[Emerging Infectious Diseases]  
[Volume 4 No. 4 /October-December 1998]

The OctoberDecember 1998 issue of the journal will contain the articles
listed below. Highlighted articles are currently available in electronic
format.

International EditorsUpdate

   * Emerging Infectious DiseasesSouth Africa, K.P. Klugman

Perspectives

   * Quasispecies Structure and Persistence of RNA Viruses, E. Domingo, E.
     Baranowski, C.M. Ruiz-Jarabo, A.M. Martn-Hernndez, J.C. Siz, and C.
     Escarms
   * Ecologic Studies of Rodent Reservoirs: Their Relevance for Human
     Health, J.N. Mills and J.E. Childs
   * Diphtheria in the Former Soviet Union: Reemergence of a Pandemic
     Disease, C.R. Vitek and M. Wharton

Synopses

   * Cell-to-Cell Signaling and Pseudomonas aeruginosa Infections, C. Van
     Delden and B.H. Iglewski
   * Rotavirus, U.D. Parashar, J.S. Bresee, J.R. Gentsch, and R.I. Glass
   * Chlamydia pneumoniae and Cardiovascular Disease, L.A.Campbell, C.C.
     Kuo, and J.T. Grayston
   * Bacterial Symbiosis in Arthropods and the Control of Disease
     Transmission, C.B. Beard, R.V. Durvasula, and F.F. Richards
   * Genetic Epidemiology of Infectious Disease in Humans: Design of
     Population-Based Studies, L. Abel and A.J. Dessein
   * Insecticide Resistance and Vector Control, W.G. Brogdon and J.C.
     McAllister

Dispatches

   * Mutators and Long-Term Molecular Evolution of Pathogenic Escherichia
     coli O157:H7, T.S. Whittam, S.D. Reid, and R.K. Selander
   * Increasing Hospitalization and Death Possibly Due to Clostridium
     difficile Diarrheal Disease, F. Frost, G.F. Craun, and R.L. Calderon
   * Introduction of Aedes albopictus into a La Crosse VirusEnzootic Site
     in Illinois, U. Kitron, J. Swanson, M. Crandell, P.J. Sullivan, J.
     Anderson, R. Garro, L.D. Haramis, and P.R. Grimstad
   * Mycobacterium canettii, the Smooth Variant of M. tuberculosis,
     Isolated from a Swiss Patient Exposed in Africa, G.E. Pfyffer, R.
     Auckenthaler, J.D.A. van Embden, and D. van Soolingen
   * Human Infections with Shiga Toxin-Producing Escherichia coli Other
     Than Serogroup O157 in Germany, L. Beutin, S. Zimmermann, and K.
     Gleier
   * New Orientia tsutsugamushi Strain from Scrub Typhus in Australia, D.M.
     Odorico, S.R. Graves, B. Currie, J. Catmull, Z. Nack, S. Ellis, L.
     Wang, and D.J Miller
   * Streptococcus parasanguinis: New Pathogen Associated with Asymptomatic
     Mastitis in Sheep, J.F. Fernndez-Garayzbal, E. Fernndez, A. Las
     Heras, C. Pascual, M.D. Collins, and L. Domnguez
   * Introduction of HIV-2 and Multiple HIV-1 Subtypes to Lebanon, D.
     Pieniazek, J. Baggs, D.J. Hu, G.M. Matar, A.M. Abdelnoor, J.E.
     Mokhbat, M. Uwaydah, A.R. Bizri, C.A. Ramos, L.M. Janini, A. Tanuri,
     C. Fridlund, C. Schable, L. Heyndrickx, M.A. Rayfield, and W. Heneine
   * Sporadic STEC O157 Infection: Secondary Household Transmission in
     Wales, S.M. Parry and R.L. Salmon
   * Ehrlichia Infection in Italy, M. Nuti, D.A. Serafini, D. Bassetti, A.
     Ghionni, F. Russino, P. Rombol, G. Macri, and E. Lillini
   * Salmonella Enteritidis PT6: Another Egg-Associated Salmonellosis? M.R.
     Evans, W. Lane, and C.D. Ribeiro
   * Reemergence of Epidemic Malaria in the Highlands of Western Kenya,
     M.A. Malakooti, K. Biomndo, and G.D. Shanks.
   * Murine Typhus in Travelers Returning from Indonesia, P. Parola, D.
     Vogelaers, C. Roure, F. Janbon, and D. Raoult
   * Differentiating Human from Animal Isolates of Cryptosporidium parvum,
     I.M. Sulaiman, L. Xiao, C. Yang, L. Escalante, A. Moore, C.B. Beard,
     M.J. Arrowood, and A.A. Lal
   * An Outbreak of Hantavirus Pulmonary Syndrome, Chile, 1997, J. Toro,
     J.D. Vega, A.S. Khan, J.N. Mills, P. Padula, W. Terry, Z. Yadn, R.
     Valderrama, B.A. Ellis, C. Pavletic, R. Cerda, S. Zaki, S. Wun-Ju, R.
     Meyer, M. Tapia, C. Mansilla, M. Baro, J.A. Vergara, M. Concha, G.
     Calderon, D. Enria, C.J. Peters, and T.G. Ksiazek

Commentaries

   * Host Genetics of Infectious Diseases: Old and New Approaches Converge,
     A.V.S. Hill
   * Does Restricted Distribution Limit Access and Coverage of of Yellow
     Fever Vaccine in the United States? T.P. Monath, J.A. Giesberg, and
     E.G. Fierros

Letters

   * Aedes Aegypti in Tucson, Arizona, T.M. Fink, B. Hau, B.L. Baird, S.
     Palmer, S. Kaplan, F.B. Ramberg, D.G. Mead, and H. Hagedorn
   * Can the Military Contribute to Global Surveillance and Control of
     Infectious Diseases? R. D'Amelio and D.L. Heymann
   * Dual Infection with Ehrlichia chaffeensis and a Spotted Fever Group
     Rickettsia: A Case Report, A.J. Sulzer
   * Dual Infection with Ehrlichia chaffeensis and a Spotted Fever Group
     Rickettsia: A Case ReportReply to Dr. Sulzer, D.J. Sexton and D.H.
     Walker
   * Reemergence of Plasmodium vivax Malaria in the Republic of Korea, P.S.
     Brachman
   * Paratyphoid Fever, M.R. Wallace
   * Hospitalizations after the Gulf War, K.M. Leisure, N.L. Nicolson, and
     G.L. Nicolson
   * Hospitalizations after the Gulf WarReply to K.M. Leisure et al., J.D.
     Knoke and G.C. Gray

Book Review

   * HIV Nursing and Sympton Management, A.C. Moorman

News and Notes

   * Overseas Medical Laboratory Conference

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Update

International Editors

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Emerging Infectious DiseasesSouth Africa
Keith P. Klugman
The South African Institute for Medical Research Johannesburg, South Africa

Infectious diseases remain a major cause of illness and death in South
Africa. The medical infrastructure is good in major cities, primary health
care is expanding in rural areas, and modern diagnostic facilities are
reasonably widely available. Urban areas have a mixture of infectious
disease problems encountered in developing as well as industrialized
countries, and in the tropical environment of the northeast, malaria and
other vector-borne diseases persist.

[Figure] An aerial view of Chris Hani Baragwanath Hospital, 
with 3,000 inpatients the largest hospital in the worldSoweto, 
South Africa. Multiple-drug resistant pneumococci were found 
here in 1978.

Emerging Infectious Disease Surveillance

National surveillance of emerging infections in South Africa is in
its infancy. A number of infectious diseases are notifiable, but no
formal laboratory-based surveillance system is in place to confirm
diagnoses or to subtype microorganisms for epidemiology purposes.
I have summarized infectious disease trends in South Africa, with
special emphasis on the antimicrobial resistance patterns of
locally circulating microorganisms.

Bacterial Diseases

Streptococcus pneumoniae

Fully resistant and multiple-drug resistant pneumococci were discovered in
South Africa in 1978. The pattern of emergence of pneumococcal resistance
in South Africa has been somewhat different from that observed in Western
Europe (and more recently in the United States). In those countries, the
emergence of resistance was explosive and associated with a limited number
of clones, particularly of serotype 23F and 6B, which are associated with
multidrug resistance and high-level penicillin resistance. The highly
resistant strains initially isolated in South Africa belonged largely to
serotype 19A and for some as yet unexplained reason, remain rare.
Antimicrobial resistance to penicillin in the pneumococcus in South Africa
has reached very high levels (approximately 45% in young children), but
most of this resistance is caused by a large number of intermediately
penicillin-resistant strains rather than a limited number of clones found
internationally. Antibiotic resistance differs in the public versus private
health-care sectors of South Africa. The most important difference may be
the relative scarcity of macrolide resistance among strains of pneumococci
in public hospitals, where these drugs are less often used. Recently, the
incidence of pneumococcal disease in both adults and children has doubled
as a result of the increased incidence of HIV infection. HIV infection is
specifically associated with antibiotic resistance and with childhood
serotypes of the pneumococcus (6A, 6B, 9V, 14, 19F, 19A, 23F).

Haemophilus influenzae

In contrast to the United States and Western Europe, South Africa has a low
incidence of betalactamase-producing H. influenzae (10%). The reason for
the contrast between the low incidence of betalactamase-producing H.
influenzae and the high incidence of penicillin resistance in the
pneumococcus is not known.

Neisseria meningitidis

Over the past few years, the distribution of meningococcal serogroups has
been fairly even between groups A, B, and C. An epidemic in Cape Town of
group B in the 1980s provided an opportunity to attempt to use an outer
membrane protein vaccine, but the epidemic ceased before enough cases could
be enrolled in the vaccine trial. An epidemic of group A meningococcal
meningitis is ongoing in the countries immediately to the north of South
Africa, particularly in Mozambique, and an increasing number of group A
strains are being identified, largely in adult refugees from Mozambique.
These group A strains have been typed by the World Health Organization
Meningococcal Reference Center in Oslo, Norway, and represent a unique
South African clone. The epidemic clone associated with the Haj (III-1) in
the 1980s was, however, discovered for the first time in South Africa in
1997.

Staphylococcus aureus

The incidence of methicillin-resistant S. aureus in South Africa is
alarming, with up to 50% of nosocomial isolates being
methicillin-resistant. No vancomycin-intermediate S. aureus strains as have
been reported in the United States and Japan have been identified in South
Africa to date. The incidence of methicillin resistance among S.
epidermidis strains in hospitals is, as expected, even higher.

Enterococcus

The first strains of enterococci resistant to vancomycin (VRE) and
teicoplanin, which were due to the presence of the van A resistance gene,
were described in South Africa only in 1996. These strains were likely
imported. The incidence of VRE in South Africa remains low but is expected
to increase.

Diarrheal Pathogens

Endogenous transmission of cholera has not been observed in South Africa
for the past decade. Epidemics of cholera have occurred in countries to the
north, particularly in Mozambique, and a number of imported cases have been
identified each summer in South Africa. The potential for an epidemic in
rural areas with poor sanitation and contaminated water exists. Shigella
dysenteriae type I producing Shiga toxin and associated with hemolytic
uremic syndrome was first identified in an epidemic in adults on the South
African/Mozambican/Swaziland border in 1993. Subsequently, the same strain
has become quite common among children and adults, particularly in
KwaZulu-Natal province, and has spread down the east coast to the western
Cape. Disease in adults as well as children suggests that this strain has
not been endemic in South Africa, at least during the past generation. The
strains are resistant to ampicillin and cotrimoxazole but are generally
susceptible to nalidixic acid and the fluoroquinolones. Occasional imports
of multidrug-resistant typhoid have been identified in KwaZulu-Natal, where
there is a large Indian community; these strains likely come from Southeast
Asia.

Nosocomial Infections

Extended spectrum betalactamases are endemic particularly among Klebsiella
species in intensive-care units throughout the country. Gram-negative
bacteria producing chromosomally mediated Beta-lactamase are common in all
of these settings and Acinetobacter-related problems abound in most
intensive-care units.

Tuberculosis (TB)

The incidence of TB is increasing in parallel with the HIV epidemic in
South Africa. Multiple-drug resistance remains <5% and is commonly
associated with noncompliance to drug regimens.

Plague

Low levels of endemic plague are present in rural South Africa, but the
last epidemic was in Namibia in the 1980s.

Viral Diseases

HIV infection was not apparent in South Africa until around 1988 when
endogenous cases began to emerge. The first reported cases occurred
predominantly in homosexual men who had sexual contacts in the United
States; in one early case, the patient presumably had heterosexual contact
with a sex worker in Central Africa when the disease was first discovered
there. The incidence of HIV infection among pregnant African women in South
Africa in 1990 was 0.4%. The number of infected persons has doubled at
approximately 9-month intervals until now when approximately 20% of adult
African men and women are infected in the northern and eastern parts of the
country. This number declines to less than 10% in the south, but HIV,
although delayed with reference to the rest of Africa, has had and will
have a staggering impact on emerging infectious diseases in South Africa.
No specific diseases peculiar to South Africa have been found to be
associated with HIV infection; major opportunistic infections include TB,
cryptococcal disease, cytomegalovirus infections, bacterial diseases such
as pneumococcal or salmonella bacteremia, and toxoplasmosis.

Viral Hemorrhagic Fevers

A case of Marburg fever was identified in the 1970s in a tourist visiting
several African countries to the north of South Africa; nosocomial
transmission to two persons was identified at that time. One case of
nosocomial transmission of Ebola has been reported (the patient with the
index case was flown down to South Africa from Gabon); however, no endemic
transmission of Ebola virus occurs in South Africa. That the nosocomial
transmission of Ebola virus was limited to a single person despite the fact
that the diagnosis of the index case was not made until a number of weeks
after the patient entered the country is a tribute to the standard of
medical care in South Africa. Congo Crimean hemorrhagic fever is endemic in
southern Africa, but as the vector tick rarely bites humans, not many human
cases have been found. Epidemics have been associated with infections in
rural communities particularly of farmers and workers who slaughtered
infected cattle. An epidemic among ostriches occurred in 1997.

Parasitic Diseases

Malaria remains a serious systemic disease in South Africa. The disease is
endemic in the northern and eastern parts of the country where the climate
is tropical. After many years of spraying huts with DDT, malaria became
rare, but the interruption of the program because of war in the 1970s and
1980s in Mozambique and Zimbabwe (and the development of drug resistance in
other insects such as bedbugs, which reduced the acceptability of the
program) has led to a rapid resurgence of malaria. Chloroquine-resistant
strains, which were rare in the 1980s, are now common, although
quinine-resistant strains have not yet been demonstrated. Schistosoma
haematobium and S. mansoni are endemic in rivers along the eastern part of
the country and pose a health risk to nonimmune visitors.

Fungi

Cryptococcus neoformans var gattii and var neoformans represent an
increasing fraction of cerebrospinal fluid isolates in South Africa because
of the HIV epidemic.

Further Reading

Additional information on emerging infectious diseases in South Africa is
available from the South African Virus Laboratories Surveillance Bulletin,
which is published by the National Institute for Virology (Private Bag X4,
Sandringham, 2131 Johannesburg, South Africa); data can be obtained from
the South African Institute for Medical Research Annual Reports (P.O. Box
1038, Johannesburg 2000, South Africa); national antimicrobial
susceptibility data are published in the South African Medical Journal by
the Antibiotic Study Group of South Africa; and clinically based notifiable
diseases are reported to the Department of Health, which produces a free
journal called Epidemiological Comments (The Director General, Department
of Health, Private Bag X828, Pretoria, 0001, South Africa.) The final
source of information on emerging infectious diseases in South Africa,
aside from the specific reports that would appear in any international
journal is the Proceedings of the Annual Meeting of the Infectious Diseases
Society of Southern Africa (membership and meeting information, Dr. Lucille
Blumberg, e-mail: lucilleb@mail.saimr.wits.ac.za).

Acknowledgment

      I thank Professor H. J. Koornhof for his review and comments on this
report.

      Dr. Klugman, director of the South African Institute for Medical
Research and and its Pneumococcal Diseases Research Unit, is professor of
microbiology and chair of the School of Pathology, University of the
Witwatersrand. He has broad interests and experience in bacterial
infections and works with WHO committees and advisory groups on acute
respiratory infections, meningitis, pneumonia, vaccine, and drug
management.

      Address for correspondence: K.P. Klugman, The South African Institute
for Medical Research, Johannesburg, South Africa; fax: 27-11-489-9012;
e-mail: KEITHK@mail.saimr.wits.ac.za.

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Perspectives

Quasispecies Structure and Persistence of RNA Viruses

Esteban Domingo,* Eric Baranowski,* Carmen M. RuizJarabo,* Ana M.
Martn-Hernndez, Juan C. Siz, and Cristina Escarms*
*Centro de Biologa Molecular "Severo Ochoa," Universidad Autnoma de
Madrid, Cantoblanco, Madrid, Spain; John Innes Center, Norwich, United
Kingdom; Universitat de Barcelona, Barcelona, Spain

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      Viral quasispecies are closely related (but nonidentical)
      mutant and recombinant viral genomes subjected to continuous
      genetic variation, competition, and selection. Quasispecies
      structure and dynamics of replicating RNA enable virus
      populations to persist in their hosts and cause disease. We
      review mechanisms of viral persistence in cells, organisms, and
      populations of organisms and suggest that the critical
      interplay between host and viral influences (including in some
      cases the quasispecies organization) is the main driving force
      for long-term survival of viruses in nature.

The emergence and reemergence of infectious diseases is influenced by the
genetics of the infectious agents, the genetics of their hosts and
potential new host species, and a considerable number of environmental
factors (1-3). The current view proposes a strong stochastic (chance)
component regarding the time, place, severity, and epidemiologic impact of
infectious disease emergences (2,3). For RNA and possibly some DNA viruses,
increasing evidence suggests that genetic variation (mutation,recombination, 
and genome segment reassortment in the case of multipartite genomes) affects 
adaptability to environmental changes (4-9). Since some types of adaptation 
involve changes in host cell specificity (5,6,10), genetic variation of 
viruses may be involved in the emergence of pathogenic viruses from 
apathogenic ancestors. Many studies over the last 2 decades have documented 
the unpredictability of genetic variation in viruses (2,5-7).

In this report, we consider viral persistence in connection with the
population structure of RNA viruses, specifically, the extension (in space
and time) of the pool of replicating genomes, which are a potential source
of variant viruses with altered biologic features. For example,
hantaviruses are apathogenic and endogenous to several rodent species. In
several geographic areas of the American continent, the unusually mild and
wet (El Nio effect) spring seasons of 1992 and 1993 resulted in abundant
food and coverage for deer mice, increased numbers of infected deer mice,
and increased risk for human infections. These factors led to the newly
recognized severe pulmonary syndrome of humans in 1993 (11). Like many
pathogenic RNA viruses, hantavirus evolved in humans, and recent
epidemiologic evidence suggests human-to-human transmission (12); whether
human-to-human transmission is exceedingly rare or is an unusual property
associated with Andes virus is not known. The number of carriers, their
ability to transport virus (because of their mobility and absence of severe
symptoms), and the viral load (number and concentration of infectious
particles) in each carrier must be involved in viral emergences and
reemergences (2-4,6,13).

Population Complexity of Replicating Genomes: Viral Quasispecies

The viral load in an infected host is both static and dynamic. The static
component can be divided into the total number of particles and the numbers
and types of mutant viral genomes present in that total. Rather than being
homogeneous, RNA virus populations consist of complex distributions of
mutant (and sometimes also recombinant) genomes, in a type of population
structure known as quasispecies (6,14-16). Assuming a random distribution
of mutations among genomes, the number of variant genomes in a viral
quasispecies increases dramatically with population size (15,17). For
example, in a genome distribution with an average of five mutations per
genome, the expected number of genomes with 20 mutations is 26 when the
population size is 1x10(sup 8) viral particles but reaches 2,600 when the
population size is 2x10(sup 11) particles, as calculated from the Poisson
distribution (17). Fitness variations of the individual mutants modify the
actual number of genomes in each mutation class (6,14-17). In a horse
infected with epizootic Venezuelan equine encephalitis, viral titers in
blood reach a high of 10 (sup 8) infectious units (i.u.)/ml, or a total of
approximately 3x10(sup 12) i.u. These high titers are probably needed for
efficient transmission of the virus to insect vectors and for completion of
the arbovirus life cycle (18). High titers are also reached in acute
infections with HIV-1, hepatitis B virus (HBV, a hepadnavirus replicated by
an error-prone polymerase through an RNA intermediate), hepatitis C virus
(HCV), influenza virus, the animal foot-and-mouth disease virus (FMDV), and
probably many others (7,8).

Increasing evidence indicates that quasispecies evolution may lead to the
selection of virulent viruses and to the emergence of new viral pathogens
(1-7,10,17,18). For example, a single site at the 5'-untranslated genomic
region of coxsackievirus B3 was associated with its cardiovirulent
phenotype. Swine vesicular disease, not reported before 1966, may represent
a human coxsackie B5 variant adapted to swine. Specific mutations in the
RNA of lymphocytic choriomeningitis virus can confer a viral tropism for
neurons or for cells of the immune system. Evolution of measles virus can
lead to hypermutated forms that have been associated with subacute
sclerosing panencephalitis. Major human influenza pandemics have been
associated with antigenic shift resulting from genome reassortment between
a human influenza virus and an animal influenza virus. Some small DNA
viruses possess considerable genetic heterogeneity within infected hosts.
Canine parvovirus was probably derived from a feline parvovirus as a result
of two amino acid replacements in the viral capsid (1-7,10,17,18).

The viral load during RNA virus replication also has a strong dynamic
component. In persons infected with HIV-1, HBV, or HCV, an estimated 10(sup 10)
to 10(sup 12) new virions are produced each day. For some microorganisms (not
only viruses), in what has been termed short-sighted evolution of
pathogenic microorganisms (19), virulence may be an inadvertent consequence
of mutation and selection in the parasite population. Models of HIV-1
pathogenesis based on the continuous production of antigenic variants have
been proposed (8,19,20). As the infection progresses the complexity of
antigenically distinct mutants may overwhelm the immune system, leading to
AIDS (20). Models of HIV-1 pathogenesis, based on the stimulation of
infected T-lymphocytes by secondary antigens (from opportunistic
infections), have also been proposed; the progression of HIV infection to
AIDS is still poorly understood. In HIV, and many other infections, the
evolving viral quasispecies are exploring new mutant variants at
astonishingly high rates. The balance between mutation rates and
replication rounds is one of the reasons for the great adaptability of RNA
viruses (6,7,14-17).

Types of Viral Persistence

"Persistence," which refers to long-term survival of viruses in their
hosts, has been described in at least three ways. 1) Long-term survival of
virus within a viable cell population occurs when cell disease and
destruction are limited and viral genomes replicate in balance with the
multiplication of the host cells. 2) Survival of viruses in entire
organisms can rarely be reduced to persistence in one cell type since
organisms are built as sets of interconnected mosaics of cell types and
cell protein effectors; persistence of viruses in organisms often means
coping with multitudes of selective forces and defense reactions while
allowing the host to survive. 3) A virus can be maintained in nature by the
continuous infection of susceptible host organisms, with or without
persistence in cells or organisms and with or without long-term stability
of viruses as free particles. Genetic, ecologic, and environmental factors
exert different influences on these types of persistence. The quasispecies
structure of RNA viruses plays an obvious, positive role in some
persistence mechanisms. In others the role is more subtle, marginal, or
nonexistent. The observations summarized here suggest, however, that
persistence is always the result of interactions between viral and host
determinants.

Persistence of Viruses in Cells

A variety of mechanisms enable viral genomes to replicate in balance with
host cell multiplication. Most of the well-studied mechanisms of
persistence of RNA viruses in primary cell cultures or established cell
lines involve genetic variation of the virus, the cell, or both. One way to
limit cell death is by generating and accumulating defective genomes.
Defective genomes depend on a complementing, standard virus for
replication; yet they may compete with that standard virus for cellular and
viral gene products (which the virus needs to complete its life cycle and
kill cells), thus increasing cell survival (21). A classic example is
provided by defective interfering (DI) particles of vesicular stomatitis
virus (VSV), and related viruses (22). During serial passage of VSV at high
multiplicity of infection, DI particles accumulate in a cyclic pattern
(23). Standard VSV and DI particles alternate in their dominance in a
continuous process of mutant generation, competition, and selection. Excess
virus leads to generation and accumulation of DI particles. With excess DI
particles, mutant viruses able to escape interference rise to dominance
(5). A feedback mechanism is established, which underlines the biologic
relevance of the rapid evolutionary potential of RNA genomes (5).

Cell disease can also be limited by infecting cells with limited
permissivity for the virus or by selecting noncytolytic variant viruses. In
a number of cell-virus systems, cells and viruses coevolve, as documented
for reovirus persisting in L cells (24) and for other RNA and DNA viruses
(25-28). FMDV illustrates how the quasispecies structure initiates and
maintains persistence (26,29). The presence in the FMDV quasispecies of
variants with decreased ability to kill BHK-21 cells was not the mechanism
(at least the prevailing mechanism) responsible for persistence. Rather,
these experiments (29), which measured the proportion of cells that
survived an initial cytolytic infection, indicated that a rapid variation
of the cells initiated persistence. Indeed, the cells rapidly became more
resistant to the infecting FMDV, and the virus became more virulent for the
host BHK-21 cells (29). The quasispecies structure and consequent
adaptability of RNA genomes do not justify any generalization on the
participation of quasispecies, rather than the seemingly more static
cellular DNA genomes, in initiation of viral persistence (29). However, the
rapid evolution of FMDV toward virulence was very likely facilitated by
mutant generation and was essential to sustain persistence (26,29). When
the carrier cultures were challenged with FMDVs of distinct degrees of
virulence (a population replacement experiment), the endogenous persistent
virus was replaced by the externally added virus only when the latter
displayed a higher virulence for BHK-21 cells. Thus, virulence can be a
positive trait in viral persistence (30), and virulent variants present in
the FMDV quasispecies helped maintain persistence when the triggering
cellular event had occurred (29,30).

Persistence of Viruses in Organisms

Viral persistence in organisms requires a supply of susceptible cells
replicating at the same pace as the virus and the ability to survive the
host immune response. With RNA viruses, the continuous production of mutant
viruses (inherent to the quasispecies dynamics [14,15]) contributes to
virus survival (2,5-7,15-17). Viruses often use alternative receptors and
coreceptors, and one or a few amino acid substitutions at exposed surface
sites may trigger a shift in receptor specificity (8,10,31). For HIV-1,
amino acid substitutions at the surface glycoprotein may effect shifts in
receptor use (10,31), and mutations at several genes may promote escape
from antibodies or cytotoxic T lymphocytes (CTLs) (8,10); the generation of
109 to 1010 viral particles per day undoubtedly facilitates escape (8,20).
Although evidence of positive (Darwinian) selection of escape mutants is
firm, the quasispecies structure and dynamics predict genetic variation in
the absence of immune selection (5,32). The two mechanisms are compatible.

Some viruses (HBV, HIV-1, FMDV, measles virus, herpesviruses) may persist
after an acute infection, and the dose of infecting virus often determines
either clearance or long-term persistence (8,33). Viruses transmitted
vertically may induce immune tolerance and persist in adults (33). Viruses
may also persist by being sequestered in some privileged sites of an
organism, such as the central nervous system, partially hidden from immune
attack (33). Ineffective antibody responses may be due to tolerance,
immunosuppression (as a result of some infection, genetic disease, or
immunosuppressive treatments), production of nonneutralizing antibodies, or
cell-to-cell spread of virus not exposed to immune recognition (33).
Viruses that infect lymphocytes or macrophages (HIV, cytomegalovirus,
measles virus) may alter immune responses and thus facilitate their own
persistence (8,33).

Persistence of Viruses in Nature

All viruses have developed common functional and adaptive strategies;
however, the strategies used by DNA and RNA viruses to evade host defenses
have distinct features. RNA viruses often exploit mutation to achieve
changes in host range and escape antibody and CTL responses (8,10,31,32).
Because of their limited genetic complexity (which can be equated with the
size of their genomes [of 3 to 30 Kb]), RNA viruses are generally tolerant
to high levels of mutagenesis (6,14,16). In contrast, large DNA viruses (of
the herpesvirus family, poxviruses, iridoviruses, adenoviruses) have
complex genetic information; the need to maintain this information limits
their tolerance to mutation. That simple genomes are generally more
tolerant to mutagenesis than more complex ones can be argued on the basis
of the higher mutation rates observed in simple replicons and the evolution
of replication to include proofreading and postreplicative repair functions
for the replication of DNA of cells and of at least some of the complex DNA
viruses (5,6,14,16). Although antibody and CTL-escape mutants (as well as
drug-resistant mutants) have also been described for DNA viruses, the
latter have evolved alternative mechanisms to counteract host defenses
(34,35). As examples, the adenovirus proteins E3/19K and E1a suppress
surface molecules (MHC class I, class II, adhesion molecules) required for
T-cell recognition. The Epstein-Barr virus BCRF1 protein is a host
interleukin (IL)-10 homologue that activates the IL-10 receptor. Human
cytomegalovirus encodes a protein structurally resembling the macrophage
inflammatory protein 1[alpha]/RANTES receptor. Cytokines regulate immune and
inflammatory responses and may trigger antiviral responses in organisms. It
is not surprising that DNA viruses causing either persistent or acute
infections have evolved to encode homologues of the extracellular binding
domains of cytokine receptors (34,35).

Phylogenetic analyses of herpesvirus genomes infecting a broad range of
host animal species (the complete genomic nucleotide sequence of 18
herpesvirus genomes is known) suggest a possible cospeciation with their
host organisms (36). The capture of cellular genes (and gene assemblies) by
DNA viruses to counteract host defense responses agrees with the proposal
of a modular origin of viruses (37,38) and has opened a new approach for
analyzing new functions related to the immune response in differentiated
organisms (35). The selective forces imposed by viral parasites may have
contributed to a more rapid diversification of cellular proteins involved
in host defense (34). In turn, coevolution may have relaxed the specificity
of viral analogues of cellular effectors: the viral chemokines vMIP I and
vMIP II of herpes simplex virus-8 bind to a broader range of cellular
chemokine receptors (although with lower affinity) than their cellular
homologues (39). Genes that have strong sequence identity with cellular
counterparts are also encoded by RNA viruses (e.g., sarcoma and leukemia
viruses) (34) that tend to exchange genetic material with their hosts.
However, evasion strategies based on gene capture and protein mimicry are
dominant in DNA viruses, and strategies based on mutant production are
widespread among RNA viruses (6-8,16,17,34,35).

Persistence of Viruses at the Population Level

To be maintained in nature and avoid extinction, viruses must have
susceptible hosts as well as adaptability to a range of biologic
environments (6). Even persistence in an individual host would not help
long-term persistence of a viral pathogen in nature, without a number of
additional influences (13,19,40), such as the possibility of transmission
within the same host species (sexual, perenteral, or respiratory routes) or
to a different host species (13,40). Human rhinoviruses do not persist in
their hosts and succeed (supported by the high frequency of common colds)
in continuous reinfections through aerosols transmission (or other
contacts). In the other extreme of life cycle complexity, arboviruses
sequentially infect a number of disparate hosts. As an example, insects can
transmit Venezuelan equine encephalitis virus between horses and other
mammalian species and can also infect humans, the dead-end point of a
complex infectious cycle (18). Evidence indicates that a few point
mutations in the viral genome may be sufficient to upset the balance of
viral loads in enzootic cycles, render the virus epizootic, and cause
severe outbreaks (18). In La Crosse virus, an important cause of pediatric
encephalitis, the virus persists in cells of the midgut epithelium of Aedes
triseriatus. The virus is transovarially transmitted and survives
transeasonally in the diapausing mosquito embryo. In quiescent ovaries
there is reduced viral replication with limitations in the host-derived
5'-mRNA sequences that prime viral transcription (41). Long-term survival
at the population level is associated with persistence and limitation of
virulence in the vector mosquito. The complex arboviral life cycle appears
to require the fine tuning of a number of factors: the amount of virus in
viremia and the duration of viremia, which are likely to contribute to the
efficient uptake of virus by the vectors (18), and replication and
stability of the virus in the vectors to ensure infection of the mammalian
host (18). Perturbations in these, and probably other factors, could lead
to viral extinction. What prevents viral extinction? Again, all evidence
points to genetic factors of the virus and the hosts together with
environmental and ecologic influences (1,3,5-7,17,18). Virus variants
unable to fulfill the required processes with the correct timing may be
generated, but they would be selected against. We can study only successful
examples. Virus variants that do not complete a complex life cycle are yet
another example of negative selection (elimination of suboptimal viruses).
Negative selection is one of the forces preserving virus variants that are
fit in relation to the interactions with their hosts (6,7,14-17,32), a
force we believe is responsible for maintaining (at least to some extent)
the identity of RNA viruses as disease-causing agents.

The stability of virus particles may also play a relevant role in
successful transmission, as documented in aerosol transmission, airborne
spread, or mechanical transport of viruses by insects (42). An example is
provided by FMDV. In spite of its lability at mildly acidic pH and at
moderate temperatures, FMDV is resistant to desiccation and can be
transported on dust particles over long distances. Most spectacular is the
case of the highly complex and host-specific baculoviruses. They form
rod-shaped virions occluded in large capsules made of a viral-coded matrix
protein termed polyhedrin. Capsules are uptaken by the insects (of the
orders Lepidoptera and Hymenoptera) and are dissolved by midgut epithelial
cells. Thus, capsule formation is responsible for the spread of the virus
in the insect population. Again, a great diversity of mechanisms (as varied
as those seen at the level of individual organisms or of the cells) operate
to ensure viral persistence at the population level.

Basic Issues

The mutant distributions that compose viral quasispecies are the raw
material on which selective forces and random sampling events act in the
molecular evolution of RNA viruses (5-7,14-18,32,38). In addition to
constituting a basic adaptive strategy, the quasispecies genetic
organization has a number of biologic implications (5-9,16,32,43), some of
which have a direct bearing on viral persistence. It has been wrongly
argued that if quasispecies distributions were involved in virus
persistence, all RNA viruses would establish persistent infections but that
on the contrary, only a minority do. We hope to have shown that persistent
infections are unavoidably, necessarily, and evidently the result of an
interplay between viruses and their hosts (44). Thus, a quasispecies
structure does not imply necessarily that the virus will produce a
persistent infection. A potent CTL response may clear a virus infection
provided that the viral load has a size amenable to clearing, and this may
occur whether the virus is a complex quasispecies or not. In contrast, a
similar CTL response confronting a high viral load may frequently fail to
clear the infection; in this failure, the presence (in a dynamic
quasispecies) of CTL-escape mutants (and many other types of mutants with
biologically deviant properties) may be crucial for virus survival,
including the establishment of persistence or chronicity. Also, increasing
evidence suggests that viruses thought not to persist, such as poliovirus,
may actually do so; late postpoliomyelitis syndromes may be one consequence
(33,42). Even with the available analytical technology, total clearing of
an infecting virus from an organism cannot be guaranteed.

The issue is clear: either we design new antiviral strategies that take
into consideration the quasispecies structure of RNA viruses (6,9,43), or
viral diseases (classical, new, or reemergent) will remain difficult to
control.

Acknowledgments

      We thank A. Alcam for valuable information on cellular homologues of
DNA viruses; an anonymous referee for clarifying the ecologic factors
involved in hantavirus reemergence; and C. Biebricher, J.J. Holland, S.
Wain-Hobson, and S. Weaver for many useful discussions.

      Work in Madrid was supported by grants DGICYT PB94-0034-C02-01, DGES
PM97-0060-C02-01, FIS 98/0054-01, Comunidad Autnoma de Madrid, and
Fundacin Ramn Areces.

      Dr. Domingo is professor of research at Centro de Biologa Molecular
"Severo Ochoa" in Madrid, Spain. He is interested in the genetic variation
of viruses, the quasispecies structure of viral populations, and new
antiviral strategies.

      Address for correspondence: Esteban Domingo, Centro de Biologa
Molecular "Severo Ochoa" (CSIC, UAM), Universidad Autnoma de Madrid.
28049-Cantoblanco, Madrid, Spain; fax 34-91-397-4799; e-mail:
edomingo@cbm.uam.es.

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Perspectives

Ecologic Studies of Rodent Reservoirs: Their Relevance for Human Health

James N. Mills and James E. Childs
Centers for Disease Control and Prevention, Atlanta, Georgia, USA

---------------------------------------------------------------------------
      Within the past few years, the number of "new" human diseases
      associated with small-mammal reservoirs has increased
      dramatically, stimulating renewed interest in reservoir ecology
      research. A consistent, integrative approach to such research
      allows direct comparisons between studies, contributes to the
      efficient use of resources and data, and increases investigator
      safety. We outline steps directed toward understanding
      vertebrate host ecology as it relates to human disease and
      illustrate the relevance of each step by using examples from
      studies of hosts associated with rodent-borne hemorrhagic fever
      viruses.

The practical importance of understanding host and vector ecology has been
recognized at least since the early 1900s. Knowledge of the
container-breeding habits of Aedes aegypti enabled early successes in the
control of yellow fever virus transmission and, ultimately, the completion
of the Panama Canal in 1914 (1). Diverse applications of vector/reservoir
ecology-based measures to prevent zoonotic disease include the prediction
of Lyme disease risk by monitoring acorn mast production and its impact on
the vertebrate hosts of the tick vectors (2), control of vector populations
for Borrelia burgdorferi and Yersinia pestis through the application of
acaricides and insecticides to rodents and deer at feeding stations (3,4),
dissemination of bait containing vaccines to control rabies in foxes (5),
and use of satellite imagery to predict the activity of Rift Valley fever
in East Africa (6,7).

The rodent-borne hemorrhagic fevers, among the most dramatic of recently
emerging infectious diseases, are caused by two distinct groups of
negative-stranded RNA viruses: the arenaviruses (family Arenaviridae) and
the hantaviruses (genus Hantavirus, family Bunyaviridae). With few
exceptions, each virus in these two groups is primarily associated with a
single species of rodent host of the family Muridae. In the specific host,
the virus establishes a prolonged infection, which rarely causes disease in
the animal. The infected host sheds virus into the environment (in urine,
feces, and saliva) for extended periods (8-10). These characteristics are
key to the transmission of the viruses to humans (by the inhalation of
aerosolized virus) and to other rodents (by horizontal and sometimes
vertical mechanisms).

Arenaviruses cause the South American hemorrhagic fevers, which produce
hundreds of cases annually, with a case-fatality ratio as high as 33%. The
best studied of these agents is Junn virus, which is carried by the corn
mouse (Calomys musculinus) and causes Argentine hemorrhagic fever (AHF).
AHF was first recognized in 1955 on the central pampas of Argentina (11),
where before the deployment of a new vaccine in 1992, hundreds of cases
occurred each year. Although arenaviral diseases of humans (other than
lymphocytic choriomeningitis associated with the introduced Old World
rodent Mus musculus) have not been recognized in North America, Tamiami
virus has been recognized in association with cotton rats (Sigmodon
hispidus) since 1969 (12), and Whitewater Arroyo virus was identified from
wood rats (Neotoma species) in the southwestern United States in 1995 (13).
The potential of Whitewater Arroyo virus for causing human disease is under
investigation.

Hantaviruses cause hundreds of thousands of cases of hemorrhagic fever with
renal syndrome (HFRS) in Europe and Asia each year. Hantaviral disease was
thought to be rare or absent in the United States, although three cases of
mild HFRS associated with rat-borne Seoul virus had been described (14). In
1993, the discovery of hantavirus pulmonary syndrome (HPS), which rapidly
kills approximately half of those infected, surprised public health
officials and virologists in the United States. The causative agent, Sin
Nombre virus (SNV), is hosted by the deer mouse, Peromyscus maniculatus,
and the disease syndrome markedly differs from HFRS. Since 1993, at least
20 additional hantaviruses have been isolated from rodents throughout the
Americas; about half are known human pathogens (Figure 1).

[Figure 1.] Distribution of World hantaviruses. * = known human pathogens.

Recognition of new rodent-borne diseases has renewed interest in reservoir
host ecology in the United States, and recognition of HPS in South America
has prompted field studies in Paraguay, Argentina, Chile, and Brazil.  
In 1997, representatives from 13 recognized, autochthonous, New
American countries, meeting in Lima, identified surveillance in humans and
rodents as a priority for combating emerging hantaviral disease. Reservoir
studies are being designed and conducted in many parts of the world by
mammalogists, ecologists, and vector-control personnel. However,
sanitarians and others with no specific training in such areas are being
asked to conduct reservoir studies in the course of investigating disease
outbreaks. Rarely are these professionals trained in the safe handling of
animals potentially infected with viruses capable of causing fatal disease.

To promote discussion of study design, data collection, and appropriate and
safe methods for reservoir studies, we review prior efforts and lessons
learned. Although specific examples are restricted to the rodent-borne
hemorrhagic fever viruses, many of the generalizations can be applied to
other pathogens maintained by small-mammal reservoirs.

An Outline for Reservoir Studies

Assuming that the primary reservoir host has already been implicated, the
following consecutive but overlapping steps can promote an understanding of
rodent reservoir ecology as it relates to human disease: 1) determination
of the geographic distribution of the host; 2) determination of the
geographic range of the pathogen within the host range; 3) determination of
the regional distribution of the host and pathogen among the distinct
biomes or habitat types; 4) determination of the relative prevalence of
infection among demographic subpopulations of the host (e.g., males versus
females and adults versus juveniles); 5) elucidation of the temporal and
fine-scale spatial patterns of host-pathogen dynamics through prospective,
longitudinal studies; and 6) development of an integrative time- and
place-specific predictive model.

Examples from studies of hantaviruses and arenaviruses illustrate how each
step is relevant to human health.

Geographic Distribution of the Host

The geographic distribution of the host(s) defines the maximum area in
which the disease can be endemic. For most small-mammal species in North
America, these distributions are known relatively precisely (15). For
example, the deer mouse is one of the best-studied small-mammal species in
the world, and its distribution throughout North America is well defined
(Figure 2). Fewer data are available on the distribution of Central and
South American species. The range of the cotton rat, S. hispidus, which is
the reservoir of both Black Creek Canal virus (a hantavirus that causes
HPS) and Tamiami virus (an arenavirus not associated with human disease) is
well defined in the United States. However, the southern limits of the
species' distribution are unclear (16).

[Figure 2.] Geographic distribution of the deer mouse, Peromyscus
maniculatus (shaded) (48).

Problems in defining species' distributions are exacerbated by
imprecise taxonomy. The multimammate rat, Mastomys natalensis, was
originally described as the reservoir for Lassa fever in West Africa.
Subsequent taxonomic studies have shown that several morphologically similar
animals in sub-Saharan Africa actually form a species complex (17). The
phylogeny of this complex is being studied by cytologic and molecular
techniques (18), but it will be years before the geographic distributions
of each species can be mapped and used to interpret the restricted
distribution of Lassa fever.

Andes virus, associated with Oligoryzomys longicaudatus, causes HPS in the
northern Patagonian regions of Argentina and Chile (19). Another
hantavirus, Oran virus, also causes HPS and is apparently associated with
the same rodent species in extreme northern Argentina (19). However, the
population of O. longicaudatus in northern Argentina appears to be disjunct
from the population that follows the Andean cordillera and includes
northwestern Patagonia (20). Careful taxonomic and distributional studies
are required to determine if these disjunct rodent populations represent
distinct species and to define the potential HPS-endemic areas associated
with each virus.

Geographic Distribution of the Pathogen within the Host Range

The extent of the geographic area in which the infected host can be found
indicates the area in which human disease can be endemic. In some cases,
host and pathogen distributions are largely coincident. SNV infection has
been found in deer mice throughout North America north of Mexico, with the
exception of Alaska (Ksiazek et al., unpub. obs.; 21,22). Rattus norvegicus
is found throughout most of the world, and infection with Seoul virus has
been reported on every continent except Antarctica (23).

However, for many arenaviruses, including Junn, Guanarito, and Tamiami,
and for some hantaviruses, the area in which the disease is endemic
apparently includes only a small portion of the range of individual host
species (Figure 3). The reservoir for Laguna Negra virus, which causes HPS
in Paraguay, is Calomys laucha, and approximately 12% of the C. laucha
tested from the Chaco region of western Paraguay are infected with Laguna
Negra virus (24). C. laucha is also one of the most common rodents in
central Argentina, yet testing of thousands of samples has provided no
evidence of Laguna Negra virus in central Argentina (25; Levis et al.,
unpub. obs.). However, the distributional data of C. laucha indicate that
populations in Paraguay are disjunct from those in Argentina and Uruguay.
Additional distributional, taxonomic, and laboratory studies are required
to determine whether genetic differences in host populations are
influencing susceptibility to infection or other factors are influencing
virus distribution in a uniformly susceptible population.

[Figure 3.] Distribution of the corn mouse, Calomys musculinus
(dots; 20), and disease-endemic area of Argentine hemorrhagic
area of Argentine hemorrhagic fever (AHF) (shaded).

The geographic range of infection by the pathogen within reservoir 
populations may be dynamic, not static. In 1955, the AHF-endemic area included
approximately 10,000 km(sup 2) centered around the town of Junn in Buenos
Aires province. By 1985, the AHF-endemic area encompassed more than
150,000 km(sup 2) in four provinces (26). Prevalence of infection in reservoir
populations may be very high in some localities and very low, or absent,
at nearby sites, for both arenaviruses (27) and hantaviruses (28,29).
It is likely that pathogens periodically become extinct in local
reservoir populations, only to be reintroduced later from neighboring
populations. The dynamics and factors controlling these processes 
are unknown but indicate that, although a single cross-sectional survey 
approximates the range of infection, repeated surveys or longitudinal 
studies are necessary to elucidate temporal and spatial patterns.

Finally, in many instances, reservoir surveillance provides a clearer
definition of potential disease-endemic areas than human disease
surveillance, even when the incidence of human disease is extremely low
(e.g., in HPS-endemic areas), and cases where the use of a vaccine may mask
human exposure (e.g., in the AHF-endemic area).

Distribution of the Pathogen among Habitats

The distribution of the host and the pathogen among different biotic
communities and habitat types indicates the risk for human infection in
various habitats within a geographic region. The deer mouse, a habitat
generalist, was found in all eight biomes studied in the four-state region
of the initial HPS outbreak in the United States (28). Prevalence of SNV
infection, however, varied among habitat types, being lowest at the
climatic and altitudinal extremes (desert and alpine tundra) and highest at
middle-altitude habitats such as pion-juniper woodland, where most HPS
cases occurred during the 1993 outbreak.

The corn mouse, reservoir for Junn virus, was described as a crop field
specialist (30), and virus transmission was said to occur when farmers
working in their fields inhaled aerosols of virus shed by infected rodents
(31). However, during detailed studies of habitat preferences in the
AHF-endemic area (Figure 4), the corn mouse was rarely found in crop
fields. Instead, it inhabited the more stable, weedy fence lines and
roadsides bordering crop fields. These results suggest that humans might
become infected with Junn virus in border habitats and that cutting or
burning vegetation in these habitats might help decrease the incidence of
AHF.

[Figure 4.] Schematic of mark-release-recapture grid in 
central Argentina. The green shaded areas are roadside (rows 1 
and 2) and fence-line (column 7)habitats. Unshaded areas are crop 
fields. Intersections of dotted lines represent the 144 trap
stations of the 12 x 12 trapping grid. The height of the flagpoles
shows the cumulative numbers of corn mouse captures at each trap
station of the 30-month sampling period. Red boxes are the
locations of antigen-positive captures. Reprinted with
permission from (32).                      

Relative Frequency of Infection in Subpopulations of the Host

The relative frequency of infection in different demographic subgroups within
the host population suggests potential mechanisms of pathogen transmission
within host populations. An association of antibody prevalence with age,
suggesting horizontal (rather than vertical) transmission, has been
documented for Norway rats infected with Seoul virus (33); deer mice,
western harvest mice, and brush mice infected with SNV or SNV-like viruses
(28); cotton rats infected with Black Creek Canal virus (29); and corn mice
infected with Junn virus (33). For Norway rats, deer mice, and cotton
rats, a J-shaped curve of antibody prevalence with age was apparent; very
young animals had prevalence rates similar to those of some adults. This
pattern may be explained by the presence of maternal antibody in
the offspring of infected females; as this apparently protective
antibody wanes, animals become infected.

Fighting and biting among adult male rodents may be a common mechanism of
virus transmission. Scars or wounds on rodents can indicate intraspecific
aggressive encounters. A significant association between the acquisition of
scars and the development of antibody to Seoul virus was demonstrated for
rats in Baltimore (34), implying that virus was transmitted by biting
during fights. In Argentina, male C. musculinus had more scars than
females, and adult males with scars were more often infected with Junn
virus than adult males without scars. In North America, the prevalence of
antibody to SNV among male deer mice, brush mice, and western harvest mice
is at least twice that among females (28), and scarring appears associated
with antibody in some of these species. However, infection patterns vary.
Male-to-female infection ratios are approximately 2:1 for deer mice and
SNV, but two studies of harvest mice found infection of 40% to 50% in males
(21,28), whereas no females were infected. Although scars and antibody to
Seoul virus were highly correlated for Norway rats in Baltimore, antibody
prevalence did not differ between male and female rats (34). These
species-specific differences are presumably related to differences in
behavior and, consequently, to differences in specific mechanisms of viral
transmission. Further study of these mechanisms may lead to an
understanding of the timing and conditions under which increased
transmission in reservoir populations takes place, which, in turn, would
lead to improved prediction of how and when humans become infected.

Temporal Dynamics of Host and Pathogen Populations

Longitudinal studies of reservoir populations are needed to identify 1) the
effects of infection on individuals and populations of host species, 2) the
seasonal and year-to-year fluctuations in incidence and prevalence as well
as the sequence and duration of infection in the host, and 3) the
environmental variables associated with changes in host density or rates of
transmission. The mark-release-recapture method, important for longitudinal
studies of small-mammal populations, establishes permanent trapping plots
operated for several nights at standard intervals (usually monthly for
disease studies) and maintained for several years. Captured animals are
anesthetized, identified, weighed, measured, sampled (e.g., blood and oral
swab), marked with a unique number, and released at the exact site of
capture. Animals may be recaptured in subsequent months so that growth
rates, movement, reproductive condition, and infection status can be
monitored over time. Environmental variables, such as temperature,
rainfall, and vegetation, relate to reservoir population dynamics.
Mark-release-recapture studies should continue long enough to monitor the
effects of unusual events, such as the El Nio southern oscillation (ENSO).

The mark-release-recapture method was used in a 3-year study of corn mouse
populations on the Argentine pampas (33). AHF epidemics are highly
seasonal, with the peak number of cases and peak rodent populations
occurring in May when autumn crops are harvested. During the first 2 years,
corn mouse population densities were relatively low, and the magnitude of
the annual AHF epidemics was also low (Figure 5). During the third year,
unusually mild weather resulted in reproduction throughout the winter, high
overwinter survival (35), and a very high population base at the beginning
of the spring reproductive season. The following summer had abundant
rainfall, vegetation remained green, and the usual midsummer decline in
rodent populations did not occur. Consequently, corn mouse populations and
numbers of infected corn mice reached extremely high levels by early
autumn; an unusually severe epidemic of AHF cases followed (Figure 5; 33).

[Figure 5.] Mean monthly trap success for Calomys musculinus
(number of captures per 100 trap nights) and number of confirmed
cases of Argentine hemorrhagic fever (AHF) in central Argentina,
March 1988 to August 1990.

Longitudinal field studies also show the effects of infection on host
populations. Contrary to indications from laboratory studies (10), Junn
virus infection had no effect on the growth, movement, or longevity of corn
mice (33), which implies that success for Calomys musculinus
laboratory infection may not mimic natural transmission and illustrates
the importance of investigating cases of Argentine hemorrhagic
transmission mechanisms under field conditions.

Because the animals are released, mark-release-recapture studies do not
provide data on diet, reproduction, precise population age structure
(using molar wear patterns or eye lens weights), and variation in genetics
of host and pathogen populations. Complementary investigations in
which animals are collected for species identification and harvesting
of tissues are therefore essential.

Predictive Models

The goal of reservoir ecology is to integrate and apply the data toward the
development or refinement of predictive models (Figure 6) that help public
health authorities identify specific times and locations that may pose a
threat to public health. In addition to early warning capability, these
models may suggest when, where, and how to intervene (e.g., in the form of
reservoir control or habitat modification) to break the transmission cycle
or otherwise decrease the incidence of human infection.

[Figure 6.] Simplified hypothetical model of interactions among
ecosystem components within disease-endemic areas for rodent-borne
zoonotic disease. Left-hand side of model demonstrates potential
use of remote sensors (satellites) for predicting relative risk
for human disease.

Figure 6 is a simplified schematic for such a model. We assume that the risk
for human disease is directly related to reservoir population density because
increased density increases the probability of human contact with
infected rodents. Data on the incidence of HFRS due to Puumala virus in Sweden
and studies of rodent population densities at HPS case and control
households in the United States support this assumption (36,37). We also assume
that reservoir population density is influenced by characteristics of the
biotic environment (e.g., habitat quality and food supply), which in turn
are influenced by characteristics of the physical environment (e.g.,
climate, weather, and edaphic conditions). Accurate predictive models
are not yet available, but recent developments are promising. The clear
correlation between reservoir population density and the risk for AHF
indicates that monitoring of rodent populations might give public health
professionals a 2- to 3-month predictive capabilityenough time to make
advisory warnings but not enough to intervene with a vaccination
program or integrated pest management. The Argentine model also provided
clues about factors contributing to the rodent irruption. The proper
model might have allowed prediction of the rodent irruption and
increased risk to humans a year before the epidemic.

Continuous measurement of environmental variables wherever rodent-borne
zoonoses are endemic may not be necessary. Remote sensing and geographic
information systems have measured vegetation indexes that help predict
changing risk for zoonotic diseases (6,38). Integration of remote sensing
with long-term studies measuring environmental conditions on the ground may
provide the key to predicting future risk for virus transmission. A more
complete understanding of generalized climatic patterns, such as ENSO, and
their effects on local environmental variables may lead to even earlier
predictive capabilities.

The Future

An otherwise complete and carefully executed study can be rendered useless
if the species from which each sample was taken cannot be confirmed. Except
in the case of mark-release-recapture studies, voucher specimens for all
captured small mammals should be archived, and museum accession numbers
should be cited in reports of these studies. Even if field identifications
are accurate according to existing nomenclature, taxonomic revisions may
leave the source of a pathogen in question unless the voucher is available
for reexamination. For example, the recent search for the reservoir of
Ebola virus near Kikwit, Republic of the Congo, resulted in the
identification of at least seven taxa of rodents and insectivores that are
preliminarily considered new species (39).

Where resources are available, a field study is enhanced by the maintenance
of specimens in a museum frozen tissue collection. These specimens can be
used by future investigators for genetic, taxonomic, and microbiologic
studies. A listing of museums (in the Western Hemisphere) maintaining
mammal collections, including frozen tissue collections, is available (40),
as are instructions for preparation and maintenance of voucher and tissue
specimens (41-43).

Safety

Although the precise circumstances of their transmission to humans are
often unclear, zoonotic pathogens are associated with direct or indirect
exposure to infected hosts. Modes of human infection include inhalation of
infectious aerosols, direct contact of contaminated fomites with broken
skin or mucous membranes, animal bites, or arthropod vectors. Mammalogists
and others who work with reservoir populations are likely at increased
risk. Safe methods and proper protective equipment must be used in handling
potentially infected rodents. In handling reservoir species for known human
pathogens such as hantaviruses and arenaviruses, protective clothing, latex
gloves, and respirators with high-efficiency particulate air filters should
be used. Animals should be anesthetized to minimize the risk for injuries,
and the use of sharp instruments and needles for collection should be
minimized. Safety guidelines are available in English (41,44) and Spanish
(45).

Investigators studying relatively benign or less easily transmitted agents
(e.g., Borrelia) should be alert to the potential presence of more virulent
agents associated with any wild animals they may be handling. Certainly not
all arenavirus- and hantavirus-host associations have been described, and
some species, such as the white-footed mouse and the cotton rat, are known
hosts for multiple agents.

Conclusions

Reservoir studies are an essential component of any integrated public
health response to established or emerging zoonotic diseases. The proposed
algorithm facilitates study of reservoir population ecology as it relates
to human disease. Each step improves understanding of the ecology and
epizootiology of hantaviruses and arenaviruses. The approach is offered as
one model to stimulate discussion of appropriate methods and as a
conceptual framework for other investigators to critique and improve. Other
techniques, such as radio-tracking (46) and the use of injectable passive
integrative transponders (PIT tags) (47), may better identify and track
movements of individual animals. Methods and approaches will evolve as our
knowledge of the subject matter increases. All the steps may not be
appropriate in all cases, and many are beyond the capability of any single
investigator or institution. For example, the delineation of the geographic
range of any mammal species requires intensive taxonomic, genetic, and
ecologic studies and could require the collaboration of investigators from
several countries. These studies will be greatly facilitated by properly
archived specimens from previous reservoir studies.

The phylogenetic relationship and distribution of pathogens may help
elucidate the taxonomic relationships between closely related reservoir
species. Although we have specifically addressed field studies, parallel
laboratory studies of hosts and pathogens are needed for maximum benefit
from these field studies. For example, studies of host and virus genetics,
host specificity, and pathogenesis and viral shedding may elucidate 1) the
roles of cospeciation and coevolution in the associations of hantaviruses
and arenaviruses with their hosts, 2) mechanisms of viral persistence, and
3) the fundamental question of defining viral species.

Acknowledgments

      We thank Barbara Ellis for helpful discussions, encouragement,
graphics, and a critique of the manuscript. C.J. Peters provided
stimulating discussion, ideas, and suggestions.

      Dr. Mills is chief of the Medical Ecology Unit, Special Pathogens
Branch, Division of Viral and Rickettsial Diseases, CDC. His research
interests include zoonotic diseases, specifically host-pathogen evolution
and interactions.

      Dr. Childs is the epidemiology section chief in the Viral and
Rickettsial Zoonoses Branch, CDC. His major areas of expertise and research
interests are the means by which zoonotic agents are maintained in animal
populations and transmitted to humans.

      Address for correspondence: James N. Mills, Centers for Disease
Control and Prevention, 1600 Clifton Road, Mail Stop G14, Atlanta, GA
30333, USA; fax: 404-639-4436; e-mail: jum0@cdc.gov.

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Perspectives

Diphtheria in the Former Soviet Union: Reemergence of a Pandemic Disease

Charles R. Vitek and Melinda Wharton
Centers for Disease Control and Prevention, Atlanta, Georgia, USA

---------------------------------------------------------------------------
      The massive reemergence of diphtheria in the Newly Independent
      States of the former Soviet Union marked the first large-scale
      diphtheria epidemic in industrialized countries in 3 decades.
      Factors contributing to the epidemic included a large
      population of susceptible adults; decreased childhood
      immunization, which compromised what had been a
      well-established childhood vaccination program; suboptimal
      socioeconomic conditions; and high population movement. The
      role of a change in the predominant circulating strains of
      Corynebacterium diphtheriae in this epidemic remains uncertain.
      Massive, well-coordinated international assistance and
      unprecedented efforts to vaccinate adults were needed to
      control the epidemic.

In the 1990s, a massive epidemic throughout the Newly Independent States of
the former Soviet Union marked the reemergence of epidemic diphtheria in
industrialized countries. Diphtheria had been well controlled in the Soviet
Union for more than 2 decades after universal childhood immunization was
initiated in the late 1950s (Figure 1). Although all of the Newly
Independent States were affected, three quarters of the more than 140,000
cases (Table 1) and two thirds of the more than 4,000 deaths reported since
1990 (1-3) were reported by the Russian Federation.

 [Figure 1.] Reported diphtheria cases in the Soviet Union and
 the Newly Independent States, 196596.

 Table 1. Diphtheria incidence in the Newly Independent States (NIS) of the
 former Soviet Union, 19911996

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

                                      Cases (per 100,000)
                 Pop.
 Country        (mil.)   1991      1992     1993     1994      1995      1996
 --------------------------------------------------------------------------------

 Russia         149.90    1,869    3,897    15,211   39,582   35,652    13,604
                         (1.25)   (2.60)   (10.15)  (26.41)  (23.78)   (9.08)

 Kazakstan      17.46      30       45        82      489      1,105     455
                         (0.17)   (0.26)   (0.47)   (2.80)    (6.33)   (2.61)

 Kyrgyzstan       4.7      10    4 (0.09)     6       299       693      412
                         (0.21)            (0.13)   (6.36)   (14.74)   (8.77)

 Tajikistan      6.02   5 (0.08)    14       680     1,912     4,455    1,464
                                  (0.23)   (11.30)  (31.76)  (74.00)   (24.32)

 Turkmenistan    4.16   4 (0.10)    22        3        60       87        80
                                  (0.53)   (0.07)   (1.44)    (2.09)   (1.92)

 Uzbekistan     22.83   9 (0.04)    29       137      224       638      160
                                  (0.13)   (0.60)   (0.98)    (2.79)   (0.70)

 Central Asia   55.17      58       114      908     2,984     6,978    2,571
                         (0.11)   (0.21)   (1.65)   (5.41)   (12.65)   (4.66)

 Armenia         3.74   0 (0.00) 0 (0.00)     1        36       29        11
                                           (0.03)   (0.96)    (0.78)    (0.29)

 Azerbaijan      7.46      36       55       141      841       883      114
                         (0.48)   (0.74)   (1.89)   (11.27)  (11.84)   (1.53)

 Georgia         5.49   7 (0.13) 3 (0.05)     28      294       419      346
                                           (0.51)   (5.36)    (7.63)   (6.30)

 Caucasus       16.69      43       58       170      1171     1,331     471
                         (0.26)   (0.35)   (1.02)   (7.02)    (7.97)   (2.82)

 Ukraine        52.47     1,103    1,567    2,987    2,990     5,280    3,156
                         (2.10)   (2.99)   (5.69)   (5.70)   (10.06)   (6.01)

 Moldova         4.36      14       22        35      376       418       97
                         (0.32)   (0.50)   (0.80)   (8.62)    (9.59)   (2.22)

 Belarus        10.33      26       66       120      230       322      179
                         (0.25)   (0.64)   (1.16)   (2.23)    (3.12)   (1.73)

 Western NIS    67.16     1,143    1,655    3,142    3,596     6,020    3,432
                         (1.70)   (2.46)   (4.68)   (5.35)    (8.96)   (5.11)

 Estonia         1.57   7 (0.45) 3 (0.19)     11       7        19        14
                                           (0.70)   (0.45)    (1.21)   (0.89)

 Latvia          2.65   5 (0.19) 8 (0.30)     12      250       369      112
                                           (0.45)    (9.43)  (13.92)   (4.23)

 Lithuania       3.77       1    9 (0.24)     8        38       43        11
                         (0.03)            (0.21)   (1.01)    (1.14)   (0.29)

 Baltics         7.99      13       20        31      295       431      137
                         (0.16)   (0.25)   (0.39)   (3.69)    (5.39)   (1.71)

 All NIS        296.91    3,126    5,744    19,462   47,628   50,412    20,215
                         (1.05)   (1.93)   (6.55)   (16.04)  (16.98)   (6.81)

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

Massive efforts to vaccinate both children and adults are bringing the
epidemic under control; in 1996, 20,215 cases were reported, a 60% decrease
from the 50,425 cases reported in 1995 (4), and, in 1997, 6,932 cases were
reported as of February 1998 (World Health Organization [WHO], unpub.
data). The European Regional Office of WHO considers the outbreak nearly
under control in Armenia, Azerbaijan, Belarus, Estonia, Latvia, Lithuania,
Moldova, the Russian Federation, Turkmenistan, and Uzbekistan; in the five
remaining republics, control is improving, but continuing efforts are
needed to stabilize the situation.

This epidemic, primarily affecting adults in most Newly Independent States
of the former Soviet Union, demonstrates that in a modern society
diphtheria can still spread explosively and cause extensive illness and
death. Intense international efforts have focused on aiding the affected
countries and understanding the reasons for the epidemic. The study of this
resurgence, especially as it relates to diphtheria resurgence in other
industrialized countries, may elucidate the potential for the reemergence
of other vaccine-preventable diseases.

Epidemiology of Diphtheria

Prevaccine Era

In the prevaccine era, diphtheria was a dreaded, highly endemic childhood
disease found in temperate climates. Despite a gradual decline in deaths in
most industrialized countries in the early 20th century, which was
associated with improving living standards, diphtheria remained one of the
leading causes of childhood death until widespread vaccination was
implemented. In England and Wales, as recently as 1937 to 1938, diphtheria
was second only to pneumonia among all causes of childhood death (5), with
an annual death rate of 32 per 100,000 in children less than 15 years of
age.

Most urban residents acquired immunity to diphtheria by the age of 15 years
(6); only a minor portion of diphtheria cases were in adults. Only
approximately 15% of children with immunity to diphtheria had had typical
clinical diphtheria; the other 85% had milder symptoms or asymptomatic
infections (6). Children of preschool and elementary school age had the
highest attack rates. The age of school entry was associated with increased
risk for disease (7). Peaks of endemic diphtheria in the fall (8) were
often attributed to the opening of schools. Crowding and low socioeconomic
conditions were other risk factors (9).

Superimposed on the high endemic disease rates was a rough periodicity of
incidence with peaks every several years (5). Epidemic waves characterized
by extremely high incidence and deaths were sporadic: Spain in the early
1600s (10), New England in the 1730s (10,11), and Western Europe from 1850
to 1890 (12,13). The factors governing diphtheria periodicity are not
understood.

Vaccine Era in Western Europe and the United States

In the United States, Canada, and many countries in Western Europe, the
widespread use of diphtheria toxoid for childhood vaccination beginning in
the 1930s and 1940s led to a rapid reduction in diphtheria incidence (14).
However, in the 1930s, gradual rises in diphtheria incidence to 200 cases
per 100,000 in the prewar period occurred in Germany and several other
central European countries with partially implemented vaccination programs.
The onset of World War II in 1939 and the occupation by German troops of
many Western European countries led to the last diphtheria pandemic in
Western industrialized countries. Although diphtheria incidence had been
very low before the war, Holland, Denmark, and Norway had severe epidemics
following occupation by German soldiers. Newly developed biotyping methods
confirmed that endemic disease in prewar Germany was associated with
strains of gravis biotype and that epidemics in occupied countries were
associated with the introduction of gravis strains (15-17).

Data from a World War II epidemic in Halifax, Nova Scotia, suggest the high
epidemic potential of these strains (18). Diphtheria was endemic in Halifax
(30 to 80 cases per year), primarily among children in poor neighborhoods.
Although not all isolates were biotyped, mitis strains seemed to be
predominant. Gravis organisms, presumably related to the strains introduced
into Norway by German troops, were introduced into Nova Scotia in September
1940 by Norwegian sailors with diphtheria, leading to an outbreak of 649
cases by July 1941. All biotyped strains were gravis. At first, secondary
cases occurred predominantly among school-aged children; following cases
were among adults, especially women. A study of more than 1,000 school
children in February 1941 found gravis carriage rates of up to 30% in
schools in poor neighborhoods. An apparently limited introduction of new
strains resulted in an epidemic while circulation of the previously endemic
strains had not.

Diphtheria incidence continued to decline steadily throughout the vaccine
era in the United States (Figure 2) and (after the immediate postwar
period) in Western Europe. Cases of clinical diphtheria have become
extremely uncommon; several European countries have not reported a case of
diphtheria in more than 20 years (12,19). Residual indigenous cases have
been concentrated among incompletely vaccinated or unvaccinated persons of
low socioeconomic status. In the United States, the decline was interrupted
in the late 1960s by a small resurgence of diphtheria lasting until 1975
(20). Until this time, the predominant biotype in the United States,
especially in the Southeast, was mitis, although gravis outbreaks were
common in the West. Intermedius cases were uncommon, and outbreaks were
rare (21). Between 1969 and 1975, outbreaks caused by intermedius strains
were reported in economically depressed populations in Chicago (22), San
Antonio (23), Phoenix (24), the Navajo reservation in Arizona and New
Mexico (25), Seattle (26), and communities in eastern Washington State
(Centers for Disease Control and Prevention [CDC], unpub. data). Since
1975, cases of respiratory diphtheria and isolation of toxigenic strains of
Corynebacterium diphtheriae have been extremely rare in the United States
(19,27).

