Emerging Infectious Diseases
[Volume 5 No.1 / January - February 1999]

The JanuaryFebruary 1999 issue of the journal will contain the articles
listed below. Highlighted articles are currently available in electronic
format. To view a 63K image of the full cover click here.

International Update

   * Emerging Viral Diseases: An Australian Perspective, John S. Mackenzie

Perspectives

   * The Economic Impact of Staphylococcus aureus Infection in New York
     City Hospitals, R. J. Rubin, C. A. Harrington, A. Poon, K. Dietrich,
     J. A. Greene, and A. Moiduddin
   * Socioeconomic and Behavioral Factors Leading to Acquired Bacterial
     Resistance to Antibiotics in Developing Countries, Iruka N. Okeke,
     Adebayo Lamikanra, and Robert Edelman
   * Campylobacter jejuniAn Emerging Foodborne Pathogen, Sean F.
     Altekruse, Norman J. Stern, Patricia I. Fields, and David L. Swerdlow

Synopses

   * Comparative Genomics and Host Resistance Against Infectious Diseases,
     Salman T. Qureshi, Emil Skamene, and Danielle Malo
   * Cyclospora: An Enigma Worth Unraveling, Charles R. Sterling and Yns
     R. Ortega
   * Using Monoclonal Antibodies to Prevent Mucosal Transmission of
     Epidemic Infectious Diseases, Larry Zeitlin, Richard A. Cone, and
     Kevin J. Whaley

Research

   * HIV-1 Dual Infections and Recombinants Are an Integral Part of the HIV
     Epidemic in Brazil, Artur Ramos, Amilcar Tanuri, Mauro Schechter, Mark
     A. Rayfield, Dale J. Hu, Maulori C. Cabral, Claudiu I. Bandea, James
     Baggs, and Danuta Pieniazek
   * Genetic Diversity and Distribution of Peromyscus-Borne Hantaviruses in
     North America, Martha C. Monroe, Sergey P. Morzunov, Angela M.
     Johnson, Michael D. Bowen, Harvey Artsob, Terry Yates, C.J. Peters,
     Pierre E. Rollin, Thomas G. Ksiazek, and Stuart T. Nichol
   * Climatic and Environmental Patterns Associated with Hantavirus
     Pulmonary Syndrome Cases in the Four Corners Region, David M.
     Engelthaler, David G. Mosley, James E. Cheek, Craig E. Levy, Kenneth
     K. Komatsu, Paul Ettestad, Ted Davis, Dale T. Tanda, Lisa Miller, J.
     Wyatt Frampton, Richard Porter, and Ralph T. Bryan

The January-March 1999 issue of Emerging Infectious Diseases will be
highlighting hantavirus research. This theme issue will include the
following cluster of articles:

   * Long-Term Studies of Hantavirus Reservoir Population in the
     Southwestern United States: Rationale, Potential, and Methods, James
     N. Mills,Thomas G. Ksiazek, C.J. Peters, and James E. Childs
   * Long-Term Hantavirus Persistence in Rodent Populations in Central
     Arizona, Ken D. Abbott, Thomas G. Ksiazek, and James N. Mills
   * A Longitudinal Study of Sin Nombre Virus Prevalence in Rodents,
     Southeastern Arizona, Amy J. Kuenzi, Michael L. Morrison, Don E.
     Swann, Paul C. Hardy, and Giselle T. Downard
   * Long-Term field Studies of Small Mammals for Hantavirus Prevalence:
     Statistical Sensitivity for Detection of Spatial and Temporal Patterns
     in Rodent Population Densities, Cheryl A. Parmenter, Terry L. Yates,
     Robert R. Parmenter, and Jonathan L. Dunnum
   * Natural History of Sin Nombre Virus in Western Colorado, Charles H.
     Calisher, William Sweeney, James N. Mills, and Barry J. Beaty
   * Long-Term Studies of Hantavirus Reservoir Populations in the
     Southwestern United States: A Synthesis, James N. Mills, Thomas G.
     Ksiazek, C.J. Peters, and James E. Childs

Dispatches

   * Proficiency of Clinical Laboratories in and Near Monterrey, Mexico to
     Detect Vancomycin-Resistant Enterococci, L. Clifford McDonald, Luis R.
     Garza, and William R. Jarvis
   * Staphylococcus aureus with Reduced Susceptibility to Vancomycin
     Isolated from a Patient with Fatal Bacteremia, Sharon S. Rotun,
     Virginia McMath, Dianna J. Schoonmaker, Peggy S. Maupin, Fred
     C.Tenover, Bertha C. Hill, and David A. Ackman
   * Candida dubliniensis Candidemia in Patients with Chemotherapy-Induced
     Neutropenia and Bone Marrow Transplantation, Jacques F.G.M. Meis,
     Markus Ruhnke, Ben E. De Pauw, Frank C. Odds, Wolfgang Siegert, and
     Paul E. Verweij
   * Household Transmission of Streptococcus pneumoniae, Alberta, Canada,
     James D. Kellner, A. Patrick Gibb, Jenny Zhang, and Harvey R. Rabin
   * Preventing Zoonotic Diseases in Immunocompromised Persons: The Role of
     Physicians and Veterinarians, Sara Grant and Christopher W. Olsen
   * Mycoplasma penetrans Bacteremia Associated with Primary
     Antiphospholipid Syndrome, Antonio Yez, Lilia Cedillo, Olivier
     Neyrolles, Encarnacin Alonso, Marie-Christine Prvost, Jorge Rojas,
     Harold L. Watson, Alain Blanchard, and Gail H. Cassell
   * Infectious Diarrhea in Tourists Staying in a Resort Hotel , Rachel M.
     Hardie, Patrick G. Wall, Patricia Gott, Madhu Bardhan, and Christopher
     L.R. Bartlett

Commentary

   * Hantavirus Pulmonary SyndromeA Mid-Course Assessment, Robert E. Shope

Letters

   * Navigational Instinct: A Reason Not to Live Trap Deer Mice in
     Residences, Charles H. Calisher, William P. Sweeney, J. Jeffrey Root,
     and Barry J. Beaty
   * Bartonella quintana in Body Lice Collected from Homeless Persons in
     Russia, Elena B. Rydkina, Vronique Roux, Eugenia M. Gagua, Alexandre
     B. Predtechenski, Irina V. Tarasevich, and Didier Raoult
   * Tick-Transmitted Infections in Transvaal: Consider Rickettsia africae,
     Pierre-Edouard Fournier, Jean Beytout, and Didier Raoult
   * Extended-Spectrum Beta-Lactamase-Producing Salmonella Enteritidis in
     Trinidad and Tobago, B.P. Cherian, Nicole Singh, W. Charles, and P.
     Prabhakar
   * New emm (M Protein Gene) Sequences of Group A Streptococci Isolated
     from Malaysian Patients, Farida Jamal, Sabiha Pit, Richard Facklam,
     and Bernard Beall
   * Mutant Chemokine Receptor (CCR-5) and its Relevance to HIV Infection
     in Arabs, Iman H. Al-Sheikh, Amjad Rahi, and Mohammed Al-Khalifa

News and Notes

   * Workshop on the Potential Role of Infectious Agents in Cardiovascular
     Disease and Atherosclerosis
   * Workshop on Risks Associated with Transmissible Spongiform
     Encephalopathies (TSEs)
   * USDA-Report on Potential for International Travelers to Transmit
     Foreign Animal Diseases to U.S. Livestock or Poultry
   * Second Annual Conference on Vaccine Research, March 1999
   * International Scientific Forum on Home Hygiene

Commentary


Update

International Editors

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Emerging Viral Diseases: An Australian Perspective
John S. Mackenzie
The University of Queensland, Brisbane, Australia

[Fig]
Figure. Dr. Mackenzie is professor and head of the Department of Microbiology
and Parasitology, University of Queensland, Brisbane, Australia. His research 
interests include the epidemiology, ecology, and molecular biology of
mosquito-borne and emerging zoonotic viruses.          

With a few exceptions, emerging diseases in Australia are similar to those
in other industrialized countries (1-8). Most exceptions are either
vector-borne or zoonotic viral diseases, the major focus of this update.
The continuing emergence of antibiotic resistance is a worldwide problem.
In Australia, antibiotic resistance is being reported from a growing number
of organisms (9-15), often necessitating new case management practices and
guidelines (16,17). Also, like other countries, Australia has had a number
of foodborne (18-22) and waterborne (23-25) epidemics in the past few
years; the major difference is that the higher incidence of
enterohemorrhagic Escherichia coli linked to outbreaks of hemolytic uremic
syndrome is associated with serotype O111:H- rather than serotype O157:H-,
which is more common in other countries. Major waterborne epidemics or
contamination of reservoirs due to Cryptosporidium parvum have occurred
over the past 3 years in the Eastern States of Australia (23-25), with the
largest and most recent being a problem of contamination (in association
with Giardia lamblia) in the Sydney water supply between July and
September, 1998. However, despite this contamination, no increases in the
number of cases of diarrheal disease were reported, perhaps because the
inhabitants of Sydney were advised to boil the water before drinking it
(25).

The distribution and incidence of most of the recently described viral
diseases (e.g., human herpesviruses 68 and hepatitis C and E viruses) in
Australia are similar to those reported in other industrialized nations
(3). Recent data for hepatitis G in selected Australian populations also
support this contention (26).

Vector-Borne Viral Diseases

Australia has more than 70 arboviruses, but relatively few cause human
disease (27,28). The most common arbovirus causing human disease is Ross
River virus, an alphavirus, which causes an epidemic polyarthritis.
Although Ross River virus incidence has increased over the past decade, the
virus is not emerging; its increased incidence is probably due to increased
awareness and recognition by general practitioners, improved diagnostic
reagents, and increasing encroachment of human habitation into or near
wetlands and other areas conducive to mosquito breeding. The only
indigenous virus that can be called "emerging" is Barmah Forest virus, also
an alphavirus and also the cause of an epidemic polyarthritis-like disease.
Associated with human disease only since 1988 and increasing in incidence
as diagnostic reagents have become available and clinicians have become
aware of it, the virus has spread into new geographic areas, such as
Western Australia (29,30). The two mosquito-borne diseases of particular
concern, however, are "imports"Japanese encephalitis (JE) and dengue
viruses.

Japanese Encephalitis Virus

The first outbreak of JE in the Australian region occurred in Torres
Strait, northern Australia, in 1995 (31). Three cases (two of which were
fatal) were reported from Badu Island in central Torres Strait, 2,000 km
from the nearest focus of JE virus activity in Bali. Seroepidemiologic
studies showed that the virus was relatively widespread in the central and
northern islands with subclinical human cases on four islands and
seropositive pigs on nine islands. Ten virus isolates were obtained during
the outbreak: two from subclinical human infections (31) and eight from
Culex annulirostris mosquitoes collected on Badu Island (32). Sequencing
studies showed that these isolates (most closely related to a 1970 isolate
from Kuala Lumpur and a 1981 isolate from Bali) (33) were almost identical,
suggesting that the outbreak originated from a single source. These studies
also showed that all isolates had the same 11 nucleotide deletion in the 3'
untranslated region immediately downstream from the stop codon of the open
reading frame (34), which provided a signature for comparing any future
isolates. After the Badu Island outbreak, inactivated vaccine was offered
to all the inhabitants of the northern and central Torres Strait islands
(35). During 1996 and 1997, JE virus activity was detected through
seroconversions in sentinel pigs on Saibai Island, which is in the north
and only about 4 km from the Papua New Guinea coast (36) (J. Lee, D.
Phillips, J. Hanna, unpub. results).

Seroepidemiologic studies found that virus activity has been widespread in
Western Province, Papua New Guinea, since at least 1989, with
seropositivity rates of 21% at that time among the Daru-speaking people.
Results also showed that seropositivity rates were increasing in the Upper
Fly River area and that the virus was spreading geographically (37).
Indeed, recent results indicate that JE may have spread to Vanimo on the
northern coast by April 1998 and to parts of Milne Bay Province in eastern
Papua New Guinea (J. Lee, J. Wangi, P. Siba, G. Tau, unpub. results). The
first four clinical cases of JE in Papua New Guinea were observed in 1997
and 1998, with two deaths. All cases were from the Kiunga area of Western
Province (J. Oakley, S. Flew, C.A. Johansen, D. Phillips, R.A. Hall, J.S.
Mackenzie, unpub. results). Anecdotal evidence suggests that the cases may
have resulted in part from the large mosquito numbers associated with the
severe drought in 1997. The first JE virus strain isolated in Papua New
Guinea was obtained from Cx. annulirostris mosquitoes collected at Lake
Murray in Western Province in 1997. Sequence studies have shown that this
isolate was almost identical to the 1995 Torres Strait isolates, including
the acquisition of the 11 nucleotide deletion (C.A. Johansen, R. Paru, S.A.
Ritchie, A. Van den Hurk, M. Bockarie, J.S. Mackenzie, unpub. results).

A second outbreak of JE occurred in Torres Strait in March 1998, with one
human case from Badu Island and sentinel pigs seroconverting on a number of
islands. Shortly afterwards, the first human case on mainland Australia was
reported in a fisherman who acquired the infection near the mouth of the
Mitchell River in southwest Cape York (38). Extensive seroepidemiologic
investigations found no further human infections in communities on Cape
York, but domestic pigs had seroconverted both near Mitchell River and near
Bamega at the northerly tip of Cape York. Two virus isolations were
obtained from pig sera collected near Bamega, and one isolate was obtained
from a sentinel pig on Mabuiag Island in Torres Strait (J. Hanna, S. Hills,
D. Phillips, J. Lee, unpub. results). Mosquitoes were collected at a number
of sites on Cape York as well as on Badu Island. No viruses were isolated
from the Cape York mosquitoes, but approximately 44 isolates were obtained
from Badu Island43 from Cx. annulirostris mosquitoes and one from Aedes
vigilax mosquitoes (S.A. Ritchie, A. Van Den Hurk, C.A. Johansen, D.
Phillips, A. Pyke, J.S. Mackenzie, unpub. results). Nucleotide sequencing
studies have shown that the mosquito and pig isolates from Mabuiag and Cape
York were closely related to each other, as well as to the 1997 Papua New
Guinea Lake Murray and the 1995 Badu Island isolates, including all
isolates sharing the 11 base "signature" deletion, which indicated a single
virus source for the virus activity in Northern Australia and Papua New
Guinea. The focus of activity is probably in Papua New Guinea (C.A.
Johansen, A. Drew, D.A. Phillips, A. Pyke, J.S. Mackenzie, unpub. results).

JE virus activity in northern Australia began in 1998. Sentinel animal
sites are being established to investigate whether the virus has become
enzootic in the wildlife. Australia has both the mosquito vectors (Cx.
annulirostris) and vertebrate hosts (ardeid birds and pigs) for the virus
to become established. In addition, large areas of wetland habitats in Cape
York would be conducive to virus enzootic cycles and would increase the
potential for the virus to move south to more populous areas of Australia
(39).

Dengue Viruses

Despite a 120-year history, dengue does not appear to be endemic in
Australia. Several epidemics over the past decade have been initiated from
virus introduced by viremic travelers (27,28,40). Imported cases of dengue
in travelers are regularly diagnosed throughout Australia, with 30 to 60
cases reported annually, and growing in number. In most parts of Australia
where the local mosquitoes are unable to transmit dengue viruses, these
cases pose no risk, but in areas of north Queensland where Ae. aegypti is
common and travel between Australia and countries in the Asian-Pacific area
is frequent, local transmission and epidemic activity are major risks. The
potential for local transmission of dengue viruses is confined to an area
of Queensland corresponding to the geographic range of Ae. aegypti,
extending from the islands of Torres Strait in the north, to Mount Isa and
Boulia in the west, possibly to Roma in the south, and to Gladstone on the
east coast (41). Despite this relatively broad geographic range, epidemic
activity over the past 2 decades has been restricted from Torres Strait
south to Cairns, Townsville, and Charters Towers. The major epidemics over
the past 5 years have included a large outbreak of dengue type 2 in 1992 to
1993, principally in Townsville and Charters Towers with more than 2,000
cases, and with the first case of dengue hemorrhagic fever this century
(42,43); an outbreak of dengue type 2 in 1996 to 1997 on a number of Torres
Strait islands and Cairns, with more than 200 serologically confirmed cases
(44,45); and an outbreak of dengue type 3 in 1997 to 1998 largely
restricted to Cairns with 239 confirmed cases (46; S. Ritchie, S. Hills,
pers. comm.) and also a few cases of dengue type 2. This latter outbreak
also included a case of dengue hemorrhagic fever and the first case of
dengue encephalopathy in Australia (J. Hanna, unpub. obs.). Nucleotide
sequencing of dengue 2 isolates from Australia and a comparison with
isolates from elsewhere in the Asian-Pacific region indicated that the
1992-93 isolates were most closely related to an Indonesian virus, whereas
the 1996-97 isolates were most closely related to viruses originally
isolated in Burkina Faso. This latter finding is of interest because a
large outbreak of dengue type 2 occurred on a number of Pacific Islands
before and during the Australian outbreak, but the South Pacific viruses
were quite distinct from the Australian viruses (45).

After the 1992-93 outbreak, a Dengue Fever Management Plan was developed to
reduce the potential for epidemic activity from imported cases. The plan
has been extremely successful, and a number of imported cases have been
recognized early and were contained before they could cause an epidemic
(47,48). However, importation, either by continual movement of people
between Papua New Guinea and Torres Strait or by movement of people for
work, education, or recreation between Papua New Guinea and north
Queensland, will always be a major route of entry of the virus.

Vector importations occur frequently, with a number of reports for both Ae.
aegypti and Ae. albopictus (27), including two recent importations of Ae.
albopictus into Townsville in 1997 (49) and Cairns in 1998 (S. Ritchie,
pers. comm.).

Novel Zoonotic Viral Diseases

In the past 4 years, three newly described zoonotic viral diseases have
been reported from Australia; two of these diseases are caused by the
paramyxoviruses Menangle and Hendra (formerly equine morbillivirus), and
the third is caused by Australian bat lyssavirus.

Menangle Virus

An apparently new virus in the family Paramyxoviridae was isolated from
stillborn piglets with deformities at a large commercial piggery in New
South Wales (51). The farrowing rate in the piggery decreased from an
expected 82% to 60%; the number of live piglets declined in 27% of the
litters born; the proportion of mummified and stillborn piglets, some with
deformities, increased; and occasional abortions occurred. Virus was
isolated from lung, brain, and heart tissues of infected piglets, and shown
to be morphologically similar to viruses in the family Paramyxoviridae. No
disease was seen in postnatal animals of any age, but a high proportion of
sera (>90%) from animals of all ages contained high titers of neutralizing
antibodies against the virus. Tests performed at the Australian Animal
Health Laboratory confirmed that the virus, named Menangle virus, was
unrelated to other known paramyxoviruses, including viruses known to infect
pigs 51).

Serum from two workersone at the affected piggery and one at an associated
piggery that had received weaned pigs from the original piggeryhad high
titer, convalescent-phase neutralizing antibodies to the new virus. Both
workers had an influenzalike illness with rash during the pig outbreak, but
extensive serologic testing showed no evidence of any alternative cause;
therefore, the illness is believed to have been caused by the new virus
(52).

A large breeding colony of gray-headed and little red fruit bats roosted
within 200 m of the affected piggery. In a preliminary study, 42 of 125
serum samples collected from fruit bats in New South Wales and Queensland
had neutralizing antibodies to the new virus. In addition, antibodies were
found in sera collected in 1996, before the outbreak, and from a colony of
fruit bats 33 km from the piggery (51). Therefore, the fruit bats are
believed to be the primary source of virus causing the outbreak. Sera
collected from wild and domestic animals near the affected piggery were
seronegative.

The geographic range, normal host species, and genetic relationship of this
new virus to other paramyxoviruses remain unknown. Nevertheless, Menangle
appears to cause fatal disease and malformations in prenatal pigs and may
be associated with influenzalike illness in humans.

Hendra Virus

Hendra virus was first recognized in 1994 after an explosive outbreak of
severe, fatal respiratory disease affecting race horses and humans. Twenty
race horses in the Brisbane suburb of Hendra were infected; 13 died. The
trainer and stable hand were also infected, and the trainer died (53-55). A
second incident occurred in Mackay, a coastal town approximately 1,000 km
north of Brisbane. Two horses and a farmer died, the latter from severe
meningoencephalitis (56-58). The death of the horses and the initial
infection of the farmer occurred in 1994 and preceded the Brisbane
outbreak; the virus is believed to have then entered a latent phase for 1
year before reactivating to cause fatal encephalitis. No connection was
found between the Brisbane and Mackay incidents (56). Experimental studies
have shown that in horses and cats, after subcutaneous, intranasal, and
oral administration, the virus causes fatal pneumonia (59). In guinea pigs,
subcutaneous administration is also fatal, but the infection is more
generalized. Black fruit bats (Pteropus alecto) infected by subcutaneous,
intranasal, or oral routes contract a subclinical infection and generate an
antibody response (M. Williamson, unpub. results). Endothelial cell tropism
and formation of syncytia in blood vessels are common pathologic findings
in both overt and subclinical infections (B.T. Eaton, M. Williamson, unpub.
obs.).

Extensive seroepidemiologic studies found no evidence of Hendra virus among
horses, other farm animals, or more than 40 species of wildlife in
Queensland (56,60; P.L. Young, K. Halpin, H. Field, unpub. results).
However, working on the hypothesis that if outbreaks at two distant sites
were connected, the most likely wildlife source would be either birds or
fruit bats, P.L. Young and colleagues subsequently showed that fruit bats
(flying foxes), members of Megachiroptera, were the natural hosts on
serologic grounds and by virus isolation, with widespread evidence of
infection in four species of fruit bat: the black (Pteropus alecto),
grey-headed (P. poliocephalus), little red (P. scapulatus), and spectacled
(P. conspicillatus) fruit bats (61,62; P.L. Young et al., unpub. results).
Indeed the virus was antigenically and genetically indistinguishable from
the earlier horse and human isolates. Thus, it is now clearly established
that Hendra virus is a fruit bat virus and is widely distributed throughout
the range of pteropid bats in Australia, with serologic evidence of
infection in an average of 42% of wild-caught bats, the number of
seropositive animals varying with species (53% of 229 P. alecto, 47% of 195
P. poliocephalus, 12% of 115 P. scapulatus, and 41% of 99 P.
conspicillatus) and age, but not with geographic distribution (H. Field,
unpub. results). Serologic evidence of infection of fruit bats has also
been reported from Papua New Guinea. Two species of antibody-positive bats
(Dobsonia moluccense, P. neohibernicus) were identified from Madang on the
north coast of Papua New Guinea (K. Halpin, H. Field, J.S. Mackenzie, M.
Bockarie, P.L. Young, P.W. Selleck, unpub. results), and bats of four more
species (D. andersoni, P. capistratus, P. hypomelanus, and P.
admiralitatum) were identified in Port Moresby and New Britain (H. Field,
S. Hamilton, L. Hall, F. Bornacosso, K. Halpin, P.L. Young, unpub.
results).

Morphologic features (63) and preliminary sequencing data of the M and F
genes (64) suggested that Hendra virus was a member of the Paramyxoviridae,
although it had unusual surface projections of two distinct lengths, 15 nm
and 18 nm (63). The entire genome of the virus has now been sequenced
(65;66; L.F. Wang, B.T. Eaton and colleagues, unpub. results) and has
revealed a gene order and P gene organization characteristic of members of
the Paramyxovirus and Morbillivirus genera (65). Comparison of its deduced
amino acid sequences with those of other family members confirm that Hendra
virus is a member of the subfamily Paramyxovirinae, more closely related to
members of the Paramyxovirus and Morbillivirus genera than the Rubulavirus
genus. Overall, homology with other members of the subfamily is lower than
that observed within an individual genus (L.F. Wang, B.T. Eaton, unpub.
results). Hendra virus has several distinguishing features, including a
genome that is 15% larger than that of other members, with each of the six
transcription units containing a very long 3' untranslated region (L.F.
Wang, B.T. Eaton, unpub. results). The P/V/C gene has a fourth open reading
frame located between those of the C and V proteins and potentially
encoding a small basic protein similar to those of some members of the
Rhabdoviridae and Filoviridae; its long 3' untranslated region is a common
feature of the Filoviridae (65). The sequence of the N gene has also
recently been described (66), and like the P/V/C gene, has a 3'
untranslated region approximately tenfold longer than other members of the
Paramyxovirinae. Although the deduced amino acid sequence of the N protein
was slightly more homologous to members of the Morbillivirus genus than to
those of other Paramyxovirinae genera, the level of identity was much lower
than that observed within the Morbillivirus genus.

Three other findings differentiate Hendra from most other members of the
Paramyxoviridae: the wide host range (59), the cleavage site of the F
protein, and the orientation of the cell surface from which virus is
released (B.T. Eaton, W. Michalski, and M. Williamson, unpub. results). An
accumulating body of evidencesize of genome, comparative sequence
analyses, coding capacity for a small basic protein in the P gene,
morphologic features, host range, and various biologic properties, together
with the wildlife host of the virussuggests that the virus had been
misnamedit was neither an equine virus nor a morbillivirusalthough the
name was relevant when the virus was first isolated. It has therefore been
suggested that the virus be renamed Hendra and be classified in a new genus
within the Paramyxovirinae (59,65,66). A number of aspects of the ecology
of the virus remain to be determined. For instance, despite the obvious
ubiquity of the virus in the fruit bat population and the extremely close
relationship between bat caregivers and bats, there is no evidence of
seroconversions among caregivers, despite their close contact with up to
1,000 bats per year (50). Specimens of persons who have died of either
pneumonia or encephalitis of unknown etiology were virus-negative (C.
Allan, J.S. Mackenzie, L.A. Selvey, unpub. results). Furthermore, all human
infections appear to have been transmitted by horses. Thus, the virus
appears to have low transmissibility to humans; it also appears to be
linked with pregnancy: the index case of the Brisbane outbreak was a
pregnant mare, a pregnant mare was involved in the Mackay incident, both
incidents occurred during the birthing season of flying foxes, and virus
was first recovered from uterine fluid of a pregnant animal (3). Thus, a
number of questions remain about the ecologic, biologic, and pathologic
characteristics of Hendra virus: 1) the infectivity and virulence of the
virus and why it seems extremely difficult to transmit naturally between
and within some susceptible host species, 2) classification of the virus,
3) role of pregnancy to transmission of the virus, 4) role of prior
infection in horses in human infection, 5) method of transmission between
fruit bats and from fruit bats to horses, 6) tropism of the virus, and 7)
potential for producing a latent infection in humans.

Australian Bat Lyssavirus

Australia had been considered free of rabies and rabieslike viruses until
1996 when a new lyssavirus, closely related to classic rabies virus, was
first identified in a fixed brain specimen from a young black flying fox
(P. alecto), with unusual neurologic symptoms. Since this original
isolation, a further 42 isolates have been obtained from all four species
of fruit bat, with most isolates from black and little red (P. scapulatus)
flying foxes, and four isolates from an insectivorous bat
(Microchiroptera), the yellow-bellied sheathtail bat (Saccolaimus
flavicentris) (P. Daniels, R. Lunt, unpub. results). The isolates were from
as far apart as Melbourne and Darwin, but most were from Queensland.
Antibodies to rabies virus (REFIT assay) have been detected in 2.6% of 345
bat nonrandom samples submitted to the Australian Animal Health Laboratory
(P. Daniels, R. Lunt, unpub. results). Antibody has been detected in both
infected and apparently healthy bats, but whether this reflects the ability
of bats to recover from infection or become latently infected with the
virus is not known.

Analysis using nucleocapsid-specific monoclonal antibodies showed a strong
relationship between this new lyssavirus and serotype 1 rabies virus (67).
Indeed, rabies vaccine may elicit a protective immune response in humans,
indicating the antigenic similarity of Australian bat lyssavirus and
classic rabies virus (P.K. Murray, pers. comm. to the Lyssavirus Expert
Committee [68]). Phylogenetic studies of the N protein sequences indicated
that the Australian virus was genetically distinct from classic rabies
(genotype 1) and was, therefore, a previously unrecognized member of the
Lyssavirus genus and represented a new genotype, genotype 7 (67).

Two human infections have been attributed to Australian bat lyssavirus. One
fatal rabieslike infection was in a female bat caregiver from Rockhampton,
Queensland (69,70). An isolate of Australian bat lyssavirus obtained
post-mortem was antigenically and genetically similar to the virus from the
insectivorous yellow-bellied sheathtail bat (A.R. Gould, R. Lunt, P.
Daniels, unpub. results). A second death has recently been reported in
Queensland (J. Hann, J. Faoagali, G. Smith, I Serafin, J. Northill, unpub.
obs.). The infection was in a 27-year-old woman from Mackay, who died 2
years after a bat bite (by a large flying fox). Polymerase chain reaction
(PCR) testing of RNA extracted from saliva and nuchal biopsy proved vital
to the antemortem diagnosis. Immunofluorescence staining of postmortem
samples confirmed the diagnosis. Preliminary sequencing of the amplicon has
indicated that the virus is very similar to other lyssaviruses isolated
from flying foxes but clearly distinct from a virus isolate from a
yellow-bellied sheathtail bat (I. Serafin, G. Smith, J. Hanna, B. Harrower,
J. Northill, A. Westcott, unpub. obs.). More extensive sequencing of the
human and bat isolates is under way. Measures to prevent further human
infection have been implemented (68,71).

As with Hendra virus, a number of questions remain about the ecology and
biology of Australian bat lyssavirus. The finding of well,
antibody-positive bats, which suggests that bats can either recover from
infection or that they can be silently infected, needs to be investigated,
particularly with respect to infectivity and possible transmissibility.
More information is needed on the geographic and host range of the virus
ecology within bat communities and risk for transmission to terrestrial
animals.

These novel zoonotic viruses appear to have frugivorous bats as their
natural vertebrate hosts. While little is known of the viral fauna of fruit
bats (or indeed of most wildlife species) in Australia, the occurrence of
these three zoonotic viruses from bats over 3 years suggests that further
prospective studies of diseases of wildlife are warranted. Indeed, two
paramyxoviruslike viruses unrelated to any other known paramyxoviruses (K.
Halpin, P.L. Young, unpub. results) have recently been isolated from flying
foxes.

Conclusions

The vector-borne and zoonotic diseases in this editorial encompass three
patterns of emergence: known diseases increasing in incidence or geographic
range (e.g., dengue and JE virus, respectively); new infectious agents as
etiologic agents of known diseases (e.g., Australian bat lyssavirus as a
cause of a rabieslike illness); and new infectious agents causing
previously unrecognized diseases (e.g., Hendra virus). All three patterns
demonstrate the need (and international responsibility) for ongoing
surveillance and monitoring. In Australia, surveillance is the legislative
responsibility of the individual states and territories. A Communicable
Diseases Network Australia-New Zealand was established in 1989 to improve
the control of communicable diseases in Australia by coordinating national
surveillance activities and responses to outbreaks and by training public
health staff. In 1996, Australia developed a National Communicable Diseases
Surveillance Strategy to provide a national framework to monitor infectious
diseases and plan and prioritize interventions. Components of the strategy
include improvements to the national surveillance infrastructure, better
monitoring of diseases and surveillance data, and better response to
outbreaks. The strategy is being implemented and may provide the mechanism
for a national response to new and reemerging diseases.

Acknowledgments

	  I thank my many colleaguesBryan Eaton, Lin-Fa Wang, Peter Daniels, 
Ross Lunt, Jeffrey Hanna, Scott Ritchie, Peter Young, Kim Halpin, Greg Smith,
Debbie Phillips, Cheryl Johansen, Hume Field, Steve Flew, and John
Oakleywhose unpublished work I have been permitted to quote.

	  Address for correspondence: John S. Mackenzie, Department of Microbiology,
The University of Queensland, Brisbane, Qld 4072, Australia; fax:
61-7-3365-4620; e-mail: jmac@biosci.uq.edu.au.

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Perspectives


Perspectives

The Economic Impact of Staphylococcus aureus Infection in New York City
Hospitals

Robert J. Rubin, Catherine A. Harrington, Anna Poon, Kimberly Dietrich,
Jeremy A. Greene, and Adil Moiduddin
The Lewin Group, Fairfax, Virginia, USA

---------------------------------------------------------------------------
      We modeled estimates of the incidence, deaths, and direct
      medical costs of Staphylococcus aureus infections in
      hospitalized patients in the New York City metropolitan area in
      1995 by using hospital discharge data collected by the New York
      State Department of Health and standard sources for the costs
      of health care. We also examined the relative impact of
      methicillin-resistant versus -sensitive strains of S. aureus
      and of community-acquired versus nosocomial infections. S.
      aureus-associated hospitalizations resulted in approximately
      twice the length of stay, deaths, and medical costs of typical
      hospitalizations; methicillin-resistant and -sensitive
      infections had similar direct medical costs, but resistant
      infections caused more deaths (21% versus 8%).
      Community-acquired and nosocomial infections had similar death
      rates, but community-acquired infections appeared to have
      increased direct medical costs per patient ($35,300 versus
      $28,800). The results of our study indicate that reducing the
      incidence of methicillin-resistant and -sensitive nosocomial
      infections would reduce the societal costs of S. aureus
      infection.

Each year approximately two million hospitalizations result in nosocomial
infections (1). In a study of critically ill patients in a large teaching
hospital, illness attributable to nosocomial bacteremia increased intensive
care unit stay by 8 days, hospital stay by 14 days, and the death rate by
35% (2). An earlier study found that postoperative wound infections
increased hospital stay an average of 7.4 days (3).

Staphylococcus aureus was the most common cause of nosocomial infections
reported in the National Nosocomial Surveillance System between 1990 to
1996 (4). The leading cause of nosocomial pneumonia and surgical site
infections and the second leading cause of nosocomial bloodstream
infections (4), S. aureus also causes community-acquired infections (e.g.,
osteomyelitis and septic arthritis, skin infections, endocarditis, and
meningitis). More than 95% of patients with S. aureus infections worldwide
do not respond to first-line antibiotics such as penicillin or ampicillin
(5). Additionally, methicillin-resistant strains of S. aureus (MRSA) are
common. First reported in the 1960s (6), MRSA has become increasingly
prevalent since the 1980s (7,8) and is now endemic in many hospitals and
even epidemic in some, with resistance in approximately 30% of all S.
aureus infections (8).

Vancomycin is the only drug that can consistently treat MRSA. However,
beginning in 1989, hospitals have reported a rapid increase in vancomycin
resistance in enterococci (VRE) (9). Increased vancomycin use helps select
for VRE, and even a small increase in incidence of VRE infection could lead
to cross-resistance in S. aureus, since genes conferring vancomycin
resistance might be transferred from VRE (10). In 1996, Japan reported the
first case of S. aureus infection with intermediate resistance to
vancomycin (11). In 1997, two unrelated cases of S. aureus infection with
intermediate resistance to vancomycin were reported in the United States
(Michigan and New Jersey) (12). In both cases, patients had been treated
with multiple courses of vancomycin for repeated MRSA infections over the 6
months before the S. aureus infection with intermediate resistance to
vancomycin; additionally, VRE colonization had been diagnosed 7 months
before the S. aureus infection with intermediate resistance to vancomycin
in the New Jersey patient. The emergence of S. aureus infection with
intermediate resistance to vancomycin in the United States suggests that S.
aureus strains are constantly evolving and full resistance may develop
(12).

The various ways of controlling MRSA (13) are still being debated. The
elimination of endemic MRSA in hospitals is difficult and costly (14-17).
In general, infection control in the United States is less stringent than
in Canada and in some European countries, where identification of known
carriers, prospective surveillance of patients and hospital workers, and
use of nasal mupirocin have helped control drug-resistant S. aureus
infection rates (18).

Knowledge of the scope of the problem is helpful for hospital
administrators, insurers, and medical personnel who make policy decisions
on control measures to prevent the spread of MRSA and the emergence of
vancomycin-resistant S. aureus. However, the economic cost of S. aureus
infections is not well known. Many studies focus on the cost of
nonorganism-specific nosocomial infections (2,19,20). Moreover, the
reported cost of a nosocomial infection varies because of the wide range of
study populations, sites of infection, and methods used (16,21). The few
investigations into the cost of S. aureus infections have focused on the
differential cost of MRSA and MSSA infections (22,23) and are case studies
of outbreaks in single hospitals. Thus, they do not provide perspective on
the scope of the problem for a population over time.

We estimated the incidence, death rate, and cost of S. aureus infections
associated with hospitalization in the New York City metropolitan area in
1995. We selected this geographic region because of its high prevalence of
multidrug-resistant infections (24,25). We also compared the relative
contributions of nosocomial versus community-acquired infections and
methicillin-sensitive (MSSA) versus methicillin-resistant S. aureus.

The Study

Data

The 1995 Statewide Planning and Research Cooperative System (SPARCS)
Administratively Releasable File was the primary source of data (26).
SPARCS is a database of all hospital discharges in New York state, as
reported by hospitals to the State of New York Department of Health, and
the Administratively Releasable File contains discharge information on
hospital location, patient characteristics (age, sex, race, ethnicity), and
visit characteristics (primary diagnosis, secondary diagnoses, primary
procedure, secondary procedures, length of stay, total charges, patient
status, and disposition). We analyzed data for hospitals in the following
New York City metropolitan area counties: Bronx, Dutchess, Kings,
Manhattan, Nassau, Orange, Putnam, Queens, Richmond, Rockland, Suffolk,
Ulster, and Westchester. Data on infection incidence or resource use not in
SPARCS were obtained through a comprehensive literature search or estimated
by a clinical panel consisting of four physicians specializing in
infectious disease. Other sources for cost information were the 1995
Medicare Fee Schedule (27) for physician fees and the 1995 Red Book (28)
for outpatient pharmaceutical average wholesale prices.

Definitions

We identified patients with the most common types of hospital-associated S.
aureus infections: pneumonia, bacteremia, endocarditis, surgical site
infections, osteomyelitis, and septic arthritis (Table 1) from SPARCS,
which uses the International Classification of Diseases, Ninth Revision,
Clinical Modification (ICD-9-CM) diagnosis codes (29). With the exception
of ICD-9-CM code 482.4 (staphylococcal pneumonia) and 038.1 (staphylococcal
septicemia), these codes are not organism-specific.

 Table 1. ICD-9-CM codes used to identify infections in Statewide Planning and
 Research Cooperative System
 ------------------------------------------------------------------------------
 Type of          ICD-9-CM(sup a)
 Infection                                         Description
 ------------------------------------------------------------------------------
 Pneumonia        482.4            Pneumonia due to staphylococcus
 Bacteremia       038.1            Staphylococcal septicemia
                  790.7            Bacteremia
                  996.62           Infection and inflammatory reaction due to
                                   internal vascular device, implant, and
                                   graft
 Endocarditis     421.0            Acute and subacute bacterial endocarditis
                  996.61           Infection and inflammatory reaction due to
                                   cardiac device, implant, and graft
 Surgical site    998.3
 infection                         Disruption of operation wound
                  998.5            Postoperative infection
 Osteomyelitis    730.01-730.09    Acute osteomyelitis
                  730.10-730.19    Chronic osteomyelitis
 Septic arthritis 711.00-711.09    Pyogenic arthritis
                  996.66           Infection and inflammatory reaction due to
                                   internal joint problems
 ------------------------------------------------------------------------------
 (sup a)International classification of diseases, 9th Revision, Clinical
 Modification, 1995.

To identify S. aureus infections, we used the nonorganismspecific codes in
conjunction with an additional ICD-9-CM code to identify the bacterial
agent (i.e., 041.11 bacterial infection due to S. aureus in conditions
classified elsewhere and of unspecified site). Patients with multiple
infections were counted only once in the overall incidence rate. Their
primary or first occurrence of a diagnosis of interest was used.

Because source of infection (nosocomial versus community-acquired) is not
reported in SPARCS, we assumed that specific types of disease were either
nosocomial or community-acquired on the basis of the clinical panel opinion
(Table 2).

 Table 2. Definitions of nosocomial or community-acquired Staphylococcus
 aureus infections
 ------------------------------------------------------------------------------
 Type of
 Infection        Nosocomial                  Community-acquired
 ------------------------------------------------------------------------------
 Pneumonia        Secondary diagnosis(sup a)  Primary diagnosis(sup a)

 Bacteremia       Catheter- or surgery-       Noncatheter- and nonsurgical-
                  associated infections(sup b)associated infections

 Endocarditis     Prosthetic valve
                  infections                  Natural valve infections
 Surgical site
 infection (SSI)  All SSIs                    None
 Osteomyelitis    None                        All
 Septic arthritis Prosthetic joint
                  infections                  Natural joint infections
 ------------------------------------------------------------------------------
 (sup a)ICD-9-CM 482.4 as the primary diagnosis vs. 482.4 as one of several
 other diagnoses.
 (sup b)ICD-9-CM 996.62, or 038.1 associated with a surgical ICD procedure
 code, or 790.7 associated with a surgical ICD procedure code.

Modeling the Incidence Rate

ICD-9-CM code 041.11 (bacterial infection due to S. aureus) is not widely
used by reporting hospitals. Therefore, the incidence of S. aureus
infections based on the counts of 041.11 in SPARCS would underestimate the
number of cases. We estimated the incidence of S. aureus infections (except
pneumonia) as follows (Table 3): the total incidence of each type of
infection (e.g., endocarditis) in SPARCS was multiplied by the estimated
percentage attributable to S. aureus (determined by research or clinical
panel opinion) to give the total number of infections due to S. aureus. The
incidence of pneumonia was equated with the occurrence of the ICD-9-CM code
482.4 (staphylococcal pneumonia). For ICD-9-CM code 038.1 (staphylococcal
septicemia), we assumed that only 50% of infections were attributable to S.
aureus (with the remainder attributable to S. epidermidis) (30).

 Table 3. Incidence of Staphylococcus aureus infections from research or
 clinical panel
 -------------------------------------------------------------------------------

 Type of
 infection       Description                        S. aureus  %    Reference
 -------------------------------------------------------------------------------

 Bacteremia      Staphylococcal septicemia               50             30

                 Bacteremia                              15           31,32

                 Infection and inflammatory              16             4
                 reaction due to internal
                 vascular device, implant, and
                 graft
 Endocarditis    Acute and subacute bacterial                        Clinical
                 endocarditis                            30           panel
                 Infection and inflammatory              14             33
                 reaction due to cardiac device,
                 implant, and graft
 Surgical site   Disruption of operation wound           20              4
 infection       and postoperative infection
 Osteomyelitis   Acute and chronic osteomyelitis         50           34,35

 Septic                                                                 33
 arthritis       Pyogenic arthritis              11 (age <5 yr)

                                                 33 (age 5-18 yr)

                                                 55 (age >18 yr)

                 Infection and inflammatory              25             33
                 reaction due to internal joint
                 prosthesis
 -------------------------------------------------------------------------------

Modeling Death Rates

The death rates attributable to bacteremia, endocarditis, or
community-acquired pneumonia were assumed to be equal to the death rates
found when these infections were coded as a primary diagnosis in SPARCS and
041.11 was used as a secondary diagnosis. For nosocomial pneumonia,
however, we assumed that the attributable death rate was a percentage of
the actual death ratefor ventilator-associated pneumonia patients, death
rate is a function of both the severity of underlying disease and the
pneumonia. A series of matched-cohort studies have demonstrated that the
death rate attributable to ventilator-associated pneumonia is 0% to 57% of
the actual death rate (36-39). On the basis of this research and expert
panel judgment, nosocomial pneumonia in ventilator-associated pneumonia
patients (identified by ICD-9-CM V46.0 or V46.1) was assumed to have an
attributable death rate of 50% of the death rate found in SPARCS (30,40).
We assumed that the attributable death rate of nonventilator-associated
pneumonia was the death rate found in SPARCS. On the basis of the low death
rate found in SPARCS (approximately 2%), we assumed that no deaths were
attributable to osteomyelitis, septic arthritis, or surgical site
infections.

Modeling Direct Medical Costs

Direct medical costs were defined as hospital costs attributable to
S.aureus infection, professional fees incurred during hospitalization, and
costs of other infection-related medical services provided after discharge.
For each infection, total direct medical costs were calculated by
multiplying the average direct medical cost per patient by the incidence of
disease. Average hospital costs attributable to S. aureus per patient were
assumed to be equal to the average hospital charge from SPARCS when the
infection (e.g., pneumonia, bacteremia) was coded as a primary diagnosis
and 041.11 was used as a secondary diagnosis. Professional fees incurred
during hospitalization include physician visits and consultations for
evaluation and management, as well as radiologic, surgical, and
anesthesiologic costs. The average frequency of physician services per
patient was based on clinical panel estimates. Costs of these services were
based on 1995 Medicare Payment Rates for the Long Island, New York, area as
an intermediate point between New York City costs and those of outlying
counties. 

Costs of medical services after discharge include those of
postdischarge complications (e.g., abscesses, aneurysms) requiring
rehospitalization, home-based intravenous antibiotic therapy, and
outpatient oral antibiotic therapy. The average frequency of other medical
services provided per patient was based on clinical panel estimates. Costs
of hospital readmission were based on SPARCS charges; costs of home-based
intravenous therapy were based on literature estimates (40,41); and costs
of outpatient medications were based on average wholesale prices (25).

Modeling MRSA and MSSA S. aureus Infections

SPARCS does not identify MRSA or MSSA infections, and a code for infection
with a drug-resistant organism (V09) is rarely used. Therefore, we modeled
the comparative incidence, death rate, and cost of MRSA and MSSA. We
computed the incidence of MRSA and MSSA infections by using the estimate
that 29% of infections were due to MRSA (8). The clinical panel estimated
that 10% of community-acquired infections were due to MRSA (includes
infections acquired at long-term care facilities). The number of deaths for
MRSA and MSSA infections was estimated as follows: the clinical panel
estimated a risk ratio for death rates of MRSA and MSSA infections, and
deaths due to MRSA and MSSA infections were calculated from the estimated
risk ratio and the overall number of deaths due to S. aureus infection. We
estimated the direct medical cost per patient for MRSA and MSSA infections
as follows: differences in resource use for those with MRSA and MSSA
infections were identified by the clinical panel; these differences were
converted into differences in cost using a method similar to that described
above for modeling direct medical costs; and average costs for MRSA and
MSSA infections were calculated by using the average cost for an S. aureus
infection and the average difference in cost between MRSA and MSSA
infections.

Incidence, Death Rate, and Attributable Costs

S. aureus Infection

Of 1,351,362 nonobstetrical hospital discharges in SPARCS for New York City
in 1995, an estimated 13,550 (1.0%) were discharges of patients with S.
aureus infections (Table 4). The total direct medical costs incurred by
these patients was an estimated $435.5 millionaverage length of stay
nearly 20 days, direct cost of infection, $32,100 (Table 4). The number of
deaths was estimated at 1,400 (a 10% death rate). In contrast, the hospital
charges for the average hospital stay in SPARCS (for all nonobstetrical
discharges) were $13,263average length of stay 9 days, death rate 4.1%.
Thus, patients with S. aureus infection had approximately twice the cost,
length of stay, and death rate of a typical hospitalized patient.

 Table 4. Incidence, length of stay, costs, and death rates of Staphylococcus
 aureus infections by type of infection
 -------------------------------------------------------------------------------
                                           Direct Medical Costs
                                          -----------------------
                                Length of                             Deaths
                                   stay     Total   Per patient
 Type of infection   Incidence   (days)     ($M)       ($)        Total      %
 -------------------------------------------------------------------------------
 Pneumonia             3,600      22.2     128.3      35,400       890       25
 Bacteremia            4,400      18.0     137.0      31,300       470       11
 Endocarditis           550       25.9      25.8      47,200        40        7
 Surgical site                                                     ND(sup a)
 infection             2,300      13.6      50.5      21,800                 ND
 Osteomyelitis         2,000      23.9      68.4      35,000        ND       ND
 Septic arthritis       700       22.0      25.5      35,100        ND       ND
 Total or average      13,550     19.8     435.5      32,100     1,400       10
 -------------------------------------------------------------------------------
 (sup a)ND=no data.

Pneumonia and bacteremia represented most S. aureus infections and
accounted for 60% of the total direct medical costs and 97% of the number
of deaths. Endocarditis caused the longest stay (26 days) and highest
direct cost per patient ($47,200); surgical site infection caused the
shortest stay (14 days) and lowest direct cost per patient ($21,810).
Hospital charges were an average of $29,000 (90% of the total costs);
professional fees were an average of $2,300 (7%); and postdischarge costs
represented $800 (3%) (Table 5).

 Table 5. Direct medical chargesaverage hospital facility charges,
 professional fees, and postdischarge costs per case
 ----------------------------------------------------------------------
                                    Profes-     Post-
                       Hospital     sional    discharge
                        charges      fees       costs
 Type of infection      $    (%)     $   (%)    $   (%)     Total $
 ----------------------------------------------------------------------
 Pneumonia            33,400 (94)  2,000 (6)     ND(sup a)  35,400
 Bacteremia           27,900 (89)  2,100 (7)  1,300 (4)     31,300
 Endocarditis         41,700 (88)  4,300 (9)  1,200 (3)     47,200
 Surgical site
 infection            20,200 (93)  1,600 (7)     ND         21,800
 Osteomyelitis        30,000 (86)  3,200 (9)  1,800 (5)     35,000
 Septic arthritis     30,600 (87)  3,100 (9)  1,400 (4)     35,100
 Average              29,000 (90)  2,300 (7)    800 (3)     32,100
 ----------------------------------------------------------------------
 (sup a)ND=no data.

Nosocomial Infection

Nosocomial infections accounted for 46% of the total incidence of S. aureus
infections (6,300 infections), while community-acquired infections
accounted for 54% (7,250 infections) (Table 6). Community-acquired
pneumonia as a primary diagnosis accounted for 12% (1,500) of the total
cases. If community-acquired pneumonia is assumed to be mostly acquired in
long-term care facilities, most infections (58%) were acquired
institutionally. The cost attributable to community-acquired infections
($35,300) was approximately $6,500 higher on a per patient basis than the
cost attributable to nosocomial infections ($28,800). The death rates
attributable to community-acquired and nosocomial infections were similar
(10.5% and 10.1%).

 Table 6. Incidence, length of stay, costs, and deaths of Staphylococcus
 aureus infections by source of infection and degree of resistance
 --------------------------------------------------------------------------
                                 Direct medical cost
                                            Per        Deaths
                                 Total     patient
 Source of infection Incidence    ($M)      ($)     Total    (%)
 --------------------------------------------------------------------------
 Nosocomial            6,300     181.0     28,800     640    10
 Community             7,250     254.5      5,300     760    11
 Pneumonia             1,500      51.7     34,900     380    25
 Non-pneumonia         5,750     202.8     35,400     380     7
 MRSA(sup a)           2,780      94.5     34,000     590    21
 MSSA(sup b)          10,770     339.4     31,500     810     8
 --------------------------------------------------------------------------
 (sup a)Methicillin-resistant strains of S. aureus.
 (sup b)Methicillin-sensitive strains of S. aureus.

MRSA Infection

MRSA infections accounted for 21% (2,780) of the total S. aureus infection
incidence (29% of 6,300 nosocomial infections plus 10% of 7,250
community-acquired infections), while MSSA infections accounted for 79%
(10,770) of total infections (Table 6). The attributable cost of a patient
with MRSA was approximately $2,500 higher than the attributable cost of a
patient with MSSA ($34,000 versus $31,500). The higher cost of MRSA
infections is due to the higher cost of vancomycin, longer hospital stay,
and the cost of patient isolation procedures. For nosocomial infections
alone, the cost attributable to MRSA was approximately $3,700 higher on a
per patient basis than the cost attributable to MSSA infections ($31,400
versus $27,700). The death rate attributable to MRSA infections was
estimated at more than 2.5 times higher than that attributable to MSSA
infections (21% versus 8%).

Sensitivity Analyses
 
Although assumed to be underused in SPARCS, the ICD-9-CM code 041.11
represents a lower boundary of the total incidence of S. aureus infection.
In SPARCS, code 041.11 was used 7,366 times associated with a diagnosis of
interest (e.g., endocarditis) and represented a total cost of $236.4
million and a death rate (740 deaths) of 2% (Table 7). The upper
boundary of the total cost of S. aureus infections was calculated by
assuming that all hospital charges and deaths of patients with S. aureus
infections were attributable to the infection, representing a total cost of
$599 million and a death rate of 14.5% (1,960 deaths). We conducted
sensitivity analyses (varying the percentage of nosocomial MRSA; percentage
of patients isolated; difference in length of stay between patients with
MRSA and MSSA; attributable length of stay for patients with ventilator-
associated pneumonia; number of S. aureus catheter infections; and
percentage of S. aureuscaused bacteremia, septicemia, and postoperative
infections) and found that the difference in cost per case between MRSA
and MSSA infections was $1,700 to $5,100.

Table 7. Sensitivity analyses.
---------------------------------------------
                       Direct               
                       medical   
                       cost ($)   Deaths       
 ---------------------------------------------
Study results          435.5      1,400       
Lower boundary:  
  only 041.11 cases    236.4        740
Upper boundary:  
  all costs 
attributable           599.0      1,960 
---------------------------------------------

                                                                                                                
Comments

Our sensitivity analysis shows that we did not vastly over- or
underestimate the direct medical costs of S. aureus infections in New York
City. However, the study had several limitations; it was retrospective, and
the data sources were not validated by other means (e.g., interviews or
chart review). Therefore, coding errors in this database may affect the
results. The clinical panel estimates we used to model differences between
MRSA and MSSA may lead to some inaccuracy in those difference estimates.
Thus, our comparison of costs and deaths between MRSA and MSSA should be
viewed as a best approximation in the absence of case-control data or a
multivariate analysis of a well-defined patient population.

Our estimates of the cost per infection are generally higher than estimates
in studies reviewed by Jarvis (19). A major reason may be our focus on New
York City, where costs are much higher than in other areas of the United
States. In addition, earlier studies have used only hospital costs. Our
perspective was societal; therefore, we included physician fees and
outpatient costs, as well as hospital charges. Finally, most of these
studies focused on nonorganism-specific nosocomial infections; S. aureus
infections may have a higher average cost per episode than infections of
other organisms (42). 

On the other hand, we used conservative estimates for
certain costs. Medicare prices for professional services are generally
lower than commercial rates. Also, we did not account for postdischarge
complications that did not lead to hospitalization. Additionally, our
societal estimates did not include the cost of dying or lost productivity
associated with these illnesses. 

Despite its limitations, this study shows
that hospitalizations associated with S. aureus are serious and have high
medical costs and death rates. The average length of stay attributable to
S. aureus infection for these patients was very high, 20 daysnearly three
times the average for any other type of hospitalization (43). The increased
length of stay in turn leads to increases in direct medical costs, with an
average cost per case of $32,100 in 1995.

Treating an MRSA infection costs 6% to 10% more than treating an
MSSA infection $2,500 to $3,700 per case). This cost difference does not
reflect MRSA's greater virulence; rather, it reflects the increased cost
of vancomycin use and isolation procedures (if used). These estimates are
slightly lower than the difference of $5,104 found by Wakefield et al. (21),
perhaps because they focused on the cost of serious S. aureus infections,
while our analysis examined all hospitalizable S. aureus infections. 

Patients with MRSA infections have a high average attributable death rate of 
21% versus 8% for an MSSA infection. Some of the death rate difference may be
related to the underlying condition of patients who become infected with
MRSA (e.g., older patients, drug users, sicker patients, patients previously
exposed to other antibiotics) (44) and to the lack of effectiveness of
vancomycin itself in curing MRSA. (Vancomycin has a narrow therapeutic index
that allows little room for increasing blood concentration without incurring
substantial losses in tolerance [45]). 

Both MSSA and MRSA infections are associated with high costs and large numbers 
of deaths in the New York City metropolitan area. The costs and deaths 
associated with S. aureus infections may dramatically increase if the newly 
isolated S. aureus infection with intermediate resistance to vancomycin spreads 
or if VRSA emerges. For example, after penicillin-resistant S. aureus appeared 
in the 1950s, the death rate of bacteremia increased from 28% to 50% at the
University of Minnesota (Figure) (46). After methicillin was introduced,
the death rate decreased (47). Efforts should be directed toward reducing
the incidence of MRSA and MSSA nosocomial infections to reduce their
economic impact on society.

[Fig.]                              
Figure. Death rate of staphylococcal bacteremia over
time. (Data from 46, 47.)                               

Acknowledgment

      We thank the clinical panel: Drs. Donald Armstrong, Donald Low, James
Rahal, and Richard B. Roberts.

      This study was sponsored by the Public Health Research Institute in
conjunction with the Bacterial Antibiotic Resistance Group and The
Rockefeller University. Funders included The New York Community Trust, The
Horace W. Goldsmith Foundation, The United Hospital Fund of New York City,
The Texaco Foundation, and the U.S. Centers for Disease Control and
Prevention.       

     Dr. Rubin, president of The Lewin Group, a Washington-based health-care 
consulting company, is a clinical professor of medicine at Georgetown 
University School of Medicine. From 1981-1984, he was assistant surgeon 
general in the U.S. Public Health Service and assistant secretary for planning 
and evaluation, U.S. Department of Health and Human Services.       

     Address for correspondence: Robert J. Rubin, The Lewin Group, 9302 Lee 
Highway, Fairfax, VA 22031-1214, USA; fax: 703-218-5501.

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Perspectives

Socioeconomic and Behavioral Factors Leading to Acquired Bacterial
Resistance to Antibiotics in Developing Countries

Iruka N. Okeke,* Adebayo Lamikanra,* and Robert Edelman
*Obafemi Awolowo University, Ile-Ife, Nigeria; and University of Maryland
School of Medicine, Baltimore, Maryland, USA

---------------------------------------------------------------------------
      In developing countries, acquired bacterial resistance to
      antimicrobial agents is common in isolates from healthy persons
      and from persons with community-acquired infections. Complex
      socioeconomic and behavioral factors associated with antibiotic
      resistance, particularly regarding diarrheal and respiratory
      pathogens, in developing tropical countries, include misuse of
      antibiotics by health professionals, unskilled practitioners,
      and laypersons; poor drug quality; unhygienic conditions
      accounting for spread of resistant bacteria; and inadequate
      surveillance.

Acquired bacterial resistance is common in isolates from healthy persons
and from patients with community-acquired infections in developing
countries, where the need for antibiotics is driven by the high incidence
of infectious disease (1). Among isolates of diarrheal, respiratory, and
commensal enteric pathogens (2-5), resistance is increasing, particularly
to first-line, inexpensive, broad-spectrum antibiotics (Table 1).
Furthermore, introduction of newer drugs (e.g., fluoroquinones) has been
followed relatively quickly by the emergence and dissemination of resistant
strains (5). The selection and spread of resistant organisms in developing
countries, which can often be traced to complex socioeconomic and
behavioral antecedents, contribute to the escalating problem of antibiotic
resistance worldwide.

Table 1. Pathogens with a steadily increasing prevalence of acquired
antibiotic resistance in developing tropical countries
-----------------------------------------------------------------------------
Pathogen                Drug(s)               Country (years)     Ref.
-----------------------------------------------------------------------------
Shigella flexneri,   ampicillin,             Bangladesh           (6)
S. dysenteriae        tetracycline,           (1983-1990)          
                      sulfonamides (alone    Brazil (1988-1993)   (7)
                      of with trimethoprim), Rwanda (1983-1993)   (8)
                      nalidixic acid         Thailand (1981-1995) (5)
Vibrio cholerae      cotrimethoxazole,       Guinea-Bissau	      (9)
                     nalidixic acid,         (1987-1995)
                     ampicillin              India (1993-1995)    (10)
Salmonella typhi     ampicillin,             Bangladesh		(3)
                     chloramphenicol,        (1989-1993)          
                     cotrimethoxazole
Salmonella           cotrimethoxazole        Thailand (1981-1995) (5)
(nontyphoidal)                               
Enterotoxigenic      cotrimethoxazole        Thailand (1981-1995) (5)
 Escherichia coli                            
Campylobacter        fluoroquinolones        Thailand (1987-1995) (5)
Mycobacterium        isoniazid,              Kenya (1981-1990)    (11)
tuberculosis         streptomycin,           Morocco (1992-1994)  (12)
                     rifampicin (primary
                     resistance)
-----------------------------------------------------------------------------

Misuse of Antibiotics by Physicians in Clinical Practice

Antibiotic use provides selective pressure favoring resistant bacterial
strains; inappropriate use increases the risk for selection and
dissemination of antibiotic-resistant bacteria, which are placed at a
competitive advantage. Therefore, one would expect that drugs more commonly
affected by bacterial resistance in developing countries are generally
inexpensive and popular broad-spectrum agents (2-5,13). However, the
relationship between antibiotic use and the emergence and spread of
resistance is complex. Antibiotic use in clinical practice alone cannot
explain the high frequency of resistant organisms in developing countries
(14,15). Nevertheless, excessive clinical use (a form of misuse) is at
least partially responsible for the escalating rates of resistance,
especially in hospital settings, worldwide. The unnecessary prescription of
antibiotics seen in industrialized nations has also been documented in many
developing countries, particularly in cases of acute infantile diarrhea and
viral respiratory infections (16-22). Clinical misuse of antibiotics may be
more common among private practitioners than among public health
personnelprivate practitioners charge higher fees, the demand for
antibiotics seen in private patients is higher, and more drugs are
available in private clinics than in public hospitals (23-25).

Several strategies have been proposed for combating the inappropriate use
of antibiotics by clinicians (26). Antibiotic monitoring systems and
hospital formularies or antibiotic treatment protocols often reduce
antibiotic prescription rates (24,27). Adoption of a national essential
drug list can limit the antibiotics available to prescribers (28,29).
However, implementation of these strategies does not guarantee optimal
antibiotic use by clinicians in developing countries because the irregular
drug supply, availability of drugs from unofficial sources, and financial
constraints also affect antibiotic choices (30-32).

Continuing medical education changes the attitude of clinicians. Studies of
antibiotic misuse in Cuba and Pakistan (33,34) recommend continuing medical
education for health workers as the single most important tool for
combating antibiotic misuse. A study in Zambia has demonstrated the
efficacy of education in reducing antibiotic prescription rates (35).
However, education has not been successfully implemented in many developing
countries, where too often, governments and health workers cannot afford
the time and money required for continuing medical education (36).

Health workers in many developing countries have almost no access to
objective health information (24). Pharmaceutical company representatives
typically outnumber practitioners and often adversely influence their
prescription habits (37), as reflected by sales of nonessential drugs and
drug combinations (38). Drug labels and package inserts often fail to
provide accurate information (39), and in industrialized countries,
patients often pressure physicians to prescribe antibiotics (19).

Misuse of Antibiotics by Unskilled Practitioners

In many developing countries, well-trained health personnel are scarce and
cannot serve the entire population, especially in rural areas. Community
health workers and others with minimal training treat minor ailments (40).
The qualifications and training of community health workers, as well as the
quality of care they provide, vary from country to country. Unskilled
personnel are less aware of the deleterious effects of inappropriate
antibiotic use. For example, pharmacy technicians in Thailand prescribed
rifampicin for urethritis and tetracycline for young children (41).
Unqualified drug sellers offer alternative drugs when the prescribed drugs
are out of stock or refill prescriptions without consulting the prescriber
(42,43). In India, traditional healers often dispense antibiotics (44). A
high proportion of patients in some developing countries are treated by
untrained practitioners simultaneously with oral and injectable antibiotics
administered with contaminated needles and syringes (45-47) for
misdiagnosed noninfectious diseases (48).

Misuse of Antibiotics by the Public

In most developing countries, antibiotics can be purchased without
prescription, even when the practice is not legal. In many African, Asian,
and Latin American countries, antibiotics are readily available on demand
from hospitals, pharmacies, patent medicine stalls (drugstores), roadside
stalls, and hawkers (17,43,46,49-53). In rural Bangladesh, for example, 95%
of drugs consumed for 1 month by more than 2,000 study participants came
from local pharmacies; only 8% were prescribed by physicians (54). People
are encouraged to buy from unofficial distributors because drugs often are
not available in government hospitals (55). Drug vendors usually have
little or no knowledge of the required dosage regimen, indications, or
contraindications (43,45,55). In markets and public transport in West
African countries such as Cameroon (49) and Nigeria (Okeke and Lamikanra,
pers. obs.), the vendor (usually a medically untrained salesman) tries to
convince potential buyers to purchase the drug, even if they are not ill.

To save time and keep drug-hunting to a minimum, a patient may start at a
source more likely to stock the desired drug, forgoing the expertise of a
doctor. Unofficial sources are generally more accessible than official
sources. For example, in Nepal, retail drug outlets are four times as
numerous as government health posts and hospitals (46). Alternate sources
offer the option of purchasing small quantities of medicines, while
hospitals require purchase of the complete 5- or 7-day antibiotic regimen
(17,43,52). The purchase of small samples is exceedingly common,
particularly for most customers, who buy without prescription (52). These
subinhibitory antibiotic regimens predispose for selection of resistant
bacterial strains.

Antibiotic use in developing countries is underestimated. The quantity of
drugs distributed within a country is calculated under the assumption that
each person purchases a complete regimen (56). However, medication can be
purchased in small aliquots from roadside stalls, and distribution of
locally produced or counterfeit antibiotics is not recorded. The motives
for self-medication and antibiotic overuse by laypersons are similar to
those for clinical abuse by health professionals: to cut costs and act
expeditiously to treat confirmed or suspected bacterial infection (57). For
example, 50% to 80% of Bangladeshi patients infected with Shigella admitted
that they had taken at least one antibiotic in the 15 days before a
hospital visit (58), as had 18% to 70% of pediatric patients with acute
respiratory infection in two Chinese studies (20,59). The proportion of
patients who self-medicate is probably higher, because patients are often
reluctant to admit having taken antibiotics before visiting a hospital
(60).

Common cultural beliefs about antibiotics include the notions that there is
a pill for every symptom; antibiotics can heal many illnesses, including
dyspepsia and headaches; and injections are more powerful than pills. The
misuse of antibiotics frequently becomes integrated into the local culture
(62) (e.g., antibiotics are used to prevent diarrhea after eating suspected
contaminated foods or [by prostitutes] to prevent sexually transmitted
diseases [52,63]).

Another cause of antibiotic abuse and selection for resistant bacteria is
poor patient compliance. First, physician-patient interactions are often
inadequate. They can be short (e.g., a mean of 54 sec was recorded in a
Bangladeshi study [16]) and of poor quality (e.g., in Mexico, poor
patient-physician communication was partially responsible for the
noncompliance of patients with antibiotic regimens [21]). Second, because
patients often travel long distances and incur large expenses for medical
care, they are unlikely to return for follow-up visits. The reverse
situationthe prescriber visiting his patientis difficult logistically,
especially in rural Africa (64). In addition, the patient may be unable to
read medicine labels. Finally, because many drugs are expensive, indigent
patients purchase incomplete regimens whenever possible and discontinue
treatment when symptoms disappear but before the pathogen is eliminated
(52).

Poor Quality of Antibiotics

Lack of Quality Compliance and Monitoring

Besides the risk for therapeutic failure, degradation products or
adulterants in poor quality antibiotics can produce subinhibitory
concentrations in vivo, which increase the selection of resistant strains.
Drugs that do not comply with minimum standards are illegal in all
countries. However, the quality of many antibiotics and other drugs in
developing countries is often below standards in the formulary. In Nigeria
for example, substandard ampicillin, ampicillin/cloxacillin, tetracycline,
and oxytetracycline capsules have been detected (53,65-67). In many cases,
therapeutic failure is the only indication of substandard drugs. Analytic
laboratories to detect substandard drugs are uncommon, and when they exist,
health workers, distributors, and consumers are often unaware of them.

Degraded Antibiotics

The shelf lives of drugs developed and marketed in temperate countries are
determined by storage temperatures. During distribution in tropical
countries, conditions of transport and storage are poorly controlled, and
the drugs may be degraded. Ballereau et al. (68) recorded temperatures of
26C to 40C and 30% and 90% humidity in Guinea-Bissau during a 2-year
period (temperatures of greater than 25C can degrade antibiotics). Many
antibiotics, being heat- and moisture-labile, are particularly vulnerable.
Of seven drugs that lost 10% or more of their active constituents when
stored in pharmacies in Guinea-Bissau for 2 years, six were antimicrobial
drugs (68). Drug consignments are exposed to such adverse conditions during
shipment (69) or at tropical ports while they await lengthy port clearance.
Drugs are often handled by untrained workers who may store them
incorrectly. Hawkers and small traders in Nigeria frequently display large
glass jars containing different types of antibiotic capsules mixed
together, fully exposed to harsh sunlight and high ambient temperature and
humidity. In a Nigerian study of eight batches of tetracycline capsules,
only the batch obtained directly from the manufacturer was not excessively
degraded and contained active drug levels within formulary limits (Table 2)
(53,70). Studies conducted in Thailand and Nigeria demonstrated similar
degradation of chloroquine and amoxicillin (67,70).

Table 2. Source and quality of tetracycline capsules in a Nigerian 
suburban town (compiled with data from [53])
----------------------------------------------------------------- 
                          Tetracycline                      
                            content                      
                            (% of   Content        Bioavail-  
                            label   of ATC(sup a)   ability   
Sample    Source            claim)    (%)          (%)(sup b)
----------------------------------------------------------------- 
C1      Manufacturer(sup c) 105.9     None            100      
                                    detected             
C2      Hospital            107.5     5.3              63.4(sup d)
C3      Roadside stall      104.5     1.1              80.5(sup d)    
C4      Pharmacy             66.1     2.4              65.2(sup d)    
C5      Patent medicine      84.5     1.9              87.6(sup d)    
        stall                                         
C6      Roadside stall       67.8     1.5              Not    
                                                        tested   
C7      Patent medicine      89.6     1.8              Not     
        stall                                           tested    
-----------------------------------------------------------------
(sup a)Anhydrotetracycline, one of four tetracycline         
degradation products.                                  
(sup b)Measured from cumulative excretion of tetracycline   
in the urine of five volunteers.                       
(sup c)Reference standard obtained from the manufacturer.    
(sup d)Significantly different from C1 (p = 0.01,            
Wilcoxon signed rank test).                           

Expired Antibiotics

Some pharmacologically active drugs produced in countries have expired when
distributed in developing countriesthey were shipped at the end of the drugs'
shelf lives or their clearance and distribution after trancontinental shipment
were delayed. Expired drugs may receive new labels, be dumped without a
label change, or be donated rather than sold (71-73). Tax deductions and
the cost of liquidation are incentives for donating expired or near-
expired drugs. Effective enforcement of the World Health Organization
(WHO) guidelines on drug donations may curtail such practices (74)

Counterfeit Drugs

Some drugs sold in developing countries do not contain the concentration of
active substances stated on their labels, even at the time of manufacture.
These counterfeit drugs flourish, despite efforts of local regulatory
agencies to stop their production and distribution (75-77). Approximately
65% of the 751 instances of counterfeit pharmaceuticals reported to WHO or
to Interpol from 28 countries in the past 15 years were produced in
developing countries (77). Counterfeit drugs include products with little
or no active ingredients (e.g., in Nigeria, Indonesia, Brazil, Thailand,
Bangladesh, Malaysia, and Francophone African countries [39,76,78,79]) or
products for which excipients have been replaced by less expensive
alternatives (e.g., substitution of ethylene glycol for propylene glycol in
pediatric paracetamol formulations, which caused many deaths in Nigeria,
Argentina, Bangladesh, India, and Haiti [76,78]). Counterfeit drugs, like
other counterfeit materials, compete favorably in the markets of developing
countries. The analytic facilities available to law enforcement agencies
often cannot detect these drugs before they reach the patient.
Multinational pharmaceutical companies, which probably possess the best
analytic facilities for in-house quality assurance in developing countries,
try to detect counterfeit drugs to protect their income and reputation;
however, such efforts are directed primarily at counterfeits of these
companies' own products. Because of the profusion of generic drugs in
developing countries, a substantial proportion of counterfeit drugs go
undetected.

Adulterated Drugs

Herbal preparations in developing countries are often adulterated with
orthodox medicaments. For example, in one study, 24% of Chinese herbal
preparations marketed in Taiwan contained one or more of such adulterants
(80). Although the adulteration of such products with antibiotics has not
been reported, such practices may be common (81). A Nigerian traditional
healer, for example, admitted to 'augmenting' herbal preparations with
tetracycline from commercially available capsules (82).

Bioinequivalent Antibiotics and Biopharmaceutic Interactions

In the last 2 decades, the importance of bioavailability has been
underscored by the recognition that chemically equivalent generic drug
formulations do not always deliver the expected amount of drug to the
bloodstream. Slowly absorbed and acid-labile antibiotics are particularly
prone to bioinequivalence and consequent therapeutic failure. In addition,
poorly absorbed antibiotics remain in the gut to facilitate the selection
of resistant organisms. The few published studies from the developing world
have found bioinequivalence in antibiotic formulations, and the problem may
be widespread (Table 2) (53,83). Inexpensive generic antibiotics commonly
used in developing countries usually are not subject to bioavailability
studies.

The bioavailability of an antibiotic formulation is modulated by conditions
surrounding its administration; conditions unique to developing countries
are rarely investigated. Drug combinations used in the tropics but rarely
elsewhere may not be optimally absorbed. For example, coadministration of
chloroquine and ampicillin lowers the bioavailability of ampicillin (84). A
Nigerian meal lowered the biologic availability of orally administered
nitrofurantoin (85). Chewing of Khat, a popular Yemeni stimulant, adversely
affected the bioavailability of ampicillin and amoxicillin (86). By
contrast, the Ayurvedic preparation Trikatu enhanced the absorption of
several drugs (87). Whether traditional medicines with antimicrobial
properties enhance antibiotic resistance is unknown.

