Perspectives
Emerging Infectious Diseases and Amphibian Population Declines
P. Daszak, L. Berger, A.A. Cunningham, A.D. Hyatt, D.E. Green, and R. Speare
735-748
Development of Orphan Vaccines: An Industry Perspective
J. Lang and S.C. Wood
749-756

Synopsis
Antimicrobial Resistance with Streptococcus pneumoniae in the United States, 199798
G.V. Doern, A.B. Brueggemann, H. Huynh, E. Wingert, and P. Rhomberg
757-765

Research

Epidemiologic Studies of Cyclospora cayetanensis in Guatemala, C. Bern, B. Hernandez, M.B.
Lopez, M.J. Arrowood, M.A. de Mejia, A.M. de Merida, A.W. Hightower, L. Venczel, B.L.
Herwaldt, and R.E. Klein
766-774
Serologic Evidence of Human Monocytic and Granulocytic Ehrlichiosis in Israel, A. Keysary, L.
Amram, G. Keren, Z. Sthoeger, I. Potasman, A. Jacob, C. Strenger, J.E. Dawson, and T. Waner
775-778
Supplementing Tuberculosis Surveillance with Automated Data from Health Maintenance
Organizations, D.S. Yokoe, G.S. Subramanyan, E. Nardell, S. Sharnprapai, E. McCray, and R.
Platt
779-787
Using Automated Pharmacy Records to Assess the Management of Tuberculosis, G.S.
Subramanyan, D.S. Yokoe, S. Sharnprapai, E. Nardell, E. McCray, and R. Platt
788-791
Hantavirus Reservoir Hosts Associated with Peridomestic Habitats in Argentina, G. Caldern, N.
Pini, J. Bolpe, S. Levis, J. Mills, E. Segura, N. Guthmann, G. Cantoni, J. Becker, A. Fonollat, C.
Ripoll, M. Bortman, R. Benedetti, M. Sabattini, and D. Enria
792-797

Dispatches

Large, Persistent Epidemic of Adenovirus Type 4-Associated Acute Respiratory Disease in U.S.
Army Trainees, K.M. McNeill, R.M. Hendrix, J.L. Lindner, F.R. Benton, S.C. Monteith, M.A.
Tuchscherer, G.C. Gray, and J.C. Gaydos
798-801

Changes in Antimicrobial Resistance among Salmonella enterica Serovar Typhimurium Isolates
from Humans and Cattle in the Northwestern United States, 19821997, M.A. Davis, D.D.
Hancock, T.E. Besser, D.H. Rice, J.M. Gay, C. Gay, L. Gearhart, and R. DiGiacomo
802-806

Toxic Shock Syndrome in the U.S.: Surveillance Update, 1979-1996, R.A. Hajjeh, A. Reingold,
A.Weil, K. Shutt, A. Schuchat, and B.A. Perkins
807-810

New Rickettsiae in Ticks Collected in Territories of the Former Soviet Union, E. Rydkina, V.
Roux, N. Fetisova, N. Rudakov, M. Gafarova, I. Tarasevich, and D. Raoult
811-814

Computer-Generated Dot Maps as an Epidemiologic Tool: Investigating an Outbreak of
Toxoplasmosis, S.B. Eng, D.H. Werker, A.S. King, S.A. Marion, A. Bell, J.L. Issac-Renton, G.S.
Irwin, and W.R. Bowie
815-819

HIV Infection as a Risk Factor for Shigellosis, J.T. Baer, D.J. Vugia, A.L. Reingold, T. Aragon,
F.J. Angulo, and W.Z. Bradford
820-823

Dengue Seroconversion among Israeli Travelers to Tropical Countries, I. Potasman, I. Srugo, E.
Schwartz
824-827

Effectiveness of Pneumococcal Polysaccharide Vaccine for Preschool-Age Children with Chronic
Disease, A.E. Fiore, O.S. Levine, J.A. Elliott, R.R. Facklam, J.C. Butler, for the Pneumococcal
Sentinel Surveillance Working Group
827-831

Commentary
Stimulating the Development of Orphan (and Other) Vaccines
B. Schwartz and N.R. Rabinovich
832-832

Letters
Swine as a Potential Reservoir of Shiga Toxin-Producing Escherchia coli O157:H7 in Japan, M.
Nakazawa, M. Akiba, and T. Sameshima
833-834
Hospitalizations for Rotavirus Gastroenteritis in Gipuzkoa (Basque Country), Spain, G. Cilla, E.
Prez-Trallero, L.D. Pieiro, a. Iturzaeta, and D. Vincente
834-835
Israeli Spotted Fever Rickettsia (Rickettsia conorii Complex) Associated with Human Disease in
Portugal, F. Bacellar, L. Beati, A. Frana, J. Poas, R. Regnery, and A. Filipe
835-836
Avoiding Misdiagnosis of Malaria: A Novel Automated Method Allows Specific Diagnosis,even
in the absence of Clinical Suspicion, T. Hnscheid, B.G. Pinto, I. Pereira, J.M. Cristino, and E.
Valadas
836-838
The First Reported Case of Aerococcus Bacteremia in a Patient with HIV Infection, J.H. Razeq,
G.M. Thomas, and D. Alexander
838-839
Proficiency in Detecting Vancomycin Resistance in Enterococci among Clinical Laboratories in
Santiago, Chile, J.A. Labarca, L.C. McDonald, M.E. Pinto, E. Palavecino, P. Gonzlez, E. Cona,
A. Fernndez, M.S. Giglio, and W.R. Jarvis
839-840
Food-Related Illness and Death in the United States, C. Hedberg
840-841
Food-Related Illness and Death in the United StatesReply to Dr. Hedberg, P.S. Mead, L.
Slutsker, P.M. Griffin, R.V. Tauxe
841-842
Specimen Collection for Electron Microscopy, J.A. Marshall and M.G. Catton
842-842

News and Notes
Upcoming Keystone Symposia, 843
Applied Epidemiology Course, 843
4th Decennial International Conference on Nosocomial and Health Care-Associated Infections,
844
Towards a Healthy Europe for the Year 2010, 844
Erratum, 844
ICEID 2000, 845

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

Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999]

   Perspectives

   Emerging Infectious Diseases and Amphibian Population
   Declines

   Peter Daszak,* Lee Berger, Andrew A. Cunningham,
   Alex D. Hyatt, D. Earl Green, and Rick Speare

   *Institute of Ecology, University of Georgia, Athens,
   Georgia, USA; Australian Animal Health Laboratory,
   Commonwealth Scientific Industrial Research
   Organization, Geelong, Victoria, Australia; School of
   Public Health and Tropical Medicine, James Cook
   University, Townsville, Queensland, Australia;
   Institute of Zoology, Zoological Society of London,
   London, United Kingdom; and National Wildlife Health
   Center, U.S. Geological Survey, Madison, Wisconsin, USA

    We review recent research on the pathology,
    ecology, and biogeography of two emerging
    infectious wildlife diseases,
    chytridiomycosis and ranaviral disease, in
    the context of host-parasite population
    biology. We examine the role of these
    diseases in the global decline of amphibian
    populations and propose hypotheses for the
    origins and impact of these panzootics.
    Finally, we discuss emerging infectious
    diseases as a global threat to wildlife
    populations.

   Emerging infectious diseases have been reported
   increasingly as causes of death in free-living wild
   animals (1). These diseases are a particular threat to
   wildlife species whose population, habitat, or range
   has been diminished or artificially manipulated to
   promote species survival (e.g., captive breeding,
   translocation, and release programs) (2-4). An early
   example of an emerging disease panzootic was the
   introduction of rinderpest in African domestic cattle
   in 1889 (5). More recently, epizootics and panzootics
   of wildlife have been increasingly reported in
   terrestrial (1) and marine (6) habitats and are
   probably underreported (1,4,7-9). Recent advances in
   theoretical and experimental host-parasite ecology have
   demonstrated a major role for infectious agents in the
   population biology of wild animals (10,11). We discuss
   recent data on two newly emerging infectious diseases
   of amphibians and, by reference to host-parasite
   ecology, propose hypotheses to explain their origin and
   impact.

   Amphibian Population Declines

   Global declines in amphibian population are perhaps one
   of the most pressing and enigmatic environmental
   problems of the late 20th century (12-19). While some
   declines are clearly due to habitat destruction, others
   are not associated with obvious environmental factors.
   Causal hypotheses include the introduction of predators
   or competitors, increased ultraviolet (UV-B)
   irradiation, acid precipitation, adverse weather
   patterns, environmental pollution, infectious disease,
   or a combination of these. Transdermal water uptake and
   gaseous exchange and a biphasic life cycle are
   important aspects of amphibian biology. These factors
   led to the hypothesis that amphibians act as sentinels
   for global environmental degradation (12,18). However,
   this role has yet to be demonstrated, and many causal
   factors may be present (12,19,20).

   Of particular concern are population declines in
   ecologically pristine areas, such as the montane
   tropical rain forests of Australia and Central America,
   where human impact from agriculture, deforestation, or
   pollution is thought to be negligible. Here, long-term
   data demonstrate recent and catastrophic amphibian
   population declines, often resulting in the complete
   loss of amphibian species (local extinction of multiple
   species) from large swaths of habitat (20-25). These
   declines include the disappearance and presumed
   extinction of the recently discovered golden toad (Bufo
   periglenes) of Costa Rica (23) and as many as seven
   Australian amphibian species, including two species of
   gastric-brooding frog (Rheobatrachus spp.) (20). These
   data, along with recent findings of amphibian mass
   deaths in these areas, suggest that such local
   extinctions are not normal population fluctuations or
   metapopulation dynamics.

   Investigations in Australia, the United Kingdom, and
   North and Central America (26-31) have repeatedly found
   two diseases as the causes of amphibian mass deaths
   globally (Table 1): chytridiomycosis in the rain
   forests of Australia and Central and South America and
   some parts of North America (28-32) and iridoviral
   infections in the United Kingdom, the United States,
   and Canada (26,27,33-36). Both diseases have been
   classified as emerging (1). The parasitic infection
   recently implicated as the cause of amphibian
   deformities in North America has not been associated
   with mass deaths or population declines (31).

Table 1. Mass deaths caused by chytridiomycosis and ranaviral disease in wild populations of amphibians
Chytridiomycosisan Emerging Panzootic Fungal Disease of Amphibians

Chytridiomycosis is a fungal disease first
described in 1998 from moribund and dead adult
amphibians collected at sites of mass deaths in 
Australia and Panama from 1993 to 1998 (28).
Here, long-term ecologic study sites reported   
catastrophic amphibian population declines in    
Big Tableland, Queensland (39,40), and Fortuna   
and Cerro Pando, Panama (24,25,28). No
significant pathogens were found on routine
parasitologic, bacteriologic, mycologic,
virologic examinations of tissue samples (28).
Fresh skin smears and histologic sections of
the epidermis, however, consistently contained
large numbers of developing and mature
sporangia of a new genus of chytrid fungus
(phylum Chytridiomycota) (Figures 1, 2).
Sporangia were also present within the keratinized mouthparts, but not the epidermis, of sympatric tadpoles (tadpoles lack epidermal keratin) (28,29). No significant morphologic differences between chytrids infecting 
Australian and Central American amphibians were
found by transmission electron microscopy, and 
the pathogen was identified as a member of the
order Chytridiales by analysis of zoospore
ultrastructure and 18s rDNA sequence data (28). Chytrids are
ubiquitous fungi that develop without hyphae and are found in aquatic habitats and moist soil, where they degrade cellulose, chitin, and keratin (41). Parasitic chytrids mainly infect plants, algae, protists, and invertebrates (41); the amphibian pathogen is the only example of a chytrid parasitizing vertebrates (28).

Figure 1. Figure 1a: Ventral abdominal skin of Bufo haematiticus from western Panama. The superficial keratinized
layer of epidermis (stratum corneum) contains numerous intracellular spherical-to-ovoid sporangia
(spore-containing bodies) of Batrachochytrium sp. The mature sporangia (sp, arrows) are 12-20 m (n =
25) in diameter and have refractile walls 0.5-2.0 m thick. Most sporangia are empty, having discharged
all zoospores, but a few sporangia contain two to nine zoospores. This stratum corneum is markedly
thickened adjacent to groups of parasitized cells and in some cases, the superficial layer has become
detached. No chytrids are present in the stratum spinosum, stratum basale, dermis, dermal glands, and
blood vessels. Note the absence of hyphae and lack of an inflammatory cell response in the deeper layers
of epidermis and the dermis. Hematoxylin and eosin stain. Bar = 35 m. 1b. Ventral skin of upper hind
limb of Atelopus varius from western Panama. Two sporangia containing numerous zoospores are visible
within cells of the stratum corneum. Each flask-shaped sporangium has a single characteristic discharge
tube (arrow) at the skin surface. Exiting zoospores are visible in the discharge tubes of both sporangia.
Hyperkeratosis is minimal in this acute infection. Tissues were fixed in neutral-buffered 10% formalin,
paraffin-embedded, sectioned at 6 m thick and stained with hematoxylin and eosin. Bar = 35 m.

Clinical signs of amphibian chytridiomycosis include abnormal posture, lethargy, and loss of righting reflex. Gross lesions, which are usually not apparent, consist of abnormal epidermal sloughing and (more rarely) epidermal ulceration; hemorrhages in the skin, muscle, or eye; hyperemia of digital and ventrum skin, and congestion of viscera (29). Diagnosis is by identification of characteristic intracellular flask-shaped sporangia and septate thalli within the epidermis (Figures 1, 2).

Its occurrence solely in keratinized tissue suggests that the chytrid uses amphibian keratin as a nutrient. A hyperkeratotic and hyperplastic response of the epidermis to infection (restricted to the stratum corneum and stratum granulosum) usually coincides with the immediate location of chytrid developmental stages. Inflammatory cell response is negligible. An isolate cultured from captive dendrobatid frogs has recently been used to fulfil Koch's postulates
as a fatal pathogen of frogs and has been described as a new genus and species, Batrachochytrium dendrobatidis (42). Three mechanisms by which chytridiomycosis causes death have been proposed (28): 1) epidermal hyperplasia impairs essential cutaneous respiration or osmoregulation; 2) a fungal toxin is absorbed systemically (although
a lack of clinical signs in infected larvae suggests otherwise); and 3) these factors are combined (28,37).

Figure 2.Scanning electron micrograph of digital skin of a wild frog (Litoria lesueuri, from Queensland,
Australia) that died of cutaneous chytridiomycosis. Many cells within this area of the superficial layer of
the epidermis contain mature sporangia, and unopened discharge tubes are visible protruding through
infected cells. The skin was fixed in 2.5% glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated,
critical-point-dried, sputter coated with gold, and examined with a JEOL JSM 840 scanning electron
microscope at 5 kV. All specimens are from animals that were naturally infected and died due to
chytridiomycosis in montane rain forest regions of Panama and Australia. Bar = 5 m. Photo courtesy of L.
Berger, reprinted with permission from A. Campbell (29).
Comparison of histologic, ultrastructural, and
18s rDNA sequence data indicates that the
chytrids found in wild Australian (28) and
captive Central American (T.Y. James, D.
Porter, J.E. Longcore, pers. comm.) amphibians  enlarged
belong to the genus Batrachochytrium, are  image
probably conspecific, and form a distinct  
monophyletic clade within the Chytridiales.

Emergence of Chytridiomycosis 
 
Retrospective histologic surveys of museum 
specimens of montane, riparian anurans from
protected sites in Central America and
Australia, conducted 1 to 10 years before the
population declines, showed no evidence
chytrid infection, which suggests that
chytridiomycosis has recently emerged on two continents (28). The relatively synchronous discovery of chytridiomyosis in Australia and Central America in association with amphibian population declines is striking. The data suggest that Batrachochytrium 1) may
be endemic to these regions and the amphibian deaths and declines attributed to it have only recently been discovered; 2) may be endemic and has recently become pathogenic (e.g., through an increase in the organism's prevalence or virulence, or a decrease in the host's defenses), or 3) may have been introduced recently into
these geographic regions and is now parasitizing novel host species.

The pattern of amphibian deaths and population declines associated with chytridiomycosis is characteristic of an introduced virulent pathogen dispersing through a nave population (7,10,39). In Australia, a distinct geographic and temporal progression in population declines has occurred (20), moving northward at a mean rate of 100 km per year (39). In Central America, a progression from northern Costa Rica to western Panama occurred from 1996 to 1998
(24,25). The uneven progression of declines in Australia (40) may reflect gaps in surveillance. Small-scale irregularities, however, characterize the epidemiology of many pandemics (43,44), within which individual epidemics progress at different rates in different areas. In amphibian populations, unevenness may be due to differences in ecologic factors (e.g., population density, habitat,
age structure); differences among pathogen strains; stochastic factors, such as the time of introduction; or a combination of these factors. In Australia and Central America, population declines have been catastrophic, occurring over a few months, with dramatic population loss and high rates of adult deaths (20,24,25,28,39,40,45). Such high depopulation rates are characteristic of introduced virulent pathogens (10). Conversely, in coevolved host-pathogen relationships, a degree of herd immunity to
the pathogen and lower virulence (infectivity and death rates) are normally observed. The low host specificity of amphibian chytridiomycosis (more than 30 species of wild amphibians from seven families in Central America and Australia [28,29]) also suggests that the disease was not enzootic in those montane rain-forest populations. Ability to infect a range of host species is a characteristic of many invading pathogens (10) and is less common in
endemic microparasites that have coevolved with their hosts.

The most parsimonious hypothesis for the origin of chytridiomycosis panzootics in Australia and Central America is the introduction of disease into populations of previously unexposed amphibians. Introduction of pathogens, termed pathogen pollution (1), is increasingly recognized as a significant threat to global biodiversity (1,6,46) and forms an integral part of human history (10,47). There are precedents for the introduction of fungal pathogens (including chytrid parasites) that cause high death rates
(41,48). Mechanisms by which wildlife pathogens can be introduced are common; for example, a consequence of the increasing mobility of humans is the global translocation of wildlife, plants, soil, and ballast water (1,4,49,50). Freshwater fish and amphibians are also transported globally. In Australia, chytridiomycosis-infected cane
toads (Bufo marinus), a recently introduced species, have been found (29), and in North America, bullfrogs (Rana catesbeiana) and other species of amphibians have been translocated or introduced widely. Some authors have suggested that tourists or fieldworkers surveying
amphibian populations may have facilitated the dissemination of Batrachochytrium (19), although this has not been demonstrated. Batrachochytrium may have coevolved with some amphibians (e.g., lowland) species, populations of which remain unaffected. Recent disturbances of rain forest habitats may have introduced this parasite into nave populations in Central America and Australia, leading to mass deaths. This range of disease outcomes parallels
many diseases of humans, e.g., measles and smallpox, which produce a range of effects on persons in disease-endemic regions and cause massive deaths when introduced into nave populations (48).

The occurrence of chytridiomycosis in free-living North American  amphibians (Table 1) suggests a less obvious pattern of dissemination than in Central America and Australia. This irregularity may be due to a paucity of data, the pathogen's being enzootic to the United States, or the pathogen's introduction a number of years before. Historical reports of declines in the United
States include postmetamorphic death syndrome, which progressed in waves through populations of amphibians, causing 90% to 100% death rates in recently metamorphosed animals and low death rates in larvae (50). Recent reports of chytridiomycosis-linked die-offs in Bufo boreas markedly resemble these previous die-offs. This observation and the finding that chytridiomycosis caused similar die-offs (of B. canorus and R. pipiens) in the 1970s (Table 1) (32),
support the last of the above hypotheses. In the United States an amphibian pathogen (histologically very similar to Batrachochytrium but identified as Basidiobolus ranarum) has been described in wild Wyoming toads (Bufo hemiophrys baxteri) (51) and captive dwarf African clawed frogs (Hymenochirus curtipes) (52). As the latter species was widely introduced in ornamental garden ponds throughout
the United States in the late 1980s, it may be involved in the dissemination of Batrachochytrium.

Chytridiomycosis as the Cause of Population Declines

The ability of a pathogen to cause local population declines
resulting in local host extinction requires a mechanism of
persistence at low host densities. In epidemiologic models, highly virulent parasites rapidly suppress the host population density below a threshold value required to maintain transmission, resulting in the pathogen's extinction and recovery of the host population
(7,10). Microparasites such as Batrachochytrium, with their
relatively short duration of infection and high death rates, have an increased threshold population density and are usually less able to persist. Many parasites have evolved life history strategies for persistence (10) and the presence of reservoir hosts may augment the impact of other introduced wildlife diseases on host populations (46). The aclinical presence of Batrachochytrium in the keratinized
mouthparts of amphibian larvae implicates this life-cycle stage as a reservoir host for the pathogen. This form of infection may enable Batrachochytrium to persist in reduced amphibian populations (Figure 3). In both Australia and Central America, chytrid infection was observed in larval mouthparts months after initial adult deaths: the
larvae of many tropical amphibian species survive 12 to 18 months and some temperate species as long as 3 years before metamorphosing. Examples of larval infection enhancing pathogen-mediated population declines and leading to host population extinctions have been reported for invertebrates (53).

Figure 3. Diagrammatic representation of the range of disease outcomes in populations of amphibians
affected by a Batrachochytrium-like pathogen. Factors that hypothetically predispose some amphibian
populations to declines are illustrated. In this model, host ecologic traits (left side of pyramid) and
parasite biologic traits (right side of pyramid) combine to produce declines in a specific group of
amphibian species that have low fecundity, are stream-breeding habitat specialists, and occur in montane
regions. These characteristics predispose them to population declines after introduction of a waterborne
pathogen with a low preferred developmental temperature and ability to persist at low host population
densities.1 
1Note that the relative number of mass death events decreases with increasing impact on population. 

Persistence may be further enhanced by
saprophytic development (Figure 3). 
Batrachochytrium can be cultured in vitro on
tryptone agar without the addition of keratin
or its derivatives (37,42), and it will grow for at least one generation on cleaned epidermal keratin or on amphibians that have died of the infection (42). Batrachochytrium 
may survive and reproduce as a saprophytic
organism in the environment, at least for
short periods. Keratin (from decaying
carcasses, shed skin, and other sources) is
widely distributed in the environment, and
chytrids that use this substrate are well 
known (42). Furthermore, the ability to
develop and reproduce saprophytically is
common to many other fungal (54) (including
chytrid [41]) and bacterial (55) pathogens. An epidemiologic model of a host-parasite system for pathogens that can reproduce saprophytically clearly shows a lowering of the host threshold population, allowing the pathogen to drive the host to extinction (55). Development of Batrachochytrium for even short periods outside its amphibian host may greatly increase its impact and accelerate population declines. Long-term presence as a saprophyte may explain
the lack of recolonization of streams from which amphibians have been extirpated in both Australia (29) and Central America (24,25).

The impact of chytridiomycosis may be enhanced by the ecologic characteristics of certain host species. In Australia, chytridiomycosis-linked deaths have occurred in both declining and nondeclining species (28,29). Species with declining populations belong to a similar ecologic guild: regionally endemic rain forest specialists with low fecundity that reproduce in streams and live at high altitudes (22). These characteristics, which are largely shared by declining Central American amphibians (24,25), are predictors of increased impact from chytridiomycosis. Species that reproduce in streams are probably more susceptible to a waterborne pathogen than terrestrial breeders. Low fecundity (56) and habitat specialization
indicate a reduced ability to recover from population declines caused by stochastic events, including disease introduction. Laurance, McDonald, and Speare (39) suggested that the relation between high-altitude populations and declines may be due to a pathogen with a lower preferred developmental temperature. Preliminary data on cultured Batrachochytrium are consistent with this hypothesis: it develops most rapidly at 23oC in culture, with slower growth at 28oC and reversible cessation of growth at 29oC
(42). The growth rate of Batrachochytrium in the skin (and therefore virulence) and the survival of zoospores outside the host (and therefore transmission rate) are likely to be lower in amphibians from the warmer lowland regions. The ability of the pathogen to survive saprophytically in the environment and for the disease to persist may also be enhanced in the cooler montane regions. These
laboratory data may explain why chytridiomycosis has been associated with population declines in North American amphibians in montane localities (31,32) and after periods of cool weather at many U.S. and Australian sites (29,31).

Potential Environmental Cofactors in the Emergence of
Chytridiomycosis 

Multiple factors (host, pathogen, environmental) may be involved in chytridiomycosis emergence. Some authors have hypothesized that infectious disease is only the proximate cause of declines and that environmental factors such as increased UV-B, chemical pollution, climate change, or stress may have predisposed amphibian populations
to opportunistic pathogens (13,32,57). Recent work at Monteverde, Costa Rica, suggests that atmospheric warming, with a resultant elevation of the average altitude of the base of the orographic cloud bank and an increase in dry periods, is causally linked to amphibian declines at this site (57). Although no pathologic studies of amphibians were undertaken, overcrowding during periods of drought may have allowed chytridiomycosis to cause substantial deaths (57). Further work is required to test this hypothesis, since
a drier climate would also predict a lower overall impact from chytridiomycosisa disease transmitted by flagellated, waterborne zoospores. The evidence suggests that cofactors are not required for chytridiomycosis to cause amphibian mass deaths. Chytridiomycosis is highly pathogenic to captive-bred amphibians exposed in captivity where control animals remained healthy (28,42). Further experimental
infections using extremely small inocula (100 zoospores) also proved fatal (29).

Figure 4. Transmission electron micrographs of iridovirus cultured from the liver of a naturally diseased
common frog (Rana temporaria) by using a fathead minnow epithelial cell line. 4a. Virus-infected cell.
Large isocahedral viruses are conspicuous within the cytoplasm (arrows). Bar = 2 m. 4b. Paracrystalline
array of iridovirus. Bar = 200 m.

Some deaths among wild amphibians have been attributed to immunosuppression, predisposing them to infectious disease (13,32). In the chytridiomycosis-related deaths,
chytridiomycosis was consistently found as the cause of death, and the range of opportunistic infections expected to occur in immunocompromised animals was not found (28,29). An increase in UV-B irradiation may influence amphibian declines (58), but in the subtropical regions of Australia and Central America, data demonstrate no significant    increase in UV irradiation (29,59). Even so, the potentional effect of increased irradiation on montane riparian rain forest amphibians is uncertain, since these animals lay eggs under rocks or in sand banks and adults are exposed to direct sunlight (24,29). Furthermore, in these regions, the species most likely to be affected by UV increases (arboreal 
amphibians, which bask or lay exposed eggs) are not in decline (20). Despite extensive research, chemical pollution (20,25), habitat destruction (22), or climate change (57) have not, so far, been causally linked either to the Australian declines or to those at Central American sites other than Monteverde. No other possible cofactors, such as sympatric pathogens, have been found.

Emerging Viral Diseases of Amphibians

Iridoviruses have been implicated as the cause of amphibian mass deaths worldwide, with novel iridoviruses of amphibians recently identified from a number of regions (Tables 1, 2). The Iridoviridae encompass five recognized genera: Iridovirus, Chloriridovirus, Ranavirus, Lymphocystivirus, and goldfish virus 1-like viruses (71). Of these, the genus Ranavirus contains pathogens of fish, amphibians, and reptiles (Table 2; Figure 4).

Table 2. Iridovirusesa,b of herpetofauna (34)
Characteristics of Ranaviral Disease

Ranaviruses are often highly virulent and cause systemic infections in amphibians. Experiments with Bohle iridovirus and Gutapo virus suggest that tadpoles are the most susceptible developmental stage for ranavirus infection, and death rates of 100% occur (72). Infected metamorphs die without overt signs of infection, and infected adults show either no overt signs or, occasionally, a general weakness. Histologically, acute necrosis of hematopoietic and lymphoid tissues and of leukocytes occurs in most organs of infected animals (72). Epizootiologic data on tadpole edema virus
infections of North American amphibians are scanty, although the virus was isolated mostly from diseased animals (60-62).
Experimental infections show that bullfrog (R. catesbeiana) tadpoles infected with tadpole edema virus develop an acute lethal disease after a short incubation. The virus invades the liver, kidneys, and digestive tract and causes hemorrhage in skeletal tissue, pathologic findings similar to those described in B. marinus infected with the
ranaviruses frog virus 3, Bohle iridovirus, or Gutapo virus (72). Tadpole edema virus was uniformly lethal to three species of experimentally infected North American toad (61,62). Experimental inoculation with tadpole edema virus caused high rates of deaths in toads (100%, 4 to 17 days postinfection in young and adult Bufo sp. and newly metamorphosed Scaphiopus sp.), 40% death rates in
metamorphosing bullfrogs (R. catesbeiana), and no deaths in newly hatched salamanders (Ambystoma sp.). For the last two species, the low virulence suggests either some prior exposure and acquired immunity or a degree of innate resistance.

Pathologic data from ranaviral infections in wild amphibians may be complicated by secondary bacterial infection. Cunningham et al. (26) described two syndromes in Rana temporaria collected at sites of mass deaths in the United Kingdom: a hemorrhagic syndrome affecting the skeletal musculature and the alimentary and reproductive tracts
and an ulcerative skin syndrome with dermal ulceration and often necrosis of the distal limbs, but without hemorrhage in the muscles or viscera. In some areas, frogs were found with lesions common to both syndromes (26). These lesions are characteristic of red-leg, a syndrome thought to be caused by bacterial infection; however, Cunningham et al. (26) concluded that the lesions they described were caused by primary iridoviral infection, with or without secondary bacterial infection. Secondary bacterial infection was
also reported in wild-collected, diseased A. t. stebbinsi infected with Ambystoma tigrinum virus (27). This infection resulted in rapid onset (5 to 7 days) of apparent epidermal hyperplasia, then dermal hemorrhage, followed by visceral hemorrhage and death rates of up to 45% (27).

Factors Associated with the Emergence of Ranaviruses

The epizootiology of ranaviral disease in amphibians is poorly understood. Data from closely related ranaviral infections of fish, however, suggest a number of factors which may explain their recent emergence. Epizootic hematopoietic necrosis virus is a ranaviral pathogen of fish and the causative agent of epizootic hematopoietic
necrosis, a notifiable disease under the Office Internationale des Epizooties (73,74). Sequencing data suggest that this disease is unique among the ranaviruses, is probably indigenous to Australia, and has a wide geographic range. Dissemination may be partly due to
the virus's ability to remain infectious under adverse conditions and for prolonged periods (Table 3).

 Table 3. Longevity of infectious epizootic
 hematopoietic necrosis virus (72,74,75)
 
The resistant nature of epizootic hematopoietic necrosis virus suggests that amphibian iridoviruses may survive long periods at the bottom of ponds, particularly during winter. Jancovich et al. (27) demonstrated that water containing Ambystoma tigrinum virus-infected Ambystoma became uninfective after 2 weeks at 25oC, and and epizootics of Ambystoma tigrinum virus and regina ranavirus often
followed periods of cold weather (27,36). Epizootic hematopoietic necrosis virus may be spread by fomites such as fishing nets, boats, and fishing rods and through artificial stocking of ponds for recreational fishing (76). Birds have the potential to mechanically transfer virus on their feathers, feet, or bills, or by regurgitation of ingested infected material (76). Similar modes of
spread are likely to occur with the amphibian ranaviruses. The occurrence of epizootic hematopoietic necrosis may be correlated with poor husbandry, including overcrowding, inadequate water flow, and fouling of local environments with feed from fish farms (77).

Recent movements of amphibians may have disseminated ranaviral diseases. The North American bullfrog (Rana catesbeiana), the host of tadpole edema virus, has been widely introduced in the western United States and South America. In the United Kingdom, ranaviruses may be disseminated by the common practice of translocation of
amphibian egg masses and larvae by humans. Zupanovic et al. (65,78) reported the existence of ranaviruses and antibodies to ranaviruses in the cane toad (Bufo marinus) in Venezuela. In Australia, where B. marinus was introduced to Queensland in 1935, ranaviral antibodies can be identified in this species throughout its range (78). The range of B. marinus has expanded rapidly in Australia, and the toad threatens environmentally sensitive wetlands in the Kakadu National
Park. The effects of toxicity, predation, and competition of this relentlessly expanding species are well documented, and its potential to disseminate novel viral pathogens to native amphibians should also be considered in evaluating its ecologic impact. So far, a ranavirus (Bohle iridovirus) has been isolated from only one native Australian amphibian, the ornate burrowing frog (Limnodynastes ornatus) (79).

Experimental infection with Bohle iridovirus causes illness and death in a range of frogs and toads, fish, and reptiles (L. Owens, pers. comm.) (80,81). Gutapo virus, isolated from Venezuelan B. marinus (65), is also able to infect other amphibian species (72). Despite Bohle iridovirus' low host specificity, current levels of surveillance have not implicated it in diseases of free-ranging Australian animals other than those from which it was originally isolated. The ability of Bohle iridovirus to infect fish implicates the widespread introduction of fish for recreational purposes as a potentially significant factor in the dissemination of amphibian ranaviruses. Similarly, Redwood Creek virus, a ranavirus found to cause death of the endangered red-legged frog (67), can infect both amphibians and fish.

Impact of Ranaviral Disease on Amphibian Populations

The link between amphibian population declines and ranaviral disease is less clear than that with chytridiomycosis. However, these highly virulent ranaviruses are a potential threat to amphibian populations, especially populations isolated from previous disease outbreaks (and thus lacking specific immunity) and species with low fecundity. In particular, the high death rates of tadpoles infected
by ranaviruses predict a negative impact on populations. Using the model of Lampo and De Leo (82), a tadpole death rate of 80% to 90% would remove 80% to 90% of the animals that would otherwise survive metamorphosis, resulting in an approximately 80% reduction in adult populations (Lampo, pers. comm.). Although such an epizootic might not endanger the long-term survival of a highly adaptable, highly
fecund species such as B. marinus, species that inhabit specialized ecologic niches or have low fecundity might be adversely affected.

Recent outbreaks of iridoviral disease have been particularly notable in Rana temporaria (United Kingdom) and Ambystoma tigrinum (Arizona, Saskatchewan). These outbreaks involved of extensive deaths and the annual recurrence of the diseases. Common ecologic themes occur for these amphibian populations. The natural habitat of A. t. stebbinsi has been entirely destroyed by drainage after human colonization of the southwestern United States, and relict populations are now confined to manmade watering holes for cattle. Other outbreaks of ranaviral disease in A. tigrinum have been associated with altered habitats and artificial ponds. Similar habitat destruction has occurred in the United Kingdom, where most outbreaks of iridovirus infection are reported from artificial ponds (16). High population densities of frogs (16) and salamanders (27)
occur in these bodies of water, facilitating the transmission of viruses and predisposing the amphibian populations to mass deaths.

Implications of Emerging Infectious Diseases for Amphibian
Population Declines and Wildlife Conservation

The geographic spread of chytridiomycosis threatens populations of endemic and endangered amphibians in rain forests of Australia and Central and South America. Chytridiomycosis has now been reported from 38 amphibian species in 12 families, including ranid and hylid
frogs, bufonid toads, and plethodontid salamanders
(28,32,37,38,42,83). Although many of these records are from captive animals, the data demonstrate that chytridiomycosis is fatal to species originating from Europe, Africa, Madagascar, the Americas, and Oceania and attest to its potential impact should the disease be introduced into these areas. The emergence of amphibian ranaviruses raises similar concerns. Their ability to infect a wide range of amphibian and fish hosts (different vertebrate classes), global
distribution, and high virulence clearly establish them as a global threat to amphibian populations. Despite the recent geographic spread and impact of chytrids and ranaviruses, these diseases probably do not account for all the reports of declining amphibian populations, and evidence exists for other causal factors, such as habitat loss (12). Although chytridiomycosis causes high death rates in a range of species and habitats, it has caused population declines of amphibians only in certain species confined to montane
rain forests, while the emergence of ranaviral disease seems to be associated with disturbed or degraded habitats.

Hypotheses regarding the origins and impact of emerging infectious diseases on amphibians are being tested. A number of Batrachochytrium and iridovirus isolates have been cultured, and analysis of their phylogenetic and pathologic features in natural and experimental infections, as well as their biologic characteristics (e.g., ability to survive in the environment, evolution of virulent strains) has already begun. Basic host and parasite ecologic data, such as prevalence and duration of infection and presence of reservoir hosts, are being collected. Data from these studies may enable the formulation of management plans to
limit the diseases' impact. Development of chemotherapeutic regimens and diagnostic tests (enzyme-linked immunosorbent assay, in situ hybridization) may enable rapid progress toward these goals (29). National and international structures for the rapid dissemination of information between scientists, politicians, and the public may be
crucial in combating the threat of these globally emergent
pathogens. However, large geographic areas (e.g., Africa and much of Asia) have not yet been surveyed for declining amphibian populations or for the occurrence of these pathogens. Raising awareness of this threat should be one of the highest priorities for the immediate future.

Many introduced pathogens have affected human populations (10,47). However, far fewer studies of introduced diseases among wildlife have been published, and usually only those producing obvious population losses are studied vigorously (1-6). The loss of amphibian populations to the point of local extinction is a striking effect of chytridiomycosis in Australia and Central America. However, the effects of introduced wildlife diseases may be more far-reaching and subtle, with knock-on (ripple) effects permeating throughout the ecosystem (1,2,7,82,84). In many tropical and some temperate areas, amphibians make up a significant proportionsometimes mostof these vertebrate biomass (85), and their loss is likely to have unpredictable effects on populations of other species. For example, loss of herbivorous amphibian larvae may cause an overgrowth of algae in montane tropical streams, with further knock-on effects. In Australia and Central America, certain species
of snakes prey exclusively on amphibians and are likely to suffer significant population declines, in the same way that the red fox population was drastically reduced in the United Kingdom after the introduction of myxomatosis (10). Such insidious effects following disease introduction and an underestimation of their historical incidence suggest that this pathogen pollution may be as serious a conservation threat as habitat destruction and chemical pollution
(1). 

Acknowledgments

The authors thank Trent K. Bollinger, Cynthia Carey, V. Greg
Chinchar, Doug Docherty, Timothy Y. James, M. Lampo, Karen Lips, Joyce E. Longcore, Don K. Nichols, David Porter, L. Owens, and Allan P. Pessier for access to unpublished data and papers in press. and Mark A. Farmer, and David Porter, University of Georgia and members of the Infectious Diseases Pathology Activity, Centers for Disease Control and Prevention, for their hospitality. 

Dr. Daszak is a parasitologist at the Institute of Ecology,
University of Georgia, Georgia, USA, investigating amphibian
chytridiomycosis and other emerging pathogens of wildlife. He is interested in the pathology, cell biology, and host-parasite ecology of emerging diseases of wildlife.

Address for correspondence: Peter Daszak, Institute of Ecology, University of Georgia, Ecology Building, Athens, GA 30602, USA; fax: 706-542-4819; e-mail:  daszak@arches.uga.edu.

References

1. Daszak P, Cunningham AA, Hyatt AD. Emerging infectious diseases of wildlifethreats to human health and biodiversity. Science 1999. (In press).

2. Lyles AM, Dobson AP. Infectious disease and intensive management: population dynamics, threatened hosts, and their parasites. J Zoo Wildl Med 1993;24:315-26.

3. Viggers KL, Lindenmayer DB, Spratt DM. The importance of disease in reintroduction programmes. WildlifeResearch 1993;20:687-98.

4. Cunningham AA. Disease risks of wildlife translocations.
Conservation Biology 1996;10:349-53.

5. Plowright W. The effects of rinderpest and rinderpest control on wildlife in Africa. Symposia of the Zoological Society of London 1982;50:1-28.

6. Harvell CD, Kim K, Bukholder JM, Colwell RR, Epstein PR, Grimes DJ, et al. Emerging marine diseasesclimate links and anthropogenic factors. Science 1999;285:1505-10.

7. Dobson AP, May RM. Disease and conservation. In: Soul M, editor. Conservation biology: The science of scarcity and diversity. Sunderland (MA): Sinauer Assoc Inc.; 1986. p. 345-65.

8. Scott ME. The impact of infection and disease on animal
populations: Implications for conservation biology. Consservation Biology 1988;2:40-56.

9. Daszak P, Cunningham AA. Wildlife disease biology comes of age. Oryx 1998;32:238-9.

10. Anderson RM, May RM. The invasion, persistence and spread of infectious diseases within animal and plant communities. Philos Trans R Soc Lond B Biol Sci 1986;314:533-70.

11. Tompkins DM, Begon M. Parasites can regulate wildlife
populations. Parasitology Today 1999;15:311-3.

12. Blaustein AR, Wake DB. Declining amphibian populations: A global phenomenon. Trends in Ecology and Evolution 1990;5:203-4.

13. Carey C. Hypothesis concerning the causes of the disappearance of boreal toads from the mountains of Colorado. Conservation Biology 1993;7:355-62.

14. Drost CA, Fellers GM. Collapse of a regional frog fauna in the Yosemite area of the California Sierra Nevada, USA. Conservation Biology1996;10:414-25.

15. Cunningham AA, Langton TES, Bennett PM, Drury SEN, Gough RE, Kirkwood JK. Unusual mortality associated with poxvirus-like particles in frogs (Rana temporaria). Vet Rec 1993;133:141-2.

16. Cunningham AA, Langton TES, Bennett PM, Lewin JF, Drury SEN, Gough RE, et al. Investigations into unusual mortalities of the common frog (Rana temporaria) in Britain. In: Proceedings of the 5th International Colloquium on the Pathology of Reptiles and Amphibians; 1995 March 3; The Netherlands. p. 19-27.

17. Gupta BK. Declining amphibians. Current Science 1998;75:81-4.

18. Weygoldt P. Changes in the composition of mountain stream frog communities in the Atlantic Mountains of Brazil: Frogs as indicators of environmental deteriorations? Studies of Neotropical Fauna and Environments 1989;24:249-55.