[Figure 2.] Diphtheria IncidenceUnited States and Russian
Federation, 19201996.

Prevaccine and Early Vaccine Era in the Soviet Union                              

Diphtheria incidence in Russia was high throughout the first half of the 20th
century (Figure 2); more than 750,000 cases were reported in Russia alone in    
the 1950s. Although immunization against diphtheria began in some areas of the
Soviet Union as early as the 1920s, it was only in 1958 that universal
childhood immunization began throughout the Soviet Union (28). By 1963, the
incidence in the Soviet Union had decreased 15-fold compared with 1958 and
elimination was thought attainable. From 1965 until 1980, the Soviet
childhood vaccination schedule mandated five doses of high antigenic
content diphtheria vaccine by school entry and a school booster (Table 2).
Diphtheria incidence levels continued to drop and in the mid-1970s
approximated levels in the United States; only 199 cases (0.08 cases per
100,000) were reported in the Soviet Union in 1975 and only 198 cases in
1976. As in the prevaccine era, most cases were in children; in 1975 and
1976, 62% of the 109 cases in the Russian Federation were in children under
15 years of age (N.M. Maksimova, pers. comm.). During this period,
diphtheria incidence was higher in the Central Asian republics (14).

 Table 2. Diphtheria vaccination schedule (Soviet Union 19651991, Russian
 Federation 199197, Ministry of Health, Russian Federation)

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

                                          Year (sup a)

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

                                      1980                     1987
             1965                                                          1994

                     ------------------------ -------------------------
                                                           Alternative
                     Alternative Alternative  Alternative
 Age                      A           B             A           B
 ----------------------------------------------------------------------------------

 3-18 mo     DTP         DTP      DT or Td        DTP       DT or Td       DTP

 (infancy)(3 doses)   (3 doses)   (2 doses)    (3 doses)    (2 doses)   (3 doses)

 18-36
 mo(sup b)   DTP         DTP      DT or Td        DTP       DT or Td       DTP

 6 yr        DTP                Td                      Td

 9 yr                                                   Td

 11-12 yr                       Td                      Td                  d

 16 yr                   Td (after 1983)                Td                 Td

 ----------------------------------------------------------------------------------
 (sup a) Antigenic content of Russian-manufactured vaccine: DTP = 15 lf units
 diphtheria toxoid per dose; DT = 30 lf units diphtheria toxoid per dose; 
 Td = 5 lf units diphtheria toxoid per dose;
 d = 5 lf units diphtheria toxoid per dose.
 (sup b)To be given 12-18 months after last infancy dose.

Resurgence and Stabilization of Diphtheria, 1977-1989

In 1977, diphtheria started to gradually make a comeback in the Soviet
Union; incidence peaked in 1984 (1,609 cases, 0.9 per 100,000 population).
The cases were concentrated in Russia, especially Central European Russia
and the Russian Far East (29,30). In the Russian Federation, the resurgence
was associated with a change in the predominant circulating biotypefrom
gravis (75% of cases 1975 to 1976) to mitis (60% of cases 1977 to 1981, 80%
of cases 1982 to 1986). The circulating gravis strains belonged to several
phage types and serotypes (31).

In 1978, for the first time most cases occurred in adults and, although
incidence rates rose in all age groups, the rates in adults rose more
rapidly and to higher levels than in children. The proportion of cases
associated with mitis strains was also lower in children, suggesting that
children were initially relatively protected or not exposed during this
epidemic (29,30). In response to the resurgence, Soviet public health
authorities intensified diagnostic and case investigation efforts, called
for widespread vaccination of adult occupational groups at high risk, and
in some localities with multiple cases, called for widespread community
adult immunization (30).

However, changes in the immunization schedule during this period encouraged
less intensive vaccination of children. Use of an alternative schedule of
fewer doses of lower antigenic content (adult formulation) vaccine was
allowed beginning in 1980; in 1986, the school entry booster dose was
dropped, lengthening to 7 years the interval between recommended childhood
booster doses after a primary series (Table 2). In addition, an increasing
number of conditions were considered temporary or permanent
contraindications to childhood vaccination. At the same time, public
support for childhood vaccination programs fell, at least in some areas,
for several reasons. Many childhood vaccine-preventable diseases had low
incidence rates. In addition, a vocal antiimmunization movement received
favorable press coverage in an atmosphere of increased distrust of
government during perestroika (1985 to 1991). Participants in focus groups
conducted in 1996 on diphtheria vaccination vividly recalled these reports
and said that the reports had made them more fearful of vaccinating
themselves or their children (N. Keith, pers. comm.). Childhood vaccination
rates declined during the 1980s, with coverage of infants with a primary
diphtheria toxoid series dropping to 70% or below for most of the decade
(14). Many children vaccinated in the Russian Federation received low
antigen content vaccine as their primary series, especially in some areas
such as Moscow and St. Petersburg. In 1989 in Russia, more than 25% (a
higher proportion than in any other republic [32]) of vaccinated children
received primarily adult formulation vaccine preparations lacking the
pertussis component.

After 1984, diphtheria incidence gradually declined to 839 cases (0.3 per
100,000) in 1989, although remaining above the nadir reached in 1976 to
1977. Most cases were reported by the Russian Federation; although most
were in adults, the proportion of cases in children rose gradually
throughout the 1980s (Figure 3).

[Figure 3.] Reported cases and proportion of cases among
children <15 years, Russian Federation. Data provided by the 
Russian Ministry of Health.

The military may have played an important role in the persistence and
spread of diphtheria during the 1980s. Military service remained universal, and
recruits were not routinely vaccinated against diphtheria until 1990. The
source of one prolonged Russian outbreak (Kovrov District, Vladimir Oblast, 1982
to 1987) was diphtheria cases among a unit of military recruits from Central
Asia that spread into the civilian population through social functions
V.A. Grigorevna, pers. comm.). Investigations in military units
in various parts of Russia between 1983 and 1987 found carrier rates
of toxigenic C. diphtheriae of up to 5.0% (33). Although cases in the
military were (and still are) not reported to the Ministry of Health or
included in the reported incidence data, some published data exist. An outbreak
in Kzyl-Orda, Kazakstan, in 1988 began among the military but spread to
the civilian population, causing 58 cases (34). From 1987 to 1990, most
adult diphtheria patients seen at the Botkin Infectious Disease Hospital,
one of two treatment units for diphtheria in Moscow, were members of
the military (35). On the basis of published data, between 1990 and 1992
the minimum incidence of diphtheria in the military was 21 cases per
100,000 service members (36), six times higher than that of the
civilian population (37). This incidence ratio was even more
disproportionate in the late 1980s (32,36).

Beginning in 1986, gravis strains accounted for an increasing proportion of
isolates, and in 1989, accounted for 52% of reported cases. One hundred and
fifty-six diphtheria strains from various sites in Russia from 1985 to 1994
were analyzed by multilocus enzyme electrophoresis (MEE) and ribotyping.
Although the mitis strains predominant in the 1980s were of multiple
electrophoretic and ribotypes, most of the gravis strains from 1986 to 1989
belonged to a group of strains closely related by both MEE and ribotyping;
this epidemic clone became predominant overall in the 1990s epidemic in
Russia (38).

Epidemic Diphtheria, 1990 to 1996

Spread from the Center: 1990 to 1992

In 1990, 1,431 cases were reported in the Soviet Union, a 70% increase over
1989 (32). Cases were heavily concentrated in the Russian Federation (1,211
cases), especially Moscow City and Oblast (541 cases combined), and the
three Pacific Coast oblasts of Khabarovsk, Primorye Krai, and Sakhalin
Island (109 cases). In 1991, 3,126 cases were reported from the now Newly
Independent States; epidemic diphtheria had reached St. Petersburg City and
Oblast (246 cases) and in the Ukraine, Kiev (372 cases), Kharkov (129
cases), and Lvov (190 cases). Further spread within Russia, the Ukraine,
and Belarus accounted for most of the 5,744 cases reported in 1992 (14).

Explosion and Spread: 1993 to 1994

In 1993, the number of reported diphtheria cases surged to 19,462; epidemic
diphtheria became established throughout urban Russia, the Ukraine, and
Belarus. Russia alone reported 15,211 cases, an increase of 290% from 1992,
with more cases reported in each succeeding month. For the first time in
the epidemic, a pronounced seasonal incidence was seen (Figure 4), and the
incidence rate in children exceeded that in adults by 60%. Elsewhere,
Azerbaijan reported 141 cases and in the aftermath of a civil war, an
epidemic sprang up in Tajikistan (680 cases) that spilled over into
neighboring areas of densely populated Uzbekistan (137 cases). Cases had
also increased in other Newly Independent States, although the incidence
was still less than 1 per 100,000.

[Figure 3.] Diphtheria cases in the Russian Federation, 1992-96. 
1994 = 19,582; 1995 = 35,652 (-10%); 1996 = 13,604 (-62%).

In 1994, epidemic diphtheria was reported from all states except Estonia,  
where most of the adult population had been vaccinated in 1985 to 1987. Russia   
had 39,582 (83%) of the 50,412 cases reported by the Newly Independent
States. In Central Asia, areas of high incidence included regions adjacent to    
Russia in Kazakstan and regions adjacent to Tajikistan in both Uzbekistan and      
Kyrgyzstan, where the outbreak reportedly began with eight cases in a
refugee family from Tajikistan (39).

In Russia, the Ukraine, Belarus, and the Baltics, most cases occurred in
adults (3). In Russia in 1993, two thirds of cases were in persons older
than 14 years. The highest incidence rates were among school-aged children
and adolescents (12.4 to 18.2 per 100,000) and in adults ages 40 to 49
years (16.7 per 100,000); 45% of all deaths and the highest death rate (1.3
per 100,000 population) were reported among persons 40 to 49 years of age.
Incidence rates dropped sharply in persons above age 50 (2.8 per 100,000)
(40). Similarly in the Ukraine, adolescents 15 to 19 years of age and
adults 40 to 49 years of age had the highest incidence rates (41). In
several areas, most reported adult diphtheria cases were in women; women
accounted for 60% of adult cases in St. Petersburg in 1991 to 1992 (42) and
for 64% in three Russian regions in 1994 to 1995 (43). In the three
regions, incidence rates among women 20 to 49 years of age were 68% higher
than the rates among men of that age.

Biotype gravis strains have been predominant in Russia in the 1990s.
Molecular studies using ribotyping and MEE demonstrated emergence of an
epidemic clone of closely related strains (38,44). In the Ukraine, Belarus,
the Baltics, and northern Kazakstan, the predominant strains were gravis
biotype; in Tajikistan, Uzbekistan, Kyrgyzstan, and southern Kazakstan, the
mitis biotype strains predominated.

Public Health Response and Control of the Epidemic: 1995 to 1996

Initial diphtheria control efforts were hindered by shortfalls in strategy
and vaccine supply. In the early years of the epidemic, public health
officials concentrated on improving childhood coverage rates and on
vaccinating adults in occupational groups perceived at high risk;
vaccination of all adults was not directed by Russian public health
authorities until 1993. The resulting unprecedented demand for adult
formulation vaccine was met with stepped-up Russian vaccine production
during 1994 to 1995 when nearly 80 million doses of adult formulation
vaccine was produced (compared with less than six million in 1992 [Russian
Federation Ministry of Health, unpub. data]).

Implementation efforts focused on vaccinating adults at work sites,
followed by intensified efforts including house-to-house visits, to reach
and vaccinate nonworking adults. Similarly, to further raise childhood
coverage, a shortened list of contraindications was adopted and the use of
full-strength vaccine preparations in the primary series was increased. In
October 1994, the school-entry booster dose was reinstituted.

By the end of 1995, considerably improved coverage in children in Russia
was reported (93% coverage with primary series at 1 year reported compared
with 68.7% in 1991). Adult coverage with one or more doses in the previous
10 years was estimated at 75%; between January 1993 and December 1995, 70
million adults were vaccinated in the Russian Federation (45). In 1995, the
incidence (24 cases per 100,000 population) decreased 10% from 1994. In
1996, 13,604 cases (9 per 100,000) were reported, a further 62% decline
(2,4). In 1997, a provisional total of 4,057 cases were reported to WHO
(WHO, unpub. data).

Unlike Russia, other Newly Independent States of the former Soviet Union
states were not producers of diphtheria vaccine. The disruption of vaccine
supply and economic difficulties associated with the dissolution of the
Soviet Union were reflected in sharp drops in childhood vaccination rates
in the early 1990s in the Central Asian and Caucasian republics (3). In
1994 to 1995, WHO, United Nations Children's Fund (UNICEF), other agencies,
and Newly Independent States' governments developed and adopted an epidemic
control strategy that aimed at rapidly raising adult and childhood coverage
through nationwide immunization campaigns. Subsequently, a massive
international effort, involving governmental and nongovernmental
organizations and United Nations agencies and coordinated by an oversight
committee, the Interagency Immunization Coordinating Committee, proved
successful in mobilizing resources, purchasing and delivering vaccine to
the Newly Independent States, and providing technical assistance to
implement the strategy (2,3).

In 1995 and 1996, the Newly Independent States raised adult and childhood
coverage and began to control the epidemic. All states made widespread use
of workplace vaccinations for adults and other intensified vaccination
tactics; these efforts resulted in increased adult coverage. The strategy
was implemented with highly successful national mass campaigns in Moldova,
Tajikistan, Latvia, Lithuania, and Azerbaijan, which achieved high adult
coverage and rapid steep declines in incidence. All countries also made
efforts to increase childhood coverage and to limit contraindications and
the use of lower antigen content vaccine; routine childhood coverage
exceeded 90% in most countries (2). Most countries reinstated a
school-entry revaccination, and many of the national mass vaccination
campaigns focused on children and adolescents. In 1996, 6,611 diphtheria
cases were reported in the Newly Independent States excluding Russia, a 55%
decrease compared to 1995; in the first quarter of 1997, 885 cases were
reported, a 57% decrease compared with the first quarter of 1996 (4). WHO
has received provisional reports of 2,875 cases for the full year of 1997,
although data are not complete for a few countries (WHO, unpub. data).

Factors Contributing to the Diphtheria Epidemic in the Newly Independent
States

The control of epidemic diphtheria by childhood vaccination has been one of
the outstanding successes of medicine in this century. Most Western
industrialized countries have nearly eliminated this disease; many
developing countries have progressively increased vaccination coverage by
introducing diphtheria toxoid into vaccination programs. Global diphtheria
incidence declined approximately 70% between the mid-1970s and the early
1990s (14). The diphtheria epidemic in the Newly Independent States raised
numerous concerns about the efficacy of diphtheria control programs and of
the diphtheria vaccine itself. However, case-control studies in the Ukraine
and in Moscow demonstrated that three or more doses of Russian-manufactured
diphtheria toxoid was highly effective in preventing disease in children
(46). The rapid decline in disease incidence with increased vaccination
coverage among both adults and children provides strong evidence of the
continued effectiveness of diphtheria vaccine.

Numerous factors appear to have contributed to the epidemic: 1) increased
adult susceptibility, which is reflected in the age distribution of cases
and deaths; 2) increased susceptibility of children; 3) a clone of closely
related strains of C. diphtheriae, gravis biotype, associated with most of
the cases in Russia, even though its role remains uncertain; 4) highly
crowded urban populations and service in the military; 5) the breakup of
the former Soviet Union, perhaps by disrupting vaccine supply to all
countries other than Russia and initiating large-scale population movements
throughout the Newly Independent States.

Increased Adult Susceptibility

Arguably the most important factor for the diphtheria epidemic was the
development of large populations of adults susceptible to the disease as a
consequence of successful childhood vaccination programs. The decreased
opportunity for naturally acquired immunity, along with the waning of
vaccine-induced immunity in the absence of routine adult revaccination, has
resulted in a high proportion of adults susceptible to diphtheria as
documented by serologic studies in many countries (12). In the United
States, a trend to increased diphtheria incidence in older age groups was
noted before the near-total disappearance of diphtheria (47). In several
developing countries that have conducted immunization programs for more
than 10 years, recent small epidemics have shown a similar increase in the
proportion of adult cases (12).

Adults born between the early 1940s and the late 1950s in the Russian
Federation and some other Newly Independent States were at the highest risk
for never having acquired immunity to diphtheria; during their childhood,
diphtheria incidence was decreasing, but not all children were reached by
newly implemented vaccination programs. The gap in immunity in this age
group, observed in serologic studies (48,49), was reflected in the very
high numbers of deaths and illnesses among persons 35 to 50 years of age.
Adults with similar high susceptibility to diphtheria in the United States
and Western Europe are likely to be older than susceptible adults in the
former Soviet Union because of the earlier implementation of vaccination
programs in the West. The only sizeable diphtheria outbreaks in the United
States that involved predominantly adults occurred in the early 1970s in
Arizona and Washington. At this time, the analogous cohort of U.S. adults
born just before widespread immunization would have been approximately 30
to 50 years old. Points of maximal population susceptibility to epidemic
adult diphtheria therefore may depend on the age distribution of adults
susceptible to the disease and the frequency of high contact rates for
these susceptible adults (e.g., military service for young adults, care of
school-aged children for young and middle-aged adults, homelessness among
young and middle-aged men).

A high rate of adult susceptibility to diphtheria is a necessary but not
sufficient precondition for the development of epidemic diphtheria in
adults. The United States and most other European countries have high rates
of adult susceptibility but have not had sustained chains of transmission,
despite documented importations. In Poland and Finland, which border on the
Newly Independent States that had diphtheria epidemics, multiple documented
importations since 1990 have led to only very limited secondary
transmission; these countries have maintained very high levels of childhood
coverage against diphtheria (50).

In the epidemic of the Newly Independent States, sustained transmission in
adults may have been limited to certain focal groups. In addition to
military units, high-level transmission between adults (clusters of
multiple cases and high rates of carriage) was demonstrated in other adult
groups characterized by crowding, low levels of hygiene, and high contact
rates, such as the homeless and patients in neuropsychiatric hospitals
(40,42); in routine work settings, clusters of cases were rare, and the
carrier rates among adult contacts of cases were very low.

Increased Childhood Susceptibility

By 1990, on-time coverage of infants and young children had fallen because
of changes in childhood vaccination recommendations and practices and
increased population skepticism regarding vaccination, which was greatly
exacerbated by an active antivaccination movement. Vaccination was
frequently delayed and temporary contraindications to vaccination were
extremely common. In addition, many children were considered to have
permanent contraindications to vaccination, large numbers of children
received lower antigen content vaccines in the primary series, and the
interval between childhood booster doses was lengthened. The lowered
coverage with pertussis-containing vaccine has been linked to a resurgence
of pertussis in the Russian Federation (51). Some data exist on the direct
impact of these changes on diphtheria incidence; in Russia and the Ukraine,
incidence was higher among unvaccinated than vaccinated children, and lack
of vaccination was a strong risk factor for severe disease (40). A
case-control study after the reinstitution of the school entry booster dose
found an interval of greater than 5 years since the last dose to be a
strong risk factor for disease (52). No data regarding the effect of the
increased use of lower antigen content vaccine are available.

Although since the mid-1970s most of the diphtheria cases in the Newly
Independent States have been in adults, throughout the 1980s and early
1990s the proportion of diphtheria cases in children increased during the
period of worsening overall control of diphtheria in all age groups (Figure
3). The increase in diphtheria cases among children suggests an important
role for the susceptibility of children in the dissemination of diphtheria.
In addition, most reported carriers in the Russian Federation were children
in both the 1980s (31) and 1990s (Russian Federation Ministry of Health,
unpub. data). Clusters of multiple cases in schools and within families
were prominent features in this epidemic, as in previous ones (40). Some
data suggest that a proportion of the disease in adults may represent
'sentinel' events and may have been transmitted from ill or asymptomatic
children; these data include the much higher disease incidence among women
compared with men (despite no evidence of lower serologic immunity [50,53])
and the large proportion of adult cases linked with multiple asymptomatic
child carriers (CDC and Russian Federation Ministry of Health, unpub.
data). In this respect, epidemic diphtheria in the era of adult
susceptibility may resemble epidemic influenza, in which studies suggest
that schoolchildren are a very important population in transmission and
spread, although the bulk of severe disease occurs in their adult contacts
(54).

Change in Biotype or Epidemic Clone

Changes in the circulating strains of C. diphtheriae could be responsible
for the cyclicity and episodic epidemic waves associated with diphtheria
incidence in the prevaccine era. The outbreaks in Western Europe during
World War II, in the United States in the 1970s, and in the Soviet
Union/Newly Independent States in the mid-1970s and the late 1980s were
associated with the emergence of a new biotype. Host factors (such as
antimicrobial immunity) could contribute to the epidemic potential of a
newly introduced strain, but microbial factors cannot be excluded. However,
the role of antibacterial immunity in preventing infection with C.
diphtheriae has not been studied since the 1930s, and no microbial factors
have been identified that distinguish epidemic from nonepidemic strains.
The molecular characterization of an epidemic clone of gravis strains
associated with the current epidemic in most parts of Russia supports a
role for a change in the agent in the development of the epidemic in the
Newly Independent States of the former Soviet Union; however, other
countries of the Newly Independent States have had epidemic diphtheria
linked to strains of both mitis and gravis biotypes.

Although the source of the epidemic strains in Russia is unclear,
persistent foci of diphtheria in Russia are a possible source. Russia was
never totally free of reported cases of diphtheria, and recent reports of
persistent disease-endemic foci in the United States (55) and Canada (56)
suggest that circulation of toxigenic strains of C. diphtheriae can occur
for prolonged periods even in the absence of recognized clinical cases, at
least in certain communities. Other suggested sources include persistent
diphtheria foci in the Central Asian countries or importation by returning
military units from the war in Afghanistan between 1979 and 1990.

Soviet Economic Development and the Post-Soviet Economic Crisis

Soviet populations were highly urbanized, but because of economic growth
lagging behind that in Western Europe and the United States, most city
dwellers lived in crowded apartments. Many of the amenities conducive to
decreasing transmission of bacteria were deficient or lacking, especially
in public areas, including routine access to functioning faucets for hand
washing. A case-control study of diphtheria cases in Georgia found an
increased risk for diphtheria associated with sharing utensils, cups,
glasses, or a bedroom and with decreased bathing (57).

The economic crisis of the post-Soviet period in all Newly Independent
States may have worsened these living conditions and contributed to the
epidemic. The crisis indisputably led to lowered childhood coverage in the
Central Asian and Caucasian republics in the early 1990s and may have
contributed to the high proportion of childhood diphtheria cases reported
from many of these republics.

Militarization

The Soviet Union was the most extremely militarized large country in the
world, with 1.4% of its population armed (58). The high level of
militarization and the lack of routine immunization of recruits resulted in
bringing large numbers of susceptible adults together from all parts of the
immense country under conditions of crowding and suboptimal hygiene. These
adverse conditions may have played a role in the development of the
epidemic as suggested by the high diphtheria incidence in the military
early in the 1990s.

Increased Travel and Mass Population Movement

Other major changes after the breakup of the Soviet Union in 1991 included
loosening of controls on movement within countries and increasing movements
of populations between the Newly Independent States. During the Soviet era,
movement was restricted by regulations and housing shortages. The success
of control measures in the epidemic of the early 1980s may have been
enhanced by slower dissemination of toxigenic strains due to movement
restrictions. Similarly, while the epidemic clone was already established
in the Newly Independent States by 1991, epidemic spread may have been
facilitated by the mass movements of populations, primarily the
repatriation of ethnic Russians from Central Asian and Caucasian countries
and the flight of refugees from fighting in Georgia, Armenia, Azerbaijan,
and Tajikistan.

Factors in Successful Epidemic Control

The epidemic was controlled by vaccination efforts that achieved very high
childhood coverage and unprecedented coverage in adults. The control
strategy was developed and refined on the basis of epidemiologic analyses
of disease incidence and population immunity. Implementation of the
strategy for all of the Newly Independent States, except Russia, required
massive international assistance; the instrumental role of the Interagency
Immunization Coordinating Committee in successfully coordinating the
multiple partners in this effort should serve as a model for future
international public health emergencies. Finally, an effective, although
underfunded, system of primary health care and public health centers had
functioned throughout the Soviet Union for decades. The health workers of
this system played a critical role in rapidly implementing the control
measures once adequate strategies and material resources were identified.

Lessons for Other Potential Reemerging Diseases

The epidemic in the Newly Independent States was unexpected; however, many
of the factors apparently contributing to the epidemic coincide with
factors important in the emergence of infectious diseases (Table 3) (59)
and are connected with other diseases and other countries. An increased
susceptibility in adults to childhood diseases is a predictable consequence
of successful childhood vaccination programs with vaccines that produce
less than lifelong immunity; this type of susceptibility has been suggested
as one factor contributing to the current increase in pertussis in the
United States. Although extreme loss of confidence in immunization may not
occur often in other countries, an increasing population resistance to
childhood vaccination as a result of adverse publicity and a diminished
perception of risk occurs commonly (60) and has contributed to outbreaks of
pertussis in England and Sweden (61,62). The role of changes in etiologic
agents contributing to emergence is under study for many diseases. A recent
outbreak of pertussis in the Netherlands may be due to a change in the
predominant circulating strains resulting in decreased vaccine efficacy in
children (63). Rapid urbanization with large segments of the population
living under suboptimal hygienic conditions is characteristic of rapidly
industrializing nations, and mass population movements are regular
accompaniments of sociopolitical instability. The reemergence of diphtheria
in the Newly Independent States of the former Soviet Union demonstrates the
continued threat of this disease and of other infectious agents that may
exploit similar social and political vulnerabilities.

 Table 3. Factors influencing the emergence of diphtheria,
 Newly Independent States (NIS), 19901996.

 -----------------------------------------------------------
 Technology and industry
     Population of susceptible adults
 Human demographics and behavior
     Population resistance to vaccinating children
     Changes in childhood vaccination schedule
     High levels of militarization
     Decreased social controls, increased travel
 Microbial adaptation and change
     Change in biotype or emergence of epidemic clones
 Economic development and land use
     Highly crowded and intense urbanization, substandard
     housing
 Breakdown of public health measures
     Decreased immunization in Central Asia and Caucasus
     due to breakup of Soviet Union
 International travel and land use
     Repatriation of Russian population from republics
     Refugees from Tajikistan, refugees in Georgia
 -----------------------------------------------------------

      Dr. Vitek is a medical epidemiologist in the National Immunization
Program, CDC. His research focuses on diphtheria and pertussis; he has
worked extensively in the Russian Federation and Kazakstan.

      Dr. Wharton is chief, Child Vaccine Preventable Diseases Branch,
National Immunization Program, CDC. Dr. Wharton's research focuses on the
epidemiology of vaccine-preventable diseases, especially pertussis,
varicella, mumps, and diphtheria.

      Address for correspondence: Charles Vitek, Centers for Disease
Control and Prevention, Mail Stop E61, Atlanta, GA 30333, USA; fax:
404-639-8616; e-mail: cxv3@cdc.gov.

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Synopses

Cell-to-Cell Signaling and Pseudomonas aeruginosa Infections

Christian Van Delden and Barbara H. Iglewski
University of Rochester School of Medicine and Dentistry, Rochester, New
York, USA

---------------------------------------------------------------------------
      Pseudomonas aeruginosa is a bacterium responsible for severe
      nosocomial infections, life-threatening infections in
      immunocompromised persons, and chronic infections in cystic
      fibrosis patients. The bacterium's virulence depends on a large
      number of cell-associated and extracellular factors.
      Cell-to-cell signaling systems control the expression and allow
      a coordinated, cell-densitydependent production of many
      extracellular virulence factors. We discuss the possible role
      of cell-to-cell signaling in the pathogenesis of P. aeruginosa
      infections and present a rationale for targeting cell-to-cell
      signaling systems in the development of new therapeutic
      approaches.

Pseudomonas aeruginosa as a Human Pathogen

Pseudomonas aeruginosa, an increasingly prevalent opportunistic human pathogen,
is the most common gram-negative bacterium found in nosocomial infections.            
 P. aeruginosa is responsible for 16% of nosocomial pneumonia cases (1), 12%    
 of hospital-acquired urinary tract infections (2), 8% of surgical wound      
infections (3), and 10% of bloodstream infections (4). Immunocompromised 
patients, such as alginate/biofilm, neutropenic cancer and bone marrow        
transplant patients, are particularly susceptible to opportunistic infections.
In this group of patients, P. aeruginosa is responsible for pneumonia and
septicemia with attributable deaths reaching 30% (5,6). P. aeruginosa is
also one of the most common and lethal pathogens responsible for
ventilator-associated pneumonia in intubated patients (7), with
directly attributable death rates reaching 38% (8). In burn patients,
P. aeruginosa bacteremia has declined as a result of better wound
treatment and dietary changes (removal of raw vegetables, which can
be contaminated with P. aeruginosa, from the diet) (3). However, P.
aeruginosa outbreaks in burn units are still associated with high (60%)
death rates (9). In the expanding AIDS population, P. aeruginosa bacteremia
is associated with 50% of deaths (10). Cystic fibrosis (CF) patients are
characteristically susceptible to chronic infection by P. aeruginosa, which
is responsible for high rates of illness and death in this population (11).
The capacity of P. aeruginosa to produce such diverse, often overwhelming
infections is due to an arsenal of virulence factors (Figure 1). Many
extracellular virulence factors secreted by P. aeruginosa have been shown
to be controlled by a complex regulatory circuit involving cell-to-cell
signaling systems that allow the bacteria to produce these factors in a
coordinated, cell-densitydependent manner (12). In this article we
describe major virulence factors of P. aeruginosa and the possible
involvement of cell-to-cell signaling in the pathogenesis of acute P.
aeruginosa infection. We also summarize data suggesting that these
regulatory systems could be exploited for the design of therapeutic
interventions.

[Figure 1.] Virulence factors of Pseudomonas aeruginosa. P.
aeruginosa has both cell-associated (flagellum,
pilus, nonpilus adhesins, lipopolysaccharide [LPS]) and
extracellular virulence factors proteases, hemolysins, exotoxin
A, exoenzyme S, pyocyanin).

Pathogenesis of P. aeruginosa Infections

P. aeruginosa (family Pseudomonadaceae), an aerobic, motile, gram-negative rod
able to grow and survive in almost any environment, lives primarily in water,
soil, and vegetation. However, despite abundant opportunities for spread, P.
aeruginosa rarely causes community-acquired infections in
immunocompetent patients. As a result, the pathogen is viewed as opportunistic.
The different phases of P. aeruginosa infection are shown in Figure 2.

[Figure 2.]  Model of the different phases of Pseudomonas aeruginosa
infection. After an initial colonization phase, mostly       
dependent on cell-associated virulence factors, the 
infectious process evolves either to a chronic infection
characterized by low production of extracellular virulence
factors or to an acute infection characterized by high production 
of cell-to-cell signaling dependent virulence factors.
During acute exacerbation of chronic infection, the
production of cell-to-cell factors is highly increased.     

Colonization: The Predominant Role of Cell-Associated Virulence Factors
 
To initiate infection, P. aeruginosa usually requires a substantial break in
first-line defenses. Such a break can result from breach or bypass of normal
cutaneous or mucosal barriers (e.g., trauma, surgery, serious burns, or
indwelling devices), disruption of the protective balance of normal mucosal
flora by broad-spectrum antibiotics, or alteration of the immunologic
defense mechanisms (e.g., in chemotherapy-induced neutropenia, mucosal
clearance defects from cystic fibrosis, AIDS, and diabetes mellitus).

The first step in P. aeruginosa infections is colonization of altered
epithelium. The pathogen colonizes the oropharynx of up to 6% and is
recovered from the feces of 3% to 24% of healthy persons (2). In contrast,
up to 50% of hospitalized patients are at high risk for P. aeruginosa
colonization (2). Adherence of P. aeruginosa to epithelium is probably
mediated by type 4 pili similar to those of Neisseria gonorrhoeae (13).
Several other nonpilus adhesins responsible for the binding to mucin have
been described, but their role in the infection process remains unclear
(14). Flagella, which are primarily responsible for motility, may also act
as adhesins to epithelial cells (15).

From Colonization to Acute Infection: The Role of Extracellular Virulence
Factors

P. aeruginosa produces several extracellular products that after
colonization can cause extensive tissue damage, bloodstream invasion, and
dissemination (Figure 1). In vivo studies have shown that mutants defective
in the production of exotoxin A, exoenzyme S, elastase, or alkaline
protease are essential for maximum virulence of P. aeruginosa; however, the
relative contribution of a given factor may vary with the type of infection
(16). Many of these factors are controlled by regulatory systems involving
cell-to-cell signaling. We will summarize the known biologic effects of the
most-studied extracellular virulence factors associated with acute P.
aeruginosa infection.

Exotoxin A is produced by most P. aeruginosa strains that cause clinical
infections. Like diphtheria toxin, P. aeruginosa exotoxin A catalyzes
ADP-ribosylation and inactivation of elongation factor 2, leading to
inhibition of protein biosynthesis and cell death (17). Exotoxin A is
responsible for local tissue damage, bacterial invasion (18), and
(possibly) immunosuppression (19). Purified exotoxin A is highly lethal for
mice which supports its role as a major systemic virulence factor of P.
aeruginosa (18).

Exoenzyme S is also an ADP-ribosyl transferase, but unlike exotoxin A, it
preferentially ribosylates GTP-binding proteins such as Ras (20). This
exoproduct is responsible for direct tissue destruction in lung infection
(21) and may be important for bacterial dissemination (22).

Two hemolysins, phospholipase C and rhamnolipid, produced by P. aeruginosa,
may act synergistically to break down lipids and lecithin. Both may
contribute to tissue invasion by their cytotoxic effects. Rhamnolipid, a
rhamnose-containing glycolipid biosurfactant, has a detergentlike structure
and is believed to solubilize the phospholipids of lung surfactant, making
them more accessible to cleavage by phospholipase C (23). The resulting
loss of lung surfactant may be responsible for the atelectasis associated
with chronic and acute P. aeruginosa lung infection (24). Rhamnolipid also
inhibits the mucociliary transport and ciliary function of human
respiratory epithelium (25). However, the relative role of rhamnolipid in
acute or chronic infection is not known.

Proteases are assumed to play a major role during acute P. aeruginosa
infection. P. aeruginosa produces several proteases including LasB
elastase, LasA elastase, and alkaline protease (12). The role of alkaline
protease in tissue invasion and systemic infections is unclear; however,
its role in corneal infections may be substantial (26,27). The ability of
P. aeruginosa to destroy the protein elastin is a major virulence
determinant during acute infection. Elastin is a major part of human lung
tissue and is responsible for lung expansion and contraction. Moreover,
elastin is an important component of blood vessels, which rely on it for
their resilience. The concerted activity of two enzymes, LasB elastase and
LasA elastase, is responsible for elastolytic activity (28). Elastolytic
activity is believed to destroy elastin-containing human lung tissue and
cause the pulmonary hemorrhages of invasive P. aeruginosa infections. LasB
elastase is a zinc metalloprotease that acts on a number of proteins
including elastin. LasB elastase is highly efficient, with a proteolytic
activity approximately 10 times that of P. aeruginosa alkaline protease and
an activity toward casein approximately four times that of trypsin (28).
The LasA elastase is a serine protease that acts synergistically with LasB
elastase to degrade elastin. LasA elastase nicks elastin, rendering it
sensitive to degradation by other proteases such as LasB elastase, alkaline
protease, and neutrophil elastase (28). Both LasB elastase and LasA
elastase have been found in the sputum of CF patients during pulmonary
exacerbation (29). However, the role of LasB elastase in tissue destruction
during the chronic phase of CF is less clear. It has been postulated that
during this phase, antibodies present in high titers neutralize LasB
elastase, and elastin damaged by minute amounts of LasA is degraded mostly
by neutrophil elastase (28). LasB elastase degrades not only elastin but
also fibrin and collagen (30). It can inactivate substances such as human
immunoglobulins G and A (31), airway lysozyme (32), complement components
(33), and substances involved in protecting the respiratory tract against
proteases such as a-1-proteinase inhibitor (34) and bronchial mucus
proteinase inhibitor (35). Therefore, LasB elastase not only destroys
tissue components but also interferes with host defense mechanisms. Studies
in animal models show that mutants defective in LasB elastase production
are less virulent than their parent strains (16,36,37), which supports the
role of LasB elastase as a virulence factor.

Cell-to-Cell Signaling: A Global Regulation System of P. aeruginosa
Extracellular Virulence Factors

Cell-to-Cell Signaling Systems

P. aeruginosa appears to control the production of many of its
extracellular virulence factors by a mechanism that monitors bacterial cell
density and allows communication between bacteria by cell-to-cell
signaling. Bacteria are able to sense their environment, process
information, and react appropriately; however, their ability to sense their
own cell density, to communicate with each other, and to behave as a
population instead of individual cells has only recently been understood
(38,39). This phenomenon, called quorum-sensing or cell-to-cell signaling,
is a generic phenomenon described in many gram-negative (40) and
gram-positive bacteria (41).

The first cell-to-cell signaling system described is the lux system,
responsible for the cell-densitydependent control of bioluminescence genes
by the marine bacteria Vibrio fischeri (38). Cell-to-cell signaling systems
of gram-positive bacteria involve peptide pheromones as signals (41). In
contrast, cell-to-cell signaling systems of gram-negative bacteria, with
the exception of Ralstonia solanacearum, in which an endogenous fatty acid
derivative has been suggested as a cell-to-cell signal (42,43), are
composed of a small molecule called an autoinducer, which is synthesized by
a LuxI-type autoinducer synthase and a LuxR-type transcriptional activator
protein (R-protein) (Figure 3) (38). The various autoinducers described in
gram-negative bacteria are homoserine lactone-based molecules that differ
between one another in length and substitutions on their acyl side chains.
At low cell density, autoinducer is synthesized at basal levels and is
thought to diffuse into the surrounding media, where it becomes diluted.
With increasing cell density, the intracellular concentration of
autoinducer increases until it reaches a threshold concentration. At this
critical concentration, the autoinducer has been proposed to bind to a
specific R-protein (40). The R-protein itself is not active without the
corresponding autoinducer, and it is the R-protein/autoinducer complex that
is proposed to bind to specific DNA sequences upstream of target genes
enhancing their transcription (44,45). The resulting increase in expression
of these genes can reach 1,000-fold. The autoinducer, therefore, allows the
bacteria to communicate with each other (cell-to-cell signaling), to sense
their own density (quorum-sensing), and together with a transcriptional
activator to express specific genes as a population instead of individual
cells.

[Figure 3.] Cell-to-cell signaling systems. Cell-to-cell signaling
systems are composed of two genes. The I gene encodes an
autoinducer synthase and the R gene encodes a transcriptional
activator protein (R-protein). The autoinducer synthase is
responsible for the synthesis of an autoinducer molecule (AI),
which crosses the cell membrane. With increasing cell-density the
intracellular concentration of AI reaches a threshold level, and
the AI then binds to the transcriptional activator. The
complex R-protein/AI activates the expression of specific target genes.

The las Cell-to-Cell Signaling System of P. aeruginosa
                                         
described in P. aeruginosa was shown to regulate expression of LasB elastase
and was therefore named the las system (46). The las cell-to-cell signaling     
system is composed of lasI, the autoinducer synthase gene 3-oxo-C12-HSL 
N-[3-oxododecanoyl]-L-homoserine lactone, previously named PAI-1 or 
OdDHL), and the lasR gene that codes for a transcriptional activator protein  
(Figure 4) (47,48). The las cell-to-cell signaling system regulates  
lasB expression and is required for optimal production of other 
extracellular virulence factors such as LasA protease and exotoxin A (49).
LasI  is the most sensitive gene to activation by LasR/3-oxo-C12-HSL (50).   
The preference for the lasI promoter allows an initial rapid rise in
autoinducer synthesis, which increases the amount of 3-oxo-C12-HSL
available to bind to LasR. This autoinduction hierarchy is responsible for
a dramatic increase of expression of virulence genes (such as lasB) once a
critical cell density has been reached. Recently, the las system has also
been shown to activate the xcpP and xcpR genes that encode proteins of the
P. aeruginosa secretory pathway (51). 3-oxo-C12-HSL alone has been
suggested to contribute to the virulence of P. aeruginosa because it has
some immunomodulatory activity (52). The las cell-to-cell signaling system
is positively controlled by GacA (53), as well as by Vfr, which is required
for the transcription of lasR (54). An inhibitor, RsaL, that represses the
transcription of lasI, has also been described (de Kievit et al., unpub.
data). The multiple regulatory levels of the las cell-to-cell signaling
system and the various genes under its control highlight the importance of
this system for P. aeruginosa.

[Figure 4.] The cell-to-cell signaling circuitry of P.   
aeruginosa. The las cell-to-cell signaling system
controls the rhl cell-to-cell signaling system in a
hierarchy cascade. The LasR/3-oxo-C12-HSL complex
activates the transcription of rhlR, and 3-oxo-C12-HSL     
blocks the activation of RhlR by C4-HSL. The las system
itself is controlled positively by Vfr and GacA,
and negatively by RsaL. 3-oxo-C12-HSL is required for
biofilm differentiation and has immunomodulatory
activity. Both cell-to-cell signaling systems regulate
the expression of numerous genes (lasB: LasB elastase,
lasA: LasA elastase, toxA: exotoxin A, aprA: alkaline
protease, xcpP and xcpR: genes of the xcp secretory
pathway, rhlAB: rhamnosyltransferase required
for rhamnolipid production, rpoS: stationary phase sigma
factor).                      
     
The rhl Cell-to-Cell Signaling System of P. aeruginosa

P. aeruginosa has a second cell-to-cell signaling system, named the rhl system
because of its ability to control the production of rhamnolipid. This system is
composed of rhlI, the C4-HSL N-butyrylhomoserine lactone, previously
named PAI-2 or BHL) autoinducer synthase gene, and the rhlR gene encoding a
transcriptional activator protein (Figure 4) (55-59). This system regulates the
expression of the rhlAB operon that encodes a rhamnosyltransferase required for
rhamnolipid production (60). The rhl system is also necessary for optimal production of
LasB elastase, LasA protease, pyocyanin, cyanide, and alkaline protease
(53,57,61,62). Therefore, like the las cell-to-cell signaling system, the rhl
system, sometimes referred to as vsm (virulence secondary metabolites),
regulates the expression of various extracellular virulence factors of P.
aeruginosa. Interestingly, the rhl system also regulates the expression of rpoS,
which encodes a stationary sigma factor [sigma] dS) involved in the regulation of various
stress-response genes (63,64).
 
 The Cell-to-Cell Signaling Hierarchy in P. aeruginosa
 
Recent data have shown that the las and rhl cell-to-cell
signaling systems of P. aeruginosa interact. Both systems are
highly specific in that their respective autoinducers are unable to
activate the transcriptional activator protein of the other system (i.e.,
3-oxo-C12-HSL cannot activate RhlR, and C4-HSL cannot activate LasR)
(57,62). It has also been shown that the R-protein/autoinducer complexes
prefer certain promoters that they will activate; LasR/3-oxo-C12-HSL
preferentially activates lasB over rhlA, and RhlR/C4-HSL preferentially
activates rhlA over lasB (57,62). However, neither system is completely
independent of the other. The LasR/3-oxo-C12-HSL complex activates the
expression of rhlR placing the las system in a cell-to-cell signaling
hierarchy above the rhl system (Figure 4) (63,65). Moreover, 3-oxo-C12-HSL
can bind to RhlR, blocking the binding of C4-HSL to its transcriptional
activator rhlR (65). The las system therefore controls the rhl system at
both a transcriptional and posttranslational level. Another yet
unidentified regulatory mechanism directly influencing the expression of
rhlRI has been suggested (63). Rhl system regulation of such important
genes as rpoS could explain why multiple levels of controls are required
for its tight regulation.

Cell-to-Cell Signaling: A Powerful System to Overcome Host Defenses during
Acute Infection

Cell-to-cell signaling systems might enable P. aeruginosa to overcome host
defense mechanisms. Isolated production of extracellular virulence factors
by a small number of bacteria would probably lead to an efficient host
response neutralizing these compounds. However, the coordinated expression
of virulence genes by an entire bacterial population once a certain density
has been reached might allow P. aeruginosa to secrete extracellular factors
only when they can be produced at high enough levels to overcome host
defenses. These factors could alter the precarious balance between host
defenses and production of bacterial toxins, leading to invasion of blood
vessels, dissemination, systemic inflammatory-response syndrome, and
finally death. Even appropriate antibiotic therapies are often unable to
stop this course; therefore, the process must be blocked early, before
virulence gene expression can be coordinated.

Biofilms and Cell-to-Cell Signaling

The most striking feature of persistent P. aeruginosa infections in CF
patients is the selection of mucoid mutants producing the exopolysaccharide
alginate (a polymer of mannuronic and guluronic acid) (11). These mutant
bacteria grow inside a biofilm (defined as microcolonies surrounded by an
exopolysaccharide) as a survival strategy because the surrounding matrix
protects bacteria from phagocytes and complement activity (11). P.
aeruginosa growing in an alginate "slime matrix" are resistant to
antibiotics (e.g., aminoglycosides, -lactam antibiotics, fluoroquinolones)
and disinfectants (11). The exact nature of the increased resistance is
unclear but has been attributed to slow growth, penetration barriers,
-lactamase production, and other factors. P. aeruginosa also produces
other less well-defined biofilms essential in the colonization of
indwelling devices such as catheters. Recently, the las cell-to-cell
signaling system has been shown to be involved in the differentiation of P.
aeruginosa biofilms (66). A mutant defective in the production of
3-oxo-C12-HSL formed an abnormal biofilm that, in contrast to the wild type
biofilm, was sensitive to low concentrations of the detergent sodium
dodecyl sulfate (SDS). Furthermore, the addition of 3-oxo-C12-HSL to the
culture media restored production of a differentiated, SDS-resistant
biofilm by the mutant (66). Whether the formation of an undifferentiated
biofilm renders this mutant more sensitive to antibiotics is still unknown.
Also unclear is whether 3-oxo-C12-HSL is required for the differentiation
of alginate biofilms. The link between 3-oxo-C12-HSL and biofilm
differentiation highlights the broad range of systems controlled by
cell-to-cell signaling in P. aeruginosa.

Interference with Cell-to-Cell Signaling: A Potential Therapeutic Approach
against P. aeruginosa

The increasing emergence of bacterial strains resistant to antibacterial
drugs is a major challenge. P. aeruginosa is one of the most problematic
human pathogens, showing intrinsic resistance to many structurally
unrelated antibiotics. Resistance mechanisms include low outer membrane
permeability or multidrug efflux pumps (tetracycline, imipenem,
fluoroquinolones, aminoglycosides) or production of antibiotic modifying
enzymes (aminoglycosides, -lactams) (67,68). Previous exposure to
antibiotics often leads to multidrug-resistant P. aeruginosa strains.
Moreover, the eradication of colonization with P. aeruginosa is almost
impossible in CF patients because of the emergence of multidrug-resistant
strains and the protective effects of alginate (69). Therefore, major
efforts have been made to find new therapeutic interventions against P.
aeruginosa. A tempting approach is to reduce the production of
extracellular virulence factors. In this regard, the interference with
cell-to-cell signaling systems is very attractive. Experimental evidence
for effective interference with cell-to-cell signaling systems has been
recently provided by the use of autoinducer analogs (70-72) or furanone
compounds (73). The LasR/3-oxo-C12-HSL complex, which plays a central role
in controlling virulence gene expression in P. aeruginosa, is essential not
only for the production of elastase and other proteases required for tissue
invasion but also for the control of the rhl cell-to-cell signaling system
in a hierarchical cascade and for the differentiation of biofilm. Mutants
with a nonfunctional las cell-to-cell signaling system are unable to
produce several virulence factors (including elastase) and have
significantly reduced virulence in a neonatal mouse pneumonia model (37).
Therefore, the LasR/3-oxo-C12-HSL complex has become a potential target for
new therapeutic interventions. Analogs of 3-oxo-C12-HSL (74) are under
investigation for their ability to act as 3-oxo-C12-HSL antagonists, which
block activation of LasR and thereby interfere with virulence gene
expression. The fact that 3-oxo-C12-HSL can block the bioactivity of C4-HSL
(65) suggests that it might be possible to interfere efficiently with
cell-to-cell signaling in P. aeruginosa. As elements essential for the
synthesis of autoinducers have recently been described, drugs inhibiting
the biosynthesis process could also be designed (75-80). These approaches
could reduce the production of virulence factors, and by interfering with
biofilm differentiation they could also render colonizing P. aeruginosa
more susceptible to antibiotics and biocides. Combination therapies,
including antibiotics and drugs reducing virulence factor production, might
have an important role in the treatment of CF and intensive-care patients.

As with any therapeutic approach against P. aeruginosa, the possible
emergence of mutations that would prevent the desired effect is a concern.
We have recently described spontaneous mutants that were restored in the
production of extracellular virulence factors (elastase and rhamnolipid) in
the absence of a functional las cell-to-cell signaling system (81). We were
unable to isolate mutants restored in virulence factor production when both
the las and the rhl cell-to-cell signaling systems were deficient. These
results suggest that therapeutic interventions directed only against the
las cell-to-cell signaling system are likely to fail because of the
emergence of resistant strains able to produce virulence factors despite an
inactive las system (81). However, if both the las and the rhl cell-to-cell
signaling systems are blocked, P. aeruginosa might be unable to restore the
production of cell-to-cell signaling dependent virulence factors; this
approach may efficiently reduce virulence factor production and high death
rates associated with P. aeruginosa.

Cell-to-Cell Signaling Systems in Other Human Gram-Negative Pathogens

Other gram-negative human pathogens including Serratia marcescens, S.
liquefaciens, Aeromonas hydrophila, Vibrio cholerae, V. parahaemoliticus,
Yersinia enterocolitica, Enterobacter agglomerans, Hafnia alvei, and
Rahnella aquatis have been shown to produce homoserine lactone-based
autoinducer molecules. These organisms are thought to contain cell-to-cell
signaling systems analogous to the las and rhl systems of P. aeruginosa
(39,40). Burkholderia cepacia (formerly known as Pseudomonas cepacia) is an
opportunistic pathogen responsible for fatal pulmonary infections in CF
patients (11). Virulence factor production of this pathogen can be enhanced
by P. aeruginosa exoproducts, and this interspecies communication may be
due to homoserine-lactone signals (82). Whether B. cepacia contains
cell-to-cell signaling systems is still unknown. As the number of bacteria
known to use cell-to-cell signaling systems increases, therapeutic
interference with cell-to-cell signaling might expand to other human
pathogens.

Conclusions

The pathogenesis of P. aeruginosa is clearly multifactorial as underlined
by the large number of virulence factors and the broad spectrum of diseases
the bacterium causes. Many of the extracellular virulence factors required
for tissue invasion and dissemination are controlled by cell-to-cell
signaling systems involving homoserine lactone-based signal molecules and
specific transcriptional activator proteins. These regulatory systems
enable P. aeruginosa to produce virulence factors in a coordinated,
cell-densitydependent manner that could allow the bacteria to overwhelm
the host defense mechanisms. Interference with cell-to-cell signaling
dependent virulence factor production is a promising therapeutic approach
for reducing illness and death associated with P. aeruginosa colonization
and infection. The growing number of human pathogens found to contain
cell-to-cell signaling systems highlights the importance of exploring
interference with bacterial cell-to-cell signaling for new therapeutic
interventions.

Acknowledgment

      We thank E.C. Pesci, T. deKievit, and J.P. Pearson for helpful
discussions.

      Supported by NIH grant R01A133713-04 (to B.H.I.), a Wilmot
Foundation, and a Swiss Research Foundation grant (to C.V.D.).

      Dr. van Delden is consulting physician, Division of Infectious
Diseases, and investigator, Swiss National Science Foundation, Department
of Genetics and Microbiology, University Hospital, Geneva, Switzerland. His
research interests include bacterial pathogenesis and the development of
new antibacterial approaches.

      Dr. Iglewski is professor and chair, Department of Microbiology and
Immunology, University of Rochester, Rochester, New York. Her research
focuses on microbial pathogenesis, cell-to-cell signaling, and gene
regulation in bacteria.

      Address for correspondence: Barbara H. Iglewski, Department of
Microbiology and Immunology, University of Rochester, 601 Elmwood Avenue,
Rochester, NY 14642, USA; fax: 716-473-9573; e-mail:
BIGL@uhura.cc.rochester.edu.

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     an autoinducer synthase. J Bacteriol 1997;179:4882-7.
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Synopses

Rotavirus

Umesh D. Parashar, Joseph S. Bresee, Jon R. Gentsch, and Roger I. Glass
Centers for Disease Control and Prevention, Atlanta, Georgia, USA

---------------------------------------------------------------------------
      Rotavirus, the most common diarrheal pathogen in children
      worldwide, causes approximately one third of
      diarrhea-associated hospitalizations and 800,000 deaths per
      year. Because natural infection reduces the incidence and
      severity of subsequent episodes, rotavirus diarrhea might be
      controlled through vaccination. Serotype-specific immunity may
      play a role in protection from disease. Tetravalent
      rhesus-human reassortant rotavirus vaccine (RRV-TV) (which
      contains a rhesus rotavirus with serotype G3 specificity and
      reassortant rhesus-human rotaviruses with G1, G2, and G4
      specificity) provides coverage against the four common
      serotypes of human rotavirus. In clinical trials in
      industrialized countries, RRV-TV conferred 49% to 68%
      protection against any rotavirus diarrhea and 61% to 100%
      protection against severe disease. This vaccine was licensed by
      the U.S. Food and Drug Administration on August 31, 1998, and
      should be cost-effective in reducing diarrheal diseases in
      industrialized countries. The vaccine's efficacy and
      cost-effectiveness in developing countries should be evaluated.

In 1973, Bishop and colleagues observed by electron microscopy, in the
duodenal epithelium of children with diarrhea, a 70-nm virus, subsequently
designated rotavirus (Latin, rota = wheel) because of its appearance
(Figure 1) (1). Before this discovery, a bacterial, viral, or parasitic
etiologic agent could be detected in only 10% to 30% of children with
diarrhea. Within 5 years, rotavirus was recognized as the most common cause
of diarrhea in infants and young children worldwide, accounting for
approximately one third of cases of severe diarrhea requiring
hospitalization (2-4). In the United States, approximately 2.7 million
children < 5 years of age are affected by rotavirus diarrhea each year,
resulting in 500,000 physician visits and 50,000 hospitalizations at an
estimated $274 million in medical care and more than $1 billion in societal
costs (5,6). The cost of rotavirus diarrhea is even higher in developing
countries, where 20% to 70% of hospitalizations and 800,000 of the three
million deaths per year from diarrhea are caused by this pathogen (Figure
2) (7,8). Recognition of rotavirus as a major cause of diarrhea in children
led to extensive research for interventions to reduce the incidence of this
disease.

 [Figure 1.] Rotavirus particles           [Figure 2.] Estimated global
 visualized by immune electron             distribution of the 800,000 annual
 microscopy in stool filtrate from child   deaths caused by rotavirus
 with acute gastroenteritis. 70-nm         diarrhea. Reprinted with
 particles possess distinctive             permission from (8).
 double-shelled outer capsid. Bar = 100
 nm.

Because rotavirus infects virtually all children < 3 to 5 years of age in
both industrialized and developing countries, improving water, food, and
sanitation appeared unlikely to reduce disease incidence. Instead, early
studies identified epidemiologic features indicating that rotaviral disease
might best be controlled through vaccination. Natural immunity was
suggested by the infrequent occurrence of more than one episode of
rotavirus diarrhea in a child and decreased incidence of disease with
increasing age (9). Furthermore, protection increased with each new
infection and was greatest against moderate to severe disease, less against
mild illness, and least against asymptomatic infection (10). These findings
implied that an attenuated rotavirus vaccine that simulates natural
infection could induce protective immunity and that more than one dose of
vaccine may be required.

Several rotavirus vaccine candidates developed in the past 3 decades have
proven safe and effective in clinical trials. On August 31, 1998, only 25
years after the discovery of rotavirus, a live, oral, tetravalent
rhesus-human reassortant rotavirus vaccine (RRV-TV) was licensed by the
U.S. Food and Drug Administration, and this vaccine may soon be available
for immunization of children. This paper reviews the biologic and
epidemiologic characteristics of rotavirus, discusses the development of
rotavirus vaccines, and identifies research needs for expediting the
introduction of rotavirus vaccines into childhood immunization programs.

Rotavirus Characteristics

Morphology and Classification

Rotavirus, an icosahedral virus in the family Reoviridae, has a distinct
morphologic appearance by negative-stain electron microscopy (3). The viral
capsid is triple-layered; the inner layer (core) contains the virus genome,
which comprises 11 segments of double-stranded RNA, each coding for
products that are either structural viral proteins (VP) or nonstructural
proteins (NSP) (Figure 3). The segmented genome of rotavirus readily
reassorts during coinfection, a property that has been used in developing
vaccines and undoubtedly plays a role in virus evolution.

                                  The major antigenic properties of
					    rotavirusesgroup, subgroup, and
                                  serotypeare determined by the viral
 					    capsid proteins. Rotavirus has seven
 					    major groups (A-G); most human strains
                                  belong to group A, although groups B and
 [Figure 3.] Gene coding            C have occasionally been associated with
 assignments and                  human illness. The product of the 6th
 three-dimensional structure of   gene of group A rotaviruses encodes VP6,
 rotavirus particles.             the most abundant viral protein, which
 Double-stranded RNA segments     is the major determinant of group
 separated on polyacrylamide gel  reactivity, the target of common
 (left) code for individual       diagnostic assays, and contains the
 proteins, which are localized in antigen used to further classify
 the schematic of virus particle  rotaviruses into subgroups I and II. The
 (center) or in different protein outer capsid proteins, VP7, the
 shells of virus (right). Outer   glycoprotein or G-protein (encoded by
 capsid proteins VP4 and VP7 are  gene 7, 8, or 9, depending on the
 neutralization antigens, which   strain), and VP4, the protease-cleaved
 induce neutralizing antibody;    or P-protein (encoded by gene segment
 protein that makes up            4), determine the serotype specificity
 intermediate protein shell, VP6, and form the basis of the binary
 is the subgroup antigen.         classification (G and P type) of
 Reprinted with permission from   rotaviruses. Both G and P proteins
 (4).                             induce neutralizing antibodies and may
                                  be involved in protective immunity.

Global Distribution of Rotavirus Strains

Fourteen G serotypes of rotavirus, 10 of which occur in humans, have been
defined by cross-neutralization studies with polyclonal animal serum
samples; these serotypes correlate with antigenic specificities of the VP7
glycoprotein. The characterization of P serotypes has been difficult
because adequate reagents are not available. Eight P serotypes of human
rotaviruses have been characterized. Additional VP4 gene variants have been
identified, so ultimately the number of P serotypes may exceed 20.
Theoretically, 80 different strains of rotavirus could result from various
combinations of the known 10 G and 8 P serotypes of human rotaviruses. For
vaccine development purposes, it is fortunate that only four common strains
(P[8]G1; P[8]G3; P[8]G4; and P[4]G2) of rotavirus predominate globally
(Figure 4) (11). However, the prevalence of rotavirus strains varies
considerably from one geographic area to another, and unusual strains are
common in several developing countries (e.g., unusual P[6] strains,
including those with serotype G9 specificity, accounted for 9.5% of all
rotaviruses from a multicenter collection in India) (12).

Epidemiology
                                          
Rotaviruses are ubiquitous; 95% of
children worldwide are infected by 3 to   
5 years of age. The prevalence of         
rotavirus infection in neonates has not
been systematically examined, but high    [Figure 4.] Distribution of
infection rates were documented in        rotavirus strains from a global
newborns in six hospitals in India (13).  collection of 2,748 strains.
These infections, frequently              "Others" includes strains that
asymptomatic, were caused by unusual      were not typable. Adapted from
strains of rotavirus. The incidence of    (11).
clinical illness peaks among children
ages 4 to 36 months, who are also at
greatest risk for severe disease requiring hospitalization. Rotavirus
infections of adults are usually subclinical but occasionally cause illness
in parents of children with rotavirus diarrhea, immunocompromised patients
(including those with HIV), the elderly, and travelers to developing
countries. In temperate climates, rotavirus diarrhea occurs predominantly
during the fall and winter; in tropical settings and in developing
countries, seasonality is less marked (14). Peak rotavirus activity in the
United States begins in the Southwest in autumn (October through December)
and ends in the Northeast during spring (March through May) (Figure 5)
(15); the reason for this pattern is unknown.

                                  Rotaviruses are shed in large numbers
 					    during episodes of diarrhea, and usually
                                  are detectable by antigen enzyme
 					    immunoassays (EIA) up to 1 week after
 					    infection or for more than 30 days in
                                  immunocompromised patients. A recent
 [Figure 5.] Average time of      study showed that as many as 30% of
 peak rotavirus activity in the   immunocompetent infants with severe
 contiguous 48 states, United     rotavirus diarrhea may have virus
 States, July 1991 to June 1997.  detectable by polymerase chain reaction
 This contour plot was derived    (PCR) for more than 25 days after
 using the median value for time  hospital admission. The predominant mode
 of peak activity for each        of rotavirus transmission is fecal-oral.
 laboratory. The surveillance     Spread through respiratory secretions,
 system and analytic methods used person-to-person contact, or
 to create this map are described contaminated environmental surfaces has
 in greater detail in (15). +     also been speculated because of the high
 indicates location of            rates of infection in the first 3 years
 laboratories participating in    of life regardless of sanitary
 the surveillance system. To view conditions, the failure to document
 an animated version of this map  fecal-oral transmission in several
 or maps through the year 1997,   outbreaks of rotavirus diarrhea, and the
 please see below.                dramatic spread of rotavirus over large
                                  geographic areas in the winter.
                                  Animal-to-human transmission does not
 					    appear to be common, although human
                                  rotavirus strains that possess a high
degree of genetic homology with animal strains have been identified (16).
Animal strains of rotavirus differ from those that infect humans.

Pathogenesis and Immunity

Rotaviruses infect the mature absorptive villous epithelium of the upper
two thirds of the small intestine. After replication in the upper small
intestine, infectious particles are released into the intestinal lumen and
undergo further replication in the distal areas of the small intestine.
Infection is generally confined to the intestinal mucosa. Although
rotaviruses can be found in the lamina propria and regional lymphatics,
replication at these sites and systemic spread usually do not occur in
immunocompetent persons (3).

Despite the superficial nature of mucosal infection, rotaviruses induce
both local intestinal and systemic immune responses (17,18). Early animal
studies suggested that the presence of rotavirus antibodies in the
intestinal lumen (but not in the serum) was correlated with protection
against disease. Oral administration of preparations containing rotavirus
antibodies has successfully treated chronic rotavirus infection and
diarrhea in immunocompromised children (19,20). In a randomized clinical
trial, a single oral dose of gamma globulin reduced the duration of illness
and the shedding of virus in infants hospitalized with rotavirus diarrhea.
These observations indicate that intestinal immunity protects against
rotavirus diarrhea and that the success of a rotavirus vaccine will depend,
in part, upon its ability to induce mucosal immune responses.

In infants and young children, neutralizing antibodies directed primarily
against the G serotype of the infecting strain (homotypic response) develop
after primary infection with rotavirus (18). Repeat rotavirus infections
elicit both a homotypic and heterotypic (against strains with different G
serotypes) antibody response. Protection against rotavirus diarrhea
correlates with serum antibody titers following natural infection of young
children, and infected children are more protected against reinfection with
similar rather than different G serotypes. A protective role of placentally
transferred maternal antibody among infants < 3 months of age has also been
speculated since rotavirus disease is uncommon in this age group. However,
serum neutralizing antibody responses among vaccine recipients have
sometimes correlated poorly with protection from disease; therefore, the
exact role of serum antibody in protection against disease remains unclear.

Rotavirus Vaccines

Monovalent "Jennerian" Vaccines

Initial development of rotavirus vaccines was based on the Jennerian
approach, which involved the use of a live, attenuated, antigenically
related virus derived from a nonhuman host (21). This approach was prompted
by studies indicating that animal and human rotaviruses shared a common
group antigen and that experimental animals immunized with animal strains
of rotavirus had a significantly lower risk for illness and viral shedding
when subsequently challenged with human rotaviruses. Furthermore,
neutralizing antibodies to human rotavirus serotypes in the animal models
indicated the potential for cross-protection.

Bovine Vaccines

The first two Jennerian vaccines were developed with bovine rotavirus
strains RIT4237 and WC3. The WC3 strain was passaged in cell culture less
than RIT4237 and was developed because of concern that excessive passaging
of the RIT vaccine might cause overattenuation and diminished efficacy.
RIT4237 and WC3 were nonreactogenic and immunogenic when administered to
infants 2 to 18 months of age. However, the protection conferred by both
vaccines varied greatly in efficacy studies, 0% to 76% against any
rotavirus diarrhea and 0% to 100% against severe disease (22-32). A
well-defined correlate of protection was not identified, and reasons for
the variable efficacy were unknown, although late age at vaccination,
timing of vaccination with respect to the onset of the rotavirus season,
and variations in the strength and number of doses of the vaccine were
proposed as contributing factors. Both vaccines performed less well in
developing than in industrialized countries, possibly because of
interference by other enteropathogens or inadequate surveillance during
follow-up.

Rhesus Vaccine

The third Jennerian vaccine was developed with rhesus rotavirus strain
MMU18006, which shares neutralization specificity with human rotavirus G3
strains. Besides sharing antigenic specificity with an epidemiologically
important human rotavirus serotype, MMU18006 was suitable for vaccine
development because it grew efficiently in cell culture. As in the bovine
rotavirus-based vaccines, MMU18006 was safe and immunogenic, although in
some trials, one third of infants became febrile 3 to 4 days after
vaccination. The reactogenicity of MMU18006 was particularly high in two
studies in Finland and Sweden in which 64% and 79% of infants,
respectively, became febrile. Most children with febrile responses were >5
months of age; lack of passively transferred maternal antibody might have
contributed to the high reactogenicity of the vaccine. As in the RIT4237
and WC3 vaccines, the protective efficacy of MMU18006 in field trials was
quite variable, 0% to 60% against any rotavirus diarrhea and 0% to 85%
against severe rotavirus diarrhea (33-39).

Reassortant "Modified Jennerian" Vaccines

The greatest efficacy of MMU18006 was observed in a Venezuelan trial in
which the rotavirus strain circulating in the community (G3) was the same
serotype as the vaccine strain, which suggested that serotype-specific
immunity against each of the epidemiologically important strains of human
rotaviruses may be required for maximum protection. Similar observations in
vaccine challenge cross-protection studies in animals initiated the
development of vaccines that used a modified Jennerian approach in which
animal-human reassortants expressing VP7 proteins of serotypes 1 through 4
were used as the immunogens.

Rhesus-Human Reassortant Vaccines

Rhesus-human reassortants were generated by coinfecting cell cultures with
rhesus rotavirus (RRV) strain MMU18006 (G serotype 3) and human rotavirus
strains D (G serotype 1), DS-1 (G serotype 2), and ST3 (G serotype 4).
Selection pressure (induced by the addition of neutralizing antibody to VP7
of RRV) produced reassortant strains D x RRV, DS-1 x RRV, and ST3 x RRV,
each of which possessed the VP7 gene from HRV serotype 1, 2, or 4 and the
other 10 genes from RRV (Figure 6) (40). Because vaccines made from the
individual reassortants were safe and immunogenic, RRV-TV was developed
incorporating each of the three reassortants and MMU18006 to provide
coverage against the four common VP7 serotypes of rotavirus.

                                       [Figure 7.]Percentage of children with
 [Figure 6.] Schematic demonstration   adverse reactions during 5 days of
 of production of rhesus rotavirus     surveillance following each of three
 (RRV), human rotavirus (HRV) x        doses of tetravalent rhesus-rotavirus
 rhesus rotavirus (RRV)reassortant     vaccine (RRV-TV) or placebo. A)
 quadrivalent vaccine with VP7         percentage with fever (as measured by
 serotype 1, 2, 3, and 4               axillary temperatures).*On day 4, 2.2%
 specificity. Reprinted with           of RRV-TV recipients vs. 0.2% of
 permission from (40).                 placebo recipients became febrile (p =
                                       0.02). B) percentage with diarrhea. C)
                                       percentage with vomiting. Adapted from
                                       (43).