Dissemination of Resistant Organisms

Crowding and Unhygienic Conditions

Residents of developing countries often carry antibiotic-resistant fecal
commensal organisms (13,88). Visitors to developing countries passively
acquire antibiotic-resistant gut Escherichia coli, even if they are not
taking prophylactic antibiotics, which suggests that they encounter a
reservoir of antibiotic-resistant strains during travel (89). Apparently
healthy people in developing countries carry potentially pathogenic,
antibiotic-resistant organisms asymptomatically (90). Several factors, such
as urban migration with crowding and improper sewage disposal, encourage
the exchange of antibiotic-resistant organisms between people and the
exchange of resistance genes among bacteria, thereby increasing the
prevalence of resistant strains. In Nigeria, resistant E. coli isolates
from persons in an urban metropolis (Lagos) were significantly more likely
to be resistant to ampicillin and streptomycin (p < 0.05), and possibly
more resistant to sulphathiazole and tetracycline (p < 0.10), than isolates
from residents of nearby smaller towns and villages (Table 3) (91).
Moreover, strains isolated from Lagos were more likely to show resistance
to 4 to 6 of 7 antibiotics tested, whereas strains from rural areas were in
most cases resistant to only 0 to 3 antibiotics (91).

Table 3. Antibiotic resistance of Escherichia coli   
strains isolated from residents of an urban area     
(Lagos) or rural/suburban areas (southwest Nigeria)  
(from [91])                                          
-------------------------------------------------------- 
                  Percentage of resistant isolates             
                                             
Antimicrobial         Urban      Rural/suburban       
agent                (n = 30)       (n = 44)          
------------------------------------------------------- 
Ampicillin(sup a)      53             27             
Chloramphenicol        13             14             
Streptomycin(sup a)    63             32             
Sulphathiazole(sup b)  73             48            
Tetracycline(sup b)    87             64            
Trimethoprim           53             41           
-------------------------------------------------------- 
(sup a)Significant differences between the two groups at p 
< 0.05 (Chi-square test).                             
(sup b)Significant differences between the two groups at p 
< 0.10 (Chi-square test.                             

In 1991, 80% of residents of developing countries had no sanitary
facilities for sewage disposal (92). Pipe-borne water, often scarce
in developing countries, is not always potable. The development of
sanitation and other facilities is not always proportionate
to the rapid rises in urban populations (93,94). As urban
migration continues, overcrowding increases and hygiene declines,
increasing the probability of spread of antibiotic-resistant and 
commensal pathogens. Potable water, well-ventilated housing and 
proper waste disposal should reduce infections, the need for
antibiotics, and subsequent development of antibiotic resistance.

Because tropical conditions encourages the survival of bacteria, more
pathogens and commensals are found in tropical environments than in
temperate climates (95). The warm and humid tropical climate and the low
levels of health care, hygiene, and sanitation contribute to a relatively
high prevalence of infectious disease in developing countries.

Inadequate Hospital Infection Control Practices

Infection control practices in many hospitals in developing countries are
rudimentary and often compromised by economic shortfalls and opposing
traditional values (96). The resulting nidus of nosocomial pathogens and
resistant organisms may be disseminated to the outside community. Improper
disposal of hospital waste accentuates such spread. Untreated hospital
waste in Uganda was often dumped into public sewers or thrown into rubbish
heaps ravaged by scavengers (97).

Inadequate Surveillance

Susceptibility Testing and Surveillance

Information from routine susceptibility testing of bacterial isolates and
surveillance of antibiotic resistance, which provides information on
resistance trends, including emerging antibiotic resistance, is essential
for clinical practice and for rational policies against antibiotic
resistance. Bacterial infections are often treated after they become
life-threatening, which encourages empirical selection of broad-spectrum
antibiotics (98,99). The antibiotic susceptibility pattern of bacterial
isolates in much of the developing world is unknown, and little guides
empirical prescribing. Susceptibility testing cannot be done readily
because equipment, personnel, and consumables are scarce and expensive
(59,100). In most all infections, no clinical specimens are cultured. Where
available, community-based antibiotic surveillance data may be useful to
prescribers in the absence of patient-specific antibiotic-susceptibility
results. For example, Ringertz et al. (101) demonstrated that resistance
among respiratory pathogens was infrequent in parts of Ethiopia. This
information would help local Ethiopian prescribers to treat such infections
with inexpensive, broad-spectrum antibiotics.

National surveillance programs for antibiotic resistance, the norm in
industrialized nations, are less common and less elaborate in developing
countries (4). Current inferences about antibiotic resistance trends in
developing countries are based on a small number of reports, generated by a
handful of microbiology laboratories in urban areasdata not representative
of a country, because wide variations in antibiotic resistance patterns may
exist within countries (Table 3). Moreover, surveillance should be
conducted regularly and continuously because resistance rates can vary in
one region of a country over time (Table 1) (102).

Defective Antibiotic Susceptibility Assays

Well-standardized antibiotic susceptibility assays provide more reliable
results (103). However, standard bacterial strains with which to assay new
batches of antibiotics or antibiotic disks are not available in
laboratories in many developing countries. Delayed transportation and
breakdown of cold storage also affects the quality of antibiotics used as
diagnostic reagents. Degraded antibiotic powders and antibiotic disks used
for susceptibility testing lead to exaggerated estimates of bacterial
resistance levels. The frequent recovery of bacteria resistant to the
beta-lactams or tetracyclines in tropical countries could reflect, in part,
the temperature and moisture lability of test reagents. Laboratory
scientists in developing countries face difficulties in obtaining research
supplies, which often require them to improvise by, for example, using
injectable antibiotic formulations to measure MICs when standard antibiotic
powders are not available. The report that clinical microbiologists in
developing countries make their own disks from "local blotting papers"
(104) illustrates how improvisation can lead to inconsistent laboratory
results and unreliable data.

Economic and Political Factors

Lack of resources hampers implementation of most strategies against
antibiotic resistance. Statistics from the World Bank show that developing
countries spent $41 per person on health in 1990, compared with the $1,500
per person spent by industrialized countries. Disease prevalence as
measured by disability-adjusted life years and by communicable disease in
particular is much greater in developing than in industrialized countries
(93,105-107). As a result of such gross underfunding, the drug supply is
chronically inadequate or at best erratic in health facilities in many
countries, including Nigeria (43,105,106).

Armed conflicts have recently led to a breakdown in health services and
sanitation and rapid dissemination of resistant pathogens, particularly in
sub-Saharan Africa and Asia (108,109,110). During an outbreak of cholera
and bacillary dysentery in Rwandan refugees, resistance to multiple
first-line antibiotics in clinical isolates of Vibrio cholerae and Shigella
dysenteriae contributed to high death rates (109).

Even in developing countries not at war, political corruption and
mismanagement of funds, personnel, and development programs have created
large populations living in abject poverty and at high risk for infection
(111). Medical expenses, days lost from work, and transportation costs
account for substantial economic loss. The cost of medical treatment, even
subsidized treatment, is beyond the means of many patients. Poorly paid
health workers sometimes extort fees from patients (111). Thus, persons
with communicable diseases, unable to afford medical treatment, may infect
others. Poverty also interferes with patient compliance, which in turn
promotes the emergence of antibiotic resistance during short-term therapy
of acute infections and long-term therapy of chronic infections, such as
tuberculosis (111).

Combating the Problem of Antibiotic Resistance

The recommendations of WHO for ensuring proper drug use (79) can be adapted
to combat the escalation of community-acquired antibiotic resistance in
developing countries. The misuse of antibiotics by health-care
professionals, unskilled practitioners, and patients can be alleviated by
auditing antibiotics, limiting antibiotic choice, developing prescription
guidelines, and emphasizing continuing medical and public education. The
quality of antibiotics can be improved by emphasizing quality compliance
and monitoring antimicrobial drugs manufactured or dispensed. Such reforms
will help control substandard drugs that are degraded, counterfeit, or
bioinequivalent. Dissemination of resistant organisms in the community can
be impeded by improved public sanitation and hygienic practices and
upgraded hospital infection control. Finally, strategies to ensure that
these recommendations are adopted and implemented under difficult economic
and political conditions can be formulated. Antimicrobial resistance will
continue to escalate in developing countries unless corrective measures are
instituted.

Acknowledgments

      We thank the International Program in the Chemical Sciences, which
supports the current research in antibiotic resistance by I.N. Okeke and A.
Lamikanra. Ms. I.N. Okeke thanks the Fulbright Commission and the United
States Information Service for a fellowship and Dr. James B. Kaper for his
support.

     Ms. Okeke, lecturer in pharmaceutical microbiology at Obafemi Awolowo
University, Ile-Ife, Nigeria, and Ph.D. candidate, is a Fulbright Fellow at
the Center for Vaccine Development in Dr. James Kaper's laboratory at the
University of Maryland School of Medicine in Baltimore, working on
molecular genetics of enteric bacteria. Her interests include clinical
pharmacology and research on the pharmacology of antibiotics and natural
products. She is studying antibiotic resistance and virulence
characteristics of enteric organisms in Nigeria.

      Address for correspondence: Robert Edelman, Center for Vaccine
Development, 685 West Baltimore St., Room 480, Baltimore, MD 21201, USA;
fax: 410-706-6205; e-mail: redelman@umppa1.ab.umd.edu.

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Perspectives

Campylobacter jejuniAn Emerging Foodborne Pathogen

Sean F. Altekruse,* Norman J. Stern, Patricia I. Fields, and David L.
Swerdlow
*U.S. Food and Drug Administration, Blacksburg, Virginia, USA; U.S.
Department of Agriculture, Athens, Georgia, USA; and Centers for Disease
Control and Prevention, Atlanta, Georgia, USA

---------------------------------------------------------------------------
      Campylobacter jejuni is the most commonly reported bacterial
      cause of foodborne infection in the United States. Adding to
      the human and economic costs are chronic sequelae associated
      with C. jejuni infectionGuillian-Barr syndrome and reactive
      arthritis. In addition, an increasing proportion of human
      infections caused by C. jejuni are resistant to antimicrobial
      therapy. Mishandling of raw poultry and consumption of
      undercooked poultry are the major risk factors for human
      campylobacteriosis. Efforts to prevent human illness are needed
      throughout each link in the food chain.

History

Awareness of the public health implications of Campylobacter infections has
evolved over more than a century (1). In 1886, Escherich observed organisms
resembling campylobacters in stool samples of children with diarrhea. In
1913, McFaydean and Stockman identified campylobacters (called related
Vibrio) in fetal tissues of aborted sheep (1). In 1957, King described the
isolation of related Vibrio from blood samples of children with diarrhea,
and in 1972, clinical microbiologists in Belgium first isolated
campylobacters from stool samples of patients with diarrhea (1). The
development of selective growth media in the 1970s permitted more
laboratories to test stool specimens for Campylobacter. Soon Campylobacter
spp. were established as common human pathogens. Campylobacter jejuni
infections are now the leading cause of bacterial gastroenteritis reported
in the United States (2). In 1996, 46% of laboratory-confirmed cases of
bacterial gastroenteritis reported in the Centers for Disease Control and
Prevention/U.S. Department of Agriculture/Food and Drug Administration
Collaborating Sites Foodborne Disease Active Surveillance Network were
caused by Campylobacter species. Campylobacteriosis was followed in
prevalence by salmonellosis (28%), shigellosis (17%), and Escherichia coli
O157 infection (5%) (Figure 1).

[Fig]
Figure 1. Cases of Campylobacter and other foodborne infections by
month of specimen collection; Centers for Disease Control and
Prevention/U.S. Department of Agriculture/Food and Drug
Administration Collaborating Sites Foodborne Disease Active
Surveillance Network, 1996. 

Disease Prevalence

In the United States, an estimated 2.1 to 2.4 million cases of human
campylobacteriosis (illnesses ranging from loose stools to dysentery) occur
each year (2). Commonly reported symptoms of patients with laboratory-
confirmed infections (a small subset of all cases) include diarrhea,
fever, and abdominal cramping. In one study, approximately    
half of the patients with  laboratory-confirmed campylobacteriosis 
reported a history of bloody diarrhea (3). Less frequently, C.
jejuni infections produce bacteremia, septic arthritis, and other 
extraintestinal symptoms (4). The incidence of campylobacteriosis in 
HIV-infected patients is higher than in the general population. For 
example, in Los Angeles County between 1983 and 1987, the
reported incidence of campylobacteriosis in patients with AIDS was 519
cases per 100,000 population, 39 times higher than the rate in the general
population. (5). Common complications of campylobacteriosis in HIV-infected
patients are recurrent infection and infection with antimicrobial-resistant
strains 6). Deaths from C. jejuni infection are rare and occur primarily in
infants, the elderly, and patients with underlying illnesses (2).

Sequelae to Infection

Guillain-Barr syndrome (GBS), a demyelating disorder resulting in acute
neuromuscular paralysis, is a serious sequela of Campylobacter infection
(7). An estimated one case of GBS occurs for every 1,000 cases of
campylobacteriosis (7). Up to 40% of patients with the syndrome have
evidence of recent Campylobacter infection (7). Approximately 20% of
patients with GBS are left with some disability, and approximately 5% die
despite advances in respiratory care. Campylobacteriosis is also associated
with Reiter syndrome, a reactive arthropathy. In approximately 1% of
patients with campylobacteriosis, the sterile postinfection process occurs
7 to 10 days after onset of diarrhea (8). Multiple joints can be affected,
particularly the knee joint. Pain and incapacitation can last for months or
become chronic.

Both GBS and Reiter syndrome are thought to be autoimmune responses
stimulated by infection. Many patients with Reiter syndrome carry the HLA
B27 antigenic marker (8). The pathogenesis of GBS (9) and Reiter syndrome
is not completely understood.

Treatment of C. jejuni Infections

Supportive measures, particularly fluid and electrolyte replacement, are
the principal therapies for most patients with campylobacteriosis (10).
Severely dehydrated patients should receive rapid volume expansion with
intravenous fluids. For most other patients, oral rehydration is indicated.
Although Campylobacter infections are usually self limiting, antibiotic
therapy may be prudent for patients who have high fever, bloody diarrhea,
or more than eight stools in 24 hours; immunosuppressed patients, patients
with bloodstream infections, and those whose symptoms worsen or persist for
more than 1 week from the time of diagnosis. When indicated, antimicrobial
therapy soon after the onset of symptoms can reduce the median duration of
illness from approximately 10 days to 5 days. When treatment is delayed
(e.g., until C. jejuni infection is confirmed by a medical laboratory),
therapy may not be successful (10). Ease of administration, lack of serious
toxicity, and high degree of efficacy make erythromycin the drug of choice
for C. jejuni infection; however, other antimicrobial agents, particularly
the quinolones and newer macrolides including azithromycin, are also used.

Antimicrobial Resistance

The increasing rate of human infections caused by antimicrobial-resistant
strains of C. jejuni makes clinical management of cases of
campylobacteriosis more difficult (11,12). Antimicrobial resistance can
prolong illness and compromise treatment of patients with bacteremia. The
rate of antimicrobial-resistant enteric infections is highest in the
developing world, where the use of antimicrobial drugs in humans and
animals is relatively unrestricted. A 1994 study found that most clinical
isolates of C. jejuni from U.S. troops in Thailand were resistant to
ciprofloxacin. Additionally, nearly one third of isolates from U.S. troops
located in Hat Yai were resistant to azithromycin (11). In the
industrialized world, the emergence of fluoroquinolone-resistant strains of
C. jejuni illustrates the need for prudent antimicrobial use in food-animal
production (12). Experimental evidence demonstrates that
fluoroquinolone-susceptible C. jejuni readily become drug-resistant in
chickens when these drugs are administered (13). After flouroquinolone use
in poultry was approved in Europe, resistant C. jejuni strains emerged
rapidly in humans during the early 1990s (12). Similarly, within 2 years of
the 1995 approval of fluoroquinolone use for poultry in the United States,
the number of domestically acquired human cases of ciprofloxacin-resistant
campylobacteriosis doubled in Minnesota (14). In a 1997 study conducted in
Minnesota, 12 (20%) of 60 C. jejuni isolates obtained from chicken
purchased in grocery stores were ciprofloxacin-resistant (14).

Pathogenesis

The pathogenesis of C. jejuni infection involves both host- and
pathogen-specific factors. The health and age of the host (2) and C.
jejuni-specific humoral immunity from previous exposure (15) influence
clinical outcome after infection. In a volunteer study, C. jejuni infection
occurred after ingestion of as few as 800 organisms (16). Rates of
infection increased with the ingested dose. Rates of illness appeared to
increase when inocula were ingested in a suspension buffered to reduce
gastric acidity (16).

[Fig]
Figure 2. Scanning electron microscope image of Campylobacter    
jejuni, illustrating its corkscrew appearance and bipolar flagella.     
Source: Virginia-Maryland Regional College of Veterinary Medicine,      
Blacksburg, Virginia.                     

Many pathogen-specific virulence determinants may contribute to the
pathogenesis of C. jejuni infection, but none has a proven role (17).
Suspected determinants of pathogenicity include chemotaxis,
motility, and flagella, which are required for attachment and
colonization of the gut epithelium (Figure 2) (17). Once colonization
occurs, other possible virulence determinants are iron acquisition,
host cell invasion, toxin production, inflammation and active secretion, 
and epithelial disruption with leakage of serosal fluid (17).

Survival in the Environment

Survival of C. jejuni outside the gut is poor, and replication does not
occur readily (17). C. jejuni grows best at 37C to 42C (18), the
approximate body temperature of the chicken (41C to 42C). C. jejuni grows
best in a low oxygen or microaerophilic environment, such as an atmosphere
of 5% O(sub 2), 10% CO(sub 2), and 85% N(sub 2). The organism is sensitive 
to freezing, drying, acidic conditions (pH < 5.0), and salinity.

Sample Collection and Transport

If possible, stool specimens should be chilled (not frozen) and submitted
to a laboratory within 24 hours of collection. Storing specimens in deep,
airtight containers minimizes exposure to oxygen and desiccation. If a
specimen cannot be processed within 24 hours or is likely to contain small
numbers of organisms, a rectal swab placed in a specimen transport medium
(e.g., Cary-Blair) should be used. Individual laboratories can provide
guidance on specimen handling procedures (18).

Numerous procedures are available for recovering C. jejuni from clinical
specimens (18). Direct plating is cost-effective for testing large numbers
of specimens; however, testing sensitivity may be reduced. Preenrichment
(raising the temperature from 36C to 42C over several hours), filtration,
or both are used in some laboratories to improve recovery of stressed C.
jejuni organisms from specimens (e.g., stored foods or swabs exposed to
oxygen) (19). Isolation can be facilitated by using selective media
containing antimicrobial agents, oxygen quenching agents, or a low oxygen
atmosphere, thus decreasing the number of colonies that must be screened
(18,19).

Subtyping of Isolates

No standard subtyping technique has been established for C. jejuni. Soon
after the organism was described, two serologic methods were developed, the
heat-stable or somatic O antigen (20) and the heat-labile antigen schemes
(21). These typing schemes are labor intensive, and their use is limited
almost exclusively to reference laboratories. Many different DNA-based
subtyping schemes have been developed, including pulsed-field gel
electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD)
analysis (22). Various typing schemes have been developed on the basis of
the sequence of flaA, encoding flagellin (23); however, recent evidence
suggests that this locus may not be representative of the entire genome
(24).

Transmission to Humans

Most cases of human campylobacteriosis are sporadic. Outbreaks have
different epidemiologic characteristics from sporadic infections (2). Many
outbreaks occur during the spring and autumn (2). Consumption of raw milk
was implicated as the source of infection in 30 of the 80 outbreaks of
human campylobacteriosis reported to CDC between 1973 and 1992. Outbreaks
caused by drinking raw milk often involve farm visits (e.g., school field
trips) during the temperate seasons. In contrast, sporadic Campylobacter
isolates peak during the summer months (Figure 1). A series of case-control
studies identified some risk factors for sporadic campylobacteriosis,
particularly handling raw poultry (25,26) and eating undercooked poultry
(27-31) (Table). Other risk factors accounting for a smaller proportion of
sporadic illnesses include drinking untreated water (29); traveling abroad
(25); eating barbequed pork (28) or sausage (27); drinking raw milk (29,32)
or milk from bird-pecked bottles (33); and contact with dogs (27) and cats
(29,31), particularly juvenile pets or pets with diarrhea (25,34).
Person-to-person transmission is uncommon (25,32). Overlap is reported
between serotypes of C. jejuni found in humans, poultry, and cattle,
indicating that foods of animal origin may play a major role in
transmitting C. jejuni to humans (35).

In the United States, infants have the highest age-specific Campylobacter
isolation rate, approximately 14 per 100,000 person years. As children get
older, isolation rates decline to approximately 4 per 100,000 person years
for young adolescents. A notable feature of the epidemiology of human
campylobacteriosis is the high isolation rate among young adults,
approximately 8 per 100,000 person years. Among middle-aged and older
adults, the isolation rate is < 3 per 100,000 person years (2). The peak
isolation rate in neonates and infants is attributed in part to
susceptibility on first exposure and to the low threshold for seeking
medical care for infants (2). The high rate of infection during early
adulthood, which is pronounced among men, is thought to reflect poor
food-handling practices in a population that, until recently, relied on
others to prepare meals (2).

Reservoirs

The ecology of C. jejuni involves wildlife reservoirs, particularly wild
birds. Species that carry C. jejuni include migratory birdscranes, ducks,
geese (36), and seagulls (37). The organism is also found in other wild and
domestic bird species, as well as in rodents (38). Insects can carry the
organism on their exoskeleton (39).

Table. Epidemiologic studies of laboratory-confirmed cases of sporadic
campylobacteriosis
--------------------------------------------------------------------------
                                              Foods
    Number                                    associated
                                              with         Animal
Cases Controls Date  Population  Location     illness      contacts  Ref.
--------------------------------------------------------------------------
52    103      1989- Residents   Norway       Poultry,     Dogs      27
               1990   of three                 sausage
                      counties
218   526      1982- HMO         Washington   Undercooked  Animals   30,
               1983   patients    State        chicken       with     34
                                                            diarrhea
29    42       1990  Residents   England      Bottled                33
                      of                       milk(sup a)
                      Manchester
45    45       1983- University  Georgia      Chicken      Cats      31
               1984   students
53    106      1982- Rural       Iowa         Raw milk               32
               1983   children
40    80       1981  Residents   Colorado     Untreated    Cats      29
                      of Denver,               water,
                      Ft. Collins              raw milk,
                                               undercooked
                                               chicken
54    54       1982  Residents   Netherlands  Chicken,               28
                      of                       pork,
                      Rotterdam                barbequed
                                               foods
10    15       1982  Residents   Colorado     Preparing              26
                      of                       chicken
                      Larimer
                      County
55    14       1980  Residents   Sweden       Preparing    Kitten,    25
                      of                       chicken      dog
                      Gteborg                              with
                                                            diarrhea
--------------------------------------------------------------------------
(sup a)Bottle tops pecked by wild birds.

The intestines of poultry are easily colonized with C. jejuni. Day-old
chicks can be colonized with as few as 35 organisms (40). Most chickens in
commercial operations are colonized by 4 weeks (41,42). Vertical
transmission (i.e., from breeder flocks to progeny) has been suggested in
one study but is not widely accepted (43). Reservoirs in the poultry
environment include beetles (39), unchlorinated drinking water (44), and
farm workers (41,42,45). Feeds are an unlikely source of campylobacters
since they are dry and campylobacters are sensitive to drying.

C. jejuni is a commensal organism of the intestinal tract of cattle (46).
Young animals are more often colonized than older animals, and feedlot
cattle are more likely than grazing animals to carry campylobacters (47).
In one study, colonization of dairy herds was associated with drinking
unchlorinated water (48).

Campylobacters are found in natural water sources throughout the year. The
presence of campylobacters is not clearly correlated with indicator
organisms for fecal contamination (e.g., E. coli)(49). In temperate
regions, organism recovery rates are highest during the cold season
(49,50). Survival in cold water is important in the life cycle of
campylobacters. In one study, serotypes found in water were similar to
those found in humans (50). When stressed, campylobacters enter a "viable
but nonculturable state," characterized by uptake of amino acids and
maintenance of an intact outer membrane but inability to grow on selective
media; such organisms, however, can be transmitted to animals (51).
Additionally, unchlorinated drinking water can introduce campylobacters
into the farm environment (44,48).

Campylobacter in the Food Supply

C. jejuni is found in many foods of animal origin. Surveys of raw
agricultural products support epidemiologic evidence implicating poultry,
meat, and raw milk as sources of human infection. Most retail chicken is
contaminated with C. jejuni; one study reported an isolation rate of 98%
for retail chicken meat (52). C. jejuni counts often exceed 10(sup 3) per 100 g.
Skin and giblets have particularly high levels of contamination. In one
study, 12% of raw milk samples from dairy farms in eastern Tennessee were
contaminated with C. jejuni (53). Raw milk is presumed to be contaminated
by bovine feces; however, direct contamination of milk as a consequence of
mastitis also occurs (54). Campylobacters are also found in red meat. In
one study, C. jejuni was present in 5% of raw ground beef and in 40% of
veal specimens (55).

Control of Campylobacter Infection

On the Farm

Control of Campylobacter contamination on the farm may reduce contamination
of carcasses, poultry, and red meat products at the retail level (27).
Epidemiologic studies indicate that strict hygiene reduces intestinal
carriage in food-producing animals (41,42,45). In field studies, poultry
flocks that drank chlorinated water had lower intestinal colonization rates
than poultry that drank unchlorinated water (42,44). Experimentally,
treatment of chicks with commensal bacteria (56) and immunization of older
birds (57) reduced C. jejuni colonization. Because intestinal colonization
with campylobacters readily occurs in poultry flocks, even strict measures
may not eliminate intestinal carriage by food-producing animals (39,41).

At Processing

Slaughter and processing provide opportunities for reducing C. jejuni
counts on food-animal carcasses. Bacterial counts on carcasses can increase
during slaughter and processing steps. In one study, up to a 1,000-fold
increase in bacterial counts on carcasses was reported during
transportation to slaughter (58). In studies of chickens (59) and turkeys
(60) at slaughter, bacterial counts increased by approximately 10- to
100-fold during defeathering and reached the highest level after
evisceration. However, bacterial counts on carcasses decline during other
slaughter and processing steps. In one study, forced-air chilling of swine
carcasses caused a 100-fold reduction in carcass contamination (61). In
Texas turkey plants, scalding reduced carcass counts to near or below
detectable levels (60). Adding sodium chloride or trisodium phosphate to
the chiller water in the presence of an electrical current reduced C.
jejuni contamination of chiller water by 2 log(sub 10) units (62). In a 
slaughter plant in England, use of chlorinated sprays and maintenance of 
clean working surfaces resulted in a 10- to 100-fold decrease in carcass
contamination (63). In another study, lactic acid spraying of swine
carcasses reduced counts by at least 50% to often undetectable levels (64).
A radiation dose of 2.5 KGy reduced C. jejuni levels on retail poultry by
10 log(sub 10) units (65).

Conclusions

C. jejuni, first identified as a human diarrheal pathogen in 1973, is the
most frequently diagnosed bacterial cause of human gastroenteritis in the
United States. Sequelae including GBS and reactive arthritis are
increasingly recognized, adding to the human and economic cost of illness
from human campylobacteriosis. The emergence of fluoroquinolone-resistant
infections in Europe and the United States, temporally associated with the
approval of fluoroquinolone use in veterinary medicine, is also a public
health concern. The consumption of undercooked poultry and
cross-contamination of other foods with drippings from raw poultry are
leading risk factors for human campylobacteriosis. Reinforcing hygienic
practices at each link in the food chainfrom producer to consumeris
critical in preventing the disease.

      Dr. Altekruse is a Public Health Service Epidemiology Fellow with the
Food and Drug Administration, Center for Veterinary Medicine. His current
research interest is antimicrobial-resistant foodborne pathogens.

      Address for correspondence: Sean Altekruse, Virginia-Maryland
Regional College of Veterinary Medicine, Duckpond Road, Blacksburg, VA,
24060, USA; fax: 540-231-7367; e-mail: saltekru@vt.edu.

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Synopses


Synopses

Comparative Genomics and Host Resistance against Infectious Diseases

Salman T. Qureshi,* Emil Skamene* and Danielle Malo*
*McGill University, Montral, Canada; Montral General Hospital,
Montral,Canada

---------------------------------------------------------------------------
      The large size and complexity of the human genome have limited
      the identification and functional characterization of
      components of the innate immune system that play a critical
      role in front-line defense against invading microorganisms.
      However, advances in genome analysis (including the
      development of comprehensive sets of informative genetic
      markers, improved physical mapping methods, and novel
      techniques for transcript identification) have reduced the
      obstacles to discovery of novel host resistance genes. Study
      of the genomic organization and content of widely divergent
      vertebrate species has shown a remarkable degree of
      evolutionary conservation and enables meaningful cross-species
      comparison and analysis of newly discovered genes. Application
      of comparative genomics to host resistance will rapidly expand
      our understanding of human immune defense by facilitating the
      translation of knowledge acquired through the study of model
      organisms. We review the rationale and resources for
      comparative genomic analysis and describe three examples of
      host resistance genes successfully identified by this
      approach.

Two major elements underlie a thorough understanding of the pathogenesis of
virtually any infectious disease: identification and characterization of
the virulence factors and in vivo survival mechanisms of the invading
microorganism (e.g., surface attachment factors, exotoxins, or enzymes that
disrupt cellular homeostasis [1]) and understanding of the components of
the host response that lead to elimination of the invading pathogen and
resolution of disease. (These include both nonspecific [or innate] immune
defense mechanisms, such as the complement cascade, and adaptive elements,
such as clonally derived lymphocytes capable of eliminating specific
targets [2]). The traditional approach to human infectious diseases has
been to focus research on the study of important pathogens. The outcome of
investigation of relevant bacteria, viruses, fungi, and parasites has led
to the production of protective vaccines, antimicrobial agents, and
effective strategies for control and elimination of disease outbreaks. A
principal advantage of microbiologic research is the relative ease with
which the organisms may be obtained, manipulated, and analyzed in the
laboratory. Because microbial genomes are smaller, complete cloning and DNA
sequencing of several microorganisms have been achieved and have paved the
way for comprehensive study of gene expression and genome organization
(3,4). In contrast are relatively limited advances in our understanding of
the molecular basis of host defense. The study of host immune defense in
humans is inherently complex; obstacles to greater understanding include
limited opportunities for controlled observation and experimental
manipulation, a large genome, and until recently, a lack of molecular
techniques capable of facilitating genomewide analysis.

Genetic Analysis

One of the principal aims of the study of host response to infectious
diseases is to uncover novel components of the host immune system critical
to robust host defense. Identification of these components at a molecular
level is the first step in understanding how the host deals with an
infectious challenge and lays a foundation upon which rational therapies
that augment host resistance may someday be designed. Despite this promise,
the interaction between host and pathogen that leads to infection is
multidimensional, dynamic, and exceedingly complex. From a genomic
perspective, a thorough understanding of the pathogenesis of a given
infection would include a complete inventory of the spatial and temporal
expression of the genes by both the host and pathogen from the time of
exposure to the final resolution of the infection. Given the potentially
large number of factors that contribute to host defense, precise gene
identification is a formidable challenge. Nevertheless, researchers have
recently made progress in dissecting and identifying the most important
individual genetic elements that govern the host response to important
pathogenslargely through the use of animal models of human disease (5). 
Of the model organisms amenable to genetic analysis, the mouse is by far the
most well-developed and physiologically relevant system for study of human
host defense (6,7). Identification of commercially available inbred strains
of mice that show a differential response to a well-defined infectious
challenge is the first requirement for study of genetically regulated host
resistance factors. Once distinct phenotypes are identified, controlled
breeding is carried out to determine the mode of inheritance of the
phenotype (simple or complex). Correlation of the inheritance of
susceptibility or resistance to a specific infectious challenge with one or
more chromosomal regions is then performed by using linkage analysis.
Finally, known genes within the genetic interval must be evaluated and
novel genes must be positionally cloned to elucidate the underlying
molecular basis of immune defense. Comparative genomic analysis is a
logical extension of these principles (8). Knowledge of the genomic
organization of human and mouse, for example, facilitates direct
localization and identification of the human orthologues of susceptibility
genes identified through experimental challenge. These genes can then be
tested as candidates for human disease susceptibility through mutation
analysis.

Genetic Linkage Maps

Genetic linkage maps provide an organizational framework for genes and
phenotypes in the genome (9). Maps, by establishing the location, order,
and relative distance of genes, anonymous DNA markers, and biologically
important traits along a species' chromosomes, are critical tools in
analyzing genetic contribution to a given disease state. Genetic maps can
help precisely localize chromosomal region(s) linked to host resistance
phenotypes and provide the starting point for identification of the
causative gene(s). During the past decade, comprehensive genetic maps
spanning the genomes of mouse and human, have been created largely through
the initiative of the Human Genome Project (10).

Mapping the Human Genome

A great deal of effort has resulted in the creation of a whole genome human
linkage map, consisting of 5,624 microsatellite markers located to 2,335
positions (11). The DNA markers in this map are highly informative and are
densely distributed, with an average interval between markers of 1.6
centimorgans (cM) (1 cM = a 1% rate of recombination during meiosis, or
approximately 1 million bp). Other comprehensive maps have been assembled
on the basis of a collection of more than 16,000 distinct transcribed
sequences (including known genes and gene fragments) or expressed sequence
tags, which are estimated to represent at least 50% of all genes in the
human genome (12). This human transcription map has been integrated with
selected microsatellite markers from the Gnthon collection, thus allowing
the position of gene-based markers to be resolved to specific intervals
measured in centimorgans. The map is available electronically (13). Work is
also under way to generate comprehensive physical maps of the human genome
in which the relative location of markers is defined by the actual length
along the chromosome, rather than by recombination events (14,15).

Mapping the Mouse Genome

Among model organisms, genetic mapping is most well established in the
mouse, having begun in 1915 with the discovery of the first linkage group
(16). Controlled crosses of common laboratory strains segregating a small
number of visible phenotypes such as coat color then became the mainstay of
genetic mapping. In the past decade, two major breakthroughs have
revolutionized the technique of mouse genetic mapping and paved the way for
generation of high-resolution whole genome maps. The first was the
development of the interspecific cross, involving a laboratory strain (Mus
musculus) and a distantly related species Mus spretus (17), allowing
literally thousands of genes to be mapped within the same cross. The second
advance was the development of abundant genetic markers rapidly typable by
polymerase chain reaction (PCR) (termed microsatellites), which amplified
polymorphisms in simple sequence length repeats such as [CA]n (Figure)
(18). Several comprehensive genetic maps of the mouse (based on genes or
microsatellites) have been developed, and in some cases, these are being
integrated. At least three are publicly available, while the others are
available for mapping in a collaborative arrangement (19). As of January
1997, more than 17,000 markers had been mapped in the mouse (one locus
approximately every 200kb), including more than 5,000 genes and more than
10,000 (mostly microsatellite) DNA markers.

[Fig]
 Figure. Schematic representation of microsatellite
 marker analysis in mice. A) Flanking forward (F)   
 and reverse (R) oligonucleotides are designed to   
 specifically amplify a simple sequence repeat by  
 polymerase chain reaction (PCR) (in this case a CA
 dinucleotide). The length of the dinucleotide (N)  
 varies among inbred mouse strains. B) Gel          
 electrophoresis of a PCR-amplified microsatellite  
 in homozygous parental strains A and B and         
 heterozygous F1 progeny. The larger microsatellite 
 from strain A migrates more slowly than that of    
 strain B. Inheritance of both parental alleles is  
 shown in the F1.                                  

Mapping in Other Species

Genetic mapping has been widely embraced by the scientific
community; more than 30 vertebrate species are the subject of
genetic mapping projects, and high-resolution maps of microsatellite
markers have been developed for humans, mice, rats, cows, sheep, 
pigs, fish, and chickens (19). Two invertebrates, Drosophila 
melanogaster (a dipteran fly) and Caenorhabditis elegans (a nematode), 
also have complete genetic and physical maps; the complete nucleotide 
sequence of the latter is expected in the near future. The status of 
individual genetic mapping projects and resources has been summarized, 
along with a compilation of databases for species-specific or comparative 
mapping reference (19,20). Integrating the data from these species-
specific projects in a form that allows relevant information from 
diverse organisms to be assembled is a major challenge to biologic 
information systems. The most extensive coverage of mammalian species 
homologies is the Mouse Genome Database of The Jackson Laboratory (21). 
Initially developed for the mouse, comparative mapping data for more than 
55 species may be searched online, with links to related genomic resources, 
such as the Human Genome Database, Ratmap, SheepBase, and PigBase.

Comparative Genetic Mapping

Because of the density of genetic markers positioned along the chromosomes
of both organisms, the comparative map of the mouse and human genomes is
the most well developed of all species. In a comprehensive summary of
mouse/human homology published in 1996, 1,416 loci were placed on both maps
by using human physical mapping data and mouse genetic maps (22). This
comparison defined 181 conserved linkage groups, approximately 90% of the
mouse genome. Further comparative mapping with newly discovered genes and
expressed sequence tags will refine the chromosomal relationships between
mouse and human.

Integrating Maps and Aligning Genomes

The integration of existing genetic maps of different species is a
formidable challenge. Accurate, comprehensive comparisons of gene
arrangements across different species will rapidly advance our
understanding of all aspects of biology by allowing rapid information
exchange across different model organisms and experimental systems. Several
approaches have been used in developing universal mapping probes for
diverse genomes (23-25). Of the two classes of loci used to construct gene
maps, coding gene sequences (Type I markers), which show conservation among
distantly related mammalian species, are most useful as landmarks for
comparing linkage and syntenic association. Highly polymorphic sequences
(Type II markers), such as microsatellites, are more abundant and are
invaluable for mapping within a pedigree but are less useful for
comparative purposes because they do not show adequate sequence
conservation to recognize locus homology between mammalian orders. In 1993,
a list of anchored reference loci for comparative genome mapping in mammals
was proposed; it consists of 321 Type I markers equivalently spaced
throughout the mammalian genome (26). This approach allowed the position of
homologous loci in the maps of four species (human, mouse, cattle, and
cat), which represent different mammalian orders, to be established.
Interspecies comparison of conserved exon sequences of homologous genes has
generated a new overlapping set of anchor loci called comparative anchor
tagged sequences (25). Large-scale mapping of these sequences in several
species may be an efficient way of developing high-resolution comparative
maps with essentially complete genome coverage.

Alternative Techniques for Comparative Genomic Analysis

Mammalian genomes may be compared at several levels by using a variety of
tools and strategies tailored to individual objectives. Although direct
sequence comparison of whole genomes will provide the highest resolution
for comparative study, this sophisticated form of analysis is at least
several years away from being realized. At a cytologic level, species may
be compared by fluorescence in situ hybridization (FISH) with single or
multiple probes (single or multicolor Zoo-FISH), producing rapid,
high-resolution chromosomal localization detectable by microscopy.
Alternatively, libraries from microdissected or individual flow-sorted
chromosomes may be constructed and used as fluorescence-labeled chromosome
"paints" to probe the chromosomes of other species and identify homologous
regions (27,28). The main advantage of chromosome painting is its rapid
overall evaluation of the extent and character of genomic conservation
among distantly related species, such as pig and cattle. In contrast to
FISH, chromosome painting does not allow determination of gene order or
high-resolution demarcation of chromosomal breakpoints. Radiation hybrid
panels, another method for physical assignment of homologous loci (29,30),
are generated by irradiation and subsequent fusion of a cell line
containing a chromosome from one species, such as human, on another
background, such as hamster. The donor DNA is fragmented at random,
resulting in a series of lines retaining only fragments of the original
chromosome. Conserved genes from other species may be mapped to the
homologous region of the human genome by comparing the PCR pattern for each
cell line to reference loci with well-established map positions.

Models of Human Disease

Identifying genetically regulated host immune responses might significantly
advance our understanding of the molecular targets and immunologic
mechanisms critical to robust defense against pathogenic microbes. To date
the number of host defense genes that have been cloned remains small;
comparative genomics has the potential to accelerate gene discovery by
allowing available data for model organisms to be rapidly applied to the
study of human disease. We summarize three examples of human host
resistance genes in the following section; in each example, genetic
analysis of mouse models of the human disease phenotype played a crucial
role in the initial discovery of the human homologue or served as a means
of validating the identity of the proposed human candidate disease gene.

Nramp 1 and NRAMP1

The Mouse Nramp1 Gene

In classic inbred strains of mice, natural resistance to infection with
Mycobacterium bovis (BCG), M. lepraemurium, Salmonella Typhimurium, and
Leishmania donovani is controlled by the Bcg locus, also known as Ity and
Lsh (31-33). The major effect of the Bcg gene is to modulate the growth
rate of these diverse pathogens in cells of the reticuloendothelial system
of the mouse during the preimmune phase of the infection (33). Resistant
and susceptible strains are distinguished by the kinetics of infection
shown by pathogen counts (CFUs or Leishmania-forming units) in liver and
spleen after infection. The susceptible phenotype is characterized by a
higher net growth rate of BCG, Salmonella, or Leishmania in the
reticuloendothelial system during the early phase of infection, followed by
specific immune responses in BCG- and L. donovaniinfected mice or by a
rapidly lethal infection with the virulent pathogen S. Typhimurium. Bcg is
inherited as a simple autosomal dominant Mendelian trait in crosses between
classical strains of laboratory mice; it was localized to mouse chromosome
1 by linkage analysis (34). Using a positional cloning strategy, Vidal et
al. (35) isolated the Nramp1 (natural resistance-associated macrophage
protein 1) gene as a strong candidate for the Bcg mutation based on its map
location, its macrophage-restricted expression pattern and a nonconservative
Gly(sup 169)Asp substitution in the protein of all susceptible strains.
Creation of a null allele at Nramp1 then provided formal proof that a 
mutation within Nramp1 is the cause of the mouse susceptibility to 
infection with M. bovis, S. Typhimurium, and L. donovani (36).

Nramp1, an integral membrane phosphoglycoprotein located in the late
endosome/lysosome compartment of resting macrophages, is recruited to the
maturing phagosomal membrane (37), consistent with its potential function
in controlling the replication of intracellular parasites by altering the
intravacuolar environment in which they reside. Nramp1 is part of an
ancient family of proteins with highly conserved members in mammals
(including humans, cows, rats, sheep), birds, invertebrates (C. elegans, D.
melanogaster), plants (Oryza sativa, Arabidopsis thaliana), fungi
(Saccharomyces cerevisiae), and even bacteria (M. leprae and Escherichia
coli) (38,39). This family is characterized by a highly conserved
hydrophobic core consisting of 10 transmembrane (TM) domains with a
structural organization typical of families of ion transporters and
channels. In addition, the most highly conserved segments of the Nramp
family (TM8-TM9 intracellular loop) show impressive similarity with the
highly conserved region of mammalian voltage-gated K(sup +) channels of the
shaker type (40).

Several issues concerning the biochemical function of Nramp1 with respect
to intracellular survival of taxonomically unrelated pathogens remain
unresolved. Studies of the function of Nramp1-related sequences (Nramp2 and
Smf1 in model organisms) provide insight into how Nramp1 confers resistance
to microbial agents. Nramp2 has been isolated in mouse and human and shows
a high degree of similarity to Nramp1 (77% overall similarity), with
identical hydropathy profiles and predicted secondary structures (41,42).
Mouse and human Nramp2 mRNA are both widely expressed in contrast with the
tissue-specific expression of Nramp1 (41,42). Recently, Nramp2 was shown to
be a metal ion transporter with broad divalent cation specificity (including 
Fe(sup 2+), Zn(sup 2+), Mn(sup 2+), Co(sup 2+), Cd(sup 2+), Cu(sup 2+), 
Ni(sup 2+), and Pb(sup 2+)), driven by the proton electrochemical gradient 
in Xenopus laevis oocytes (43). Studies using the yeast double mutant SMF1/
SMF2 provided additional support concerning the function of Nramp2 as a 
divalent cation transporter. Inactivation of SMF1 and SMF2, two yeast Nramp 
homologues encoding divalent cation transporters (44), is specifically 
complemented by Nramp2 (45). In vivo, Nramp2 plays an important role in normal 
iron transport. Mutation within Nramp2 causes microcytic anemia in mk mutant 
mice because of severe defects in intestinal iron uptake (46). Interestingly, 
the missense mutations in mutant Nramp1 and Nramp2 alleles introduce a charged 
amino acid in two adjacent positions of TM4, confirming the importance of this
region of both proteins for normal function. It has been suggested that
Nramp1 may also be a divalent cation transporter; its role in
reticuloendothelial cells remains unexplored (40,44).

The Chicken NRAMP1 Gene

The discovery of Nramp1 allowed the study of its role in susceptibility to
related infections in other species. Salmonellosis, one of the most common
causes of food poisoning in humans, is frequently caused by ingestion of
contaminated poultry products; efforts to identify salmonella resistance
genes in poultry could lead to more efficient poultry control strategies,
thereby reducing secondary human morbidity. Genetic regulation of chicken
host resistance exists, as inbred poultry lines differ in their
susceptibility to infection with several strains of Salmonella. Segregation
analysis with a combination of Salmonella-resistant and
Salmonella-susceptible lines has shown that resistance to infection is
fully dominant and is not sex-linked or associated with the major
histocompatibility complex (47). The candidacy of the chicken Nramp1
homologue was tested in the differential resistance of inbred chicken lines
to infection with S. Typhimurium by using sequencing and linkage analyses
(48). Through the use of a mouse cDNA, the chicken homologue Nramp1 has
been cloned and shown to share 68% identity with the mouse gene (49). As
demonstrated in mice, the macrophage is a major site of NRAMP1 mRNA
expression in chickens (49). NRAMP1 mRNA transcripts from S.
Typhimurium-resistant or-susceptible chickens were analyzed to identify
amino acid sequence variants that could be associated with the disease
phenotype. Eleven sequence variants in Nramp1 mRNA were obtained from three
Salmonella-resistant and three Salmonella-susceptible chicken lines; almost
all (10) resulted in silent mutations or conservative changes (to amino
acids with similar physical properties) that were detected both in
resistant and susceptible chicken lines, while only one sequence variant
resulted in a non-conservative substitution of a positively charged residue
(Arg(sup 223) by a polar residue (Gln[sup 223] ). This allelic variant was specific to
the susceptible line C and was clearly associated with survival to
infection (a resistance allele at NRAMP1 improved survival rate from 13% to
27%) (48). Taken together, these data strongly suggest a direct role of
NRAMP1 in susceptibility to infection in chickens.

The Human NRAMP1 Gene

Work in inbred strains of mice has established unambiguously that Nramp1
has an important role in determining resistance to mycobacterial infections
and has encouraged several research groups to test the association of
NRAMP1 with corresponding human infections. Host genetic factors play a
major role in determining the outcome of mycobacterial infections in
humans, as shown by racial variation in susceptibility to infection and
higher concordance of tuberculosis and leprosy among monozygotic twins
compared with dizygotic twins and siblings (50,51). Segregation analysis in
a population from Desirade Island (French West Indies) has demonstrated
that susceptibility to leprosy (regardless of the clinically defined
subtype) is controlled by a major gene not linked to the major
histocompatibility complex (52). Through use of a candidate gene approach,
population association studies, and linkage analysis, several genes
(HLA-linked genes, tumor necrosis factor, collectin, vitamin D receptor,
interferon gamma receptor) have each been associated with susceptibility to
mycobacterial infections (53,54).

The chromosomal region surrounding Nramp1 on mouse chromosome 1 has been
conserved on the telomeric end of human chromosome 2q35 and contains the
human NRAMP1 orthologue (55). Sequence comparison of the mouse and human
Nramp1/NRAMP1 proteins showed a high degree of conservation between the two
species (85% identity, 92% similarity); the most conserved region was the
intracellular loop containing the consensus sequence transport motif (56).
In humans, the highest sites of NRAMP1 expression are peripheral blood
leukocytes and lungs (56). The high degree of sequence homology between
mouse and human NRAMP1, the presence of similar regulatory elements within
the promoter regions of the genes, and similar tissue expression patterns
support the notion that the NRAMP1 protein exerts similar roles in vivo in
both mouse and humans.

A number of polymorphic variants have been used to study the association of
NRAMP1 and susceptibility to leprosy and tuberculosis (57-60). One study
based on the segregation analysis of certain NRAMP1 haplotypes in 20
multiplex families involving 168 individuals from South Vietnam clearly
showed that NRAMP1 was involved in predisposition to leprosy (61). Another
large study measuring the association of NRAMP1 with clinical tuberculosis
in a population of Gambia (West Africa) demonstrated that polymorphic
variations within the human NRAMP1 gene affect susceptibility to the
disease (62). Nevertheless, susceptibility to either leprosy or
tuberculosis appears to be genetically heterogeneous since the role of
NRAMP1 was observed only in certain ethnic groups (63,64).

Identification of Nramp1 illustrates the value of comparative genomics for
identification and characterization of the biologic basis for differences
between susceptible and resistant hosts. Genetic dissection of the mouse
model of M. bovis infection was crucial to the identification of similar
mechanisms governing the human response to medically important pathogens
such as tuberculosis and leprosy. Comparative genomics was also important
in accelerating the identification of an important host resistance gene for
salmonellosis in the chicken (a species of significant agricultural
importance), where the available genetic tools are modest, relative to mice
and humans.

Chediak-Higashi Syndrome (CHS)

CHS is a rare autosomal recessive disorder characterized by partial ocular
and cutaneous albinism, a mild bleeding diathesis, and peripheral
sensorimotor neuropathy. The most serious phenotype among CHS patients,
however, is a marked increased in susceptibility to bacterial infection
that may lead to death during the first 2 decades of life. These clinical
features are attributable to dysfunctional granule-containing cells
including melanocytes, platelets, Schwann cells, neurons, and granulocytes
(65,66). On the basis of phenotypic similarity, the beige (bg) mutation in
mice has long been regarded as a model for CHS (67). Several components of
the immune system are affected in Beige/CHS. Neutrophils exhibit defective
chemotaxis and reduced intracellular killing for up to 90 minutes after
bacterial phagocytosis, and their granules lack the serine proteases
cathepsin G and elastase because of a failure of normal protein sorting
(68,69). Natural killer cell activity is defective, causing impaired
cytolysis of tumors and virally infected cells; cytotoxic T-cell responses
against allogeneic tumor cells are also abnormal (70,71). Mice with the bg
mutation have increased susceptibility to a variety of pathogens, including
cytomegalovirus, Leishmania donovani, Candida albicans, and a variety of
pathogenic bacteria (E. coli, Klebsiella pneumoniae, Staphylococcus aureus,
Streptococcus pneumoniae) (72-74).

To identify the genetic basis of this host resistance defect, the bg gene
was localized to a 0.24 cM interval of proximal mouse chromosome 13 by
genetic mapping of three mouse back-crosses segregating this phenotype
(75). A DNA contig of this region spanning 2,400 kb was constructed from
large-capacity yeast artificial chromosomes and P1 bacteriophage clones
(76). Using yeast artificial chromosome complementation and direct cDNA
selection, two groups subsequently identified portions of a candidate gene
for bg, named Lyst (lysosomal trafficking regulator) (77,78). Lyst,
ubiquitously expressed in the mouse, has a maximum transcript size of
approximately 12kb and possible complex alternative splicing. Several
mutations predicted to severely truncate the Lyst polypeptide were
identified within each transcript. Through the use of partial sequence data
for mouse Lyst, 27 cDNAs corresponding to the human gene were identified
and assembled into a complete human gene sequence of 13,499 bp, with an
open reading frame of 11,403 bp (79). Comparison of the partial 3' mouse
cDNA to the human sequence demonstrated 77.2% nucleotide identity and 87.9%
amino acid identity, indicating that human and mouse genes are highly
homologous, and sequence analysis of three CHS patients identified
pathologic mutations in all.

Comparative genetic mapping between the region of mouse chromosome 13 with
the bg mutation and the human genome indicates homology with distal
chromosome 1q. Consistent with this alignment, genetic mapping of the human
CHS locus in affected families localized it to 1q42-1q44 as part of a
conserved linkage group shared with mouse chromosome 13 (80,81). Radiation
hybrid mapping also assigned the human CHS candidate gene to 1q43,
confirming that the bg phenotype in mouse and human CHS are both caused by
mutations in orthologous genes (79). Database searches with the complete
nucleotide sequence of the CHS gene showed significant homology to open
reading frames from S. cerevisiae and C. elegans, as well as a human cell
division control protein-4 (CDC4L) (82). The modular architecture of the
CHS protein is similar to Vps15, a yeast serine/threonine kinase protein
kinase thought to be part of a membrane-associated signal transduction
complex regulating intracellular protein trafficking (83). To date, the
function of the CHS gene remains unknown, although it may be similar to
Vps15 and may be part of a novel gene family.

X-Linked Agammaglobulinemia (XLA)

XLA, one of the first primary immunodeficiency disorders described in
humans, is the prototypic example of the protective role of humoral
immunity against common bacterial pathogens (84). XLA is characterized by a
profound deficiency of B-lymphocyte development at two sequential stages of
maturation within the bone marrow (85). This defect results in marked
reductions in the serum levels of all three major classes of
immunoglobulins and a profound decrease in the number of B lymphocytes in
the peripheral blood as well as in the lymphoid follicles and germinal
centers of lymph nodes. The clinical manifestations generally begin by the
end of the first year of life, once the level of maternally derived
antibodies has declined. Bacterial infections with organisms such as S.
pneumoniae, Haemophilus influenzae, S. aureus, and Pseudomonas species are
most common, with the respiratory tract being most frequently affected.
Gastrointestinal infection with Salmonella or Campylobacter have also been
reported, as have urogenital infections with Mycoplasma or Chlamydia. XLA
patients have defective host resistance to enteroviruses, since
neutralizing antibody is important in controlling these pathogens during
their passage through the blood stream. Resistance to other infections for
which intact T lymphocyte function is required (e.g., tuberculosis or
histoplasmosis) remains intact.

Recognition of the familial occurence of this rare disorder and pedigree
analysis demonstrated an X-linked recessive inheritance pattern of the
trait (86). Carrier females could not be detected because they are
phenotypically normal, with normal serum levels of immunoglobulin. Linkage
studies of over 500 individuals from 60 families mapped the gene for XLA to
the midportion (Xq22) of the X chromosome, cosegregating with the
polymorphic genetic marker DXS178 (87,88). By using complementary
strategies of positional cloning and low-stringency cDNA library screening,
two groups identified a novel src-like cytoplasmic tyrosine kinase, named
Btk (Bruton agammaglobulinemia tyrosine kinase) as a strong candidate gene
for XLA (89,90). Btk was mapped to the XLA locus by FISH and somatic cell
hybrid analysis and was expressed in cell lines representing all stages of
B cell development, myelomonocytic cell lines, and a macrophage cell line;
it was not detectable in T lineage cell lines (90). In transformed B-cell
lines from individuals affected with XLA, the expression level of Btk mRNA
and protein, and consequently its kinase activity, was reduced or absent.
Southern blot analysis of DNA from pedigrees with XLA cases showed
restriction fragment length alterations that segregated in an X-linked
recessive pattern; detailed analysis disclosed either genomic DNA deletions
in the region encompassing Btk or missense point mutations resulting in
nonconservative amino acid substitutions at important residues in the
putative protein-tyrosine kinase domain (89). These findings provide strong
evidence that the failure of normal B-cell growth and differentiation in
XLA is caused by abnormal function of an intracellular protein tyrosine
kinase.

The CBA/N inbred mouse strain's X-linked immunodeficiency (xid) has been
regarded as an experimental model for human XLA since it was first
described in 1972 (91). B lymphocytes from these mice exhibit pleiotropic
defects in development and function. Normal numbers of pro-B, pre-B, and
surface immunoglobulin-positive B cells exist in the bone marrow, while
peripheral B-cell numbers are significantly reduced (30% of normal). The B
lymphocytes that are present have an abnormal surface marker phenotype, and
B-cell proliferation triggered through the surface immunoglobulin M (IgM)
receptor or surface immunoglobulin cross-linking is impaired, as are
responses to a number of other mitogenic stimuli including
lipopolysaccharide, interleukins IL-5 and IL-10, CD38 receptors, and CD40
ligands. Consistent with these defects, CBA/N mice have reduced serum IgM
and IgG3 antibody levels and cannot make antibody responses when challenged
with type-2 thymus-independent antigens (e.g., polysaccharides and
hapten-polysaccharide conjugates). As with human XLA, impaired humoral
immunity can result in increased susceptibility to bacterial pathogens,
including S. Typhimurium (92). Inheritance of the susceptibility trait was
linked to the xid locus by using back-cross and F2 progeny derived from
crosses of CBA/N and DBA/2N parental strains.

To determine whether XLA and xid were caused by mutations in homologous
genes, two groups performed genetic mapping of xid and Btk. The Btk gene
was closely linked to the xid locus in the distal region of the mouse X
chromosome by using an interspecific back-cross mapping panel (93), and
precise co-localization of Btk and xid was observed in 1,114 segregating
back-cross progeny (94). Normal and mutant mouse strains did not differ in
Btk expression or in vitro kinase activity. Sequence analysis of the mouse
Btk transcript in CBA/N and several immunocompetent mouse strains
(including the CBA/CaHN progenitor) demonstrated a point mutation within
the first coding exon that is predicted to convert a highly conserved
arginine residue to cysteine. This amino acid substitution occurs within
the pleckstrin homology domain in the amino-terminal region of the protein
and is presumed to alter normal B-cell signaling by disrupting
protein-protein interactions. To unequivocally confirm that mutations in
Btk were responsible for the xid phenotype, targeted gene disruption (a
gene knockout experiment) was performed in embryonic stem cells (95,96).
Complete elimination of Btk protein production identically reproduced the
xid phenotype, indicating that the naturally occurring point mutation
produces a complete loss-of-function phenotype or results in a protein with
dominant negative properties (presence of a single mutant allele is
sufficient to block normal gene function). The severe early B-lymphocyte
developmental arrest of human XLA was not observed, which suggests that Btk
function in mice is accompanied by a compensatory mechanism operating
during early B-cell development to rescue B-cell maturation.

On the basis of comparative mapping and sequence analysis, human XLA and
the mouse xid phenotype are clearly homologous disorders caused by
mutations in orthologous genes. Nevertheless, although the underlying
genetic alteration in both species was successfully identified, a number of
issues remain unresolved. First, the phenotypes observed in these two
disorders are not identical; the more severe block of early lymphocyte
development in XLA results in a greater deficiency of peripheral B cells
relative to the CBA/N mouse strain, suggesting that the requirement for Btk
in early murine B-cell development is less stringent than that for humans.
Second, the range of pathogens to which humans are are highly susceptible
appears more diverse than the range for mice. Finally, the exact role of
Btk in normal B-cell physiology remains to be demonstrated. Thus far,
identification of BTK has led to carrier detection and prenatal
counselling; additional characterization of a mouse model with great
similarity to the human condition could advance our understanding of the
fundamental processes underlying B-lymphocyte development and function.

Conclusions

Complete understanding of infectious disease pathogenesis requires
identification and characterization of host genes that regulate the
response to virulent microorganisms. Through evolutionary selection, a
series of innate immune defense mechanisms have evolved to protect the host
against the constant threat of microbial injury and direct the development
of specific adaptive immune responses. Genetic analysis of naturally
occurring variation in the host response among model organisms has
successfully identified novel genes such as Nramp1, Lyst, and Btk, thus
providing new insights into the molecular nature of host resistance. Rapid
advances are now being made in the creation and integration of dense
genetic maps of model organisms and humans. Comparative genomics will play
an increasingly important role in facilitating the transfer of new
knowledge from experimental models to a more complete understanding of
human host resistance.

      This work was supported by grants to Danielle Malo from the Medical
Research Council of Canada (MRC), and the Canadian Bacterial Diseases
Network.

      Dr. Qureshi is a research fellow at the Centre for the Study of Host
Resistance, McGill University, and assistant physician (infectious
diseases) at Montral General Hospital. His research focuses on genetic
analysis of host resistance against bacterial diseases.

      Address for correspondence: Danielle Malo, L11-144 Montral General
Hospital, 1650 Cedar Avenue, Montral, Canada H3G 1A4; fax: 514-934-8261;
e-mail: mc76@musica.mcgill.ca.

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Synopses

Cyclospora: An Enigma Worth Unraveling

Charles R. Sterling and Yns R. Ortega
University of Arizona, Tucson, Arizona, USA

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      In part, Cyclospora cayetanensis owes its recognition as an
      emerging pathogen to the increased use of staining methods for
      detecting enteric parasites such as Cryptosporidium. First
      reported in patients in New Guinea in 1977 but thought to be a
      coccidian parasite of the genus Isospora, C. cayetanensis
      received little attention until it was again described in 1985
      in New York and Peru. In the early 1990s, human infection
      associated with waterborne transmission of C. cayetanensis was
      suspected; foodborne transmission was likewise suggested in
      early studies. The parasite was associated with several disease
      outbreaks in the United States during 1996 and 1997. This
      article reviews current knowledge about C. cayetanensis
      (including its association with waterborne and foodborne
      transmission), unresolved issues, and research needs.

Cyclospora Overview

Cyclospora cayetanensis is a protozoan parasite (subphylum Apicomplexa,
subclass Coccidiasina, order Eucoccidiorida, family Eimeriidae). The
organism's link to the Eimeriidae extends to the genus level through use of
molecular phylogenetic analysis techniques (1). Collected data link
infection to a single hosthumans. In 1993, asexual meronts were described
from jejunal enterocytes of humans (2). In 1997, two types of meronts and
sexual stages were observed in jejunal enterocytes of biopsy specimens from
infected patients excreting oocysts, confirming that the entire life cycle
could be completed within a single host (3); infected persons excrete
unsporulated oocysts. In the laboratory, oocysts are induced to sporulate
in potassium dichromate in a petri dish at ambient temperatures (25C to
30C). After 1 week and up to 2 weeks, approximately 40% of oocysts contain
two sporocysts with two sporozoites in each (4). Excystation of sporulated
oocysts occurs in vitro when oocysts are subjected to bile salts and sodium
taurocholate and mechanical pressure from a glass tube mortar and pestle
(5). These findings suggest that direct person-to-person transmission is
unlikely.

Oocysts measure 8 m to 10 m in diameter and stain variably acid-fast.
Without the use of an ocular micrometer, oocysts of Cyclospora might be
easily confused with those of Cryptosporidium or other fecal artifacts that
stain acid-fast positive, as was the case in a pseudo-outbreak of
cyclosporiasis reported in Florida (6). Cyclospora oocysts are easily
observed by phase contrast microscopy, and the algal-like morula appearance
is evident in fresh stool specimens. A useful and distinguishing feature is
oocyst autofluorescence, which appears blue by Epi-illumination and a
365-nm dichroic exciter filter and green by a 450-nm to 490-nm dichroic
filter.

Susceptible humans are infected by ingesting sporulated oocysts. While
unknown, the infectious dose is presumed to be low. Symptoms of infection
may include watery diarrhea, mild to severe nausea, anorexia, abdominal
cramping, fatigue, and weight loss. Diarrhea can be intermittent and
protracted (3,7,8). Persons with no previous immunity as well as very young
children in developing countries are likely to exhibit symptoms. Limited
data suggest that in disease-endemic countries, frequent exposure may
predispose to asymptomatic infection in children and absence of infection
in adults (9). Symptomatic infections can be treated with
trimethoprim-sulfamethoxazole (Bactrim) (9-11).

Cyclospora infections have been confirmed in North, Central, and South
America, the Caribbean, England, eastern Europe, Africa, the Indian
subcontinent, Southeast Asia, and Australia (12). In the United States,
England, and Australia, most cases were first observed in travelers
returning from the areas listed above (7,13,14). As more indigenous cases
are reported from all areas, however, a cosmopolitan distribution of
Cyclospora appears possible. A seasonal distribution of infection,
coinciding with wet or warm months of the year, has also been suggested
(15).

Association with Waterborne Transmission

While the organism causing Cyclospora infection was still being identified,
an outbreak occurred in the staff of a Chicago hospital in 1990 (16).
Infection was confirmed in 11 of 21 persons exhibiting diarrheal symptoms
and lasted up to 9 weeks with alternating cycles of disease and remission.
Epidemiologically, infections were associated with drinking tap water (in a
resident's dormitory) possibly contaminated with stagnant water from a
rooftop storage reservoir. In an isolated incident (also in Chicago), an
8-year-old child became ill and passed oocysts in the feces 1 week after
swimming in Lake Michigan (7). In another isolated incident, a man from
Utah became ill with severe watery diarrhea and passed oocysts after
cleaning his basement, which had been flooded by sewage backup following
heavy rains (8). The man's house was located near a dairy farm and much of
the sewage backup was attributed to water runoff from this site. In yet
another isolated incident in the United States, consumption of well water
was implicated in the infection of one of three patients in Massachusetts
(17).

Two outbreaks of Cyclospora infection in Nepal have also been linked to
waterborne transmission (18,19). In the first outbreak in 1992,
expatriates, who were more likely to drink untreated water or milk
reconstituted with water, became ill with diarrhea and passed oocysts. The
infections occurred during the summer, which coincided with annual
epidemics among the expatriates. The second waterborne disease outbreak
occurred in 12 of 14 British soldiers, despite chlorination of the water
involved. In this outbreak, Cyclospora oocysts were demonstrated for the
first time in drinking water, which consisted of a mixture of river and
municipal water.

Though not directly connected with water-associated disease outbreaks,
Cyclospora oocysts have been isolated from wastewater in sewage lagoons
adjacent to an area of endemic disease in Lima, Peru (20); their presence
was confirmed by microscopy and PCR. Water from these sewage lagoons is
used to irrigate pasture land, corn fields, and trees. In other parts of
Lima, water from such lagoons is used to irrigate vegetable crops.

Association with Foodborne Transmission

While the Nepal study conducted in 1992 strongly suggested waterborne
transmission of Cyclospora, only 28% of infected patients reported drinking
untreated water or milk possibly contaminated with untreated water (18).
Therefore, other modes of transmission were likely, although none was
identified. Foodborne transmission was suspected when consumption of raw or
undercooked meat and poultry products was reported as part of case
histories before the infectious organism was identified as Cyclospora
(21,22). Foodborne transmission was first suggested in 1995 when the
illness of an airline pilot was associated with food prepared in a Haitian
kitchen and brought on board the airplane (23). Cyclospora is endemic in
Haiti; this study underscored that this type of illness could be acquired
from meals brought on board without visiting the country in which infection
originated.

Foodborne transmission of Cyclospora in the United States, first reported
in 1995, was widely reported in 1996 and 1997 (24-28). Some reports early
in 1996 implicated strawberries, but as more epidemiologic information was
gathered, attention shifted to raspberries. In 1996, a total of 1,465 cases
of cyclosporiasis were reported from 20 states (predominantly east of the
Rocky Mountains), the District of Columbia, and two Canadian provinces
(24). Almost half (725 cases) were event associated; the remaining (740
cases) were sporadic (i.e., not epidemiologically linked to other cases);
978 (67%) cases were laboratory confirmed; 55 clusters of cases were
associated with social events. A total of 3,035 persons attended these
events; 1,339 (44.1%) were interviewed, and of these 735 (54.1%) were
designated case-patients. Cyclospora infection was laboratory confirmed in
238 (32.8%) cases. Raspberries were definitely served at 50 events and
possibly at four more. Even in the documented 740 sporadic cases in 1996,
many patients recalled eating some type of berries. Of the 54 cluster
events at which raspberries were or may have been served, well-documented
traceback data as to the source were uncovered for 29; of these, 21 were
definitely traceable to raspberries imported from Guatemala, and an
additional eight may have originated there. Twenty-five (86%) of the 29
well-documented events were traceable to one (versus more than one)
exporter per event. Further tracings showed that as few as five Guatemalan
farms could have accounted for the 25 events traceable to a single exporter
per event. In part because of previous links with waterborne transmission,
it was postulated that the berries were contaminated when sprayed with
insecticides or fungicides mixed with water containing sporulated oocysts.

As of August 1997, 1,450 cases of cyclosporiasis (550 laboratory confirmed)
were reported (28). Many cases were cluster-associated and involved
raspberries linked to Guatemala. In addition, 25 confirmed and 20 possible
clusters of cases of cyclosporiasis were associated with consumption of
food that contained fresh basil. An additional two clusters of cases in
Florida were linked with eating mesclun lettuce (28). In each situation,
the outbreaks were linked to non-Guatemalan fresh produce.

Cyclospora oocysts have been isolated from vegetables from a
disease-endemic area of Lima, Peru, and from Nepal (29,30). Although the
number of oocysts recovered was small, encountered in only a few samples,
and not associated with any known disease outbreak, the implication was
clear: foodborne transmission by this route could occur. In addition,
oocysts experimentally seeded on vegetables could not easily be removed by
washing (30). Washing of vegetables, even though highly recommended as a
means of reducing risk for infection, may therefore not totally eliminate
the risk.

Unresolved Issues

Unresolved issues concerning Cyclospora fall into three broad categories:
environmental survival, transmission to humans, and epidemiology. The
boundaries of these categories frequently overlap.

Environmental Survival

The biggest issues of concern in this category are oocyst distribution in
the environment, oocyst survival under changing conditions, and oocyst
sporulation times under changing environmental conditions. All these
factors affect transmission.

Because of technologic limitations, Cyclospora oocysts have only been
recovered in very limited numbers from water sources and vegetables
(19,20,29,30). A heavy reliance has been placed on techniques used for
isolating Cryptosporidium, which are inadequate (31). Very little is known
about conditions that may favor the survival of Cyclospora. Preliminary
studies have shown that oocysts subjected to -20C for 24 hours and
exposure to 60C for 1 hour cannot be induced to sporulate. Oocyst storage
at 4C or 37C for 14 days retards sporulation (32). The most intriguing
environmental issue is oocyst sporulation time. The report that confirmed
the identity of Cyclospora indicates that the organism requires 1 to 2
weeks to completely sporulate and become infectious under ambient
conditions of 25C to 30C (5). Oocysts maintained at 4C can sporulate
within 6 months (4). These periods are longer than those reported for most
coccidia; therefore, direct person-to-person transmission is unlikely.
Also, (if confirmed under changing conditions) a prolonged sporulation time
would imply that oocysts favor a moist environment, ideally water. Early in
the Guatemalan berry investigations, water used to irrigate plants was
thought to play a role in contaminating raspberries with oocysts. This
notion, which would likely apply only to berries grown with spray
irrigation, however, has largely been discarded since direct contact
exposure to excessive moisture promotes rapid fruit deterioration and most
raspberries grown in Guatemala rely on drip irrigation. The exact method of
contamination is not known, and even though use of insecticides and
fungicides made with oocyst-contaminated water has been hypothesized, its
role has yet to be confirmed. If this hypothesis is true, how these agents
might affect oocyst viability is also not known. Another unresolved issue
is how the water might have become contaminated.

Transmission to Humans

The primary issues concerning transmission of Cyclospora to humans are
infectious dose and species specificity. For most coccidia that infect
humans and animals (e.g., Cryptosporidium [33]), the infectious dose is
presumed to be low (34). What we know about the waterborne transmission of
Cryptosporidium and how few of its oocysts are usually isolated from water
is likely true for Cyclospora (35). However, only two foodborne outbreaks
of cryptosporidiosis have been reported (one involved fresh pressed cider
and the other chicken salad) (36,37). Cryptosporidium is immediately
infectious upon passage from an infected person, and oocysts are usually
passed in large numbers if the person is symptomatic. Unlike what has been
reported for Cyclospora to date, Cryptosporidium oocysts are ubiquitous in
the environment and could easily contaminate foods, especially vegetables.
In one study, Cryptosporidium oocysts were recovered more frequently from
vegetables than Cyclospora oocysts (30). In addition, Cryptosporidium
infectious to humans has many known animal hosts (38).

The issue of potential animal hosts for Cyclospora has not been resolved.
Cyclospora-like organisms have been recovered from ducks, chickens, dogs,
and primates (39-41). Only in primates has there been any concrete evidence
identifying the agent as a species of the genus Cyclospora, and whether it
is the same as C. cayetanensis is not known (41). For the other animal
species mentioned, recovered oocysts, if they were oocysts of Cyclospora,
may have been passing through these hosts. Attempts at finding animal hosts
infected with Cyclospora-like organisms in human disease-endemic areas have
largely failed, as have preliminary attempts at infecting conventionally
used laboratory animals. Some researchers have convincingly shown on the
basis of molecular data that Cyclospora and Eimeria are closely related
(1): others have even suggested that Cyclospora should be considered a
mammalian Eimeria species (42). To clarify the taxonomic issue, small
subunit rRNA sequences from Isospora should be compared with those of C.
cayetanensis and with Cyclospora isolates from nonhuman primates. In
addition, conventional and molecular taxonomists should name the species on
the basis of combined phenotypic and genotypic characteristics.

Epidemiology

Even though epidemiologic investigations of Cyclospora have been thorough
and convincing, they raise environmental and transmission issues that
require further investigation. The two areas we will consider are the
relative geographic restriction of cases and attendant traceback issues
associated with clusters of cyclosporiasis cases and potential indigenous
infections within the United States and elsewhere.