19. Halliday TR. A declining amphibian conundrum. Nature
1998;394:418-9.

20. Mahony M. The decline of the green and golden bell frog Litoria aurea viewed in the context of declines and disappearances of other Australian frogs. Australian Zoologist 1996;30:237-47.

21. Richards SJ, McDonald KR, Alford RA. Declines in populations of Australia's endemic tropical forest frogs. Pacific Conservation Biology 1993;1:66-77.

22. Williams SE, Hero J-M. Rainforest frogs of the Australian Wet Tropics: Guild classification and the ecological similarity of declining species. Proc R Soc Lond B Biol Sci 1998;265:597-602.

23. Pounds JA, Fogden MPL, Savage JM, Gorman GC. Tests of null models for amphibian declines on a tropical mountain. Conservation Biology 1997;11:1307-22.

24. Lips KR. Decline of a tropical montane fauna. Conservation Biology 1998;12:106-17.

25. Lips KR. Mass mortality and population declines of anurans at an upland site in Western Panama. Conservation Biology 1999;13:117-25.

26. Cunningham AA, Langton TES, Bennett PM, Lewin JF, Drury SEN, Gough RE, et al. Pathological and microbiological findings from incidents of unusual mortality of the common frog (Rana temporaria). Philos Trans R Soc Lond B Biol Sci 1996;351:1529-57.

27. Jancovich JK, Davidson EW, Morado JF, Jacobs BL, Collins JP. Isolation of a lethal virus from the endangered tiger salamander Ambystoma tigrinum stebbinsi. Diseases of Aquatic Organisms 1997;31:161-7.

28. Berger L, Speare R, Daszak P, Green DE, Cunningham AA, Goggin CL, et al. Chytridiomycosis causes amphibian mortality associated with population declines in the rainforests of Australia and Central America. Proc Natl Acad Sci U S A 1998;95:9031-6.

29. Berger L, Speare R, Hyatt AD. Chytrid fungi and amphibian declines: Overview, implications and future directions. In: Campbell A, editor. Declines and disapparances of Australian frogs. Environmental Australia, Canberra, Australia: Environmental Australia; 2000. p.21-31.

30. Daszak P, Cunningham AA. Extinction by infection. Trends in Ecology and Evolution 1999;14:279.

31. Morell V. Are pathogens felling frogs? Science 1999;284:728-31.

32. Carey C, Cohen N, Rollins-Smith L. Amphibian declines: An immunological perspective. Dev Comp Immunol 1999;23:459-72.

33. Drury SEN, Gough RE, Cunningham AA. Isolation of an
iridovirus-like agent from common frogs (Rana temporaria). Vet Rec 1995;137:72-3.

34. Hyatt AD, Gould AR, Zupanovic Z, Cunningham AA, Hengstberger S, Whittington RJ, et al. Characterisation of piscine and amphibian iridoviruses. Arch Virol 2000. In press.

35. Docherty D, Chinchar VG, Meteyer C, Brannian R, Hansen W, Wang J, Mao J. A diagnostic evaluation of three salamander mortality events associated with an iridovirus and subsequent genomic comparison of the virus isolates obtained. In: Proceedings of the 48th Annual Wildlife Disease Association Conference; 1999 Aug 8-12;
Athens, Georgia.

36. Bollinger TK, Mao J, Schock D, Brigham RM, Chinchar VG.
Pathology, isolation and preliminary molecular characterization of a novel iridovirus from tiger salamanders in Saskatchewan. Journal of Wildlife Diseases 1999;35:413-29.

37. Pessier AP, Nichols DK, Longcore JE, Fuller MS. Cutaneous chytridiomycosis in poison dart frogs (Dendrobates spp.) and White's tree frogs (Litoria caerulea). J Vet Diagn Invest 1999;11:194-9.

38. Milius S. Fatal skin fungus found in U.S. frogs. Science News 1998;July 4th:7.

39. Laurance WF, McDonald KR, Speare R. Epidemic disease and the catastrophic decline of Australian rain forest frogs. Conservation Biology 1996;10:406-13.

40. Alford RA, Richards SJ. Lack of evidence for epidemic disease as an agent in the catastrophic decline of Australian rain forest frogs. Conservation Biology 1997;11:1026-9.

41. Powell MJ. Looking at mycology with a Janus face: A glimpse at chytridiomycetes active in the environment. Mycologia 1993;85:1-20.

42. Longcore JE, Pessier AP, Nichols DK. Batrachochytrium
dendrobatidis gen. et sp. nov., a chytrid pathogenic to amphibians. Mycologia 1999;91:219-27.

43. Garnett GP, Homes EC. The ecology of emergent infectious
disease. Bioscience 1996;46:127-35.

44. Quinn TC. Global burden of the HIV pandemic. Lancet
1996;348:99-106.

45. Laurance WF, McDonald KR, Speare R. In defense of the epidemic disease hypothesis. Conservation Biology 1997;11:1030-4.

46. McCallum H, Dobson A. Detecting disease and parasite threats to endangered species and ecosystems. Trends in Ecology and Evolution 1995;10:190-4.

47. Dobson AP, Carper ER. Infectious diseases and human population history. Bioscience 1996;46:115-26.

48. Burdon JJ. Fungal pathogens as selective forces in plant
populations and communities. Australian Journal of Ecology
1991;16:423-32.

49. Vitousek PM, D'Antonio CM, Loope LL, Westbrooks R. Biological invasions as global environmental change. American Scientist 1996;84:468-78.

50. Scott NJ. Postmetamorphic death syndrome. Froglog 1993;7:1-2. 

51. Taylor SK, Williams ES, Thorne ET, Mills KW, Withers DI, Pier AC. Causes of mortality of the Wyoming toad. Journal of Wildlife Diseases 1999;35:49-57.

52. Groff JM, Mughannam A, McDowell TS, Wong A, Dykstra MJ, Frye FL, et al. An epizootic of cutaneous zygomycosis in cultured dwarf African clawed frogs (Hymenochirus curtipes) due to Basidiobolus ranarum. J Med Vet Mycol 1991;29:215-23.

53. Richard A, Cory J, Speight M, Williams T. Foraging in a pathogen reservoir can lead to local host population extinction: A case study of lepidoptera-virus interaction. Oecologia 1999;118:29-38.

54. Cox RA. Coccidioidomycosis. In: Fungal infections and immune responses. Murphy JW, Friedman H, Bendinelli M, editors. New York: Plenum Press; 1993. p. 173-211.

55. Godfray HCJ, Briggs CJ, Barlow ND, O'Callaghan M, Glare TR, Jackson TA. A model of insect-pathogen dynamics in which a pathogenic bacterium can also reproduce saprophytically. Proc R Soc Lond B Biol Sci 1999;266:233-40.

56. Bennett PM, Owens IPF. Variation in extinction risk among birds: Chance or evolutionary predisposition? Proc R Soc Lond B Biol Sci 1997;264:401-8.

57. Pounds JA, Fogden MPL, Campbell JH. Biological response to climate change on a tropical mountain. Nature 1999;398:611-5.

58. Kiesecker JM, Blaustein AR. Synergism between UV-B radiation and a pathogen magnifies amphibian embryo mortality in nature. Proc Natl Acad Sci U S A 1995;92:11049-52.

59. Madronich S, de Gruijl FR. Skin cancer and UV radiation. Nature 1993;366:23.

60. Granoff A, Came PE, Rafferty KA. The isolation and properties of viruses from Rana pipiens: Their possible relationship to the renal adenocarcinoma of the leopard frog. Ann N Y Acad Sci 1965;126:237-55.

61. Clark HF, Brennan JC, Zeigel RF, Karzon. Isolation and
characterization of viruses from the kidneys of Rana pipiens with renal adenocarcinoma before and after passage in red eft (Triturus viridescens). J Virol 1968;2:629-40.

62. Wolf K, Bullock GL, Dunbar CE, Quimby MC. Tadpole edema virus: A viscerotrophic pathogen for anuran amphibians. J Infect Dis 1968;118:253-62.

63. Fijan N, Matasin Z, Petrinec Z, Valpotic I, Zwillenberg LO. Isolation of an iridovirus-like agent from the green frog Rana esculenta L. Veterinarski Archiv Zagreb 1991;61:151-8.

64. Hengstberger SG, Hyatt AD, Speare R, Coupar BEH. Comparison of epizootic haematopoietic necrosis and Bohle iridoviruses, recently isolated Australian iridoviruses. Diseases of Aquatic Organisms 1993;15:93-107.

65. Zupanovic Z, Musso C, Lopez G, Louriero C-L, Hyatt AD,
Hengstberger S, et al. Isolation and characterization of
iridoviruses from the giant toad Bufo marinus in Venezuela. Diseases of Aquatic Organisms 1998;33:1-9.

66. Mao J, Green DE, Fellers G, Chinchar VG. Molecular
characterization of iridoviruses isolated from sympatric amphibians and fish. Virus Res 1999;63:45-52.

67. Mao J, Hedrick RP, Chinchar VG. Molecular characterization, sequence analysis and taxonomic position of newly isolated fish iridoviruses. Virology 1997;229:212-20.

68. Zhang QY, Li ZQ, Gui JF. Studies on morphogenesis and cellular interactions of Rana grylio virus in an infected fish cell line. Aquaculture 1999;175:185-97.

69. Westhouse RA, Jacobsen ER, Harris RK, Winter KR, Homer BL. Respiratory and pharyngo-esophageal iridovirus infection in a gopher tortoise (Gopherus polyphemus). J Wildl Dis 1996;32:682-6.

70. Chen Z-X, Zheng J-C, Jiang Y-I. A new iridovirus from
soft-shelled turtle. Virus Res 1999;63:147-51.

71. Murphy FA, Fauquet CM, Bishop DHL, Ghabrial SA, Jarvis AW, Martelli P, et al. Iridoviridae. In: Murphy FA, Fauquet CM, Bishop DHL, Ghabrial SA, Jarvis AW, Martelli P, et al., editors. Virus taxonomy: The classification and nomenclature of viruses. The sixth report of the international committee on the taxonomy of viruses. New York, Wien: Springer-Verlag; 1995. p. 95-9.

72. Hyatt AD, Parkes H, Zupanovic Z. Identification,
characterisation and assessment of Venezuelan viruses for potential use as biological control agents against the cane toad (Bufo marinus) in Australia. Report to the Australian Federal Government and Environment Australia. 1998.

73. Ahne W, Bremont M, Hedrick RP, Hyatt AD, Whittington RJ.
Iridoviruses associated with epizootic haematopoietic necrosis (EHN) in aquaculture. World Journal of Microbiol ogy and Biotechnology 1997;13:367-73.

74. Hyatt AD, Whittington RJ. National diagnostic tests for the detection of epizootic haematopoietic necrosis virus (EHNV) and certification of EHNV-free fish. Final report (project 92/66). Fish Research Development Council of Australia; 1997.

75. Langdon JS. Experimental transmission and pathogenicity of epizootic haematopoietic necrosis virus (EHNV) in redfin perch, Perca fluviatilis L., and 11 other teleosts. Journal of Fish Diseases 1989;12:295-310.

76. Whittington RJ, Kearns C, Hyatt AD, Hengstberger S, Rutzou T. Spread of epizootic haematopoietic necrosis virus (EHNV) in redfin perch (Perca fluviatilis) in Southern Australia. Aust Vet J 1996;73:112-4.

77. Whittington RJ, Hyatt AD. Diagnosis and prevention of epizootic haematopoietic necrosis virus infection. Proceedings of the National Research Institute of Aquaculture International Workshop: New approaches to viral diseases of aquatic animals; 1997 Jan 21-24; Kyoto, Japan. National Research Institute of Aquaculture; 1997.

78. Zupanovic Z, Lopez B, Hyatt AD, Green B, Bartran G, Parkes H, et al. Giant toads Bufo marinus in Australia and Venezuela have antibodies against "ranaviruses". Dieases of Aquatic Organisms 1998;32:1-8.

79. Speare R, Smith JR. An iridovirus-like agent isolated from the ornate burrowing frog Limnodynastes ornatus in northern Australia. Diseases of Aquatic Organisms 1992;14:51-7.

80. Cullen BR, Owens L, Whittington RJ. Experimental infection of Australian anurans (Limnodynastes terraereginae and Litoria latopalmata) with Bohle iridovirus. Diseases of Aquatic Organisms 1995;23:83-92.

81. Moody NJC, Owens L. Experimental demonstration of the
pathogenicity of a frog virus, Bohle iridovirus, for a fish species, barramundi Lates calcarifer. Diseases of Aquatic Organisms 1994;18:95-102.

82. Lampo M, De Leo GA. The invasion ecology of the toad Bufo marinus: From South America to Australia. Ecological Applications 1998;8:388-96.

83. Nichols DK, Pessier AP, Longcore JE. Cutaneous chytridiomycosis: an emerging disease? Proceedings American Association of Zoo Veterinarians 1998:269-71.

84. Lessios HA. Mass mortality of Diadema antillarum in the
Caribbean: What have we learned? Annual Reviews in Ecological Systematics 1988;19:371-93.

85. Burton, TM, Likens GE. Salamander populations and biomass in the Hubbard Brook Experimental Forest, New Hampshire. Copeia 1975;3:541-6.

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

URL: http://www.cdc.gov/ncidod/eid/vol5no6/ascii/daszak.txt

Please note that figures, equations, and most tables are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively.  Greek symbols are spelled out. >/= is used for greater than or equal to.

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

Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999] 

  Perspectives

   Development of Orphan Vaccines:  An Industry
   Perspective

   Jean Lang and Susan C. Wood
   Pasteur Mrieux Connaught, Lyon, France

    The development of vaccines against rare
    emerging infectious diseases is hampered by
    many disincentives. In the face of growing
    in-house expenditures associated with
    research and development projects in a
    complex legal and regulatory environment,
    most pharmaceutical companies prioritize
    their projects and streamline their product
    portfolio. Nevertheless, for humanitarian
    reasons, there is a need to develop niche
    vaccines for rare diseases not preventable
    or curable by other means. The U.S. Orphan
    Drug Act of 1983 and a similar proposal
    from the European Commission (currently
    under legislative approval) provide
    financial and practical incentives for the
    research and development of drugs to treat
    rare diseases. In addition, updated
    epidemiologic information from experts in
    the field of emerging diseases; increased
    disease awareness among health
    professionals, patients, and the general
    public; a list of priority vaccines;
    emergence of a dedicated organization with
    strong leadership; and the long-term
    pharmacoeconomic viability of orphan
    products will be key factors in overcoming
    the complexity of orphan status and the
    limited need for vaccine.

   The Problem

   In considering the development of a new vaccine,
   preventive immunization, generally considered the most
   cost-effective health intervention, should be ranked
   against other strategies for disease control, such as
   case management (treatment of disease) or control of
   environmental factors linked to vector prevalence and
   dynamics (e.g., overpopulation, rural-to-urban
   migration, economic status, vector control, inadequate
   domestic water supply or sewage disposal) (1).
   Evaluating vaccination options and economic impact is
   particularly important for vaccines against
   low-prevalence or geographically contained emerging
   infectious diseases with limited demand, for which
   development costs may not be recovered. Thus, consensus
   should be reached on the mid- to long-term public
   health significance (e.g., vector dynamics and
   potential control, age prevalence and targets, risk
   categories, case-fatality rates, and possible future
   epidemiologic scenarios) of any vaccine-preventable
   disease. Without clear premises and long-term
   commitment, the development of vaccines for rare
   infectious diseases or those of narrow scope (e.g.,
   geographically limited but regionally important
   diseases such as arboviral or diarrheal diseases) or
   for which development costs offset the market
   potential, called here orphan vaccines, may be
   considered a precarious venture that most organizations
   would hesitate to pursue.

   Disincentives for Orphan Vaccine Development

   Competing Costs

   Vaccine development involves a substantial investment
   in time, effort, and resources. Any private- or
   public-sector vaccine research and development process
   involves choices concerning the allocation of resources
   at all levels, including personnel and management. The
   costs from research to licensure, the risks inherent in
   vaccine development (e.g., technological constraints,
   regulatory approval) and the short- and long-term
   market financial evaluations (e.g., net present value,
   return on investment [2]) are key factors in the
   decision to develop a vaccine against a rare disease.
   In addition, long-term market evaluation and return on
   investment are often difficult to estimate because of
   the unpredictable nature of disease outbreaks and
   vector dynamics. Growing in-house expenditures
   associated with research and development projects in a
   complex legal and regulatory environment prompt most
   pharmaceutical companies to prioritize their projects
   and streamline their product portfolio (3). The same is
   true in the public health sector where the appearance
   of an orphan vaccine would increase the already tough
   competition for resources, as evidenced by the present
   shortcomings in developing countries' use of current
   and candidate Expanded Program of Immunization (EPI)
   vaccines (hepatitis B, measles, yellow fever,
   Haemophilus influenzae type b).

   Vaccine Pricing

   It has been repeatedly shown that one of the most
   accurate predictors of the successful use of an EPI
   vaccine, such as hepatitis B, is not necessarily the
   endemicity of the disease but instead the vaccine cost
   per dose (4,5). Thus, the research, development,
   production, marketing, and distribution of a safe and
   effective vaccine should be assessed to determine if
   its potential cost per dose would be acceptable in an
   already difficult marketplace (4). The limited economic
   prospects and size of the market, with probably no
   prospect for economies of scale in production, are
   particularly relevant in the vaccine industry. Economic
   models of vaccine production have shown an inverse
   relationship between the number of doses produced and
   the cost per dose (6). As a consequence, a tiered
   pricing strategy has been endorsed by the World Health
   Organization (WHO), in which high-cost but low-volume
   vaccine sales in industrialized countries could
   subsidize the low cost and larger volume of sales in
   developing countries, although this may not be feasible
   if the quantity of vaccines needed in developing
   countries is low (6,7).

   Patent Protection and Product Liability

   Introduction of new vaccines relies heavily on the
   strategic use of intellectual property rights to
   reassure investors that a candidate vaccine will
   provide a fair return on invested funds. The lack of
   patent protection or legal framework for intellectual
   property rights in some developing countries interferes
   with the long-term viability of a vaccine. In Western
   countries, liability issues associated with a candidate
   vaccine and its intended population (8) also affect
   development costs.

   Orphan Drugs and Vaccines Situation in the United
   States

   The United States was the first nation to propose a
   legal framework to overcome the disincentives to
   developing orphan drugs and encourage their development
   and availability (9,10). The Preamble on Orphan Drugs
   to the legislation passed by the U.S. Congress
   contained the following points: 1) Many diseases and
   conditions (so-called orphan diseases) exist that
   affect very small numbers of persons; however, the
   overall group of patients affected by such diseases
   totals 20 million or more in the United States; 2)
   adequate drugs for orphan diseases have not been
   developed; 3) pharmaceutical companies may reasonably
   expect to generate relatively small sales in comparison
   to the cost of developing an orphan product; and 4)
   costs of developing such drugs should be reduced, and
   financial incentives should be provided.

   The legislation defines two classes of orphan diseases.
   The first class comprises diseases that affect fewer
   than 200,000 Americans. In this case, sales of a drug,
   vaccine, diagnostic test, or blood product intended for
   use in such a disorder would be insufficient to offset
   the costs incurred during development and marketing of
   the product. This program is directed at public health
   needs beyond the U.S. borders, providing a stimulating
   factor for the U.S. pharmaceutical community to develop
   products to meet the needs of populations elsewhere.
   The second class of orphan diseases affects more than
   200,000 Americans but has no potential recovery costs
   from U.S. sales. Thus, the program may also apply to
   specific subpopulations of patients with a more common
   disease for which the sponsor does not expect to offset
   development and marketing costs in the first 7 years of
   sales.

   Concerning vaccines, the U.S. Food and Drug
   Administration (FDA) stipulated that, when establishing
   the claim for orphan status, the intended population
   should reflect the number of persons who would receive
   the vaccine annually as of the date of designation.

   Orphan Drug Incentives

   To further encourage orphan drug availability,
   accompanying market-oriented incentives for orphan drug
   development were issued by the Office of Orphan
   Products Development, under the auspices of FDA (11).
   The sponsor makes the request for orphan drug status
   (before filing a New Drug Application or a Product
   License Application) on the basis of information and
   circumstances at the time the request is submitted.

   Funds for research through Orphan Products Grants
   Programs benefit from a 50% deduction tax credit for
   clinical trial expenses (9) and a market exclusivity of
   7 years. Protocol assistance in the form of written
   recommendations from the secretary of the Department of
   Health and Human Services for the nonclinical and
   clinical investigations needed for marketing approval
   are provided to accelerate the approval process. In
   this respect, a flexible approach has been adopted for
   the development of orphan drugs. For example, the
   preclinical dossier (i.e., the pharmaceutical and
   pharmacotoxologic data included in the registration
   file) may not have to include data on animal toxicity,
   and teratogenicity or carcinogenicity results may be
   waived in some cases (12). This flexibility in the
   registration requirements can be applied in certain
   cases to expedite the approval process but cannot be
   used in instances where it could compromise the safety
   of the consumer.

   The legislation states that the clinical dossier of an
   orphan drug or vaccine should be built on a realistic
   assessment of the qualitative and quantitative nature
   of the studies that can be performed. This measure is
   relevant because the orphan nature of the disease and
   its prevalence in regions with limited medical
   facilities and services may make it difficult to
   recruit a large enough number of qualified participants
   for a clinical trial. On the other hand, the drawback
   of basing a clinical dossier on a limited amount of
   data is the obvious difficulty in evaluating the safety
   profile of an orphan product with sufficient
   statistical confidence. On average, orphan drugs may be
   associated with greater hazard than other products. For
   example, during clinical testing, 31% of orphan drugs
   on the market had more pronounced adverse effects than
   nonorphan medicinal products (13). Likewise, after FDA
   approval, 13% of orphan products provoked more side
   effects than anticipated.

   To encourage development of novel orphan compounds, FDA
   stipulated that two products would be considered the
   same (and thus the latter one would not qualify for the
   incentives in the Orphan Drug Act), unless the second
   product was shown to be clinically superior to the
   first. This stipulation provides a clear incentive for
   the original manufacturer of a product likely to be
   reproduced, who funds the full costs of research and
   development. For example, in the case of two live,
   attenuated viral vaccines, only the first would be
   granted orphan status for a given preventive
   indication, unless the second vaccine proved clinically
   superior.

   Liability Coverage

   Although not definitely clarified, proposals have been
   made to solve some specific liability issues, including
   design defects, duty to warn, negligence in testing or
   manufacturing, and defining responsibility for no-fault
   injury (13). The National Vaccine Compensation Program
   (issued in 1986), which provides no-fault compensation

   for vaccine-related injuries, is financed by a trust
   fund created by an excise tax on every dose of vaccine
   sold (14).

   Orphan Drugs and Vaccines in Europe

   In 1994, the European Commission (the legislative body
   of the European Union [EU]) stated its interest in
   orphan diseases. In 1998, with close collaboration of
   the French Ministry of Health (15) and the European
   Medicine Evaluation Agency, a text was approved
   recommending the creation of a European Office for
   Orphan Drugs along the same lines as the U.S. Office of
   Orphan Products Development.

   The proposed European criteria for classification of a
   drug as an orphan drug (including vaccines) are almost
   identical to the U.S. criteria, except that they are
   based on a disease prevalence of 5 per 10,000 Europeans
   (falling between the United States [7 per 10,000] and
   Japan [2.5 per 10,000]), when no current methods of
   diagnosis, prevention or treatment, or major
   contribution to current patient care exist.

   The legislation will provide incentives to the European
   pharmaceutical industry in terms of research and
   development assistance (protocol assistance, normal
   evaluation, possible form of centralized but fast-track
   approval procedures), fee waiver, tax credits, funds
   from the European Orphan Product Grant Program, and
   market exclusivity for 10 years (interim period 6
   years) and will encourage national policies (subsidiary
   principle, e.g., the French compassionate use
   authorization [Autorisation Temporaire Utilisation])
   (16).

   The role of patient groups in increasing awareness of
   orphan drug development has been widely recognized for
   pharmaceutical orphan drugs in the United States and
   has been emphasized in the European project. The
   potential end-users of an orphan product may not be
   aware of therapeutic or preventive options. The
   European Office of Orphan Products Development will
   therefore support the establishment of groups of
   persons with the same rare conditions to play a role in
   increasing awareness of the disease within the
   population and will coordinate their activities at
   national and community levels. It remains to be seen
   how this initiative will apply to vaccine-preventable
   infectious diseases in communities where individuals or
   groups may not be aware of the risk for infection and
   thus the value of the vaccine.

   To clarify the extent of patent protection and the
   right to benefit from the orphan incentive package, the
   European Commission (DG24 committee) recently defined
   "similarity" between orphan products as the same
   substance, or a substance that differs from the
   original substance in molecular structure, source
   material, or manufacturing process, or an organism
   (living or nonliving) that is comparable with the
   original substance or organism in terms of biologic
   action and properties (including efficacy and safety)
   and ability to act through the same mechanism. In the
   same way as the U.S. legislation, this would favor the
   development of novel orphan products by the innovative
   company.

   In June 1999, the European Parliament's committee on
   the environment, public health, and consumer protection
   adopted a report by one of its senior members in favor
   of the Policy on Orphan Drugs and proposed some
   amendments to widen the scope of the legislation. Among
   other changes, the committee requested more flexibility
   in the proposed provisions for clinical trials,
   allowing (under specific conditions) availability of
   the product before final authorization is granted. The
   committee proposed extending the definition of orphan
   drug status to cover products intended for serious and
   chronic diseases. It also recommended, as in the United
   States, additional incentives for developing medicinal
   or biologic products for diseases that occur mainly in
   tropical regions but rarely within EU territory.
   Finally, the committee called for an Orphan Medicinal
   Product Innovation Promotion Fund to be financed from
   the sales of orphan drugs after the proposed 10-year
   period of market exclusivity. The European Orphan Drug
   Policy could be enacted early in the year 2000.

   Orphan Drugs in Other Industrialized Countries

   After the U.S. Orphan Drug Act, similar legislation was
   enacted in Japan in 1993. An Australian orphan drugs
   program based on the U.S. program began in 1998 (17).
   Since then, the Therapeutic Goods Administration has
   designated two biological drugs as orphansrabies
   immunoglobulin and recombinant enzyme imiglucerase for
   replacement therapy in patients with Gaucher disease. A
   cross-national comparison of orphan drug and vaccine
   policies has been made for different countries,
   including Japan, Canada, France, Sweden, and the United
   Kingdom (12).

   Orphan Vaccines in Developing Countries

   The availability and use of orphan vaccines in
   developing countries are complex since these countries
   have yet to ensure optimum use of existing priority
   vaccines (17). The limitations and obstacles involved
   in expanding the use of these priority vaccines are
   further multiplied for orphan vaccines of limited need.
   Within the framework of WHO, the Children's Vaccine
   Initiative set the development of vaccines with
   commercial prospects as a priority measure (7). This
   cost-oriented definition reflects mainly the difficulty
   of developing vaccines and drugs for tropical diseases,
   even those as prevalent as malaria (19,20). The
   Children's Vaccine Initiative's role has been
   problematic for various reasons (21), and this
   structure has faced increasing difficulties in
   maintaining its visibility.

   Nevertheless, other noneconomic factors (3) could
   justify an industry's decision to develop and market an
   orphan vaccine: desire to enhance the company's ethical
   profile by fulfilling a medical or social need;
   capacity to develop, produce, and market the drug; a
   larger company strategy (e.g., part of a product
   range); and possible additional uses that would
   increase the drug's future economic viability. The
   latter point may be less relevant for vaccines, which
   are usually tailor-made to their infectious agents.

   In the development of any vaccine against an emerging
   infectious disease, certain general rules apply
   (4,6,18,22), for example, developing strong research
   and development capacity, obtaining reliable scientific
   results and training in industrialized and developing
   countries; bulk filling arrangements; licensing
   technology; negotiating partnerships for specific
   products; joint venture agreements with western
   research and development manufacturers (economic value
   of the alliance); identifying the neediest countries on
   the basis of a banding strategy that classes countries
   according to their gross national product per capita,
   thereby allowing tiered pricing among them (6,23); and
   creation of funding mechanisms. Some could argue that
   from an industry perspective, if all of these criteria
   cannot be met, the vaccine should not be developed.

   No trade-off on the quality of an orphan vaccine is
   ethically justified or accepted. For the pharmaceutical
   industry, therefore, the costs incurred in development,
   ensuring tight quality controls, and establishing
   industrial good manufacturing procedures for an orphan
   vaccine are similar to those incurred with a
   traditional vaccine. For this reason, the development
   of any orphan vaccine should be broadly supported by
   measures to increase the awareness of immunization
   benefits at three levelsthe decision-makers, the
   caregivers, and the patients.

   Increasing Orphan Vaccine Availability

   Development of orphan vaccines is guided by the limited
   need for or market potential of the product, with the
   accompanying regulations, as well as the specific
   characteristics of the vaccine and those who need it
   (24). Because of the pitfalls related to these
   limitations, few orphan vaccines have reached the
   neediest populations. For example, in the United
   States, by the end of 1997, 837 medicinal products had
   been designated orphan drugs; 152 of these obtained
   authorization. This number was a clear improvement over
   that of the previous 14 years, during which 34
   medicinal orphan products obtained authorization.
   However, our website review found only eight vaccines
   registered with orphan status (seven for therapeutic
   indications [e.g., cancer and sickle cell anemia], one
   to prevent an Asiatic infectious diseaseJapanese
   encephalitis virus) and, to our knowledge, none has yet
   obtained final authorization. In addition, "It is not
   vaccines that save lives but vaccination." Even when
   orphan vaccines are available, we have to examine the
   feasibility of getting them to the intended population.

   Various strategies, proposals, and recommendations for
   overcoming limitations inherent in orphan vaccine
   development and availability are listed in the Table.

Table. Solutions and proposals for accelerating
orphan vaccine availability

1. Provide information, prioritize, and secure demand

  * Increase awareness of disease: set-up of special
    interest groups (patients, parents,
    professionals), expert groups, and national
    forums.
  * Acquire epidemiologic data on selected
    infectious diseases to guide decision-making:
    obtain access to data registries with comparable
    case-definitions across countries, and obtain
    information from specialized units and experts,
    scientific literature, patient organizations,and
    pharmaceutical manufacturers associations.
  * Establish the suitability of vaccine prevention
    vs. other options: realistic comparisons of
    vaccination with patterns and costs of other
    alternatives, such as treatment or vector
    control.
  * Ensure political support for orphan vaccine
    initiatives and organize tripartite partnerships
    between public, private, and nongovernmental
    sectors.

2. Facilitate vaccine research and development and
national/regional approvals

  * Promote innovative research and development
    technologies that could be applied to
    blockbuster vaccines or, alternatively, promote
    low-cost traditional vaccine technologies.
  * Encourage public/private sector links:
    academic/industrial research groups.
  * Set international standards of quality, safety,
    and efficacy and define minimum amount of data
    required for licensure.
  * Make recommendations on appropriate schedules,
    target ages.
  * Promote national and regional ex-U.S. and
    European Community incentives on Orphan Drug
    Policies (Latin America, Asia).
  * Expand and harmonize orphan drug policies as
    part of the ICH process (decrease time to
    regulatory approval).

3. Ensure market/funding visibility, production and
distribution

  * Reduce investment risks for manufacturers by
    providing realistic demand estimates.
  * Fund development of orphan vaccines for
    developing countries through various
    institutional bodies, such as CVI, WHO, UNICEF,
    PAHO, WB, USAID, NIH, CDC, PATH, other donor
    bodies, and nongovernmental organizations and
    foundations (e.g., Gates Foundation) on the
    basis of target assistance for the neediest
    countries based on total gross national product.
  * Strengthen political and public health
    collaboration between orphan programs (European
    Community, United States) and other countries to
    create a supranational office dedicated to
    orphan vaccines (World Office of Orphan Vaccine
    Development or CVI) that could harmonize and
    coordinate funding (from research to
    manufacturing) from various sources.
  * Identify and expand the pool of the committed
    purchasers based on expected coverage criteria.
  * Promote and support protection of intellectual
    property.
  * Clarify compensation programs that may assume
    responsibility for liability.
  * Evaluate tiered pricing (high/low) feasibility
    at two levels:
  * Multinational: traveler or military vaccines in
    industrialized countries, endemic community
    vaccines in developing countries.
  * National: a private market for the high GNP per
    capita subgroup, a public market for the low GNP
    per capita subgroup.
  * Establish manufacturing strategies, such as
    campaigning to subsidize orphan vaccine cost
    investments by large volume sales of EPI
    vaccines.
  * Strengthen the vaccine distribution network for
    the targeted population.

    ICH, International Conference on Harmonization; CVI,
    Children's Vaccine Initiative; WHO, World Health
    Organization; UNICEF, United Nation's Children's
    Fund; PAHO, PanAmerican Health Organization; WB,
    World Bank; USAID, U.S. Agency for International
    Development; NIH, National Institutes of Health; CDC,
    Centers for Disease Control and Prevention; PATH,
    Program for Appropriate Technology in Health; EPI,
    Expanded Program of Immunization.

   Providing Information, Prioritizing, and Securing
   Demand

   Although funding is a major obstacle to orphan vaccine
   development, it may not be the only impediment to the
   introduction of new vaccines (25). Reliable information
   on the epidemiology, disease severity, and effect on
   public health is essential to substantiating the need
   for a vaccine and may not be available to support the
   development decision. Market forces may not always be
   good cultivators of vaccines, which, unlike some
   chemical drugs, are not big money-making products. For
   this reason, the public and decision makers should know
   about the benefits of immunization, to increase disease
   awareness, and support an orphan vaccine initiative.

   Facilitating Vaccine Research and Development and
   National and Regional Approvals

   An accelerated procedure for final authorization and
   exemptions from all or part of the registration fee can
   reduce development costs, staffing requirements, and
   time to market and render the development of an orphan
   vaccine more attractive for the sponsor. Local
   initiatives may also speed the authorization process.

   Ensuring Market and Funding Visibility, Production, and
   Distribution

   Finally, increasing patent protection and the defined
   period of market exclusivity reduces investment risks
   for manufacturers. Furthermore, funding for orphan
   projects may be advanced from private bodies looking to
   capitalize on an ethical business image. Indeed, the
   private sector looks to take on an increasingly
   important role in international health development,
   especially in poorer countries (26). Increased world
   travel and the risk for transport of pathogens across
   borders (27) support tiered pricing between the western
   traveler and the disease-endemic country. In addition,
   an orphan infectious agent in a remote developing
   country requiring an orphan vaccine with limited need
   could, over time, become an emerging disease worldwide.
   HIV is a case in point: a disease originating in Africa
   that has successfully spread to the industrialized
   world.

   In Argentina, strong political and governmental
   support, aided by the U.S. Army Medical Research
   Institute of Infectious Diseases collaboration,
   ultimately culminated in a successful Candid 1
   vaccination campaign against Argentinean hemorrhagic
   fever in agricultural workers (28). Close collaboration
   between the pharmaceutical sector, WHO, and the Chinese
   government resulted in the development of the
   antimalarial drug artemisin (29).

   Conclusions

   The dilemmas intrinsic to the development and
   distribution of orphan vaccines against emerging
   infectious diseases reflect many of the issues faced by
   policy makers worldwide with regards to cost, quality
   of care, access to care, and the role of government
   intervention in regulating the health-care market (30).
   In view of the current globalization of trades and
   markets, worldwide orphan vaccine policies and a
   specialized organization with a strong leadership and
   commitment similar to the Children's Vaccine Initiative
   project for a National Vaccine Authority may be needed
   (6,18). This kind of organization could be responsible
   for establishing a list of priority orphan vaccines and
   indicating reasons for not including other vaccines.
   The organization could also oversee all stages of
   vaccine development and have access to funds that could
   rapidly be mobilized. Such a global structure could
   serve as a forum for discussing the current limitations
   on orphan vaccine development and availability.
   Nevertheless, the problems recently faced by the
   Children's Vaccine Initiative indicate the difficulties
   in mounting and maintaining such a worldwide
   initiative.

   Acknowledgments

   The authors thank Stanley Plotkin and the two reviewers
   whose constructive criticism and advice helped to
   improve this manuscript.

   Dr. Lang is a researcher at Pasteur Mrieux Connaught,
   the vaccine manufacturer. His clinical research
   interests include rabies, dengue, Japanese
   encephalitis, yellow fever, malaria, and antivenoms as
   part of the Vector Borne Vaccine Clinical Research and
   Development Program.

   Dr. Wood is responsible for education in vaccinology
   and vaccine policy for the international business unit
   of Pasteur Mrieux Connaught, which covers all
   countries outside Western Europe and North America.

   Address for correspondence: Jean Lang, Vector-Borne
   Diseases Clinical Program, R&D Clinical Development
   Department, Pasteur Mrieux Connaught, Lyon, France;
   fax: 33-47-273-7928; e-mail: jlang@fr.pmc-vacc.com.

   References

   1. Dodet B, Saluzzo J. Factor in the emergence of
   arbovirus diseases. (Les Pensires): Veyrier du Lac
   Elsevier Science 1997; 8-10 Decembre 1996.

   2. Brealey RA, Myers SC. In: Principles of corporate
   finance. 3rd ed. Singapore: McGraw-Hill; 1988.

   3. Stucki J. The development of orphan drugsa
   pharmaceutical company perspective. Cooperative
   approaches to research and development of orphan drugs
   1985;95-104.

   4. Aylward B, Kane M, Batson A, Scott R. A framework
   for the evaluation of vaccines for use in expanded
   programme on immunization. Vaccine 1994;12:1155-9.

   5. Shepard DS, Walsh JA, Kleinau E, Stansfield S,
   Bhalotra S. Setting priorities for the Children's
   Vaccine Initiative: a cost-effectiveness approach.
   Vaccine 1995;13:707-14.

   6. Milstien J, Batson A. Accelerating availability of
   new vaccines: the role of the international community.
   Drug Inf J 1998;32:175-82.

   7. CVI (Children's vaccine initiative). The CVI
   strategic planmanaging opportunity and change: a vision
   of vaccination for the 21st century. OMS
   1997;CVI/GEN(97-04):1-92.

   8. Trannoy E. Will ethical and liability issues and
   public acceptance allow maternal immunization? Vaccine
   1998;16:1482-5.

   9. Haffner M. Orphan products: origins, progress, and
   prospects. Annu Rev Pharmacol Toxicol 1991;31:603-20.

   10. Haffner M, Kelsey J. Evaluation of orphan products
   by the U.S. Food and Drug Administration. Int J Tech
   Assess Health Care 1992;8:647-57.

   11. Cramer R. Orphan drugs improved medical treatment
   of rare diseases. Alabama J Med Sci 1998;25:257-67.

   12. Thamer M, Brennan N, Semansky R. A cross-national
   comparison of orphan drug policies: implications for
   the U.S. Orphan Drug Act. J Health Pol Policy Law
   1998;23:265-90.

   13. Scharf S. Orphan drugs: the question of product
   liability. Am J Law Med 1985;10:491-513.

   14. Bloom B. The United States needs a national vaccine
   authority. Science 1994;265:1378-80.

   15. Brandissou S, Yagoubi N, Hasselot N. Les
   mdicaments orphelins: problme de sant publique et
   enjeux conomiques. Thrapie 1996;51:647-53.

   16. Benzi G, Ceci A. Drugs trying to get the patents:
   there will be incentives for the European scientific
   community to develop research in the field of the
   orphan drugs. Pharmacol Res 1997;35:89-93.

   17. Hillcoat BL. Rare diseases and "orphan" drugs
   [editorial]. Med J Aust 1998;169:69-70.

   18. Mahoney RT, Maynard JE. The introduction of new
   vaccines into developing countries. Vaccine
   1999;17:646-52.

   19. Local initiative aids in fight against malaria
   [editorial]. Lancet 1998;352:212.

   20. Ollario P. Will the fight against tropical diseases
   benefit from orphan drug status? Trop Med Int Health
   1997;2:113-5.

   21. Muraskin W. The politics of international health:
   the children's vaccine initiative and the struggle to
   develop vaccines for the third world. Albany (NY):
   State University of New York Press; 1998.

   22. Milstien J, Batson A, Meaney W. A systematic method
   for evaluation of the potential viability of local
   vaccine producers. Vaccine 1997;15:1358-63.

   23. Batson A. Sustainable introduction of affordable
   new vaccines: the targeting strategy. Vaccine
   1998;16:S93-8.

   24. Bloom B. Bumps on the vaccine road. Science
   1994;265:1371-3.

   25. Hausdorff WP. Prospects for the use of new vaccines
   in developing countries: cost is not the only
   impediment. Vaccine 1996;14:1179-86.

   26. Public-private partnershipsbusiness as usual
   [editorial]. Lancet 1998;352:212.

   27. Spier R. Report on a meeting of a working party
   sponsored by the European Commission to discuss the
   ethical, legal and social aspects of research on
   vaccines and vaccination. Vaccine 1999;17:400-2.

   28. Maiztegui J, MacKee K, Barrera Oro J, Harrison L,
   Gibbs P, Feuillade M, et al. Protective efficacy of a
   live attenuated vaccine against Argentine hemorrhagic
   fever. J Infect Dis 1998;177:277-83.

   29. Helenport J, Roche G. What have been the strategies
   for the registration, positioning and control of
   medical information for im artemether? The rational use
   of Qinghaosu and its derivatives. 1998; Les Pensires
   (19-22 April):141-7, Fondation Merieux Eds.

