Volume 2,  Number 1,  January-March 1996

EMERGING INFECTIOUS DISEASES, VOLUME 2, NUMBER 1

Editors

Editor
Joseph E. McDade, Ph.D.
National Center for Infectious Diseases
Centers for Disease Control 
  and Prevention (CDC)
Atlanta, Georgia, USA

Perspectives Editor
Stephen S. Morse, Ph.D.
The Rockefeller University
New York, New York, USA

Synopses Editor
Phillip J. Baker, Ph.D.
Division of Microbiology and Infectious Diseases
National Institute of Allergy and Infectious Diseases
National Institutes of Health (NIH)
Bethesda, Maryland, USA

Dispatches Editor
Stephen Ostroff, M.D.
National Center for Infectious Diseases
Centers for Disease Control and Prevention (CDC)
Atlanta, Georgia, USA

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National Center for Infectious Diseases
Centers for Disease Control and Prevention (CDC)
Atlanta, Georgia, USA

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Robert Shope, M.D.
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Yale University School of Medicine
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Natalya B. Sipachova, M.D., Ph.D.
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Russian Republic Information & Analytic Centre
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Bonnie Smoak, M.D.
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Walter Reed Army Institute of Research
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Robert Swanepoel, B.V.Sc., Ph.D.
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National Institute for Virology
Sandrinham 2131, South Africa

Roberto Tapia, M.D.
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Direccin General de Epidemiologa
Secretara de Salud
Mxico

Emerging Infectious Diseases

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Table of Contents
EMERGING INFECTIOUS DISEASES
Volume 2,  Number 1,   January-March 1996

Perspective

   * Molecular Population Genetic Analysis of Emerged Bacterial Pathogens:
     Selected Insights, James M. Musser

Synopses

  * Emergence of the Ehrlichioses as Human Health Problems, David H.
    Walker, J. Stephen Dumler

  * Surveillance for Pneumonic Plague in the United States During an
    International Emergency: A Model for Control of Imported Emerging
    Diseases, Curtis L. Fritz, David T. Dennis, Margaret A. Tipple, Grant
    L. Campbell, Charles R. McCance, Duane J. Gubler

  * Changing Patterns of Autochthonous Malaria Transmission in the United
    States: A Review of Recent Outbreaks, Jane R. Zucker

Dispatches

  * Cluster of Lyme Disease Cases at a Summer Camp in Kent County,
    Maryland, G. Thomas Strickland, Leena Trivedi, Stanley Watkins,
    Margaret Clothier, John Grant, John Morgan, Edward Schmidtman, Thomas
    Burkot

  * Unexplained Deaths Due to Possibly Infectious Causes in the United
    States: Defining the Problem and Designing Surveillance and Laboratory
    Approaches, Bradley A. Perkins, Jennifer M. Flood, Richard Danila,
    Robert C. Holman, Arthur L. Reingold, Laura A. Klug, Michael Virata,
    Paul R. Cieslak, Sherif R. Zaki, Robert W. Pinner, Rima F. Khabbaz,
    and the Unexplained Deaths Working Group

  * Trends in Bacteremic Infection Due to Streptococcus pyogenes (Group A
    Streptococcus), 1986-1995, Daniel M. Musher, Richard J. Hamill,
    Charles E. Wright, Jill E. Clarridge, Carol M. Ashton
 
Letter                                                                                                                                    

* PHLS Surveillance of Antibiotic Resistance, England and Wales:
  Emerging Resistance in Streptococcus pneumoniae, David C. E. Speller,
  Alan P. Johnson, Barry D. Cookson, Pauline Waight, Robert C. George

Commentary

 * Infectious Diseases in Latin America and the Caribbean: Are They
   Really Emerging and Increasing? A. David Brandling-Bennett, Francisco
   Pinheiro

 * Microbial Threats and the Global Society, Stuart B. Levy

 * Xenotransplantation: Risks, Clinical Potential, and Future Prospects,
   Robert E. Michler

News and Notes

   * Another Human Case of Equine Morbillivirus Disease in Australia

   * Social Science and the Study of Emerging Infectious Diseases, Johannes
     Sommerfield, Sandra Lane

  *  WHO Establishes New Rapid-Response Unit for Emerging Infectious
     Diseases, Philippe Stroot

  * Rotavirus Vaccine Workshop Held, Roger I. Glass

  * International Conference Addresses Preparedness for Emerging Strains
    of Pandemic Influenza, Dominick A. Iacuzio

  * Course Offered on Clinical and Pathologic Features of Emerging
    Infections, C. Robert Horsburgh, Jr.
  