Unraveling the first issue involves tracing imported fruits or vegetables
in a forward direction (possible distribution sites) as well as tracing
them back (to their originating sites). In the raspberry-associated
outbreaks of 1996, good traceback data were obtainable for 29 of 55
clusters. All sites (except one) were east of the Rocky Mountains. For the
25 events traceable to one (versus more than one) exporter per event, 33
(85%) of 36 shipments entered through Miami, Florida (24). If berries were
also being distributed in large quantities to other, largely western
regions of the country during this period, would we not expect more
infections in western regions? This point, along with the attendant
epidemiologic investigations, helped dissociate strawberries from reported
Cyclospora infections. California strawberry growers were as likely or more
likely to ship strawberries within their own region of the United States as
they were to ship them elsewhere, yet most infections occurred in eastern
regions of the country. In addition, Guatemalan raspberries are imported
into the United States in large quantities twice a year, yet no outbreaks
occurred during the winter months when this importation occurs, which
indicates that the epidemiology of this infection in countries such as
Guatemala where the berries are grown needs further study.

The issue of indigenous U.S. infections should be investigated. Waterborne
and sporadic cases have occurred in which no association could be made to
raspberry consumption (7,8,16-19,24,29). Preliminary data (in one region of
the United States) have linked Cyclospora infection to gardening and
working with soil (43).

      Dr. Sterling is professor and head of the Department of Veterinary
Science and Microbiology, University of Arizona, Tucson. His laboratory
research focuses on cryptosporidiosis in immunologically naive and
immunocompromised persons, monoclonal and polyclonal antibodies in
diagnostic parasitology and immunotherapy, and Cyclospora and the
microsporidia as emerging pathogens.

      Dr. Ortega is assistant research scientist in the Department of
Veterinary Science and Microbiology, University of Arizona, Tucson. She was
first to identify taxonomically Cyclospora, and her research focuses on the
molecular biology and epidemiology of this organism.

      Address for correspondence: Charles R. Sterling, Department of
Veterinary Science and Microbiology, University of Arizona, Tucson, AZ
85721, USA; fax: 520-621-2799; e-mail: csterlin@u.arizona.edu.

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 27. Chambers J, Somerfeldt S, Mackey L, Nichols S, Ball R, Roberts D, et
     al. Outbreaks of Cyclospora cayetanensis infectionUnited States,
     1996. MMWR Morb Mortal Wkly Rep 1996;45:549-51.
 28. Pritchett R, Gossman C, Radke V, Moore J, Busenlehner, Fischer K, et
     al. Outbreak of cyclosporiasis. Northern Virginia-Washington,
     DC.-Baltimore, Maryland, Metropolitan Area, 1997. MMWR Morb Mortal
     Wkly Rep 1997;46:689-91.
 29. Kocka F, Peters C, Dacumos E, Azarcon E, Kallick C, Langkop C.
     Outbreaks of diarrheal illness associated with cyanobacteria
     (Blue-green algae)-like bodies-Chicago and Nepal, 1989 and 1990. MMWR
     Morb Mortal Wkly Rep 1991;40:325-7.
 30. Ortega YR, Roxas CR, Gilman RH, Miller NJ, Cabrera L, Taquiri C, et
     al. Isolation of Cryptosporidium parvum and Cyclospora cayetanensis
     from vegetables collected from markets of an endemic region in Peru.
     Am J Trop Med Hyg 1997;57:683-6.
 31. Steiner TS, Thielman NM, Guerrant RL. Protozoal agents: what are the
     dangers for the public water supply? [review] Annu Rev Med
     1997;48:329-40.
 32. Smith HV, Paton CA, Mtambo MMA, Girdwood RWA. Sporulation of
     Cyclospora sp oocysts. Appl Envir Microbiol 1997;63:1631-2.
 33. DuPont H, Chappell CL, Sterling CR, Okhuysen PC, Rose JB, Jakubowski
     W. The infectivity of Cryptosporidium parvum in healthy volunteers. N
     Engl J Med 1995;332:855-9.
 34. Jackson GJ, Leclerc JE, Bier JW, Madden JM. Cyclosporastill another
     new foodborne pathogen. Food Technology 1997;51:120.
 35. Rose JR, Lisle JT, LeChevallier M. Waterborne cryptosporidiosis:
     incidence, outbreaks, and treatment strategies. In: Fayer R, editor.
     Cryptosporidium and cryptosporidiosis. Boca Raton (FL): CRC Press,
     Inc.; 1997. p. 93-109.
 36. Besser-Wiek JW, Forfang J, Hedberg CW, Korlath JA, Osterholm MT,
     Sterling CR, et al. Foodborne outbreak of diarrheal illness associated
     with Cryptosporidium parvumMinnesota, 1995. MMWR Morb Mortal Wkly Rep
     1996;45:783-4.
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     Hudson A. An outbreak of cryptosporidiosis from fresh pressed apple
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 38. Meng J, Doyle MP. Emerging issues in microbiological food safety
     [review]. Annu Rev Nutr 1997;17:255-75.
 39. Garcia-Lopez HL, Rodriguez-Tovar LE, Medina-de la Garza CE.
     Identification of Cyclospora in poultry [letter]. Emerg Infect Dis
     1996;2:356-7.
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     first two cases of Cyclospora in dogs, Sao Paulo, Brasil. Rev Inst Med
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     human associated Cyclospora a mammalian Eimeria species? Emerg Infect
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     infectionFlorida. 1996. Annual Epidemic Intelligence Service
     Conference 1996;45:60.

Synopses

Using Monoclonal Antibodies to Prevent Mucosal Transmission of Epidemic
Infectious Diseases

Larry Zeitlin,* Richard A. Cone,* and Kevin J. Whaley *
*ReProtect, LLC, Baltimore, Maryland, USA; and The Johns Hopkins
University, Baltimore, Maryland, USA

---------------------------------------------------------------------------
        Passive immunization with antibodies has been shown to
        prevent a wide variety of diseases. Recent advances in
        monoclonal antibody technology are enabling the development
        of new methods for passive immunization of mucosal
        surfaces. Human monoclonal antibodies, produced rapidly,
        inexpensively, and in large quantities, may help prevent
        respiratory, diarrheal, and sexually transmitted diseases
        on a public health scale.

In 1975, Khler and Milstein noted that monoclonal antibodies (MAbs)
"...could be valuable for medical and industrial use" (1). Since then, the
use of MAbs has become routine in the research and diagnostic laboratory,
but antibodies have yet to be used to their maximum potential in medical
and public health applications. Two recent reviews of the therapeutic use
of antibodies suggest that systemically administered antibodies may play an
important role in treating infections by drug-resistant pathogens as well
as pathogens for which no antimicrobial drugs are available (2,3). However,
the greatest potential for MAbs probably lies in prevention since
antibodies are in general more effective for prophylaxis than for therapy
(3,4). From a public health perspective, prevention is especially important
(5). In particular, direct application of MAbs to mucosal surfaces blocks
the entry of pathogens into the body.

We review here the evidence of antibody efficacy in preventing disease and
recent advances that have facilitated the development of MAbs for mucosal
applications in humans. Finally, we consider the public health potential of
topical delivery of MAbs for preventing mucosal transmission of infections.

Immunologic Strategies for Preventing Mucosal Transmission

Vaccines that stimulate systemic immunity can prevent systemic disease, but
generally fail to prevent mucosal disease. Vaccines that stimulate active
mucosal immunity have demonstrated good efficacy in animal models, but with
few exceptions (polio and influenza vaccines), have not been as effective
as they could be in humans. Some of the discrepancies between study results
in animals and humans are probably due to a failure of studies in animals
to model immune evasion strategies of pathogens (6) that occur in humans.
These strategies include rapid evolution of variable strains (7), pathogens
that coat themselves with host antigens (8), and pathogens that are
transmitted to a new host by hiding inside cells shed by the infected host
(cell vectors) (9). Furthermore, most vaccines successful in stimulating
mucosal immunity in animals contain irritating adjuvants or attenuated
pathogens, which are generally considered unacceptable for use in humans;
vaccines with human-safe adjuvants have not generated high concentrations
of protective antibody in the mucosa. Current research is investigating
improved immunogens, delivery vehicles, and adjuvants, as well as exploring
the best inductive sites for generating a protective mucosal immune
response at a specific mucosal surface (10).

In contrast to vaccines, passive immunizations can deliver protective
levels of antibodies immediately and directly to the susceptible
mucosal surface (Figure 1-top). Also, with passive mucosal immunization,
it may be possible to defeat some key immune evasion strategies by
using antibodies directed against host cell vectors, host antigens
that coat the pathogen, or receptors used by pathogens to enter
target cells (11). In addition, new methods for the sustained release
of antibodies offer the possibility of long-term protection (12).

[Fig]
Figure 1. Topical delivery of pathogen-specific MAbs can protect 
the mucosal epithelium. (Top) Protective MAbs (in this figure, 
secretory immunoglobulin A; SIgA) can be topically applied to the 
mucosa in various ways. (Bottom) In mucus, MAbs are believed to act 
by a number of mechanisms to prevent penetration of the mucous layer 
and subsequent infection of target cells (62). MAbs can trap pathogens 
in the mucous gel by forming low affinity bonds with mucin fibers 
and can agglutinate pathogens into clusters too large to diffuse 
through the mucous gel.

Efficacy of Antibodies in Preventing Disease
   
The first use of immune serum for preventing disease by passive immunization
was reported more than 100 years ago by von Behring and Kitasato (13).
Subsequently, systemic passive immunization with antibodies has been proven
effective in preventing many diseases. By binding to a pathogen,
systemically delivered antibodies can inhibit attachment to and fusion with
target cells, inhibit internalization by target cells, inhibit uncoating
inside a cell, aggregate pathogens thereby preventing them from reaching
target cells, interact with complement to lyse the pathogen, induce
phagocytosis of the pathogen, and cause killer cells to lyse the pathogen
by antibody-dependent cellular cytotoxicity (14). Table 1 lists the highest
efficacy reported for systemically delivered antibodies in preventing
disease in mammalian species and against a wide range of pathogens that
infect humans. No antiviral treatments are available for most viruses
listed in the table, yet antibodies can prevent the diseases caused by all
of these viruses.

Table 1: Examples of highly effective systemic passive immunization    
 -----------------------------------------------------------------------
                                                                   
                                  Anti-                        
                         Species  body    Preven- 
Pathogen                  (sup a) (sup b)  tion(%)  DRS(sup c)  Ref.
 -----------------------------------------------------------------------
Viruses                                                                
  Chikungunya              mou     p      100                  (15)         
  Cytomegalovirus          hum     p       50          X       (16)         
  Dengue                   mou     p      100                  (17)         
  Ebola                    bab     p       80                  (18)         
  Hantavirus               mou     m      100                  (19)         
  Herpes simplex (genital) mou     m      100          X       (20)         
                 (ocular)                                                       
                           mou     m      100                  (21)         
  HIV                      mou     m      100          X       (22)         
  Hepatitis A              hum     p       90                  (23)         
  Hepatitis B              hum     p       92                  (24)         
  Influenza                mou     m      100                  (25)         
  Lassa                    mon     p      100                  (26)         
  Measles                  mou     m      100                  (27)         
  Polio                    hum     p       58                  (28)         
  Rabies                   mou     m      100                  (29)                                                                                 antibodies
  Reovirus                 mou     m      100                  (30)         
  Rift Valley fever        ham     p      100                  (31)         
  Respiratory  syncytial   hum     m      100                  (32)         
                                   p       40                  (33)         
  Rubella                  hum     p       57                  (34)         
  Varicella zoster         hum     p      100                  (35)         
  Venezuelan equine        mou     p      100                  (36)         
   encephalomyelitis                                                   
                                                                       
Bacteria                                                               
  Borrelia burgdorferi     ham     p      100                  (37)         
  Bordetella pertussis     mou     m      100           X      (38)         
  Chlamydia pneumoniae     mou     p      100                  (39)         
  Chl. trachomatis         mou     m       90                  (40)         
  Escherichia coli         rat     m      100           X      (41)         
  Francisella tularensis   mou     p      100                  (42)        
  Group B Streptococcus    mou     m      100           X      (43)         
  Haemophilus influenzae   rat     p      100           X      (44)         
  Mycoplasma pneumoniae    ham     p       80                  (45)         
  Neisseria meningitis     mou     m       90           X      (46)         
  Proteus mirabilis        mou     m      100           X      (47)         
  Pseudomonas aeruginosa   mou     p      100           X      (48)         
  Salmonella Typhimurium   mou     p      100           X      (49)         
  Shigella flexneri        rab     p      100           X      (50)         
  Staphylococcus aureus    rab     m      100           X      (51)         
  Streptococcus pneumoniae mou     p       90           X      (52)
  Treponema pallidum       ham     p      100                  (53)         
  Yersinia pestis          mou     p      100                  (54)         
                                   m      NR(sup d)            (55)         
                                                                       
Fungi                                                                 
  Candida albicans         mou     p     > 67           X      (56)         
  Cryptococcus neoformans  mou     m       70           X      (57)        
                                                                       
Parasites                                                             
  Plasmodium falciparum    mon     p       75           X      (58)         
  Toxoplasma gondii        mou     m      100                  (59)        
-----------------------------------------------------------------------
(sup a)Species: mou=mouse; hum=human; bab=baboon; mon=monkey;                
ham=hamster; rat= rat; rab=rabbit.                                     
(sup b)Antibody: m=monoclonal; p=polyclonal.                                
(sup c)DRS=Drug-resistant strains reported (from Ref. 60).                   
(sup d)NR = not reported                                                     
                                                                        

Although less studied than systemic passive immunization, the prophylactic
use of mucosal antibodies predates the therapeutic use of immune sera.
Antibodies delivered in mother's milk have been protecting the
gastrointestinal tract of nursing infants since the mammary gland first
evolved approximately 50 million years ago. Most infections begin in
mucosal surfaces (approximately 400 m(sup 2)in an adult human); supplementing
the antibody repertoire in a mucous secretion (Figure 1-top) thus offers an
effective method for protecting a mucosal surface against pathogens to
which the host has not been exposed or become immune. In addition to the
protective mechanisms described above, antibodies delivered to mucosal
surfaces can trap pathogens in the mucous gel, make them mucophilic, and
prevent their diffusion and motility (Figure 1-bottom); as a result,
pathogens trapped in mucus are shed from the body with the normal flow of
mucous secretions or are digested if these secretions enter the digestive
tract (61-63). Topical passive immunization of mucosa can block
transmission of bacteria, viruses, fungi, and parasites that infect humans
(Table 2).

Table 2: Examples of highly effective topical passive      
immunization of mucosa                                                                                                 herpes simplex
-----------------------------------------------------------
                                       Anti-  Pre-          
                        Species Route  body   ven-          
Pathogen                (sup a) (sup b)(sup c)tion   Ref.      
-----------------------------------------------------------
Viruses                                                            
 Herpes simplex           mou      v     m    100%   (64,65)  
                                   r     m    100%      (66)      
 Influenza                fer      o     p    100%      (67)      
                          mou      n     p    >4(sup d) (68)      
 Rotavirus                hum      o     p    100%   (69,70)  
 Respiratory syncytial    mon      n     m   3-4(sup d) (71)      
                                                            
Bacteria                                                   
 Chlamydia trachomatis    mou      v     m     90%      (72)     
 Clostridium difficule    ham      o     p    100%      (73)     
 Escherichia coli         hum      o     p    100%      (74)     
 Porphyromonas gingivalis hum      o     m    100%      (75)      
 Shigella flexneri        hum      o     p    100%      (76)      
 Staphylococcus aureus    mou      n     p   3-4(sup e) (77)     
 Streptococcus mutans     hum      o     m    100%      (78)     
 Vibrio cholerae          mou      o     m    100%      (79)      

Fungi                                                      
 Candida albicans         mou      v     p    >50(sup f)(80)      

Parasites                                                                                                             
 Cryptosporidium parvum   mou      o     m     77(sup g)(81)      
 -----------------------------------------------------------                                                            
(sup a)Species tested in: mou=mouse; fer=ferret; hum=human;      
mon=monkey; ham=hamster.                                   
(sup b)Delivery route of pathogen and antibody: v=vaginal;      
r=rectal; o=oral; n=nasal.                                 
(sup c)Antibody: m=monoclonal; p=polyclonal.
(sup d) log10 reduction in virus titer.                          
(sup e) log10 reduction in cfu.                                                                                             
(sup f) % reduction in cfu.                                      
(sup g) % reduction in number of parasites.                     
                                                           
The predominant (and perhaps the most appropriate for mucosal delivery)
antibody isotype on most human mucosal surfaces is secretory immunoglobulin
A (SIgA); efficient methods for producing SIgA have been reported (82,83).
SIgA, a tetravalent dimer of monomeric IgA associated with two polypeptides
(joining chain and secretory component), is especially stable and well
suited to function in the enzymatically hostile environment that prevails
at mucosal surfaces (84). SIgA, the least phlogistic class of antibody
(84), is the least likely to induce inflammatory responses that can make it
easier for toxins and pathogens to breach the mucosal surface. Immune
exclusion of antigens, enzymes, and toxins has been repeatedly demonstrated
in vivo, and protection generally correlates with levels of SIgA antibodies
in the relevant mucous secretions. Finally, the protective role of SIgA has
been demonstrated in many systems (85).

Recent Advances in mAb Technology

Generating High-Affinity Human MAbs

Since the advent of cloning of human antibodies from combinatorial
libraries constructed from seropositive persons (86,87), generation of
fully human MAbs against human pathogens has become routine (Figure 2)
(88). For example, from a single bone marrow donor, human MAbs were
prepared against HIV, respiratory syncytial virus cytomegalovirus,
herpes simplex virus types 1 and 2, varicella zoster virus, and rubella
virus (88). MAbs can even be obtained from naive libraries prepared from
unexposed persons (if the large enough repertoire) (89); therefore,
antibodies against pathogens lethal to humans can be generated. Alternatively,
human MAbs can be generated by traditional immunization of commercially
available mice that have been genetically engineered to contain human
immunoglobulin loci in their germline (Figure 2)(90,91). 

[Fig]
Figure 2. Generation of human monoclonal antibodies. (Phage display) 
Heavy and light chain cDNA isolated from human B-cells is used to 
generate a combinatorial library in which random heavy (H) and light 
chain (L) pairings are expressed on the surface of phage. These phage 
can then be screened for antigen binding by traditional techniques 
(e.g., ELISA). Since only the antigen binding region is used in the 
phage display process, the selected clone is then placed into an 
appropriate expression vector to produce a full antibody molecule.
(Transgenics) Genetically manipulated mice have been produced with 
inactivated endogenous immunoglobulin genes, and with unrearranged 
human immunoglobulin gene segments introduced (90,91). These mice 
are then immunized with antigen, and hybridomas are produced by 
traditional routes. (See refs. 88, 89 for more technical information 
on these two methods and refs. 92, 93 for comparisons of these two methods).

Dramatic enhancement of the affinity of an mAb has been demonstrated by
molecular biologic techniques in which mutants of an antibody are generated 
and then screened for higher affinity or higher neutralization activity 
(93-95). For example, the affinity of one anti-HIV mAb has been enhanced 
420-fold, and this matured antibody neutralizes more HIV strains than the 
original mAb (94). Furthermore, expressing a mAb as a multivalent isotype, 
such as SIgA or IgM, can dramatically enhance the potency of an antibody by 
increasing the avidity (96) or agglutination activity (14). For example, an
anti-Escherichia coli IgM was 1,000-fold more effective in protecting
neonatal rats than its class-switched IgG (both in vitro and in vivo)(41).
From a commercial standpoint, a 1,000-fold increase in avidity could
translate into a 1,000-fold decrease in dose and subsequent cost. Also, a
large dose of a highly potent mAb can substantially increase the duration
of protection (97).

Production Systems

MAbs have traditionally been produced in cell culture and have been
prohibitively expensive for most preventive uses. Over the years, however,
the cost has continually dropped; MAbs are now being produced in cell
culture for $200 to $1,000 per gram (98,99). Production of MAbs has
recently been reported in both transgenic plants and animals (82,100,101).
Both of these systems are expected to lower costs dramatically. Indeed,
transgenic plants can be scaled up in agricultural fields to produce tons
of "plantibody," and plant-produced antibody is predicted to cost less than
U.S. $1/g (102). The actual cost, however, will remain unknown until
large-scale batches are produced, purified, and formulated in accordance
with Good Manufacturing Practices.

Safety and Regulatory Status  
        
More than 80 MAbs are now in clinical trials (most for cancer imaging or
therapy) and more than one quarter of these are in phase III trials (103).   
Few safety problems have been reported for systemic applications; antibodies 
are now considered "biotechnology-derived pharmaceuticals" by the U.S. 
Food and Drug Administration (FDA)enabling a more straightforward regulatory        
process than in the past (92,104). Even though MAbs have often been       
evaluated for systemic applications, only recently have they been evaluated 
in humans for mucosal applications. This new interest in mucosal           
antibodies may be partially due to the increasing recognition of the          
importance of mucosal immunity. Only two clinical trials have evaluated 
topically delivered MAbs: intranasally delivered anti-RSV in infants at 
high risk (105) and orally delivered anti-Streptococcus mutans in 
adults (106); no major adverse effects were reported in these studies.

Safety concerns, such as peptide and glycosylation immunogenicity, are
important when MAbs are delivered systemically but are likely to be of less
concern when MAbs are applied to the mucosa, a surface that has evolved to
interact with the external environment. Indeed, antibodies delivered to the
lumen of a mucosal surface have minimal interaction with circulating immune
cells. Although proteins, and even antibodies, can be absorbed through
mucosal surfaces (107,108), generally only small quantities are absorbed
(109,110). The inability of SIgA to activate complement by the classic
pathway is likely involved in maintaining the integrity of mucosal surfaces
(63); therefore, SIgA may be preferable to IgG or IgM for many mucosal
applications.

The FDA "Points to Consider" for characterization of antibodies produced in
cell-culture and transgenic animals (111) are better defined than for
characterization of antibodies produced in transgenic plants; however,
plant-derived antibodies are free of animal viruses and may therefore not
require rigorous viral inactivation processing steps. In addition, although
glycosylation patterns of MAbs produced in mammalian cell-culture and
transgenic animals are closer phylogenetically to humans than glycosylation
patterns in plants, given our repeated exposure to plant sugars in food and
personal care products, it is unlikely that any of these patterns are novel
to human immune systems (112). In fact, in a recently completed clinical
trial with repeated applications of plant-produced antibody for the
prevention of oral colonization by S. mutans, no safety problems were
encountered, nor were there any detectable human anti-plant antibody
responses (113).

Selection for resistant organisms by widespread and repeated use of
antibiotics is a serious health concern (60). Drug-resistant strains 
of a wide variety of pathogens have already been reported (Table 1). 
Antibiotic or antiviral treatment of infected persons in which 
pathogens are actively replicating provides a strong evolutionary 
selection process for developing drug-resistant pathogens. In
contrast, MAbs are less likely to create resistant organisms 
when used in a preventive context at a mucosal surface against 
a pathogen that is not yet actively replicating. Even if a
systemic infection does occur during topical use of MAbs, resistant
organisms will likely not be created since the pathogen will not be
replicating and evolving in the presence of the mAb applied to the
mucosal surface. This is in marked contrast to the settings in which
antibiotics and antiviral drugs select for resistant strains (60). 
If MAbs are used frequently on a population level, the risk of 
selecting for resistant organisms may increase. When the emergence 
of resistant strains is of particular concern, the tendency to
select mAb-resistant organisms could be minimized by using 
cocktails of mucosal antibodies directed at multiple antigenic 
targets (2,114). Because new MAbs can be produced with a rapid
turnaround time (discussed below), the emergence of an antibody-resistant
strain could be countered by producing a new mAb directed toward the
mutated epitope or another antigenic target of the resistant strain.
Indeed, the flexibility of the antibody structure to create a virtually
inexhaustible repertoire of antigen binding specificities suggests that
immunoglobulins evolved in part as a means to cope rapidly with new
pathogens.

Turnaround Time for Developing a New mAb

Since human MAbs can be identified quickly by cloning variable regions from
specific antigen-binding human lymphocytes (115) or panning combinatorial
libraries (87), antibodies could be used as a rapidly developed method for
defending against new pathogens. The time required for collecting
lymphocytes from a seropositive person, screening for an appropriate
antibody, cloning, and expressing the antibody in culture in a
well-equipped laboratory is 1 to 3 months; quantities sufficient for
protecting persons at high risk or those at the focal point of an outbreak
could be available in fewer than 6 months. High-capacity production in
quantities sufficient for broad public health application could be
available in several years, assuming that the safety of antibodies as a
class of molecules is established and an infrastructure is in place for
producing these antibodies. While in rare instances vaccines can be
developed this quickly (e.g., the 1976 influenza vaccine [5]), new
vaccines, antibiotics, and antiviral therapies usually take considerably
longer to develop. Moreover, even though passive immunization may require
repeated applications, MAbs delivered to a mucosal surface can provide
immediate protection against infection.

Potential Preventive Uses for Topically Delivered MAbs

From a public health perspective, MAbs are most promising for preventing
gastrointestinal, respiratory, and reproductive tract infections. These
infections cause almost 11 million deaths annually worldwide, accounting
for more than 50% of the deaths caused by communicable diseases and 22% of
deaths by all causes (116). Sexually transmitted diseases (STDs) accounted
for 87% of all cases reported among the top ten most frequently reported
diseases in 1995 in the United States; more than 12 million Americans are
infected with STDs each year at an estimated annual cost of more than $12
billion (117).

If a track record of safety and efficacy can be achieved, mucosal
antibodies will probably be most useful as over-the-counter products that
could reach populations not well integrated into the health-care system.
The condom, a nonmedical over-the-counter personal protection product, has
played an important preventive role in the HIV epidemic. Personal
protection provided by over-the-counter antibody-based technology could
play a similar role in future emerging disease epidemics.

Diarrheal Disease

Studies in animal models have demonstrated that orally delivered antibodies
were 100% effective in preventing rotavirus (70) and cholera (79)
infections. In humans, orally delivered bovine antibodies were 100%
effective in preventing rotavirus (118), enterogenic E. coli (74), Shigella
infection (76), and necrotizing enterocolitis (119).

For orally delivered MAbs, digestive degradation is a potential concern.
However, significant levels of functional antibody survive treatment with
pepsin at pH 2 or with a pool of pancreatic enzymes at pH 7.5 in vitro
(120). In addition, most ingested IgA in milk survives passage through the
gastrointestinal tract of infants (121); intact antibody delivered orally
with an antacid survived passage through the gastrointestinal tract of
adults (74,76). Assuming that a 10-mg dose of antibody is protective (i.e.,
assuming that the mAb is only 100-fold more potent than polyclonal
preparations [118]), the production costs for the amount of plantibody
needed for 100 days of protection could be approximately one cent (102).

Since diarrheal diseases are most prevalent in developing countries,
preventive strategies must be extremely inexpensive; therefore, MAbs
produced in plants or in the milk of animals are likely most suitable for
these countries. Because of the speed with which MAbs pass through the
gastrointestinal tract, antibodies delivered orally will need to be
delivered frequently, perhaps more than once a day. In endemic-disease
regions, MAbs could be delivered orally as a supplement with food or water.

Respiratory Disease

Animal studies have demonstrated the efficacy of nasal delivery of
antibodies for the prevention of RSV infection (71) and influenza (68). In
one study, topical application was approximately 100 times more effective
than systemic delivery (122). Another study found an anti-RSV mAb
(MEDI-493) to be approximately 100 times more effective than an equal
quantity of a polyclonal preparation (32). These results suggest that
10,000 times less anti-RSV mAb would be required for topical applications
than for systemically delivered polyclonal preparations. Protective
systemic doses of MEDI-493 are approximately 100 mg (15 mg/kg) (32), so <1
mg might suffice for protection if this mAb were applied topically.
Intranasally applied mAb has a residence half-time of a little under one
day in the monkey (71), suggesting that once-a-day applications that
deliver several-fold more than a protective dose can provide continuous
protection. MAbs for protecting the respiratory tract could be delivered in
nose drops or by aerosol once a day to those at particular risk (e.g.,
infants and the elderly during influenza season) or to everyone living near
the epicenter of an epidemic.

STDs

With the exception of hepatitis B, no vaccines are available for the 
prevention of STDs (Table 3). Until effective and safe vaccines
are developed, vaginal delivery of a cocktail of anti-STD
pathogen MAbs might make an effective new method for broad 
spectrum protection against STDs (11). In animal models, MAbs     
have been shown to protect against transmission of C. albicans, 
C. trachomatis, HSV, HIV, and syphilis (Tables 1, 2) (11).
Antibodies have been delivered experimentally to the vagina in solution,
gels, and more recently, by sustained release devices for long-term
delivery of protective MAbs (123,124). Antibodies were found to be stable
when stored in seminal fluid or cervical mucus for 48 hours at 37C (125);
no significant inactivation occurred over the pH range of the human vagina
(pH 4 to 7) for at least 24 hours at 37C (Zeitlin et al., unpub. obs.).
Since the effective half-life of antibodies applied topically depends on
the turnover time of mucus, a single vaginal application may thus provide
protection for at least 1 day, and probably several days (97). If so,
passive immunization of the vagina may extend protection to the occasional
days when the user forgets to apply the mAb. Considering there are an
estimated 5 billion acts of sexual intercourse per year in the United
States (11), large-scale production of MAbs in plants may offer the best
system for the low costs needed for such a public health initiative. In
addition, because the most common class of infection in the first month of
life is primarily caused by STD pathogens present in the birth canal (126),
the same mucosal antibodies could be used in a predelivery cervicovaginal
lavage or applied to newborns' eyes for studies in the prevention of
ophthalmia neonatorum. Indeed, in some cultures the mother's colostrum, a
fluid rich in SIgA, is applied to the newborns' eyes (127).

 Table 3: Preventive vaccines or cures for major
sexually transmitted disease pathogens
----------------------------------------------------------
Pathogen                      Vaccine Cure     DRS(sup a)
----------------------------------------------------------
Chlamydia trachomatis           no     yes
Haemophilus ducreyi             no     yes        X
Hepatitis B                     yes    no
Herpes simplex 1 and 2          no     no         X
HIV-1 and 2                     no     no         X
Human papilloma virus (HPV)     no     yes(sup b)
Neisseria gonorrhoeae           no     yes        X
Treponema pallidum              no     yes
Trichomonas vaginalis           no     yes        X
----------------------------------------------------------
(sup a)Drug-resistant strains reported.
(sup b)Surgical removal of HPV-infected tissue is performed.
HPV-related cervical cancer identified early has a high
cure rate; however, in the United States, for every three
new cases, there is approximately one death (117).

Conclusions

In animal models and human studies, antibodies have been shown to prevent a
wide variety of infectious human diseases. Recent advances allow
development of a new era of mucosal mAb-based products. These advances
include the development of combinatorial libraries for rapid selection of
human MAbs, the ability to increase dramatically the potency of a specific
mAb, and the marked reduction in the cost of cell-cultureproduced MAbs as
well as the ability to produce MAbs inexpensively and at high capacity in
transgenic animals and plants. In addition, since MAbs can be developed
considerably more rapidly than most vaccines and antimicrobial drugs, MAbs
may prove useful for combating emerging pathogens. Mucosal infections
account for a large percentage of infectious disease-related illness and
deaths; hence topical passive immunization with MAbs may offer a new
opportunity for improving public health. Finally, many of the remaining
safety issues regarding the human use of mucosal MAbs are likely to be
addressed by clinical trials now under way.

Acknowledgments

      The authors thank the Rockefeller Foundation Bellagio Study and
Conference Center and Drs. Polly F. Harrison, Mich B. Hein, and Thomas R.
Moench for their review of drafts.

      Dr. Zeitlin is a research scientist at ReProtect, LLC. His interests
focus on the development of monoclonal antibodies for contraception and the
prevention of sexually transmitted diseases.

      Address for correspondence: Kevin Whaley, 3400 North Charles St.
Jenkins Hall, Baltimore, MD 21218, USA; fax: 410-516-6597; e-mail:
whale@jhu.edu.

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Research


Research

Dual and Recombinant Infections: An Integral Part of the HIV-1 Epidemic in
Brazil

Artur Ramos,* Amilcar Tanuri, Mauro Schechter, Mark A. Rayfield,* Dale
J. Hu,* Maulori C. Cabral, Claudiu I. Bandea,* James Baggs, and Danuta
Pieniazek*
*Centers for Disease Control and Prevention, Atlanta, Georgia, USA;
Universidade Federal do Rio de Janeiro, Brazil; Emory University,
Atlanta, Georgia, USA

---------------------------------------------------------------------------
      We systematically evaluated multiple and recombinant infections
      in an HIV-infected population selected for vaccine trials.
      Seventy-nine HIV-1 infected persons in a clinical cohort study
      in Rio de Janeiro, Brazil, were evaluated for 1 year. A
      combination of molecular screening assays and DNA sequencing
      showed 3 dual infections (3.8%), 6 recombinant infections
      (7.6%), and 70 (88.6%) infections involving single viral
      subtypes. In the three dual infections, we identified HIV-1
      subtypes F and B, F and D, and B and D; in contrast, the single
      and recombinant infections involved only HIV-1 subtypes B and
      F. The recombinants had five distinct B/F mosaic patterns:
      B(sup gag-p17)/B(sup gag -p24)/F(sup pol(sup)/B(sup env) , 
	F(sup gag-p17)/B(sup gag -p24)/F(sup pol)/F(sup env) ,
      B(sup gag-p17)/B-F(sup gag -p24)/F(sup pol)/F(sup env) , 
	B(sup gag-p17)/B-F(sup gag -p24)/F(sup pol)/B(sup env)
      , and F(sup gag-p17)/B-F(sup gag -p24)/F(sup pol)/F(sup env). 
	No association was found between dual or recombinant infections 
	and demographic or clinical variables. These findings indicate 
	that dual and recombinant infections are emerging as an integral 
	part of thE HIV/AIDS epidemic in Brazil and emphasize the 
	heterogenous character of epidemics emerging in countries where 
	multiple viral subtypes coexist.

Our understanding of the global molecular epidemiology of HIV-1 infections
has improved substantially in recent years. Remarkable antigenic diversity,
especially in the viral envelope, has emerged among HIV isolates worldwide
(1). As the HIV/AIDS pandemic grows, viral strains are becoming more
geographically dispersed, and the simultaneous presence of multiple
subtypes in a given region is now common (2). Mixed infections and
recombinants involving sequences of distinct HIV-1 subtypes (mosaics) are
being recognized, but their prevalence and effect on the pandemic have not
been fully evaluated (3). Consequently, the distribution of dual infections
and mosaic viruses among different populations and the changes of this
distribution over time are still relatively unknown. In addition,
scientists are increasingly interested in possible differences in the
transmission, epidemiologic patterns, and natural history of HIV-1
infections caused by more than one viral subtype and recombinant genomes.
Finally, the efficacy of HIV-1 vaccines, primarily developed against
subtype B viruses, may differ against more divergent recombinant variants
and mixed infections of distinct HIV-1 subtypes. Such knowledge is vital to
understanding the relevant role of mixed infections as a prerequisite for
recombination and could be applied immediately in molecular epidemiology
and immunotherapy.

Studies using convenience samples first documented mixed infections caused
by viruses of subtypes B and E in Thailand (4). Subsequently, such studies
have identified cases of dual infections with subtypes B, F, C, and D in
Brazil (5-7), B and F in Puerto Rico (8), A and C in Rwanda (9), and triple
HIV-1 infection with groups O and M of different clades in a single
Cameroonian AIDS patient (10). In addition, potential dual infections have
been detected by molecular screening assays among HIV-1 infected
populations in Uganda and Kenya, where subtypes A and D coexist (11). The
consequences of HIV-1 mixed infections may profoundly influence the dynamic
of the pandemic through altered patterns of viral transmission and
pathogenesis. Moreover, the resulting genetic variation may lead to the
emergence of new HIV variants, including those with altered antigenicity
and reduced sensitivity to detection by current diagnostic assays. The
global emergence of such variants is exemplified in the impact of two HIV-1
recombinants of subtypes A/E and G/A on epidemics in Thailand and in
certain parts of Central Africa, respectively (1,12-14). Interestingly, the
presumptive parental subtypes E and G have not been identified.

Because Brazil has been selected as a World Health Organization field site
for HIV-1 vaccine evaluation programs, priority has been given to extensive
molecular examination of the prevalence and genetic diversity of HIV-1
strains circulating in the country. By November 1997, 116,277 AIDS cases
had been reported to the Brazilian AIDS Control Program of the Ministry of
Health (15). The 293 HIV-1 strains that have been molecularly characterized
document that, although four HIV-1 subtypesB, F, C, and D arecirculating
in Brazil, only subtypes B and F are common (16-21). Moreover, five HIV-1
dual infections were identified in 21 HIV-infected patients during testing
of molecular techniques that would discriminate between HIV-1 infections
caused by single and multiple subtypes (5-7). Also, two cases of B/F
recombinants have been found accidentally through sequence analysis of the
env region (22). These data only indicate the potential for dual and
recombinant infections in Brazil, where multiple subtypes circulate; they
cannot, however, be used to assess the frequency of these infections among
the HIV-infected population. In this study, we evaluated the proportion of
HIV-1 dual and recombinant infections among 79 patients enrolled in a
prospective clinical cohort study in Rio de Janeiro (an area where HIV-1
subtypes B and F are common).

The Study

Population

Part of an ongoing prospective clinical cohort study established in 1991 by
the AIDS program of the Federal University of Rio de Janeiro, the
HIV-1-infected patients have been continuously enrolled in the cohort for
consultation, treatment, evaluation of different clinical parameters during
the progression of the disease, and assessment of the genetic variation of
HIV-1 strains (23). With informed consent, blood samples were obtained from
the 79 patients, who consecutively attended the clinic between October and
December 1994. For the purposes of this study, samples collected in 1994
were compared as needed, and follow-up specimens were collected 1 year
later. The clinical profile of patients was based on the medical evaluation
at the time of blood collection. Because the patients were randomly
selected, the findings presented in this article likely reflect the trends
in the HIV/AIDS epidemic in the Rio de Janeiro area. Epi Info, Version 6
program (CDC, Atlanta, GA, USA) was used to calculate frequencies, means,
and analyses of variance of demographic information, clinical stages, and
laboratory parameters.

Design

To investigate the proportion of mixed infections, we must distinguish
between those involving two or more distinct HIV-1 strains and infection
with a single intersubtype recombinant strain. By definition, the mixed 
infection occurs when multiple phylogenetically distinct copies of the 
same gene representing different viral genomes are present within one 
patient (24). In contrast, in the mosaic strain, different viral regions 
within the same genome are classified by phylogenetic analysis into 
different subtypes (3). Potential HIV-1 mixed infections involving distinct 
HIV-1 variants were segregated from single infections caused by only 
one subtype by using a restriction fragment length polymorphism (RFLP) 
screening assay of the prt gene (5,7). The restriction map profiles of 
the viral prt allow the segregation of HIV-1 strains to subtypes A, B, 
C, D, and F (Figure 1A). AluI digestion patterns separate subtypes A, 
C, and F from subtypes B and D. Sequential restriction analysis of prt 
using HinfI, BclI, and ScaI restriction enzymes further differentiates 
among these two subtype groups. The simultaneous occurrence of more 
than one digestion pattern indicates a potential mixed infection 
(Figure 1B). We selected recombinants among RFLP-subtyped single
infections by additionally subtyping the C2-V3 env region with the
heteroduplex mobility assay (25). Subtype discrepancies between prt and env
regions were considered potential HIV-1 recombinants. All potential
multiple infections and recombinants were analyzed by sequence analysis.
The search for recombinants was further expanded by sequence analysis of
the entire p17 gag or a 311-bp of the p24 gag fragment or both among
preselected prt-env recombinants, all prt-env subtype F, and some prt-env
subtype B viruses.

[Fig]
Figure 1. Differentiation between single (A) and dual (B) HIV-1 infections 
by the restriction fragment length polymorphism analysis of the polymerase 
chain reactionamplified prt. A: Three AluI digestion patterns represent 
subtypes A, C, and F (pattern 1) and subtypes B and D (patterns 2 and 3); 
two HinfI patterns represent subtypes D (pattern 1) and B (pattern 2); 
two BclI patterns represent subtypes F (patterns 1) and A and C (pattern 2); 
two ScaI patterns represent subtypes A (pattern 1) and C (pattern 2). B: 
Two AluI digestion patterns (1 and 2) in the dually infected patient with 
HIV-1 subtypes F and B; two HinfI patterns (1 and 2) in the patient infected 
with subtypes B and D viruses.

Polymerase Chain Reaction (PCR)

Uncultured or cultured peripheral blood mononuclear cells from patients
were used for nested-PCR of the entire HIV-1 protease gene (prt, 297-bp),
p24 gag fragment (311-bp), and C2-V3 domain of env (565-bp) (6,7). The
outer primers for amplification of a 717-bp gag fragment spanning the
entire 17 gag region and a 311-bp of p24 gag were LTRF:
5'GGGCTAATTTGGTCAAAAAGAAG; nucleotide position: 6-28, HIV-1(sub MN), and P24RA:
5'ATGTCACTTCCCCTTGGTTCT; nucleotide position: 1482-1502. The inner primers
were P17F: 5'GCAAGAGGCGAGGGGCAGCAGCCG; nucleotide position: 716-739, and
P17R: 5'CCCATTCTGCAGCTTCATTGA; nucleotide position: 1413-1433. The outer
primers for amplification of a 1,444-bp fragment from p24 gag to the
reverse transcriptase region were P24F1: 5'ATAGAGGAAGAGCAAAACAAAA;
nucleotide position: 1,099 to 1,120, and MOPR1: 5'AAAATTGGAGTATTGTATGGATT;
nucleotide position: 2,724 to 2,746. The inner primers were P24FA:
5'CAAAATTACCCTATAGTGCA; nucleotide position: 1,177 to 1,196, and MOPR2:
5'GGTCCATCCATTCCTGGTTT; nucleotide position: 2,601 to 2,620. PCR conditions
were the same for amplification of all viral regions (7).

Sensitivity of Detection of Dual Infections

To determine the sensitivity of detection of dual infections by the RFLP      
assay, we mixed 5, 10, 25, 50, and 100 copies of subtypes B and F cloned      
proviral DNA in equal proportions or in ratios 1:2, 1:4, 1:10, and 1:20 for    
nested PCR amplification of prt (Table 1). For each combination, one to 
four independent PCR reactions were run. The simultaneous amplification 
of the viral prt of subtypes B and F was evaluated by the RFLP assay.
PCR controls included DNA templates of single HIV-1 subtypes. 

Table 1. Sensitivity of detection of HIV-1 dual infections caused 
by viruses of subtypes and F
-----------------------------------------------------------------
No. of viral           
  subtypes	             No. of          
 F       B        F:B  experiments    B         F            B&F
-----------------------------------------------------------------
100     100       1:1      1                               +
 50      50       1:1      4                               +
 25      25       1:1      4                   + (1)        +(3)
 10      10       1:1      4                               +
  5       5       1:1      4                   + (3)        + (1)
100       5      20:1      2                   + (1)        + (1)
100      10      10:1      2                   + (1)        + (1)
100      25       4:1      2                   +            
100      50       2:1      2                               +
  5     100       1:20     1                               +
 10     100       1:10     1                               +
 25     100       1:4      1                               +
 50     100       1:2      1                               +
100       0                3                   +            
 50       0                3                   +            
  5       0                3                   +            
 10       0                3                   +            
  5       0                2                   +            
  0     100                3          +                     
  0      50                2          +                     
  0      25                3          +                     
  0      10                3          +                     
  0       5                3          +                     
------------------------------------------------------------------
Absence (-) and presence (+) of AluI digestion pattern of prt 
characteristic for subtype B, subtype F, and combination of 
subtypes B and F (Fig. 1). The cloned proviral DNA of HIV-1
subtypes B and F spanning a 1444-bp fragment from p24 gag to 
rt was used for the nested PCR amplification of prt.
Amplified products were digested with AluI restriction enzyme, 
and the presence of two digestion patterns was analyzed on a 
10% polyacrylamide gel by ethidium-bromide staining.
                     
Cloning, Sequencing, and Phylogenetic Analysis

The PCR-amplified proviral prt sequences from potential dual infections 
were cloned by using the Original TA Cloning Kit (Invitrogen, Carlsbad, 
CA, USA). DNA from 30 clones of each specimen Was screened for distinct
HIV-1 sequences by the RFLP assay (7). Double-stranded viral DNA from 
selected clones or from direct PCR-amplified prt, p17 and p24 gag, and 
C2-V3 env products was cycle-sequenced in both directions with 
fluorescent dyelabeled sequencing terminators (26). Sequencing 
reactions were run in an automated DNA sequencer (Applied Biosystems, 
Foster City, CA, USA). The sequences were aligned by the CLUSTAL
multiple sequence alignment program (27). After gaps were eliminated, 
the aligned sequences were analyzed by the maximum likelihood method, 
with the fastDNAml program, which uses randomized data input and
global rearrangement (28). Additionally, the neighbor joining method 
(PHYLIP package version 3.5c [29]) was used, with or without
bootstrapping. The stability of the tree's topology was tested by 
pruning (removing one sequence from the alignment and rerunning the  
phylogenetic analysis). The SIV-cpz sequences (GenBank accession no. 
X52154) were used as outgroups. HIV-1 sequences generated in this  
study have been submitted to GenBank. 

Results

Detection of Mixed Infections

RFLP analysis of the prt gene showed the simultaneous presence of two
different digestion patterns in specimens from 10 of 79 patients. A complex
pattern composed of elements of AluI patterns #1 (subtypes A, C, or F) and
#2 (subtypes B or D) was identified in nine patients, and a combination of
two HinfI digestion patterns (subtypes B and D) was found in one patient
(Figure 1B). These data suggest that each of 10 patients could be infected
with multiple distinct HIV-1 subtypes. The remaining 69 samples were
classified as single infections caused by viruses of prt subtypes B (59)
and F (10).

The PCR-amplified viral prt products from these 10 patients were cloned and
sequenced to confirm the presence of mixed infections. Sequence analysis
showed the simultaneous presence of two distinct HIV-1 variants in only
three patients: BR45, BR62, and BR83. The nucleotide divergence between the
two prt sequences within the patients was 6.8% for BR45, 7.4% for BR62, and
7.1% for BR83, indicating two distinct HIV-1 variants in each of these
patients (6). Phylogenetic analysis confirmed these findings and
demonstrated that the divergent HIV-1 prt sequences segregated into
subtypes B and F (BR45), subtypes D and F (BR62), and subtypes B and D
(BR83) (Figure 2a). In the remaining seven specimens, the observed RFLP
results were consequences of either point mutations in AluI restriction
site (five cases) or G A hypermutation (two cases), which occurred across
one of the sequences within each specimen and destroyed the defining AluI
sites. These changes in AluI restriction sites gave rise to genetically
distinct quasispecies within the patients but did not represent distinct
subtypes, as further confirmed by phylogenetic analysis (e.g., BR55-1 and
BR55-2, and BR99-1 and BR99-2 in Figure 2a). Thus, despite the presence of
mixed AluI digestion patterns in these seven specimens, they were
classified as single infections of subtype B variants.

[Fig]
Figure 2. Phylogenetic classification of HIV-1 sequences from Brazilian 
patients (denoted with BR prefix). The trees were constructed on the basis 
of DNA sequences of prt (a), env (b),  gag-p24 (c), and gag-p17 (d) by the 
neighbor-joining method. Numbers at the branch nodes connected with 
subtypes indicate bootstrap values. An arrow indicates dually infected 
specimens; an asterisk shows viral sequences, which clustered into 
different lineages depending on which parts of viral genome were analyzed; 
! represents hypermutated sequences; ? represents unclassified subtype 
of p24 gag sequences. The distinct HIV-1 subtypes are delineated. 
The scale bar indicates an evolutionary distance of 0.10 nucleotides 
per position in the sequence. Vertical distances are for clarity only. 
GenBank accession numbers: prt [AF099155-99171;AF079986-79989;AF079991; 
and AF079994-79996]; env [AF113560-113576]; p17gag [AF115443-115451]; 
p24gag [AF115780-115797].

To address the issue of potential laboratory contamination, we
collected repeat blood samples from dually infected patients
approximately 1 year later and processed them on separate occasions. 
(Blood was unlikely to be contaminated during collection because a 
disposable vacutainer system was used to obtain each blood sample.) 
The sequence data from the first and second blood samples of
each person showed a 98% to 99% similarity. Also, the viral
sequences from these patients were distinct from those of laboratory
strains (Figure 2a; 855M, 8,986, and 9,001) commonly used as standards.

 Sensitivity of Detection of Dual Infections

To investigate the sensitivity of the RFLP screening method, we
performed reconstruction  experiments, in which two distinct
viral DNA templates of subtypes B and F were analyzed in the same
reaction mixture (Table 1). When equal proportions of 10 to 100 HIV-1
DNA template copies were used for prt amplification, two viral
subtypes could be simultaneously detected in all but one of 19
experiments. However, when five or fewer copies of each viral subtype were
used, two subtypes were identified in only one of four assays; in the
remaining three assays either subtype B or subtype F amplicons, but not
both, were found. Similarly, in experiments containing varying proportions
of subtypes B and F DNA templates, the simultaneous presence of two viral
subtypes could be detected in only 8 (66%) of 12 experiments. In
comparison, 5 to 100 copies of a single viral subtype (B or F) were
routinely amplified and identified in all control reactions.

Detection of Intersubtype HIV-1 Recombinants

Parallel RFLP/heteroduplex mobility assay screening for HIV-1 subtypes in
the prt and env regions identified two potential recombinants among 76
single infections. Subtype F prt and subtype B env were found in both
specimens BR43 and BR60 during this initial screening. The remaining
specimens were classified into subtypes F (n = 8) and B (n = 66) in both
prt and env regions. These prt-env potential recombinants, all subtype F
specimens, and eight selected subtype B samples were further evaluated by
sequence analysis, which confirmed the results of the screening assays
(Figure 2a, 2b).

The search for the HIV-1 recombinant genome in these 18 samples was further
expanded to the gag region. Mosaic sequences of subtypes B and F were found
within the p17 or p24 gag (Table 2, Figure 2, discussion below) in four
prt-env subtype F variants (BR46, BR57, BR59, and BR97). Similarly, in one
of two prt-env recombinants (BR60), the gag sequences had a mosaic pattern.
In contrast, the p24 gag sequences of eight prt-env subtype B specimens
were homogeneous and also classified as subtype B (Figure 2). Taken
together, the comparative molecular analysis of gag, pol, and env regions
allowed the identification of six specimens that carried HIV-1 recombinant
genomes, representing five distinct mosaic structures: B(sup gag-p17)/B(sup gag
-p24)/F(sup pol)/B(sup env) , F(sup gag-p17)/B(sup gag -p24)/F(sup pol)/F(sup 
env) , B(sup gag-p17)/B-F(sup gag -p24)/F(sup pol)/F(sup env) , B(sup gag-p17)/
B-F(sup gag -p24)/F(sup pol)/B(sup env) , and F(sup gag-p17)/B-F(sup gag
-p24)/F(sup pol)/F(sup env) .

Table 2. p17/p24 gag, prt, and C2-V3 genetic subtyping of HIV-1 DNA 
sequences(sup a) from  peripheral blood mononuclear cells collected                         
from 18 patients in Rio de Janeiro                                   
-------------------------------------------------------------------                         
                                        Genotypes                              
Specimen         No.       gag p17    gag p24     prt   C2-V3                           
------------------------------------------------------------------                         
Group 1(sup b)   8         ND(sup c)    B          B      B                             
Group 2(sup d)   4         F            F          F      F                            
(BR46)                    (NA[sup e])  (B)        (F)    (F)                            
(BR59)                    (F)          (B)        (F)    (F)                            
(BR57)                    (F)          (B/F)      (F)    (F)                            
(BR97)                    (B)          (B/F)      (F)    (F)                            
(BR60)                    (B)          (B/F)      (F)    (B)                            
(BR43)                    (B)          (B)        (F)    (B)                            
 ------------------------------------------------------------------                         
(sup a)Recombinant specimens are shown in parentheses; B/F mosaic 
structure within a 311-bp of the p24 gag fragment consisting of
subtypes B and F sequences.
(sup b)Group 1: BR34, BR52, BR55, BR64, BR65,BR71, BR75, and BR92.                       
(sup c)ND=not done.                               
(sup d)Group 2: BR41, BR54, BR58, and BR112.      
(sup e)NA = not available due to negative PCR.  

[Fig]
Figure 3. Analysis of the putative recombination within the gag 
region. The aligned sequences were classified into subtype B, 
subtype F, and recombinant subtype B/F on the basis of linearity 
of subtype assignment for the p17-p24 gag region. Asterisks show 
characteristic nucleotide patterns for subtypes B and F sequences; 
dots represent nucleotides homologous to the MN gag sequence; 
dashes indicate gaps introduced to maintain the alignment; and 
arrows indicate the potential recombination regions within the 
p24 gag fragment. The nucleotide position is marked.  
                                             
The potential crossover breakpoints within 717-bp of the p17-p24 
gag mosaic sequences were examined by comparison with nucleotide
signature patterns characteristic for subtypes B and F viruses 
(Figure 3). We performed comparative analyses with aligned DNA 
sequences of recombinants (BR57, BR59, BR60, and BR97), subtype B 
(MN and BR43), and subtype F variants (BR41, BR54, BR58, and BR112). 
This analysis confirmed an intragene recombination within p24 gag 
in specimens BR57, BR60, and BR97a finding consistent with our failure 
to phylogenetically assign these gag sequences to any known subtype 
(Figure 2c).

The putative breakpoints within the intragene recombinant sequences were
located between nucleotides 97 and 137 in sample BR57 and between
nucleotides 173 and 213 in BR60 and BR97 (Figure 3). The exact breakpoint
position could not be determined because of extensive sequence homology
between subtypes B and F in this viral region. This analysis also revealed
putative crossover breakpoints for variants BR57 and BR59 in proximity to
the coding region for the p17-p24 protein-processing site. The potential
breakpoints between the second half of gag and the beginning of pol region
in variants BR43, BR46, and BR59 are being investigated.

To examine the possibility of in vitro recombination during PCR
amplification (30), we performed PCR amplification of the long fragments
covering the gag and prt area in the endpoint- diluted lysates (31). To
ensure that endpoint PCR products were amplified from single copy
templates, we used samples only from dilutions at which 1 of 10 PCR
amplifications were productive for further sequence analysis. The
comparative analysis of the entire p17 gag, p24 gag, and prt sequences
demonstrated 98% homology between the undiluted and diluted lysatesstrong
evidence that the recombinant sequences were not a result of the PCR
amplification process.

Epidemiologic and Clinical Characteristics

The mean ages of patients infected with HIV-1 of single subtype B or F were
not different from those with dual or recombinant infections (p = 0.77)
(Table 3). In addition, the patients did not differ significantly by
gender, risk group, and clinical stage of disease (p = 0.44 to p = 0.48).
The patients infected with HIV-1 subtype B (403) and subtype F (854) did,
however, differ (p = 0.04) by mean CD4 counts. Although dates of
seroconversion were not known for all patients, the earliest HIV-positive
results were reported among patients infected with subtype B (in agreement
with previous observations that the spread of subtype B viruses occurred
earlier than other HIV-1 subtypes in Brazil [16,17]), which might explain
the difference in CD4 counts.

Table 3. Characteristics of the study population
--------------------------------------------------------------------------
                        Dual      Recombinant
                     infection     infection     Subtype B     Subtype F
Characteristic         n=3           n=6          n=66           n=4
--------------------------------------------------------------------------
Gender (Female/male)    1/2           3/3          24/42          3/1
Mean age in years       36 (30-44)    34 (25-45)   37 (23-60)     34 (26-43)
 (range)
Clinical stage(sup a)
      1                  3             5            29             4
      2                               1            17             
      3                                           14             
      4                                            6             
Mean CD4 cells          527          484           403           823
 (range)             (404-743)     (190-821)  (59-1281)(sup b)  (570-1270)
Years of 1st serologic tests
      1985-1987                                    2             
      1988-1991          2             2            30             2
      1992-1994          1             4            34             2
Heterosexual             2             5            27             4
Homosexual               1                         19             
Bisexual                                           8             
Blood transfusion		                           3             
 recipient
Intravenous drug user                              1             
Multiple factors                      1             2             
Unknown                                            6             
--------------------------------------------------------------------------
(sup a)WHO staging system [32].
(sup b)Based on data available for 54 patients.

Conclusions

HIV-1 infections caused by dual and intersubtype-recombinant genome may be
relatively common among HIV-1-infected Brazilians. Using both heteroduplex
mobility assay and RFLP screening methods, as well as sequencing, we
identified three dual (3.8%) and six recombinant (7.6%) infections
involving distinct viral subtypes among 79 HIV-1-infected persons from Rio
de Janeiro.

We chose viral prt to screen for dual infections because this highly
conserved region provides the best opportunity for simultaneous
amplification of distinct HIV-1 variants in the same PCR reaction. Proviral
prt sequences can be routinely amplified from approximately 95% of all
analyzed seropositive samples collected from the Americas, Asia, Africa,
and Europe (data not shown). Moreover, the RFLP assay of the prt gene is
convenient for screening a large number of samples (5). The detection of
B/F, B/D, and F/D dual infections is in agreement with our 1993 study among
patients from the same Rio de Janeiro cohort, which showed five dual HIV-1
infections involving subtypes F and B (one case), F and D (one case), and B
and C (three cases of familial clustering) among 21 HIV-infected persons
(5-7). Interestingly, dual infections involving HIV-1 subtype D continue to
be detected in patients from Rio de Janeiro, an area with a high
predominance of HIV-1 subtypes B and F, but rare subtype D, single
infections. Because retrospective specimens (peripheral blood mononuclear
cells) were not available for these patients, we could not confirm whether
the acquisition of mixed strains was sequential (superinfection) or
simultaneous. However, combination of subtypes F or B with rare subtype D
viruses among some dual infections may suggest that in these cases two
viral strains might be acquired through cotransmission rather than through
superinfection.

All naturally occurring HIV-1 dual infections are likely not detected by
current methods. First, quantitative differences in two distinct viral DNA
templates in the sample can lead to selective PCR amplification of only one
subtype. Second, despite targeting conserved genes such as prt, some
divergent viral strains may escape PCR amplification because of primer
mismatches. Finally, a single nucleotide mutation in the endonuclease
restriction site can abrogate the recognition pattern and distort detection
of dual infections in the RFLP analysis. These observations suggest that
the rate of HIV-1 mixed infections within this Brazilian cohort might be
even higher than 4%. Our previous findings of five dual infections among 21
patients from the same cohort support this assumption. If we take into
account these five cases, the percentage of mixed infections caused by
viruses of distinct subtypes circulating between 1993 and 1994 among 100
patients analyzed from this Rio de Janeiro cohort would increase to 8%.

The potential underestimate of mixed infections is highlighted by the
additional detection of 6 (7.6%) distinct recombinants within the cohort.
This finding is consistent with the estimated 5% to 10% intersubtype
mosaics among HIV-1 genomes in the Los Alamos database (3). Interestingly,
all recombinant or mosaic genomes described in this report involved only
subtypes B and F viral regions, although dual infections caused by other
subtypes were also circulating in this cohort. Moreover, our results
indicate that recombination between gag and pol (prt) regions is more
frequent than between pol and env and lead to speculation that such stable
B/F mosaics have selective advantage. Such observations support the
assumption that recombination within the gag gene occurs more often than
within other viral regions (33) and emphasize the need for rapid subtyping
methods specific to the gag region.

To investigate the potential impact of dual and recombinant infections on
the clinical status of patients, we compared the clinical and demographic
characteristics of these patients with those of patients infected with one
nonrecombinant viral subtype. Epidemiologic information for all 79 patients
was available only at the first draw of blood. Although the results did not
show significant differences between the two groups, the possibility of
differences exists.

Our findings provide the first baseline measure of the range of HIV
variability in Rio de Janeiro from 1993 to 1994 and the proportion of dual
and recombinant infections among the HIV-infected Brazilian population.
Future systematic molecular epidemiologic surveys of HIV heterogeneity in
Rio de Janeiro may show the potential changes in the molecular profile of
the HIV/AIDS epidemic over time; the laboratory tools we used to identify
single, dual, and recombinant infections may be useful in such
investigations. Nevertheless, our study on genetic variation of HIV-1
subtypes among blood donors from the state of Rio de Janeiro documented the
presence of mosaic viruses of subtypes B and F and subtypes B and D in
blood units collected in 1996 (34). Moreover, recent genetic analysis of
viral strains collected in 1997 from HIV-1-infected patients living in
Manaus (a city in Brazil's Amazon region) showed the presence of dual
infections and recombinants caused by subtypes B and F viruses (A. Tanuri,
pers. comm.). Therefore, these data indicate that the heterogenic pattern
of HIV-1 infections, first observed in Rio de Janeiro, also exists in other
regions of Brazil.

Our findings indicate that mixed infections and mosaic viruses may be more
common in worldwide epidemics than previously thought and, therefore, may
provide a basis for developing AIDS vaccines and predicting the global
evolution of HIV.

Acknowledgments

      We thank Priscilla Swanson for her help in sequencing and Renu Lal
for critical review of the manuscript.

      Artur Ramos is a graduate fellow at the Federal University of Rio de
Janeiro, Rio de Janeiro, Brazil. His laboratory research focuses on HIV-1
genetic variability worldwide. His research interests include application
of molecular biology techniques to epidemiology of HIV.

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

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Research

Genetic Diversity and Distribution of Peromyscus-Borne Hantaviruses in
North America

Martha C. Monroe,* Sergey P. Morzunov, Angela M. Johnson,* Michael D.
Bowen,* Harvey Artsob, Terry Yates, C.J. Peters,* Pierre E. Rollin,*
Thomas G. Ksiazek,* and Stuart T. Nichol*
*Centers for Disease Control and Prevention, Atlanta, Georgia, USA;
University of Nevada, Reno, Nevada, USA; Laboratory Centre for Disease
Control, Federal Laboratories, Winnipeg, Manitoba, Canada; and University
of New Mexico, Albuquerque, New Mexico, USA

---------------------------------------------------------------------------
      The 1993 outbreak of hantavirus pulmonary syndrome (HPS) in the
      southwestern United States was associated with Sin Nombre
      virus, a rodent-borne hantavirus; The virus' primary reservoir
      is the deer mouse (Peromyscus maniculatus). Hantavirus-infected
      rodents were identified in various regions of North America. An
      extensive nucleotide sequence database of an 139 bp fragment
      amplified from virus M genomic segments was generated.
      Phylogenetic analysis confirmed that SNV-like hantaviruses are
      widely distributed in Peromyscus species rodents throughout
      North America. Classic SNV is the major cause of HPS in North
      America, but other Peromyscine-borne hantaviruses, e.g., New
      York and Monongahela viruses, are also associated with HPS
      cases. Although genetically diverse, SNV-like viruses have
      slowly coevolved with their rodent hosts. We show that the
      genetic relationships of hantaviruses in the Americas are
      complex, most likely as a result of the rapid radiation and
      speciation of New World sigmodontine rodents and occasional
      virus-host switching events.

Hantaviruses, rodent-borne RNA viruses, can be found worldwide. The Old
World hantaviruses, such as Hantaan, Seoul, and Puumala, long known to be
associated with human disease, cause hemorrhagic fever with renal syndrome
of varying degrees of severity (1). After hantavirus pulmonary syndrome
(HPS) was discovered in the southwestern United States in 1993 (2-4),
intensive efforts were begun to detect and characterize hantaviruses in
North America and determine their public health importance (5). As of
January 1999, 205 HPS cases had been confirmed in 30 states of the United
States, and 30 cases had been confirmed in three provinces of Canada; most
cases occurred in the western regions of both countries. While Sin Nombre
virus (SNV) has been identified as the cause of most HPS cases in North
America, an increasingly complex array of additional hantaviruses has
appeared (Table 1).

 Table 1. Hantaviruses in the New World
 ---------------------------------------------------------------------------

 Virus(sup a)Disease(sup b)Known or suspected host   Location       Virus
                                                                   isolate
 ---------------------------------------------------------------------------
 Sigmodontinae
 associated
                                                      West &
   Sin Nombre HPS      Peromyscus maniculatus         Central         Y
                       (grassland form)               U.S.and
                                                      Canada
                                                      Eastern U.S.
 Monongahela  HPS      P. maniculatus (forest form)   and Canada      N
       New             P. leucopus (eastern
 York         HPS      haplotype)                     Eastern U.S.    Y
       Blue
 River                 P. leucopus (SW/NW haplotypes) Central U.S.    N

   Bayou      HPS      Oryzomys palustris             Southwestern    Y
                                                      U.S.
   Black Creek         Sigmodon hispidus (eastern
 Canal        HPS      form)                          Florida         Y

   Muleshoe            S. hispidus (western form)     Southern        N
                                                      U.S.
   Cao
 Delgadito             S. alstoni                     Venezuela       Y

   Andes      HPS      Oligoryzomys longicaudatus     Argentina       Y
                                                      and Chile

      Oran    HPS      O. longicaudatus               Northwestern    N
                                                      Argentina
                                                      Central
 Lechiguanas  HPS      O. flavescens                  Argentina       N

      Bermejo          O. chacoensis                  Northwestern    N
                                                      Argentina

      Hu39694 HPS      Unknown                        Central
                                                      ArgentinaN

   Pergamino           Akadon azarae                  Central         N
                                                      Argentina

   Maciel              Bolomys obscurus               Central         N
                                                      Argentina
   Laguna                                             Paraguay and
 Negra        HPS      Calomys laucha                 Bolivia         Y
   Juquitiba  HPS      Unknown                        Brazil          N

   Rio Mamore          O. microtis                    Bolivia and     Y
                                                      Peru
   El Moro                                            Western U.S.
 Canyon                Reithrodontomys megalotis      and Mexico      N
   Rio Segundo         R. mexicanus                   Costa Rica      N
 Arvicolinae
 associated
   Prospect
 Hill                  Microtus pennsylvanicus        N. America      Y

 Bloodland             M. ochrogaster                 N. America      N
 Lake
      Prospect         M.
 Hill-like             pennsyl./montanus/ochrogaster  N. America      N

   Isla Vista          M. californicus                Western U.S.    N
                                                      and Mexico
 Murinae
 associated
   Seoul      HFRS     Rattus norvegicus              Worldwide       Y
 ---------------------------------------------------------------------------
 (sup a)Major virus types or species are in bold and indented below the rodent
 subfamilies with which they are associated; related genetically distinct
 virus lineages that may represent additional species or subspecies are
 indented below virus types and species.
 (sup b)HPS = hantavirus pulmonary syndrome; HFRS = hemorrhagic fever with 
 renal syndrome.

Surveys of rodents for hantavirus antibody have shown hantavirus-infected
rodents in most areas of North America (3;6-9; Ksiazek et al., unpub. data;
Artsob et al., unpub data). Serologic evidence of hantavirus infection has
been found in North American rodents of the family Muridae. Most North
American hantaviruses are associated with the subfamily Sigmodontinae; only
a small number are associated with the subfamilies Arvicolinae or Murinae.
To determine the number and distribution of hantaviruses in North America,
we conducted a nucleotide sequence analysis of a polymerase chain reaction
(PCR) fragment amplified from a large number of representative HPS patient
and hantavirus-infected rodent samples from throughout the region. We
focused on the North American viruses (particularly those associated with
Peromyscus species rodents), although the nucleotide sequences of many
hantaviruses from South America and elsewhere were included as outgroups to
increase the resolution of the analysis.

Genetic Detection and Phylogenetic Analysis of New World Hantaviruses

The nucleotide sequences of 139 bp fragments of the G2 encoding region of
virus M segments amplified by reverse transcriptase-PCR (RT-PCR) from 288
hantavirus-infected rodent and human samples were compiled from Genbank
sources or from data reported here. Details of the specimen selection and
methods of genomic analysis are provided in the Appendix. The Genbank
accession numbers of those sequences published earlier (bigtree.xls) can be
accessed here. The entire aligned dataset (bigtree.nex), including 130
newly presented sample sequences, is also available here. These sequences
include those derived from 229 SNV-like viruses associated with Peromyscus
species rodents from throughout North America. Maximum parsimony analysis
of the aligned sequences was conducted with PAUP (12; Appendix), which
resulted in a reasonably well-defined tree topology with several distinct
lineages of SNV-like viruses and other clearly discernable hantaviruses
(Figures 1, 2). Bootstrap analysis showed that while several of the major
nodes of the tree were not well supported (values of 50% or less), many
others were robust (values of 70% or higher) (Figures 1, 2). In most
phylogenetic analyses, bootstrap values provide highly conservative
estimates of the probability of correctly inferring the corresponding
clades (13). Bootstrap values of 70% or higher corresponded to a
probability of 95% or higher that the corresponding clade was correctly
identified. Values of 50% or lower corresponded to a probability of 65% or
lower that the clade was correctly identified (13).

[fig 1]
Figure 1. Overall hantavirus phylogenetic tree based on analysis of a 139 
nucleotide fragment of the G2 coding region of the virus M segment. All 
newly reported sequences are shown bolded. The three virus groups 
corresponding to the hantaviruses carried by rodents of the subfamilies 
Murinae, Sigmodontinae and Arvicolinae are indicated. P indicates the clade 
containing the Sin Nombre- like viruses found in Peromyscus species rodents, 
the details of which are shown in figure 2. Horizontal branch lengths are 
proportional to the nucleotide step differences between taxa and predicted
nodes. No scale bar is indicated as the actual number values are arbitary 
due to the weighting used in the successive approximations method (see 
appendix for details). Bootstrap values greater than 50% are indicated above 
branches. Virus labels include the virus or virus lineage name (ISLA, Isla 
Vista; TULA, Tula; PH, Prospect Hill or Prospect Hill-like; KBR, Khabarovsk; 
PUU, Puumala; SN, Sin Nombre; ELMC, El Moro Canyon; CDG, Cao Delgadito;
BCC, Black Creek Canal; BAY, Bayou; JUQ, Juquitiba; AND, Andes; LN, Laguna 
Negra; THAI, Thailand; DOB, Dobrava; HTN, Hantaan; SEO, Seoul), species source 
of material (Mcalif, Microtus californicus; Mross, Microtus rossiaemeridionalis; 
Marv, Microtus arvalis; Mpenn, Microtus pennsylvanicus; Mmont, Microtus montanus,
Mochro, Microtus ochrogaster; Mfort,Microtus fortis; Cg, Clethrionomys
glareolus; Rm, Reithrodontomys megalotis;Salst, Sigmodon alstoni; Shisp, 
Sigmodon hispidus; Op, Oryzomys palustris; Cal, Calomys laucha; Bi, Bandicota 
indica; Apfl, Apodemus flavicollis; Apag, Apodemus agrarius; Rn Rattus 
norvegicus), identifier, and state, region or country of origin. For spreadsheet 
containing the details of all samples, click on (bigtree.xls)

[fig 2]
 Figure 2. Phylogenetic tree of hantaviruses associated with Peromyscus
 species rodents. Figure provides a detailed view of clade P in figure 1.
 S indicates clade containing classical SN virus samples detected in
 humans or P. maniculatus. See figure 1 legend for overall tree
 description. Additional species source of material abbreviations
 include: Pm, Peromyscus maniculatus; Pl, Peromyscus leucopus, Prg.fasc,
 Perognathus fasciatus; Tam.quad, Tamias quadrimaculatus; Pt, Peromyscus
 truei; Mus musc., Mus musculus, and Tam.dors., Tamias dorsalis. Samples
 from historic materials are followed by an H.

Diversity of New World Hantaviruses

As expected on the basis of earlier nucleotide sequence analysis of a
limited number of complete S or M hantavirus genome segments or virus
genome fragments (5), the evolutionary relationships among hantaviruses
were closely correlated with those of their known or suspected primary
rodent reservoirs (Figure 1; Table 1). Hantaviruses associated with
subfamily Murinae rodents (Hantaan, Dobrava, Seoul, and Thailand viruses)
are clearly separated from those associated with Arvicolinae and
Sigmodontinae rodents. The Arvicolinae-associated viruses (Puumala,
Khabarovsk, Tula, Isla Vista, Prospect Hill [PH], and PH-like viruses])
form a reasonably well-supported clade, but the phylogenetic position of
this group relative to the Murinae- and Sigmodontinae-associated viruses is
not well resolved.

The New World hantaviruses of the Arvicolinae group, primarily associated
with Microtus species voles, include not only the classic PH virus (labeled
PHV-1), originally isolated from M. pennsylvanicus in Maryland (14,15), and
two other distinct PH-like virus lineages recently found in this vole
species in North Dakota (R737 and R731; R742), but also Isla Vista virus in
M. californicus, PH-like hantavirus lineages in M. ochrogaster in North
Dakota (R812 and R789), and M. montanus in Wyoming and Nevada (3485;
LY-R2312) (16,17). Virus phylogenetic placement is not clearly correlated
with Microtus species of origin, indicating that either spill-over
infection or host switching may occur with these viruses. An apparent
example exists in the Ohio rodent samples of spill-over of a PH-like virus
infection from Microtus species rodents to a deer mouse Peromyscus
maniculatus (Pm1047). These viruses have not been associated with HPS
cases.