   30. Beutels P. Economic evaluations applied to HB
   vaccination: general observations. Vaccine
   1998;16:S84-92.

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

URL: http://www.cdc.gov/ncidod/eid/vol5no6/ascii/lang.txt

Please note that figures, equations, and most tables are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively.  Greek symbols are spelled out. >/= is used for greater than or equal to.

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

Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999]

Synopsis

   Antimicrobial Resistance with Streptococcus pneumoniae in
   the United States, 199798

   Gary V. Doern, Angela B. Brueggemann, Holly Huynh,
   Elizabeth Wingert, and Paul Rhomberg
   University of Iowa College of Medicine, Iowa City, Iowa,
   USA

    From November 1997 to April 1998, 1,601
    clinical isolates of Streptococcus pneumoniae
    were obtained from 34 U.S. medical centers.
    The overall rate of strains showing
    resistance to penicillin was 29.5%, with
    17.4% having intermediate resistance.
    Multidrug resistance, defined as lack of
    susceptibility to penicillin and at least two
    other non--lactam classes of antimicrobial
    drugs, was observed in 16.0% of isolates.
    Resistance to all 10 -lactam drugs examined
    in this study was directly related to the
    level of penicillin resistance. Penicillin
    resistance rates were highest in isolates
    from middle ear fluid and sinus aspirates of
    children <5 years of age and from patients in
    ambulatory-care settings. Twenty-four of the
    34 medical centers in this study had
    participated in a similar study 3 years
    before. In 19 of these 24 centers, penicillin
    resistance rates increased 2.9% to 39.2%.
    Similar increases were observed with rates of
    resistance to other antimicrobial drugs.

   Before 1990, most clinical isolates of Streptococcus
   pneumoniae in the United States were susceptible to a
   variety of antimicrobial drugs, including penicillin
   (1,2). In the early 1990s, however, antimicrobial
   resistance began to emerge (3-7), and -lactam resistance
   as a result of altered penicillin binding proteins was
   recognized (8-11). Resistance to other non--lactam
   drugs, such as the macrolides, clindamycin, tetracycline,
   chloramphenicol, and trimethoprim/sulfamethoxazole
   (TMP/SMX), began to increase (4-6). Therapeutic failures
   in patients with pneumococcal infections treated with
   previously effective drugs were reported (12).

   During November 1994 through April 1995, we assessed the
   prevalence of antimicrobial resistance with S. pneumoniae
   at 30 U.S. medical centers (4). Among 1,527 isolates of
   S. pneumoniae, 14.1% had intermediate resistance, and
   9.5% were fully resistant to penicillin. Aggregate rates
   of intermediate and high penicillin resistance were 2.1%
   to 52.9%. In addition, high rates of resistance were
   noted with other antimicrobial drugs.

   From November 1997 to April 1998, we surveyed 34 U.S.
   medical centers to assess changes in antimicrobial
   resistance rates with S. pneumoniae during the 3 years
   since the 1994-95 study. Twenty-four of these centers had
   also participated in the earlier investigation. Similar
   patient populations were sampled, and identical test
   methods were used. In addition, we assessed the
   relationship between various demographic factors and
   resistance and undertook a systematic analysis of
   multidrug resistance. Finally, macrolide and
   fluoroquinolone resistance was characterized at a
   molecular level.

   The Study

   From November 1, 1997, to April 30, 1998, 1,601 isolates
   of S. pneumoniae were recovered in 34 U.S. medical
   centers. All isolates included in this study were from
   consecutive patients. With the exception of specimens
   from the lower respiratory tract, all isolates were from
   normally sterile body sites (i.e., blood, cerebrospinal
   fluid, middle ear fluid, sinus aspirates, pericardial
   fluid, and pleural fluid). Isolates from lower
   respiratory tract specimens were included only if they
   were of clinical significance.

   In the study centers, isolates were subcultured onto 5%
   sheep blood agar plates and incubated overnight at 35C
   to 37C in 5% to 7% CO2. Colony growth was collected on a
   rayon swab and immediately immersed in a transport tube
   containing 12 ml of semisolid Ames transport medium with
   charcoal (Difco Laboratories, Detroit, MI). Transport
   tubes were then shipped overnight to the University of
   Iowa College of Medicine for additional analysis
   (Appendix). The recovery rate from this transport system
   was 100%. Twelve concentrations each of 23 antimicrobial
   drugs were tested against 1,601 isolates of S.
   pneumoniae.

   Overall, 17.4% of isolates had intermediate and 12.1% had
   full resistance to penicillin (Table 1). Overall
   nonsusceptible rates with ceftriaxone and cefuroxime
   (intermediate plus fully resistant) were 14.9% and 23.3%,
   respectively. Because National Committee for Clinical
   Laboratory Standards (NCCLS)-approved breakpoints are
   lacking for the six other cephalosporins examined in this
   study (cefpodoxime, cefixime, ceftibuten, cefprozil,
   cefaclor, and loracarbef), rates of resistance were not
   determined for these drugs. However, when MIC values were
   compared, cefpodoxime was the most active.

   Comparison of MIC values of the three macrolides we
   examined showed that clarithromycin was consistently
   twice as active as erythromycin, which in turn was
   consistently twice as active as azithromycin (Table 1).
   Overall rates of resistance, however, based on NCCLS
   breakpoints, which differ for these agents, were similar
   (18%-19%).

Table 1. In vitro activity of 23 antimicrobial agents for 1,601 isolates of Streptococcus pneumoniae
Compared with erythromycin as an indicator of macrolide activity, 302 (18.9%) isolates had MICs >/=1 g/ml
and thus were classified as resistant (Table 1). Among these, 217 (71.9%) had erythromycin MICs </=32 g/ml;
the remaining 85 strains (28.1%) had erythromycin MICs >/=64 g/ml. Of the 217 strains with erythromycin MICs
</=32, 214 had clindamycin MICs </=0.25 and thus were categorized as clindamycin susceptible. Thirty-five of
these strains, randomly selected, were examined by polymerase chain reaction (PCR) for the presence of
ermAM and mefE genes. Of the 85 strains with erythromycin MICs >/=64 g/ml, 83 had clindamycin MICs >/=8.
Thirty-eight of these isolates, chosen randomly, were ermAM positive; 12 were also positive for mefE (Table
2). Five resistant strains were also characterized for the presence of macrolide resistance determinants
(Table 2). Finally, the three isolates (Table 2) negative for both the ermAM and mefE genes were also
negative by PCR for other known determinants of macrolide/lincosamide resistance in gram-positive bacteria
(ermA, ermC, ereA, ereB, msrA, and linA genes).

   Table 2. Characterization of 302 Streptococcus
   pneumoniae isolates that were erythromycin
   resistant (MICs 1>/= g/ml)
   
Of the 1,601 isolates examined, one had intermediate resistance to trovafloxacin with an MIC 2 g/ml; three
strains (0.2%) were resistant, two with trovafloxacin MICs 4 g/ml and one with a trovafloxacin MIC 8 g/ml. The single strain with intermediate resistance had a ciprofloxacin MIC of 16 and an asp83-->tyr substitution in the C subunit of topoisomerase IV, as well as a ser84-->tyr substitution in the A subunit of DNA gyrase. The three trovafloxacin-resistant strains had ciprofloxacin MICs >/=32 g/ml and a
ser79-->phe substitution in the C subunit of topoisomerase, as well as a ser84-->phe substitution in the A subunit of DNA gyrase. None of these four strains had detectable mutations in the QRDR of par E.

Overall rates of resistance, expressed as the percentage of isolates intermediate or resistant, for selected other agents are described in Table 1: tetracycline, 13.2%, TMP/SMX, 31.1%, chloramphenicol, 7.2%, and rifampin, 0.2%. Linezolid was uniformly active over a narrow range of MICs (i.e., 0.12 to 2 g/ml). Two strains among the 1,601 examined in this study were resistant to quinupristin/dalfopristin; one had an MIC
4 g/ml and the other 8 g/ml. Vancomycin was uniformly active against the 1,601 isolates of S. pneumoniae
in this survey, with MICs </=0.5 g/ml.

The in vitro activity of all -lactams (penicillins, -lactamase inhibitor combinations and cephalosporins), macrolides, clindamycin, tetracycline, TMP/SMX, and chloramphenicol was lowest with high-level penicillin-resistant strains of S. pneumoniae and greatest with penicillin-susceptible isolates. This trend was not apparent with linezolid, trovafloxacin, rifampin,  quinupristin/dalfopristin, or vancomycin.

The prevalence of resistance to selected agents was assessed according to the specimen source of isolates,
the patient's age, and the health-care setting (Table 3). In general, the highest resistance rates for all
antimicrobial drugs were observed in middle ear fluid and sinus aspirate isolates and in isolates from
patients </=5 years old and from patients seen in ambulatory-care settings.

Table 3. Recovery of Streptococcus pneumoniae strains with
intermediate and high levels of resistance, by specimen source and patient characteristics

When patterns of multidrug resistance were analyzed, coresistance to penicillin, the macrolides,
tetracycline, TMP/SMX, and chloramphenicol was observed in 5.4% of isolates (Table 4). Resistance to the
first four of these drugs but not to chloramphenicol was seen with 3.7% of strains. The most common pattern
of multidrug resistance (intermediate level or resistant to penicillin and resistant to TMP/SMX, but
susceptible to the macrolides, tetracyclines, and chloramphenicol) was observed in 6.9% of isolates.

   Table 4. Frequency of isolation of strains of
   Streptococcus pneumoniae with various patterns
   of antimicrobial resistancea

In the 34 participating medical centers, the lowest and highest overall rates of penicillin resistance
(intermediate plus resistant) were 12.8% and 64.6% (Table 5). Eight medical centers had 10% to 18%
penicillin resistance; 11 centers had 19% to 26%; 5 had 27% to 36%; 7 had 37% to 45%; 3 had >46%. Although
no distinct geographic clustering was identified, the highest overall rates of penicillin resistance were
in the South and Southeast. The lowest overall rate of ceftriaxone resistance was 2.9%; the highest was
48.1%. With erythromycin, overall rates of resistance were 3.8% to 43.8%. With tetracycline,
chloramphenicol, and TMP/SMX, the rates were 5.7% to 29.6%, 0.0% to 25.9%, and 11.9% to 63.2%,
respectively. In general, the highest rates of resistance with most antimicrobial classes were observed in
the same centers.

Table 5. Resistance rates of selected antimicrobial drugs, by study center

Among the 34 medical centers, 24 had participated in a similar study 3 years before (4). Because the sampling period, the nature of the patients, and the test methods were the same, resistance rates obtained with selected antimicrobial drugs were compared at the 24 centers for the two study periods (Table 6). In 19 of 24 centers, penicillin resistance rates (intermediate and resistant) increased by at least 2% during the 3-year interval. In six cases, the rate of increase was statistically significant (Table 6). The number of centers in which rates of resistance increased by >2% with other selected antimicrobial drugs over the
3-year period were ceftriaxone 13, erythromycin 21, tetracycline 20, chloramphenicol 12, and TMP/SMX 16. In
most cases, resistance rates increased; however, with certain antimicrobial agents (i.e., ceftriaxone,
TMP/SMX, and chloramphenicol), resistance rates remained the same or decreased. Three centers (Texas Children's Hospital, UNC Hospital, and the University of South Alabama Medical Center) had statistically significant increased rates of resistance to nearly every class of antimicrobial drug.

Table 6. Resistance rate comparison of selected antimicrobial drugs for 24 medical centers, by study
period

Conclusions

A total of 1,601 clinical isolates of S. pneumoniae obtained from November 1997 to April 1998 from patients in 34 U.S. medical centers were characterized with respect to the in vitro activity of 23 antimicrobial drugs. The overall rate of penicillin resistance was 29.5% (17.4% with intermediate resistance; 12.1% fully resistant). Penicillin, amoxicillin, and amoxicillin/clavulanate had essentially equivalent MICs for the pneumococcal isolates examined. Among the  cephalosporins tested, the rank order of activity based on a comparison of MICs was ceftriaxone > cefpodoxime = cefuroxime > cefprozil > cefixime > cefaclor = loracarbef > ceftibuten. NCCLS has developed MIC interpretive breakpoints for two of these compounds, ceftriaxone and cefuroxime. On the basis of these breakpoints, overall resistance rates were 14.9% with ceftriaxone (10.9% intermediate, 4.0% resistant) and 23.3% with cefuroxime (1.3% intermediate and 22.0% resistant). Although no NCCLS-approved breakpoints have been developed for cefaclor, loracarbef, and ceftibuten versus S. pneumoniae, the high MICs we obtained with these drugs indicate that they would be poor choices for treating pneumococcal infections.

We observed a direct relationship between penicillin activity and the activity of all the other -lactam
drugs we examined. This relationship, which has been observed by others, results from the principal
mechanism of penicillin resistance in S. pneumoniae, alterations in penicillin binding proteins (4-6). The
same proteins to which penicillin binds are necessary for the expression of the activity of all other
-lactam antimicrobial drugs (8-11).

Among the macrolides examined, clarithromycin was generally one dilution more active than erythromycin,
which in turn was one dilution more active than azithromycin. However, since NCCLS MIC interpretive
breakpoints differ for these agents and given the actual distribution of MICs, overall rates of resistance
for these three agents were similar (19.0%).

Two previous studies have indicated that macrolide resistance with S. pneumoniae exists primarily in one of
two forms: strains with altered ribosomal targets due to expression of the ermAM gene and strains with an
active efflux pump due to expression of the mefE gene (13,14). ErmAM-positive isolates have constitutive
expression of resistance and typically have high levels of resistance to both clindamycin (/= >8 g/ml)
and erythromycin (MICs >/=64 g/ml). Efflux mutants are susceptible to clindamycin (MICs </=0.25 g/ml) and
typically have erythromycin MICs 1 to 32 g/ml.

In a recent survey in Canada, approximately 60% of macrolide-resistant strains of S. pneumoniae appeared to
be efflux mutants (19). In our study, 214 (70.8%) of 302 macrolide-resistant strains were characterized by
the efflux phenotype. When a random sample of 35 of these isolates was examined by PCR, 31 (88.6%) lacked
the ermAM gene but had the mefE gene responsible for efflux. Among the remaining four strains in this group
that were characterized at a molecular level, one was ermAM positive and mefE negative, two were positive
for both ermAM and mefE, and one was negative for all macrolide-resistance markers. Based on their
phenotype (i.e., erythromycin MICs >/=64 g/ml, with clindamycin MICs >/=8 g/ml), 83 (27.5%) of the 302
macrolide-resistant strains appeared to have constitutive expression of ermAM-mediated methylation of
ribosomal targets. All 38 randomly selected isolates from this group were ermAM positive; 12 were also mefE
positive by PCR.

Five macrolide-resistant strains were not readily placed into either the efflux or the target modification
groups based on phenotype. Two strains with high-level clindamycin resistance but erythromycin MICs 2 and
32 g/ml were ermAM positive but mefE negative. Three strains with erythromycin MICs >/=64 had clindamycin
MICs 0.5, 1, and 4 g/ml. One of these strains, which had a clindamycin MIC of 4 and a high erythromycin
MIC, appeared to be an efflux mutant since it was mefE positive but ermAM negative. The mechanism of
resistance in the last two isolates in this group is unclear. Both isolates were negative by PCR for ermAM
and mefE, as well as other genetic markers for macrolide and lincosamide resistance in gram-positive
bacteria.

Several conclusions can be drawn from these observations. Macrolide resistance in S. pneumoniae is nearly always characterized either by efflux or ribosomal target modifications. The phenotype of isolates in both categories is highly predictable. Efflux mutants usually have  erythromycin MICs 1 - 32 g/ml and
clindamycin MICs </=0.25 g/ml. Target modified strains are characterized by constitutive expression of resistance and typically have erythromycin MICs >/=64 g/ml and clindamycin MICs >/=8 g/ml. Although rare exceptions to these patterns exist, clindamycin activity, as assessed by an MIC determination, is a reliable indicator of the nature of macrolide resistance.

These results show that therapeutic efficacy can be predicted for patients with pneumococcal infections who
are treated with macrolides. Using current NCCLS interpretive criteria, 18% to 19% of clinical isolates of
S. pneumoniae in the United States would be categorized as resistant. Approximately three-fourths of the
resistant strains, however, are efflux mutants and have mid-range resistant macrolide MICs. Patients
infected with mefE versus ermAM-positive strains of S. pneumoniae may differ in their response to macrolide
therapy. The answer to this important question would define true clinical rates of macrolide resistance. If
mefE strains respond to therapy with macrolide drugs, then true macrolide resistance rates may not be 18%
to 19%, but closer to 4% to 5%, and high-level clindamycin MICs could be used to accurately identify
strains highly resistant to macrolide drugs.

Multidrug resistance was noted with 16.0% of 1,601 isolates, a substantial increase over the 9.1% prevalence of multidrug resistant S. pneumoniae reported in our 1994-95 study (4). Among multidrug resistant strains, four resistance patterns were the most common: penicillin and TMP/SMX (n = 111);
penicillin, macrolide, and chloramphenicol (n = 75); penicillin, macrolide, tetracycline, and TMP/SMX (n = 57); and penicillin, macrolide, tetracycline, TMP/SMX, and chloramphenicol (n = 87). No obvious clustering
of multidrug resistant strains was observed in either individual medical centers or geographic regions.

That we found resistance to -lactam antimicrobial agents and numerous non--lactam agents in the same organism is of interest. The mechanisms of resistance to the different antimicrobial classes are distinct and do not appear to be linked genotypically. One explanation, therefore, for the increasing prevalence of multidrug resistant strains is clonal spread of organisms resistant to more than one class of antimicrobial drugs. Under such circumstances, regardless of the antimicrobial drug used to select for resistance,
multidrug resistant organisms may become more prevalent. Clonal spread warrants further study using appropriate techniques for establishing clonal relationships among multiple geographically distinct isolates of S. pneumoniae.

Acknowledgments

We thank Kay Meyer for excellent secretarial assistance. We also thank the following for providing clinical isolates of Streptococcus pneumoniae: Carla Clausen, Susan Rossmann, Paul Southern, Michael Wilson, Michael Saubolle, John Washington, Michael Dunne, Gerald Denys, Melodie Beard, Tom Thompson, Sue Kehl, Frank Cockerill, Pat Murray, Ann Robinson, Betz Forbes, Dwight Hardy, Phillis Della-Latta, Allan Truant, Joe Campos, Paul Bourbeau, Peter Gilligan, Bob Jerris, Kim Chapin, and Sue Sharp.

This study was funded by a grant from Abbott Laboratories, which produces clarithromycin.

Dr. Doern is Director of the Clinical Microbiology Laboratories at the University of Iowa College Hospital
and Clinics and Professor in the Department of Pathology in the medical school. His research interests focus on antibiotic resistance among bacterial pathogens. He has been Chair of the Clinical Microbiology Division of the American Society for Microbiology and a member of the Antimicrobial Subcommittee of the National Committee for Clinical Laboratory Standards.

Appendix

Isolates were frozen at -70C at the University of Iowa College of Medicine until further characterization.
After two subcultures, the identity of isolates was confirmed as S. pneumoniae by conventional tests and
criteria. MICs were determined in Mueller-Hinton broth supplemented with 3% lysed horse blood, according to
the broth microdilution method recommended by the National Committee for Clinical Laboratory Standards
(NCCLS) (15). Microdilution trays (final volume 100 l per well) were inoculated with approximately 5 x 105
colony-forming units (CFU)/ml (final concentration) of test organism and incubated 22 to 24 hours at 35C
in ambient air before MICs were determined.

S. pneumoniae ATCC 49619 and Haemophilus influenzae ATCC 49247, ATCC 49766, and ATCC 10211 were used as
controls. Calculations of the percentages of isolates resistant to individual agents were based on the most
recent MIC interpretive criteria published by NCCLS (16).

Selected isolates were examined for mutations in the quinolone resistance-determining region of the parC
and par E genes encoding the C and E subunits of topoisomerase IV, respectively, and the quinolone
resistance-determining region of the gyrA gene encoding for the A subunit of DNA gyrase. Polymerase chain
reaction (PCR) amplification with subsequent sequencing of PCR products was done by using the method and
primers described by Pan et al. (17) and Perichon et al. (18). Other isolates were screened for various
macrolide resistance determinants by PCR amplification as described by Shortridge et al. (13). Primers were
chosen by using Oligo 5.0 software (NBI, Plymouth, MI) from sequences deposited in Genbank. Strains
negative for both mefE (efflux mutants) and ermAM (ribosome methylase) were characterized further for the
presence of other methylase genes (ermA and ermC), erythromycin esterase (ereA and ereB), MS efflux (msrA)
and the gene for lincosamide resistance (linA), again as described by Shortridge et al. (13).

Address for correspondence: Gary V. Doern, Medical Microbiology Division, C606 GH, Department of Pathology,
University of Iowa College of Medicine, Iowa City, IA 52242, USA; fax: 319-356-4916; e-mail:
gary-doern@uiowa.edu.

References

1. Jorgensen JH, Doern GV, Maher LA, Howell AW, Redding JS. Antimicrobial resistance among respiratory
isolates of Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae in the United
States. Antimicrob Agents Chemother 1990;34:2075-80.

2. Spika JS, Acklam RR, Plikaytis BD, Oxtoby MG, the Pneumococcal Surveillance Working Group. Antimicrobial
resistance of Streptococcus pneumoniae in the United States, 1979-1987. J Infect Dis 1991;163:1273-8.

3. Barry AL, Pfaller MA, Fuchs PC, Packer RR. In vitro activities of 12 orally administered antimicrobial
agents against four species of bacterial respiratory pathogens from U.S. medical centers in 1992 and 1993.
Antimicrob Agents Chemother 1994;38:2419-25.

4. Doern GV, Brueggemann A, Holley HP Jr, Rauch AM. Antimicrobial resistance of Streptococcus pneumoniae
recovered from outpatients in the United States during the winter months of 1994 to 1995: results of a
30-center national surveillance study. Antimicrob Agents Chemother 1996;40:1208-13.

5. Doern GV, Pfaller MA, Kugler K, Freeman J, Jones RN. Prevalence of antimicrobial resistance among
respiratory tract isolates of Streptococcus pneumoniae in North America: 1997 results from the SENTRY
Antimicrobial Surveillance Program. Clin Infect Dis 1998;27:764-70.

6. Jones RN, Pfaller MA, Doern GV. Comparative antimicrobial activity of trovafloxacin tested against 3,049
Streptococcus pneumoniae isolates from the 1997-1998 respiratory infection season. Diagn Microbiol Infect
Dis 1998;32:119-26.

7. Thornsberry C, Brown SD, Yee C, Bouchillon SK, Marler JK, Rich T. Increasing penicillin resistance in
Streptococcus pneumoniae in the U.S. Infections in Medicine Supplement 1993;93:15-24.

8. Grebe T, Hakenbeck R. Penicillin binding proteins 2b and 2x of Streptococcus pneumoniae are primary
resistance determinants for different classes of -lactam antibiotics. Antimicrob Agents Chemother
1996;40:829-34.

9. Jamin M, Hakenbeck R, Frere JM. Penicillin binding protein 2x as a major contributor to intrinsic
-lactam resistance of Streptococcus pneumoniae. FEBS 1993;331:101-4.

10. Laible G, Hekenback R. Five independent combinations of mutations can result in low-affinity penicillin-binding protein 2x of Streptococcus pneumoniae. J Bacteriol 1991;173:6986-90.

11. Markiewicz Z, Tomasz A. Variation in penicillin-binding protein patterns of penicillin-resistant
clinical isolates of pneumococci. J Clin Microbiol 1989;27:405-10.

12. Kaplan SL, Mason EO Jr. Management of infections due to antibiotic-resistant Streptococcus pneumoniae.
Clin Microbiol Rev 1998;11:628-44.

13. Shortridge VD, Flamm RK, Ramer N, Beyer J, Tanaka SK. Novel mechanism of macrolide resistance in
Streptococcus pneumoniae. Diagn Microbiol Infect Dis 1996;26:73-8.

14. Sutcliffe J, Tait-Kamradt A, Wondrack L. Streptococcus pneumoniae and Streptococcus pyogenes resistant
to macrolides but sensitive to clindamycin: a common resistance pattern mediated by an efflux system.
Antimicrob Agents Chemother 1996;40:1817-24.

15. National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility
tests for bacteria that grow aerobically, 4th ed. Approved standard M7-A4. Wayne (PA): The Committee; 1997.

16. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial
susceptibility testing. Eighth informational supplement, M100-S8. Wayne (PA): The Committee; 1997.

17. Pan X-S, Ambler J, Mehtar S, Fisher LM. Involvement of topoisomerase IV and NDA gyrase as ciprofloxacin
targets in Streptococcus pneumoniae. Antimicrob Agents Chemother 1996;40:2321-6.

18. Perichon B, Tankovic J, Courvalin P. Characterization of a mutation in the par E gene that confers
fluoroquinolone resistance in Streptococcus pneumoniae. Antimicrob Agents Chemother 1997;41:1166-7.

19. Johnston NJ, De Azavedo JC, Kellner JD, Low DE. Prevalence and characterization of the mechanisms of
macrolide, lincosamide, and streptogramin resistance in isolates of Streptococcus pneumoniae. Antimicrob
Agents Chemother 1998;42:2425-6.

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

URL: http://www.cdc.gov/ncidod/eid/vol5no6/ascii/doern.txt

Please note that figures, equations, and most tables are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively.  Greek symbols are spelled out. >/= is used for greater than or equal to.

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

Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999]

Research

Epidemiologic Studies of Cyclospora cayetanensis in
Guatemala

Caryn Bern,* Beatriz Hernandez, Maria Beatriz Lopez,
Michael J. Arrowood,* Maricruz Alvarez de Mejia, Ana
Maria de Merida, Allen W. Hightower,* Linda Venczel,
Barbara L. Herwaldt,* and Robert E. Klein

*Centers for Disease Control and Prevention, Atlanta,
Georgia, USA; Universidad del Valle, Guatemala City,
Guatemala; and University of North Carolina, Chapel
Hill, North Carolina, USA

In 1996 and 1997, cyclosporiasis outbreaks
in North America were linked to eating
Guatemalan raspberries. We conducted a
study in health-care facilities and among
raspberry farm workers, as well as a
case-control study, to assess risk factors
for the disease in Guatemala. From April 6,
1997, to March 19, 1998, 126 (2.3%) of
5,552 surveillance specimens tested
positive for Cyclospora; prevalence peaked
in June (6.7%). Infection was most common
among children 1.5 to 9 years old and among
persons with gastroenteritis. Among 182
raspberry farm workers and family members
monitored from April 6 to May 29, six had
Cyclospora infection. In the case-control
analysis, 62 (91%) of 68 persons with
Cyclospora infection reported drinking
untreated water in the 2 weeks before
illness, compared with 88 (73%) of 120
controls (odds ratio [OR] 3.8, 95%
confidence interval [CI] 1.4, 10.8 by
univariate analysis). Other risk factors
included water source, type of sewage
drainage, ownership of chickens or other
fowl, and contact with soil (among children
younger than 2 years).

Before 1995, the coccidian parasite Cyclospora
cayetanensis was primarily reported as a cause of
gastroenteritis among children living in poor sanitary
conditions (1,2) and adults from industrialized
countries who lived or traveled in developing countries
(3-5). From 1990 to 1995, three small Cyclospora
outbreaks were reported in North America, at least one
of them epidemiologically associated with drinking
water (6-8). However, in the spring and early summer of
1996, the largest ever reported outbreak of
cyclosporiasis, affecting more than 1,400 persons in
North America (9), was associated with eating fresh
raspberries; trace-back data indicated that the source
of the berries was Guatemala (9). A year later, despite
improvements in water quality and sanitary practices
instituted on berry farms after the 1996 outbreak,
Guatemalan raspberries were again implicated in
cyclosporiasis outbreaks (10).

The source of contamination of the implicated
raspberries has not been established. Most likely,
fecal contamination of water used for spraying
fungicides and other substances directly on fruit was
the source (9). Another possibility is that berries
were contaminated with sporulated organisms during
hand-sorting and packing; however, because Cyclospora
may require 1 to 2 weeks in the environment to become
infectious (2), direct fecal contamination from the
hands of an infected worker is less likely. The
possibility that berries might have been contaminated
by bird droppings before being picked has also been
raised (11), although no natural (12) or experimental
infection with C. cayetanensis has been confirmed in
any animal (M. Eberhard, pers. comm.). Studies of
Cyclospora have been limited by this lack of an animal
model; by dependence on microscopy for detection; by
the lack of sensitivity of the available methods in
water, fruit, and other environmental specimens; and by
inability to culture the organism.

Cyclospora in Guatemala was first reported in 1994
(13). Since then, the organism has been reported among
AIDS patients (14) and in several case series in
hospital outpatient departments in Guatemala City
(15-17). However, diagnosis of Cyclospora infection is
not part of routine stool examination in health-care
facilities in Guatemala.

In 1997, we initiated a study whose objectives were to
provide the first comprehensive description of the
epidemiology of Cyclospora in Guatemala and explore
potential environmental sources of contamination. The
study included surveillance among patients in
outpatient departments and among raspberry farm
workers, a case-control study to identify risk factors
for Cyclospora infection, and screening of surface
water specimens for fecal contamination and for
Cyclospora oocysts.

Methods

Outpatient Surveillance

In two government health centers (both in the
Department of Guatemala) and three hospital outpatient
departments (two in the Department of Guatemala and one
in the Department of Sacatepequez), we interviewed
outpatients whose health-care providers had ordered
stool specimens for bacterial pathogen testing or
screening for helminth eggs. In each center, we
screened up to 25 specimens submitted each day. The
study was explained to potential participants and
written consent was requested. For each person who
agreed to participate, part of the stool specimen was
collected, and information was recorded about age, sex,
and current diarrhea (defined as 3 or more loose or
liquid stools in 24 hours) or other gastrointestinal
symptoms. For data analysis, we defined gastroenteritis
as diarrhea, other gastrointestinal symptoms, or both.
Persons with symptomatic Cyclospora infection were
treated with trimethoprim-sulfamethoxazole unless they
had a history of allergy to sulfa drugs. Asymptomatic
persons were not treated, as the risk for side effects
was thought greater than the benefits of treating a
self-limited asymptomatic infection.

Raspberry Farm Cohorts

On three raspberry farms (two in the Department of Guatemala and one in the Department of Chimaltenango), cohorts of workers and their family members were recruited in early April 1997. All participating farms, except one, had been implicated in the 1996 outbreak and were still exporting raspberries. The farms included at least one from each of the two main areas where raspberries are cultivated. The
study was described in detail to participants, and written consent was solicited. A stool specimen was collected every other week from workers (less regularly from family members) and each time a participant reported having gastroenteritis. Persons found to have Cyclospora infection were treated with trimethoprim-sulfamethoxazole unless they had a history of allergy to sulfa drugs; they were monitored until their stool specimens were negative. Because of concerns about potential contamination of berries, all workers and
their family members with Cyclospora infection, whether symptomatic or asymptomatic, were treated. Surveillance was intended to continue throughout the spring and fall berry harvest seasons.

Case-Control Study

From May 7 to September 3, 1997, we recruited participants for the case-control study. Because surveillance on raspberry farms ended earlier than anticipated, only four case-control participants were from the farms; all others were recruited from health-care facilities. All persons with symptomatic Cyclospora infection detected through the surveillance system were sought in their homes;
however, some could not be located because of incorrect addresses. The purpose of the visit was to offer antibiotic treatment; at the same time, we solicited participation in the case-control study. We also requested stool specimens from family members of case-patients; however, family members were not included in the case-control study,
and no rigorous attempt was made to include all family members. 

We recruited controls from persons who had a stool specimen screened in any of the surveillance system's health centers or hospitals during the period in which we were enrolling cases. Participants were eligible to be controls if they reported no gastrointestinal symptoms and had negative stool results. We chose the controls to permit a final age distribution similar to that seen for Cyclospora
cases in the surveillance system and a roughly equal distribution by surveillance site; individual cases and controls were not matched. Thus, each week we rotated the surveillance site from which we recruited controls, and we attempted to enroll 10 controls per week: one to two children younger than 1.5 years of age, four children 1.5
to 4 years of age, three 5 to 14 years of age, and one to two persons older than 14 years of age.

We administered a questionnaire that focused on potential risk factors for Cyclospora, including drinking water source and handling, household sanitation, presence of animals, socioeconomic variables, and consumption of specific fresh fruits, vegetables, and herbs. Several key variables were defined: drinking untreated water was defined as having drunk other than commercially bottled water that had not been boiled, chlorinated, or filtered in the household;
water source was divided into high risk (public standpipe, well, spring, or water bought from a vendor) and low risk (in-house piped municipal water or commercially bottled water). Among children <5 years of age, we defined breastfed as having been breastfed at all, whether exclusively or partially, at the time the child provided the specimen.

Laboratory Methods

Stool specimens were processed by using a standard formalin-ethyl acetate concentration procedure and screened by two methods: light-microscopy examination of a modified acid-fast stained smear and UV epifluorescence examination of a wet mount (18). To confirm the identity of the parasite as C. cayetanensis, three positive specimens stored in 2.5% dichromate at ambient temperature (approximately 23C) were examined at regular intervals over a 2-week period, starting from the time the specimen was provided (18). We observed characteristic sporulation in all three.

Analysis of Water Samples

From May 13 to July 17, 1997, we collected a weekly 10-liter water sample from each of three rivers (Villa Lobos, Los Verdes, and Guacalate). Two sampling sites were located in the Department of Guatemala and one in the Department of Sacatepequez; these rivers all contain raw sewage outfalls. Standard methods were used to quantify the level of fecal contamination with Escherichia coli (19). The specimens were concentrated by the flocculation method developed for detection of Cryptosporidium in water (20), with the following modifications for logistical reasons: the specimens were left to flocculate overnight; the centrifugation speed was 600 g, the maximum for our centrifuge, rather than 3,000 g; and we used
only one washing with Tween. The concentrated sediments were
examined by bright-field and UV epifluorescence microscopy for Cyclospora oocysts.

Data Analysis

All data were entered in Epi-Info 6.04b. Surveillance data were analyzed in Epi-Info and in Excel spreadsheets. The case-control data were analyzed in SAS 6.12. Potential risk factors were assessed in a series of univariate analyses; unless otherwise specified, the significance of differences was tested by Mantel-Haenszel chi-square test. We evaluated potential interactions among variables through a series of stratified analyses before constructing models that included interaction terms. Factors that were significant at p<0.10
level were then examined in stepwise regression models. The final multivariable model was chosen from alternative models resulting from the stepwise regressions, including interaction terms when indicated.

Results

Hospital and Health-Center Surveillance

From April 6, 1997, to March 19, 1998, we enlarged screened 5,552 specimens from health-care facilities. Overall, Cyclospora was detected in 126 (2.3%) specimens. The rate of Cyclospora detected began to rise (3.8%)in May and peaked 
at 6.7% in June (Figure). This period corresponds to the first 2 months of the rainy season in Guatemala City. The prevalence remained above 3% in July and August and fell
to undetectable levels after mid-November. In January and February, Cyclospora was detected in fewer than 1% of specimens.

Figure. Surveillance for Cyclospora cayetanensis infection
in stool specimens from three hospital outpatient departments and two health centers in Guatemala, April 6,
1997, to March 19, 1998. From the bottom, the three graphs demonstrate the Cyclospora detection rate, mean temperature
in centigrade, and rainfall in mm by month. Median number
of specimens per month 444 (324-638).

Cyclospora infection was more frequent among 
children and among persons with gastroenteritis
(Table 1). The prevalence of Cyclospora was 
3.5% in specimens from children 1.5 to 4 years
of age and 3.8% among children 5 to 9 years of age, irrespective of symptom status. Children 
in these age groups were five times more likely
than adults screened in the surveillance system 
to have Cyclospora infection. Persons with
gastroenteritis were two to three times more
likely than asymptomatic persons of the same
age to have Cyclospora. Cyclospora infection
was relatively uncommon among children <18
months of age (1.0%). A substantial proportion
of gastroenteritis cases during the early rainy 
season was associated with Cyclospora
infection: from May 1 to June 30, 30 (19%)of
160 children 1.5 to 9 years old with
gastroenteritis had Cyclospora detected in their stool. The overall rate of infection did not differ significantly by sex (44 [2.1%] of 2,067 male versus 57 [1.7%] of 3,266 female patients [p = 0.32]).

Although we did not collect data concerning HIV status, one hospital submitted specimens for screening from a clinic that served the HIV-infected population in Guatemala City. Of 32 specimens from adult patients attending this clinic, 9 (28%) were positive for Cyclospora, a much higher rate than that detected among other adults at the same hospital (2 [0.4%] of 448; RR 87, 95% CI 16.2, 847). Seasonality among these patients was the same as that observed for other participants in the surveillance system: eight of the nine
Cyclospora detections occurred during May to August. These patients were not included in the case-control study.

Raspberry Farm Cohort

The raspberry farm cohort comprised 164 workers and 18 family members of workers who submitted specimens from April 6 to May 29, 1997. All 176 workers on the three farms agreed to participate; however, not all submitted specimens. We collected 269 specimens from workers and 31 from their family members, a median of one specimen per person (1 to 4). Six of 182 persons had an episode of infection with Cyclospora. Of the six infections, two occurred in family members, ages 1 and 10 years; both had gastroenteritis. The
other four Cyclospora infections occurred in farm workers; all but one were asymptomatic. On May 29, 1997, the Guatemalan Berry Exporters Commission required us to suspend surveillance.

Case-Control Study

During the case-control study period, we recruited 69 persons with Cyclospora infection and 125 controls. Five controls were excluded because they had gastrointestinal symptoms when their specimens were submitted, leaving 69 cases and 120 controls for the analysis. Of 69 cases, 33 (48%) were in patients < 5 years of age and 26 (38%) were
in male patients; among 120 controls, 56 (47%) were <5 years of age and 56 (47%) were male (p = 0.23).

Table 1. Prevalence of Cyclospora in specimens from outpatients attending three hospitals and two health centers. April 6, 1997, to March 19, 1998

In addition to specimens from the case-control study, we screened stool specimens from 182 family members belonging to 56 families of case-patients. Of these, 14 persons from 11 families tested positive for Cyclospora. The detection rate was highest among children 1.5 to 9 years (10 [11.6%] of 86). There were no cases among 14 family members <18 months of age and few among adults >14 years of age (2
[4%] of 57). The rate among family members ages 1.5 to 9 years did not differ from that among children of the same age in surveillance data from the same period (64 [9%] of 714; p = 0.42).

Table 2. Characteristics of illness
reported by 62 persons with Cyclospora
infection who participated in the
case-control study, Guatemala, May 7-
September 3, 1997
--------------------------------------------
                       Age group of
                        participants
                      <5 years >/=5 years
Characteristic         (n = 32) (n = 30)    p
--------------------------------------------
Days of illness          15	     10
	                  (3-76)a  (2-90)a  0.05b
Stools per day            5         3
                        (1-10)a  (1-20)a  0.04b

Stool consistency                          nsc
Liquid/watery             22 (69)  18 (60)
Semisolid                 10 (31)   6 (20)
Solid                      0 (0)    4 (13)
Blood in stool             7 (22)   1 (3)   0.05
Mucus in stool            29 (91)  11 (37)  <0.001
Fever                     24 (75)   9 (30)  <0.001
Vomiting                  16 (50)   8 (27)   ns
Abdominal pain            10 (31)  13 (43)   ns
Anorexia                   8 (25)   7 (23)   ns
Headache                   2 (6)    7 (23)   ns
Treated before
visiting
health-care
facility with
Any antibiotic            22 (69)  15 (50)   ns
Metronidazole             11 (34)   9 (30)   ns
Cotrimoxazole              1 (3)    4 (13)   ns
Mebendazole                7 (22)   2 (7)    ns
or albendazole
--------------------------------------------
aMedian (range).
bBy Wilcoxon 2-sample test
cns = not significant.

Data were available on characteristics of illness in 62 patients in the case-control study (Table 2). The median duration of illness before diagnosis was 15 days in those <5 years of age and 10 days in those >/=5 years of age. Young children also tended to have a higher median number of stools per day than older patients and were significantly more likely to have fever and mucoid stools. Of the 62
patients, 40 (65%) reported predominantly watery or liquid stools, but 33 (53%) reported that diarrhea was intermittent. Of the 62 for whom data were available, 37 (60%) had been treated with an antibiotic, most often metronidazole, before Cyclospora was diagnosed.

Table 3. Univariate analysis of factors associated with risk for Cyclospora infection among 69 cases and 120 controls in Guatemala

A number of variables were associated with risk for Cyclospora infection in univariate analyses; all were related to water, sanitation, or presence of animals in the household (Table 3). Persons with Cyclospora infection were significantly more likely than controls to report having drunk untreated water in the 2 weeks before illness, having obtained drinking water from a high-risk source, or having swum in or drunk water from a river or spring. In addition, having a septic tank rather than municipal sewage drainage
and having had direct contact with soil were associated with an elevated risk for infection. Persons with Cyclospora infection were twice as likely as controls to own dogs, chickens, or other fowl; other animals such as cats and pigs were not associated with increased risk for infection. We asked about eating 16 kinds of fresh, uncooked produce in the 2 weeks before illness, including raspberries, blackberries, lettuce, cabbage, mint, and cilantro;
none was associated with an elevated risk for Cyclospora infection.

Among children <5 years of age, maternal education was protective: 10 of 33 children with cyclosporiasis (compared with 31 of 55 controls) had mothers with 6 or more years of education (OR 0.34, 95% CI 0.12, 0.92). Breastfeeding also had a protective effect, although not of statistical importance: 3 of 33 case patients were breastfed, compared with 13 of 52 controls (OR 0.30, 95% CI 0.05, 1.25).