  * NASA Sponsors Symposium on Remote Sensing and Control of
    Insect-Transmitted Diseases, Michael Braukus, Raj Khanna

  * CDC Convenes Meeting to Discuss Strategies for Preventing Invasive
    Group A Streptococcal Infections, The Working Group on Prevention of
    Severe Group A Streptococcal Infections

  * Regional Conference on Emerging Infectious Diseases Sparks Plan for
    Increased Collaboration, Bradford A. Kay
                                                                                                                                          News 

PERSPECTIVE

(Pages 1-17)

Molecular Population Genetic Analysis of Emerged Bacterial Pathogens:
Selected Insights

James M. Musser, M.D., Ph.D.
Department of Pathology, Baylor College of Medicine, Houston, Texas, USA

ABSTRACT

Research in bacterial population genetics has increased in the last 10
years. Population genetic theory and tools and related strategies have been
used to investigate bacterial pathogens that have contributed to recent
episodes of temporal variation in disease frequency and severity. A common
theme demonstrated by these analyses is that distinct bacterial clones are
responsible for disease outbreaks and increases in infection frequency.
Many of these clones are characterized by unique combinations of virulence
genes or alleles of virulence genes. Because substantial interclonal
variance exists in relative virulence, molecular population genetic studies
have led to the concept that the unit of bacterial pathogenicity is the
clone or cell line. Continued new insights into host-parasite interactions
at the molecular level will be achieved by combining clonal analysis of
bacterial pathogens with large-scale comparative sequencing of virulence
genes.


To avert the threat of resurgent and new microbial diseases, it is critical
to gain insight into the molecular mechanisms contributing to temporal
variation in disease frequency and severity. Although comprehensive,
unambiguous understanding of the host and parasite factors mediating these
processes is not available for any infectious agent, population genetic
research in the last 10 years has provided noteworthy new information about
the bacterial side of the equation. This review will summarize the insights
accrued from population genetic analysis of bacteria responsible for
disease outbreaks or increases in infection frequency and severity. One of
the primary themes emerging from this research is that distinct bacterial
clones have been responsible for several infection outbreaks (Table 1).
Moreover, the distinct clones are frequently characterized by unique
combinations of virulence genes or alleles of virulence genes. These
observations have important implications for our understanding of
infectious diseases and the public health measures required to reduce their
detrimental and potentially devastating effect on society.

Population Genetics and Clonal Analysis of Bacterial Pathogens: Basic
Concepts

Population genetic study of bacterial pathogens arose largely as an
offshoot of research designed to address questions of longstanding interest
to students of the molecular evolutionary processes in higher eukaryotic
organisms. Bacteria were an attractive group of experimental organisms
because of their phenotypic diversity, short generation times, haploid
chromosomal genomes, and accessory genetic elements. Hence, bacterial
population genetic research was originated by population geneticists
interested in bacteria, rather than bacterial geneticists or medical
microbiologists interested in population genetics (1, 2). In spite of its
important implications for how the field has developed over the last
decade, this ontogeny will not be discussed in detail here. However, the
reader should recognize that bacterial population genetics is a discipline
separate and distinct from the study of the molecular epidemiology of
infectious agents. The research tools, methods of data analysis, and
general thought processes are very different from the typological thinking
used by investigators of disease outbreaks or microbial pathogenesis (3-6).