RRV-TV testing was initiated at an inoculum of 10(sup 4)PFU of each of the four
viruses (i.e., at 4 x 10(sup 4)PFU) and completed at 10(sup 5)PFU, the dose 
submitted for licensure (i.e., at 4 x 10(sup 5)PFU). In most trials, vaccine 
was administered orally in three doses separated by at least 3 weeks to
optimize the immune response to the component antigens; immunization was
completed by the age of 6 to 7 months. Because the vaccine virus strains
are acid labile, they are administered with 2.5 ml of citrate-bicarbonate
buffer.

Safety. In clinical trials involving approximately 10,000 infants, the
tetravalent vaccine has not been associated with any major adverse
reactions. In a large multicenter trial in the United States, RRV-TV
recipients had a small increase in febrile reactions (axillary temperature
>38C) on day 4 after the first dose (2.2% vs. 0.2%, p = 0.02) compared
with placebo recipients; no significant differences were observed in rates
of fever after doses 2 and 3 and of vomiting and diarrhea after any dose
(Figure 7). However, the fevers were mild compared with those associated
with other childhood vaccines (e.g., diphtheria-tetanus-pertussis vaccine).
The results of clinical trials of the monovalent parent RRV strain indicate
that febrile reactions may be more frequent or severe among infants who
receive the first dose of RRV-TV at >5 months of age.

In the Venezuelan trial of RRV-TV (41), PCR detected vaccine strains of
rotavirus (in concentrations too low to be detected by routine methods such
as EIA or polyacrylamide gel electrophoresis) in fecal samples from 15% of
vaccinated and 13% of unvaccinated children. Although these data suggest
the spread of vaccine virus to unvaccinated children, it is not known
whether such spread would result in "herd protection" through induction of
protective antibody responses in unvaccinated persons. Despite the
theoretical risk for transmission of vaccine virus from RRV-TV recipients
to household members and close contacts with known contraindications for
receipt of RRV-TV (such as immunocompromised persons), this risk must be
weighed against the benefit of protecting these immunocompromised persons
by immunizing young children at risk for wild rotavirus infection.

Efficacy. Seven large efficacy trials have been completed using three doses
of RRV-TV: four with the 4 X 10(sup 5) PFU dose submitted for licensure and
three with a lower dose (4 X 10(sup 4) PFU) (Table) (4147). The four trials 
at the 4 X 10(sup 5) PFU dose yielded similar results: the vaccine demonstrated 
significant protection against any rotavirus diarrhea (49% to 68%), greater 
protection against severe rotavirus diarrhea (61% to 91%), and 50% to 100% 
efficacy in preventing doctor visits for diarrhea. The efficacy of the vaccine 
in developing countries has been variable, but in a recent trial involving
more than 2,200 underprivileged urban children in Venezuela, vaccine
efficacy approached levels seen in industrialized countries (48% against
all episodes of diarrhea, 70% against episodes requiring hospitalization,
75% against dehydrating illness, and 88% against severe episodes of
rotavirus diarrhea) (41). This large trial is the first to clearly show the
potential usefulness of rotavirus vaccines in developing countries, where
they are most needed.

 Table. Efficacy of the tetravalent rhesus-human reassortant rotavirus vaccine
 (RRV-TV) in clinical trials
 ---------------------------------------------------------------------------------------
                                                                     Vaccine efficacy (%)
                                                                     --------------------
                         No.enrolled     Vaccine
 Country         Age of    vaccine/       dose     No.  Circulating    All       Severe
 (ref)         vaccinees    placebo      (PFU)    doses  strains     disease    disease
 ----------------------------------------------------------------------------------------
Industrialized
 Finland (42)   3-5 mo    1,191/1,207 4 x 10(sup 5)  3      G1,G2        68       61-100

 United 
 States (43)    5-25 wk    403/400    4 x 10(sup 5)  3      G1,G3        49       80-100

 United                                
 States (44)    2-6 mo     347/348    4 x 10(sup 5)  3         G3        50          64

 United                                
 States (45)    4-26 wk    332/330    4 x 10(sup 4)  3      G1,G3        57          82
 
Developing
 Venezuela                             
 (41)           8-18 wk   1,112/1,095 4 x 10(sup 5)  3         G1        48          88
 Peru (46)      2-4 mo     219/209    4 x 10(sup 4)  3      G1,G2        24        0-40

 Brazil (47)    1-5 mo     233/233    4 x 10(sup 4)  3      G1,G2        35          46

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

Bovine-Human Reassortant Vaccines

Bovine-human reassortant rotavirus vaccines include a tetravalent WC3
rotavirus reassortant vaccine with genes coding for the VP7 of three major
serotypes of rotavirus (G1, G2, and G3) and W179-4, a human VP4 reassortant
with P[8] specificity. Theoretically, this vaccine should induce antibodies
broadly reactive to the three common serotypes of rotavirus sharing P[8]
specificity, thereby increasing the protective efficacy of this vaccine. In
an efficacy trial of a three-dose regimen of the WC3 reassortant vaccine,
protection was 67% against all rotavirus diarrhea and 69% against severe
rotavirus diarrhea (48).

Other Candidate Vaccines

In clinical trials, no Jennerian vaccine has provided complete protection
against rotavirus diarrhea; as a result, several non-Jennerian candidate
vaccines are being developed. Vaccines based on neonatal, cold-adapted, and
attenuated human strains of rotavirus are under evaluation (49). Other
approaches, such as the use of baculovirus-expressed viruslike particles or
naked DNA vaccines, are also being used to develop candidate rotavirus
vaccines (50,51).

Implementing Rotavirus Vaccines

Introduction of rotavirus vaccines as routine childhood immunizations will
be predicated on their safety, efficacy, expected impact, and cost. These
aspects need to be addressed separately for industrialized and developing
countries because of the difference in the epidemiology of rotavirus, the
performance of rotavirus vaccines in clinical trials, and the logistics of
implementing new vaccines.

In industrialized countries, the death rate from diarrheal illness has
declined to a level that it no longer poses a serious threat to child
survival. In the United States, diarrheal illnesses cause approximately 300
deaths per year, accounting for less than 2% of all childhood deaths.
However, diarrhea still causes 170,000 hospitalizations each year,
resulting in high medical and societal costs. Rotavirus is the most common
pathogen identified in children hospitalized with diarrhea and is estimated
to account for one third of all diarrheal illnesses. Large multicenter
trials in the United States have convincingly demonstrated that RRV-TV can
safely prevent half of all mild rotavirus illnesses, 80% of severe
episodes, and nearly all hospitalizations associated with rotavirus
diarrhea. A routine, universal immunization program against rotavirus would
prevent an estimated 227,000 physician visits, 95,000 emergency room
visits, and 34,000 hospitalizations from diarrhea among children in the
first 5 years of life (6). Therefore, even at a relatively high operational
cost, an immunization program against rotavirus is likely to be
cost-effective and can be recommended for the United States and probably
for other industrialized countries on the basis of similar considerations.

In developing countries, recommending rotavirus vaccines for universal
childhood immunization is more complex. With approximately 2,000 children
dying each day from rotavirus diarrhea, the potential benefit of rotavirus
vaccines is much greater in developing than in industrialized countries.
Yet several questions remain unanswered.

Will rotavirus vaccines be as efficacious in developing countries as in
industrialized countries? Several factorsyounger age at infection,
potentially larger inoculum of infection, presence of unusual strains of
rotavirus, interference by other enteropathogens, and poorer nutritional
status of childrencould adversely affect the efficacy of rotavirus
vaccines in developing countries. While the high efficacy observed in
Venezuela is encouraging, further efficacy studies are needed in other
parts of the developing world.

How many doses of vaccine are required for maximum protection against
rotavirus diarrhea? In all clinical trials of the tetravalent rotavirus
vaccine, three doses were administered to children 4 weeks to 6 months of
age. For this reason and because of a lack of good correlation between
neutralizing antibody response and protection from disease, it is difficult
to determine whether fewer vaccine doses would confer the same level of
protection. While fewer vaccine doses are appealing for programmatic and
economic reasons, multiple vaccine doses may be required to overcome the
factors that could reduce the efficacy in developing countries.

Finally, can the developing world afford another childhood vaccine? A
vaccine priced in the $10- to $30-per-dose range may be cost-effective for
industrialized countries but unaffordable for developing countries where
the total per capita health-care expenditure may be $10 to $20 per year or
less. Nonetheless, because the vaccine is prepared from virus grown in
tissue culture, the price may be much lower once the developmental costs
are recovered and the vaccine is produced and marketed locally. In
addition, by recovering a major portion of manufacturing costs from the
middle- and upper-class population, vaccine manufacturers may be able to
provide vaccine to the underprivileged at a nominal price. Ultimately, the
rate at which rotavirus vaccines are incorporated into immunization
programs will depend not only on economic considerations but also on their
perceived value by national governments and international and bilateral
agencies (52).

Acknowledgments

      We thank the following for contributing illustrations: Dr. Charles
Humphrey (Fig. 1), Dr. Mark Miller (Fig. 2), Drs. Mary Estes, Bidadi
Prasad, and A.L.Shaw (Fig. 3), Dr. Thomas Trk and Michael Bosley (Fig. 5
and the rotavirus map animation), and Dr. Albert Kapikian (Fig. 6). We
thank Dr. Linda Han for helpful discussions and Anne Mather for editorial
assistance.

      Dr. Parashar is a medical epidemiologist with the Respiratory and
Enteric Viruses Branch, DVRD, NCID, CDC. His research focuses on the
epidemiology of viral gastroenteritis and methods for its prevention and
control.

      Address for correspondence: Umesh D. Parashar, Viral Gastroenteritis
Section, Mail Stop G04, Centers for Disease Control and Prevention, 1600
Clifton Road, NE, Atlanta, GA 30333; fax: 404-639-3645; e-mail:
uap2@cdc.gov.

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Synopses

Chlamydia pneumoniae and Cardiovascular Disease

Lee Ann Campbell, Cho-Chou Kuo, and J. Thomas Grayston
University of Washington, Seattle, Washington, USA

---------------------------------------------------------------------------
      Chlamydia pneumoniae is a ubiquitous pathogen that causes acute
      respiratory disease. The spectrum of C. pneumoniae infection
      has been extended to atherosclerosis and its clinical
      manifestations. Seroepidemiologic studies have associated C.
      pneumoniae antibody with coronary artery disease, myocardial
      infarction, carotid artery disease, and cerebrovascular
      disease. The association of C. pneumoniae with atherosclerosis
      is corroborated by the presence of the organism in
      atherosclerotic lesions throughout the arterial tree and the
      near absence of the organism in healthy arterial tissue. C.
      pneumoniae has also been isolated from coronary and carotid
      atheromatous plaques. To determine whether chronic infection
      plays a role in initiation or progression of disease,
      intervention studies in humans have been initiated, and animal
      models of C. pneumoniae infection have been developed. This
      review summarizes the evidence for the association and
      potential role of C. pneumoniae in cardiovascular disease.

Chlamydia pneumoniae, a common cause of human respiratory disease, was
first isolated from the conjunctiva of a child in Taiwan in 1965 and was
established as a major respiratory pathogen in 1983 when it was isolated
from the throat of a college student at the University of Washington. C.
pneumoniae causes approximately 10% of community-acquired pneumonia and 5%
of pharyngitis, bronchitis, and sinusitis (1). The clinical symptoms of C.
pneumoniae pulmonary infections are similar to those caused by other
respiratory pathogens, except for a few distinguishing features (1).
Subacute onset and pharyngitis are common. Often a biphasic pattern is
observed, with pharyngitis resolving before bronchitis or pneumonia
develops. Cough is very common and prolonged. Although pneumonia is often
relatively mild, recovery is slow, even with antibiotic therapy; and cough
and malaise may persist for many weeks.

Considerable knowledge of the epidemiology of C. pneumoniae infection has
been derived from serologic studies using the C. pneumoniaespecific
microimmunofluorescence test. C. pneumoniae infection is ubiquitous.
Virtually everyone is infected at some point in life, and reinfection
occurs commonly. Antibodies against C. pneumoniae are rare in children
under the age of 5, except in developing and tropical countries. Antibody
prevalence increases rapidly at ages 5 to 14, reaches 50% at the age of 20,
and continues to increase slowly to 70% to 80% at ages 60 to 70 (1).

C. pneumoniae has been associated with other acute and chronic respiratory
diseases (e.g., otitis media, chronic obstructive pulmonary disease,
pulmonary exacerbation of cystic fibrosis, and asthma) as well as other
clinical syndromes (e.g., erythema nodosum, Reiter syndrome, and
sarcoidosis [1]). These associations are determined by seroepidemiologic
observations, case reports, isolation or direct detection of the organism
in specimens, successful response to antichlamydial antibiotics, or a
combination of these methods.

The expanding spectrum of C. pneumoniae infection has been extended to
atherosclerosis and related clinical manifestations such as coronary heart
disease, carotid artery stenosis, aortic aneurysm, claudication (occlusion
of the arteries of the lower extremities), and stroke. This overview
summarizes the studies associating C. pneumoniae infection with
atherosclerosis and discusses preliminary in vitro and in vivo studies
suggesting the plausibility of a causative role.

Is C. Pneumoniae Present in Atherosclerotic Tissues?

Patients with coronary artery disease are significantly more likely to have
serologic evidence of past infection with C. pneumoniae (2,3). Immune
complexes containing C. pneumoniae lipopolysaccharide have also been
associated with 42- and 98-kDa C. pneumoniae species-specific antigens (4).
These associations have been confirmed and extended to carotid artery
disease and cerebrovascular disease (5,6). Although the high prevalence of
C. pneumoniae antibodies in the population leaves a very narrow window for
demonstrating statistically significant differences between cases and
controls, seroepidemiologic studies have shown a consistent association. C.
pneumoniae antibodies have been associated with coronary heart disease (16
studies) and cerebrovascular disease (2 studies) in 17 of 18 published
studies; most had an odds ratio of 2.0 or greater (6). Most were
statistically significant, and the risk is independent of other
atherosclerosis risk factors (i.e., hypercholesterolemia, cigarette
smoking, hypertension, diabetes, and family history). Studies of 2,700
patients and 5,000 controls have demonstrated a serologic association of C.
pneumoniae antibody and cardiovascular disease (6).

Compelling evidence of the association between C. pneumoniae and
atherosclerosis has been obtained by polymerase chain reaction (PCR),
immunocytochemical (ICC) staining, and electron microscopy, which have
detected C. pneumoniae in atherosclerotic lesions (Table). Structures found
within coronary atheromas were remarkably similar to the pear-shaped
elementary body morphologic characteristics described for C. pneumoniae
(Figure 1) (25). By ICC staining using a C. pneumoniaespecific monoclonal
antibody (Mab), C. pneumoniae was demonstrated within the atherosclerotic
lesion in 5 of 7 tissues. By PCR or ICC stain, the organism was detected in
20 of 36 coronary artery tissues from autopsy (7). At the University of
Washington, the organism has been detected in coronary, carotid, aortic,
femoral, and popliteal atheromas in both early lesions and fibrolipid
plaques (7-10,15,18,22,26,27). In all studies using ICC stain, control
tissues were stained with control antibody to rule out background staining
(Figure 2). The organism was found in tissues of male and female study
participants of different ages and ethnic groups. Other investigators have
confirmed these findings and have also found the organism in
atherosclerotic lesions in iliac arteries and tissues from abdominal aortic
aneurysms and aortic valve stenosis (4-14,16, 17,19-21,23,24,28). The
organism has been found in atherosclerotic lesions in 257 (52%) of 497
tissue specimens (6). In contrast, the organism has been found in only 5%
of tissues that appear normal. Remarkably, the detection in diseased
arterial tissue compared to normal arterial tissue represents an odds ratio
of 10 [95% Confidence Interval (CI) 5-22]. In our studies the organism was
not found in cardiovascular tissue that appeared histologically normal (5).

Table. Studies of Chlamydia pneumoniae in atherosclerotic tissue
------------------------------------------------------------------

Source of                                          Atherosclerotic
specimens                                           tissue(sup a)
(reference)       Artery         Type of specimen   (% positive)

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

South Africa (7)  Coronary       Autopsy 20/36 (56)   20/36 (56)
                                 
PDAY9(sup b)
study (8)   	Coronary       Autopsy              8/18  (44)

Univ. Washington
(9)               Coronary       Atherectomy          20/38 (53)
Alaskan Natives
(10)              Coronary       Autopsy              23/59 (39)
Louisville,
Kentucky (11)     Coronary       Vascular surgery     7/12  (58)
Japan (12)        Coronary       Atherectomy          20/29 (69)
Salt Lake City,
Utah (13)         Coronary       Atherectomy          71/90 (79)

India (14)        Coronary       Coronary artery      4/40  (10)
                                 bypass
California &      Carotid        Endarterectomy       37/61 (61)
Univ. Washington
(15)
Germany (16)      Carotid        Endarterectomy        7/50 (14)
Canada (17 )      Carotid        Endarerectomy        54/76 (71)
Univ. Washington
(18)              Aorta          Autopsy              7/21  (33)
Finland (19)      Aorta          Vascular surgery    12/12 (100)
Italy (20)        Aorta          Vascular surgery     26/51 (51)
United Kingdom    Aorta,         Vascular surgery     15/33 (45)
(21)              femoral, iliac
California (22)   Popliteal      Vascular bypass      10/23 (43)
                  femoral
Finland (23)      Aortic valve   Autopsy              25/46 (54)
Sweden (24)       Aortic Valve   Surgery              19/39 (49)
--------------------------------------------------------------------
(sup a)Number positive by immunocytochemical staining and/or 
polymerase chain reaction over number tested.
(sup b)PDAY, pathobiological determinants of atherosclerosis in youth.

 Figure 1. Ultrastructural
 evidence of Chlamydia pneumoniae
 in coronary atheroma. This
 transmission electron micrograph
 demonstrates the presence of
 endosomes containing C.
 pneumoniae pear-shaped elementary
 bodies within a foam cell in
 tissue from coronary artery
 atheroma. Arrows in inset point
 to the elementary bodies. Bar=0.5
 m. (Reprinted from Journal of
 Infectious Diseases (8) with
 permission from the publisher,
 University of Chicago Press).

 Figure 2. Immunocytochemical
 staining demonstrating
 Chlamydia pneumoniae in
 fibrolipid plaque from coronary
 artery atheroma. Panel A
 illustrates positive staining
 of foam cells in the plaque
 with the C. pneumoniaespecific
 monoclonal antibody TT-401.
 Panel B shows negative staining
 of the adjacent section using
 normal ascites fluid as the
 control.
                                    

C. pneumoniae has been difficult to isolate from atheromatous tissues, not
surprising as isolation from infected tissues in chronic chlamydial
infection or after repeated experimental inoculation of animal models is
rare. However, the organism is often demonstrated by DNA and antigen
detection methods, and a chronic inflammatory response persists. The
immunologic characteristics of atherosclerosis are similar to the
inflammatory response resulting from chronic infection. In two recent
cases, C. pneumoniae has been isolated from atheromatous plaques (11,15).
The first isolate was obtained during a multicenter study that examined
coronary arteries of patients (with or without coronary artery disease)
undergoing heart transplants. The organism was found in the coronary artery
of 7 of 10 patients with atherosclerosis; it was not found in two patients
without evidence of coronary atherosclerosis. C. pneumoniae was isolated
from the atherosclerotic lesion of one patient with severe coronary artery
disease (11).

The second isolate was from carotid atheroma obtained in a study of
patients undergoing carotid endarterectomy in Seattle. C. pneumoniae was
detected in 11 of 16 specimens and was cultured from a patient undergoing
elective carotid endarterectomy (15).

In addition to C. pneumoniae, other infectious agents including herpes
simplex virus (HSV), cytomegalovirus (CMV), and Helicobacter pylori have
been associated with cardiovascular disease. Only a few studies have
concurrently investigated the presence of C. pneumoniae and these
infectious agents within lesions. In a study of risk factors for
atherosclerosis in young persons, 6 of 7 samples with atheromas and 2 of 11
with intimal thickening were positive for C. pneumoniae; all 31 unaffected
tissues were negative (8). Two specimens from atheromas and two from normal
tissues were positive for CMV. Only one coronary atheroma was positive for
both CMV and C. pneumoniae. In a study investigating the presence of C.
pneumoniae, CMV, and HSV in atherosclerosis of the carotid artery, C.
pneumoniae was also detected more frequently (71%) than CMV and HSV (35%
and 10%, respectively). Infection with two or three of these agents
occurred in 23.7 and 7.9%, respectively, and C. pneumoniae and CMV were
independently associated with an increased risk for thrombosis (17). In a
study addressing the presence of C. pneumoniae or H. pylori or both in
aortic aneurysm, C. pneumoniae was found in 26 of 51 patients with plaques
(20). Despite the fact that 47 of 51 patients were seropositive for H.
pylori, this agent was not detected in any atherosclerotic plaques (20).
Thus, although C. pneumoniae and HSV or CMV may be found in the same
lesion, C. pneumoniae has been more frequently found as the only infectious
agent.

Does C. pneumoniae Play a Role in Atherogenesis?

Despite solid evidence that C. pneumoniae exists in atherosclerotic
lesions, evidence that the presence of the organism is related to disease
pathogenesis is circumstantial. Three possibilities can be examined. The
organism 1) persists in vascular cells but does not contribute to
pathologic abnormality, 2) causes the initial injury and induces the
atherosclerotic process, or 3) accelerates the severity or progression of
the disease. If the organism is involved, its role must fit within the
context of events in atherogenesis. The early events in lesion development
include endothelial injury or activation resulting in monocyte/macrophage
adherence to the endothelium, migration to the subendothelium, uptake of
oxidized low-density lipoproteins transforming them into foam cells, and
release of cytokines. These cytokines upregulate endothelial cell adhesion
molecules leading to increased leukocyte adhesion. Platelet aggregation at
the site of endothelial damage results in the release of platelet-derived
growth factor, which stimulates smooth muscle cell proliferation.
Dedifferentiated smooth muscle cells secrete collagen, elastin, and
proteoglycans leading to the formation of fibrous tissue. The mature
fibrolipid plaque consists of a lipid/cholesterol-rich core surrounded by a
fibrous cap composed of matrix elements (29).

Human Tissue Studies

To investigate the hypothesis that C. pneumoniae is an "innocent
bystander," one study explored how frequently the organism was found in
tissues from different anatomic sites. Multiple tissues, including coronary
artery, lung, liver, spleen, and bone marrow, were obtained at autopsy from
38 patients, half of whom had cardiovascular disease recognized before
death. Twenty-one of the patients had C. pneumoniae in one or more tissues.
Eighteen had the organism in cardiovascular tissue (11 in cardiovascular
tissue only) and seven in both cardiovascular and noncardiovascular tissue.
Of the remaining three C. pneumoniaepositive patients, two had the
organism only in lung tissue and one only in the spleen. While this study
shows that C. pneumoniae could be detected in noncardiovascular tissue, it
also shows the organism is much more frequently found in cardiovascular
tissue (27).

Another possible explanation for the frequent presence of C. pneumoniae in
atheroma is that infected lung macrophages disseminate to any granulomatous
tissue. Of 33 surgical or autopsy specimens of granuloma from patients with
tuberculosis, leprosy, coccidioidomycosis, Crohn disease, Wegner disease,
rheumatoid nodule, giant cell tumor, or sarcoidosis, three contained C.
pneumoniae, all sarcoid skin granulomas (27). Sarcoidosis is a disease of
unknown etiology for which a serologic association with C. pneumoniae has
been reported (30). These studies suggest that C. pneumoniaeinfected cells
are found preferentially within atheromas.

In Vitro Studies

Consistent with observations in animal models and humans, in vitro studies
have demonstrated that vascular cells are susceptible to C. pneumoniae
infection and that C. pneumoniae produces productive infection in human
macrophages, endothelial cells, and artery smooth muscle cells (31,32), key
cellular components in atherosclerosis. In vitro studies have also measured
whether infection leads to the production of immunomodulators. The primary
host cells are the epithelial cells that line the trachea and nasopharynx.
The first in vitro study addressing a potential role of C. pneumoniae in
atherogenesis found that exposure of human monocyte-derived macrophages to
C. pneumoniae followed by the addition of low-density lipoprotein resulted
in foam cell formation and the accumulation of cholesteryl esters (33).
Foam cell formation is an early event in the atherosclerotic process.

Ultrastructural studies have demonstrated C. pneumoniae in ciliated
bronchial cells in mice, in lung macrophages in mice and rabbits, and in
human foam cells (which are macrophages) and smooth muscle cells that take
up lipids (7,34,35); by double ICC staining with chlamydia-specific and
cell-specific MAbs, C. pneumoniae was found in both smooth muscle cells and
macrophages within aortic atheroma lesions (18). Similarly, in specimens
removed from patients with symptomatic coronary artery disease, C.
pneumoniae was found within macrophages in atherectomy tissue was also
found in tissue removed at atherectomy, C. pneumoniae bacteremia, as
determined by PCR positivity of buffy coat specimens, was found in 13% of
patients with symptomatic coronary atherosclerosis; control specimens were
negative (13,53). In mouse models of C. pneumoniae infection, C. pneumoniae
was detected by culture and PCR in peripheral blood mononuclear cells but
not in plasma after intranasal inoculation (36).

The ability of C. pneumoniae infection to induce production of
proinflammatory and procoagulant activities was investigated to determine
its putative role in eliciting immune responses consistent with
atherosclerotic processes. C. pneumoniae infection of human vascular
endothelial cells results in production of tissue factor, increased levels
of monocyte chemotactic protein-1, and increased platelet adhesion to
infected cells (37,38). Infection of endothelial cells also results in
expression of adhesion molecules, including E-selectin, intercellular
adhesion molecule-1, and vascular adhesion molecule-1, which are important
in leukocyte adhesion (38,39). Lastly, infection of macrophages results in
the production of proinflammatory cytokines, tumor necrosis factor alpha,
interleukin-1 beta, interleukin-6, and interleukin-8 as well as in
expression of CD14 molecules (38,40).

Animal Models

Although studies support a potential role for C. pneumoniae in
atherogenesis, etiology can be established only through animal models or
intervention studies. Rabbits and mice are susceptible to C. pneumoniae
infection and provide well-defined models of atherosclerosis. Respiratory
disease in both species is characterized by multifocal interstitial
pneumonia. The disease is more severe and longer lasting in mice, and
organisms are reisolated more readily from the lungs and aorta.

Rabbit models have been used to determine whether C. pneumoniae respiratory
infection leads to vessel wall infection and inflammatory changes
characteristic of atherosclerosis. In one study, New Zealand White rabbits
received a single nasopharyngeal inoculation of C. pneumoniae at 1 month of
age. Two of ten rabbits demonstrated atherosclerotic changes. The changes
were observed 7 and 14 days postinoculation. One rabbit had an accumulation
of foam cells in the aortic arch (characteristic of an early lesion) and
focal perioaortitis in the abdominal aorta (41). A second rabbit
demonstrated spindle cell proliferation of smooth muscles cells in the
aorta. Both rabbits had bronchiolitis and pneumonitis (41). In another
study, atherosclerotic-li ke changes were found in the aortas of six of
nine New Zealand White rabbits 2 to 4 weeks after they received two
inoculations of C. pneumoniae (42). In both studies, rabbits were fed
normal diets, and atherosclerotic-like changes were not observed in any of
the controls.

We used two mouse models, C57BL/6J and apolipoprotein E (apoE)-knockout, to
test the hypothesis that following upper respiratory tract infection, lung
macrophages are infected, disseminate to the aorta, and contribute to
atherogenesis. C57BL/6J mice, the background strain of apoE-knockout mice,
get atherosclerosis only if fed a diet high in fat and cholesterol. In
contrast, apoE-knockout mice get atherosclerotic lesions, with some
characteristics of human disease, spontaneously on a regular chow diet in a
time- and age-dependent manner. Using C57Bl/6J mice, we demonstrated that
after intranasal or intraperitoneal infection, C. pneumoniae infects
alveolar and peritoneal macrophages, respectively (36). Additionally, the
organism was found in blood monocytes and not in plasma cells, which
indicates that a cell-associated bacteremia followed acute infection.
Passive transfer by intraperitoneal inoculation of alveolar or peritoneal
macrophages obtained from mice inoculated intranasally or intraperitoneally
with C. pneumoniae resulted in dissemination of infection to the lung,
thymus, spleen, and abdominal lymph nodes (36).

To determine whether infection disseminates to the aorta and is found
within atherosclerotic lesions as in human disease, apoE-knockout mice
received single or multiple intranasal inoculations. C. pneumoniae was
detected for up to 20 weeks postinfection in the aorta and within the
lesion in apparent foam cells by ICC staining (43). When the aorta was
positive, the percentage of C. pneumoniaepositive mice was 33% to 100%.
Controls remained negative for C. pneumoniae (43). In contrast, 1 (0.8%) of
12 animals of the background strain (C57BL6/J mice) fed a nonatherogenic
diet contained the organism in the aorta for up to (but not later than) 2
weeks postinfection. Like studies of human granulomatous tissues and
tissues from different anatomic sites (27,43), these studies suggest that
the organism has a tropism to atherosclerotic lesions.

Computer-assisted morphometric analysis of lesion size has been used to
determine if C. pneumoniae infection alters disease progression.
Comparisons of 10 infected and 10 uninfected mice at different times
following three intranasal inoculations of C. pneumoniae suggest that
infection significantly augments the progression of lesion size (44). Thus,
early studies in rabbit and mouse models indicate that C. pneumoniae
infection can induce inflammatory changes similar to those of atherogenesis
and augment the progression of the atherosclerotic lesion.

Persistent Infection

Although C. pneumoniae antigen and DNA are often found in atheromas,
isolation is rare. Similarly, in the mouse model of C. pneumoniae
infection, lungs remain PCR positive and pathologic lesions persist after
the organism can no longer be cultured. In the apoE model, the organism can
be cultured from the lung and aorta in a few mice for up to 3 weeks
postinfection but is detected by PCR and ICC up to 20 weeks postinfection
at both sites. Does PCR positivity in the absence of culture positivity
reflect persistent infection or undegraded DNA? Two lines of evidence
strongly support the presence of viable organisms. Two independent studies
using two different strains of mice have demonstrated that lung infection
could be reactivated by treatment with cortisone (45,46), specifically
after intranasal inoculation, when the organism could no longer be cultured
from lungs. Animals were treated with cortisone or saline. With saline
tratment, C. pneumoniae was not cultured, although animals were frequently
PCR positive. In contrast, with cortisone treatment, reactivated infection
was demonstrated by culture in 46% and 60% of infected mice. The second
line of evidence came from mice acutely infected intranasally with live or
UV-inactivated organisms; alveolar macrophages were isolated at various
times postinfection. When live organisms were used, the organism could be
cultured and detected by PCR up to 7 days postinfection. In contrast, when
mice were inoculated with UV-inactivated organisms, the organism could only
be detected by PCR immediately after inoculation in isolated macrophages
(36). These experiments demonstrated that DNA from dead organisms is
rapidly degraded, whereas live organisms survive within macrophages.

Drug efficacy studies of chlamydia in animal models or humans must be
interpreted with caution because chlamydial infections can persist after
antibiotic therapy. The efficacy of two antibiotic therapies was
investigated in the mouse model of pneumonitis. After infection, mice were
treated with a single dose of doxycycline each day for 3 days or with a
single dose of azithromycin. After either treatment, when the organism
could no longer be cultured, the infection appeared to be cleared. However,
C. pneumoniae DNA could be detected by PCR isolation in 25% or 77% of mouse
lungs, depending on the infecting dose. No differences were observed in the
lungs of treated and untreated mice (47). These results suggest that the
organism can persist after single-dose treatment regimens and that
prolonged treatment may be needed.

Intervention Studies

Animal Models

Animal models of C. pneumoniae infection and atherosclerosis further define
a causative role by determining whether intervention with antimicrobial
agents can alter disease progression and by identifying successful
treatment regimens. New Zealand White rabbits were fed a diet with 0.25%
cholesterol and were inoculated intranasally three times with C. pneumoniae
(48). Infected rabbits and controls were treated for 7 weeks with
azithromycin. Three months after the final inoculation, the maximal intimal
thickness (MIT) of the thoracic aortas increased in infected rabbits but
not in controls. The MIT of azithromycin-treated rabbits was less than that
of untreated infected rabbits and similar to that of controls. However, the
organism was detected by immunofluorescence in the aorta of treated rabbits
as frequently as it was detected in the aorta of untreated rabbits (48).

Human Studies

Three small pilot studies on the potential use of antibiotics against C.
pneumoniae have yielded promising results. The first intervention study
focused on male patients at least 6 months after myocardial infarction.
Titers were arbitrarily classified as seronegative (8), intermediately
positive (>8, 32), or highly positive (64). Those who had serum
antibodies that persisted 3 months later were treated with one or two
courses of azithromycin (500 mg per day orally for 3 days). Treated
patients had a fivefold decrease in cardiovascular events and a reduction
in immunoglobulin (Ig)G titer (49).

The second study included 202 patients hospitalized with unstable angina or
non-Q wave infarctions. The study was randomized with half of the patients
receiving roxithromycin (150 mg twice a day for 30 days) and the other half
receiving a placebo. Serology was not considered for inclusion or exclusion
in the study. After 30 days, a statistically significant decrease in
cardiovascular events was observed in treated patients, while those
receiving the placebo were treated only when a combined endpoint of severe
recurrent angina, acute myocardial infarction, or ischemic death was used
(50).

In the third (randomized) study, 88 patients with percutaneous coronary
revascularization procedures were treated with azithromycin (500 mg per day
for 2 days and 250 mg per day for 28 days) or received placebo (51). After
6 months, patients receiving azithromycin had lower frequencies of both
angiographically confirmed restenosis (9% versus 16%) and recurrent angina
(40%) than patients receiving placebo (60%). No changes in antibody titers
were observed after azithromycin treatment. Despite study limitations (52)
and antiinflammatory effects of the antibiotic on atherosclerosis, the
results are encouraging and warrant carefully designed larger-scale
intervention studies with longer observation times.

Conclusions

A causative role of C. pneumoniae infection in cardiovascular disease has
not yet been firmly established. However, the high frequency of infection
found in human atherosclerotic tissue in comparison to normal tissue, the
induction and progression of atherosclerotic-like inflammatory changes in
infected animal models of atherosclerosis, and the early results from
antichlamydial intervention studies in humans are consistent with a
causative role of C. pneumoniae in the disease process.

      Dr. Campbell is a professor, Department of Pathobiology, School of
Public Health and Community Medicine, University of Washington. Her overall
research focus is on chlamydial pathogenesis with an emphasis on Chlamydia
pneumoniae infection.

      Address for correspondence: Lee Ann Campbell, Department of
Pathobiology, Box 357238, University of Washington, Seattle, WA 98195, USA;
fax: 206-543-3873; e-mail: lacamp@u.washington.edu.

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Synopses

Bacterial Symbiosis in Arthropods and the Control of Disease Transmission

Charles B. Beard,* Ravi V. Durvasula, and Frank F. Richards
*Centers for Disease Control and Prevention, Atlanta, Georgia, USA; and
Yale University School of Medicine, New Haven, Connecticut, USA

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      Bacterial symbionts may be used as vehicles for expressing
      foreign genes in arthropods. Expression of selected genes can
      render an arthropod incapable of transmitting a second
      microorganism that is pathogenic for humans and is an
      alternative approach to the control of arthropod-borne
      diseases. We discuss the rationale for this alternative
      approach, its potential applications and limitations, and the
      regulatory concerns that may arise from its use in interrupting
      disease transmission in humans and animals.

For more than 80 years, insecticides have been one of the primary means of
controlling insect-borne diseases. Recently, however, the control of insect
pests and vectors of disease has become increasingly difficult for various
reasons, including the emergence of insecticide resistance, changes in the
environment, and reduction in public health interventions due to social and
economic problems in countries where insect-borne diseases are endemic.
According to the World Health Organization (WHO), approximately 125
arthropod species are resistant to at least one, and often two or more,
insecticides (1). In many parts of the world where insect-borne diseases
cause illness and death, insecticides are available; however, sustaining
insecticide vector control long term can be extremely costly and may be
unachievable.

Alternative Approaches to Controlling Disease Transmission by Arthropods

Concerns related to the use of insecticides for vector control have led to
alternative approaches for reducing disease transmission by arthropods. One
approach focuses on the use of transgenic methods, i.e., the insertion and
expression of a gene derived from one organism in a second, heterologous
organism. Major components of the transformation system include the
identification of 1) potential DNA vectors (transposable elements or
viral-transducing agents) for genome integration (2-8); 2) selectable
phenotypic marker genes, such as eye color mutants or various enzymes
(e.g., -galactosidase, neomycin phosphotransferase, or
organophosphate-degrading enzyme), as indicators of stable germ line
transformation (9-11); and 3) specific refractory genes that express the
desired phenotype (i.e., a factor that would inhibit transmission of the
pathogen) (12-14). Additional studies have focused on the identification of
regulatory sequences (e.g., stage-specific, tissue-specific, and
constitutive promoters [15-20]), vector population genetics (21-23), and
the development of mathematical models that can be used for predicting the
behavior of genes once they are introduced into wild populations (24,25).
The accomplishments and future prospects of efforts to produce transgenic
arthropods have been discussed extensively (26-31).

Another approach for reducing disease transmission by arthropods is to
genetically modify symbiotic bacteria of arthropod vectors to prevent the
arthropods from transmitting human pathogens. With this approach, the
arthropod is not transformed, but the symbiotic bacteria that it harbors
are (32). Such arthropods are called paratransgenic. This approach is
guided by the following basic concepts: 1) many arthropods (especially
those that throughout their entire developmental cycle feed on restricted
food sources such as blood, cellulose, phloem, stored grains) harbor
bacterial symbionts; 2) in some cases, these symbionts can be cultured and
genetically transformed to express a gene whose product kills a pathogen
that the arthropod transmits; 3) normal arthropod symbionts can be replaced
with genetically modified symbionts, resulting in a population of arthropod
vectors that can no longer transmit disease. While not applicable to all
groups of arthropods, this approach has worked in three species of Chagas
disease vectors and holds promise in a number of other arthropods.

Bacterial Symbionts and Control of American Trypanosomiasis

American trypanosomiasis (Chagas disease) is a parasitic illness that
affects 16 to 18 million people in regions of South and Central America,
according to current WHO estimates. Neither a cure for chronic Chagas
disease nor a vaccine for preventing infection is available. The etiologic
agent, the flagellate protozoon Trypanosoma cruzi, is transmitted by
blood-sucking triatomine bugs, which become infected while feeding on an
infected host. Transmission to humans occurs as the insect feeds when the
engorged triatomine bug, while feeding, deposits on the skin a fecal
droplet that contains infective trypanosomes, which then get rubbed either
into the bite lesion or a mucous membrane (Figure 1).

Rhodnius prolixus is the chief vector of       
Chagas disease in certain regions of Central
America and northern South America.            
Rhodococcus rhodnii, a soil-associated         
nocardiform actinomycete, residing
extracellularly in the gut lumen of R.         [Figure 1.] A triatomine bug
prolixus in close proximity to T. cruzi (33),  vector of Chagas disease in
is transmitted effectively from adult          the process of feeding. The
triatomid bugs to their progeny through        fecal droplet contains
coprophagy (ingestion of fecal material from   infective trypanosomes and
other bugs). The vital role of R. rhodnii in   bacterial symbionts.
the growth and development of R. prolixus has  (Photographs courtesy of
been demonstrated repeatedly under laboratory  Robert B. Tesh).
conditions (34-36). Aposymbiotic nymphs of R.
prolixus (insects that have been cured of
symbionts) do not reach the sexually mature adult stage; most deaths occur
after the second developmental molt. Introduction of the bacteria to first
or second instar nymphs permits normal growth and maturation.

Scientists have exploited this symbiotic association to introduce and
express a series of foreign gene constructs in R. prolixus (37,38). DNA
shuttle plasmids capable of replication in both Escherichia coli and in R.
rhodnii have been constructed and used to genetically modify R. rhodnii
(Figure 2). The genetically altered symbionts can then be introduced into
aposymbiotic first instar nymphs of R. prolixus, where, like unmodified
microorganisms, they allow normal growth and reproduction of the insect
while expressing specific gene products of interest.

                                     In our laboratory, a selectable
                                     genetic marker coding for resistance
   						 to the antibiotic thiostrepton was
                                     expressed by transgenic symbionts
 [Figure 2.] Shuttle plasmid         within the gut of insects colonized
 for genetic transformation          experimentally (37). The insects could
 of Rhodococcus rhodnii.             be fed on blood that contained
                                     thiostrepton, and the bacteria
                                     survived and persisted throughout the
insect's development to adulthood. Furthermore, we showed that when
thiostrepton was omitted from the blood, the symbionts maintained
resistance to the antibiotic, which indicates that the plasmid was stable
in its bacterial host. By sterilizing the surface of the eggs with a
topical iodine solution and colonizing the resulting aposymbiotic (sterile)
insects with genetically modified symbionts (delivered to the insects
orally), we developed a line of paratransgenic arthropods in which
individual insects were refractory to infection with T. cruzi (38).
Refractoriness was conferred after the antimicrobial peptide L-Cecropin A
was expressed and secreted. The gene for this peptide was contained in a
shuttle plasmid expression vector used to transform R. rhodnii, which
subsequently expressed and secreted the gene product within the gut of
experimentally colonized insects (38). Cecropin A, a 38 amino acid peptide,
belongs to a family of small channel-forming peptides with potent
antimicrobial activity; these peptides insert into biologic membranes,
forming channels that ultimately lead to cell lysis and death (39-41). This
family of peptides has been isolated from several insect and vertebrate
tissues, which they defend against bacterial pathogens (41). Cecropin A has
strong lytic activity against T. cruzi but negligible deleterious effects
on R. rhodnii or gut tissues of R. prolixus. Laboratory colonies of R.
prolixus that carried genetically altered R. rhodnii transformed to express
mature Cecropin A were completely refractory to infection with T. cruzi
strain DM28 in approximately 70% of the insects. In the remaining insects,
numbers of T. cruzi were reduced to less than 1% of the numbers seen in
control R. prolixus, which carried untransformed R. rhodnii.

                                     These studies demonstrate that
                                     symbiotic bacteria of
                                     disease-transmitting triatomine bugs
                                     can be genetically modified to express
                                     biologically active molecules and then
 	 					 can be reintroduced into the host
                                     insect, expressing the desired
                                     phenotype (Figure 3). The genetically
                                     altered R. rhodnii allowed normal
                                     development and sexual maturation of
 						 the host insect.

 [Figure 3.] Symbionts can           Delivery Mechanisms/Field Applications
 be genetically altered and used 
 to replace native symbionts,        For the bacterial symbiont approach to
 resulting in insects that can no    be used in a field intervention
 longer transmit disease.            program, a mechanism must be developed
 (Illustration courtesy of Mark Q.   for spreading the genetically altered
 Benedict).                          bacteria through an insect population.
                                     The delivery system must allow
                                     dispersal of recombinant genetic
material without adverse environmental effects. Using bacteria that have
specialized symbiotic associations with specific insect hosts to spread
transgenes greatly reduces the chance of unwanted gene spread; the natural
insect-symbiont association can be used for this purpose. In R. prolixus,
early instar nymphs acquire the symbiont R. rhodnii by coprophagy. When
they first emerge, instar nymphs are transiently aposymbiotic; they pick up
the required bacteria by probing the eggshell or fecal droplets of other
bugs (Figure 1). Scientists have observed that triatomine bugs actively
probe small dots of black ink on paper, apparently because they resemble
the black fecal droplets shed by triatomine bugs after digestion of the
bloodmeal. When live bacterial cultures and a small amount of India ink are
added to an inert carrier, a formulation of the genetically modified
bacteria is produced that resembles bug feces and can be ingested by
immature insects; in this way, modified bacteria can be established
uniformly and effectively in laboratory colonies of R. prolixus (37,38).
Studies to test this approach in the laboratory use a design that simulates
a field application. Wooden frames composed of housing materials common in
the rural tropics (primarily mud and thatch) are treated with the bacterial
formulation. Field-collected R. prolixus are added to these frames, which
are then enclosed in large plexiglass containers. The progeny of the
field-collected insects are examined for colonization with the modified
symbiont, ability of the symbiont to compete with native symbionts
established in the field-collected specimens, and expression of the desired
transmission phenotype.

A possible strategy for using vector-symbiont intervention for the control
of Chagas disease transmission would require that in disease-endemic areas,
individual houses likely to become infested with triatomines be treated
with the bacterial formulation, either when they are new and uncolonized or
after insecticide treatment that kills any resident insects, especially in
corners and cracks where they are most likely to hide. In homes treated
with the bacterial formulation, which likely contain wild-type symbionts,
the genetically modified symbionts could be given a competitive advantage
of being applied in greater concentrations than the native symbionts. As
triatomine bugs from adjacent untreated areas reinfest the house, they
would lay their eggs, which would hatch within days. The digestive tract of
the new immature bugs would be colonized through coprophagy with
genetically altered symbionts, which would keep them from subsequently
becoming infected with the Chagas disease trypanosome. These progeny would
then amplify and disperse the altered symbionts through natural coprophagic
routes. Since triatomine bugs involved in domiciliary transmission must
invade and become established in the homes, peridomestic or sylvatic
habitats would not need to be treated to affect domestic transmission.
Because of the labor and expense involved in repeated insecticide
treatment, reinfestation is a potential major obstacle in Chagas disease
control. Vector-symbiont intervention could play an important role in an
integrated control approach that used a combination of insecticidal and
molecular genetic interventions.

Bacterial Symbionts in Tsetse

Bacterial symbionts have also been evaluated  
in other insect disease vectors. The tsetse
vectors of African sleeping                   
sickness(trypanosomiasis) harbor as many as
three distinct populations of bacterial       [Figure 4.] The tsetse
flora (33,42-45). Like triatomine bugs,       secondary symbiont GP01
tsetse are obligate bloodfeeders, and at      growing intracellularly and
least one population of bacteria (found in    extracellularly in culture.
uterine secretory cells) is presumed to be
nutritional mutualist symbionts. The
primary, or P-symbionts, are highly specialized intracellular bacteria
apparently essential for the flies' survival (33,45). These bacteria have
not been cultured or transformed. The secondary, or S-symbionts, comprise
another population of gram-negative bacteria found in various tissues of
tsetse, including the salivary glands, where they reside in large numbers,
especially in older flies (S. Aksoy, pers. comm.). S-symbionts can be
isolated from hemolymph and cultivated in vitro, where they have been shown
to grow both intra- and extracellularly (Figure 4) (46-48). A potential
transformation system has been developed for these bacteria with the
recombinant plasmid pSUP204 (47). This DNA vector contains the broad host
range replicon oriV, derived from a Pseudomonas aeruginosa plasmid,
RSF1010, and ligated into the E. coli cloning vector pBR325 (49,50). Recent
studies indicate that genetically transformed tsetse S-symbionts can be
microinjected into recipient flies and express a reporter gene (S. Aksoy,
pers. comm.). This area of research will likely yield new approaches for
controlling tsetse transmission of trypanosomes.

Intracellular Insect Reproductive System AgentsWolbachia

Obligate intracellular bacteria found in many species of arthropods
(51,52), the Wolbachia are maternally transmitted from parent to offspring
and are often involved in a variety of reproductive anomalies, such as
cytoplasmic incompatibility (reproductive incompatibility due to maternal,
nongenetic factors), sex ratio determination and distortions, and
parthenogenesis (53-56). Although extremely fastidious, these microbes
could be used in the transgenic alteration of arthropod vectors in two ways
(32): 1) direct transformation and subsequent expression of a gene product
by the Wolbachia in the arthropod, and 2) use of Wolbachia-induced
cytoplasmic incompatibility to drive a second, maternally inherited factor
into a population of vectors. The first approach entails the use of either
episomal plasmids or DNA integration vectors for transformation of the
Wolbachia. Although theoretically possible, such a transformation system
(i.e., a suitable DNA vector and methods for introducing the DNA and
selecting for transformants) does not now exist. Recent successful genetic
transformation of rickettsial agents, however, suggests that this
limitation may soon be overcome (57).

Since Wolbachia are frequently observed in reproductive tissues of
arthropods (Figure 5), one potential approach that they might be used to
disrupt transmission of a pathogen is suggested by studies performed using
a viral transduction system in the mosquito Aedes aegypti (14). In these
experiments, antisense DNA sequences corresponding to a membrane protein of
the dengue type 2 virus were expressed somatically by using a recombinant
Sindbis virus vector. Mosquitoes that were coinfected with the two viruses
were shown not to be able to transmit dengue virus. Similar work has been
done with Aedes triseriatus mosquitoes, which are potential vectors of the
LaCrosse virus (58). These types of experiments could be done by using
genetically modified Wolbachia, with the goal of blocking transovarial
transmission of an arboviral agent, such as La Crosse encephalitis virus or
Rift Valley Fever virus, which is dependent on vertical transmission from
the adult mosquito to its progeny for maintenance of the virus in nature.
Genetically transformed Wolbachia could be utilized in a manner similar to
that of viral transducing agents to express antisense DNA sequences that
interfere with replication of transovarially transmitted virus pathogen.

                                     The disruption of transovarial
                                     transmission is an application that
                                     could potentially be adapted for use
                                     in a number of species of mosquitoes,
                                     ticks, and mites in which transovarial
 				             transmission of pathogens is
                                     important. Very recent studies report
                                     the distribution of Wolbachia
  [Figure 5.] Wolbachia-like         throughout somatic and germ-line
  would not be limited simply to     tissues in various insects. These
  organisms in insect reproductive   observations suggest that potential
  tissues.                           transmission-blocking applications
                                     using genetically modified Wolbachia
                                     transovarially transmitted agents but
                                     potentially applicable to microbial
pathogens that reside and/or replicate in other insect tissues as well,
such as the hemolymph or salivary glands (59).

Alternatively, Wolbachia could be used to render a population of arthropod
disease vectors incapable of transmitting a disease agent:
Wolbachia-mediated cytoplasmic incompatibility could spread a second
maternally inherited gene into the vector population. In its simplest form
(Figure 6), cytoplasmic incompatibility results when a Wolbachia-negative
female mates with a Wolbachia-positive male; no offspring are produced from
such an incompatible cross. Since the reciprocal cross between an infected
female and an uninfected male results in Wolbachia-infected progeny, the
net effect in matings between the two strains, other things being equal, is
that Wolbachia-infected progeny will be more prevalent than Wolbachia-free
progeny. This phenomenon has been observed in natural populations of
Wolbachia-infected and -uninfected Drosophila simulans in California,
demonstrating the rapid spread of Wolbachia and the corresponding
cytoplasmic incompatibility phenotype across broad geographic regions (61).
How this phenomenon might be utilized has been discussed in great detail
(32,60).

As Wolbachia spread through the population by 
cytoplasmic incompatibility, other maternally
inherited organisms (or organelles) in the    
Wolbachia-infected strains are transmitted.
"Cage studies," using mosquitoes with         [Figure 6.] Wolbachia-mediated
different mitochondrial haplotypes, have      cytoplasmic incompatibility.
found that haplotype frequencies changed so
quickly that within two generations, one
haplotype was completely replaced by the other as a result of
Wolbachia-driven cytoplasmic incompatibility (62).

In an actual scheme for genetic modification of a vector population, the
gene blocking pathogen transmission could be carried by a second,
nonpathogenic species of bacteria, such as the tsetse S-symbiont (32,,63)
or a rickettsialike commensal that is maternally inherited (64), or even
possibly an organelle such the mitochondrion. The transformed agent could
be introduced into a Wolbachia-infected laboratory colony, and the females
could be released. Over time, because of the driving mechanism of
cytoplasmic incompatibility, a gene product critical for transmission of an
arthropod-borne disease agent could be blocked by a second product
expressed and secreted by the genetically transformed, maternally inherited
microorganism, resulting in the modification of the vector population's
capacity to transmit the disease agent.

Limitations

The application of these methods is limited primarily by the occurrence of
suitable microorganisms in specific arthropod vectors. In general, many
arthropods (e.g., ticks, mites, triatomine bugs, bed bugs, lice, some
species of fleas, and tsetse) that feed on single food sources throughout
their entire developmental cycle contain microbial symbionts that produce
nutritional supplements lacking in the restricted diet (so called
nutritional mutualists) (33,65,66). Mosquitoes and many other dipterans,
however, do not fall into this category because they do not feed on blood
strictly as a source of nutrition for basal metabolism; rather, the females
alone feed on blood, which is used directly to produce progeny. Although
other symbionts may be present in mosquitoes, nutritional mutualists are
altogether lacking.

Reported from numerous and diverse species of arthropods (51), Wolbachia
may occur naturally in more than 15% of all insect species (67). Wolbachia
have been transferred in the laboratory from one arthropod species to
another, successfully conferring the cytoplasmic incompatibility phenotype
to the recipient species (68,69). Other "passenger" microorganisms, such as
tsetse S-symbionts (33,42) and Rickettsia-like organisms (70,71), are
common in arthropods, and although the nature of their symbiotic
relationship with the arthropod host is often unclear, their consistent
association suggests their potential as vehicles for expressing a transgene
in the arthropod.

Other limitations to the use of arthropod bacteria include the development
of appropriate transformation methods and reagents. Because many
nonpathogenic microorganisms in arthropods have been poorly studied and
characterized, accurate identification of the agent is a prerequisite to
genetic transformation studies. These microorganisms can be characterized
either through traditional bacteriologic characterization or by a
polymerase chain reactionbased approach that uses common eubacterial
primers to amplify the 16S rRNA locus that can be sequenced and used for
identifying the microbe (72,73). DNA vectors and transformation methods
have been developed for diverse groups of both gram-positive and -negative
microorganisms; these tools and approaches can often be adapted for use
with a bacterial symbiont or passenger. We have efficiently transformed
several species of coryneform bacteria, common agents of various triatomine
bugs that transmit Chagas disease, by using transformation vectors and
regulatory sequences developed for use in Mycobacterium spp.

Release of Paratransgenic Insects: Regulations and Safety

In addition to questions regarding science and efficacy, numerous safety
and regulatory concerns pertaining to laboratory and field research with
genetically modified arthropod vectors of human disease must be addressed.
These concerns include potential environmental and ecologic hazards
associated with release; potential public health risks; and overall public
perception, which can determine the future of entire programs (74,75).

In the United States, federal guidelines and regulations address a broad
range of issues pertaining to research, transport, and release of
microorganisms, plants, and insects. The U.S. Department of Agriculture
Animal and Plant Inspection Service (APHIS) regulates the release of
transgenic arthropods of agricultural importance. None of these guidelines
or regulations, however, directly address genetically modified arthropod
vectors of human disease agents. Nevertheless, the work that has been done
and the policy that is in place set a precedent.

The basic principles of containment regarding research with transgenic and
paratransgenic insect disease vectors are similar to those regarding human
pathogens and biologic insecticide agents (insects and microorganisms).
Guidelines that discuss established standards for work with human disease
agents, typically classified as biosafety level (BSL)-2, BSL-3, or BSL-4,
on the basis of the assessment of virulence and transmissibility have been
published (76), and a permitting process oversees the import and transport
of these agents into and throughout the United States. The Environmental
Protection Agency (EPA) and USDA-APHIS share oversight with regard to the
development and use of biologic insecticides (77). USDA-APHIS also
regulates the use of insects and arthropods that are predators of insect
pests. The containment procedures and facilities associated with the use of
these organisms in new geographic regions in the United States might be
adapted as the minimum standards for work with transgenic arthropods (75).

With respect to regulation of the release of transgenic arthropods of
agricultural importance and risk assessment, USDA-APHIS has formed an
interagency "virtual" committee charged with the responsibility of
evaluating proposals to release transgenic arthropods that could affect
plants and animals (excluding humans); efforts are under way to form an
interagency committee with the authority to regulate the release of
arthropods that could affect humans. Information on the program that APHIS
has assembled, including details regarding permits and current applications
that have been submitted under several plant pest statutes, can be accessed
at http://www.aphis.usda.gov/biotech/arthropod/; risk assessment
information is also available at this site.

Assessment of the environmental risks of releasing transgenic and
paratransgenic arthropods shares principles with environmental impact
analysis of biologic insecticides (78). The same arguments used to promote
the potential benefits of recombinant organisms are equally effectively
used to argue the potential hazards (79). To assess risk, Hoy (74)
addresses four basic concerns: 1) the attributes of the unmodified
organism; 2) attributes of the genetic alteration, i.e., only of the gene
transferred; 3) phenotype of the modified organism compared with the
unmodified organism; and 4) attributes of the accessible environment. These
concerns can guide formulation of research plans to assess environmental
risks associated with the release of transgenic arthropods.

Transgenic and paratransgenic research with arthropods raises several
public health concerns. Because the arthropods may transmit human disease
agents, ethical issues must be considered. For example, consider that a
transgenic or paratransgenic insect is released under the assumption that
its ability to transmit disease has been demonstrated to be significantly
decreased or eliminated, as determined by laboratory studies in carefully
defined conditions. Then suppose that when pilot field studies are
conducted, for some reason, it is shown that under true field conditions
the ability to transmit disease is not reduced as much as had been
anticipated on the basis of the controlled laboratory studies. The result
is that the human population present where the release takes place could
actually be at increased risk for disease due to the release of large
numbers of competent or partially competent vectors. The assessment of this
type of risk is extremely important, as is the related human subjects issue
of informed consent and how it would be obtained in a study where the
population that is potentially at risk cannot be accurately determined.

Public perception, frequently not considered until a potential product is
ready for field testing, is extremely important (e.g., public response to
genetic control studies in India in the 1970s, including the release of
sterile mosquitoes [80] and to the ongoing controversy surrounding the
labeling of foods that are, or contain, the products of genetic engineering
in agriculture [81-83]). The most effective biotechnology programs are
those in which the public is informed and brought into discussions at the
earliest possible time (75,84).

The efficacy of a biologic agent to prevent or reduce the impact of a
disease is of paramount importance, yet most of this information can be
obtained definitively only after the method has been tested on a large
scale and under different environmental conditions. On the other hand,
information on safety, as well as on the behavior of the agent in the
field, is required before we know how well it works and what other effects
it has on the environment. Thus, the scientist developing a new agent must
make an honest, imaginative leap into the future and try to predict any
possible dangerous consequencesthe responsibility for risk assessment must
be shouldered by the scientist, together with the appropriate regulatory
agencies. Because the safety questions are numerous, those of the greatest
potential significance, both in the short term and in the long term, should
be examined first. The risks associated with the intervention must be
assessed against the risk for disease, the risk to health and the
environment from pesticides, and the risk of doing nothing.

Conclusions

Decreasing effectiveness of controlling diseases transmitted by arthropods
has led to evaluation of new control strategies, including the use of
transgenic methods. A strategy that relies on producing paratransgenic
arthropods has been applied with initial success to the triatomine bugs
that transmit Chagas disease, resulting in vector strains incapable of
being infected with the pathogenic agent T. cruzi. This approach may also
prove feasible for a number of other vector-borne diseases, using either
nutritional symbionts of arthropods that feed exclusively on blood,
Wolbachia that have been successfully transferred between species in the
laboratory, or other microbial passengers consistently associated with
their arthropod host. As the tools and methods that allow broader
applications of this approach are developed, and actual products arrive at
the point of field testing, permitting and regulation will be required.

The regulatory process must begin with the individual investigators, who
must thoroughly evaluate the product being developed and work with
regulatory agencies and others to ensure that if and when genetically
modified organisms are used for the control of insect borne diseases, they
will be both safe and efficacious.

Acknowledgments

      The authors thank Drs. Orrey P. Young, Suzanne Binder, and Mark
Benedict, for their critical review of this manuscript.

      Dr. Beard is research entomologist and chief, Vector Biology
Activity, Division of Parasitic Diseases, 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: Charles B. Beard, Centers for Disease
Control and Prevention, 1600 Clifton Road, Mail Stop F12, Atlanta, GA
30333, USA; fax: 770-488-7469; e-mail: cbb0@cdc.gov.

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Synopses

Genetic Epidemiology of Infectious Diseases in Humans: Design of
Population-Based Studies

Laurent Abel* and Alain J. Dessein
*Institut National de la Sant et de la Recherche Mdicale Unit 436, Paris,
France; and Institut National de la Sant et de la Recherche Mdicale Unit
399, Marseille, France

---------------------------------------------------------------------------
      The spread and clinical manifestations of an infection in human
      populations depend on a variety of factors, among them host
      genetics. Familial linkage studies used in genetic epidemiology
      to identify host genes test for nonrandom segregation of a
      trait with a few candidate chromosomal regions or any regions
      in the genome (genomewide search). When a clear major gene
      model can be inferred and reliable epidemiologic information is
      collected (e.g., in schistosomiasis), parametric linkage
      studies are used. When the genetic model cannot be defined
      (e.g., in leprosy and malaria), nonparametric linkage studies
      (e.g., sibling-pair studies) are recommended. Once evidence of
      linkage is obtained, the gene can be identified by
      polymorphisms strongly associated with the trait. When the
      tested polymorphism is in strong linkage disequilibrium with
      the disease allele or is the disease allele itself (e.g., in
      HIV infection and malaria), association studies can directly
      identify the disease gene. Finally, the role of the detected
      polymorphism in causing the trait is validated by functional
      studies.

The profound influence of the host's genetic makeup on resistance to
infections has been established in numerous animal studies (1,2) in which
disease phenotypes, environmental factors, and crosses can be controlled.
Furthermore, recent developments (e.g., use of gene knockout or mutant and
transgenic mice) allow genetic analysis of complex traits involved in
susceptibility or resistance to infectious pathogens (2,3). As a result of
these new developments, the Lsh/Ity/Bcg gene was isolated on mouse
chromosome 1, which controls innate early susceptibility to several
Mycobacterium species, as well as other intracellular pathogens (e.g.,
Salmonella Typhimurium, Leishmania donovani) (2,4), and was further
identified and designated natural resistance-associated macrophage protein
1 (Nramp1) (5). Involvement of a gene in an experimental infection does not
imply that differences in susceptibility or resistance to that infection in
human populations can be accounted for by polymorphisms in the human
homologue of this gene. Genetic epidemiology studies (6,7) combine
epidemiologic and genetic information to identify the genes that influence
substantially the expression of human complex phenotypes, such as
infectious disease-related traits. Epidemiologic information includes
measured risk factors that could influence the trait under study (e.g.,
contamination by the infectious agent, age). Genetic information is derived
from familial relationships between study participants (collection of
families) or from the typing of genetic markers. Recent maps of the human
genome established on the basis of highly polymorphic markers (8) are a
fundamental tool for studies involving genetic markers, and two strategies
can be used in this context. The first, the candidate gene method, is the
typing of a few markers in a limited number of chromosomal regions
containing genes related to the phenotype under study. The second is a
random search along the whole genome (genomewide search) for chromosomal
regions that could be involved in the control of the phenotype.

The genetic epidemiology of human infectious diseases differs from the
genetic study of other complex phenotypes in three ways. 1) Environmental
factors influencing the risk for infection are generally known and when
accurately measured, can be included in the analysis; 2) Choice of
candidate genes is strongly determined by the gene's function and response
to the studied pathogen or by mouse-human chromosome tests that exploit the
identification of murine resistance loci; and 3) Major genes involved in
the response to a given pathogen can be identified by characterizing
phenotypic response to pathogen exposure, such as clinical response,
biologic response (intensity of infection), and immunologic response
(levels of antibodies or cytokines). The role of genetic factors in the
control of these phenotypic responses is generally suggested by twin
studies, by strong ethnic differences, or by the great variability of
individual phenotypes within their familial aggregation. Specific
statistical methods are used to identify these genetic factors and to
distinguish them from environmental factors causing the familial
resemblance. All these statistical methods search for one or more genes
that influence the studied phenotype and are classically divided into
parametric and nonparametric. Parametric, or model-based, methods
(segregation analysis and linkage analysis by the classical lod-score
method) require defining the model and specifying the relationship between
the phenotype and factors (mainly a putative gene and environmental
covariates) that may influence its expression. Nonparametric or model-free
methods (nonparametric linkage analysis and association studies) study the
genetic factors influencing a phenotype without specifying the model. Each
method has advantages and disadvantages; however, the two methods
complement each other. The choice of a design for a particular study
depends on several factors related to the phenotype (e.g., nature,
frequency), population, accurate measurement of environmental factors, and
known genetic background. Both methods have led to successful gene
localizations and identifications in the analysis of several infectious
disease phenotypes (9,10).

Parametric (Model-Based) Studies

Parametric studies require explicit specification of the model, i.e., the
definition of the relationship between the observed phenotype and the
putative genotype. In a simple monogenic disease due to a diallelic gene
(D,d), the model is specified by the frequency of the deleterious allele (D
for example) and the three probabilities for a person to have the disease,
given the presence of genotype DD, Dd, or dd (penetrances). For complex
instances, such as susceptibility/resistance, the susceptibility (or the
resistance) depends not only on a putative genotype but also on
environmental factors that may influence exposure. In such cases, the
phenotype/genotype model includes, in addition to the frequency of the
deleterious allele, all the parameters that describe and quantify the
relationship between susceptibility and the relevant genetic and
environmental factors. This relationship can be mathematically expressed in
several ways, most recently regression methods that define model parameters
in terms of regression coefficients. Furthermore, regression methods could
be used to analyze binary (11) as well as quantitative (12) phenotypes. In
quantitative phenotypes, the effect of a genotype is defined in terms of
three different phenotypic means depending on the genotypes of the study
participants. Parametric methods are based on two kinds of complementary
analyses, segregation analysis and linkage analysis by the classical
lod-score method (13). Both require epidemiologic information (i.e., the
measure of the phenotype and of all relevant environmental factors) for
each family member. Linkage analysis needs the typing of genetic markers.

Parametric Segregation Analysis

Segregation analysis is the first step in determining from family data how
a given phenotype was inherited. Familial aggregation of infection-related
phenotypes can result from genetic relationships, shared environment, and
cultural habits. The goal of segregation analysis is to discriminate
between these factors, primarily to test for the existence of a single
gene, called a major gene. The major gene is not the only gene involved in
the expression of the phenotype; rather, of all involved genes, this one
has an effect important enough to distinguish it from the others. For a
binary clinical phenotype (affected/unaffected by the disease), this effect
can be expressed in terms of relative risks, e.g., the ratio of the
probability for being infected given a DD genotype to the probability of
being infected given a dd genotype. For a quantitative phenotype, this
effect is measured by the proportion of the phenotypic variance explained
by the major gene (heritability due to the gene). Primarily, segregation
analysis uses maximum likelihood methods to test whether the observed
familial distributions of the phenotype fit the distributions expected
under different hypotheses of familial transmission (in particular the
segregation of a major gene). When evidence indicates a major gene,
segregation analysis estimates the measurements for the phenotype/genotype
model, which are required for parametric linkage analysis.

Parametric Linkage Analysis

Linkage analysis by the classical lod-score method (13) confirms and
locates the gene, detected by segregation analysis (denoted as the
phenotype locus). Linkage analysis tests whether, in families, the
phenotype locus is transmitted with genetic markers of known chromosomal
location. The lod score is a likelihood ratio testing the hypothesis of
linkage (against the hypothesis of no linkage) for different genetic
distances (or recombination fractions) between the phenotype locus and the
marker locus (14). Classically, two conclusions can be reached with a
lod-score analysis: 1) linkage between the two loci when the lod score is
above a given threshold, and 2) exclusion of linkage between the two loci
when the lod score is below a given threshold. Linkage with the phenotype
locus can be tested marker by marker (two-point analysis) or by a set of
linked markers (multipoint analysis). In linkage, as in segregation
analysis, all inferences for individual genotypes at the phenotype locus
are made from individual phenotypes and the specified phenotype/genotype
model; the lod-score method is most powerful when this model is well
defined. A mispecification of the phenotype/genotype model, however, can
lead to both inability to detect linkage (and therefore to false exclusion
of the region containing the phenotype locus) and to a bias in the
recombination fraction estimate (i.e., the genetic distance) between the
phenotype locus and the marker locus (15). Nevertheless, such a
mispecification does not affect the robustness of the method; i.e., it does
not lead to false conclusions in favor of linkage, as long as only one
phenotype/genotype model is tested. Correction for multiple testing should
accompany the use of several phenotype/genotype models. Similar problems
occur when several markers are tested, and guidelines have been proposed to
adapt lod-score thresholds to the context of genomewide search (16).
Another problem arises when marker data are missing for some family
members. In this case, linkage analysis also depends on marker allele
frequencies; mispecification of these frequencies can affect both the power
and robustness of the method. Multiple marker testing and mispecification
of marker allele frequencies are also common problems to the nonparametric
methods.