Table 4. Multivariable logistic regression model
of risk factors for Cyclospora infection among 68
cases and 120 controls
--------------------------------------------------
Characteristic    OR (95% CI)a p
--------------------------------------------------

Drank untreated water  4.2 (1.4,    0.009
    12.5)

Chickens1.9 (1.0,    0.054
    3.7)

Contact with soil among19.8 (2.2,   0.008
    182)
[Image]children <2

years old

Contact with soil among1.4 (0.6,    nsc
    2.9)
persons > 2 years oldb
--------------------------------------------------
aModel adjusted for age category (<2 years, >2
years).
bOdds ratio for persons 2 years or older
calculated as (OR for persons <2) *(OR for
interaction term for soil contact with age <2). p
value for the interaction term = 0.03.
cns = not significant.

In stepwise regression analyses, the variables that remained
significant were drinking untreated water and, among young children, contact with soil (Table 4). Drinking untreated water was associated with a fourfold increase in risk. For contact with soil, there was a significant interaction with age, carrying a 20-fold increased risk for Cyclospora infection among children <2 years of age but not among those 2 years of age or older (p for interaction term = 0.03).
The presence of chickens in the household was associated with a twofold increased risk but did not reach statistical significance in the multivariable model (p = 0.054). The presence of chickens, poultry, or other birds in the household could be substituted for chickens in the model with similar results (OR 1.7; p = 0.09). The results of the model were unchanged when data were adjusted for  socioeconomic variables, including educational level (for adult participants and of mother for children <15 years of age), housing type, and ownership of items such as radio, television, bicycle, or motor vehicle.

River Water Analysis

All three rivers had evidence of heavy fecal contamination
throughout the period of study, with mean E. coli counts of 4.7x105, 1.9x107, and 4.5x106 colonies per 100 cc in the Department of Guatemala sampling sites and the Department of Sacatepequez site, respectively. We detected both sporulated and unsporulated Cyclospora oocysts in two of the 30 specimens, one specimen from each of the two rivers in the Department of Guatemala; both positive specimens were collected on May 26, 1997. The number of oocysts seen
was small (2 oocysts and 3 oocysts, respectively). However,
calculating from the amount of sediment obtained in each specimen (40 and 20 ml), the amount of sediment examined (20 and 40 l), and the recovery rate of oocysts (5% to 10%; estimated from studies of seeded specimens, M. Alvarez de Mejia, M.B. Lopez, unpub. results), we estimate that these findings may represent actual concentrations of 15,000 or more oocysts per 10-liter specimen.

Conclusions

This study advances our understanding both of the epidemiology of C. cayetanensis in a disease-endemic area and of the context in which repeated outbreaks of foodborne cyclosporiasis have occurred in North America. We now know that Cyclospora is commonly associated with pediatric gastroenteritis in Guatemala, as it is in Peru, Nepal, and other developing countries (1,2,5,21). Moreover, as in
Peru and Nepal, the organism displays a marked seasonality. In Guatemala, the prevalence of Cyclospora in specimens submitted to health-care facilities increased around the time of the spring raspberry harvest in April-June (9). In June, we recorded weekly detection rates as high as 10%. In our surveillance data from May through June, 19% of cases of pediatric gastroenteritis were associated with Cyclospora. We were also able to detect oocysts in surface water specimens, despite the fact that the available methods
for detection in water have very low sensitivity (22). By October, the detection rate in human surveillance specimens had dropped below 1%. These data suggest that the association of Cyclospora outbreaks with raspberries from the spring but not the fall harvest may be related to the inherent seasonality of the organism.

A major limitation of our study was our inability to test for other diarrheal pathogens. We cannot be certain that every gastroenteritis episode during which we detected Cyclospora was due to Cyclospora. However, in the aggregate data, we observed a consistent epidemiologic and clinical pattern associated with detections of Cyclospora.

Although 1 year of surveillance is insufficient to say with
certainty what the pattern of Cyclospora infections will be from year to year, the data so far suggest that the seasonality of Cyclospora in Guatemala City is similar to that in Kathmandu, another subtropical city at approximately the same altitude (1,500 m) above sea level (3,23). In Guatemala as in Kathmandu, the rainy season starts in May or June, with warmer temperatures in the preceding months; in both places, Cyclospora appears around this time. Nevertheless, the organism's seasonality cannot be explained
by rainfall alone, since in Lima an equally marked pattern is seen in the near absence of rain (21). Several years of surveillance in multiple sites with varying climates, as well as a better understanding of the organism's biology in the environment, will be necessary to explain why Cyclospora infections apparently fluctuate with the seasons.

Cyclospora has a number of other remarkable characteristics. It tends to cause prolonged diarrhea (2,3,24). Because all our patients were treated once the diagnosis was made, we could not estimate a total duration of illness; nevertheless, among children <5 years of age, the median length of illness before diagnosis was 15 days. Among children with diarrhea in developing countries, the proportion of persistent episodes (>14 days long) is 3% to 23% depending on the study cited. These episodes, however, carry a higher risk for malnutrition and death than shorter episodes (25-27). Perhaps because of the long duration of illness, persons with Cyclospora gastroenteritis are likely to receive various antibiotics before the correct diagnosis is made: in our study, 70% of children <5 years of age had received at least one antibiotic, and a third of all our
patients had received metronidazole. Prompt diagnosis would avert such inappropriate use of antibiotics.

In North America, patients have been infected with Cyclospora through eating fresh raspberries, pesto dishes, and mesclun lettuce (9,28-30). In Guatemala, the main vehicle of infection appears to be untreated water. That water truly is an important vehicle for Cyclospora in Guatemala is underlined by the other water-related
factors that were associated with infection. Our results support the findings in other studies that contaminated water is a likely source of infection (1,3,31). However, multiple routes of transmission for Cyclospora in Guatemala almost certainly exist. Among very young children, soil contact was a strong risk factor; an outbreak   investigation in the United States also raised the possibility that soil might be a potential source of infective oocysts (6). Family members of patients had a rate of infection similar to that of persons in the same age group screened in our surveillance system at the same time of year, a finding consistent with the postulated lack
of direct person-to-person transmission.

The fact that the presence of chickens or other domestic fowl was a significant risk factor is intriguing but difficult to interpret. Scientists have failed to establish experimental C. cayetanensis infection in any bird; a limited survey of 110 wild birds captured in May 1997 in Guatemala did not demonstrate natural infection,although other coccidia were observed (M. Eberhard, pers. comm.). Although the association remains when the data are adjusted for socioeconomic status, we cannot rule out the possibility that ownership of fowl is a marker for some other unidentified factor. The role of birds in Cyclospora transmission merits further investigation.

C. cayetanensis is a pathogen commonly associated with pediatric gastroenteritis in Guatemala, especially from May through August. The seasonality of Cyclospora in Guatemala follows a pattern similar to that seen in Kathmandu. Our case-control analysis suggests that contaminated water and, for young children, soil, are likely vehicles of transmission. More sensitive diagnostic tools areurgently needed to help establish how fresh produce becomes
contaminated.

Acknowledgments

The authors thank the following members of the Cyclospora
surveillance team in Guatemala for their contributions to the study: Dolores del Rio, Miriam Galicia Guzman, Norma Patricia Lopez Sierra, Liliana Godoy de Alvarez, Nazario Lopez, Jorge Sincal, Mirna Elisa Ochoa, Eduardo Ibaez, Olga Yolanda Flores, Gilberto Davila, Rudinio Acevedo, Cedric Heriberto Lopez, Katherine Alvarado Illescas, Efrain
de Paz, Silvia Ramirez, Lorena de Monserrat Mart, and Gladys Chali de Samayoa; and Enriqueta Parinello, Mildred Chavez, Laura Hall, and Monica Santos for secretarial support. We also thank Mark Eberhard, Sue Binder, Tom Navin, Bob Gilman, and Greg Sturbaum for advice on scientific aspects of the study.

The study protocol was reviewed and approved by the Centers for Disease Control Institutional Review Board, as well as by local review committees required in Guatemala. In addition, for the part of the study conducted on raspberry farms, agreements were signed with the Guatemalan Berry Exporters Commission and the three participating farm owners regarding the terms of work, including human subjects protection and confidentiality.

Dr. Bern is a medical epidemiologist in the Division of Parasitic Diseases, CDC. Her research interests include the epidemiology of the enteric parasites Cyclospora, Cryptosporidium, and microsporidia.

Address for correspondence: Caryn Bern, National Center for
Infectious Diseases, Centers for Disease Control and Prevention, Mail Stop F22, Atlanta, GA 30333, USA; fax: 770-488-7761; e-mail: cxb9@cdc.gov.

References

1. Hoge CW, Echeverria P, Rajah R, Jacobs J, Malthouse S, Chapman E, et al. Prevalence of Cyclospora species and other enteric pathogens among children less than 5 years of age in Nepal. J Clin Microbiol 1995;33:3058-60.

2. Ortega YR, Sterling CR, Gilman RH, Cama VA, Diaz F. Cyclospora species--a new protozoan pathogen of humans. N Engl J Med 1993;328:1308-12.

3. Hoge CW, Shlim DR, Rajah R, Triplett J, Shear M, Rabold JG, et al. Epidemiology of diarrhoeal illness associated with coccidian-like organisms among travellers and foreign residents in Nepal. Lancet 1993;341:1175-9.

4. Soave R. Cyclospora: an overview. Clin Infect Dis 1996;23:429-37.

5. Soave R, Herwaldt BL, Relman DA. Cyclospora. In: Hughes JM, Conte JE, editors. Infectious disease clinics of North America. Vol 12, No. 1. Philadelphia: W.B. Saunders Company; 1998.

6. Koumans EHA, Katz DJ, Malecki JM, Kumar S, Wahlquist SP, Arrowood MJ, et al. An outbreak of cyclosporiasis in Florida 1995: a harbinger of multistate outbreaks in 1996 and 1997. Am J Trop Med Hyg 1998;59:235-42.

7. Huang P, Weber JT, Sosin DM, Griffin PM, Long EG, Murphy JJ, et al. The first reported outbreak of diarrheal illness associated with Cyclospora in the United States. Ann Intern Med 1995;123:409-14.

8. Carter R, Guido F, Jacquette G, Rapoport M. Outbreak of
cyclosporiasis associated with drinking water. Proceeding of the 30th Interscience Conference on Antimicrobial Agents and
Chemotherapy; 1996 Sep 15-18; New Orleans, Louisiana. Washington: American Society for Microbiology, 1996.

9. Herwaldt B, Ackers ML, the Cyclospora Working Group. An outbreak in 1996 of cyclosporiasis associated with imported raspberries. N Engl J Med 1997;336:1548-56.

10. Herwaldt BL, Beach MJ, the Cyclospora Working Group. The return of Cyclospora in 1997: another outbreak of cyclosporiasis in North America associated with imported raspberries. Ann Intern Med 1999;130:210-20.

11. Osterholm MT. Cyclosporiasis and raspberrieslessons for the future [editorial]. N Engl J Med 1997;336:1597-8.

12. Ortega YR, Roxas CR, Gilman RH, Miller NJ, Cabrera L, Taquiri C, et al. Isolation of Cryptosporidium parvum and Cyclospora cayetanensis from vegetables collected in markets of an endemic region in Peru. Am J Trop Med Hyg 1997;57:683-6.

13. Pratdesaba RA, Velaquez T, Torres MF. Occurrence of Isospora belli and cyanobacterium-like bodies in Guatemala. Ann Trop Med Parasitol 1994;88:449-50.

14. Arathoon E, Velasquez T, Estrada y Martin RM, Mayorga R.
Cyclospora cayetanensis, un nuevo patgeno causante de diarrea en pacientes infectados por el VIH. Revista del Colegio de Mdicos y Cirujanos de Guatemala 1994;4:36-7.

15. Merida SC. Prevalencia de Cyclospora en nios menores de 60 meses. Doctor of Medicine Thesis, School of Medicine, Universidad Francisco Marroquin, Guatemala City; 1998, 59 pages.

16. Cuellar NS. Prevalencia de infecciones intestinales causadas por coccidios: Cryptosporidium spp, Cyclospora cayetanensis, e Isospora belli en pacientes con SIDA. Masters Thesis, Faculty of Laboratory Sciences and Pharmacy, Universidad de San Carlos de Guatemala, Guatemala City, 1997, 71 pages.

17. Alvarado KM. Cyclospora cayetanensis como agente causal de diarrea en pacientes de la consulta externa del Hospital General San Juan de Dios. Masters Thesis, Faculty of Laboratory Sciences and Pharmacy, Universidad de San Carlos de Guatemala, Guatemala City, 1997, 41 pages.

18. Eberhard ML, Pieniazek NJ, Arrowood MJ. Laboratory diagnosis of Cyclospora infections. Arch Pathol Lab Med 1997;121:792-7.

19. Environmental Protection Agency. Test Methods for Escherichia coli and enterococci in water by the membrane filter procedure. Manual EPA-600/4-85/076: Environmental Monitoring and Support Laboratory, 1985.

20. Vesey G, Slade JS, Byrne M, Shepherd K, Fricker CR. A new method for the concentration of Cryptosporidium oocysts from water. J Appl Bacteriol 1993;75:82-6.

21. Madico G, McDonald J, Gilman RH, Cabrera L, Sterling CR.
Epidemiology and treatment of Cyclospora cayetanensis infection in Peruvian children. Clin Infect Dis 1997;24:977-81.

22. Sturbaum GD, Ortega YR, Gilman RH, Sterling CR, Cabrera L, Klein DA. Detection of Cyclospora cayetanensis in wastewater. Appl Environ Microbiol 1998;64:2284-6.

23. Connor BA, Shlim DR, Scholes JV, Rayburn JL, Reidy J, Rajah R. Pathologic changes in the small bowel in nine patients with diarrhea associated with a coccidia-like body. Ann Intern Med 1993;119:377-82.

24. Baqui AH, Sack RB, Black RE, Haider K, Hossain A, Alim ARMA, et al. Enteropathogens associated with acute and persistent diarrhea in Bangladeshi children under 5 years of age. J Infect Dis 1992;166:792-6.

25. Lima AAM, Fang G, Schorling JB, de Albuquerque L, McAuliffe JF, Mota S, et al. Persistent diarrhea in Northeast Brazil: etiologies and interactions with malnutrition. Acta Paediatr Supple 1992;381:39-44.

26. Black RE. Persistent diarrhea in children of developing
countries. Pediatr Infect Dis J 1993;12:751-61.

27. Cruz JR, Bartlett AV, Mendez H, Sibrian R. Epidemiology of persistent diarrhea among Guatemalan rural children. Acta Paediatr Suppl 1992;381:22-6.

28. Centers for Disease Control and Prevention. Update: Outbreaks of cyclosporiasisUnited States and Canada, 1997. MMWR Morb Mortal Wkly Rep 1997;46:521-2.

29. Centers for Disease Control and Prevention. Update: Outbreaks of cyclosporiasisUnited States, 1997. MMWR Morb Mortal Wkly Rep 1997;46:461-2.

30. Centers for Disease Control and Prevention. Outbreaks of
cyclosporiasisUnited States, 1997. MMWR Morb Mortal Wkly Rep 1997;46:451-2.

31. Rabold JG, Hoge CW, Shlim DR, Kefford C, Rajah R, Echeverria P. Cyclospora outbreak associated with chlorinated drinking water [letter]. Lancet 1994;344:1360.
Emerging Infectious Diseases National Center for Infectious Diseases Centers for Disease Control and Prevention
Atlanta, GA URL: http://www.cdc.gov/ncidod/eid/vol5no6/ascii/bern.txt

Please note that figures, equations, and most tables are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively.  Greek symbols are spelled out. >/= is used for greater than or equal to.

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

Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999]  

 Research

   Serologic Evidence of Human Monocytic and Granulocytic
   Ehrlichiosis in Israel

   Avi Keysary,* Lili Amram, Gershon Keren, Zev
   Sthoeger, Israel Potasman, Amir Jacob,# Carmella
   Strenger,* Jacqueline E. Dawson,** and Trevor Waner*

   *Israel Institute for Biological Research, Ness Ziona,
   Israel; Asaf Harofe Medical Center, Tzrifin, Israel;
   Sheba Medical Center, Tel Hashomer, Israel; Kaplan
   Hospital, Rehovot, Israel; Bnei-Zion Medical Center,
   Haifa, Israel; #Schneider Children's Medical Center of
   Israel, Petach Tikvah, Israel; and **Centers for
   Disease Control and Prevention, Atlanta, Georgia, USA

    We conducted a retrospective serosurvey of
    1,000 persons in Israel who had fever of
    undetermined cause to look for Ehrlichia
    chaffeensis antibodies. Four of five cases
    with antibodies reactive to E. chaffeensis
    were diagnosed in the summer, when ticks
    are more active. All patients had
    influenzalike symptoms with high fever.
    None of the cases was fatal. Three serum
    samples were also seroreactive for
    antibodies to E. canis, and one was also
    reactive to the human granulocytic
    ehrlichiosis (HGE) agent. The titer to the
    HGE agent in this patient was higher than
    the serum titer to E. chaffeensis, and the
    Western blot analysis also indicated that
    the HGE agent was the primary cause of
    infection. We present the first serologic
    evidence that the agents of human monocytic
    ehrlichiosis (HME) and HGE are present in
    Israel. Therefore, human ehrlichiosis
    should be included in the differential
    diagnoses for persons in Israel who have
    been exposed to ticks and have
    influenzalike symptoms.

   Human ehrlichiosis (HME) and human granulocytic
   ehrlichiosis (HGE), two emerging infectious diseases
   transmitted by ticks, are caused by Ehrlichia
   chaffeensis and the HGE agent of the E. phagocytophila
   genogroup, respectively. In the United States, HME and
   HGE were first described in 1987 and 1994, respectively
   (1). Since then, seroepidemologic studies have shown
   that these infections are also present in other parts
   of the world.

   The first cases of HME and HGE were reported in Europe
   in 1991 and 1995, respectively (2,3). Serologic
   evidence of HGE has been found in Norway and Sweden
   (4). In South America, a case of E. canis infection was
   reported in Venezuela (5). One clinical case of HME has
   been reported in Mali, Africa (6). A serosurvey for HME
   of 756 patients from eight African countries suggested
   that the disease is rare in Africa (7).

   We describe the first serologic survey in Israel for
   HME and HGE, which documents the detection of
   antibodies reactive with HME and HGE agents.

   Materials and Methods

   Sera

   One thousand serum samples from patients in Israel with
   fever of undetermined cause from 1994 to 1997 were
   received by the Israel National Reference Laboratory
   for Rickettsial Diseases. All specimens were
   serologically negative for Mediterranean spotted fever,
   murine typhus, and Q fever.

   Serology

   The sera were tested retrospectively for immunoglobulin
   (Ig) G antibodies to E. chaffeensis and E. canis by
   indirect immunofluorescence antibody (IFA) (8).
   Briefly, DH82 cells heavily infected with the Israeli
   strain of E. canis (#611) (8) or the Arkansas strain of
   E. chaffeensis were pelleted and resuspended in growth
   medium. Five microliters of the suspension were placed
   in each well of eight-well teflon-coated slides. The
   slides were dried at room temperature for approximately
   30 minutes, fixed in acetone for 15 minutes, and then
   stored at 40C. Serum samples were assayed for IgG by
   preparing and testing serum dilutions in PBS at their
   cutoff points of 1:64 for E. chaffeensis and 1:40 for
   E. canis. Positive sera were subsequently assayed at
   twofold dilutions. Positive control sera were provided
   by the Centers for Disease Control and Prevention
   (CDC).

   Serum samples were sent to CDC for confirmation of
   results of the HME titers and for testing for HGE.

   Western Blot Analysis

   One patient (#3) sample found positive for HGE was
   tested by Western blot at Johns Hopkins University,
   Baltimore, Maryland.

   Results

   Of the 1,000 sera tested, five were found seropositive
   with HME (Table). During validation of the sera for HME
   antibodies, the CDC laboratory also found that one
   patient (#3) had an antibody titer of 1:2,048 to HGE.
   This sample was confirmed positive for HGE by Western
   blot.

   None of the patients documented in this study had
   traveled overseas before their illness. All five cases
   occurred in persons, three male and two female, who
   lived on the coastal plain. Two of the three men lived
   in agricultural settlements. The average age of
   patients was 34.2 years (8 to 77 years). Four of the
   five patients were ill during summer.

   The disease lasted up to 14 days. None of the cases was
   fatal. None of the patients reported being bitten by a
   tick. All patients had fever from 38.5C to 40.2C.
   Clinical signs were inconsistent: macular rash was
   present in only three patients and lymphomegaly in two.
   Four patients were leukopenic, and two were also
   thrombocytopenic. No changes in liver enzymes were
   detected in any of the patients.

   The antibody titers to E. chaffeensis were 1:128 to
   1:1,024. Similar results were obtained by CDC. Three of
   the five sera were also seropositive for E. canis
   antibodies; however, their titers were lower than those
   to E. chaffeensis.

Table. Clinical and serologic data for patients in Israel with antibodies to Ehrlichia chaffeensis and the human granulocytic ehrlichiosis agent
Conclusions

We retrospectively looked for E. chaffeensis antibodies in human patients with fever of undetermined cause. Four cases of HME were found, as well as a possible case of HGE with cross-reacting antibodies to HME.

Four of the five cases were diagnosed in summer, during peak tick activity. Patients' ages were 8 to 77 years. Symptoms were nonspecific, as has been described (1). All patients had influenzalike symptoms with high fever. Leukopenia was seen in four patients and thrombocytopenia in two; both these hematologic changes are typical of HME.

In our study three sera positive for E. chaffeensis were also seropositive for E. canis, unlike the African study in which all E. chaffeensis-positive sera were seronegative to E. canis in spite of the known strong cross-reactivity between the strains (7). The reason for this lack of cross-reactivity is unknown; however, reactivity in E. chaffeensis patients to E. canis antigens may develop only after prolonged exposure to the Ehrlichia, allowing expression of common antigens to be revealed.

In one seropositive E. chaffeensis case (#3), the titer to the HGE agent was higher than to E. chaffeensis and Western blot analysis for HGE was positive, which indicates that the HGE agent was the primary cause of infection. Serologic reactions with E. chaffeensis have been demonstrated after HGE infection (9). Cross-reaction between the two species of Erhlichia has been found in a small proportion of all HGE patients tested, which suggests that the causative Ehrlichiae share antigenic determinants.

Several tick species of the genus Ixodes are found in Israel, including the I. ricinus, which is the vector of the disease in Europe, and I. redikorzevi, which often bites humans (10,11). Rhipicephalus sanguineus, which is abundant in Israel, is a potential vector of HME agent. Coinfection by a number of tickborne diseases is not uncommon, and persons may be infected by both HME and HGE agents simultaneously.

A recent serosurvey of jackals in Israel tested against E. canis, E. chaffeensis, and E. phagocytophila genogroup antigens has shown that some jackals were seroreactive only to the E. phagocytophila genogroup antigen (12). The latter group of Ehrlichia consists of E. equi, E. phagocytophila, and the HGE agent (13). A close serologic and genetic relationship has been shown to exist among these three
members, suggesting that they may be strains of a single species (1). The finding in jackals adds further evidence of one or more of the E. phagocytophila genogroup of Ehrlichiae in Israel.

In conclusion, we have presented the first serologic evidence that the agents of HME and HGE are present in Israel. Human ehrlichiosis should therefore be included in the differential diagnoses for persons in Israel who have been exposed to ticks and have influenzalike symptoms.

Acknowledgments

We thank J.E. Dawson for her help in confirming the titers, and J.S. Dumler for the Western blot analysis.

Dr. Keysary is head of the Israel National Reference Laboratory of Rickettsial Diseases. His interests include diagnosis of infections caused by Rickettsia, Coxiella, and Ehrlichia.

Address for correspondence: Trevor Waner, Israel Institute for Biological Research, P.O. Box 19, Ness Ziona 70400, Israel; fax: 972-8-940-1443; e-mail: wanertnt@shani.net.

References

1. Walker DH, Babour AG, Oliver JH, Lane RS, Dumler JS, Dennis DT, et al. Emerging bacterial zoonotic and vector-borne diseases. JAMA 1996;275:463-9.

2. Morais JD, Dawson JE, Greene C, Filipe AR, Galhardas LC, Bacellar F. First European case of ehrlichiosis [letter]. Lancet 1991;338:633-4.

3. Brouqui P, Dumler JS, Leinhard R, Brossard M, Raoult D. Human granulocytic ehrlichiosis in Europe. Lancet 1995;346:782-3.

4. Bakken JS, Krueth J, Tilden RL, Dumler JS, Kristiansen BE. Serological evidence of human granulocytic ehrlichiosis in Norway. Eur J Clin Microbiol Infect Dis 1996;15:829-32.

5. Arraga-Alvarado C, Montero-Ojeda M, Bernardoni A, Parra 0. Human ehrlichiosis: report of the first case in Venezuela. Invest Clin 1996;37:35-49.

6. Uhaa IJ, MacLean JD, Greene CR, Fishbein DB. A case of human ehrlichiosis acquired in Mali: clinical and laboratory findings. Am J Trop Med Hyg 1992;46:161-4.

7. Brouqui P, Lecam C, Kelly PJ, Laurens R, Tounkara A, Sawadogo S, et al. Serologic evidence for human ehrlichiosis in Africa. Eur J Epidemiol 1994;10:695-8.

8. Keysary A, Waner T, Rozner M, Dawson JE, Zass R, Warner CK, et al. The first isolation, in vitro propagation, and genetic characterization of Ehrlichia canis in Israel. Vet Parasitol 1996;62:331-40.

9. Dumler JS, Brouqui P. Human granulocytic ehrlichiosis. In: Anderson B, Friedman H, Bendinelli M, editors. Rickettsial infection and immunity. New York and London: Plenum Press; 1997. p. 149-61.

10. Reed KD, Mitchell PD, Persing DH, Cameron V. Transmission of human granulocytic ehrlichiosis. JAMA 1995;273:23.

11. Theodor O, Costa M. In: Ectoparasites. A survey of the parasites of wild mammals and birds in Israel. Part 1. Jerusalem: The Israel Academy of Sciences and Humanities; 1997. p. 92-103.

12. Waner T, Beneth G, Strenger C, Keysary A, King R, Harrus S. Antibodies reactive with Ehrlichia canis, Ehrlichia phagocytophila genogroup antigens and the spotted fever group antigens, in free-ranging jackals (Canis aureus syriacus) from Israel. Vet Parasitol 1999;82:121-8.

13. Dumler JS, Asanovich KM, Bakken JS, Richter P, Kimsey R, Madigan JE. Serologic cross-reactions among Ehrlichia equi, Ehrlichia phagocytophila, and human granulocytic ehrlichia. J Clin Microbiol 1995;33:1098-103.

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

URL: http://www.cdc.gov/ncidod/eid/vol5no6/ascii/keysary.txt

Please note that figures, equations, and most tables are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively.  Greek symbols are spelled out. >/= is used for greater than or equal to.

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

Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999]

Research

Supplementing Tuberculosis Surveillance with Automated Data from Health Maintenance Organizations

Deborah S. Yokoe,* Girish S. Subramanyan,* Edward Nardell, Sharon Sharnprapai, Eugene McCray, and Richard Platt*

*Brigham and Women's Hospital, Boston, Massachusetts, USA; Massachusetts Department of Public Health, Jamaica Plain, Massachusetts, USA; Centers for Disease Control
and Prevention, Atlanta, Georgia, USA; and Harvard Medical School, Harvard Pilgrim Health Care, Boston, Massachusetts, USA

  Data collected by health maintenance organizations (HMOs), which provide care for an increasing number of persons with tuberculosis TB), may be used to complement traditional TB surveillance. We evaluated the ability of HMO-based surveillance to contribute to overall TB reporting through the use of routinely collected automated data for approximately 350,000 HMO members. During approximately 1.5 million person-years, 45 incident cases were identified in either HMO or public health department records. Eight
(18%) confirmed cases had not been identified by the public health department. The most useful screening criterion (sensitivity of 89% and predictive value positive of 30%) was dispensing of two or more TB drugs. Pharmacy dispensing information routinely collected by many HMOs appears to be a useful adjunct to traditional TB surveillance, particularly for identifying cases without positive microbiologic results that may be missed by traditional public health surveillance methods.

As more persons move into managed health-care organizations, traditional tuberculosis (TB) surveillance methods, which rely heavily on information collected and channeled through the public health system, may need to be supplemented.
Accurate, complete surveillance information is important for identification and follow-up of persons with TB, as well as for accurate assessment of the impact of TB on public health, the effectiveness of control activities, and the planning and prioritizing of interventions.

Automated data routinely collected by managed care organizations may complement TB surveillance obtained through reporting to local and state health departments. In
this study, we evaluated the use of pharmacy dispensing information and other inpatient and ambulatory-patient data routinely collected by managed care organizations for identifying TB cases.

Methods

Study Population

The study population consisted of approximately 350,000 persons with pharmacy coverage who received their care at one of the 14 Harvard Pilgrim Health Care centers with automated full-text medical records for ambulatory patients and 100,000 persons with pharmacy coverage at 17 practices without such records within Massachusetts from January 1, 1992, to June 30, 1996. Automated pharmacy and billing
data, however, were available for the entire study population. 

Identification of TB Cases from HMO Records

Ambulatory care, hospital, and emergency room claims for the entire study population were screened for any of 60 International Classification of Diseases,, 9th Revision
Clinical Modification diagnosis codes or current procedures terminology codes suggestive of TB. Automated pharmacy records were searched for dispensing of any of 10 antituberculosis medications during the study period (Table 1). The automated ambulatory-patient record, available for approximately 250,000 persons within our study population, has been described in detail (1). The automated medical record system uses standardized forms that are completed for every patient encounter at specific Harvard Pilgrim Health Care centers, including telephone calls, office visits, urgent care visits, and hospitalizations. For each encounter, the provider either writes in or selects from a list of all coded diagnoses, tests, procedures,
and prescriptions and enters additional comments as free text. The automated ambulatory-patient records were also screened for any one of 17 coded diagnoses, tests, and procedures suggestive of TB (Table 1).

Table 1. Components of the health maintenance organization-based screening criteria for tuberculosis (TB)
Twelve combinations of screening codes suggestive of active TB were used for automated ambulatory-patient records, and five combinations of screening codes were used for other records (Table 2). To limit the number of persons meeting screening criteria, we focused on combinations of codes likely to have the highest yield of TB cases. Cases that met any of these screening criteria were assessed further.

Full-text ambulatory-patient medical records were reviewed for all persons identified by screening criteria who had automated ambulatory records. For individuals identified through screening who did not have automated ambulatory
records, a modified version of the Centers for Disease Control and Prevention's (CDC) Report of Verified Case of Tuberculosis form was sent to the primary-care physicians. The form is routinely used to report to CDC individual TB case information, including clinical characteristics and laboratory results. Our modified form included the question "While under your care, did this patient have suspected
and/or confirmed ACTIVE tuberculosis?" If "Yes" was checked, the full-text medical records of the person were reviewed. In addition, the medical records of a random sample of 10% of the patients with questionnaires returned by providers were reviewed to validate the use of data obtained from questionnaire results. A case of TB was defined according to the CDC surveillance definition (2). A culture-positive
case is defined as isolation of Mycobacterium tuberculosis from a clinical specimen. A smear-positive case is defined as demonstration of acid-fast bacilli (AFB) in a specimen if either a culture was not obtained or results were unknown. In the absence of laboratory evidence of disease, a clinical case is one that meets the following criteria: a positive tuberculin skin test, a completed diagnostic work-up,
clinical evidence and signs and symptoms compatible with TB, an abnormal and unstable (worsening or improving) chest radiograph if intrathoracic disease is present, and treatment with two or more antituberculosis drugs. All cases without a positive culture for M. tuberculosis that were not known to the public health department were verified by review with the Massachusetts State Tuberculosis Control
Officer, using all available primary patient data from the ambulatory-patient medical record, public health records, and hospital records.

Table 2. Performance of health maintenance organization-based screening criteria for tuberculosis (TB)
Identification of TB Cases from Public Health Department Records

Reporting of confirmed or clinically suspected TB cases to the Massachusetts Department of Public Health by health-care providers, laboratories, boards of health, or administrators of hospitals is mandatory. In addition, the Massachusetts
State Laboratory Institute performs susceptibility testing on most M. tuberculosis isolates in Massachusetts and provides the public health department with direct
access to microbiology information about virtually all persons in Massachusetts with culture-positive M. tuberculosis. All verified cases are entered into the public
health TB registry. 

The entire HMO population was matched to the public health TB registry by using limited patient identifiers (first two letters of last name, first two letters of first name, month and year of birth, and sex) to maintain patient  confidentiality. Potential matches were confirmed by using full identifiers. This method for matching registries with minimal disclosure of individual identities is described elsewhere (3).

Analysis

The sensitivity, defined as the proportion of TB cases detected by either HMO-based screening criteria or routine public health surveillance, was determined by comparison with any verified TB case identified through public health or HMO records. Positive predictive value was defined as the proportion of persons with verified TB meeting screening criteria. Exact binomial confidence intervals were
calculated for sensitivity and positive predictive value (4). The performance of the different screening rules for detecting TB was compared.

Results

In approximately 1.5 million person-years, 768 persons met at least one of the HMO-based screening criteria, with a positive screening criteria rate of 0.4 per 10,000 person-years among persons with automated ambulatory-patient records and 0.7 per 10,000 person-years among those without such records. Thirty-nine (9%) incident TB cases were identified among the 415 persons with automated ambulatory-patient records who met screening criteria, and 4 (1%) incident TB cases were identified among the 353 persons without automated ambulatory records who met screening
criteria. The response rate to the provider questionnaire was 100%, as was the agreement rate between classification of TB cases based on provider questionnaire results and on-site medical record review.

Thirty-five (81%) of the 43 incident TB cases detected by HMO-based screening had been identified previously by the public health department. Of these 35 cases, 32 were culture-positive, and three met the clinical case definition. Two additional TB cases, both of which were culture-positive, were known to the public health
department but did not meet HMO-based screening criteria. These two patients received treatment and medication from state-funded TB clinics. Thus, 45 cases were identified through either HMO-based screening or public health department records. All 45 cases met the CDC surveillance definition. Eight (18%) of these cases were unknown to the public health system. Most cases (41 of 45) were diagnosed at one of the HMO centers with automated ambulatory-patient records, a proportion consistent with the concentration of urban regions within their catchment areas. The rates were
approximately 11.7 TB cases per 100,000 population among HMO members with automated ambulatory-patient records and four TB cases per 100,000 population among those without such records.

The sensitivity of each of the screening criteria was 0% to 100%, and the positive predictive value was 0% to 52% (Table 2). Screening criteria based on pharmacy dispensing information had the best combinations of sensitivity and positive predictive value. Two or more dispensed  antituberculosis drugs, combining the results for persons with and without automated ambulatory-patient records, had an overall sensitivity of 89% (95% confidence interval [CI] = 76%, 96%) and positive predictive value of 30% (95% CI = 22%, 39%). Three or more antituberculosis drugs had an overall sensitivity of 84% (95% CI = 71%, 94%) and positive predictive value of 50% (95% CI = 38%, 62%), and two or more antituberculosis drugs dispensed on the same date had an overall sensitivity of 87% (95% CI = 73%, 95%) and positive
predictive value of 36% (95% CI = 27%, 50%). The differences between the performance of two or more dispensed antituberculosis drugs among persons with automated
ambulatory-patient records (sensitivity = 90%, positive predictive value = 34%) and persons without automated records (sensitivity = 75%, positive predictive value =
12%) were not statistically significant, although the small number of TB cases in each group limits the power to detect a difference.

Among the 71 persons with automated ambulatory-patient records who received two or more antituberculosis drugs but did not have incident TB, 9 (13%) had active TB diagnosed outside the study period, 23 (32%) were treated for other mycobacterial infections, 11 (15%) received more than one drug during TB prophylaxis, 2 (3%) received drugs for multiple unrelated conditions (e.g., rifampin for eradication of Staphylococcus aureus; ethambutol for M. avium complex prophylaxis), and the remaining 26 (37%) were suspected of having active TB without subsequent confirmation (Table 3).

Of the 118 persons with automated ambulatory-patient records who met screening criteria involving a diagnosis code for TB but did not have incident TB, 57 received the diagnosis code as an indication of routine prenatal screening for TB, 12 had a previous history of TB, 31 were suspected of having active TB without subsequent confirmation, and 18 had the diagnosis code documented in their HMO ambulatory medical record for no apparent reason (Table 3).

Of the eight patients whose cases had not been identified by the public health department, seven were culture-negative and met the TB clinical case definition, and one did not have a microbiology culture and met the smear-positive TB case definition. Three of the patients had AFB smear-positive pathology specimens; of these, two had negative cultures for M. tuberculosis, and one did not have a culture
performed. Of the eight cases, one involved pulmonary TB, and the remaining seven were extrapulmonary. One patient was 2 years old at the time of diagnosis; the remaining seven were 18 years of age or older. All cases were confirmed by review with the Massachusetts State Tuberculosis Control Officer. Of these eight cases, seven were detected by the two or more antituberculosis drug screening criterion.

Conclusions

Since the establishment of a national surveillance system  for TB in 1953, TB surveillance has depended on laboratories, public health clinics, and reporting by
private practitioners. Several retrospective studies performed by local TB programs suggest that TB cases may be underreported (5-7). Although ascertainment of culture-positive cases is likely to be nearly complete, since laboratories are required by law in most states to report isolation of M. tuberculosis to the state health department, surveillance for cases lacking positive cultures depends largely on reporting by health-care providers or referrals to public health clinics for treatment. Underreporting of TB cases without positive cultures may contribute to
incomplete surveillance. A study assessing the completeness of TB case reporting in Puerto Rico (6) found that 19.5% of patients with TB were not reported, partly because of underreporting of cases without positive cultures for M. tuberculosis. 

The recent shift into managed care of populations at high risk for TB, including Medicaid and Medicare recipients, has raised additional concern about the continued completeness of reporting. However, HMOs routinely collect information that can be used to identify persons likely to have TB. McCray et al. (6) noted that, according to pharmacy prescription data in Maryland, the cases of 19% of patients receiving two or more antituberculosis drugs had not been reported to the public health department; however, the patients' medical records were not reviewed to verify a
diagnosis of active TB. Maggini et al. (8) evaluated the use of Italy's National Health Service pharmacy dispensing information to identify TB cases in the province of Rome and found that pharmacy screening detected seven times more new TB cases than routine passive surveillance. Hripcsak et al. (9) evaluated a number of screening rules based on automated information available at an urban medical center in New York City and found that inpatient use of antituberculosis drugs had a sensitivity of 68% and a positive predictive value of <1% for detecting TB cases based on their health  department's TB registry. These investigators did not,
however, have access to records of antituberculosis drugs received by ambulatory patients and did not specifically evaluate the use of more than one antituberculosis
drug as a screening criterion. No previous study has compared the utility of pharmacy data with that of other automated administrative or health-care data.

Of the screening criteria evaluated in our study, dispensing of two or more antituberculosis drugs was the most useful, with an overall sensitivity of 89%. The most common reasons for dispensing of two or more antituberculosis drugs to persons without TB were the empiric use of antituberculosis drugs for suspected active TB (37%) and the use of antituberculosis drugs for treatment of mycobacterial
infections other than TB (32%). In addition, 15% of patients without TB received more than one drug for TB prophylaxis, which can occur, for example, when isoniazid is switched to another antituberculosis drug because of adverse drug reactions. A possible strategy for improving the positive predictive value of screening criteria based on pharmacy dispensing information is the use of more rigorous criteria, such as dispensing of three or more antituberculosis drugs (positive predictive value = 50%), or restricting the timing of drug dispensing, such as requiring that two or more antituberculosis drugs be dispensed on the same date (positive predictive value = 36%). The improvement in positive predictive value for these more rigorous
criteria, however, must be weighed against loss of sensitivity in identifying TB cases. For our HMO study population, requiring three or more antituberculosis drugs
missed two TB cases, and requiring that two or more drugs be dispensed on the same date missed one case detected by the less stringent criterion. The choice of the screening criterion with the most useful balance between sensitivity and specificity depends in part on the surveillance strategy used.

Surveillance based on HMO pharmacy dispensing information can be used to identify HMO enrollees most likely to have active TB, so that efforts can be focused on additional evaluation of these persons. As with traditional TB surveillance methods, surveillance based on pharmacy dispensing information requires information from the
patients' medical records to verify whether the TB case definition is satisfied. Using this surveillance strategy, screening for two or more antituberculosis drugs
would require reviewing the medical records of approximately three patients to identify each case of incident active TB. We feel that the positive predictive value of 30% is sufficient to make this surveillance screening method practical if it can be applied in other managed care settings.

The positive predictive values of screening criteria that include TB diagnosis codes are limited by a number of factors. TB diagnosis codes, for example, were frequently
used for patients with suspected active TB during the weeks required for diagnostic work-up or observation for clinical response to therapy. These codes were also frequently used to indicate that routine TB skin testing had been performed rather than to indicate the presence of active disease or prior history of TB. The difference in the TB case rates between HMO members with automated ambulatory-patient records (approximately 11.7 TB cases per 100,000 population) and members without such records (approximately four TB cases per 100,000 population) in our study could either reflect a true difference in the underlying risk for TB in
the two populations or case ascertainment bias resulting from differences in the methods used to identify TB cases. The former explanation is more likely for several
reasons. First, the HMO health centers with automated ambulatory-patient records serve a largely urban population concentrated in the Boston area, while the HMO-affiliated practices without such records serve a largely suburban population. The difference in the rates found in our study mirrors the difference in the 1992 to 1998 TB case rate averages reported by the Massachusetts Department of Public Health for the city of Boston (17.7 TB cases per 100,000 population) compared with the rest of the state of Massachusetts (4.1 TB cases per 100,000 population). Second, the match between the health department's TB registry and the HMO membership list did not identify any TB patients who had not previously been detected through screening
criteria and record review based on our modified RVCT results among HMO members without automated ambulatory patient records. This argues against inadequate case
finding resulting in apparent lower TB case rates in this group.