 -------------------------------------------------------------------------------------
 Table 1. Representative Emerged Bacterial Pathogens 
 Investigated with Molecular Population Genetic Strategies
 --------------------------------------------------------------------------------------

 Organism                    Disease                             Reference
----------------------------------------------------------------------------------------
 Borrelia species            Lyme disease                      146-149,150,151
 Escherichia coli            hemorrhagic colitis;              33-35
     O157:H7                   hemolytic uremic syndrome 
 Haemophilus influenzae      Invasive disease                  156
 H. influenzae
    biogroup aegyptius       Brazilian purpuric fever          25,26
 Listeria monocytogenes      Food-borne invasive disease       157, 158
 Mycobacterium
   tuberculosis              Tuberculosis                      91,105
 Neisseria meningitidis      Sepsis, meningitis                52-63,66,69-73
 Salmonella species           Food-borne illness               159-161
 Staphylococcus aureus       Toxic shock syndrome;     
			      methicillin-resistant strains    47,143,144
 Streptococcus pyogenes      Invasive disease,                 82,83,85
			      toxic-shock-like syndrome
 S.pneumoniae                Otitis, pneumonia,                125-131,136
			      sepsis, meningitis
 Vibrio cholerae             Cholera                           153-155
 ----------------------------------------------------------------------------------------------------------

Early work on the clonal nature of bacterial pathogens was conducted
largely with Escherichia coli, through a framework supplied by serotyping
of one or a few polymorphic surface antigens (7, 8). Only a few of the many
possible O and H antigen serotypes were frequently associated with
outbreaks of infantile diarrhea in the United Kingdom and other countries,
which suggested that isolates expressing these traits had special virulence
properties (7-9). Because serotype analysis of relatively few surface
structures does not provide robust data for estimating overall levels of
chromosomal diversity and relationships among strains, the primary research
tool used to examine the population genetics of emerging bacterial
pathogens has been multilocus enzyme electrophoresis (10, 11). This
technique indexes allelic variation in sets of randomly selected structural
genes located on the chromosome and provides a basis for estimating overall
levels of genotypic variation in populations, i.e., the sample of bacteria
chosen for analysis. The key concept underlying use of starch gel-based
protein electrophoresis in population genetics is that electromorphs
(mobility variants) of an enzyme can be directly equated with alleles of
the corresponding structural gene. Moreover, electromorph profiles over a
sample of different enzymes, therefore, correspond to multilocus enzyme
genotypes and are frequently referred to as electrophoretic types or ETs.
The proteins analyzed are usually metabolic enzymes expressed by virtually
all isolates of a species under the growth conditions used. The allelic
variation detected is unaltered by environmental conditions such as culture
conditions, laboratory storage, anatomic site of recovery, or specific
clinical disease. Allelic variation in these metabolic enzymes is
selectively neutral, or nearly so, which means that convergence to the same
allele through adaptive evolution is unlikely (4, 12-14). As a consequence,
this approach to the study of bacterial and other microbial pathogens
provides a convenient strategy for indexing overall levels of chromosomal
diversity in the sample and for inferring genetic relationships among
strains. Because of the strong correlation of chromosomal divergence
indexed by multilocus enzyme electrophoretic data and DNA-DNA hybridization
studies (15-18), several cryptic species have been identified once their
existence was initially discovered by population genetic analyses that
employed starch gel electrophoresis (16, 19-21). In most bacterial species,
the number of allelic variants is large, and it is unlikely that
recombinational processes would, by chance, frequently generate strains
with the identical electromorph profile. Hence, organisms with the same
electromorph profile are generally thought to be similar by descent, rather
than by convergence through lateral gene flow.

Recently, convenient, rapid, and relatively inexpensive large-scale DNA
sequencing techniques have also been adopted by several laboratories.
Large-scale automated DNA sequencing has been used to rapidly and
unambiguously identify a causative infectious agent and confirm or refute
the identity of isolates recovered from temporally-linked patients thought
to be involved in a disease outbreak. In addition, sequence-based studies
have been employed to define the nature and extent of allelic variation in
toxin and other virulence factor genes and to rapidly identify mutations
associated with antimicrobial agent resistance (22).

Unless noted, data on the population genetic analysis of emerging bacterial
pathogens summarized in this article were generated by multilocus enzyme
electrophoresis, sometimes performed in concert with automated DNA
sequencing.