The viruses associated with the subfamily Sigmodontinae rodents are highly
diverse and are made up of several distinct viruses and lineages in North
and South America. All viruses associated with Peromyscus species rodents
form a well-supported distinct monophyletic clade (labeled P in Figure 1);
these viruses constitute the major cause of HPS cases in North America.
Other HPS-associated viruses in this group include Black Creek Canal virus,
associated with Sigmodon hispidus. This virus, the cause of a single HPS
case, has been genetically detected in cotton rats throughout southern
Florida but, so far, nowhere else in the United States. Another genetically
distinct virus, Muleshoe virus, has been identified in S. hispidus from the
western part of its range (18), but sequences were not available for
comparison at the time of our analysis. Cao Delgadito virus, found in S.
alstoni in Venezuela (19), appears to be monophyletic with Black Creek
Canal viruses. However, bootstrap support for this relationship is low
(lower than 50%). Reasonable support is found for the clade containing both
these Sigmodon sp.-associated viruses and the Bayou viruses, present in
Oryzomys palustris throughout the southeastern United States from the
Atlantic coast to Texas (20-22). Bayou viruses have been associated with
three HPS cases (20-22). El Moro Canyon virus has been found in numerous
harvest mice (Reithrodontomys megalotis) throughout the southwestern United
States but has also been found in other rodents (e.g., WA-R2025, in M.
montanus), presumably indicating spill-over infections (16,18,23,24). So
far, these viruses have not been associated with human disease. The current
phylogenetic analysis places these viruses in a distinct supported clade.

We analyzed hantaviruses that are also associated with HPS cases in South
America and form a well-supported clade that encompasses viruses from
Brazil, Argentina, and Paraguay, including the original Juquitiba virus
detected in a human autopsy sample from an HPS patient in Brazil in 1993
(25-27). The rodent host for this virus is unknown. Two additional
hantavirus lineages have been detected in more recent Brazilian HPS cases
(Johnson and Nichol, unpub. data), suggesting that at least three
genetically distinct hantaviruses are associated with HPS cases in Brazil.
One of these lineages (b9618005) is phylogenetically closer to the Andes
virus found in Argentina (28). Andes virus has recently been associated
with several HPS cases in Patagonia; its likely host is Oligoryzomys
longicaudatus (5,28,29). Finally, Laguna Negra viruses form a
well-supported monophyletic lineage. This virus, associated with a large
HPS outbreak in the Chaco region of Paraguay, is found in Calomys laucha
rodents (10,30).

SNV-Like Viruses of Peromyscus Species Rodents

We analyzed 229 SNV-like viruses associated with Peromyscus species
rodents; they form a well-supported (83%) clade (labeled P in Figure 1;
details shown in Figure 2) and are distinct from other
Sigmodontinae-associated hantaviruses. These SNV-like viruses include many
classic SNVs, which are the major causes of HPS cases throughout the
western and central United States and Canada, and are primarily associated
with P. maniculatus. These viruses form a distinct, well-supported (78%)
clade (labeled S in Figure 2), separate from other SNV-like viruses (Figure
2). Classic SNV 139 bp G2 fragments show up to an 18% nucleotide sequence
divergence. Despite a number of exceptions, different genetic variants of
SNV are grouped, generally speaking, by geographyan approximate geographic
progression is apparent from the north and west toward the south and east,
from the top of the tree down toward the node connecting these SNVs
(labeled S in Figure 2). For instance, all samples from western Canada,
including the Yukon, British Columbia, Alberta, Saskatchewan, and Manitoba
are in the upper portion of this clade; two major lineages in California
and Nevada (16,31) are also in this clade region. The lower part of the
clade is dominated by viruses associated with the original Four Corners
outbreak (New Mexico, Colorado, Utah, and Arizona) and other viruses from
the Southwest, such as Kansas and Texas. Human HPS cases are represented
throughout the SNV clade, indicating that these SNV variants can be
associated with HPS illness.

In addition to recent samples, 30 SNV-like virus samples from the 1980s
were included in the analysis to examine stability of the various SNV
genetic lineages and their distribution (labeled H in Figure 2). Only small
numbers of nucleotide differences, if any, were observed between old and
recent virus sequences from the same geographic areas. The most striking
example is the detection of identical viral G2 fragment sequences in
rodents captured 12 years apart in New Mexico (Pm434) and Arizona (Pt AZ
R29). Similarly, identical viral G2 sequences were found in rodents
captured in eastern California in 1983 (our Pm435 and the previously
published Sweetwater Canyon sequence [32]) and in human and rodent
materials from eastern California and western Nevada sampled 10 or more
years later (e.g., Humans CAH19 and NY-H575, and Pm LY-758, 786, and 792).
Other examples include 1 of 139 and 2 of 139 nucleotide sequence
differences between Washington rodent Pm432 (captured in 1980) and Pm206
and HPS case 0669 (sampled 16 years later), respectively; only 2 of 139
nucleotides are different between Pm428 from southern Oregon and Pm
LY-R2302 from northern Nevada, despite capture 12 years apart. These and
other data (6,7,32,33) suggest that SNV has been present in North America
for a considerable time and has been relatively stably maintained in rodent
populations.

The next most closely related viruses are those detected in the
northeastern United States, referred to as New York virus (34). These
viruses have been detected in two human HPS cases and in P. leucopus in New
York and Rhode Island (Figure 2). The 139 nucleotide fragments of these
viruses have up to 10.1% nucleotide variation, and they differ from classic
SNVs by at least 11.5% at the nucleotide level. The next closest group
contains viruses associated with several "forest form" subspecies of P.
maniculatus throughout the eastern United States and Canada, including the
cloudland deer mouse (P. maniculatus nubiterrae), which inhabits the
Appalachian mountain region (35). These viruses can also be found in some
P. leucopus in this region (e.g., rodent Pl 313 from Pennsylvania). Up to
17.3% nucleotide variation can be seen among the 139 nucleotide fragments
of these viruses. The name Monongahela has been suggested for this virus
lineage (36), which differs from New York and SN viruses by at least 8.6%
and 10.8% nucleotide differences, respectively. Another distinct hantavirus
lineage can be seen in P. maniculatus in Tennessee and has been associated
with an HPS case (0027) in eastern North Carolina. These viruses are 7.9%
different from one another at the nucleotide level for the 139 nucleotide
fragment analyzed, and at least 12.2%, 14.4%, and 15.8% different from New
York, Monongahela, and SN virus lineages, respectively. Additional distinct
virus lineages, recently referred to as Blue River virus (37), can be
detected in P. leucopus in Oklahoma (Pl 707), Indiana (e.g., Pl 9436372 and
Pl 9436378), and Missouri (e.g., Pl 170). The Oklahoma lineage virus is
10.1%, 10.8%, 15.8% different from the viruses in the Missouri, Indiana,
and Tennessee lineages.

In addition to identifying the distinct SNV-like viruses and virus genetic
lineages throughout North America, our study provides data suggesting the
likely site of infection and minimum incubation time for some HPS cases. As
reported earlier (2), the HPS case labeled CO H5 was originally described
as an Arizona case because the person was residing near Springerville,
Arizona, when the illness began. However, the person had been living in
Hesperus, Colorado, 11 days before disease onset. The PCR fragment
amplified from the case autopsy specimen and from the P. maniculatus
trapped at the household in Hesperus matched exactly and differed from
those amplified from P. maniculatus in the Arizona location (Figure 2).
Similarly, a patient (labeled human 0038) whose symptoms began in Los
Angeles, California, had been in the Santa Fe, New Mexico, area 28 to 35
days before illness onset. Analysis of PCR fragments linked the source of
infection to New Mexico, rather than to California (Figure 2).

Virus and Host Genetic Relationships and Evolution

The genetic data we present indicate a broad spectrum of genetic variants
of SNV-like viruses throughout North America, associated primarily with
Peromyscus rodents. Recent analysis of rodent mitochondrial DNA sequence
differences suggests that the different SNV-like virus lineages are
primarily associated with different Peromyscus species, and in some cases,
with phylogenetically distinct subspecies or mitochondrial DNA haplotypes
(Morzunov and Nichol, unpub. data; 37). For instance, the classic SNV and
the Monongahela virus lineages are found associated with the "grassland
form" and "forest form" of P. maniculatus, respectively (they represent
different subspecies and appear phylogenetically distinct with respect to
their mitochondrial DNA [Morzunov and Nichol, unpub. data]). The New York
virus, and the Blue River virus lineages found in Indiana and Oklahoma,
appear associated with genetically distinct P. leucopus populations (37).
This pattern likely reflects microadaptation of the virus to the rodent
host and not just geographic isolation of the virus variants. This view is
supported by the observation that even in areas such as the eastern United
States (particularly the Appalachian Mountain region), where P. maniculatus
(forest form) and P. leucopus (eastern form) are sympatric and share
microhabitat, extensive virus mixing between species is not seen; the
Monongahela virus lineage is found predominantly in P. maniculatus, and the
New York virus lineage in P. leucopus. Such data suggest that the broad
correlation clearly evident between virus evolutionary relationships and
those of their primary rodent reservoirs likely exists even at the finer
level of closely related species and subspecies. However, the fact that the
P. leucopusassociated New York virus appears phylogenetically closer to
the P. maniculatusassociated viruses (SN and Monongahela) than to other P.
leucopusassociated viruses (Blue River) suggests that this coevolutionary
relationship is not absolute and that some species jumping (host-switching)
may also have occurred. While the exact phylogenetic relationship of the
SNV lineages to Monongahela, New York, and the other P. leucopus virus
lineages is not well resolved by using the 139-bp G2 fragment we analyzed,
analysis of more complete sequence data strongly supports a similar
topology, placing New York virus firmly within the clade of P. maniculatus
borne viruses (37). This evidence, together with significant spill-over
infection that sometimes occurs between sympatric rodents, illustrates the
complexity of the hantavirus-host interactions.

This observation leads into another area of complexity, namely, the
definition of distinct hantavirus serotypes or species. In the past, a
newly identified arbovirus would be considered a distinct virus or virus
serotype if a fourfold or greater two-way difference between this virus and
previously recognized closely related viruses was obtained in virus
neutralization assays. Despite the obvious biologic limitation (a single
amino acid change can allow virus to escape from neutralization), this
traditional criterion correlates remarkably well with more recent molecular
data. One problem is that hantaviruses are generally difficult to isolate
in tissue culture and are frequently noncytopathic, often making plaque
assay analysis impractical (Table 1).

An attempt to define distinct virus species by using more widely used
general criteria for the definition of biologic species is under way. Most
defined species could be described as the lowest taxonomic unit that is
geographically and ecologically contained, reproductively isolated,
phylogenetically distinct, and self-sufficient. The apparent long-term
maintenance and coevolution of phylogenetically distinct hantaviruses with
different primary rodent reservoir species provides a foundation on which
to build a hantavirus species definition. That is, if little host switching
has occurred and if instead hantaviruses are associated with specific
primary rodent reservoir species for many thousands of years,
identification of a hantavirus in a unique primary rodent reservoir species
would strongly suggest that in further analyses (e.g., nucleotide and amino
acid sequence, cross-neutralization), it will be found to represent a new
virus species. Hantaviruses maintained in rodent hosts from different
genera (e.g., SNV in Peromyscus species rodents compared with Black Creek
Canal virus in Sigmodon species rodents) will clearly meet the broad
criteria for separate species status. This view is reinforced by recent
data showing that stable reassortant viruses of different SNV genetic
lineages can be readily detected in nature (31,38) and in tissue-culture
mixed infections (39), but not in virus pairs such as SNV and Black Creek
Canal virus (39). Difficulty can arise when trying to determine the species
status of viruses maintained within rodent hosts of the same genera or
species. So far, SN, New York, Monongahela, and Blue River viruses have
been suggested as distinct hantaviruses with independent species names
(5,36,37). The genetic analysis we present suggests that, as more
hantavirus-infected Peromyscus species samples are analyzed, it is
increasingly difficult to draw clear lines separating these virus species.
The decision regarding whether to lump these viruses together as SNV-like
viruses or to split them into separate species status will require the
availability of neutralization data for several representatives of each
virus, more detailed identification of the virus-host relationships, and
more complete genetic characterization of both viruses and their hosts.

Appendix

Rodent and HPS Case Materials

The newly described nucleotide sequences were derived from rodent materials
collected as part of a nationwide survey of rodents for hantavirus
antibodies (Ksiazek et al., unpub. data). Most of the human HPS-case blood
and tissue autopsy samples were obtained and examined during the original
investigation of an HPS outbreak in the Four Corners area of the
southwestern United States in 1993 and as part of national surveillance for
hantavirus disease throughout the United States from 1993 to 1997. Canadian
rodent and HPS case materials were provided by the Laboratory Centre for
Disease Control, Canada. Historic rodent samples were obtained from the
Division of Biological Materials of the Museum of Southwestern Biology
(Albuquerque, NM), University of New Mexico.

RNA Extraction, RT-PCR Amplification and Sequencing

Total RNA was extracted from human and rodent tissues, blood, or serum
(2,10). Because of the hazardous nature of the virus, homogenization of
rodent and human autopsy materials and extraction of RNA were performed in
a certified class IIb laminar flow biosafety hood in Biosafety Level 3
containment. RNA was extracted from tissue or blood products by using acid
guanidinium thiocyanate and phenol-chloroform and purified by using the
RNaid Kit (Bio 101, La Jolla, CA). Nested RT-PCR assays were used to
amplify DNA products containing a small fragment of the G2 coding region of
M segment (2,10). Rodent and human samples were amplified separately, and
all manipulations that might result in possible cross-contamination of
samples were avoided. PCR products of correct size were sequenced with the
same primers used for second-round PCR amplification in conjunction with
various generations of sequencing kits available from Applied Biosystems,
Inc. (Perkin Elmer, Foster City, CA). Sequences 139 nucleotides in length
determined from each PCR product were used in phylogenetic analysis.

Oligonucleotide Primer Design

Oligonucleotide primers were used to generate DNA fragments from the G2
region of hantavirus M RNA (Table 2). In the initial phase of this project,
amplification of hantavirus sequences from autopsy tissues of fatal HPS
cases and hantavirus antibody-positive rodents in the southwestern United
States used primers designed on the basis of nucleotide sequences conserved
among PH and Puumala viruses (2). On the basis of SNV nucleotide sequences
derived from these materials, new primers were designed and optimized for
detection of SNV-like viruses associated with P. maniculatus (11). As more
sequence data became available, additional generations of primers were
refined that would detect hantaviruses from other geographic regions of the
United States. The development of broadly reactive primers designed to
detect hantaviruses associated with subfamily Sigmodontinae rodents (10)
has eliminated the effort of amplifying RNA samples with many sets of
primers.

 Table 2. PCR and sequencing primers

                                                                   Basis of
                                                                primer design  Amplicon
 1st-round primers (5' to 3')     2nd-round primers (5' to 3')      (ref.)       size

 TTTAAGCAATGGTG(C/T)ACTAC(T/A)AC  AGAAAGAAATGTGCATTTGC          Puumala/         278
                                                                Prospect Hill/

 CCATAACACAT(A/T)GCAGC            CCTGAACCCCATGC(A/T/C)CCATC    Arvicolinae      (2)

 TTTAAGCAATGGTG(C/T)ACTAC(T/A)AC  AAGGTAACACAGT(G/C)TCTGGATTC   Sin Nombre/      185
                                                                Western

 CCATAACACAT(A/T)GCAGC            GGTTATCACTTAGATC(C/T)TGAAAGG  U.S. 1st         (2)
                                                                generation

 AGAAAGATCTGTGGGTTTGC             AAGGTAACACAGT(G/C)TCTGGATTC   Sin Nombre/      185
                                                                Western

 CCTGAACCCCAGGCCCCGT              GGTTATCACTTAGATC(C/T)TGAAAGG  U.S. 2nd         (11)
                                                                generation

 TGTGTGTTTGGAGACCCTGG             ATGTCAACAAC(A/G)AGTGGGATG     Sin Nombre       185
                                                                Nevada/

 TC(A/G)ATAGATTGTGTATGCA          CATGGGTTATCACTTAG(G/A)TC      E. California    (31)

 CAGAAAGATCTGCGGGTTTGC            CAAGGGAATACTGTCTCTGGATTT      Bayou            185
                                                                virus/LA/

 CCCGAGCCCCATGCACCAT              GATTGTCACTCAGATCTTGAAATG      East Coast/
                                                                S. American
                                                                (19,20)

 TGTGAITATCAAGGIAAIAC             TGTGAITATCAAGGIAAIAC          General          242
                                                                Sigmodontinae

 ACIG(A/T)IGCICCATAICACAT         CCCCAIGCICCITCAAT             (10)

Nucleotide Sequence and Phylogenetic Analyses

Compilation, alignment, and comparative nucleotide sequence analysis was
carried out by using the Wisconsin Sequence Analysis Package, version 8.1
(Genetics Computer Group, Inc., Madison, WI) on a DEC 3000-500X AXP
workstation (Digital Equipment Corp., Maynard, MA). Phylogenetic analysis
was performed by maximum parsimony analysis using PAUP version 4.0 d52 (12)
on a Power PC 9500. The size and complexity of the dataset prevented the
use of branch and bound search method and weighting of the data matrix
based on transition:transversion bias. Maximum parsimony analysis of the
hantavirus G2 fragment nucleotide sequences was carried out by using the
heuristic search option. The initial unweighted analysis showed
considerable homoplasy within the dataset. A successive approximations
method was used in which characters were weighted by using the maximum
value of their rescaled consistency index (12), and the heuristic search
repeated. Bootstrap analysis was carried out by 500 replicates of the
heuristic search with random resampling of the data. The analysis required
several months of computer time; thus, it was not possible to include some
recently published additional hantaviruses sequences. The nucleotide
sequence dataset (bigtree.nex) we used is available in NEXUS format
(compatible with most phylogenetic analysis software packages). A brief
description of all samples analyzed is available in  EXCEL 2.1 spreadsheet
format (bigtree.exl). The phylogenetic tree of Figures 1 and 2 can also be
found at the same location on the website.

Acknowledgments

      We thank Ali Khan, Jim Mills, Jamie Childs, John Krebs, Tom Mather,
Joe Camp, Fred Jannett, and many others for their efforts in collecting
hantavirus infected materials. We also thank Kent Wagoner and Laura Morgan
for assistance with graphics and database manipulations.

      Ms. Monroe is a biologist with the Special Pathogens Branch, Division
of Viral and Rickettsial Diseases, CDC. Her research focuses on molecular
phylogenetics of hantaviruses and reverse genetic studies of Ebola virus.

      Address for correspondence: Stuart Nichol, Centers for Disease
Control and Prevention, Special Pathogens Branch, Mail Stop G14, 1600
Clifton Rd., N.E., Atlanta, Georgia 30333, USA; fax: 404-639-1118; e-mail:
stn1@cdc.gov.

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Hantavirus

Climatic and Environmental Patterns Associated with Hantavirus Pulmonary
Syndrome, Four Corners Region, United States

David M. Engelthaler,* David G. Mosley,* James E. Cheek, Craig E. Levy,*
Kenneth K. Komatsu,* Paul Ettestad, Ted Davis, Dale T. Tanda, Lisa
Miller, J. Wyatt Frampton, Richard Porter,* and Ralph T. Bryan#
*Arizona Department of Health Services, Phoenix, Arizona, USA; Indian
Health Service, Albuquerque, New Mexico, USA; New Mexico Department of
Health, Santa Fe, New Mexico, USA; Colorado Department of Public Health
and Environment, Denver, Colorado; Utah Department of Health, Salt Lake
City, Utah, USA; and #Centers for Disease Control and Prevention,
Albuquerque, New Mexico, USA

---------------------------------------------------------------------------
      To investigate climatic, spatial, temporal, and environmental
      patterns associated with hantavirus pulmonary syndrome (HPS)
      cases in the Four Corners region, we collected exposure site
      data for HPS cases that occurred in 1993 to 1995. Cases
      clustered seasonally and temporally by biome type and
      geographic location, and exposure sites were most often found
      in pinyon-juniper woodlands, grasslands, and Great Basin desert
      scrub lands, at elevations of 1,800 m to 2,500 m. Environmental
      factors (e.g., the dramatic increase in precipitation
      associated with the 1992 to 1993 El Nio) may indirectly
      increase the risk for Sin Nombre virus exposure and therefore
      may be of value in designing disease prevention campaigns.

Relationships between environmental characteristics (i.e., climate, biome,
elevation, habitat structure, and microhabitat), time, and hantavirus
pulmonary syndrome (HPS) cases in the United States have not been
systematically evaluated. We describe environmental factors associated with
probable exposure sites for all diagnosed HPS cases in the Four Corners
region (Arizona, New Mexico, Colorado, Utah) of the United States before
1996 and evaluate the strength of the associations between climate, time,
and HPS cases.

Case and Exposure Site Identification

All cases met the surveillance case definition for HPS, which requires the
presence of clinically compatible symptoms and laboratory confirmation (1).

Fifty-nine sites in Arizona, New Mexico, Colorado, and Utah were identified
as probable exposure sites for HPS cases (sites at which HPS patients were
most likely infected with Sin Nombre virus (SNV), according to previously
collected data from environmental assessments and individual patient
questionnaires [2]) occurring from 1985 to 1995. Rodent trapping and
testing data were also used to determine exposure sites, including those
identified by linking hantavirus genome sequences between patients and
rodents (3,4). Our inability to determine precisely where persons were
exposed to HPS may have created a selection bias.

Climate Investigations

Climate analyses were limited to 52 probable exposure sites of HPS cases (n
= 53 cases) (two case-patients were exposed to SNV at the same site); onset
of illness was between 1993 and 1995. Climate data for probable exposure
sites were collected from the nearest weather station monitored by the
National Oceanic and Atmospheric Administration's Western Regional Climate
Center in Reno, Nevada. Ten years (1986 to 1995) of monthly precipitation
sums and monthly averages of daily ambient temperature were obtained for
each station. To be included, sites had to be located within a 30-km radius
and a 300-m elevation range from their closest corresponding weather
station and without a mountain range between the exposure site and the
identified weather station. Twelve sites did not meet these inclusion
criteria, six were removed from precipitation analyses, and five were
removed from temperature analyses because of missing weather data. Data
from 24 weather stations were used for the precipitation (n = 34 exposure
sites) and temperature analyses (n = 35 exposure sites). Four weather
stations represented multiple exposure sites because they were the closest
available weather stations. Twenty-two (85%) exposure sites included in the
analyses were within 15 km (mean = 13.2 km, standard deviation [SD] = 7.9
km) and 150 m of elevation (mean = 78.0 m, SD = 79.2 m) of their
corresponding weather station. Data were analyzed with the SPSS and
Epi-Info statistical software programs (5,6).

The use of climatic data collected from weather stations as far as 30 km
away from probable exposure sites, with as much as a 300-m difference in
elevation, may have created an ascertainment bias. Weather patterns,
however, generally cover large regions. In addition, a withdrawal bias may
have been introduced by eliminating exposure sites and weather stations
that did not meet the inclusion criteria.

Precipitation Analysis

Precipitation totals from the 24 identified weather stations for each of
the 48 months during 1992 through 1995 were compared with the corresponding
calendar month's mean precipitation total for 1986 to 1991. Each month's
mean precipitation difference was plotted against the number of HPS cases
with onset of symptoms during that month for cases between 1993 and 1995.
The Wilcoxon matched-pairs signed rank test was used to test the
statistical significance of precipitation during two periods of substantial
departure from normal precipitation patterns (i.e., 6-year mean sum
precipitation data for the same calendar months). These weather stations,
representing the 1993 to 1995 cases, reported above average (p < 0.01)
precipitation totals during December 1992 through March 1993 and below
average (p < 0.01) precipitation totals during June 1993 through July 1993
(Figure 1).

 					        By using a Spearman's correlation, we
                                      found a negative correlation between
						  the number of cases per month (onset
[Fig 1]   					  date) and the number of months after
 Figure 1. Mean difference in monthly the 1992 to 1993 El Nio weather
 precipitation and temperature        pattern (r(sub s)= -0.70; p < 0.01)
 between month of interest and 6-year
 mean (19861991) at the study sites  (Figure 2). For this portion of the
 and number of cases by month,        precipitation analysis, the months of
 19931995.                           onset were used for all 53 cases
                                      between 1993 and 1995. The end of the
                                      1992 to 1993 El Nio was described as
 						  the month (March 1993) in which the
 						  abnormally high precipitation totals
[Fig 2]   					  for November 1992 through March 1993
 Figure 2. Correlation of hantavirus  returned to normal, on the basis of
 pulmonary syndrome (HPS) cases per   the data in Figure 1.
 month and number of months from end
 of El Nio period (n = 53 cases).    The 1993 outbreak of HPS in the Four
                                      Corners region followed a dramatic
                                      increase in precipitation associated
with the 1992 to 1993 El Nio phenomenon and peaked in the middle of a
drought. Because the 1992 to 1993 El Nio resulted in an abundance of
rodent food resources (e.g., vegetation and insects) and a 20-fold rodent
population increase over the previous year at the Sevilleta National
Wildlife Refuge in central New Mexico, increased rainfall from El Nio was
associated with the 1993 HPS outbreak (7). A similar pattern of above
average rainfall followed by drought was observed preceding an outbreak of
HPS in western Paraguay in 1995 to 1996 (8). Our study shows that the
number of HPS cases per month in the Four Corners region during 1993 to
1995 was negatively correlated with the number of months after the 1992 to
1993 El Nio. The data suggest that the association between the 1992 to
1993 El Nio and the number of HPS cases in the Southwest may have lasted
for as long as 2 years.

The association between El Nio and the HPS outbreak is probably complex.
The above average precipitation during the winter and spring of 1992 to
1993 may have increased rodent populations and thereby increased the
likelihood of more rodent-to-rodent contact, rodent-to-human contact, and
viral transmission, thereby resulting in the large number of cases in 1993
and 1994. In addition, as rodent populations surpassed the carrying
capacity of their local environments and precipitation plummeted, available
food sources may have been depleted, resulting in rodent population stress.
Increased stress likely increased rodent-to-rodent contact, as rodents
competed for food and water, and increased rodent-to-human contact, as
rodents moved into new, potentially less stressful environments, such as
homes and peridomestic structures.

During 1995, no cases occurred in the original outbreak area near the
Arizona-New Mexico border and in western Colorado, possibly because the
effect of the 1992 to 1993 El Nio had dissipated. Preliminary data from
longitudinal trapping studies in the Southwest suggest that the relative
rodent densities decreased to normal levels in 1995 (J. Mills, T. Yates,
pers. comm.). Hantavirus infection rates in rodents dramatically decreased
at case sites in Arizona and Colorado 3 years after the outbreak (9).

Temperature Analysis

Monthly ambient temperature means from the 24 identified weather stations
for each of the 48 months during 1992 through 1995 were compared to the
corresponding calendar month's ambient temperature mean for 1986 to 1991.
The mean difference was plotted against the number of HPS case onsets per
month for cases occurring between 1993 and 1995. With all 24 weather
stations included, the Wilcoxon matched-pairs signed rank test was used to
test the statistical significance of two periods with substantial
departures from normal temperature patterns (i.e., 6-year mean daily
temperature averages for the same calendar months): 1) November 1992
through December 1992 and 2) January 1993. The 24 weather stations reported
below average (p < 0.01) temperatures during November 1992 through December
1992, which corresponded with the onset of El Nio (Figure 1). Conversely,
the mean temperature for January 1993 was above average (p < 0.01).

While interpreting variations in precipitation totals is fairly
straightforward, interpreting variations in ambient temperature is far more
difficult. Whether the lower-than-average ambient temperature during
November 1992 through December 1992 affected rodent population dynamics is
unknown; however, the higher-than-average temperatures during January 1993
may have promoted rodent survival during what is normally the coldest month
of the year. (Figure 1 indicates that the 24 weather stations reported the
most substantial mean temperature extremes in 1995.)

To examine the relationship between ambient temperature and month of
exposure for HPS cases, we compared the distribution of daily ambient
temperatures of probable month (defined as the month in which onset date
minus 14 days [hypothesized mean incubation period] occurred) of exposure
to daily ambient temperatures during the other 11 months of the same
calendar year. Exposure months had higher ambient temperatures and a
smaller temperature range than the other months in the same calendar year.
The mean temperature for the probable exposure months was 15C (SD = 7C;
range = 0C - 24C) (Figure 3), and the mean throughout the rest of the
year was 11C (SD = 9C; range = -7C - 36C).

Spatial, Temporal, and Environmental   
Investigations
                                       
Spatial and environmental data were    [Fig 3]   
collected for 59 of the 64 known HPS   Figure 3. Distribution of monthly
cases that occurred before 1996 in the ambient temperatures for months
Four Corners region: Arizona (20       estimated as the month SNV
sites), New Mexico (25 sites),         exposure occurred and the 11
Colorado (7 sites), and Utah (7        nonexposure months for the same
sites). For five cases (in 1959, 1975, calendar year (n = 35 cases and 24
1984, 1985, and 1993), the exposure    weather stations).
site was not known. Habitat data
(e.g., dominant biome type as
described by Brown [10] within 200 m) were collected at each of the 59
probable exposure sites. Elevations were taken from U.S. Geological Service
quadrant maps. All analyses were conducted with the SPSS and Epi-Info
statistical software programs (5,6).

Spatial Analysis

Figures 4, 5, and 6 display the spatial and temporal distribution of
probable hantavirus exposure sites in the Four Corners region, from 1993
through 1995 (n = 52). In 1993, most case-patients were exposed in
northeastern Arizona and northwestern New Mexico; in 1994, HPS
case-patients were exposed in all four states, including two new areas,
central Utah and southern Arizona; and in 1995, HPS case-patients were
exposed only in a small geographic area near the border of southcentral
Colorado and northcentral New Mexico (on the eastern slope of the Rocky
Mountains).

						    From 1993 through 1995, HPS cases
                                        in the Four Corners region shifted
 						    geographically. The apparent
 [Fig 4]   					    spatial and temporal movement of
 Figure 4. Hantavirus pulmonary         HPS foci in this region mirrors the
 syndrome cases in the Four Corners     "focal nidality" exhibited by
 region, by probable exposure site      hemorrhagic fever with renal
 location, 19931995 (n = 53 cases and  syndrome (HFRS), the Eurasian
 52 exposure sites).                    hantaviral manifestation (11-15).
                                        HFRS cases can occur sporadically
                                        throughout the year, but outbreaks
occur seasonally (12,16). Seasonal prevalence varies by locality and
meteorologic and climatic conditions that favor activity of rodents
associated with viruses causing HFRS (12). Niklasson et al. (17)
hypothesize that large outbreaks of HFRS occur in geographic "hot spots,"
which may depend on certain ecologic characteristics correlating with
rodent populations, e.g., rodent habitats and environmental or meteorologic
conditions (17). In the Four Corners states, seroprevalence of hantaviruses
in rodents displays a similar focality (18). Our data suggest that HPS
cases may have occurred in similar hot spots in this region between 1993
and 1995.

 Temporal Analysis

 The 1993 to 1995 HPS onset months were unevenly distributed
 (Kolmogrov-Smirnov test, p = 0.01), displaying a spring-summer
 seasonality, as has been reported previously (2,3,15) (Figure 6). While
 cases occurred throughout all seasons, probable exposures occurred during
 the time of year when the monthly mean ambient temperature was 0C to
 24C.

[Fig 5]   
Figure 5. Hantavirus pulmonary syndrome cases in the Four Corners
region by state, 19931995 (n = 53 cases and 52 exposure sites).

[Fig 6]   
Figure 6. Hantavirus pulmonary cases in the Four Corners region,
by month of onset, 19931995 (n = 53 cases and 52 exposure sites).

The seasonality of HPS cases varies by location, elevation, and biome
(Figures 4, 5, and 7). The clustering of all four Sonoran Desert HPS cases
during the late winter and early spring and the two montane conifer cases
during the late spring and summer, when the mean temperatures for both
biomes are mild (12C - 21C), are the best examples of this trend. Most
grassland, Great Basin scrub, pinyon-juniper, and montane conifer cases
occurred during spring and summer (Figure 7).

Environmental Analysis

Probable exposure sites occurred in seven biomes, most often in
pinyon-juniper woodland, grassland, and Great Basin desert scrub biomes
(33.9%, 28.8%, and 23.7%, respectively) (Table). Approximately 66% (39/59)
of case-patients were exposed at elevations of 1,800 m to 2,500 m; none was
exposed at elevations higher than 2,500 m (Table). This description may
reflect the typical biomes in which the 1993 outbreak took place but also
holds for cases in Utah and Colorado. Mills et al. (18) reported the same

habitat and elevation for sites with the highest Peromyscus densities (as
well as the highest rodent SNV antibody prevalence) in the Four Corners
states. Unpublished case-control data (J. Cheek and R. Bryan) show that
significantly more HPS cases occurred in Great Basin pinyon-juniper than in
any other biome on the Navajo reservation in the Southwest; the data also
show that HPS cases are more likely at higher elevations. Environmental
conditions surrounding HPS exposure sites appear similar to those
surrounding plague exposure sites on the Navajo reservation (K. Gage, R.
Enscore, unpub. data).

Probable exposure sites were most      
frequently found in rural areas, with
five or fewer homes within a 1-km      
radius (64.4%); most exposure sites      [Fig 1]   
(88.1%) were found in areas with 25 or   Figure 7. Number of hantavirus
fewer homes. An almost equal number of   pulmonary syndrome cases occurring
exposure sites (44.1%) was more than 2   in specific biomes by season of
km away from a permanent water source    onset (19931995) (n = 53 cases).
as exposure sites (42.4%) within 1 km
of a permanent water source. Overall,
104 plant species belonging to 72 genera were identified at exposure sites.
The three most common dominant herbaceous species were Artemisia tridentata
(big sagebrush), Gutierrezia sarothrae (snakeweed), and Bromus tectorum
(downy chess grass); and the three most common dominant overstory and
understory species were Juniperus monosperma (one-seed juniper), J.
osteosperma (Utah juniper), and Pinus edulis (two-needle pinyon pine). For
21 of 59 sites with 5% slope or greater, more than twice as many had a
northward aspect (9 of 21) than any other aspect (south = 4 of 21; east = 4
of 21; and west = 4 of 21).

While the habitat may have changed in minor ways between the time of
exposure and data collection, no major habitat changes (e.g., fire, timber
harvesting) were identified. Because these environmental findings are
descriptive, caution should be used when drawing conclusions on the basis
of these data.

Conclusions               Table. Frequency of probable Sin Nombre virus
                          exposure sites in selected biomes and elevation
Previous studies have     ranges
examined behavior and
environmental risk        -------------------------------------------------
factors for the                                            Frequency
acquisition of HPS        Biome type                        (n = 59)   %
(19-22). Certain behavior -------------------------------------------------
and occupations may
increase the likelihood   Sonoran Desert                       4       6.8
of exposure to the        Chihuahuan Desert                    1       1.7
excreta of infected       Great Basin Desert scrub             14     23.7
rodents; however, the
overall level of risk     Great Basin and plains grassland     17     28.8
seems low (21,22).        Great Basin pinyon-juniper           20     33.9
Environmental risk
factors may be divided    Riparian woodland                    1       1.7
into endemic and          Montane conifer forest               2       3.4
epidemic. Endemic         Elevation range
environmental risk
factors are tied to the   -------------------------------------------------
static habitat structure  0 - 619                              3       5.1
of a geographic area. For 620 - 1,239                          1       1.7
example, the habitat of
the Four Corners region   1,240 - 1,859                        13     22.0
allows population levels  1,860 - 2,480                        42      1.2
of Peromyscus maniculatus
great enough to maintain  -------------------------------------------------
SNV transmission (18,23). Some level of risk will likely always exist for
exposure to SNV in the region; however, the level seems low (9). Epidemic
environmental risk factors, also tied to the static habitat structure of a
geographic area (i.e., they can only occur in disease-endemic areas), are
primarily dynamic events associated with large-scale environmental changes
of limited duration. For example, the precipitation pattern during the 1992
to 1993 El Nio, associated with increased rodent populations in the
Southwest (7) and consequently with the 1993 outbreak of HPS in the Four
Corners region, greatly increased the level of risk for human SNV infection
in the outbreak area. By 1995, rodent populations (J. Mills, T. Yates,
pers. comm.) and the number of HPS cases in this area dramatically
decreased, and the level of risk for human SNV infection may have returned
to endemic levels in 1995. Early recognition of these endemic and epidemic
environmental risk factors might allow public health agencies to predict
where and when the next HPS outbreak may occur and thus to effectively
target prevention efforts.

While this report does not address data related to cases after 1995, the
1997 to 1998 El Nio may not have displayed the same precipitation pattern
in the Four Corners region as the 1992 to 1993 El Nio. Limited data from
Arizona's primary weather station for northern Arizona indicate 4.70 cm of
precipitation in December 1997the 98-year mean (18991997) for December is
4.88 cm (Office of the State ClimatologistArizona State University, pers.
comm.). In December 1992, the same station reported 17.22 cm of
precipitation (Office of the State ClimatologistArizona State University,
pers. comm.). Future studies should examine and compare the two
precipitation events, their lasting effects on rodent populations, and the
subsequent effects on hantavirus epidemiology.

Acknowledgments

      We thank Rosalyn Curtis, Herman Shorty, Rusty Enscore, Dave Tibbs,
Chuck Freeman, Benny Joe, Bobby Villines, Eric Faist, Charlie Irland, Tim
Doyle, and Dorothy Miller for their assistance in data collection and
Charles Calisher, T. Michael Fink, Nicole Rossi, Mira Leslie, and Thomas G.
Engelthaler for their critical review and comments.

      This study was supported in part by CDC cooperative agreements with
Arizona, Colorado, New Mexico, and Utah.

      David Engelthaler is a visiting fellow, Division of Vector-Borne
Infectious Diseases, Centers for Disease Control and Prevention, Fort
Collins, CO. Mr. Engelthaler is involved in plague research. His primary
research interest is pathogen-vector relationships.

      Address for correspondence: David G. Mosley, Bureau of Epidemiology
and Disease Control, Arizona Department of Health Services, 3815 North
Black Canyon Highway, Phoenix, Arizona 85015, USA; fax: 602-263-4956;
e-mail: dmosley@hs.state.az.us.

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Hantavirus

The following articles on hantavirus contain preliminary results of 3 years
of longitudinal mark-release-recapture studies in the southwestern United
States, provide an analysis of the sensitivity of the field techniques and
statistical analyses for detecting changes in rodent population densities,
and discuss the role of longitudinal studies in understanding reservoir
host ecology as it relates to human disease. Although the data presented in
the articles are overall standardized, methods used to extract information
vary by research institution.

Long-Term Studies of Hantavirus Reservoir Populations in the Southwestern
United States: Rationale, Potential, and Methods

James N. Mills, Terry L. Yates, Thomas G. Ksiazek, C.J. Peters, and James
E. Childs
Centers for Disease Control and Prevention, Atlanta, Georgia, USA

---------------------------------------------------------------------------
      Hantaviruses are rodent-borne zoonotic agents that cause
      hemorrhagic fever with renal syndrome in Asia and Europe and
      hantavirus pulmonary syndrome (HPS) in North and South America.
      The epidemiology of human diseases caused by these viruses is
      tied to the ecology of the rodent hosts, and effective control
      and prevention relies on a thorough understanding of host
      ecology. After the 1993 HPS outbreak in the southwestern United
      States, the Centers for Disease Control and Prevention
      initiated long-term studies of the temporal dynamics of
      hantavirus infection in host populations. These studies, which
      used mark-recapture techniques on 24 trapping webs at nine
      sites in the southwestern United States, were designed to
      monitor changes in reservoir population densities and in the
      prevalence and incidence of infection; quantify environmental
      factors associated with these changes; and when linked to
      surveillance databases for HPS, lead to predictive models of
      human risk to be used in the design and implementation of
      control and prevention measures for human hantavirus disease.

Hantaviruses (genus Hantavirus, family Bunyaviridae) are rodent-borne
zoonotic agents that cause mild to severe hemorrhagic fevers throughout
most of Europe, Asia, and the Americas. The epidemiology of these
hemorrhagic fevers is largely defined by the distribution and ecology of
the rodent hosts of the viruses. Hantaviruses have been identified at a
dramatically increased rate in recent years; some 30 hantaviruses are now
recognized throughout the world (1,2). With very few exceptions, each virus
is associated with a single primary rodent host of the family Muridae. The
rodent, in which the virus establishes a chronic infection, sheds
infectious virus into the environment in urine, feces, and saliva (3,4);
these characteristics are key to the transmission of the virus, both to
humans (most frequently by inhalation of infectious aerosols [5]) and among
rodents (frequently by aggressive encounters and biting [6,7]).

Human diseases due to hantaviruses, which have been recognized at least
since World War I and probably occurred much earlier (8), were unknown in
the Americas until recently. Although Rattus norvegicus infected with Seoul
virus is common in many cities throughout the Americas (9), human disease
associated with a rat-borne hantavirus was not documented in a U.S. city
until 1994 (10). Prospect Hill virus, an indigenous North American
hantavirus, was isolated from the meadow vole (Microtus pennsylvanicus) as
early as 1982 (11) but has never been associated with human disease.

Since 1993, when hantavirus pulmonary syndrome (HPS) was recognized and its
etiologic agent, Sin Nombre virus (SNV), was isolated and associated with
the deer mouse (Peromyscus maniculatus) (12,13), at least 20 New-World
hantaviruses, all associated with the same group of indigenous American
rodents (family Muridae, subfamily Sigmodontinae) have been described, and
HPS has been diagnosed from Canada to Patagonia. The severity
(approximately 50% death rate) and wide geographic distribution of this
rodent-borne zoonotic disease has prompted intensive collaboration between
public health investigators and ecologists to elucidate the ecologic and
epizootiologic features of infection in host populations and the factors
that lead to human infection.

Because no specific treatment is yet available, prevention measures are
essential in decreasing HPS-related illness and death. Developing effective
preventive measures requires a detailed knowledge of the ecology and
epizootiology of hantavirus infection in reservoir populations and the
specific situations and mechanisms that result in the transfer of
hantaviruses from hosts to humans.

Reservoir Studies

Reservoir studies, whose role in understanding, controlling, and preventing
human disease has been reviewed, have resulted in a series of research
goals that may facilitate the collection of data concerning reservoir
ecology, a subject pertinent to human health (14). The first goal is to
identify the reservoir host; others are a) to determine the area in which
the disease may be endemic by identifying the geographic range of the host
and the range of infection by the pathogen within the host range; b) to
more precisely define relative risk to humans by determining the
distribution of the host and pathogen among distinct habitats regionally;
c) to investigate potential mechanisms of pathogen transmission within host
populations by conducting cross-sectional surveys to define the prevalence
of infection among various subpopulations of the host (e.g., male versus
female, juvenile versus adult); d) to conduct long-term prospective studies
to explain the temporal patterns of infection in host populations; and e)
to integrate the data from these studies in a predictive model that will
allow early identification of specific times and places where conditions
can increase rodent populations or infection in rodent populations and
elevate the risk for human disease. This model could be used to minimize
the incidence of human disease through public education, habitat
modification, or reservoir control.

Investigations of HPS cases in the United States have resulted not only in
studies of the deer mouse and SNV but also in the identification of three
additional host-virus relationships that maintain hantaviruses responsible
for human disease (New York virus carried by the white-footed mouse,
Peromyscus leucopus [Figure 1] [15]; Black Creek Canal virus carried by the
cotton rat, Sigmodon hispidus [16]; and Bayou virus carried by the rice
rat, Oryzomys palustris [17]). Most HPS cases in the United States have
been caused by SNV.

Intensive studies of deer mouse       
populations have addressed most of
the proposed goals. One of the most   
common and most extensively studied   [fig 1]
small mammals in North America, the   Figure 1. White-footed mouse
deer mouse has a well-known           (Peromyscus leucopus). Photo by R.B.
geographic distribution (18).         Forbes, Mammal Image Library of the
Antibody screening of deer mouse      American Society of Mammalogists.
populations throughout North America
has provided evidence of SNV infection throughout most of the species'
range (Ksiazek et al., unpub. data). Regional studies have shown
differences in the prevalence of hantavirus infection among deer mouse
populations in different habitats and helped define the varying disease
risk to humans in these habitats (7). Finally, studies of the age- or
size-specific prevalence of hantavirus infection among reservoir
populations have shown that SNV, and other hantaviruses, are transmitted
horizontally within reservoir populations, and that one important specific
mechanism of transfer may be aggressive encounters and bites, most
frequently among male animals (6,7,19). Although these cross-sectional
studies have increased our understanding of host-virus ecology as it
relates to human disease, they have not explained the temporal dynamics of
host-virus ecology nor have they identified the environmental factors
associated with these dynamics; only long-term prospective studies can
provide this additional information.

Long-Term Studies

Long-term studies, widely regarded by ecologists as indispensable for
understanding the temporal dynamics of vertebrate communities (20), are
especially useful for assessing the effects of rare events (e.g., El Nio
southern oscillation) and for detecting and observing processes that unfold
slowly in communities or populations (e.g., establishment or disappearance
of a reservoir species from part of its range; changes in reservoir
population density; changes in community composition; introduction or
extinction of a pathogen in a specific host population; and changes in the
incidence or prevalence of infection within the host population).

Long-term studies of reservoir populations have helped elucidate the
temporal dynamics of hantavirus infection in host populations for Seoul and
Prospect Hill viruses (21) and identify characteristics of reservoir
ecology associated with outbreaks of human disease. The numbers of cases of
hemorrhagic fever with renal syndrome due to Puumala virus in Scandinavia
and Argentine hemorrhagic fever due to Junn virus (an arenavirus with many
epidemiologic similarities to hantaviruses) were correlated with cyclic
changes in the density of reservoir host populations (22,23). Increases in
population density are associated with improved reproductive success and
survivorship that may be due to improved habitat. Changes in the
environment may be associated with favorable weather patterns, accelerated
vegetation growth, and availability of plant and small-animal foods
(14,23). The 1993 HPS outbreak in the southwestern United States may have
resulted from improvements in the quality of deer mouse habitat caused by
the 1991-92 El Nio southern oscillation (24). When the environmental
variables associated with increasing reservoir population densities are
identified and quantified, a key component of a predictive model of human
risk will be in place.

Despite their importance and utility, long-term studies of reservoir
populations associated with zoonotic agents are rare. By definition, they
require stable funding for many years, they are labor intensive, expensive,
and may not produce significant results in the short term. The periodic
shifts in environmental conditions that change host populations and
increase risk for human disease may take many years.

The most common method for conducting long-term studies of small-mammal
populations is the mark-release-recapture (MRR) technique. Animals trapped
live on permanent trapping plots are measured, sampled (blood or oral
swab), identified with a permanent mark or number, and released at the
exact site of capture. The trapping plots are operated at predetermined
intervals for several days. Animals recaptured in subsequent trappings are
measured and sampled again so that changes in numbers of animals, body
growth rates, movement, reproductive condition, and infection status can be
monitored. Environmental variables such as weather conditions and
vegetative cover also may be monitored on the trapping plots. Control
plots, where invasive procedures are minimized, may be necessary for
determining or correcting for the influence of sampling methods on animal
survival or population size.

After the 1993 HPS outbreak, the Centers for Disease Control and Prevention
(CDC) established a network of hantavirus and rodent monitoring sites in
the southwestern United States to 1) monitor and quantify the seasonal and
year-to-year changes in host population density and the prevalence and
incidence of hantavirus infection, 2) identify and quantify the biotic and
abiotic environmental factors associated with and likely influencing these
dynamics, 3) identify mechanisms of virus transmission within reservoir
populations, and 4) identify and measure any effects of infection on
individuals and populations of the host.

The studies should lead to predictive models of human risk for hantavirus
infection and should facilitate prevention and control of human hantavirus
disease.

Study Sites

						  Arizona, Colorado, and New Mexico
                                      were chosen as the general study
						  area because of their high numbers
 [fig 2]                              of HPS cases at the time the study
 Figure 2. Geographic locations of    was being designed. In addition,
 nine sites where mark-release-       four sigmodontine rodent species
 recapture webs are being identified  as hantavirus reservoirs
 operated to study rodent reservoirs  inhabit at least parts of the
 of hantaviruses in a three- state    three-state area (P. maniculatus, P.
 area of the southwestern United      boylii, Reithrodontomys megalotis,
 States. PCMS=Pinyon Canyon Maneuver  and S. hispidus). Longitudinal MRR
 Site (U.S. Army).                    studies are being conducted on 24
                                      trapping webs at nine sites in the
three states (Figure 2): 10 webs at four sites in New Mexico, operated by
the University of New Mexico; six webs at three sites in Colorado, operated
by Colorado State University; four webs at one site in northern Arizona,
operated by Yavapai College; and four webs at one site in southern Arizona,
operated by the University of Arizona. Site selection criteria included
presence of populations of Peromyscus spp. and evidence of infection by SNV
or related viruses in these populations. The location of each trapping web
was fixed precisely by global positioning system technology. A sketch of
each trapping web site, including a description of the vegetation, was
prepared.

Placing Permanent Trapping Webs

Small-mammal populations were monitored through the use of permanent
trapping webs (25) (Figure 3A). Each web covered 3.14 ha and contained 12
100-m transects radiating from a central point and resembling the spokes of
a wheel (Figure 3B). Each web contained 148 Sherman (8 x 9 x 23 cm; H.B.
Sherman Trap Company, Tallahassee, FL) and 24 Tomahawk (14 x 14 x 40 cm;
Tomahawk Live Trap Company, Tomahawk, WI) live-capture traps, at 12 trap
stations along each radiating spoke. The first four trap stations were at
5-m intervals and the remaining eight at 10-m intervals. Four Sherman traps
were placed around the central point. In addition to a Sherman trap, one
Tomahawk trap was placed at trap stations 7 and 12 in each radiating arm
(Figure 3B). Two to four webs were located at each sampling site. At least
one web at each site was designated a control web. At this web, rodent
populations were monitored but not sampled by blood and oral swab
collection so that the effects of sampling on small-mammal survivorship
could be assessed. At the remaining webs, virus activity in small-mammal
populations was monitored through monthly blood and oral swab samples from
captured animals. After the second year of the study, the purpose of the
control webs was achieved, so sampling of captured small mammals from these
webs was initiated (25,26).

Trapping Schedules                            
                                     
All trapping web sites were visited  
monthly, except those in Colorado,   [fig 3]
which were visited every 6 weeks,    Figure 3. A. Characteristics of
as weather permitted. Webs were      landscape and vegetation near Fort
operated for 3 consecutive nights on Lewis trapping web "A," southwestern
each trapping occasion, generally    Colorado. Photo courtesy of C.
coinciding with the new moon. Traps  Calisher.  B. Schematic representation
were set out in the evening of the   of a trapping web showing the relative
first day and baited with peanut     locations of the 148 trap stations.
butter and rolled oats, cracked      Small circles indicate the location of
corn, or mixed grain. In cold        one Sherman trap, larger circles, one
weather, cotton or polyester         Sherman plus one Tomahawk trap.
fiberfill was placed in the traps to Diameter of the web was 200 m. After
provide nesting material and reduce  Parmenter et al. (25).
trap-associated deaths.

Captured rodents were collected, transported, and sampled according to
standardized procedures (27,28). Briefly, traps were checked for captures
early each morning. Investigators wearing rubber gloves collected the traps
containing captured animals, labeled them with the web and trap station
number, and placed them in double plastic bags for transport to a
centralized outdoor processing station. Before opening the bags containing
captured small mammals, investigators donned protective clothing, including
latex gloves, disposable surgeon's gowns, and respirators fitted with HEPA
filters. Each captured animal was processed individually. The animal was
first shaken from the trap into a plastic bag containing cotton or gauze
soaked with inhalant anesthesia (methoxyflurane, Pitman-Moore, Mundelein,
IL; or isoflurane, Abbott Laboratories, North Chicago, IL). To prevent
potential cross-infection between animals, each was anesthetized in a clean
plastic bag, and the anesthesia-soaked cotton was contained in a tea
strainer that allows diffusion of the anesthesia, yet between animals can
be wiped with a disinfectant. In one case, investigators used a specially
adapted "nose cone" for anesthesia (Abbott et al., this issue, pp.
102-112). After being anesthetized, the animal was removed from the bag and
placed on a clean surface. A standardized form was used at all trapping
sites to collect the following data (28): unique capture number; date of
capture; exact location of capture on the trapping web (trap station
number); ear tag number; fate (first capture, recapture [different trapping
session], or repeater [within same 3-day trapping session]); species; age
(juvenile, subadult, or adult); mass; lengths of tail plus body, tail only,
ear, and right hind foot; reproductive status including position of the
testes (scrotal or abdominal) for males and condition of the vagina (closed
or perforate) and description of the nipples (enlarged or small, lactating
or not) for females; and the presence or absence of scars or wounds. For
animals from the sampling webs, oral swabs were taken with Dacron-tipped
applicators cut with scissors at the level of the Dacron and inserted into
0.5 ml of virus medium (phosphate-buffered saline containing 20% fetal
bovine serum, 2% penicillin and streptomycin, and 0.1% Fungizone) in a 2-ml
cryovial. Approximately five drops of whole blood were collected into a
second cryovial by a capillary tube inserted into the retro-orbital
capillary plexus. Whole blood and oral swab samples were immediately placed
in liquid nitrogen or on dry ice until transferred to -70C freezers for
storage. Animals recaptured on days 2 or 3 of the trapping session were not
bled a second time to avoid trauma. Animals newly captured were marked with
a uniquely numbered ear tag (some smaller animals were marked by toe
clipping). The animal was then replaced in the original trap or in a clean,
ventilated one-quart, screw-capped jar, and was allowed to recover fully
from the effects of anesthesia and was released at the exact site of
capture. A clean, baited trap was replaced at the site, and the original
trap was returned to the processing site to be decontaminated before reuse.
Animals from the control webs were treated similarly, except that blood and
oral swab samples were not taken.

Investigators recorded environmental data, including a general description
of the vegetation and depending on resources available, more detailed
descriptions of vegetation at individual webs and weather conditions during
the trapping session (detailed rainfall and temperature data were available
from meteorologic stations near each trapping site).

Laboratory Analysis

Serologic testing was conducted at CDC, Atlanta, or at the Arthropod-Borne
and Infectious Diseases Laboratory, Colorado State University, Fort
Collins, CO, USA. Samples of whole blood were tested for antibody reactive
with SNV recombinant nucleocapsid protein antigen by enzyme-linked
immunosorbent assay according to a standardized protocol (29). Briefly,
blood specimens were initially diluted 1:25 in 5% skim milk in 0.01 M
phosphate-buffered saline with 0.5% Tween-20 and subsequently diluted to
1:100 through 1:6,400 in fourfold dilutions in microtiter plates. Samples
were tested against the recombinant nucleocapsid antigen and a recombinant
control antigen (29). A conjugate mix of anti-Rattus norvegicus and
anti-Peromyscus leucopus (heavy and light chains) immunoglobulin G (IgG)
(Kirkegaard and Perry, Gaithersburg, MD) was used to detect bound
immunoglobulin. Adjusted optical densities (OD) for each dilution were
calculated by subtracting the OD(sup 410) of the control antigen from the 
OD(sup 410) of the SNV antigen. Titers were assigned on the basis of an 
adjusted OD value exceeding 0.20 for each dilution. A second measure 
consisting of the sum of the adjusted OD values for all four dilutions was 
also calculated. Serum specimens were considered SNV-positive if their titer 
was 1:400 or their sum-adjusted OD was 0.95. The cut-off values were determined 
by assessment of rodents found to be SNV-positive by several serologic tests
during the initial investigation of the 1993 outbreak (13) and have been
reassessed periodically among large populations of rodents collected in
North and South America. Antibodies to other North American hantaviruses
are cross-reactive with SNV antigen. This assay would detect (but not
distinguish among) infections by New York virus (from the white-footed
mouse [15]), Prospect Hill-like viruses (from arvicoline rodents [30]), El
Moro Canyon virus (from the Western harvest mouse [31]), Black Creek Canal
virus (from the cotton rat [16]), and Bayou virus (from the rice rat [17]).

At this writing, analyses on oral swab specimens (antigen or antibody
detection, polymerase chain reaction) have not been conducted. Blood and
oral swab samples are archived in -70C freezers at CDC, Atlanta, and at
the Museum of Southwestern Biology, Albuquerque, New Mexico.

Acknowledgments

      We are indebted to many investigators at CDC for laboratory analyses
and database management. Special thanks to R. Meyer, M.L. Martin, V.
Semenova, G. Gallucci, M. Curtis, A.J. Williams, and P. Stockton in the
laboratory; B. Farrar in the stock rooms; and L. Morgan and K. Schmidt at
the computer. K. Wagoner, K. Colbert, and B. Ellis prepared the figures. K.
Abbott, C. Calisher, B. Ellis, and M. Morrison provided helpful comments on
an earlier version of the manuscript.

      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.

      Address for correspondence: James N. Mills, Centers for Disease
Control and Prevention, 1600 Clifton Road, Mail Stop G14, Atlanta, GA
30333, USA; fax: 404-639-1118; e-mail: jum0@cdc.gov.

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 29. Feldmann H, Sanchez A, Morzunov S, Spiropoulou CF, Rollin PE, Ksiazek
     TG, et al. Utilization of autopsy RNA for the synthesis of the
     nucleocapsid antigen of a newly recognized virus associated with
     hantavirus pulmonary syndrome. Virus Res 1993;30:351-67.
 30. Yanagihara R, Daum CA, Lee PW, Baek LJ, Amyx HL, Gajdusek DC, et al.
     Serological survey of Prospect Hill virus infection in indigenous wild
     rodents in the USA. Trans R Soc Trop Med Hyg 1987;81:42-5.
 31. Hjelle B, Chavez-Giles F, Torrez-Martinez N, Yates T, Sarisky J, Webb
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Hantavirus

Long-Term Hantavirus Persistence in Rodent Populations in Central Arizona

Ken D. Abbott,* Thomas G. Ksiazek, and James N. Mills
*Yavapai College, Prescott, Arizona, USA; and Centers for Disease Control
and Prevention, Atlanta, Georgia, USA

---------------------------------------------------------------------------
      For 35 months, we monitored hantavirus activity in rodent
      populations in central Arizona. The most frequently captured
      hantavirus antibodypositive rodents were Peromyscus boylii and
      P. truei. Antibody-positive P. boylii were more frequently male
      (84%), older, and heavier, and they survived longer on trapping
      web sites than antibody-negative mice. The number of
      antibody-positive P. boylii was greater during high population
      densities than during low densities, while antibody prevalence
      was greater during low population densities. Virus transmission
      and incidence rates, also related to population densities,
      varied by trapping site. The spatial distribution of
      antibody-positive P. boylii varied by population density and
      reflected the species preference for dense chaparral habitats.
      The focal ranges of antibody-positive P. boylii also
      demonstrated a patchy distribution of hantavirus.

We report results of the initial 35 months of one of several longitudinal
hantavirus studies begun in the southwestern United States after the 1993
outbreak of hantavirus pulmonary syndrome (HPS) (Mills et al., this issue,
pp. 95-101). This study monitors and quantifies the seasonal and
year-to-year changes in rodent populations and the prevalence and incidence
of hantavirus infection, identifies environmental factors associated with
these dynamics, explores aspects of temporal and spatial viral transmission
within reservoir populations, and examines the characteristics of infected
animals.

Trapping and Processing

In January 1995, we established four 3.14-ha mark-recapture trapping webs
in northcentral Arizona, elevation 1,648 m (Mills et al., this issue, pp.
95-101). The webs were located north of Prescott in Limestone Canyon
(3531'N, 12129'W). All sites were in juniper-pinyon and interior
chaparral communities (1), although each site varied in physiognomy,
aspect, slope, and plant species composition and distribution. Trapping web
sites S-1 and C-1 were separated by a valley 150 m wide and were .6 km
north of sites S-2 and C-2, which were set apart by a 100-m ravine and
creek bed. All webs were operated from January 1995 to September 1996.
Serologic samples were taken from rodents captured at S-1 and S-2, while
C-1 and C-2 were initially operated as control sites to determine the
effects of sampling on rodent survivorship. In October 1996, trapping was
discontinued at C-2 (since our field data and others' [2] indicated that
sampling had no effect on rodent survival), and blood collection and
antibody testing were initiated at C-1 because of its microhabitat
uniqueness and high rodent densities.

Web design and placement, trapping periods, mark-recapture techniques,
animal processing, and serologic sampling procedures are described in Mills
et al. (this issue, pp. 95-101). We anesthetized animals by securing the
dorsal skin behind the head and slipping a nose cone with cotton wetted
with isoflorane over the nose. Between animals, the nose cone was cleaned
with disinfectant. When clearly anesthetized, the animal was placed on a
clean table, measured, ear-tagged, and bled.

Serologic testing was conducted at the Centers for Disease Control and
Prevention, Atlanta, Georgia. Samples of whole blood were tested for
antibody reactive with Sin Nombre virus (SNV)-recombinant nucleocapsid
protein antigen by enzyme-linked immunosorbent assay (ELISA) (3). The
laboratory methods we used are described in Mills et al.; (this issue, pp.
95-101).

Data Analysis

Peromyscus boylii (brush mouse) and P. truei (pinyon mouse) were assigned
to three categories on the basis of body mass at first capture. Body mass
classes (derived from our field data and other sources [4]) were used as an
indication of relative age: 6.0 g to 19.0 g (juvenile), 19.1 g to 22.0 g
(young adult), and 22.1 g to >30.0 g (adult). We estimated the survival of
trappable populations by using mark-recapture data to assess the number of
times an animal was caught between the first and last capture. While not a
measure of actual life span, average survival provides some indication of
population turnover and longevity (5). The minimum number alive (the number
of rodents captured in a month plus the number of rodents captured on at
least one prior and one subsequent occasion) was used to estimate
population sizes (5-7). The minimum number infected was calculated for
antibody-positive rodents by using the same technique. Estimated standing
prevalence was calculated by dividing the monthly minimum number infected
by minimum number alive. These methods provide an estimate of the number of
rodents alive and population sizes for a period, an estimate of the number
of infected rodents, and comparisons of antibody prevalence between
trapping web locations.

Field data were transferred to a computer database by using Excel
(Microsoft Corp., Redmond, WA) and Lotus 1-2-3 for Macintosh (Lotus
Development Corporation, Cambridge, MA). Statistical analyses were
performed by using MINITAB (Minitab Inc, State College, PA) statistical
software, the Mann-Whitney and two-sample t tests, one-way analysis of
variance, and linear trend model (8).

Trapping Results

During 35 months of trapping at three grids, 844 rodents were captured
3,552 times. Blood samples were obtained from 553; from these rodents,
1,418 samples were collected (as a result of subsequent captures of the
same rodents during progressive trapping sessions) and tested for
hantavirus antibody (Table 1).

 Table 1. Sin Nombre virusantibody-positive mice and hantavirus prevalence
 at three mark-recapture webs, December 1995November 1997(sup a)
 --------------------------------------------------------------------------
                                  Trapping webs
                       -----------------------------------
 Species                      S-1          S-2      C-1(sup b)   Totals
 --------------------------------------------------------------------------
 Peromyscus boylii       76/286/109   74/516/178   3/56/22     153/858/309
   (Brush mouse)            (26.6%)      (14.3%)    (5.4%)         (17.8%)
 Peromyscus truei          3/165/67     5/133/55    0/15/8       8/313/130
   (Pinyon mouse)            (2.0%)       (3.8%)    (0.0%)          (2.6%)
 Tamias dorsalis            0/73/40      0/83/29    0/19/9        0/175/78
   (Cliff chipmunk)                                                 (0.0%)
 Dipodomys ordii              0/3/2      0/33/13     0/7/3         0/43/18
   (Ord's kangaroo rat)								  (0.0%)
 Onychomys leucogaster            0       0/10/3         0          0/10/3
   (Northern grasshopper							  (0.0%)
    mouse)
 Neotoma stephensi            0/3/1        0/2/2     0/3/2           0/8/5
   (Stephen's woodrat)                                              (0.0%)
 Neotoma albigula             0/1/4        0/4/1     0/2/1           0/7/6
   (White-throated								  (0.0%)
    wood rat)
Reithrodontomys megalotis         0        0/4/4         0           0/4/4
   (Western harvest mouse)                                          (0.0%)
All species              79/531/223   79/785/285  3/102/45    161/1,418/553
 --------------------------------------------------------------------------
 (sup a)Positive samples/number of samples tested/number individuals tested.
 Values in parentheses are hantavirus antibody prevalences for 35 months
 based on the number of samples tested.
 (sup b)C-1 was initially a control web; serologic sampling began in October
 1996.

P. boylii was the most commonly captured species (70%), followed by P.
truei (18%), Tamias dorsalis (9%), and Dipodomys ordii (2%). Irregular
species (Neotoma albigula, N. stephensi, Onychomys leucogaster, and
Reithrodontomys megalotis) accounted for 1% of the total captures. The
highest rodent densities occurred at webs S-2 and C-1 (40% and 33% of all
captures, respectively), while S-1 accounted for 27% of the total captures.

Population Dynamics

Population levels of the two most frequently captured rodent species, P.
boylii and P. truei, were relatively high through the winter of 1995 to
1996 and then declined (p <0.05) during the subsequent summer and autumn,
remaining at low levels through 1996 to 1997 (Figure 1). The P. boylii
population had the most persistent decline (76%) followed by T. dorsalis
(64%) and P. truei (34% short-term reduction). Population levels of P.
boylii were consistently higher than those of P. truei, except for the
summer of 1997 (May through August); during this period P. boylii densities
were at their lowest, 6.5 animals per 6.2 ha per month, while the P. truei
populations increased to near high density levels (12.2 animals per 6.2 ha
per month). For 4 months, far more P. truei were captured than P. boylii
(Figure 1).

 [Fig]          
Figure 1. Minimum number of Peromyscus boylii and P. truei alive (MNA) and 
the minimum number infected (MNI) with Sin Nombre virus (antibody-positive)
at two mark-recapture webs (6.2 ha).*
*Because of adverse weather conditions, we only trapped for 2 nights
in January and May 1995.
                    
During the first 5 months, adverse weather conditions (rain, snow, high
winds) hampered trapping efforts. Strong wind and wind gusts seemed the
main factor contributing to reduced periodic capture rates (Figure 1).
 
Characteristics of Antibody-Positive Captured Rodents
 
Although data from C-1 were not included in comparative analysis
because serologic sampling was not initiated at this site until October
1996 during low population densities (4.0 samples per month, range 0 to
8), of the 21 P. boylii captured and tested, 2 were hantavirusantibody-
positive (10%); 0 (0%) of 7 females and 2 (28%) of 14 males. After samples
were collected from one antibody-positive P. boylii in October 1996, no
antibody-positive samples were collected until the following October, 
when another P. boylii, which had survived for 12 months, became antibody-
positive for the first time.

The 62 hantavirus antibodypositive rodents captured at the two sites
represented two species: 58 P. boylii and 4 P. truei (Table 2). The
prevalence of hantavirus antibody differed considerably by species: P.
boylii had a prevalence of 20%, P. truei 3%. All four antibody-positive P.
truei were trapped before September 1996 when population densities were
high for all rodent species.

Table 2. Antibody-positive and antibody-negative Peromyscus boylii and P.
truei at two mark-recapture webs,(sup a) December 1995November 1997
--------------------------------------------------------------------------
                            No. (%) P. boyii       No. (%) P. truei
                   --------------------------- ----------------------------
 Characteristic    Positive  Negative  Totals  Positive  Negative  Totals
 --------------------------------------------------------------------------
 Sex
    Male           49 (32)   106 (68)  155 (54)  3 (4)     63 (96)   66 (56)
    Female          9  (7)   123 (93)  132 (46)  1 (2)     50 (98)   51 (44)
    Totals         58 (20)   229 (80)  287       4 (3)    113 (97)  117
 Body mass class(sup b)
      I             2  (3)    75 (97)   77 (27)  0         31 (100)  31 (26)
     II             9 (15)    51 (85)   60 (21)  1 (5)     18 (95)   19 (16)
    III            47 (31)   103 (69)  150 (52)  3 (4)     64 (96)   67 (58)
 Web-site
 longevity
 [months](sup c)
    Male           4.4[1-16] 2.9[1-26]           2.3[1-5] 3.2[1-18]
    Female         3.3[1-13] 3.5[1-18]           1  [1]   3  [1-15]
 --------------------------------------------------------------------------
(sup a)S-1 and S-2 webs.
(sup b)Classes assigned at first capture. I = 6.0g-19.0g; II = 19.1g-22.0g; 
III = 22.1g to >30.0g.
(sup c)Longevity is the mean number of months animals were captured, from 
first to last capture. Values in brackets are ranges.

Antibody-positive Peromyscus were more often male and within the heaviest
mass class (Table 2). Although approximately half of the P. boylii tested
were male, 84% of the antibody-positive mice were male. The male-to-female
ratio was similar to that of P. truei, despite the small sample size. We
found more adults and fewer young among the antibody-positive Peromyscus,
even though young-to-adult capture ratios were similar among seronegative
mice.

Longevity of antibody-positive mice was considerably different between the
two species, while longevity of antibody-negative mice was similar (Table
2). Antibody-positive male P. boylii tended to survive longer than
antibody-positive female. Furthermore, antibody-positive male P. boylii
lived longer than antibody-negative male P. boylii (4.4 months and 2.9
months, respectively; t = 2.58, df = 48, p = 0.007).

P. boylii Population Dynamics and Temporal Patterns of Infection

The number of captures per month and the number of samples per month were
usually not the samesome animals were not sampled because of death,
weakened physical condition, hypothermia, or escape. The number of animals
tested for antibody to hantavirus, however, mirrored population trends. The
P. boylii population declined dramatically during summer and autumn 1996,
stabilized at low levels during winter 1996 and 1997, and fell to minimal
levels in spring 1997 (Figure 1) (Table 3).

 Table 3. Population densities and hantavirus-antibody prevalence in
 Peromyscus boylii at two mark-recapture trapping webs, by period
 ------------------------------------------------------------------------------
           Dec 1995-Nov 1997      High density(sup a)    Low density(sup b)
         ---------------------- ---------------------  ------------------------
         Density/ Prevalence/  Density/  Prevalence/   Mean/  Prevalence/
 Web     month     month        month     month        month    month
 sites   (sup c)   (sup d)      (sup c)   (sup d)      (sup c)  (sup d)
 ------------------------------------------------------------------------------

 S-1 &     26.1       20.2       43.6       18.4        11       25.4
 S-2      (4-52)     (0-43)     (32-52)    (10-22)    (4-22)   (12-43)

 S-1       9.7        28.5       15.2       26.4       4.5       37.0
          (1-20)     (0-75)     (10-20)    (15-38)    (1-9)     (0-75)

 S-2       16.4       14.2       28.4       14.3       6.5       15.0
          (3-42)     (0-33)     (13-42)    (6-19)     (3-13)    (0-33)
 ------------------------------------------------------------------------------
 (sup a)June 1995 to June 1996
 (sup b)September 1996 to September 1997
 (sup c)Population density (number of individuals per 6.2 hectares), determined by
 minimum number alive. Values in parentheses are ranges.
 (sup d)Antibody prevalence to hantavirus (%), determined by estimated standing
 prevalence. Values in parentheses are ranges.

[Fig]
Figure 2. Minimum number of living Peromyscus boylii and the estimated standing
prevalence of hantavirus antibodypositive mice at two mark-recapture webs (6.2
ha).*
*Because of adverse weather conditions, we only trapped for 2 nights in January
and May 1995.

For the 35-month sampling period, the mean number of antibody-positive P.
boylii was 5.0 animals per 6.2 ha per month, range 0 to 11 (Figure 1). The
number of antibody-positive P. boylii was higher during high population
densities than during low densities (8.0 and 2.8 animals per 6.2 ha per
month, respectively; t = 4.83, df = 21, p < 0.001). Numbers of antibody-positive
animals were similar during 35 months at S-1 and S-2 (2.7 and 2.4 animals per 
6.2 ha per month, respectively), even though population densities at S-2 were 
regularly higher than at S-1.

The mean antibody prevalence for the sampling period was 20.2% (range 0% to
43%) and was higher during low densities than high densities (Figure 2). At
each site, antibody prevalence rates were also higher during low densities,
but not significantly different from rates during high population
densities. However, antibody prevalence varied between sites and was
consistently higher at S-1 (Table 3). The highest mean monthly antibody
prevalence occurred on S-1 during low population densities (37.0%) and was
higher than prevalence on S-2 during the same period. The highest monthly
antibody prevalence occurred at S-1 during minimal population densities,
May 1997, when three of four captured P. boylii were antibody-positive
(75%).

Maximum and minimum antibody prevalence occurred during similar periods at
both sites. During low population densities, S-2 had 4 months without an
antibody-positive sample, while antibody-positive animals were not captured
from S-1 for 2 months. The months when no antibody-positive animals were
captured were not the same for both sites; at least one positive sample was
recorded each month, even during low population densities.

Longevity and Seroconversion of Infected Mice

Hantavirus antibody-positive P. boylii tended to survive longer (mean 4.2
months) than seronegative mice (mean 3.2 months) (t = 1.77, df = 138, p =
0.04) (Table 4). At site S-2, survival was similar between
antibody-positive and antibody-negative mice, but at S-1, antibody-positive
mice lived longer (4.8 months) than seronegative mice (3.0 months) (t =
2.58, df = 48, p = 0.007). At both sites, survival among male and female
mice was not significantly different.