A substantial number of TB cases in our study were unknown to the public health department (18% of cases among our HMO study population). This proportion is comparable with the fraction described in the studies cited above.  Underreporting of these cases compromises the usefulness of TB surveillance. Screening for dispensing of antituberculosis drugs may be a particularly useful method for identifying cases without positive cultures for M. tuberculosis that might otherwise be missed by routine surveillance methods dependent on laboratory- and provider-based reporting.

The positive predictive value of screening criteria based on the dispensing of antituberculosis drugs may also be limited to some degree in clinical settings where many patients receive these medications for other indications, including other mycobacterial infections (e.g., in cases of HIV infection). Strategies that could be applied in such settings include excluding those persons also receiving medications frequently used for treatment of M. avium complex infections (e.g., clarithromycin). During our study period, however, more than 1,000 known HIV-infected patients were treated in HMO centers with automated ambulatory-patient records, of whom only 23 (Table 3) had false-positive cases identified by the two or more TB drug criterion. In addition, widespread implementation of new CDC recommendations for use of multidrug therapy for TB prophylaxis may require modification of the screening
criteria. One possible strategy would be to require that antituberculosis drugs be dispensed over a minimum time interval (e.g., >4 months).

Although TB surveillance based on pharmacy dispensing information depends upon availability of automated pharmacy data, such data are available for most of the U.S. population, including most Medicaid and Medicare recipients. Our results indicate that pharmacy dispensing information routinely collected by many HMOs has high sensitivity and reasonable positive predictive value and is particularly useful for identifying TB cases without positive cultures, which may be missed by traditional public health surveillance.

Acknowledgments

We thank Matthew McKenna for his contributions to the planning of this study, Liz Martino and Ralph Blair for their help in evaluating the Harvard Vanguard data, and
Claire Canning, Linda Lacke, and Shirley Golberg for their assistance in this study. 

Supported by the American Association of Health Plans and CDC Contract 200-95-0957-010.

Dr. Yokoe is an associate physician at Brigham and Women's Hospital in Boston, Massachusetts, and an instructor in Medicine at Harvard Medical School.

 Address for correspondence: Deborah S. Yokoe, 181 Longwood Ave., Boston, MA 02115, USA; fax: 617-731-1541; e-mail: deborah.yokoe@channing.harvard.edu.

References

1. Platt R. Studies of prescription drugs at Harvard Community Health Plan. In: Strom B, editor. Pharmacoepidemiology. 2d ed. New York: John Wiley and Sons;
1994. p. 278-87.

2. Centers for Disease Control and Prevention. Case efinitions for infectious conditions under public health surveillance. MMWR Morb Mortal Wkly Rep 1997;46(RR-10):40-1.

3. Subramanyan G, Yokoe D, Sharnprapai S, Tang Y, Platt R. An algorithm to match registries with minimal disclosure of individual identities. Public Health Reports 1999;114:91-3.

4. Stata statistics and data analysis [computer program]. Version 5.0. College Station (TX): Stata Corporation; 1997.

5.  Marier R. The reporting of communicable diseases. Am J Epidemiol 1977;105:587-90.

6. McCray E, Weinbaum CM, Braden CR, Onorato IM. The epidemiology of tuberculosis in the United States. Clin Chest Med 1997;18:99-113.

7. Driver C, Braden CR, Nieves R, Navarro AM, Rullan JV, Valway SE, et al. Completeness of tuberculosis case reporting, San Juan and Caguas Regions, Puerto Rico, 1992. Public Health Rep 1996;111:157-61.

8. Maggini M, Salmaso S, Alegiani SS, Caffari B, Raschetti R. Epidemiological use of drug prescriptions as markers of disease frequency: An Italian experience. J Clin Epidemiol 1991;44:1299-307.

9. Hripcsak G, Knirsch C, Jain N, Pablos-Mendez A. Automated tuberculosis detection. JAMIA 1997;4:376-81.

 10. Winickoff RN, Barnett GO, Morgan M, Coltin KL. Quality assurance in a prepaid group practice. Journal of Ambulatory Care Management 1979;2:19-28.

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

URL: http://www.cdc.gov/ncidod/eid/vol5no6/ascii/yokoe.txt

Please note that figures, equations, and most tables are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively.  Greek symbols are spelled out. >/= is used for greater than or equal to.

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

Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999]

   Research

   Using Automated Pharmacy Records to Assess the
   Management of Tuberculosis

   Girish S. Subramanyan,* Deborah S. Yokoe,* Sharon
   Sharnprapai, Edward Nardell, Eugene McCray, and
   Richard Platt*

   *Brigham and Women's Hospital, Boston, Massachusetts,
   USA; Massachusetts Department of Public Health,
   Jamaica Plain, Massachusetts, USA; Centers for Disease
   Control and Prevention, Atlanta, Georgia, USA; Harvard
   Medical School, Harvard Pilgrim Health Care, Boston,
   Massachusetts, USA

    We used automated pharmacy dispensing data
    to characterize tuberculosis (TB)
    management for 45 health maintenance
    organization (HMO) members. Pharmacy
    records distinguished patients treated in
    HMOs from those treated elsewhere. For
    cases treated in HMOs, they provided useful
    information about appropriateness of
    prescribed regimens and adherence to
    therapy.

   Health-care coverage, especially from health
   maintenance organizations (HMOs), often includes
   pharmacy benefits. Pharmacy dispensing records can
   identify cases of tuberculosis (TB) unknown to the
   public health system (1). In this article, we examine
   the utility of automated pharmacy dispensing data in
   assessing the quality of management of active TB and
   patients' compliance with recommended therapy.

   Methods

   We used automated pharmacy dispensing records to
   characterize therapy in 45 cases of active TB diagnosed
   from January 1, 1992, through June 30, 1996, at Harvard
   Pilgrim Health Care, a mixed model HMO in New England.
   These were all known cases of TB in a sample of 350,000
   HMO members (1,2); all met the Centers for Disease
   Control and Prevention's (CDC) surveillance case
   definition (3). Cases were drawn from the 90% of HMO
   members with pharmacy benefits.

   We identified all dispensings of isoniazid, rifampin,
   pyrazinamide, ethambutol, streptomycin, ethionamide,
   kanamycin, cycloserine, capreomycin, and
   para-aminosalicylic acid (PAS). We also reviewed the
   full medical records. Empiric regimens, i.e., those
   dispensed before susceptibility results were known, and
   final treatment regimens were graded for consistency
   with American Thoracic Society (ATS) and CDC guidelines
   in effect at the time of diagnosis (4,5). Two measures
   of the adequacy of therapy were calculated. 1) The
   standard regimen dispensed is a percentage calculated
   by comparing the cumulative dose of each drug dispensed
   with the total recommended. Each drug received equal
   weight to a maximum of 100% per drug, as noted in the
   following formula for a three-drug regimen: percent
   standard regimen = ([D1/SR1] + [D2/SR2] + [D3/SR3]) x
   (100/3), where Dx is the cumulative dose dispensed of
   drug X and SRx is the recommended total dose. Patients
   with a score >/=80% were considered to have received an
   appropriate amount of antituberculosis medications (6).
   2) The days without medication (identical to the
   "MED-OUT" adherence index validated for other
   medications) (7), for isoniazid or another drug
   required for the entire duration of treatment, is a
   percentage calculated by dividing the total number of
   days without medication (based on medication refill
   intervals and quantities dispensed) by the number of
   days between the first and last dispensing. The last
   refill does not influence this calculation. All the
   preceding calculations included all medicine dispensed
   to a patient, from all pharmacies required to report
   dispensing to the HMO to be reimbursed.

   Results

   Medical records indicated that 27 (60%) of 45 TB cases
   were treated solely by HMO providers (Table 1); nearly
   all remaining patients received their non-HMO care in
   public health-funded TB programs. Thirty-seven (82%)
   cases received empiric regimens through pharmacies
   reimbursed by the HMO. In 34 (92%) instances, the
   empiric regimen dispensed was appropriate; for the
   remainder, empiric regimens contained too few drugs.
   Twenty-six (96%) of the 27 solely HMO-treated cases
   were prescribed a final antituberculosis regimen that
   was adequate in agents used, doses prescribed, and
   duration of treatment.

Table 1. Characteristics of tuberculosis cases

In 15 of the 18 cases treated at least
partially outside the HMO, patients received
some antituberculosis medication from
pharmacies reimbursed by the HMO. In 14 of   
these cases, patients received medications only   
once or twice. A cutoff value of 70 days of  
drug dispensing through HMO-reimbursed  
pharmacies differentiated HMO-treated cases  
from cases treated in other settings. Among  
HMO-treated cases, 26 (96%) of 27 patients   
received medications for 70 days, compared with   
1 (6%) of 18 who were at least partially
treated outside the HMO (Figure 1) (RR = 17, 
95% confidence interval [CI] 3 to 117, p<0.0001) (8). In HMO cases, median duration of dispensing from HMO pharmacies was 189 days (interquartile range: 148 days to 291 days) and  
the median standard regimen dispensed score was   
99% (interquartile range: 86% to 100%). In   
cases outside the HMO, median duration of
dispensing through HMO-reimbursed pharmacies was 1 day
(interquartile range: 0 days to 32 days), and the median standard regimen dispensed score was 24% (interquartile range: 17% to 40%). Figure 2 shows the relationship between the appropriateness of the amount of medications dispensed and the timeliness of medication refills. In 4 (15%) of 26 HMO-treated cases, standard regimen dispensed scores were <80%, and days without medication scores were >30%. In only one of these four undertreated cases did the treating
physician document nonadherence. Two other patients who received 100% of a standard regimen with unremarkable refill intervals were noted as noncompliant in physicians' medical records.

Figure 1. Pharmacy dispensing profiles of tuberculosis (TB) cases treated in the health maintenance organization (HMO) and outside the HMO. Standard regimen (percentage) and duration of dispensing of antituberculosis medication dispensed for TB cases. A cutoff value of >/=70 days of medication dispensed from HMO-reimbursed pharmacies, as assessed from automated pharmacy records, differentiated
HMO-treated cases from cases at least partially treated outside the HMO. 

Figure 2. Appropriateness of the amount of antituberculosis medications dispensed and the timeliness of
medication refills. Percentage of standard regimen dispensed is plotted against percentage of days
without antituberculosis medication for tuberculosis cases treated in the health maintenance
organization.

Conclusions

Automated pharmacy data provided useful
information both about physicians' intended 
management of TB and about patients' adherence   
to prescribed therapy. The ability to monitor    
these aspects of TB care efficiently is
particularly important when care is    
decentralized or a substantial proportion of
patients receive care from more than one    
provider. We were able to determine that in 
nearly all cases appropriate initial or empiric
regimens were prescribed, and that in most  
cases managed by HMO providers full 
ATS/CDC-recommended regimens were dispensed.
This approach was thus more informative and
efficient than prior study methods that assessed prescribed regimens by reviewing patients' medical records (9). Many HMOs and other insurers routinely monitor pharmacy dispensing records for various reasons. While the initial cost of creating a routine monitoring report varies, the marginal cost of running it periodically is usually negligible. This allows 100% surveillance of therapy,
compared with manual record review, which is more expensive even when only a sample of records is reviewed. Additional investigation would determine whether this method will be helpful for other infectious diseases, such as pelvic inflammatory disease.

In using a pharmacy-based system to monitor adherence to therapy, it is important to identify cases treated solely within a delivery system, since automated pharmacy information is reliably complete only for these persons. Restricting to patients with at least 70 days of therapy accomplishes this, since it excludes those who receive empiric regimens within one health system and then complete
their care at another. Most care delivered by non-HMO providers was provided by public health clinics. Potential physician incentives for transfer of care included closer monitoring of therapy by experts in TB treatment,  particularly in difficult-to-manage cases. Patients' incentives included free medications provided by the
department of public health. Although all patients described here had pharmacy benefits, the cost to the patients of copayments for a standard treatment regimen would have been $75 to $200.

Monitoring automated pharmacy records of patients treated for TB was an efficient adjunct for monitoring adherence but did not entirely replace providers' assessments documented in the medical records. It also provided no advantage in settings that used directly observed therapy, which is not routinely used in Massachusetts.

Pharmacy records may be used to contribute to management of TB in two ways: in real time, to identify suboptimal regimens and noncompliant patients and periodically, to assess overall appropriateness of care. In real time, one might monitor the dispensing of antituberculosis medications for confirmed cases of TB, intervening if necessary with physicians to ensure that appropriate regimens are used and with patients to minimize gaps in dispensing. Identifying noncompliant patients in a regular and timely manner could allow for interventions (e.g., directly observed therapy) to improve adherence to the treatment regimen. Such oversight could be coordinated with or overseen by public health
agencies. Coordination between delivery systems and public health agencies is expected to become an important element of TB control (10). Periodic assessment of dispensing records can also provide a simple, efficient measure of the overall appropriateness of TB care in a wide array of settings. These measures would allow targeting of
resources to improve organizations' management of TB. If these findings are confirmed in other settings, routine monitoring of dispensing of antituberculosis medications may be useful as an adjunct to other methods of assessing and ensuring appropriate therapy.

Acknowledgment

We thank Claire Canning for her assistance with this study.

This study was supported by CDC Contract 200-95-0957-010 and the Harvard Pilgrim Health Care Foundation.

Dr. Subramanyan was a research fellow at the Channing Laboratory, Brigham and Women's Hospital, Boston, Massachusetts, and a student at Harvard Medical School when he performed this work. He is currently a resident at the University of California, San Francisco.

Address for correspondence: Deborah Yokoe, 181 Longwood Ave., Boston, MA 02115, USA; fax: 617-731-1541; e-mail:
deborah.yokoe@channing.harvard.edu.

References

1. Yokoe DS, Subramanyan GS, Nardell E, Sharnprapai S, McCray E, Platt R. Supplementing tuberculosis surveillance with automated data from health maintenance organizations. Emerg Infect Dis 1999;5:779-87.

2. Subramanyan GS, Yokoe DS, Sharnprapai S, Tang Y, Platt R. An algorithm to match registries with minimal disclosure of individual identities. Public Health Rep 1999;114:91-3.

3. Centers for Disease Control and Prevention. Case definitions for infectious conditions under public health surveillance. MMWR Morb Mortal Wkly Rep 1997;46:40-1.

4. Bass JB Jr, Farer LS, Hopewell PC, O'Brien R, Jacobs RF, Ruben F, et al. Treatment of tuberculosis and tuberculosis infection in adults and children. Am J Respir Crit Care Med 1994;149:1359-74.

5. American Thoracic Society. Treatment of tuberculosis and
tuberculosis infection in adults and children. American Review of Respiratory Disease 1986;134:355-63.

6. Fox W. Whither short-course chemotherapy? British Journal of Diseases of the Chest 1981;75:331-57.

7. Steiner JF, Koepsell TD, Fihn SD, Inui TS. A general method of compliance assessment using centralized pharmacy records: description and validation. Med Care 1988;26:814-23.

8. EpiInfo [computer program]. Version 6.04b. Atlanta (GA): Centers for Disease Control and Prevention; 1997.

9. Migliori GB, Spanevello A, Ambrosetti M, Neri M. Surveillance of tuberculosis treatment prescriptions in Italy. The Varese TB Study Group. Monaldi Arch Chest Dis 1998;53:37-42.

10. Halverson PK, Mays GP, Miller CA, Kaluzny AD, Richards TB. Managed care and the public health challenge of TB. Public Health Rep 1997;112:22-8.

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

URL: http://www.cdc.gov/ncidod/eid/vol5no6/ascii/subramanyan.txt

Please note that figures, equations, and most tables are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively.  Greek symbols are spelled out. >/= is used for greater than or equal to.

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

Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999]

   Research

   Hantavirus Reservoir Hosts Associated with Peridomestic
   Habitats in Argentina

   Gladys Caldern,* Noem Pini,* Jorge Bolpe, Silvana
   Levis,* James Mills, Elsa Segura,* Nadia Guthmann,
   Gustavo Cantoni, Jos Becker,* Ana Fonollat,# Carlos
   Ripoll,** Marcelo Bortman, Rosendo Benedetti, Marta
   Sabattini, and Delia Enria*

   *Instituto Nacional de Enfermedades Virales Humanas
   "Dr. Julio I. Maiztegui," ANLIS, "Dr. Carlos G.
   Malbrn," Pergamino, Buenos Aires, Argentina;
   Departamento de Zoonosis Rurales de Azul, Azul, Buenos
   Aires, Argentina; Centers for Disease Control and
   Prevention, Atlanta, Georgia, USA; Universidad
   Nacional del Comahue, S.C. de Bariloche, Rio Negro,
   Argentina; Consejo Provincial de Salud Pblica, Rio
   Negro, Argentina; #Fundacin Lillo, San Miguel de
   Tucumn, Argentina; **Departamento de Chagas y
   Patologas Regionales, San Salvador de Jujuy,
   Argentina; Subsecretara de Salud, Neuqun,
   Argentina; Zona Sanitaria Noroeste, Esquel, Chubut,
   Argentina; and the Hantavirus Study Group1

    Five species of sigmodontine rodents have
    been identified in Argentina as the
    putative reservoirs of six circulating
    hantavirus genotypes. Two species of
    Oligoryzomys are associated with the
    genotypes causing hantavirus pulmonary
    syndrome, Oligoryzomys flavescens for
    Lechiguanas and O. longicaudatus for Andes
    and Oran genotypes. Reports of human cases
    of hantavirus pulmonary syndrome prompted
    rodent trapping (2,299 rodents of 32
    species during 27,780 trap nights) at
    potential exposure sites in three
    disease-endemic areas. Antibody reactive to
    Sin Nombre virus was found in six species,
    including the known hantavirus reservoir
    species. Risk for peridomestic exposure to
    host species that carry recognized human
    pathogens was high in all three major
    disease-endemic areas.

   Hantaviruses, a genus in the family Bunyaviridae, are
   rodentborne pathogens producing chronic persistent
   infections in their reservoir hosts. Although the exact
   mechanism of transmission from rodents to humans is
   unknown, strong evidence suggests that these viruses
   are infectious by aerosols. Inhalation of aerosolized
   virus from rodent excreta is thought to be the main
   route of transmission to humans (1).

   Although hantaviruses have been
   reported in the Americas since the
   1980s (2,3), before 1993 human
   illnesses caused by hantaviruses,
   grouped under the name of
   hemorrhagic fever with renal
   syndrome, were thought to be
   limited to Europe and Asia. After
   hantavirus pulmonary syndrome
   (HPS) was described as a clinical
   form of hantavirus illnesses in
   the New World, outbreaks of HPS as
   well as isolated cases were
   recognized in many parts of the
   Americas. In Argentina, where
   cases of HPS were identified
   retrospectively as early as the 1980s (4), three
   geographically and ecologically distinct HPS-endemic
   areas have been recognized (5): the northern zone, a
   subtropical area bordering the Bermejo River; the
   central zone, a region of humid plains and temperate
   climate; and the southern zone, a cold, forested region
   bordering the Andean range (Figure).

   The common rodents in populated areas of Argentina
   belong to two groups of the family Muridae. The most
   common rodents in natural, as well as disturbed
   habitats outside urban and peridomestic areas, are
   numerous species of the Murid subfamily, Sigmodontinae
   (the New World rats and mice) (6). All hantaviruses
   known to cause HPS are associated with sigmodontine
   rodents. The common rodents in towns, cities, and
   peridomestic (in and around homes) environments are
   three introduced species of the subfamily Murinae:
   Rattus rattus (black rat), R. norvegicus (Norway rat),
   and Mus musculus (house mouse) (6).

   In South America, hantaviruses are associated with
   several species of indigenous sigmodontine rodents. In
   Argentina, seven viral genotypes have been described:
   Bermejo and Oran in the northern zone; Lechiguanas,
   Hu39694, Maciel, and Pergamino in the central zone; and
   Andes in the southern zone (7,8). Andes, Lechiguanas,
   Hu39694, and Oran have been associated with human
   disease, and the putative reservoirs of three of these
   genotypes are two species of Oligoryzomys: O.
   longicaudatus from southern Argentina for Andes, O.
   longicaudatus from northern Argentina for Oran, and O.
   flavescens for Lechiguanas. O. longicaudatus (reservoir
   of Oran and Andes genotypes) may represent two species
   (8). The putative reservoir for the Bermejo genotype,
   not yet associated with human disease, is reported to
   be O. chacoensis. The reservoir for Hu39694 is unknown,
   although its close genetic similarity to Andes, Oran,
   and Bermejo suggests that it may be another
   Oligoryzomys species from central Argentina. In the
   central zone, two genotypes not yet associated with HPS
   were identified from other sigmodontine species:
   Maciel, from Necromys benefactus (previously designated
   Bolomys obscurus), and Pergamino, from Akodon azarae
   (8).

   Since 1996, follow-up investigations have been
   conducted when HPS cases in Argentina were confirmed.
   As of January 20, 1999, 210 cases of HPS had been
   confirmed in Argentina (Ministerio de Salud y Accin
   Social). This investigation includes rodent studies to
   identify areas in which HPS poses a high risk and to
   determine the spatial distribution of rodent reservoir
   populations in relation to the suspected sites of
   exposure for persons with HPS.

    Figure. Sites of rodent trapping
    and human cases in three
    hantavirus pulmonary
    syndrome-endemic zones in
    Argentina.

Identification of HPS Cases and

Study Areas

Confirmed cases of HPS were defined as having the following
characteristics: 1) a compatible clinical illness and 2) laboratory evidence of acute hantavirus infection, such as a positive enzyme-linked immunosorbent assay (ELISA) hantavirus immunoglobulin (Ig) M or a fourfold rise in ELISA IgG; a positive reverse transcription-polymerase chain reaction (RT-PCR) for hantavirus RNA; or positive immunohistochemistry for hantavirus antigen. When an HPS
case was confirmed, small mammals were trapped in collaboration with the local health authorities at the patient's home or work sites and neighboring areas (Figure).

Selection and Classification of Potential Exposure Sites

The potential exposure sites were chosen by selecting all places where patients had been living or working or had visited during the 6 weeks before onset of symptoms. Rodents were trapped in all these sites, which were classified into six categories: domestic and peridomestic urban, domestic and peridomestic rural, other urban, and other rural. Peridomestic urban and rural categories were all
sites in the immediate vicinity of homes or buildings, including yards, parks, driveways, adjoining lands, outbuildings, vegetable gardens, and fence lines. The peridomestic rural category includes ponds, natural or planted woodlots, weeds, sugar cane or plantain
plantations, and corn stubble in the immediate vicinity of the house. All other trapping sites distant from the previously mentioned settings were considered other urban or other rural. Other urban includes sites from the outskirts of towns and natural and artificial corridors that could allow the access of sigmodontine rodents to urban areas, such as railroad rights-of-way and roadsides inside the perimeter of the town. In other rural sites rodents were
captured in open fields, where the representative habitats of each area were sampled, including natural and modified land, such as cultivated areas and weeds.

Small-Mammal Trapping and Processing

In the southern and central zones, rodents were trapped as soon as HPS case reports were received. In the remote northern zone, three expeditions were organized to trap rodents at sites frequented by six persons with HPS reported in previous months, and only rarely was trapping conducted inside houses. The three expeditions took place in July 1995, October 1996, and May 1998; rodents were trapped
at 18 sampling sites. 

From August 1994 to April 1998, 46 sampling sites were selected in the central zone. In the southern zone, we included 51 sampling sites from November 1996 to April 1998 (Table 1).

Table 1. Relative density (as indicated by trap successa) for frequently captured rodent species in three hantavirus pulmonary syndrome-endemic zones in Argentina

Each site was sampled with Sherman (8 x 9 x 23 cm) and Tomahawk (14 x 14 x 40 cm) live-capture traps. The number of traps depended on the area available for trap placement at each site. Animals were trapped and sampled according to established safety guidelines (9) and were anesthetized with Isoflurane (Abbott Laboratories) before blood was drawn from the retroorbital sinus. Carcasses were tentatively identified in the field and kept in a solution of 10%
formalin for confirmation of identification at the Museum of Natural Sciences "Bernardino Rivadavia," Buenos Aires.

Structure of Small-Mammal Communities

During 26,458 Sherman and 1,322 Tomahawk trap-nights, 2,299 small mammals belonging to two orders (Rodentia and Didelphimorphia) and three families (Muridae, Caviidae, and Didelphidae) were captured. These animals belonged to 32 species, with the murid subfamily Sigmodontinae representing 86.3% of the total sample.

The introduced murine rodents R. rattus and M. musculus, as well as Cavia aperea (Caviidae), were captured in all three areas. Sigmodontine rodents were represented by different species in the three regions.

Distribution of Species by Site of Capture

In all three regions, M. musculus was found in domestic urban sites (Table 1). In two of the three areas, we also observed rodents inside urban homes; this is the first documented occurrence of sigmodontine species entering homes in Argentina. 

We also found sigmodontine rodents inside rural homes: one Calomys laucha and one O. flavescens in the central zone and one Akodon spegazzinii in the northern zone. Sigmodontine rodents, including the reservoirs for Lechiguanas and Andes viruses, were also captured in the peridomestic urban sites, especially in the central and southern zones. In peridomestic rural habitats next to open fields,
captures of sigmodontines were expected. The trap success values for hantavirus reservoir species in peridomestic rural sites were similar or higher than those in open fields represented by other rural sites. The relative proportion of rodent species among site categories includes all species antibody positive and the species that were numerically dominant but antibody negative in each zone. The category "others" includes species that were less representative
in each zone; the high values observed in PU and OU sites in the northern zone were due to the low number of captures and in OR to the high diversity of species captured (Table 2). The relative proportion was compared by chi-square test with Epi Info version 6.04. Only site categories with >30 captures could be tested. An increase in the relative proportion of O. flavescens (host of the genotype Lechiguanas, associated with human disease) in the central
zone and O. longicaudatus (putative reservoir of the genotype Orn, also associated with human disease) in the northern zone was seen in peridomestic rural settings in comparison with other rural. O. longicaudatus (proposed reservoir for Andes virus) was captured in similar relative proportions in both peridomestic and other rural sites. In all cases, these findings emphasize the risk linked to
peridomestic settings.

Table 2. Relative proportiona of rodent species in each site
category, by site Hantavirus Infection in Rodents

We tested 2,159 (93.9%) rodents in IgG ELISA by using Sin Nombre virus antigen (CDC, SPR293). We used a recombinant nucleocapsid protein as antigen applied to the solid phase of a microtiter plate. Hantavirus-specific IgG in test samples of rodent whole blood was allowed to bind to the antigen. A mixture of two conjugates (anti-Peromyscus leucopus and anti-Rattus norvegicus, Kirkegaard and
Perry) was used to detect immune globulins from various murid rodent phyla. This was followed by 2,2'-azin o-di(3-ethybenthiazonline sulfonate) substrate (Kirkegaard and Perry Laboratories, Inc.) and read with a Bio-Tek Microplate autoreader at 405 and 450 nm. A titer >/=1:400 was considered positive (10).

Of 330 rodents tested in the north, 5 (1.5%) were positive (Table 3). In the central zone, we found 35 (2.6%) positives among 1,326 rodents, associated with eight HPS cases. In the south, 27 (5.4%) of 503 rodents tested had positive results. In the northern zone, the presence of infected O. longicaudatus was associated with HPS cases in peridomestic rural habitats. The importance of detecting infected
O. chacoensis and Akodon varius associated with an HPS case in peridomestic urban and rural sites cannot be assessed until data on the viral genotypes of the rodents and the case patients are available.

In the central zone, apart from O. flavescens, already shown to be associated with HPS, another species found infected was A. azarae, the putative reservoir of the Pergamino genotype, which has not yet been associated with human disease. Spatial and temporal association between an HPS case and an infected A. azarae does not confirm this
species as the source of infection. Further genetic studies are under way to determine if Pergamino virus was responsible for the HPS cases.

In the southern zone, human cases were associated with O.
longicaudatus captured in peridomestic and other rural settings (Table 3). In the three zones, in all other site categories, no seropositive animals were found. Nevertheless, because of small sample sizes, any conclusions concerning lack of infection in these site categories are tentative.

Table 3. Antibody distribution in rodents, by province, species, and site category

Conclusions

Infected hantavirus reservoir hosts (as evidenced by antibody positivity) were found within peridomestic environments in all three HPS-endemic zones in Argentina. Reservoir species were captured inside urban houses in two of the three endemic zones. Although host species were not captured in homes in the northern zone, sampling
was not sufficient to exclude the possibility that they enter homes occasionally.

The presence of hantavirus reservoir species in peridomestic
environments indicates risk for human inhabitants. The primary measure for reducing the risk is preventing access of rodents to homes (11). The efficacy of proposed and currently used exclusion methods in Argentina needs to be evaluated (12).

Sigmodontine rodents, including known hantavirus reservoir species, were frequently captured in the rural and small-town peridomestic environments we studied. At many of the case sites, the level of hygiene was suboptimal. The widespread presence of such conditions underscores the importance of local habitat management to prevent
wild (sigmodontine) rodents from entering domestic areas in towns, villages, and urban centers and of health education for the local population to reduce the risk for hantavirus infection.

Acknowledgments

We thank Horacio Lopez, Diego Olivera, Mario Palmigiano, Felix May, Oscar Gallicchio, Anibal Hirsch, Horacio Larraburu, Mariana Lozada, Pablo Sandoval, Federico Bianconi, Mario Diaz, Malcom Elder, Carmelo
Saavedra, and Omar Fuentes for the rodent trapping effort; T. Ksiazek for providing antigen for the enzyme-linked immunosorbent assay; and Marta Piantanida, Elio Massoia, and Jaime Polop for identification of rodent specimens.

This work was supported in part by grant N US 1181199 from the World Health Organization and by Administracin Nacional de Laboratorios e Institutos de Salud (ANLIS) "Dr. Carlos G. Malbrn," Ministerio de Salud Pblica de la Nacin.

Dr. Caldern is chief of the Quality Control Division at the
Instituto Nacional de Enfermedades Virales Humanas "Dr. Julio I. Maiztegui." Her research interests include rodentborne diseases, specifically those caused by arenaviruses and hantaviruses.

Address for correspondence: Gladys Caldern, Instituto Nacional de Enfermedades Virales Humanas "Dr. Julio I. Maiztegui," Monteagudo 2510, (2700) Pergamino, Buenos Aires, Argentina; fax: 54-2477-433045; e-mail: gladys@inevh.sld.ar.

References

1. McKee KT Jr, LeDuc JW, Peters CJ. Hantaviruses. In: Belshe RB, editor. Textbook of human virology. St. Louis (MO): Mosby Year Book; 1991.

2. LeDuc JW, Smith GA, Childs JE, Pinheiro FP, Maiztegui JI,
Niklasson B et al. Global survey of antibody to Hantaan-related viruses among peridomestic rodents. Bull WHO 1986;64:139-44.

3. Weissenbacher MC, Cura E, Segura E, Hortal M, Back LJ, Yong K, et al. Serological evidence of human hantavirus infection in Argentina, Bolivia and Uruguay. Medicina (Buenos Aires) 1996;56:17-22.

4. Parisi M, Enria D, Pini N, Sabattini MS. Deteccin retrospectiva de infecciones clnicas por hantavirus en la Argentina. Medicina (Buenos Aires) 1996;56:1-13.

5. Levis SC, Briggiler AM, Cacase M, Peters CJ, Ksiazek TG, Corts J, et al. Emergence of hantavirus pulmonary syndrome in Argentina. Proceedings from the 44th Annual Meeting; 1995 Nov 17-21; San Antonio, Texas. American Society of Tropical Medicine and Hygiene; 1995. p. 233.

6. Redford K, Eisenberg J. Mammals of the neotropics. The southern cone. Vol 2. The University of Chicago Press; 1992.

7. Lopez N, Padula R, Rossi C, Lzaro ME, Franze-Fernandez MT. Genetic identification of a new hantavirus causing severe pulmonary syndrome in Argentina. Virology 1996;220:223-6. 

8. Levis S, Morzunov S, Rowe J, Enria D, Pini N, Caldern G, et al. Genetic diversity and epidemiology of hantaviruses in Argentina. J Infect Dis 1998;177:529-38.

9. Mills J, Childs J, Ksiazek T, Peters CJ, Velleca W. Mtodos para trampeo y muestreo de pequeos mamferos para estudios virolgicos. Washington: Organizacin Panamericana de la Salud; 1998. Report #OPS/HPS/HCT98.104.

10. Ksiazek T, Peters CJ, Rollin PE, Zaki S, Nichol S, Spiropoulou C, et al. Identification of a new North American hantavirus that causes acute pulmonary insufficiency. Am J Trop Med Hyg 1995;52:117-23.

11. Centers for Diseases Control and Prevention. Hantavirus
infection-southwestern United States: interim recommendations for risk reduction. MMWR Morb Mortal Wkly 1993;42(RR-11):1-13.

12. Glass G, Johnson J, Hodenbach G, Disalvo C, Peters CJ, Childs J, 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.

1Eduardo Herrera and Edmundo Larrieu, Consejo Provincial de Salud Pblica, Rio Negro, Argentina; Mara Cacace, Hospital San Vicente de Paul, Orn, Salta, Argentina; Roberto Gonzalo, Ricardo Fernandez, Gustavo Martinez, and Alberto Suzzi, Zona Sanitaria Noroeste, Esquel, Chubut, Argentina.
Emerging Infectious Diseases National Center for Infectious Diseases Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/eid/vol5no6/ascii/calderon.txt

Please note that figures, equations, and most tables are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively.  Greek symbols are spelled out. >/= is used for greater than or equal to.

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

Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999]

Dispatches

   Large, Persistent Epidemic of Adenovirus Type
   4-Associated Acute Respiratory Disease in U.S. Army
   Trainees

   K. Mills McNeill,* Rose M. Hendrix, Jane L. Lindner,
   F. Ridgely Benton,* Susan C. Monteith,* Margaret A.
   Tuchscherer,* Gregory C. Gray, and Joel C. Gaydos

   *Dwight David Eisenhower Army Medical Center, Fort
   Gordon, Georgia, USA; University of South Carolina
   School of Medicine, Columbia, South Carolina, USA;
   Moncrief Army Community Hospital, Fort Jackson, South
   Carolina, USA; Naval Health Research Center, San
   Diego, California, USA; Department of Defense Global
   Emerging   Infections System, Washington, D.C., USA

   --------------------------------------------------------
    In May 1997, a large, persistent epidemic
    of adenovirus type 4-associated acute
    respiratory disease began at Fort Jackson,
    South Carolina, the largest army basic
    training center. The epidemic lasted until
    December and declined when vaccine
    administration resumed. More than 1,000
    male and female trainees were hospitalized;
    66.1% of those hospitalized had an
    adenovirus type 4 isolate.

   Nonvaccine interventions have proven unreliable in the
   control of adenovirus-associated acute respiratory
   disease (1). Before live, oral, enteric-coated
   adenovirus types 4 and 7 vaccines were introduced in
   1971, adenovirus-associated acute respiratory disease
   produced high attack rates and excessive illness in
   soldiers during service-entry basic combat training
   (2). The subsequent policy to vaccinate all male
   trainees during basic combat training drastically
   reduced adenovirus type 4- and type 7-associated acute
   respiratory disease (3).

   In 1996, the sole manufacturer of the vaccines ceased
   production (4). To conserve the remaining vaccine lots,
   the army restricted use of adenovirus vaccines to the
   period of September 1 through March 31, the peak season
   for acute respiratory disease. Administration of
   adenovirus vaccines to military trainees was suspended
   on March 31, 1997, at all army basic training centers.
   To monitor the impact of this modified policy,
   intensive, laboratory-based surveillance was initiated
   in late April 1997 at Fort Jackson, South Carolina, the
   army's largest basic combat training center. An
   epidemic of adenovirus type 4-associated acute
   respiratory disease occurred among male and female
   soldiers in basic combat training at Fort Jackson after
   the vaccination was suspended. The first case appeared
   in late May, and the outbreak lasted until December,
   after vaccination was resumed in November.

   The Outbreak

   During May through December 1997, a monthly average of
   6,847 soldiers from all geographic regions of the
   United States and its protectorates were engaged in the
   8-week basic combat training program at Fort Jackson.
   During this period, 38.2% of the trainees were women.
   The mean age of soldiers enrolled in this study was
   19.7 years.

   All trainees who report to sick call with fever of
   100.5 F or higher are admitted to a minimal-care
   hospital ward at the Fort Jackson Army Hospital for
   observation and self-care. Soldiers from this group who
   had an oral temperature of 100.5 F or higher, plus at
   least one sign or symptom of an upper respiratory
   infection, were enrolled in the surveillance program.
   Approximately 80% of soldiers hospitalized with acute
   respiratory disease were enrolled since patients
   admitted on weekends were not included. A pharyngeal
   swab was taken from each trainee and immediately placed
   into virus transport medium (Viromed Laboratories,
   Inc., Minneapolis, MN). Specimens were stored at 2C to
   8C for an average of 72 hours before overnight express
   shipment on wet ice to the laboratory for virus
   isolation.

   Virus isolations and identifications were performed by
   the Department of Pathology Laboratory at Dwight David
   Eisenhower Army Medical Center. Adenoviruses were
   isolated in human lung carcinoma (A-549) cells (Viromed
   Laboratories, Inc., Minneapolis, MN). The serotype of
   isolated adenoviruses was determined by virus
   neutralization with type-specific antisera (Centers for
   Disease Control and Prevention, Atlanta, GA); the
   Reed-Muench method was used for calculating the LD50
   titer (5). Each culture was also tested for influenza A
   and B; parainfluenza 1, 2, and 3; herpes simplex virus;
   and enteroviruses by cell culture, enzyme immunoassay,
   and fluorescent antibody staining.

   The first isolate of adenovirus
   type 4 came from a patient who was
   hospitalized on May 22, 1997,
   approximately 7 weeks after
   administration of the adenovirus
   vaccines ended. During May through
   December 1997, 1,018 basic
   trainees whose illness met the
   case definition were examined. Of
   these, 673 (66.1%) were positive
   for adenovirus type 4. No other
   respiratory disease agent was
   identified as an important cause
   of illness in this epidemic. The
   monthly case distribution was
   calculated by sex for all
   hospitalized acute respiratory disease patients (Figure
   1) and for adenovirus type 4-positive patients (Figure
   2). Of the total acute respiratory disease cases, 35.3%
   (only slightly less than their representation in the
   total trainee population of 38.2%) were in women.
   Similarly, 31.2% of all adenovirus type 4 isolates were
   from women. During May, June, and July, the monthly
   rates of acute respiratory disease admissions were
   higher for women than for men (Figure 1). These higher
   rates were not due to adenovirus type 4 (Figure 2) or
   any other agent tested for in this study.

   The percentage of cases from which adenovirus type 4
   was isolated increased as the epidemic progressed. At
   its peak, almost all patients had an adenovirus type 4
   isolate (Figure 3). Isolation rates of more than 90%
   were seen in both male and female patients toward the
   end of the outbreak. At the peak of this epidemic,
   approximately 70 soldiers per week were hospitalized at
   Fort Jackson Army Hospital. This corresponded to a
   weekly hospitalization rate for the entire post of
   approximately 1.0 admission per 100 soldiers. However,
   cases were not uniformly distributed throughout the
   trainee population but tended to occur in clusters. The
   outbreak intensified until November 1997, when the
   adenovirus vaccines were reintroduced. (Vaccination was
   to resume on September 1, 1997, according to the
   modified army policy. However, the vaccines did not
   reach Fort Jackson until November.) Resumption of
   adenovirus immunizations was associated with a rapid
   decline in both new cases of acute respiratory disease
   and isolations of adenovirus type 4. The last isolate
   of adenovirus type 4 was seen in December 1997. No
   additional isolates were reported during January
   through April 1998, except for a single isolate in a
   vaccinated trainee in February 1998. Administration of
   adenovirus types 4 and 7 vaccines continued through
   March 31, 1998.

Figure 1. Patients with acute respiratory disease, by sex, MayDecember 1997, Fort Jackson, South
Carolina.

Figure 2. Patients with adenovirus type 4-associated acute respiratory disease, by sex, MayDecember 1997, Fort Jackson, South Carolina.

Figure 3. Percentage of total patients with acute respiratory disease who also had an adenovirus
type 4 isolate, MayDecember, 1997, Fort Jackson, South Carolina.

Conclusions

Traditionally, adenovirus vaccines were
administered to male trainees entering basic
combat training between October and April, but
outbreaks due to types 4 and 7 occurred in the
absence of vaccines. The year-round use of the
two vaccines beginning in 1984 resulted in the
elimination of outbreaks due to both the type 4
and type 7 viruses (3).

In 1994, administration of the vaccines was
temporarily interrupted, and a limited outbreak
occurred among male and female basic trainees
at Fort Jackson (6). This outbreak, which began
in 1995 approximately 5 to 6 weeks after the
administration of the adenovirus vaccines was
reinstated, was centered in one military unit
that had not received the vaccines; the outbreak lasted only a few weeks, probably because the pool of susceptible soldiers was small. Concern about the threat of adenoviruses was validated by serologic data, which revealed that the adenoviral susceptibility of personnel entering the military in the 1990s was similar to that of personnel who entered in the 1970s (4).