Representative Insights

Brazilian Purpuric Fever

Brazilian purpuric fever (BPF), a serious invasive disease of children, was
first characterized in 1984 after an outbreak in Promissao, Sao Paulo
State, Brazil. Children with BPF have acute onset of fever and usually die
within 48 h with disseminated purpura, vascular collapse, and hypotensive
shock (23). BPF is caused by Haemophilus influenzae biogroup aegyptius, an
organism associated with sporadic or epidemic conjunctivitis (24).
Multilocus enzyme electrophoresis and other molecular techniques have
demonstrated that isolates recovered from BPF patients represent a distinct
clone (25).

As a first step toward identifying the evolutionary origin of this
pathogenic H. influenzae biogroup aegyptius clone, chromosomal variation
and genetic relationships were indexed among 17 biogroup aegyptius
isolates, and 2,209 encapsulated H. influenzae strains were recovered
worldwide (26). Biogroup aegyptius isolates form three distinct
evolutionary lineages of the species H. influenzae. Isolates of the case
clone are only very distantly related to other isolates classified as
biogroup aegyptius; that is, the case clone was no more related to other
biogroup aegyptius isolates than are (for example) two H. influenzae
isolates selected at random from the species. The BPF case clone was
genetically allied with H. influenzae isolates expressing serotype c
polysaccharide capsule, a result that explains an earlier observation (27)
that BPF isolates, like serotype c strains, produce type 2 IgA1 protease,
whereas other isolates of biogroup aegyptius express type 1 IgA1 protease.
Thus, the population genetic evidence showed that biogroup aegyptius is
polyphyletic and that the BPF organism is a genetically distinct clone
unrelated to other isolates with the phenotypic criteria of biogroup
aegyptius.

The genetic diversity in the sample of all biogroup aegyptius strains was
approximately equal to that recorded for entire species of certain
pathogenic bacteria (16, 17). Therefore, the effective population size of
aegyptius must be large; however, this interpretation is difficult to
reconcile with the observation that strains in the biogroup are rare
pathogens associated only with human disease. A possible explanation for
the relatively extensive genetic diversity among biogroup aegyptius strains
is that they represent cell lineages spawned from a much larger base
population of diverse nonpathogenic precursor clones. According to this
hypothesis, acquisition or loss of one or more genes (or, perhaps, a shift
in ecological niche) may produce a pathogenic form with the characteristic
viscerotropism for human conjunctivae.

Although population genetic analysis did not provide a simple reason for
the BPF outbreak, the demonstration that the causative clone of biogroup
aegyptius was highly differentiated from other phenotypically similar
organisms provided an explanation for the unique infection manifestations
and the unique group of characters associated with the clone (28-30).
Moreover, population genetic analyses demonstrated distinct medical
correlates to isolates classified as biogroup aegyptius. The results of
numerous subsequent studies have confirmed that, as a population, H.
influenzae biogroup aegyptius strains vary in their behavior, as one would
expect of a genetically diverse set of organisms.

Escherichia coli O157:H7

Strains of E. coli expressing serotype O157:H7 were recognized in the early
1980s as important causes of hemorrhagic colitis and hemolytic uremic
syndrome in North America (31). Disease usually occurs after consumption of
contaminated beef or other food. Several large outbreaks have occurred, and
more than 60 case clusters have been reported in the United States (32).
Because several E. coli reference laboratories rarely identified organisms
expressing this serotype before the early 1980s, reporting of these
isolates has increased dramatically. Because of the medical and economic
importance of E. coli strains considerable effort has been directed toward
elucidating genetic relationships among and between them as well as between
them and other members of the species; as a result extensive information is
now available about clonal relationships among these important bacteria
(33-35).

The observation that O157:H7 strains synthesize one or more Shiga-like
toxins and lack the ability to rapidly ferment sorbitol initially suggested
that strains of this serotype had shared a recent common ancestor. To
directly test this idea, multilocus enzyme electrophoresis was used to
assess genetic relatedness of 100 strains of E. coli serotypes recovered
from patients with hemorrhagic colitis or hemolytic uremic syndrome (33).
Although 25 distinct multilocus enzyme genotypes were identified, cluster
analysis found that O157:H7 isolates are closely related organisms. The
results were interpreted to mean that O157:H7 organisms recovered from
epidemiologically unassociated North American outbreaks belong to a single
geographically widespread pathogenic clone with specific virulence
properties (33). Subsequent analysis of O157:H7 strains by pulsed-field gel
electrophoresis has supported this idea (36).