Table 4. Frequency of intervals between first and last capture of individual Peromyscus
boylii, December 1995November 1997
------------------------------------------------------------------------------------------
                             No. months in interval between first and last captures
                             --------------------------------------------------------
                   No.
 Web sites         P.boylii 1   2  3  4  5   6 7 8 9 10 11 12 13  14 15 16 18 19 26 Mean
                   (sup a)       									(sup b)
 ------------------------------------------------------------------------------------------
 Antibody-positive mice
 S-1 & S-2         58        22  4  6  5  7   2   2 2 3  2     1   1     1           4.2
 S-1               30        10  2  2  1  6   1   1 2 2  1     1         1           4.8
 S-2               28        12  2  4  4  1   1   1   1  1         1                 3.5

 Antibody negative mice
 S-1& S-2          250       117 48 24 11 11  6 7 7 3 4  3  3  1      2     1  1  1  3.2
 S-1               90        47  15 8  3  4   2 2 2 2    3  1                     1  3.0
 S-2               160       70  33 16 8  7   4 5 5 1 4     2  1      2     1  1     3.3
------------------------------------------------------------------------------------------
(sup a)Total number of individual P. boylii.
(sup b)Mean number of months in interval.

[Fig]
Figure 3. Initial antibody acquisition in Peromyscus boylii at two mark- recapture webs, 
by month, December 1995November 1997.

Initial acquisition of hantavirus antibody (seroconversion) was observed in 33% of the
antibody-positive P. boylii. P. boylii acquired hantavirus antibody in all months except 
December, January, and March (Figure 3). Two transmission peaks, accounting for
79% of seroconversions, took place during the typical 7-month reproductive
period, April through October (37% during April, May, and June; 42% during
September and October). Seroconversions at S-2 were directly related to
population levels, with 9 (90%) of 10 S-2 seroconversions taking place
during high population densities in 1995. This relationship did not appear
at S-1, where the number of seroconversions was similar during high and low
population densities.

Incidence of Infection

The incidence rate for seroconversion per 100 mice per month was twice as
high at S-2 (34.5) as at S-1 (17.0). The greater number of mice at risk and
the number of months before seroconversion accounted for the higher
incidence rate at S-2 (Table 5). S-1 had fewer P. boylii at risk, which did
not seroconvert for an average of 5.4 months; S-2 had a larger number of
mice at risk, which seroconverted after 2.2 months.

Table 5. Incidence rates of hantavirus infection in Peromyscus boylii that 
were recaptured and sampled at least twice, December 1995November 1997, 
two web sites
------------------------------------------------------------------------
            No.                      Mouse-mo.               Mean mo.  
            at              (Cumu    of obser-               before  
           risk   Serocon-   lative   vation     Incidence   serocon- 
Sites     (sup a) versions    %)      (sup b)    (sup c)     version                        
------------------------------------------------------------------------ 
S-1         43       9      (20.9)      53.0       17.0        5.4    
S-2         90      10      (11.1)      29.0       34.5        2.2    
S-1 & S-2  133      19      (14.3)      82.0       23.2        3.7    
------------------------------------------------------------------------ 
(sup a)Antibody-negative at time of first capture.                             
(sup b)Includes all time intervals between successive captures when mice were  
seronegative, and half the interval between captures when mice became seropositive.    
(sup c)Seroconversions per 100 mice per month.                                 
                                                                          
Spatial of Patterns of Infected Mice
 
Distribution and movement of antibody-negative and antibody-positive P. boylii
varied by population density and availability of sheler and food resources. At
both trapping web sites, P. boylii distributions were associated with brushy 
chaparral plant species. The ranges of high density mice outline, in general, 
the distribution of thick chaparral stands (Figure 4).

Because plant species diversity and belts of chaparral stands were greater
at S-2, P. boylii distribution was relatively continuous and widespread.
Chaparral stands at S-1 were discontinuous, and P. boylii lived in rocky
pockets of vegetation and were seldom trapped in different chaparral
pockets if separated by open terrain (Figure 4). At both sites, P. boylii
avoided open juniper-pinyon areas.

[Fig] 
Figure 4. Ranges and trap stations of hantavirus antibodypositive
Peromyscus boylii during high and low population densities. Each web  
covered 3.1 ha. Trap stations within ranges were occupied by
antibody-negative and antibody-positive mice at various times.
High densities represent 13 months (June 1995 to June 1996), and low 
densities represent 13 months (September 1996 to September 1997).

During periods of high population density, antibody-positive mice
occupied scattered chaparral habitats of undergrowth areas of the sites and
moved freely between web transects. Movement, however, appeared to be
directly influenced by chaparral cover. During low population densities, 
antibody-positive mice withdrew to a few, well-defined refuges (Figure 4).
The movement of antibody-positive P. boylii during low densities was 
also restricted; mice seldom moved between web transects.

Hantavirus Prevalence Rates and Patterns

The prevalence rates of P. boylii (20.2%) and P. truei (3%) in our study
were similar to those found in other studies carried out in pinyon-juniper
habitats (4). The short-term infection in P. truei may have been caused by
spillover from syntopic P. boylii (the four antibody-positive P. truei were
found only during spring and summer 1995, when P. boylii densities and the
potential of interspecies contact were greatest). Six other rodent species
coexisting with P. boylii should have had similar risks for hantaviral
infection since they had been captured at trap stations used by P. boylii
at one time or another (capturing two or three different species at one
station during a single trapping session was not uncommon). The evident
rarity of hantavirus infection in P. truei and the absence of infection in
other sympatric rodents suggests that P. boylii is the primary hantavirus
host in this area and that transmission to other rodent species may be
unlikely during periods of average population densities. Similar
relationships have been demonstrated in southern Arizona, where only
species of Peromyscus had immunoglobulin G (IgG) antibody reactive with SNV
(Kuenzi et al., this issue, pp. 113-117).

On the basis of long-term infection patterns and persistent virus shedding
(9-11), we assume that hantavirus antibodypositive P. boylii are
chronically infected and infectious (Mills et al., this issue, pp.
135-142). Studies using reverse transcription-polymerase chain reaction
(RT-PCR) on blood samples from field-caught P. maniculatus from Nevada (12)
mirror other studies of host-hantavirus associations in suggesting initial
viremia, followed by a relatively rapid immune response that cleared virus
from blood in approximately 1 month (animals remained antibody-positive for
at least 7 months). However, as numerous studies have shown (9-11;13), the
short duration of hantavirus RNA in blood does not reflect its residence in
organs. Another study demonstrated that 97% of antibody-positive P.
maniculatus were PCR-positive for viral RNA in organ tissues (13), which
implies chronic infection, as has been demonstrated for other
hantavirus-host associations. Nevertheless, the crucial experiments to
demonstrate chronic infection and persistent shedding have not been done
for P. boylii. We are attempting to develop methods to reliably and
consistently collect urine from mark-release-capture animals in the field
to address this problem.

Slightly more male than female mice (1.2:1) were tested for antibody to
hantavirus; however, fewer male than female mice (1:1.2) were
antibody-negative. The higher antibody prevalence in males may be due to
territoriality, aggression toward other males during breeding periods,
longer survival, and breadth of travel (4,14).

Factors Affecting Population Density

The population densities and distributions of rodents were related to
seasonal and year-to-year availability of acorns, seeds, and juniper
berries (mast). Acorns, pinyon seeds, juniper berries, and grasses were
abundant throughout the study sites during summer and autumn 1995,
reflecting surplus winter precipitation the previous 3 years (15).
Population levels of the rodent communityrelatively high during autumn and
winter 1995-96may have been related to this abundance of seed crops.
During the winters of 1995-96 and 1996-97, precipitation was well below
normal, and the first winter drought resulted in complete mast failures by
all chaparral species and pinyon pine. Juniper crops, evident in autumn
1996, were depleted soon after. The second winter drought resulted in the
mast failure of oak species, pinyon, and juniper, but other chaparral
species produced minimal crops during the spring and late summer (Abbott et
al., unpub. data).

P. boylii population fluctuations (Figure 1), related to year-to-year mast
resources and variations in seasonal female reproductive efforts, are
consistent with fluctuations of mast-consuming Peromyscus and Tamias
rodents, which show a positive correlation between mast production and
breeding behavior (16-18). Comparable regional population fluctuations
occurred during this same period in Colorado and southern Arizona (Calisher
et al., this issue, pp. 126-134; Kuenzi et al., this issue, pp. 113-117).
Female reproductive activity was consistently absent during the colder
winter months of November through February. The reproductive period, April
through October, typically unimodal and coinciding with seed development of
syntopic vegetation, started at low levels in April and peaked in late
summer and autumn. Mast of oak species, pinyon pine, and juniper usually
ripen in late summer and early autumn while summer monsoons may cause other
chaparral species to produce seeds in both spring and summer (Abbott et
al., unpub. data).

This pattern of reproduction and food supply was evident during the 1995
breeding season; 40% of the female mice captured in spring showed signs of
reproductive activity, compared with 76% of those captured in summer and
86% of those captured in autumn. The subsequent breeding season began
normally, with 43% of females pregnant, but new pregnancies nearly halted
during the summer months, decreasing 96% from the previous year. Only a few
of the 64 female mice captured in June and July 1996 had a perforate
vagina, and none showed signs of lactation or pregnancy. The autumn
breeding effort declined by 56% from the previous year. During the 1997
breeding season, there were 84% fewer female mice than during the 2
previous years, but most were reproductively active, suggesting that a
population recovery was under way.

Factors Affecting Hantavirus Prevalence

The number of hantavirus-infected mice was higher during the high
population densities of 1995-96 (Figure 1). Month-to-month numbers of
antibody-positive mice appeared more stable than those of antibody-negative
mice. The number of high densityantibody-positive P. boylii was stable
during the winter, with small peaks proportional to monthly capture
success. The numbers of antibody-positive mice remained stable (though
lower) during the subsequent precipitous 7-month population decline. Even
during low population densities, antibody-positive mice were persistent at
minimal, yet stable levels. This consistent presence of at least a few
infected mice may reflect the resident nature of antibody-positive mice,
characteristically older and able to survive for longer periods. Fifty-five
percent of the antibody-positive mice survived on trapping web sites 3
months or longer and were considered resident, while 34% of the
seronegative mice were resident.

The proportion of hantavirus antibodypositive P. boylii varied by
population density and trapping web site (Figure 2) (Table 3). S-1
maintained the highest mean antibody prevalence; during low population
densities, prevalence increased. Almost half of the P. boylii captured at
S-1 tested positive during low density months when at least one mouse was
antibody-positive. Population densities at S-2 were consistently greater
than at S-1 and were associated with lower overall prevalence rates.
Approximately 23% of the P. boylii captured at S-2 were antibody-positive
during low density months when at least one mouse was antibody-positive.
Positive linear correlations between population density and antibody
prevalence have not been found in other species of Peromyscus (Calisher et
al., this issue; pp. 126-134;11;19).

We observed that one third of the antibody-positive P. boylii acquired
antibody. No mice reverted from antibody-positive to antibody-negative.
Transmission of hantavirus was bimodal and associated with spring and
autumn reproductive activity (Figure 3). Thirty-seven percent of P. boylii
seroconverted in the spring, and 42% in the autumn reproductive period.
Mice that seroconverted were more frequently male, within the heaviest mass
class, and survived longer than mice that remained antibody-negative. The
trend for bimodal transmission may reflect intraspecific competition,
greater movement, and aggressive behavior by resident antibody-positive
males during peak reproductive periods (20). Similar transmission trends
have been reported in rat populations (6). Consequently, risks of
horizontal transmission may increase during the more active seasons.

Incidence of infection varied with population densities, recapture rates,
and population dynamics. Rates of P. boylii seroconversion varied by site,
but collectively, both sites had an average 14.3% incidence of infection
among the population at risk during the study period (Table 5). The number
of seroconversions at both sites was similar, but the number of mice at
risk at S-2 was much larger, since population densities were regularly
higher. Consequently, the cumulative proportion of mice seroconverting at
S-2 was 47% lower than at S-1, whereas the incidence of seroconversions per
100 mice per month was 103% greater. Characteristics of the S-1 population
(longer survival as antibody-negative animals, more restricted centers of
activity, and continuous infection during periods of high and low
population densities) may have been contributing factors to the difference
in incidence rates between sites.

The focal ranges of antibody-positive P. boylii were patchy; they expanded
and contracted over time (Figure 4). Hantavirus infection and distribution
patterns were influenced by habitat structure, seasonal food availability,
and the behavioral characteristics of infected mice. At both sites, P.
boylii were associated with corridors and patches of chaparral understory
within the juniper-pinyon woodland, and especially with dense stands of
chaparral associated with rocky substrates and downed trees that provided
optimal shelter. These favored sites were usually located on slopes and
along creek channels. In southern Arizona, P. boylii were found in
analogous habitat distributions; the species favored oak riparian
vegetation, and most were captured in one portion of one trapping web
(Kuenzi et al., this issue, pp. 113-117).

Diverse chaparral stands were more widespread and continuous at S-2. During
high population densities, P. boylii occupied scattered chaparral areas
throughout most of the web and were often trapped at sites several meters
apart. The relatively high abundance of mice over a large area may explain
the greater incidence of infection and lower antibody prevalence at S-2.
The greater number of mice during high population densities and the greater
turnover rate seemed to dilute the prevalence of infection and, at the same
time, increase the risk for infection because of intensified encounters.

The patchiness of hantavirus infection was more evident and focalized at
S-1. Chaparral stands were discontinuous; P. boylii occupied discrete
chaparral pockets, seldom migrating from one pocket to another (Figure 4).
During this study, S-1 had three prominent centers of hantavirus infection
and three associated centers of P. boylii activity. The structure and
disjunct nature of the activity centers (and associated centers of
antibody-positive animals) may have contributed to higher antibody
prevalences and greater cumulative seroconversion since the mice occupying
these restricted habitats had a greater chance of encountering each other.
During low population densities, the higher prevalence rates of 50% to 75%
were related to antibody-positive male mice that were older, heavier, and
able to reside for a longer period within the activity centers. Similar
patterns of clustering or patchiness and hantavirus infection have been
documented for cotton rats, Sigmodon hispidus, in Florida (21).

Along with high population densities, the longer stay of dominant male mice
in optimal and reliable habitats may be a primary variable contributing to
hantavirus infection. This assumption is based on three trends: animals
that became antibody-positive survived longer than those that did not
seroconvert; antibody-positive tended to survive longer than
antibody-negative mice; and in patchy optimal habitats, resident mice
tended to be dominant, male, and antibody-positive. Consequently, resident
male mice may provide a reliable reservoir during low population densities
and therefore ensure the survival of hantavirus within rodent communities.

Conclusions

Our preliminary results, and those of other recent studies (Kuenzi et
al.,this issue, pp. 113-117;18), have implicated precipitation, habitat
structure, and food resources as ultimate environmental factors that
influence reservoir population dynamics, viral transmission, and hantavirus
persistence. The results of this and other recent studies have raised
questions concerning proximate patterns of hantavirus maintenance,
seroconversions, and transmission within specific reservoir species
occupying different western regions (Mills et al., this issue, pp.
135-142). Additional data suggesting that sex ratios, size, and social
organization affect temporal and spatial seroconversion relationships will
be addressed in forthcoming articles. We hope that this ongoing study will
collect sufficient data to explain the interplay of habitat resources,
social hierarchies, intraspecific competition, and dispersal behavior and
how these proximate factors influence hantavirus ecology and human risk.

Acknowledgments

      Special recognition is due to field crew regulars who endured extreme
field conditions: Lisa Gelczis, Nathan Zorich, Tyler Williams, Samantha
Yazzi, Henry Provencio, Dan Carroll, Heather Shane, Chris Davis, Jon Mock,
Karen Mock, and Romey Haberle. Thanks to E. Chambers for veterinary
guidance, C. Levy for initial assistance, B. Farrar for shipping supplies
when requested, J. Dunnum and C. Parmenter for sample management, C.J.
Peters, J.E. Childs, and two anonymous reviewers for manuscript
suggestions, D. Dailey for valuable support throughout this study, and the
Chino Ranger District, Prescott National Forest, for necessary permits.

      This work was supported by grants 08-5071 from the Arizona Department
of Health Services and U50/CCU913429-02 from the Department of Health and
Human Services, Public Health Service, Centers for Disease Control and
Prevention. Partial support was provided by the Yavapai College Foundation.

      Dr. Abbott is chair of the Biology Department, Yavapai College,
Prescott, Arizona. His research focuses on small mammal population ecology
and hantavirus associations, vertebrate metapopulation distributions and
ecology, and riparian ecology. His areas of expertise include desert
ecology and the physiologic ecology of vertebrates; he serves as an
ecologic consultant to federal, state, and private agencies.

      Address for correspondence: Ken D. Abbott, Department of Biology,
Yavapai College, 1100 E. Sheldon, Prescott, AZ 86301, USA; fax:
520-776-2315; e-mail: sm_ken@yavapai.cc.az.us.

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Hantavirus

A Longitudinal Study of Sin Nombre Virus Prevalence in Rodents,
Southeastern Arizona

Amy J. Kuenzi, Michael L. Morrison, Don E. Swann, Paul C. Hardy, and
Giselle T. Downard
University of Arizona, Tucson, Arizona, USA

---------------------------------------------------------------------------
      We determined the prevalence of Sin Nombre virus antibodies in
      small mammals in southeastern Arizona. Of 1,234 rodents (from
      13 species) captured each month from May through December 1995,
      only mice in the genus Peromyscus were seropositive. Antibody
      prevalence was 14.3% in 21 white-footed mice (P. leucopus),
      13.3% in 98 brush mice (P. boylii), 0.8% in 118 cactus mice (P.
      eremicus), and 0% in 2 deer mice (P. maniculatus). Most
      antibody-positive mice were adult male Peromyscus captured
      close to one another early in the study. Population dynamics of
      brush mice suggest a correlation between population size and
      hantavirus-antibody prevalence.

We examined the role of rodent species as natural reservoirs for
hantaviruses in southeastern Arizona to identify the species infected with
hantavirus, describe the characteristics of infected animals, and assess
temporal and intraspecific variation in infection rates.

Trapping Procedures

Beginning in May 1995, we established four permanent trapping webs on the
Santa Rita Experimental Range in the Santa Rita Mountains of southeastern
Arizona (Pima County). The design of these webs, as well as details on
mark-recapture trapping procedures, are described by Mills et al. (this
issue, pp. 95-101). Elevations of the trapping webs are approximately 1,250
m to 1,379 m. All trapping webs contained approximately equal amounts of
two main vegetation types, semidesert grassland (characterized by Lehmann
lovegrass [Eragrostis lehmanniana], three-awn [Aristida spp.], prickly pear
cactus [Opuntia spp.], and mesquite [Prosopis velutina]) and oak riparian
(characterized by deciduous trees including Arizona white oak [Quercus
arizonica] and netleaf hackberry [Celtis reticulata]; occurs in drainage
areas where water flow is seasonally intermittent), occur at these
elevations. Web 1 was operated from May 1995 through September 1996, when
trapping was discontinued because of low trap success, and webs 2, 3, and 4
were operated from May 1995 through December 1997.

From May 1995 through September 1996, webs 1 and 4 were considered
controls. Captured mice from these webs were identified, marked, weighed,
and measured, but not bled. Beginning in November 1996, we began collecting
blood samples from mice on web 4. The bleeding process had little effect on
survival (1). The methods for obtaining blood samples and the serologic
testing of samples for hantavirus antibodies are described in Mills et al.
(this issue, pp. 95-101).

We examined population dynamics of common species infected with Sin Nombre
virus (SNV) using data from three webs that were trapped continuously from
May 1995 through December 1997. Using the minimum number of rodents known
to be alive during a 3-day trapping session, we calculated an index of
population size by taking the total number of rodents captured during each
3-day trapping session and adding to that sum the number of rodents
captured on at least one previous and one subsequent session (2). The
minimum number of hantavirus antibodypositive rodents was calculated in
the same way. We estimated standing prevalence for each trapping session by
dividing the minimum number of antibody-positive rodents by the minimum
number of rodents known to be alive.

Capture histories were used to estimate survivorship of the trappable
population. These estimates were calculated as the percentage of rodents
known to be alive a given number of months after initial capture. Although
we refer to these estimates as survival rates, they are more accurately
described as trapping web residency rates, as deaths cannot be
distinguished from emigration.

Trapping Results

Between May 1995 and December 1997, 1,234 rodents were captured a total of
3,226 times, and 1,231 blood samples were obtained (Table 1). Bailey's
pocket mouse (Chaetodipus baileyi) was the most common species captured
(57% of rodents captured). Common murid rodents captured included
white-throated wood rat (Neotoma albigula) (10%) and four species in the
genus Peromyscus (27%). The cactus mouse (P. eremicus) was the most common
Peromyscus species captured (12%) followed closely by the brush mouse (P.
boylii) (11.5%). Deer mice (P. maniculatus) and white-footed mice (P.
leucopus) were also captured but in low numbers (<3% each). Other species
captured infrequently (<1%) included the fulvous harvest mouse
(Reithrodontomys fulvescens), yellow-nosed cotton rat (Sigmodon
ochrognathus), desert pocket mouse (C. penicillatus), and Merriam's
kangaroo rat (Dipodomys merriami).

 Table 1. Prevalence of antibodies to Sin Nombre virus among wild rodents
 in southeastern Arizona, May 1995December 1997

 --------------------------------------------------------------------------
                                          No. rodents
                                           trapped
                                         and released               No.
                                            (total         No.    positive
 Family/Species      Common name       captures)(sup a)  tested     (%)
 --------------------------------------------------------------------------
 Heteromyidae

   Dipodomys merriamiMerriam's             1        (2)      0     0 (0.0)
                     kangaroo rat
   Chaetodipus spp.  Pocket mice

     C. baileyi      Bailey's pocket      704     (715)     329    0 (0.0)
                     mouse

     C. penicillatus Desert pocket         25      (27)       7    0 (0.0)
                     mouse
 Subtotal                                 730     (744)     336    0 (0.0)
 Muridae

   Neotoma albigula  White-throated       126     (126)      51    0 (0.0)
                     wood rat

   Onychomys torridusSouthern               7       (7)       7    0 (0.0)
                     grasshopper mouse
   Peromyscus spp.   White-footed mice
     P. boylii       Brush mouse          137     (142)      98  13 (13.3)
     P. eremicus     Cactus mouse         151     (152)     118    1 (0.8)

     P. leucopus     White-footed           29     (30)      21   3 (14.3)
                     mouse
     P. maniculatus  Deer mouse             6       (6)       2    0 (0.0)
   Reithrodontomys   Fulvous harvest
 fulvescens          mouse                 16      (16)      12    0 (0.0)
   Sigmodon          Yellow-nosed           11
 ochrognathus        cotton rat           (11)     (11)       5    0 (0.0)
 Subtotal                                 483     (490)     314   17 (5.4)
 Total                                  1,213   (1,234)     650    17 (2.6)
 --------------------------------------------------------------------------
 aTotal captures include rodents trapped and released and those that died
 during handling.

Prevalence of Antibody-Positive Rodents

Only rodents in the genus Peromyscus had antibodies reactive with SNV;
however, antibody prevalence varied considerably among species within this
genus (Table 1). Most (13 of 17) antibody-positive rodents were brush mice.
One cactus mouse and three white-footed mice were also antibody positive.
With the exception of one white-footed mouse, all antibody-positive rodents
were captured in oak riparian vegetation. Antibody-positive rodents were
captured on all three webs from which animals were bled; however, most
(65%) were first captured on web 2 early in the study (May to June 1995).
The farthest distance between trap stations where these web 2rodents were
captured was approximately 190 m, and half were captured at three adjacent
trap stations along one transect line.

All antibody-positive rodents were positive upon first capture, and most
(58%) were never recaptured. Antibody-positive animals that were recaptured
were caught an average of 3.8 times (standard deviation = 2.03, n = 7,
range 2 to 8). All but one of the recaptured animals remained antibody
positive on subsequent captures. The exception, a male brush mouse, was
antibody negative on its three recaptures.

Characteristics of Infected Populations

Antibody-positive rodents were more likely to be male than female and were
predominately adult (Table 2). The ratio of male to female among
antibody-positive brush mice was significantly higher than that among the
total sample (chi-square with Yates' correction = 7.97, degrees of freedom
= 1, p = 0.005), and significantly more adults were antibody positive than
would be expected from the distribution of age classes among the total
sample (chi-square = 9.69, df = 2, p = 0.002). Although the sample size is
too small for significance testing, these patterns hold for white-footed
mice as well (Table 2).

 Table 2. Distribution of antibody-positive versus all brush mice, cactus
 mice, and white-footed mice, by sex and age

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

                    Brush mice        Cactus mice     White-footed mice

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

                No. (%)  Total     No. (%)  Total    No. (%)       Total
 Characteristic positive no. (%)   positive no. (%)  positive     no. (%)
 ------------------------------------------------------------------------------

 Sex

    Male        12 (92)  51 (52)   0        59 (50)  3 (100)     10 (48)

    Female      1 ( 8)   47 (48)   1 (100)  59 (50)  0           11 (52)

 Age

   Juvenile     0        12 (12)   0        22 (18)  0            2 (10)

   Young adult  1 (8)    38 (40)   0        48 (41)  0            3 (14)

   Adult        12 (92)  48 (48)   1 (100)  48 (41)  3 (100)      16 (76)

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

Brush Mice Population Dynamics and Temporal Pattern of Infection

The number of brush mice varied both by season and by year. The minimum
number known to be alive was relatively high during the first 10 months of
the study, May 1995 through March 1996 (Figure 1). The number of brush mice
declined during the spring of 1996 and remained low until the fall, when
the numbers increased but never reached the levels of the previous year.
Captures for the next year followed a similar pattern with increased
numbers during fall and winter (October through March), followed by a
steady spring decline and summer low.

 						  The minimum number of brush mice
                                      known to be infected was highest
 						  during the initial part of our study
 [fig 1]                              (Figure 1). Eleven of the 13
 Figure 1. Population trends of brush hantavirus antibodypositive mice
 mice, as determined by the minimum   were first captured between May and
 number known to be alive, Santa Rita September 1995, gradually
 Experimental Range, southeastern     disappearing from the population. By
 Arizona, May 1995December 1997.     October 1996, no animals were known
                                      to be infected on any of our trapping
 						  webs. One new antibodypositive brush
						  mouse was captured in November 1996
 [fig 2]                              and another in November 1997.
 Figure 2. Survivorship functions     Similarly, the estimated standing
 (percentage of brush mice known to   prevalence of hantavirus antibody
 be alive after initial capture)      ranged from 40% in May 1995 to 0% in
 based on recapture data, Santa Rita  both October 1996 and April through
 Experimental Range, southeastern     October 1997 (mean= 8.25%).
 Arizona, May 1995December 1997.
                            		  Male and female brush mice showed
                                      similar rates of survivorship with an
                                      approximately 50% turnover rate
around 2 months after initial capture (Figure 2). Hantavirus
antibodypositive mice did not survive quite as long; the 50% turnover rate
occurred approximately 1 month after initial capture. By 6 months after
first capture, approximately 80% of all rodents had disappeared. A small
percentage of brush mice continued to be captured for more than 1 year
after tagging.

Conclusions

The overall prevalence of antibodies reactive with SNV antigen varied
considerably among wild rodents captured in southeastern Arizona between
May 1995 and December 1997, from 0% for Heteromyidae to 5.4% for Muridae.
Low prevalence within the heteromyids has been commonly documented (3-5).
Of mice, only Peromyscus were seropositive at our study site. The mean
antibody prevalence of 7% for all Peromyscus was similar to the mean
prevalence reported from Kansas (6) and Montana (7), although lower than
that at many other sites in Arizona and New Mexico (3,4). The low
hantavirus-antibody prevalence at our site may be related to its location
in Sonoran Desert semigrassland and its relatively low rainfall; Mills et
al. (4) found that prevalence of SNV was lowest at altitudinal and climatic
extremes.

The primary Peromyscus species with evidence of hantavirus infection at the
Santa Rita Experimental Range was the brush mouse, recently shown to be an
important carrier of SNV or an SNVrelated virus throughout the
southwestern United States (4). Even within a single species, overall
prevalence of hantavirus antibodies has been reported to vary widely among
different regions and habitats and in different seasons and years. In
samples of deer mice from sites throughout the southwestern United States,
Mills et al. (4) found antibody prevalence of 0% to 50%. Within states,
overall prevalence in deer mice was 9.5% to 38.6% at 10 sampled sites in
New Mexico (3) and 0% to 50% in 34 counties in California (5).

Several studies have indicated, as does ours, that the presence and number
of antibody-positive mice are not evenly distributed. Although Peromyscus
were trapped in both vegetation types within our study site, all but one of
the antibody-positive mice were trapped in oak riparian vegetation, and
most were trapped in one portion of one web. Similarly, Mills et al. (4)
captured antibody-positive deer mice in only 21 of 41 sites where deer mice
were captured, and hantavirus antibody-positive brush mice in only 9 of 17
sites. Our results suggest that the prevalence of antibody-positive animals
may be correlated with different habitats and provide additional evidence
for focality of hantavirus in "reservoir" populations (4).

While our sample sizes are too small to determine statistical significance,
they suggest a correlation between population size and prevalence of
hantavirus antibody. The number of antibody-positive animals was highest
when the population was decreasing from an abundance of Peromyscus in the
spring of 1995, the most recent peak. This finding is in contrast to local
studies in the Channel Islands (8), Montana (7), and the regional study of
Mills et al. (4), which found no relationship between antibody prevalence
and density of deer mice. However, Childs et al. (3) found higher antibody
prevalence in pinyon-juniper vegetation in 1993, when evidence suggests
that rodent densities were unusually high (9).

Additional data from our long-term study and other studies should help
determine whether any relationship between density and antibody prevalence
exists and if so, what the related temporal patterns are. Population sizes
of rodents in the Sonoran Desert of southeast Arizona, as in other areas
with climatic extremes, are highly variable. The number of P. boylii at
Santa Rita Experimental Range was initially high but declined over the
course of our study (perhaps because of changes in annual rainfall). To
reproduce, many desert rodents require green vegetation (10), often not
available in semidesert grasslands and xeroriparian areas. Total annual
rainfall at Santa Rita Experimental Range was higher than normal in the 2
years before the start of our study. Since 1995, annual rainfall has been
approximately 8 cm to 10 cm below the norm (unpub. data). Petryszyn (11)
has linked high variability of Peromyscus populations in the Sonoran Desert
with extreme fluctuation in winter rainfall. Others (12) have indicated
local population expansion and retraction in response to wetter and drier
conditions.

Finally, our results are consistent with those of other studies that show a
higher prevalence of infection (as indicated by antibody) in male and
sexually mature rodents. However, we did not observe direct signs of
aggressive encounters or fighting among infected males, as observed by
Childs et al. (13) for hantaviral infection in Rattus norvegicus.

Field studies of hantavirus infection and wild rodent populations provide a
rare opportunity for public health officials, virologists, and ecologists
to better understand the dynamics of rodent populations and the
interactions between disease, humans, small mammals, habitat, and climatic
factors. The few long-term datasets in ecology are invaluable for their
contributions to the understanding of processes that vary in complex ways
over time but are also relevant to management of both the natural
environment and human health.

Acknowledgments

      We thank T. Abeloe, C. Boal, M. Bucci, T. Cutler, L. Hall, C.
Johnson, A. McLuckie, J. Martin, I. Rodden, and S. Simpson for assistance
in the field. We also thank C. Levy, D. Engelthaler, J. Mills, T. Ksiazek,
C. J. Peters, and J. Dunnum for assistance. R. Sanderson and C. Plumb at
the Santa Rita Experimental Range provided logistical support.

      Funding for this study was provided by the Centers for Disease
Control and Prevention and the Arizona Department of Health Services.

      Dr. Kuenzi is an assistant research professor at Montana Tech, the
University of Montana. Her research focuses on small mammal population
ecology.

      Address for correspondence: Amy J. Kuenzi, Department of Biology,
Montana Tech of the University of Montana, Butte, MT 59701, USA; fax:
406-496-4650; e-mail: Akuenzi@mtech.edu.

References

  1. Swann DE, Kuenzi AJ, Morrison ML, DeStefano S. Effects of sampling
     blood on survival of small mammals. Journal of Mammalogy
     1997;78:908-13.
  2. Krebs CJ. Demographic changes in fluctuating populations of Microtus
     californicus. Ecological Monographs 1966;36:239-73.
  3. Childs JE, Ksiazek TG, Spiropoulou CF, Krebs JW, Morzunov S, Maupin
     GO, et al. Serologic and genetic identification of Peromyscus
     maniculatus as the primary rodent reservoir for a new hantavirus in
     the southwestern United States. J Infect Dis 1994;169:1271-80.
  4. Mills JN, Ksiazek TG, Ellis BA, Rollin PE, Nichol ST, Yates TL, et al.
     Patterns of association with host and habitat: antibody reactive with
     Sin Nombre virus in small mammals in the major biotic communities of
     the southwestern United States. Am J Trop Med Hyg 1997;56:273-84.
  5. Jay M, Ascher MS, Chomel BB, Mason M, Sesline D, Enge BA, et al.
     Seroepidemiologic studies of hantavirus infection among wild rodents
     in California. Emerg Infect Dis 1997;3:183-90.
  6. Kaufman GA, Kaufman DW, McMillan BR, Brillhart DE. Prevalence of
     hantavirus antibodies in natural populations of deer mice in north
     central Kansas. Prairie Naturalist 1994;26:209-16.
  7. Douglass RJ, Van Horne R, Coffin KW, Zanto SN. Hantavirus in Montana
     deer mouse populations: preliminary results. J Wildl Dis
     1996;32:527-30.
  8. Graham TB, Chomel BB. Population dynamics of the deer mouse
     (Peromyscus maniculatus) and Sin Nombre Virus, California Channel
     Islands. Emerg Infect Dis 1997;3:367-70.
  9. Parmenter R, Virgil R. The hantavirus epidemic in the southwest: an
     assessment of autumn rodent densities and population demographics in
     central and northern New Mexico. Department of Biology, University of
     New Mexico, Albuquerque, New Mexico; 1993. Sevilleta Long-Term
     Ecological Research Program (LTER); Publication No.: 45.
 10. Beatley JC. Dependence of desert rodents on winter annuals and
     precipitation. Ecology 1969;50:721-4.
 11. Petryzyn Y. Population dynamics of nocturnal desert rodents: a nine
     year study [dissertation]. Tucson (AZ): University of Arizona; 1982.
 12. Brown JH, Heske EJ. Temporal changes in a Chiuahuan Desert rodent
     community. Oikos 1990;59:290-302.
 13. Childs JE, Glass GE, Korch GW, LeDuc JW. Prospective seroepidemiology
     of hantaviruses and population dynamics of small mammal communities of
     Baltimore, Maryland. Am J Trop Med Hyg 1987;37:648-62.

Hantavirus

Statistical Sensitivity for Detection of Spatial and Temporal Patterns in
Rodent Population Densities

Cheryl A. Parmenter, Terry L. Yates, Robert R. Parmenter, and Jonathan L.
Dunnum
University of New Mexico, Albuquerque, New Mexico, USA

---------------------------------------------------------------------------
      A long-term monitoring program begun 1 year after the epidemic
      of hantavirus pulmonary syndrome in the U.S. Southwest tracked
      rodent density changes through time and among sites and related
      these changes to hantavirus infection rates in various
      small-mammal reservoir species and human disease outbreaks. We
      assessed the statistical sensitivity of the program's field
      design and tested for potential biases in population estimates
      due to unintended deaths of rodents. Analyzing data from two
      sites in New Mexico from 1994 to 1998, we found that for many
      species of Peromyscus, Reithrodontomys, Neotoma, Dipodomys, and
      Perognathus, the monitoring program detected species-specific
      spatial and temporal differences in rodent densities;
      trap-related deaths did not significantly affect long-term
      population estimates. The program also detected a short-term
      increase in rodent densities in the winter of 1997-98,
      demonstrating its usefulness in identifying conditions
      conducive to increased risk for human disease.

We analyzed the statistical capabilities of the long-term rodent monitoring
program begun in 1994 to detect spatial and temporal changes in rodent
densities and determine if the low death rates at all study sites resulted
in biased (underestimated) rodent population density estimates. We also
examined a short-term subset of the data (mid-1997 to early 1998) to test
whether the program design could statistically detect a sudden rodent
increase in density that may precede a hantavirus pulmonary syndrome
outbreak.

Study Sites

We selected two monitoring sites in New Mexico for analysis because they
provided the longest period of field sampling, the greatest range in rodent
species richness, and the largest difference in habitat types. The first, a
desert grassland site 90 km south of Albuquerque, on the Sevilleta National
Wildlife Refuge, Socorro County, New Mexico, USA (34 21.3'N, 106 53.1'W,
elevation 1,465 m), was dominated by one-seed juniper (Juniperus
monosperma), honey mesquite (Prosopis glandulosa), and various desert
grasses (Sporobolus spp. and Bouteloua eriopoda). The second site
(Placitas), a pinyon-juniper woodland site located in Sandoval County in
the foothills of the Sandia Mountains, Cibola National Forest,
approximately 30 km north of Albuquerque, New Mexico, USA (35 16.7'N, 106
18.6'W, elevation 1,830 m), was dominated by pinyon pine (Pinus edulis),
one-seed juniper, and blue grama grass (Bouteloua gracilis).

Statistical Methods

The overall experimental design and field sampling procedures are described
by Mills et al. (pages , this issue). The rodent density estimates we
analyzed were based on mark-recapture trapping data from three permanently
marked trapping webs at each site (1,2). We used monthly trapping data
collected from August 1994 to February 1998. Trapping and rodent handling
methods were described by Parmenter et al. (3), and safety procedures
during animal processing followed Mills et al. (4). Blood samples collected
from Peromyscus maniculatus were analyzed by the Centers for Disease
Control and Prevention for Sin Nombre virus (SNV). Rodent densities were
calculated by the program DISTANCE (2). Each dataset was analyzed by three
models (uniform, half-normal, and hazard) with three possible model
adjustments (cosine, polynomial, and hermite). Akaike's information
criterion was then used to select the model that best fit the particular
dataset (2). The three web density estimates for each trapping period were
then partitioned into "proportional" densities representing each species,
on the basis of the relative proportion of each in the total web sample.
These species-specific densities (in numbers of mice per hectare) were
analyzed by a repeated measures analysis of variance (RMANOVA) to test for
differences between sites and through time and for site and time
interactions. If significant F values were observed, we conducted
within-trapping-period tests to detect differences among site means; we
used Fisher's least-significant-difference method.

The effect of low death rates among rodents on estimates of population size
was analyzed by minimum number alive (MNA) methods (5). For this analysis,
we calculated the "observed" MNA values for each species during each
sampling period on the basis of actual field data, which included
occasional trap deaths of rodents. We then constructed a hypothetical MNA,
assuming that no trap deaths occurred in the sampled populations. The
hypothetical death-free MNAs were computed by extending the projected life
span of each animal that died in the trap. The length of the extensions
differed by species and site and was determined by the mean number of
trapping periods during which each species would normally have been present
on each site (this figure was based on the lifespans of all other mice of
that species that did not die in the traps or during handling). For
example, if Neotoma albigula at the Sevilleta National Wildlife Refuge
site, Web 1, had a mean lifespan of three trapping periods (i.e., its
initial capture period [time zero] plus three additional sampling periods),
and a mouse died in a trap on its first capture, we would add one mouse to
the observed MNA values for three additional trapping periods to arrive at
the hypothetical MNA values. If the mouse died during its second trapping
period, we would add two trapping periods to the MNA estimates, and so on.
In addition, if the dead mouse was pregnant or lactating, we increased the
hypothetical MNA by the average number of offspring that would have been
produced; mean numbers of offspring for each species were determined from
specimen databases at the University of New Mexico's Museum of Southwestern
Biology. These offspring were included for the duration of the expected
lifespan on each study site. Thus, if a pregnant female N. albigula died
during the study, we would add two offspring (the mean litter size for this
species in New Mexico) to the MNA estimates for the full three trapping
periods of their life expectancy. This process created species-specific
hypothetical MNA values that were either equal to or greater than the
observed MNA values. The two MNA datasets were then compared by RMANOVA.

Spatial Differences in Rodent Densities

RMANOVA          Table 1. Repeated-measures analysis of variance testing
successfully     for differences among representative rodent population
distinguished    densities(sup a), Sevilleta National Wildlife Refuge and
rodent densities Placitas, 1994-1998
between sites
for a number of  ----------------------------------------------------------
species (Table   Species            Source           DF   F value    p
1). Ord's
kangaroo rat and ----------------------------------------------------------
the Plains       Dipodomys          Site               1   7.52    0.0517
pocket mouse       ordii            Error (Site)       4
(Heteromyidae)
were much more     (Ord's kangaroo
abundant at the     rat)            Time              35   2.19    0.0007
Sevilleta                           Time x Site       35   2.21    0.0006
National                            Error (Time)     140
Wildlife Refuge
site than at     Perognathus        Site               1   25.69   0.0071
Placitas (Figure   flavescens       Error (Site)       4
1A, B) and had
greater            (Plains pocket
statistical         mouse)          Time              35   2.91    0.0001
differences in                      Time x Site       35   2.89    0.0001
the RMANOVA                         Error (Time)     140
results (Table
1). In contrast, Peromyscus         Site               1   0.28    0.6233
other rodent       maniculatus      Error (Site)       4
species            (Deer mouse)     Time              35   2.80    0.0001
(Muridae) had no
overall                             Time x Site       35   1.18    0.2513
differences by                      Error (Time)     140
site (Table 1)
and usually had  Peromyscus         Site               1   0.11    0.7609
similar            leucopus         Error (Site)       4
densities,         (White-footed
except for          mouse)          Time              35   7.25    0.0001
occasional
episodes                            Time x Site       35   4.56    0.0001
(Figures 1C-G).                     Error (Time)     140
Although we      Peromyscus truei   Site               1   1.98    0.2328
observed no
overall effect     (Pinyon mouse)   Error (Site)       4
of site on                          Time              35   3.77    0.0001
density for
these species,                      Time x Site       35   3.29    0.0001
Fisher's                            Error (Time)     140
least-significant-difference        Site               1   6.65    0.0614
methods showed     Neotoma
significant        albigula         Error (Site)       4
differences        (White-throated
between sites       wood rat)       Time              35   1.53    0.0431
during certain                      Time x Site       35   1.65    0.0221
periods (Figures
1C,D,F,G),                          Error (Time)     140
demonstrating    Reithrodontomys    Site               1   0.34    0.5906
that within a
particular         megalotis        Error (Site)       4
species,         (Western harvest
intersite          mouse)           Time              35   4.04    0.0001
differences                         Time x Site       35   3.57    0.0001
could be
discerned in                        Error (Time)     140
both long-term   ----------------------------------------------------------
sequences and    (sup a)Density=number of mice per hectare.
during episodic,
site-specific population irruptions.

Temporal Changes in Rodent Densities

The analyses also detected changes in rodent densities through time in all
species examined (Table 1). Several species with generally low densities
(e.g., the harvest mouse [Figure 1D], the white-footed mouse [Figure 1E],
and the deer mouse [Figure 1G]) occasionally became locally extinct but
periodically recolonized the sites. Other species (e.g., the pinyon mouse
[Figure 1F] and the white-throated wood rat [Figure 1C]) were found
consistently on both sites, although their densities fluctuated
considerably. In all cases, RMANOVA found significant differences in these
temporal patterns.

Short-Term Rodent Population Increases

To determine the capability of the analyses to show statistically
significant short-term increases in rodent densities (e.g., a rodent
population "explosion"), we selected May 1997 to February 1998, a period
characterized by a rodent density increase in some species (Figures 1A-G).
We then tested the datasets for this period alone; RMANOVA results
indicated significant increases in densities for the Plains pocket mouse,
deer mouse, white-footed mouse, pinyon mouse, and Western harvest mouse
(Table 2; Figure 1 B,D-G) during the winter of 1997 to 1998. Therefore, the
monitoring program was capable of showing sudden, short-term increases in
rodent densities that may precede a disease outbreak in humans.

Blood tests to determine the presence of SNV in deer mice showed generally
low infection rates (Figure 2), with a maximum of only one mouse testing
positive per trapping period at Placitas, and none at Sevilleta. The
SNV-positive rodents were detected during periods of moderate abundance in
1994 and 1995 but not in the early stage of the population increase during
the winter of 1997-98.

Low Death Rates among Rodents and Population Estimates

Four species had sufficient sample sizes for the MNA analyses, which were
based on 28 trapping periods between August 1994 and January 1997 and
included 7,024 rodent captures (Table 3). During field sampling, trap death
rates were generally lower than 10% (3). A breakdown of the number of new
animals, recaptured resident animals, and trap deaths indicated that
immigration and reproduction rates were consistently higher than trap death
rates (Figure 3A-D). Species showing territorial behavior (e.g., Merriam's
kangaroo rat [Figure 3A] and the white-throated wood rat [Figure 3C]) had
higher ratios of residents to immigrants than species without strongly
defended territories.

 [fig 1]                                                                          
 Figure 1. Mean densities of rodents at the Sevilleta National Wildlife     
 Refuge (solid circles) and Placitas (open circles) study sites. Asterisks  
 indicate significantly different means between sites (Fisher's             
 least-significant-difference tests, p < 0.05). A. Ord's kangaroo rat       
 (Dipodomys ordii). B. Plains pocket mouse (Perognathus flavescens). C.     
 White-throated wood rat (Neotoma albigula). D. Western harvest mouse       
 (Reithrodontomys megalotis). E. White-footed mouse (Peromyscus leucopus).  
 F. Pinyon mouse (P. truei). G. Deer mouse (Peromyscus maniculatus).        
                                                                            
In constructing the hypothetical MNA values, we used the following mean
life expectancy values (number of trapping periods after initial capture): 
D. merriami = 2.16; P. flavescens = 1.11; P. truei = 0.68; N. albigula = 2.75. 
For female rodents of reproductive age, the following mean numbers of offspring 
were used (on the basis of University of New Mexico's Museum of Southwestern 
Biology data): D. merriami = 2.03; P. flavescens = 2.75; P. truei = 3.46; 
N. albigula = 1.83. When analyzed with RMANOVA, observed MNA values were not 
different from hypothetical (Table 3). While highly significant temporal 
differences in MNA values were observed (Figures 4A-D), no treatment-by-time
interactions were produced, which demonstrated that the low death rates during 
the monitoring program did not affect rodent population estimates.

Conclusions

Our analyses indicated that the field experimental statistical design was
sufficiently sensitive to detect a range of differences in densities of
rodent species across study sites and through time. Even relatively
moderate levels of increases were detectable by our methods, although they
were far less dramatic than those observed during the 1993 SNV outbreak
(6). While rodent densities of certain species significantly increased
during this study (1994 to 1998), the maximum densities were considerably
lower than those observed at the Sevilleta National Wildlife Refuge site in
1993 during the SNV outbreak (6). In addition, seroprevalence in deer mice
dropped to zero in 1996 (Figure 1G) and did not return, despite the higher
densities observed in this species at these sites. Clearly, the population
dynamics observed at this time were not equivalent to the rodent "outbreak"
of 1993. Continued monitoring of these populations will be needed to
determine the extent and importance of this apparent trend in rodent
population ecology.

 Table 2. Short-term repeated-measures analysis of         The statistical
 variance for differences among representative rodent      sensitivity
 population densities, Sevilleta National Wildlife Refuge  demonstrated in
 and Placitas , May 1997 to February 1998                  this study is
                                                           critical to the
 --------------------------------------------------------- success of field
 Species            Source          DF F value      p      monitoring
 --------------------------------------------------------- programs,
                                                           particularly
 Dipodomys          Site             1   5.75     0.0745   those that
   ordii            Error (Site)     4                     function as
   (Ord's                                                  early warning
    kangaroo rat)   Time             8   2.13     0.0616   systems to alert
                                                           health-care
                    Time x Site      8   2.13     0.0616   workers and
                    Error (Time)    32                     researchers of
                                                           impending
 Perognathus        Site             1  27.09     0.0065   outbreaks of
   flavescens       Error (Site)     4                     disease (7,8).
   (Plains pocket                                          In the case of
    mouse)          Time             8   4.61     0.0008   SNV, the
                                                           monitoring
                    Time x Site      8   4.61     0.0008   program serves
                    Error (Time)    32                     as both an early
 Peromyscus         Site             1   2.70     0.1759   warning system
                                                           and as a
   maniculatus      Error (Site)     4                     research
   (Deer mouse)     Time            35   2.45     0.0432   database to
                                                           which numerous
                    Time x Site     35   1.57     0.1868   environmental
                    Error (Time)   140                     variables and
 Peromyscus         Site             1   2.25     0.2084   the prevalence
                                                           of SNV
   leucopus         Error (Site)     4                     infections in
   (White-footed                                           rodents can be
    mouse)          Time             8   7.83     0.0001   correlated.
                    Time x Site      8   5.79     0.0001
                                                           In addition to
                    Error (Time)    32                     the need for
 Peromyscus         Site             1   5.09     0.0870   statistical
                                                           power in
   truei            Error (Site)     4                     distinguishing
   (Pinyon mouse)   Time             8   7.09     0.0001   spatial and
                    Time x Site      8   4.94     0.0005   temporal
                                                           patterns, the
                    Error (Time)    32                     monitoring
 Neotoma            Site             1   1.06     0.3618   program must
   albigula         Error (Site)     4                     provide study
                                                           site population
   (White-throated                                         estimates that
    wood rat)       Time             8   1.17     0.3443   can be directly
                    Time x Site      8   0.82     0.5944   compared.
                                                           Standardization
                    Error (Time)    32                     of techniques
 Reithrodontomys    Site             1   4.58     0.0990   used by
   megalotis        Error (Site)     4                     collaborating
                                                           research groups
 (Western harvest                                          ensures such
   mouse)           Time             8   4.67     0.0007   comparability.
                    Time x Site      8   1.64     0.1530   In these
                    Error (Time)    32                     hantavirus
                                                           studies, the use
 --------------------------------------------------------- of trapping webs
 Density = numbers of mice per hectare.                    and distance
                                                           sampling theory
(2) also allows for direct comparisons of rodent densities among species
and across widely varying ecosystems. Trapping grids, and their associated
population estimators, often produce results that may be suitable for local
studies (internally consistent within a single experiment), but cannot be
compared across ecosystem types and taxa because of site-specific
characteristics or species-specific assumptions of capture probabilities.
Trapping webs and distance sampling density estimators, however, have
produced reasonably accurate density estimates in both a computer
simulation study (9) and a field study (10). The accuracy of trapping webs
and grids in estimating rodent densities is being evaluated more fully at
the Sevilleta National Wildlife Refuge site.

All these methods require an appropriate sampling design and an
understanding of the basic biology of the rodents. For example, the Plains
pocket mouse has MNA values of four animals per month for the months of
November, 1994 to March 1995 (Figure 4B). In contrast, its density for the
same period is zero (Figure 1B), which indicates that no animals were
captured during field sampling. The species' habit of becoming inactive and
remaining in underground burrows during cold winter weather accounts for
this discrepancy. Thus, in ecologic and perhaps disease transmission terms,
density data accurately reflect which species of rodents are active on a
site, whereas MNA data show the combined survivorship of active and
inactive species.

A potential source of estimation bias in rodent monitoring programs is the
inadvertent influence of trapping and handling of rodents during field
sampling. Capturing, anesthetizing, measuring, and collecting blood and
saliva samples traumatizes small animals and may affect future trapping
success, which, in turn, could bias the accuracy of the density or
population estimators. Previous studies have shown various effects of
trapping and handling on rodent body mass, ability to trap rodents in the
future, and survival (3,11,12-17), but none have addressed the effect of
low death rates on long-term population trends. While precautions are taken
to ensure survival of sampled animals, occasionally a few die during
sampling, especially those with a lower tolerance to the physical,
physiologic, and psychologic stress of being captured and handled. Chronic
loss of study animals from trapping or handling could underestimate their
densities when compared to those of "natural" or undisturbed populations
nearby. Results of our study indicate that death rates from trapping at
these sites had no significant effect on long-term rodent population
estimates.

The existing network of rodent population study sites seems successful in
identifying local species-specific fluctuations in densities. Data from
these sites can be used in addressing hypotheses on the relationships among
environmental factors, rodent abundance, and SNV infections, as well as in
providing an early warning for potential rodent population explosions that
may increase the risk for other hantavirus pulmonary syndrome outbreaks in
the southwestern United States.

[fig 2]
 Figure 2. Seroprevalence of Sin
 Nombre virus (SNV) in the deer
 mouse populations at Sevilleta
 National Wildlife Refuge and
 Placitas study sites.

[fig 3]
 Figure 3. Composition of sampled
 populations at the Sevilleta
 National Wildlife Refuge study
 site. The number of immigrants
 greatly exceeds incidental trap
 deaths. A. Merriam's kangaroo rat
 (Dipodomys merriami). B. Plains
 pocket mouse (Perognathus
 flavescens). C. White-throated wood
 rat (Neotoma albigula). D. Pinyon
 mouse (Peromyscus truei).
                                     
[fig 4]
Figure 4. Mean minimum number alive
(MNA) values of "observed" (solid
circles) and "hypothetical" (open
circles) for the following:
A. Merriam's kangaroo rat (Dipodomys
merriami)
B. Plains pocket mouse (Perognathus
flavescens)
C. White-throated wood rat (Neotoma
albigula) at the Sevilleta National
Wildlife Refuge study site
D. Pinyon mouse (Peromyscus truei)
at the Placitas study site.
Values are means of three replicate
trapping sites; error bars represent
one standard deviation.

 Table 3. Repeated-measures analysis of variance for
 differences between actual minimum number alive (MNA)
 estimates and hypothetical MNAs (zero deaths from trapping),
 New Mexico, 1994 to 1997
 -------------------------------------------------------------
 Species          Source             DF F value     p
 -------------------------------------------------------------
 Dipodomys
   merriami       MNA Treatment       1   0.03   0.8672
   (Merriam's     Time               27  21.19   0.0001
   kangaroo       Time x MNA         27   0.30   0.9997
    rat)          Error (Time)      108
 Perognathus      MNA treatment       1   0.08   0.7858
   flavescens     Error (MNA)         4
   (Plains        Time               27   7.99   0.0001
    pocket        Time x MNA         27   0.01   1.0000
    mouse)        Error (Time)      108
 Peromyscus       MNA treatment       1   0.03   0.8661
   truei          Error (MNA)         4
   (Pinyon        Time               27   6.24   0.0001
    mouse)        Time x MNA         27   0.13   1.0000
                  Error (Time)      108
 Neotoma          MNA treatment       1   0.57   0.4914
   albigula       Error (MNA)         4
   (White-        Time               27  11.82   0.0001
    throated      Time x MNA         27   0.15   1.0000
    wood rat)     Error (Time)      108
 -------------------------------------------------------------

Acknowledgments

      We thank the U.S. Fish and Wildlife Service (Sevilleta National
Wildlife Refuge) and the U.S. Forest Service (Cibola National Forest) for
their cooperation in the study.

      This research was supported by a collaborative agreement between the
Department of Biology, University of New Mexico, the U.S. Centers for
Disease Control and Prevention, the Indian Health Service, and the State of
New Mexico Health Department. Additional support was provided by the
Sevilleta Long-Term Ecological Research Program (NSF Grant DEB-9411976),
the Museum of Southwestern Biology, and the National Institutes of Health
(Grant 1 PO1 AI39780).

      Cheryl A. Parmenter is a research associate in the University of New
Mexico's Museum of Southwestern Biology and data manager for the New Mexico
Hantavirus Monitoring Team.

      Address for correspondence: Cheryl A. Parmenter, Department of
Biology, The University of New Mexico, 167 Castetter Hall, Albuquerque, NM
87131-1091, USA; fax: 505-277-0304; e-mail: cparment@unm.edu.

References

  1. Anderson DR, Burnham KP, White GC, Otis DL. Density estimation of
     small-mammal populations using a trapping web and distance sampling
     methods. Ecology 1983;64:674-80.
  2. Buckland ST, Anderson DR, Burnham KP, Laake JL. Distance sampling.
     Estimating abundance of biological populations. New York: Chapman and
     Hall, 1993.
  3. Parmenter CA, Yates TL, Parmenter RR, Mills JN, Childs JE, Campbell
     ML, et al. Small mammal survival and trapability in mark-recapture
     monitoring programs for hantavirus. J Wildl Dis 1998;34:1-12.
  4. Mills JN, Yates TL, Childs JE, Parmenter RR, Ksiazek TG, Rollin PE, et
     al. Guidelines for working with rodents potentially infected with
     hantavirus. Journal of Mammalogy 1995;76:716-22.
  5. Krebbs CJ. Demographic changes in fluctuating populations of Microtus
     californicus. Ecological Monographs 1966;36:239-73.
  6. Parmenter RR, Brunt JA, Moore DL, Ernest SM. The hantavirus epidemic
     in the Southwest: rodent population dynamics and the implications for
     transmission of Hantavirus-associated adult respiratory distress
     syndrome (HARDS) in the Four Corners region. Report to the Federal
     Centers for Disease Control and Prevention, July 1993. Publication No.
     41. Sevilleta Long-Term Ecological Research Program (LTER) 1993:1-45.
  7. Mills JN, Ellis BA, McKee KT Jr, Calderon GE, Maiztegui JI, Nelson GO,
     et al. A longitudinal study of Junin virus activity in the rodent
     reservoir of Argentine hemorrhagic fever. Am J Trop Med Hyg
     1992;47:749-63.
  8. Mills JN, Ellis BA, Childs JE, McKee KT Jr, Maiztegui JI, Peters CJ,
     et al. Prevalence of infection with the Junin virus in rodent
     populations in the epidemic area of Argentine hemorrhagic fever. Am J
     Trop Med Hyg 1994;51:554-62.
  9. Wilson KR, Anderson DR. Evaluation of a density estimator on a
     trapping web and distance sampling theory. Ecology 1985;66:1185-94.
 10. Parmenter RR, MacMahon JA, Anderson DR. Animal density estimation
     using a trapping web design: field validation experiments. Ecology
     1989;70:169-79.
 11. Swann DE, Kuenzi AJ, Morrison ML, DeStefano S. Effects of sampling
     blood on survival of small mammals. Journal of Mammalogy
     1997;78:908-13.
 12. Slade NA, Iskjaer C. Daily variation in body mass of free-living
     rodents and its significance for mass-based population dynamics.
     Journal of Mammalogy 1990;71:357-63.
 13. Wood MD, Slade NA. Comparison of ear-tagging and toe-clipping in
     prairie voles, Microtus ochrogaster. Journal of Mammalogy
     1990;71:252-5.
 14. Slade NA. Loss of body mass associated with capture of Sigmodon and
     Microtus from northeastern Kansas. Journal of Mammalogy 1991;72:171-6.
 15. Kaufman GA, Kaufman DW. Changes in body mass related to capture in the
     prairie deer mouse (Peromyscus maniculatus). Journal of Mammalogy
     1994;75:681-91.
 16. Douglass RJ, Van Horn R, Coffin KW, Zanto SN. Hantavirus in Montana
     deer mouse populations: Preliminary results. J Wildl Dis
     1996;23:527-30.
 17. Vanblankenstein T, Botzler RG. Effect of ectoparasite removal
     procedures on recapture of Microtus californicus. J Wildl Dis
     1996;32:714-5.

Hantavirus

Natural History of Sin Nombre Virus in Western Colorado

Charles H. Calisher,* William Sweeney,* James N. Mills, and Barry J.
Beaty,*
*Colorado State University, Fort Collins, Colorado, USA; and Centers for
Disease Control and Prevention, Atlanta, Georgia, USA

---------------------------------------------------------------------------
      A mark-recapture longitudinal study of immunoglobulin G (IgG)
      antibody to Sin Nombre virus (SNV) in rodent populations in
      western Colorado (1994results summarized to October 1997)
      indicates the presence of SNV or a closely related hantavirus
      at two sites. Most rodents (principally deer mice, Peromyscus
      maniculatus, and pinyon mice, P. truei) did not persist on the
      trapping webs much beyond 1 month after first capture. Some
      persisted more than 1 year, which suggests that even a few
      infected deer mice could serve as transseasonal reservoirs and
      mechanisms for over-winter virus maintenance. A positive
      association between wounds and SNV antibody in adult animals at
      both sites suggests that when infected rodents in certain
      populations fight with uninfected rodents, virus amplification
      occurs. At both sites, male rodents comprised a larger
      percentage of seropositive mice than recaptured mice, which
      suggests that male mice contribute more to the SNV epizootic
      cycle than female mice. In deer mice, IgG antibody prevalence
      fluctuations were positively associated with population
      fluctuations. The rates of seroconversion, which in deer mice
      at both sites occurred mostly during late summer and midwinter,
      were higher than the seroprevalence, which suggests that the
      longer deer mice live, the greater the probability they will
      become infected with SNV.

To monitor Sin Nombre virus (SNV) dynamics in natural rodent communities,
we established longitudinal studies at two sites in western Colorado, each
near a location where human hantavirus infections occurred in 1993. This
article provides a summary of the data collected during the first 3 years
of the studies. The results indicate that rodent populations in western
Colorado have decreased since 1993; SNV or an SNV-like hantavirus persists
at these sites; and prevalence of immunoglobulin G (IgG) antibody to SNV
fluctuates with time and perhaps with weather patterns that modify the
ecosystem.

The Study

Selected Sites

Each study area was selected on the basis of its proximity to residences of
hantavirus pulmonary syndrome (HPS) case-patients, convenience for field
work, and guaranteed cooperation by land managers. Sites at Fort Lewis (La
Plata County, southwest Colorado) (N 37 13' 30.9" latitude, W 108 10'
51.1" longitude, altitude 2,438 m) and Molina (Mesa County, west central
Colorado) (N 39 09' 45.8" latitude, W 108 03' 18.4" longitude, altitude
1,951 m) were within a few kilometers of case-patient residences.

Fort Lewis (approximately 22 km west of Durango, 8 km south of Hesperus) is
10 km north of Red Mesa, Southern Ute Indian Reservation, Colorado, where
rodent trapping in 1993 showed that deer mice had an antibody prevalence
rate of 50% to SNV and near where one of the persons who later died of HPS
had been infected with SNV (1). Seroprevalence in Peromyscus maniculatus,
the principal rodent reservoir of SNV, was approximately 50% to 19%,
respectively, near study sites in La Plata and Mesa Counties (1).

We established trapping webs (Mills et al., this issue, pp. 95-101) in two
protected areas (Fort Lewis A and Fort Lewis B) of the 2,550 ha-Colorado
State University San Juan Basin Research Center, which serves as a model
for cattle breeders and livestock geneticists. The natural characteristics
of these sites have been preserved.

Fort Lewis is in the drainage of the La Plata River, south of Mount
Hesperus in the La Plata Mountains. The general ecosystem of the area is
montane shrubland (2) superimposed on intrusive igneous rocks forming
laccoliths (3). The overstory vegetation at Fort Lewis A is predominately
ponderosa pine (Pinus ponderosa) and Gambel's oak (Quercus gambeli);
understory vegetation is primarily blue grama (Bouteloua gracilis), black
grama (B. eriopoda), and floral components also seen at Fort Lewis B. At
Fort Lewis B, 500 m from Fort Lewis A, overstory is essentially all
Gambel's oak; understory is composed of blue and black grama or there is no
overstory, with the microcommunity composed primarily of blue and black
grama, small soapweed (Yucca glauca), tree cholla (Opuntia imbricata), and
pasture sagebrush (Artemesia frigida).

The trapping sites near Molina (approximately 60 km east of Grand Junction)
are within 2 km of the home of a 1993 case-patient. In 1993, deer mice had
an antibody prevalence rate of 19% to SNV (4).

At Molina we established webs in two areas (Molina A and Molina B, 500 m
apart) that are privately owned and have not been grazed by cattle for many
years. The sites have no standing water sources, but an irrigation ditch,
containing rapidly running water, flows during the summer at the west and
north edges of Molina A.

The general ecosystem of the area is semidesert shrubland (2) superimposed
on Mancos shale (3). At Molina A, we found principally Rocky Mountain
juniper (Juniperus scopulorum), pinyon pine, small soapweed, and pasture
sagebrush. Molina B is characterized by pasture sagebrush, Rocky Mountain
juniper, Parry's rabbitbrush (Chrysothamnus paryii), and pinyon pine at the
periphery.

All field data were recorded on hard copy and entered into EPI-5, a
database and statistical program available from the Centers for Disease
Control and Prevention (CDC) (5).

Sampling Methods

All materials were transported to the study sites or were available in
towns near the sites. Under license of the State of Colorado's Department
of Natural Resources, sampling was done every 6 weeks, weather permitting.
Trapping webs were established according to methods agreed upon by
collaborating groups (Mills et al., this issue, pp. 95-101). In brief, each
web comprised 12 rows of 12 Sherman traps (7.6 cm x 8.9 cm x 22.9 cm; H.B.
Sherman Traps, Inc., Tallahassee, FL) each, the first four traps in each
row being placed 5 m apart, the next eight placed 10 m apart; rows were 30
degrees from each other. The location of each trap was marked with a
construction flag. Rodents were anesthetized with Metaphane
(methoxyflurane, Pitman-Moore, Mundelein, IL) during processing, marked
with sequentially numbered stainless steel ear tags, and released at the
capture site.

Webs A and B at each location were sampled for 2 or 3 consecutive nights,
but rodents were neither bled nor swabbed at webs B until October 1996,
when animals from both sites were sampled. The original intent had been to
not take blood or oropharyngeal swab samples at either web B to determine,
by comparison with data from the corresponding web A, the impact of these
invasive procedures on the rodent populations. Because the death rates at
webs A and B were essentially the same after 2 years (6; C.H. Calisher and
B.J. Beaty, unpub. data), in October 1996, we began to take blood samples
from rodents at both webs and to no longer collect oropharyngeal swabs.
Rodents, principally deer mice, were processed and samples were placed on
dry ice (-70C), returned to the laboratory in Fort Collins, and placed in
a mechanical freezer (-80C) until they were tested for IgG antibody.

Sampling was conducted according to standardized protocols (Mills et al.,
this issue, pp. 95-101). To compare age categories, in the field we
empirically classified captured animals as juvenile, subadult, or adult,
according to Fitzgerald, Meaney, and Armstrong (2). For final
determination, we separated animals into weight classes (10% to 40% of
adult mean weight = juvenile, 41% to 80% = subadult, and 81% to 100% =
adult).

After being tested at Colorado State University, blood samples and
oropharyngeal swabs were shipped to Atlanta, Georgia, where confirmatory
testing for IgG antibody to SNV was conducted with blood samples, and
oropharyngeal swabs were stored for possible future testing.

Enzyme-Linked Immunosorbent Assays (ELISA) for IgG Antibody to SNV

ELISA was performed at Colorado State University as described (Mills et
al., this issue, pp. 95-101). Results presented here were obtained at
Colorado State University; testing at CDC provided confirmation. We
initially screened whole blood samples at 1:100; antibody-positive samples
were titrated to determine end points.

Population Densities

We estimated the population size at each sampling period by calculating the
minimum number of rodents alive (7). The minimum number of rodents alive
for a given trapping session was calculated by taking the total number of
rodents captured during that session and adding to that sum all rodents
that had been captured on at least one previous and one subsequent
occasion. The minimum number of antibody-positive rodents was calculated
similarly, and the estimated standing prevalence was calculated as minimum
number of antibody-positive rodents/minimum number of rodents alive.

Findings

Over the 41-month trapping period at Fort Lewis and the 37-month trapping
period at Molina, antibody reactive with SNV was detected in 29 (9.6%) of
302 deer mice at Fort Lewis and 36 (9.4%) of 385 at Molina; 4 (2.6%) of 155
of pinyon mice at Molina also had antibody (Table 1). For comparison, in
1993, prevalence of antibody to SNV in P. maniculatus was approximately 50%
near Fort Lewis (La Plata County) and 19% near Grand Junction (Mesa County)
(1). Of 112 least chipmunks (Tamias minimus), two Colorado chipmunks (T.
quadrivittatus), and two western harvest mice (Reithrodontomys megalotis),
none had antibody to SNV.

Table 1. Antibody (enzyme-linked immunosorbent
assay for immunoglobulin G) to Sin Nombre virus,
Fort Lewis and Molina, Colorado, 1994-1997
--------------------------------------------------
                               No.         %
				    positive/  Antibody-
Location	Species	   No. tested  positive
--------------------------------------------------
Fort       Peromyscus        29/302       9.6
Lewis       maniculatus
	     Tamias minimus     0/48        0
	     P. truei           1/3        33

Molina     P. maniculatus    36/385       9.4
	     P. truei           4/155       2.6
	     P. leucopus        1/2        50
           Reithrodontomys    0/2         0
		megalotis
 	     T. minimus         0/64        0
	     T. quadrivittatus  0/2         0
--------------------------------------------------

At Fort Lewis, trapping success (number of animals per total number 
of trap nights) was 0.3% to 7.6%, depending on the season (lowest 
rates, AprilJune; highest, AugustOctober). Antibody-positive deer  
mice were found in 13 of 21 trapping intervals Antibody prevalence     
(calculated when more than four deer mice were caught in a given
trapping period) was 0%  Molina to 42.9% with a mean of           
29 (9.5%) of 302. Antibody to SNV was detected in adult (10.5%), 
subadult (9.8%), and juvenile (12.5%) deer mice; the stages represented                
63.9%, 13.1%, and 23%, respectively, of the deer mice captured.
Males represented 48.8% of the deer mice (and 47.8% of recaptured deer
mice) but 58.3% of the antibody-positive rodents.

At Molina, trapping success was 2.6% to 17.9% and, as at Fort Lewis,
depended on the season (lowest rates, MayJune; highest, JulyOctober).
Antibody-positive deer mice were found in 12 of 17 trapping intervals.
Antibody prevalence was 0% to 33% in deer mice (mean 9.4%) and 0% to 18.2%
in pinyon mice (mean 2.6%). Antibody to SNV was detected in adult (11.3%),
subadult (1.7%), and juvenile (4.4%) deer mice; the stages represented
73.1%, 15.3%, and 11.7%, respectively, of the mice captured. Males
represented 45.5% of the deer mouse population, 46.3% of the recaptured
deer mice, and 60% of the antibody-positive mice. Antibody was detected in
four adult (three male, one female) pinyon mice (P. truei). Of 118 pinyon
mice collected, 62 (50.8%) were female and 56 (49.4%) were male. We
detected seropositive pinyon mice only during May and June 1995 and April
1996.

Wounds and Antibody

Because we were working with a large number of anesthetized rodents, we did
not closely examine each animal for wounds, as had been done by Glass et
al. (8). However, we noted the most obvious wounds (ear nicks, torn ears,
scarred tail) and those likely not to have been caused by trapping,
tagging, or processing, and we evaluated the data for deer mice at webs A
for Fort Lewis and Molina.

Of 233 adult deer mice at Fort Lewis, 20 had both antibody and wounds, 76
had no antibody but had wounds, 4 had antibody and no wounds, and 133 had
neither antibody nor wounds; thus, wounds were associated with antibody to
SNV among adult deer mice (Yates-corrected chi-square 17.71, p = <0.001).
At Molina, of 339 adult deer mice, 8 had antibody and wounds, 23 had no
antibody but had wounds, 21 had antibody and no wounds, and 287 had neither
antibody nor wounds; again wounds were associated with antibody to SNV
(Yates-corrected chi-square 10.67, p = <0.001).

Seroconversion

Fifteen deer mice and one pinyon mouse seroconverted (i.e., seronegative to
seropositive or a fourfold or greater increase in titer) between captures
(Figure 1). At Fort Lewis, 302 deer mice (150 female and 152 male) were
captured. Of these, 37 female and 37 male mice were recaptured at least
once. Five male and three female deer mice at Fort Lewis seroconverted. One
deer mouse had antibody for the first time 14 months after it was initially
captured. At Molina, 385 deer mice (212 female, 173 male) and 155 pinyon
mice (85 female, 70 male) were captured. Of these, 33 female and 30 male
deer mice and 12 female and 10 male pinyon mice were recaptured at least
once. Five male and two female deer mice and one male pinyon mouse
seroconverted. An additional three deer mice (two male, one female) at
Molina were recaptured and had significant (3,200 to 25,600) but stable IgG
antibody titers; we did not consider these as having seroconverted. The
five male mice seroconverted at Fort Lewis during the summer (one between
July and September 1994, two between July and September 1995, one at
[estimated] midsummer 1995, and one between June and September 1997); two
female mice seroconverted between October 1994 and May 1995, and one female
mouse seroconverted during late summer (September to October) 1997. At
Molina, one male deer mouse seroconverted in late spring (estimated May)
1995, one in late fall 1995, two male deer mice and a male pinyon mouse
during the winter or early spring of 1995 to 1996, and one male deer mouse
during late spring 1996; one female deer mouse seroconverted in late summer
1995 and one during the winter 1995 to 1996. Seropositive samples were
titrated by IgG ELISA with fourfold dilutions. Titers were 100 to 102,400,
with most of them at 6,400 to 25,600.