Model-Based Studies and Infectious Diseases

Leprosy Studies

Several segregation analyses have been performed in infectious diseases;
some suggest that a recessive major gene may play a role in leprosy
subtypes (lepromatous or nonlepromatous) (17-19). A recessive major gene
was also found to influence leprosy regardless of the clinical defined
subtype, in pedigrees of large families from a small Caribbean island (17);
the frequency of the deleterious allele was estimated to be 0.3 (9% of
homozygous persons predisposed to leprosy); by age 60, the penetrance was
approximately 0.6 for predisposed homozygous, whereas it remained below
0.02 for others. Lod-score analysis could not find any linkage between this
leprosy susceptibility locus and five markers (including HLA) that were
typed in this population (20).

Malaria Studies

In malaria, segregation analyses have focused on a quantitative phenotype
measuring the intensity of infection, i.e., parasitemia levels. Although
one study showed the role of a recessive major gene controlling levels of
parasitemia (21), two subsequent studies found evidence of a more complex
genetic mechanism (22,23). The discrepancies in these results can be
explained by several factors related to the host, the parasite, and
mosquito transmission. However, all studies showed correlations between
siblings and between age and infection (children becoming more often
infected than adults). Further genetic analyses such as sibling-pair
(sib-pair) study designs should focus on infection in young children.

Schistosomiasis Studies

Model-based studies have been particularly successful in finding
susceptibility genes in schistosomiasis. Several reports indicated that
infection intensity was largely determined by the susceptibility/resistance
of infected persons (24). In a Brazilian population, segregation analysis
showed that the intensity of infection by Schistosoma mansoni was
controlled by a major gene (25). This gene, SM1, accounts for 66% of the
infection intensity variance that remains after other covariate effects
(water contact levels, age, gender) have been taken into account. Under
this major gene model, approximately 3% of the population is homozygous and
predisposed to very high infection levels, 68% is homozygous resistant, and
29% is heterozygous with intermediate levels of resistance (Figure 1).
Parametric linkage analysis using the model estimated from segregation
analysis was used to locate the gene. A genomewide search was carried out,
and SM1 was mapped to human chromosome 5q31-q33, a genetic region that
contains several genes encoding molecules that control T-lymphocyte
differentiation (26). More recently, a study in a Senegalese population
confirmed the presence of a locus influencing S. mansoni infection levels
on chromosome 5q31-q33 (27). Furthermore, this region has been linked with
loci related to immunoglobulin E (IgE) and eosinophilia production, i.e., a
locus regulating IgE levels (28,29), a locus controlling bronchial
hyperresponsiveness in asthma (30), and a locus involved in familial
hypereosinophilia (31). This genetic localization, together with
observations that human resistance to schistosomiasis is regulated by
lymphokines characteristic of Th2 subsets (32) and that resistant
homozygotes mount a Th0/2 response while susceptible homozygotes exhibit a
Th0/1 response against schistosomes (V. Rodrigues, A. Dessein, unpub.
data), argues strongly that differences in human susceptibility to
schistosomiasis are influenced by polymorphisms in a gene controlling
T-lymphocyte subset differentiation. In this regard, a segregation analysis
showed that interleukin 5 (IL-5) levels are also under the control of a
major gene in the same Brazilian population used in the study on infection
intensity(33), raising the possibility that IL-5 might play a critical role
in resistance, a view consistent with the known role of IL-5 in the defense
against schistosome infections.

 							  Another trait of interest in
                                            schistosomiasis is the
 [Figure 1.] Distribution of the adjusted   phenotype of severe hepatic
 standardized infection intensities by      fibrosis due to S. mansoni
 Schistosoma mansoni predicted by the major infection for which the role of
 gene model obtained from segregation       genetic factors has been
 analysis and used for linkage analysis.    suggested. Segregation analysis
 The frequency of allele A predisposing to  conducted in a Sudanese village
 high infection levels was estimated at     found evidence of major gene
 0.16 (70% of aa, 27% of Aa, and 3% of AA   involvement in severe hepatic
 persons), and the three means              periportal fibrosis (A.
 (corresponding to vertical lines) were     Dessein, L. Abel, unpub. data).
 -0.43, 0.78, and 3.96 for aa, Aa, and AA   Whether this gene and SM1 are
 persons, respectively, with a residual     one and the same is under
 variance equal to 0.33.                    investigation.

Nonparametric (Model-Free)
Studies

Nonparametric or model-free studies (nonparametric linkage analysis and
association studies) examine the genetic factors influencing a phenotype
without specifying the phenotype/genotype model. These studies are strongly
recommended when little is known about the relationship between the
phenotype and a putative gene as in the study of complex traits (e.g.,
infectious disease-related traits) when either no segregation analysis has
been performed or no clear major gene model can be inferred from
segregation analysis. Nonparametric studies test whether or not the alleles
of a given marker are distributed at random in persons having a certain
phenotypic resemblance. Nonparametric linkage analyses study the
distribution of marker alleles inherited from a same ancestor, i.e.,
alleles identical by descent (IBD), in persons from the same family (e.g.,
siblings), whereas association studies examine the distribution of a given
marker allele, e.g., HLA-DR2, in persons not from the same family.

Nonparametric Linkage Analysis

The most commonly used nonparametric linkage analysis is the sib-pair
method. Two siblings can share 0, 1, or 2 parental IBD alleles of any
locus, and the respective proportions of this sharing under random
segregation are simply 0.25, 0.5, and 0.25 (Figure 2). When the phenotype
under study is a clinical disease (affected/unaffected), the method tests
whether affected sib-pairs share more parental alleles than expected under
random segregation. This excess allele sharing can be tested by a simple
chi-square, in particular when all parental marker data are known. Maximum
likelihood methods have also been developed to analyze data from affected
sib-pairs data, such as the maximum likelihood score (34) and a maximum
likelihood binomial approach (35), and can lead to more powerful tests.
When the phenotypic response under study is quantitative, the method tests
whether siblings with close phenotype values share more IBD alleles than
siblings with more distant values. This is the basis of the classical
approach proposed by Haseman and Elston (36), which regresses the squared
difference of the sib-pair phenotypic values on the expected proportion of
alleles shared IBD by the sib-pair. Many recent studies have used other
methods not detailed here (37-39). Some of these methods are implemented in
popular packages, such as MAPMAKER/SIBS (40), which also allow multipoint
analysis of sib-pair data. Sib-pair methods have the same problems as
parametric linkage analysis with respect to missing parental marker data
and testing with multiple markers; in particular, the number of comparisons
made influences the significance levels of the tests, and suspected linkage
should be confirmed by replication studies. However, affected sib-pair
methods have been effective for several diseases, e.g., insulin-dependent
diabetes mellitus (41,42), in genomewide searches for human susceptibility
genes in a multifactorial phenotype.

Leprosy Studies                      

Sib-pair methods in infectious      [Figure 2.] Principle of sib-pair
diseases have focused on candidate  analysis. Two siblings can share 0,
regions and have not yet resulted   1, or 2 parental marker alleles
in published genome scans. In       identical by descent (IBD) at any
leprosy studies using the HLA       locus with respective probabilities
complex, sib-pair analyses have     0.25, 0.5, and 0.25 under random
shown a nonrandom segregation of    segregation.
parental HLA haplotypes in sets of
children with tuberculoid leprosy
and in siblings with lepromatous leprosy, respectively (18,43,44). However,
the observed random segregation of HLA haplotypes in all leprosy patients
and in healthy siblings in families with multiple cases of leprosy argued
against any involvement of HLA-linked factors in susceptibility to leprosy
(44,45). The human gene NRAMP1 (46), homologue of the mouse gene Nramp1,
has provided an excellent candidate gene for the study of susceptibility to
leprosy. A recent sib-pair study in Vietnam has found linkage between
leprosy and NRAMP1 haplotypes consisting of six intragenic variants of
NRAMP1 and four polymorphic flanking markers (47) and provided the first
evidence that NRAMP1 could be a susceptibility locus for leprosy.
Furthermore, this study, combined with segregation analysis performed in
the same population (18), suggested genetic heterogeneity according to the
ethnic origin of the families (Vietnamese or Chinese), which may explain,
at least in part, the results of two previous reports that showed no
association between leprosy and distal chromosome 2q where NRAMP1 is
located (48,49). Overall, these studies suggest genetic control on at least
two levels: a first dependent on nonHLA-linked factors, among which NRAMP1
could play a role, and a second influenced by HLA-linked genes.

Malaria Studies

Two sib-pair studies focusing on candidate genes have been reported in
malaria-related phenotypes. In one (50), nonrandom segregation of the MHC
region was found in pairs of dizygous twins with mild clinical malaria. In
another (51), the 5q31-q33 region, previously shown to be linked to S.
mansoni infection levels (26), may be involved in the control of
parasitemia due to Plasmodium falciparum, although the sample size was too
small for definitive conclusion; larger studies are ongoing.

Mycobacterium Studies

The recent demonstration that mutations in the interferon [gamma] receptor 1
(IFN[gamma]R1) gene cause disseminated infection due to weakly pathogenic
mycobacteria (52,53) was first based on homozygosity mapping (54), a
nonparametric linkage method, which locates a rare recessive mutation in
consanguineous families by searching for chromosomal regions for which all
affected family members are homozygous IBD; i.e., they have received two
copies of the same ancestral mutation. In consanguineous infected children
from two families, two groups located the genetic defect on chromosome
region 6q22-q23 and identified mutations in the IFN[gamma]R1 gene leading to 
the absence of expression of the receptor at the cell surface (52,53). In 
vitro experiments established the causative relationship between the presence 
of two mutated IFN[gamma]R1 alleles and impaired response to IFN by the cells 
of these patients (55). Although inherited IFN[gamma]R1 deficiency was found in
additional families, IFN[gamma]R1 mutations were not found in other families 
with infected patients (J.L. Casanova, pers. comm.), which suggests that other
genetic defects may be involved.

Association Studies

Classic association studies are population-based case-control studies that
compare the frequency of a given allele marker in unrelated persons with
the phenotype and controls without the phenotype (6,7). G is the disease
locus influencing the trait, and M is the marker locus under consideration;
G is assumed to be diallelic (D,d) with D being the deleterious allele, and
M has several alleles (M(sub 1), M(sub 1), ..., M(sub n)). Association 
studies examine the role of a particular allele of M. As an example, 
M(sub 1)is said to be associated with the disease under study if it is found 
at a significantly higher or lower frequency in case-patients than in controls 
by a simple 2 x 2 contingency table. The simplest explanation for the association 
is that allele M(sub 1)is the deleterious allele D itself. Another explanation 
is that M(sub 1)has no direct effect on the phenotype but is in linkage 
disequilibrium with allele D. Linkage disequilibrium means two conditions: 
1) linkage between locus M and locus G (generally close linkage) and 2) 
preferential association of allele M(sub 1)with allele D; i.e., the DM(sub 1)
haplotype is more frequent than expected by the respective frequencies of D and 
M(sub 1)(e.g., many present cases are due to one D allele from an ancestor 
bearing the DM(sub 1) haplotype). Even very close linkage alone (only the first 
condition is fulfilled) does not lead to association, and therefore, the 
absence of association does not exclude linkage. On the basis of these two
explanations, association studies best use the candidate gene approach when
they consider markers that are either within or in close linkage with a
gene that is related to the phenotypic response. A final explanation for
association is the existence of an artifact due to population admixture.
For example, a case-control study conducted in a mixture of two
subpopulations of which one has a higher disease prevalence and a higher M(sub 1)
frequency than the second will show a positive association of allele M(sub 1)
with the disease. To avoid population admixture, family-based association
methods have been developed (56), such as the transmission disequilibrium
test (TDT) (57). The sampling unit in these methods consists of two parents
with an affected child; parental alleles not transmitted to affected
children are used as controls. More specifically, the TDT considers
affected children of parents heterozygous for M(sub 1), e.g., M(sub 1)M(sub 1), 
and simply tests whether these children have received M(sub 1)with a probability 
different from 0.5, the value expected under random segregation (Figure 3). 
The TDT is a very efficient method of detecting the effect of allele M(sub 1)
when M(sub 1)is the deleterious allele D itself (58). Under this hypothesis 
that the tested allele M1 is the deleterious allele, TDT was more powerful 
than even the sib-pair method in the context of a genomewide search involving 
500,000 diallelic polymorphisms (5 polymorphisms per gene for an assumed 100,000
genes) (58). However, in the more common situation where M(sub 1)is different
from D, the power of TDT is highly dependent on the respective frequencies
of M(sub 1)and D and the strength of the linkage disequilibrium between M(sub 1)
and D (59). These results indicate that linkage methods are still useful for
identifying genes involved in infectious diseases, at least until molecular
resources become available for full genomic screening of human genes.

Leprosy Associations

Most reported associations between leprosy and different HLA alleles could
be due to population admixture and statistical problems (multiple testing);
therefore, replication studies are very important. In tuberculoid leprosy,
the most consistent associations were found with HLA-DR2 (43,45). With HLA
molecular typing, a recent study (60) associated Indian tuberculoid leprosy
patients and alleles DRB1*1501, DRB1*1502 (both DR2 alleles), and
DRB1*1404, which are characterized by arginines at position 13 or 70-71.
Lepromatous leprosy was associated with HLA-DR3 in several studies (43,45).
One report (44) analyzed the transmission of the parental DR3 allele to
lepromatous children by a method (similar to TDT) presented several years
later (57).

                                               
							     Malaria Associations
 [Figure 3.] Principle of the transmission     
 disequilibrium test (TDT) for investigating   In malaria, population-based
 association between a disease and allele M1.  association studies have
 The sample consists of x+y families with one  been used to test the
 affected child and two parents. For ease of   hypothesis that certain
 presentation, we assume that only one parent  genetic red cell defects,
 is heterozygous for M(sub 1) (e.g., M(sub 1)  found more frequently in
 M(sub 2)),although the second parent could    malaria-endemic areas than
 be used for  in nonendemic-disease areas,     had a protective effect
 the test if he were himself heterozygous for  against severe malaria
 M(sub 1). There are x affected children       (cerebral malaria, severe 
 who have received allele M1 from their        anemia); the results
 M(sub 1)M(sub 2)parent and y who have         supported the hypothesis       
 received M(sub 2). The TDT statistic is       that persons with certain                 
 simply (x-y)(sup 2)/(x+y), which is           abnormal hemoglobins (61) or                 
 distributed as a chi-square with one          glucose-6-phosphate- 
 degree of freedom.                            deshydrogenase deficiency
                                               (62) had a reduced risk of      
developing severe malaria. More recently, a study in Gambia (63) showed that 
an HLA class I antigen and an HLA class II haplotype were independently 
associated with protection from severe malaria when a two-stage strategy 
was used to avoid the problem of multiple testing. In the same population, 
persons homozygous for a variant of the TNF-[alpha] gene promoter, denoted 
as TNF2, were found to have an increased risk (independent of their HLA alleles) 
for cerebral malaria (64). A recent work showing that TNF2 is a much stronger
transcriptional activator than the more common allele TNF1 65) indicates
that TNF2 affects TNF-[alpha] expression and may be directly responsible for the
reported association of TNF2 with cerebral malaria. These genetic findings
are consistent with immunologic reports showing high TNF-[alpha] blood levels in
cerebral malaria. Although these genetic polymorphisms (genetic defects of
the red cell HLA-TNF polymorphisms) have certainly played a role in
selection among populations exposed to malaria infection (61,63), they
cannot entirely explain the large interindividual variable responses to the
parasite; likely only a minority of genes influencing malaria resistance
have been identified (66). This view is supported by a recent report that a
coding polymorphism in the intercellular adhesion molecule-1 (ICAM-1), a
molecule that affects adherence of infected red blood cells to small vessel
endothelium, is associated with an increased susceptibility to cerebral
malaria (67).

HIV Associations

A major advance in the involvement of host factors in HIV-1 infection came
when infection status (seropositive/seronegative) was associated with the
gene encoding the CC-chemokine receptor 5 (CCR5), the coreceptor of
macrophage-tropic HIV-1 strains (68). Two persons exposed many times to
HIV-1, yet uninfected, were shown to be homozygous for a defective CCR5
allele containing an internal 32 base-pair deletion ([delta]32) (69), and 
several large cohort studies found HIV-1 infected patients not to be 
CCR5[delta]32 homozygous, whereas exposed HIV-1 seronegative persons did 
have the defective allele (70-72). Subsequent reports showed that this 
protection was not complete since some CCR5[delta]32 homozygous persons 
were found to be HIV-1 infected (10). Furthermore, several studies in HIV-1 
infected persons found CCR5[delta]32 heterozygous status may protect against 
disease progression (71,72), depending on virus strain (73). However, it is 
clear that CCR5[delta]32 does not alone explain HIV-1 infection status, 
especially in African populations where [delta]32 is absent (70,74), and the 
search for other host genes involved in susceptibility/resistance to HIV 
infection will be of major interest.

Conclusions

Recently developed genetic epidemiology methods and dense human genetic
maps, together with the growing availability of candidate genes, are
essential for identifying genes that influence human infectious diseases.
Nevertheless, investigating the role of genetic factors in a given
phenotypic response depends on many different factors related to the
phenotype, population, accurate measurement of environmental factors, and
previous knowledge; no unique optimal design can be applied for most
phenotypic responses related to infectious agents. Among possible study
designs, familial linkage studies search for a chromosomal region showing a
nonrandom segregation with the phenotype by either focusing on a few
candidate regions or using a genomewide search. The main goals of the
genome approach are to ensure that all major loci involved in the control
of a phenotype are identified and to provide the opportunity to discover
new major genes (and consequently physiopathologic pathways) involved in
phenotypic responses. Parametric linkage studies are powerful when a clear
major gene model can be inferred from segregation analysis. Nonparametric
linkage studies are strongly recommended when little is known about the
relationship between the studied phenotype and a putative gene, and
sib-pair studies have led to successful gene localizations in the analysis
of several complex traits, including infectious disease-related traits.
Once evidence for linkage is obtained, fine genetic and physical mapping is
performed to narrow down the genetic interval. The next step is the search,
by molecular methods, of polymorphisms in candidate genes located within
the identified interval. These candidate genes are selected from gene
databanks or are obtained by a systematic characterization of the genes of
the region (positional cloning). On the other hand, association studies
performed with candidate genes can directly identify the disease gene when
the tested polymorphism is in strong linkage disequilibrium with the
disease allele or is the disease allele itself. Finally, evidence for an
association should be completed by functional analysis, which will test
whether the detected polymorphism modifies the gene expression or the gene
product in a manner that can affect susceptibility to the disease.

Progress in the genetic dissection of infectious diseases will also come
from the integrated analysis of different phenotypic responses (clinical
response, intensity of infection, immunologic response), which can all
contribute to the pathologic process, as illustrated in malaria and
schistosomiasis studies. The identification of host genes in human
infectious diseases will provide new understanding of disease pathogenesis.
How this genetic information will modify our approach to prevention and
treatment of infectious diseases cannot yet be fully appreciated. However,
the identification of susceptibility/resistance genes in schistosomiasis,
mycobacterial, and HIV infections has already opened new avenues for the
screening of genetically predisposed persons and the development of
vaccines.

Dr. Abel is a senior researcher in INSERM (Institut National de la Sant et
de la Recherche Mdicale) Unit 436, Mathematical and Statistical Modeling
in Biology and Medicine, where he heads the group working on the genetic
epidemiology of infectious diseases.

Dr. Dessein is professor at the Facult de Mdecine de Marseille-Universit
de la Mditerrane and head of INSERM Unit 399, Immunology and Genetic of
Parasitic Diseases.

Address for correspondence: Laurent Abel, INSERM U.436, Mathematical and
Statistical Modeling in Biology and Medicine, CHU Piti-Salptrire, 91 Bd
de l'Hpital, 75013 Paris, France; fax 33-1-45-85-15-29; e-mail:
abel@biomath.jussieu.fr.

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Synopses

Insecticide Resistance and Vector Control

William G. Brogdon and Janet C. McAllister
Centers for Disease Control and Prevention, Atlanta, Georgia, USA

---------------------------------------------------------------------------
      Insecticide resistance has been a problem in all insect groups
      that serve as vectors of emerging diseases. Although mechanisms
      by which insecticides become less effective are similar across
      all vector taxa, each resistance problem is potentially unique
      and may involve a complex pattern of resistance foci. The main
      defense against resistance is close surveillance of the
      susceptibility of vector populations. We describe the
      mechanisms of insecticide resistance, as well as specific
      instances of resistance emergence worldwide, and discuss
      prospects for resistance management and priorities for
      detection and surveillance.

Many new and reemerging diseases are transmitted by arthropod vectors.
Mosquitoes transmit malaria (1,2), dengue-dengue hemorrhagic fever (DHF)
(3-5), yellow fever (6), Venezuelan equine encephalitis (3,7), and
filariasis (8); sand flies transmit leishmaniasis (7); ticks transmit Lyme
disease and ehrlichiosis (9,10); fleas and lice transmit Bartonella (11);
and fleas, lice, and ticks transmit various rickettsioses (12-14).
Resistance to insecticides has appeared in the major insect vectors from
every genus. As of 1992, the list of insecticide-resistant vector species
included 56 anopheline and 39 culicine mosquitoes, body lice, bedbugs,
triatomids, eight species of fleas, and nine species of ticks (15). Other
insects of public health importance, such as certain flies and cockroaches,
show resistance in all genera.

Resistance has developed to every chemical class of insecticide, including
microbial drugs and insect growth regulators. Despite decades of
international efforts, a detailed practical description of insecticide
resistance that would allow control strategies to be adjusted to specific
needs remains the exception rather than the rule.

Insecticide resistance is expected to directly and profoundly affect the
reemergence of vector-borne diseases (1), and where resistance has not
contributed to disease emergence, it is expected to threaten disease
control (15). However, careful scrutiny of current information about vector
resistance (e.g., the World Health Organization [WHO] resistance database
and records of control programs) shows that the full effect of resistance
on control efforts is not known. Many instances of resistance reported for
vector species and their regional or countrywide distribution are based on
single datasets from a single point within a country and may be years, if
not decades, old. Researching every resistance problem and every
application of vector control is not practical. Yet control measures have
to be selected for use, often at times of emergency. Although alternatives
to vector control with insecticides are available, drug resistance problems
(e.g., malaria) or vaccine cost and availability (e.g., Japanese
encephalitis) make vector control an important option (1,16). Shrinking
availability of insecticides as a result of resistance is exacerbated by
removal from the market of insecticides no longer registered for public
health use, especially in the past decade; the cost to keep certain
compounds on the market is higher than can be recouped from such use. In
addition, insecticide use is also monitored and restricted by regulatory
agencies.

The potential of resistance to interfere with emergency use of insecticides
first became apparent in 1993 when flooding in nine midwestern states
increased the threat over the next 2 years of arboviral disease
transmission (17). Most of the nine states affected had no public health
entomologic or vector control resources, and none had susceptibility data
for their vector mosquitoes. Preliminary data showed that resistance to the
insecticides proposed for emergency use was widespread throughout the
Midwest. As a result of these findings, a resistance surveillance
laboratory was established at the Centers for Disease Control and
Prevention (CDC), Atlanta, Georgia. Data collected by this laboratory in
the last 3 years confirm that states vary enormously in their resources to
deal with insecticide resistance. At present, 26 states participate in the
Emerging Infectious Disease insecticide resistance surveillance project.

We provide an update on resistance of disease vectors to insecticides, use
specific instances of emerging resistance to illustrate this complex,
worldwide problem, and offer strategic priorities for combating it.

Resistance Mechanisms

Insecticide resistance mechanisms (as opposed to insecticide avoidance
behaviors important in the control of malaria vectors) have a biochemical
basis (Figure 1). The two major forms of biochemical resistance are
target-site resistance, which occurs when the insecticide no longer binds
to its target, and detoxification enzyme-based resistance, which occurs
when enhanced levels or modified activities of esterases, oxidases, or
glutathione S-transferases (GST) prevent the insecticide from reaching its
site of action. An additional mechanism based on thermal stress response
has been proposed (18), but its importance has not been assessed.

Target-Site Mechanisms

Alterations of amino acids responsible 
for insecticide binding at its site of
action cause the insecticide to be     
less effective or even ineffective.
The target of organophosphorus (OPs)   [Figure 1.] Examples (drawn from
(e.g., malathion, fenitrothion) and    references cited in the text) of
carbamate (e.g., propoxur, sevin)      biochemical resistance mechanisms
insecticides is acetylcholinesterase   on the molecular level. A. Single
in nerve synapses, and the target of   amino acid mutation in the IIS6
organochlorines (DDT) and synthetic    membrane-spanning region of the
pyrethroids are the sodium channels of sodium channel gene that confers
the nerve sheath. DDT-pyrethroid       target-site DDT-pyrethroid
cross-resistance may be produced by    resistance in Anopheles gambiae.
single amino acid changes (one or both The same mutated codon produces
of two known sites) in the axonal      resistance in insects as diverse as
sodium channel insecticide-binding     mosquitoes, cockroaches, and flies.
site (19,20). This cross-resistance    B. Regulatory element (found
appears to produce a shift in the      upstream of coding sequence) termed
sodium current activation curve and    the "Barbie Box" that allows
cause low sensitivity to pyrethroids   induction of insecticide
(21). Similarly, cyclodiene (dieldrin) detoxifying oxidase and esterase
resistance is conferred by single      resistance genes. Many such
nucleotide changes within the same     putative control elements have been
codon of a gene for a [gamma]-         found associated with vector
aminobutyric acid (GABA) receptor      At least resistance enzymes. C. Esterase
(22). five point mutations in the      A2-B2 amplicon. These resistance
acetylcholinesterase                   esterase genes lie 5' end to 5' end
insecticide-binding site have been     within the same amplification unit.
identified that singly or in concert   More than 100 copies of this
cause varying degrees of reduced       amplicon may be present in a single
sensitivity to OPs and carbamate       mosquito. This is one example of a
insecticides (23).                     family of amplified esterase genes.

Detoxification Mechanisms

The enzymes responsible for detoxification of xenobiotics in living
organisms are transcribed by members of large multigene families of
esterases, oxidases, and GST. Perhaps the most common resistance mechanisms
in insects are modified levels or activities of esterase detoxification
enzymes that metabolize (hydrolyze ester linkages) a wide range of
insecticides. These esterases comprise six families of proteins belonging
to the [alpha]/ hydrolase fold superfamily (24,25). In Diptera, they occur as a
gene cluster on the same chromosome (26,27). Individual members of the gene
cluster may be modified in instances of insecticide resistance, for
example, by changing a single amino acid that converts the specificity of
an esterase to an insecticide hydrolase (28) or by existing as
multiple-gene copies that are amplified in resistant insects (the best
studied examples are the B1 [29] and A2-B2 [30] amplicons in Culex pipiens
and C. quinquefasciatus).

The cytochrome P450 oxidases (also termed oxygenases) metabolize
insecticides through O-, S-, and N-alkyl hydroxylation, aliphatic
hydroxylation and epoxidation, aromatic hydroxylation, ester oxidation, and
nitrogen and thioether oxidation (31). The cytochrome P450s belong to a
vast superfamily. Of the 62 families of P450s recognized in animals and
plants, at least four (families 4,6,9,18) have been isolated from insects.
The insect P450 oxidases responsible for resistance have belonged to family
6, which, like the esterases, occur in Diptera as a cluster of genes (32).
Members of the cluster may be expressed as multiple (up to five) alleles
(33). Enhanced levels of oxidases in resistant insects result from
constitutive overexpression rather than amplification (34,35). The
mechanisms of oxidase overproduction in resistance are under extensive
investigation and appear to result from both cis- and trans-acting factors,
perhaps associated with the phenomenon of induction (36-38).

Most organisms possess multiple GST from two or more classes (39). GST
implicated in DDT insecticide resistance exist as clusters of genes that
have been further shuffled through the genome by recombination (40). A
number of resistance GST genes, including multiple forms in the same
insect, have been characterized in vectors (41-43).

Resistance to Growth Regulators, Ivermectins, and Other Microbial Agents

Because of their more recent introduction to vector control programs, we
discuss growth regulators, ivermectins, and other microbial agents as a
group. The initial mechanisms that conferred resistance to insect growth
regulators were oxidase-based (44). Resistance to ivermectins has resulted
from a number of factors, including oxidase, conjugation, and altered
target-site mechanisms (45). Vectors have not yet demonstrated resistance
to these compounds in the field.

Microbial agents such as Bacillus sphaericus and B. thuringiensis are
considered insecticides because the principal active agents are crystal
toxins produced by the bacteria. The mechanisms of resistance to B.
sphaericus are not yet defined (46,47), but more than one mechanism seems
to be involved (48). Resistance to B. thuringiensis has resulted from
reduced binding of the toxin to the brush border in the lumen of the insect
gut (49,50) or by enhanced digestion of toxin by gut proteases (51). The
six different toxin types in the B. thuringiensis israelensis strain used
for vector control were expected to retard or prevent development of a
comprehensive resistance mechanism; however, multitoxin resistance to B. T.
israelensis has already appeared (52,53).

Detecting and Monitoring Resistance

The initial step in identifying a potential problem is to detect
changes in the susceptibility of a population of vectorsthrough
biochemical assay, or molecular assay (Figure 2).

[Figure 2.] Cross-resistance bioassay, 
relationships of commonly used      
classes of insecticides.
                                     
Bioassays

WHO has developed susceptibility bioassay tests (available in kit form for
purchase from WHO) for mosquitoes, lice, bedbugs, reduviid bugs,
cockroaches, blackflies, houseflies, ticks, and fleas (15). Our laboratory
found that time-mortality bioassays were more sensitive than dose-mortality
bioassays in detecting changes in susceptibility and had better correlation
with microplate-based biochemical assays for resistance mechanisms (54,55).
Time-based bioassays have been further modified through the use of
insecticide-coated glass bottles and solutions of standard-grade
insecticides or synergists; this approach simplifies the bioassay process
and increases the amount of information that can be obtained from a limited
pool of mosquitoes (56).

Biochemical and Molecular Assays

Biochemical and molecular methods can detect resistance mechanisms in
individual insects; therefore, they can confirm resistance with the use of
only a small number of insects. Identification of resistance mechanisms
helps determine the cross-resistance spectrum (Figure 3), facilitates the
choice of alternative insecticides, and allows detailed mapping of areas
with resistant populations. Specific biochemical assays have been developed
for all known resistance mechanisms, except the modified sodium and GABA
receptor mechanisms (57-59).

Molecular information on resistance    
mechanisms will increasingly be
incorporated into resistance           
diagnostic procedures. One type of
mechanism that will become much        [Figure 3.] Examples of simple
easier to detect will be the point     diagnostic assays for insecticide
mutations that cause target-site       resistance include bioassays run in
resistance or changes in               treated bottles (upper) and
detoxification enzyme specificity.     biochemical detection and
Thus far, target-site mechanisms have  measurement of resistance enzyme
been detected by polymerase chain      activity in microplates (lower).
reaction-restriction enzyme (PCR-REN)
and PCR amplification of specific
alleles (60,61).

Features of Resistance Emergence

Innumerable genetic, biologic, and operational factors influence the
development of insecticide resistance. In many respects, resistance is a
chaotic problem, with different outcomes possible in a particular area,
depending on the influence of diverse factors on initial conditions. Even
so, certain factors affect resistance development throughout the world. We
discuss major resistance characteristics and show why each manifestation of
resistance is potentially unique and therefore must be independently
evaluated.

Focal Nature of Resistance

Vector control personnel frequently assume that resistance in a particular
species occurs throughout their control area, but in reality, insecticide
resistance is focal. In Guatemala, sampling sites for Anopheles albimanus
only a few kilometers apart varied not only by presence or absence of
resistance, but also by level of resistance and by dominant mechanism
responsible for resistance (55).

Surveillance data (Brogdon and McAllister, unpub. data) from the United
States show that resistance to OPs in Culex mosquito species is focal in a
number of statesgenerally high in urban areas and absent in rural sites.
The higher levels of resistance are in areas of ongoing control activities.
When resistance levels in adjacent counties were compared, levels were
higher in areas of intensive mosquito control.

While the relative importance of agricultural versus public health use of
insecticides to resistance development has been widely argued (62),
resistance has been associated with both uses. Agricultural use caused
resistance in Central American An. albimanus (55). However, in Haiti (54)
and in Sudan (62), the impact of public health spraying on development of
resistance is clear. In Sri Lanka, resistance in one vector, An.
cuilifacies, is characteristic of public health spraying, while resistance
in another, An. nigerrimus, has a profile that indicates agricultural
chemicals (62). Resistance appears rapidly in areas where bed nets are used
to control mosquitoes (Western Kenya) or sandflies (Colombia) (63; Brogdon
and McAllister, unpub. data).

Resistance and Disease-Endemic Areas

Vector control is affected not only by the focal nature and distribution of
resistance but also by disease incidence. Resistance detection could
actually interfere with disease control programs if adequate surveillance
data are not also collected. In Ecuador (57) we observed OPs resistance in
the malaria vector An. albimanus in the central agricultural provinces of
Guayas, Manabi, and Los Rios, the sources of the country's resistance
surveillance data. However, in the northern province of Esmeraldas, where
insecticide use had been limited and an epidemic of Plasmodium falciparum
malaria was under way, populations were completely susceptible. Lack of
insecticide use does not preclude immigration of resistance genes (e.g.,
the movement of esterase resistance to OPs in C. pipiens into certain areas
of France [64]).

Resistance and Disease Control

To compromise insecticide vector control, the level of resistance must be
high enough to adversely affect disease transmission. In many cases, vector
control may not be affected by the level of resistance. For example, an
activity may be controlling only 75% of the vector population. If, for
example, the level of resistance is lower than 10%, resistance will not
affect disease control efforts; in this situation, increasing surveillance
and monitoring level and frequency of resistance would be sufficient. No
change in control methods would be needed. Western Kenya is a good
operational example of the coexistence of resistance and disease control.
Pyrethroid resistance appeared soon after bed nets were introduced (64).
After 2 years, the resistance level had not changed significantly, possibly
because of the continual massive introduction of susceptible genes (65).
Other reasons may explain why the presence of insecticide resistance genes
in vectors in a control area does not mean that effective control is not
being achieved. For example, resistance genes may not be expressed, they
may be expressed in an alternative stage of development to that being
controlled by insecticide, or the gene detected may be a member of an
alternative gene subfamily to one that can affect the compound being used.
We have observed in An. albimanus and An. gambiae that resistance enzymes,
especially esterases and GST, may be expressed only in freshly emerged
adult anophelines and may be absent in older mosquitoes, those potentially
infective for malaria (Brogdon and McAllister, unpub. data).

Pyrethroid Resistance

Pyrethroid resistance is emerging despite early optimism that because of
its rapid toxicologic action this newest large class of insecticides would
not produce resistance (66). Resistance is not evolving through unique new
mechanisms; rather, existing mechanisms are being enhanced, and
cross-resistance is occurring. In Guatemala, pyrethroid resistance was
first reported in an An. albimanus population resistant to fenitrothion.
When deltamethrin was used, the esterase conferring fenitrothion resistance
was enhanced by selective pressure to produce deltamethrin cross-resistance
(67). Additionally, we are now finding DDT-permethrin cross-resistance due
to oxidase cross-resistance in the same mosquito. A similar pattern of
cross-resistance has been documented for C. pipiens in Ohio.
Multiresistance (two or more resistance mechanisms in the same insect) is
becoming widespread as control programs make sequential use of one chemical
class after another.

A far more threatening development in pyrethroid resistance is the
appearance of target-site resistance (also termed knockdown resistance) to
pyrethroids in several important vectors in multiple locations. We have
detected the knockdown resistance mechanism in the dengue and yellow fever
vector Aedes aegypti from Puerto Rico and Indonesia and in the encephalitis
vector C. quinquefasciatus from Louisiana. French researchers have detected
the mechanism in An. gambiae, the primary African vector of malaria, in
several countries of West Africa (68,69). This resistance mechanism may be
a legacy of similarities in the site of action of pyrethroids and DDT.

Prospects for Resistance Management

A National Research Council report (70) on strategies and tactics for
pesticide resistance management described insecticide susceptibility as a
resource and resistance surveillance as an essential step in resistance
management. Resistance surveillance has three objectives: 1) Provide
baseline data for program planning and pesticide selection before the start
of control operations; 2) Detect resistance at an early stage so that
timely management can be implemented (even detection of resistance at a
late stage can be important in elucidating the causes of failure of disease
control; however, in such cases, any management other than replacement of
the pesticide may not be possible); 3) Continuously monitor the effect of
control strategies on resistance.

Resistance to insecticideseven to pyrethroidsin disease vectors is
widespread. With the availability of more sensitive and easy-to-use
surveillance techniques (56,58,59), the means for managing resistance are
at hand. A challenge to resistance management is that efforts to control
vector-borne diseases are becoming more diversified; the global effort to
control malaria is a case in point. In the 1950s, WHO mounted a global
effort to eradicate malaria. Failure of this effort was caused by many
factors, including insecticide resistance. Now the best prospect for a
worldwide malaria vector control strategy appears to be the use of
pyrethroid-impregnated bed nets, because they are less expensive than
spraying walls with residual insecticide, are effective in reducing child
deaths, and can be better administered through a horizontal,
community-based program (71). This type of vector control means that
resistance surveillance will also have to be handled and interpreted
locally. Decisions will have to be made within local programs by the end
user. Moreover, as urbanization increases around the world, some horizontal
programs are better able financially to contract out control and
surveillance activities. In the United States, local control programs use
diverse methods for vector control to meet their specific, often unique
needs (72). Diverse methods will likely become the characteristic approach
to vector control worldwide, given the prohibitive economics of mounting a
worldwide disease control campaign based on vertical programs and the
availability of entrepreneurs that increasingly contract for vector control
on a city-by-city basis internationally (73-75). In vertical programs,
resistance surveillance was a component more in theory than in fact. The
challenge will be to maximize the exposure of vector control personnel and
entrepreneurs to management principles and to make widely available the
surveillance tools required.

Priorities for the Future

Sufficient means exist to detect and manage resistance at a higher level
and with greater effectiveness. A number of training courses and
consultations have been conducted to broaden the base of resistance
surveillance in the United States. However, the need for resistance
surveillance information is global. Perhaps the most cost-effective way to
disseminate this information will be the Internet.

Internationally, the increasing diversity of vector control measures will
require continued development of simple and informative methods. How can we
use the most sensitive and informative molecular and biochemical methods in
concert with bioassay techniques? How can these be best simplified so that
relatively untrained personnel can use them?

While initial detection and field surveillance for resistance will likely
continue to be based upon simple bioassay, biochemical, and molecular
tools, the deeper understanding of how resistance arises and maintains
itself in populations requires molecular genetics studies.

The most complete understanding of insecticide resistance mechanisms in
disease vectors has come from studies with mosquitoes. Much more attention
is needed for resistance detection and surveillance methods in other vector
groups, such as sand flies, triatomids, lice, fleas, and ticks. Most
important, however, is that resistance detection be made an integral part
of all control programs. The resources for vector control, even under
emergency situations, are limited and must be used as effectively as
possible.

      Dr. Brogdon is a research entomologist with the National Center for
Infectious Diseases, CDC. His research interests include insecticide
resistance in disease vectors; vector biochemistry, physiology, and
molecular biology; and mosquito behavior.

      Dr. McAllister is a visiting fellow funded by the Emerging Infections
Program, CDC.

      Address for correspondence: William G. Brogdon, Centers for Disease
Control and Prevention, F22, 1600 Clifton Road, NE, Atlanta, GA 30333, USA;
fax: 770-488-7794; e-mail: wgb1@cdc.gov.

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Dispatches

Mutators and Long-Term Molecular Evolution of PathogenicEscherichia coli
O157:H7

Thomas S. Whittam, Sean D. Reid, and Robert K. Selander
Pennsylvania State University, University Park, Pennsylvania, USA

---------------------------------------------------------------------------
      It has been proposed that an increased mutation rate (indicated
      by the frequency of hypermutable isolates) has facilitated the
      emergence of Escherichia coli O157:H7. Analysis of the
      divergence of 12 genes shows no evidence that the pathogen has
      undergone an unusually high rate of mutation and molecular
      evolution.

Escherichia coli O157:H7, a highly virulent organism first linked to
infectious disease in 1982 (1) and now found worldwide, has caused serious
foodborne epidemics in the United States, Japan, and Europe (2). One
hypothesis for the emergence and rapid spread of this organism is that
strong mutator alleles enhance genetic variability and accelerate adaptive
evolution (3). LeClerc et al. (3) found that more than 1% of O157:H7
strains had spontaneous rates of mutation that were 1,000-fold higher than
those of typical E. coli. These mutator strains were defective in
methyl-directed mismatch repair (MMR) as a result of deletions in the
intergenic region between the mutS and rpoS genes (3). According to the
mutator hypothesis, a pathogen able to enter a transient hypermutable state
could overcome the fitness costs of deleterious mutations by accruing new
genetic variation at times critical for survival and colonization of new
hosts.

 [Figure.] Evolutionary distance in terms of synonymous and nonsynonymous
 changes per 100 sites (4) for 12 genes sequenced from Escherichia coli
 O157:H7, E. coli K-12, and Salmonella enterica Typhimurium. The points for
 synonymous sites are (left to right): gap, crr, mdh, icd, fliC (conserved
 5' and 3' ends), trpB, putP, aceK, mutS, trpC, tonB, and trpA. Under the
 mutator hypothesis, the genetic distance between the pathogenic O157:H7
 strain (or the closely related strain ECOR37) and the outgroup
 (Typhimurium) is expected to exceed the distance between the commensal
 K-12 and the outgroup. Prolonged periods of enhanced mutation rate should
 drive the points above the dotted line marking equal rates of molecular
 evolution. Two loci (tonB and trpA) show departure from the equal rate
 line, but neither has evolved differently from that expected by the
 molecular clock. The sequences of 12 genes were obtained from GenBank or
 the original sources as follows: aceK (5), crr (6-8), fliC (9), gap (10),
 icd (11), mdh (12), mutS (13,14), putP (15), tonB (16,17), trp (17-20).

Adaptive evolution by transient or prolonged states of hypermutation can
cause neutral mutations to rapidly accumulate throughout the genome. To
detect possible elevation in the rate of molecular evolution in the
emergence of E. coli O157:H7, we compared 12 genes with housekeeping
functions (Figure) that have been sequenced in both E. coli O157:H7 and E.
coli K-12 (a commensal organism), as well as in an outgroup species,
Salmonella enterica serotype Typhimurium. The evolutionary distance
(expressed in point mutations per 100 sites) between Typhimurium and K-12
is shown against the distance between Typhimurium and O157:H7 for
synonymous and nonsynonymous sites separately (Figure). The line indicates
equal rates of evolution in the two lineages. An elevated mutation rate in
O157:H7 over evolutionary time should result in greater divergence from
Typhimurium than from K-12 and in the distribution of points above the
equal-rate line. For both synonymous and nonsynonymous sites, most genes
fall below or very near the equal-rate line with only two exceptions: tonB
and trpA deviate in the direction expected under the mutator hypothesis. To
test the significance of these deviations, we compared the observed degrees
of divergence of K-12 and O157:H7 from Typhimurium and the expectations of
the molecular evolutionary clock hypothesis (21). The basis of this test is
that a constant rate of mutation results in equal numbers of substitutions
in two sequences from an outgroup (21). Considering synonymous and
nonsynonymous changes together with Typhimurium as an outgroup, we found
that 11 of the 12 loci, including tonB (m(sub 1)= 0, m(sub 2)= 3, X(sup 2)= 3.00, 
p > 0.05) and trpA (m(sub 1)= 4, m(sub 2)= 10, X(sup 2)= 2.57, p > 0.05), 
did not deviate significantly from a uniform rate of evolution predicted by 
a molecular clock. Only mdh exhibited a significant departure from the 
molecular clock (m(sub 1)= 14, m(sub 2)= 5, X(sup 2)= 4.26, p < 0.05); 
however, the direction was away from that predicted by the mutator hypothesis 
(the TyphimuriumK-12 distance exceeded the Typhimurium-O157:H7 distance).

Our findings do not conflict with the observation that MMR defects occur in
relatively high frequency in emerging pathogens; however, the findings
indicate no evidence of a genomewide elevation of the mutation rate in
pathogenic E. coli O157:H7. The uniform rate of divergence of O157:H7 and
K-12 suggests several possibilities. One is that the mutator state is
transient and so brief that the impact on long-term rates of evolution is
undetectable. This possibility is consistent with the view that mutators
may generate favorable mutations in periods of intense selection and then
revert to a nonmutator phenotype (22,23). Another possibility is that all
bacterial populations experience brief episodes of adaptive evolution
driven by hypermutation. Matic and co-workers (24) found equivalent
frequencies of mutators among strains of commensal bacteria and both
emerging and classical pathogenic E. coli.

Finally, defects in MMR that produce the mutator phenotype also relax the
normal barriers to recombinational exchange between bacterial species (25).
The enhanced recombination that accompanies the mutator phenotype may
explain why E. coli O55:H7, the immediate ancestor of O157:H7 (26) that
also carries the same defective MMR allele (3), harbors such an
extraordinary variety of plasmid and chromosomal virulence factors (27).
Together with our finding of clock-like divergence of E. coli O157:H7
housekeeping genes, these observations indicate that the main evolutionary
benefit of the mutator phenotype is the enhanced ability to acquire useful
foreign DNA (3), not an increased rate of point mutation over the long
term.

Dr. Whittam is professor of biology at the Institute of Molecular
Evolutionary Genetics, Pennsylvania State University. His research focuses
on understanding how evolutionary forces operate to determine the amount
and organization of genetic variation in natural populations of bacteria.
Specific studies include the evolution of pathogenic forms of Escherichia
coli associated with intestinal and extraintestinal infections, the
evolution of virulence and resistance in a host-parasite interaction using
an amoeba-Legionella system, and the ecologic determinants and evolution of
host specificity in Rhizobium-legume associations.

Address for correspondence: Thomas S. Whittam, Institute of Molecular
Evolutionary Genetics, Pennsylvania State University, University Park, PA
16802, USA; fax: 814-865-9131; e-mail: tsw1@psu.edu.

References

  1. Riley LW, Remis RS, Helgerson SD, McGee HB, Wells JG, Davis BR, et al.
     Hemorrhagic colitis associated with a rare Escherichia coli serotype.
     N Engl J Med 1983;308:681-5.
  2. Doyle MP, Zhao T, Meng J, Zhao S. Escherichia coli O157:H7. In: Doyle
     MP, Beuchat LR, Montville TJ, editors. Food microbiology: fundamentals
     and frontiers. Washington: American Society for Microbiology; 1997. p.
     171-91.
  3. LeClerc JE, Li B, Payne WL, Cebula TA. High mutation frequencies among
     Escherichia coli and Salmonella pathogens. Science 1996;274:1208-11.
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  6. Hall BG, Sharp PM. Molecular population genetics of Es-cherichia coli:
     DNA sequence diversity at the celC, crr, and gutB loci of natural
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  7. Nelson SO, Schuitema AR, Benne R, van der Ploeg LH, Plijter JS, Aan F,
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  8. Saffen DW, Presper KA, Doering TL, Roseman S. Sugar transport by the
     bacterial phosphotransferase system. Molecular cloning and structural
     analysis of the Escherichia coli ptsH, ptsI, and crr genes. J Biol
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  9. Li J, Nelson K, McWhorter AC, Whittam TS, Selander RK. Recombinational
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 10. Nelson K, Whittam TS, Selander RK. Nucleotide polymorphism and
     evolution in the glyceraldehyde-3-phosphate dehydrogenase gene (gapA)
     in natural populations of Salmonella and Escherichia coli. Proc Natl
     Acad Sci U S A 1991;88:6667-71.
 11. Wang FS, Whittam TS, Selander RK. Evolutionary genetics of the
     isocitrate dehydrogenase gene (icd) in Escherichia coli and Salmonella
     enterica. J Bacteriol 1997;179:6551-9.
 12. Boyd EF, Nelson K, Wang F-S, Whittam TS, Selander RK. Molecular
     genetic basis of allelic polymorphism in malate dehydrogenase (mdh) in
     natural populations of Escherichia coli and Salmonella enterica. Proc
     Natl Acad Sci U S A 1994;91:1280-4.
 13. Haber LT, Pang PP, Sobell DI, Mankovich JA, Walker GC. Nucleotide
     sequence of the Salmonella typhimurium mutS gene required for mismatch
     repair: homology of MutS and HexA of Streptococcus pneumoniae. J
     Bacteriol 1988;170:197-202.
 14. Schlensog V, Bock A. The Escherichia coli fdv gene probably encodes
     mutS and is located at minute 58.8 adjacent to the hyc-hyp gene
     cluster. J Bacteriol 1991;173:7414-5.
 15. Nelson K, Selander RK. Evolutionary genetics of the proline permease
     gene (putP) and the control region of the proline utilization operon
     in populations of Salmonella and Escherichia coli. J Bacteriol
     1992;174:6886-95.
 16. Hannavy K, Barr GC, Dorman CJ, Adamson J, Mazengera LR, Gallagher MP,
     et al. TonB protein of Salmonella typhimurium. A model for signal
     transduction between membranes. J Mol Biol 1990;216:897-910.
 17. Milkman R. Recombinational exchange among clonal populations. In:
     Neidhardt FC, Curtiss IR, Ingraham JL, Lin ECC, Low KB, Magasanik B,
     et al, editors. Escherichia coli and Salmonella: cellular and
     molecular biology. 2nd ed. Washington: American Society for
     Microbiology; 1996. p. 2663-84.
 18. Crawford IP, Nichols BP, Yanofsky C. Nucleotide sequence of the trpB
     gene in Escherichia coli and Salmonella typhimurium. J Mol Biol
     1980;142:489-502.
 19. Horowitz H, Van Arsdell J, Platt T. Nucleotide sequence of the trpD
     and trpC genes of Salmonella typhimurium. J Mol Biol 1983;169:775-97.
 20. Nichols BP, Yanofsky C. Nucleotide sequences of trpA of Salmonella
     typhimurium and Escherichia coli: an evolutionary comparison. Proc
     Natl Acad Sci U S A 1979;76:5244-8.
 21. Tajima F. Simple methods for testing the molecular evolutionary clock
     hypothesis. Genetics 1993;135:599-607.
 22. Rosenberg SM, Thulin C, Harris RS. Transient and heritable mutators in
     adaptive evolution in the lab and in nature. Genetics
     1998;148:1559-66.
 23. Taddei F, Radman M, Maynard-Smith J, Toupance B, Goupon PH, Godelle B.
     Role of mutator alleles in adaptive evolution. Nature 1997;387:700-2.
 24. Matic I, Radman M, Taddei F, Picard B, Doit C, Bingen E, et al. Highly
     variable mutation rates in commensal and pathogenic Escherichia coli.
     Science 1997;277:1833-4.
 25. Rayssiguier C, Thaler DS, Radman M. The barrier to recombination
     between Escherichia coli and Salmonella typhimurium is disrupted in
     mismatch-repair mutants. Nature 1989;342:396-401.
 26. Whittam TS, McGraw EA, Reid SD. Pathogenic Escherichia coli O157:H7: a
     model for emerging infectious diseases. In: Krause RM, editor.
     Emerging infections. New York: Academic Press; 1998. p. 163-83.
 27. Rodrigues J, Scaletsky ICA, Campos LC, Gomes TAT, Whittam TS, Trabulsi
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     coli of the classic serogroup O55. Infect Immun 1996;64:2680-6.
---------------------------------------------------------------------------------
---------------------------------------------------------------------------------

Dispatches

Increasing Hospitalization and Death Possibly Due to Clostridium difficile
Diarrheal Disease

Floyd Frost,* Gunther F. Craun, and Rebecca L. Calderon
*Southwest Center for Managed Care Research, Albuquerque, New Mexico, USA;
Gunther F. Craun and Associates, Staunton, Virginia, USA; and U.S.
Environmental Protection Agency, Research Triangle Park, North Carolina,
USA

---------------------------------------------------------------------------
      This study calculated yearly estimated national hospital
      discharge (1985 to 1994) and age-adjusted death rates (1980 to
      1992) due to bacterial, viral, protozoal, and ill-defined
      enteric pathogens. Infant and young child hospitalization (but
      not death) rates in each category increased more than 50%
      during 1990 to 1994. Age-adjusted death and hospitalization
      rates due to enteric bacterial infections and hospitalizations
      due to enteric viral infections have increased since 1988. The
      increases in hospitalization and death rates from enteric
      bacterial infections were due to a more than eightfold increase
      in rates for specified enteric bacterial infections that were
      uncoded during this period (ICD9 00849). To identify bacterial
      agents responsible for most of these infections, hospital
      discharges and outpatient claims (coded with more detail after
      1992) were examined for New Mexico's Lovelace Health Systems
      for 1993 to 1996. Of diseases due to uncoded enteric pathogens,
      73% were due to Clostridium difficile infection. Also, 88% of
      Washington State death certificates (1985 to 1996) coded to
      unspecified enteric pathogen infections (ICD0084) listed C.
      difficile infection.

Infectious diarrhea remains a major cause of death worldwide (1). In the
United States, enteric pathogens are estimated to cause 25 to 99 million
episodes of diarrhea and vomiting each year, resulting in 2.2 million
physician visits (2). U.S. residents at highest risk for severe illness or
death from diarrhea are young children (3-5) and the elderly (5). Race,
socioeconomic status, and residence in a nursing home are also risk factors
for death due to diarrhea (1-6).

Increasing concern over waterborne   Table 1. International Classification
transmission of enteric pathogens    of Disease (ICD9)
(7-10) prompted this study. Since
hospitalization or death from
infectious diarrhea is uncommon,     -------------------------------------
state- or hospital-specific studies  Categories
are unlikely to include enough
cases to accurately estimate the
incidence or describe the temporal   -------------------------------------
trends in hospitalization or death   Bacterial
rates. Therefore, this study
focused on national hospitalization  001(sup a)Cholera
and death data to determine if the
incidence of hospitalizations or     002(sup a)Typhoid and paratyphoid
deaths due to infectious diarrhea              fevers
has changed during the past decade   003(sup a)Other salmonella infections
and, if so, to identify specific
pathogens responsible for the        004(sup a)Shigellosis
changes.
                                     005(sup a)Other food poisoning
Data Sources                                   bacterial
Computerized data on U.S. death      008(sup a)Intestinal infections due to
rates (1980 to 1992), which                    other organisms
included coded underlying cause of
death, and National Hospital         0080    Escherichia coli
Discharge Survey (NHDS) data (1985
to 1994) were obtained from the      0081    Arizona group of paracolon
National Center for Health                   bacilli
Statistics (NCHS). NHDS data came    0082    Aerobacter aerogenes
from yearly surveys of hospital              (Enterobacter)
discharges conducted by NCHS         0083    Proteus
through a multistage sampling
scheme (11). Yearly national         0084    Other specified bacteria
estimates of discharges by
diagnosis, age, and gender were      0085    Bacterial enteritis
obtained by using multipliers                (unspecified)
provided by NCHS. Yearly midyear     041(sup a)Bacterial infection in
Bureau of the Census population                conditions classified
estimates were used for calculating            elsewhere and of unspecified
national death and hospitalization             site
rates.                                 For hospital coding

Deaths Due to Diarrhea, 19801992
                                       00841    Staphylococcus

The number of diarrhea deaths in       00842    Pseudomonas
1980 to 1992 was determined by
using the coded underlying causes      00849    Other
of death. Deaths are coded to only
four digits of the International        After 1992
Classification of Disease 9th
revision (ICD9). Causes of death        00843   Campylobacter

were grouped into four categories       00844   Yersinia enterocolitica
according to type of pathogen:
bacterial, parasitic, viral, and        00845   Clostridium difficile
ill-defined (Table 1).
                                        00846   Other anaerobes
Hospitalizations Due to Diarrhea,
19851994                               00847   Other gram-negative
                                                bacteria
Hospitalizations due to diarrhea     Viral
(coded to five digits of the ICD9)
were ascertained for each year from  074(sup a)Specified disease due to
1985 to 1994 by selecting the same             coxsackie virus
groups of ICD9 codes from the first  045(sup a)Acute poliomyelitis
seven discharge diagnoses recorded
for each hospital discharge. For     047(sup a)Meningitis due to
the few hospital discharges with               enterovirus
multiple infectious diarrhea codes,
priority was given to the code that  048     Other enterovirus diseases
appeared first.                              of central nervous system

Hospitalization and Death Rates      0086    Enteritis due to specified
Adjusted by Age                              virus
                                     0088    Other viral organism not
Age-adjusted hospitalization and             elsewhere classified
death rates for each year,
standardized to the 1990 U.S.        0700(sup a)Viral hepatitis A with
population, were calculated for                 hepatic coma
each of the four ICD9 enteric        0701(sup a)Viral hepatitis A without
pathogen categories (bacterial,                 mention of hepatic coma
parasitic, viral, and ill-defined).          

A ratio of deaths to                 Parasitic
hospitalizations for each category
was calculated by dividing the
number of deaths caused by           006(sup a)Amebiasis
pathogens in each category by the
number of hospital discharges with   007(sup a)Other protozoal intestinal
the first infectious enteric                   diseases
disease discharge diagnosis          127(sup a)Other intestinal
included in that category.                     helminthiases
                                     128     Other and unspecified
Hospitalizations and Deaths                  helminthiases
Bacterial Causes                     129(sup a)Intestinal parasitism,
                                               unspecified
Hospitalizations and deaths due to
bacterial causes (including          Other
cholera, typhoid and paratyphoid
fever, other salmonella infections,  009(sup a)Ill-defined intestinal
shigellosis, other food poisoning              infections
bacteria, and infections due to      -------------------------------------
other specified bacteria) were also  (sup a)Diarrhea codes selected for study.
analyzed (Table 1). Age-adjusted     
death rates (each year) from "other
specified enteric bacteria" (ICD9 0084) and age-adjusted hospitalization
rates (each year) due to "uncoded but specified enteric bacteria" (ICD9
00849) were calculated. Additional cause codes were added in 1992 (ICD9
00843-00847 for hospital ICD coding, including 00845 for Clostridium
difficile (Table 1). However, these codes were not yet incorporated for
this analysis of national hospital discharge data. During this time, cause
of death was only coded to the fourth ICD9 digit.

Deaths (19851996) and Hospitalizations (19931996)

To make use of enhanced ICD9 coding available after 1992, inpatient
health-care records for 1993 to 1996 from the Lovelace Health Systems in
Albuquerque, New Mexico, were reviewed to identify bacterial pathogens
responsible for most hospitalizations that would have been coded (before
1992) to specified but uncoded bacterial pathogens. Since death records are
only coded to the fourth digit, Washington State deaths (with any multiple
cause of death code of ICD9 0084) occurring between 1985 and 1996 were also
reviewed to identify the specific pathogen that resulted in this ICD9 code
assignment.

Findings

Death Rates

                              Age-adjusted death rates for protozoal,
					viral, and other causes remained relatively

 					stable from 1980 to 1992 (Figure 1), but
 	                        age-adjusted death rates for bacterial causes
                              increased from 0.060 per 100,000 population
 [Figure 1.] Age-adjusted     in 1980 to 0.104 per 100,000 in 1994, more
 death rates per 100,000      than 60% (p < 0.00001) (Figure 1). To
 population by grouped        ascertain the specific bacterial agents
 underlying cause of death    responsible for the increase, deaths were
 for selected enteric         initially categorized by each three- and
 pathogens, United States     four-digit ICD9 code included in the
 1985-94. (Standardized to    bacterial category.
 the 1970 U.S. population).
                              The age-adjusted death rate due to other
                              specified bacterial pathogens (ICD9
0080-0084) increased from 0.0102 per 100,000 population to 0.0821 per
100,000 population (p < 0.000001), more than eightfold (Table 2). This
increase accounted for the overall increase in the age-adjusted death rate
for deaths due to enteric bacterial pathogens, as the age-adjusted death
rate for other enteric bacterial causes remained stable or declined.

Most of the increase in the age-adjusted death rate for ICD9 0080-0084 was
due to a statistically significant (p < 0.00001) increase in the death rate
of persons age 45 years and older (Table 2). For both older and younger age
groups, the increase was most apparent from 1988 to 1992. During this time,
approximately 96% of U.S. deaths coded to ICD9 0080-0084 were coded to ICD9
0084, which includes other specified but uncoded enteric bacterial
pathogens.

Table 2. Age-adjusted death rates for bacterial causes of diarrhea
-------------------------------------------------------------------------------------------            
          ICD9 0080-0084(sup a)                   Remainder of bacterial causes(sup b) 
         -----------------------------------  ---------------------------------------------                        
                   <45        >45                              >45          >45                              
Year AARATE(sup c)yrs(sup d) yrs(sup d)(Deaths) AARATE(sup 3) yrs(sup d) yrs(sup d)(Deaths)
-------------------------------------------------------------------------------------------

1980   0.0102    0.0013     0.0090      ( 21)    0.0500       0.0133      0.0368     (107)

1981   0.0135    0.0035     0.0100      ( 29)    0.0559       0.0107      0.0453     (120)

1982   0.0107    0.0022     0.0085      ( 23)    0.0463   	  0.0087  	  0.0365     (100)

1983   0.0144    0.0040     0.0104      ( 32)    0.0426       0.0092      0.0334     ( 94)

1984   0.0101    0.0026     0.0075      ( 23)    0.0433       0.0068      0.0365     ( 98)

1985   0.0092    0.0016     0.0075      ( 21)    0.0587       0.0131      0.0456     (135)

1986   0.0119    0.0029     0.0090      ( 28)    0.0485       0.0108      0.0347     (105)

1987   0.0175    0.0029     0.0146      ( 41)    0.0506       0.0097      0.0409     (120)

1988   0.0241    0.0008     0.0232      ( 57)    0.0305       0.0071      0.0234     ( 74)

1989   0.0342    0.0016     0.0326      ( 83)    0.0465       0.0111      0.0354     (114)

1990   0.0520    0.0043     0.0478      (129)    0.0374       0.0070      0.0303     ( 93)

1991   0.0547    0.0027     0.0520      (139)    0.0254       0.0055      0.0190     ( 65)

1992   0.0821    0.0046     0.0775      (213)    0.0220       0.0049      0.0171     ( 57)

-------------------------------------------------------------------------------------------
(sup a) Causes include Escherichia coli (0080), Arizona group (0081), Aerobacter aerogenes 
(Enterobacter) (0082), Proteus mirabilus and morganii (0083), other specified enteric 
bacterial infections (0084).
(sup b) Causes include cholera (001), typhoid and paratyphoid fever (002),other salmonella 
infections (003), shigellosis (004), bacterial enteritis, unspecified (0085), bacterial 
infection in conditions classified elsewhere (041).
(sup c) Age-adjusted death rates per 100,000 population, adjusted to the 1990 U.S. population.
(sup d) Contribution to age-adjusted death rate from persons younger than age 45 years and 
from persons age 45 years and older.

Hospitalization Rates

                              The estimated number of hospitalizations in
					the United States coded to infectious enteric
				      agents increased from 131,252 to 337,178 from
 	                        1985 to 1994, a 2.5-fold increase.
                              Age-adjusted hospitalization rates for viral
 [Figure 2.] Age-adjusted     causes increased more than twofold, whereas
 hospital discharge rates     rates for bacterial causes increased more
 per 100,000 population by    than fourfold (Figure 2). Hospitalization
 grouped discharge diagnosis  rates for protozoal and ill-defined causes
 for selected enteric         remained stable or fluctuated from year to
 pathogens, United States     year (Figure 2). In 1985, bacterial causes
 1980-92. (Standardized to    accounted for 21% of all hospitalizations for
 the 1970 U.S. population).   infectious enteric agents, while in 1994,
 Discharges were included if  they accounted for 38% (Figure 2). A large
 a selected enteric pathogen  increase was apparent in the age-adjusted
 was among the first seven    hospitalization rate for other specified
 discharge diagnoses. The     bacterial pathogens (ICD9 00800-00849)
 pathogen group was assigned  (Figure 2).
 according to the first
 pathogen listed.             The increased age-adjusted rate of hospital
                              discharge coded to "other specified bacterial
                              pathogen" infections (ICD9 0080-00849) was
statistically significant (p < 0.00001) (Table 3). The age-adjusted
hospitalization rate for bacterial causes, other than ICD9 0080-00849,
remained stable (Table 3). As with deaths, the increase in hospitalizations
for bacterial causes appears to have begun in 1988, but unlike the increase
in death rates, hospitalization rates increased for persons both younger
and older than 45 years of age (p < 0.001) (Table 3). For discharges coded
to ICD9 0080-00849, 88% were coded to ICD9 00849, which includes "other
uncoded but specified enteric bacterial pathogens." Discharge rates due to
bacterial causes of diarrhea increased in each age group, with greater
increases seen in children and persons more than 45 years of age (Table 4).

Infant hospitalization rates increased for bacterial, viral, protozoal, and
ill-defined conditions (p < 0.001) (Table 4). Hospitalization rates also
increased for children ages 1 to 4 years of age for bacterial, viral, and
ill-defined conditions (p < 0.001) (Table 4); however, no increases in
children's death rates were observed.

Table 3. Age-adjusted hospitalization rates for bacterial causes of diarrhea
---------------------------------------------------------------------------------------------
                ICD9 0080-0084(sup a)             Remainder of bacterial causes(sup b)
      ------------------------------------      -----------------------------------------
                    <45        >45       (Dis-                   <45         >45     (Dis-
Year AARATE(sup c) yrs(sup d) yrs(sup d) charges) AARATE(sup c) yrs(sup d) yrs(sup d)charges)
---------------------------------------------------------------------------------------------

1985    3.72       1.71       2.02      ( 8,716)   7.76            4.06       3.70    (18,286)

1986    4.79       2.01       2.74      (11,316)   5.56     	 2.60       2.96    (13,174)

1987    6.05       1.95       4.10      (14,433)   4.55     	 2.14       2.41    (11,037)

1988    11.36      3.20       8.14      (27,492)   9.07            6.04        3.02   (22,219)

1989    16.09      5.64      11.05      (38,894)   8.10      	 4.96        3.14   (19,931)

1990    25.23      6.40      18.82      (62,750)   8.28     	 4.29        4.00   (21,037)

1991    30.67    	 7.55      23.12      (78,100)   6.05     	 3.52        2.53   (15,620)

1992    41.96      9.71      32.25     (107,874)   5.56     	 3.15        2.42   (14,568)

1993    33.91      8.32      25.60      (88,715)   5.51      	 3.54        1.98   (14,454)

1994    42.66     10.48      30.18     (113,411)   5.22     	 3.44        1.78   (13,944)

-----------------------------------------------------------------------------------------------
(sup a)Causes include Escherichia coli (0080), Arizona group (0081), Aerobacter aerogenes
(Enterobacter) (0082), Proteus mirabilus and morganii (0083), Staphylococcus (00841), 
Pseudomonas (00842), Campylobacter (00843), Yersinia enterocolitica (00844), Clostridium
difficile (00845), other anaerobes (00846), other gram-negative (00847), other (00849). 
Codes 00843-00847 were added in 1992.
(sup b)Causes include cholera (001), typhoid and paratyphoid fever (002), other salmonella 
infections (003), shigellosis (004), bacterial enteritis, unspecified (0085), bacterial 
infection in conditions classified elsewhere (041).
(sup c)Age-adjusted discharge rates per 100,000 population, adjusted to the 1990 U.S. population.
(sup d)Contribution to age-adjusted discharge rate from persons younger than age 45 years 
and from persons age 45 years and older.

Death-to-Discharge Ratio

The ratio of deaths to hospitalizations for protozoal and ill-defined
causes of diarrhea remained stable between 1985 and 1992. However, despite
the increasing incidence of enteric bacteria-caused deaths, the ratio of
deaths to hospitalizations declined from 6.6 per 1,000 to 2.7 per 1,000
hospitalizations (p < 0.001). The ratio of hospitalizations to deaths in
the viral cause category declined from 6.0 per 1,000 to 2.9 per 1,000
hospitalizations (p < 0.001).