The temporal relationship of this epidemic to suspension of
adenovirus vaccination in March 1997 demonstrates the effectiveness of type-specific vaccine in controlling and preventing military epidemics. Trainees began and ended the 8-week basic combat training program each week, so it would take approximately 2 months for all trainees who received the vaccines to complete basic combat training
and leave Fort Jackson. The first case of adenovirus type
4-associated acute respiratory disease occurred in May,
approximately 7 weeks after vaccination was suspended. The epidemic continued until December 1997, when it was interrupted by the administration of adenovirus types 4 and 7 vaccines to all new soldiers and to all soldiers (men and women) already enrolled in basic combat training at Fort Jackson. The army hospital managed the acute respiratory disease patient workload during the 1997 epidemic,
but the unexpected inpatient census proved stressful for staff and facilities.

When men and women had separate basic combat training programs, adenovirus-associated acute respiratory disease outbreaks were never documented in women, and the adenovirus
vaccines were not administered to women (2,4). When the gender-integrated program started at Fort Jackson  approximately 5 years ago, the decision was made to continue providing the vaccine only to men. In the limited adenovirus
type 4-associated acute respiratory disease outbreak at Fort Jackson in 1995, women were hospitalized and had adenovirus type 4 isolated (6). During this large 1997 epidemic, women and men in the integrated basic combat training program were similarly affected by adenovirus type 4-associated acute respiratory disease. Early in this outbreak, admission rates for acute respiratory disease were higher in women. However, an etiologic agent was not identified. Acute respiratory disease in women in integrated basic combat training programs cannot be assumed to be the same as that observed in men and must be studied further.

Historically, adenovirus types 4 and 7 have been the major causes of acute respiratory disease in the military (2). We cannot state why type 4 caused this outbreak rather than type 7. However, documented military outbreaks demonstrate that both vaccines are necessary, since prevention of type 4-associated acute respiratory disease alone will result in the emergence of type 7-associated disease (2). This epidemic demonstrates that adenovirus type 4 is a major threat to both male and female soldiers in basic combat training. Additionally, it confirms that large outbreaks due to adenoviruses should be expected in unvaccinated basic combat trainees.

Acknowledgment

The authors thank Johnnie Conolly, Moncrief Army Community Hospital, Fort Jackson, SC, for her invaluable and expert technical assistance.

Dr. McNeill is a District Health Director with the South Carolina Department of Health and Environmental Control. He is a public health physician with doctoral training in Tropical Medicine. His major research interest is laboratory-based surveillance for communicable diseases.

References

1. Foy HM. Adenoviruses. In: Evans AS, Kaslow RA, editors. Viral infections in humans. 4th ed. New York: Plenum Press; 1997. p. 119-38.

2. Gaydos CA, Gaydos JC. Adenovirus vaccines in the U.S. military. Mil Med 1995;160:300-4.

3. Brundage JF, Gunzenhauser JD, Longfield JN, Rubertone MV, Ludwig SL, Rubin FA, et al. Epidemiology and control of respiratory diseases with emphasis on group A beta-hemolytic streptococcus: a decade of U.S. Army experience. Pediatrics 1996;97:964-70.

4. Ludwig SL, Brundage JF, Kelley PW, Nang R, Towle C, Schnurr DP, et al. Prevalence of antibodies to adenovirus serotypes 4 and 7 among unimmunized U.S. Army trainees: results of a retrospective nationwide seroprevalence survey. J Infect Dis 1998;178:1776-8.

5. Hawkes RA. General principles underlying laboratory diagnosis of viral infections. In: Lennette EH, Schmidt NJ, editors. Diagnostic procedures for viral, rickettsial, and chlamydial infections. 5th ed. Washington: American Public Health Association; 1979. p. 32-5.

6. Barraza EM, Ludwig SL, Gaydos JC, Brundage JF. Reemergence of adenovirus type 4 acute respiratory disease in military trainees: report of an outbreak during a lapse in vaccination. J Infect Dis 1999;179:1531-3.

Address for correspondence: K. Mills McNeill, Catawba Health
District, P.O. Box 817, Lancaster, SC 29721, USA; fax: 803-286-5418; e-mail: mcneilkm@lncstr60.dhec.state.sc.us.

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

URL: http://www.cdc.gov/ncidod/eid/vol5no6/ascii/mcneill.txt

Please note that figures, equations, and most tables are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively.  Greek symbols are spelled out. >/= is used for greater than or equal to.

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

Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999]

Dispatches

   Changes in Antimicrobial Resistance among Salmonella
   enterica Serovar Typhimurium Isolates from Humans and
   Cattle in the Northwestern United States, 19821997

   Margaret A. Davis, Dale D. Hancock, Thomas E. Besser,
   Daniel H. Rice, John M. Gay, Clive Gay, Lynne Gearhart,
   and Ronald DiGiacomo
   Washington State University, Pullman, Washington, USA

    We compared antimicrobial resistance
    patterns of Salmonella enterica serovar
    Typhimurium (ST) of isolates from humans (n
    = 715) and cattle (n = 378) in the Pacific
    Northwest from 1982 through 1997. The major
    changes in antimicrobial resistance can be
    attributed to the widespread clonal
    dissemination of multidrug-resistant
    definitive phage type 104 ST.

   Enteritidis is the most frequent Salmonella serovar in
   all regions of the United States, except the Pacific
   Northwest where Typhimurium is the most frequent. In
   1996, 31.1% of all Salmonella isolates from human
   sources in Washington, Oregon, and Idaho were serovar
   Typhimurium, while 14.6% were serovar Enteritidis (1).
   Typhimurium is also one of the most common Salmonella
   serovars from animal sources in the United States (2).

   Use of antimicrobial drugs in food animals may lead to
   resistant strains of pathogens, which may be
   transmitted to humans through food (3,4). Although
   there is evidence that this transmission occurs, the
   contribution of antimicrobial use in food animals to
   resistance in bacteria infecting humans is the subject
   of debate (5-8).

   We compared antimicrobial resistance patterns of
   Salmonella enterica serovar Typhimurium (ST) from human
   and cattle sources over a 15-year period and examined
   how these patterns relate to antimicrobial use in
   livestock and humans.

   The Study

   We used all ST (including S. Typhimurium var
   Copenhagen) isolates from clinical bovine samples
   submitted to the Washington Animal Disease Diagnostic
   Laboratory from 1986 through 1997 and from cattle herds
   tested by the Field Disease Investigation Unit during
   salmonellosis outbreaks over the same period (n = 378).
   For herds sampled repeatedly over time, all but the
   first ST isolate (per year) were excluded.
   Antimicrobial resistance data were also available for
   ST isolates from cattle for 1982 through 1986 from
   clinical submissions to the Washington Animal Disease
   Diagnostic Laboratory. Isolates from human clinical
   specimens (n = 715) were obtained from the Washington
   State Department of Health Public Health Laboratory for
   1989, 1994, 1996, and 1997, and from the Idaho Division
   of Health, Bureau of Laboratories for 1997.

   Isolates from other laboratories were subcultured onto
   solid brain heart infusion agar. All isolates were
   maintained in a -70 C bank freezer in brain heart
   infusion broth containing 25% to 30% buffered glycerol.
   Isolates were streaked for isolation on sheep blood
   agar prior to susceptibility testing. Susceptibility
   testing for the antimicrobial drugs listed in Table 1
   was done by a disk diffusion method (9) on
   Mueller-Hinton agar prepared according to National
   Committee for Clinical Laboratory Standards (NCCLS)
   guidelines (10,11). Ciprofloxacin susceptibility was
   tested on a subset of isolates systematically selected
   to include five per species per year.

   Table 1. Resistance to individual antibiotics among
   Salmonella Typhimurium isolates from cattle and humans

   
Data for the analysis were divided into periods 1982-1986,
1987-1990, 1991-1994, and 1995-1997. These periods were chosen to compare isolates from cattle with isolates from humans for the years for which isolates from humans were available. Each isolate was classified as resistant or susceptible to each antimicrobial drug tested by the threshold zone size for resistance, as recommended by
NCCLS (10,11). The proportions of isolates resistant to individual drugs and having each antimicrobial resistance pattern were computed by species and period. Significance testing of differences in proportions was done with Epi Info (12) using the chi-square test and the chi-square test for trend.

Marked changes in resistance to chloramphenicol were observed for isolates from both cattle and humans (Tables 1, 2). Before 1991, fewer than 5% of isolates from cattle and only 3% of the 1989 isolates from humans were resistant to chloramphenicol; by the mid-1990s, more than 70% (90 of 123) of isolates from cattle (p <0.01) and almost 50% (92 of 187) of isolates from humans (p <0.01) were resistant to chloramphenicol. Most (79%) isolates from cattle
were resistant to ampicillin, streptomycin, tetracycline, and sulfonamides throughout the study period. Among isolates from humans, the proportion resistant to these drugs was significantly lower in 1989 than in 1997 (p <0.01). The proportion of isolates from cattle resistant to kanamycin was significantly less (p <0.01) in 1990 to 1994 than in 1986 to 1990. All isolates tested were susceptible to ciprofloxacin, and average zone sizes showed no evidence of decline during the period (data not shown).

Table 2. Antimicrobial resistance patterns for Salmonella
Typhimurium isolates from cattle and humans

Among isolates from both cattle and humans, the ACSSuT resistance pattern was the most frequent and increased in frequency oer the study period (Table 2). Before 1991, ACSSuT accounted for fewer than 4 (2%) of 255 of isolates from both species, and by the mid-1990s it accounted for more than 55 (40%) of 123 of isolates from cattle and
more than 156 (35%) of 436 of isolates from humans. Isolates with the ACSSuT resistance pattern together with ACKSSuT-resistant isolates accounted for 79 (64%) of 123 isolates from cattle and 191 (44%) of 436 isolates from humans by the mid-1990s. As previously reported, 57 (95%) of 60 of these isolates were phage typed and found to be DT104 with a single pulsed-field gel electrophoresis pattern (13).

Isolates susceptible to all drugs tested were more common from human than cattle sources (166 [23.2%] of 715 vs. 19 [5.0%] of 378, p <0.01). The proportion of isolates from humans susceptible to all drugs tested decreased substantially from 1989 to 1997 (chi-square test for trend, p <0.01), while that of isolates from cattle was 10%
or less for all periods studied (Table 2).

Conclusions

Antimicrobial resistance has been commonly observed in human and bovine ST isolates since the earliest days of antimicrobial use. This study provides a longitudinal perspective on resistance in ST from cattle and humans in a region and allows insight as to the mechanism of changes in antimicrobial resistance. The greatest changes were in chloramphenicol and kanamycin resistance in isolates
from cattle and ampicillin, chloramphenicol, streptomycin,
sulfonamides, and tetracycline resistance in isolates from humans. Changes in resistance in isolates from both species were primarily due to the sharply increased occurrence of isolates displaying the ACSSuT resistance pattern, a reliable marker for multidrug-resistant definitive type 104 ST (MR-DT104) (14). This has been shown to be so
in the Pacific Northwest through a subset of isolates from this study (13).

MR-DT104 was first detected almost simultaneously in several
geographic areas, including the United Kingdom (15), the United States (13), and Canada (16). Molecular genetic studies indicate that the same gene cassette accounts for multiple resistance in isolates from these and other diverse geographic areas (17,18). This study not only provides supporting evidence that MR-DT104 from different regions are clonal in origin, but refutes the notion that the multiple antimicrobial resistance of this clone was due to acquisition of new resistance genes by indigenous ST in each region.

It is possible that local antimicrobial selection pressure played an important role in dissemination of MR-DT104 through cattle populations into the human population. However, several observations argue against this hypothesis. First, the rise in the percentage of resistance to chloramphenicol in isolates from cattle occurred after
the withdrawal of the drug for use in food animals in the mid-1980s (19) and before the 1996 approval of florfenicol (a chloramphenicol analog that shares resistance loci with chloramphenicol in MR-DT104 [20]) for therapeutic use in cattle. Second, before the dissemination of MR-DT104, most isolates from cattle were resistant to ampicillin, streptomycin, sulfonamides, and tetracycline. It is
not evident how, in the absence of chloramphenicol use,
antimicrobial selection pressure would favor R-type ACSSuT over ASSuT, although it is possible that an unmeasured resistance factor favored the dissemination of MR-DT104 over ASSuT strains. Third, early in its global dissemination, MR-DT104 was isolated from several species of wildlife, which are not exposed to substantial amounts of antimicrobial drugs (13). Finally, reports of broad dissemination of Salmonella clones susceptible to antimicrobial drugs commonly used in livestock provide evidence that agent factors other than antimicrobial resistance are necessary for broad dissemination (21,22).

Nevertheless, some selection pressure that likely involved
antimicrobial use must explain the high prevalence of antimicrobial resistance among ST. There is strong evidence that livestock are the main reservoir for human salmonellosis in industrialized countries (23); however, it would be an error to assume that the emergence of
a globally disseminated clone can be attributed to antimicrobial use in livestock. A human reservoir exists for nontyphoidal Salmonella, including serovar Typhimurium, in developing countries (24,25), and there is strong evidence that antimicrobial use in humans has not only driven the emergence of multidrug-resistant clones in these regions but has resulted in an increasingly high prevalence of
multiple resistance (26-29). Dissemination of multidrug-resistant Salmonella from developing countries, through human traffic, is well documented (30,31) and seems a more likely mode of international transport than the far more limited international livestock traffic.

Multidrug-resistant clones capable of global dissemination can emerge as a result of antimicrobial selection pressure in either livestock or humans; simply restricting antimicrobial use in livestock populations cannot prevent broad dissemination. The problem of globally distributed multidrug-resistant bacterial clones can be compared to the nosocomial scenario: prudent antimicrobial use is a sensible step, but the main effort must go toward preventing dissemination if the program is to be effective.

Acknowledgments

The authors thank Jay Lewis, Donna Green, and Beth Mamer for their help in providing Salmonella isolates for this project.

Dr. Davis is a Ph.D. candidate in the Washington State University College of Veterinary Medicine Field Disease Investigation Unit. Formerly she was an epidemiologist at the Seattle-King County Department of Public Health. Dr. Davis is interested in the epidemiology and farm ecology of zoonotic enteric diseases, including Escherichia coli O157:H7 infection, salmonellosis, and campylobacteriosis.

Address for correspondence: Margaret A. Davis, Field Disease
Investigation Unit, Department of Veterinary Clinical Sciences, Washington State University, Pullman, WA 99164-6610, USA; fax: 509-335-0880; e-mail: madavis@vetmed.wsu.edu.

References

1. Centers for Disease Control and Prevention. U.S. Department of Health and Human Services. Salmonella surveillance. Annual Tabulation Summary 1996.

2. Ferris KE, Miller DA. Salmonella serotypes from animals and related sources reported during July 1996-June 1997. Proceedings of the 101st Annual Meeting of the United States Animal Health Association; 1997 Oct 18-24; Louisville, Kentucky. 1997. p. 423-43.

3. Oosterom J. Epidemiological studies and proposed preventive measures in the fight against human salmonellosis. Int J Food Microbiol 1991;12:41-51.

4. Khachatourians GG. Agricultural use of antibiotics and the evolution and transfer of antibiotic-resistant bacteria. CMAJ 1998;159:1129-36.

5. Piddock LJV. Does the use of antimicrobial agents in veterinary medicine and animal husbandry select antibiotic-resistant bacteria that infect man and compromise antimicrobial chemotherapy? J Antimicrob Chemother 1996;38:1-2.

6. Van den Bogaard AE. Antimicrobial resistancerelation to human and animal exposure to antibiotics. J Antimicrob Chemother 1997;40:453-4.

7. Vernon R. Ciprofloxacin-resistant Salmonella typhimurium DT104 [letter]. Vet Rec 1998;142:287.

8. Institute of Medicine. Antimicrobial resistance: Issues and options. Workshop report, Forum on Emerging Infections. Washington: National Academy Press; 1998.

9. Bauer AW, Kirby MMW, Sherris JC, Turck M. Antibiotic
susceptibility testing by a standard single disk method. Am J Clin Pathol 1966;45:493-6.

10. National Committee for Clinical Laboratory Standards.
Performance standards for antimicrobial disk susceptibility tests. 5th ed. Approved standard; M2-A5, Vol. 13, No. 24. Villanova (PA): The Committee; 1993.

11. National Committee for Clinical Laboratory Standards.
Performance standards for antimicrobial disk susceptibility tests. 5th informational supplement; M100-S5, Vol. 14, No. 16. Villanova (PA): The Committee; 1994.

12. Dean AG, Dean JA, Coulombier D, Brendel KA, Smith DC, Burton AH, et al. Epi Info, Version 6: A word-processing, database, and statistics program for public health on IBM-compatible microcomputers. Atlanta (GA:) Centers for Disease Control and Prevention; 1995.

13. Besser TE, Gay CC, Gay JM, Hancock DD, Rice D, Pritchett LC, et al. Salmonellosis associated with S. typhimurium DT104 in the USA. Vet Rec 1997;140:75.

14. Glynn MK, Bopp C, Dewitt W, Dabney P, Mokhtar M, Angulo FJ. Emergence of multidrug-resistant Salmonella enterica serotype Typhimurium DT104 infections in the United States. N Engl J Med 1998;338:1333-8.

15. Threlfall EJ, Frost JA, Ward LR, Rowe B. Epidemic in cattle and humans of Salmonella typhimurium DT104 with chromosomally integrated multiple drug resistance. Vet Rec 1994;134:577.

16. Poppe C, Smart N, Khakhria R, Johnson W, Spika J, Prescott J. Salmonella typhimurium DT104: a virulent and drug-resistant pathogen. Can Vet J 1998;39:559-64.

17. Ridley A, Threlfall EJ. Molecular epidemiology of antibiotic resistance genes in multiresistant epidemic Salmonella typhimurium DT104. Microb Drug Resist 1998;4:113-8.

18. Casin I, Breuil J, Brisabois A, Moury F, Grimont F, Collatz E. Multidrug-resistant human and animal Salmonella typhimurium isolates in France belong predominantly to a DT104 clone with the chromosome- and integron-encoded -lactamase PSE-1. J Infect Dis 1999;179:1173-82.

19. Knapp WA Jr. Report of the Committee on Pharmaceuticals,
Pesticides, and Related Toxicology. Proceedings of the 88th Annual Meeting of the United States Animal Health Association. 1984 Oct 21-26; Fort Worth, Texas.

20. Bolton LF, Kelley LC, Lee MD, Fedorka-Cray PJ, Maurer JJ. Detection of multidrug-resistant Salmonella enterica serotype typhimurium DT104 based on a gene which confers cross-resistance to florfenicol and chloramphenicol. J Clin Microbiol 1999;37:1348-51.

21. Khakhria R, Bezanson G, Duck D, Lior H. The epidemic spread of Salmonella typhimurium phage type 10 in Canada (1970-1979). Can J Microbiol 1983;29:1583-8.

22. Passaro DJ, Reporter R, Mascola L, Kilman L, Malcolm GB, Rolka H, et al. Epidemic Salmonella enteritidis infection in Los Angeles County, Californiathe predominance of phage type 4. West J Med 1996;165:126-30.

23. Hancock DD, Lynn TV, Besser TE, Wikse SE. Feasibility of
preharvest food safety control. Compendium on Continuing Education for the Practicing Veterinarian 1997;19:S200-7.

24. Gracey M, Iveson JB, Sunoto, Suharyono. Human salmonella
carriers in a tropical urban environment. Trans R Soc Trop Med Hyg 1980;74:479-82.

25. Vahaboglu H, Dodanli S, Eroglu C, ztrk R, Soyletir G, Yildirim I, et al. Characterization of multiple-antibiotic-resistant Salmonella typhimurium strains: molecular epidemiology of PER-1-producing isolates and evidence for nosocomial plasmid exchange by a clone. J Clin Microbiol 1996;34:2942-6. 

26. Kariuki S, Gilks C, Corkill J, Kimari J, Benea AP, Hart CA. Multi-drug resistant non-typhi salmonellae in Kenya. J Antimicrob Chemother 1996;38:425-34.

27. Agarwal KC, Garg RK, Panhotra BR, Verma AD, Ayyagari A, Mahanta J. Drug resistance in Salmonellae isolated at Chandigarh (India) during 1972-1978. Antonie Van Leeuwenhoek 1980;46:383-90.

28. Sharma KB, Bhat MB, Pasricha A, Vaze S. Multiple antibiotic resistance among salmonellae in India. J Antimicrob Chemother 1979;5:15.

29. Ling J, Chau PY, Rowe B. Salmonella serotypes and incidence of multiply-resistant salmonellae isolated from diarrhoeal patients in Hong Kong from 1973-1982. Epidemiol Infect 1987;99:295-306.

30. Centers for Disease Control. Multiresistant Salmonella and other infections in adopted infants from India. MMWR Morb Mortal Wkly Rep 1982;31:285-7.

31. Seyfarth AM, Wegener HC, Frimodt-Mller. Antimicrobial
resistance in Salmonella enterica subspecies enterica serovar typhimurium from humans and production animals. J Antimicrob Chemother 1997;40:67-75.

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

URL: http://www.cdc.gov/ncidod/eid/vol5no6/ascii/davis.txt

Please note that figures, equations, and most tables are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively.  Greek symbols are spelled out. >/= is used for greater than or equal to.

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

Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999]
   Dispatches

   Toxic Shock Syndrome in the United States: Surveillance
   Update, 197919961

   Rana A. Hajjeh,* Arthur Reingold, Alexis Weil,*
   Kathleen Shutt,* Anne Schuchat,* and Bradley A.
   Perkins*

   *Centers for Disease Control and Prevention, Atlanta,
   Georgia, USA; and School of Public Health, University
   of California, Berkeley, California, USA

    Menstrual toxic shock syndrome (TSS)
    emerged as a public health threat to women
    of reproductive age in 197980. We reviewed
    surveillance data for the period 1979 to
    1996, when 5,296 cases were reported, and
    discuss changes in the epidemiologic
    features of TSS.

   Toxic shock syndrome (TSS) emerged as a result of
   changes in industry and personal behavior but responded
   to rapid public health action, including active
   surveillance (1). This illness received national
   attention in 1980 when unexplained febrile illness
   associated with shock, multiorgan dysfunction, and high
   death rates was reported in healthy young women from
   several states (2,3). This clinical syndrome had been
   described sporadically since the 1920s (4). The
   dramatic increase in the number of cases in 1979-80
   spurred epidemiologic, clinical, and laboratory studies
   that resulted in better understanding of the
   association between high-absorbency tampons and TSS
   (5). These studies led to recommendations that
   substantially decreased the risk for TSS (6,7).

   TSS became a nationally notifiable disease in 1980 (8).
   After the initial epidemic, the number of reported
   cases decreased significantly. In 1986, active
   surveillance was conducted in many areas in the United
   States (total population 34 million) to confirm that
   trend (9). The cumulative incidence (0.5 per 100,000
   population) confirmed the substantial decrease in the
   incidence of menstrual TSS observed in the passive
   surveillance system. Incidence rates decreased from 6
   to 12 per 100,000 among women 12 to 49 years of age
   (10,11) in 1980 to 1 per 100,000 among women 15 to 44
   years of age in 1986. These data also demonstrated that
   passive surveillance accurately described the
   demographic characteristics and case-fatality ratio of
   TSS cases (12,13).

   Ongoing surveillance in the United States allows us to
   estimate the current incidence of TSS and monitor
   whether new menstrual or vaginal products affect the
   risk for disease. Other products, including
   high-absorbency disposable diapers, have raised similar
   questions regarding TSS in children. To address these
   questions and describe the current epidemiologic
   characteristics and recent temporal trends of the
   syndrome, we reviewed data from the ongoing national
   surveillance for TSS from 1979 through 1996, focusing
   on three periods: 1979 to 1980 (the epidemic years),
   1981 to 1986 (a period of increased TSS awareness,
   culminating in active surveillance in 1986), and 1987
   to 1996.

   The Study

   The national TSS surveillance system has been described
   (8). Cases of TSS are reported to the Centers for
   Disease Control and Prevention (CDC) by state health
   departments in standardized case reports that include
   information on demographic and clinical
   characteristics, hospitalization status, outcome,
   laboratory data, products used during menses, and
   recurrence of menstruation-associated cases.

   We used the surveillance case definition for TSS
   revised in 1981, which requires five clinical criteria:
   fever, hypotension, rash, desquamation, and
   abnormalities in three or more organ systems (8). A
   definite case fulfilled all five criteria (unless the
   patient died before desquamation), and a probable case
   fulfilled four of the five criteria. For the purposes
   of this analysis and because information on
   desquamation was often unavailable because of death or
   early discharge, we included all TSS reports meeting
   either definition. A similar change regarding exclusion
   of desquamation was adopted in a retrospective study
   (14).

A TSS case was considered menstrual if onset of symptoms
occurred within 3 days of the beginning or end of menses;    all other cases were considered nonmenstrual and were classified into three categories: surgical
wounds, postpartum or postabortion, and other.
Information on recurrent episodes of TSS was collected only from women with menstrual TSS, by asking them about a similar previous illness during menstruation. Data were analyzed by using SAS 6.1 (SAS, Cary, NC). The chi-square test was used to test for statistical significance.

   From 1979 to 1996, 5,296 TSS cases were reported; 1,035
   of these were reported from 1987 to 1996 (Figure).
   Overall, 93% of all TSS cases reported from 1979 to
   1996 were among women. Although the proportion of
   menstrual cases decreased (Table), TSS affected mostly
   women. The median age was 22 years (3 days to 87
   years); 91% were white. No significant seasonal
   variation was noted. For cases in which the source of
   the report was known (n = 2,118), 64% were reported by
   infection control practitioners and 28% by physicians.

Figure. Toxic shock syndrome cases,* menstrual vs. nonmenstrual, United States, 19791996.

Table. Demographic characteristics, outcome, and proportion of menstrual cases of toxic shock syndrome, United States,
19791996

Menstrual TSS accounted for 74% of TSS cases during 1979 to 1996 (n = 5,296); this proportion, however, declined from 91% during 1979 and 1980 to 71% during 1981 to 1986 and 59% during 1987 to 1996. The median age of patients with menstrual TSS was 21 years, but 41% (n = 1,591) of menstrual TSS cases occurred in female patients 13 to 19 years of age. No significant change in the age distribution of menstrual cases was noted between the three periods (median age: 21
years, 1979 to 1980; 20 years, 1981 to 1986; 25 years, 1987 to 1996). Most (98%) patients with menstrual TSS cases for which the menstrual product was known (n = 3,457) reported tampon use, a proportion that did not change; 89% used tampons only, while 5.0% used both tampons and pads and 3% used tampons and minipads. The level of tampon absorbency was reported in only 41% (n = 1,385) of cases: 28% of patients used regular tampons, while 71% used super-absorbency tampons. Staphylococcus aureus was isolated from
90% of women with menstrual TSS who had vaginal cultures performed (n = 2,536). Of all the women with menstrual TSS, 1,606 (30%) responded to the question regarding recurrent disease, and 10% reported a previous illness similar to their TSS episode.

Nonmenstrual cases also occurred mostly in women (73%) and whites (87%). During 1987 to 1996, the proportion of all TSS cases that were nonmenstrual averaged 41% (30% to 55%). Of nonmenstrual cases, 18.3% were reported after surgical procedures, 11.5% were postpartum or postabortion, and 23.1% had nonsurgical cutaneous lesions. The proportion of all nonmenstrual cases reported after surgical procedures increased from 14% during 1979 to 1986 to 27% during 1987
to 1996 (p <0.05). Among female nonmenstrual case-patients, 12% reported using barrier contraceptives (sponges and diaphragms); this proportion was, however, significantly less (p <0.05) in 1987 to 1996 (6%) than in 1979 to 1986 (14%). Fifty cases of TSS in children < 5 years of age were reported during the 17-year period; more than half of these occurred in children < 2 years of age, and most (61.7%) were associated with nonsurgical cutaneous lesions. The percentage of TSS associated with nonsurgical cutaneous lesions (23.1%) was higher among younger patients (overall case-fatality ratio 4% [n = 2 deaths]) than among patients with other nonmenstrual cases.

Hospitalization status was known for 2,930 patients; 98% were hospitalized. The overall TSS case-fatality ratio was 4.1% (3% for menstrual cases, 5% for nonmenstrual cases) and was significantly higher among nonmenstrual cases (p <0.005). Among menstrual cases, the case-fatality ratio decreased significantly with time (from 5.5% in 1979 and 1980, to 2.8% in 1981 to 1986, to 1.8% in 1987 to 1996,
chi-square for linear trend [p = 0.0001]). This trend was not observed among nonmenstrual cases (for the three time periods: 8.5%, 5.3%, and 6%, respectively).

Conclusions

Our review of recent passive surveillance data confirms the
declining trend previously noted by active surveillance in 1986. Changes observed in the epidemiologic characteristics of TSS include an increase in the proportion of nonmenstrual cases and the difference in the risk for death between menstrual and nonmenstrual cases.

A number of factors could account for the observed decline,
including the decrease in tampon absorbency, the standardized labeling required by the U.S. Food and Drug Administration; greater awareness of TSS among women; and the proliferation of educational materials for women, including tampon package inserts (12). However,
at least 40% of menstrual TSS cases continue to affect women 13 to 19 years old, an age group not as likely to be aware of the risk for TSS and for whom further education may be needed.

Over the last few years, two changes have occurred in tampon use and composition: 1) All-cotton tampons have been introduced and marketed as an alternative product; in vitro studies have not, however, found any differences in the effects of these new tampons on the production of toxic shock syndrome toxin-1 (TSST-1) or its adsorption (15). 2) Tampons marketed specifically for overnight use have also been introduced. TSST-1 toxin production is not the only
indicator of the potential risk for TSS. Previous case-control studies found that exclusive use of tampons was associated with a higher risk for TSS than use in conjunction with pads (13). Continued surveillance will monitor the effect of these changes on TSS occurrence. However, because the syndrome is rare, only large
changes in the use of higher absorbency products would be likely to come to public health attention.

One of the important changes in the epidemiology of TSS is the increasing proportion of nonmenstrual cases, in particular, cases reported after surgical procedures. The factors contributing to this increase may include changes in delivery of surgical health-care services, with an increase in both outpatient procedures and the use of prosthetic devices. Hospitalizations caused by infections from
prosthetic devices and postoperative infections increased
significantly from 1980 to 1994 in the United States (16). In addition, the case-fatality ratio of nonmenstrual TSS cases did not decline, although the case-fatality ratio of menstrual TSS did. This difference in death rates could be due to several factors: nonmenstrual TSS may occur in less healthy (e.g., older) persons; diagnostic and reporting biases may result in more sensitive detection of severe cases of nonmenstrual TSS; decreased awareness
of nonmenstrual TSS among health-care professionals may lead to increased deaths because of late treatment; and the nonspecific signs and symptoms of postoperative TSS may delay diagnosis (17). Further studies are needed to validate the difference in deaths between the two types of TSS and to clarify the risk factors for nonmenstrual TSS. Recent molecular studies of the gene that carries toxic shock toxin (18) may contribute to a better understanding of the emergence of TSS and transmission of toxin-producing strains of S. aureus and could improve prevention. Physicians, in particular surgeons, and other health-care professionals may need further education about the risks for nonmenstrual TSS.

Dr. Hajjeh is a medical epidemiologist in the Division of Bacterial and Mycotic Diseases Branch, CDC. Her areas of expertise and research interests include epidemiology of mycotic diseases, bacterial meningitis, and unexplained critical illnesses of possible infectious etiology.

Address for correspondence: Rana A. Hajjeh, Centers for Disease Control and Prevention, Division of Bacterial and Mycotic Diseases, 1600 Clifton Road, Mail Stop C09, Atlanta, GA 30333, USA; fax: 404-639-0817; e-mail: rfh5@cdc.gov.

References
 
1. U.S. Public Health Services. Addressing emerging infectious disease threats. A prevention strategy for the United States. Atlanta: Centers for Disease Control and Prevention; 1994.

2. Todd JK, Kapral FA, Fishaut M, Welch TR. Toxic shock syndrome associated with phage group 1 staphylococci. Lancet 1978;2:1116-8.

3. Centers for Disease Control and Prevention. Toxic-shock
syndromeUnited States. MMWR Morb Mortal Wkly Rep 1980;29:229-30.

4. Broome CV. Epidemiology of toxic shock syndrome in the United States: Overview. Reviews of Infect Diseases 1989:2 Suppl 1:S14-21.

5. Shands KN, Schmid GP, Dan BB, Blum D, Guidotti RJ, Hargrett NT, et al. Toxic-shock syndrome in menstruating women: association with tampon use and Staphylococcus aureus and clinical features in 52 cases. N Engl J Med  1980:303:1436-42.

6. Centers for Disease Control and Prevention. Toxic shock
syndromeUnited States. MMWR Morb Mortal Wkly Rep 1980:2979.

7. Centers for Disease Control and Prevention. Follow-up on
toxic-shock syndrome. MMWR Morb Mortal Wkly Rep 1980;29:441-5.

8. Reingold AL, Hargrett NT, Shands KN, Dan BB, Schmid GP,
Strickland BY, et al. Toxic shock syndrome surveillance in the United States, 1980 to 1981. Ann Intern Med 1982;96 (part 2):875-80.

9. Gaventa S, Reingold AL, Hightower AW, Broome CV, Schwartz B, Hoppe C, et al. Active surveillance for toxic shock syndrome in the United States. 1986. Reviews of Infectious Diseases 1989:2 Suppl 1:S28-34.

10. Davis JP, Chesney PJ, Wand PJ, LaVenture M. Toxic-shock
syndrome: epidemiologic features, recurrence, risk factors and prevention. N Engl J Med 1980:303:1429-35.

11. Latham RH, Kehrberg MW, Jacobson JA. Toxic shock syndrome in Utah: a case-control and surveillance study. Ann Intern Med 1982:96:906-8.

12. Schuchat A, Broome CV. Toxic shock syndrome and tampons.
Epidemiol Rev 1991:13:99-112.

13. Centers for Disease Control and Prevention. Reduced incidence of menstrual toxic shock syndromeUnited States, 1980-1990. MMWR Morb Mortal Wkly Rep 1990;39:421-4.

14. Petitti DB, Reingold AL. Update through 1985 on the incidence of toxic shock syndrome among members of a prepaid health plan. Reviews of Infectious Diseases 1989;11 Suppl 10:S22-7.

15. Schlievert PM. Comparison of cotton and cotton/rayon tampons for effect on production of toxic shock syndrome toxin. J Infect Dis 1995;172:1112-4.

16. Simonsen L, Conn LA, Pinner RW, Teutsch S. Trends in infectious disease hospitalizations in the United States, 1980-1994. Arch Intern Med 1998;158:1923-8.

17. Hughes D, Stapleton J. Postoperative toxic shock syndrome. Iowa Med 1991;81:55-8.

18. Lindsay J, Ruzin A, Ross HF, Kurepina N, Novick RP. The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus. Mol Microbiol 1998;29:527-43.

1Presented in part at the European Conference on Toxic Shock
Syndrome, Royal Society of Medicine, London, September 1997, and at the International Conference on Emerging Infectious Diseases, Atlanta, March 1998. (Arbuthnott J, Furman B, editors. European conference on toxic shock syndrome, 1997. London [UK]: The Royal Society of Medicine Press Limited; 1998.)

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

URL: http://www.cdc.gov/ncidod/eid/vol5no6/ascii/hajjeh.txt

Please note that figures, equations, and most tables are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively.  Greek symbols are spelled out. >/= is used for greater than or equal to.

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

Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999]

   Dispatches

   New Rickettsiae in Ticks Collected in Territories of
   the Former Soviet Union

   Elena Rydkina,* Vronique Roux,* Natalia Fetisova,
   Nikolai Rudakov, Mouniver Gafarova, Irina
   Tarasevich, and Didier Raoult*

   *Facult de Mdecine, Universit de la Mditerrane,
   Marseille, France; Russian Academy of Medical
   Sciences, Moscow, Russia; Research Institute of
   Endemic Infectious Diseases, Omsk, Russia; and Medical
   University, Simferopol, Crimea

    Dermacentor nuttallii from Siberia,
    Rhipicephalus sanguineus from Crimea, and
    Rh. pumilio from the Astrakhan region were
    infected with Rickettsia sibirica (12%), R.
    conorii (8%), and the Astrakhan fever agent
    (3%), respectively. Three new Rickettsiae
    of the R. massiliae genogroup were
    identified in ticks by 16S rDNA, gltA, and
    ompA sequencing.

   Rickettsiae are obligate intracellular gram-negative
   bacteria associated with arthropod vectors, ticks,
   mites, and insects that, while feeding, can transmit
   Rickettsiae to animals and humans. The rickettsioses
   have characteristic clinical features, including fever,
   headache, maculopapular eruption, and sometimes eschar
   formation (primary lesion). The number of
   representatives of the genus Rickettsia has increased
   over the last decades as a result of improved cell
   culture isolation and agent identification techniques
   (1). Sequence comparison of gene coding for citrate
   synthase (gltA) (2), rOmpA outer membrane protein
   (ompA) (3), and 16S rRNA (4) has become the most
   reliable method of identifying Rickettsiae (5-8). We
   describe polymerase chain reaction (PCR) amplification
   and sequence determination to identify Rickettsiae in
   naturally infected ixodid ticks in three regions of
   Russia endemic for tickborne rickettsioses.

   Rhipicephalus pumilio ticks (65
   adults) were collected in 1996
   from dogs in the Astrakhan region.
   Dermacentor nuttallii ticks (101
   adults) were collected in 1994 in
   the village of Verhnyi Kouus, the
   Altay Mountains, Siberia. In 1997,
   Rh. sanguineus ticks (2 adults and
   35 nymphs) were collected in the
   town of Saki, Crimea region, from
   dogs whose owners had serologic
   evidence of Mediterranean spotted fever (Figure 1). The
   ticks were kept at room temperature before being washed
   in iodized alcohol (10 minutes) just before testing,
   rinsed in distilled water, and dried on sterile filter
   paper. DNA was extracted from ticks by using the QIAmp
   Tissue Kit (QIAGEN, Hilden, Germany). Rickettsial DNA
   was detected by PCR with primers specific for
   Rickettsiae: RpCS.877p-RpCS.1273r, which amplify a
   396-bp fragment of gltA (2), and Rr190.70p-190-701 (3),
   which amplify a fragment of ompA from 629 to 632 bp.
   For all positive ticks, 587 to 590 bp of ompA were
   sequenced by using the ABI PRISM Dye Terminator Cycle
   Sequencing Kit with Amplitap Polymerase FS (PE Applied
   Biosystems, Warrington WA1 4SR, UK). Sequences were
   analyzed with the Applied Biosystem 377 automatic
   sequencing system. For newly detected genotypes,
   sequences of 16S rRNA encoding gene, gltA, and ompA
   were determined as previously described (2-4) (see
   Figure 2 for GenBank codes).

   We detected two different Rickettsiae in Rh. pumilio
   (Astrakhan fever agent and RpA4 genotype); two
   Rickettisiae from D. nutallii in Siberia (Rickettsia
   sibirica and DnS14 and DnS 28 genotypes); and R.
   conorii from Rh. sanguineous ticks in Crimea (Table).

    Figure 1. Areas from which ticks in the study were collected. 

   Table. Ticks infected in regions of the former
   territories of the USSR

Our results confirm previous data of high epidemic activity of the Altay focus for North Asian tick typhus and the crucial role of D. nuttallii as a reservoir of R. sibirica infection (9). Our results are also consistent with those of a study in 1991 based on hemolymph testing (10), in which 3.2% of ticks from the Astrakhan region were demonstrated to be infected with Rickettsiae.

An outbreak of Mediterranean spotted fever due to infection with R. conorii occurred in Crimea from 1947 to 1957. Only sporadic cases of the disease were reported (11) until 1995, when the incidence of Mediterranean spotted fever increased in central Crimea, with 40 cases in 1996 and more than 70 in 1997. Most cases occurred in the summer, when the Rh. sanguineus nymphs (principal vectors of R. conorii) (1) were active. Our results, showing that 8% of the Rh. sanguineus studied contained R. conorii DNA, provide further evidence of the Mediterranean spotted fever outbreak in the region.
To date, only the R. conorii strain M-1, isolated in the territories of the former Soviet Union (the Black Sea coast of Georgia),has been genetically characterized. This strain is genetically distinct from the other strains of R. conorii, i.e., Indian tick typhus and the Moroccan and Malish strains (3). Our detection of the R. conorii
strain identical to the Malish strain is the first evidence of the genetic heterogeneity of R. conorii in the region.

The ompA sequences obtained from PCR-amplified products were
different from those described for the known Rickettsiae for one DNA sample extracted from Rh. pumilio from the Astrakhan region (RpA4) and four DNA samples from D. nuttallii collected in Siberia (DnS14, DnS28, DnS79, DnS94). The sequences for the samples from D. nuttalii
(DnS28, DnS79, and DnS94) were identical but differed from those of DnS14 and Rh. pumilio RpA4/2.

The three new rickettsial agents were closely
related and branched with members of the R.
massiliae group, together with R. rhipicephali,
Bar 29, R. aeschlimannii, and R. montanensis
(Figure 2). Comparison of the sequences
obtained by using the program BLAST
demonstrated that they also differed from those
of the Cadiz agent characterized from Ixodes
ricinus in Spain (6), those of the Cooleyi
genotype characterized from I. scapularis (5),
MOA and WB-8-2 isolated from Amblyomma
americanum and I. scapularis, respectively (8),
and R. peacockii (7) in the United States.