To delineate clonal relationships among O157:H7 organisms and other E. coli
strains that cause hemorrhagic colitis and infantile diarrhea, 1,300
isolates representing 16 serotypes from patients with these diseases were
studied by multilocus enzyme electrophoresis and probing for genes encoding
Shiga-like toxins (34). The O157:H7 clone was closely related to a clone of
O55:H7 strains that has a long history of worldwide association with
outbreaks of infantile diarrhea (34). The data strongly suggested that the
O157:H7 and O55:H7 clones have recently radiated from a common ancestral
cell. The O157:H7 clone arose from an O55:H7-like ancestor, perhaps through
horizontal transfer and recombination events adding Shiga-like toxin genes
and adhesion genes to an E. coli genome preadapted for causing diarrheal
disease (34, 35). If, as the multilocus enzyme electrophoretic data
indicate, O157:H7 and O55:H7 organisms have shared a recent common
ancestor, it is likely that the close genetic affiliation would be
reflected at the nucleotide level. To test this notion, the gene (eae) (34)
encoding intimin, a protein involved in bacterial attachment to enterocytes
and subsequent effacement of the microvilli, was sequenced from
representative isolates of these two serotypes. The resulting sequence data
were consistent with the hypothesis that O157:H7 and O55:H7 organisms share
a close genetic affinity and thereby provide a plausible explanation for
the observation that these bacteria cause similar attaching and effacing
lesions in cells grown in culture (38) and in animal models (39). Because
conventional serotyping of E. coli does not provide a reliable basis for
analyzing population structure and can be grossly misleading as to genetic
relationships among isolates (40-42), many important medical correlates of
the population structure will not be recognized and understood fully until
E. coli isolates are sorted out along clonal lineages.

Staphylococcus aureus Toxic Shock Syndrome

Toxic shock syndrome (TSS) was described in 1978 (43) as a severe acute
illness (characterized by high fever, erythematous rash, hypotension or
shock, multiorgan involvement, and desquamation of the skin) of young
children associated with infection with Staphylococcus aureus. Two years
later, it was recognized that TSS is a geographically widespread disease
affecting mainly young, healthy, menstruating women, especially those using
certain high absorbency tampons (44). Most vaginal isolates of S. aureus
recovered from patients with TSS produce a chromosomally encoded toxin,
designated as toxic shock syndrome toxin-1 (TSST-1) (45). Evidence
implicating TSST-1 as a major virulence factor in the pathogenesis of TSS
has accumulated (46). Almost all strains recovered from patients with
menstrual TSS, which account for approximately 90% of TSS cases, synthesize
TSST-1, whereas only 50%-60% of isolates from cases of nonmenstrual TSS and
5%-25% of strains causing other diseases produce this protein.

Several questions of importance to both medical bacteriology and
evolutionary genetics were addressed in a study of 315 TSST-1-producing
strains of S. aureus (47). It was discovered that the organisms responsible
for most cases of TSS with a female urogenital focus are members of a
single distinctive clone (designated as ET 41), a result that explains the
observation that isolates recovered from patients with TSS share many
traits (48, 49). The investigation also showed that TSST-1 is expressed by
isolates of a great variety of clones representing virtually the full
breadth of genotypic diversity in the species as a whole. In addition,
isolates of ET 41 represented 24% of a sample of TSST-1-producing strains
recovered before 1978, which meant that the tst gene encoding the toxin
neither evolved nor was acquired recently by this species. The failure to
recover isolates of ET 41 from non-human hosts effectively eliminated the
likelihood that animals are important in the transmission of this clone.