Clostridium difficile Infections

Additional disease codes for
hospital discharge coding were      Table 4. Age-specific rates of
added in 1992, reducing the number  hospitalizations (estimated
of discharges coded to ICD9 00849.  discharges) by diagnostic group
Examination of all computerized
health-care billing records (1993   --------------------------------------
to 1996) with an ICD9 of
00843-00849 from the Lovelace                 Discharge rate/100,000
Hospital and Lovelace Health Care             population (discharges)
Systems in Albuquerque, New Mexico,               
found 94 inpatients with these
codes; C. difficile (ICD9 00845)            ------------------------------
accounted for 73%. This suggests
that C. difficile infection was     Age        1985-89        1990-94
likely to have been the most common
pathogen previously coded to ICD9   --------------------------------------
00849. Eighty-six percent of        Bacterial causes(sup a)
patients diagnosed with C.
difficile were younger than age 60
with 65% younger than age 40. In
addition, the records of 22 (88%)   1       57.5 (10,374)  100.8 (19,536)                                                           
of 25 Washington State deaths
occurring between 1985 and 1996     1-4     7.5 ( 5,416)   21.5 ( 16,696)
with a multiple cause of death code 5-14    4.6 ( 7,988)   8.2 ( 14,988)
ICD9 0084 cited C. difficile.
                                    15-24   7.6 (14,739)   10.6 ( 19,216)

Conclusions                         25-34   13.0 (26,955)  13.5 ( 28,583)

Infectious diarrhea remains an      35-44   10.1 (17,089)  18.5 ( 36,871)
uncommon cause of hospitalization
and accounted for almost the same   45-54   11.1 (13,551)  26.4 ( 36,152)
number of deaths in 1992 as in      55-64   18.1 (19,302)  41.7 ( 43,774)
1980. Increases in death rates for
bacterial causes offset stable or   65-74   34.9 (30,182)  95.4 ( 87,902)
declining death rates for viral,
parasitic, and ill-defined causes   75      65.9 (39,903)  211.8(145,965)
of diarrhea. The increase in death
rates for "other specified enteric  Protozoal causes(sup b)
bacteria" was due to increases in
deaths associated with ICD9 code
0084, "uncoded but specified        1       3.8 ( 680)     9.1 ( 1,767)
bacterial pathogens." Increases in  1-4     3.0 (2,157)    2.6 ( 2,006)
hospitalization rates for these
"uncoded but specified bacterial    5-14    1.4 (2,508)    1.3 ( 2,426)

pathogens" correspond temporally to 15-24   3.3 (6,337)    1.8 ( 3,228)
the increase in death rates coded
to ICD9 0084. A review of recent    25-34   4.3 (8,934)    5.6 (11,941)

hospitalization records from a New  35-44   5.7 (9,723)    5.0 (10,044)
Mexico health maintenance
organization and death records from 45-54   4.8 (5,902)    2.8 ( 3,894)

Washington State show that C.       55-64   4.5 (4,786)    3.4 ( 3,611)
difficile was the most common
pathogen in this coding group.      65-74   9.0 (7,783)    5.0 ( 4,640)
These findings suggest that C.
difficile was likely to have been   75      12.4 (7,507)   6.6 ( 4,561)
responsible for the increase in     
and death rates from enteric
both age-adjusted hospitalization   Viral causes(sup c)
bacterial pathogens.                1       167.6 (30,234) 387.7 (75,120)

Increases in death and              1-4     8.9 ( 6,400)   67.1 (52,021)
hospitalization rates due to
bacterial causes may simply         5-14    8.3 (14,566)   16.3 (29,617)
indicate improved ICD9 coding for
both hospitalization and death or   15-24   10.4 (20,142)  13.5 (24,577)

increased diagnostic accuracy. New  25-34   15.3 (31,694)  17.4 (36,913)
kits for detection of C. difficile
toxins in stool samples may have    35-44   8.8 (14,837)   11.4 (22,707)

resulted in increased C. difficile  45-54   6.7 ( 8,209)   8.3 (11,312)
diagnoses. If so, the increase in
enteric bacterial infections,       55-64   10.6 (11,231)  5.4 ( 5,658)

likely due to C. difficile, may not 65-74   10.4 ( 9,020)  8.1 ( 7,481)
be a true increase in illness from
this pathogen. If this were the     75      18.8 (11,390)  15.8 (10,893)
case, however, one might predict a  
corresponding reduction in		Ill-defined diarrheal causes(sup d)
hospitalizations and deaths from
ill-defined diarrheal causes. Death
rates for ill-defined causes of     1       178.9 (32,272) 283.9 (55,006)
diarrhea presumed to be infectious
actually rose somewhat during 1990  1-4     27.0 (19,452)  62.8 (48,694)

to 1992, and hospitalization rates  5-14    13.3 (23,301)  17.3 (31,594)
have remained stable. Further
studies are needed to determine if  15-24   24.4 (47,242)  18.2 (32,966)
this increased age-adjusted         25-34   30.3 (62,701)  21.7 (45,966)
hospitalization and death rate is
due to increased C.                 35-44   25.7 (43,618)  19.4 (38,516)

difficileassociated disease and,   45-54   29.5 (35,972)  28.9 (39,459)
if so, to identify risk factors for
infection and disease.              55-64   46.8 (49,773)  32.6 (34,245)

Additional information about the    65-75   75.5 (65,336)  50.2 (46,257)

causes of increased hospitalization 75      114.7 (69,476) 93.1 (64,163)
and death from enteric bacterial
pathogens could be provided by a    --------------------------------------
review of computerized health-care
delivery records. C. difficile can  (sup d)Discharges with an enteric bacteria
be associated with almost any       diagnosis (ICD9 001-005, 0080-0085,
antibiotic therapy, but it has been 041).
particularly associated with        (sup b)Discharges with an enteric parasite
aminopenicillins, cephalosporins,   diagnosis (ICD9 006-007, 127, 129).
and clindamycin, which have greater (sup c)Discharges with an enteric virus
effects on the intestinal flora     diagnosis (ICD9 0086, 0088, 045, 047,
(12). Other factors trigger C.      048, 0700, 0701, 074).
difficile toxinassociated colitis
(13). Records from health
maintenance organizations containing prior diagnoses and pharmaceutical
treatments may provide better understanding of the risk factors.

In this study, data from one hospital system in one state and deaths
occurring in one state suggest that a likely cause of the national increase
in hospitalizations and deaths due to enteric bacteria may be C. difficile
infection. It is possible that C. difficile is a relatively more important
cause of hospitalization at Lovelace Health Systems in New Mexico and of
death in Washington State than elsewhere in the United States.

The age-specific rates of hospital discharge coded to enteric bacterial,
viral, and ill-defined conditions increased for children under 5 years of
age. Reasons for these increases merit further inquiry. No increases in
death rates from these pathogens were observed in these age groups.
However, since adverse outcomes of infectious diarrhea that requires
hospitalization may indicate problems with access to health care,
information on the socioeconomic characteristics of the families of these
children would be of interest.

      Dr. Frost is an epidemiologist and the director of the Southwest
Center for Managed Care Research in Albuquerque, New Mexico. His research
interests include the study of waterborne diseases and the use of
health-care data for promoting public health programs within managed care
organizations.

      Address for correspondence: Floyd Frost, Southwest Center for Managed
Care Research, 2425 Ridgecrest Drive, S.E., Albuquerque, NM 87108, USA;
fax: 505-262-7598; e-mail: ffrost@lrri.org.

References

  1. Murray CJL, Lopex AD. The global burden of disease: a comprehensive
     assessment of mortality and disability from diseases, injuries and
     risk factors in 1990 and projected to 2020. Geneva: World Health
     Organization; 1996.
  2. Garthright WE, Archer DI, Kvenberg JE. Estimates of incidence and cost
     of intestinal infectious diseases in the United States. Public Health
     Rep 1988;103:107-15.
  3. Gibson JJ, Alexander GR. Correlates of infant death from infectious
     diarrhea in the southeastern United States. South Med J 1985;78:26-30.
  4. Mel-Shang H, Glass RI, Pinsky PF, Young-Okoh N, Sappenfield WM,
     Buehler JW, et al. Diarrhea deaths in American children: are they
     preventable? JAMA 1988;260:3281-5.
  5. Lew JF, Glass RI, Gangarosa RE, Cohen IP, Bern C, Moe CL. Diarrhea
     deaths in the United States, 1979 through 1987. JAMA 1991;265:3280-4.
  6. Cheney CP, Wong RKH. Acute infectious diarrhea. Gastrointestinal
     Emergencies 1993;77:1169-90.
  7. Moore AC, Herwaldt BL, Craun FG, Calderon RL, Highsmith AK, Juranek
     DD. Surveillance for waterborne disease outbreaksUnited States
     1991-92. MMWR CDC Surveill Summ 1993;42(5):1-22.
  8. Calderon RL, Johnson CC, Craun GF, Dufour AP, Karlin RK, Sinks T, et
     al. Health risks from contaminated water: do class and race matter?
     Toxicol Ind Health 1993;9:879-900.
  9. Kramer MH, Herwaldt BL, Craun GF, Calderon RL, Juranek DD.
     Surveillance for waterborne-disease outbreaksUnited States 1993-94.
     MMWR CDC Surveill Summ 1996;45:1-33.
 10. Solo-Gabriele H, Neumeister S. U.S. outbreaks of cryptosporidiosis.
     Journal of the American Water Works Association 1996;(Sept):76-84.
 11. Simmons WR. Development of the design of the NCHS hospital discharge
     survey. Vital Health Statistics 1970;2(39).
 12. Job ML, Jacobs NF. Drug-induced Clostridium difficile-associated
     disease. Drug Saf 1997:17:37-46.
 13. Caputo GM, Weitekamp MR, Bacon AE, Whitener C. Clostridium difficile
     infection: a common clinical problems for the general internist. J Gen
     Intern Med 1994;9(9):528-33.
---------------------------------------------------------------------------------
---------------------------------------------------------------------------------

Dispatches

Introduction of Aedes albopictus into a La Crosse VirusEnzootic Site in
Illinois

Uriel Kitron,* Jack Swanson, Michael Crandell, Patrick J. Sullivan,*
Justin Anderson, Robert Garro,* Linn D. Haramis, and Paul R. Grimstad
*University of Illinois, Urbana, Illinois, USA; Illinois Department of
Public Health, Peoria, Illinois, USA; Peoria City/County Health
Department, Peoria, Illinois, USA; and University of Notre Dame, Notre
Dame, Indiana, USA; Illinois Department of Public Health, Springfield,
Illinois, USA

---------------------------------------------------------------------------
      In late summer and fall 1997, Aedes albopictus mosquitoes were
      found in Peoria, Illinois, a long recognized focus of La Crosse
      virus transmission. Larvae were found in tires and other
      artificial containers, biting adults were recovered, and eggs
      were collected in oviposition traps within a 25-ha area. One
      chipmunk trapped < 0.25 km from the infested area tested
      positive for neutralizing antibodies against La Crosse virus.

Aedes albopictus (Skuse) was probably introduced into the United States in
used tires from northern Asia (1) and has spread throughout much of the
southern United States, often displacing Ae. aegypti (L.) (2,3). Although
Ae. albopictus mosquitoes have a short flight range (200 m to 600 m), they
are readily transported in containers and vehicles (3,4).

In laboratory studies, Ae. albopictus was a more efficient vector of La
Crosse (LAC) virus than the natural vector, Ae. triseriatus (Say) (5), and
could readily transmit LAC virus to chipmunks (6). In field and laboratory
studies of larval competition in tires and artificial containers, Ae.
albopictus outnumbered Ae. triseriatus (7,8). LAC virus has not been
isolated from field-caught Ae. albopictus females in an endemic-disease
area.

LAC virus infection, the most common cause of pediatric arboviral
encephalitis in the United States (9), is endemic in several midwestern and
mid-Atlantic states (10). In Illinois, human clinical cases of LAC virus
encephalitis are concentrated around Peoria, often near discarded tires and
containers (11,12). LAC virus encephalitis is one of the main threats
associated with the introduction of Ae. albopictus to the midwestern United
States (1,2).

Mosquito Surveillance

Peoria City/County Health Department and Illinois Department of Public
Health personnel annually conduct surveillance of container-breeding
mosquitoes in Peoria County by searching for and monitoring artificial
containers, collecting adult females, and monitoring oviposition activity
(13).

During an investigation of a suspected LAC virus encephalitis case, Ae.
albopictus larvae and adults were collected on August 20, 1997, from
approximately 10 tires and a steel box. During annual surveillance in
August and September 1997, Ae. albopictus larvae and pupae were collected
from tires and other containers. Adult mosquitoes were collected near most
breeding sites and in several additional locations (Figure). Approximately
160 Ae. albopictus and one Ae. triseriatus adult females were collected.
All artificial containers with mosquito larvae were treated with Abate
larvicide. During the first week of October, larvae were also collected
from a small boat containing water, and adults were collected in the
immediate vicinity.

Nine oviposition traps were placed in the infested site starting August 21,
1997 (Figure). The Aedes eggs found in seven traps were hatched, and the

 [Figure.] Locations in Peoria,       larvae were reared to adults. Ae.
 Illinois, where Aedes albopictus     albopictus were identified from four
 larvae and adults were found and     traps, with two traps each yielding
 where a La Crossepositive chipmunk  more than 100 mosquitoes. Ae.
 was trapped. Location of             triseriatus were identified from four
 oviposition traps are shown,         traps, with the total numbering less
 including traps where no Ae.         than half of Ae. albopictus. No LAC
 albopictus eggs were deposited.      virus was isolated from these
 Insets show location of Peoria and   field-collected mosquitoes.
 of the study sites.
                                      
Reservoir Host Trapping and Sampling

Fifteen Tomahawk live traps (model 102/rat: 40.6 x 12.7 x 12.7 cm.;
Tomahawk Live Trap Company, Madison, WI) were placed 5 to 15 m apart in an
east-west transect in St. Joseph Cemetery, Peoria, Illinois, on September
20, 1997 (Figure). To attract chipmunks and squirrels, traps were baited
with sunflower seeds and small pieces of peanut butter sandwiches. Trapped
animals were processed and released after recovering from anesthesia.
Animal trapping and handling procedures were conducted in accordance with a
protocol approved by the Laboratory Animal Care Advisory Committee at the
University of Illinois (protocol #V6R131).

In St. Joseph Cemetery, 10 chipmunks (Tamias striatus) were caught in 15
traps opened one morning (Figure). This 67% trapping success rate is high
compared with the rate at Forest Park Nature Center (Figure), a
long-recognized LAC virusenzootic transmission focus in Peoria (11), where
on the same date, chipmunks and squirrels (Sciurus carolinensis and S.
niger) were collected in 15 (30%) of 50 traps.

LAC Antibody Detection

Blood (0.2 ml) was taken from anesthetized chipmunks by a suborbital sinus
puncture behind the right eye with a 100-l capillary tube (14) and
absorbed onto two Nobuto (Toyo Roshi Kaisha, Tokyo, Japan) filter strips,
which were tested for antibody at the University of Notre Dame.
Neutralizing (N) antibodies against LAC virus were detected by a cell
culture assay and the serum-dilution neutralization test (SDNt) (15).
Titers, calculated by the method of Reed and Muench (16), were expressed as
the highest dilution showing < 50% cytopathic effects. Homologous (LAC
virus) and heterologous (Jamestown Canyon, Trivittatus, eastern equine
encephalitis, St. Louis encephalitis virus) mouse hyperimmune ascitic
fluids served as positive and negative N antibody controls, respectively.

Of the 10 chipmunks trapped in St. Joseph Cemetery, 1 (10%) was positive
(titer = 8) for N antibodies against LAC virus. Two (22%) of nine chipmunks
trapped on the same date in Forest Park Nature Center were N-antibody
positive (titers 4, 16). Low titers may be associated with the differing
amounts of sera present in the Nobuto strips (all sera were eluted in the
same amount of media and then diluted as if all were equal). Low titers may
also be caused by animals having been infected earlier in the season.
Because little serum was available, we conducted only SDNt, our most
sensitive and specific serologic procedure for detecting California
serogroup positives.

Mapping

The spatial distribution of mosquito collections (adults and larvae),
locations and catches of oviposition traps, and locations of chipmunk traps
were overlaid on a street map of the city of Peoria (Figure). The LAC
virusseropositive chipmunk caught in St. Joseph Cemetery was trapped
approximately 150 m from sites where Ae. albopictus eggs, larvae, and
adults were collected (Figure). These data were compared with data on human
cases (with known addresses) (12); all epidemiologic and entomologic data
were stored in a geographic information system (MapInfo GIS, Troy, NY) as
part of an ongoing study. The system allows for ready management of
georeferenced data, continuous updating of entomologic and epidemiologic
data, and production of custom maps.

Conclusion

The detection of Ae. albopictus mosquitoes, coupled with the trapping of an
LAC virusseropositive chipmunk within 150 m of larval and adult Ae.
albopictus collection sites (well within the flight range of Ae.
albopictus), is noteworthy. Human cases were reported within 1.5 km of
these sites in the 1970s and in 1994. Even within Peoria, the spatial
distribution of enzootic foci and human cases is patchy. These findings are
the first evidence that Ae. albopictus has been introduced into the heart
of an urban and suburban area where LAC virus has long been endemic and has
caused disease in humans. A focus in rural southeastern Indiana has been
described (17) in a highly LAC virus-endemic area of the state (18).

Studies in Indiana and an estimate of the northern limits of the
distribution of Ae. albopictus (19-21) suggest that Ae. albopictus may
become entrenched in central Illinois, especially after mild winters. Ae.
albopictus eggs stored outdoors at the University of Notre Dame in northern
Indiana were hatched successfully in late February 1998, following several
days of record warm temperatures, and in mid-April after cold weather had
passed. In addition, Ae. albopictus have long been detected in
Indianapolis, Indiana, at a latitude close to that of Peoria, Illinois (M.
Sinsko, Indiana State Board of Health, pers. comm.).

The detection of this Ae. albopictus focus marks the beginning of a natural
experiment to test the ability of Ae. albopictus to overwinter in central
Illinois, displace Ae. triseriatus from artificial and natural containers,
and survive intensive control efforts. If this population of Ae. albopictus
reemerges in 1998, its vertebrate host feeding preference and its ability
to act as a vector for enzootic and endemic transmission of LAC virus
should also be evaluated.

Acknowledgment

      M. Guerra and M. Lancaster provided fieldwork assistance.

      This study was supported by the Used Tire Management Fund, State of
Illinois, U.S. Department of Agriculture Animal Health and Disease Funds, a
UIUC Research Board award to U. Kitron, and NIH/NIAID grant AI02753 to P.
Grimstad.

      Uriel Kitron is professor and chair of the Division of Epidemiology
and Preventive Medicine in the Department of Veterinary Pathobiology,
College of Veterinary Medicine, University of Illinois. He studies the
ecology and epidemiology of vector-borne diseases, including malaria, Lyme
disease, and mosquito-borne arboviruses. He is particularly interested in
landscape ecology, spatial analysis, and risk assessment of infectious
diseases.

      Address for correspondence: Uriel Kitron, College of Veterinary
Medicine, University of Illinois, 2001 S. Lincoln, Urbana, IL 61801, USA;
fax: 217-244-7421; e-mail: u-kitron@uiuc.edu.

References

  1. Hawley WA, Reiter P, Copeland RS, Pumpuni CB, Craig GB Jr. Aedes
     albopictus in North America: probable introduction in used tires from
     northern Asia. Science 1987;236:1114-6.
  2. Rai KS. Aedes albopictus in the Americas. Annu Rev Entomol
     1991;36:459-84.
  3. Moore CG, Mitchell CJ. Aedes albopictus in the United States: ten-year
     presence and public health implications. Emerg Infect Dis
     1997;3:329-34.
  4. Estrada-Franco JG, Craig GB Jr. Biology, disease relationships, and
     control of Aedes albopictus. Pan American Health Organization
     Technical Paper No. 42; 1995.
  5. Grimstad PR, Kobayashi JF, Zhang M, Craig GB Jr. Recently introduced
     Aedes albopictus in the United States: potential vector of La Crosse
     virus (Bunyaviridae: California serogroup). J Am Mosq Control Assoc
     1989;5:422-7.
  6. Cully JF Jr, Street TG, Heard PB. Transmission of La Crosse virus by
     four strains of Aedes albopictus to and from the eastern chipmunk
     (Tamias striatus). J Am Mosq Control Assoc 1992;8:237-40.
  7. Ho BC, Ewert A, Chew LM. Interspecific competition among Aedes
     aegypti, Ae. albopictus and Ae. triseriatus (Diptera: Culicidae):
     larval development in mixed cultures. J Med Entomol 1989;26:615-23.
  8. Novak MG, Higley LG, Christianssen CA, Rowley WA. Evaluating larval
     competition between Aedes albopictus and A. triseriatus (Diptera:
     Culicidae) through replacement series experiments. Environmental
     Entomology 1993;22:311-8.
  9. Calisher CH. Medically important arboviruses of the United States and
     Canada. Clin Microbiol Rev 1994;7:89-116.
 10. Grimstad PR. California group viruses. In: Monath TP, editor. The
     arboviruses: epidemiology and ecology. Vol II. Boca Raton (FL): CRC
     Press; 1988. p. 99-136.
 11. Clark GG, Pretula HL, Rohrer WH, Harroff RN, Jakubowski T. Persistence
     of La Crosse virus (California encephalitis serogroup) in
     north-central Illinois. Am J Trop Med Hyg 1983;32:175-84.
 12. Kitron U, Michael J, Swanson J, Haramis L. Spatial analysis of the
     distribution of La Crosse encephalitis in Illinois, using a geographic
     information system and local and global spatial statistics. Am J Trop
     Med Hyg 1997;57:469-75.
 13. Moore CG, McLean RG, Mitchell CJ, Nasci RS, Tsai TF, Calisher CH, et
     al. Guidelines for arbovirus surveillance programs in the United
     States. Fort Collins (CO): U.S. Department of Health and Human
     Services; 1993.
 14. McLean RG, Ubico SR, Cooksey LM. Experimental infection of the eastern
     chipmunk (Tamias striatus) with the Lyme disease spirochete (Borrelia
     burgdorferi). J Wild Dis 1993;29:527-32.
 15. Pantuwatana S, Thompson WH, Watts DM, Hanson RP. Experimental
     infection of chipmunks and squirrels with La Crosse and trivittatus
     viruses and biological transmission of La Crosse virus by Aedes
     triseriatus. Am J Trop Med Hyg 1972;21:476-81.
 16. Reed LJ, Muench HA. A simple method of estimating fifty per cent
     endpoints. Am J Trop Med Hyg 1938;27:493-7.
 17. Cully JF Jr, Heard PB, Wesson DM, Craig GB Jr. Antibodies to La Crosse
     virus in eastern chipmunks in Indiana near an Aedes albopictus
     population. J Am Mosq Control Assoc 1991;7:651-3.
 18. Grimstad PR, Barrett CL, Humphrey RL, Sinsko MJ. Serologic evidence
     for widespread infection with La Crosse and St. Louis encephalitis
     viruses in the Indiana human population. Am J Epidemiol
     1984;119:913-30.
 19. Hawley WA, Pumpuni CB, Brady RH, Craig GB Jr. Overwintering survival
     of Aedes albopictus (Diptera: Culicidae) eggs in Indiana. J Med
     Entomol 1989;26:122-9.
 20. Nawrocki SJ, Hawley WA. Estimation of the northern limits of
     distribution of Aedes albopictus in North America. J Am Mosq Control
     Assoc 1987;3:314-7.
 21. Hanson SM, Craig GB Jr. Aedes albopictus (Diptera: Culicidae) eggs:
     field survivorship during northern Indiana winters. J Med Entomol
     1995;32:599-604.
---------------------------------------------------------------------------------
---------------------------------------------------------------------------------

Dispatches

Mycobacterium canettii, the Smooth Variant of M. tuberculosis, Isolated
from a Swiss Patient Exposed in Africa

Gaby E. Pfyffer,* Raymond Auckenthaler, Jan D. A. van Embden, and Dick
van Soolingen
*University of Zurich, Zurich, Switzerland; University Hospital, Geneva,
Switzerland; and National Institute of Public Health and the Environment
(RIVM), Bilthoven, The Netherlands

---------------------------------------------------------------------------
      An exceptionally smooth and glossy morphotype of Mycobacterium
      tuberculosis complex was isolated from a 56-year-old Swiss
      patient with mesenteric tuberculosis. Direct 16S rRNA sequence
      analysis of the hypervariable signature gene regions revealed a
      100% homology to the specific M. tuberculosis complex sequence.
      Spoligotyping and restriction fragment length polymorphism
      analyses using the insertion sequences IS6110 and IS1081 and
      the polymorphic GC-rich sequence as additional genetic markers
      identified the isolate as the novel taxon M. canettii. Like a
      Somali child with a similar case, this patient probably
      contracted the infection in Africa, which raises questions
      about the geographic distribution of M. canettii.

A novel pathogenic taxon of the Mycobacterium tuberculosis complex was
described (1) in 1997 in a highly exceptional strain, designated So93,
isolated in 1993 from a 2-year-old Somali child with lymphadenitis.
Although on the basis of biochemical tests and the 16S rRNA gene sequence
this isolate was clearly a member of the M. tuberculosis complex, it
exhibited an unusually smooth and glossy colony morphology. Genetically,
strain So93 differed from all known M. tuberculosis complex strains (nearly
2,000 M. tuberculosis isolates have been examined) (1). It contained a
single copy of the insertion sequence IS1081, had a direct repeat region
with an exceptional composition, and differed in the sequence of the recA
gene. In 1969, Canetti had isolated from a French patient an M.
tuberculosis strain with a smooth colony morphology. Daff et al. (2) had
demonstrated that this particular strain differed from the commonly rough
strains by having large amounts of lipooligosaccharides. Isolate So93 and
the Canetti strain from the 1960s shared virtually all phenotypic and
genetic characteristics. Therefore, these two smooth M. tuberculosis
variants were designated "M. canettii" (1).

We report here a third tuberculosis (TB) case caused by M. canettii. The
mycobacterial isolate NZM 217/94 was cultured from pus aspirated from the
ileocecal region of a 56-year-old male patient born in Switzerland.
Phenotypic and genetic characteristics were compared with both the isolate
So93 and the Canetti strain.

The patient had been working for an international organization since 1963,
in Uganda for 20 years, then in Kenya for 1 year. HIV infection was
detected in this patient in 1993. That same year, he contracted pneumonia
of unknown etiology, which was successfully treated with cotrimoxazole. Two
short episodes of severe diarrhea followed in 1994. The patient was
transferred from Kenya to the University Hospital in Geneva, where a
partial occlusion of the ileum caused by an extrinsic compression was
diagnosed. A right hemicolectomy was performed. An extensive inflammatory
mass of mesenteric lymph nodes was strongly positive for acid-fast bacilli
by direct microscopy. After as little as 1 week, culture (BACTEC 12B
medium; Becton Dickinson Diagnostic Instrument Systems, Sparks, MD) yielded
mycobacteria, which were identified as M. tuberculosis complex. The patient
was already in stage C2 of AIDS, which favored a rapid development of
mesenteric disease. No pulmonary signs of the disease were observed (3).
Response to quadruple antituberculotic chemotherapy was excellent. However,
within 3 months, extensive cutaneous lesions developed due to a generalized
highly malignant non-Hodgkin lymphoma, another complication of AIDS, known
as fulminant disease with poor prognosis. A cytostatic therapy was
initiated. The patient returned to Kenya but did not follow any treatment
regimens. He was hospitalized in Geneva in 1995 with severe cachexia and
died 9 months later. The postmortem findings showed massive invasion of the
abdominal cavity with malignant lymphoma but no further evidence of active
TB, neither by histology nor by culture.

Although the isolate exhibited abundant smooth and glossy colonies on
Middlebrook 7H10 agar (Figure 1), the M. tuberculosis complex gene probe
(Accuprobe; Gen-Probe Inc., San Diego, CA) was positive. Growth was
strictly aerobic. The strain reduced nitrate, but niacin production and
68C catalase were negative. Analysis of cellular fatty acids was done by
gas-liquid chromatography in conjunction with the Microbial Identification
System (Microbial ID Inc., Newark, DE) (4) and yielded M. tuberculosis
complex. Cellular fatty acid esters ranged from C12:0 to C20:0, the major
components being C16:0 (35.5%), C18:1 (24.9%), C18:0 (11.2%), and
tuberculostearic acid (10 Me-18:0 TBSA; 12.1%). Mycolic acids consisted of
[alpha], methoxy, and keto mycolates, which are typical for
tubercle bacilli. Guanine plus cytosine content of the DNA was 66.4 mol%.
The polymerase chain reaction/restriction enzyme pattern of the amplified
65-kDa heat shock protein gene fragment (65-kDa-HSP-PRA) (5) yielded bands
of 245/125/80 bp and of 160/140/70 bp, respectively, following BstEII and
HaeIII digests, a pattern compatible with M. tuberculosis complex (5).
Direct 16S rDNA sequence analysis (6) showed that the hypervariable
signature gene regions were identical to M. tuberculosis complex.

                                  Strain NZM 217/94 was susceptible to
                                  p-nitro-[alpha]-acetylamino-
                                  -hydroxypropiophenone (NAP) (7) and
                                  thiophene carboxylic acid (T(sup 2)H) 
                                  as to all primary antituberculotic drugs
[Figure 1.] Colony morphology of  as well(isoniazid 0.2 and 1g/ml, 
strain NZM 217/94 on Middlebrook  rifampin 0.2 g/ml, ethambutol 2.5 g/ml), 
7H10 agar.                        except pyrazinamide (100 g/ml). 
                                  Susceptibility was observed against the 
                                  second-line drugs streptomycin (10 g/ml),
ethionamide (5 g/ml), rifabutin (1 g/ml), sparfloxacin (1 g/ml),
ofloxacin (1 g/ml), ciprofloxacin (1 g/ml), and p-amino salicylic acid (2
g/ml). Susceptibility testing of the primary antituberculotics was done
radiometrically (BACTEC 460TB System, Becton Dickinson) (8). Second-line
drugs have been tested by the method of proportion (indirect test) on
Middlebrook 7H10 agar (9).

To determine whether the isolate from the Swiss patient represented the M.
canettii genotype, restriction fragment length polymorphism (RFLP) analyses
and spoligotyping were performed (1,10). The IS6110 RFLP patterns of the
Swiss (NZM 217/94) and the Somali isolates (So93) were identical (Figure
2a). As previously shown, the Canetti isolate contains an additional copy
of this insertion sequence. Furthermore, the Swiss isolate contains a
single copy of IS1081 on a similarly sized PvuII restriction fragment, as
previously found in the So93 strain (Figure 2b). The patterns generated
with the polymorphic GC-rich repetitive sequence (PGRS) of strains NZM
217/94 and So93 were identical; whereas for the Canetti strain, 7 out of 20
PGRS-containing AluI restriction fragments were found at other positions
(data not shown). By the recently developed spoligotyping technique (10) we
could demonstrate that the Swiss isolate contained the same two spacer
sequences found in both the Canetti strain and the So93 isolate (Figure
2c).

These data demonstrate the first TB case
caused by M. canettii in a Swiss          
patient. RFLP analyses using IS6110,      
IS1081, and PGRS as probes as well as     
spoligotyping revealed the same DNA
fingerprint patterns for the Swiss        [Figure 2.] IS6110 (a) and IS1081
isolate as previously found for strain    (b) hybridization patterns of
So93 isolated from a Somali child. With   PvuII-digested genomic DNA.
its highly exceptional smooth and glossy  Lanes 1 to 3: clinical M.
colony morphology and the unusual,        tuberculosis isolates; lane 4:
abundant growth, the Swiss isolate        the Somali isolate, So93; lane
belongs to the same taxon of the M.       5: the Canetti strain isolated
tuberculosis complex as the Somali        in France in 1969; lane 6: the
isolate and the Canetti strain. The       Swiss isolate, NZM 217/94; lane
Canetti strain isolated in France about   7: M. tuberculosis Mt14323
30 years ago (from a patient of whom no   (reference strain). Figure c
further details are available) showed     represents spoligotyping
RFLP patterns that were highly similar    patterns. Lane 1: M.
but not identical to the ones of the      tuberculosis H37Rv (reference
Swiss and Somali isolates. Our data       strain); lane 2: the Canetti
indicate 1) that the M. canettii taxon    strain; lane 3: the Somali
comprises strains with some degree of     isolate, So93; lane 4: the Swiss
evolutionary divergence and 2) that this  isolate, NZM 217/94.
evolutionary lineage already existed but
was likely overlooked because the lack
of adequate genetic characterization techniques.

Although the exact source of infection in this latest clinical case of M.
canettii could not be established with certainty, the patient was
repeatedly exposed to TB patients in East Africa. Since the patient had no
known exposure to pets or wild animals, a human source of infection seems
very likely. This report indicates that M. canettii may be of wider
prevalence than previously thought; therefore, further studies are needed
to determine its geographic distribution (i.e., its reservoir) and
eventually the extent of genetic variation and presence of virulence
factors associated with this particular taxon of the M. tuberculosis
complex. In addition, this study demonstrates the value of applying a
combination of several subtyping methods by which future studies can be
directed to determine whether M. canettii is emerging as an important
pathogen.

Acknowledgments

      We thank A. Strssle, K. Kremer, P. de Haas, J. Kwakkel, and R. Wirth
for technical assistance.

      Financial support was obtained from the Belport Foundation (Zurich),
the Hartmann Mller Foundation (Zurich), the Swiss Federal Office of Public
Health (Berne), the European Community Programme for Science Technology and
Development and Biomed 2, and the Dutch Foundation for Technical Sciences.

      Dr. Pfyffer is professor of medical microbiology at the Department of
Medical Microbiology and head of the Swiss National Center for
Mycobacteria, both of the University of Zurich. Areas of expertise are in
the diagnosis of TB and nontuberculous mycobacteria, molecular epidemiology
of TB and other mycobacteria, direct detection of mycobacteria in clinical
specimens by nucleic acid amplification, and external quality control in
mycobacteriology. Research interests include the chemical and molecular
taxonomy of mycobacteria, DNA fingerprinting and other typing methods, and
new techniques for growth and detection of mycobacteria and for
susceptibility testing.

Address for correspondence: Gaby E. Pfyffer, Swiss National Center for
Mycobacteria, Department of Medical Microbiology, University of Zurich,
Gloriastrasse 30, 8028 Zurich, Switzerland; fax: 41-1-634-49-18; e-mail:
pfyffer@immv.unizh.ch.

References

  1. Van Soolingen D, Hoogenboezem T, de Haas PEW, Hermans PWM, Koedam MA,
     Teppema KS, et al. A novel pathogenic taxon of the Mycobacterium
     tuberculosis complex, Canettii: characterization of an exceptional
     isolate from Africa. Int J Syst Bacteriol 1997;47:1236-45.
  2. Daff M, McNeil M, Brennan PJ. Novel type-specific
     lipooligosaccharides from Mycobacterium tuberculosis. Biochemistry
     1991;30:378-88.
  3. Jakubowski A, Elwood RK, Enarson DA. Clinical features of abdominal
     tuberculosis. J Infect Dis 1988;158:687-92.
  4. Smid I, Salfinger M. Mycobacterial identification by computer-aided
     gas-liquid chromatography. Diagn Microbiol Infect Dis 1994;19:81-8.
  5. Telenti A, Marchesi F, Balz M, Bally F, Bttger EC, Bodmer T. Rapid
     identification of mycobacteria to the species level by polymerase
     chain reaction and restriction enzyme analysis. J Clin Microbiol
     1993;31:175-8.
  6. Kirschner P, Springer B, Vogel U, Meier A, Wrede A, Kieckenbeck M, et
     al. Genotypic identification of mycobacteria by nucleic acid sequence
     determination: report of a 2-year experience in a clinical laboratory.
     J Clin Microbiol 1993;31:2882-9.
  7. Laszlo A, Siddiqi S. Evaluation of a rapid radiometric differentiation
     test for Mycobacterium tuberculosis, Mycobacterium bovis, and other
     mycobacteria. J Clin Microbiol 1984;19:694-8.
  8. Siddiqi SH. BACTEC 460TB System. Product and Procedure Manual.
     MA-0029. Sparks (MD): Becton Dickinson Diagnostic Instrument Systems;
     1995.
  9. Inderlied C, Nash KA. Antimicrobial agents: in vitro susceptibility
     testing, spectra of activity, mechanisms of action and resistance, and
     assays for activity in biologic fluids. In: Lorian V, editor.
     Antibiotics in laboratory medicine. Baltimore: Williams & Wilkins;
     1996. p. 127-75.
 10. Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D,
     Kuijper S, et al. Simultaneous detection and strain differentiation of
     Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin
     Microbiol 1997;35:907-14.
---------------------------------------------------------------------------------
---------------------------------------------------------------------------------

Dispatches

Human Infections with Shiga Toxin-Producing Escherichia coli Other Than
Serogroup O157 in Germany

Lothar Beutin, Sonja Zimmermann, and Kerstin Gleier
Robert Koch-Institut, Berlin, Germany

---------------------------------------------------------------------------
      We investigated different types of Shiga toxin-producing
      Escherichia coli (STEC) not belonging to serogroup O157 for
      their role as human pathogens. Non-O157 STEC isolated from 89
      human patients in Germany were characterized according to
      serotypes, virulence markers, and association with human
      illness. EaeA-positive STEC were isolated from 54 (60.7%) of
      the patients and were frequently associated with severe
      diarrheal disease, hemolytic uremic syndrome, and young age.
      EaeA-negative STEC were found in 35 (39.3%) of the patients and
      were more associated with clinically uncomplicated cases and
      adult patients. For pediatric patients, a serotype-independent
      diagnosis of STEC is recommended.

Certain strains of Escherichia coli belonging to different O-(LPS) and
H-(flagellar) serotypes produce potent cytotoxins called Shiga toxins (Stx)
(1). The natural hosts of Shiga toxin-producing E. coli (STEC) are farm and
wildlife ruminants. In humans, STEC can cause disease, although the
clinical picture may vary from uncomplicated diarrhea, to hemorrhagic
colitis (HC), to hemolytic uremic syndrome (HUS) (2,3). Large outbreaks and
cases of HC and HUS in humans were mainly associated with STEC strains
belonging to serogroup O157. Human infections with STEC O157 are under
nationwide surveillance in a number of countries, but the detection of
non-O157 STEC infections is often limited to a small number of specialized
laboratories because STEC O157 colonies are more easily detectable on some
culture media than non-O157 STEC types, which are thus often missed in
laboratory diagnosis of stool specimens (4,5). However, humans are likely
more exposed to non-O157 STEC because these strains are more prevalent in
animals and as contaminants in foods than STEC O157 (4,6). Infections with
some non-O157 STEC types, such as O26 and O111, are associated with illness
in humans, but the role of other non-O157 STEC types in human disease needs
further examination (3,7). In Germany, the number of clinical laboratories
performing diagnosis of STEC from stool samples has increased since
immunologic assays (Stx-enzyme-linked immunosorbent assay [ELISA]) specific
for detection of Stx1 and Stx2 became commercially available. The
Stx-specific ELISA kits are useful for detection of different serotypes of
STEC; consequently, more non-O157 STEC-positive stool specimens and
bacterial isolates were sent to our laboratory for confirmation and typing.
In this study, we report on non-O157 STEC infections in 89 human patients
infected during 1996 in Germany. We investigated the relationship between
STEC serotype, STEC virulence factors (such as the eaeA gene, production of
enterohemolysin), Stx1 and Stx2, and the clinical signs in the patients.

Isolation of Non-O157 STEC Strains

Of 89 non-O157 STEC isolates from 89 patients, 46 were isolated in the
collaborating laboratories and 43 in our laboratory from stool samples.
STEC isolates or stool specimens were sent together with patient data (age,
gender, and illness) from 37 private, hospital, and public health
laboratories from rural and urban areas of Germany. In our laboratory, a
small sample of stool was injected into 5 ml of Tryptic Soy Broth and
incubated overnight at 37C (8). Aliquots from the grown stool culture were
tested for toxicity and for Stx-specific DNA sequences by polymerase-chain
reaction (PCR) (9,10). STEC were isolated from Stx-positive stool cultures
by combined use of enterohemolysin agar and the STEC-RPLA test (8).
Enterohemolysin-negative STEC were identified by testing different coliform
colonies with the STEC-RPLA assay. All stool cultures were additionally
plated on Sorbitol MacConkey Agar (SMAC) for identification of
sorbitol-nonfermenting STEC O157 strains.

Most of our collaborating laboratories in Germany used Stx-specific ELISA
for routine examination of stool samples. Examination for STEC was
performed in cases of diarrhea, HUS, or on special request; diagnostic
approaches differed among laboratories. Some supplied only
Stx-ELISA-positive stool specimens for further examination; others
attempted to isolate STEC colonies by using combinations of commercially
available diagnostic tools such as Stx-ELISA, STEC-RPLA, agglutinating
rabbit antisera for enteropathogenic E. coli (EPEC) O-groups including
serogroup O157, enterohemolysin agar, and SMAC. Of the 37 collaborating
laboratories, two used their own methods, such as the Stx-toxicity test or
an Stx-specific PCR for identification of STEC from stool.

In our laboratory, all 89 non-O157 STEC isolates were tested for
cytotoxicity by the Stx test and for production of Stx1 and Stx2 by the
STEC-RPLA assay. The enterohemolytic phenotype was determined on
enterohemolysin agar (8). The presence of the eaeA gene was determined by
PCR using two different primer pairs specific for the conserved region of
the eaeA gene (11,12). Serotyping of O- and H-antigens of E. coli strains
was performed as described (13).

Characterization of Non-O157 STEC Strains

Only one STEC serotype was isolated from each of the 89 patients except for
one patient with HC who excreted simultaneously STEC types O157:H7 and
O26:H11. Of the 89 STEC strains, 69 (77.5%) could be typed to 15 different
O-groups, and 20 were O-untypable or O-rough (Table 1). The STEC strains
varied in H-antigens, which were conserved within some of the O-groups, and
31 STEC strains (34.8%) were nonmotile. Most isolates (n = 59) produced
only Stx1 (66.3%) and 11 (12.4%) only Stx2. Production of Stx1 and Stx2 was
found in 19 (21.3%) strains and was associated with strains of O-groups 113
and 145. Fifty-four (60.7%) of the STEC strains were found eaeA-positive.
The eaeA-gene was present in all STEC strains belonging to some common
serogroups (O26, O103, O111, O118, and O145) and in 8 of 17 STEC O-rough
strains. The enterohemolytic phenotype was expressed in 82 strains (92.1%)
and was highly associated with eaeA positive STEC (53 [98.2%] of 54 were
positive). All STEC strains were positive for fermentation of sorbitol.

Relationship Between STEC Types, Virulence Markers, and Clinical Status of
the Patients

Fifty-one (60.7%) of the patients were female, and 33 (39.3%) were male;
the gender of five patients was not reported. Of 85 patients whose ages
were known, 46 (54.1%) were 3 months to 6 years of age, 10 (11.8%) were 6
to 15 years of age, and 29 (34.1%) were older than 15 years (Table 2).
Patients were divided into four groups according to case history (Table 1).
Six cases (6.7%) of asymptomatic excretors of STEC were detected by routine
screening of stool specimens. Nonbloody diarrhea, in some cases accompanied
with abdominal pain and vomiting, was reported for 72 (80.9%) patients.
Bloody diarrhea was reported in 7 (7.9%) and HUS in 4 (4.5%) cases.
Episodes of protracted, nonbloody diarrhea for 2 weeks or more were
reported for 11 (12.4%) patients. Double infections with STEC and other
diarrheal pathogens were not reported except for 6 (6.7 %) patients who
were additionally infected with Salmonella enterica sp. (Table 1).

The clinical disease was not associated with the Stx types produced by the
infecting strains. In contrast, the eaeA-gene was closely associated with
severe illness and young age (Tables 1, 2). Thus, 9 (81.8%) of the 11
patients with bloody diarrhea or HUS were infected with eaeA-positive STEC
of serogroups O26, O103, O111, O118, or O145; 8 (72.7%) of 11 patients with
protracted, nonbloody diarrhea carried eaeA-positive STEC of serogroups
O118, O145, O163, and O-rough. Of 54 patients with eaeA-positive STEC
isolates, 41 (75.9%) were younger than 6 years of age with a peak age of 1
to 3 years (Table 2) and a mean age of 8 years and 9 months. In contrast,
eaeA-negative STEC were more prevalent in adults and were isolated from 35
patients; age was known in 31 (Table 2). Only five (16.1 %) were younger
than 6 years of age, and the mean age in this group was 27 years and 10
months.

Table 1. Non-O157 STEC infections in human patients in Germany, 1996

----------------------------------------------------------------------------------------------------
    STEC serotypes             No. of STEC strains  with properties    Clinical status
 --------------------------------  --------------------------------  -------------------------------
O-group(sup a)                                                     Entero-Asymp-   Non-
(no. of                                        Stx1+         hemo-   to-   bloody  Bloody
strains)         H-types(sup b)   Stx1   Stx2  Stx2    eaeA  lytic  matic  diarrhe diarrhea HUS(sup c) 
----------------------------------------------------------------------------------------------------

O5 (1)           NM                 0     0      1       0     1      0      1       0      0

O18 (1)          H15                0     0      1       0     1      0      1d      0      0

O26 (20)         H11, NT,  NM      16     4      0      20    19      1      17  2(sup d,e) 0       
O76 (3)          H19                3     0      0       0     3       0      3      0      0

O78 (1)          NM                 0     1      0       0     1      1       0      0      0

O84 (1)          NT                 1     0       0      1     1      0    1(sup d)  0      0

O91 (4)          H14, NM            3     0      1       0    4       0       4      0      0

O103 (7)         H2, H7, NT         6     1      0       7    7       0     5(sup d) 1      1

O111 (3)         NT                 1     1      1       3    3       1       1      0      1

O113 (3)         H32, NT            0     0      3       0    3       0       3      0      0

O118 (8)         H12, H16 NT        7     1      0       7    7       0     6(sup d) 1      1

O128 (5)         H2, NM             3     0      2       0    5       1      4       0      0

O145 (6)         H28, NM            1     0      5       6    6       0       5      0      1

O146 (5)         H21,H28            3     1      1       0    5       0      4    1(sup d)  0

O163 (1)         H19                0     1      0       0    1       0      1       0      0

ONT (3)          ND                 2     0      1       1    3       1       2      0      0

O-rough (17)     ND                13     1      3       8   12       1      14      2      0

Total (89)                         59    11     19      54   82       6      72      7      4

----------------------------------------------------------------------------------------------------
(sup a)ONT= O-antigen not typable; O-rough= rough LPS, O-antigen not typable.
(sup b)NM= nonmotile; ND= H-antigen not determined; NT= H-antigen not typable.
(sup c)HUS= hemolytic uremic syndrome.
(sup d)A case of double infection with Salmonella enterica sp.
(sup e)A case of a double infection with STEC O26:H11 and O157:H7.

Most of the non-O157 STEC infections were sporadic, and the sources of
infection were not identified. However, outbreak investigations were not
routinely performed, and a number of these infections could be part of
unidentified outbreaks. In four cases, stool specimens from persons who
were in contact with the patients were examined, and two outbreaks in
families with diarrheal episodes were detected. One outbreak occurred in a
family living in Berlin; diarrheal symptoms developed in the parents and
their two children after they returned from vacation on a farm in Northern
Germany. STEC O145:H- (Stx2 and eaeA positive STEC) could be isolated from
two family members. The second outbreak was detected in a family living in
the region of Augsburg (Bavaria). The parents had no diarrheal symptoms,
but their children had diarrhea over a period of 6 weeks. STEC O-rough,
Stx1, and eaeA-positive STEC were isolated from stool samples of both
infants. All patients from both outbreaks recovered.

Conclusions and Recommendations

Human pathogenicity of E. coli strains belonging to the STEC group varied
according to serotypes, virulence attributes, and other unknown factors
(7). Besides Shiga toxins, the eaeA gene product intimin contributed to
diarrheal disease in humans, and typical human virulent STEC (e.g.,
enterohemorrhagic E. coli O157 strains) were found to carry genes for Shiga
toxin(s), intimin, and enterohemolysin (7). Two major groups of non-O157
STEC strains were isolated from humans. Group I consisted of 54 strains
(60.7%), which were all positive for eaeA and with one exception for
enterohemolysin, and group II of 35 (39.3%) STEC strains lacking the eaeA
gene. Group I STEC strains were more frequently associated with severe
diarrheal disease, HUS, and with young age than group II strains, which
were more frequently found in clinically uncomplicated cases (asymptomatic
carriers, abdominal pain, uncomplicated diarrhea) and in older patients.
These findings indicate that some or all of the group II strains are less
virulent or nonpathogenic for humans, although they can colonize the human
intestine. Many eaeA-negative non-O157 STEC strains isolated from healthy
animals have been found to adhere to HEp-2 cells in culture without
carrying DNA sequences specific for diffuse adherence (daa), local
adherence (eaf), or for enteroaggregative E. coli (14). Characterizing the
colonization mechanisms of eaeA-negative STEC could help to further define
their role as human pathogens.

Table 2. Age of patients and STEC type (eaeA)

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

               No. of patients with STEC-isolate

Patient       ------------------------------------
age 
(years)(sup a) eaeA-positive     eaeA-negative
--------------------------------------------------

0-1                   6                 1

1-2                   13                1

2-3                   11                1

3-4                   5                 2

4-5                   2                 1

5-6                   4                 0

6-10                  2                 2

10-15                 2                 4

15-20                 0                 4

20-30                 2                 2

30-40                 3                 5

40-50                 4                 2

50-60                 0                 6

60-72                 0                 1

Total                 54                31

--------------------------------------------------
(sup a)Patient's age was known in 85 cases. The
youngest patient was 3 months of age; the oldest,
72 years of age.

Our finding that infections with eaeA-positive non-O157 STEC were more
frequent in young infants resembles EPEC infection-related finding. EPEC
cause gastroenteritis in very young children and occur rarely in adults.
Humans exposed to EPEC show an immune response against bacterial antigens
such as LPS, intimin, and fimbriae and may thus develop protective immunity
to these pathogens (15). Similarly, a Canadian study has shown that
non-O157 STEC-infections at young ages may confer protective immunity to
subsequent infections in humans (16). It is possible that EPEC infections
in early childhood confer cross-reacting protective immunity against STEC
types that share common antigens (such as LPS and intimin) with classical
EPEC strains. This could explain why infections with eaeA-positive STEC
occur less frequently in older patients.

Our data support previous findings that non-O157 STEC types are more
frequently involved in nonbloody diarrhea than STEC-O157, whereas the
latter are more frequently associated with HC and HUS (7). In 1996, 89
non-O157 STEC and 33 STEC O157 from 121 human patients were investigated in
our laboratory. The proportion of patients with HUS or HC was 45.5% in the
group of STEC O157 infected compared with 12.4% in the group of non-O157
STEC-infected patients. Only one of the 33 STEC O157 strains from human
patients belonged to the sorbitol-fermenting, -glucuronidase-positive type
of O157:H- strains reported in Germany (17).

Humans of all age groups can be infected with STEC belonging to different
serotypes. Because the association between serotype and human pathogenicity
is not always certain, a serotype-independent diagnosis of STEC is
required. Although it is not possible for economic reasons to examine all
patients with diarrhea for STEC, these organisms should be sought in
pediatric patients, who are at higher risk for serious infection with these
pathogens.

      Dr. Beutin is head of the Pathogenic E. coli and Enterobacteria Unit
at the Robert Koch-Institut. He is also a microbiologist and lecturer at
the veterinary department of the Free University of Berlin. His expertise
is in microbiology and molecular biology with particular interest in
virulence markers of intestinal pathogenic bacteria, ecology of pathogenic
bacteria, and interactions between host and pathogens.

      Address for correspondence: Lothar Beutin, Robert Koch-Institut, Abt.
Mikrobiologie, Nordufer 20, D-13353 Berlin, Germany; fax: 47-304547-2673;
e-mail: BeutinL@rki.de.

References

  1. Calderwood SB, Acheson DWK, Keusch T, Barrett TJ, Griffin PM,
     Strockbine NA, et al. Proposed new nomenclature for SLT (VT) family.
     American Society for Microbiology News 1996;62:118-9.
  2. Karmali MA. Infection by verocytotoxin-producing Escherichia coli.
     Clin Microbiol Rev 1989;2:15-38.
  3. Griffin PM, Tauxe RV. The epidemiology of infections caused by
     Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the
     associated hemolytic uremic syndrome. Epidemiol Rev 1991;13:60-98.
  4. World Health Organization, Food Safety Unit Consultations and
     Workshops. Prevention and control of enterohaemorrhagic Escherichia
     coli (EHEC) infections. Report of a WHO consultation; Geneva,
     Switzerland; 1997 28 Apr-1 May. Geneva: The Organization; 1997. Report
     No.: WHO/FSF/FOS/97.6.
  5. Tarr PI. Escherichia coli O157:H7: Clinical, diagnostic, and
     epidemiological aspects of human infection. Clin Infect Dis
     1995;20:1-10.
  6. World Health Organization. Veterinary Public Health Unit. Report on a
     WHO working group meeting on shiga-like toxin producing Escherichia
     coli (SLTEC) with emphasis on zoonotic aspects; Bergamo, Italy; 1994 1
     Jul. Geneva: The Organization; 1995. Report No.: WHO/CDS/VPH/94.136.
  7. Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol
     Rev 1998;11:142-201.
  8. Beutin L, Zimmermann S, Gleier K. Rapid detection and isolation of
     Shiga-like toxin (Verocytotoxin)-producing Escherichia coli by direct
     testing of individual colonies from washed sheep blood agar plates in
     the VTEC-RPLA assay. J Clin Microbiol 1996;34:2812-4.
  9. Karch H, Meyer T. Single primer pair for amplifying segments of
     distinct shiga-like toxin genes by polymerase chain reaction. J Clin
     Microbiol 1989;27:2751-7.
 10. Beutin L, Horbach I, Zimmermann S, Gleier K. Comparative evaluation of
     different diagnostic methods for the detection of verotoxin
     (shiga-toxin) producing strains of Escherichia coli (VTEC) in human
     clinical stool specimens. J Lab Med 1997;21:537-46.
 11. Beebakhee G, Louie M, De Azavado J, Brunton J. Cloning and nucleotide
     sequence of the eae homologue from enterohemorrhagic Escherichia coli
     serotype O157:H7. FEMS Microbiol Lett 1992;91:63-8.
 12. Schmidt H, Plaschke B, Franke S, Rssmann H, Schwarzkopf A, Heesemann
     J, et al. Differentiation in virulence patterns of Escherichia coli
     possessing eae genes. Med Microbiol Immunol (Berl) 1994;183:23-31.
 13. Orskov F, Orskov I. Serotyping of Escherichia coli. Methods in
     Microbiology 1984;14:43-112.
 14. Beutin L, Geier D, Zimmermann S, Karch H. Virulence markers of
     shiga-like toxin-producing Escherichia coli strains originating from
     healthy domestic animals of different species. J Clin Microbiol
     1995;33:631-5.
 15. Donnenberg MS. Enteropathogenic Escherichia coli. In: Blaser MJ, Smith
     PD, Ravdin JI, Greenberg HB, Guerrant RL, editors. Infections of the
     gastrointestinal tract. New York: Raven Press; 1995. p. 709-26.
 16. Wilson JB, Clarke RC, Renwick SA, Rahn K, Johnson RP, Karmali MA, et
     al. Verocytoxigenic Escherichia coli infection in dairy farm families.
     J Infect Dis 1996;174:1021-7.
 17. Gunzer F, Bhm H, Rssmann H, Bitzan M, Aleksic S, Karch H. Molecular
     detection of sorbitol-fermenting Escherichia coli O157 in patients
     with hemolytic-uremic syndrome. J Clin Microbiol 1992;30:1807-10.
---------------------------------------------------------------------------------
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Dispatches

New Orientia tsutsugamushi Strain from Scrub Typhus in Australia

Dimitri M. Odorico,* Stephen R. Graves, Bart Currie, Julian Catmull,*
Zoltan Nack, Sharon Ellis, Ling Wang, and David J. Miller*
*James Cook University, Townsville, Queensland, Australia; The Geelong
Hospital, Geelong, Victoria, Australia; and Royal Darwin Hospital,
Casuarina, Northern Territory, Australia

---------------------------------------------------------------------------
      In a recent case of scrub typhus in Australia, Orientia
      tsutsugamushi isolated from the patient's blood was tested by
      sequence analysis of the 16S rDNA gene. The sequence showed a
      strain of O. tsutsugamushi that was quite different from the
      classic Karp, Kato, and Gilliam strains. The new strain has
      been designated Litchfield.

                                                

                                       
                                       
Scrub typhus has been recognized as an
important cause of fever among
inhabitants of tropical Queensland      [Figure.] Geographic distribution of
(northeast Australia) for the past 60   rickettsial diseases in Australia.
years (1-6). All three groups of
rickettsial diseases occur in
Australia (Figure). 1) Spotted fever
group (SFG): i) Rickettsia australis (Queensland tick typhus) occurs along
the eastern side of the continent to the east of the Great Dividing Range
(7,8); ii) R. honei (Flinders Island spotted fever) is only now recognized
on Flinders Islanda small island off the southeast corner of Australia
(9,10); iii) an unidentified SFG rickettsial infection in Tasmania, a large
island state to the southeast of the Australian mainland, has been
identified through serologic evidence, but no isolate has been obtained. 2)
Typhus group (TG): R. typhi (murine typhus) occurs throughout Australia,
with foci recognized in southwest Australia, southcentral Australia (first
described by Hone [11] in Adelaide, South Australia), and central
Queensland (northeast Australia). 3) Scrub typhus: Orientia tsutsugamushi
infection, well recognized in tropical coastal Queensland, has probably
long been endemic in this area. As Europeans gradually settled the area,
the etiologies of numerous febrile illnesses were determined. Serologic
evidence (the Weil-Felix serologic test) of scrub typhus became available
(1-3) and later isolates of O. tsutsugamushi were made by inoculating mice
with patients' blood (4). A serologic analysis of the Queensland strains of
O. tsutsugamushi suggested that most strains were Karp-like (12). (The Karp
strain was isolated from Papua New Guinea [13].) The geographic
distribution of scrub typhus in the rest of Australia is less clear. The
disease was not considered to occur west of coastal Queensland. However, in
the tropical region of the Northern Territory, six possible cases from 1937
to 1939 and one in 1957 were reported (all presumptively diagnosed). Since
1990, however, nine cases of scrub typhus have been reported in Litchfield
Park, a discrete area of rain forest in the Northern Territory opened as a
park in 1986 (16). All patients had eschars, and all cases were confirmed
by specific O. tsutsugamushi serologic tests. A case of scrub typhus was
also reported from a similar rain forest pocket in the tropical Kimberley
region of northwestern Australia (17).

Case Report

In mid-August 1996, a 38-year old man working on the construction of a
tourist path in the rain forest fringe of Litchfield Park (15) became ill
with fever, sweats, headache, sore throat, cough, lethargy, and confusion
during his second week on the job. His condition worsened over at least a
week. He eventually received amoxycillin and clavulanate from a local
medical practitioner but contracted diarrhea and was admitted to Royal
Darwin Hospital with fever, rigors, but no evident focus of infection. A
diagnosis of septicemia was made, and he was started on ceftriaxone and
gentamicin.

Over the next day, he became increasingly confused, and his fever
persisted. He became hypotensive, hypoxemic, and oliguric and was
transferred to the intensive care unit. On day 3 after admission, he
required fluid loading, inotrope therapy, intubation, and ventilation. At
that time, his work history was obtained from relatives, and a 6-mm sore
with a necrotic dark center was noted on his upper right buttock. Scrub
typhus was suspected, and he was given intravenous doxycycline. However,
his condition continued to deteriorate with hypotension, renal failure, and
adult respiratory distress syndrome. Mucosal and gastrointestinal bleeding
developed, and the patient died 6 days after admission.

Laboratory Investigation

Rickettsial Serologic Tests

Paired sera, 6 days apart, showed a greater than fourfold rise in antibody
titer to O. tsutsugamushi by microimmunofluorescence (15). Sera did not
react to SFG rickettsiae or TG rickettsiae. All three classic strains of O.
tsutsugamushi (Karp, Kato, Gilliam) reacted with the patient's serum, at
titers of >1/1024 (negative <1/64).

Mouse Inoculation

Three female outbred white mice were inoculated with blood taken from the
patient 4 days before he died. The mice were inoculated intraperitoneally
with 0.1 ml, 0.2 ml, and 0.5 ml of whole blood in EDTA. The mice were
housed in a flexible film isolator in a high security microbiology
laboratory at the Australian Animal Health Laboratory, Geelong, Australia.
None of the mice died.

Subsequently three additional mice were inoculated with very concentrated
preparations of the O. tsutsugamushi strain isolated from the patient; none
died. This strain of O. tsutsugamushi appears not to be virulent for mice
despite being highly virulent for the patient. No attempt was made to
isolate the strain from mouse organs.

Seroconversion of the inoculated mice to O. tsutsugamushi was confirmed by
micro-immunofluorescence.

Rickettsial DNA Detection by Polymerase Chain Reaction (PCR)

One ml of the patient's blood, taken 4 days before death, was lysed; DNA
was extracted (18); and part of the 56kDa-type specific antigen (an outer
membrane protein) gene was amplified by PCR. Primers 'a'
(5'-TACATTAGCTGCGGGTATGACA-3') and 'b' (5'-CCAGCATAATTCTTCAACCAAG-3') were
used as previously described (19), except that a nested procedure was not
used. The PCR product was electrophoresed on agarose gel, stained with
ethidium bromide, and detected by UV fluorescence. Positive and negative
controls were run with the unknown samples. The PCR product from the
patient's blood had the same molecular weight as a PCR product from a known
O. tsutsugamushi strain.

Tissue Culture Infection

Three 25-cm(sup 2) flasks containing a confluent monolayer of Vero cells were
infected with whole blood (in EDTA) from the patient (0.1 ml, 0.5 ml, and
1.0 ml, respectively). After sitting at 35C for 4 hours without
centrifugation, the blood was removed, and the monolayers were washed twice
with Hank's balanced salt solution. The medium used for culture was RPMI
164O with 10% fetal calf serum (heat-inactivated) at 35C [without CO(sub 2)].
Cultures were examined weekly for abnormalities, and the pH was adjusted as
necessary. On day 60 postinfection, a 3+ cytopathogenic effect was observed
in one flask, with most Vero cells floating in the supernatant. These cells
were tested for bacterial contamination (Gram stainnegative; growth on
horse blood agarnegative) and for rickettsiae (positive for Giminez stain;
positive for scrub typhus, as determined by immunofluorescence staining
with convalescent-phase human serum; and positive as determined by PCR for
56 kDa antigen gene). The isolated strain was subcultured and grown for DNA
extraction.

16S rDNA Sequencing

The 16S rDNA was amplified by PCR with primers A and H*(20), and the
products were cloned into pGEM-T (Promega). The complete nucleotide
sequences of both strands of several clones were determined by using
internal primers (20,21); sequencing reactions were analyzed on an ABI 310
Genetic Analyser by using the ABI Big-Dye chemistry. The sequence has been
submitted to GenBank under the accession number AF062074.

Comparison with the databases confirmed that the sequence from the novel
isolate was O. tsutsugamushi but indicated that it differed greatly from
the four type strains (Karp, Kuroki, Gilliam, Kato). The novel isolate has
five unique base substitutions in the 16S rDNA and thus differs more from
the reference strains than they do from each other. On this basis, it was
designated a new strain and was called Litchfield.

The Litchfield strain of O. tsutsugamushi is the first isolated in culture
from the Northern Territory of Australia, although previous isolates have
been made from Queensland (4,12). O. tsutsugamushi has probably been
present in the mites and native mammals of northern Australia for
millennia. One of the most common mite vectors, Leptotrombidium deliense,
has been detected on several species of native rat in Litchfield Park (22),
although O. tsutsugamushi has not yet been isolated from native mammals
from the Northern Territory. In Queensland, however, O. tsutsugamushi has
been isolated from native mammals and mites (23-25).

The emergence of scrub typhus in Litchfield Park in the Northern Territory
of Australia is probably due to increasing contact between humans and this
scrub and rain forest environment and its animals. Tourism is a major
industry in Australia, and increasing numbers of visitors are traveling to
remote parts to experience the tropical "out-back." Thus, public health
officials and local health providers need to be aware of foci of endemic
diseases in remote parts of Australia. One problem with rickettsial
diseases is that they do not respond to -lactam antibiotics (e.g.,
penicillins and cephalosporins) because of their unusual bacterial cell
wall or to aminoglycoside antibiotics (e.g., gentamicin) because of their
intracellular location. Treatment of patients with undiagnosed sepsis in
Australian hospitals is very likely to involve the use of a cephalosporin
plus gentamicin, a combination that is not effective against scrub typhus
or any other rickettsial disease. The travel history of a febrile patient
is essential if tropical infections are to be included in the differential
diagnosis of febrile illness.

The 16S rDNA evolves relatively slowly. Hence, despite geographic
proximity, the key difference between the sequences from the Karp strain
(isolated from a patient in New Guinea) and the Litchfield isolate implies
a long period of isolation between these strains. Few general conclusions
can be drawn from one sequence for a single Australian isolate. However,
the prediction is that at least some Australian strains of O. tsutsugamushi
will differ substantially from type strains.

Dr. Odorico is a molecular biologist with a broad range of interests,
including molecular phylogenetics. His present research involves the
functional analysis of eukaryotic promoters by use of site-directed
mutagenesis and reporter gene techniques.

Acknowledgment

      We thank Letka Kociska and Lyn Merkelbach for assistance with
preparation of this report.

      Address for correspondence: Stephen R. Graves, Australian Rickettsial
Reference Laboratory, The Geelong Hospital, Geelong, Victoria 3220,
Australia; fax: 61-3-5221-2232; e-mail:stepheng@gh.vic.gov.au.