Figure 2.1 Phylogenetic tree of Rickettsiae inferred from comparison of the ompA sequences. The known tick vectors for the bacteria presented on the dendrogram are indicated on the right. The ompA sequences were aligned by the multisequence alignment program CLUSTAL in the
BISANCE software package. Phylogenetic relationships were inferred by using version 3.4 of the PHYLIP software package. Evolutionary distance matrices, generated by DNADIST, were determined by the Kimura method. Matrices were used to construct dendrograms by the neighbor-joining
method. Data were also examined by using parsimony analysis (DNAPARS in PHYLIP), and bootstrap analyses were performed to investigate the stability of the trees obtained. Bootstrap values were obtained for a consensus tree based on 100 randomly generated trees by using SEQBOOT and CONSENSE in the same package. The percentage of similarity between strains was determined by using the PC/GENE software  package. 

1The received sequences of the new rickettsial agents have been deposited in GenBank. Sequences of ompA were deposited as two parts under accession numbers: DnS28, AF120018 and AF120019; DnS14, AF120021, and AF120020; RpA4, AF120022, and AF120023. The 16S rRNA gene and gltA sequences have been deposited in GenBank under accession numbers DnS28, gltA - AF120027, 16S rRNA gene AF120024; DnS14, gltA AF120028, 16S rRNA gene AF120025; RpA4, gltA - AF120029, 16S rRNA gene AF120026. 

The pathogenicity of the members of the R.
massiliae group is unknown, and their main reservoirs are regarded as ticks of the genus Rhipicephalus for R. massiliae and Bar 29. R. aeschlimannii has been isolated from Hyalomma marginatum and R. montanensis from ticks of the genus Dermacentor. R. rhipicephali has been demonstrated in ticks of the genus Dermacentor and in Rh. sanguineus (1). The similarity of gltA, ompA, and 16S rRNA gene sequences indicates that these three new agents are close to each
other (from 99.7% to 99.9%) and could constitute a new rickettsial species.

In the United States, various tickborne Rickettsiae occur in areas endemic for R. rickettsii, the agent of Rocky Mountain spotted fever (7). Similarly, in Mediterranean countries, several recently described Rickettsiae have been found in ticks of the Rh. sanguineus complex in the regions endemic for Mediterranean spotted fever caused by R. conorii (6). The effects of the presence of different Rickettsiae on the prevalence of infection rates of ticks with individual Rickettsiae and on the epidemiology of infections in
humans have yet to be determined. R. sibirica and the Astrakhan fever agent are prevalent in Siberia and the Astrakhan region, respectively, but the pathogenicity of the new rickettsial genotypes has yet to be investigated.

Acknowledgments

We thank P.J. Kelly and E. Birtles for reviewing the article.

Dr. Rydkina is a senior researcher at the Gamalaya Institute in Moscow. She was trained as a postdoctoral fellow in Marseille, France. Her main interest is in rickettsial diseases.

Address for correspondence: Didier Raoult, Unit des Rickettsies. CNRS UPRES-A 6020, Facult de Mdecine, Universit de la Mditerrane, 27 bd. J. Moulin 13385 Marseille, Cedex 05, France; fax: 33-49-183-0390; e-mail: Didier.Raoult@medecine.univ-mrs.fr.

References

1. Raoult D, Roux V. Rickettsioses as paradigms of new or emerging infectious diseases. Clin Microbiol Rev 1997;10:694-719.

2. 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.

3. Fournier P-E, Roux V, Raoult D. Phylogenetic analysis of spotted fever group rickettsiae by study of the outer surface protein rOmpA. Int J Syst Bacteriol 1998;48:839-49.

4. Roux V, Raoult D. Phylogenetic analysis of the genus Rickettsia by 16S rDNA sequencing. Res Microbiol 1995;146:385-96.

5. Billings AN, Teltow GJ, Weaver SC, Walker DH. Molecular
characterization of a novel Rickettsia species from Ixodes
scapularis in Texas. Emerg Infect Dis 1998;4:305-9.

6. Marquez FJ, Muniain MA, Soriguer RC, Izquierdo G, Rodriguez-Bao J, Borobio M. Genotypic identification of an undescribed spotted fever group Rickettsia in Ixodes ricinus from southwestern Spain. Am J Trop Med Hyg 1998;58:570-7.

7. Niebylski ML, Shrumpf ME, Burgdorfer W, Ficher ER, Gage KL, Schwan TG. Rickettsia peacockii sp. nov., a new species infecting wood ticks, Dermacentor andersoni, in Western Montana. Int J Syst Bacteriol 1997;47:446-52.

8. Weller SJ, Baldridge GD, Munderloh UG, Noda H, Simser J, Kurtti T. Phylogenetic placement of Rickettsiae from ticks Amblyomma americanum and Ixodes scapularis. J Clin Microbiol 1998;36:1305-17. 

9. Rudakov NV. Tick-borne rickettsiosis in Russia (epidemiology and current conditions of natural foci). In: Kazar J, Toman R, editors. Rickettsiae and rickettsial diseases. Proceedings of the 5th International Symposium; 1996; Veda, Bratislava. p. 216-20.

10. Eremeeva ME, Beati L, Makarova VA, Fetisova NF, Tarasevich IV, Balayeva NM, et al. Astrakhan fever rickettsiae: antigenic and genotypic analysis of isolates obtained from human and Rhipicephalus pumilio ticks. Am J Trop Med Hyg 1994;51:697-706.

11. Kulagin SM, Tarasevich IV, Rubakin PE, Nikitin AM, Krupina ZN. On eradication of Marseilles fever (in Russian). J Microbiol Epidemiol Immunobiol 1960;8:117-21.

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

URL: http://www.cdc.gov/ncidod/eid/vol5no6/ascii/rydkina.txt

Please note that figures, equations, and most tables are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively.  Greek symbols are spelled out. >/= is used for greater than or equal to.

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

Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999]

   Dispatches

   Computer-Generated Dot Maps as an Epidemiologic Tool:
   Investigating an Outbreak of Toxoplasmosis

   Steven B. Eng,* Denise H. Werker, Arlene S. King,
   Stephen A. Marion, Alison Bell, Judith L.
   Issac-Renton, G. Stewart Irwin,# and William R.
   Bowie

   *Capital Regional District Health Department, Victoria,
   B.C., Canada; Health Canada, Ottawa, Ontario, Canada;
   British Columbia Centre for Disease Control,
   Vancouver, B.C., Canada; University of British
   Columbia, Vancouver, B.C., Canada; Centers for Disease
   Control and Prevention, Atlanta, Georgia, USA; and
   #Greater Victoria Water District, Victoria, B.C.,
   Canada

    We used computer-generated dot maps to
    examine the spatial distribution of 94
    Toxoplasma gondii infections associated
    with an outbreak in British Columbia,
    Canada. The incidence among patients served
    by one water distribution system was 3.52
    times that of patients served by other
    sources. Acute T. gondii infection among
    3,812 pregnant women was associated with
    the incriminated distribution system.

   Epidemiologists have traditionally used maps to examine
   the spatial distribution of disease incidence.
   Computer-generated dot maps have facilitated
   identification of case clusters (1), formulation of
   hypotheses about the source of infection or spatially
   distributed risk factors (2,3), and analysis of data.
   Because most populations are served by identifiable
   water systems, waterborne disease outbreaks lend
   themselves to being plotted on dot maps (1,4-6). We
   describe an automated address-matching and base map
   system in a geographic information system structure
   used to assess information related to an outbreak of
   toxoplasmosis associated with a municipal water system.

   The Capital Regional District (population 321,585) is
   located on Vancouver Island, British Columbia, Canada.
   The district includes the City of Victoria, surrounding
   municipalities and districts, and the Gulf Islands.

   In March 1995, an outbreak of toxoplasmosis was
   suspected when 15 residents of this district were
   identified as having acute infection with Toxoplasma
   gondii. Investigation of these and subsequent cases
   confirmed an outbreak but identified no common food,
   beverage, or event source. A hand-drawn dot map of the
   initial 47 cases showed clustering in the Greater
   Victoria area. The municipal water supply system was
   considered a possible explanation for this spatial
   distribution. To examine this hypothesis,
   computer-based geographic mapping was used to study the
   distribution of all outbreak-related acute cases and
   data collected from a population-based serologic
   screening program to detect T. gondii infection in
   women who were or had been pregnant (7).

   Classification of Cases and the Water Distribution
   Systems

   Patients were classified as having acute, equivocal, or
   nonacute cases or as never infected on the basis of
   serologic tests performed at the Provincial Laboratory
   (British Columbia Centre for Disease Control) and the
   Toxoplasma Serology Laboratory, Research Institute,
   Palo Alto Medical Foundation (8-11). Cases were further
   classified on the basis of clinical symptoms, outbreak
   relatedness, patient's residence, and pregnancy status
   (pregnant, nonpregnant) (7).

   At the time of the outbreak, the Greater Victoria Water
   District operated two disinfection plants supplying
   unfiltered, chloraminated surface water to
   approximately 292,000 residents of the Capital Regional
   District. The higher-pressure Japan Gulch distribution
   system supplied water from the Japan Gulch Reservoir to
   73,000 residents, as well as a one-way transfer of
   water into the other distribution system. The Humpback
   distribution system supplied 219,000 residents (12).

   District residents were further classified as receiving
   high, intermediate, or no exposure to water from the
   Humpback Reservoir.

   Geographic Mapping

   Geographic mapping analysis was performed by using
   MapInfo Version 3.0 for Windows (MapInfo Corporation,
   Troy, NY, 1992-94). MapInfo, which enables data
   containing geographic information to be placed on a
   map, was used for geocoding (i.e., placing markers into
   the database, like pins onto a map). Street address
   data for each record in the datafile were matched
   against an electronic street map of the Capital
   Regional District. Geographic coordinates were taken
   from the electronic street map of the district, which
   is based on the Transportation Centerline Network of
   the British Columbia Ministry of Transportation and
   Highways (13). Lotus Approach 3.0 for Windows (Applied
   Software Corporation, subsidiary of Lotus Development
   Corporation) was used to prepare and validate the data
   before mapping. On the basis of information from the
   Greater Victoria Water District, a street-level scale
   map was drawn in MapInfo to delineate the geographic
   area in the Capital Regional District that receives
   municipal drinking water from the Humpback Reservoir.

Geographic Distribution of Acute Cases

All 94 persons with outbreak-related acute cases
who lived in the Capital Regional District were
grouped by geographic area as being served by the
Humpback Reservoir or other water sources. 
Incidence rates were calculated by using estimated   
population figures (1994). Information on 
residential addresses was obtained from laboratory
requisitions, physicians' offices, hospital
records, the Medical Services Plan of British
Columbia, and direct communication with patients.
For each case, the residential street address at
the time the first specimen was drawn for testing
for T. gondii antibodies was used to produce a
computer-generated dot map. Of the 94 persons with outbreak-related acute cases who lived in the Capital Regional District, 83(88%) lived in the area served by the Humpback  
Reservoir. The incidence rate of acute infection
among persons residing in the area served by the Humpback Reservoir was more than three times that for areas served by other sources (RR = 3.53; 95% confidence interval [CI]: 1.88-6.63; p = 0.0003) (Figure 1).

Figure 1. Geographic distribution of
outbreak-related acute cases of toxoplasmosis
in the Capital Regional District, Vancouver
Island, British Columbia, 1995 (n = 94).

Geographic Distribution of Women Screened during Pregnancy

Data from a population-based screening program were used to determine whether residents served by the Humpback Reservoir were more likely to have acute infection with T. gondii. Serologic screening was offered to an estimated 4,500 women living in the capital regional district who were pregnant between October 1, 1994, and April 30, 1995. To offer
screening to as many of these women as possible, information regarding the screening program was extensively distributed to women, physicians, and the public.

Serologic results were available from the Provincial Laboratory database at the British Columbia Centre for Disease Control. Residential street addresses were obtained by linking the Provincial Laboratory database with the Medical Services Plan database, using the unique personal
health number assigned by the Province. A computer datafile containing the serologic results of screened pregnant women and their addresses was provided to the Capital Regional District Health Department. The data were then prepared and validated, geocoded and mapped, and statistically analyzed.

Three dot maps were generated by MapInfo, showing
the geographic distribution of the screened
population according to their laboratory
classification of never infected (i.e., no
serologic evidence of immunoglobulin [Ig] G or IgM
antibody to T. gondii), nonacute (i.e., typically 
IgG but not IgM antibody to T. gondii), and acute 
(i.e., serologic evidence of acute infection by a
battery of tests) (7-11). Several data subsets were
generated on the basis of two variables: 1)the
residence in the area served by the Humpback 
Reservoir and 2) municipality of residence. Odds
ratios were then calculated by StatCalc in Epi Info
(version 6.03) to test the hypothesis that living
in the area served by the Humpback Reservoir was
associated with infection with T. gondii.
Of 3,982 laboratory records for screened women,
3,962 records were available for coding. The 3,812
women with successfully coded addresses comprise
85% of the estimated 4,500 women eligible for
screening. Of these women, 36 (0.9%) were
classified according to their laboratory results as
having acute infection, 216 (5.7%) as having a
nonacute infection, 3,558 (93.3%) as never having
been infected, and 2 (0.1%) as having equivocal
cases. The age distribution of the women (15 to 45
years [mean 29 years]) did not differ by infection
status (women with equivocal status were excluded).  
Women acutely infected with T. gondii were more the 
than three times as likely as uninfected women 
live in the area served by the Humpback distribution system (odds ratio [OR] 3.05, and exact 95% CI: 1.08-11.91). In contrast, distribution of women with serologic evidence of nonacute infection with T. gondii was not associated with the municipal water distribution system (OR 0.91, exact 95% CI: 0.67-1.25).

Figure 2. Geographic distribution of the residences of women screened during pregnancy and classified as never
infected with toxoplasmosis (n = 3558).

Figure 3. Geographic distribution of the residences of women
screened during pregnancy and classified as having
nonacute cases of toxoplasmosis (n = 216).

Computer-generated dot maps (Figures 2, 3, 4) provided visual information, as well as data for 
statistical analysis. These maps are characteristic 
of an outbreak associated with a one-time event.
The geographic distribution of pregnant women who 
were never infected (Figure 2) indicates that the
susceptible population is similar to the
distribution of the whole population. The
geographic distribution of pregnant women who had 
nonacute cases (Figure 3) is similar to the
population distribution, indicating that before the
outbreak, residence was not associated with
antibody. Visually comparing the geographic
distribution of pregnant women with acute cases
(Figure 4) with that of pregnant women who were
never infected (Figure 2) suggests that most of the recently infected pregnant women lived in the area served by the Humpback Reservoir. The difference between the distributions seen in Figures 3 and 4 suggests that this event was new and unusual, rather than an ongoing or intermittent exposure
that had not been recognized.

Figure 4. Geographicdistribution of the
residences of women screened during pregnancy and
classified as having acute cases of toxoplasmosis (n = 36).

Well-defined geographic areas of the Capital Regional District were classified as receiving high, intermediate, and no exposure to water from the Humpback Reservoir (exposure scores 3, 2, and 1, respectively). An analysis for linear trend in proportions was performed by using StatCalc in Epi Info to demonstrate whether a dose-response relationship was present.

If water from the Humpback Reservoir was the source of infection, the rate of infection would be expected to increase with increased exposure. The Table shows the geographic distribution of women according to the estimated concentration of water from Humpback Reservoir received at
their residence. When acutely infected women were compared with seronegative women, the trend in linear proportions across the three exposure scores was significant (chi-square = 6.67; p = 0.01). In contrast, a significant linear trend in proportions was not demonstrated when women with nonacute cases were compared with seronegative women (chi-square = 1.30; p = 0.25).

Table. Linear trend analysis of acute and nonacute infections with Toxoplasma gondii among screened pregnant women, by degree of exposure to Humpback Reservoir water

Conclusions 

The conclusions drawn from geographic mapping depend on the accuracy and validity of the datasets and the availability of denominator data to avoid making false associations that are a function of population densities. For the overall population, denominators were available from census data. Firm denominator data were available from the serologic
screening study.

With respect to accurate placement on the maps, the addresses and the delineation of the water distribution systems were critical to the evaluation. Although extensive efforts were made to verify addresses, there was some potential for misclassifying exposure. However, any
misclassification resulting from the use of the Medical Services Plan database should not have systematically biased our analyses.

The Greater Victoria Water District engineers delineated the boundaries of the Humpback distribution system and ranked the zones in the Capital Regional District according to the proportion of water received from the Humpback Reservoir. As the engineers had no prior knowledge of the distribution of outbreak-related cases of toxoplasmosis, it is unlikely
that the determination of these geographic boundaries was biased. 

A limitation of the geographic mapping analysis of cases is that rates were not adjusted for age or other covariates. However, the subsequent analyses using data from the population-based screening of pregnant women were not confounded by age. The age distribution of the women in
these analyses did not differ when grouped by infection status.

Computer mapping software had the advantages of 1) facilitating the verification and correct placement of addresses, 2) reducing the time required to map the location of large datasets, 3) enabling queries and statistical analysis of the data after mapping, 4) allowing several sets
of mapped data to be analysed simultaneously for potential
relationships, and 5) generating printouts or overheads for
presentations.

Acknowledgments

We thank Timothy Johnstone, Louise Egan, Michael Aeberhardt, Kevin Kirkwood, Bill Keenan, and Jenny Cadman for invaluable technical assistance and critical information. We also thank Jack S. Remington and the Toxoplasma Serology Laboratory, Research Institute, Palo Alto Medical Foundation.

This investigation was supported by the Ministry of Health, British Columbia; the Capital Regional District Health Department, Victoria, British Columbia; and the Laboratory Centre for Disease Control, Health Canada, Ottawa, Ontario.

Mr. Eng is with the Health Protection and Environmental Program for Greater Victoria, British Columbia. His interests focus on environmental epidemiology, manipulation and analysis of computer data, and computer-based mapping.

Address for correspondence: Steven B. Eng, Health Protection and Environmental Services, Capital Health Region, #201-771 Vernon Avenue, Victoria, B.C., Canada, V8X 5A7; fax: 250-475-5130; e-mail: steven.eng@caphealth.org.

References

1. Barreto ML. The dot map as an epidemiological tool: a case study of Schistosoma mansoni infection in an urban setting. Int J Epidemiol 1993;22:731-41.

2. Glass GE, Schwartz BS, Morgan JM, Johnson DT, Noy PM, Israel E. Environmental risk factors for Lyme disease identified with geographic information system. Am J Public Health 1995;85:944-8.

3. Skorpanich MA, Taylor TH, Anton-Culver H. Mapping disease and risk factors to identify public health concerns. In: URISA 88. Urban & Regional Information Systems Association, Los Angeles, CA 1988;4:310-9.

4. Timmreck TC. An introduction to epidemiology. Boston: Jones and Bartlett Publishers, Inc.; 1994.

5. Rosenberg ML, Haziet KI, Schaefer J, Wells JG, Pruneda RC. Shigellosis from swimming. JAMA 1976;236:1849-52.

6. Merson MH, Goldmann DA, Boyer KM, Peterson NJ, Patton C, Everett LG, et al. An outbreak of Shigella sonnei gastroenteritis on Colorado River raft trips. Am J Epidemiol 1974;100:186-95.

7. Bowie WR, King AS, Werker DH, Issac-Renton JL, Bell A, Eng SB, et al. Outbreak of toxoplasmosis associated with municipal drinking water. Lancet 1997;350:173-7.

8. Remington JS, McLeod R, Desmonts G. Toxoplasmosis. In: Remington JS, Klein JO, editors. Infectious diseases of the fetus and newborn. 4th ed. Philadelphia (PA): W.B. Saunders Company; 1995. p. 140-267.

9. Wong S-Y, Remington JS. State of the art clinical article: Toxoplasmosis in pregnancy. Clin Infect Dis 1994;18:853-62.

10. Montoya JG, Remington JS. Studies on the serodiagnosis of toxoplasmic lymphadenitis. Clin Infect Dis 1995;20:781-9.

11. Dannemann BR, Vaughan WC, Thulliez P, Remington JS. Differential agglutination test for diagnosis of recently acquired infection with T. gondii. J Clin Microbiol 1990;28:1928-33.

12. Irwin GS, Zanette M, Morris B. Greater Victoria's drinking water system 1994 bacteriological summary. Greater Victoria Water District, Victoria, British Columbia, Canada; 1995.

13. British Columbia Roads and Rivers (TcN data), based on
Transportation Centerline Network (1993), Planning Services Branch, Ministry of Transportation and Highways, Province of British Columbia.

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

URL: http://www.cdc.gov/ncidod/eid/vol5no6/ascii/eng.txt

Please note that figures, equations, and most tables are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively.  Greek symbols are spelled out. >/= is used for greater than or equal to.

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

Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999]

Dispatches

HIV Infection as a Risk Factor for Shigellosis1

Jefferson T. Baer,* Duc J. Vugia,* Arthur L. Reingold,* Tomas Aragon, Frederick J. Angulo,# and Williamson
Z. Bradford***

 *California Emerging Infections Program, San Francisco,
 California, USA; California Department of Health
 Services, Berkeley, California, USA; University of
 California, San Francisco, California, USA; School of
 Public Health, University of California, Berkeley,
 California, USA; San Francisco Department of Public
 Health, San Francisco, California, USA; #Centers for
 Disease Control and Prevention, Atlanta, Georgia, USA;
 and **IntraBiotics Pharmaceuticals, Mountain View,
 California, USA

  We investigated cases of shigellosis in San
  Francisco and Alameda Counties identified
  during 1996 by active laboratory
  surveillance to assess the role of HIV
  infection as a risk factor for shigellosis.
  Dramatically elevated rates of shigellosis
  in HIV-infected persons implicate HIV
  infection as an important risk factor for
  shigellosis in San Francisco.

 Shigella infections are responsible for an estimated
 300,000 illnesses and 600 deaths per year in the United
 States and more than 600,000 deaths per year worldwide
 (1). Shigella species are typically transmitted by
 direct or indirect fecal-oral contact; as a result,
 shigellosis has long been associated with outbreaks in
 day-care centers, nursing homes, and institutionalized
 populations (2-4). However, several studies have
 demonstrated an increased frequency of shigellosis
 cases in young adult men residing in urban settings who
 have little, if any, exposure to these traditionally
 recognized risk groups (5-9). These investigations also
 suggest that Shigella infection occurs during the
 practice of gay sex; however, since most of these
 studies occurred before the HIV epidemic, the
 relationship between HIV infection and gay sex and the
 subsequent risk for shigellosis has yet to be evaluated
 (5-9).

 HIV-infected persons are at increased risk for
 infection by several common enteric pathogens (10).
 Previous investigations have demonstrated that
 HIV-infected patients are at 20 times greater risk for
 infection with Salmonella species and 39 times greater
 risk for infection with Campylobacter species than the
 general population (11,12). To determine the rate of
 shigellosis in HIV-infected persons, we investigated
 all cases of shigellosis in San Francisco, a county
 with a high prevalence of HIV infection and a high
 incidence of shigellosis. Alameda, a neighboring county
 with lower rates of shigellosis and HIV infection, was
 used as a comparison area for our investigation because
 of its proximity and differing shigellosis and HIV
 epidemiology.

 During 1996, cases of culture-confirmed shigellosis
 were identified in San Francisco and Alameda Counties
 by active surveillance in 28 laboratories for isolates
 of Shigella species cultured from any anatomic site as
 part of the California Emerging Infections Program. The
 program comprises one of five sites in the Foodborne
 Diseases Active Surveillance Network (FoodNet), which
 is part of the Centers for Disease Control and
 Prevention's Emerging Infections Program. All available
 medical records of patients were reviewed by a
 standardized data collection instrument detailing
 demographic and medical information. Data concerning
 sexual activity and orientation and foreign travel were
 obtained from routine telephone interviews of patients
 with shigellosis, conducted by the San Francisco
 Department of Public Health. This information was not
 available for Alameda residents because no telephone
 interviews were conducted in Alameda County.

 Patients were considered HIV-infected if their medical
 record contained a physician's note or a laboratory
 report documenting HIV infection. In the absence of
 such documentation, patients were considered
 HIV-negative. San Francisco patients were classified as
 gay if they were male and had identified themselves as
 gay or bisexual during telephone interviews. Recent
 sexual contact for San Francisco patients was assessed
 during the telephone interviews and was defined as
 having had a sexual encounter within 10 days of the
 onset of shigellosis symptoms. Foreign travel exposure
 was defined as travel to an area where shigellosis was
 endemic 7 days before the onset of symptoms.

 Postcensus data for San Francisco and Alameda Counties
 were obtained for 1996 (13). Estimates of the
 prevalence of HIV infection in San Francisco by groups
 at risk were obtained from the 1997 HIV Consensus
 Report on HIV Prevalence and Incidence in San Francisco
 (14). This report is based on the findings of a
 consensus panel that systematically reviewed numerous
 sources of published and unpublished data. FoodNet
 incidence rates for culture-confirmed cases of
 shigellosis were based on aggregated data collected by
 active laboratory-based surveillance during 1996 in
 four urban areas in Connecticut, Georgia, Minnesota,
 and Oregon (15). National incidence rates were based on
 culture-confirmed cases of shigellosis reported through
 passive laboratory-based surveillance in 1996 (15).

Data were managed and analyzed by Stata 4.0 (Stata Corporation, College Station, TX) and EpiInfo 6.04b 
(CDC, Atlanta, GA) software. Univariate analyses of proportions were performed by chi-square test. 
Incidence rates and incidence rate ratios were
compared by using the exact method to calculate 
confidence intervals and statistical significance. 
Temporal trends of shigellosis were assessed by
conducting chi-square for trend analysis on the number
of cases of shigellosis diagnosed by month.

During 1996, 228 and 140 culture-confirmed cases of shigellosis were identified in San Francisco and
Alameda, respectively. In San Francisco, 142 (62%)
of these cases were caused by S. sonnei, 73 (32%) by S. flexneri, 7 (3%) by S. boydi, 2 (1%) by S. dysenteriae,
and 4 (2%) were not speciated. In Alameda, 93 (66%) 
of the cases were caused by S. sonnei, 28 (20%) by S. flexneri, 6 (4%) by S. boydi, 2 (2%) by S. dysenteriae,
and 11 (8%) were not speciated. No difference was 
observed in the proportion of cases caused by
different species in the two counties (p = 0.16).

An analysis of the month of diagnosis for all
patients with S. sonnei infections demonstrated
a distinct trend in both San Francisco (p = .001) and Alameda (p = .03). The number of infections per 
month was highest in San Francisco between January
and May and in Alameda between August and November. 
No temporal trend was apparent for S. flexneri 
infections in San Francisco (p = .77) or Alameda 
(p = .36).

San Francisco patients were significantly more
likely than Alameda patients to be male, adult, 
white, and HIV-infected (Table 1). Sixty-six 
(39%) of 168 shigellosis patients and 30 (54%) 
of 56 S. flexneri-infected patients in San 
Francisco were HIV-infected; 11 (15%) of 
75 HIV-negative patients and 1 (2%) of 56 
HIV-infected patients in San Francisco 
reported recent travel to a shigella-endemic
area outside the United States (p = 0.01).

Table 1. Persons with shigellosis, by county
of residence, 1996
-------------------------------------------------
 San Francisco  Alameda    (n =           p
Characteristic (n = 228)   140)         value
-------------------------------------------------
Male        157/228 (69%)  60/140 (43%) <0.001
Age >18     181/227 (80%)  57/140 (41%) <0.001
Race
 White      117/200 (59%)  15/92 (16%)  <0.001

 Black       39/200 (20%)  44/92 (48%)  <0.001

 Hispan      37/200 (19%)  22/92 (24%)   0.285

Hospitalized 22/201 (11%)  12/140 (9%)   0.472
HIV
 infection    66/168 (39%)  9/125 (7%)  <0.001
Foreign   NAa
travel        25/185 (14%)
Gay male      96/190 (51%)  NAa
Recent sex    70/136 (51%)  NAa
-------------------------------------------------
aNA = not available

The annual incidence rates of shigellosis for various population groups in San Francisco, Alameda, other 
FoodNet sites, and the United States are shown in Table 2. San Francisco had higher overall rates, particularly among men and persons ages 25 to 64 years, than Alameda, other FoodNet sites, and the United States.

Table 2. Annual incidence rates of culture-confirmed
shigellosis per 100,000 population for selected groups,
1996
----------------------------------------------------------
        San
      Francisco Alameda FoodNet   United
Group	  County  County  sitesa    States
----------------------------------------------------------
Overall 30.9    10.5b     7.3       5.3
 rate
Male    43.2     9.2b     7.4       3.3
Female  19.1    11.8b     7.1       3.9
Age
groups
(yrs)
 <5     82.5    47.9b    36.7      16.7

5-14    22.1    17.1     12.6       6.8

15-24   19.9     9.3b     5.3       2.4
25-39   49.9     7.6b     5.6       2.7

40-64   25.9     3.3b     2.4       1.0

65+      2.5     4.0      0.5        0.7

----------------------------------------------------------
aDoes not include California.
bComparison of rates in San Francisco and Alameda
Counties, p value <.05.

In San Francisco, an analysis of the annual incidence rates of shigellosis per 100,000 population by sexual orientation and HIV status showed rates of 12.4 in heterosexual and HIV-negative persons, 60.1 in gay and HIV-negative persons, 378 in not gay and HIV-infected persons, and 442 in gay and HIV-infected persons. Incidence rate ratios for these groups, relative to the not gay and HIV-negative population, were as follows: gay and HIV-negative 4.9 (95% CI 2.7-8.1); not gay and HIV-infected 30.6 (95% CI 12.8-63.0); and gay and HIV-infected 35.7 (95% CI 25.1-50.4).

Thirty-four (10%) of 341 of patients were hospitalized for
shigellosis (median hospital stay 3 days). Furthermore, in San Francisco 13 (22%) of 60 HIV-infected shigellosis patients were hospitalized, while 7 (8%) of 86 HIV-negative persons were hospitalized (p = .02). Twelve (13%) of 93 S. flexneri and 19 (9%) of 216 S. sonnei patients were hospitalized (p = .27).

These population-based data demonstrate a high overall annual incidence rate of shigellosis in San Francisco compared with neighboring Alameda County, other FoodNet sites, and the United States, and dramatically elevated rates in HIV-infected San Francisco residents. The high proportion of cases in San Francisco in both the gay and the HIV-infected populations suggests that these groups play a major role in the epidemiologic features of endemic
shigellosis in San Francisco. Furthermore, the greatly elevated incidence rates of shigellosis in the HIV-infected population suggest that HIV may be a important risk factor for Shigella infection. These data also demonstrate that shigellosis is associated with extensive illness and increased health-care expenditures, particularly in the HIV-infected population, as evidenced by the frequency of hospitalization in these persons.

There are several possible explanations for the high rates of shigellosis observed in HIV-infected patients in this study. The compromised host immunity of HIV-infected persons may increase their risk for clinical infection after exposure. A recent study found that 75% of asymptomatic household contacts of symptomatic shigellosis patients had evidence of Shigella infection by polymerase chain reaction, which suggests that host immunity may play an important role in determining which exposed persons progress to clinical infection (16). Increased susceptibility to shigellosis
among HIV-infected persons could be mediated through different mechanisms, including compromised cell-mediated immunity or achlorhydria. Alternatively, the high rate of shigellosis in HIV-infected patients may be related to factors other than host immunity, such as sexual or behavioral practices, which were not thoroughly investigated in our study.

Our investigation suggests that HIV infection is an important risk factor for shigellosis. This finding has not been previously described on a population level. This investigation also suggests that HIV infection is an important determinant of the epidemiologic features of shigellosis in San Francisco and that public health
prevention strategies in areas with a large HIV-infected and gay male population may need to be revisited. A diagnosis of shigellosis in young adult men who are not part of a recognized outbreak and have not recently traveled to a Shigella-endemic area may serve as a marker for HIV infection and may indicate a need for counseling and
HIV testing.

Due to the methods of data collection, misclassification of HIV infection status and sexual orientation could have occurred in either shigellosis patients or in the San Francisco Department of Public Health population estimates, thereby altering the incidence rates in an unpredictable manner. Moreover, the propensity of HIV-infected patients with shigellosis to seek medical attention and the likelihood of their health-care providers to obtain cultures may differ from that of HIV-negative patients. This bias, if present, could influence the observed risk associated with HIV infection. However, it is unlikely that such a bias could account for the entire difference between groups, given the magnitude of the elevation in incidence rates in the HIV-infected population.

Acknowledgments

The authors thank Sue Shallow, Gretchen Rothrock, Pam Daily,
Nandeeni Murkerjee, and Lisa Gelling for their disease surveillance work.

This work was funded in part by Centers for Disease Control and Prevention cooperative agreement number U50/CCU915546-01.

Mr. Baer is a 3rd-year medical student at the University of North Carolina at Chapel Hill. His current research includes examining a possible molecular pathway for the Chlamydia pneumoniae and atherosclerosis association.

Address for correspondence: Williamson Z. Bradford, IntraBiotics Pharmaceuticals, Inc., 1255 Terra Bella Ave., Mountain View, CA 94080, USA; fax: 650-969-0663; e-mail: BBradford@intrabiotics.com.

References

1. Bennett JV, Holmberg SD, Rogers MF, Solomon SL. Infectious and parasitic diseases. Am J Prev Med 1987;3:102-14.

2. Weissman JB, Schmerler A, Weiler P, Filice G, Godbey N, Hansen I. The role of preschool children and day-care centers in the spread of shigellosis in urban communities. J Pediatr 1974;84:797-802.

3. Ryan MJ, Wall PG, Adak GK, Evans HS, Cowden JM. Outbreaks of infectious intestinal disease in residential institutions in England and Wales 1992-1994. J Infect 1997;34:49-54.

4. DuPont HL, Gangarosa EJ, Reller LB, Woodward WE, Armstrong RW, Hammond J,et al. Shigellosis in custodial institutions. Am J Epidemiol 1970;92:172-9.

5. Dritz SK, Back AF. Shigella enteritis venereally transmitted [letter]. N Engl J Med 1974;291:1194.

6. Bader M, Petersen AH, Williams R, Spearman J, Anderson H.
Venereal transmission of shigellosis in Seattle-King County. Sex Transm Dis 1977;4:89-91.

7. Dritz SK, Ainsworth TE, Garrard WF, Back A, Palmer RD, Boucher LA, et al. Patterns of sexually transmitted enteric diseases in a city. Lancet 1977;2:3-4.

8. Tauxe RV, McDonald RC, Hargrett-Bean N, Blake P. The persistence of Shigella flexneri in the United States: Increasing role in adult males. Am J Public Health 1988;78:1432-5.

9. Quinn TC, Stamm WE, Goodell SE, Mkrtichian E, Benedetti J, Corey L, et al. The polymicrobial origin of intestinal infections in homosexual men. N Engl J Med 1983;309:576-82.

10. Angulo FJ, Swerdlow DL. Bacterial enteric infections in persons infected with human immunodeficiency virus. Clin Infect Dis 1995;21 Suppl 1:S84-93.

11. Sorvillo FJ, Lieb LE, Waterman SH. Incidence of
campylobacteriosis among patients with AIDS in Los Angeles County. J Acquir Immune Defic Syndr Hum Retrovirol 1991;4:598-602.

12. Celum CL, Chaisson RE, Rutherford GW, Barnhart JL, Echenberg DF. Incidence of salmonellosis in patients with AIDS. J Infect Dis 1987;156:998-1002.

13. U.S. Census Bureau. Annual time series of county population estimates by age, sex, race, and Hispanic origin; 1996; [accessed 1998 Aug 1]. Available from: URL
http://www.census.gov/population/estimates/countypop.html.

14. Shafer KP, McFarland W, Katz MH. 1997 consensus report on HIV prevalence and incidence in San Francisco. San Francisco Department of Public Health HIV Seroepidemiology Unit 1997.

15. Centers for Disease Control and Prevention. Revised FoodNet 1996 final report. 1998 Sep.

16. Gaudio PA, Sethabutr O, Echeverria P, Hoge CW. Utility of a polymerase chain reaction diagnostic system in a study of the epidemiology of shigellosis among dysentery patients, family contacts, and well controls living in a shigellosis-endemic area. J Infect Dis 1997;176:1013-8.

1Presented in part at the International Conference on Emerging Infections, March 1998, Atlanta, Georgia.

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

URL: http://www.cdc.gov/ncidod/eid/vol5no6/ascii/baer.txt

Please note that figures, equations, and most tables are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively.  Greek symbols are spelled out. >/= is used for greater than or equal to.

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

Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999]

  Dispatches

   Dengue Seroconversion among Israeli Travelers to
   Tropical Countries

   Israel Potasman,* Isaac Srugo,* Eli Schwartz

   *Bnai Zion Medical Center, Haifa, Israel; Bruce
   Rappaport Faculty of Medicine Technion-Israel Institute
   of Technology, Haifa, Israel; Center for Geographical
   Medicine, Sheba Medical Center, Tel Hashomer, Israel

    We tested for dengue seroconversion among
    104 Israeli young adults who traveled to
    tropical countries for at least 3 months.
    Seven (6.7%) seroconverted during travel;
    four (3.8%) had immunoglobulin (Ig) M
    antibodies; one was symptomatic with
    borderline IgM and a rise in IgG; two
    others (1.9%) had a rise in IgG titers,
    without detectable IgM. All four
    IgM-positive patients had traveled to
    Southeast Asia.

   Dengue fever is a rapidly spreading mosquito-borne
   viral disease. Some 40% of the world's population lives
   in disease-endemic areas, and dengue outbreaks occur in
   more than 100 countries (1). Infected persons usually
   have high fever, chills, frontal headache, rash, severe
   myalgia, and malaise. Sometimes, the disease goes
   unrecognized (2).

   According to surveillance data, nearly 200,000 cases of
   dengue fever occur in 31 countries in Central and South
   America (3); the attack rate among disease-endemic
   populations may be as high as 6,400 per 100,000 persons
   exposed. In contrast to data regarding the rate of
   dengue among populations in disease-endemic areas, data
   on the attack rate among travelers are scarce.

   The rate of dengue fever has been examined in selected
   Japanese, Spanish, Swiss, and German travelers and in
   U.S. troops deployed in Somalia (4-8). The rate of
   dengue in these febrile patients was 6.9% to 65%. To
   the best of our knowledge, the rate of dengue fever has
   never been examined prospectively in a cohort of
   healthy, long-term travelers to disease-endemic areas.

   The Study

   The study was approved by the Helsinki Committee of the
   Bnai Zion Medical Center, which provides service to
   approximately 1,500 travelers per year. Each traveler
   is requested to fill out a questionnaire including
   demographic, itinerary, and vaccination data, which are
   stored in a computerized database. The purpose of the
   study was explained to the travelers, and informed
   consent was obtained upon enrollment. Serum was drawn
   from random volunteers before starting the recommended
   vaccinations. Eligibility for inclusion was based on a
   minimum length of travel of at least 3 months and
   donation of a serum sample before and after travel. One
   hundred and four travelers fulfilled the inclusion
   criteria. The second serum sample was taken 1 to 4
   months after returning home. Travelers who had positive
   dengue immunoglobulin (Ig) G serologic results were
   sent a questionnaire. In addition to demographics, the
   patients were asked to indicate their destination,
   season and length of stay, mosquito bites, use of
   repellents, fever, chills, nausea or vomiting, muscle
   aches, headache, cough, rash, or arthralgia. A case of
   dengue fever was defined according to Centers for
   Disease Control and Prevention (CDC) criteria (9). An
   asymptomatic dengue infection was one that met the
   laboratory criteria for diagnosis without clinical
   signs. Confidence intervals (CIs) were calculated with
   Statmate (GraphPad Software, San Diego, CA).

   After thawing, 104 posttravel serum samples were tested
   for antibodies to dengue by an IgG enzyme-linked
   immunosorbent assay (ELISA) (Pan-Bio Pty, Ltd.,
   Queensland, Australia). All positive sera were then
   tested in parallel with pretravel sera to confirm
   seroconversion. Seroconverting pairs were sent to the
   CDC laboratories in San Juan, Puerto Rico, for
   confirmation and dengue IgM and IgG antibody
   determination.

   IgM antibody was measured as a qualitative ELISA. An
   optical density of 0.2 or greater compared with a
   negative control was considered positive. The test may
   show some cross-reactivity to other flaviviruses (10).
   IgG antibody was measured as a quantitative ELISA using
   mixed dengue antigens. This test may also show
   cross-reactivity with other flaviviruses (11).

   The mean age of the 104 study participants was 22.4 
   2.2 years. The average length of stay abroad was 6.1
   months (3 to 16 months), and the total time abroad for
   all 104 travelers was 53 person-years. The average time
   between serum samples was 11.1 months (4 to 22 months).
   The destinations were Southeast Asia (70%), South
   America (24%), Africa (4%), and both Southeast Asia and
   South America (2%).

   Seven travelers (6.7%; 95% CI = 2.7-13.3%) had either
   an IgM antibody or a fourfold (or greater) rise in IgG
   titers after the trip. The median age of this group was
   22 years, and four travelers were women. All seven
   stayed at their destinations (Table) during the summer
   months (some also spent the spring or autumn there).
   Their mean length of stay abroad was 5.3 months. The
   rates of conversion per month of exposure by continent
   were as follows: Southeast Asia, 5 (1.1%) of 451 (95%
   CI = 0.36-2.6%); South America, 1 (0.6%) of 159 (95% CI
   = 0.02-3.5%); and Africa, 1 (4%) of 25 (95% CI =
   0.1-20.3%).