[Fig]
Figure 1. Approximate month of seroconversion in Peromyscus
species at Fort Lewis and Molina, Colorado, June 1994October 1997, 
by sex.
                           
Incidence Rates

We calculated incidence rates of IgG antibody to SNV in deer mice recaptured
and sampled at least twice at Fort Lewis and Molina (Table 2). At Fort Lewis A,
the overall incidence was 4.6 new infections per 100 mice per month (4.8
for male, 4.4 for female); at Fort Lewis B, the overall incidence was 10.91 (9.1
for male, 18.2 for female); and at the two sites combined, the overall 
incidence was 6.1 (6.3 for male, 5.8 for female). At Molina A, the overall 
incidence rate in deer mice was 2.8 new infections per 100 mice per month 
(3.2 for male, 2.3 for female); at Molina B, no new infections were detected 
during the observation period; the incidence at the two sites combined was 2.6 
(3.0 for male, 2.2 for female). Because sufficient numbers of pinyon mice were 
captured and a seroconversion was detected at Molina A, we were able to calculate 
the incidence of seroconversion: 0.6 overall (1.4 for male, 0 for female); the
incidence at the two sites combined was 0.5 (1.3 for male, 0 for female).

Table 2. Incidence of immunoglobulin G antibody reactive with Sin Nombre
virus in Peromyscus maniculatus (deer mice) recaptured and sampled at
least twice at Fort Lewis (June 1994October 1997) and P. maniculatus and
P. truei (pinyon mice) recaptured and sampled at least twice at Molina,
western Colorado (October 1994October 1997)
---------------------------------------------------------------------------
Species/       No. at     No.of new     Cum. %     Mouse mos. of
location Sex  risk(sup a)infections antibody-pos.observation(sup b)Incidence(sup c)
 ---------------------------------------------------------------------------
Deer    Grid A    38        4           10.5          88           4.6
mice/    male     17        2            5.9          42           4.8
Ft.      female   21        2            9.5          46           4.4
Lewis   Grid B    11        3           27.3          27.5        10.9
         male      6        2           33.3          22           9.1
         female    5        1           20.0          5.5         18.2
        Grids A+B 49        7           14.3         115.5         6.1
         male     23        4           17.4          64           6.3
         female   26        3           11.5          51.5         5.8

Deer    Grid A    59        5            8.5         179.5         2.8
mice/    male     33        3           11.0          94           3.2
Molina  female    26        2            7.7          85.5         2.3
        Grid B     7        0            0            12.0          0
         male      4        0            0             7.5          0
         female    3        0            0             4.5          0
        Grids A+B 66        5            7.6         191.5         2.6
         male     37        3            8.1         101.5         3.0
         female   29        2            6.9          90           2.2

Pinyon  Grid A    22        1           4.5           157          0.6
mice/    male     10        1           10.0           71          1.4
Molina   female   12        0            0             86           0
        Grid B    15        0            0            33.5          0
         male      7        0            0             9            0
         female    8        0            0            24.5          0
        Grids A+B 37        1            2.7         190.5         0.5
         male     17        1            5.9          80           1.3
         female   20        0            0           110.5          0
---------------------------------------------------------------------------
(sup a)No. of mice antibody-negative at first capture.
(sup b)Total time intervals between successive captures when mice were
antibody-negative, plus half the interval when mice changed from
antibody-negative to antibody-positive.
(sup c)New infections per 100 mice per month.

Longevity

By recapturing animals, we were able to estimate the longevity of infected
and uninfected mice at the sites. Most Peromyscus spp. (75.7% at Fort
Lewis, 66.2% at Molina) were not recaptured after they were first caught.
At Fort Lewis, of 118 female and 117 male deer mice, 79 and 83,
respectively, were caught only once; 61 were recaptured only within a
5-month period, eight within 6 to 9 months, and four 11 to 14 months after
they were first captured. At Molina, of 164 female and 150 male deer mice,
115 and 93, respectively, were caught only once; 89 were recaptured only
within a 4-month period, 15 only within 5 to 9 months, and 2 as long as 10
months after they were first captured. Of 63 female and 59 male pinyon
mice, 49 and 43, respectively, were caught only once; 21 were recaptured
only within a 4-month period, 5 only within 5 to 9 months, and 1 each for
10, 11, 19, and 20 months after they were first captured.

Longevity data of seropositive and seronegative deer mice at Fort Lewis and
Molina are summarized in Table 3. Eighteen deer mice had antibody at two or
three bleeding intervals from 1 to 7 months after first capture (mean = 2.4
months). Totals do not match the text above because blood samples were not
collected from captured rodents at sites B until October 1996 and because
we included separately periods of seronegativity and seropositivity for
deer mice that seroconverted. Differences between mean longevities by sex,
site, or antibody status were not statistically significant
(Yates-corrected chi-square, p = >0.2).

 Table 3. Longevity of hantavirus-infected and -uninfected male and female
 Peromyscus maniculatus at Fort Lewis and Molina, Colorado, June
 1994October 1997 expressed as number of months between first and last
 capture
 -----------------------------------------------------------------------
                                       No. months between first
                                          and last capture
                   Sero-    Total
 Site       Sex    status    No.   1   2 3  4 5  6 7 8  9 10  11  Mean
 -----------------------------------------------------------------------
 Fort Lewis F        +        3    2               1              3
            M        +        6    3     2         1              2.7
 Molina     F        +        3    2     1                        1.7
            M        +        6    4     1         1              2.3
 Fort Lewis F                27   15  5 2       2 1 1    1       2.5
            M                22   7   4 2  2 1  1 2    2 1       3.7
 Molina     F                37   24  2 4  2    1 2    1     1   2.4
            M                27   13  1 4  2    3 1 1  2         3.2
 -----------------------------------------------------------------------

[Fig]
Figure 2. Minimum number of deer mice alive (MNA) (the number of
individual mice captured in a month plus those mice captured 
on at least one previous and one subsequent occasion) and
estimated standing prevalence (ESP) (minimum number infected
divided by MNA), Fort Lewis, June 1994October 1997.

[Fig]
Figure 3. Minimum number of deer mice alive (MNA) (the number of
individual mice captured in a month plus those mice captured
on at least one previous and one subsequent occasion) and
estimated standing prevalence (ESP) (minimum number infected
divided by MNA), Molina, June 1994October 1997.

Population Densities

Deer mouse populations and prevalence of antibody to SNV at Fort Lewis 
(Figure 2)were relatively low throughout this study, except in May and 
June 1995 when samples included only four deer mice and one deer mouse, 
respectively. Mean minimum number of rodents alive was 28 in 1994 but 
lower from 1995 to 1997(10.8, 13.4, and 16.4, respectively). At Molina, 
populations were relatively stable between 1995 and 1997 (only one
collection made in 1994), with mean minimum number of rodents alive values
of 31.2, 20.4, and 25.4, respectively. As at Fort Lewis, estimated standing
prevalence values were commensurately low (Figure 3).

Conclusions

On the basis of the high antibody titers of these seropositive samples, 
our findings elsewhere in Colorado (Calisher, Beaty, and Mills, unpub.
data), and the findings of others studying hantaviruses in the Southwest
(9), we presumed that IgG antibody to SNV in deer mice indicated infection
with SNV and not with El Moro Canyon or another hantavirus. Although 
we did not attempt to isolate or detect hantaviral RNA in blood or other 
tissues from mice with antibody, the only hantavirus specifically identified 
in deer mice in western Colorado has been SNV (10).

The presence of IgG antibody to hantaviruses in rodents is presumed to
indicate past infection and present infection, at least in the primary
vertebrate hosts of hantaviruses (Mills et al., this issue, pp. 135-142).
That is, rodents infected with hantaviruses with which they appear to be
closely associated coevolutionarily (e.g., deer mice and SNV, Western
harvest mice and El Moro Canyon virus, rice rats [Oryzomys palustris] and
Bayou virus, Black Creek Canal virus and cotton rats [Sigmodon hispidus])
do not appear ill or otherwise affected by hantaviruses specific to them.
In host-virus associations that have been studied, the specific hosts
become infected early or later in life, are viremic for a short period, and
excrete virus in their saliva, urine, and feces, perhaps for life (11-14).

Fighting (including exchange of blood and saliva) between infected and
uninfected adult rodents has been suggested as the primary mechanism by
which hantaviruses are amplified epizootically (8). Infected rodents become
viremic and viruric and serve as subsequent sources of infection for others
in the population. Earlier studies using Seoul virus and laboratory rats as
a model system had indicated that while in newborn rats infection became
persistent, in older rats it was transient (15). However, evidence using
Black Creek Canal virus and adult hispid cotton rats, Hantaan virus and
Apodemus agrarius, and Puumala virus and Clethrionomys glareolus indicates
that whereas viremia may diminish over time, virus can still be detected in
various organs, including the salivary gland, for several months after
infection (11-14). Given the relatively brief life span of rodents,
infection and concomitant infectivity for a few weeks or months would
provide a mechanism for seasonal, albeit not transseasonal, persistence of
hantaviruses. Passive acquisition of maternal antibody may protect the
offspring of infected dams early in their lives, but when antibody wanes,
they enter the adult population as susceptibles. Infected later in life,
they can become persistent shedders of virus and sources of infection for
others in the population.

Deer mice infected with SNV when very young likely are able to serve as
reservoirs of the virus for the remainder of their lives. Although our
studies do not distinguish between death and dispersal, the life span of
many deer mice at these sites may not be much more than a month. However,
because some deer mice live for 1 or 2 years, longevity of even a small
proportion of the deer mouse population may provide a transseasonal
mechanism for virus persistence.

A second mechanism of virus transmission, an epizootic one, depends on
short-term infections of deer mice infected as subadults or as adults. At
periods of deer mouse population peaks (e.g., at the end of the breeding
season, in late summer and fall, and during period of decreased
availability of food), male mice fight one another for breeding partners,
food, and territory. This premise is supported by results of serologic
tests of recaptured deer mice at Fort Lewis and at Molina. At Fort Lewis,
48.8% of the deer mice and 47.8% of the recaptured deer mice were male, but
58.3% of the seropositive deer mice were male. At Molina, 45% of the deer
mice and 46.3% of the recaptured deer mice were male, but 60% of the
seropositive deer mice were male. These data support the hypothesis that
male deer mice contribute more to the epizootic cycle of SNV than female
deer mice. However, the lack of association between sex, wounds, and
antibody at either Fort Lewis or Molina indicates that individual mice of
either sex may fight and, through this mechanism or another, become
infected with a hantavirus. That most mice with antibody to SNV are male
supports the suggestion that fighting among mice, biting, and scratching
can lead to hantavirus transmission from an infected to an uninfected,
wounded mouse (4). The limited time these mice may be able to transmit
virus might be sufficient to maintain virus infection in the population.

When deer mouse populations decrease precipitously because of decreased
availability of food and water, the likelihood that SNV will disappear from
the population increases. However, a few long-lived, persistently infected
deer mice can serve as reservoirs until conditions are suitable for the
populations to recover.

Our data appear to support such a unified hypothesis. Fluctuations in IgG
antibody prevalence in deer mice at Fort Lewis and at Molina have lagged
somewhat behind but have been similar to fluctuations in deer mouse
population. In male deer mice at Fort Lewis and at Molina, most
seroconversions (recent infections) occurred during the summer or fall,
whereas in female deer mice, most occurred between fall and spring. During
winter, Colorado deer mice reduce their home range, aggregate in nests, and
enter short-term torporstrategies that together temper reduced food
availability and energy loss due to cold (2). Although we did not find deer
mice that had been infected for more than 3 months, we recovered a few more
than 1 year (some nearly 2 years) after they first were trapped; thus,
under natural conditions and despite the usual declines caused by
predation, cold, heat, and decreases in food, deer mice that reach
adulthood can live as long as 2 years (2), a period sufficient to allow SNV
to survive adverse conditions of low populations and the resulting
decreased number of susceptibles. Furthermore, whereas the overall
seroprevalence of IgG antibody to SNV in deer mice at Fort Lewis was 6.8%
(12 of 165) and in deer mice and pinyon mice, respectively, at Molina 7.2%
(15 of 193) and 5.5% (3 of 52), the rate of seroconversion among deer mice
at Fort Lewis was 16.3% (8 of 41) recaptures, and among deer mice and
pinyon mice, respectively, at Molina 9.9% (7 of 64) and 3.3% (1 of 29).
These results suggest that the longer deer mice live, the greater the
cumulative probability they will become infected with SNV.

The deer mouse, the most numerous mammal in North America, often described
as a "quintessential generalist," can survive on any dry land habitat in
its range and invade and exploit areas disturbed by flood, fire, avalanche,
landslides, mining, construction, extreme grazing, or land development. In
ecologically stable areas, deer mice may be limited by the presence of more
specialized rodent species (2), but they are found from forests to
grasslands, canyons to deserts, farmlands to farm houses and suburban
homes, moving into the latter more often in fall but able to take up
residence whenever an opportunity presents itself. Thus, the movement of
SNV-infected deer mice into human residences itself creates a risk factor
for HPS. The Fort Lewis and Molina sites have not been affected by obvious
ecosystem perturbations in recent years, and deer mouse populations at
these sites are not high, yet are considerably decreased from the
apparently inordinately high levels of 1993 (J. Mills, pers. comm. 1997).
Each site seems ecologically stable, but subtle changes may have gone
unnoticed.

Deer mice are omnivores, storing food for winter consumption but known to
feed on acorns, nuts, insects, other small invertebrates, carrion, fungi,
bone, and various plant parts, including seeds, leaves, and bark, roots,
and tubers (2,16). In one study, seeds accounted for 69% to 76% of stomach
contents of deer mice in Colorado, insects for 14% to 25% (2). This
proportion depends on the season (i.e., availability of food supply); deer
mice are more likely to feed on insects and insect larvae in spring, seeds
and berries in fall.

Notwithstanding the nature of deer mice to consume a variety of foods, they
rely heavily on acorns, when oaks (Quercus sp.) comprise a significant
proportion of their habitat (16). Further, in the northeastern United
States, the quantity of mast seems directly related to population size of
white-footed mice (P. leucopus) and eastern chipmunks (T. striatus) (17).
Although Gambel's oak is abundant at the Fort Lewis trapping sites and
copious numbers of acorns were attached to the trees and on the ground in
1994, we did not observe acorns on trees there between spring 1995 and fall
1997. The chipmunk population at this site was, with the exception of a
transient, moderate increase in June 1996, never high during our study
period (June 1994 to October 1997). At the Molina sites, which do not have
oaks, the chipmunk population declined considerably after June 1996 and did
not return to its previous level. Chipmunks may serve as an indicator for
the ready availability of acorns and other nuts or food in general.

The continued low population densities of deer mice at Fort Lewis and
Molina are puzzling. If, for example, deer mouse population densities in
surrounding areas are higher than at the study sites, one might expect deer
mice from those areas to move into the area with the low population.
However, at Fort Lewis 59% of adult and 5% of subadult deer mice were
recaptured; at Molina 86% of adults and no subadult or juvenile deer mice
were recaptured as adults. These data indicate that few, if any, young deer
mice are immigrating to these sites or if they are, they did not survive
long enough to be captured, and the survival rate of this species' young is
not high.

Temperature fluctuations that affect habitat characteristics can influence
rodent breeding seasons (e.g., rate of plant growth, total available
nutritional biomass). However, analyses of available data (not presented)
did not provide obvious evidence for such direct relationships. In
contrast, a paucity of precipitation at Fort Lewis, between March 1995 and
October 1996, and at Molina between May 1995 and April 1997, coincided with
the usual breeding season of deer mice, least chipmunks, and other rodents
at these sites, and with lower rodent population densities between the end
of 1995 and the end of 1997; antibody prevalence fluctuated in parallel.
Whether the two consecutive relatively wet years 1996 and 1997 will bring
about conditions suitable to increase rodent populations near Fort Lewis
and Molina and lead to an increase in HPS in the near term has not been
determined. Availability of water may be the sine qua non of plant food
availability, reproductive preparedness, gravidity rates, and attendant
intraspecific fighting among individual mice within an increased
population. From data (not shown) collected at these sites since October
1997, indications are that both rodent population densities and antibody
prevalence are increasing at both sites. If precipitation excess correlates
with rodent population, density increases, and the prevalence of
hantaviruses, we will be able to predict increases in risk for hantavirus
infection in the human population.

Acknowledgments

      We thank the following for directly assisting us in the field or with
logistics, encouragement, and the odd cool drink: David W. Schafer, Mr. and
Mrs. Roger Szczecinski, Catherine Crabb, Robin Carns, and Marcia Patterson,
Heather Clifton, Ted Davis, Ed Kuhn and Gordon Smith, Brendan Wolff, and
Edgar C. de Van III.

      Funding for this work was provided by the U.S. Centers for Disease
Control and Prevention, Atlanta, GA, under cooperative agreement No.
U50/ccu809862-03.

      Dr. Calisher is professor of microbiology, Arthropod-Borne and
Infectious Diseases Laboratory, Department of Microbiology, Colorado State
University. His areas of expertise are arboviruses, hantaviruses, and other
rodent-borne viruses. His research focuses on hantaviruses, arboviruses,
arenaviruses, and epidemiology.

      Address for correspondence: Charles H. Calisher, Arthropod-borne and
Infectious Diseases Laboratory, Foothills Campus, Colorado State
University, Fort Collins, CO 80523, USA; fax: 970-491-8323; e-mail:
calisher@usa.healthnet.org.

References

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     Hantaviruses and their rodent reservoirs in the United States. In:
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     Pest Conference; 1994. p. 188-91.
  2. Fitzgerald JP, Meaney CA, Armstrong DM. Mammals of Colorado. Niwot
     (CO): Colorado University Press of Colorado; 1994.
  3. Baars DL. Navajo country: a geology and natural history of the Four
     Corners region. Albuquerque (NM): University of New Mexico Press;
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  4. Mills JN, Ksiazek TG, Ellis BA, Rollin PE, Nichol ST, Yates TL, et al.
     Patterns of association with host and habitat: antibody reactive with
     Sin Nombre virus in small mammals in the major biotic communities of
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 10. Nichol ST, Spiropoulou CF, Morzunov S, Rollin PE, Ksiazek TG, Feldmann
     H, et al. Genetic identification of a novel hantavirus associated with
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     et al. Modes of Seoul virus infections: persistency in newborn rats
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     1996;46:323-30.

Hantavirus

Long-Term Studies of Hantavirus Reservoir Populations in the Southwestern
United States: A Synthesis

James N. Mills, Thomas G. Ksiazek, C.J. Peters, and James E. Childs
Centers for Disease Control and Prevention, Atlanta, Georgia, USA

---------------------------------------------------------------------------
      A series of intensive, longitudinal, mark-recapture studies of
      hantavirus infection dynamics in reservoir populations in the
      southwestern United States indicates consistent patterns as
      well as important differences among sites and host-virus
      associations. All studies found a higher prevalence of
      infection in older (particularly male) mice; one study
      associated wounds with seropositivity. These findings are
      consistent with horizontal transmission and transmission
      through fighting between adult male rodents. Despite very low
      rodent densities at some sites, low-level hantavirus infection
      continued, perhaps because of persistent infection in a few
      long-lived rodents or periodic reintroduction of virus from
      neighboring populations. Prevalence of hantavirus antibody
      showed seasonal and multiyear patterns that suggested a delayed
      density-dependent relationship between prevalence and
      population density. Clear differences in population dynamics
      and patterns of infection among sites, sampling periods, and
      host species underscore the importance of replication and
      continuity of long-term reservoir studies. Nevertheless, the
      measurable associations between environmental variables,
      reservoir population density, rates of virus transmission, and
      prevalence of infection in host populations may improve our
      capacity to model processes influencing infection and predict
      increased risk for hantavirus transmission to humans.

A series of ongoing studies of the natural history of hantavirus-host
associations in the southwestern United States was conducted by four
independent investigative teams in a variety of ecosystems. The studies,
which have a common experimental design, describe several patterns common
to all study sites; provide insight into hantavirus maintenance in natural
reservoir populations; highlight differences among geographic regions,
ecosystems, and closely related host-virus associations; and illustrate
that different sigmodontine rodent species (even within the genus
Peromyscus) may respond differently to the same environmental conditions at
the same site.

Sin Nombre virus (SNV), whose host is the deer mouse (Peromyscus
maniculatus), has been responsible for most, if not all, cases of
hantavirus pulmonary syndrome (HPS) in the southwestern United States since
1993. Deer mouse population density and prevalence of SNV infection in deer
mouse populations in the arid Southwest have declined sharply since the
high levels documented in 1993 (1-3). Nevertheless, moderate population
densities of deer mice persisting at the higher altitude web trapping sites
in Colorado provided an opportunity to look at the natural history of this
species over a wide range of conditions (Calisher et al., this issue, pp.
126-134).

The high prevalence of SNV-reactive antibody in brush mouse (P. boylii)
populations observed during these and previous studies in the Southwest (2)
led to the investigation and identification of a distinct hantavirus
carried by brush mice (S. Nichol and A. Johnson, unpub. data). Before these
studies were undertaken, it was not known whether antibody in brush mice
represented spillover of SNV from the deer mouse reservoir (as may have
been the case during the initial 1993 outbreak), unusual maintenance of the
same hantavirus by two species of rodents, or (as molecular evidence now
indicates) another example of cospeciation leading to a unique hantavirus
maintained in a single rodent species. Although the status of the virus
associated with P. boylii as a human pathogen is unknown, P. boylii is a
common species in the Southwest, and its population density fluctuates
dramatically with environmental conditions. Data on the brush mouse
host-virus association can contribute to our understanding of hantavirus
reservoir ecology.

We summarize major conclusions from the first 3 years of hantavirus
reservoir studies in the southwestern United States, examine consistent
patterns and salient differences, and discuss the implications of the
studies for understanding reservoir host ecology.

Temporal Patterns

In Host Populations

    Multiyear Patterns

In most rodent communities examined, sampling methods using trapping webs
demonstrated periodic fluctuations in population densities; many
populations were simultaneously adversely affected by periods of low
rainfall. In the southwestern United States, unusually dry conditions
directly followed the wet, exceptionally favorable conditions believed to
have contributed to the increase in rodent numbers and the HPS outbreak of
1993-94.

Specific habitat characteristics critical to some hantavirus reservoir
species are the availability of thick chaparral cover for brush mice
(Abbott et al., this issue, pp. 102-112) and food supply, including acorns
and other fruits and seeds (this issue, Calisher et al. pp. 126-134 and
Abbott et al., pp. 102-112). As illustrated by the different effects of
drought on pinyon mice (P. truei) and brush mice in northern Arizona
(Abbott et al., this issue, pp. 102-112), the response of a rodent
population to ecologic conditions depends on its specific requirements for
food, water, and habitat. A complete understanding of the ecologic
requirements and adaptability of each reservoir species is required before
the species' response to specific environmental conditions and its
potential contribution to future outbreaks of HPS can be predicted.

Many species of murid rodents typically exhibit year-to-year fluctuations
in population density. The Arvicolinae are a panarctic group containing
several species that undergo fairly regular population cycles with a 3- to
4-year periodicity. Periodic fluctuations in populations of the bank vole
(Clethrionomys glareolus), an arvicoline rodent and the reservoir for
Puumala virus (a hantavirus that causes a mild form of hemorrhagic fever
with renal syndrome [nephropathia epidemica] in Europe), have been related
to nephropathia epidemica incidence in Sweden (4). The causes of these
population cycles in arvicoline rodents are not well understood (5,6). All
viruses known to cause HPS are carried by rodents of the New World murid
subfamily Sigmodontinae. Although sigmodontine rodent populations do not
fluctuate on a regular, cyclic basis, periodic, sometimes dramatic
increases ("irruptions") occur in population density; these increases may
be tied to unusual climatic events that result in highly favorable (if
temporary) conditions for nutrition and reproduction. Such an increase
involving deer mouse populations may have been associated with the first
recognized outbreak of HPS in the southwestern United States in 1993 (7).
Understanding the factors that control or influence the population dynamics
of sigmodontine reservoir species is central to understanding the
epidemiology of HPS.

    Seasonal Patterns

In addition to the overall year-to-year trends in rodent population
dynamics, some populations demonstrated seasonal patterns that varied by
ecosystem: the size of brush mice and deer mice populations at desert
grassland sites peaked in winter and waned in midsummer, while at high
altitude sites in Colorado, it was generally highest in the fall. Models of
disease risk to humans must consider altitude and biome, as well as
regional weather patterns.

In Prevalence of Infection

   Assumptions Concerning Antibody Analyses

In these studies, we assume that antibody-positive hosts (P. maniculatus,
P. boylii, and Reithrodontomys megalotis) are chronically infected and
infectious. Studies of other specific hantavirus-host associations,
including Hantaan virus in Apodemus agrarius (8), Puumala virus in C.
glareolus (9,10), and Black Creek Canal virus in Sigmodon hispidus (11),
show a similar pattern: infection is followed by a brief period of viremia
and then by the development of antibody and clearing of virus from blood.
Nevertheless, in spite of the continuous presence of circulating antibody,
high-titer virus could be isolated from organs, and infectious virus was
shed persistently or sporadically in urine, feces, and saliva for extended
periods, probably the life of the host. The quantities of virus shed may be
greatest during the early phases (2 to 4 weeks postinoculation) of
infection (8). In a field study of SNV, 97% of antibody-positive P.
maniculatus had viral RNA in organ tissue (1), which implies a similar
pattern of chronic infection for the deer mouseSNV association.

These studies confirm the cited laboratory investigations demonstrating the
maintenance of antibody for the expected life span (approximately 1 year or
less) of the host. Antibody was detected by enzyme-linked immunosorbent
assay (ELISA) for up to 16 months in individual rodents, and no mice
reverted from antibody-positive to antibody-negative. Nevertheless, loss of
antibody may be observed in animals born with transient maternal antibody
(2,12).

Finally, these studies used antibody as the only marker of infection. Mice
sampled between infection and development of detectable antibody (probably
3 to 4 weeks [11]) are not recognized as infected; these may represent 2%
to 7% of animals sampled (13,14). In one study (1), 55% of seronegative
animals had viral RNA in blood samples; however, this study was unusualit
was conducted during epizootic conditions, which presumably involved very
high rates of transmission in the reservoir population. In addition, the
use of a heterologous antigen (Prospect Hill virus) to detect SNV antibody
in the ELISA format may have slightly decreased the sensitivity of
serologic tests. Thus, although the actual correction factor is imprecisely
known and probably variable, the seroprevalence provided in these reports
underestimate the true prevalence of infection in host populations.

   Multiyear Patterns

These studies support previous investigations (2,12,15) demonstrating that
rodents do not acquire hantavirus infection vertically but instead become
infected (and presumably infectious) and develop antibody in an age- or
size-related manner. Infection appears associated with life history and
behavioral events surrounding the maturation of animals into sexually
mature adults. Given the horizontal transmission of hantavirus within
reservoir populations, increasing population densities should result in
increased rodent-to-rodent contact, opportunities for virus transmission
(to susceptible mice), and overall incidence and cumulative prevalence of
infection within host populations. Such findings would be consistent with
the mass action principle of disease transmission, which assumes that
transmission is a function of density (16). Nevertheless, clear evidence of
increased population densities leading to increased prevalence of infection
in hantavirus host populations is lacking (2,14,17,18). Indeed, many
datasets, such as that presented by Abbott et al. (this issue, pp.
102-112), show an inverse relationship between population density and
antibody prevalence over time.

These same data, however, can provide insights into the interaction of
temporal patterns of reproduction, changing population age structure, and
virus transmission. For example, the successful breeding seasons for brush
mice in northern Arizona (spring through fall 1995 and spring 1996)
resulted in a population with a high proportion of juvenile and young mice
not yet infected (as evidenced by antibody). Increasing population density
resulted in increasing incidence of virus transmission (as evidenced by the
high rate of seroconversion during this period), but the overall antibody
prevalence in the population was continuously diluted and offset by the
addition of uninfected juvenile mice. By summer 1996, however, local
environmental conditions caused breeding to end and production of young to
subsequently decline. Until noninfected susceptible young mice began to
enter the population again during the summer of 1997, the population
consisted of older residents that, by virtue of their age and cumulative
life experiences, were commonly infected with hantavirus. Thus, the
relatively high prevalence of infection during this period reflects the
high rate of transmission during the previous fall, the subsequent decline
in new births, and the resultant older age structure and accumulated life
experience of the population.

    Seasonal Patterns

The dynamics of changing population structure and virus transmission may
also result in predictable seasonal patterns in the prevalence of
infection. In strongly seasonal climates, the interplay of host demography
and horizontal virus transmission may result in a strongly seasonal
alternation of peaks in population density and prevalence of infection. As
an example of other hantavirus-host associations, in Sweden, bank vole
population density was highest in the fall, but the prevalence of Puumala
virus antibody was highest in the spring and correlated with vole density
the previous fall and spring (4).

This delayed density-dependent prevalence of infection occurs in other
reservoir populations in strongly seasonal environments, such as the
high-altitude grids near Fort Lewis, Colorado (Calisher et al., this issue,
pp. 126-134). Every year, except 1994, when populations may have been
recovering from El Nio southern oscillation conditions and thus showed an
atypical pattern, population density of P. maniculatus was lowest in the
early spring (presumably because of the high number of winter deaths) and
increased throughout the breeding season, into summer and fall.
Furthermore, in 1995 and 1997 (no antibody-positive animals were captured
in 1996), antibody prevalence was highest in the early spring and lower in
the fall; this pattern could be the result of reproduction resulting in
highest population density in the fall but with the juvenile dilution
effect, which leads to low antibody prevalence. The spring population,
consisting largely of overwintering adult mice, reflects the relatively
high antibody prevalence expected in older animals. The high prevalence in
spring presumably reflects virus transmission in the high density
population of the previous autumn.

A study of hantavirus in rodent communities in Argentina provides
additional evidence for the broad applicability of this pattern in
temperate ecosystems. Several hantavirus reservoir species on the central
Argentine pampa displayed the same spring-fall alternation of peaks in
population density and antibody prevalence (19). Thus, the temporal
asynchrony between reservoir population density and prevalence of infection
on both the year-to-year and within-year scales can be explained by the
interaction of seasonal changes in population structure and horizontal
transmission of virus.

The explanations for this pattern suggest three corollary hypotheses: virus
"overwinters" in temperate rodent communities as persistent infections in
older adult animals, which serve as a reservoir for reintroducing virus
into susceptible young animals in the spring; spring antibody prevalence is
a function of the population density (infectious and susceptible) the year
before (the high fall population densities and higher spring antibody
prevalence at the Colorado trapping webs in spring 1995 provide tentative
support for this hypothesis); and deviations from typical environmental
conditions alter the pattern of infection in potentially predictable
directions. For example, a mild winter might prolong the period of
reproduction and transmission, simultaneously increasing population
densities and improving overwinter survival. Such conditions might result
in an atypically high prevalence of infection, as well as a
higher-than-usual population base in the spring. Such a pattern might help
account for the conditions of high population densities and high prevalence
of infection during the initial phases of the HPS outbreak in the
southwestern United States in the spring of 1993 (1). Expected changes in
population density, antibody prevalence, and population age structure over
a hypothetical multiyear cycle are shown in the Figure.

 [mills2.gif (10790 bytes)]                                     
Figure. A hypothetical schematic of seasonal changes in        
 hantavirus prevalence, rodent host population density, and     
 population age structure.In the first autumn, after a normal   
 breeding season, the high density population consists          
 primarily of young not exposed to virus or recently exposed    
 before development of antibody. Because of deaths in winter,   
 populations decrease to a spring nadir. However, antibody      
 prevalence is high in this population of overwintered adults   
 exposed during the previous breeding season or during winter   
 communal huddling. Unusually favorable conditions during the   
 following spring and summer (first horizontal bar) result in   
 higher population density the second fall, and the increased   
 influx of young of the year (juvenile dilution effect) results 
 in an even lower antibody prevalence than the previous fall. A 
 typical winter results in a high number of winter deaths and a 
 typical low density spring population, but seroprevalence the  
 second spring is higher because of increased opportunities for 
 transmission events in the high density population of the      
 previous fall. The second extended favorable season (second    
 horizontal bar) leads again to high population density and low 
 seroprevalence in autumn. The reservoir population of the      
 third spring demonstrates high antibody prevalence, because of 
 high rates of exposure during the "crowded" conditions of the  
 previous fall, and unusually high population density, because  
 of extended breeding and high overwinter survival. Depending   
 on environmental conditions, the population may abruptly       
 "crash" (if it has exceeded the carrying capacity of the       
 environment) or might continue to increase (e.g., if growing   
 conditions the previous spring and summer resulted in abundant 
 food supplies, such as a mast crop of acorns or pinyon nuts).  
                                                                

Prevalence of infection in reservoir populations, however, is only one of
several factors that may be useful in predicting risk for human disease.
The highest absolute numbers of infected rodents (but not prevalence)
coincided with high population density (Abbott et al., this issue, pp.
102-112);thus, all other factors being equal, the highest risk for human 
contact with infected rodents would be during the period of highest rodent 
population density. Factors of the host-virus interaction (e.g., time course 
of infection and periods of maximum virus shedding), rodent behavior (e.g., 
entering human habitations), and human behavior (e.g., planting or harvesting 
in the spring and fall and opening and cleaning rodent-infested sheds or cabins 
in the spring) interact to modify specific temporal risk patterns.

In Virus Transmission

The two reports that documented a high incidence of infections as evidenced
by first acquisition of antibody (this issue, Calisher et al., pp. 126-134
and Abbott et al., pp. 102-112) provide evidence for seasonal patterns in
transmissionone (Abbott et al., this issue, pp. 102-112) clearly documented
that the highest rates of seroconversion corresponded with highest
population density. The apparently different seasonal patterns of
seroconversion of male and female animals in Colorado were unexpected
(Calisher et al., this issue, pp. 126-134). The Colorado study suggests
that winter transmission of virus occurs during communal "nesting." This
may help explain why brush mice, living in desert and brushland habitats
with milder winters, have a higher ratio of male to female
antibody-positive mice. Virus transmission among brush mice may be more
restricted to aggressive encounters, which would favor male infection;
virus transmission among deer mice at high altitudes might also include
opportunities for transmission during communal nesting (e.g., by aerosol or
mutual grooming), which could diminish differences in transmission ratios
between male and female mice.

These studies indicate that hantavirus infection was resilient in the face
of population fluctuations. Even when rodent populations were very low,
some foci of infection were apparently sustained, presumably through
persistent infection in a few long-lived residents (Abbott et al., this
issue, pp. 102-112). However, other data indicate that infection may
disappear completely from a population during periods of low density, only
to reappear sporadically in a few infected mice (Kuenzi et al., this issue,
pp. 113-117). The latter phenomenon could indicate that low levels of
infection were continuously present in the population, but the sampling
method was not sensitive enough to detect it; or it may indicate that virus
periodically becomes extinct in semi-isolated populations that have
declined in numbers. Virus might be reintroduced into a population through
contact with, or dispersal of infected rodents from, adjacent populations,
which suggests that reservoir species should be considered metapopulations
in maintaining hantavirus infection.

Spatial Patterns

Several reports in this series provide evidence for spatial restrictions in
the geographic distribution of rodent reservoir populations, as well as for
focality of infection within populations. Although focality has been
observed on a regional scale (2), the data by Kuenzi et al. (this issue,
pp. 113-117) and Abbott et al. (this issue, pp. 102-112) demonstrated
distinct "islands" of hantavirus infection apparently associated with
preferred microhabitat for brush mice on web trapping sites, a pattern
reminiscent of the concept of natural "nidality" of zoonotic disease as
expounded by Pavlovsky (20). Characterizing preferred habitat types may
help identify areas at increased risk for virus transmission to humans.
However, these pockets of reservoir activity become blurred during periods
of high reservoir population density (Abbott et al., this issue, pp.
102-112). Not only would it be more difficult to identify foci that pose a
high risk for virus infection during reservoir population irruptions, but
also the increased movement of individual rodents may lead to transfer of
virus among previously distinct subpopulations, increasing the overall risk
for human exposure.

Characteristics of Infected Populations

At all sites and for both brush and deer mice, infected animals were more
frequently older males. These data are consistent with horizontal
transmission of infection and suggest that the (or a) specific mode of
transmission involves male more frequently than female animals. An
alternative hypothesis, that males live longer than females (which would
lead to greater cumulative probability of infection), is inadequate (Kuenzi
et al., this issue, pp. 113-117); therefore, behavioral differences (e.g.,
greater home range, increased aggression, bites, wounding) may be the most
likely explanation. Indeed male murid rodents may be more likely to have
scars or wounds (indicators of aggressive encounters) than female rodents
(19,21); the presence of scars has been associated with increased
prevalence of infection for hantaviruses (12;19; Calisher et al., this
issue, pp. 126-134).

Nevertheless, patterns of antibody prevalence differed distinctly by site
and species. For instance, the male bias among infected animals was much
greater for brush mice in Arizona (85% to 90% of infected animals were male
[this issue, Abbott et al., pp. 102-112 and Kuenzi et al., pp. 113-117])
than for deer mice in Colorado (approximately 60% [Calisher et al., this
issue, pp. 126-134]). These differences presumably relate to intersite or
interspecies differences in social structure or behavior that influence
hantavirus transmission. Are female deer mice more likely to fight or
otherwise interact than female brush mice? Does winter communal nesting
facilitate transmission among both male and female deer mice and not brush
mice? Could venereal transmission be involved for deer mice? Some of these
questions can be addressed only by continued data collection at the
long-term trapping webs. For instance, collection and analysis of data
concerning wounding and scars should document the relative frequency of
aggressive encounters among males and females of all species. As
demonstrated by Abbott et al. (this issue, pp. 102-112), comparing data on
interactions of same sex and opposite sex mice involved in dual captures
may also yield insights. In fact, preliminary analysis of scar frequency by
Calisher et al. (this issue, pp. 126-134) indicates that male deer mice in
Colorado may not experience more aggressive encounters than female mice;
dual-capture results by Abbott et al. (this issue, pp. 102-112) show that
male-male interactions among brush mice can be considerably more aggressive
than female-female interactions. If communal nesting increases viral
transmission between deer mice, the pattern of antibody prevalence among
male and female mice may differ for deer mice captured at trapping webs in
lower altitude sites in New Mexico and eastern Colorado. These data are
being collected. Venereal transmission, which would be difficult to address
in field studies, will require parallel studies in the laboratory.

Comparison of SNV Prevalence with Prevalence of Other Rodent-Borne Agents

The prevalence of infection with hantavirus shown by these studies is 0% to
approximately 25%. Even under conditions of high rodent density in the
areas of human disease outbreaks, the prevalence of SNV infection in deer
mice reached only 30% (1). The high rate of population turnover and
relatively short life span of most sigmodontine hosts results in
populations frequently dominated by young mice not yet infected with
hantavirus; the delay between infection and development of antibody further
decreases the apparent prevalence of infection. However, when data are
stratified by age and sex, antibody prevalence can be high. For example,
90% of male Norway rats >500 g in Baltimore, Maryland, had antibody to
Seoul virus (15), and 88% of male cotton rats >200 g in southern Florida
had antibody reactive with SNV (12). These prevalences are comparable to
the highest prevalences for agents reported to be vertically transmitted
such as some arenaviruses including Lassa (22) and lymphocytic
choriomeningitis viruses (23).

The Future

Preliminary results from these studies indicate that some patterns, such as
age- and male-associated infection, are clear. Nevertheless, upon closer
inspection, the patterns differ between sites and species. Reservoir
studies at one site, in one ecosystem, during 1 year, or of one host-virus
system cannot provide the data necessary to piece together the natural
history of hantavirus infection in North American reservoirs. Environmental
conditions cannot be controlled in the field; therefore, adequate
replication of field studies across time, space, and host-virus systems is
critical. Although extensive, the studies reported in this series are
preliminary. Three years is a very brief period for detecting effects due
to environmental changes (e.g., weather and landscape) and for detecting
the impact of extremely rare events (e.g., a 20-year flood). The conditions
that lead to rodent population irruptions may be infrequent, and there may
be thresholds for either environmental conditions or population densities
that lead to the increased numbers of infected rodents that are indicators
of risk of virus transmission to humans. The ultimate usefulness of these
studies depends upon their long-term maintenance.

The methods used in these studies appear sensitive enough to detect changes
in reservoir populations associated with increased virus transmission. The
sampling methods did not significantly increase deaths among study animals
(this issue, Calisher et al., pp. 126-134 and Abbott et al., pp. 94-104;
24;25). The few unavoidable deaths associated with periodic bleeding of
animals and mark-recapture studies do not affect most population estimates;
the statistical analyses are sufficiently sensitive to detect intersite and
temporal differences in population densities (Parmenter et al., this issue,
pp. 118-125).

But can these studies provide early warning of conditions that predate and
predict an increase in virus transmission and HPS? Data from the last few
months of the study period show an abrupt increase in the population
density of some reservoir species that coincides with habitat improvements,
most likely resulting from increases in rainfall associated with an El Nio
southern oscillation event beginning in 1997. The current environmental
changes may provide a rare opportunity to document the weather and ecologic
conditions associated with demographic changes in reservoir host
populations that increase risk for virus transmission to human populations.
Recent increases in reservoir populations have been associated with
increased numbers of HPS cases in the southwestern United States. As of
August 1998, approximately 14 cases have been reported in Arizona,
Colorado, New Mexico, and Utah, in comparison to 2, 2, and 4, for the same
period in 1995, 1996, and 1997 (A. Khan, unpub. data). The qualitative and
quantitative data on reservoir populations and environmental variables
collected during this period may also provide the necessary
habitat-specific correlations so that satellite images can be related to
specific environmental clues. When these links are established, the wide
coverage offered by remote sensing platforms may provide the capability to
predict increased risk in areas without direct reservoir monitoring.

Even though the variety of ecosystems and host-virus systems included in
these studies may lead to models with broad applicability, they still
represent a relatively small geographic area and a small percentage of the
known hantavirus-host associations in the world. Similar studies in other
areas of the United States provide comparisons (17), but similar studies of
other sigmodontine reservoirs in South America and arvicoline and murine
reservoirs in Europe and Asia are needed.

Finally, the value of these longitudinal studies will increase when these
data are integrated with data from complementary field and laboratory
studies. These mark-recapture studies are restricted to wild populations in
natural environments, while most human cases of HPS are acquired in the
peridomestic environment. Although the dynamics of natural populations
ultimately influence the density and behavior of peridomestic deer mice,
for example, the specific factors of human and rodent behavior that lead to
peridomestic exposure can be elucidated only through studies in the
specific environment of exposure.

The presence of IgG antibody reactive with SNV antigen is used as the
marker of infection in these studies. Given the pattern of chronic
infection and long-term shedding of virus in hantavirus-host associations
(8,9,11), antibody is probably a good marker. Nevertheless, the specific
dynamics and timing of infection, antibody development, and timing of
maximum viral shedding are unknown for most hantavirus reservoir species.
These data must be provided by controlled laboratory studies using
artificially infected animals and (because laboratory infections may not
always mimic natural infections [26]) field studies involving naturally
infected animals. Natural or manipulative field studies might use captive
populations in seminatural enclosures or excretory products collected (by
use of metabolic chambers) from wild-caught animals in mark-recapture
studies; the success of these studies will depend on the development of
assays for infectious virus.

Acknowledgment

      Barbara Ellis provided the graphics and helpful suggestions that
improved the manuscript.

      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.

      Address for correspondence: James N. Mills, Centers for Disease
Control and Prevention, Mailstop G14, 1600 Clifton Road, N.E., Atlanta, GA
30333, USA; fax: 404-639-1118; e-mail: jum0@cdc.gov.

References

  1. Childs JE, Ksiazek TG, Spiropoulou CF, Krebs JW, Morzunov S, Maupin
     GO, et al. Serologic and genetic identification of Peromyscus
     maniculatus as the primary rodent reservoir for a new hantavirus in
     the southwestern United States. J Infect Dis 1994;169:1271-80.
  2. Mills JN, Ksiazek TG, Ellis BA, Rollin PE, Nichol ST, Yates TL, et al.
     Patterns of association with host and habitat: antibody reactive with
     Sin Nombre virus in small mammals in the major biotic communities of
     the southwestern United States. Am J Trop Med Hyg 1997;56:273-84.
  3. Engelthaler DM, Levy CE, Fink M, Tanda D, Davis T. Short report:
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     1993-1994 hantavirus pulmonary syndrome case sites. Am J Trop Med Hyg
     1998;58:737-8.
  4. Niklasson B, Hornfeldt B, Lundkvist A, Bjorsten S, LeDuc J. Temporal
     dynamics of Puumala virus antibody prevalence in voles and of
     nephropathia epidemica incidence in humans. Am J Trop Med Hyg
     1995;53:134-40.
  5. Krebs CJ, Myers JH. Population cycles in small mammals. Advances in
     Ecological Research 1974;8:267-399.
  6. Hornfeldt B. Delayed density dependence as a determinant of vole
     cycles. Ecology 1994;75:791-806.
  7. Parmenter RR, Brunt JW, Moore DI, Ernest S. The hantavirus epidemic in
     the southwest: rodent population dynamics and the implications for
     transmission of hantavirus-associated adult respiratory distress
     syndrome (HARDS) in the four corners region. University of New Mexico.
     Sevilleta LTER Publication. No. 41:1993.
  8. Lee HW, Lee PW, Baek LJ, Song CK, Seong IW. Intraspecific transmission
     of Hantaan virus, etiologic agent of Korean hemorrhagic fever, in the
     rodent Apodemus agrarius. Am J Trop Med Hyg 1981;30:1106-12.
  9. Yanagihara R, Amyx HL, Gajdusek DC. Experimental infection with
     Puumala virus, the etiologic agent of nephropathia epidemica, in bank
     voles (Clethrionomys glareolus). J Virol 1985;55:34-8.
 10. Bogdanova SB, Gavrilovskaya IN, Boyko VA, Prokhorova NA, Linev MB,
     Apekina NS, et al. Persistent infection caused by hemorrhagic fever
     with renal syndrome in red mice (Clethrionomys glareolus), natural
     hosts for the virus (in Russian, translated by SCITRAN). Mikrobiol Zh
     1987;49:99-106.
 11. Hutchinson KL, Rollin PE, Peters CJ. Pathogenesis of a North American
     hantavirus, Black Creek Canal virus, in experimentally infected
     Sigmodon hispidus. Am J Trop Med Hyg 1998;59:58-65.
 12. Glass GE, Livingstone W, Mills JN, Hlady WJ, Fine JB, Rollin PE, et
     al. Black Creek Canal virus infection in Sigmodon hispidus in southern
     Florida. Am J Trop Med Hyg 1998;59:699-703.
 13. Otteson EW, Riolo J, Rowe JE, Nichol ST, Ksiazek TG, Rollin PE, et al.
     Occurrence of hantavirus within the rodent population of northeastern
     California and Nevada. Am J Trop Med Hyg 1996;54:127-33.
 14. Boone JD, Otteson EW, McGwire KC, Villard P, Rowe JE, St Jeor SC.
     Ecology and demographics of hantavirus infections in rodent
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     hantavirus infections in Baltimore: isolation of a virus from Norway
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     1987;126:55-68.
 16. Dobson AP, Hudson PJ. Microparasites: observed patterns in wild animal
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Dispatches


Dispatches

Proficiency of Clinical Laboratories in and near Monterrey, Mexico, To
Detect Vancomycin-Resistant Enterococci

L. Clifford McDonald,* Luis R. Garza, and William R. Jarvis*
*Centers for Disease Control and Prevention, Atlanta, Georgia, USA; and
Hospital Jose A. Muguerza, S.A. De C.V., Monterrey Nuevo Len, Mexico

---------------------------------------------------------------------------
      Early detection of vancomycin-resistant enterococci is
      important for preventing its spread among hospitalized
      patients. We surveyed the ability of eight hospital
      laboratories in and near Monterrey, Mexico, to detect
      vancomycin resistance in Enterococcus spp. and found that
      although laboratories can reliably detect high-level vancomycin
      resistance, many have difficulty detecting low-level
      resistance.

Since vancomycin-resistant enterococci (VRE) were first reported in the
late 1980s, their geographic distribution and importance as nosocomial
pathogens have continued to increase worldwide. In the United States, the
percentage of states with a National Nosocomial Infections Surveillance
System hospital reporting one or more patients with VRE infection increased
from 27% (19891993) to 44% (19941995) (1). Among enterococci causing
infection in these hospitals, the percentage resistant to vancomycin
increased from 0.4% (1989) to 10.8% (1995) in intensive care unit patients
and from 0.3% (1989) to 10.4% (1995) in nonintensive care unit patients
(1).

Although early detection of VRE is important for preventing its spread
among hospitalized patients, clinical laboratories in the United States
have difficulty detecting VRE, especially those with intermediate or
low-level resistance, characteristic of VRE with the VanB phenotype (2,3).
Certain automated and manual antimicrobial susceptibility test systems are
associated with the inability of laboratories to detect VRE. Laboratories
outside the United States (e.g., in Argentina [4]) may have difficulty
detecting VRE for largely the same reasons: limitations of susceptibility
test systems. In addition, laboratories outside the United States, Canada,
and Europe may face language and financial barriers to accessing
information important for updating their methods and optimizing VRE
detection.

No VRE have been reported from hospitals in Mexico. However, because VRE
have been reported from hospitals in Texas, and VanB has been described as
the predominant phenotype in at least one hospital in the Houston area (5),
we assessed the ability of clinical laboratories in and near Monterrey (in
northeastern Mexico) to detect VRE.

Study Protocol

The laboratory survey and data collection were performed in July-August
1997. Five strains of enterococci (two Enterococcus faecium, two E.
faecalis, one E. gallinarum), with or without resistance to vancomycin,
were coded as isolates 1 through 5 (Table). Four of these had been used in
proficiency surveys in the United States (3) and Argentina (4). The fifth
isolate came from the American Type Culture Collection (ATCC 29212). The
isolates were distributed, along with standardized susceptibility test
results forms, to eight clinical laboratories (seven within and one near
Monterrey).

Laboratories were    
blinded to the       
susceptibility       
patterns of the
isolates and asked   Table. Characteristics of enterococcal study isolates 
to test the five     and results of enterococcal susceptibility testing,                                       
isolates for         by category
resistance to        ------------------------------------------------------
vancomycin with the						No. of labs(sup a)                                           
antimicrobial								 (n = 8)
susceptibility       Isolate no.       MIC    Vancomycin   ---------------
testing method they  and species     (g/ml)   phenotype    S     I   R
routinely used. The  ------------------------------------------------------
laboratorians
recorded disk zone   1. E. faecium     512        VanA      0     0   8(sup b)

sizes or MICs and    2. E. faecium      64     VanB-like    2     1   5(sup b)
their interpretation
of the results       3. E. faecalis   16-32       VanB      2     1b  5(sup b)
(susceptible,
intermediate, or     4. E. gallinarum         8         VanC      1       2                                       5(sup b)
resistant), in       
addition to species  5. E. faecalis     <4    Susceptible 7(sup b)1    0
identification       ------------------------------------------------------
methods used, zone   (sup a)Laboratories reporting susceptibility to vancomycin;
size breakpoints     S = susceptible, I = intermediate, R = resistant.
used for disk        (sup b)Correctly identified.
diffusion, existence 
of an antimicrobial  
control program, and
hospital demographic characteristics. No information was collected
regarding the version of software program used with automated
susceptibility test systems. The Centers for Disease Control and Prevention
(CDC) analyzed the forms and classified errors in vancomycin susceptibility
testing as very major (reporting a resistant strain as susceptible), major
(reporting a susceptible strain as resistant), minor (reporting an
intermediate or resistant strain as susceptible or intermediate,
respectively), or very minor (reporting a susceptible or intermediate
strain as intermediate or resistant, respectively).

MICs were determined by broth microdilution and disk diffusion testing (3).
In addition, a polymerase chain reaction assay was used to confirm the
presence of the vanA resistance determinant in organism 1 (3).

Study Findings

The eight participating laboratories each serviced one hospital with a
median bed count of 148 (70 to 185). All but one hospital had neonatal,
pediatric, and adult intensive care units; half were teaching hospitals.
Only two laboratories reported an antimicrobial use control program in
place in their hospital.

The antimicrobial susceptibility testing methods used were the Sceptor
system (Becton-Dickinson Microbiology Systems, Cockeysville, MD) (three
laboratories); Vitek (bioMerieux, St. Louis, MO) (two); standard disk
diffusion (two); and Microscan Autoscan (Dade International, West
Sacramento, CA) (one). Of the two laboratories that used disk diffusion,
one used outdated breakpoint zone sizes; the other used breakpoints
currently recommended by the National Committee for Clinical Laboratory
Standards (NCCLS).

Vancomycin Resistance Detection

All laboratories correctly detected the high-level vancomycin resistance in
isolate 1 (high-level vancomycin resistance [MIC 512 g/ml] of the VanA
phenotype) (Table). Only five laboratories reliably detected the low-level
resistance (MIC 64 g/ml) typical of the VanB-like phenotype possessed by
isolate 2; laboratories made two very major errors reporting this resistant
isolate as susceptible. Six laboratories correctly categorized isolate 3
with the VanB phenotype (MIC 16-32 g/ml) as intermediate or resistant; two
laboratories committed minor errors by reporting this isolate as
susceptible. Five laboratories correctly categorized isolate 4; one
laboratory committed a minor error, and two laboratories committed very
minor errors. Of the eight laboratories, seven correctly identified
vancomycin susceptibility in isolate 5.

Of the 15 test results from the three hospitals using the Sceptor system, 3
(20%) were errors (all minor). Of the 10 results from the two hospitals
using disk diffusion, four (40%) were errors. The laboratory using outdated
NCCLS zone-size breakpoints committed one very major and one very minor
error; the laboratory using the current NCCLS breakpoints committed two
very minor errors. The two hospitals using the Vitek automated system
generated 10 vancomycin susceptibility test results; two were errors (one
very major and one minor).

Species Identification

Of the participating laboratories, three used the Sceptor system to
identify species, two used the Vitek system, one used Microscan Autoscan,
one used the Pasco system (Difco, Wheatridge, CO), and one did not report
its identification method. Seven laboratories correctly identified isolates
1 and 2, both E. faecium; five laboratories correctly identified isolate 3,
an E. faecalis; none correctly identified isolate 4, an E. gallinarum (six
laboratories identified it as E. faecalis and two as E. faecium); and all
laboratories correctly identified isolate number 5, an E. faecalis.

Conclusions

Because VRE may be transmitted easily by health-care workers from infected
to uninfected patients, who may become colonized and serve as reservoirs
for transmission, the delay caused by failure of the laboratory to detect
VRE from an infected patient may facilitate the emergence of VRE in a
hospital or a group of hospitals in a geographic region. Early studies
suggested laboratories were more likely to miss intermediate- or low-level
vancomycin resistance in enterococci if they relied on commercial automated
test systems (3,6). More recent studies suggest persistent problems with
the Vitek instrument (2). In addition, disk diffusion is not sufficiently
sensitive in detecting low- and intermediate-level resistance to vancomycin
when used as the sole method (2,4). Despite revision of NCCLS-recommended
breakpoint zone sizes (7), disk diffusion remains relatively insensitive,
possibly because susceptible and resistant zone sizes cluster around
breakpoints. In addition, laboratory personnel may not consistently follow
recommendations to read plates with transmitted light after a full 24
hours' incubation (7). Microscan has shown improved performance (2), likely
due to software and hardware revisions (6).

Of the 40 test results in our study, 9 (23%) were erroneous compared with
results at CDC; 2 (5%) were very major errors. In comparison, of 335
vancomycin susceptibility test results in 67 New Jersey laboratories, 111
(33%) were erroneous, with 20 (6%) very major errors (3). Of 25 vancomycin
susceptibility test results in four Argentine laboratories, 11 (44%) were
erroneous, with 2 (8%) very major errors (4). Although our results compared
favorably with those of the New Jersey and Argentina studies, which used
four test strains we used, the rate of very major errors is unacceptably
high, especially for low-level resistance.

Some errors may have been caused by inadequately skilled personnel. One
laboratory was using outdated zone-size breakpoints, despite 1992
revisions, a reminder that instituting contemporary methods in some
laboratories may be difficult and lead to incorrect epidemiologic data.
However, the overall lower error rate compared with previous studies and
the association of errors with certain test methods suggest that
limitations of test methods were primarily responsible for inaccuracies.
Too few vancomycin susceptibility tests were performed to ascertain
relative performance of test methods. However, the 20% error rate and lack
of major errors obtained by the Sceptor system, compared with higher error
rates and very major errors from laboratories that used the Vitek and disk
diffusion methods, are consistent with previous results (8).

NCCLS recommends the use of an agar screen plate consisting of brain heart
infusion agar containing 6 g/ml of vancomycin to detect low- and
intermediate-level vancomycin resistance (9). We recommend that
laboratories in the Monterrey area use a supplemental brain heart infusion
screening agar to test for vancomycin resistance in enterococci isolated
from selected clinical specimens (e.g., blood, urine, sterile body sites).
Because screening agar has a sensitivity of 100% and specificity of 96% to
99% (2), we recommend that isolates that grow on the screening agar be
reported as resistant unless repeat testing with a reference MIC method
suggests otherwise.

Some enterococci (e.g., E. gallinarum and E. caseliflavus), which have
intermediate-level vancomycin resistance known as the VanC phenotype,
rarely cause human infection. Isolation of this form of VRE, therefore,
does not have the same public health importance as that of
vancomycin-resistant E. faecium or E. faecalis. Therefore, to focus efforts
on controlling antimicrobial resistance in species likely to cause serious
human infection, laboratories must correctly identify VRE to the species
level. Both E. gallinarum and E. casseliflavus can be differentiated from
other enterococci on the basis of their motility. E. casseliflavus may be
easily differentiated from E. faecium or E. faecalis by its yellow pigment;
in contrast, E. gallinarum may not be reliably identified unless a motility
test is performed with the appropriate media (10). A new conventional
biochemical test for methyl-[alpha]-D-glycopyranoside (MDG) may be even more
reliable than motility in differentiating E. gallinarum from E. faecium and
E. faecalis (11).

The laboratories' difficulty in detecting low- and intermediate-level
vancomycin resistance and in correctly identifying enterococci to the
species level, especially E. gallinarum, suggests a need for additional
tests to aid in the early detection and correct identification of VRE.
Although no VRE have been reported from Mexico, the study area is close to
areas in the United States where VRE have been reported and the VanB
phenotype may be predominant (5).

      Dr. McDonald is an associate investigator in the division of Clinical
Research, National Health Research Institute, Taipei, Taiwan. He is
assisting with the establishment of the Microbial Infections Reference
Laboratory to coordinate surveillance and conduct research in antimicrobial
resistance in Taiwan and elsewhere in Southeast Asia. His research
interests include the epidemiology of antimicrobial resistance and methods
to control its spread.

      Address for correspondence: William R. Jarvis, Hospital Infections
Program, Centers for Disease Control and Prevention, 1600 Clifton Road,
Mail Stop E69, Atlanta, GA 30333, USA; fax: 404-639-6459;
e-mail:wrj1@cdc.gov.

References

  1. Gaynes R, Edwards J, the NNIS system. Nosocomial vancomycin-resistant
     enterococci in the United States, 1989-1995: the first 1000 isolates
     [abstract]. Infect Control Hosp Epidemiol 1996;17:P18.
  2. Rosenberg J, Tenover FC, Wong J, Jarvis W, Vugia DJ. Are clinical
     laboratories in California accurately reporting vancomycin-resistant
     enterococci? J Clin Microbiol 1997;35:2526-30.
  3. Tenover FC, Tokars J, Swenson J, Paul S, Spitalny K, Jarvis W. Ability
     of clinical laboratories to detect antimicrobial agent-resistant
     enterococci. J Clin Microbiol 1993;31:1695-9.
  4. Cookson ST, Lopardo H, Marin M, Arduino R, Rial MJ, Altschuler M, et
     al. Study to determine the ability of clinical laboratories to detect
     antimicrobial-resistant Enterococcus spp. in Buenos Aires, Argentina.
     Diagn Microbiol Infect Dis 1997;29:107-9.
  5. Coque TM, Tomayko JF, Ricke SC, Okhyusen PC, Murray BE.
     Vancomycin-resistant enterococci from nosocomial, community, and
     animal sources in the United States. Antimicrob Agents Chemother
     1996;40:2605-9.
  6. Iwen PC, Kelly DM, Lindner J, Hinrichs SH. Revised approach for
     identification and detection of ampicillin and vancomycin resistance
     in Enterococcus species by using MicroScan panels. J Clin Microbiol
     1996;34:1779-83.
  7. Swenson JC, Ferraro MJ, Sahm DF, Charache P, the National Committee
     for Clinical Laboratory Standards Working Group on Enterococci,
     Tenover FC. New vancomycin disk diffusion breakpoints for enterococci.
     J Clin Microbiol 1992;30:2525-8.
  8. Tenover FC, Swenson JM, O'Hara CM, Stocker SA. Ability of commercial
     and reference antimicrobial susceptibility testing methods to detect
     vancomycin resistance in enterococci. J Clin Microbiol 1995;33:1524-7.
  9. National Committee for Clinical Laboratory Standards. Methods for
     dilution antimicrobial susceptibility tests for bacteria that grow
     aerobically. 4th ed. Approved Standard. M7-A4. Villanova (PA): The
     committee; 1997.
 10. Facklam RR, Sahm DF. Enterococcus. In: Murray PR, editor. Manual of Clinical
     Microbiology. 6th ed. Washington: American Society for Microbiology; 1995. 
	  p. 308-14. 
 11. Turenne CY, Hoban DJ, Karlowsky JA, Shanel GG, Kabani AM. Screening
     of stool samples for identification of vancomycin-resistant enterococcus 
	  isolates should include the methyl-[alpha]-glycopyranoside test to differentiate
     nonmotile Enterococcus gallinarum from E. faecium. J Clin Microbiol
     1998;36:2333-5.

Dispatches

Staphylococcus aureus with Reduced Susceptibility to Vancomycin Isolated
from a Patient with Fatal Bacteremia

Sharon S. Rotun,* Virginia McMath,* Dianna J. Schoonmaker, Peggy S.
Maupin, Fred C.Tenover, Bertha C. Hill, and David M. Ackman
*United Hospital Medical Center (UHMC), Port Chester, New York, USA; New
York State Department of Health, Albany, New York, USA; and Centers for
Disease Control and Prevention (CDC), Atlanta, Georgia, USA

---------------------------------------------------------------------------
      A Staphylococcus aureus isolate with reduced susceptibility to
      vancomycin was obtained from a dialysis patient with a fatal
      case of bacteremia. Comparison of the isolate with two
      methicillin-resistant S. aureus (MRSA) isolates obtained from
      the same patient 4 months earlier suggests that the S. aureus
      with reduced susceptibility to vancomycin emerged from the MRSA
      strain with which the patient was infected. Atypical phenotypic
      characteristics, including weak or negative latex-agglutination
      test results, weak or negative-slide coagulase test results,
      heterogeneous morphologic features, slow rate of growth, and
      vancomycin susceptibility (by disk diffusion test) were
      observed.

Three cases of infection by Staphylococcus aureus with reduced
susceptibility to the glycopeptide antibiotics vancomycin and teicoplanin
(glycopeptide intermediate S. aureus, GISA) have been reported from Japan,
Michigan, and New Jersey (1,2). We report the third case in the United
States of S. aureus with reduced susceptibility to vancomycin. We found
that this GISA apparently emerged from a methicillin-resistant S. aureus
(MRSA) isolate that had infected/colonized the patient during the 3 months
before his death. This report highlights key phenotypic characteristics of
interest to both clinicians and microbiologists.

The first (in New York State) S. aureus isolate with reduced susceptibility
to vancomycin was obtained from the blood of a 79-year-old man with a
history of coronary artery disease, obstructive pulmonary disease,
hypothyroidism, and renal failure requiring chronic hemodialysis.
Bacteremia developed in December 1997, and an MRSA was isolated from the
patient's blood on December 31. He received vancomycin while in the
hospital, improved clinically, and was discharged. He was readmitted to the
hospital in January 1998 with an infected jugular catheter, which was
removed. An MRSA isolate was obtained from the catheter on January 10, and
MRSA was also isolated from the patient's blood. He improved clinically and
was subsequently discharged on vancomycin therapy. From December 1997 to
February 1998 he received 3.5 g of intravenous vancomycin, followed by a
7-day course of 3.5 g of oral ciprofloxacin. In March 1998, the patient was
admitted to the dialysis center with signs of congestive heart failure and
sepsis and was transferred to the hospital. He received vancomycin (1 g),
ceftriaxone (2 g), and tobramycin (200 mg) but died the following day. Over
the last 4 months of his life, the patient received 8.0 g of vancomycin for
various infections or for surgical prophylaxis of thrombectomy repairs. An
autopsy was not performed.

The vancomycin MIC of the isolate, obtained from blood collected before the
administration of antibiotics on March 20 at the patient's last hospital
admission, was determined by the hospital laboratory to be 8 g/ml by Etest
(3) and MicroScan conventional panels (Dade Behring, Inc., MicroScan). This
MIC was confirmed by broth microdilution at the New York State Department
of Health and the Centers for Disease Control and Prevention (4). The MRSA
isolates obtained from the patient in December and January were susceptible
to vancomycin by broth microdilution (MIC <2 g/ml) and Etest (3 g/ml and
4 g/ml, respectively). All three isolates appeared to be susceptible to
vancomycin when tested by the disk diffusion method, with zones of 17 mm to
18 mm (5), and were susceptible to teicoplanin by broth microdilution (MIC
2-4 g/ml). Broth microdilution and Etest results showed that in addition
to vancomycin and methicillin, the GISA isolate is also resistant to
penicillin, erythromycin, ciprofloxacin, and rifampin; the two MRSA
isolates from December and January were also resistant to the latter four
antibacterial agents, although at higher levels than the GISA isolate for
penicillin (>32 g/ml and 6 g/ml, respectively) and erythromycin (256
g/ml and 16 g/ml, respectively). All three isolates were susceptible to
clindamycin, trimethoprim/sulfamethoxazole, gentamicin, chloramphenicol,
and tetracycline. Although the susceptibilities of the recently described
GISA isolates to antimicrobial agents vary, all are resistant to
clindamycin (6), whereas the GISA isolate was susceptible.

The GISA isolate has two colony types, one of which is smaller than the
typical S. aureus colony. The MRSA obtained from the patient in January
also demonstrated colony size heterogeneity, unlike the MRSA obtained in
December, which had only the larger typical colony type. Coagulase activity
of the GISA isolate was detected at 24 hours by tube test but was only very
weakly detected by slide test (NYSDOH) or was negative (UHMC, CDC).
Atypical (weakly positive [Staphyloslide, BBL] or negative [Staphaurex,
Murex]) results were obtained by latex-agglutination tests.

As part of a contact investigation, cultures were obtained from the nares
and hands of dialysis center and hospital employees, health-care aides,
dialysis center patients, and family members. Nares of 45 persons and the
hands of 19 of the 45 were cultured; 14 methicillin-susceptible S. aureus
(MSSA) and two MRSA isolates (one from a nephrologist and one from a
dialysis patient) were obtained. However, no other GISA isolates were
found. There was no evidence that the GISA was spread to other patients or
employees.

The S. aureus isolates obtained from the patient in December, January, and
March, as well as the MRSA from the nephrologist and the dialysis patient,
were compared by pulsed-field gel electrophoresis (PFGE). The three
isolates from the patient had the same PFGE type (Figure). The isolates
from the nephrologist and the dialysis patient (not shown) had PFGE types
unrelated to the GISA type and unrelated to each other. This suggests that
GISA emerged from the MRSA strain that had infected the patient in
December, presumably as a result of vancomycin therapy. Although the
patient was admitted to the hospital in March with a high fever and
evidence of pneumonia and the GISA isolate was obtained from blood
specimens during hospitalization, it was not possible to determine whether
vancomycin resistance in this organism was responsible for the patient's
death.

                          				    The third reported in
 [fig]                                             the United States, this
 Figure. Pulsed-field gel electrophoresis (PFGE)   case of GISA is similar
 profiles of SmaI digested DNA. Case patient MRSA  to previously reported
 isolates from December 1997 and January 1998,     cases. The patient
 GISA isolate from March 1998. Nephrologist MRSA   described above had been
 isolate from March 1998 and unrelated MRSA        infected with MRSA for
 isolates.                                         several months and had
                                                   received prolonged
                                                   treatment with
vancomycin. Laboratories should retain MRSA isolates because they may be
valuable in outbreak investigations. As in this case, MRSA isolates,
together with subsequent isolates, can be used in studying the emergence of
glycopeptide resistance in MRSA. The GISA isolate grew slowly, had
heterogeneous morphologic features, weak or negative slide-coagulase test
results, and weak or negative latex-agglutination test results. GISA
appeared to be susceptible to vancomycin by disk diffusion, as was reported
for the previous isolates (6). The disk diffusion test does not appear to
be accurate for determining vancomycin susceptibility in these organisms.
Microbroth dilution or the Etest method should be used instead. Atypical
phenotypic characteristics have been reported for every other GISA,
although not all isolates exhibited the same atypical characteristics
(1,2,6,7). Laboratorians should be aware of these atypical characteristics
so that GISA are not misidentified. Evidence for cell-wall reorganization
has been reported for GISA isolates (1,7). These changes in cell-wall
structure may be responsible for the atypical phenotypic characteristics
and decreased susceptibility to vancomycin.

Acknowledgments

      We thank the staff of the United Hospital Microbiology Laboratory for
their technical assistance and Larry Bopp, Dale Morse, Harry Taber, and
Mehdi Shayegani for their assistance and support.

      Dr. Rotun is microbiology manager at Our Lady of Mercy Health Care
System, New York, NY. Her research interests include developing procedures
to detect and monitor antimicrobial resistance in clinically important
bacteria.

      Address for correspondence: Dianna J. Schoonmaker, Wadsworth Center,
New York State Department of Health, 120 New Scotland Avenue, Albany, NY
12208, USA; fax: 518-486-7971; e-mail: djs03@health.state.ny.us.

References

  1. Hiramatsu K, Hanaki H, Ino T, Yabuta K, Oguri T, Tenover FC.
     Methicillin-resistant Staphylococcus aureus clinical strain with
     reduced vancomycin susceptibility. J Antimicrob Chemother
     1997;40:135-6.
  2. Staphylococcus aureus with reduced susceptibility to vancomycinUnited
     States. MMWR Morb Mortal Wkly Rep 1997;46:765-6.
  3. Etest package insert. AB Biodisk North America., Piscataway, NJ. 1996.
  4. National Committee for Clinical Laboratory Standards. Methods for
     dilution antimicrobial susceptibility tests for bacteria that grow
     aerobically. 4th ed. Vol 17, no. 2, Approved Standard M7-A4, National
     Committee for Clinical Laboratory Standards, Wayne, PA. 1998.
  5. National Committee for Clinical Laboaratory Standards. Performance
     standards for antimicrobial disk susceptibility tests. 6th ed. Vol.
     17, no. 1. Approved Standard M2-A6, National Committee for Clinical
     Laboratory Standards, Wayne, PA. 1998.
  6. Tenover FC, Lancaster MV, Hill BC, Steward CD, Stocker SA, Hancock GA,
     et al. Characterization of Staphylococci with reduced susceptibilities
     to vancomycin and other glycopeptides. J Clin Microbiol
     1998;36:1020-7.
  7. Daum RS, Gupta S, Sabbagh R, Milewski WM. Characterization of
     Staphylococcus aureus isolates with decreased susceptibility to
     vancomycin and teicoplanin: isolation and purification of a
     constitutively produced protein associated with decreased
     susceptibility. J Infect Dis 1992;166:1066-72.

Dispatches

Candida dubliniensis Candidemia in Patients with Chemotherapy-Induced
Neutropenia and Bone Marrow Transplantation

Jacques F. G. M. Meis,* Markus Ruhnke, Ben E. De Pauw,* Frank C. Odds,
Wolfgang Siegert, and Paul E. Verweij*
*University Hospital Sint Radboud, Nijmegen, The Netherlands; Humbold
University, Berlin, Germany; and Janssen Research Foundation, Beerse,
Belgium

---------------------------------------------------------------------------
      The recently described species Candida dubliniensis has been
      recovered primarily from superficial oral candidiasis in
      HIV-infected patients. No clinically documented invasive
      infections were reported until now in this patient group or in
      other immunocompromised patients. We report three cases of
      candidemia due to this newly emerging Candida species in
      HIV-negative patients with chemotherapy-induced
      immunosuppression and bone marrow transplantation.

Although Candida albicans remains the most common opportunistic yeast
pathogen in patients with AIDS and other immunocompromised persons, species
less susceptible to fluconazole are becoming more common (1). Recently, a
newly described species, Candida dubliniensis, was isolated from
oropharyngeal lesions in patients with AIDS living in Dublin, Ireland (2).
C. dubliniensis, phenotypically very similar to C. albicans in producing
both germ tubes and chlamydospores, has since been recovered from the oral
washings of approximately 25% of 94 HIV-positive Irish patients with or
without AIDS and 3% of 150 HIV-negative Irish persons (3,4), which suggests
that this species belongs to the indigenous microflora of the oral cavity,
albeit in a minority of healthy persons. Subsequent reports indicate that
the species has a worldwide distribution (4). The role of C. dubliniensis
as a pathogen has been limited to oral candidiasis. We now report three
cases of candidemia due to C. dubliniensis in patients not infected with
HIV. The yeasts were initially identified as C. albicans because each
produced germ tubes and chlamydospores; this identification became suspect
when equivocal carbohydrate assimilation patterns were obtained.

