suite 750, Bethesda, MD 20814-5228, USA, tel: 301-656-0003, fax:
301-907-0878, http://www.nfid.org/nfid, e-mail: kkantelo@aol.com.

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Erratum

Vol. 4, No. 1

In the article, "Emerging Infectious Diseases-Brazil," by Hooman Momen, on
page 3 the last sentence of the next-to-the-last paragraph should read,
"The existing, generally passive epidemiologic surveillance system produces
information that arrives too late to be effective; however, a number of
measures, if implemented immediately, can mitigate the impact of any future
epidemic: a containment laboratory (biosafety level 4) that can handle
known and unknown microbes of high virulence; at least one infirmary,
properly designed and fully equipped, to treat highly contagious and
virulent diseases (the current lack of this facility poses a great danger
to the population should an outbreak of such a disease occur); and a mobile
multidisciplinary task force, including epidemiologists, microbiologists,
entomologists, and clinicians, ready to investigate suspected disease
outbreaks on short notice."


Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/newsnote.htm