   Four patients (3.8%) tested positive for dengue IgM
   after travel. All four had negative IgM dengue
   serologic test results before travel, and all but one
   had negative IgG titers. All four (two of them male)
   visited Southeast Asia, with Thailand being the only
   common destination. Two of these patients, who had
   traveled separately, indicated that they became ill on
   the island of Ko-Pangan. All four were engaged in
   extensive outdoor activities and consequently had
   mosquito exposure. All four used mosquito repellents
   (containing 20% to 25% DEET), but only three recalled
   being bitten by mosquitoes. Three travelers had fever,
   which in two (both female) was also accompanied by
   chills, headache, and protracted fatigue. One patient
   had an apparently asymptomatic infection.

Table. Dengue serologic results in seroconverting Israeli travelers to tropical countries
Only two of the four IgM-positive patients had received Japanese B encephalitis vaccine after the initial blood collection, which could have interfered with after-travel testing. Of the three travelers who had a fourfold IgG rise, two were asymptomatic, and one had a clinical picture compatible with dengue fever 1 month after arriving in
Thailand. This traveler also had a marginal IgM test. Among these three travelers, two had received yellow fever vaccine and one Japanese B encephalitis vaccine, which could have interfered with after-travel testing.

Conclusions

Researchers have found that the younger the age, the higher the rate of illness among travelers, but dengue fever has not been thoroughly investigated (12,13). Dengue fever has not been reported in Israel over the last 50 years, which made our group of young travelers particularly
suitable for this study. In addition, there has been a dramatic increase in the number of Israeli travelers to tropical areas during the past decade. Approximately 40,000 Israelis travel to the tropics each year, more than 25,000 of whom are backpackers who travel for 3 to 12 months off the beaten track (14) and are exposed to the same diseases as travelers from other countries. Dengue fever has not been
mentioned as a real hazard to Israeli travelers, despite an incidence in our study that may be as high as the rate of malaria without prophylaxis, and higher than the rates of hepatitis A, giardiasis, or typhoid fever (15). Nevertheless, it would be premature to extrapolate
from our group of youngsters traveling on prolonged journeys, to groups with other travel characteristics. Older and perhaps short-term travelers may, for example, choose other tracks or adhere more closely to recommendations regarding insect repellents (16).

Four travelers (3.8%) of our group had IgM antibodies, indicating acute infection. Another traveler had symptoms of dengue fever with borderline IgM and a rising titer of IgG, which most probably reflected a recent infection. This traveler could have been infected earlier during her 6-month trip, and by the time the serum was taken, the IgM
level might have dropped. Two additional travelers had a rise in IgG titers without detectable IgM.

Our results are tentative, as the serologic tests for dengue are not devoid of cross-reactivity (11). Both IgM and IgG may cross-react with other flaviviruses, such as Japanese B encephalitis, West Nile encephalitis, or yellow fever. The rate of IgG cross-reactivity between dengue infection and Japanese B encephalitis or yellow fever vaccine may be 17% to 40%; however, IgM cross-reactivity was not found after
vaccination (E. Schwartz, pers. comm.). As none of our travelers had signs of encephalitis and yellow fever does not exist in Southeast Asia, the five travelers with IgM antibodies (including the one with a borderline case) contracted dengue fever. Indeed, four of them had
clinical symptoms compatible with dengue fever. The diagnosis in the two travelers who had a fourfold rise in IgG titers was uncertain, since both were asymptomatic and had received yellow fever vaccines, which may have caused a cross-reaction in IgG assays. Dengue infection may be asymptomatic. Only three of the four IgM-positive patients were febrile; two of these also had chills. Asymptomatic dengue, which was found in three of our patients, has been described in populations of disease-endemic areas but never in travelers (2,17).

Two additional points in our study deserve comment. First, all four IgM-positive cases and the borderline IgM case occurred in travelers to Southeast Asia. A higher density of the vector and virus in Southeast Asia or visits by many of our travelers to Thai destinationsknown for their high rate of dengue (18-20) may have played a major role in this
trend (our data are insufficient to permit conclusions regarding travel to Africa and South America). Secondly, all seven cases occurred during the summer, a season known for its high rate of mosquito activity and dengue transmission. The rate of dengue fever among travelers has been
studied by four groups of researchers (4-7); an additional group described dengue in U.S. troops deployed in Somalia (8). The rate of dengue in their cohorts was 6.9% to 65%. However, all of these researchers focused on febrile patients with either fever of unknown origin, suspected dengue, or malaria. Hence, it is impossible to
extrapolate from their data the actual risk of acquiring dengue during travel to the tropics.
 
Other travel clinics in Israel also have indicated that (after malaria) dengue is the second most frequent cause of hospitalization of returning travelers (20). Based on a minimum figure of the four IgM-positive patients, the calculated risk for dengue during a 1-month trip is thus 630 out of 100,000 travelers, which puts dengue high on
the list of diseases contracted in the tropics. This statement holds true at least for Israeli travelers to the Far East. As patients may be evacuated because of dengue fever or may become sick after returning home, physicians need to be all the more vigilant in the face of this
diagnostic possibility.

We conclude that dengue fever is perhaps the most common mosquito-borne disease of long-term young travelers, particularly those visiting Southeast Asia. The present results should serve as a further impetus toward the development of a vaccine for dengue fever.

Acknowledgments

The authors thank Gary Clark and Vance Vorndam for performing the serologic tests and for their comments, and to R. Singer for excellent secretarial assistance.

This work was supported by the Research and Infrastructure Foundation, Israel Ministry of Health.

Dr. Potasman is head of Infectious Diseases at the Bnai Zion Medical Center, which is part of the Faculty of Medicine, Technion, Haifa. His area of expertise is travel medicine, with special interest in diseases among travelers, antimalarial drugs, vaccines, and Helicobacter pylori.

Address for correspondence: Israel Potasman, Infectious Disease Unit, Bnai Zion Medical Center, P.O. Box 4940, 31048 Haifa, Israel; fax: 972-4-835-9755; e-mail: ipotasma@netvision.net.il.

References

1. World Health Organization/CTD. Dengue and DHF prevention and control. Geneva: The Organization; 1998.

2. Burke DS, Nisalak A, Johnson DE, Scott RM. A prospective study of dengue infections in Bangkok. Am J Trop Med Hyg 1988;38:172-80.

3. Lifson AR. Mosquitoes, models, and dengue [commentary]. Lancet 1996;347:1201-2.

4. Yabe S, Nakayama M, Yamada K, Kitano T, Arai Y, Horimoto T, et al. Laboratory virological diagnosis of imported dengue cases. Kansenshogaku Zasshi 1996;70:1160-9 (In Japanese).

5. Lopez-Velez R, Perez-Casa C, Vorndam A, Rigau J. Dengue in Spanish travelers returning from the tropics. Eur J Clin Microbiol Infect Dis 1996;15:823-6.

6. Settah SG, Vernazza PL, Morant R, Schultze D. Imported dengue fever in Switzerlandserological evidence for a hitherto unexpectedly high prevalence. Schweiz Med Wochenschr 1995;125:1673-8 (In German).

7. Jelinek T, Dobler G, Holscher M, Loscher T, Nothdurft H-D. Prevalence of infection with dengue virus among international travelers. Arch Intern Med 1997;157:2367-70.

8. Sharp TW, Wallace MR, Hayes CG, Sanchez JL, DeFraites RF, Arthur RR, et al. Dengue fever in U.S. troops during Operation Restore Hope, Somalia, 1992-1993. Am J Trop Med Hyg 1995;53:89-94.

9. Centers for Disease Control. Dengue fever. MMWR Morb Mortal Wkly Rep 1990;39:10-1.

10. Burke DS, Nisalak A, Ussery MA. Antibody capture  immunoassay detection of Japanese encephalitis virus immunoglobulin M and G antibodies in cerebrospinal fluid. J Clin Microbiol 1982;16:1034-42.

11. Makino Y, Tadano M, Saito M, Maneekam N, Sittisombut N,
Sirisanthana V, et al. Studies on serological cross-reaction in sequential flavivirus infections. Microbiol Immunol 1994;38:951-5.

12. Steffen R, Rickenbach M, Wilhelm U, Helminger A, Schar M. Health problems after travel to developing countries. J Infect Dis 1987;156:84-91.

13. Reid D, Grist NR, Najera R. Illness associated with "package tours": a combined Spanish-Scottish study. Bull World Health Organ 1978;56:117-22.

14. Berger SA, Giladi M, Shapira I. Health concerns of Israelis traveling to third world countriesexperience of a travel advisory clinic. Harefuah 1994;126:410-2 (In Hebrew).

15. Reid D, Keystone J. Health risks abroad: general considerations. In: DuPont H, Steffen R, editors. Textbook of travel medicine and health. Vol 1. Hamilton, Canada: BC Decker; 1997. p. 3-9.

16. Fradin MS. Mosquitoes and mosquito repellents: a clinician's guide. Ann Intern Med 1998;128:931-40.

17. da Cunha RV, Dias M, Nogueira RM, Chagas N, Miagostovich MP, Schatzmayr HG. Secondary dengue infection in schoolchildren in a dengue endemic area in the state of Rio de Janeiro, Brazil. Rev Ins Med Trop Sao Paulo 1995;37:517-21.

18. Pick N, Potasman I. Dengue fever. Harefuah 1995;129:30-2 (In Hebrew).

19. Thavara U, Tawatsin A, Phan-Urai P, Ngamsuk W, Chansang C, Liu M, et al. Dengue vector mosquitos at a tourist attraction, Ko Samui, in 1995. Southeast Asian J Trop Med Public Health 1996;27:160-3.

20. Schwartz E, Mendelson E, Sidi Y. Dengue fever among travelers. Am J Med 1996;101:516-20.

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

URL: http://www.cdc.gov/ncidod/eid/vol5no6/ascii/potasman.txt

Please note that figures, equations, and most tables are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively.  Greek symbols are spelled out. >/= is used for greater than or equal to.

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

Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999]

   Dispatches

   Effectiveness of Pneumococcal Polysaccharide Vaccine
   for Preschool-Age Children with Chronic Disease

   Anthony E. Fiore, Orin S. Levine, John A. Elliott,
   Richard R. Facklam, Jay C. Butler, for the Pneumococcal
   Sentinel Surveillance Working Group

   Centers for Disease Control and Prevention, Atlanta,
   Georgia, USA

    To estimate the effectiveness of
    pneumococcal polysaccharide vaccine, we
    serotyped isolates submitted to the
    Pneumococcal Sentinel Surveillance System
    from 1984 to 1996 from 48 vaccinated and
    125 unvaccinated children 2 to 5 years of
    age. Effectiveness against invasive disease
    caused by serotypes included in the vaccine
    was 63%. Effectiveness against serotypes in
    the polysaccharide vaccine but not in a
    proposed seven-valent protein conjugate
    vaccine was 94%.

   Streptococcus pneumoniae is a leading cause of
   pneumonia, meningitis, bacteremia, and death in young
   children. A polysaccharide vaccine has been recommended
   for use in chronically ill children and adults 2 to 64
   years of age, as well as all adults >/=65 (1). While many
   studies have assessed the immunogenicity of the
   polysaccharide vaccine, scant data exist on its
   effectiveness in younger children.

   More than 90 serotypes of S. pneumoniae have been
   described (2); however, most invasive infections in the
   United States are caused by </=10 serotypes (3).
   Pneumococcal vaccines available since 1978 consist of a
   mixture of capsular polysaccharides from the most
   common serotypes causing invasive disease. This vaccine
   is recommended for children >/=2 years of age with
   underlying diseases or immunosuppressive medical
   treatments that are risk factors for invasive
   pneumococcal disease (1,3).

   Clinical trials of pneumococcal polysaccharide vaccine
   effectiveness in children have shown conflicting
   results (4-7). Vaccine failure in immunized children
   has been reported (8), and one study comparing
   immunization with antibiotic prophylaxis in children
   with sickle cell disease concluded that the vaccine was
   inferior to penicillin prophylaxis (9). Uncertainty
   regarding the effectiveness of vaccination may
   contribute to low vaccination rates among persons at
   risk for pneumococcal disease (1).

   In indirect cohort analysis (10), the distribution of
   pneumococcal serotypes causing invasive disease among
   vaccinated and unvaccinated groups is compared. If the
   vaccine is effective, vaccinated persons have fewer
   infections with serotypes represented in the vaccine
   than unvaccinated persons. This method has been used to
   calculate an overall effectiveness of 57% in persons >5
   years of age, based on serotypes of invasive isolates
   obtained through a national, voluntary sentinel
   surveillance system (11). Using data from national
   surveillance, we examined vaccine effectiveness for
   children 2 through 5 years of age.

   The Study

   Since 1978, a national, hospital laboratory-based
   surveillance system has collected data on invasive
   pneumococcal disease (12). Participating institutions
   are requested to report all pneumococcal isolates
   obtained from normally sterile body sites, along with
   information on the patient's age, sex, symptoms,
   underlying diseases, and vaccination history. The
   specifics of how demographic and vaccination
   information is collected are the responsibility of
   participating institutions. Isolates are serotyped at
   the Centers for Disease Control and Prevention on the
   basis of capsular swelling with serotype-specific
   antisera (Quellung reaction).

   Children included in the analysis were 24 to 59 months
   of age with one or more chronic illnesses, had
   vaccination status and date indicated on the
   surveillance form, received vaccine between January
   1984 and April 1996, and had onset of invasive
   pneumococcal disease between January 1984 and April
   1996. Only isolates from cerebrospinal fluid (CSF) or
   blood were considered in the analysis. Information on
   antibiotic prophylaxis was not collected. Chronic
   illness was defined as an underlying illness considered
   a risk factor for invasive pneumococcal disease and an
   indication for vaccination (3).

Vaccine effectiveness was defined as the percentage of reduction in the risk for infection from serotypes included in the vaccine (vaccine-type serotypes) among vaccinated persons compared with unvaccinated persons. Infections with vaccine-related serotypes (6A, 9A, 9L, 18B, 18F, 23A) not specifically included in the vaccine were categorized as infections with nonvaccine serotypes, except where
noted. Effectiveness was expressed as 1 minus the odds ratio x 100%; the 95% confidence intervals (also x 100%) were calculated by the methods of Cornfield when cell sizes were all greater than five subjects and by exact methods otherwise. Calculations were performed with Epi-Info version 6.02 (CDC/World Health Organization, Atlanta, GA) with the EXACT supplemental program (David O. Martin).

We performed a preliminary analysis of all pneumococcal isolates from children in the database to determine the proportion of vaccine-type organisms in unvaccinated persons by sex, underlying illness, or state of residence. Proportions of vaccine-type serogroups did not differ by underlying illness or by sex. Because the proportion of vaccine-type isolates from children from Alaska was 92.3%, compared with the 85.4% of isolates from children from
other states (chi-square = 6.3; p <0.02), children from Alaska were excluded from the analysis.

The analysis included 173 children, 52% male,
median age 3 years; 48 children (28%) had
received vaccine before acquiring invasive
pneumococcal disease. Isolates were obtained
from blood only from 156 children (90%), from
CSF only from 10 children (6%), and from both
sites from 7 children (4%). The median time
between date of vaccination and date of
specimen collection was 338.5 days (33 days
1,341 days), and no child had been vaccinated
within 30 days of invasive pneumococcal infection. Of serotypes from the 173 invasive
infections, serotypes 4, 6A, 6B, 14, 23F, 19F, 
9V, and 18C accounted for 81% of the isolates
(Figure 1).

Figure 1. Invasive pneumococcal infections among 173 children ages 2 through 5 years (24-59 months), by
serotype. Bottom bar represents proportion of total invasive infections in the cohort caused by each
serotype. Top bar depicts cumulative proportion of invasive infections caused by serotypes represented by
the bars to the left. Serotypes in the "other" category included 19 serotypes with three or fewer isolates.
Two isolates could not be serotyped.

Forty-six (27%) of children in the study had sickle cell disease (Figure 2). The "other" category included children with congenital anomalies such as congenital heart or lung defects, children with anatomic asplenia, and children on immunosuppressive medication regimens. Thirty-three (69%) of 48 vaccinated children had sickle cell disease.

The Table presents vaccine effectiveness estimates for the overall cohort and for children with and without sickle cell disease. For children with the disease, the lower bound of the 95% confidence interval included 0%. The estimate of vaccine effectiveness for children without sickle cell disease was higher than the estimate for children with the disease. Point estimates of effectiveness for children with nephrotic syndrome or HIV infection were 80%; however,
the 95% confidence intervals included 0% (data not shown). Other chronic diseases reported in this cohort included leukemia, nonhematopoetic malignancy, and organ transplant; however, none of the children with these underlying diseases were vaccinated, and effectiveness could not be calculated.

Protein conjugate vaccines offer the advantage
of being effective in the first 2 years of
life, when response to polysaccharide vaccines
is poor. However, the number of serotypes that
can be represented in these vaccines is
limited. To evaluate polysaccharide
effectiveness for serotypes not represented in
a protein conjugate vaccine under evaluation
for license (13), we excluded children infected
with serotypes 4, 6B, 9V, 14, 18C, 19F, and 
23F. Polysaccharide vaccine was highly 
effective in preventing invasive disease due to
serotypes included in the polysaccharide
vaccine but not in the conjugate vaccine
(Table). If the 14 children with serotypes 6A,
9A, 9L, 18B, 18F, and 23A are also excluded
(because of potential protection conferred by
the proposed conjugate vaccine for these vaccine-related serotypes), the vaccine effectiveness estimate is 92% (exact 95% confidence intervals 17% to 100%).

Figure 2. Frequency of various underlying chronic illnesses among 173 children with invasive pneumococcal disease. The category "malignancy" excluded hematopoetic malignancies, which are included in the leukemia category. Organ transplant includes both solid organ and bone marrow
transplants. CSF is cerebrospinal fluid.

Table. Estimates of pneumococcal polysaccharide vaccine
effectiveness among 173 children 2 through 5 years of age, using the indirect cohort method

Conclusions

Case-control studies have demonstrated that pneumococcal capsular polysaccharide vaccines are effective (14-16) and cost-effective (17,18) in the prevention of invasive pneumococcal disease among elderly and chronically ill adults. We used data from a national sentinel surveillance system for invasive pneumococcal disease to determine whether children ages 2 to 5 years were also protected. An
overall vaccine effectiveness of 63% was demonstrated by indirect cohort analysis (15). The indirect cohort analysis presented here strengthens the case for the use of pneumococcal polysaccharide vaccine for children with underlying conditions. For children with sickle cell disease, penicillin prophylaxis remains the most
effective preventive measure for reducing pneumococcal disease.

Accuracy of vaccine history is critical to this analysis and may vary between surveillance sites. To minimize inaccuracies, patients with no indication of vaccine history were excluded. For those with a reported vaccine history, misclassification due to inaccurate history should be as likely among patients with vaccine-type as among nonvaccine-type infections because the serotype of patient isolates was not known when vaccine status was determined (serotyping was done at CDC). Bias due to this nondifferential misclassification will be towards the null hypothesis (no effect of vaccination) (19).

Newly developed pneumococcal protein conjugate vaccines are safe and immunogenic for infants and young children (13,20,21). Preliminary results from a large, Phase-III trial of a heptavalent conjugate vaccine among healthy children indicate substantial efficacy in preventing invasive disease (13). However, the expense and technical
difficulty of creating conjugates for each serotype will likely limit the number of serotypes represented in a polyvalent conjugate vaccine to fewer than 12. Available data suggest that polysaccharide vaccine, when administered after primary immunization with a conjugate vaccine, elicits a significant booster effect in healthy infants (22) equivalent to the booster response engendered by a
second conjugate vaccine series (23). These results and the level of effectiveness seen with pneumococcal polysaccharide vaccine in our study suggest that the polysaccharide vaccine will still be a useful adjunct to conjugate vaccine, by providing additional protection to children >/=2 years of age for whom polysaccharide vaccine is currently indicated.

Acknowledgments

The authors thank A.R. Franklin, D. Jackson, L. LaClaire, and N. Pigott for serotyping of pneumococcal isolates and the members of the Pneumococcal Sentinel Surveillance Working Group: Carol Camp, Patricia Charache, Mel Jackson, W. Keith Hadley, Joan Hoppe-Bauer, Michael R. Jacobs, Phyllis Tyler, Janet Monahan, Harold Moore, Jane
D. Siegel, David Sherer, and David Welch.

Dr. Fiore is a medical epidemiologist in the Hepatitis Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases.

Address for correspondence: Anthony E. Fiore, National Center for Infectious Diseases, Centers for Disease Control and Prevention; 1600 Clifton Road, Mail Stop G37, Atlanta, GA 30333, USA; fax: 404-639-1538; e-mail: abf4@cdc.gov.

References

1. Centers for Disease Control and Prevention. Prevention of
pneumococcal disease: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Morb Mortal Wkly Rep 1997;46(RR-8).

2. Henrichsen J. Six newly recognized types of Streptococcus
pneumoniae. J Clin Microbiol 1995;33:2759-62.

3. American Academy of Pediatrics. Pneumococcal infections. In: Peter G, editor. 1997 Red book: report of the Committee on Infectious Diseases. 24th ed. Elk Grove Village (IL): American Academy of Pediatrics; 1997. p. 410-9.

4. Riley ID, Everingham FA, Smith DE, Douglas RM. Immunisation with a polyvalent pneumococcal vaccine: Effect on respiratory mortality in children living in the New Guinea highlands. Arch Dis Child 1981;56:354-7.

5. Rosn C, Christensen P, Hovelius B, Prellner K. Effect of
pneumococcal vaccination on upper respiratory tract infections in children: Design of a follow-up study. Scand J Infect Dis 1983;Suppl 39:39-44.

6. Douglas RM, Miles HB. Vaccination against Streptococcus
pneumoniae in childhood: lack of demonstrable benefit in young Australian children. J Infect Dis 1984;149:861-9.

7. Ammann AJ, Addiego J, Wara DW, Lubin B, Smith WB, Mentzer WC. Polyvalent pneumococcal-polysaccharide immunization of patients with sickle-cell anemia and patients with splenectomy. N Engl J Med 1977;297:897-900.

8. Ahonkhai VI, Landesman SH, Fikrig SM, Smalzer EA, Brown  K, Cherubin CE, et al. Failure of pneumococcal vaccine in children with sickle-cell disease. N Engl J Med 1979;301:26-7.

9. John AB, Ramlal A, Jackson H, Maude GH, Waight-Sharma, A,
Serjeant GR. Prevention of pneumococcal infection in children with homozygous sickle cell disease. BMJ 1984;288:1567-70.

10. Broome CV, Facklam RR, Fraser DW. Pneumococcal disease after pneumococcal vaccination: an alternative method to estimate the efficacy of pneumococcal vaccine. N Engl J Med 1980;549-52.

11. Butler JC, Breiman RF, Campbell JF, Lipman HB, Broome CV, Facklam RR. Pneumococcal vaccine efficacy: an evaluation of current recommendations. JAMA 1993;270:1826-31.

12. Broome CV. Efficacy of pneumococcal polysaccharide vaccines. Revv Infect Dis 1981;Suppl 3:S82-8.

13. Black SB, Shinefield H, Ray P, Lewis E, Fireman P, et al. Efficacy of heptavalent conjugate pneumococcal vaccine (Wyeth Lederle) in 37,000 infants and children: results of the Northern California Kaiser Permanente Efficacy Trial. In: Programs and abstracts of the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy; 1998; San Diego, California. Washington: American Society for Microbiology, 1998.

14. Shapiro ED, Berg AT, Austrian R, Schroeder D, Parcells V, Margolis A, et al. The protective efficacy of polyvalent
pneumococcal polysaccharide vaccine. N Engl J Med  1991;325:1453-60.

15. Sims RV, Steinmann WC, McConville JH, King LR, Zwick WC,
Schwartz JS. The clinical effectiveness of pneumococcal vaccine in the elderly. Ann Intern Med 1988;108:653-7.

16. Farr BM, Johnston BL, Cobb JK, Fisch MJ, Germanson TP, Adal KA, et al. Preventing pneumococcal bacteremia in patients at risk: Results of a matched case-control study. Arch Intern Med 1995;155:2336-40.

17. Gable CB, Holzer SS, Engelhart L, Friedman RB, Smeltz F,
Schroeder D, et al. Pneumococcal vaccine: efficacy and associated cost savings. JAMA 1990;264:2910-5.

18. Sisk JE, Moskowitz AJ, Whang W, Lin JD, Fedson DS, McBean AM, et al. Cost-effectiveness of vaccination against pneumococcal bacteremia among elderly people. JAMA 1997;278;1333-9.

19. Copeland KT, Checkoway H, Holbrook RH, McMichael AJ. Bias due to misclassification in the estimate of relative risk. Am J Epidemiol 1977;105:488-95.

20. Kyhty H, hman H, Rnnberg P-R, Tillikainen R, Eskola J. Pneumococcal polysaccharide-meningococcal outer membrane protein complex conjugate vaccine is immunogenic in infants and children. J Infect Dis 1995;172:1273-8.

21. Rennels MB, Edwards KM, Keyserling HL, Reisinger KS, Hogerman DA, Madore DV, et al. Safety and immunogenicity of heptavalent pneumococcal vaccine conjugated to CRM197 in United States infants. Paediatrics 1998;101:604-11.

22. hman H, Kyhty H, Lehtonen H, Leroy O, Froeschle J, Eskola J. Streptococcus pneumoniae capsular polysaccharide-diphtheria toxoid conjugate vaccine is immunogenic in early infancy and able to produce immunologic memory. Pediatr Infect Dis J 1998;17:211-6.

23. Obaro SK, Huo Z, Banya WAS, Henderson DC, Monteil MA, Leach A, et al. A glycoprotein conjugate vaccine primes for antibody responses to a pneumococcal polysaccharide vaccine in Gambian children. Pediatr Infect Dis J 1997;16:1135-40.

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

URL: http://www.cdc.gov/ncidod/eid/vol5no6/ascii/fiore.txt

Please note that figures, equations, and most tables are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively.  Greek symbols are spelled out. >/= is used for greater than or equal to.

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

Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999]

   Commentary

   Stimulating the Development of Orphan (and Other)
   Vaccines

   Drs. Lang and Wood (this issue, pp. 749-756) highlight
   factors that affect vaccine development decisions at
   large pharmaceutical companies and suggest measures to
   make development of orphan vaccines more attractive.
   Because of the importance of economic assessment in
   corporate decision-making, development of vaccines for
   rare diseases is usually problematic. Exceptions may
   include vaccines for potential bioterrorism agents (the
   government may support development and production) and
   therapeutic vaccines for chronic or deadly diseases
   (the price of a vaccine could be high, commensurate
   with the cost of therapy). Of seven vaccines defined by
   the Institute of Medicine as being "most favorable" for
   development, three were therapeutic vaccines (for
   diabetes mellitus, rheumatoid arthritis, and multiple
   sclerosis) (1).

   In the developing world, price has been a major
   impediment to the introduction of new vaccines. Whether
   this reflects limitations in ability or willingness to
   pay, the end result is that a company could not expect
   sales in developing countries to provide the desired
   return on investment (2). Clearly, novel solutions are
   needed if vaccines that could save millions of
   children's lives are to be used effectively. Support
   for vaccination from the Bill and Melinda Gates
   Foundation and the promotion of vaccines as an
   acceptable component of bilateral loans by the World
   Bank may begin shifting the balance between market
   imperatives and public health needs.

   Drs. Lang and Wood propose a package of incentives that
   may help promote development of orphan vaccines by
   major manufacturers. But will these measures be enough
   to alter vaccine development priorities? Lowering the
   risks or costs of vaccine development may be much less
   important than increasing the potential for profit. The
   vaccine development pipeline is full of products that
   will never come to market, not because they cost more
   to develop but because the company projects
   insufficient profit from their eventual use. Promoting
   a greater appreciation of the benefits of prevention in
   both developing and industrialized countries and
   enhancing the size of the market and the willingness to
   pay will likely have a greater impact on investment
   decisions than an incremental decrease in vaccine
   development costs.

   If large manufacturers shift vaccine development
   priorities on the basis of incentives and other
   measures so that the total number of products brought
   to market is not increased but one set of priorities is
   substituted for another, the overall impact on disease
   prevention may be not change. The greatest increase in
   disease prevention and in the development of orphan
   vaccines would occur by increasing the total number of
   vaccines produced. The therapeutics industry differs
   from the vaccine industry in that it includes a
   substantially greater number of players that can bring
   a new product to market. In the United States
   currently, 194 drugs and biologics have been brought to
   market as orphan products, but none are vaccines. Thus,
   incentives that draw new companies to invest in vaccine
   development may be extremely useful for the development
   of orphan vaccines.

   Vaccines prevent more than 3.2 million deaths per year
   (3). Developments in biotechnology have created the
   promise of prevention for many more infectious and
   chronic diseases (4). Realizing this promise will
   require bringing to licensure more of the vaccines now
   in development. Finally, our credibility in designating
   disease areas as priorities for vaccine development
   rests on our ability to use the new vaccines already in
   hand.

   Benjamin Schwartz* and N. Regina Rabinovich

   *Centers for Disease Control and Prevention, Atlanta,
   GA, USA; National Institute of Allergy and Infectious
   Diseases, National Institutes of Health, Bethesda, MD,
   USA

   References

   1. Institute of Medicine. Vaccines for the 21st
   century: a tool for decision-making. Washington:
   National Academy Press, 1999.

   2. Hausdorf WP. Prospects for the use of new vaccines
   in developing countries: cost is not the only
   impediment. Vaccine 1996;14:1179-86.

   3. World Health Organization, United Nations Children's
   Fund. State of the world's vaccines and immunization.
   Geneva, Switzerland: The Organization, 1996.

   4. Jordan Report. Accelerated development of vaccines
   1998. Washington: National Institute of Allergy and
   Infectious Diseases, 1998.

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

URL: http://www.cdc.gov/ncidod/eid/vol5no6/ascii/schwartz.txt

Please note that figures, equations, and most tables are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively.  Greek symbols are spelled out. >/= is used for greater than or equal to.

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

Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999]

   Commentary

   Stimulating the Development of Orphan (and Other)
   Vaccines

   Drs. Lang and Wood (this issue, pp. 749-756) highlight
   factors that affect vaccine development decisions at
   large pharmaceutical companies and suggest measures to
   make development of orphan vaccines more attractive.
   Because of the importance of economic assessment in
   corporate decision-making, development of vaccines for
   rare diseases is usually problematic. Exceptions may
   include vaccines for potential bioterrorism agents (the
   government may support development and production) and
   therapeutic vaccines for chronic or deadly diseases
   (the price of a vaccine could be high, commensurate
   with the cost of therapy). Of seven vaccines defined by
   the Institute of Medicine as being "most favorable" for
   development, three were therapeutic vaccines (for
   diabetes mellitus, rheumatoid arthritis, and multiple
   sclerosis) (1).

   In the developing world, price has been a major
   impediment to the introduction of new vaccines. Whether
   this reflects limitations in ability or willingness to
   pay, the end result is that a company could not expect
   sales in developing countries to provide the desired
   return on investment (2). Clearly, novel solutions are
   needed if vaccines that could save millions of
   children's lives are to be used effectively. Support
   for vaccination from the Bill and Melinda Gates
   Foundation and the promotion of vaccines as an
   acceptable component of bilateral loans by the World
   Bank may begin shifting the balance between market
   imperatives and public health needs.

   Drs. Lang and Wood propose a package of incentives that
   may help promote development of orphan vaccines by
   major manufacturers. But will these measures be enough
   to alter vaccine development priorities? Lowering the
   risks or costs of vaccine development may be much less
   important than increasing the potential for profit. The
   vaccine development pipeline is full of products that
   will never come to market, not because they cost more
   to develop but because the company projects
   insufficient profit from their eventual use. Promoting
   a greater appreciation of the benefits of prevention in
   both developing and industrialized countries and
   enhancing the size of the market and the willingness to
   pay will likely have a greater impact on investment
   decisions than an incremental decrease in vaccine
   development costs.

   If large manufacturers shift vaccine development
   priorities on the basis of incentives and other
   measures so that the total number of products brought
   to market is not increased but one set of priorities is
   substituted for another, the overall impact on disease
   prevention may be not change. The greatest increase in
   disease prevention and in the development of orphan
   vaccines would occur by increasing the total number of
   vaccines produced. The therapeutics industry differs
   from the vaccine industry in that it includes a
   substantially greater number of players that can bring
   a new product to market. In the United States
   currently, 194 drugs and biologics have been brought to
   market as orphan products, but none are vaccines. Thus,
   incentives that draw new companies to invest in vaccine
   development may be extremely useful for the development
   of orphan vaccines.

   Vaccines prevent more than 3.2 million deaths per year
   (3). Developments in biotechnology have created the
   promise of prevention for many more infectious and
   chronic diseases (4). Realizing this promise will
   require bringing to licensure more of the vaccines now
   in development. Finally, our credibility in designating
   disease areas as priorities for vaccine development
   rests on our ability to use the new vaccines already in
   hand.

   Benjamin Schwartz* and N. Regina Rabinovich

   *Centers for Disease Control and Prevention, Atlanta,
   GA, USA; National Institute of Allergy and Infectious
   Diseases, National Institutes of Health, Bethesda, MD,
   USA

   References

   1. Institute of Medicine. Vaccines for the 21st
   century: a tool for decision-making. Washington:
   National Academy Press, 1999.

   2. Hausdorf WP. Prospects for the use of new vaccines
   in developing countries: cost is not the only
   impediment. Vaccine 1996;14:1179-86.

   3. World Health Organization, United Nations Children's
   Fund. State of the world's vaccines and immunization.
   Geneva, Switzerland: The Organization, 1996.

   4. Jordan Report. Accelerated development of vaccines
   1998. Washington: National Institute of Allergy and
   Infectious Diseases, 1998.

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

URL: http://www.cdc.gov/ncidod/eid/vol5no6/ascii/schwartz.txt

Please note that figures, equations, and most tables are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively.  Greek symbols are spelled out. >/= is used for greater than or equal to.

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

Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999]

   News and Notes

   Upcoming Keystone Symposia

   "Pathogen Discovery: From Molecular Biology to
   Diseases"

   Organizers: Georg Hess and Helen H. Lee

   "Genetics, Pathogenesis and Ecology of Emerging Viral
   Diseases"

   Organizers: Michael J. Buchmeier and Clarence J. Peters

   The two symposia will be held concurrently at the Civic
   Center, Taos, New Mexico, January 24-30, 2000. For
   information on registration and abstract submission,
   see the Keystone Symposia Web Site at:
   http://www.symposia.com.

   Summary of Symposia Contents

   There is still a long list of serious diseases whose
   causes remain undetermined. Recently, new technologies
   have allowed the discovery of a number of new and
   emerging pathogens. Elucidation of pathogenesis and
   disease associated with these agents has been
   challenging and controversial.

   The first of these concurrent symposia, "Pathogen
   Discovery: From Molecular Biology to Diseases," seeks
   to foster discussion of important subjects related to
   pathogen discovery, including the assessment of
   evidence implicating candidate infectious agents in
   disease, the value of animal models, technologies used
   to search for candidate pathogen sequences, and
   approaches to the study of disease pathogenesis.
   Interactions between the host and the infectious agent
   will be addressed, with reference to susceptibility,
   viral persistence, and disease expression.

   The second symposium will explore these issues in
   detail as they apply to emerging viruses. Emerging
   viruses have attracted attention in the scientific and
   popular press, and their recognition as serious human
   disease threats has resulted in a flood of reports
   describing their appearance and spread. Examination of
   this body of literature during the second symposium
   will allow the identification of several themes central
   to the understanding and control of emerging viruses.
   These themes include interpreting the role of genetic
   variation in animal and human disease, understanding
   the ecologic relationships of the virus to its natural
   host and vectors, and delineating how these
   relationships affect humans.

   The mechanisms of viral pathogenesis must be understood
   so that therapeutic and vaccination strategies can be
   designed. The combination of a rapidly expanding world
   population and the ease of travel on a global scale
   provide opportunities for transport of viral vectors or
   infected persons from disease-endemic areas to other
   regions, heightening the urgent need for detection and
   prevention strategies. Key investigators in each of
   these areas will present recent findings in a setting
   that facilitates comparisons with other presentations
   highlighting the social and economic problems posed by
   emerging viral diseases.

   Applied Epidemiology Course
   Wellington, New Zealand,
   February 14-18, 2000

   This course, offered by the Wellington School of
   Medicine as part of its Third International Summer
   School, provides participants with core skills in
   applied epidemiology: the ability to analyze data from
   an epidemiologic perspective, explore data using
   geographic information systems (GIS), set up
   surveillance systems, and investigate disease outbreaks
   and clusters. The course also teaches analytical
   skills, including practical use of EpiInfo and GIS
   software.

   Highly interactive teaching and case studies provide
   the knowledge and skills needed for work in public
   health. The course is specifically designed for medical
   and nonmedical staff who undertake field
   investigations, for infection control staff, and for
   policy analysts who interpret data.

   The course is convened by Michael Baker, a public
   health physician at New Zealand's national communicable
   disease surveillance center (ESR) `and senior lecturer
   at the Department of Public Health, Wellington School
   of Medicine. Other contributors, from ESR, the
   Wellington School of Medicine and the Wellington public
   health service, include epidemiologists with training
   and experience in New Zealand, Australia, and the
   United States.

   Cost for the 5-day course is NZ$1,150. For further
   information or application, contact the Department of
   Public Health, Wellington School of Medicine, P.O. Box
   7343, Wellington South; telephone: 64-4-385-5999, ext.
   6052; fax: 64-4-389-5139; e-mail: comhtw@wnmeds.ac.nz;
   homepage: www.wnmeds.ac.nz/academic/dph.

4th Decennial International Conference on Nosocomial and Health Care-Associated Infections
Atlanta, Georgia, USA, March 59, 2000

On-Line Abstract Submission Deadline--November 15, 1999

Every 10 years, the Centers for Disease Control and Prevention sponsors the International Conference on Nosocomial and Health Care-Associated Infections in Atlanta, Georgia. The 1970, 1980, and 1990 conferences set the agenda for research and infection control practices for the respective decades; the Fourth Decennial Conference will set the agenda for the new millennium. 

Call for abstracts

The deadline for on-line submission of abstracts is November 15, 1999. The deadline for abstracts submitted by mail is November 1, 1999. Selected late-breaker abstracts must be submitted by Monday, January 17, 2000. Abstracts can be submitted on-line starting in June at  http://www.decennial.org/. Abstract submission forms can be
downloaded from the Internet site or mailed upon request. Abstract submission forms will be automatically distributed to all Association for Professionals in Infection Control and Epidemiology (APIC), Infectious Disease Society of America (IDSA), and Society for Health Care Epidemiology of America (SHEA) members, as well as attendees at the Interscience Conference on Antimicrobial Agents and
Chemotherapy (ICAAC).

Program Registration

Program and registration information will be distributed to all members of APIC, IDSA, and SHEA, as well as to ICAAC attendees. If you are not a member of one of these organizations, please contact the 4th Decennial Conference (see below) and ask to be placed on the mailing list. The conference will be held in conjunction with the 10th annual meeting of SHEA; separate registration is required for
SHEA events.

For more information contact the 4th Decennial Conference, 6220 Montrose Road, Rockville, MD 20852, USA; telephone: 301-984-9450; fax: 301-984-9441; e-mail: info@decennial.org; Internet: www.decennial.org.

Towards a Healthy Europe for the Year 2010

V European Conference on Health Promotion and Health Education Santander, Spain, May 10-13, 2000

The theme of cultural diversity has been chosen for this conference, which will deal with professional practices, communication in social settings, and health policies that should be developed to achieve a healthy Europe for the next millennium.

We believe that the coexistence of different cultures is sometimes the cause of health problems but also increasingly the source of solutions and opportunities for health promotion and for the achievement of a healthy Europe.

The subjects related to this central theme will be addressed in plenary sessions, open debates, oral presentations, and poster sessions. There will also be workshops which will serve as a forum for professionals with common interests in specific areas.

For additional information, please contact Asociacin de Educacin para la Salud (ADEPS), Servicio de Medicina Preventiva, 4a Norte, Hospital Clnico San Carlos, c/o Profesor Martn Lagos, 28040 Madrid, Spain; telephone: 34-91-330-3422; fax: 34-91-543-7504; e-mail: msainz@hcsc.es.

Erratum Vol. 5 No. 3

In the article "Use of Antimicrobial Growth Promoters in Food Animals and Enterococcus faecium Resistance to Therapeutic Antimicrobial Drugs in Europe, " by H.C. Wegener et al., reference 3 is incorrect. The correct reference is

3. Centers for Disease Control and Prevention. Summary of Notifiable Diseases, United States, 1997. MMWR Morb Mortal Wkly Rep 1998; 46:1-87.

We regret any confusion this error may have caused.

ICEID 2000  Call for Abstracts

Abstract submission to ICEID 2000 is now possible through the Conference Web site, and we strongly encourage you to make use of this electronic method. Not only will you be able to submit your abstract in a finished form (prepared using your own word processing program such as Word or WordPerfect, on PC or Macintosh platforms),
but you can check back to learn acceptance status, presentation location, and time information. In addition, it will save you both the costs of postage and the worry of a lost submission.

ICEID 2000 is using the American Society for Microbiology's
Web-based Abstract Submission System. If you have used the system to submit to either the 1999 or 2000 ASM General Meetings or the 1999 ICAAC Meeting, you will be able to reenter the system as a returning user.

For more information on ICEID abstract submission, see the ASM Web site: www.asmusa.org. Follow the links to Meetings and ICEID 2000. 

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

URL: http://www.cdc.gov/ncidod/eid/vol5no6/ascii/newsandnotes.txt

Please note that figures, equations, and most tables are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively.  Greek symbols are spelled out. >/= is used for greater than or equal to.