Case 1

Graft-versus-host disease of the skin, liver, and digestive tract developed
in a 39-year-old woman with chronic myelogenous leukemia after an
allogeneic hematopoietic stem cell transplant in September 1995, during
which she was treated with cyclosporine and high-dose prednisolone. Germ
tubeproducing Candida spp., later identified as C. dubliniensis, were
isolated from stool samples obtained for routine testing. The white-cell
count was 2.7 x 10(sup 9)/L (72% granulocytes); 4 days later fever and ascites
developed, and C. dubliniensis was cultured from three separate blood
cultures (two sets obtained by venipuncture and one by the central venous
line) taken on the same day (MIC fluconazole, 0.25 g/ml). Ascitic fluid
obtained by a sterile puncture also grew C. dubliniensis. Ascites was
probably related to hypoalbuminemia. An echogram showed no radiologic
evidence of liver candidiasis, although alkaline phosphatase was elevated
(222 U/L; normal < 120 U/L). Treatment was started intravenously with
fluconazole, 800 mg/day; 3 days later, C. dubliniensis were still recovered
in one of five blood cultures taken over 2 days, but from then on, blood
cultures were yeast-negative. Because the patient was in stable condition,
the central line was not removed. Cytomegalovirus (CMV) disease also
developed, which could explain the elevated alkaline phosphatase, with
severe thrombopenia (< 20 x 10(sup 9)/L). The patient was given ganciclovir and
hyperimmune gammaglobulin (Cytotect); Staphylococcus epidermidis bacteremia
also developed, and the patient died 3 weeks after the onset of candidemia,
with severe graft-versus-host disease stage IV, complicated by candidemia
and CMV disease. Permission for autopsy was not granted.

Case 2

In July 1995, a 5-year-old boy was treated with cytotoxic chemotherapy for
relapsed nasopharyngeal rhabdomyosarcoma. Two episodes of bacteremia caused
by Streptococcus mitis and S. epidermidis followed, and the boy was treated
with ceftazidime and later ciprofloxacin (combined with vancomycin) for 3
weeks. Cultures of stools and oral specimens yielded germ tubeproducing
Candida spp. later identified as C. dubliniensis. Four days before the
onset of candidemia the patient became febrile; Staphylococcus aureus and
C. dubliniensis were cultured from sputum. At this time, the child was not
aplastic (leukocyte count 2 x 10(sup 9)/L). Flucloxacillin and ceftazidime were
started. When fungal blood cultures were taken, the patient was very ill,
had profuse diarrhea and high fever, and was leukopenic with a total
leukocyte count of 0.3 x 10(sup 9)/L (granulocytes <0.1 x 10(sup 9)/L; 
thrombocytes 12 x 10(sup 9)/L); 1 day later, three blood cultures, taken 
over 24 hours through a central line, yielded C. dubliniensis (MIC fluconazole, 
0.5 g/ml). Treatment with 12 mg/kg fluconazole was started immediately. C.
dubliniensis were still being recovered from two blood cultures 2 days
after treatment began, but after that, cultures remained sterile, and the
patient gradually improved. The central line was removed 20 days after the
last positive blood culture but was not submitted for culture. The patient
was treated with fluconazole for 1 month (3 weeks intravenously, and 1 week
orally) and was discharged 2 months after the onset of candidemia. No
yeasts were recovered from fecal cultures and oral washes, but 1 month
after discharge, oral washes again sporadically grew C. dubliniensis. The
patient received further radiotherapy without any evidence of candidemia
but died 1 year later of relapsed rhabdomyosarcoma.

Case 3

An 8-year-old girl with sickle cell disease combined with -thalassemia and
recurrent hemolytic crisis received an allogeneic hematopoietic stem cell
transplant in January 1995. One year before, she had a splenectomy because
of hypersplenism. The conditioning regimen for the transplant consisted of
busulfan, cyclophosphamid, and antithymocyte globulin administered with a
Hickman catheter. Cyclosporine and methotrexate were given as prophylaxis
against graft-versus-host disease. A suspension of cotrimoxazole and
amphotericin B was given as antiinfective prophylaxis. Seventeen days after
transplant, sepsis syndrome and renal failure developed while the patient
was still profoundly granulocytopenic (< 0.1 x 10(sup 9)/L). Germ tubeproducing
Candida spp. later identified as C. dubliniensis were isolated from two
sets of blood cultures drawn 6 hours apart from a peripheral vein and from
two sets through the central venous catheter (MIC fluconazole, 0.25 g/ml).
The patient had no signs of oral candidiasis, and yeasts were not recovered
from cultures (oral washes and stools). At this time the patient had
already been treated for 72 hours with imipenem and vancomycin. Because of
persistent fever unresponsive to broad-spectrum antibacterial agents,
intravenous amphotericin B (30 mg) was empirically added. Once the results
of the positive blood cultures became known, 5-flucytosin (100 mg/kg) was
added to the regimen. After initiation of amphotericin B, later blood
cultures remained negative for yeasts. The Hickman catheter was removed 14
days later when the patient had recovered from neutropenia. Catheter tip
cultures remained negative. However, low grade fever persisted.
Nonetheless, because the patient's condition was stable, treatment was
changed to oral fluconazole (50 mg t.i.d.) for another 2 weeks and the
patient was discharged. The cause of persistent fever was not identified,
but approximately 6 months later, the patient recovered.

Microbiologic Results

All yeast isolates were initially identified by germ tube and chlamydospore
formation as C. albicans, but carbohydrate assimilation patterns by
commercial test kits (Auxacolor, Sanofi Pasteur, Paris and API 20C,
Analytab Products, Plainview, New York) gave equivocal results.
Furthermore, the isolates did not elaborate -glucosidase, grew very weakly
at 42C, and failed to grow at 45C (6); they produced dark green colonies
on CHROMagar Candida plates (Becton Dickinson, Etten-Leur, The Netherlands)
typical of C. dubliniensis (4,5) and abundant chlamydospores on rice-cream
agar after 24 hours (2). In contrast with C. albicans, the yeasts isolated
from our patients' specimens hybridized poorly with the C.
albicansspecific Ca3 fingerprinting probe (5) and gave characteristic
arbitrary primer phosphatase-polymerase chain reaction patterns for C.
dubliniensis with primer RP02 (5'-GCGATCCCCA-3'). Each C. dubliniensis
isolate yielded two major bands at 0.4 kb and 1.0 kb, with up to five weak
bands ranging from 0.9 kb to 1.3 kb. In contrast, with C. albicans, the two
major bands were never observed. Instead, each C. albicans isolate yielded
approximately 15 bands of various intensity, ranging from 0.65 kb to 2.4
kb. Furthermore, banding patterns obtained with RP02 were clearly different
from C. glabrata, C. krusei, C. tropicalis, and C. parapsilosis. In vitro
susceptibility testing for fluconazole (powder provided by Pfizer BV,
Capelle a/d IJssel, The Netherlands) was performed by the broth
microdilution method with RPMI-1640 with L-glutamine, buffered with MOPS
incubated at 35C, and read after 48 hours according to NCCLS M-27A (7). C.
parapsilosis ATCC 22019 and C. krusei ATCC 6258 were included as quality
control strains. The isolates from patients 1 and 2 were deposited as CBS
8500 and CBS 8501 at the yeast division, Centraalbureau voor
Schimmelcultures (CBS), Delft, The Netherlands.

C. dubliniensis was first described 3 years ago (2) and is genetically and
phylogenetically distinct from C. albicans (8). Hitherto, its pathogenic
role has been mainly restricted to oropharyngeal infections in HIV-infected
persons and AIDS patients (3,5). In a recent study of C. dubliniensis, one
isolate recovered from a blood culture and one from postmortem lung tissue
was examined (6); however, no clinical data were described to allow
determination of the pathogenic role. The cases we have described show that
C. dubliniensis can cause candidemia in immunocompromised patients.
However, these may not be the first cases of invasive disease due to this
yeast. Identification and differentiation from other germ tubeproducing
yeasts on the basis of phenotypic characteristics has been problematic (8);
therefore, the incidence and prevalence of this organism and its role in
invasive disease have been difficult to determine. For instance, a strain
of C. stellatoidea originally isolated in 1957 from the sputum of a patient
with bronchopneumonia and deposited in the British Culture Collection of
Pathogenic Fungi has been shown to be C. dubliniensis (2,3), and an isolate
of C. albicans (from sputum of a Dutch patient) deposited in the culture
collection of CBS in 1952 has been shown to be C. dubliniensis (Meis,
unpub. obs.). In both cases, it has not been established whether the C.
dubliniensis isolates were responsible for invasive infections.

Fluconazole appears to be less active against C. dubliniensis than against
C. albicans (4) since C. dubliniensis is usually associated with recurrent
episodes of candidiasis and protracted exposure to azole antifungal drugs
in patients with AIDS. Fluconazole showed excellent in vitro activity
against each of the C. dubliniensis isolated from the blood cultures of our
patients; each patient responded well clinically. Nevertheless, it is too
early to estimate the true susceptibility of this species to fluconazole.
This requires the correct identification of the species, which now seems
necessary, given its ability to cause invasive disease in patients treated
for malignant diseases.

Acknowledgments

      We thank T. Rijs, L. Van Nuffel, and G. Dams for excellent technical
assistance and J.P. Donnelly for discussion.

      Dr. Meis is head of the Division of Bacteriology and Mycology,
University of Nijmegen, The Netherlands, and consultant for medical
microbiology and infectious diseases, University Hospital and Clinics. He
is a member of the Nijmegen Mycological Research Group, and his research
focuses on infections in immunocompromised patients, with particular
interest in the management and diagnosis of invasive fungal infections.

      Address for correspondence: Jacques F.G.M. Meis, Department of
Medical Microbiology (440), University Hospital Nijmegen, P.O. Box 9101,
6500 HB Nijmegen, The Netherlands; fax: 31-24-354-0216; e-mail:
j.meis@mmb.azn.nl.

References

  1. Abi-Said D, Anaissie E, Uzun O, Raad I, Pinzcowsli HM, Vartivarian S.
     The epidemiology of hematogenous candidiasis caused by different
     Candida species. Clin Infect Dis 1997;24:1122-8.
  2. Sullivan DJ, Westerneng TJ, Hayes KA, Bennett DE, Coleman DC. Candida
     dubliniensis sp. nov.: phenotypic and molecular characterization of a
     novel species associated with oral candidosis in HIV-infected
     individuals. Microbiology 1995;141:1507-21.
  3. Sullivan DJ, Coleman DC. Candida dubliniensis: characteristics and
     identification. J Clin Microbiol 1998;36:329-34.
  4. Coleman DC, Sullivan DJ, Bennett DE, Morgan GP, Barry HJ, Shanley DB.
     Candidiasis: the emergence of a novel species, Candida dubliniensis.
     AIDS 1997;11:557-67.
  5. Schoofs A, Odds FC, Colebunders R, Ieven M, Goossens H. Use of
     specialised isolation media for recognition and identification of
     Candida dubliniensis isolates from HIV infected patients. Eur J Clin
     Microbiol Infect Dis 1997;16:296-300.
  6. Pinjon E, Sullivan D, Salkin I, Shanley D, Coleman D. Simple,
     inexpensive, reliable method for differentiation of Candida
     dubliniensis from Candida albicans. J Clin Microbiol 1998;36:2093-5.
  7. National Committee for Clinical Laboratory Standards. Reference method
     for broth dilution antifungal susceptibility testing of yeasts.
     Approved standard M27-A. Wayne (PA): The Committee; 1997.
  8. Sullivan D, Coleman D. Candida dubliniensis: an emerging opportunistic
     pathogen. Curr Top Med Mycol 1997;8:15-25.

Dispatches

Household Transmission of Streptococcus pneumoniae, Alberta, Canada

James D. Kellner,* A. Patrick Gibb, Jenny Zhang, and Harvey R. Rabin*
*Foothills Medical Centre and Alberta Children's Hospital, Calgary,
Alberta, Canada; University of Calgary, Alberta, Canada; Calgary
Laboratory Services, Alberta, Canada; and Provincial Laboratory of Public
Health, Calgary, Alberta, Canada

---------------------------------------------------------------------------
      Proven or presumptive multidrug-resistant Streptococcus
      pneumoniae pneumonia was diagnosed simultaneously in three
      married couples in Alberta, Canada. The pair of isolates from
      each couple had identical antibiotic resistance profiles,
      serotypes, and pulsed-field gel electrophoresis patterns. One
      or more of these cases could have been prevented by S.
      pneumoniae vaccine.

Outbreaks of Streptococcus pneumoniae (antibiotic resistant and
nonresistant) have been reported from child-care centers, nursing homes,
hospitals, military camps, homeless shelters, and penal institutions (1-6).
Simultaneous cases within households have rarely been reported (7-11); such
cases require common exposure and transmission, as well as similar
likelihood of disease in the hosts or increased virulence in the pathogen.

In December 1996 and January 1997, three married couples with
multidrug-resistant S. pneumoniae (MDRSP) were admitted to Foothills
Medical Centre in Calgary. The couples were not admitted on the same day.
None of the couples lived with children, although couple C had daily
contact with children. All patients received appropriate antibiotic therapy
after their culture and antibiotic sensitivity results were known. We
reviewed each patient's health record (Table) and were able to contact two
of the three couples for further information.

Table. Clinical and laboratory features of three couples with Streptococcus 
pneumoniae pneumonia
----------------------------------------------------------------------------
                                Couple A      
                ----------------------------------------------------------- 
Feature         Patient 1                    Patient 2                
----------------------------------------------------------------------------
Age (yrs)       62                            61                      
chronic         Hypertension, diabetes        Gout 3 previous MIs(sup a) 
conditions
Smoker          No                            No         
S. pneumoniae   No                            No                                   
vaccine
Recent          None                          None  
antibiotics                                               
Others in home  None                          None                                                  
Initial         URTI(sup d) symptoms,         URTI(sup d) symptom, 
complaint	    cough, fever                  cough, fever        
Physical exam   Febrile,                      Febrile,                         
                [arrowup]HR(sup e),		    [arrowup]HR(sup e)
                [arrowup]RR(sup f),           [arrowup]RR(sup f),                                          
                severe distress,              [arrowdwn]breath sounds
                [arrowdwn] breath sounds
Chest X-ray     Right upper lobe              Right lower lobe
(admission or   consolidation                 consolidation
as noted) 
Admitting       Right lobe pneumonia          Bilateral pneumonia     
diagnosis
Discharge       Right upper lobe              Right lower lobe 
diagnosis       Pneumonia                     pneumonia
Complications   Empyema, osteomyelitis        None       
Source of       Day 1blood                   Day 1blood 
Gram stain      Not applicable                Not applicable

Other potential None                          None     
pathogens when
pneumonia
diagnosed
Antibiotic
susceptibility(sup l)
 Penicillin            2       R              1.5     I                         
 Cefuroxime            4       R              6       R 
 Ceftriaxone           1       I              0.5     S    
 TMP/SMXm              >/= 32  R              >/= 32  R                           
 Erythromycin           0.25   S              0.25    S         
  Serotype             14                     14          
PFGE pattern(sup n)    AA                     AA     

                                   Couple B
                ------------------------------------------
Feature         Patient 1                     Patient 2
-------------------------------------------------------------------
Age (yrs)       72                             71                               
Chronic         Hypertension CAD(sup b)        COPD(sup c)                      
conditions		    
Smoker          Yes                            Yes                             
S. pneumoniae   Unknown                        Unknown                          
vaccine
Recent          Unknown                        Unknown   
antibiotics
Others in home  None                           None  
Initial         URTI(sup d) symptoms, cough,   URTI(sup d) symptoms, cough,
complaints      fever, chest pain              fever, chest pain,
                                               eye discharge
Physical exam   Febrile,                       Febrile,
                [arrowup]HR (sup e),           [arrowup]HR(sup e)      
                [arrowup]RR(sup f),            [arrowup]RR(sup f),  
                [arrowdwn]O(sub 2)saturation   [arrowdwn] breath sounds
Chest X-ray     Bibasilar consolidation        Extensive right-sided
(admission or					     consolidation
 as noted)               
Admitting       Pneumonia			     Lobar pneumonia
diagnosis
Discharge       Pneumonia                      Lobar pneumonia
diagnosis
Complications   None                           None                          
Source of       Day 1sputum (4+[sup i])       Day 1sputum (3+[sup i])
isolate
Gram stain      GPC resembling S. pneumoniae   GPC resembling S. pneumoniae
		    (sup j)                        (sup j), GNB(sup k)                                                                                             moniaej, GNBk
Other potential None                           H. influenzae (3+[sup i])
pathogens when
pneumonia
diagnosed
Antibiotic
susceptibility(sup l)
  Penicillin              1.5     I            2       R
  Cefuroxime              3       R            4       R
  Ceftriaxone             0.75    S            0.38    S    
  TMP/SMXm                >/= 32  R            >/= 32  R
  Erythromycin            16      R            16      R 
  Serotype                9V                   9V
  PFGE pattern(sup n)     BB                   BB

		                          Couple C 
                ----------------------------------------------------------- 
Feature         Patient 1                       Patient 2                 
---------------------------------------------------------------------------
Age (yrs)       39				      37
Chronic         Recurrent	                  Recurrent
conditions      sinustis			      sinustis
Smoker          Yes                             Yes     
S. pneumoniae   No	                        No
vaccine
Recent           >3 courses in	            >3 courses in
antibiotics      previous year			previous year
Others in home  None                            None        
Initial         Burn, recent				Burn, recent 
complaints      URTI(sup d)				URTI(sup d)
		    symptoms, cough, fever		symptoms, cough, fever
Physical exam   Febrile, [arrowup]distress      Febrile, [arrowup]distress
                on ventilator,			on ventilator,
		    [arrowdwn]breath sounds,		[arrowdwn]breath sounds,
		    crepitations				crepitations
Chest X-ray     Day 3 - extensive bilateral	Day 2 - extensive bilateral
(admission or   consolidation                   consolidation
as noted)
Admitting       Burn					Burn
diagnosis
Discharge       Burn, complicated by		Burn, complicated by
diagnosis       pneumonia				pneumonia, fatal sepsis
Complications   None                            Died
Source of       Day 3 - ETT(sup g)			Day 2 - BAL(sup h)
isolate	    (4+[sup i])				(10[sup 5] CFU/mL[sup i])
Gram stain     GPC resembling S. 		      GPC resembling S. 
               pneumoniae(sup j),               pneumoniae (sup j)
Other potential GNB(sup k)				H. influenzae
pathogens when						(10[sup 3] CFU/mL[sup i])
pneumonia
diagnosed
Antibiotic
susceptibility(sup l)
  Penicillin              1.5    I		       1      I
  Cefuroxime              6      R			 4      R
  Ceftriaxone             0.75   S			 0.75   S
  TMP/SMXm                >/= 32 R               >/= 32 R   
  Erythromycin            0.25 S                 0.25   S
  Serotype                9V                     9V 
  PFGE pattern(sup n)     BC			       BC

------------------------------------------------------------------------
(sup a)Myocardial infarction.
(sup b)Coronary artery disease.
(sup c)Chronic obstructive pulmonary disease.
(sup d)Upper respiratory tract infection.
(sup e)Heart rate.
(sup f)Respiratory rate.
(sup g)Endotracheal tube.
(sup h)Bronchoalveolar lavage.
(sup i)For sputum or ETT aspirates, 3+ & 4+ reflect growth on the third and fourth set of streaks, respectively, on the culture plate; for BAL, sample fluid is an approximately 100-fold dilution of lung fluid
(sup j)Gram-positive lancet-shaped cocci found singly, in pairs or in short chains.
(sup k)Gram-negative coccobacilli.
(sup l)Antibiotic susceptibilities reported as MIC (micrograms/mL) and as S (susceptible), I (intermediate) or R (resistant) (NCCLS criteria).
(sup m)TMP/SMX (trimethoprim/sulfamethoxazole).
(sup n)Pulsed-field gel electrophoresis.

S. pneumoniae were identified by standard methods. MICs were determined by
E-Test (AB Biodisk, Solna, Sweden) and classified as susceptible (S),
intermediate resistant (I), or fully resistant (R) to each antibiotic,
according to National Committee for Clinical Laboratory Standards
guidelines (12). Serotyping of S. pneumoniae was performed by the Quellung
reaction technique at the National Centre for Streptococcus, Edmonton.
Electrophoretic fingerprinting of S. pneumoniae was performed by
pulsed-field gel electrophoresis (PFGE) of DNA digested with Sma1 (BRL,
Gaithersburg, MD). The PFGE patterns were classified as indistinguishable,
related, or different according to criteria suggested by Tenover (13).

The diagnosis of S. pneumoniae pneumonia in couple A was confirmed by
positive blood cultures, chest X-ray lobar pneumonia, and
disease-compatible clinical findings. Patient 1 in couple A was a health
records clerk at Foothills Medical Centre. Her illness was complicated soon
after admission by empyema, which was drained; the fluid was S.
pneumoniae-negative. Vertebral osteomyelitis was suspected from clinical
evidence 18 days after admission and was confirmed by bone scan; no
diagnostic culture was obtained. Osteomyelitis in this patient was
presumably caused by S. pneumoniae. The initial 7-day course of cefuroxime
(to which S. pneumoniae was resistant) may not have cleared the infection
and thus allowed secondary seeding to bone.

Couple B (who could not be reached for further information) had had recent
visitors from Texas (one a hospital worker) with upper respiratory tract
infections. S. pneumoniae pneumonia was presumptively diagnosed in this
couple on the basis of symptoms, signs, and chest X-rays compatible with
the diagnosis of pneumonia, as well as sputum samples, which had
gram-positive lancet-shaped cocci identified on Gram stain and grew S.
pneumoniae. From the sputum of patient 2 in couple B, gram-negative bacilli
were identified on Gram stain; Haemophilus influenzae was also isolated.
Thus, this patient may have been coinfected, or primarily infected, with H.
influenzae. The patient's blood cultures were negative; a blood culture was
not performed on patient 1 in couple B.

Couple C was admitted with severe burns and inhalation injuries after the
stove in their two-room trailer exploded. They had had recurrent sinusitis
and other respiratory infections in the previous year since moving to their
trailer, which had poor air circulation. Patient 1 of this couple was
taking antibiotics at the time of admission, and patient 2 had recently
completed a course of antibiotics. The diagnosis of pneumonia (patient 1 on
day 3 of admission and patient 2 on day 2) was made on the basis of recent
upper respiratory symptoms and fever, diminished breath sounds,
crepitations, and disease-compatible chest X-ray findings (previous films
had been normal), which made pneumonia more likely than noninfectious
conditions such as acute lung syndrome. The presumptive diagnosis of S.
pneumoniae as the etiologic agent in the case of patient 1, couple C, was
made on the basis of the initial endotracheal tube aspirate, which had
gram-positive lancet-shaped cocci identified on Gram stain and grew S.
pneumoniae. Only gram-positive lancet-shaped cocci were identified from the
initial bronchoalveolar lavage of patient 2 on Gram stain, and S.
pneumoniae grew in much greater numbers than H. influenzae. Blood cultures,
performed for couple C only after antibiotic therapy was started, were
negative. Patient 2 died of septic shock 20 days after admission, with
Candida albicans in his blood. The bronchopneumonia never resolved
clinically, although S. pneumoniae was not isolated from any further
cultures. Thus, S. pneumoniae may have been a contributing factor to, but
not likely the direct cause, of this patient's death.

The identical susceptibility patterns, serotypes, and PFGE patterns
indicate that both partners in each couple were infected with the same
multidrug-resistant S. pneumoniae strain. Couples A and B apparently had
community-acquired pneumonia. Although couple C contracted pneumonia 48 to
72 hours after admission, each partner entered the hospital already
infected with MDRSP; the infecting organisms were identical, and no other
recognized cases of nosocomial MDRSP occurred at Foothills Medical Centre
at the time of their admission (they were admitted 1 month before couple B,
who were also infected with serotype 9V MDRSP). Couple A may have been
exposed to MDRSP as a result of one partner's work in a tertiary-care
hospital; couple B as a result of one partner's exposure to a health-care
worker with respiratory symptoms. At the time of these cases, the
prevalence of penicillin-nonsusceptible S. pneumoniae infections in Calgary
was approximately 10% (A.P. Gibb, unpub. data).

None of these patients had received S. pneumoniae vaccine, yet each had one
or more risk factors for infection (advanced age, exposure to young
children, smoking, and chronic lung or heart disease). Couple C had a
history of recent antibiotic use, the predominant risk factor for
antibiotic-resistant infections.

In Canada, the S. pneumoniae vaccine is recommended for all persons >/=65
years old and persons >/=2 years with identified risk factors (14). Despite
the vaccine's reasonable effectiveness, its use has been very low in Canada
until recently (fewer than 12 doses per 10,000 population distributed
annually [15,16]). The vaccine has been provided free of charge to persons
with medical indications, but not to healthy persons 65 years of age and
older and not as part of a routine vaccination schedule (17). Some
provinces (including Alberta, beginning in 1998) have begun to routinely
provide the vaccine to all persons at risk. The current incidence of
invasive S. pneumoniae infections in Calgary is 20 per 100,000 per year
overall and 87 per 100,000 per year in those older than 64 years of age
(J.D. Klein, unpub. data).

Outbreaks of S. pneumoniae disease occur in institutions with crowding,
poor air quality, or increased host susceptibility (2,4,6). These factors
may also exist within households (9,11). Couple C, for example, lived in a
very crowded space with poor air circulation.

The rate at which secondary S. pneumoniae infections occur in household
contacts of index patients with invasive disease is not known, but rare
cases have been reported (7-11). Factors contributing to secondary
infections include the likelihood of nasopharyngeal infection due to
exposure to the index patient or a common source, susceptibility to the
strain of the index infection, and likelihood that colonization will lead
to disease rather than to development of asymptomatic immunity. Data on
contemporaneous nasopharyngeal carriage of the outbreak strain by household
contacts are limited. A recent study from Gambia found carriage in 8.5% of
household contacts, compared with 21% in an older U.S. study (18,19). In
healthy adults, the prevalence of circulating S. pneumoniae antibodies is
low (4% to 34%, depending on the serotype); however, two thirds of adults
have protective antibody within 1 month of colonization (20). Approximately
15% of children who acquire a new S. pneumoniae strain nasopharyngeally in
a nonoutbreak setting acquire clinical disease (usually otitis media); this
rate is unknown for adults (21). In contrast, during a recent nursing-home
pneumonia outbreak, 23% of residents were infected with the S. pneumoniae
outbreak strain, and 4% became ill (22). The median age of residents was 85
years; only 4% had received S. pneumoniae vaccine.

Increased use of S. pneumoniae vaccine may prevent MDRSP pneumonia within
households and among persons living in crowded conditions.

Acknowledgments

      We thank Sheila Robertson for performing the chart reviews, James
Talbot and Marguerite Lovgren for serotyping, and Kevin Fonseca for
directing the pulsed-field gel electrophoresis.

      Dr. Kellner is an assistant professor of Pediatrics and Microbiology
and Infectious Diseases at the University of Calgary, Canada. His research
interests include S. pneumoniae infections and antimicrobial resistance.

      Address for correspondence: James D. Kellner, Division of Infectious
Diseases, Alberta Children's Hospital, 1820 Richmond Road, SW, Calgary,
Alberta T2T 5C7, Canada; fax: 403-229-7665; e-mail:
jim.kellner@crha-health.ab.ca.

References

  1. Cherian T, Steinhoff MC, Harrison LH, Rohn D, McDougal LK, Dick J. A
     cluster of invasive pneumococcal disease in young children in day
     care. JAMA 1994;271:695-7.
  2. Hoge CW, Reichler MR, Dominguez EA, Bremer JC, Mastro TD, Hendricks
     KA, et al. An epidemic of pneumococcal disease in an overcrowded,
     inadequately ventilated jail. N Engl J Med 1994;331:643-8.
  3. Quick RE, Hoge CW, Hamilton DJ, Whitney CJ, Borges M, Kobayashi JM.
     Underutilization of pneumococcal vaccine in nursing homes in
     Washington State: report of a serotype-specific outbreak and a survey.
     American Journal of Medicine 1993;94:149-52.
  4. Mercat A, Nguyen J, Dautzenberg B. An outbreak of pneumococcal
     pneumonia in two men's shelters. Chest 1991;99:147-51.
  5. Musher D, Groover J, Reichler M, Riedo F, Schwartz B, Watson D, et al.
     Emergence of antibody to capsular polysaccharides of Streptococcus
     pneumoniae during outbreaks of pneumonia: association with
     nasopharyngeal colonization. Clin Infect Dis 1997;24:441-6.
  6. Mandigers CMPW, Diepersloot RJA, Dessens M, Mol SJM, van Klingeren B.
     A hospital outbreak of penicillin-resistant pneumococci in the
     Netherlands. Eur Respir J 1994;7:1635-9.
  7. Asmar BI, Dajani A. Concurrent pneumococcal disease in two siblings.
     Am J Dis Child 1982;136:946-7.
  8. Fenton PA, Spencer RC, Savill JS, Grover S. Pneumococcal bacteremia in
     mother and son. Brit Med J 1983;287:529-30.
  9. Collingham KE, Littlejohns PD, Wiggins J. Pneumococcal meningitis in a
     husband and wife. J Infect 1985;10:256-8.
 10. Tilghman RC, Finland M. Pneumococcic infections in families. J Clin
     Invest 1936;15:493-9.
 11. Heffron R. Pneumonia: with special reference to pneumococcus lobar
     pneumonia. Cambridge: Harvard University Press; 1939.
 12. National Committee for Clinical Laboratory Standards. Table 2G. MIC
     Interpretive Standards (g/mL) for Streptococcus pneumoniae. Villanova
     (PA): National Committee for Clinical Laboratory Standards; 1998. p.
     68-9.
 13. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing
     DH, et al. Interpreting chromosomal DNA restriction patterns produced
     by pulsed-field gel electrophoresis: criteria for bacterial strain
     typing. J Clin Microbiol 1995;33:2233-9.
 14. National Advisory Committee on Immunization. Canadian Immunization
     Guide. 4th ed. Ottawa: Health and Welfare Canada, 1993.
 15. Fedson DS. Influenza and pneumococcal vaccination in Canada and the
     United States, 1980-1993: what can the two countries learn from each
     other? Clin Infect Dis 1995;20:1371-6.
 16. Fedson DS. Pneumococcal vaccination in the United States and 20 other
     developed countries, 1981-1996. Clin Infect Dis 1998;26:117-23.
 17. Epidemiology CDCA. Alberta immunization manual. Edmonton: Alberta
     Health; 1996.
 18. Lloyd-Evans N, O'Dempsey TJ, Baldeth I, Secka O, Demba E, Todd JE, et
     al. Nasopharyngeal carriage of pneumococci in Gambian children and
     their families. Pediatr Infect Dis J 1996;15:866-71.
 19. Smillie WG, Jewett OF. The relationship of immediate family contact to
     the transmission of type-specific pneumococci. American Journal of
     Hygiene 1940;32:79-88.
 20. Musher DM, Groover JE, Rowland JM, Watson DA, Struewing JB, Baughn RE,
     et al. Antibody to polysaccharides of Streptococcus pneumoniae:
     prevalence, persistence and response to revaccination. Clin Infect Dis
     1993;17:66-73.
 21. Gray BM, Converse III GM, Dillon HC. Epidemiologic studies of
     Streptococcus pneumoniae in infants: acquisition, carriage, and
     infection during the first 24 months of life. J Infect Dis
     1980;142:923-33.
 22. Nuorti JP, Butler JC, Crutcher JM, Guevera R, Welch D, Holder P, et
     al. An outbreak of multidrug-resistant pneumococcal pneumonia and
     bacteremia among unvaccinated nursing home residents. N Engl J Med
     1998;338:1861-8.

Dispatches

Preventing Zoonotic Diseases in Immunocompromised Persons: The Role of
Physicians and Veterinarians

Sara Grant and Christopher W. Olsen
University of Wisconsin, Madison, Wisconsin, USA

---------------------------------------------------------------------------
      We surveyed physicians and veterinarians in Wisconsin about the
      risk for and prevention of zoonotic diseases in
      immunocompromised persons. We found that physicians and
      veterinarians hold significantly different views about the
      risks posed by certain infectious agents and species of animals
      and communicate very little about zoonotic issues; moreover,
      physicians believe that veterinarians should be involved in
      many aspects of zoonotic disease prevention, including patient
      education.

The bond between humans and animals has been recognized for many years, and
pet ownership has been associated with both emotional and health benefits
(1-4). However, pet ownership may also pose health risks through the
zoonotic transmission of infectious diseases, especially in the
immunocompromised (5). Animal-associated pathogens of concern to
immunocompromised persons include Toxoplasma gondii, Cryptosporidium spp.,
Salmonella spp., Campylobacter spp., Giardia lamblia, Rhodococcus equi,
Bartonella spp., Mycobacterium marinum, Bordetella bronchiseptica,
Chlamydia psittaci, and zoophilic dermatophytes (2,6). However, with the
exception of Bartonella henselae and zoophilic dermatophytes, infections in
humans are more commonly acquired from sources other than pets, and the
infectious disease risk from owning pets is considered low (2,7).
Nonetheless, HIV-infected persons may still be advised not to own pets (8).

Since human medicine often does not delve deeply into the role of animals
in the transmission of zoonotic agents (7,9) and veterinary medicine does
not cover the clinical aspects of human disease, zoonotic disease control
requires involvement of both physicians and veterinarians. We examined how
frequently physicians and veterinarians encounter zoonotic diseases, what
role physicians think veterinarians should play in zoonotic disease
prevention, how often physicians and veterinarians communicate about
zoonoses issues, and what physicians and veterinarians perceive as the
disease risk of immunocompromised persons from pets.

Our sample populations were drawn from membership lists of the Wisconsin
Veterinary Medical Association (WVMA) and the State Medical Society of
Wisconsin (excluding retired practitioners). Veterinarians (n = 526) were
chosen by a systematic sampling of every third name on the WVMA membership
list. Since veterinarians in all types of practice may encounter zoonotic
problems, sampling was not stratified by specialty. Physicians (n=698) were
chosen by specialty most likely to involve both zoonotic diseases and
immunocompromised patients (all physicians who listed infectious disease [n
= 38] or hematology/oncology [n = 103] as specialties), as well as randomly
selected cohorts of pediatricians (n = 100), and general internal medicine
physicians (n = 500). Duplicate names were removed.

Each participant was mailed a cover letter; a number-coded, postage-paid
return envelope; and a physician- or veterinarian-specific survey.
Nonresponders received a second survey 3 weeks after the first. For
questions with a response scale of 1 to 5, the sample size was large enough
for a 2-sample Z-test to statistically compare mean responses between
physicians and veterinarians. Additionally, responses were analyzed by
veterinary practice type and physician specialty. Statistically significant
differences in responses by specialty are noted in the text. A 2-sample
Z-test employing the standard large sample approximation to binomial data
was used to compare the proportions of responses by physicians and
veterinarians to the question about zoonotic pathogens of concern to
immunocompromised patients.

Surveys were completed by 327 veterinarians and 322 physicians (overall
response rate of 53%). Responses from veterinarians were as follows: 142
(43%) small-animal practice, 65 (20%) large-animal practice, 98 (30%)
mixed-animal practice, and 22 (7%) exotic-animal practice. The distribution
of respondents by practice type is very similar to the distribution by
practice type across the State of Wisconsin (46% small animal, 19% large
animal, 35% mixed, and 0.3% exotic [data courtesy of M. Mardock, Wisconsin
Veterinary Medical Association]), except for an overrepresentation of
exotic-animal practitioners. Nationally, 8% of veterinarians are in large
animal practice exclusively, 29% in mixed practice, 58% in small animal
practice and 5% "other" (data from the Veterinary Economics Statistics
Brochure of the AVMA, September 1998). The distribution of responses from
physicians included 24 (7%) in infectious diseases, 48 (15%) in
hematology/oncology, 53 (16%) in pediatrics and 197 (61%) in general
internal medicine (including 16 who specifically categorized themselves as
pulmonologists and 11 as rheumatologists). Among our random selection of
pediatricians and general internists, the respondent ratio of 3.7
internists for every 1 pediatrician is slightly higher than the statewide
ratio of 2.5 (Wisconsin physician data courtesy of M. O'Brien, State
Medical Society of Wisconsin) and the national ratio of 2.0 (10).

The survey results indicate that veterinarians (Table 1) encounter zoonotic
diseases in their practices or discuss them with their clients more
frequently (p < 0.00001) than physicians (Table 2). Among veterinarians,
small-animal practitioners encounter zoonoses more frequently than
veterinarians as a whole (mean = 2.80, p = 0.05), and large-animal
veterinarians less frequently (mean = 3.41, p = 0.001). Among physicians,
infectious disease specialists encounter zoonoses more frequently than the
overall population of physician respondents (mean = 3.44, p = 0.001), but
these specialists still encounter zoonoses problems less frequently than
veterinarians (p = 0.05).

 Table 1. Survey of veterinarians

 ------------------------------------------------------------------------------
 Questions                                  Responses
 ------------------------------------------------------------------------------
 How often do you encounter or discuss
 zoonotic diseases in your patient
 population?
      1=Several times/day;
      2=Daily; 3=Weekly;               
      4=Occasionally; 5=Never         X = 3.02(sup a) (0.05)(sup b)

 How often do physicians contact you
 for advice on the animal aspects of
 transmission and risks of zoonotic
 diseases?
      1=Several times/week;
      2=Several times/month;
      3=Several times/year;
      4=Rarely; 5=Never                   X = 4.30 (0.04)
 How often do you contact physicians
 regarding a zoonotic disease?
      1=Several times/week;
      2=Several times/month;
      3=Several times/year;
      4=Rarely; 5=Never                   X = 4.21 (0.04)
 If you know that a client is
 immunocompromised, do you offer
 consultation on zoonotic disease
 prevention?
      - Yes                                   n=96(sup c)

      - No                                       n=9
      - The situation has never
      arisen						 n=205
 How much risk to immunocompromised
 patients is associated with owning or
 having contact with the following
 animals?
      1=Highest risk to 5=Lowest
      risk

           - Reptile                   	X = 2.28 (0.09)

           - Bird                      	X = 2.49 (0.07)
           - Kitten (<6 months of age)    X = 2.81 (0.07)
           - Puppy (<6 months of age)     X = 3.02 (0.07)

           - Farm animals              	X = 3.05 (0.07)

           - Cat                       	X = 3.28 (0.06)

           - Dog                       	X = 3.86 (0.06)
 ------------------------------------------------------------------------------
 (sup a)Mean of all respondents.
 (sup b)Standard error of the mean.
 (sup c)Absolute number of veterinarians answering "yes", "no" or "the situation has
 never arisen".

When physicians were asked (on a scale of 1 to 5 with 1 = very comfortable
and 5 = not comfortable) how comfortable they felt about advising patients
on the role of animals in the transmission of zoonotic agents and
associated risks, with the exception of infectious disease specialists
(whose mean comfort level = 1.92 was significantly [p < 0.0001] better than
that of the overall population of physicians), they responded that they
were not very comfortable in this role (mean = 3.69, Table 2); moreover,
physicians indicated that veterinarians should play an equal or greater
role in advising patients about zoonotic diseases (Table 2). In particular,
they suggested that veterinarians should be involved not only in
controlling zoonotic disease pathogens in animals, but also in providing
information for patients and physicians (Table 2). However, the survey
demonstrated a nearly complete lack of communication between physicians and
veterinarians about zoonotic disease issues (Tables 1,2). In addition,
patients themselves do not appear to view veterinarians as a source of
zoonotic disease information. Of 310 veterinarians, 96 indicated that they
offer special consultation about additional steps for zoonotic disease
prevention if they are aware of the fact that a client is
immunocompromised; however, for 205 of 310 respondents, the client's health
was never discussed (Table 1).

 Table 2. Survey of physicians

 ------------------------------------------------------------------------------
 Questions                                 Responses
 ------------------------------------------------------------------------------
 How often do you encounter or
 discuss zoonotic diseases in your
 patient population?
      1=Several times/day;
      2=Daily; 3=Weekly;              
      4=Occasionally; 5=Never		     X = 4.16(sup a) (0.03)(sup b)

 How comfortable do you feel in
 advising patients specifically on
 the animal aspects of transmission
 and the risks for zoonotic diseases?
      1=Very comfortable to
      5=Not 						X = 3.69 (0.05)
 Should veterinarians be involved in
 advising clients about the potential
 for zoonotic disease?
      1=Veterinarian should have
      primary responsibility;
      3=Responsibility should be
      equal; 5= Physician should
      have primary
      responsibility                  		X = 2.77 (0.05)
 How involved should veterinarians be
 in the following areas in reducing
 transmission of zoonotic disease
 agents to immunocompromised
 patients, providing that client
 confidentiality is maintained?
      1=Very involved to 5=Not
      involved

           - General
           maintenance of
           animal health              		X = 1.62 (0.06)
           - Additional
           zoonotic disease
           screening of
           animals                    		X = 1.78 (0.06)
           - Zoonoses
           education for
           patients                   		X = 2.08 (0.06)
           - Consultation
           for physicians             		X = 2.12 (0.06)
 How often do veterinarians contact
 you regarding zoonotic diseases?
      1=Several times/week;
      2=Several times/month;
      3=Several times/year;
      4=Rarely; 5=Never               		 X = 4.74 (0.03)
 How often do you contact
 veterinarians for advice on the
 animal aspects of transmission and
 risks of zoonotic diseases?
      1=Several times/week;
      2=Several times/month;
      3=Several times/year;
      4=Rarely; 5=Never               		X = 4.55 (0.03)
 How much risk to immunocompromised
 patients is associated with owning
 or having contact with the following
 animals?
      1=Highest risk to 5=Lowest
      risk

           - Bird                     		X = 2.37 (0.07)
           - Kitten (<6 months of age)          X = 2.47 (0.08)

           - Cat                      		X = 2.58 (0.07)

           - Reptile                  		X = 2.64 (0.09)

           - Farm animals             		X = 2.94 (0.08)
           - Puppy (<6 months of age)           X = 3.28 (0.08)

           - Dog                      		X = 3.69 (0.06)
 ------------------------------------------------------------------------------
 (sup a)Mean of all respondents.
 (sup b) Standard error of the mean.

In the second portion of the survey, we examined the views of physicians
and veterinarians on the possible disease risks (from specific animals or
pathogens) to immunocompromised persons. Various animals were ranked on a
risk scale of 1 to 5 (1 = highest risk to 5 = lowest risk, with an option
to respond "unsure"). Veterinarians assigned a higher risk than physicians
to reptiles (p = 0.004) and puppies (p = 0.01); physicians assigned a
higher risk than veterinarians to cats (p < 0.00001) and kittens (p =
0.001) (Tables  1,2). Physicians and veterinarians were also asked to list
the two zoonotic pathogens of greatest concern for immunocompromised
persons (Table 3). The two most frequently named pathogens were Salmonella
spp. and Toxoplasma gondii. Within this ranking, Salmonella spp. were
listed more frequently (p = 0.001) by veterinarians than physicians, and
this concern may explain why veterinarians thought that reptiles pose the
greatest risk to the immunocompromised (Table 1). (Because of the high
prevalence in reptiles of Salmonella infection, immunocompromised persons
are advised not to own or handle reptiles [2,11]). In contrast, T. gondii
was listed as a potential disease risk more often by physicians (p =
0.001), which is consistent with physician's concern about
immunocompromised persons owning kittens and cats as pets (Table 2).

 Table 3. Responses of physicians and veterinarians when In summary, our
 asked to "List the two zoonotic pathogens you believe   survey results
 should be of greatest concern for immunocompromised     indicate that
 individuals"                                            physicians and
                                                         veterinarians hold
                                                         very different
 --------------------------------------------------------views about the
 Pathogen              Physicians   Veterinarians        disease risks from
 --------------------------------------------------------certain animals
                                                         and infectious
 Toxoplasma gondii  n(sup a) = 144 n(sup b) = 74(sup c)  agents and
 Salmonella spp.         n = 61        n = 111(sup c)    communicate very
                                                         little about
 Cryptosporidium                                         zoonotic disease
 parvum                  n = 54         n = 86           prevention. The
 Mycobacterium spp.      n = 29         n = 18           perceived risks
                                                         posed by specific
 Chlamydia psittaci      n = 24         n = 31           pathogens raise
 Bartonella spp.         n = 15         n = 10           some questions.
 Histoplasma                            n = 1c           First, for both
 capsulatum              n = 13                          Salmonella spp.
                                                         and T. gondii,
 Giardia lamblia         n = 12         n = 14           contact with pets
 Pasteurella spp.         n = 9         n = 6            is not the only,
                                                         or even the most
 Borrelia burgdorferi     n = 8         n = 6            important, source
 Pneumocystis carinii     n = 8         n = 2            of infection for
 Cytomegalovirus          n = 8       n = 0(sup C)       humans.
                                                         Contaminated foods
 Blastomyces                                             are the most
 dermatitidis             n = 7         n = 9            common vehicle of
 Rabies virus             n = 6         n = 2            Salmonella spp.
                                                         infection (12);
 Campylobacter spp.       n = 5         n = 10           undercooked meat
 Escherichia coli         n = 2        n = 11(sup d)     is also a common
                                                         vehicle of T.
 Streptococcus spp.       n = 1        n = 9(sup d)      gondii infection.

 Dermatophytes            n = 1        n = 24(sup C)     Up to 25% of lamb
                                                         and pork samples
 "Unsure"                n = 29         n = 9            contain Toxoplasma
 Total number of         n = 259       n = 271           tissue cysts (13).
 participants                                            Therefore,
 responding                                              although cats are
 to this question                                        the definitive
                                                         hosts for T.
 --------------------------------------------------------gondii, cat
 (sup a)Total number of times each agent was listed by   ownership is not
 physicians.                                             associated with an
 (sup b)Total number of times each agent was listed by   increase in
 veterinarians.                                          among HIV-infected                                                        Toxoplasma seroconversion
 (sup c,d) The number of times these organisms were 	   persons (14).
 physicians and veterinarians were significantly         of the infectious   
 different. (sup c)p < 0.001; (sup d)p=0.02 ).           disease agents (e.g., 
 Secondly, a number						   Borrelia burgdorferi,
                                                         Histoplasma capsulatum,
 								         Blastomyces dermatitidis,
and Pneumocystis carinii) listed as zoonotic disease risks by both physicians 
and veterinarians are not truly zoonotic, but rather shared infections. Both 
animals and humans are infected, but animals are not the direct vehicles of 
infection for humans. In addition, cytomegaloviruses of humans and animals are 
not infectious across species. Finally, it is surprising that infection with 
B. henselae, the causative agent of cat scratch disease, which also causes 
bacillary angiomatosis, peliosis hepatis, and other conditions in immunocompromised 
persons, was listed relatively infrequently by both physicians and veterinarians
(Table 3). Exposure to kittens has been clearly implicated as a significant risk 
factor in the epidemiology of B. henselae (15). Both physicians and veterinarians 
need to recognize the role of this pathogen in the zoonotic infection of 
immunocompromised persons and the role of cats in its transmission.

Our finding that 205 of 310 veterinarians never knew a client's
immunocompromised condition is consistent with a previous study in which
only 21% of HIV patients felt most comfortable in asking their veterinarian
about the health risks of pet ownership (16). Through approaches such as
small signs in exam rooms, zoonotic disease brochures in reception areas,
comments in practice newsletters, and affiliation with support groups in
the community, veterinarians can encourage immunocompromised persons to
avail themselves of the diagnostic and preventive measures that can be
provided for zoonotic agents.

Our results suggest that communication between physicians and veterinarians
about zoonotic diseases is largely absent. Enhancing such communication
could help prevent transmission of zoonotic agents. In addition to directly
contacting veterinary practitioners in their community, physicians can also
contact their state health departments for information, since some health
departments have public health veterinarians on staff. Links between the
professions on a broader scale (e.g., through combined veterinary/medical
student training and continuing education) to foster a broader consensus
about zoonotic disease risks and prevention should also be encouraged.

Acknowledgments

We thank Linda Sullivan, George Mejicano, Ken Felz, Barbara Burell, and
Winifred Grant for assistance in designing or distributing the surveys and
Chet Thomas, Rick Nordheim, Brian Aldridge, Sandra Martin, and Tom Tabone
for assisting in data analysis.

      The Geraldine R. Dodge Foundation and the Bernice Barbour Foundation
supported this research.

      Dr. Grant is a staff veterinarian at the New Haven Central Hospital
for Veterinary Medicine in New Haven, Connecticut. She conducted this
research during her studies at the University of Wisconsin School of
Veterinary Medicine.

      Dr. Olsen is an assistant professor of public health at the
University of Wisconsin School of Veterinary Medicine. His research
interests include DNA vaccine development and immunity to influenza viruses
in horses and pigs, as well as the molecular epidemiology of swine
influenza viruses as zoonotic agents.

      Address for correspondence: Christopher W. Olsen, Department of
Pathobiological Sciences, School of Veterinary Medicine, University of
Wisconsin, 2015 Linden Drive West, Madison, WI 53706, USA; fax:
608-263-0438; e-mail: olsenc@svm.vetmed.wisc.edu.

References

  1. Fitzgerald FT. The therapeutic value of pets. West J Med
     1986;144:103-5.
  2. Angulo FJ, Glaser CA, Juranek DD, Lappin MR, Regnery RL. Caring for
     pets of immunocompromised persons. J Am Vet Med Assoc 1994;205:1711-8.
  3. Burton BJ. Pets and PWAs: claims of health risk exaggerated. AIDS
     Patient Care 1989;3:34-7.
  4. Beck AM, Meyers NM. Health enhancement and companion animal ownership.
     Annu Rev Public Health 1996;17:247-57.
  5. AIDS patients can acquire some infections from animals [News]. J Am
     Vet Med Assoc 1990;197:1268-9.
  6. Greene CE. Pet ownership for immunocompromised people. In: Bonagura
     JD, editor. Kirk's current veterinary therapy XII. Philadelphia: WB
     Saunders Company; 1995. p. 271-6.
  7. Glaser CA, Angulo FJ, Rooney JA. Animal-associated opportunistic
     infections among persons infected with the human immunodeficiency
     virus. Clin Infect Dis 1994;18:14-24.
  8. Spencer L. Study explores health risks and the human animal bond. J Am
     Vet Med Assoc 1992;201:1669.
  9. Tan JS. Human zoonotic infections transmitted by dogs and cats. Arch
     Intern Med 1997;157:1933-43.
 10. Seidman B, Pasko T, editors. Physician characteristics and
     distribution. Chicago: American Medical Association; 1998. p 57-60.
 11. Centers for Disease Control and Prevention. 1997 USPHS/IDSA guidelines
     for the prevention of opportunistic infections in persons infected
     with human immunodeficiency virus. MMWR Morb Mortal Wkly Rep
     1997;46:4-44.
 12. Angulo FJ, Swerdlow DL. Bacterial enteric infections in persons
     infected with human immunodeficiency virus. Clin Infect Dis
     1995;21:S84-93.
 13. Dubey JP. A review of toxoplasmosis in pigs. Vet Parasitol
     1986;19:181-223.
 14. Wallace MR, Rosetti RJ, Olson PE. Cats and toxoplasmosis risk in
     HIV-infected adults. JAMA 1993;269:76-7.
 15. Zangwill KM, Hamilton DH, Perkins BA, Regnery RL, Plikaytis BD, Hadler
     JL, et al. Cat scratch disease in Connecticutepidemiology, risk
     factors, and evaluation of a new diagnostic test. N Engl J Med
     1993;329:8-13.
 16. St Pierre LA, Kreisle RA, Beck AM. Role of veterinarians in educating
     immunocompromised clients on the risks and benefits of pet ownership.
     In: Proceedings of the Geraldine R. Dodge Foundation Gathering and
     Reports of 1996 Veterinary Student Fellows; 1996 Sep 27-29; Ithaca,
     New York. Morristown (NJ): The Foundation; 1996.

Dispatches

Mycoplasma penetrans Bacteremia and Primary Antiphospholipid Syndrome(ft 1)

(ft 1)Presented in part at the 11th International Congress of the International
Organization for Mycoplasmology. July 1419, 1996. Orlando, FL, USA.

Antonio Yez,* Lilia Cedillo, Olivier Neyrolles, Encarnacin Alonso,*
Marie-Christine Prvost, Jorge Rojas,* Harold L. Watson, Alain
Blanchard, and Gail H. Cassell
*Centro de Investigacin Biomdica de Oriente-IMSS, Puebla City, Mexico;
Benemrita Universidad Autnoma de Puebla, Puebla City, Mexico; Institut
Pasteur, Paris, France; and University of Alabama at Birmingham,
Birmingham, Alabama, USA

---------------------------------------------------------------------------
      Mycoplasma penetrans, a rare bacterium so far only found in
      HIV-infected persons, was isolated in the blood and throat of a
      non-HIV-infected patient with primary antiphospholipid syndrome
      (whose etiology and pathogenesis are unknown).

Antiphospholipid syndrome (APS), first described in 1983 to 1986, is
characterized by a wide variety of hemocytopenic and vaso-occlusive
manifestations and is associated with antibodies directed against
negatively charged phospholipids. Features of APS include hemolytic anemia,
thrombocytopenia, venous and arterial occlusions, livedo reticularis,
pulmonary manifestations, recurrent fetal loss, neurologic manifestations
(stroke, transverse myelitis, Guillain-Barr syndrome); and a positive
Coombs test, anticardiolipin antibodies, or lupus anticoagulant activity
(1). The factor(s) causing production of the antiphospholipid antibodies in
primary antiphospholipid syndrome (PAPS) remain unidentified (2).

A substantial number of patients with Mycoplasma pneumoniaeinduced
respiratory disease have anticardiolipin antibodies (3). Furthermore, many
clinical criteria for APS have also been well documented in patients with
M. pneumoniae infection, including Guillain-Barrlike illness and other
central nervous system manifestations, hemolytic anemia, positive Coombs
test, thrombocytopenia, and arthritis (4).

In this report, we describe the case of a patient with clinical features of
PAPS and a documented bacteremic infection due to M. penetrans (5).

Case

One month before hospital admission, a previously healthy non-HIV-infected
17-year-old woman (blood group O Rh+) who had not previously received a
blood transfusion and had not had sexual experience had acute onset of
arthritis of both ankles, generalized arthralgias, fever, progressive
asthenia, and hemolytic anemia (hemoglobin 87 g/L, unconjugated bilirubin
25.1 mol/L). In the 30 days before hospital admission she had not received
medications other than nonsteroidal antiinflammatory drugs for 5 days.
Three days before hospital admission, she became ill with respiratory
distress, generalized weakness, anorexia, and inability to walk.

On admission to the hospital (day 1), physical examination showed severe
pallor, a swollen cervical lymph node, slight edema of both legs,
tachycardia, and no hypertension. Laboratory data showed severe hemolytic
anemia (hemoglobin 34 g/L, lactate dehydrogenase 5.1 mol/s/L),
leukocytosis (24.5 x 10[sup 9]/L), thrombocytopenia (28.0 x 10[sup 9]/L), and normal
renal function. A Coombs test was positive at 4C, 22C, and 37C. Blood
and bone marrow smears did not show a neoplasic process. The nonsteroidal
antiinflammatory drugs were suspended, and treatment was started with a
combination of methylprednisolone (1 gm bolus q24h intravenously [i.v.] for
3 days) and trimethoprim/sulfamethoxazole (80/400 mg q12h orally) on day 2,
but her condition deteriorated. On day 3, the antibiotic treatment was
changed to ceftazidime (1 gm q8h i.v.). Transfusion was not attempted
because serologic tests indicated the lack of compatibility; (there was a
strong positive mismatch incompatibility in 55 different blood samples and
a mild mismatch in one sample). Another transfusion was partially rejected
because of unidentified nonspecific antibodies. On day 4 severe respiratory
distress and hypoxemia developed, requiring a ventilator, and the patient
was admitted to the intensive care unit. Livedo reticularis was noted, and
methylprednisolone (125 mg q8h i.v.) was administered. Venereal Disease
Research Laboratory tests were negative as were tests for lupus
erythematosus. The patient had anti-dsDNA antibodies (Kallestad Quantafluor
Crithidia lucilae Sanofi Diagnostic Pasteur, Inc.) but positive
anticardiolipin antibodies by enzyme-linked immunosorbent assay (ELISA)
(100 GPL units) (negative test = <10 GPL units) (ImmunoWell, Cardiolipin
Antibody Immunoglobulin [Ig]G ELISA; and Reaads Medical Products, Inc.),
which remained positive 4 and 12 months later, and antiplatelet antibodies
by immunofluorescence (Anti-Human IgG H-chain Fluorescein conjugated,
OTIY-05 Behring Diagnostics). Laboratory data showed hemoglobin 33 g/L,
leukocyte 23.6 x 10[sup 9]/L, a prolonged activated partial thromboplastin time
>150 seconds (control <42 seconds) and prothrombin time of 26.1 seconds
(control 14.0 seconds), International Normalized Ratios value = 3D 2.09,
and the presence of lupus anticoagulant (LA) antibodies (prolonged Russell
viper venom time and confirmed by the STACLOT LA ELISA test, Reaads Medical
Products, Inc.). Respiratory secretions were culture-negative and negative
by immunofluorescence for respiratory syncytial virus, adenovirus,
influenza A, influenza B, parainfluenza 1,2,3, and Chlamydia. Serologic
analysis indicated that the patient had no antibodies against HIV,
hepatitis B surface and core antigens (HbsAg, HBc), or hepatitis C virus.
No acid-fast bacilli or other bacteria were observed on blood and tracheal
aspirate smears. In addition, thoracic radiography showed only bilateral
diffuse pulmonary infiltrates, which was not suggestive of an anaerobic
infection.

On day 2 of hospital admission, blood and throat samples were cultured for
aerobic flora and mycoplasma. M. penetrans in pure culture was isolated
from the patient's blood (isolate HF-1) and throat (isolate HF-3). Later M.
penetrans was isolated from tracheal aspirate in pure culture (isolate
HF-2). Treatment was initiated on day 6 with clindamycin 600 mg q8h i.v.
and vancomycin 500 mg q6h i.v. The patient also received transfusion of two
units of washed red blood cells.

By the microbroth dilution method (6), the HF-1 isolate was sensitive to
clindamycin, clarithromycin, azithromycin, erythromycin, tetracycline,
doxycycline, ofloxacin, and chloramphenicol but resistant to vancomycin and
gentamicin. After 3 days of treatment, the patient improved clinically and
was released from the intensive care unit on day 9; thoracic radiographs
were clear.

The unique evidence of thrombosis was a low-degree paresthesia of both legs
while the patient was receiving anticoagulant therapy; when the condition
developed, anticoagulant therapy was increased. The patient received
physiotherapy to correct paresis and reduced sensation in the left foot and
ankle region. She left the hospital after 26 days, with minimal evidence of
peripheral neuropathy as a sequela.

M. penetrans infection was detected in the patient's specimens prior to
culture and was confirmed by specific polymerase chain reaction (PCR) (7)
(Figure 1A, 1B). Similar results were obtained by another pair of PCR
primers also within the 16S rRNA gene and designed for the specific
detection of M. penetrans (data not shown). Samples from both original
specimens and broth cultures were tested by PCR for other human mycoplasmas
(8,9), but none were detected (data not shown).

[Fig] 
Figure 1. Polymerase chain reaction (PCR)
detection of Mycoplasma penetrans in clinical samples. A. M. penetrans 
PCR genomic amplification with the primers MYCPENET-P and MYCPENET-N (7) 
and analyzed by electrophoresis in 2% agarose gel. Lysates from the 
following original samples: throat swab (lane 1); tracheal aspirate 
(lane 2); blood (lane 3); first blood subculture (HF-1 isolate) (lane 4); 
M. penetrans GTU-54-6A1 (lane 5), showing the amplification product of 
407-bp; and negative control (lane 6). B. Southern blotting of the same 
material. Hybridization with the internal oligonucleotide (MYCPENET-S) probe 
confirmed the specific amplification of M. penetrans genetic sequences.

The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
protein patterns of different extracts (whole cell lysate, Triton X-114
extracts) for the isolate HF-1 and the type strain GTU-54-6A1 were almost
identical (Figure 2A). Upon close examination, minor differences were
found, in particular for antigens approximately 38 kDa in both SDS and
Triton X-114 extracts. Ultrastructural examination of the HF isolates by
transmission electron microscopy showed mycoplasma cells with morphologic
features typical of M. penetrans (Figure 2B).

[Fig]
Figure 2. HF isolates belong to the species Mycoplasma penetrans. A. 
Comparison of protein patterns from the type strain of M. penetrans and the 
isolate HF-1. Mycoplasma cells were directly lysed with SDS (cell lysate), 
or antigens were extracted with the neutral detergent Triton X-114 (total
extract). Antigens were further separated after partitioning between the 
aqueous and detergent phases. The two mycoplasmas compared are the M. 
penetrans type strain GTU-54-6A1 (GTU) and the isolate HF. B. Ultrastructural 
features of the M. penetrans isolates HF-1, HF-2 and HF-3. The HF isolates
were passaged four times in SP-4 broth without antibiotics and processed for
transmission electron microscopy (TEM). Ultrathin sections were stained 
with osmiun tetroxide and ruthenium red and observed by TEM. The three 
isolates show the typical elongated, flask-shaped morphology of M.
penetrans, with the two divided internal compartments. The cytoplasm is 
limited by a single triple-layered unit membrane that is covered with 
capsular material.

Serologic assays (ELISA and Western blot) with Triton X-114-extracted
antigens and other M. penetrans polypeptides from whole-cell lysates both
from the type strain GTU-54-6A1 and from our isolate HF-1 were done as
previously described (10). A lack of reactivity against the Triton
X-114extracted antigens of M. penetrans was observed by both methods.
However, with whole-cell extracts from both type strain and the HF-1
isolate, a 20-kDa polypeptide was immunodetected by Western blotting with
three serum samples collected on days 2, 4, and 9 of hospitalization. The
20-kDa polypeptide is an M. penetrans product, but whether the observed
reaction corresponds to a cross-reacting epitope is not known. The
patient's samples were also negative for antibodies against M. pneumoniae,
M. genitalium, and M. fermentans by ELISA (11) (data not shown).

Conclusions

Since M. penetrans was first reported in 1993 as an emerging infectious
agent, M. penetransspecific antibodies have been detected more frequently
(18.2% to 35.4%) in HIV-infected than in non-HIV-infected persons (0.4% to
1.3%) (10). Until this case, M. penetrans had only been isolated eight
times (5,10), always from the urine of HIV-infected persons (10).

The results indicating that the isolates HF-1, HF-2, and HF-3 belong to the
M. penetrans species are as follows: 1) clinical samples and the
mycoplasmal isolates obtained from them were positive in the M.
penetrans-specific PCR assay; 2) protein patterns of the HF isolates and
the type strain of M. penetrans GTU-54-6A1 were almost identical; 3) serum
samples from different patients (10), which contained M. penetrans-specific
antibodies on the basis of a reaction with the p35 antigen from the type
strain of M. penetrans also reacted with a similar Triton X-114extracted
polypeptide from the HF-1 isolate; and 4) HF isolates exhibited typical
morphologic features of M. penetrans, which are unique among mycoplasmas
isolated from humans. The fact that serum from other patients reacted with
a similar polypeptide from HF isolates indicates that this protein is
produced by this strain of M. penetrans. The lack of M. penetrans strong
humoral response in the HF patient was a factor in favor of dissemination
of the mycoplasma, hence its isolation from the blood. A possible
association between M. penetrans and PAPS should be considered.

Snowden et al. (1990) found antiphospholipid antibodies in more than 50% of
patients with M. pneumoniae pneumonia, especially those with severe
infections requiring hospitalization (3). Catteau et al. (1995) described
two cases of Stevens-Johnson syndrome associated with M. pneumoniae
infection and the presence of antiphospholipid antibodies (12). Our patient
had manifestations typical of PAPS (2). Thus, this report is the first of
M. penetrans isolation in a non-HIV-infected patient and the first of a
blood and respiratory tract infection with M. penetrans.

Acknowledgments

      The authors thank Constantino Gil, M. Ran Nir-Paz, Donna C. Crabb,
Lynn B. Duffy, Padma Patel, Blanca Tobn, Angeles Ros, and Eduardo Aguirre
for technical contributions.

      This work was supported in part by grant R01 AR 42469, National
Institute of Arthritis and Metabolism, National Institutes of Health; the
Institut Pasteur; and FOSIZACONACYT grant 960202008.

      Dr. Yez is researcher in the Eastern Biomedical Research Center
(Centro de Investigacin Biomdica de Oriente), IMSS. He has worked with
human mycoplasmas for the last 12 years and is interested in bacterial
pathogenesis, in particular in the field of autoimmune diseases triggered
by mycoplasmas.

      Address for correspondence: Antonio Yez, Centro de Investigacin
Biomdica de Oriente-IMSS, 2 Piso Ala Sur, Hospital de Especialidades, 2
Norte 2004, 72000 Puebla City, Mexico; fax: 52-22-46-00-57; e-mail:
ayanez@gemtel.com.mx.

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  8. Blanchard A, Gautier M, Mayau V. Detection and identification of
     mycoplasmas by amplification of rDNA. FEMS Microbiol Lett
     1991;81:37-42.
  9. Blanchard A, Yez A, Watson H, Griffiths G, Cassell GH. Evaluation of
     intraspecies genetic variation within the 16S rRNA gene of Mycoplasma
     hominis and detection by polymerase chain reaction. J Clin Microbiol
     1993;31:1358-61.
 10. Grau O, Slizewicz B, Tuppin P, Launay V, Bourgeois E, Sagot N, et al.
     Association of Mycoplasma penetrans with immunodeficiency virus
     infection. J Infect Dis 1995;172:672-81.
 11. Cassell GH, Gambil G, Duffy LB. ELISA in respiratory infections of
     humans. In: Molecular and diagnostic procedures in mycoplasmology. Vol
     II. Tully JG, Razin S, editors. Academic Press 1996:123-36.
 12. Catteau B, Delaporte E, Hachulla E, Piette F, Bergoend H. Infection 
     mycoplasme avec syndrome de Stevens-Johnson et anticorps
     antiphospholipides:  propos de deux cas. Rev Med Interne
     1995;16:10-4.

Dispatches

Infectious Diarrhea in Tourists Staying in a Resort Hotel

Rachel M. Hardie,* Patrick G. Wall, Patricia Gott, Madhu Bardhan, and
Christopher L.R. Bartlett*
*PHLS, Communicable Disease Surveillance Centre, London, United Kingdom;
Food Safety Authority of Ireland, Dublin, Ireland; and Department of
Public Health, Coventry Health Authority, Coventry, United Kingdom

---------------------------------------------------------------------------
      An outbreak of infectious diarrhea with 70 laboratory-confirmed
      cases (58 with Giardia lamblia) and 107 probable cases occurred
      in U.K. tourists who stayed in a hotel in Greece. After a
      cluster of six cases in persons who had stayed at the hotel was
      reported, the Communicable Disease Surveillance Centre began
      active case ascertainment. This outbreak illustrates the value
      of an approach to surveillance that integrates routine
      surveillance data with active case ascertainment.

A large outbreak of infectious diarrhea in residents of the United Kingdom
who became infected while staying at a hotel in Greece was investigated in
the month after their return. The outbreak was first identified by the
Communicable Disease Surveillance Centre (CDSC) in June 1997 when a cluster
of six microbiologically confirmed cases of giardiasis (all in patients who
had stayed at the same hotel in Greece) were reported by a local public
health epidemiologist. The cluster was identified after a school sought
advice about excluding a child with diarrhea. These six patients reported
that many other hotel guests had also been ill.

British guests who arrived at the hotel between 22 May and 9 June 1997 (the
period of reported illness) were included in the investigation. The names
of these guests were obtained from the tour operator (who sent, through
travel agents, a letter to each booking group advising them of the outbreak
and asking them to contact CDSC); other local public health epidemiologists
and environmental health officers; and a list of ill people compiled by one
particularly concerned guest.

In July, a standard questionnaire was administered by telephone to a member
of each group (with contact information available) that had booked their
holiday together. If the group consisted of more than one family, attempts
were made to interview one member from each family. Fact sheets about
giardiasis were distributed.

Of 86 booking groups on the tour operator's list, 51 groups (59%) (239
persons) were contacted and interviewed; 35 groups (41%) (138 persons)
could not be contacted. Those contacted resided throughout the United
Kingdom; 128 (54%) were male, and 132 (55%) were 16 years or older. Of the
239, 224 (94%) were ill while on holiday or within a few days of return;
diarrhea was more commonly reported (95%) than stomach cramps (82%) or
vomiting (64%). The median duration of diarrhea (13 days, interquartile
range 4 to 27) exceeded that of vomiting (1 day, interquartile range 1 to
2).

Of the 224 persons who were ill, 70 (31%) were categorized as definite
cases (those with a pathogen identified in stool specimen reported by
mid-July); 107 (48%) as probable cases (those with no pathogen identified
by mid-July but diarrhea lasting 4 or more days); and 47 (21%) as possible
cases (those with no pathogen identified by mid-July and diarrhea lasting
less than 4 days). Of the definite cases, Giardia lamblia was identified in
58. Other pathogens were identified, and some cases had dual infections
(Table 1).

 Table 1. Pathogens identified in definite        Epidemic curves for both
 cases                                            diarrhea and vomiting
                                                  suggested a point-source
 ----------------------------------------------   outbreak with peak onset
                              No. of               from 5 to 7 June (Figure
                             definite             1). Without knowing the
 Pathogen                   cases(sup a)  (%)      
date of exposure, we
                                                  could not calculate the
 ----------------------------------------------   exact incubation period;
 Giardia lamblia                58       (83)     instead, we estimated the
 Cryptosporidium parvum         11       (16)     upper limit of the
                                                  incubation period by
 Campylobacter spp.             4         (6)     calculating the interval
 Salmonella spp.                3         (4)     between arrival at the
 Entamoeba histolytica          2         (3)     hotel and onset of
                                                  illness (Figure 2). The
 Rotavirus                      1         (1)     short incubation period
 ----------------------------------------------   (most persons became ill
                                                  within 3 days) and high
(sup a) Sum exceeds 70 because of co-infections.  proportion of cases
                                                  reporting vomiting at an
early stage suggest that some initial illness was caused by a viral
gastroenteropathic pathogen. Supporting this hypothesis was the isolation
of small round-structured virus (Norwalk virus) from one stool specimen
obtained in Greece and of rotavirus in the United Kingdom. 

No stool specimens were examined for G. lamblia in Greece. During telephone
interviews in the United Kingdom, members of 20 groups were advised to
provide further stool specimens for testing for G. lamblia. By the end of
the investigation, at least one stool specimen had been examined for ova,
cysts, and parasites for 142 (63%) of the cases. Of the 58 confirmed cases
of giardiasis, 51 were treated with metronidazole; 40 cases were treated
empirically.

[fig 1]					 [fig 2]
Figure 1. Epidemic curve for         Figure 2. Interval between arrival at 
diarrhea, by category of case.       hotel and onset of illness (vomiting
                                     or diarrhea), by category of case.
                                     
During the telephone interviews, travelers were asked about problems with
the food or water in Greece: 54 reports were documented from 51 groups. Of
the reports, 32 (59%) identified problems with food (tasted strange,
inadequately cooked or heated, left out uncovered); 27 (50%) identified
problems with room water (sewage smell and discoloration, principally
around 4 June); and none identified problems with drinking water (travelers
drank bottled water). However, 7 (13%) identified problems with drinks
reconstituted with tap water. None identified problems with swimming pool
water. Statistical analysis showed that attendance at children's activities
and problems with food were not associated with certainty of diagnosis.
However, date of departure, consumption of reconstituted orange juice,
consumption of raw vegetables and salads, and reports of problems with
water in the hotel room were significantly associated with illness (Table
2).

 Table 2. Possible risk factors for illness
 -------------------------------------------------------------------------
                                        Number (%) of guests
                            ----------------------------------------------
                             Definite    Probable   Possible
                              cases,      cases       cases     Not ill
                              (n=70,     (n=107,     (n=47,      (n=15,
                    		 36          39          21          11
Risk factor,                children)   children)   children)  children)
chi-square,			    ----------  ----------  ---------- -----------
and p values                yes   no   yes    no   yes   no    yes    no
--------------------------------------------------------------------------

 Departure after 5 June      65  5 (7)   99  8 (7)   38    9    11     4
                            (93)        (93)        (81) (19)  (73)  (27)
    (chi-square=7.3,
 p=0.007)
 Attendance at children's    23    13    25    14    15    6     6     5
   activities               (64)  (36)  (64)  (36)  (71) (29)  (55)  (45)
    (chi-square=0.01,
 p=0.92)
 Consumption of raw          52    18    78    29    33   14     6     9
   vegetables and salads    (74)  (26)  (73)  (27)  (70) (30)  (40)  (60)
    (chi-square=3.9,
 p=0.04)
 Consumption of              67  3 (4)  100  7 (7)   43  4 (9)  10     5
   reconstituted orange     (96)        (93)        (91)       (67)  (33)
 juice
    (chi-square=8.4,
 p=0.004)

 Problem with water in room  49    21    54    53    17   30     9     6
                            (70)  (30)  (50)  (50)  (36) (64)  (60)  (40)

 (chi-square=7.2, p=0.007)

 Problem with food           43    27    76    31    26   21     9     6
                            (61)  (39)  (71)  (29)  (55) (45)  (60)  (40)
    (chi-square=0.3,
 p=0.58)
 --------------------------------------------------------------------------

This outbreak of giardiasis was the largest reported in the United Kingdom
since 1985 (1) and the first identified in tourists returning from abroad.
The investigation, which relied on active case finding, was complicated by
occurrence of illness in more than one country and multiplicity of
pathogens identified. Evidence suggests that two principal illnesses, both
due to sewage contamination of the water supply, were responsible for the
outbreak: an epidemic viral gastroenteritis and giardiasis.