References

  1. Langan AM, Mathew RY. The establishment of "Mossman," "Coastal" and
     other previously unclassified fevers of north Queensland as endemic
     typhus. Med J Aust 1935; Aug 3:145-8.
  2. Unwin ML. "Coastal fever" and endemic tropical typhus in north
     Queensland: recent investigation, clinical and laboratory findings.
     Med J Aust 1935; Sept 7:303-8.
  3. Heaslip WG. Tsutsugamushi fever in north Queensland, Australia. Med J
     Aust 1941; March 29:380-92.
  4. Carley JG, Doherty RL, Derrick EH, Pope JH, Emanuel ML, Ross CJ. The
     investigation of fevers in north Queensland by mouse inoculation with
     particular reference to scrub typhus. Australasian Annals of Medicine
     1955;4:91-9.
  5. Doherty RL. A clinical study of scrub typhus in north Queensland. Med
     J Aust 1956; Aug 11:212-20.
  6. Derrick EH. The incidence and distribution of scrub typhus in north
     Queensland. Australasian Annals of Medicine 1961;10:256-67.
  7. Andrew R, Bonnin JM, Williams S. Tick typhus in Queensland. Med J Aust
     1946; Aug 24;253-8.
  8. Sexton DJ, Dwyer B, Kemp R, Graves S. Spotted fever group rickettsial
     infections in Australia. Reviews of Infectious Diseases
     1991;13:876-86.
  9. Stewart RS. Flinders Island spotted fever: a newly recognised endemic
     focus of tick typhus in Bass Strait. Part 1. Clinical and
     epidemiological features. Med J Aust 1991;154:94-9.
 10. Graves SR, Dwyer BD, McColl D, McDade JE. Flinders Island spotted
     fever: a newly recognised endemic focus of tick typhus in Bass Strait.
     Part 2. Serological investigations Med J Aust 1991;154:99-104.
 11. Hone FS. A series of cases closely resembling typhus fever. Med J Aust
     1922; Jan 7:1-13.
 12. Shirai A, Campbell RW, Gan E, Chan TC, Huxsoll DL. Serological
     analysis of Rickettsia tsutsugamushi isolates from north Queensland.
     The Australian Journal of Experimental Biology and Medical Science
     1982;60:203-5.
 13. Derrick EH, Brown HE. Isolation of the Karp strain of Rickettsia
     tsutsugamushi. Lancet 1949;July 23:150-1.
 14. Cook GC, editor. Manson's tropical diseases. 20th ed. Philadelphia: WB
     Saunders Company Ltd.; 1996. p. 809.
 15. Currie B, Lo D, Marks P, Lum G, Whelan P, Krause V. Fatal scrub typhus
     from Litchfield Park, Northern Territory. Commun Dis Intell
     1996;20;420-1.
 16. Currie B, O'Connor L, Dwyer B. A new focus of scrub typhus in tropical
     Australia. Am J Trop Med Hyg 1993;49:425-9.
 17. Quinlan ML, Chappell T, Golledge CL. Scrub typhus in Western
     Australia. Commun Dis Intell 1993;17:570-1.
 18. Wyman AR, White R. A highly polymorphic focus in human DNA. Proc Natl
     Acad Sci U S A 1980;77;6754-8.
 19. Kawamori F, Akiyama M, Sugieda M, Kanda T, Akahane S, Yamamoto S, et
     al. Two-step polymerase chain reaction for diagnosis of scrub typhus
     and identification of antigenic variants of Rickettsia tsutsugamushi.
     J Vet Med Sci 1993;55:749-55.
 20. Edwards U, Rogall T, Blocker H, Emde M, Bottger EC. Isolation and
     direct complete nucleotide determination of entire genes.
     Characterisation of a gene coding for 16S ribosomal RNA. Nucleic Acids
     Res 1989;17(19):7843-53.
 21. Wilson KH, Blitchington RB, Greene RC. Amplification of bacterial 16S
     ribosomal DNA with polymerase chain reaction. J Clin Microbiol
     1990;28:1942-6.
 22. Bell PJ, Whelan PI. A scrub typhus vector Leptotrombidium deliense
     (Walch) (Acari: Trombiculidae) on rats in Litchfield Park, Northern
     Territory, Australia. Journal of the Australian Entomological Society
     1993;32:207-8.
 23. Cook I, Scott W, Campbell RW. Scrub typhus and other infections in
     north Queensland animals. Trans R Soc Trop Med Hyg 1967;61:343-50.
 24. Campbell RW, Domrow R. Rickettsioses in Australia: isolation of
     Rickettsia tsutsugamushi and R. australis from naturally infected
     anthropods. Trans R Soc Trop Med Hyg 1974;68:397-402.
 25. Glazebrook JS, Campbell RSF, Hutchinson GW, Stallman ND. Rodent
     zoonoses in North Queensland, the occurrence and distribution of
     zoonotic infections in north Queensland rodents. The Australian
     Journal of Experimental Biology and Medical Science 1978;56:147-56.
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Dispatches

Streptococcus parasanguinis: New Pathogen Associated with Asymptomatic
Mastitis in Sheep

J.F. Fernndez-Garayzbal,* E. Fernndez,* A. Las Heras,* C. Pascual, M.D.
Collins, and L. Domnguez*
*Universidad Complutense, Madrid, Spain; and BBSRC Institute of Food
Research, Reading, United Kingdom

---------------------------------------------------------------------------
      We describe two unusual cases in sheep of subclinical mastitis
      caused by Streptococcus parasanguinis. This bacterium has been
      associated with the development of experimental endocarditis;
      its presence at relatively high concentrations in apparently
      healthy sheep milk may pose a health risk in persons with
      predisposing heart lesions.

Ovine mastitis represents a major sanitation and economic problem for both
milking sheep farmers and the sheep-milk cheese industry because it reduces
milk production and quality. Many microorganisms produce asymptomatic, or
subclinical, mastitis in milking sheep. Coagulase-negative staphylococci,
particularly Staphylococcus epidermidis, are most prevalent (1,2,3),
although other bacterial groups have recently emerged as clinically
important (4). Streptococci are also responsible for a significant
proportion of cases; however, while a large number of species of
staphylococci cause subclinical mastitis, the number of streptococci
species found to do so is limited, usually to Streptococcus agalactiae, S.
uberis, and S. dysgalactiae (3,5).

We report two unusual cases of subclinical mastitis in sheep involving S.
parasanguinis (6,7). This atypical viridans Streptococcus bacterium has
been isolated from humans (throat, blood, and urine) (7) but has not been
associated with mastitis. The viridans group streptococci are the most
common pathogens associated with native valve endocarditis, a disease of
increasing medical importance in industrialized countries (8). In patients
with this disease, eating and teeth brushing cause low-grade bacteremia,
which allows circulating bacteria to adhere to the damaged endocardium (9).
Although S. parasanguinis has not yet been established as a human pathogen,
it has been clearly associated with the development of experimental
endocarditis (10). Therefore, the excretion of S. parasanguinis at
relatively high concentrations in milk from apparently healthy sheep is of
concern. Indeed, its presence in certain dairy products, such as
nonpasteurized handmade cheeses, may pose a health risk in persons with
predisposing heart lesions.

The two isolates in our study were recovered in 1997 from two sheep of an
Assaf flock during a bacteriologic survey for determining the prevalence of
subclinical mastitis in Madrid (central region of Spain). Affected sheep
did not show signs of clinical mastitis or milk abnormalities but had
California Mastitis Test (CMT) scores of 2+. CMT estimates the degree of
inflammation of the mammary gland by detecting increased numbers of
leukocytes in milk (11). The bacteriologic counts of the milk samples were
1 x 10(sup 4) CFU/ml in one of the sheep and 1.7 x 10(sup 3) CFU/ml in the 
other. Mammary glands with these characteristics (no clinical abnormalities,
apparently normal milk secretion, positive for CMT, and bacteriologically
positive) are routinely considered to have subclinical mastitis (1,12).
Milk samples were collected and analyzed as described previously (13).
After 48 hours of incubation at 37C under aerobic conditions on Columbia
blood agar, pure cultures of [alpha]-hemolytic colonies were recovered from 
both milk samples. The isolates are clinically important because both sheep 
fit the criteria for subclinical mastitis and the isolates were recovered 
in pure culture.

Both strains were catalase-negative gram-positive cocci, and biochemical
identification was attempted with the commercial rapid ID 32 Strep system
(Bio-Mrieux S.A.). Both isolates had an identical biochemical profile,
which did not correlate with any of the species identified with the ID 32
multisubstrate strip. Sequencing of 16S rRNA genes provided a definitive
identification of the streptococci isolates as S. parasanguinis. This
molecular technique is a powerful method for determining the identity of
bacteria that cannot be properly identified by conventional physiologic or
biochemical approaches and has proven powerful for describing new bacterial
pathogens causing mastitis (13,14). The 16S rRNA gene of each isolate was
amplified by polymerase chain reaction (PCR) and was sequenced to determine
genotypic identity (13,14). The determined sequences consisted of more than
1,400 nucleotides (representing more than 95% of the total 16S rRNA gene)
and were compared with the sequences of other streptococcal species
available in the European Molecular Biology Laboratory Database Library.
The 16S rRNA gene sequence analysis showed both isolates to be
genotypically identical and to display 99.7% sequence similarity with the
strain 85-81 of S. parasanguinis, which on the basis of DNA-DNA
hybridization studies was shown to be a true member of this species (7).

The two S. parasanguinis isolates represented 7% of our total streptococci
isolated from cases of subclinical mastitis. According to these data, only
sporadic cases of mammary gland infections due to S. parasanguinis would be
expected. However, because many streptococcal isolates are usually
identified only to the genus level (2,3,11,15), the diversity of species
involved and the incidence of infection due to particular streptococcal
species are difficult to know. The same difficulty is true with
corynebacteria (4).

The recognition of S. parasanguinis as a new animal pathogen causing
subclinical mastitis in sheep and the recent recognition of new
Corynebacterium species associated with this disease (13,14) suggest the
need for epidemiologic surveys to determine the range of species diversity,
other than coagulase-negative staphylococci, involved in subclinical sheep
mastitis. According to recent results, this range appears higher than
previously considered. Particular attention should be paid to species
potentially pathogenic for humans.

Acknowledgment

      We thank F. Vilas, Director General de Salud Pblica de la Comunidad
de Madrid, for his encouragement.

      Dr. Las Heras is recipient of a long-term predoctoral grant from the
Ministerio de Educacin y Cultura de Espaa. This work has been partially
supported by the Cooperativa Castellana de Ganaderos (Campo Real, Madrid)
and by a project from the Comunidad Autnoma de Madrid (Consejera de
Educacin y Cultura, No. 06G/026/96).

      Address for correspondence: J.F. Fernndez-Garayzbal, Departamento
de Patologa Animal I (Sanidad Animal), Universidad Complutense, Facultad
de Veterinaria, 28040 Madrid, Spain; fax: 34-91-394-3908; e-mail:
garayzab@eucmax.sim.ucm.es.

References

  1. Stefanakis A, Boscos C, Alexopoulos C, Samartzi F. Frequency of
     subclinical mastitis and observations on somatic cell counts in ewes'
     milk in northern Greece. Animal Science 1995;61:69-76
  2. Watkins GH, Burriel AR, Jones JET. A field investigation of
     subclinical mastitis in sheep in southern England. British Veterinary
     Journal 1991;147:413-20.
  3. Watson DL, Franklin NA, Davies HI, Kettlewell P, Frost AJ. Survey of
     intramammary infections in ewes on the New England Tableland of New
     South Wales. Aust Vet J 1990;67:6-8.
  4. Fernndez-Garayzbal JF, Dominguez L. Corinebacterias como agentes
     responsables de mastitis subclnicas. In: Abstracts of the XVI
     Congreso de la Sociedad Espaola de Microbiologa; 1997 Jul 14-17;
     Barcelona, Espaa. Abstract No. P5.
  5. Bor A, Winkler M, Gootwine E. Nonclinical intramammary infection in
     lactating ewes and its association with clinical mastitis. British
     Veterinary Journal 1989;145:178-84.
  6. Truper HG, Clari L. Taxonomic note: necessary correction of specific
     epithets formed as substantives (nouns) "in apposition". Int J Syst
     Bacteriol 1997;47:908-9.
  7. Whiley RA, Fraser HY, Douglas CWI, Hardie JM, Williams AM, Collins MD.
     Streptococcus parasanguinis sp. nov., an atypical viridans
     Streptococcus from human clinical specimens. FEMS Microbiol Lett
     1990;68:115-22.
  8. Scheld W, Sande M. Endocarditis and intravascular infections. In:
     Mandell G, Bennett J, Dolin R, editors. Principles and practice of
     infectious diseases. 4th ed. New York: Churchill Livingstone, Inc.;
     1995. p. 740-83.
  9. Kaye D, Abrutyn E. Prevention of bacterial endocarditis. Ann Intern
     Med 1991:114:803-4.
 10. Burnette-Curley D, Wells V, Viscount H, Munro CL, Fenno JC,
     Fives-Taylor P, et al. FimA, a major virulence factor associated with
     Streptococcus parasanguinis endocarditis. Infect Immun
     1995;63:4669-74.
 11. Fthenakis GC. California mastitis test and Whiteside test in diagnosis
     of subclinical mastitis of dairy ewes. Small Ruminant Research
     1995;16:271-6.
 12. Gonzlez-Rodrguez MC, Gonzalo C, San Primitivo F, Crmenes P.
     Relationship between somatic cell count and intramammary infection of
     the half udder in dairy ewes. J Dairy Sci 1995;78:2753-9.
 13. Fernndez-Garayzbal JF, Collins MD, Hutson RA, Fernndez E,
     Monasterio R, Marco J, et al. Corynebacterium mastitidis sp. nov.,
     isolated from milk of sheep with subclinical mastitis. Int J Syst
     Bacteriol 1997;47:1082-5.
 14. Fernndez-Garayzbal JF, Collins MD, Hutson RA, Gonzlez I, Fernndez
     E, Domnguez L. Corynebacterium camporealensis sp. nov., associated
     with subclinical mastitis in sheep. Int J Syst Bacteriol
     1998;48:463-8.
 15. Cruz M, Serrano E, Montoro V, Marco J, Romero M, Baselga, R, et al.
     Etiology and prevalence on subclinical mastitis in the Manchega sheep
     at mid-late lactation. Small Ruminant Research 1994;14:175-80.
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Dispatches

Introduction of HIV-2 and Multiple HIV-1 Subtypes to Lebanon

Danuta Pieniazek,* James Baggs, Dale J. Hu,* Ghassan M. Matar, Alexander
M. Abdelnoor, Jacques E. Mokhbat, Marwan Uwaydah, Abdul Rahman Bizri,
Artur Ramos,* Luiz M. Janini,* Amilcar Tanuri,* Carol Fridlund,* Charles
Schable,* Leo Heyndrickx,, Mark A. Rayfield,* and Walid Heneine*
*Centers for Disease Control and Prevention, Atlanta, Georgia, USA; Emory
University, Atlanta, Georgia, USA; American University of Beirut, Beirut,
Lebanon; Universidade Federal de Rio de Janeiro, Rio de Janeiro, Brazil;
and Institute of Tropical Medicine, Antwerp, Belgium

---------------------------------------------------------------------------
      HIV genetic variability, phylogenetic relationships, and
      transmission dynamics were analyzed in 26 HIV-infected patients
      from Lebanon. Twenty-five specimens were identified as HIV-1
      and one as HIV-2 subtype B. The 25 strains were classified into
      six env-C2-V3 HIV-1 subtypes: B (n = 10), A (n = 11), C (n =
      1), D (n = 1), G (n = 1), and unclassifiable. Potential
      recombinants combining parts of viral regions from different
      subtypes A(sup env)/D(sup pol)/A(sup gag), G(sup env)/A(sup pol) 
      and the unclassifiable-subtype(sup env)/ unclassifiable-
      subtype(sup pol)/A(sup gag) were found in three patients. 
      Epidemiologic analysis of travel histories and behavioral risks 
      indicated that HIV-1 and HIV-2 subtypes reflected HIV strains 
      prevalent in countries visited by patients or their sex partners. 
      Spread of complex HIV-subtype distribution patterns to regions 
      where HIV is not endemic may be more common than previously 
      thought. Blood screening for both HIV-1 and HIV-2 in Lebanon is 
      recommended to protect the blood supply. HIV subtype data provide 
      information for vaccine development.

Understanding the global genetic diversity of HIV is important for
monitoring the spread of infection and developing effective vaccines.
Currently, two principal genetic groups, designated M (main) and O
(outlier), have been identified. M, which is highly prevalent, is further
classified into 10 established envelope subtypes, A through J (1). Mosaic
forms, which combine the genetic material from two distinct subtypes, have
also been identified (1,2). HIV-1 subtype B predominates in Europe and the
Americas, whereas HIV-1 non-subtype B strains dominate in sub-Saharan
Africa. In contrast to HIV-1 infection, which is spread through all
continents, HIV-2 is primarily restricted to West Africa and to population
movements from or through this region. HIV-2 strains are classified into
five subtypes, A through E; only subtypes A and B viruses are predominant
(3).

HIV-subtype distribution patterns in regions where HIV is not endemic are
not well known (4). Information about these distribution patterns is
important for 1) implementing proper diagnostic assays to protect the blood
supply; 2) providing basic information on the prevalence of viral sub-types
for vaccine development; and 3) monitoring trends of viral spread. Two
distribution patterns of HIV subtypes have been recognized in areas where
the disease had not been endemic. The first pattern consists of a
restricted number of HIV-1 viral subtypes or their combination with HIV-2.
Examples include HIV-1 subtypes E and B in Thailand (5) and HIV-1 subtype C
and HIV-2 in India (6). In contrast, the second pattern includes a large
number of only HIV-1 subtypes as documented in Cyprus (A, B, C, F, and I)
and the Philippines (A, B, C, D, and E) (7,8). However, information is
limited as to how common the spread of this HIV-subtype pattern is to areas
in which the disease is not endemic.

Lebanon was chosen for a genetic variability study for three reasons.
First, HIV is not endemic in Lebanon, but its presence is slowly increasing
(9,10). From 1984 (when the first case of AIDS was identified) (11) to
1996, 400 cases of HIV infection were reported to the Lebanon National AIDS
Control Program (J.E. Mokhbat, pers. comm.). Second, Lebanon has a central
geographic location, strong historic links, and frequent population
movements to and from other countries. Third, the potential for
introduction of multiple HIV subtypes is present given the large number of
Lebanese expatriates who return to Lebanon after living and working in
HIV-endemic regions such as Africa (mainly West Africa).

Study Population

With informed consent, interviews and blood samples were obtained from 26
HIV-seropositive Lebanese citizens (17 men and 9 women) living in Lebanon
at the time of the study. They were referred (in 1996) from both urban and
rural areas to the Medical Center of the American University of Beirut or
the Saint George Hospital in Beirut, Lebanon.

Study Design

HIV-1 antibodies were discriminated from HIV-2 antibodies by a synthetic
slot immunoblot composed of recombinant viral proteins and synthetic
peptides (Genelavia Mixt, Peptilav 1-2, HIV Blot 2.2-Diagnostic
Biotechnology and New LAV Blot II, Sanofi Diagnostic Pasteur, France).

Double-stranded viral DNA of nested polymerase chain reaction
(PCR)-amplified env(C2-V3), pol (protease-prot), and gag (p24 fragment)
from peripheral blood mononuclear cells were direct sequenced (12,13).
Comparative phylogenetic analysis of two or three independent viral regions
enables identification of a possible mosaic viral genome (2). The sequences
were aligned by use of the CLUSTAL multiple-sequence alignment program
(14). After regions containing gaps were eliminated, the aligned sequences
were analyzed with the maximum likelihood method by using the fastDNAml
program, which uses randomized data input and global rearrangement (15).
Additionally, the neighbor-joining method (PHYLIP package version 3.5c [16]
or TREECON software [17]) was used, with or without bootstrapping. The
stability of the tree's topology was tested by pruning, which consists of
removing one species from the alignment and rerunning the phylogenetic
analysis. SIV-cpz sequences were used as outgroup. All specimens were also
screened for HIV-2 DNA by nested-PCR amplification of viral HIV-2 prot
gene. The outer PCR primers were DP20: 5'GACAGAGGACTTGCTGCA; nucleotide
position: 2068-2083, HIV-2ROD, and DP21: 5'GGCCATTGTCTCAGTTTTGG; nucleotide
position: 2435-2454. The inner primers were DP26a: 5'CACCTCAATTCTCTCTTTGGA;
nucleotide position: 2082-2102; and DP27: 5'TAGATTTAATGACATGCCTAA;
nucleotide position: 2360-2380. Analysis based on HIV-2 pol region is
highly specific and allows classification of HIV-2 sequences into
phylogenetic subtypes (3,18,19).

Molecular Characteristics of HIV Infection

Of 26 patients, 25 were HIV-1 PCR-positive; the remaining patient (LE25)
was HIV-2 PCR-positive and was classified as having subtype B infection
(Figure 1). No dual infections with HIV-1 and HIV-2 strains were found.

The comparative phylogenetic analysis of 25
HIV-1 specimens identified 23 cases with the      
same subtype assignment for the env and pol
sequences and classified 22 as known subtypes A   
(n = 10), B (n = 10), C (n = 1), D (n = 1)        
(Figure 2). In contrast, the remaining specimen
(LE7) consisted of distinctively divergent pol    [Figure 1.] Phylogenetic
and env sequences (Figure 2a, b) as they failed   classification of HIV-2
to cluster with known standard HIV-1 sequences,   sequence from Lebanese
including divergent env subtypes I and J (Figure  patient LE25 (arrow)
2c). Intragene recombination within pol and env   basing on the prot gene
sequences of specimen LE7 was not observed when   (GenBank accession no.
nucleotide signature patterns in HIV-1 subtypes   AF026912). The tree was
were compared. Thus, this strain might represent  generated by the
a potential novel HIV-1 subtype. However, gag     maximum-likelihood
sequence of this virus grouped into HIV-1         method. Numbers at the
subtype A, and this association was supported by  branch nodes connected
the bootstrap value of 90% (Figure 2d). Taken     with subtypes indicate
together, these results indicate that LE7 viral   bootstrap values. The
genome has a mosaic pattern involving elements    distinct HIV-2 subtypes
of an unclassifiable-divergent strain and         are delineated. The
subtype A (unclassifiable(sup env)/               scale bar indicates an
unclassifiable(sup pol)/A(sup gag)).              evolutionary distance of
In addition, potential recombinant viruses        0.10 nucleotides per
combining viral env, pol,and gag from different   position in the
subtypes were identified in two other specimens.  sequence. Vertical
Two known recombinant patterns, G(sup env)/       distances are for
A(sup pol) and A(sup env)/D(sup pol)/A(sup gag), clarity only.
have been found in patients LE24 and LE9, 
respectively. However, a potential recombinant 
is sometimes difficult to recognize, especially between subtypes B and D
viruses, which are relatively closely related; specimen LE22 illustrates
such a situation. The protease gene sequence, the most outside branch in
this clade, grouped weakly with subtype B (Figure 2a). This association,
however, was not stable by analysis that used pruning techniques and not
supported by bootstrap analysis (only 48% value as opposed to 72% without
this sequence). These data suggest that it is unlikely that LE22 prot is
subtype B, and because both env and gag sequences clustered into subtype D,
we have classified this sample as subtype D.

Epidemiologic Characteristics

To better understand the transmission dynamics and temporal introduction of
these HIV variants, we analyzed patients' data (demographics, behavioral
risk, modes of transmission, and travel histories) (Table).

Of eight male patients with subtype B HIV-1 infection, six traveled to
Europe, the Americas, Asia (Thailand), or different locations in the Middle
East. All reported sexual contacts abroad either with commercial sex
workers or homosexual or bisexual men. Of the remaining two patients, one
traveled selectively to Africa and had sexual contacts in Cte d'Ivoire and
Nigeria, whereas the other reported no travel outside Lebanon and had no
sexual contacts with commercial sex workers. In contrast, all nine male
patients infected with non-subtype B HIV-1, including three mosaic viruses,
traveled to Africa or the United Arab Emirates and reported sexual contacts
with commercial sex workers.

                                                      The behavioral risks
                                                      of the female
                                                      patients differed
                                                      substantially from
                                                      those of the male
 [Figure 2.] Phylogenetic   [Figure 2.] continued.    patients. All female
 classification of HIV-1    Arrows indicate Lebanese  patients were
 sequences from Lebanese    samples that clustered in married, and none
 patients denoted with LE   different lineages        reported high-risk
 prefix (GenBank accession  depending on which parts  activities or
 nos.  AF025691-            of their genome were      contacts with
 AF025745). The trees were  analyzed. The distinct    commercial sex
 constructed on the basis   HIV-1 subtypes are        workers. Moreover,
 of the DNA sequences of    delineated. ? represents  all female patients
 prot (a), env (b and c),   the unclassified position (except one [LE12],
 and gag (d) by the         of prot and env sequences who had acquired
 maximum-likelihood        of sample LE7. The scale   HIV-1 infection from
 method. Numbers at the     bar indicates an          blood transfusion)
 branch nodes connected     evolutionary distance of  reported that their
 with subtypes indicate     0.10 nucleotides per      spouses were
 bootstrap values. Env      position in the sequence. HIV-infected and
 trees: (b) includes all    Vertical distances are    traveled abroad with
 25 Lebanese sequences of   for clarity only.         them or alone.
 325 bp and reference
 sequences from eight                                 To determine if the
 subtypes A through H; and                            female patients
 (c) includes only                                    acquired HIV
 Lebanese LE7 sequence of                             infection from their
 216 bp and marker                                    spouses, we analyzed
 sequences from subtypes A                            available
 through J.                                           epidemiologic data on
                                                      the spouses of three
                                                      female patients LE17,
LE19, and LE13. The female patients LE17 and LE19 were spouses of patients
LE18 and LE23, respectively. These two couples, LE17/LE18 and LE19/LE23,
were infected with HIV-1 subtype A and subtype B viruses, respectively
(Figure 2b). In further detailed pairwise analysis of viral DNA isolated
from the husband and wife, a small nucleotide divergence of 3.1% within env
and of 0.6% for both prot and gag (13,20) showed that in the first couple
viral strains were closely related. These data are concordant with
epidemiologic findings, which strongly indicate that the husband was the
source of infection because of his high-risk sexual activities with
commercial sex workers during travels to the United Arab Emirates. His wife
did not travel outside Lebanon and did not report activities that would
have placed her at risk for HIV-1 infection. In the second couple
(LE19/LE23), however, only weak relation between viral strains was observed
(nucleotide divergence of 9.8% within env and 2% within prot). This could
be due to many factors including 1) the time that elapsed from infection;
2) transmission of different quasispecies to the spouse; 3) detection of
only major quasispecies by direct sequencing, which differed in the couple;
and 4) a different source of HIV-1 infection in the sex partner. The latter
hypothesis was not supported by epidemiologic data, which indicated that
only the husband was involved in high-risk sexual activities in and outside
Lebanon. The third female patient, LE25, was infected with HIV-2 subtype B
strain. Her husband, who died of AIDS-related complications, had traveled
to Cte d'Ivoire, a region in which this HIV variant is endemic (21). These
data suggest that very likely the female patients acquired infections from
their HIV-infected spouses.

Table. Demographic, clinical, and behavioral information

------------------------------------------------------------------------------------
                                      Sex partner 
                                     characteristic
                                  --------------------        
        Gen-               CD4/   WHO        Bi/homo-            
Patient der   Age  Status  mm    class  AIDS/ sexual  CSW   Travel/sexual         
no.  (sup a) (yrs)(sup b)(sup 3)(sup c) HIV   male  (sup d) contacts abroad  Subtype   
-------------------------------------------------------------------------------------
LE1      M    37     M    172      3    No     No     Yes   West Africa (Cte    A
                                                            d'Ivoire)(sup e)
LE5      M    42     M    NA       3    NKf    No     Yes   West Africa(sup e)   A
LE6      M    29     M    168      2    Yes    No     Yes   West Africa(sup e)   A
LE11     M    26     S    44       3    Yes    No     Yes   Africa(sup e)        A
LE18
(sup g)  M    34     M    400      1    Yes    No     Yes   Middle East          A
                                                            (United Arab
                                                            Emirates)(sup e)
LE22     M    42     S    NA       3    No     No     Yes   West Africa (Cte    D
                                                            d'Ivoire)(sup e)
LE9      M    57     M    253      1    No     No     Yes   Middle East      A/D/A(sup h)
                                                            (United Arab       
                                                            Emirates)e, Asia,
                                                            Europe, North
                                                            America
LE24     M    54     M    180      1    NK      No    Yes   West Africa      G/A(sup i)
                                                            (Nigeria)(sup e)
LE7      M    54     M    177      3    No      No    Yes   West Africa (Cte NC/A(sup j)
                                                            d'Ivoire)(sup e),        
                                                            Middle East
LE16     M    32     M    86       2    NK      No    Yes   Europe                B
LE26     M    36     M    86       1    No      No    Yes   Europe (France)(sup e)B
LE2      M    27     S   396       3   Yes     Yes     No   Europe (France,       B
                                                            Germany, UK)(sup e),
                                                            North America
                                                           (USA)(sup e), Middle
                                                            East
LE8      M    43     S   98        3    No     Yes     No   Europee, South        B
                                                            America, Middle
                                                            East (Bahrain)(sup e)
LE10     M    35     S   56        4    No     Yes     No   Europe (France)e      B
                                                            Middle East,
LE14     M    45   D/SP   7        3    No      No     Yes  Europe (Spain,        B
                                                            Germany)e Asia
                                                            (Thailand)(sup e),
                                                            South, America
LE23
(sup k)  M   47     M    NA       1     Yes     No    Yes   West Africa (Cte     B
                                                            d'Ivoire,
                                                            Nigeria)(sup e)
LE21     M    31     S  340       1     No      No     No   Lebanon only          B
LE3      F    31     M  378       1     Yes     No     No   Lebanon only          A
LE17
(sup g)  F    32     M  300       3     Yes     No     No   Lebanon only          A
LE20     F    32     M   NA       1     Yes     No     No   Europe                A
LE4      F    36     M  233       1     Yes     No     No   Africa                A
LE13     F    35     M  350       1     Yes     No     No   West Africa           A
LE15     F    34     M  171       1     Yes     No     No   Asia                  C
LE12
(sup l)  F    31     M  109       3     NK      NK     NK   Africa                B
LE19
(sup k)  F    42     M   30       4     Yes     No     No   West Africa           B
LE25     F    28     M  200       2     Yes     No     No   West Africa         HIV-2                                                                         
-------------------------------------------------------------------------------------
(sup a)M=male, F=female.
(sup b)M=married, S=single, D/SP=divorced/separated.
(sup c)World Health Organization classification based on opportunistic infections.
(sup d)Commercial sex worker.
(sup e)Sexual contact reported in region, country listed in parentheses.
(sup f)Not known.
(sup g)Couple number 1.
(sup h)Subtype A -env, subtype D -pol, subtype A -gag.
(sup i)Subtype G -env, subtype A -pol.
(sup j)Unclassified subtype for env and pol, subtype A - gag.
(sup k)Couple number 2.
(sup l)Reported to be infected by blood transfusion.

The molecular and epidemiologic data indicate multiple HIV-1 introductions
to Lebanon. First, most male patients were infected abroad with the HIV-1
strains prevalent in the geographic region they visited. Second, the
topology of HIV-1 env subtype A demonstrated that viral sequences of
patients who had sexual contacts with persons from West Africa clustered in
the branch with marker sequences from Cte d'Ivoire. In contrast, HIV-1
sequences of patients who reported contacts with persons from the United
Arab Emirates (LE9 and LE18) clustered in another branch with
Ugandan/Kenyan marker sequences (Figure 2b). Independent branching of HIV-1
env sequences of subtype A from West and East Africa had been observed
(22). Third, the structures of two mosaic viral genomes and epidemiologic
data of patients suggested that they were infected with potential
recombinant viruses, which had been identified in two geographic regions.
Briefly, patients LE24 and LE9, who reported sexual contacts with
commercial sex workers in Nigeria or the United Arab Emirates, respectively,
have been infected with mosaic viruses G(sup env)/A(sup pol) and A(sup env)/
D(sup pol)/A(sup gag). Such recombination patterns were found in West-Central
Africa and East Africa, respectively (23,24). Finally, analysis of env
sequences from Lebanese patients revealed a relatively high intra-subtype
genetic diversity within subtype B (average 14.9%, range 4.2% to 25.6%) and
subtype A (15.5%, range 2.5% to 28.9%. Epidemiologic data and a low
prevalence of HIV-1 infection suggest multiple introductions (rather than
long existence) of genetically diverse HIV-1 strains to Lebanon.

Conclusions

The results presented in this report provide new information on HIV genetic
variation in Lebanon. The identification of HIV-2 and 6 env HIV-1 subtypes,
including three distinct HIV-1 mosaics, confirms high heterogeneity of HIV
variants in Lebanon. Given the limited number of samples tested, finding
both HIV-2 and multiple HIV-1 subtypes is remarkable and indicates that the
distribution pattern of HIV variants resembled that of Cyprus or the
Philippines rather than that of Thailand or India. These data indicate that
the spread of complex HIV-subtype distribution patterns involving a large
number of distinct HIV-1 and HIV-2 strains to countries without endemic HIV
may be more common than previously thought.

The HIV-2 infection detected in this study, the first documented in
Lebanon, reiterates the importance of appropriate screening methods for
detecting both HIV-1 and HIV-2 to protect the blood supply. Our findings
provide preliminary information for monitoring the spread of HIV variants
in Lebanon and developing subtype-specific subunit vaccines.

Acknowledgment

We thank Priscilla Swanson for her help in sequencing, Eve Lackritz for
critical review of the manuscript, and Mr. Issam Khneisser, American
University of Beirut, for processing blood specimens .

Part of this work was done at the Institute of Tropical Medicine, Antwerp,
Belgium, and was sponsored by grant numbers G 3301.96 and G 30134.97 of the
Belgian Nationaal Fonds voor Wetenschappelijk Onderzoek.

Danuta Pieniazek is a molecular biologist in the HIV/AIDS and Retroviral
Branch, CDC. Her research interests include implications of mixed HIV
infections for the evolution of the HIV/AIDS epidemic and for vaccine
development; her areas of expertise include developing molecular assays for
detection of novel HIV strains and the molecular epidemiology of HIV.

Address for correspondence: Danuta Pieniazek, HIV/Retrovirus Diseases
Branch, Division of AIDS, STD, and TB Laboratory Research, CDC, 1600
Clifton Road, Mail Stop G19, Atlanta, GA 30333, USA; fax: 404-639-1010;
e-mail: dxp1@cdc.gov.

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

Dispatches

Sporadic STEC O157 Infection: Secondary Household Transmission in Wales

Sharon M. Parry* and Roland L. Salmon
*Welsh Combined Centres for Public Health, Cardiff, United Kingdom; and
PHLS Communicable Disease Surveillance Centre, Cardiff, United Kingdom

---------------------------------------------------------------------------
      We conducted a study to quantify and characterize household
      transmission of Shiga toxin (Vero cytotoxin)producing
      Escherichia coli O157 (STEC O157) following sporadic infection
      in Wales. Through total population surveillance, we identified
      83 index case-patients and their household contacts. We
      screened fecal samples submitted from household contacts for
      STEC O157 and calculated the household transmission rate for
      sporadic STEC O157 infection to be 4% to 15%. Household
      contacts in groups at high risk (particularly children under 5
      years of age) present a risk of spreading the infection in the
      wider community.

Shiga toxinproducing Escherichia coli O157 (STEC O157) infection causes
severe enteric symptoms (hemorrhagic colitis with diarrhea, severe
abdominal pain, and blood in the stools) (1) and hemolytic uremic syndrome
(2% to 7% of cases) (2). The bacteria, which are from a predominantly
bovine reservoir, are found in food or water (particularly beef products
and milk) and are transmitted through zoonotic or environmental spread or
person to person by the fecal-oral route (3). Fecal-oral transmission would
explain why children with hemolytic uremic syndrome are seven times more
likely than otherwise healthy children to have had close contact with a
person with diarrhea in the 2 weeks before onset of illness (4).

Outbreaks attributed to person-to-person transmission have been confined
mainly to children's day-care facilities and institutions providing care
for the elderly or for those with physical or mental disabilities
(5-8)facilities in which hygiene is difficult to maintainalthough an
outbreak among siblings in a large family and a playmate has been described
(9).

Most (90%) of STEC O157 cases reported in England and Wales are sporadic
(10). Evidence suggests that the infectious dose is very low, perhaps as
few as 10 organisms (11,12); hence the potential for person-to-person
transmission among sporadic STEC O157 cases might be expected to be far
greater than among sporadic cases of other common gastrointestinal
pathogens. Although secondary spread of STEC O157 by sporadic cases has
been reported (13), such transmission has not been accurately quantified or
characterized on a population basis. This study aimed to quantify and
characterize the secondary household transmission of STEC O157 from
sporadic cases.

Cases (in persons residing in Wales and the English Borders) in which STEC
O157 was isolated from initial fecal specimens and was subsequently
confirmed by the Public Health Laboratory Service Laboratory of Enteric
Pathogens were included in the study. Only the index case (i.e., the first
to be recognized in a household) and sporadic cases (i.e., not part of a
general outbreak) were included. Index case-patients were interviewed, face
to face, by using a standard structured questionnaire to record age group,
gender, onset, and hospitalization details. The names, as well as the age,
gender, and occupation, of all household contacts were recorded in a
confidential database.

Any person staying in the same household as a person with STEC O157
infection for one or more nights during the 7 days before disease onset and
up to the interview date was considered a household contact. Diarrheal
illness (defined as three or more loose stools in any 24-hour period during
the 7 days before the onset of disease in the index patient and up to the
date of the interview with the index patient) in the contact and the date
of onset of the symptoms were also recorded. All household contacts were
requested to provide one fecal specimen, which was examined as described
(Table).

 Table. Probability of isolating Shiga toxin-producing Escherichia coli
 (STEC) O157 from household contacts, by age group of index case-patient
 and age group of household contact, for all contacts(sup a)
 ------------------------------------------------------------------------------

 Age group of                 Age group of index case-patient (%)
 household contact   < 1 yr     1-4 yr      5-14 yr    15 + yr      Total
 ------------------------------------------------------------------------------
 < 1 yr             0/0  (0)   0/3   (0)   0/1   (0)  0/0  (0)   0/4     (0)
 1-4 yr             1/2 (50)   3/8  (38)   1/3  (33)  0/3  (0)   5/16   (31)                                                     
 5-14 yr            0/0  (0)   1/11  (9)   0/7   (0)  0/8  (0)   1/26    (4)
 15 + yr            2/6 (33)   5/49 (10)   2/19 (10)  0/61 (0)   9/135   (7)
 Total              3/8 (40)   9/71 (13)   3/30 (10)  0/72 (0)   15/181  (8)
 ------------------------------------------------------------------------------
 (sup a)All initial fecal samples submitted to laboratories serving the
 resident population of Wales between 1 January 1994 and 31 December
 1995 were cultured on sorbitol MacConkey agar (Oxoid) and incubated
 at 37C for 18 hours. All types of morphologic appearance of
 nonsorbitol-fermenting colonies were tested. Five colonies of each
 type were picked and tested for latex agglutination with O157
 antiserum (Oxoid) and biochemically confirmed as E. coli by API 20E
 (bioMerieux sa69280 Marcy L'Etoile, France). E.coli O157 isolates
 were referred to the Public Health Laboratory Service Laboratory of
 Enteric Pathogens for confirmation and testing for Shiga cytotoxin
 production.

To determine whether the household contact cases were secondary or
coprimary, the dates of disease onset for index case-patients were compared
with those of their symptomatic household contacts. Also, the rates of
infection in the households of index case-patients who had been
hospitalized were compared with rates in households of patients who
remained at home. We constructed matrices to determine the frequency of
STEC O157 isolation for each age group of household contacts and index
case-patients. The frequency of isolation of the organism from household
contacts in the households of male index case-patients was compared with
that from households of female index case-patients by calculating relative
risks (RR) and Mantel-Haenszel chi squares. To determine any bias in the
household contacts screened, we quantified factors affecting an individual
contact's likelihood of submitting a fecal specimen (i.e., age, gender, and
symptomatic status), as well as age and gender of index case-patient. We
examined the delay between disease onset in the index case-patient and date
of submission of specimens by contacts and compared the delays for
household contacts of different ages. Household contacts infected with STEC
O157 who belonged to certain groups (food handlers, health-care workers,
children under 5 years of age, older children or adults unable to implement
good hygiene) were considered a risk to the community, in accordance with
current guidelines (14,15). The number of contacts falling into these
groups was recorded.

Eighty-three persons considered index case-patients had 181 household
contacts. Of the 181 contacts, 101 (56%) submitted fecal samples. Samples
were submitted a median of 12 days after disease onset in the index
case-patient (mean = 13.46 days; mode = 12 days; range = 6-40 days);
timeliness of sample submission did not differ by age group. Of the 101
contacts examined, 15 had STEC-positive samples, giving a crude secondary
household transmission rate of 15%. If it is assumed that contacts who did
not submit fecal samples were not excreting STEC O157, the rate is 8% (15
of 181).

 
 [Figure.] Time interval between onset of index case and contacts.

Of the 181 household contacts, 32 reported diarrhea: of these, 3 became ill
on the same day as the index case-patient (none had confirmed STEC O157), 8
became ill before the index case-patient (2 had confirmed STEC O157), and
21 became ill after the index case-patient (7 had confirmed STEC O157)
(Figure). If only the cases of household contacts who became ill after the
index case-patient are considered secondary cases, the household
transmission rate is 4% (7 of 181 cases).

STEC O157 were isolated from 15 of 109 contacts of case-patients under 14
years of age compared with 0 of 72 contacts of case-patients 15 years of
age and older (p < 0.01) (Table). STEC O157 was no more likely to be
isolated from male than female household contacts (8 of 95 versus 7 of 86,
RR = 1.03, confidence interval [CI] = 0.39-2.73, p = 0.95). The contacts of
male index case-patients were no more likely to have STEC-positive samples
than those of female index case-patients (11 of 100 versus 4 of 81, RR =
2.23, CI = 0.74-6.73, p = 0.14). Among 53 parents of children with STEC
O157, 2 (8%) of 24 fathers and 5 (17%) of 29 mothers had STEC-positive
samples. Household contacts were more likely to be STEC positive if the
index case-patient had not been hospitalized (12 of 102 versus 3 of 79, RR
= 3.10, CI = 0.90-10.61, p = 0.05).

Seven household contacts were food handlers; five submitted samples, and
none was STEC positive. Of 12 staff members at health-care facilities, 11
submitted samples, and none was STEC positive. Of 13 contacts who were
children under 5 years of age attending a day-care facility, 11 submitted
samples, and 2 were STEC positive. No household contacts were categorized
as adults unable to implement good hygiene. Female household contacts were
as likely as male to submit fecal samples (RR = 1.13, CI = 0.87-1.46, p =
0.37). Household contacts of female index case-patients were equally as
likely as contacts of male patients to submit fecal specimens (RR = 0.81,
CI = 0.62-1.06, p = 0.12).

Symptomatic contacts were more likely to submit specimens than asymptomatic
contacts (81% [26/32] versus 50% [75/149], RR = 1.61 [1.28-2.03], p =
0.001) and also more likely to excrete STEC O157 than asymptomatic contacts
(35% [9/32] versus 4% [6/149], RR = 6.98, CI = 2.67-18.24, p < 0.001 Fisher
exact 2-tailed test).

The study showed that 8% to 15% of household contacts of persons with
sporadic cases of STEC O157 were STEC positive. However, the household
contacts could have had cases coprimary to the index case or even to the
primary cases in the household. By excluding contacts who had symptoms
before the index case-patient did and those who had no symptoms, we arrived
at a household transmission rate of 4%.

The method of identifying secondary cases had certain biases. First,
household contacts who had diarrheal symptoms were more likely to submit a
stool sample, and their cases were therefore more likely to be recognized.
As 40% of the secondary cases were asymptomatic, the true level of
asymptomatic and mild infection in household contacts and overall secondary
transmission were probably underestimated. This statement, however, does
not take into account any role preexisting immunity might play in
determining the severity of symptoms and the likelihood of fecal carriage.
Persons with higher titers of immunoglobulin (Ig) G antibodies to VT1 in
two family outbreaks of STEC have been more likely to be both asymptomatic
and not excreting STEC than other family contacts with lower titers (16).
Certain household contacts thus might not be able to contract secondary
cases. Secondly, the age of the household contact is clearly associated
with the likelihood of submitting a fecal sample. Younger contacts were
more likely to be screened; therefore, their cases were more likely to be
recognized. Nevertheless, the pattern of household transmission
demonstrated using as the denominator 101 screened contacts was similar to
that using all 181 contacts. The timeliness of specimens did not vary with
age.

Overall, household contacts of 1 to 4 years of age and adults of 15 to 34
years of age are most likely to contract STEC O157 from an index
case-patient. Children of 1 to 4 years of age explore the domestic
environment and frequently put their hands (and other objects) in their
mouths, and adults 15 to 34 years of age are likely to be looking after
children with diarrhea. When we compared contacts who are parents of an
infected child with the remainder of contacts, we observed an elevated risk
of contracting STEC O157 in the home (not significant at the 10% level).
The pattern of transmission suggests person-to-person spread by the
fecal-oral route rather than by food.

Our findings have several implications for the control of STEC O157.
Current U.K. guidelines (14,15) advise the prompt investigation of sporadic
cases of STEC and the identification of other persons with symptoms in the
household. Since STEC O157 is readily transmitted in the home, contacts of
the index case-patient may excrete the organism and present a risk to the
wider community through attendance at child day-care centers or through
their occupations. Among household contacts, 2 of 13 children under 5 years
of age who attended day care had STEC-positive samples. Our data support
information from published outbreaks in day-care centers showing that
children attending with diarrhea pose a major risk (8) and reinforcing U.K.
guidelines (14,15) that, irrespective of symptoms, young siblings of an
index case-patient under 5 years of age be excluded from day care until
they have two negative fecal specimens at intervals not smaller than 48
hours.

Secondary spread of STEC O157 both during outbreaks and by sporadic cases
has been documented (13,17,18). In a large hamburger-associated outbreak in
the United States in 1993, an estimated 11% of all cases resulted from
secondary spread (19). Whatever the source of the initial infection,
secondary transmission, particularly by children, contributes significantly
to the incidence of STEC O157 infection. Advice on the risk and on
necessary hygienic precautions in the home when a family member has a
diarrheal illness should be provided promptly to all affected persons.

Acknowledgments

We gratefully acknowledge the help and support of the following
laboratories and their link personnel: Bronglais, Aberystwyth; Caerphilly
Miners; East Glamorgan, Pontypridd; Llandough, Cardiff; Nevill Hall,
Abergavenny; Prince Charles, Merthyr; Prince Phillip, Llanelli; Princess of
Wales, Bridgend; Royal Gwent, Newport; Withybush, Haverfordwest; Wrexham
Maelor; Bangor Public Health Laboratory (PHL); Cardiff PHL; Carmarthen PHL;
Chester PHL; Hereford PHL; Rhyl PHL; Shrewsbury PHL; Swansea PHL;
Laboratory of Enteric Pathogens, Central Public Health Laboratory,
Colindale, London. We also thank the Welsh Office Standing Specialist
Advisory Group (Microbiology): Local Authority Environmental Health
Departments, health authority consultants in communicable disease control,
and general practitioners throughout Wales and the Borders.

This work was funded by Department of Health (Grant 145).

      Dr. Parry worked as an environmental health officer (sanitarian) for
Cardiff City Council in infectious diseases and food safety. Since 1993 she
has been a research officer with the Welsh Combined Centres for Public
Health.

      Dr. Salmon is a medical epidemiologist with the U.K. Public Health
Laboratory Service Communicable Disease Surveillance Centre in Cardiff. He
has a particular interest in zoonoses and has worked on the epidemiology of
STEC infections for 10 years; in 1990 he created in Wales a system of total
population surveillance for STEC O157.

      Address for correspondence: R.L. Salmon, PHLS-CDSC (Wales), Abton
House, Wedal Road, Cardiff CF4 3QX, United Kingdom; fax: 44-0-1222-521987;
e-mail: roland.salmon@cdsc.wales.nhs.uk.

References

  1. Riley LW, Remis RS, Helgerson SD, McGee HB, Wells J, Davis BR, et al.
     Hemorrhagic colitis associated with a rare Escherichia coli serotype.
     N Engl J Med 1983;308:681-5.
  2. Griffin PM. Escherichia coli O157:H7 and other enterohemorrhagic
     Escherichia coli. In: Blaser MJ, Smith PD, Ravdin JI, Greenberg HB,
     Guerrant RL, editors. Infections of the Gastrointestinal Tract. New
     York: Raven Press; 1995. p. 1-11.
  3. Griffin PM, Tauxe RV. The epidemiology of infections caused by
     Escherichia coli O157:H7, other entero-hemorrhagic E. coli, and the
     associated hemolytic uremic syndrome. Epidemiol Rev 1991;13:60-98.
  4. Rower PC, Orrbine E, Lior H, Wells GA, McLaine PN, Canadian Pediatric
     Kidney Disease Reference Centre (CPKDRC) co-investigators. Diarrhea in
     close contacts as a risk factor for childhood haemolytic uraemic
     syndrome. Epidemiol Infect 1993;110:9-16.
  5. Kohli HS, Chaudhuri AKR, Todd WTA, Mitchell AAB, Liddell KG. A severe
     outbreak of E.coli O157 in two psychogeriatric wards. J Public Health
     Med 1994;16:11-5.
  6. Carter AO, Borczyk AA, Carlson JAK, Harvey B, Hockin JC, Karmali MA,
     et al. A severe outbreak of Escherichia coli O157:H7-associated
     hemorrhagic colitis in a nursing home. N Engl J Med 1987;317:1496-500.
  7. Allaby MAK, Mayon-White R. Escherichia coli O157: outbreak in a day
     nursery. Commun Dis Rep CDR Rev 1995;5:R4-6.
  8. Belongia EA, Osterholm MT, Soler JT, Ammend DA, Braun JE, MacDonald
     KL. Transmission of Escherichia coli O157:H7 infection in Minnesota
     child day-care facilities. JAMA 1993;26:883-8.
  9. Ludwig K, Ruder H, Bitzan M, Zimmermann S, Karch H. Outbreak of
     Escherichia coli O157 infection in a large family. Eur J Clin
     Microbiol Infect Dis 1997;16:238-41.
 10. Thomas A, Cheasty T, Frost JA, Chart H, Smith HR, Rowe B. Vero
     cytotoxin-producing Escherichia coli, particularly serogroup O157,
     associated with human infections in England and Wales. Epidemiol
     Infect 1996;117:1-10.
 11. Tilden J Jr, Young W, McNamara AM, Custer C, Boesel B, Lambert-Fair
     MA. A new route of transmission for Escherichia coli: infection from
     dry fermented salami. Am J Public Health 1996;86:1142-5.
 12. Willshaw GA, Thirlwell J, Jones AP, Parry S, Salmon RL, Hickey M. Vero
     cytotoxin-producing Escherichia coli O157 in beefburger linked to an
     outbreak of diarrhoea, haemorrhagic colitis and haemolytic uraemic
     syndrome in Britain. Letters in Applied Microbiology 1994;19:304-7.
 13. E. coli O157 infections. SCIEH Weekly Report 1995;29:57.
 14. Subcommittee of the Public Health Laboratory Service Working Group on
     Vero cytotoxin producing Escherichia coli (VTEC). Interim guidelines
     for the control of infections with Vero cytotoxin producing
     Escherichia coli (VTEC). Commun Dis Rep CDR Rev 1995;5(Review
     6):R77-81.
 15. Working Party of the Public Health Laboratory Service Salmonella
     Committee. The prevention of human transmission of gastrointestinal
     infections, infestations, and bacterial intoxications. Commun Dis Rep
     CDR Rev 1995;5(Review 11):R158-72.
 16. Karmali M, Petric M, Winkler M, Bielaszewska M, Brunton J, van de Kar
     N, et al. Enzyme-linked immunosorbent assay for detection of
     immunoglobulin G antibodies to Escherichia coli Vero cytotoxin 1. J
     Clin Microbiol 1994;32:1457-63.
 17. Davis BS, Brogan RT. A widespread community outbreak of E. coli O157
     infection in Scotland. Public Health 1995;109:381-8.
 18. Upton P, Coia JE. Outbreak of Escherichia coli O157 infection
     associated with pasteurised milk supply [Letter]. Lancet
     1994;344:1015.
 19. Centers for Disease Control and Prevention. Update: multistate
     outbreak of Escherichia coli O157:H7 infections from
     hamburgersWestern United States, 19921993. JAMA 1993;269:2194-6.
---------------------------------------------------------------------------------
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Dispatches

Ehrlichia Infection in Italy

M. Nuti,* D.A. Serafini, D. Bassetti, A. Ghionni, F. Russino, P.
Rombol,# G. Macri,# and E. Lillini#
*1st Rome University, Rome, Italy; US Navy Medical Services, Colorado
Springs, Colorado, USA; Public Health Department, Trento, Italy; Pieve di
Cadore Hospital, Belluno, Italy; Multizone Prevention Service, Belluno,
Italy; and #Zooprophylaxis Institute, Rome, Italy

---------------------------------------------------------------------------
      Immunoglobulin M seroconversion to Ehrlichia chaffeensis was
      documented in U.S. citizens bitten by ticks in Sardinia. Seven
      cases of suspected ehrlichiosis in local residents were not
      confirmed by laboratory tests. In Alpine areas antibodies to E.
      phagocytophila were detected in persons at high risk, i.e.,
      foresters (8.6%) and hunters (5.5%), and in controls (l.5%). Of
      153 persons bitten by ticks, only one was Ehrlichia
      antibody-positive after 6 months.

A newly recognized rickettsial disease, human ehrlichiosis is emerging in
tick-infested areas. First described in the United States (1), the disease,
in both monocytic and granulocytic forms, is usually found where Lyme
disease is endemic (2,3). Sporadic cases of ehrlichiosis have been reported
in southern and northern Europe. Recently, cases were reported in Slovenia,
not far from the Italian border (4). Preliminary studies show that 17.1%,
5%, 6.3%, and 11.4% of forestry workers in Switzerland, United Kingdom,
Italy, and Sweden, respectively (5-8), have antibodies to Ehrlichia (E.
phagocytophila and E. equi).

Ehrlichiosis in Southern Italy

In 1995 through 1996,    Table. Immunoglobulin (Ig) M antibodies to
four U.S. citizens (two  Ehrlichia chaffeensis in four U.S. citizens
boys and two women)      bitten by ticks in Sardinia (La Maddalena)
living on a U.S. Navy
base in Sardinia (La
Maddalena) became ill    --------------------------------------------------
with acute fever,                          Date of           No.
headache, malaise, and   Case               onset    S1    weeks   S2
cytopenia after a tick
bite. Once rickettsial   --------------------------------------------------
disease was excluded     8-yr-old boy(sup a)8/95     Neg     1   1:128
(Sardinia is a
boutonneuse              12-yr-old boy      7/96     Neg     1   1:64(sup b)
feverendemic focus),
ehrlichia infection was  25-yr-old
suspected. Coupled serum woman              7/96     Neg     1   1:128
samples (S1 and S2 a     24-yr-old
week apart) from each    woman              7/96     Neg     1   1:128(sup c)
patient were processed
by immunofluorescence    --------------------------------------------------
assay (IFA). All S1 were (sup a)Evacuated to U.S. Army Hospital in Landsthul,
negative for both			  Germany.
species of Ehrlichia,    (sup b)Not diagnostically significant.    
while three S2 were      (sup c) Third IgM control 1 month later = 1:256.  
positive for             
immunoglobulin (Ig) M
(1:128) to E. chaffeensis and negative for IgG (Table), meeting the case
definition criteria for ehrlichiosis (Olson, pers. comm.). Subsequently,
seven cases of ehrlichiosis-like illness following a tick bite were
reported in Sardinian residents. The cases were not confirmed by serologic
tests in Rome and Atlanta (E. Lillini, unpub. data).

Ehrlichiosis in Alpine Areas of Italy

In the last 10 years, tick-borne diseases, especially Lyme disease and
tick-borne encephalitis (TBE), have become more widespread in Alpine
mountain areas (9). Although new diagnostic tests aid clinical diagnosis,
some febrile illnesses in persons bitten by ticks remain unexplained. Human
ehrlichiosis must now be considered an emerging problem in tick-infested
areas. Pioneering studies in the northern Veneto region (7,10) have
recorded Ehrlichia antibodies in residents of the Alpine area, where Ixodes
ticks are present and Lyme disease appears widespread (with more than 500
clinical cases reported annually). This study included Cadore, Bellunese
(Veneto region), and Trento Districts, neighboring zones located around an
area of 80 to 100 miles in the upper northeastern Alpine region (Figure),
which pose equal risk of acquiring tick-borne pathogens.

									     In 1995 through
                                                           1996, 544 serum
 [Figure.] Areas under study, Sardinia and northeastern    samples were
 Alpine areas.                                             collected from
                                                           inhabitants of
                                                           the
northeastern Alpine area presumably at risk for ehrlichiosis because they
lived in wooded areas and spent time outdoors. A simplified classification
system subdivided the participants according to job or primary vocation:
healthy residents (controls, n = 193), rangers and forestry workers from
Belluno Regional Park and Forestry Services of Trento District (n = 242),
and hunters (n = 109) from both areas. Ehrlichia IgG antibodies were
detected by IFA using E. phagocytophilainfected ovine neutrophils as
antigen (courtesy of Dr. K.J. Sumption, Royal School of Veterinary Studies,
Edinburgh University, UK). An IFA titer >1:80 was considered positive (all
analyses contained controls of conjugate antibodies and positive and
negative sera). Antibodies to Borrelia burgdorferi sensu lato were
routinely detected by a commercial standard immunoenzyme test kit (Behring,
Marburg, Germany). IgG and IgM were considered positive at values (optical
density) of >0.6 for IgG and >0.50 for IgM. The TBE antibody (positive
titer >150) was performed by immunozyme and confirmed by immunoblot
(Immuno AG, Wien, Austria). This retrospective serologic study of a cohort
of persons at high risk showed an IFA positivity (without benefit of
immunoblot confirmation) to E. phagocytophila (highest titer 1:160) in 21
(8.6%) of 242 foresters, 6 (5.5%) of 109 hunters, and 3 (1.5%) of 193
controls, without significant difference between the three wooded areas
studied. Concerning the Borrelia positivity, the mean prevalence value
among the 541 persons tested was 14.6%, with a lower value (10%) in
foresters from Trento District. Seropositivity was most frequent in persons
ages 40 to 60 years.

In summer and autumn of 1997, we followed for 6 months 153 Cadore residents
bitten by ticks; we detected Borrelia, Ehrlichia, and TBE antibodies
immediately after the tick bite and 2 and 6 months later. At the
preliminary examination, serologic evidence indicated exposure to multiple
tick-borne agents in 26 (16.9%) of 153 patients, 15% to Borrelia, 0.6% to
E. phagocytophila, 1.3% to TBE virus. At 2 months, four of the 131 patients
tested had IgM antibodies to Borrelia (overall incidence 3%); two of these
had a clinical pattern of borreliosis. The mean IgG titer to Borrelia was
46 (range 7 to >375); the IgM titers were 0.511 to >1.892. Concomitantly,
one patient showed typical clinical features of TBE infection, followed by
a diagnostic IgM and IgG titer rise (>400); a febrile flulike illness in
another patient remained undiagnosed. In the only E.
phagocytophilapositive person, the titer remained unchanged (1:80). Among
the 128 patients tested at 6 months, the four previously recorded as IgM
positive to Borrelia were confirmed with increased titers (>1,892), and a
concomitant IgG positivity (titer >375) was observed in one of these four
cases. This study may be one of the largest prospective serologic
evaluations for tick-borne pathogens; previous studies have been
single-sample serosurveys.

Our data confirm the well-documented high incidence of Borrelia infections
in upper Alpine areas and show remarkably lower antibody prevalence rates
(1%) for TBE and Ehrlichia. The different values observed between foresters
and healthy controls (8.6% versus 1.5%; p <0.050) from the same zones could
be justified by different lifestyles. Foresters spent most of their time in
potentially infected areas, changing worksites daily, coming into frequent
contact with ticks, and reporting one or more tickbites per year. Controls,
on the contrary, had limited outdoor activity, generally restricted to the
vicinity of their homes, and less contact with ticks.

Seroconversion to either Borrelia or TBE virus were strongly
associated with a history of tick attachment; this association and the
origin of the persons from a Lyme diseaseendemic area support the
likelihood that the observed seroconversions were due to a tick-borne
agent. The local residents, mainly living in small villages, have close
contact with nature but often do not remember tick bites, perhaps because
ticks are small and may remain attached for a long time without being
noticed. In fact, in approximately 30% of the Lyme disease patients, tick
bites are not detectable or reported; consequently, it is difficult to
divide these persons into well-defined groups according to tick bite
history (9).

Since Lyme disease and ehrlichiosis are zoonoses transmitted by the same
tick species (Ixodes) and perpetuated in the same reservoir (rodents,
deers, sheep), human infections by these pathogens are likely to occur in
the same wooded environments. Hunters and rangers, who frequently come in
direct contact with animal (deer) carcasses infested by Ixodes ticks, or
who camp overnight, are at high risk for tick-borne infections (9). Hunters
may be infected directly by deer blood (11). Ehrlichia seropositivity in
persons at risk suggests that mild or subclinical illness due to an
infection by Ehrlichia species is possible, as recently confirmed (Caruso
and Brouqui, pers. data). The low titers observed until now (maximum 1:160)
are probably related to a past, mild infection or to the use of an antigen
with low specificity.

Conclusions

Our findings indicate the presence of ehrlichiosis in southern Italy
(Sardinia) and of subclinical infections in the upper Alpine northeastern
areas. In these forested areas, where most patients and physicians are
still unaware of the disease, clinical cases of ehrlichiosis had not been
documented, although we can presume that some cases of unexplained febrile
illness, especially in areas where Lyme disease is endemic, could be
related to Ehrlichia infection. Further epidemiologic surveillance and
controls, now in progress, may better clarify the emergence of this new
infection in Italy.

      Dr. Nuti is associate professor of tropical and infectious diseases
at the La Sapienza University of Rome. His research has focused on leprosy,
viral hepatitis, malaria, dengue, Hantaan virus, and tick-borne diseases.

      Address for correspondence: E. Lillini, Istituto Zooprofilattico, Via
Appia Nuova 1411, 00178 Roma; fax: 39-6-79340724.

References

  1. Maeda K, Markowitz N, Hawley RC, Ristic M, Cox D, McDade JE. Human
     infection with Ehrlichia canis, a leukocytic rickettsia. N Engl J Med
     1987;316:853-6.
  2. Magnarelli LA, Dumler JS, Anderson JF, Johnson RC, Fikrig E.
     Coexistence of antibodies to tick-borne pathogens of babesiosis,
     ehrlichiosis, and Lyme borreliosis in human sera. J Clin Microbiol
     1995;33:3054-7.
  3. Schwartz I, Fish D, Daniels TJ. Prevalence of rickettsial agent of
     human granulocytic ehrlichiosis in ticks from a hyperendemic focus of
     Lyme Disease. N Engl J Med 1997;337:49-50.
  4. Petrovec M, Furlan SL, Avsic-Zupanc T, Strle F, Brouqui P, Roux V, et
     al. Human disease in Europe caused by granulocytic Ehrlichia species.
     J Clin Microbiol 1997;35:1556-9.
  5. Brouqui P, Dumler JS, Lienhard R, Brossard M, Raoult D. Human
     granulocytic ehrlichiosis in Europe. Lancet 1995;346:782-3.
  6. Sumption KJ, Wright DJM, Cutler SJ, Dale BAS. Human ehrlichiosis in
     the UK. Lancet 1995;346:1487-8.
  7. Lillini E, Macri G, Rombol P, Grazioli D, Russino F, Nuti M. Ehrlichia
     seropositivity in Italian foresters. In: Kazar J, Toman R, editors.
     Rickettsiae and rickettsial diseases. Bratislava, Slovakia: Slovak
     Academy of Science; 1996. p. 335-9.
  8. Dumler JS, Dotevall L, Gustafson R, Granstrom M. A population-based
     seroepidemiologic study of human granulocytic ehrlichiosis and Lyme
     borreliosis on the west coast of Sweden. J Infect Dis 1997;175:720-2.
  9. Nuti M, Amaddeo D, Crovatto M, Ghionni A, Polato D, Lillini E, et al.
     Infections in an Alpine environment: antibodies to hantavirus,
     leptospira, rickettsiae, and Borrelia burgdorferi in defined Italian
     population. Am J Trop Med Hyg 1993;48:20-5.
 10. Nuti M, Russino F, Grazioli D, Rombolo P, Macri G, Lillini E.
     Anticorpi anti-ehrlichia in soggetti a rischio di zone pedemontane del
     Veneto. Microbiologia Medica 1996;11:492-5.
 11. Bakken JS, Krueth JK, Lund T, Malkovitch D, Asanovich K, Dumler JS.
     Exposure to deer blood may be a cause of human granulocytic
     ehrlichiosis. Clin Infect Dis 1996;23:198.
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Dispatches

Salmonella Enteritidis PT6: Another Egg-Associated Salmonellosis?

Meirion R. Evans,* Will Lane, and C. Donald Ribeiro
*Bro Taf Health Authority, Cardiff, United Kingdom; Cardiff Environmental
Protection, Cardiff, United Kingdom; and Cardiff Public Health Laboratory,
Cardiff, United Kingdom

---------------------------------------------------------------------------
      Salmonella Enteritidis phage type 6 (PT6) increased
      dramatically in the United Kingdom during 1997. The sharp rise
      suggests that PT6 contamination has spread rapidly throughout a
      basic food commodity; however, the source and food vehicle
      remain unknown. We present evidence from three outbreaks
      suggesting a possibile link between PT6 and eggs. Poor
      documentation of the egg supply network continues to pose
      problems for public health investigators. Thorough
      investigation of all future PT6 outbreaks and case-control
      studies of sporadic infections are needed to confirm the
      etiology of PT6 infection. .

Nearly 10 years ago, evidence from both sides of the Atlantic first
implicated eggs in the sharp increase during the 1980s of food poisoning
due to Salmonella Enteritidis (1,2). In the United Kingdom and Western
Europe, the predominant phage type (PT) responsible for eggborne Salmonella
infection is PT4 (3). In the United States, although PT8 and PT13a are the
most common phage types, no single phage type is more likely to be
associated with eggborne infection (4). We report the possible emergence of
a new egg-associated Salmonella phage type in the United Kingdom.

During 1997, we investigated three Salmonella Enteritidis PT6 food
poisoning outbreaks in Cardiff, Wales. In outbreak 1, five of approximately
200 staff of an education establishment who regularly used a staff canteen
reported diarrhea; three were positive for S. Enteritidis PT6. All five had
eaten fish cakes from a batch of 18 served at the canteen; no illness was
reported by other staff, nearly all of whom had eaten the alternative meal.
Fish cakes, made with fish and reconstituted dried mashed potatoes, were
shaped by hand, coated with raw shell egg, dipped in breadcrumbs, and
shallow-fried in oil. When we reproduced the cooking process, as described
in a previous outbreak (5), we found that fish cakes were too thick to be
properly cooked by the shallow-frying method. The temperature varied
greatly both between fish cakes and between parts of the same fish cake.
Although the top and bottom of the fish cake were cooked, the egg coating
on the sides had insufficient contact with the frying oil to achieve
adequate cooking temperature.

Outbreak 2 occurred in a nursing home accommodating 99 residents on four
floors. Two residents from different floors and two staff (a cook and a
health-care worker) were initially confirmed with S. Enteritidis PT6
infection. The only common factor linking the residents was a pureed food
diet. When we screened all 10 residents on pureed food diets and a sample
of 30 residents on normal diets, we found four other S. Enteritidis PT6
cases, all in patients on pureed food diets. The health-care worker was
probably infected by secondary spread. The cook's illness began 4 days
after the index patient's, which suggested that the cook was infected from
a common food source and had not in fact been the cause of the outbreak.
Raw shell egg was regularly used in the kitchen; pureed food may have been
cross-contaminated from a mixing bowl used for both raw and pureed food.

In outbreak 3, eight customers of the same small Italian restaurant had S.
Enteritidis PT6 food poisoning. All had eaten the dessert tiramisu during a
5-day period. Two batches of tiramisu containing raw shell egg were
implicated, but neither was available for microbiologic examination.
However, a layered cassata ice cream also containing raw egg was tested,
and the top layer, made the same day as the first batch of tiramisu, was S.
Enteritidis PT6positive. Kitchen inspection indicated that the second
batch of tiramisu was probably cross-contaminated when the same whisks and
mixing bowls used to make the first batch were reused.

PT6 is a hitherto uncommon phage type in the United Kingdom. In Cardiff,
one case not associated with foreign travel was reported in 1994 and in
1995, and two in 1996. During 1997, in addition to the outbreaks described,
33 sporadic cases were reported in Cardiff. In Wales as a whole, reports of
S. Enteritidis PT6 rose from 23 in 1995 and 43 in 1996 to 279 in 1997,
while reports of PT4 remained constant (PHLS Communicable Disease
Surveillance Centre [Wales], unpub. data) (Figure). More than 1,000 cases
of S. Enteritidis PT6 were reported in England and Wales in 1996, almost
twice the number in 1995, making it the second most common phage type after
PT4 (6). Most cases appear sporadic and acquired within the United Kingdom.
Poultry meat and shell eggs were implicated in three of the six outbreaks
reported in 1996 (2). The largest outbreak involved 49 persons, of whom 13
had laboratory confirmed S. Enteritidis PT6 (7). Eating egg sandwiches
served at a buffet meal was strongly statistically associated with illness,
and S. Enteritidis PT6 was isolated from several environmental samples from
the kitchen where the food had been prepared. However, the investigation
did not show whether or not shell eggs used in the sandwiches were the
original source or had been cross-contaminated.