The epidemic curve suggests a point-source outbreak (although some of the
viral gastroenteritis may have been transmitted person to person before the
peak incidence). If exposure to G. lamblia occurred at the same time as
that to the pathogen responsible for the viral gastroenteritis, then with
its longer incubation period (5 to 25 days vs. 24 to 48 hours for viral
gastroenteritis [2]), illness due to giardiasis would be expected to merge
with recovery from the viral illness. A biphasic epidemic curve would arise
with the second peak due to cases in persons who developed symptoms due
only to giardiasis.

The following evidence supports the water-borne nature of the outbreak: 1)
the epidemic curve suggests a point-source outbreak; 2) several water-borne
pathogens were identified in cases (although environmental investigations
in Greece at the time of the outbreak found no pathogens, G. lamblia was
not specifically sought); 3) 129 guests reported that their room water
smelled of sewage or was discolored around 4 June, and such reports were
more likely from guests in groups with definite cases; 4) consumption of
reconstituted orange juice was associated with certainty of diagnosis; 5)
although all guests reported avoiding tap water, exposure was almost
universal through reconstituted drinks and wet glasses; and 6) the water
supply was chlorinated but not filtered, except at certain points within
the hotel.

Most nationwide outbreaks of gastrointestinal disease in the United
Kingdom, as well as additional cases, are identified through nationally
collated laboratory reports. In 1996, of the 5,517 laboratory reports of G.
lamblia to CDSC from England and Wales, 995 (18%) were associated with
foreign travel. This outbreak, however, was initially identified by a local
public health epidemiologist; most cases were ascertained largely through
the tour operator. Laboratory reports resulted in identification of only 10
cases; a nationwide request for information on related cases resulted in
identification of cases from only six groups. Tour operators are likely to
collaborate with outbreak investigations, particularly since a European
Directive has made them liable for acts and omissions of those with whom
they contract to care for their clients (3). In this instance, the tour
operator organized an inspection and environmental sampling of the hotel,
initiated a survey of food exposure of hotel guests, offered alternative
accommodation, informed their clients of the outbreak after their return to
the United Kingdom, advised them to obtain an appropriate stool
examination, withdrew from assigning clients to the hotel for 1 month while
the situation was monitored, and offered guests financial compensation.

Ill travelers may have been more likely than healthy travelers to respond
to the tour operator's request to contact CDSC. The number of definite
cases, however, was likely to be underrecognized. Guests were classified as
having a protozoal illness only if stool specimens were examined
specifically for ova, cysts, and parasites; 36% of cases did not have stool
specimens examined in this way, and many others had only one stool specimen
tested (intermittent excretion of cysts means that the estimated
sensitivity of one stool specimen is 50% to 70% [4]). Only half of the
laboratories in the Public Health Laboratory Service routinely examine
stool specimens for ova, cysts, and parasites (5); most doctors must
specifically request the investigation to ensure it is performed.

Given limited health-care resources, the benefits of such an outbreak
investigation must be weighed against the costs. In this outbreak, the
benefits included identification and appropriate management of cases, as
well as provision of information to the tour operator to direct
preventative action (which is important, because the investigating body in
the country of residence of outbreak patients cannot act directly).
Increasing international collaboration in surveillance and investigation of
communicable disease outbreaks, particularly within the European Union (EU)
(e.g., Enter-Net [6]), may facilitate action by public health professionals
in the host country after illness in visitors. The United States/EU Task
Force on Communicable Disease is committed to extending Enter-Net beyond
Europe, and several countries including the United States, Canada,
Australia, South Africa, and Japan are interested in joining.

Acknowledgments

      We acknowledge the information and support provided by consultants in
communicable disease control and environmental health officers in the
United Kingdom. We thank Mrs. F. Ryan for data entry and the tour operator
for collaboration.

      Dr. Hardie is a senior registrar in public health medicine at the
PHLS Communicable Disease Surveillance Centre in London. In addition to
giardiasis, her current research interests include evaluation of health
services for tuberculosis and strategies for prevention and control of
hepatitis C and methicillin-resistant Staphylococcus aureus.

      Address for correspondence: Rachel Hardie, PHLS, Communicable Disease
Surveillance Centre, 61 Colindale Avenue, London NW9 5EQ, United Kingdom;
fax: 44-181-200-7868; e-mail: R.Hardie@cdsc.nthames.nhs.uk.

References

  1. Jephcott AE, Begg NT, Baker IA. Outbreak of giardiasis associated with
     mains water in the United Kingdom. Lancet 1985;29:730-2.
  2. Benenson AS, editor. Control of communicable diseases manual. 16th ed.
     Washington: American Public Health Association; 1995.
  3. European Community Council. Council on package travel, package
     holidays and package tours. Official Journal of the European
     Communities, 23 Jun 1990. Directive 90/314/EEC.
  4. Hill D. Giardiasis. Issues in diagnosis and management. Inf Dis Clin
     North Am 1993;7:503-25.
  5. Pugh C, Morris I. Giardiasis: the epidemiological and resource
     implications of standardisation. PHLS Microbiology Digest
     1997;14:100-1.
  6. Yang S. FoodNet and Enter-net: emerging surveillance programs for
     foodborne diseases. Emerg Infect Dis 1998;4:457-8.

Commentary


A Midcourse Assessment of Hantavirus Pulmonary Syndrome

Robert E. Shope
University of Texas Medical Branch, Galveston, Texas, USA

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

Our understanding of infectious diseases follows a natural coursethe
initial discovery of the cause, the exploration of the natural history and
biology of the etiologic agent, and finally the cure or solution. The
discovery of Sin Nombre virus (SNV), one of the viruses causing hantavirus
pulmonary syndrome (HPS), is unparalleled in terms of the rapid progress of
the scientific investigation leading to its description. A cluster of cases
of fatal adult respiratory syndrome was recognized in the Four Corners
region of the United States in May 1993, and within a few days serologic
evidence confirmed hantavirus infection (1). The outbreak occurred in the
wake of the Institute of Medicine's report Emerging Infections: Microbial
Threats to Health in the United States, in which acute respiratory disease
was first on the list of clinical syndromes requiring high-priority
surveillance (2).

The scientific community has now entered the midcourse phase of HPS
research, including the exploration of the natural history of the American
hantaviruses. Seven articles in this issue describe studies of rodent
reservoirs of Sin Nombre and related viruses. They illustrate the
multidisciplinary nature of such studies, which require ecologic methods,
in addition to the newer molecular biology techniques that have helped
hantavirologists detect and characterize the viruses.

These and other studies have elucidated much about the natural host
relationships of hantaviruses. Multiple hantavirus genotypes exist in
virtually all parts of North and South America; each hantavirus genotype
has a single rodent species as its principal reservoir. Evidence exists
that the virus and rodent have evolved together. Each hantavirus variant is
focal in distribution. Prevalence of antibody is high in some regions, and
low or absent in others, even when the same species of rodent is found in
both places. Rodents are not infected at birth; they acquire the virus from
other rodents. Once infected, the animals develop antibody, but many, or
maybe all, infected rodents remain infected. Because of rapid turnover of
the rodent population (as older antibody-carrying animals die and nonimmune
animals are born), antibody prevalence can vary greatly, from 0% to 50%,
with prevalence often lower than 20%.

The studies also have raised new questions. Why and by what route are
animals infected? The articles in this issue suggest that contamination of
wounds when animals fight may be an important route of infection, while
allowing for the possibility of secondary mechanisms, such as venereal
transmission or close association during communal nesting. Although
textbooks state that people become infected by inhaling aerosols created by
the rodents, we need better information. If the animals create infectious
aerosols, why are only a small proportion of susceptible animals infected,
even in enclosed colonies of the rodents (3)? Why is human infection so
rare, even among forest workers and mammalogists (4)? Are some animals
supersecretors of virus? Are individual virus-carrying rodents infectious
for life or only periodically? If periodically, what makes them shed and
stop shedding virus?

Classic pathogenesis studies are required to answer these questions. Two
technologic advancesreverse transcription-polymerase chain reaction
(RT-PCR) and enzyme-linked immunosorbent assay (ELISA)have revolutionized
science but at the same time lured us away from other time-tested methods.
Before the RT-PCR technique was developed, virologists measured infectious
virus by isolating it in cell cultures or laboratory animals. Although we
need information about the infectivity of rodent blood, urine, throat
secretions, feces, and organs, few investigators attempt to isolate
infectious hantaviruses. The reasons are obvious. Virus isolation is
tedious, technically difficult, and (without biosafety level three or four
facilities) dangerous. Because detecting nucleic acid by RT-PCR is safe and
easily accomplished, most investigators are satisfied with that method.
Safe isolation of hantaviruses in the Americas will require additional
investment in physical biocontainment facilities for university and
government laboratories.

Many compelling reasons exist for isolating hantaviruses. How else will
serotype specificity, animal models, pathogenic potential, replication,
transmission, and other phenotypic properties be studied? I cannot
emphasize too strongly the importance of propagating and preserving this
biologic material. To isolate hantaviruses from rodent tissues will require
new approaches. The technical difficulty now in isolating hantaviruses is
analogous to that encountered 30 years ago with dengue viruses. The
solution for dengue was to use a natural host, the mosquito (5). Colonized,
virus-free rodent reservoir hosts of hantaviruses could possibly provide
the increased sensitivity to infection needed to facilitate routine
isolation of hantaviruses. This approach has been successful on a limited
scale in Europe (6).

Only two serologic methodsELISA and, to a lesser extent,
immunofluorescence assayare used on a large scale in the Americas for
testing rodent sera for hantavirus antibody. CDC developed a recombinant
antigen product of the small RNA segment of SNV (7), which was produced in
large quantity and distributed gratis to state health departments and to
collaborators in both North and South America for use in ELISA. The antigen
is broadly cross-reactive and entirely safe. Before the advent of ELISA,
classic virologists surveyed for antibody with one test and then confirmed
a portion of the antibody-positive and -negative serum specimens with a
different test, often the neutralization test. Such confirmatory tests are
desirable in the studies of rodents for hantavirus antibody but are rarely
done. The neutralization test, used to a limited extent (8), is not
practical for study of large numbers of rodent serum specimens or for work
with biosafety level four agents. An alternate to this test is the
hemagglutination-inhibition test. Asian and European hantaviruses
agglutinate goose cells (9,10); it should be feasible, therefore, to
develop the hemagglutination-inhibition test for American hantaviruses.

What will it take to cure or prevent HPS? The only proposed antihantavirus
drug is ribavirin, which although still under trial, has not been
efficacious in treating HPS (11). Supportive emergency care can save lives,
but diagnosis must be made early, clinical expertise is concentrated in
only a few medical centers, and HPS cases are often dispersed. Approaches
to hantavirus human vaccines have been developed (12), but such vaccines
are probably not commercially feasible in the Americas, which have a low
incidence of disease caused by focal genotypes. Rodent control does not
seem practical because of the immense geographic range of hantaviruses.
Ongoing rodent studies may eventually determine whether a wildlife vaccine
analogous to that used successfully for rabies will be practical (13).

For the immediate future, we must depend on education to prevent human
exposure. To design an effective education program, we must know much more
about the rodent reservoir and the mode of virus transmission, both among
rodents and to people. The publication of these ecologic studies in a
medical journal represents important changes in the medical and ecologic
sciences. These studies have required collaboration of ecologists,
epidemiologists, and virologists. Their successful continuation, as well as
the conception, design, and conduct of future studies, requires the spirit
of innovation best achieved in a multidisciplinary atmosphere, as well as a
long-term commitment to collect data for several years.

References

  1. Nichol ST, Spiropoulou CF, Morzunov S, Rollin PE, Ksiazek TG, Feldmann
     H, et al. Genetic identification of a hantavirus associated with an
     outbreak of acute respiratory illness. Science 1993;262:914-7.
  2. Lederberg J, Shope RE, Oaks SC. Emerging infections: microbial threats
     to health in the United States. Washington: National Academy Press;
     1992. p. 136.
  3. Otteson EW, Riolo J, Rowe JE, Nichol ST, Ksiazek TG, Rollin PE, et al.
     Occurrence of hantavirus within the rodent population of northeastern
     California and Nevada. Am J Trop Med Hyg 1996;54:127-33.
  4. Vitek CR, Ksiazek TG, Peters CJ, Breiman RF. Evidence against
     infection with hantaviruses among forest and park workers in the
     southwestern United States. Clin Infect Dis 1996;23:283-5.
  5. Rosen L, Gubler D. The use of mosquitoes to detect and propagate
     dengue viruses. Am J Trop Med Hyg 1974;23:1153-60.
  6. Vapalahti O, Lundkvist A, Kukkonen SK, Cheng Y, Gilljam M, Kanerva M,
     et al. Isolation and characterization of Tula virus, a distinct
     serotype in the genus Hantavirus, family Bunyaviridae. J Gen Virol
     1996;77:3063-7.
  7. Feldmann H, Sanchez A, Morzunov S, Spiropoulou C, Rollin PE, Ksiazek
     TG, et al. Utilization of autopsy tissue RNA for the synthesis of the
     nucleocapsid antigen of a newly recognized virus associated with
     hantavirus pulmonary syndrome. Virus Res 1993;30:351-67.
  8. Chu Y-K, Jennings G, Schmaljohn A, Elgh F, Hjelle B, Lee HW, et al.
     Cross neutralization of hantaviruses with immune sera from
     experimentally infected animals and from hemorrhagic fever with renal
     syndrome and hantavirus pulmonary syndrome patients. J Infect Dis
     1995;172:1581-4.
  9. Tsai TF, Tang YW, Hu SL, Ye KL, Chen GL, Xu ZY.
     Hemagglutination-inhibiting antibody in hemorrhagic fever with renal
     syndrome. J Infect Dis 1984;150:895-8.
 10. Brummer-Korvenkontio M, Manni T, Ukkonen S, Vaheri A. Detection of
     hemagglutination-inhibiting antibodies in patients with nephropathia
     epidemica and Korean hemorrhagic fever by using Puumala virus cell
     culture antigen. J Infect Dis 1986;997-8.
 11. Mertz GJ, Hjelle BL, Bryan RT. Hantavirus infection. Adv Intern Med
     1997;42:369-421.
 12. Chu YK, Jennings GB, Schmaljohn CS. A vaccinia virus-vectored Hantaan
     virus vaccine protects hamsters from challenge with Hantaan and Seoul
     viruses but not Puumala virus. J Virol 1995;69:6417-23.
 13. Wandeler AI. Oral immunization of wildlife. In: Baer GM, editor. The
     Natural History of Rabies. Boca Raton (FL): CRC Press; 1991. p.
     485-503.

Letters


Navigational Instinct: A Reason Not to Live Trap Deer Mice in Residences

To the Editor: Although the rodent that most often invades homes in North
America is the house mouse, Mus musculus, the deer mouse, Peromyscus
maniculatus, principal vertebrate host of Sin Nombre virus (SNV) (1), also
invades homes (2), particularly in rural areas. Barring deer mice from
human habitations would prevent domiciliary acquisition of SNV. Current
recommendations (3) are to prevent wild rodents from entering homes or to
snap trap (kill) them should they enter.

To conduct longitudinal studies of hantaviruses in southeastern Colorado on
a former cattle ranch now returning to its natural condition as short-grass
prairie, we often stay in an old bunkhouse, used by many research groups at
irregular intervals. The house, furnished with beds and full kitchen
facilities, is well maintained but has openings through which mice can pass
to and from the outside. For safety and cleanliness, we removed mice we
found inside the house, but between April 1996 and April 1998, we live
trapped and released them rather than snap trapping them. Before release
the rodents were identified to species; were measured and assessed
regarding general appearance and health, sexual preparedness, and presence
of wounds; were bled for antibody tests; and were ear-tagged. Nineteen deer
mice and one pinyon mouse (a P. truei, which did not return) were examined
and tagged. At first, we simply released these animals approximately 50 m
from the house, but when we realized that they were returning, we released
them at increasing distances (50 m to 1,500 m) from the house; the
distances were measured by pace counts by at least two investigators.

Three deer mice had been captured multiple times in our test grid (as far
as 250 m from the house) before they were first captured in the house. Once
captured in the house, however, they were not captured in traps of the grid
(i.e., outside the house). The mean distance traversed by the five deer
mice that returned to the house was at least 394 m; one mouse returned
after being released 500 m and 1,000 m, then 750 m, and 1,200 m from the
house at consecutive daily trapping sessions of 3 days. Sometime within the
subsequent 6 weeks, this mouse returned to the house from the 1,000-m
release point and then from 750 m and 1,200 m away on consecutive days
within our 3-day trapping period. Each of the mice returning to the house
did so within 24 hours of release, two as few as 6 hours after release from
500 m and 750 m away. Nine mice were captured once; six of eight mice
captured twice were captured at least once more; one was captured 10 times,
one 7 times, one 6 times, one 4 times, and two 3 times. Equal numbers of
male and female, adult and juvenile mice were captured in the house, but
only adult mice (5 of 5) returned to the house. Returning deer mice
maintained or gained weight between captures and grew in length at
approximately the same rate as deer mice captured in the test grid.

Some rodents have been documented to move similar distances (e.g., 1,200
m), but they took more than 2 weeks to complete the trek (4). Homing
ability, site fidelity, and navigational proficiency of rodents are well
documented (5,6). Teferi and Millar (7) studied the homing ability of deer
mice in Alberta, Canada; 50% of deer mice in that study returned to their
home sites (a short-grass prairie habitat). The mice traveled 650 m to
1,980 m (mean 1,500 m) and had to cross a river and pass optimal habitat
patches to reach their home sites. Deer mice with previous homing
experience were more successful in returning home (100%) than inexperienced
mice (60%) and faster in doing so (8). Teferi and Millar (7) suggest that
these deer mice were able to navigate in a direct route to their home
sites. We released mice in locations where they had no direct route to the
house; they had to follow a winding road, climb over rocky outcroppings
nearly 17 m high, or otherwise surmount obstacles and dangers, such as

predators (7).

None of the mice we captured had immunoglobulin G (IgG) antibody to SNV.
However, infected deer mice released and then returning to a house or
uninfected deer mice released, infected, and then returning to a house
would increase the likelihood of human contact with an SNV-infected mouse.
The risk would be the same for other hantaviruses infecting other
peridomestic rodents. Against current recommendations that rodents in homes
be snap trapped, some homeowners live trap and release them outside their
homes. Our data strongly support snap trapping mice in homes and provide
evidence that released wild mice return and may place the residents at
risk.

Acknowledgments

      We thank T. Davis, S.B. Calisher, and E. Kuhn for their assistance in
completing these studies.

      This work was partially funded by contract U50-CCU-813420-01 from the
U.S. Centers for Disease Control and Prevention.

Charles H. Calisher,* William P. Sweeney, J. Jeffrey Root,* and Barry J.
Beaty*
*Colorado State University, Fort Collins, Colorado, USA; and University of
Texas Medical Branch, Galveston, Texas, USA

References

  1. Childs JE, Ksiazek TG, Spiropoulou CF, Krebs JW, Morzunov S, Maupin
     GO, et al. Serologic and genetic identification of Peromyscus
     maniculatus as the primary rodent reservoir for a new hantavirus in
     the southwestern United States. J Infect Dis 1994;169:1271-80.
  2. Glass GE, Johnson JS, Hodenbach GA, DiSalvo LJ, Peters CJ, Childs JE,
     et al. Experimental evaluation of rodent exclusion methods to reduce
     hantavirus transmission to humans in rural housing. Am J Trop Med Hyg
     1997;56:359-64.
  3. Centers for Disease Control and Prevention. Hantavirus
     infectionsouthwestern United States: interim recommendations for risk
     reduction. MMWR Morb Mortal Wkly Rep 1993;42(RR-11):1-13.
  4. Ostfeld RS, Manson RH. Long-distance homing in meadow voles, (Microtus
     pennsylvanicus). Journal of Mammalogy 1996;77:870-3.
  5. August PV, Ayvazian SG, Anderson JGT. Magnetic orientation in a small
     mammal, Peromyscus leucopus. Journal of Mammalogy 1989;70:1-9.
  6. Fluharty SL, Taylor DH, Barrett GW. Sun compass orientation in the
     meadow vole, Microtus pennsylvanicus. Journal of Mammalogy
     1976;57:1-9.
  7. Teferi T, Millar JS. Long distance homing by the deer mouse,
     Peromyscus maniculatus. Canadian Field-Naturalist 1993;107:109-11.
  8. Robinson WL, Falls JB. A study of homing of meadow mice. American
     Midland Naturalist 1965;73:188-224.

Bartonella quintana in Body Lice Collected from Homeless Persons in Russia

To the Editor: Lice are obligate blood-feeding insects; three lice species
(Pediculus humanus var capitatis, P. humanus var corporis, and Phtirus
pubis) have been connected with humans throughout history. The body louse
(P. humanus corporis) is the vector for three infectious diseases: epidemic
typhus caused by R. prowazekii, trench fever caused by B. quintana, and
relapsing fever caused by Borrelia recurrentis (1-3). Infestation with the
body louse is associated with cold weather, poverty, and poor hygiene. In
Russia, louse-transmitted diseases have caused more deaths than any other
infectious disease in recent centuries (4). During the last decade,
pediculosis (infestation with P. humanus) has increased markedly throughout
the world (5,6), especially in developing countries and in areas (e.g.,
Eastern Europe, Russia) that have undergone vast social and economic
changes. The incidence of pediculosis in Russia is approximately 220 to 300
cases per 100,000 inhabitants(7). Social and economic upheavals in the
former Soviet Union have increased the number of homeless people, among
whom pediculosis is highly prevalent (6).

A disease of the past, epidemic typhus, has reemerged as a public health
concern after a 1996 outbreak in Burundi, the largest outbreak of the
disease since World War II (5,8). During World War II, a huge typhus
epidemic caused illness in more than 20,000,000 people in Russia. R.
prowazekii infection can persist in a latent form in convalescent typhus
patients, remanifesting itself in a recrudescent form (Brill-Zinsser
disease) in patients under stress (1). Sporadic cases of Brill-Zinsser
disease are reported every year in all regions of the former Soviet Union
(9) and because most of the population has no immunity to R. prowazekii,
the risk for a typhus outbreak is increased. In a recent outbreak in the
Lipetsk region, 360 km from Moscow, 24 louse-infested, febrile patients in
an unheated psychiatric institution had serologically diagnosed typhus
(10).

The great epidemics of trench fever in Europe took place during World War I
(2). However, recently a large outbreak of trench fever associated with
epidemic typhus has been reported in Burundi (5). Sporadic cases of B.
quintana infection have occurred during the last decade in Europe and the
United States, mainly in HIV-infected patients, the homeless, and persons
with chronic alcoholism; the infection has manifested itself as trench
fever, bacteremia, bacillary angiomatosis, or endocarditis (11-16).
Relapsing fever has not been reported in Russia for more than 50 years,
despite a high prevalence after the 1917 revolution and during World War II
(17).

We studied the presence of typhus, trench fever, and relapsing fever agents
in body lice collected from homeless persons in Moscow. The lice were
collected at the Moscow Municipal Disinfection Center, where the homeless
wash and delouse themselves, as well as disinfect or change their clothes.
Only participants who gave informed consent were included. Lice were
collected from the participant's clothing (from the inner surface and seams
of t-shirts, shirts, and sweaters); 3 to 25 lice were found on each
volunteer. Lice were collected from May to October 1996 (214 samples) and
from June to September 1997 (54 samples).

From June to September 1997, 300 homeless male attendees were examined; 57
(19%) had body lice or louse eggs (three had only eggs) on their clothing.
Lice were identified as P. humanus corporis, according to standard
taxonomic keys (6,18). Lice from each person were split into pools of three
to eight insects, and DNA was extracted from each pool and tested for R.
prowazekii, B. quintana, and B. recurrentis by polymerase chain reaction
(PCR) analysis. Primers used for PCR analysis and conditions for DNA
amplification have been described (5,19-22). Uninfected, laboratory-reared
lice served as negative controls, and DNA of R. rickettsii, B. elizabethae,
and B. burgdorferi were used as positive controls.

Results of each amplification were resolved in 1% agarose gels (type LE;
Sigma-Aldrich Chimie, St. Quentin Fallavier, France) and were visualized
under UV light after ethidium bromide staining. The sizes of amplicons were
determined by comparison with the DNA molecular weight marker VI
(Boehringer, Mannheim, Germany). To confirm the identity of amplicons,
their nucleotide base sequence was determined by using an AmpliCycle
sequencing kit (Perkin-Elmer Corp., Foster City, CA) according to the
manufacturer's instructions.

PCRs incorporating rickettsia- and borrelia-specific primers did not yield
products from any DNA extracts derived from the louse samples. Positive
controls in both reactions yielded bands of the expected size. Thus, louse
samples were not infected with R. prowazekii or B. recurrentis. Initial
screening with PCR incorporating nonspecific primer pairs for Bartonella
species yielded products of the estimated amplicon size of approximately
1,200 bp for 33 (12.3%) of the 268 louse samples. These results were
confirmed by PCR incorporating primers (CS.443pCS.979n) specific for the
gltA gene. Products of this reaction were characterized by base-sequence
determination. All 33 Bartonella-positive samples yielded a partial gltA
sequence identical to that of B. quintana (22). Persons infested with
infected lice were younger than 30 years to older than 60 years of age.

A recent report indicates that 11% of the homeless in Russia are infested
with lice (23); in our limited study, we observed a prevalence as high as
19%. With widespread louse infestation and overcrowding, a single case of
Brill-Zinsser disease can cause an outbreak of epidemic typhus. A patient
more than 50 years of age with Brill-Zinsser disease was the suspected
primary source of typhus infection during the 1997 Lipetsk outbreak.
Presence of lice in the hospital permitted disease dissemination (10).
Although our data showed that none of the 268 louse pools were infected
with R. prowazekii, the serious threat of an outbreak requires continued
surveillance. No samples were found to contain B. recurrentis DNA, yet
dissemination of body lice could also cause relapsing fever to reemerge.

Interest in bartonellosis has recently increased, particularly in
association with HIV infection, because Bartonella species can cause
bacteremia in the immunocompromised (15). Recent investigations have
demonstrated that B. quintana cause bacillary angiomatosis, lymphadenopathy
(16), endocarditis (24), and infections of the central nervous system
(25,26) in healthy persons. Recent reports of B. quintana infection
outbreaks in the United States (14,27), Africa (5), and Europe (11,13,28)
suggest either greater awareness or a reemergence of this infection.
Persons who are homeless or alcoholic are particularly at risk
(11-13,27,29). In all recently reported cases, the role of a possible
arthropod vector has remained unclear (30,31), although lice exposure,
together with homelessness, is a risk factor for B. quintana-induced
bacillary angiomatosis (15). The fact that 12.3% of studied lice samples
were B. quintana-positive confirms the role of this arthropod vector in the
contemporary life cycle of the agent. A similar prevalence of B. quintana
in body lice was reported in Burundi (5) and has been observed in France
(D. Raoult, unpub. data). On the basis of data from our study, Moscow
should be considered an area at high risk for an outbreak of bartonellosis.

Elena B. Rydkina,* Vronique Roux,* Eugenia M. Gagua, Alexandre B.
Predtechenski, Irina V. Tarasevich, and Didier Raoult*
*Universit de la Mditerrane, Marseille, France; Russian Academy of
Medical Sciences, Moscow, Russia; Moscow Municipal Disinfection Center,
Moscow, Russia; and Research Center of Virology,Russia

References

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     infectious diseases. Clin Microbiol Rev 1997;10:694-719.
  2. Maurin M, Raoult D. Bartonella (Rochalimaea) quintana infections. Clin
     Microbiol Rev 1996;9:273-92.
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     Douglas RG, Bennet JE, editors. Principles and practice of infectious
     diseases. 3rd ed. Edinburgh: Churchill Livingstone; 1990. p. 1816-8.
  4. Patterson KD. Typhus and its control in Russia, 1870-1940. Med History
     1993;37:361-81.
  5. Raoult D, Ndihokubwayo JB, Tissot-Dupont H, Roux V, Faugere B,
     Abegbinni R, et al. Outbreak of epidemic typhus associated with trench
     fever in Burundi. Lancet 1998;352:353-8.
  6. Tarasevich IV, Zemskaya AA, Dremova VP, Frolova AI, Hudobin VV, Lange
     AB. Human lice (diagnosis, medical significanse, methods of
     elimination) [in Russian]. Moscow: Medzdrav USSR; 1990. p. 5-7.
  7. Tarasevich IV, Fetisova NF. Classical typhus [in Russian]. ZniSO
     (Health of Population and Environment) 1995;2:9-13.
  8. Raoult D, Roux V, Ndihokubwayo JB, Bise G, Baudon D, Martet G, et al.
     Jail fever (epidemic typhus) outbreak in Burundi. Emerg Inf Dis
     1997;3:357-60.
  9. Eremeeva ME, Balayeva NM, Raoult D. Serological response of patients
     suffering from primary and recrudescent typhus: comparison of
     complement fixation reaction, Weil-Felix test,
     microimmunofluorescence, and immunoblotting. Clin Diagn Lab Immunol
     1994;1:318-24.
 10. Tarasevich IV, Rydkina E, Raoult D. An outbreak of epidemic typhus in
     Russia. Lancet 1998;352:1151.
 11. Brouqui P, Houpikian P, Tissot Dupont H, Toubiana P, Obadia Y, Lafay
     V, et al. Survey of the seroprevalence of Bartonella quintana in
     homeless people. Clin Infect Dis 1996;23:756-9.
 12. Comer JA, Flynn C, Regnery RL, Vlahov D, Childs JE. Antibodies to
     Bartonella species in inner-city intravenous drug users in Baltimore,
     MD. Arch Intern Med 1996;156:2491-5.
 13. Drancourt M, Mainardi JL, Brouqui P, Vandenesch F, Carta A, Lehnert F,
     et al. Bartonella (Rochalimaea) quintana endocarditis in three
     homeless men. N Engl J Med 1995;332:419-23.
 14. Jackson LA, Spach DH, Kippen DA, Sugg NK, Regnery RL, Sayers MH, et
     al. Seroprevalence to Bartonella quintana among patients at a
     community clinic in downtown Seattle. J Infect Dis 1996;173:1023-6.
 15. Koehler JE, Sanchez MA, Garrido CS, Whitfeld MJ, Chen FM, Berger TG,
     et al. Molecular epidemiology of bartonella infections in patients
     with bacillary angiomatosis-peliosis. N Engl J Med 1997;337:1876-83.
 16. Raoult D, Drancourt M, Carta A, Gastaut JA. Bartonella (Rochalimaea)
     quintana isolation in patient with chronic adenopathy, lymphopenia,
     and a cat. Lancet 1994;343:977.
 17. Gromashevski LB, Vaindrach GM. Relapsing typhus [in Russian]. Moscow:
     Medgiz; 1946. p. 78-96.
 18. Kim KC, Ludwig HW. The family classification of the Anoplura.
     Systematic Entomology 1978;3:249-84.
 19. Regnery RL, Spruill CL, Plikaytis BD. Genotypic identification of
     rickettsiae and estimation of intraspecies sequence divergence for
     portions of two rickettsial genes. J Bacteriol 1991;173:1576-89.
 20. Roux V, Rydkina E, Eremeeva M, Raoult D. Citrate synthase gene
     comparison, a new tool for phylogenetic analysis, and its application
     for the Rickettsiae. Int J Syst Bacteriol 1997;47:252-61.
 21. Roux V, Raoult D. The 16S-23S rRNA intergenic spacer region of
     Bartonella (Rochalimaea) species is longer than usually described in
     other bacteria. Gene 1995;156:107-11.
 22. Birtles RJ, Raoult D. Comparison of partial citrate synthase gene
     (gltA) sequences for phylogenetic analysis of Bartonella species. Int
     J Syst Bacteriol 1996;46:891-7.
 23. Mitko E. To count homeless in autumn [in Russian]. Vechernyaya Moskva
     (Evening Moscow) Newspaper 1997; 137.
 24. Raoult D, Fournier PE, Drancourt M, Marrie TJ, Etienne J, Cosserat J,
     et al. Diagnosis of 22 new cases of Bartonella endocarditis. Ann
     Intern Med 1996;125:646-52.
 25. Parrott JH, Dure L, Sullender W, Buraphacheep W, Frye TA, Galliani CA,
     et al. Central nervous system infection associated with Bartonella
     quintana: a report of two cases. Pediatrics 1997;100:403-8.
 26. Case Records of the Massachusetts General Hospital. N Engl J Med
     1998;338:112-9.
 27. Spach DH, Kanter AS, Dougherty MJ, Larson AM, Coyle MB, Brenner DJ, et
     al. Bartonella (Rochalimaea) quintana bacteremia in inner-city
     patients with chronic alcoholism. N Engl J Med 1995;332:424-8.
 28. Stein A, Raoult D. Return of trench fever. Lancet 1995;345:450-1.
 29. Jackson LA, Spach DH. Emergence of Bartonella quintana infection among
     homeless persons. Emerg Infect Dis 1996;2:141-4.
 30. Relman DA. Has trench fever returned? N Engl J Med 1995;332:463-4.
 31. Walker DH, Barbour AG, Oliver JH, Lane RS, Dumler JS, Dennis DT, et
     al. Emerging bacterial zoonotic and vector-borne diseases. Ecological
     and epidemiological factors. JAMA 1996;275:463-9.

Tick-Transmitted Infections in Transvaal: Consider Rickettsia africae

To the Editor: We report a case of African tick-bite fever (ATBF) in a
54-year-old French hunter returning to France on 21 April 1997, after a
15-day visit to Transvaal, South Africa. While traveling in the veld, the
hunter removed (but did not keep) two ticks from his left leg. Two days
later, he observed eschars at the bite sites. Within 5 days, he had high
fever (39.5C) and headache and decided to fly back to France, where he was
admitted to the Infectious Diseases Department in the Hotel Dieu Hospital
in Clermont-Ferrand. The patient's clinical symptoms were persistent fever,
severe headache, and two inflammatory eschars on the left leg. Laboratory
results were normal. On 22 April, an acute-phase serum sample and eschar
biopsy were sent to our laboratory. The patient was treated with 200 mg per
day doxycycline for 10 days. His symptoms resolved. A second serum sample
was collected on 13 May.

Microimmunofluorescence was performed as previously described (1). Although
the acute-phase serologic results were negative, the convalescent-phase
serum exhibited antiR. africae and antiR. conorii titers of 16 for
immunoglobulin (Ig) G and 8 for IgM. Sera were adsorbed with R. conorii and
R. africae antigens (2), and serologic testing and Western blot analysis
(1) were performed on the resultant supernatants. Cross-adsorption of the
convalescent-phase serum caused the homologous and heterologous antibodies
to disappear when adsorption was performed with R. africae antigens; only
homologous antibodies disappeared when adsorption was performed with R.
conorii. Western immunoblot, performed with the same adsorbed serum,
indicated R. africae infection by demonstrating a specific reactivity
pattern with R. africaespecific antigens in the 110-kDa to 145-kDa region
(2). An inoculation eschar biopsy specimen was injected into human
embryonic lung fibroblasts, according to the centrifugation shell-vial
technique (3). After 6 days' incubation at 32C, a Gimenez staining of
methanol-fixed human embryonic lung fibroblasts showed rickettsialike
bacilli. The strain was identified by direct immunofluorescence performed
on the cells with an antiR. africae monoclonal antibody (4). Moreover, DNA
was extracted from the ground eschar biopsy specimen and from 200 L of
shell-vial supernatant, by using a QIAmp Tissue kit (QIAGEN GmbH, Hilden,
Germany), according to the manufacturer's instructions. These extracts were
used as templates with primers complementary to a portion of the coding
sequence of the rOmpA encoding gene in a polymerase chain reaction (PCR)
assay (5), and the base sequences of the resulting PCR products were
determined (5). The sequence obtained by both methods was the same as the
R. africae sequence in Genbank (100% similarity).

Since first described in Africa in 1910, tick-transmitted rickettsioses
have been imputed to a single rickettsial species, Rickettsia conorii,
although two distinct clinical illnesses have been observed (6): an urban
form in patients in contact with dogs and their ticks (Rhipicephalus spp.)
characterized by fever, headache, myalgia, cutaneous rash, and a lesion at
the site of the tick bite (7), and a rural form in patients in contact with
cattle or game and their ticks (Amblyomma spp.) characterized by mild signs
and frequent lack of rash (8).

Although R. africae was initially isolated from Amblyomma cattle ticks in
1973, the first evidence of its pathogenic role in humans was seen in 1992
in a patient who, after a tick bite, had fever, an inoculation eschar,
regional lymphadenopathy, but no cutaneous rash (9). Since then, an
additional 20 cases of R. africaerelated infections have been reported in
travelers returning from Zimbabwe and South Africa (2,10).

R. conorii has long been considered the only African spotted fever group
rickettsia, responsible for both Mediterranean spotted fever and ATBF.
Since the first case was described (9), most of the 20 reported cases of
ATBF occurred as outbreaks (2,10) in Europeans returning from Zimbabwe and
South Africa. The occurrence of concomitant ATBF cases is unusual since
Mediterranean spotted fever is generally sporadic and is likely related to
the biologic characteristics of the recognized vector of R. africae,
Amblyomma spp. ticks. While both are nonnidicolous ticks, Amblyomma spp.
and Rhipicephalus spp. exhibit very different host-seeking behavior (11).
Amblyomma spp. are ticks of cattle and wild ungulates, are not
host-specific, and can readily feed on humans; they are "hunter ticks" and
exhibit an "attack strategy" (in response to stimuli they specifically
converge on nearby hosts). Rhipicephalus spp. are dog ticks and vectors of
R. conorii; very host-specific, they exhibit an "ambush strategy" (they are
passive and remain quiescent in their habitat until a vertebrate host
passes). Up to 72% of A. hebraeum are infected with Rickettsia-like
organisms, in particular R. africae (12); Amblyomma spp. are widely
distributed in rural areas in sub-Saharan Africa (13) and prevalence of A.
hebraeum ticks, incidence of ATBF cases, and prevalence of R. africae
antibodies have been strongly linked (14). Rural Africans are also commonly
infected with R. africae, usually at a young age (14). In Zimbabwe, Kelly
et al. (15) demonstrated that 55% of the tested human sera had antibodies
against R. africae.

ATBF usually has specific clinical features: shorter incubation period than
for Mediterranean spotted fever, multiple inoculation eschars (related to
the host-seeking behavior and host-specificity of Amblyomma spp. ticks,
which are "attack ticks" [15]), regional lymphadenopathies, frequent lack
of cutaneous rash or a pale vesicular eruption, and absence of
complications (2). Although only 22 proven cases have been described so far
(including the present case), ATBF has been recognized as a commonly
encountered disease in southern Africa since 1900 (8,16). Epidemiologic and
clinical features indicate that several cases previously diagnosed on the
basis of serology results only as R. conorii-caused may have been caused by
R. africae.

Given the serologic cross-reactivity among spotted fever group rickettsiae,
microimmunofluorescence, the easiest serologic method, may not be
sufficient for the etiologic diagnosis of a rickettsial spotted fever. A
definitive diagnosis of ATBF requires either additional serologic
procedures, such as cross-adsorption or Western blot, or the use of PCR or
culture. As for PCR, rOmpA-amplification possesses sufficient sequence
heterogeneity among the spotted fever group rickettsiae to be used as an
identification tool (5). The centrifugation-shell vial-cell culture (3),
used routinely in our laboratory, reliably isolates strictly intracellular
bacteria, including rickettsia, from blood and tissue specimens, especially
eschar biopsies (the specimen of choice for isolation procedures or genomic
detection). We noted cross-reactions between R. africae and R. conorii.
Cross-adsorption between antiR. africae and antiR. conorii antibodies and
Western blots confirmed that the antibodies we detected were directed
specifically at R. africae. Furthermore, both PCR and cell culture
confirmed the diagnosis of R. africae infection.

ATBF appears to be an important emerging disease in visitors to rural areas
of southern Africa. R. africae should be considered a potential pathogen in
patients returning from such areas who have fever, headache, multiple
inoculation eschars, or regional lymphadenopathy after a tick bite.

Pierre-Edouard Fournier,* Jean Beytout, and Didier Raoult*
*Universit de la Mditerrane, Marseille, France; and Centre Hospitalier
Rgional Hotel Dieu, Clermont-Ferrand, France

References

  1. Teysseire N, Raoult D. Comparison of Western immunoblotting and
     microimmunofluoresence for diagnosis of Mediterranean spotted fever. J
     Clin Microbiol 1992;30:455-60.
  2. Brouqui P, Harle JR, Delmont J, Frances C, Weiller PJ, Raoult D.
     African tick bite fever: an imported spotless rickettsiosis. Arch Int
     Med 1997;157:119-24.
  3. Marrero M, Raoult D. Centrifugation-shell vial technique for rapid
     detection of Mediterranean spotted fever rickettsia in blood culture.
     Am J Trop Med Hyg 1989;40:197-9.
  4. Xu W, Beati L, Raoult D. Characterization of and application of
     monoclonal antibodies against Rickettsia africae, a newly recognized
     species of spotted fever group rickettsia. J Clin Microbiol
     1997;35:64-70.
  5. Roux V, Fournier PE, Raoult D. Differentiation of spotted fever group
     rickettsiae by sequencing and analysis of restriction fragment length
     polymorphism of PCR amplified DNA of the gene encoding the protein
     rOmpA. J Clin Microbiol 1996;34:2058-65.
  6. Conor A, Bruch A. Une fivre ruptive observe en Tunisie. Bull Soc
     Pathol Exot Filial 1910;8:492-6.
  7. Sant'Anna JF. On a disease in man following tick-bites and occurring
     in Loureno Marques. Parasitology 1912;4:87-8.
  8. Troup JM, Pijper A. Tick-bite fever in Southern Africa. Lancet
     1938;ii:1183-6.
  9. Kelly P, Matthewman LA, Beati L, Raoult D, Mason P, Dreary M, et al.
     African tick-bite fevera new spotted fever group rickettsiosis under
     an old name. Lancet 1992;340:982-3.
 10. Fournier PE, Roux V, Caumes E, Donzel M, Raoult D. An outbreak of
     Rickettsia africae infections among participants in an adventure race
     from South Africa. Clin Infect Dis. In press 1998.
 11. Sonenshine DE. Ecology of non-nidicolous ticks. In: Sonenshine DE,
     editor. Biology of ticks. Oxford (NY): Oxford University Press; 1993.
     p. 3-65.
 12. Beati L, Kelly PJ, Matthewman LA, Mason P, Raoult D. Prevalence of
     Rickettsia-like organisms and spotted fever group Rickettsiae in ticks
     (Acari: Ixodidae) from Zimbabwe. J Med Entomol 1995;32:787-92.
 13. Kelly PJ, Beati L, Mason PR, Matthewman LA, Roux V, Raoult D.
     Rickettsia africae sp nov, the etiological agent of African tick bite
     fever. Int J Syst Bacteriol 1996;46:611-4.
 14. 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.
 15. Kelly PJ, Mason PR, Matthewman LA, Raoult D. Seroepidemiology of
     spotted fever group rickettsial infections in human in Zimbabwe. Am J
     Trop Med Hyg 1991;94:304-9.
 16. Gear JHS, Bevan C. An outbreak of tick-bite fever. S Afr Med J
     1936;10:485-8.

Extended-Spectrum Beta-Lactamase-Producing Salmonella Enteritidis in
Trinidad and Tobago

To the Editor: Salmonella Enteritidis, a predominantly localized pathogen
of the human gastrointestinal tract, can become invasive in very young,
very old, malnourished, and immunocompromised patients. In recent years, S.
Enteritidis has emerged as a major intestinal pathogen in Trinidad and
Tobago (population 1.2 million); in 1997, S. Enteritidis caused 79 (66%) of
119 culture-confirmed salmonella infections, in contrast to 18 (18%) of 99,
48 (47%) of 102, and 107 (61%) of 178 in 1994, 1995, and 1996,
respectively. Increased incidence of S. Enteritidis infections has been
reported worldwide (1,2). Of 216 human S. Enteritidis isolates tested for
antimicrobial susceptibility between 1994 and 1996 in Trinidad, none were
resistant to cephalosporins, aminoglycosides, ampicillin,
trimethoprim-sulphamethoxazole, chloramphenicol, and
norfloxacin/ciprofloxacin by the Kirby-Bauer disk diffusion method, which
uses the National Committee for Clinical Laboratory Standards (NCCLS)
breakpoints (3).

Here we report an unusual isolate of S. Enteritidis resistant to all
penicillins and cephalosporinsincluding third-generation cephalosporins,
gentamicin, tobramicin, and trimethoprim-sulphamethoxazoleby the
Kirby-Bauer disk diffusion method. Amoxicillin-clavulanate and
piperacillin-tazobactam disks gave zone sizes of 15 mm and 19 mm,
respectively, which are classified as intermediate in the NCCLS guidelines.
This isolate was recovered from the blood culture of a febrile,
nonneutropenic patient with multiple myeloma on two occasions 24 hours
apart in March 1998. The isolate was sensitive only to ofloxacin and
imipenem. Admitted to the hospital with compressed fracture of the spine
for physiotherapy in December 1997, the patient had several febrile
episodes and received several courses of multiple empirically prescribed
antibiotics (cefotaxime, gentamicin, and piperacillin). The patient had not
traveled abroad during the previous 6 months.

Because cephalosporin resistance in salmonellae has not been reported
before in the Caribbean, we investigated the mechanism behind this
third-generation cephalosporin resistance further. Using
amoxicillin-clavulanate in combination with ceftazidime, ceftriaxone, and
aztreonam, we performed the double disk synergy test to determine whether
this strain was an extended-spectrum beta-lactamase producer as described
elsewhere (3); augmentation of the zone at the junction of
amoxicillin-clavulanate and aztreonam/ceftriaxone/ceftazidime zones
confirmed that indeed it was.

In the past few years, third-generation cephalosporin resistance in S.
Enteritidis has been described in Europe (4), the United States (5), Turkey
(6), India (7,8), and Argentina (9). Few reports exist of extended-spectrum
beta-lactamasemediated third-generation cephalosporin resistance in
Salmonella spp. To our knowledge, this is the first report of this type of
resistance among S. Enteritidis in the Caribbean. This patient was treated
with ciprofloxacin for 1 week; subsequent blood cultures were negative.

This unusual isolate highlights the need to establish an antimicrobial
resistance surveillance network for Salmonella isolates, including S.
Enteritidis, to monitor the trends and new types of resistance mechanisms
in the Caribbean. An epidemiologic study of S. Enteritidis infections is
being planned to describe the extent of the problem and to define risk
factors and vehicles of human infections in three Caribbean countries,
including Trinidad and Tobago.

B.P. Cherian,* Nicole Singh,* W. Charles,* and P. Prabhakar*
*Port of Spain General Hospital, Port of Spain, Trinidad; and Caribbean
Epidemiology Center (CAREC), Port of Spain, Trinidad

References

  1. Centers for Disease Control and Prevention. Salmonella surveillance
     annual tabulation summary, 1993-1995. Atlanta: U.S. Department of
     Health and Human Services; 1997.
  2. Communicable Disease Surveillance Center. Salmonella in humans: PHLS
     salmonella data set, England and Wales, 1981-1996. London: The Center;
     1997.
  3. National Committee for Clinical Laboratory Standards. Performance
     standards for the anti-microbial disk susceptibility tests for
     bacteria that grow aerobically. Approved standard M7 - A4. Villanova
     (PA): The Committee; 1997.
  4. Fantin B, Pangon B, Potel G, Caron F, Vallee E, Vallois JM, et al.
     Activity of sulbactam in combination with ceftriaxone in vitro and in
     experimental endocarditis caused by Escherichia coli producing
     SHV-2-like beta- lactamases. Antimicrob Agents Chemother
     1990:34;581-6.
  5. Morosini MI, Blasquez J, Negri MC, Canton R, Loza E, Baquero F.
     Characterization of a nosocomial outbreak involving an epidemic
     plasmid encoding for TEM-27 in Salmonella enteritidis. J Infect Dis
     1996;174:1015-20.
  6. Herikstad H, Hayes PS, Hogan J, Floyd P, Snyder L, Augulo FJ.
     Ceftriaxone resistant Salmonella in the United States. Pediatr Infect
     Dis J 1997;16:904-5.
  7. Vahaboglu H, Hall LM, Mulazimoglu L, Dodanli S, Yildirim I, Livermore
     DM. Resistance extended spectrum cephalosporins caused by PER-1 beta
     lactamase, in Salmonella typhimurium in Istanbul, Turkey. J Med
     Microbiol 1995;43:294-9.
  8. Kumar A, Nath G, Bhatia BD, Bhargava V, Loiwal V. An outbreak of
     multidrug resistant Salmonella typhimurium in a nursery. Indian
     Pediatr 1995;(3)980:881-5.
  9. Wattal C, Kaul V, Chigh TD, Kler N, Bhandari SK. An outbreak of multi
     drug resistant Salmonella typhimurium in Delhi (India). Indian J Med
     Res 1994;100:266-7.
 10. Rossi A, Lopardo H, Woloj M, Picanet A, Marino M, Galds M, et al.
     Non-typhoid Salmonella spp. resistant to cefotaxime. J Antimicrob
     Chemother 1995;36:697-702.

New emm (M Protein Gene) Sequences of Group A Streptococci Isolated from
Malaysian Patients

To the Editor: We analyzed the M-typespecific emm gene sequences of 24
random Streptococcus pyogenes isolates from sterile- and nonsterile-site
clinical specimens of Malaysian patients. In contrast to isolates in the
United States, which rarely have new emm sequences, 6 of these 24 Malaysian
isolates had new emm gene sequences, which suggests a large reservoir of
group A streptococci expressing new M-type specificities in Malaysia.

The M protein is a surface-exposed principal virulence factor of group A
streptococci (GAS) and a potential vaccine candidate. The hypervariable
M-typespecific N-terminal portion of the M molecule extends from the cell
wall and evokes protective antibodies. Approximately 75 M antigenic types
of GAS are recognized, and several provisional types have been proposed
(1). Formulation of a universally effective vaccine is complicated by the
M-typespecific nature of protective anti-GAS antibodies, temporal and
geographic variations in GAS M-type prevalence (2), and lack of information
on GAS M types from areas where rheumatic fever and rheumatic heart
disease, sequelae of GAS pharyngitis, are endemic (3). The lack of specific
M-typing antisera is also a limiting factor in determining type
distribution. Recently, Beall and colleagues (4,5) demonstrated that
sequence analysis of the hypervariable portion of the emm gene encoding
M-type specificity (emm typing) was an alternative when M-typing antisera
were not available.

Attempts to type selected Malaysian strains of GAS by M protein status have
yielded poor results. Fewer than 16% of strains were typable with standard
M-typing antisera (6). The existence of new M types in Southeast Asia was
suggested as an explanation. To investigate this possibility, we subjected
27 selected strains (6 from blood, 15 from pharyngitis, 3 from pus, and 3
pharyngeal carrier cultures) collected between January 1994 and December
1996 to emm typing. Initial isolation, serogrouping, T typing, and
determination of opacity factor production were performed in Kuala Lumpur,
by standard techniques, commercial media, reagents, and antisera (7).
Strains were transported to the Centers for Disease Control and Prevention
in Atlanta, Georgia, USA, for emm typing, where serogrouping, T typing, and
opacity factor determinations were repeated, and emm typing was performed
(4,5). DNA sequences were subjected to homology searches against all known
emm sequences by Genetics Computer Group Software, Version 9. (Most
sequences in this database were found in strains isolated from patients
living in Europe and North America.)

Of the 27 cultures analyzed, 24 were GAS, 2 were group G streptococci, and
1 was nongroupable Streptococcus. Ten of the 24 GAS strains were standard
emm types emm3, emm12, emm22, emm60, and emm76 (encoding the classic M
types M3, M12, M22, M60, and M76, respectively); 4 were the provisional emm
types pt180, pt2841, and pt5757; and 3 were previously identified emm
sequence types st64/14 and st2034 (GenBank accession numbers X72932 and
U74320, respectively). The st2034 sequence, originally identified in
children from Papua New Guinea, has also been found in Brazil, California,
and Hawaii (B. Beall, R. Facklam, unpub. data). One GAS had a sequence
previously found in group G streptococci (emmLG6, accession number U25741).
Finally, 6 were of five new emm sequence types discovered in this study
(st4529, st4547, st4532, st4545, and st3018, with accession numbers
AF060368, AF052426, AF077666, AF077668, and AF077669, respectively). The
newly found group A st4545 sequence was more similar to various group G
streptococcal emm sequences than to known group A emm sequences. One group
G isolate had a previously found group G 5' emm sequence (stLG6, accession
number U25741). The nongroupable Streptococcus had an emm sequence
previously associated with group L Streptococcus (Beall and Facklam, unpub.
data). These results demonstrate the usefulness of emm typing in areas
where specific M-typing antisera are not available.

Identifying 6 (25%) of 24 GAS with new emm types provides further evidence
of new M serotypes of GAS in Malaysia. The deduced amino acid sequences of
the mature hypervariable N termini of ST4529, ST4532, ST4547, and ST3018
ranged from 43% to 82% identity to M proteins of known sequence (data not
shown). The M nontypability of these isolates suggests unique serologic
specificity. ST4547, ST4532, and ST3018 had 70% to 82% identity over the
first 55-variable-region amino acids, with their closest matching known M
proteins (M70, M27, and M22, respectively), but whether antibodies against
any of these proteins would cross-protect against strains expressing these
M proteins is unknown. Even though the M70 protein is 70% identical over
its first 50 variable N terminal amino acids to the M33 protein, antibodies
against the M70 and M33 proteins do not cross-protect, which suggests that
no cross-protection would occur. The new deduced M protein with the lowest
similarity to any known M protein was ST4529, whose closest match had a 43%
identity over the N-terminal 55 residues of the M5 protein. st4529 likely
encodes a new serospecifically unique M protein.

These findings potentially affect vaccine development. Although new emm
sequences were identified in a survey in the United States (5), the
percentage of new strains with new emm sequences was much lower (6%) than
was found with these Malaysian isolates. emm typing of a larger number of
strains from rheumatic feverand rheumatic heart diseaseendemic areas is
required to deduce amino acid sequences for the development of a suitable M
protein-based vaccine.

Acknowledgments

      We thank Sukeri Kasni and Theresa Hoenes for technical assistance.

      This work was supported by University Kebangsaan Malaysia sabbatical
leave grant and Fulbright Fellowship Award (1997) to Farida Jamal.

Farida Jamal,* Sabiha Pit,* Richard Facklam, and Bernard Beall
*University Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, Kuala Lumpur,
Malaysia; and Centers for Disease Control and Prevention, Atlanta,
Georgia, USA

References

  1. Fraser CAM, Colman G. Some provisional M-types among Streptococcus
     pyogenes (Lancefield group A). In: Recent advances in streptococci and
     streptococcal diseases. Kimura Y, Kotami S, Shiokawa Y, editors.
     Bracknell (UK): Reedbooks Ltd; 1985. p. 35-6.
  2. Musser JM, Kapur V, Kanjilal S, et al. Geographical and temporal
     distribution and molecular characterization of highly pathogenic
     clones of Streptococcus pyogenes expressing allelic variants of
     pyogenic exotoxin A (scarlet fever toxin). J Infect Dis
     1993;167:337-46.
  3. Kaplan EL. Global assessment of rheumatic fever and rheumatic heart
     disease at the close of the century. Circulation 1993;88:1964-72.
  4. Beall B, Facklam R, Thompson T. Screening emm-specific PCR products
     for routine and accurate typing of group A streptococci. J Clin
     Microbiol 1996;34:953-8.
  5. Beall B, Facklam R, Hoenes T, Schwartz B. Survey of emm gene sequences
     and T-antigen types from systemic Streptococcus pyogenes infection
     isolates collected in San Francisco, CA, Atlanta, GA and Connecticut
     in 1994 and 1995. J Clin Microbiol 1997;35:1231-5.
  6. Jamal F, Pit S, Johnson DR, Kaplan EL. Characterization of group A
     streptococci isolated in Kuala Lumpur. J Trop Med Hyg 1995;98:343-6.
  7. Johnson DR, Sramsk J, Kaplan EL, Bicova R, Havlicek J, Havlickova H,
     et al. Laboratory diagnosis of group A streptococcal infection.
     Geneva: World Health Organization; 1996.

Mutant Chemokine Receptor (CCR-5) and Its Relevance to HIV Infection in
Arabs

To the Editor: Approximately 10% of HIV-infected patients may remain
AIDS-free for a long time; moreover, some persons do not become infected
with HIV despite multiple high-risk sexual exposures (1,2). Factors
responsible for this relative resistance to infection and disease include
cytotoxic T cells, neutralizing antibodies, high concentrations of certain
chemokines (e.g., RANTES, MIP-1), human leukocyte antigen haplotype,
mannose-binding protein, and tumor necrosis factor alpha, C4, and TAP
polymorphism (2-4). One of the chemokine receptors, CCR-5, which along with
CD4 acts as co-receptor for HIV entry into macrophages, provides upon
mutation a genetic restriction to HIV infection in homozygous persons and
control of disease progression in heterozygous persons (5,6). Thus a 32bp
deletion in the open reading frame of the region encoding the second
extracellular loop of this receptor causes synthesis of a highly truncated
protein that fails to express on the cell surface, leading to loss of HIV-1
co-receptor activity.

Studies in healthy Caucasian Europeans and North Americans show that
approximately 1% of the population are homozygous for this deletion 
([delta]32), whereas 15% to 20% are heterozygous (5-9); surprisingly, 
a higher percentage (up to 20%) of persons at high risk for HIV but HIV-
negative are homozygous for this deletion. However, no such mutation is 
seen in Japanese, Native Americans, Chinese, Africans, and Tamil Indians, 
which suggests that in these groups either resistance to HIV infection 
is not present or factors other than mutated CCR-5 are in operation.
African-Americans and Hispanics show a low rate of mutation, possibly
because of intermarriage with Caucasians (4). The low frequency of CCR-5
mutation in Arabs with close contacts with Turks in the Eastern Province of
Saudi Arabia may also be due to intermarriage. However, certain persons
with mutated CCR-5 can become HIV-infected (10); in such cases other
chemokine receptors (e.g., CXCR-4, CCR-2, and CCR-3) are believed to
facilitate infection.

HIV infection in Saudi Arabia (population 18 million) is uncommon; the
World Health Organization has so far (1985 to 1997) documented 334 cases of
AIDS in this region (11). We, therefore, studied for the first time the
mutation of CCR-5 in Arabs residing in Saudi Arabia. DNA was isolated from
the peripheral blood mononuclear cells of 105 male blood donors not
infected with HIV and nine HIV-infected patients (seven male and two
female). The latter were divided into three groups, according to published
criteria (2): four persons whose infection did not progress over the long
term and who showed only modest decline of CD4 count after several years of
infection, one person whose infection progressed normally, and four persons
whose infection progressed rapidly and CD4 count fell below 100/l within 2
years. Primers flanking 32 nucleotide deletion of CCR-5 were used to
generate wild type (W) and deleted ([delta]32) fragments of 189 bp and 157 bp,
respectively (5). Amplification was done in a Perkin-Elmer thermal cycler
9,600, by a 20 l reaction mixture that contained 0.25mM of dNTPs, 20 pM of
each primer ('5-CAAAAAGAAGGTCTTCATTACACC-3, 5-CCTGTGCCTCTTCTTCTCATTTCG-3'),
and 0.5 units of Taq polymerase in 1x reaction buffer. All reagents were
obtained from Pharmacia (Sweden). The amplified product was separated on 2%
agarose at 120 V for 45 min and examined under UV light. Of the uninfected
blood donors, 104 (99%) were homozygous for the wild type, and 1 (0.96) was
heterozygous for the mutation. None of the HIV-infected patients had the
mutation. Thus, the mutation is present, albeit infrequently, in Arabs. A
review of 68 HIV-infected patients in our files showed that, as in
Caucasians, infection progressed rapidly in 8%, did not progress over the
long term in 6%, and progressed normally in 86% (2). Therefore, other
hitherto unknown protective factors must be operative in Arabs.

Iman H. Al-Sheikh, Amjad Rahi, and Mohammed Al-Khalifa
King Faisal University and Regional Laboratory, Ministry of Health, Dammam,
Saudi Arabia

References

  1. Malkovsky M. HLA and natural history of HIV infection. Lancet
     1996;348:142-3.
  2. Haynes BF, Pantaleo G, Fauci AS. Toward an understanding of the
     correlates of protective immunity to HIV infection. Science
     1996;271:324-8.
  3. Cocchi F, DeVico AL, Garzine-Demo A, Arya SK, Gallo RC, Lusso P.
     Identification of RANTES, MIP-1a and MIP-1b as the major HIV
     suppressive factors produced by CD8+ T-cells. Science 1995;270:1811-5.
  4. McNicholl JM, Smith DK, Qari SH, Hodge T. Host genes and HIV: the role
     of the chemokine receptor gene CCR-5 and its alleles (32 CCR-5). Emerg
     Infect Dis 1997:3:261-71.
  5. Huang VY, Paxton WA, Wollinsky SM, Neumann AU, Zhang L, He T, et al.
     The role of mutant CCR-5 allele in HIV-1 transmission and disease
     progression. Nature Med 1996;2:1240-3.
  6. Dean M, Carrington M, Winkler C, Huttley GA, Smith MW, Allikmets R, et
     al. Genetic resistance of HIV infection and progression to AIDS by
     deletion of the CKR5 structural gene. Science 1996;273:1856-62.
  7. Liu R, Paxton WA, Choe S, Ceradini D, Martin SR, Horuk R, et al.
     Homozygous defect in HIV co-receptor accounts for resistance of some
     multiply-exposed individuals to HIV-1 infection. Cell 1996;86:367-77.
  8. Samson M, Libert F, Duranz BJ, Rucker J, Liesnard C, Farber CM, et al.
     Resistance to HIV-1 infection in caucasian individuals bearing mutant
     alleles of the CCR-5 chemokine receptor gene. Nature 1996;382:722-5.
  9. Zimmerman PA, Bucklewhite A, Alkhatib G, Spalding T, Kubofcik J,
     Combadiere C. Inherited resistance to HIV-1 conferred by an
     inactivating mutation in CC chemokine receptor 5- studies in
     populations with quantified risk. Mol Med 1997;3:23-36.
 10. O'Brien TR, Winkler C, Dean M, Nelson, JAE, Carrington M, Michael NL,
     et al. HIV-1 infection in man homozygous for CCR-5 (delta)32. Lancet
     1997;349:1219-20.
 11. World Health Organization. AIDS update: reported AIDS cases in East
     Mediterranean Region. EMR AIDS News 1997;1:8.

News and Notes


Meeting Summaries

Workshop on the Potential Role of Infectious Agents in Cardiovascular
Disease and Atherosclerosis

Cardiovascular and cerebrovascular disease currently exact a substantial
human and economic toll in the established market economies and are
expected to become increasingly more prevalent in developing countries. The
noncommunicable nature of coronary artery disease, myocardial infarction,
stroke, and atherosclerotic plaques has now been questioned; Chlamydia
pneumoniae, human cytomegalovirus, periodontal disease, Helicobacter
pylori, and herpes simplex virus-1 have all been associated, to some
extent, with these conditions. The strongest evidence links C. pneumoniae,
then human cytomegalovirus, to coronary artery disease, but direct
causation has not been established. Nevertheless, large antibiotic trials
employing broad spectrum macrolides are now in progress, intended to treat
C. pneumoniae infection in symptomatic cardiac atherosclerosis. In the
future, if even a portion of vascular disease can be prevented with
antimicrobial drugs, vaccines, and health education, the public health
impact of these findings will be imposing.

To address this global public health issue, the National Center for
Infectious Diseases and the National Center for Chronic Disease Prevention
and Health Promotion, Centers for Disease Control and Prevention (CDC),
Atlanta, Georgia, USA, cosponsored a multidisciplinary workshop of invited
national and international researchers, clinicians, and state public health
officials August 31September 1, 1998. The workshop had two primary goals:
1) define the basic science and clinical applied research studies required
to verify or refute the associations, within the context of other
cardiovascular risk factors (emphasizing C. pneumoniae and human
cytomegalovirus); 2) outline a preliminary public health strategy (noting
the potential involvement of CDC) that will correctly address the role of
infection in cardiovascular disease prevention and treatment.

Discussions of reviewed and new data reaffirmed that evidence of causality
between any organism and atherothrombotic disease is sufficient neither to
establish antibiotic treatment guidelines nor to generate a public health
strategy that incorporates infection into the cardiovascular disease
paradigm. To change the chronic course of disease, treatment must be
specifically directed at the responsible agent and applied to the
appropriate stage of pathogenesis in the population at risk. Thus, the
pathobiologic interaction between infection, inflammation and free
radicals, lipids, genetics, and other cardiovascular risk factors must be
clarified with more sensitive, specific, and standardized tools. Reagants
and methods must uniformly differentiate past exposure from active
infection; they must examine how acute, recurrent, and persistent infection
could initiate or aggravate atherosclerosis and acute arterial thrombotic
events. Expansion of animal and in vitro models will add valuable
information to human treatment trials and to epidemiologic studies that
address the multifactorial nature of cardiovascular disease and focus on
groups at risk. Laboratory innovations are needed to maximize
interpretation of results and evaluate the impact of antimicrobial and
non-antimicrobial interventions. Only in this way can a public health plan
based upon sound scientific evidence be developed.

The following were among the recommendations of the workshop: 1) the basic
epidemiology of C. pneumoniae should be defined, including the prevalence
of past and active infection, risk factors for infection and the
interaction of these determinants with traditional cardiovascular and
cerebrovascular disease risk factors; 2) sensitive and specific diagnostic
assays (both new and revised) for laboratory, animal, and clinical studies
should be standardized to assess the true long- and short-term effects of
infection and intervention; 3) pathobiologically relevant animal and in
vitro models should be used to investigate the pathogenesis of single and
multiple agents at different stages in the disease process, enhance the
standardization of tools, and evaluate interventions; 4) activities should
be linked to judicious antimicrobial useexamining baseline knowledge and
use of antibiotics in cardiovascular disease, monitoring changes in disease
incidence and prevalence with increasing antibiotic prescription, and
investigating the effects of treatment on other microbes; 5) a close
alliance should be formed between CDC, the National Institutes of Health,
and investigators from diverse fields to advance these objectives in a
timely fashion, accumulating high quality scientific evidence that will
define the true role of infections and inflammation in atherosclerotic
disease and the appropriateness of antimicrobial therapy.

For more information on the conference, contact Siobhn O'Connor, Centers
for Disease Control and Prevention, 1600 Clifton Rd., NE, Mailstop C12,
Atlanta, GA 30333, USA; tel: 404-639-1454; fax: 404-639-3039; e-mail:
sbo5@cdc.gov.

Workshop on Risks Associated with Transmissible Spongiform Encephalopathies
(TSEs)

A workshop on the evaluation of risks posed by TSEs was held June 8-9,
1998, at the University of Maryland College Park, under the auspices of the
Joint Institute for Food Safety and Applied Nutrition (JIFSAN), a
cooperative venture of the University of Maryland and the U.S. Food and
Drug Administration. The workshop, attended by 300 representatives from 17
countries, evolved in response to the need voiced by governmental agencies,
industry groups, international organizations, and academic experts to
evaluate TSE risks related to source materials, processing, and end
products for animal and human use. Support for the workshop came from
government agencies, industry groups, international organizations, and
JIFSAN.

One goal of the workshop was to develop a framework of guiding principles
for evaluating TSE risks. An initial draft outline of critical elements
applicable to evaluations of TSE risk was presented. The next steps are to
provide definitions for the critical elements and to develop an annotated
outline that explains the relevance of each key element. A set of
information tools (under development) to facilitate risk evaluation and
access to TSE knowledge was demonstrated at the workshop.

The workshop 1) identified research needs and reviewed risks associated
with sources of raw materials (material collection and procurement are
primary control points for ensuring low risk of TSE transmission); 2)
focused on inactivation schemes for TSE agents, which may be the most
reliable way of reducing the level of TSE agent; 3) examined use of several
categories of end products: foods, pharmaceuticals, cosmetics, blood and
blood products, biologics, and tissues of animal and human origin; and 4)
proposed strategies for TSE risk evaluation.

Representatives of governments, private organizations, and industry groups
presented their approaches to the evaluation of TSE risks and found common
themes: 1) zero risk does not exist; 2) decisions concerning public health
issues must often be made in the absence of complete information; 3) risk
analysis may be useful in understanding the key elements of a problem or
situation; 4) the risk evaluation process must be responsive to rapidly
changing information and new scientific data; 5) the risk evaluation
process should be transparent, involving all partners (general public,
regulated industry, government); 6) disagreements still exist concerning
the appropriateness of models, assumptions, and methods; and 7) although
both qualitative and quantitative approaches to TSE risk evaluation have
merit, no single approach is applicable to all situations.

Workshop documents, including illustrated transcripts of the presentations,
a draft outline of critical elements in TSE risk evaluations, observations
on human and animal issues, and workshop overviews, are available at
http://www.life.umd.edu/jifsan/tse.html).

USDA-Report on Potential for International Travelers to Transmit Foreign
Animal Diseases to U.S. Livestock or Poultry

The U.S. Department of Agriculture has published a new reportThe Potential
for International Travelers to Transmit Foreign Animal Diseases to U.S.
Livestock or Poultrywhich explores the issue of animal disease transmission
by human travelers. Millions of people travel internationally each year,
for both pleasure and business, and thus may be at risk for being infected
by animals and causing infections in animals.

The report highlights and summarizes what is currently known about the
potential for human infection and human-to-animal transmission of foreign
animal diseases. It focuses on the International Office of Epizootics List
A diseases. These transmissible animal diseases, which may spread rapidly
and have serious socioeconomic or public health consequence, are important
in the international trade of animals and animal products. These diseases
include avian influenza, Newcastle disease, Rift Valley fever, foot and
mouth disease, swine vesicular disease, vesicular stomatitis, classical
swine fever, African horse sickness, African swine fever, bluetongue,
contagious bovine pleuropneumonia, lumpy skin disease, peste des petits
ruminants, rinderpest, and sheep and goat pox.

The report includes in-depth discussion on the qualitative risk of
human-to-animal transmission, by both biologic and mechanical transmission
modes, and assigns a qualitative risk rating to each List A disease.
Because many of these diseases can survive outside the host for extended
periods of time, mechanical transmission is entirely possible. The report
concludes that humans can transmit Office of Epizootics List A diseases to
animals in the United States. Risk of either biologic or mechanical
transmission was found to be negligible to none for most List A diseases.
However, the risk of mechanical human-to-animal transmission is high for
two diseases (Newcastle disease, swine vesicular disease), moderate for
three diseases (avian influenza, foot and mouth disease, and African swine
fever), and low, but not negligible, for one disease (vesicular
stomatitis).

The full report can be found at
www.aphis.usda.gov/vs/ceah/cei/travrisk.pdf.

Second Annual Conference on Vaccine Research, March 1999

The Second Annual Conference on Vaccine Research: Basic Science, Product
Development, and Clinical and Field Studies will be held March 28-30, 1999,
in Bethesda, Maryland. Sponsored by the Centers for Disease Control and
Prevention, National Foundation for Infectious Diseases (NFID), National
Institute of Allergy and Infectious Diseases, Center for Biologics
Evaluation and Research of the Food and Drug Administration, U.S.
Department of Agriculture, World Health Organization, Children's Vaccine
Initiative, International Society for Vaccines, and Albert B. Sabin Vaccine
Institute, the conference will focus on scientific data from diverse
disciplines involved in vaccine research and development and on associated
technologies for disease control through immunization.

To obtain program announcements and forms for registration, hotel
reservation, and abstract submission, send name, address, telephone, fax,
and e-mail address to: NFID, Attention: Kip Kantelo, Suite 750, 4733
Bethesda Ave., Bethesda, MD 20814-5228, USA; fax: 301-907-0878; tel.:
301-656-0003; e-mail: kkantelo@aol.com. Program information is available
at: http://www.nfid.org/conferences/.

International Scientific Forum on Home Hygiene

Experts in health and hygiene announce the inauguration of the
International Scientific Forum on Home Hygiene (IFH). A nonprofit
organization, IFH comprises scientists and professionals in hygiene policy
and research. It was formed to raise awareness of the role of domestic
hygiene in the prevention of home-acquired infections. Through its
international activities, IFH focuses on home hygiene in situations where
infection risk exists: food, personal cleanliness, and community medical
care.

For more information on the organization, its aims, and scientific research
on home hygiene, access their web site: http://www.ifh-homehygiene.org.

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

URL: ftp://ftp.cdc.gov/pub/EID/vol5no1/ascii/vol5no1.txt

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