The three S. Enteritidis PT6 outbreaks
described occurred in very different     

settings: a work canteen, a nursing      
home, and a public restaurant. Although
the evidence is circumstantial, our      Figure. Salmonella isolates
reports suggest a possible link between  reported in Wales by serotype and
S. Enteritidis PT6 and eggs. Traceback   phage type, 1995-1997. Source:
of eggs was particularly difficult,      PHLS Communicable Disease
both because of poor egg labeling and    Surveillance Centre (Wales).
because of the complex network of
suppliers and distributors involved. As
far as could be ascertained, the eggs associated with the three outbreaks
were from different sources, which suggests a general egg contamination
problem; if this is the case, the egg contamination signals a complete
failure of measures to eradicate salmonellae from poultry-breeding flocks.
Alternatively, egg contamination may be restricted to one particular sector
of the egg industry. Salmonella-contaminated eggs imported into the United
Kingdom have been associated with particular packing stations (8).
Traceback investigations of eggs in the United Kingdom continue to pose
difficulties for public health investigators because of deficiencies in
documentation kept on the egg distribution network. Identifying the source
of egg-related outbreaks requires better egg labeling and documentation at
all levels of the supply chainfrom farmer through distributor to the point
of retail sale.

The increase in S. Enteritidis PT6 could also be explained by molecular
change in the previously dominant PT4. However, PTs 4 and 6 are well
established distinct types whose type strains have remained unchanged for
many years (5); PT6 is probably not replacing PT4 since no concomitant
decrease in PT4 incidence has been observed. The sharp rise in PT6
incidence in the United Kingdom over the past 18 months therefore suggests
that this phage type has found a new ecologic niche and that contamination
has spread rapidly throughout a basic food commodity. The source and food
vehicle remain unknown. All S. Enteritidis PT6 outbreaks should be
throughly investigated, and case-control studies are needed to establish
the etiology of sporadic PT6 infections.

      Dr. Evans is a consultant in communicable disease control, Public
Health Directorate, Bro Taf Health Authority, Cardiff, and honorary
consultant epidemiologist, Public Health Laboratory Service Communicable
Disease Surveillance Centre (Wales). His research interests include the
epidemiology of foodborne disease, childhood immunization, and travel
health.

Address for correspondence: Meirion R. Evans, Public Health Directorate,
Bro Taf Health Authority, Cathays Park, Cardiff, CF1 3NW, United Kingdom;
fax: 44-1222-371104; e-mail: meirion.evans@bro-taf-ha.wales.nhs.uk.

References

  1. Coyle EF, Palmer SR, Ribeiro CD, Jones HI, Howard AJ, Ward L, et al.
     Salmonella enteritidis phage type 4 infection: association with hen's
     eggs. Lancet 1988;2:1295-7.
  2. St. Louis ME, Morse DL, Potter ME, De Melfi TM, Guzewich JJ, Tauxe RV,
     et al. The emergence of grade A eggs as a major source of Salmonella
     enteritidis infections. JAMA 1988;259:2103-7.
  3. Rampling A. Salmonella enteritidis five years on. Lancet
     1993;342:317-8.
  4. Mishu B, Koehler J, Lee LA, Rodrigue D, Brenner FH, Blake P, et al.
     Outbreaks of Salmonella enteritidis infections in the United States,
     19851991. J Infect Dis 1994;169:547-52.
  5. Evans MR, Hutchings PG, Ribeiro CD, Westmoreland D. A hospital
     outbreak of salmonella food poisoning due to inadequate deep-fat
     frying. Epidemiol Infect 1996;116:161-7.
  6. PHLS Communicable Disease Surveillance Centre. Salmonella infections,
     England and Wales: reports to the PHLS (salmonella data set). Commun
     Dis Rep CDR Wkly 1997;7:208.
  7. Holtby I, Tebbitt GM, Grunert E, Lyle HJ, Stenson MP. Outbreak of
     Salmonella enteritidis phage type 6 infection associated with food
     items provided at a buffet meal. Commun Dis Rep CDR Rev 1997;7:87-90.
  8. de Louvois J. Salmonella contamination of eggs. Lancet 1993;342:366-7.
  9. Stanley J, Jones CS, Threlfall EJ. Evolutionary lines among Salmonella
     enteritidis phage types are identified by insertion sequence IS200
     distribution. FEMS Microbiol Lett 1991;82:83-90.
---------------------------------------------------------------------------------
---------------------------------------------------------------------------------

Dispatches

Reemergence of Epidemic Malaria in the Highlands of Western Kenya

M. A. Malakooti*, K. Biomndo, and G. D. Shanks
*University of the Health Sciences, Bethesda, Maryland, USA; Brooke Bond
Central Hospital, Kericho, Kenya; US Army Medical Research Unit, Nairobi,
Kenya

---------------------------------------------------------------------------
      Hospital records (1990-1997) of a tea company in the Kericho
      district, western Kenya, showed malaria epidemics almost
      annually from May to July, with an annual attack rate of 50%,
      857 hospitalizations per 100,000 per year, and 42 deaths per
      100,000 per year; 32% of deaths in hospitalized patients were
      caused by malaria. A questionnaire survey (June 1997) of 244
      patients hospitalized for malaria showed that only 8% had
      traveled to an area with known malaria transmission 30 days
      before diagnosis. The increasing malaria incidence may be due
      to drug resistance.

This study investigated reports of epidemic malaria in a highland area of
Kenya traditionally thought to be free of malaria to 1) formulate
recommendations for reducing disease incidence and preventing future
epidemics and 2) determine whether malaria was imported or was due to local
transmission.

The factors necessary for malaria transmission are the Plasmodium parasite,
the Anopheles mosquito vector, and the human host. Both parasite and vector
are affected by temperature and rainfall. Although temperature extremes
kill Anopheles mosquitoes, higher temperatures (15C30C) increase their
rate of development. The gonotrophic cycle (interval between blood meals)
shortens with increasing temperature, and the effect of a small temperature
increase is greatest at the cooler ambient temperatures. Thus, a small rise
in temperature from 19C to 21C shortens the gonotrophic cycle from 4 to 3
days and increases the vectorial capacity of the mosquito (1). Altitude is
thought to be a proxy for temperature, so the actual limiting factor for
malaria at high altitude is the effect of the lower temperature on the
parasite (2). Despite the effect of altitude on ambient temperature,
microclimatic factors (e.g. heated houses) can play an important role in
facilitating malaria transmission and epidemics at higher elevations.
Unlike the parasite, the mosquito vector can commonly be found at altitudes
from >1,600 m (3,4) to 3,000 m, demonstrating that the limiting factor for
malaria transmission at high altitude is the survival of the Plasmodium
parasite. The Anopheles population is very sensitive to rainfall, which
increases the availability of mosquito breeding sites. A. gambiae, the
primary malaria vector of western Kenya, lays its eggs in small pools and
puddles; in this region, rainfall of 150 mm per month leads to rapid
expansion of the A. gambiae population (3,5) and hence increased risk for
malaria transmission. Finally, epidemic malaria requires sufficient numbers
of human gametocyte carriers (to infect Anopheles mosquitoes) and nonimmune
human hosts (to acquire clinical infection).

Malaria did not exist in the western Kenya highlands until the second
decade of the 20th century (6). In 1901, completion of a railway from the
Kenya coast across the highlands and down to Lake Victoria and increased
road transport facilitated the gradual spread of infective mosquitoes into
the highlands from the low-lying hyperendemic-disease areas (5). The
development of tea estates and agriculture in the highlands, with the
concomitant clearing of the forests, provided suitable mosquito breeding
grounds. Finally, importation of infected laborers completed the conditions
necessary for malaria transmission.

The first reported epidemic was in 1918 to 1919 when local soldiers
returned from Tanzania after World War I (6). Two epidemics were recorded
in the 1920s and four in the 1930s. Garnham (2) reported epidemics of
malaria in the Londiani area of western Kenya from 1941 to 1944, close to
the site of the present study, at an altitude of approximately 2,200 m.
After the military camp in the area was disbanded in 1944, the local
outbreaks ceased, but highland malaria continued to be a serious public
health problem until the late 1950s, when an extensive control program
essentially eliminated the disease (7). The highlands were considered free
of malaria through the 1960s, but since the 1980s malaria has been
increasing (8).

More than 90% of malaria in Kenya is caused by P. falciparum (4,5) and
transmitted most often by A. gambiae, with A. funestus as a secondary
vector. It has always been assumed that malaria in the highlands of western
Kenya is not due to local transmission but is imported from the nearby
holoendemic-disease areas around Lake Victoria by the frequent travel of
the tea plantation workers and their families.

The Study

Geography

This study was conducted in the Kericho tea-growing area at 0 22' south
and 35 17' east in the Rift Valley highlands of western Kenya, 80 km from
the holoendemic malaria area of the Lake Victoria basin. The altitude of
the tea estates is 1,780 m to 2,225 m; the mean monthly temperature is
18.7C. February is the warmest month of the year (Figure 1), with a mean
maximum daily temperature of 28.3C and mean minimum daily temperature of
10.8C; July is the coolest month, with a mean maximum daily temperature of
25.4C and mean minimum daily temperature of 10.2C. Annual rainfall is
1.79 m with a March-to-June rainy season, and relative humidity is
generally slightly below 60% except for May to July. April is the wettest
month, with mean rainfall of 252 mm; December is the driest with a mean
rainfall of 78 mm.

Demographics
                                         

The study data were obtained from the    
health-care system of a major tea
company, which has 18 estates with       [Figure 1.] Mean monthly rainfall
22,000 workers and approximately         (197197), maximum and minimum
100,000 persons eligible for health      temperatures (198497), Kericho
care; 32% of company employees belong    area.
to ethnic groups whose traditional home
areas have holo/hyperendemic malaria.
Given that marriage between ethnic groups is not common, it follows that
about 30,000 workers and dependents are from malarious areas; most people
travel back to their home regions at least once per year. Approximately 31%
of employees originate from the highland areas and the remaining 37% from
nonendemic-disease areas around the country. This labor composition has
remained stable for at least 20 to 30 years. The employees and families
generally live in cement-walled, corrugated metal-roofed duplex style
houses, situated close together in large housing areas. The houses usually
have 1 rooms, with a wood or charcoal-burning fireplace, one door, and two
unscreened windows. The Central Hospital has 67 beds, two physicians, and
two clinical officers, and averages 50 to 80 hospitalized patients per day
and 20,000 outpatients per year. Three outlying medical centers, each with
a clinical officer, see 15,000 to 20,000 patients per year; and 26
dispensaries, each with a nurse provider, see approximately 180,000 to
240,000 outpatients per year.

Data Sources

Retrospective study data were extracted from the Central Hospital monthly
reports, which include inpatient and outpatient illness, death, and
laboratory data for the whole health-care system. To determine local risk
factors for malaria infection, we used daily logbooks on the inpatient
wards to identify patients admitted with malaria during June 1997; a simple
questionnaire was field tested in consultation with hospital staff and
administered to 244 of the 257 identified malaria patients hospitalized
during June 1997. The questionnaire asked for demographic and recent travel
history and was administered by the primary investigator with a local field
worker/translator or by one of the hospital clinical officers.

Findings

Central Hospital illness and death data were available from 1990 through
1997, except for outpatient and death data from October to December 1992.
Tea company temperature data were available from 1984 through 1997, and
rainfall data from 1971 through 1997 (Figures 1 and 2). The retrospective
illness data clearly show the nearly annual occurrence of epidemic malaria
(Figures 2 and 3) and the importance of known factors in highland malaria
transmission; rainfall >150 mm per month in this area promotes epidemic
malaria, and average temperature <18C appears to extinguish epidemics. In
the Kericho area, malaria epidemics occur during May to July, after the
long rains and before the temperature drops in July (Figure 3). Figure 4
shows outpatient and inpatient malaria and malaria deaths from 1991 through
1997. For outpatient malaria, the mean annual rate was 48,112 per 100,000
(range 33,914 to 56,040 per 100,000), i.e., each year malaria was diagnosed
in approximately 50% of company health-care beneficiaries; for malaria
admissions the mean annual rate was 857 per 100,000 (range 361 to 1,058 per
100,000); and for malaria deaths the mean annual rate was 42 per 100,000
(range 14 to 64 per 100,000). In hospitalized patients malaria diagnosis
was laboratory confirmed, but in most outpatients malaria was diagnosed
purely on a clinical basis. Of the 764 inpatient deaths documented from
1991 through 1997, 254 (32%) were caused by malaria, the leading cause of
death in this highland population. Of the 254 malaria deaths, 37% were
recorded as being due to severe anemia, 31% cerebral malaria, and 32%
malaria with gastroenteritis or pneumonia.

 [Figure 2.] Monthly cases of            [Figure 3.] Positive slides, rainfall,
 slide-positive malaria versus maximum,  and average temperature, per month.
 minimum, and mean temperature.

 [Figure 4.] Malaria outpatients,
 inpatients, and deaths, versus
 time.
                                       
Questionnaire results were available for 244 (95%) of the 257 patients
admitted for malaria in June 1997. The remaining 13 patients were
either discharged before interview and could not be traced to their
houses on the estates, died before interview and the family was not
available for questioning, or were minors whose parents were not available
for interview. These malaria hospitalized patients were 48% female, 52%
male, with mean age 13.2 years (range 1 month to 58 years); 20% were tea
company employees; 80% were dependents. Only 16% of malaria hospitalized
patients belonged to ethnic groups originating from areas with
holo/hyperendemic malaria, versus 32% of all company employees (relative
risk = 0.4 [95% confidence interval 0.29 to 0.57] for hospitalization for
malaria if ethnic origin was from a holoendemic-disease area versus a
nonendemic disease area). Forty-three (18%) of the patients hospitalized
with malaria had traveled away from the Kericho area in the 30 days before
admission, with an average of 19 days away (range 1 to 90 days). Of the 43
hospitalized patients who had traveled, 19 had been to malarious areas;
therefore, only 8% of the 244 malaria hospitalized patients had recently
traveled to an area with known malaria transmission.

Conclusions

These data document the reemergence of recurrent epidemic malaria due to
local transmission in the highlands of western Kenya. Although malaria in
the highlands was a serious problem after World War II, because of improved
transportation and increased immunity, recurrent epidemic malaria had not
been seen in a generation. Several pertinent factors have remained stable
and do not appear to explain the increase in malaria in this area. Firstly,
climate data from the Kericho area show no obvious change in average
temperature or rainfall over the last 10 to 20 years that would explain the
present almost annual epidemics in the highlands. Although the climate data
used in this study may not be sensitive enough to detect small changes in
environmental temperature, global warming would also not explain the
epidemics seen in the 1940s to 1950s. Deforestation may be relevant,
providing more breeding sites for vector mosquitoes in sunlit pools and
perhaps leading to localized changes in weather patterns and increased
microenvironmental temperatures (1).

Secondly, travel of infected persons from adjacent malaria-endemic areas is
not new. The main road from the malaria-holoendemic Lake Victoria basin to
this highland area was built in the 1950s; in fact, people had been making
the same journey using ox wagons decades before the road was paved (5,6).
According to the tea company's central office, the tribal/ethnic
composition of the employees has remained stable at least since the 1970s,
as has the total number of health-care system beneficiaries. Therefore, the
increase in malaria does not seem to be explained by an increase in the
number of nonimmune relative to semiimmune/immune persons nor simply by an
increase in the total population.

A third factor that could lead to an impression of increasing malaria would
be the degradation of the health-care infrastructure: if the system became
overwhelmed by other diseases and lack of funding, it would be unable to
cope with the same number of malaria cases as before, leading to increased
hospitalizations and deaths. Indeed, the public health sector in much of
sub-Saharan Africa has been overwhelmed by the AIDS epidemic, lack of
funding, and other institutional problems; however, the tea company in this
study continues to provide excellent care.

One hypothesis for the increase in highland malaria is the failure of the
drugs used to cure malaria infections. The period during which epidemic
malaria was absent from the highlands corresponds to the time when
pyrimethamine and chloroquine were still effective malaria treatments. In
1953, pyrimethamine was used successfully as the first step of a large
malaria control campaign in the Kenya highlands, but by 1954, rapid
development of resistance was reported in several areas (9,10).
Nevertheless, widespread use of chloroquine as well as incorrect use of
other malaria medications continue. Clinical control of symptoms (but not
cure) creates a large number of semiimmune gametocyte carriers, who can
infect mosquitoes when transmission is favored at midyear. In this way, the
failure to cure falciparum malaria infections leads to an increase in the
human gametocyte-carrier pool, resulting in the rapid spread of epidemic
malaria among the largely nonimmune highland population. Studies are under
way to further investigate this hypothesis. While increased drug resistance
may be the major factor in the documented increase of epidemic malaria, the
causes are undoubtedly multifactorial and include vector, host, and
environmental components.

Several possible interventions to stop the cycle of epidemic malaria and
the associated high costs may be worth exploring. Spraying of houses on the
tea estates with residual insecticide could be effective against the
endophilic and endophagic A. gambiae (11), which appears to be the primary
vector in these highlands (5,7). Indoor spraying campaigns in the past in
western Kenya with DDT (12) and Dieldrin (7) have effectively combated
malaria epidemics. Even now, worker acceptability would likely not be an
issue as houses are regularly sprayed for other public health reasons.
Insecticide-impregnated bed nets may also be effective with proper
education and follow-up. Finally, improved treatment regimens to cure
malaria infections, gametocytocidal drugs to prevent transmission, and
chemoprophylaxis of workers during travel to malaria-holoendemic areas
should be evaluated. Protection of a well-defined, economically important,
and less immune population may make effective certain strategies of drug
use, such as intermittent chemoprophylaxis, which would not normally be
considered in areas with higher levels of malaria endemicity.

      Dr. Malakooti is a preventive medicine physician, Epidemiology
Department, Navy Environmental and Preventive Medicine Unit No. 2, Norfolk,
Virginia, USA. His main interests are in tropical medicine and
international health, particularly as related to malaria.

      Address for correspondence: M. A. Malakooti, MD, MTM&H, Epidemiology
Department, Navy Environmental and Preventive Medicine Unit #2, 1887
Powhatan Street, Norfolk, VA 23511-3394, USA; fax: (757) 444-1191; e-mail:
malakooti@pol.net.

Acknowledgments

We thank the Management of Brooke Bond Kenya Ltd.; Florence Chepkwony and
the staff of Brooke Bond Central Hospital; Hussein Sijenje; Aggrey Oloo of
the Kenya Medical Research Institute; and Robert Snow and other colleagues
who have commented on the manuscript. This paper is published with
permission of the director, Kenya Medical Research Institute.

References

  1. Lindsay SW, Birley MH. Climate change and malaria transmission. Ann
     Trop Med Parasitol 1996;90:573-88.
  2. Garnham PCC. Malaria epidemics at exceptionally high altitudes in
     Kenya. BMJ 1945;2:45-7.
  3. Githeko AK, Service MW, Nbongo CM, Atieli FK. Resting behaviour,
     ecology and genetics of malaria vectors in large scale agricultural
     areas of Western Kenya. Parassitologia 1996;38:481-9.
  4. Khaemba BM, Mutani A, Bett MK. Studies of Anopheline mosquitoes
     transmitting malaria in a newly developed highland urban area: a case
     study of Moi University and its environs. East Afri Med J
     1994;71:159-64.
  5. Garnham PCC. The incidence of malaria at high altitudes. Journal of
     the National Malaria Society 1948;7:275-84.
  6. Matson AT. The history of malaria in Nandi. East Afr Med J
     1957;34:431-41.
  7. Roberts JMD. The control of epidemic malaria in the highlands of
     western Kenya, Part III. After the campaign. J Trop Med Hyg
     1964;59:230-7.
  8. Oloo AJ, Vulule JM, Koech DK. Some emerging issues on the malaria
     problem in Kenya. East Afr Med J 1996;70:50-3.
  9. Clyde DF. Observations on monthly pyrimethamine ("Daraprim")
     prophylaxis in an East African village. East Afr Med J 1954;31:41-6.
 10. Avery Jones S. Resistance of P. falciparum and P. malariae to
     pyrimethamine (Daraprim) following mass treatment with this drug. East
     Afr Med J 1954;31:47-9.
 11. Gilles HM, Warrell DA, editors. In: Bruce-Chwatt's Essential
     Malariology. 3rd ed. London: Edward Arnold; 1993.
 12. Roberts JMD. The control of epidemic malaria in the highlands of
     western Kenya, Part I. Before the campaign. J Trop Med Hyg
     1964;59:161-71.
---------------------------------------------------------------------------------
---------------------------------------------------------------------------------

Dispatches

Murine Typhus in Travelers Returning from Indonesia

Philippe Parola,* Dirk Vogelaers, Chantal Roure,Franois Janbon, and
Didier Raoult*
*Universit de la Mditerrane, Marseille, France; Universitair
Ziekenhuis, Gent, Belgium; Hpital de la Croix-Rousse, Lyon, France; and
Hpital Guy de Chauliac, Montpellier, France

---------------------------------------------------------------------------
      We report the first three documented cases of murine typhus
      imported into Europe from Indonesia, discuss clues for the
      diagnosis of the disease, and urge that murine fever be
      considered in the diagnosis of febrile disease in travelers.

One of the oldest recognized, most common, but least reported
arthropod-transmitted zoonoses (1,2), murine virus is caused by Rickettsia
typhi, formerly named R. mooseri, a gram-negative obligate intracellular
bacterium (1,2). The main vector of murine typhus is the rat flea
Xenopsylla cheopis, which maintains R. typhi in rodents. Flea bites and
contamination of excoriated skin or the respiratory tract with infected
flea feces are the major sources of human infection throughout the world
(2). The classic reservoir and vector are the rat and rat flea,
respectively (2); however, the role of cat fleas and opossums in
maintaining this microorganism has been suggested in some areas of the
United States where rats and rat fleas are absent (2,3). Although murine
typhus has a worldwide distribution, it is often unrecognized, and
documented cases are rarely reported, particularly in tropical countries.
Three recent cases of murine typhus in 2 months in patients returning to
Europe from Indonesia indicate that murine typhus should be considered a
possible cause of imported fever from Indonesia.

Case 1

In October 1997, a 29-year-old man living in France was hospitalized with a
2-day history of fever. On admission, his temperature was 40C and his
pulse, at 70/min, was dissociated. No signs of eruption or other
abnormalities were observed. The patient had returned from a 3-week trip to
Bali, Indonesia, 2 weeks before his hospitalization but reported no
arthropod bites during his trip. Increases of alanine aminotransferase (110
U/L), aspartate aminotransferase (120 U/L), lactate dehydrogenase (1000
U/L) and reactive protein C (130 mg/L) associated with a low white blood
cell count were noted. Repeated blood smears did not disclose any malaria
parasites. The patient received a 21-day treatment with doxycycline plus
rifampin and became afebrile on day 3. He was discharged from the hospital
and remained well. An indirect immunofluorescence assay (IFA) for
antibodies reactive with R. typhi antigens (4) showed raised levels of
immunoglobulin (Ig) M (1:512) and IgG (1:256).

Case 2

In November 1997, a 28-year-old man living in France was hospitalized with
a 6-day history of fever. On admission, he had a low-grade fever (38C).
Physical examination found no abnormalities except for a few skin
ulcerations of the inguinal area. The patient had returned from Bali 3
weeks before his hospitalization but reported no arthropod bites during his
trip. Biologic findings were unremarkable. Blood smears did not disclose
malaria parasites. The blood cell count, hemoglobin level, and blood
chemistry values were within normal limits. Serologic testing for
antibodies reactive with R. typhi antigens showed raised levels of IgM
(1:512) and IgG (1:256).

Case 3

The third patient, a 28-year-old man from Belgium, became acutely ill
during a trip to Indonesia (Bali, Lava, Lombok), with a fever (40C),
shivers, diffuse myalgia, but no rash. He was treated in Indonesia with
amoxicillin, followed by cefuroxim-axetil (500 mg twice a day for 10 days),
without success. The patient recalled multiple insect bites during his
trip. Clinical examination on arrival in Belgium showed severe disease with
bilateral conjunctivitis and splenomegaly, but no adenopathies. Biologic
findings were unremarkable except raised levels of alanine aminotransferase
(681 U/L), aspartate aminotransferase (659 U/L), and reactive protein C (17
mg/L). Analysis of the cerebrospinal fluid showed 20 leukocytes/ml with 75%
polymorphonuclear cells. Repeated blood smears did not disclose any
parasites. The patient received empirical treatment with quinine,
doxycycline, and a quinolone to treat suspected malaria or salmonellosis.
The fever rapidly resolved, and the patient recovered. Serologic testing
with IFA was positive for R. typhi (IgG, 512; IgM, 1024). Antibodies to R.
typhi were also detected in the cerebrospinal fluid (IgM, 2).

All patients exhibited serologic cross-reactions (low titers) with R.
prowazekii, the agent of epidemic typhus, and R. conorii, the agent of
Mediterranean spotted fever (Table;1,4). Serologic findings were completed
by cross-absorption. When absorption was performed with R. typhi,
homologous and heterologous antibodies disappeared, but when absorption was
performed with R. prowazekii, only homologous antibodies disappeared;
therefore, murine typhus was diagnosed as the cause of illness in the three
patients (4).

Murine typhus is a mild disease with nonspecific signs (1). The incubation
period is 7 to 14 days. The classic triad of fever, headache, and skin rash
is observed in fewer than 15% of cases (5). While fever and headache are
frequent, a rash occurs in only half of the patients and is often transient
or unimpressive. This is illustrated in these three cases, where no rash
was noted. Arthralgia, myalgia, or respiratory and gastrointestinal
symptoms are frequent (5). Leukocytosis and mild leukopenia, anemia, and
thrombopenia occur frequently; hyponatremia, hypoalbuminemia, hepatic
abnormalities, or renal dysfunction may occur (5). Fewer than half of the
patients report exposure to fleas or flea hosts. Of the cases reported
here, only one involved insect bites. Untreated patients may have fever and
symptoms for 7 to 14 days and then rapidly convalesce. The treatment of
choice is antibiotic therapy with tetracyclines. A single 200-mg dose of
oral doxycycline usually leads to defervescence during the first 72 hours
(6).

Like our patients, most murine typhus patients may have isolated fever and
nonspecific biologic abnormalities. Diagnosis may be missed because the
rash, the hallmark of rickettsial diseases, is absent (1). For example, in
a recent review in the United States, 22 different diagnoses were proposed
for 80 patients with murine typhus (5). Consequently, the diagnosis of the
disease is based on serologic testing. The reference method is
immunofluorescence; however, a latex test, a dot blot enzyme-linked
immunosorbent assay, and an immunoperoxydase assay have also been described
(4). The fact that travel-associated cases of murine typhus have been
identified and may represent a source of imported fever indicates that
serologic testing should be considered in febrile patients returning from
disease-endemic areas (Figure).

 Table. Immunofluorescence testing for antibodies to Rickettsia typhi
 (Wilmington), R. prowazekii (Breinl), R. conorii (Moroccan), and Orientia
 tsutsugamushi (Kato, Gilliam, Karp, Kawazaki) in febrile patients
 returning from Indonesia
 ---------------------------------------------------------------------------
                                              Titer (IgG/IgM)
                         -------------------------------------------------                                                                 
 Antigens              R. typhi  R. prowazekii  R. conorii  O. tsutsugamushi
 ---------------------------------------------------------------------------

 Patient 1

  Acute serum           512/256      128/128      <16/128       <16/<16
  Convalescent serum    512/64        128/32      <16/<16       <16/<16

 Patient 2
  Acute serum           256/512      128/512      <16/128       <16/<16
  Convalescent serum    256/512      <16/<16       <16/64       <16/<16

 Patient 3
  Acute serum          512/1024     256/2048       <16/64       <16/<16
  Convalescent serum  1024/1024     512/1024       <16/32       <16/<16

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

 [Figure.] Areas in which murine typhus poses a risk according to
 seroepidemiologic studies, case series, or imported cases in
 travelers (1,2,6-13).

Tissot-Dupont et al. recently reported that the prevalence of antibodies
against R. typhi in humans in Africa was higher in coastal areas where rats
are prevalent (Figure; 7). Other seroepidemiologic studies demonstrated
substantial seroprevalence in Asia (Thailand, Vietnam, Japan, Indonesia,
China), Europe (Croatia, Greece), and Australia (6,8). Cases have recently
been reported in Greece, Spain, Portugal, Israel, and Thailand (6). The
disease is known to be endemic in the United States (particularly
California, Hawaii, and Texas) and Mexico (6). Some authors have reported
the disease in Rumania, Yugoslavia, Albania, Italy, areas around the
Caspian Sea, in Kuwait, Latin America (Venezuela, Argentina, Brazil, Chile,
Ecuador, Panama, Guatemala, Puerto Rico), and French Guyana (9). However,
serologic cross-reactivity may occur between R. typhi and R. prowazekii,
the agent of epidemic typhus (1,4). Epidemic typhus, transmitted by body
lice, occurs more frequently in cool areas where clothes are infrequently
changed and particularly during human conflicts (1). The disease is
prevalent in countries at high altitudes in Central America or Africa
(10,14); interpretation of serologic results in these countries has at
times been difficult (7,10). However, cross-absorption is a useful
technique for identifying the infecting rickettsia to species level as
reported in the present cases (4).

Cases of murine typhus have been reported in travelers returning from
India, Nepal, Morocco, Canary Islands, Spain, and Africa (6,11-13).
Although a case of murine typhus was reported in a patient after a trip to
Malaysia and Indonesia (11), the cases reported here are the first
serologically documented cases clearly imported from Indonesia. In Malang,
East Java, humans are highly exposed to R. typhi, and murine typhus appears
to be endemic in the region (8).

Fever is a common problem in patients returning from travel abroad.
Although malaria is often suspected, rickettsial diseases (including murine
typhus) are generally not. Our cases indicate that murine typhus should be
considered in the diagnosis of febrile illness in travelers returning from
disease-endemic areas, particularly if fever is present and blood smears
show no malaria parasites. Rickettsial diseases were recently cited as the
third most frequent cause of imported fever in Switzerland (12). On the
basis of this study and two previous reports from France (13) and the
United States (11), murine typhus is a frequently imported rickettsial
disease. Increased development of tourism and travel to tropical countries
should lead to an increase in the documentation of cases.

      Dr. Raoult is affiliated with the hospital of Universit de la
Mditerrane, in Marseille, France. His research interest is medical
entomology.

      Address for correspondence: Didier Raoult, Facult de Mdecine,
Universit de la Mditerrane, CNRS UPRES A 6020, 27 Bd Jean Moulin, 13385
Marseille Cedex 5, France; fax: 33-4-91-83-0390; e-mail:
raoult@medecine.univ-mrs.fr.

References

  1. Raoult D, Roux V. Rickettsioses as paradigms of new or emerging
     infectious diseases. Clin Microbiol Rev 1997;10:694-719.
  2. Azad AF, Radulovic S, Higgins JA, Noden BH, Troyer JM. Flea-borne
     rickettsioses: ecologic considerations. Emerg Infect Dis
     1997;3:319-27.
  3. Azad AF, Sacci JB, Nelson WM, Dasch GA, Schmidtmann ET, Carl M.
     Genetic characterization and transovarial transmission of a
     typhus-like rickettsia found in cat fleas. Proc Natl Acad Sci U S A
     1992;89:43-6.
  4. Lascola B, Raoult D. Laboratory diagnosis of rickettsioses: current
     approaches to diagnosis of old and new rickettsial diseases. J Clin
     Microbiol 1997;35:2715-27.
  5. Dumler JS, Taylor JP, Walker DH. Clinical and laboratory features of
     murine typhus in South Texas, 1980 through 1987. JAMA
     1991;266:1365-70.
  6. Walker DH. Advances in understanding of typhus group rickettsial
     infections. In: Kazar J, Toman R, editors. Rickettsiae and rickettsial
     diseases. Bratislava, Slovak Republic: VEDA Press; 1996. p. 16-25.
  7. Tissot-Dupont H, Brouqui P, Faugere B, Raoult D. Prevalence of
     antibodies to Coxiella burnetii, Rickettsia conorii, and Rickettsia
     typhi in seven African countries. Clin Infect Dis 1995;21:1126-33.
  8. Richards AL, Soeatmadji DW, Widodo MA, Sardjono TW, Yanuwiadi B,
     Hernowati TE, et al. Seroepidemiologic evidence of murine typhus and
     scrub typhus in Malang, Indonesia. Am J Trop Med Hyg 1997;57:91-5.
  9. Traub R, Wisseman CL Jr, Azad AF. The ecology of murine typhus: a
     critical review. Tropical Disease Bulletin1978;75:237-317.
 10. Perrine PL, Chandler BP, Krause DK, McCardle P, Awoke S, Habte-Gabr E,
     et al. A clinico-epidemiological study of epidemic typhus in Africa.
     Clin Infect Dis 1992;14:1149-58.
 11. McDonald JC, MacLean JD, McDade JE. Imported rickettsial disease:
     clinical and epidemiologic features. Am J Med 1988;85:799-805.
 12. Raeber PA, Winteler S, Paget J. Fever in the returned traveller:
     remember rickettsial diseases. Lancet 1994;334:331.
 13. Edlinger E, Navarro P. Rickettsioses. A disease of tourism. Semaine
     des Hpitaux de Paris 1983;59:2053-4.
 14. Raoult D, Roux V, Ndihokubwayo JB, Bise G, Baudon D, Marte G, et al.
     Jail fever (epidemic typhus) outbreak in Burundi. Emerg Infect Dis
     1997;3:357-60.
---------------------------------------------------------------------------------
---------------------------------------------------------------------------------

Dispatches

Differentiating Human from Animal Isolates of Cryptosporidium parvum

Irshad M. Sulaiman, Lihua Xiao, Chunfu Yang, Lilian Escalante, Anne Moore,
Charles B. Beard, Michael J. Arrowood, and Altaf A. Lal
Centers for Disease Control and Prevention, Atlanta, Georgia, USA

------------------------------------------------------------------------
      We analyzed 92 Cryptosporidium parvum isolates from humans and
      animals by a polymerase chain reaction/restriction fragment
      length polymorphism method based on the thrombospondin-related
      anonymous protein 2 gene sequence. Used as a molecular marker,
      this method can differentiate between the two genotypes of C.
      parvum and elucidate the transmission of infection to humans.

Cryptosporidium parasites cause infection in humans and other vertebrates,
including mammals, birds, reptiles, and fish. More than 20 species of
Cryptosporidium have been reported, of which six are considered valid
species on the basis of oocyst morphologic features and site of infection
(1,2). Cryptosporidium parvum, the species that infects humans and most
mammals, has a monoxenous life cycle in which all stages of asexual and
sexual development occur within one host. The parasite generates large
numbers of viable oocysts in feces. Cross-infection studies in various
mammalian systems have indicated zoonotic transmission to humans (1,3). C.
parvum has caused waterborne outbreaks of cryptosporidiosis and (in AIDS
patients) life-threatening diarrhea for which no effective treatment exists
(4). A waterborne outbreak of cryptosporidiosis in Milwaukee, Wisconsin, in
1993 affected more than 400,000 people (5).

Molecular characterization techniques used to detect intraspecific
variations in C. parvum include isozyme profiles (6); random amplified
polymorphic DNA (RAPD) analyses (7); nucleotide sequence studies of the 18S
rRNA (8,9) and DHFR gene (10); and polymerase chain reaction-restriction
fragment length polymorphism (PCR-RFLP) analysis of the undefined
repetitive sequence (11), polythreonine motifs, and oocyst wall protein
(12,13). Two distinct genotypes of C. parvum parasites have been detected
in humans.

In a previous article, we identified several mutations in the gene
thrombospondin-related anonymous protein 2 of C. parvum (TRAP-C2) that
differentiate between anthroponotic and zoonotic infection in humans (14).
Our objective in the present study was to develop a simple, rapid protocol
that can be used as a diagnostic tool to differentiate between the two
genotypes of C. parvum and elucidate the transmission of infection in
humans. We analyzed 92 C. parvum isolates from humans, calves, deer, dogs,
and monkeys and found that this new PCR/RFLP method based on the TRAP-C2
gene sequence can be used as a molecular marker to differentiate between
the two genotypes of C. parvum.

Analytic Approach

Isolates

We summarized data for 92 isolates of C. parvum, 50 from human and 42 from
animal sources (Tables 1, 2). Twenty-one of the 50 human isolates were from
AIDS patients; the rest were primarily from cryptosporidiosis outbreak
case-patients. Seven of the human isolates came from a previous TRAP-C2
sequencing study (14), but because of the lack of DNA, other isolates we
used in the previous study were not used in this study. Fecal samples were
stored at 4 C in 2.5% potassium dichromate before oocysts were isolated.
Oocysts were purified from fecal samples by first using the discontinuous
density sucrose gradient centrifugation and then the Percoll gradient
centrifugation (15,16).

 Table 1. Cryptosporidium parvum human isolates, restriction pattern, and
 sequence type

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

                                                 Restric- Se-
                                                 tion     quence
 Source                      Isolate    Host     pattern  type
 ------------------------------------------------------------------------
 1993 Milwaukee              HM3     Hum(sup a)  Hum      Hum(sup b)     

 1993 Milwaukee              HM5        Hum      Hum      Hum

 1993 Milwaukee              HM7        Hum      Hum      ND(sup c)     

 1995 Florida                HFL1       Hum      Hum      Hum(sup b)     

 1995 Florida                HFL5       Hum      Hum      Hum(sup b)     

 1995 Florida                HFL6       Hum      Hum      ND

 1995 Atlanta                HGA1       Hum      Hum      Hum(sup b)     

 1995 Atlanta                HGA4       Hum      Hum      Hum

 1995 Atlanta                HGA5       Hum      Hum      ND

 1995 Atlanta                HGA6       Hum      Hum      ND

 1996 Canada                 HCAN9      Hum    Bov(sup d) Bov(sup b)     

 1995 Canada                 NC30       Hum      Hum      ND

 1994 Nevada                 HCNV2      Hum      Hum      ND

 1994 Nevada                 HCNV4      Hum      Hum      ND

 1997 Pennsylvania           PA41       Hum      Bov      Bov(sup b)     

 1997 Pennsylvania           PA46       Hum      Bov      Bov(sup b)     

 1997 HIV-Guatemala          HGMO7      Hum      Hum      Hum

 1997 HIV-Guatemala          HGMO8      Hum      Hum      ND

 1997 HIV-Guatemala          HGMO9      Hum      Hum      Hum

 1997 HIV-Guatemala          HGMO10     Hum      Hum      Hum

 1997 Minnesota              HMOB1      Hum      Bov      Hum

 1997 Minnesota              HMOB3      Hum      Bov      Bov

 1997 Minnesota              HMOB4      Hum      Bov      Bov

 1997 Minnesota              HMOB5      Hum      Bov      Bov

 1997 HIV-New Orleans        HNO2       Hum      Hum      Hum

 1997 HIV-New Orleans        HNO3       Hum      Hum      Hum

 1997 HIV-New Orleans        HNO4       Hum      Hum      ND

 1997 HIV-New Orleans        HNO5       Hum      Bov      Bov

 1997 HIV-New Orleans        HNO6       Hum      Hum      Hum

 1997 HIV-New Orleans        HNO7       Hum      Hum      Hum

 1997 HIV-New Orleans        HNO8       Hum      Hum      Hum

 1997 HIV-New Orleans        HNO10      Hum      Hum      Hum

 1997 HIV-New Orleans        HNO11      Hum      Bov      ND

 1997 HIV-New Orleans        HNO12      Hum      Hum      Hum

 1997 HIV-New Orleans        HNO13      Hum      Hum      Hum

 1997 HIV-New Orleans        HNO14      Hum      Hum      Hum

 1997 HIV-New Orleans        HNO15      Hum      Hum      Hum

 1997 HIV-New Orleans        HNO16      Hum      Hum      Hum

 1997 HIV-New Orleans        HNO17      Hum      Hum      Hum

 1997 HIV-New Orleans        HNO18      Hum      Hum      Hum

 1997 HIV-New Orleans        HNO19      Hum      Hum      ND

 1997 India                  HIND4      Hum      Hum      Hum

 1997 India                  HIND5      Hum      Hum      ND

 1998 Washington State       HWA1       Hum      Hum      Hum

 1998 Washington State       HWA2       Hum      Hum      ND

 1998 Washington State       HWA3       Hum      Hum      ND

 1998 Washington State       HWA4       Hum      Hum      ND

 1998 Washington State       HWA5       Hum      Hum      Hum

 1998 Washington State       HWA6       Hum      Hum      Hum

 1998 Washington State       HWA7       Hum      Hum      Hum

 ------------------------------------------------------------------------
 (sup a)Hum=human
 (sup b)Sequencing data reported earlier (14)
 (sup c)ND= Not done.
 (sup d)Bov=bovine.

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

 Table 2. Cryptosporidium parvum bovine isolates, restriction
 pattern, and sequence type

 ---------------------------------------------------------------
                                          Restric-   Se-
                                          tion       quence
 Source                Isolate   Host     pattern    type
 ---------------------------------------------------------------
 1996 Alabama          AAL35     Calf   Bov(sup a)   Bov
 1996 Georgia          AGA43     Calf     Bov        Bov
 1996 Georgia          AGA44   Mon(sup b) Bov        Bov
 1997 Georgia          AGA75     Calf     Bov        Bov
 1996 Idaho            AID21     Calf     Bov        Bov
 1996 Kansas           AKA19     Calf     Bov        Bov
 1996 Maryland         AMD36     Calf     Bov        Bov
 1996 Maryland         AMD38     Deer     Bov        Bov
 1996 Massachusetts    AMA61-    Calf     Bov        Bov
                       GCH1
 1997 Iowa             AIO62     Calf     Bov        Bov
 1996 Ohio             AOH6      Calf     Bov        Bov
 1996 Ohio             AOH7      Calf     Bov        Bov
 1996 Ohio             AOH8      Calf     Bov        Bov
 1996 Ohio             AOH9      Calf     Bov        Bov
 1996 Ohio             AOH10     Calf     Bov        Bov
 1996 Ohio             AOH11     Calf     Bov        Bov
 1996 Ohio             AOH12     Calf     Bov        Bov
 1996 Ohio             AOH13     Calf     Bov        Bov
 1996 Ohio             AOH14     Calf     Bov        Bov
 1996 Ohio             AOH15     Calf     Bov        Bov
 1996 Ohio             AOH16     Calf     Bov        Bov
 1996 Ohio             AOH17     Calf     Bov        Bov
 1997 Ohio             AOH45     Calf     Bov        Bov
 1997 Ohio             AOH47     Calf     Bov        Bov
 1997 Ohio             AOH48     Calf     Bov        Bov
 1997 Ohio             AOH49     Calf     Bov        Bov
 1997 Ohio             AOH50     Calf     Bov        Bov
 1997 Ohio             AOH52     Calf     Bov        Bov
 1997 Ohio             AOH53     Calf     Bov        Bov
 1997 Ohio             AOH54     Calf     Bov        Bov
 1997 Ohio             AOH55     Calf     Bov        Bov
 1997 Ohio             AOH56     Calf     Bov        Bov
 1997 Ohio             AOH57     Calf     Bov        Bov
 1997 Ohio             AOH58     Calf     Bov        Bov
 1997 Ohio             AOH59     Calf     Bov        Bov
 1997 Ohio             AOH107    Dog      Bov        Bov
 1996 Oklahoma         AOK3      Beef     Bov        Bov
                                 cattle
 1996 Oklahoma         AOK29     Calf     Bov        Bov
 1997 Pennsylvania     APE89     Calf     Bov        Bov
 1996 Utah             AUT37     Calf     Bov        Bov
 1996 Washington       AWA5      Beef     Bov        Bov
                                 cattle
 1997 West Virginia    AWV65     Calf     Bov        Bov
 ---------------------------------------------------------------
 (sup a)Bov=bovine.
 (sup b)Mon=monkey.

Extraction of Genomic DNA and PCR Amplification

We followed the protocol of Kim et al. in isolating the total genomic DNA
from the purified oocyst (17). A 369 base pair (bp) fragment of the TRAP-C2
gene of C. parvum was amplified by using a forward (cua cua cua cua CAT ATT
CCC TGT CCC TTG AG) and a reverse (cau cau cau cau TGG ACA ACC CAA ATG CAG
AC) primer (lower case represents nucleotide used for cloning); these
primers correspond to positions 848-867 (positive strand) and 1,180-1,199
(negative strand) of the GenBank sequence X77586, respectively. The PCR
reaction consisted of 50 ng genomic DNA, 200 mM of each dNTP (Perkin Elmer,
Foster City, CA), 40 ng of primer, 1X PCR buffer, and 0.5 units of Taq
polymerase (GIBCO BRL, Frederick, MD) in a total volume of 100 ml. DNA
amplification was carried out for 35 cycles, each consisting of denaturing
(94 C, 45 sec), annealing (48 C, 45 sec), and elongating (72 C, 60 sec),
with an initial hot start at 94 C for 5 min in a Perkin Elmer Gene Amp PCR
9600 thermocycler. An additional cycle of 7 min at 72 C was done for final
extension. Each experiment used three negative controls (reaction mixtures
without Taq polymerase, primers, or template DNA) and a positive control.

DNA Sequencing and Analysis

PCR products were purified by the Wizard PCR Preps DNA purification system
(Promega, Madison, WI) and cloned by the CLONEAMP pAMP1 System for Rapid
Cloning of Amplification Products (GIBCO BRL, Frederick, MD) according to
the manufacturer's protocol. DNA sequencing of recombinant clones that had
the correct size insert was carried out on an ABI 377 Automated Sequencer
by the dRhodomine Terminator Cycle Sequencing Kit (Perkin Elmer-Applied
Biosystems).

RFLP

To develop an RFLP technique for differentiating between the two genotypes
of C. parvum, the TRAP-C2 sequences were aligned and mapped for restriction
enzyme sites by the Genetics Computer Group program (18). Enzymes with
predicted exclusive cutting in each genotype were used in RFLP development
and analysis. For RFLP analysis, 10 ml of amplification products was
digested in a 30-ml reaction mix consisting of 10 units of BfaI (New
England BioLabs, Beverly, MA), BsetEI (Boehringer Mannheim, Indianapolis,
IN), Eco571 (MBI Fermentas, Gariciuno, Vilnius, Lithuania), HaeIII (New
England BioLabs), HphI (New England BioLabs), MaeIII (Boehringer Mannheim,
Germany), NruI (New England BioLabs), PacI (New England BioLabs), or Tsp45I
(New England BioLabs), and 3 ml of respective restriction buffer for 1 hr,
under conditions recommended by the supplier. The digested products were
fractionated on 2.0% agarose gel and visualized by ethidium bromide
staining.

Findings

Sequence Analysis of Human and Bovine Isolates

 Table 3. Human and bovine Cryptosporidium
 parvum isolates based on multiple alignment(sup a)

 -----------------------------------------------
     Position        Human           Bovine
      (nt)          genotype        genotype
 -----------------------------------------------
       51              G                A
       78              C                T
       100             T                G
       147             C                T
       280           T or C             C
 -----------------------------------------------
 (sup a)Representative sequences have been 
 deposited in the GenBank, with accession 
 numbers AF082521 to AF082524.

Two genotypes of C. parvum exist in humans,as shown by the primary sequence
of the TRAP-C2 gene (14). Nucleotide sequences differed at five positions
between most human and bovine isolates. To confirm and extend this
observation, we sequenced additional human and bovine isolates, as well as
isolates from dogs, deer, and monkeys. We obtained 42 additional sequences
of the TRAP-C2 gene from animal sources and 27 additional sequences of the
TRAP-C2 gene from human sources; results of DNA sequencing confirmed that
C. parvum is highly conserved at the TRAP-C2 locus. All animal isolates,
including those from nonbovine animals, showed bovine genotype
characteristics (Table 2). Differences between the two genotypes are shown
in Table 3. Of the additional 23 human isolates showing human genotype
pattern, four isolates (HGMO7, HGMO9, HGMO10, and HNO18) showed "C" at the
fifth place, whereas the rest showed "T".

PCR-RFLP Method To Discriminate between Human and Bovine Genotype Isolates

 [Figure.] Human- and bovine-specific restriction enzymes
 showed distinct banding pattern for genotypes of
 Cryptosporidium parvum isolates. The different lanes
 represent the TRAP-C2 PCR-amplified products belonging
 to AGA43, AMD36, AOH6, HM3, and HM5 isolates of C.
 parvum, respectively, after digestion with HaeIII
 (Lanes 1-5, human-specific marker) and BstE II (Lanes
 7-11, bovine-specific marker) restriction enzymes and
 agarose gel electrophoresis. Lane 6 is the 100 bp
 marker. Samples AGA43, AMD36 and AOH6 are bovine
 (bovine genotype) and samples HM3 and HM5 are human
 (human genotype). Human- or bovine-specific restriction
 enzyme markers can cut only the TRAP-C2 amplified
 product of the respective genotype of C. parvum
 isolates.

To avoid expensive and lengthy DNA sequencing when determining the genotype
of C. parvum isolates, we developed a simpler, quicker methodPCR
amplification of the TRAP-C2 gene followed by RFLP. Restriction enzyme
mapping on the aligned sequences of both genotypes showed five
human-genotypespecific (HaeI, HaeIII, NruI, PacI, and ThaI) and six
bovine-genotypespecific (BfaI, BsetEI, Eco571, HphI, MaeIII, and Tsp45I)
restriction enzymes. All human-genotype and bovine-genotypespecific
restriction enzymes except HaeI and ThaI were tested for the TRAPC-2
PCR-amplified products of genomic DNA of C. parvum. After restriction and
gel electrophoresis, the resulting bands were the size predicted by the
mapping analysis (Figure). Digestion of PCR products with these enzymes
resulted in a distinct band pattern for the human genotype and bovine
genotype isolates. In all cases, the DNA sequencing and PCR-RFLP mapping
data matched.

Using PCR-RFLP in Outbreak Investigations

We validated the PCR-RFLP technique by using isolates from outbreaks and
sporadic cases of human cryptosporidiosis. Human genotype characteristics
were evident in all samples from HIV-infected patients from Guatemala and
most patients with sporadic clinical cases, as well as samples from the
following outbreaks: Milwaukee (1993), Florida (1995), Atlanta (1995),
Canada (1995), Nevada (1994), and Washington (1998). Of the 17 samples from
HIV-infected patients in New Orleans, two demonstrated bovine-genotype
pattern, while the rest were similar to human genotype. However,
bovine-genotype characteristics were evident in the human isolates from
outbreaks in British Columbia, Canada (1996), Minnesota (1997), and
Pennsylvania (1997).

Conclusions

We examined a large number of C. parvum isolates (92) from human and animal
sources from patients in outbreak and nonoutbreak settings to determine the
two transmission routes of the parasite in humans. Molecular markers were
generated by restriction digestion of PCR-amplified TRAP-C2 products with
one of the 12 enzymes to differentiate the two genotypes of C. parvum. The
results based on TRAP-C2 gene PCR-RFLP showed that this method could also
be used in future cryptosporidiosis outbreak investigations.

Results of our characterization of outbreak and nonoutbreak cases of human
cryptosporidiosis indicate that anthroponotic organisms account for most
cases. We find a large number of human genotype parasites in sporadic cases
and in HIV-infected patients. Most cryptosporidiosis outbreaks examined are
caused by anthroponotic (human genotype) parasites. Our results suggest
similar epidemiologic features of cryptosporidiosis in HIV-infected persons
from New Orleans and Guatemala because both were infected with human
genotype parasites.

The results of this study confirm the polymorphic nature of C. parvum. As
we showed in a previous study, two alleles of the TRAP-C2 gene exist, each
representing a distinct genotype of C. parvum with different transmission
cycles in humans. The simple PCR-RFLP technique we developed can
effectively differentiate between these two genotypes and transmission
cycles and can be used as a tool in outbreak investigations of
cryptosporidiosis. Information generated from these investigations will be
useful not only in identifying the sources of contamination but also in
controlling the disease.

Acknowledgments

      We thank Barbara Herwaldt, Corinne S. L. Ong, P. Vijyalakshmi, Bruce
Anderson, William Shulaw, A. Morse, and J. Inungu for providing
Cryptosporidium isolates.

      This work was supported in part by funding from Environmental
Protection Agency (DW 75937984-01-1).

      Dr. Sulaiman is a postdoctoral research associate in the Molecular
Vaccine Section, Division of Parasitic Diseases, National Center for
Infectious Diseases, CDC. For the last 7 years, he has focused on the
genetic polymorphism of various organisms. He is now conducting molecular
typing of Cryptosporidium to understand the transmission routes of the
parasite.

      Address for correspondence: Altaf A. Lal, Division of Parasitic
Diseases, National Center for Infectious Diseases, Centers for Disease
Control and Prevention, Mail Stop F-12, 4770 Buford Highway, Atlanta, GA
30341-3717, USA; fax: 770-488-4454; e-mail: aal1@cdc.gov.

References

  1. O'Donoghue PJ. Cryptosporidium and cryptosporidiosis in man and
     animals. Int J Parasitol 1995;25:139-95.
  2. Dubey JP, Speer CA, Fayer R. General biology of Cryptosporidium. Dubey
     JP, Speer CA, Fayer R, editors. Cryptosporidiosis of man and animals.
     Boca Raton (FL): CRC Press; 1990. p. 1-29.
  3. Smith HV. Environmental aspects of Cryptosporidium species: a review.
     J R Soc Med 1990;83:629-31.
  4. Colford JM, Tager IB, Hirozawa AM, Lemp GF, Aragon T, Petersen C.
     Cryptosporidiosis among patients infected with human immunodeficiency
     virus. Am J Epidemiol 1996;144:807-16.
  5. Mac Kenzie WR, Hoxie NJ, Proctor ME, Gradus MS, Blair KA, Peterson DE,
     et al. A massive outbreak in Milwaukee of Cryptosporidium transmitted
     through the public water supply. N Engl J Med 1994;331:161-7.
  6. Awad-El-Kariem FA, Robinson HA, Dyson DA, Evans D, Wright S, Fox MT,
     et. al. Differentiation between human and animal strains of
     Cryptosporidium parvum using isoenzyme typing. Parasitology
     1995;110:129-32.
  7. Morgan UM, Constantine CC, O'Donoghue P, Meloni BP, O'Brien PA,
     Thompson RCA. Molecular characterization of Cryptosporidium isolates
     from humans and other animals using random amplified polymorphic DNA
     analysis. Am J Trop Med Hyg 1996;52:559-64.
  8. Carraway M, Tzipori S, Widmer G. Identification of genetic
     heterogeneity in the Cryptosporidium parvum ribosomal repeat. Appl
     Environ Microbiol 1996;62:712-6.
  9. Morgan UM, Constantine CC, Forbes DA, Thompson RCA. Differentiation
     between human and animal isolates of Cryptosporidium parvum using rDNA
     sequencing and direct PCR analysis. J Parasitol 1997;83:825-30.
 10. Vasquez JR, Gooze L, Kim K, Gut J, Petersen C, Nelson RG. Potential
     antifolate resistance determinants and genotypic variation in the
     bifunctional dihydrofolate reductase-thymidylate synthase gene from
     human and bovine isolates of Cryptosporidium parvum. Mol Biochem
     Parasitol 1996;79:153-65.
 11. Bonnin A, Fourmaux MN, Dubremetz JF, Nelson RG, Gobet P, Harly G, et
     al. Genotyping human and bovine isolates of Cryptosporidium parvum by
     polymerase chain reaction-restriction fragment length polymorphism
     analysis of a repetitive DNA sequence. FEMS Microbiol Lett
     1996;137:207-11.
 12. Carraway M, Tzipori S, Widmer G. A new restriction fragment length
     polymorphism from Cryptosporidium parvum identifies genetically
     heterogeneous parasite populations and genotypic changes following
     transmission from bovine to human hosts. Infect Immun 1997;65:3958-60.
 13. Spano F, Putignani L, McLauchlin J, Casemore DP, Crisanti A. PCR-RFLP
     analysis of the Cryptosporidium oocyst wall protein (COWP) gene
     discriminates between C. wrairi and C. parvum, and between C. parvum
     isolates of human and animal origin. FEMS Microbiol Lett
     1997;150:209-17.
 14. Peng MP, Xiao L, Freeman AR, Arrowood MJ, Escalante A, Weltman AC, et
     al. Genetic polymorphism among Cryptosporidium parvum isolates
     supporting two distinct transmission cycles. Emerg Infect Dis
     1997;3:567-73.
 15. Arrowood MJ, Sterling CR. Isolation of Cryptosporidium oocysts and
     sporozoites using discontinuous sucrose and isopycnic Percoll
     gradients. J Parasitol 1987;73:314-9.
 16. Arrowood MJ, Donaldson K. Improved purification methods for
     calf-derived Cryptosporidium parvum oocysts using discontinuous
     sucrose and cesium chloride gradients. J Eukaryot Microbiol
     1996;43:89.
 17. Kim K, Gooze L, Petersen C, Gut J, Nelson RG. Isolation, sequence and
     molecular karyotype analysis of the actin gene of Cryptosporidium
     parvum. Mol Biochem Parasitol 1992;50:105-14.
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---------------------------------------------------------------------------------
---------------------------------------------------------------------------------

Dispatches

An Outbreak of Hantavirus Pulmonary Syndrome, Chile, 1997

Jorge Toro,* Jeanette D. Vega,  Ali S. Khan, James N. Mills, Paula
Padula, William Terry, Zaida Yadn, Rosa Valderrama, Barbara A. Ellis,
Carlos Pavletic,* Rodrigo Cerda, Sherif Zaki, Shieh Wun-Ju, Richard
Meyer, Mauricio Tapia,# Carlos Mansilla,# Michel Baro,** Jose A.
Vergara,** Marisol Concha,* Gladys Calderon, Delia Enria, C.J. Peters,
and Thomas G. Ksiazek
*Ministry of Health, Santiago, Chile; Pan American Health Organization,
Santiago, Chile; Centers for Disease Control and Prevention, Atlanta,
Georgia, USA; Instituto Nacional de Enfermedades Infecciosas ANLIS "Carlos
G. Malbran," Buenos Aires, Argentina; Aysen Region XI Health Service,
Coyhaique, Chile; #Coyhaique Regional Hospital, Coyhaique, Aysen, Chile;
**Llanchipal Health Services, Puerto Montt, Chile; Instituto Nacional de
Enfermedades Virales Humanas, Pergamino, Argentina; and Catholic
University of Chile, Santiago, Chile.

---------------------------------------------------------------------------
      An outbreak of 25 cases of Andes virus-associated hantavirus
      pulmonary syndrome (HPS) was recognized in southern Chile from
      July 1997 through January 1998. In addition to the HPS
      patients, three persons with mild hantaviral disease and one
      person with asymptomatic acute infection were identified.
      Epidemiologic studies suggested person-to-person transmission
      in two of three family clusters. Ecologic studies showed very
      high densities of several species of sigmodontine rodents in
      the area.

Hantavirus pulmonary syndrome (HPS), first recognized in 1993 after a
cluster of acute respiratory distress syndrome deaths in the southwestern
United States, is now considered a pan-American zoonosis (1,2). HPS is
characterized by fever, myalgia, gastrointestinal symptoms, and headache,
with subsequent characteristic cardiopulmonary dysfunction and a 40% to 60%
case-fatality rate (3). Although the etiologic agent, Sin Nombre virus
(SNV), causes clinical symptoms that appear different from those that
Eurasian hantaviruses cause in hemorrhagic fever with renal syndrome, SNV
shares many features with these Old World hantaviruses (4,5), including an
association with a single primary rodent host (Peromyscus maniculatus [deer
mouse] in the case of SNV), which acts as the natural reservoir (6).

After SNV was identified, numerous other New World hantaviruses were
rapidly identified throughout the Americas by reverse
transcription-polymerase chain reaction (RT-PCR) amplification of viral RNA
from captured antibody-positive rodents and, occasionally, from infected
patients (2). HPS was first identified in Chile in 1995 in the Cochamo, Los
Lagos region. Genetic sequencing of RT-PCR products from the autopsy
tissues of another patient presumptively infected in this region in 1996
confirmed infection with Andes virus (7), which had previously been
identified in southern Argentina as a cause of HPS, with Oligoryzomys
longicaudatus postulated as the reservoir host (8). Sporadic cases were
reported in Chile until an outbreak that included two family clusters was
recognized in the Coyhaique Health District, Aysen region, in August 1997;
a third cluster was reported in January 1998. This outbreak prompted a
joint investigation by the National Administration of Laboratories and
Institutes of Health (Argentina), Pan American Health Organization, Centers
for Disease Control and Prevention (CDC, USA), in collaboration with the
Ministry of Health of Chile, to define the epidemiology and ecology of the
syndrome in Chile.

The Investigation

Epidemiologic Surveillance and Case Identification

National and local surveillance for HPS was reinforced with explicit case
definitions for HPS, asymptomatic hantavirus infection, and mild hantaviral
disease. HPS was defined as an acute febrile illness (temperature >38.3C)
characterized by unexplained acute respiratory distress syndrome or
bilateral interstitial pulmonary infiltrates, with respiratory compromise
requiring supplemental oxygen, or an unexplained illness resulting in
death, with autopsy results showing noncardiogenic pulmonary edema without
an identifiable specific cause of death. In addition to a compatible
clinical illness, the following laboratory evidence of infection was
required: 1) hantavirus-specific immunoglobulin M (IgM) or a fourfold rise
in IgG titer, or 2) positive RT-PCR results for hantavirus RNA, or 3)
positive immunohistochemical results for hantavirus antigen.

Asymptomatic hantavirus infection was defined as laboratory evidence of
acute hantavirus infection (presence of IgM antibodies) in persons with no
documented concurrent illness. Mild hantaviral disease was defined as
IgM-positive results and a febrile illness without objective pulmonary
dysfunction (i.e., hypoxemia and radiographic abnormalities). All other
persons with isolated detectable hantavirus-specific IgG antibodies were
classified as "other seropositives."

All available clinical charts and case reports from the Aysen region were
reviewed in conjunction with interviews of family members from the two
described clusters and survivors. A third cluster was identified in January
1998 and was also reviewed.

Ecologic Studies

Rodents were trapped at two principal sites near the residence sites of the
first two reported family clusters, as well as in several areas of less
disturbed native vegetation in Lago Atravesado. Traps were placed at each
of the two case-households, as well as at two neighboring controls for each
case-household (one within 500 m and the other 500 m to 1,000 m). Each
evening, 150 to 200 live-capture traps were placed within and around
residences, outbuildings, gardens, and woodpiles, along fence lines, and
within remnant patches of native vegetation. Traps were checked in the
early morning, and samples of blood and organ tissues were collected
according to standardized protocols (9). For comparison, we used similar
trapping techniques at additional sites near Valdivia (700 km north of the
outbreak area) and the capital city of Santiago (1,400 km north). Rodent
taxonomy is as described by Musser and Carleton, 1993 (10).

Laboratory Testing

Human serum specimens were tested for IgG antibodies reactive with SNV and
Andes antigens by an enzyme-linked immunosorbent assay (ELISA) (11). Rodent
whole-blood specimens were similarly tested for IgG antibodies to SNV. An
IgM-capture ELISA was performed on human sera by using inactivated Laguna
Negra virus at CDC and an IgM-capture ELISA with recombinant Andes
nucleocapsid antigen at the National Institute for Infectious Disease,
Buenos Aires, Argentina (P. Padula, unpub. data). Immunohistochemical
analysis for hantaviral antigens was performed by using a cross-reactive
monoclonal antibody directed against conserved hantaviral nucleocapsid
epitopes (12). Specimens from HPS patients were also examined for viral
genetic materials (M and S segment) by nested and heminested RT-PCR from
either autopsied tissues or blood clot samples (13,14). Genetic sequencing
of RT-PCR products derived from patient samples was also performed.

The Outbreak

						 From October 1995 when HPS was first
                                     recognized in Chile through June 1997,
 						 seven additional cases were identified
                                     (Figure 1). Between July 1, 1997, and
[Figure 1.]Temporal distribution of  January 22, 1998, 25 HPS patients and
 hantavirus pulmonary syndrome       three family clusters were identified
 cases, Chile, 1995-1998.            in Chile. By RT-PCR we confirmed that
                                     16 of these patients had been infected
                                     with Andes virus. Except for six
cases, the recent outbreak was centered in Regions X (Llanchipal) and XI
(Aysen) (combined population = 1.1 million) (Figure 2). The areas where
these patients lived are sparsely populated, with numerous lakes, in a
Nothofagus (southern beech) forest ecosystem. From October 4, 1995, to
January 22, 1998, a total of 33 cases were identified in Chile, with a
case-fatality rate of 54%. The mean age was 31.4 years (range: 1 year 11
months to 60 years); five (15.2%) of the patients were children under 17
years of age; and 76% of the patients were male. No cases were reported
among health-care workers.

No significant difference in mean age, case-fatality rate, or proportion of
male patients was evident between the July to January outbreak and previous
cases; however, all the pediatric cases occurred during the July to January
outbreak.

Family Clusters

The first family cluster (Cluster 1) of  
HPS cases was reported from Cisne Medio,
Lago Verde community, Aysen region. The  
39-year-old male head of the family
became ill with an acute febrile illness [Figure 2.]Geographic distribution
on July 15 and died in transit to the    of hantavirus pulmonary syndrome
regional hospital on July 21; HPS was    cases, Chile, 1995-1998.
subsequently confirmed by
immunohistochemistry and RT-PCR (M and S
segment), with a genetic sequence consistent with Andes virus. The family
moved to the village of Villa Amengual, 7 km away, on July 22 and remained
there except for a brief visit to Cisne Medio on July 27 to retrieve
personal belongings. The wife of the index patient became ill with HPS on
August 2 and died on August 8. Her 2-year-old and 12-year-old sons became
ill 1 week and 16 days, respectively, after she became ill. HPS was
confirmed in both children by serologic testing; both children survived.
The brother-in-law of the index patient, who continued to reside
intermittently in the original household, became ill 33 days after his
sister became ill and died (Figure 3). Thus, the immediate family members
became ill 12, 19, and 28 days after leaving the family homestead, and the
intervals between the onset of the index and later cases were 18, 25, and
34 days.

In contrast, the second family cluster (Cluster 2) included all four
members of a household in Lago Atravesado, Coyhaique community, Aysen
region, who became ill within 5 days of each other; one of them, a
2-year-old child with no respiratory symptoms, was classified as having a
mild hantaviral infection. The third family cluster (Cluster 3) included a
husband, who worked in a rural area, and his wife, who remained in the
family home in urban Coyhaique, a city of 60,000 inhabitants. The husband
became ill with symptoms suggestive of HPS 12 days after returning to his
family home. He was hospitalized and died on December 19. His wife became
ill 22 days after the onset of the husband's symptoms. She had not traveled
outside the town of Coyhaique during the previous 12 months and reported no
exposure to rodents or their excreta. The only known exposures for the wife
were washing her husband's clothing and caring for him while he was ill.
She survived and was discharged from the hospital on January 28.
						  
						  Genetic sequencing of viral RNA from
                                      cases of immediate family members
 						  from Clusters 1 and 2 demonstrated
                                      3.6% divergence in a 167-nucleotide
 [Figure 3.]Time-line listing for     G2 fragment between the clusters.
 hantavirus pulmonary syndrome cases  However, the genetic sequence was
 in Cluster 1, Chile, 1997.           identical within each of the two
                                      clusters. Genetic sequencing of
                                      viral RNA from an additional patient
from Cisne Medio not related to the family members in Cluster 1, and the
brother-in-law in Cluster 1, demonstrated a distinct G2 sequence, with
1%-4% sequence divergence between them (P. Padula, unpub. data).

Clinical and Histopathologic Features

In general, the clinical description of HPS cases in Aysen was similar to
that of the cases in North America. However, included in the Aysen cases
were three children with petechiae; one of them died rapidly with
hemorrhagic pulmonary secretions and bleeding from puncture sites. Six of
the seven patients on whom urinanalysis was performed had microscopic
hematuria and casts; three had proteinuria of >100 mg/dl. In addition, one
child in the Llanchipal region had concurrent acute rubella infection. One
of the most recent cases occurred in Maule, Region VII, in a 23-week
pregnant woman who had typical HPS symptoms and fetal death. Subsequently,
disseminated intravascular coagulopathy developed and the woman died of
multisystem failure.

The histopathologic features in the lung of HPS case-patients included an
interstitial mononuclear infiltrate and intraalveolar edema.
Immunohistochemical staining showed hantaviral antigens in the
microvascular endothelial cells of the lung and other tissuestypical
SNV-associated HPS cases. Prominent immunostaining of pulmonary macrophages
was also noted in some cases. A unique feature in the series was fine
granular immunostaining in the hepatic cells in some cases, a pattern not
observed in a large series of North American HPS cases previously examined
(Figure 4) (12).

Laboratory Test Results

Serologic testing was conducted for all patients and contacts of patients
with confirmed HPS cases in Aysen. Among 53 contacts of 14 patients, two
(3.8%) had serologic evidence of an acute infection (one had no illness,
and another [described previously in cluster 2] had a mild febrile illness
without pulmonary disease that did not meet the HPS case definition), and
one was IgG positive. In addition, two hospitalized patients had mild
hantaviral disease (M. Tapia, unpub. data). Test results of specimens (SNV
and Andes antigens were used) were 100% concordant. Nested and heminested
RT-PCR assays were conducted for 20 (71.4%) of 28 HPS cases; all 20 were
positive. Sixteen (57.1%) were sequenced and characterized as Andes virus.

Ecologic Studies

A total of 253 rodents were captured during 3 nights of trapping (574 trap
nights) in the vicinity of Coyhaique. Overall trap success (captures per
100 trap nights) at case and control home sites was 37% at Cisne Medio and
50% at Lago Atravesado. Trap success at Cisne Medio within and adjacent to
the case-household was 66%, compared with only 18% at control homes; this
difference may reflect the use of rodenticides in and around the control
households. Trap success at distances greater than 100 m from the
households was more similar between case and controls (63% versus 43%). The
most frequently captured rodent species was O. longicaudatus, which
comprised 47% of the captured rodents in the area; 13 (12.7%) of 102 tested
were hantavirus-antibodypositive. Akodon olivaceus comprised a further 33%
of the captures (6 [7.5%] of 80 were antibody-positive), and 16% were
Akodon longipilis (1 [2.7%] of 36 antibody-positive). Eight of 10 rodents
captured inside the case-home at Cisne Medio were O. longicaudatus, and one
was hantavirus-antibodypositive. High trap success in a forested area near
Lago Atravesado (22 captures in 40 trap nights, or 55%) indicated that high
rodent population densities were not restricted to peridomestic areas.

						  Trap success was moderate near
                                      Valdivia (52 captures from 660 trap
 						  nights, or 8% trap success) and very
                                      low near Santiago (8 captures from
 [Figure 4.] A) Low-power             453 trap nights, or <2%). The species
 photomicrograph of lung showing      composition was also very different
 interstitial pneumonitis and         from that encountered in the
 intraalveolar edema. B) Andes virus  Coyhaique area: despite trapping in
 antigenpositive intraalveolar       rural areas, 31% of rodents captured
 macrophages. C) Fine granular        near Valdivia and Santiago were the
 immunostaining of hantaviral         murine species, Rattus rattus, R.
 antigens in endothelial cells of     norvegicus, and Mus musculus. No
 pulmonary microvasculature. (A,      hantavirus-antibodypositive rodents
 Hematoxylin and eosin; B,C,          were found from Valdivia or Santiago.
 napthol-fast red with hematoxylin
 counterstain; original               Conclusions
 magnifications A x 50, B and C x
 250.)                                This outbreak of HPS and the presence
                                      of endemic disease in Chile support
                                      the assumption that HPS will continue
to occur throughout the Americas (range of the sigmodontine rodents that
host the genetic group of HPS-causing hantaviruses). In Latin America,
endemic HPS and epidemics have been reported in Argentina, Brazil, and
Paraguay (15-19). Sporadic cases and SNV-like viral sequences have been
reported in these countries and in Bolivia and Uruguay (P. Padula, pers.
comm.,7,20). Retrospective studies have verified that HPS cases occurred in
the United States as early as 1959 (21). The factors responsible for the
outbreaks in the Americas have not been well defined and may not be the
same in all regions. However, periodic climatic and ecologic events cause
dramatic local increases in rodent reservoir populations, known as
"irruptions" or "ratadas" (22) and may lead to an increase in the number of
rodent-human contacts.

Small-mammal trap success in the Coyhaique area was 5 to 30 times higher
than in rural areas 700 km to 1,400 km further north. These extremely high
trap success values indicate a rodent irruption in southern Chile whose
causes may be related to the flowering of a species of bamboo and a
relatively benign preceding winter (22). Regardless of causes, however, the
extremely high rodent densities and evidence of hantavirus infection in the
two most common species strongly implicate contact with rodents as the
primary mode of disease transmission to humans. The rodent species with the
highest antibody prevalence, O. longicaudatus, is the reservoir for Andes
virus in Argentina and Chile (8,23). No hantavirus has been associated with
A. olivaceus. RT-PCR conducted on tissue samples from seropositive animals
yielded amplifiable viral RNA from seven of seven O. longicaudatus, two of
four A. olivaceus, and one of one A. longipilis. Sequencing showed that all
PCR products represented Andes virus, and sequences were nearly identical
among all three species (S. Morzunov, unpub. data). The near identity
between sequences from all three species and the fact that two of four
samples from A. olivaceus were PCR negative suggest that hantavirus
infection in A. olivaceus and A. longipilis represents spillover of virus
from the primary reservoir, O. longicaudatus. The absence of viral RNA in
two of the four samples from A. olivaceus also suggests that the infection
is of shorter duration in this presumably nonhost species.

A number of features may eventually differentiate HPS caused by Andes virus
from that caused by other New World viruses. Several mild and asymptomatic
hantavirus infections have been reported in Argentina and Chile, whereas in
North America the case-to-infection ratio is approximately 1:1 (24,25). In
this investigation, four (11%) of the patients with serologic evidence of
acute infection did not have HPS. More mild and asymptomatic disease in
some South American hantavirus infections, in addition to infection with
nonpathogenic hantaviruses, may be among the factors explaining antibody
prevalence as high as 40% among indigenous persons of Argentina (C.
Johnson, unpub. data). Similar case-to-infection ratios may explain the
high prevalence (35% to 57%) of hantavirus antibodies in some regions of
Paraguay (16,26).

Disease in Chile has also been characterized by an increased propensity
toward bleeding, with petechiae in 50% of the pediatric cases and increased
renal involvement with microscopic hematuria and cellular casts in the
three patients whose urinanalysis results were available. Flushing of the
head and upper thorax is indicative of vascular dysregulation and is a
well-described sign in hemorrhagic fever with renal syndrome. It has also
been described in Andes virus-infected patients from the adjacent areas of
Argentina (17,27), but not in Chile.

The relatively large proportion of children with HPS reported in Chile is
unexplained. Infection in children is relatively rare in the United States,
where only 8 (4.5%) of 179 reported cases were in children 16 years old or
younger. Moreover, this dearth of pediatric cases has also been reported
for hemorrhagic fever with renal syndrome (28). It is unclear if this
greater incidence of pediatric cases is Andes virus- or outbreak-specific.
However, Argentina has also seen a larger proportion of pediatric HPS cases
than the United States, caused by three different genotypes: Andes, Oran,
and Lechiguanas.

Immunostaining showed hantaviral antigens in the microvascular endothelial
cells of the lung and other tissues with prominent staining of pulmonary
macrophages in some cases. A unique feature not previously described in
SNV-associated HPS cases was fine, granular immunostaining in the hepatic
cells of three patients. The specificity of this hepatic staining is being
investigated. In Argentina, person-to-person transmission has been
demonstrated in at least one epidemic of Andes virus infection (17).
Although the mechanism of that transmission is unknown, the increased
staining of intraalveolar pulmonary macrophages suggests the presence of
virus within the alveolar space with the potential for small-particle
aerosolization. A comparison of disease variants that may be
hantavirus-strainspecific will be required to address this hypothesis.

No discrepancies were observed in serologic results from testing with SNV
or Andes antigens, which underlines the current recommendation that
diagnosis be based on serologic testing, especially given the difficulties
in handling and transporting fresh tissues for RT-PCR and the possibility
of cross-contamination. Formalin-fixed tissues from case-patients who died
should be used for immunohistochemical tests, particularly if no serum or
blood was obtained. However, even in fatal cases, an attempt should be made
to obtain heart blood for serologic tests.

Person-to-person transmission cannot be excluded in the first or third
family cluster. We did not trap rodents within the town limits at Villa
Amengual or in the city of Coyhaique. However, although large numbers of
rodents were seen in rural areas, rodent infestation in Villa Amengual or
Coyhaique had not been reported. Further, since O. longicaudatus is a
sylvatic species not generally associated with urban habitats, subsequent
exposure to infected O. longicaudatus was likely not responsible for the
wife's illness in family cluster 3. However, as in Cluster 2, Andes and
other associated hantaviruses can cause clustering attributable to a single
pointsource contact with infected rodents.

Person-to-person transmission of Andes virus-associated HPS has been
documented (17,29), although this type of transmission has not been
observed with SNV-associated HPS (24,30). The lack of cases among
health-care workers suggests that current protective methods are adequate;
however, standard precautions should be reinforced. Until the propensity of
Andes virus to be transmitted from person to person is clarified, patients
with suspect cases should be isolated in separate rooms, contact and
droplet precautions should be used, and respiratory precautions should be
considered.

Acknowledgments

      We thank Drs. Alex Figueroa, Roberto Belmar, Hugo Salinas, and J.
Montecinos for their support to complete these studies, and John O'Connor
for editorial review of this manuscript. Dr. Roberto Murua provided
valuable support of rodent trapping efforts, and Dr. Milton Gallardo
confirmed the rodent species identifications.

      Dr. Toro is an epidemiologist with the Chilean Ministry of Health,
professor of public health at the Universidad de Chile in Santiago, and
director of the Chilean Society of Epidemiology.

      Address for correspondence: Ali S. Khan, CDC, Mail Stop A26, 1600
Clifton Road, Atlanta, GA, 30333, USA; fax: 404-639-1509, e-mail:
ask0@cdc.gov.

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Commentaries

Host Genetics of Infectious Diseases: Old and New Approaches Converge

Adrian V.S. Hill
University of Oxford, Oxford, United Kingdom

The increasing interest in infectious disease genetics over the last 5
years reflects several trends in the biomedical sciences, not least the
explosion of knowledge in human genomics. However, infectious disease
genetics is not a new field; some of the largest twin studies were
performed more than 50 years ago, and the first malaria resistance gene was
identified soon afterwards. The current excitement reflects a recent
increase in power in several of the approaches being used to map and
identify the genes responsible for variable susceptibility to many major
infectious diseases. Four distinct approaches have been used to identify
infectious disease susceptibility and resistance genes; these approaches
may now be converging.

Approaches

Candidate Genes

The most popular type of study in the host genetics of infectious disease
compares the allele or genotype frequencies of so-called "candidate genes"
in clinical cases with matched controls. Candidate genes are selected on
the basis of their function and likely relevance to the disease of interest
and on the possession of one or more genetic variants (1). This approach
has been very successful. Starting with the globin genes and
glucose-6-phosphate deficiency, several candidate genes were found in
case-control studies to be associated with malaria resistance. A different
approach associated the Duffy blood group with Plasmodium vivax malaria
resistance many years before the blood group was known to be a chemokine
receptor. In many infectious diseases, HLA variation has been studied, and
several associations have been established. The early associations between
HLA-DR2 and susceptibility to leprosy and tuberculosis (TB) in India have
been reconfirmed in recent years. Most recently, chemokine receptors and a
deletion variant of CCR5 have been found to affect both risk for HIV-1
infection and rate of progression to AIDS.

Despite these successes, problems remain with the candidate gene approach.
Most sample sizes have been adequate to detect only very strong
associations; however, less marked associations may be more common.
Matching controls to cases may be problematic and has led to the recent
trend for family-based association studies in other types of polygenic
disease. Frequent geographic heterogeneity in HLA associations has
complicated the interpretation of studies of these important genes.
Finally, the limited effects observed in most studies suggest that this
approach might be missing some other genes with more major effects.
Nonetheless, with more promising candidate polymorphisms appearing each
month, the popularity of this approach will likely increase.

Mouse Genetics

A quite different approach to finding human infectious disease resistance
and susceptibility genes has been identifying relevant murine genes. The
relative ease of mapping susceptibility genes in inbred strains of mice has
led to the mapping of numerous named loci in mice by linkage analysis. In
some cases, such as with the murine Mx influenza resistance locus, the gene
has actually been identified (2). Far more genes have been mapped in this
way than have been identified. A well-studied exception is the murine
Nramp1 gene that was positionally cloned on chromosome 1 (3) after this
locus was shown to influence susceptibility to certain leishmanial,
mycobacterial and salmonella infections in mice. Another important recent
approach has been the use of gene knockout mice to determine whether
particular genes affect susceptibility to various infectious diseases.

A difficulty with the mouse genetics approach is that a gene identified in
mouse studies may turn out to lack polymorphism in humans. This difficulty
is particularly relevant for artificially generated knockout mice;
inactivating mutations of key genes may be too deleterious to humans. Such
mutations, like mutations of the interferon-gamma receptor gene (4), may,
however, turn up rarely in immunodeficient children. However, even
mutations that are polymorphic in inbred mouse strains, such as the Nramp1
variant, may be nonpolymorphic in wild mouse populations. Given the
differences in selection pressure that mice and humans have been exposed to
over tens of millions of years, the major susceptibility genes in the two
species are unlikely to be the same. Another difficulty is, of course, the
extent to which a murine model may mimic human infectious disease. The
human malarias, for example, cannot be studied in mice.

Complex Segregation Analysis

Complex segregation analysis models the transmission of the disease in
multicase families and compares the likelihood of different ways of
inheriting the disease. Several parameters may be optimized, including the
contribution of a potential single major gene, polygenic and environmental
components, allele frequencies, and dominance. The remarkable result is the
regularity with which a single major gene is reported. For example, a
single major gene has thus been implicated in leprosy, schistosomiasis, TB,
and even malaria (regulating parasite densities) (5). Even allowing for
some publication bias, these remarkable results are surprising in view of
the highly polygenic nature of infectious disease susceptibility suggested
by candidate gene analysis. A major gene, as proposed in these models,
would need both a high frequency and very high odds ratio to account for a
substantial proportion of the overall genetic effect. One explanation might
be that such single major genes remain to be identified in these and
perhaps many other infectious diseases.

The usefulness of complex segregation analysis is difficult to assess
because in no case has the single major gene proposal been confirmed by
mapping and identifying such a gene. However, this difficulty may be ending
as genome scanning technology is increasingly applied to infectious
diseases.

Human Genomewide Analysis

The most recent approach applied to human infectious diseases is mapping
and subsequently identifying major genes affecting susceptibility or
resistance through genomewide scans. This approach entails an initial
linkage analysis in large numbers of multicase families, which is followed
by association study analysis for gene identification. Typically, a few
hundred microsatellite markers search for evidence of increased sharing of
parental alleles identical by descent in affected sibling pairs. This
nonparametric approach does not require information on how the disease was
inherited, which is almost always unknown. However, in schistosomiasis the
results of complex segregation analysis have been used in a parametric, or
model-based, analysis to map a susceptibility gene to chromosome 5 in a
small number of Brazilian families (6), as reviewed by Abel and Dessein in
this issue. The degree to which this genomewide approach can be used to map
major susceptibility genes in reasonable numbers of families remains
uncertain. However, the genes with the largest effects are those that can
be mapped with most power and may be of greatest interest.

The main limitation of a genomewide linkage scan in polygenic infectious
diseases is that its power is lower than that of association studies.
Recruiting the required large numbers of clinically well-defined multicase
families may require multicenter collaborations. Technical advances may
make genomewide association studies feasible, which would remove the need
for the use of multicase families, although the requirement for large
numbers of cases would remain.

Approach Convergence

Until recently, these various genetic approaches were typically pursued by
different research groups with relatively little synergistic interaction.
The major candidate genes studied in humans were different from those
mapped in mice, and none of the associations found appeared strong enough
to fit the single major genes being proposed by complex segregation
analysis. However, researchers working on the Nramp1 gene in mice suggested
that the human homologue, NRAMP1, could correspond to a major mycobacterial
susceptibility gene suggested by segregation analysis in humans. This
theory has now become testable with the identification of numerous
polymorphisms in NRAMP1. A clear association emerges between variation in
this gene and TB susceptibility, at least in a West African population (7).
Thus a gene identified through whole genome analysis in mice, potentially
the major gene suggested by complex segregation analysis, is associated
with susceptibility when investigated as a candidate gene in a human
case-control study. The separate approaches have converged, we hope for the
first of many meetings.

However, little in complex disease genetics is simple. The effect observed
in Gambia is different from BCG susceptibility in mice in that in human TB,
susceptibility appears dominant (7), while in mice it is recessive. Also,
the effect in humans, though highly statistically significant, is
relatively modest, perhaps a few percentage points of the overall genetic
component of susceptibility. Although not the elusive major gene, this
effect is well worth investigating for the light it may throw on mechanisms
of disease susceptibility. As other candidate genes are identified from
mouse studies, they may add to the long list of candidate genes available
for human association studies. Meanwhile, the hunt for elusive major
susceptibility genes continues. Ongoing genome scans should soon help
determine whether these genes really exist in particular infectious
diseases; if they do, their mechanisms of action as well as the forces
underlying their evolutionary maintenance should be of great interest.

References

  1. Hill AVS. The immunogenetics of resistance to human infectious
     diseases. Ann Rev Immunol. In press 1998.
  2. Staeheli P, Haller O, Boll W, Lindenmann J, Weissmann C. Mx protein:
     constitutive expression in 3T3 cells transformed with cloned Mx cDNA
     confers selective resistance to influenza virus. Cell 1986;44:147-58.
  3. Vidal SM, Malo D, Vogan K, Skamene E, Gros P. Natural resistance to
     infection with intracellular parasites: isolation of a candidate for
     Bcg. Cell 1993;73:469-85.
  4. Newport MJ, Huxley CM, Huston S, Hawrylowicz CM, Oostra BA, Williamson
     R, et al. A mutation in the interferon-gamma-receptor gene and
     susceptibility to mycobacterial infection. N Engl J Med
     1996;335:1941-9.
  5. Abel L, Cot M, Mulder L, Carnevale P, Feingold J. Segregation analysis
     detects a major gene controlling blood infection levels in human
     malaria. Am J Hum Genet 1992;50:1308-17.
  6. Marquet S, Abel L, Hillaire D, Dessein H, Kalil J, Feingold J, et al.
     Genetic localization of a locus controlling the intensity of infection
     by Schistosoma mansoni on chromosome 5q31-q33. Nat Genet
     1996;14:181-4.
  7. Bellamy R, Ruwende C, Corrah T, McAdam KJ, Whittle HC, Hill AVS.
     Variation in the NRAMP1 gene and susceptibility to tuberculosis in
     West Africans. N Engl J Med 1998;338:640-4.
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Commentaries

Does Restricted Distribution Limit Access and Coverage of Yellow Fever
Vaccine in the United States?

Thomas P. Monath,* Judith A. Giesberg, and Edward Garcia Fierros,
*OraVax Inc., Cambridge, Massachusetts, USA and Harvard School of Public
Health, Boston, Massachusetts, USA; Boston College, Chestnut Hill,
Massachusetts, USA; Harvard Graduate School of Education, Cambridge,
Massachusetts, USA

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

Yellow fever, the original "viral hemorrhagic fever," is a mosquito-borne
infection characterized by hepatic, renal, and myocardial injury, bleeding,
and a high case-fatality rate. The disease occurs in tropical South America
and sub-Saharan Africa. Maintained by zoonotic transmission between sylvan
mosquitoes and nonhuman primates, the virus cannot be eradicated, and
prevention of human infection requires high vaccination coverage. Recently
the incidence of yellow fever has increased dramatically (1), with 23,543
cases and 6,421 deaths officially reported to the World Health Organization
(WHO) between 1985 and 1996. The true incidence is believed to far exceed
the reported incidence. Yellow fever remains a public health problem
because of failure to implement effective immunization, particularly in
Africa.

The recent upsurge in yellow fever activity has been associated with an
increased risk for infection among unimmunized travelers. In 1996, two
tourists infected in the Amazon region of Brazil died after returning to
the United States (2) and Switzerland (3). Fatal and other cases in
tourists were also reported in previous years (4-8). Other cases were
likely missed, misdiagnosed as viral hepatitis, treated abroad, or not
reported.

Since viremic humans are the source of infection for the epidemic mosquito
vector Aedes aegypti, unimmunized travelers may import yellow fever to Ae.
aegypti-infested regions, including the United States, West Indies, Central
America, Mediterranean region, and Asia. The expansion of international
travel, including adventure travel to remote and tropical areas, has
increased exposure to yellow fever and other exotic infections (9). Each
year eight to nine million tourists arrive in yellow fever-endemic
countries in South America and Africa (10,11); the number of tourists
visiting regions within countries in which yellow fever transmission occurs
is unknown but probably exceeds three million. In the United States,
150,000 to 300,000 civilian travelers are immunized annually (12)likely
only a fraction of the population requiring the vaccine. However, few data
exist on vaccine coverage in travelers to yellow feverendemic regions. One
survey in the United States found that 30% of travelers to developing
countries between 1980 and 1986 received yellow fever vaccine (13). Yellow
fever vaccine coverage of European travelers varied from 0% to 90% (14,15).

WHO and the Centers for Disease Control and Prevention strongly recommend
vaccination for travel outside urban areas in the yellow feverendemic zone
(16,17). Yellow fever 17D, a live, attenuated, vaccine developed in 1936,
has been used in more than 300 million persons and has a remarkable record
of safety, tolerance, and efficacy (18). Immunity develops rapidly after a
single dose and is extraordinarily durable, probably lifelong. Yellow fever
vaccine can be administered simultaneously with most other vaccines or
immune serum globulin recommended for international travel. Cost of the
vaccine is approximately $48 for a single-dose vial in the United States.
Thus, the attributes of the vaccine itself do not constitute a barrier to
the immunization of travelers.

Yellow fever is the only human vaccine subjected to regulations regarding
distribution. The International Health Regulations (19) require that yellow
fever vaccine be given in approved vaccinating centers, listed in a 1991
WHO publication (20) and updated in the WHO Weekly Epidemiological Record.
The restriction was instituted to ensure that the relatively thermolabile
vaccine is properly stored and delivered within the specified time after
reconstitution and that a signed vaccination certificate is properly
completed (specifying the date of immunization and the vaccine lot) and
bears an official stamp. Only authorized medical facilities may order
vaccine directly from the manufacturer, and the vaccine is not available
through the normal distribution channels for drugs and biologic products.

In the United States, registration and approval of yellow fever vaccinating
centers has been delegated by the U.S. Public Health Service to the state
health departments. With the expansion of international travel in recent
years and a growing number of travel clinics, this system has become
unwieldy, and its listing of active centers is inaccurate.

The Problem, the Risks, a Solution

The United States currently has 800 to 1,000 yellow fever vaccinating
centers. Many are county health departments or clinics specializing in
travel medicine. However, most travelers first seek pretravel medical
advice from their primary-care providers. Immunizations and prophylactic
measures against many travel-related threats, such as hepatitis A, typhoid,
diphtheria, and measles, are commonly given by primary-care providers; even
more specialized travel vaccines, such as rabies, meningococcal, and
Japanese encephalitis vaccines, are readily available to general
practitioners. Specific recommendations for the use of travel vaccines are
in publications (19,20) and on the Internet.

Yellow fever is the only travel vaccine for which the primary-care
physician must refer the patient to another provider. Compliance with
recommendations for immmunization is affected by multiple factors (21),
including inconvenient access to a vaccine. A Tennessee resident who died
of yellow fever in 1996 was not vaccinated before going to Brazil because
the official vaccinating center nearest his home was 25 miles away (2). In
contrast, he had received other medical advice, hepatitis A vaccine, and a
prescription for malaria chemoprophylaxis from his primary-care physician.
Since requests for yellow fever vaccine may be few, most physicians do not
initiate the complicated process of obtaining authorization as a designated
vaccinating center. In the United Kingdom, however, where general
practitioners have become proactive in providing travel advice to their
patients, the number of physicians who obtained yellow fever vaccinating
center status rose from 181 to 1,231 between 1990 and 1992 (22).

The disadvantages of general practitioners' engaging in travel medicine,
including the immunization of travelers against exotic infections and
prescription of malaria chemoprophylaxis, have been examined in several
studies, which suggest that improvements are needed in the quality of
advice provided to patients (23,24). Among Swiss and German general
practitioners, only 11% and 1%, respectively, provided correct
recommendations to travelers to Thailand and Kenya regarding malaria
prophylaxis and vaccinations (25). County health departments in the United
States responsible for yellow fever vaccination have limited expertise in
travel medicine. While extending the distribution of yellow fever vaccine
to primary health-care providers would increase access to the vaccine and
improve coverage, additional steps are required to improve the quality of
medical advice by giving practitioners accurate and simple information to
guide use of the vaccine. Public health department nurses and inspectors in
Ontario, Canada, provided correct recommendations for yellow fever
vaccination 96% of the time (26), reflecting the value of education in this
field.

Travelers requiring yellow fever and other live virus vaccines face special
problems. The current recommendation is for either simultaneous
administration or separation of the vaccines by 4 weeks to avoid potential
interference (16). Compliance with this recommendation is difficult, and
the recommendation is probably ignored when the patient receives some
vaccines from the primary-care physician and yellow fever vaccine at a
designated center. The recommendation provides a further incentive for a
single health professional to administer a schedule of immunizations
appropriate for the individual traveler.

We sampled 15 states, which contained a very high proportion of the U.S.
population (44%) and were representative with respect to geography,
urban-rural distribution, and race. Although we could not estimate whether
these states were different from the United States as a whole with respect
to international travel, we believe that it is reasonable to extrapolate
the findings with respect to access to vaccinating centers to the general
population. We found that the current system of designated yellow fever
vaccinating centers in the United States provides reasonable access to
approximately 95% of the population, where "reasonable access" is defined
as residence within a 25-mile radius of the vaccinating center (Table).
Although this would appear sufficient, in fact there may be substantial
obstaclesfrom the perspective of the patientto utilization of centers
within geographic proximity (the 25-mile catchment area), including extra
costs, inconvenience of access to centers within congested urban areas, and
unfamiliarity with the provider. Approximately 5% to 6% of the total U.S.
population (12 to 14 million) resides in rural areas that are relatively
inaccessible to vaccinating centers (Table). In some states, such as
Montana, Wyoming, Oklahoma, and Vermont, approximately 25% of the
population resides outside 25-mile catchment areas, and very long distances
must be traveled to reach a center (Figure), with the result that only
highly motivated patients are vaccinated. Although it is possible that
urban residents with better access to vaccinating centers are more likely
to internationally travel than rural residents, our analysis clearly
demonstrated that restricted access to yellow fever vaccine by populations
in rural or sparsely populated areas.

 Table. Populations (1990 census[sup a]) inside and outside 25-mile radius
 'catchment areas' of intrastate authorized yellow fever vaccinating centers
 --------------------------------------------------------------------------------
                                                    Population x10(sup 3)
				      	---------------------------------------------
                                                     Inside             Outside
                                                  catchment           catchment
 Region      State                  Total         area (%)             area (%)
 --------------------------------------------------------------------------------
 East        New York             17,991      17,679 (98.3)           312 (1.7)
             Massachusetts         6,016       5,965 (99.2)            51 (0.8)
             Connecticut           3,287        3,287 (100)               0 (0)
             Vermont                 563         418 (74.2)          145 (25.8)
 South       Florida              12,938      12,410 (95.9)           528 (4.1)
             Louisiana             4,220       3,767 (89.3)          453 (10.7)
 Central     Illinois             11,431      10,125 (88.6)        1,306 (11.4)
             Wisconsin             4,892       4,331 (88.5)          561 (11.5)
             Nebraska              1,578       1,293 (81.9)          285 (18.1)
             Oklahoma              3,146       2,317 (73.6)          829 (26.4)
 Mountain    Montana                 799         595 (74.5)          204 (25.5)
             Wyoming                 454         350 (77.1)          104 (22.9)
 Pacific     California           29,758      29,138 (97.9)           620 (2.1)
             Oregon                2,842       2,676 (94.2)           166 (5.8)
             Hawaii                1,108         983 (88.7)          125 (11.3)
 15 states                       101,023      95,334 (94.4)         5,689 (5.6)
 --------------------------------------------------------------------------------
 (sup a)U.S. Bureau of the Census, 1990 Decennial Census.

In the past, a principal concern underlying the restricted distribution of
yellow fever vaccine was the thermolability of the vaccine and the need to
ensure its proper storage and handling. However, other vaccines in wide use
have similar or even more stringent storage requirements. Tetanus toxoid
must be stored refrigerated away from the freezer compartment,
measles-mumps-rubella must be stored refrigerated and protected from light,
and varicella vaccine must be stored frozen. Yellow fever vaccine
manufacturers now include stabilizers in the product that render the
lyophilized vaccines quite resistant to degradation (27-29). Yellow fever
vaccine is stored in the refrigerator at the point of use, as are most
other vaccines. Yellow fever vaccine is discarded 1 hour after
reconstitution; in contrast, varicella vaccine must be discarded within 30
minutes. Thus, the storage and handling of yellow fever vaccine do not pose
special difficulties.

The International Health Regulations,
adopted nearly 30 years ago (19),
require a valid certificate of          
vaccination only for yellow fever
(17,19). This regulation recognizes     [Figure.] Location of yellow fever
the risk for importation and spread of  vaccinating centers in three
yellow fever across international       western states. The circles
boundaries by viremic travelers.        indicate the area within a 25-mile
However, enforcement is problematic     radius from each center (catchment
and lax. For example, in the Calcutta   areas). Only names of selected
airport, closure of the health          locations are shown.
inspection desk in 1986 virtually
eliminated verification of vaccination
status of disembarking passengers (30). A number of yellow feverreceptive
(Ae. aegypti-infested) countries, including the United States, do not
require vaccination certificates (16,17), and many countries require but do
not enforce the inspection of certificates. The United States abandoned
inspection of vaccination certificates (because of the high cost and
questionable public health value of yellow fever quarantine regulations) in
favor of a system of surveillance and optimization of health information to
the traveling public. The difficulty in controlling yellow fever
vaccination through designated centers and the lack of a clear
justification on the basis of thermostability of the vaccine led a WHO
working group on revisions to the International Health Regulations to
recommend reconsideration of designation procedures of vaccination centers
(31). We think the link between the requirement for certification of yellow
fever vaccination imposed by some national health authorities and the
distribution of vaccine through designated centers should be the focus of
future discussions between parties to the International Health Regulations.
Certification of vaccination could be provided by any licensed physician,
as was the case for smallpox and cholera vaccines at the time that these
were required for international travel. The blank certificate (available
from the Government Printing Office) could, for example, be shipped
together with the vaccine from the manufacturer or distributor to the
physician. A change would be required in the International Health
Regulations to allow validation of the certificate by physician signature
alone without application of the uniform stamp currently used on the
certificate.

The risk of acquiring yellow fever during travel is related to the level of
virus transmission at the time and in the location visited. During yellow
fever epidemics, the risk to an unimmunized person is very high, even
during a short visit to an endemic-disease area. In recent outbreaks
affecting indigenous populations in West Africa, the incidence of infection
during the 3-month epidemic period was 20% to 30% (32,33). The risk of
developing severe disease with jaundice if infected has been estimated at
1:3.8 to 1:7.4 and the risk of dying if ill with jaundice is 20% to 60%
(18). In areas where the indigenous population is protected by vaccination,
yellow fever virus may circulate silently between monkeys and mosquitoes
without any reported human cases as a guide to risk. Moreover, the southern
United States is infested with Ae. aegypti, and importation of yellow fever
poses a considerable threat (34,35). The potential for importation by
persons infected with yellow fever has increased with the establishment of
direct links between the United States and disease-endemic areas, such as
Manaus, Brazil, and Iquitos, Peru (9). These factors provide a strong
rationale for immunizing all persons who visit disease-endemic zones.

The current system of designated yellow fever vaccinating centers and
relative inaccessibility to yellow fever vaccine are impediments to full
vaccination coverage of travelers. Extension of vaccinating privileges to
primary-care providers, together with clear and concise information
regarding recommendations for use of the vaccine, may improve protection of
travelers at risk and enhance the barrier to importation of the disease.

References

  1. Robertson SE, Hull BP, Tomori O, Bele O, LeDuc JW. Yellow fever. A
     decade of reemergence. JAMA 1996;276:1157-62.
  2. McFarland JM, Baddour LM, Nelson, Elkins SK, Craven RB, Cropp BC, et
     al. Imported yellow fever in a United States citizen. Clin Infect Dis
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  3. Barros ML, Boecken G. Jungle yellow fever in the central Amazon.
     Lancet 1996;348:969.
  4. Bendersky N, Carlet J, Ricomme JL, Souied G, Belaiche J, Lange F, et
     al. Deux cas de fivre jaune observs en France et contracts au
     Sngal. Aspects pidmiologiques et cliniques. Bull Soc Pathol Exot
     1980;73:54-61.
  5. Digoutte JP, Plassart H, Salaun JJ, Heme G, Ferrara L, Germain M. A
     propos de trois cas de fivre jaune contracte au Sngal. Bull World
     Health Organ 1981;59:759-66.
  6. World Health Organization. Yellow fever in 1981. Wkly Epidemiol Rec
     1982;59:297-301.
  7. World Health Organization. Yellow fever in 1985. Wkly Epidemiol Rec
     1986;61:377-80.
  8. Nolla-Salas J, Sadalls-Radresa J, Bada JL. Imported yellow fever in
     vaccinated tourist. Lancet 1989:2:1275.
  9. Monath TP. Epidemiology of yellow fever: current status and
     speculations on future trends. In: Saluzzo J-F, Dodet B, editors.
     Factors in the emergence of arbovirus diseases. Paris: Elsevier 1997.
     p. 143-56.
 10. Steffen R. Hepatitis A in travelers: the European experience. J Infect
     Dis 1995;171 Suppl:S24-8.
 11. World Health Organization. World Health Report 1996. Geneva: The
     Organization; 1996. p. 137.
 12. Centers for Disease Control and Prevention. Biologics surveillance.
     Atlanta (GA): The Centers; 1996. Report No. 94.
 13. Jong EC, Gurung K. Observations on obtaining immunizations for
     international travel and the need for routine immunizations. In:
     Steffen R, Lobel HO, Haworth J, Bradley DJ, editors. Travel medicine.
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     1988 Apr 5-8; Zurich, Switzerland. Berlin: Springer Verlag; 1989. p.
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     al. Statut vaccinal des voyageurs francais et europens: etude de 9156
     sujets au dpart de Paris vers 12 destinations tropicales. Rev
     Epidemiol Sante Publique 1998;46:64-72.
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     international travel 1996-97. Atlanta (GA): U.S. Department of Health
     & Human Services; 1997. p. 1-208.
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 18. Monath TP. Yellow fever. In: Plotkin SA, Mortimer EA, Orenstein W,
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     international travel. Geneva: The Organization; 1991.
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     travellers to Turkey. Journal of the Royal College of General
     Practitioners 1989;39:148-50.
 24. Mott A, Kinnersley P. Overprescription of cholera vaccine to
     travellers by general practioners. BMJ 1990;300:25-6.
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     German general practitioners. In: Abstracts of the 4th International
     Conference on Travel Medicine; 1995 Apr 23-29; Acapulco, Mexico.
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 26. MacPherson DW, Stephenson BJ, Keystone JS, Sawyer L. Travel health
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     Apr 23-29; Acapulco, Mexico. Abstract 23.
 27. Monath TP. Stability of yellow fever vaccine. Dev Biol Stand
     1996;87:219-25.
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 29. Barme M, Bronnert C. Thermostabilisation du vaccin antiamaril 17D
     lyophilis. I. Essai de substances protectrices. Journal of Biological
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 30. Sinha MK, Majumder NM. Will the present health check-up system invite
     yellow fever in India? Indian J Public Health 1990;34:119-21.
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     et al. Yellow fever in the Gambia, 1978-1979: epidemiologic aspects
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     Trop Med Hyg 1980;29:912-28.
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     diseases. Yellow fever and typhus and the possibility of their
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Letters

Aedes aegypti in Tucson, Arizona

To the Editor: The highly domestic mosquito species Aedes aegypti, a
tropical, nonnative vector of dengue and yellow fever, has been identified
in several desert communities, including the city of Tucson and the border
towns of Douglas, Naco, and Nogales (1). Ae. aegypti has now been found in
the southern Arizona communities of Benson and Sahuarita Heights, which
indicates that its distribution is probably expanding. Moreover, mosquito
surveillance indicating that Ae. aegypti populations in Tucson are
established throughout the city supports local concerns that the mosquito
poses a public health risk in Arizona's second largest metropolitan area.

Since it was first detected in 1994 on Tucson's west side, the mosquito has
been found in the central downtown and university districts, northern
foothills, and the east and south sides (both in residential areas,
including schools and parks, and in business districts). Between 1995 and
1997, almost 200 adult Ae. aegypti (female and male) were recovered in CDC
CO(sub 2) traps from 26 sites in Tucson by vector biologists from the Arizona
Department of Health Services, Pima County, and the University of Arizona
Veterinary Science Department.

These trapping events occurred between May and October of each year and
were associated with either routine arbovirus surveillance or nonsystematic
Ae. aegypti "spot-checks." Many of the sites were sampled more than once
and 30% contained Ae. aegypti on multiple occasions. Catches from
individual sites contained 1 to 40 adult mosquitoes (mode = 1).

A larval survey of the city has not yet been conducted because of limited
staff and problems associated with oviposition trapping, an important part
of Ae. aegypti larval surveillance (2). Initial attempts at ovitrapping by
state, county (1), and later university personnel were not successful
because the hay infusion water in ovitraps evaporated rapidly in Tucson's
arid climate. However, the few larvae recovered from household containers
suggest that Tucson's urban environment is providing a breeding habitat.
Since the local climate requires full-time vigilance of ovitraps, this
method of surveillance appears too labor intensive for the present.

In 1997, the University of Arizona Entomology Department initiated a
mosquito survey of the city. This multiyear project is funded by the
Entomology Department, the city of Tucson, and the Pima County Health
Department. Neighborhood associations in Tucson were surveyed for their
perceptions of the magnitude of the mosquito problem in their areas. On the
basis of survey results, the city was divided into regions: north, east,
south, west, and central. Four trapping stations were established in each
region for a total of 20 sites spanning the metropolitan Tucson and
outlying areas. The five regions were surveyed for mosquitoes through use
of CO(sub 2) traps approximately every 10 days starting July 1, 1997; traps 
were set in the late afternoon and collected in the late morning. Daytime 
CO(sub 2) trappings were not effective.

The Entomology Department's 1997 surveillance data suggest that the central
part of the city is the most heavily infested. Of 95 adult Ae. aegypti
trapped, 49.5% were from the central region of Tucson, 18.9% from the west
side, 17.9% from the east side, 10.5% from the north side, and 3.2% from
the south side. The mosquito populations appeared to fluctuate with the
weather, increasing in size after rainfall. Long-term trapping and future
larval surveys should shed more light on this association.

Pima County and the University of Arizona Veterinary Science Department
trapping activities in 1997 also produced evidence of Ae. aegypti in two
communities near Tucson. Six adult mosquitoes were recovered in the town of
Benson, 30 miles southeast of Tucson, and seven were trapped in Sahuarita
Heights, 15 miles south of Tucson. The presence of the mosquitoes in these
communities, as well as in Douglas, Naco, and Nogales, demonstrates that
Arizona's smaller desert communities are also susceptible to Ae. aegypti
infestations. The humidity emitted by home evaporative coolers may be
crucial for the survival of tropical mosquitoes, such as Ae. aegypti, in
Arizona's arid climate (N. Monteny, pers. comm.).

Genetic analysis of the Ae. aegypti collected from southeastern Arizona,
Texas, and Mexico is under way at the University of Arizona Ecology and
Evolutionary Biology Department to determine the structure, history, and
origin(s) of the reemergent mosquito populations. Preliminary findings from
mitochondrial DNA sequences suggest that Ae. aegypti in Arizona represent a
single (panmictic) population, which indicates frequent local migration.
More extensive sampling is necessary to confirm these results and determine
a point of origin.

A community outreach program has been developed to inform the public about
Ae. aegypti breeding and control in Tucson. Public involvement will be a
key factor in the control of these urban breeders. Major emphasis will also
be placed on programs for children and teachers as both groups can be
instrumental in maintaining long-term interest in this problem. As these
programs are developed, they can be expanded and amended to meet the needs
of other infested communities in southern Arizona. A mosquito control
abatement district is under consideration in a central part of Tucson. The
primary purpose of this district would be to provide approximately 10,000
homeowners with information on controlling Ae. aegypti breeding on their
property.

Just how long the Ae. aegypti infestation will last is difficult to assess.
Records of the city's earlier infestation indicate the mosquito was present
for at least a 15-year period (1931 to 1946) (1,3,4). Since their
identification in early 1998 summer mosquito samples from Tucson, adult Ae.
aegypti have been part of the city's local environment for at least 5
consecutive years (1994 to 1998). Their continued presence and the abundant
breeding habitat provided by the expansion of Tucson's urban landscape
suggest that Ae. aegypti could survive for an extended period.

T. Michael Fink,* Bosun Hau, Branford L. Baird, Sarah Palmer, Susanne
Kaplan, Frank B. Ramberg, Daniel G. Mead, and Henry Hagedorn
*Arizona Department of Health Services, Phoenix, Arizona, USA; University
of Arizona, Tucson, Arizona, USA; and Pima County Community Prevention and
Public Health Department,Tucson,Arizona, USA

References

  1. Engelthaler DE, Fink TM, Levy CE, Leslie MJ. The reemergence of Aedes
     aegypti in Arizona. Emerg Infect Dis 1997;3:241-2.
  2. Reiter P, Amador MA, Colon N. Enhancement of the CDC ovitrap with hay
     infusions for daily monitoring of Aedes aegypti populations. J Am Mosq
     Control Assoc 1991;7:52-5
  3. Murphy D. Collection records of some Arizona mosquitoes. Entomological
     News 1953;14:233-8.
  4. Bequaert J. Aedes aegypti, the yellow fever mosquito, in Arizona.
     Bulletin of the Brooklyn Entomological Society 1946;41:157.

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Can the Military Contribute to Global Surveillance and Control of
Infectious Diseases?

To the Editor: Numerous networksboth formal (e.g., Ministries of Health
and WHO Collaborating Centers and collaborating laboratories) and informal
(e.g., nongovernmental and humanitarian organizations, the media, and
electronic discussion groups)contribute to WHO's network of networks for
the global surveillance of infectious diseases (1).

A potential source of additional information on infectious diseases is the
network of military health facilities and laboratories throughout the
world. In addition to health facilities serving populations at high risk
for infectious diseases, the military also has laboratories, often among
the better-equipped, in developing countries. To evaluate the feasibility
and potential usefulness of including military laboratories in the WHO
global surveillance network, we conducted three surveys.

The first survey identified military laboratories willing to participate in
global surveillance activities and obtained information about their
infectious diseases reporting systems. Of the 107 countries surveyed, 76
replied. Among them, 53 (70%) reported having a central military laboratory
that coordinates laboratory activities throughout the military, and 62
(82%) reported that military clinical facilities had a reporting system for
infectious diseases.

The second survey quantified laboratory capabilities in the 53 laboratories
identified in the first survey and obtained details about the 62 reporting
systems. Among the 39 (74%) laboratories that replied, all can perform at
least one of the following activities: isolating and identifying bacterial,
viral, or parasitic agents. Twenty-nine (55%) have the capacity for
specialized immunologic or molecular study. In addition, one of these
laboratories has a biosafety level 4 facility, six have a biosafety level 3
facility, and 10 have a biosafety level 2 facility. Twenty-seven (51%) of
the laboratories perform compulsory screening of new recruits for HIV, 17
(33%) for hepatitis B, 7 (13%) for hepatitis C, 39 (74%) for tuberculosis,
35 (67%) for syphilis, 18 (34%) for intestinal parasites, 13 (25%) for
schistosomiasis, 12 (23%) for malaria, and 2 (4%) for Chagas disease.

Among the 54 reporting systems for which further information was obtained,
clinical diagnoses (in some countries laboratory confirmed) are reported
through the hierarchical chain, normally by mail or facsimile, but in two
countries by electronic links. Almost all military reporting systems are
parallel to civilian systems. Thirty-four (63%) of 54 systems feed into the
civilian system, with a built-in mechanism to avoid duplicate reporting; 16
(30%) systems feeding into the civilian system have no such mechanism in
place; and four have no link with the civilian system.

The third survey addressed vaccination policies. Among 52 countries that
replied, 47 (90%) have a compulsory military vaccination schedule: 45 (87%)
for tetanus, 30 (58%) for diphtheria, 23 (44%) for typhoid, 16 (31%) for
bacillus Calmette-Gurin and polio, 12 (23%) for meningococcal meningitis,
and 10 (19%) for measles, mumps, and rubella.

These surveys show that military populations are protected against many
infectious diseases and that a wealth of information is obtained by
military laboratories and health-care facilities on populations at high
risk for infectious diseases. While most of the information collected from
the health-care facilities is reported through civilian systems as well,
incorporating the military network of laboratories into the WHO global
surveillance network could ensure broader coverage.

Raffaele D'Amelio* and David L. Heymann
*Ministero della Difesa, Direzione Generale Sanit Militare, Roma, Italy;
and World Health Organization, Geneva, Switzerland

Reference

  1. Heymann DL, Rodier GG. Global surveillance of Communicable Diseases.
     Emerg Infect Dis 1998;4:362-5.

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Dual Infection with Ehrlichia chaffeensis and a Spotted Fever Group
Rickettsia: A Case Report

To the Editor: In their article, Daniel J. Sexton et al. state, "Well-
documented cases of simultaneous human infections with more than one
tick-borne pathogen are rare" (1) and mention only two reports of such
cases. However, another report should be mentioned because of its
historical interest and the lessons it may teach.

In 1900 to 1905, in the Bitter Root Valley, a tick-borne disease emerged,
which became known as Rocky Mountain spotted fever. Although Ricketts et
al. later published a report (2), which identified the causative agent, in
1904 L.B. Chowning and W.M. Wilson published Studies on Pyroplasma hominis
(3). They reported finding Pyroplasma (since changed to Babesia) in the
blood of approximately 20 patients with spotted fever. They studied this
organism in detail and even found the reservoir for it in the local rodent
species. Wilson et al. thought that the organism was the causative agent of
spotted fever. On the basis of their excellent plates and descriptions, it
is clear that the organism they were describing was what we later came to
know as Babesia microti.

The work of Wilson and Chowning was ignored and forgotten for many years.
They had incorrectly concluded that spotted fever was caused by a parasite.
For many years it was "well known" that Babesia infections became apparent
in human patients only on removal or inactivation of the spleen. That
persons with functional spleens were subject to infection with B. microti
was finally established by the so-called Nantucket outbreak (4) and
subsequent publications.

Therefore, Wilson and Chowning's work reports several cases of simultaneous
infections of humans by two tickborne pathogens; i.e., patients had spotted
fever and B. microti in the blood. More poignant was that an "emerging"
disease of humans was missed and not discovered again for some 70 years.

Alexander J. Sulzer
Fellow, American Academy of Microbiology; Fellow, emeritus, Royal Society
of Tropical Medicine and Hygiene; Member, emeritus, American Society of
Tropical Medicine and Hygiene

References

  1. Sexton DJ, Corey GR, Carpenter C, Kong LQ, Gandhi T, Breitschwerdt E,
     et al. Dual infection with Ehrlichia chaffeensis and a spotted fever
     group rickettsia: a case report. Emerg Infect Dis 1998;4:311-6.
  2. Ricketts HT. Some aspects of Rocky Mountain spotted fever. Rev Infect
     Dis 1909;1227-40.
  3. Wilson LB, Chowning WM. Studies on Pyroplasma hominis. Rev Infect Dis
     1904;1:31-57.
  4. Ruebush TK, Juranek DD, Chisholm ES, Snow PC, Healy GR, Sulzer AJ.
     Human babesiosis on Nantucket Island. N Engl J Med 1977;297:825-87.

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Dual Infection with Ehrlichia chaffeensis and a Spotted Fever Group
Rickettsia: A Case ReportReply to Dr. Sulzer

To the Editor: Several investigators have suggested that some of Wilson and
Chowning's patients may have had coinfection with Babesia and Rickettsia
rickettsii (1-4). Furthermore, the organisms that Wilson and Chowning
observed in red cells of 20% of the local Columbian ground squirrels are
consistent with later reports of various species of Babesia in the
erythrocytes of other species of squirrels (4). However, most
rickettsiologists who have commented on Wilson and Chowning's paper have
concluded that intraerythrocytic organisms observed in blood samples did
not contribute substantially to the illnesses of the 23 patients described.
Although Stiles, Wenyon, and Brumpt concluded that the organisms in human
blood samples observed by Wilson and Chowning were artifacts or malarial
parasites (5-7), contemporary experts who have reviewed the colored plates
that accompanied Wilson and Chowning's 1904 paper believe that there is
"little" or "no doubt" that Wilson and Chowning actually described
organisms of the genus Babesia (1,2,8).

In a commentary that followed the republication of Wilson and Chowning's
landmark paper in 1979 (9), Richard Ormsbee reviewed the sequence of events
that followed the publication of Wilson and Chowning's report in 1904 (10).
After more than 200 hours of careful microscopy, C.W. Stiles could find no
evidence of Pyroplasma in the blood of 12 patients with Rocky Mountain
spotted fever (RMSF). He refuted Wilson and Chowning's findings (5) and
challenged Chowning, who was also in the Bitter Root Valley, to demonstrate
the presence of organisms in the blood of a person with a typical case of
RMSF. Chowning was unable to find Pyroplasma in blood smears from these
patients (10). Ricketts did not arrive in the Bitter Root Valley to begin
his studies of RMSF until 1906 (11); thus he could not have published his
classic paper on the etiology of RMSF in volume 1 of the Journal of
Infectious Diseases.

To our knowledge, ecologic studies done in the Bitter Root Valley have not
demonstrated endemic foci of babesial infection. A serologic survey of 246
Bitter Root Valley residents in 1978 showed no antibabesial antibodies
(12). Although it is possible that 4 of the 23 patients with RMSF described
by Wilson and Chowning had incidental preexisting latent babesial
infection, the clinical and autopsy data they presented suggest that the
patients had typical R. rickettsii infection. There is no proof that any of
the patients described by Wilson and Chowning had simultaneous acute
babesial and rickettsial infection, and we agree with Ormsbee that the
significance of the "Pyroplasma hominis" described in the blood smears of
several of Wilson and Chowning's patients is "... a mystery that persists
to this day" (10).

Daniel J. Sexton* and David H. Walker
Duke University Medical Center, Durham, North Carolina, USA; and University
of Texas Medical Branch at Galveston, Galveston, Texas, USA

References

  1. Gorenflot A, Mourbri K, Precigout E, Carcy B. Human babesiosis. Ann
     Trop Med Paristol 1998;92:489-501.
  2. Hoare CA. Comparative aspects of human babesiosis. Trans R Soc Trop
     Med Hyg 1980;74:143-8.
  3. Ruebush TK II, Cassaday PB, Marsh HJ, Lisker SA, Voorhers D, Mahoney
     EB, et al. Human babesiosis on Nantucket Island. Clinical features.
     Ann Intern Med 1977;86:6-9.
  4. Pinsky RL, Lutwick LI. Spotted fever and babesiosis. Rev Infect Dis
     1979:1:895.
  5. Stiles CW. A zoological investigation into the cause, transmission,
     and source of Rocky Mountain "spotted fever." Hygienic Laboratory
     Bulletin . Washington: Government Printing Office; 1905. Publication
     No 20:7-119.
  6. Wenyon CM. Protozoology. Vol 2. London: W. Wood; 1926.
  7. Brumpt E. Precis de parasitologie. 5th ed. Paris: Masson et Cie; 1936.
     p. 497.
  8. Pruthi RK, Marshall WF, Wiltsie JC, Persing DH. Human babesiosis. Mayo
     Clin Proc 1995:70:853-62.
  9. Wilson LB, Chowning WM. Studies in Pyroplasmosis hominis (`spotted
     fever" or "tick fever of the Rocky Mountains). Rev Infect Dis
     1979;1:540-58.
 10. Ormsbee RA. Studies in Pyroplasmosis hominis ("spotted fever" or "tick
     fever" of the Rocky Mountains) by Louis B. Wilson and William A.
     Chowning. Rev Infect Dis 1979;1:559-62.
 11. Harden VA. Rocky Mountain spotted fever research and the development
     of the insect vector theory, 1900-1930. Bull Hist Med 1985;59:449-66.
 12. Chisholm ES, Ruebush TK II, Sulzer AJ, Healy GR. Babesia microti
     infection in man: evaluation of an indirect immunofluorescent antibody
     test. Am J Trop Med Hyg 1978:27:14-9.

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Reemergence of Plasmodium vivax Malaria in the Republic of Korea

To the Editor: In "Reemergence of Plasmodium vivax malaria in the Republic
of Korea" (1), the term eradication was, in my judgment, inappropriately
used. In 1981, Yekutiel proposed that eradication is "The purposeful
reduction of specific disease prevalence to the point of continued absence
of transmission within a specified area by means of a time limited
campaign" (2). In 1984, Hinman proposed an important addition that
eradication must have followed a "deliberate effort" (3). At the Dahlem
Workshop in 1997 (4), a more comprehensive definition was proposed. This
definition states that eradication is "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" (4). At the
same conference, two other terms were also defined. Elimination of disease:
"Reduction to zero of the incidence of a specified disease in a defined
geographic area as a result of deliberate efforts; continued intervention
measures are required." Elimination of infection: "Reduction to zero of the
incidence of infection caused by a specific agent in a defined geographic
area as a result of deliberate efforts; continued measures to prevent
reestablishment of transmission are required."

These definitions promote unanimity in using the term eradication and avoid
misconceptions over accomplishments.

Philip S. Brachman
The Rollins School of Public Health of Emory University, Atlanta, Georgia,
USA

References

  1. Feighner BH, Pak SI, Novakoski WL, Kelsey LL, Strickman D. Reemergence
     of Plasmodium vivax malaria in the Republic of Korea. Emerg Infect Dis
     1998;2:295-7.
  2. Yekutiel P. Lessons from the big eradication campaigns. World Health
     Forum 1981;2:465-81.
  3. Himnan AR. Prospects for disease eradication or elimination. New York
     State General Medicine 1984;84:502-6.
  4. Ottesen EA, Dowdle WR, Fenner F, Habermehl KO, John TJ, Koch MA et al.
     In: Eradication of infectious diseases. Dowdle WR, Hopkins DR,
     editors. New York: John Wiley and Sons; 1997. p. 48.

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Paratyphoid Fever

To the Editor: The letter on paratyphoid fever by Kapil et al. (1) stated
that an outbreak of enteric fever due to Salmonella paratyphi A has never
been reported. A large (227 cases) outbreak of enteric fever secondary to
S. paratyphi A occurred in the Arabian Gulf nation of Bahrain in 1987. The
clinical and epidemiologic details of the outbreak were reported in a local
medical society journal (2). Like the outbreak described by Kapil et al.,
the Bahraini outbreak was associated with sewage leaking into the water
supply.

Mark R. Wallace
Naval Medical Center San Diego, San Diego, California, USA

References

  1. Kapil A, Sood S, Reddaiah VP, Das B, Seth P. Parayphoid fever due to
     Salmonella enterica serotype paratyphi A. [Lett]. Emerg Infect Dis
     1997;3:407.
  2. Al-Madani R. Paratyphoid Outbreak in children. Journal of the Bahrain
     Medical Society 1989;1:60-3.

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Hospitalizations After the Persian Gulf War

To the Editor: Knoke et al., Naval Health Research Center, San Diego,
California, published two articles on military hospitalizations in Persian
Gulf War veterans, the most recent in Emerging Infectious Diseases (1,2).

Although the titles of both articles indicated general hospitalizations,
Knoke et al. studied just military hospitalizations among selected, mostly
healthy, active-duty Persian Gulf War veterans enlisted as of 1994. They
compared military hospitalizations of active-duty Gulf War veterans (cases)
with military hospitalizations of active-duty era veterans not in the
Persian Gulf between 1990 and 1991 (controls). "Healthy warrior" effects
would have predicted low military hospitalization rates for both cases and
control populations (3), but both were high.

The studies were "restricted to active-duty personnel" hospitalized in
military facilities because active-duty personnel were "rarely hospitalized
outside of DoD facilities" (1). However, of 150 surgical procedures, mostly
intestinal and skin biopsies, performed on 85 sick active-duty and
reservist Persian Gulf veterans from Pennsylvania between 1991 and 1995,
more than one third, 58 (39%), were performed in private facilities (4).
Most of the federal procedures were done in Veterans Administration (VA),
not military, hospitals. Many active-duty, sick Persian Gulf veterans in
Pennsylvania, Texas, and California deliberately avoided military, and some
VA, hospitals between 1991 and 1997 because of concerns about competence,
convenience, confidentiality, and career opportunities during this era of
downsizing and closing of military bases (3,5).

In addition, Knoke et al. excluded at least five groups of sick veterans
from their limited case studies: 1) those treated in VA and private
hospitals, 2) those from the Reserves and National Guard, 3) those who
retired early largely because of illness, 4) those who consented to long
military hospitalizations within the DoD Comprehensive Clinical Evaluation
Program (CCEP) for Gulf War Veterans, and 5) those who had obstetric
complications after returning home from the Gulf War. Thus, many sick
veterans were excluded from the case studies.

If we hypothesize that one or more new infectious agents like Leishmania
tropica, Brucella species, Bacillus anthracis, Mycoplasma fermentens
(incognitus), Coxiella burnetti, or obscure fungi or molds might be
involved, comprehensive research studies in the future would do better to
include all workers from the Arabian desert, reservists as well as
active-duty personnel.

Few Gulf veterans with Gulf-related illnesses were welcomed by military
hospitals and about half of 452 Persian Gulf veterans surveyed by the U.S.
General Accounting Office sought health care outside the VA for health
problems they believed were related to service in the Persian Gulf (5). An
alternative interpretation of Knoke's hospitalization study might be that
admitting officers in military facilities prevented sick Persian Gulf War
veterans from obtaining medical care within their facilities.

Not only were the case populations studied unusual; recent workers and
travelers to the Middle East were not excluded from the control population.
"Nondeployed" controls included recently deployed Persian Gulf military
personnel as long as nondeployed personnel worked in the Gulf after 1991.
Some of those late-deployed Persian Gulf workers also fell ill with the
same illnesses as veterans deployed between August 1990 and 1991. Illnesses
from late Persian Gulf deployments might explain excess hospitalizations
seen in nondeployed controls. All late-deployed personnel from the Middle
East should also have been excluded from the nondeployed control
population.

Finally, medical ICD-9 diagnoses, while interesting, were incomplete and
nonspecific. Medical diagnoses common to Gulf veterans should have been
listed in addition to unexplained illnesses. Knoke's condensed diagnostic
list, like patient charts we have seen from DoD hospitalizations, may have
failed to capture common clinical and laboratory abnormalities seen in many
sick Gulf veterans, including (but are not limited to) ulcerative colitis,
Crohn colitis, inflammatory bowel disease, intestinal bleeding due to
inflammatory colonic polyps, skin acne, nodules, plaques, psoriasiform skin
rashes, nose ulcers, nose bleeds, leukocytosis, neutropenia, elevated
alanine transaminase (SGPT/ALT) liver enzymes, hepatosplenomegaly,
thrombocytopenia, nephrolithiasis (kidney stones), and fevers of unknown
origin (4,6). In addition, more than one unexplained illness category
should have been tabulated per patient, because "Gulf War Syndrome" is a
multisystem illness (4,6-9).

More research is needed on hospitalizations in addition to deaths and new
diseases found in Persian Gulf War veterans (3). Civilian scientists and
physicians must collaborate closely with other diverse federal and
nonprofit organizations to study Gulf War illnesses objectively (5,9). The
health problems seen in Gulf War veterans may be part of a new complex of
emerging desert-associated illnesses (9-14).

Katherine Murray Leisure,* Nancy L. Nicolson, and Garth L. Nicolson
*Infectious Diseases, Travel Medicine, Lebanon, Pennsylvania, USA; and
Institute for Molecular Medicine, Huntington Beach, California, USA

References

  1. Gray GC, Coate BD, Anderson CM, Kang HK, Berg SW, Wignall RS, 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.
  2. Knoke JD, Gray GC. Hospitalizations for unexplained illnesses among
     U.S. veterans of the Persian Gulf War. Emerg Infect Dis 1998;4:211-19.
  3. Haley RW. Bias from the "healthy warrior effect" and unequal follow-up
     in three government studies of health effects of the Gulf War. Am J
     Epidemiol 1998;148:315-23.
  4. Murray-Leisure KA, Sees J, Zangwill B, Suguitan E, Legaspi C, Bagheri
     S, Mucha P, Brinser E, Kimber R, Kurban R, Greene W.
     Mucocutaneous-intestinal-rheumatic Desert Syndrome (MIRDS): Incubation
     & histopathology findings. Abstract K 48, American Society for
     Microbiology, ICAAC, Sept. 18, 1995.
  5. U.S. General Accounting Office. Gulf War Illnesses: Improved
     Monitoring of Clinical Progress and Reexamination of Research Emphasis
     are Needed. GAO/NSIAD-97-163. June 1997.
  6. Sostek MB, Jackson S, Linevsky JK, Schimmel EM, Fincke BG. High
     prevalence of chronic gastrointestinal syndromes in a National Guard
     unit of Persian Gulf Veterans. Am J Gastroenterol 1996;91:2494-7.
  7. Centers for Disease Control. Unexplained illness among Persian Gulf
     War veterans in an Air National Guard unit: Preliminary report, Aug
     1990-March 1995. MMWR Morb Mortal Wkly Rep 1995;44:443-7
  8. Fukada K, Nisenbaum R, Stewart G, Thompson WW, Robin L, Washko R, et
     al. Chronic multisystem illnesses affecting Air Force veterans of the
     Gulf War. JAMA 1998;280:981-8.
  9. Nicolson G, Nicolson N. Gulf War illnesses. Medicine, Conflict, and
     Survival 1998;14:156-65.
 10. Magill AJ, Grogl M, Gasser RA, Wellington S, Oster CN. Viscerotropic
     leishmaniasis caused by Leishmania tropica in soldiers returning from
     Operation Desert Storm. N Engl J Med 1993;328:1383-7.
 11. Magill AJ, Grogl M, Johnson SC, Gasser RA. Visceral infection due to
     Leishmania tropica in a veteran of Operation Desert Storm who
     presented two years after leaving Saudi Arabia. Clin Infect Dis
     1994;19:805-6.
 12. Ferrante MA, Dolan MJ. Q Fever meningoencephalitis in a soldier
     returning from the Persian Gulf War. Clin Infect Dis 1993;16:489-96.
 13. Seaman J, Mercer AJ, Sondorp E. Epidemic of visceral Leishmaniasis in
     western upper Nile, southern Sudan: Course and impact from 1984 to
     1994. Int J Epidemiol 1996;25:862-71.
 14. Zilinskas RA. Iraq's biological weapons. JAMA 1997;278:418-24.

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Hospitalizations after the Gulf WarReply to K.M. Leisure et al.

To the Editor: We studied all active-duty Persian Gulf Warera veterans who
remained on active duty at the conclusion of deployment (July 31, 1991),
not as Leisure et al. stated in their letter "selected, mostly healthy,
active-duty Persian Gulf War veterans enlisted as of 1994."

Our study was restricted to hospitalizations of active-duty service members
because these were the only service members whose records were available on
computerized files. No one was excluded from the defined target population.
However, there are "sick Gulf War veterans" and healthy Gulf War veterans
not in the target population. The difficulty is in studying either a random
sample or the entire population of Gulf War veterans. The only published
study we know of the entire population is the mortality report of Kang and
Bullman (1).

The suggestion that we should have excluded from the control group service
members who had ever been in the Gulf War area would have been appropriate
for a report of exposure to the Persian Gulf region; ours was a report of
exposure to the Persian Gulf War. That we should have studied a different
collection of ICD-9 diagnoses also suggests a different report.

While our study may have limitations, we have not seen objective data that
support the anecdotal observations of Leisure et al.

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

Reference

  1. Kang HK, Bullman TA. Mortality among U.S. veterans of the Persian Gulf
     War. N Engl J Med 1996;335:1498-504.
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---------------------------------------------------------------------------------

Book Review

HIV Nursing and Symptom Management. ME Ropka, AB Williams, Editors. Jones
and Bartlett Publishers, Sudbury, Massachusetts, 1998, 806 pages.

The need for updated texts on nursing care of patients with HIV is critical
for many reasons: the rapidly expanding availability of new medications and
need for adherence to complex therapeutic regimens, the requirements of
opportunistic infection prophylaxis, the broad constellation of symptoms
caused by many opportunistic diseases from early HIV infection through
long-term end-stage care, and the vulnerable populations involved. This
volume edited by nurses from the University of Virginia School of Medicine
and the Yale University School of Nursing gives a cogent overview of these
topics, broken into four overarching units.

Unit one provides introductory information on HIV infection including
disease pathogenesis and epidemiology, related opportunistic infections and
malignancies, and pharmacologic therapy. The second unit, the strongest of
this volume, reviews the nursing management of common clinical problems
such as neurologic manifestations, nutrition-related changes, fecal
incontinence, respiratory changes, psychosocial problems, and pain
management. The third unit, HIV Special Treatment Considerations, addresses
adherence to therapy, continuity of care, and ethical and legal issues.
Unit four gives guidance for approaching HIV care in some of the diverse
populations particularly affected by the HIV epidemic: women, children,
substance abusers, Hispanics, and African-Americans.

This volume, which contains more than 100 well-organized explanatory tables
and figures, provides a good overview of essential topics and allows easy
retrieval of information on nursing care as well as basic epidemiology and
pathogenesis of frequently encountered HIV-related conditions. HIV Nursing
and Symptom Management provides comprehensive background information and
practical clinical guidance for nurses in HIV patient care or research.

Anne C. Moorman
Centers for Disease Control and Prevention, Atlanta, Georgia, USA
---------------------------------------------------------------------------------
---------------------------------------------------------------------------------

News and Notes

Meeting Summary

Overseas Medical Laboratory Conference

Military Infectious Disease Research Program, U.S. Army Medical Research
and Development Command, Ft. Detrick, Maryland, USA

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The Military Infectious Disease Research Program, U.S. Army Medical
Research and Materiel Command, and the Walter Reed Army Institute of
Research (WRAIR) hosted a workshop on overseas activities in infectious
disease research in December 1997, at WRAIR, Washington, D.C. Approximately
150 Army and Navy medical research scientists met to discuss progress
toward specific goals designed to produce products and information
applicable to protection of military personnel in the field.

The Department of Defense has developed a network of six overseas
laboratories that perform research on infectious diseases of public health
and military importance (Armed Forces Research Institute of Medical
Sciences in Bangkok, Thailand; the Naval Medical Research Units No. 2 in
Jakarta, Indonesia, and No. 3 in Cairo, Egypt; the U.S. Army Medical
Research Units in Nairobi, Kenya, and Rio de Janeiro, Brazil; and the Naval
Medical Research Institute Detachment in Lima, Peru). The combination of
laboratories in the United States (WRAIR, U.S. Army Institute for
Infectious Diseases, and the Naval Medical Research Institute) and overseas
forms an effective international infectious diseases research program, in
which domestic laboratories are positioned to maintain contact with
academia and other branches of the government and overseas laboratories
detect new trends in naturally occurring endemic and epidemic infectious
diseases of potential military relevance (through their own surveillance or
close contact with foreign health authorities and conducting risk
assessment). The overseas laboratories also conduct trials of new products
for disease prevention and control.

For more information on the workshop, contact LTC Daniel Strickman, U.S.
Army Medical Research and Development Command, (Attn: MCMR-PLA), 504 Scott
Street, Fort Detrick, MD 21702-5012, USA; tel: 301-619-7567; fax:
301-619-2416; e-mail: COL_Charles_Hoke@ftdetrck-ccmail.army.mil.

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

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