Twenty-eight percent of isolates of S. aureus cultured from the introitus,
vagina, or cervix of unassociated healthy carriers or women with non-TSS
urogenital symptoms were ET 41 or closely allied clones; no other single
multilocus enzyme genotype accounted for more than 12% of normal vaginal
isolates. These observations led to the hypothesis that isolates of ET 41
are more readily able to colonize the human vagina and, hence, are widely
dispersed in an ecological niche of great consequence in TSS. Under this
"adapted clone" hypothesis, isolates of ET 41 are responsible for most
vaginal cases because this clone has a special affinity for the
cervicovaginal milieu, perhaps (but not necessarily) as a consequence of
variation in regulation of toxin- or other virulence-gene expression. In
summary, data derived from clonal analysis of TSST-1-producing S. aureus
are consistent with the notion that the "bloom" in TSS cases happened
because of a change in the character of catemenial products (perhaps
associated with decreasing levels of anti-TSST-1 antibody in human
populations), not because of a new S. aureus strain.

Two additional points are noteworthy regarding population genetic analysis
of S. aureus strains producing TSST-1. First, if the gene encoding TSST-1
were evolutionarily old, allelic variants differing in nucleotide and,
perhaps, amino acid sequence would exist in natural populations. This
prediction was borne out by the identification of a variant of TSST-1
associated with goat, sheep, and occasionally bovine mastitis that is
encoded by a gene which differs from the "human" form by 14 nucleotides,
resulting in 9 amino acid changes. The variant toxin retains mitogenic
activity for mouse splenocytes but differs significantly in other functions
ascribed to TSST-1, including ability to induce a TSS-like disease in
rabbits (50). Second, if the rapid increase in TSS cases were caused by a
change in host character rather than by the rapid spread of a single, new,
hypervirulent clone, subclonal heterogeneity would be present among
isolates classified as ET 41. Examination of RFLP patterns for the gene
(coa) encoding coagulase has shown at least three distinct subclones of ET
41 (51).

Neisseria meningitidis

Extensive work in the last 10 years has examined the molecular population
genetics of Neisseria meningitidis, predominantly by clonal analysis, and
more recently by DNA sequencing of putative virulence genes. This work
suggests that temporal variation in disease frequency and severity is
usually associated with clonal replacement much like influenza epidemics
are driven by antigenic shift.

Serogroup B ET-5 Complex Organisms

Caugant et al. (52) demonstrated that an epidemic of serogroup B
meningococcal disease that began in the 1970s in Norway and subsequently
spread through much of Europe was caused by a group of 22 very closely
related clones, designated as the ET-5 complex, that have no close genetic
relationship to other clone groups. Clones of this complex were traced
intercontinentally to Chile and South Africa, where they also caused
contemporary outbreaks of invasive disease. Clonal analysis also showed
that a severe epidemic of meningococcal disease in Cuba (characterized by a
high attack rate and incidence of septicemia) was due to ET-5 complex
organisms. The recovery of these same bacteria from outbreaks in Miami,
Florida, in 1980 and 1981, strongly suggested that Cuban refugees imported
the clones to Miami. Members of the ET-5 complex have seldom been
recognized as important pathogens in the United States. However, ET-5
complex organisms were responsible for a recent increase in meningococcal
disease rates in Washington and Oregon (53). In addition, a serogroup B
epidemic in greater Sao Paulo, Brazil, was also caused by ET-5 complex
members (54).

Recently, clonal analysis has been used to study serogroup B meningococcal
isolates that caused invasive disease in The Netherlands between 1958 and
1986 (55). Significant temporal variation in the clonal composition of
meningococcal populations was identified. Recent disease episodes were
caused predominantly by isolates of three clonal lineages (designated I,
III, and VI) that were not represented in samples collected before 1975. In
addition, an epidemic in 1966-1967, and a hyperendemic disease wave in 1972
were caused mainly by two closely related clones (ET-11 and ET-17)
expressing serotype 2b protein. Strong statistical deviation in the sex
ratio was recorded for disease caused by clones of two lineages. Clones of
lineage V were cultured far more frequently from female than for male
patients; whereas, clones of lineage IX were recovered from disease in male
patients approximately four times more often than average. The cause(s) of
these differences are unknown but warrant further investigation.

For most bacterial pathogens, few data are available regarding the
frequency with which distinctive clones are recovered in asymptomatic
persons. Caugant et al. (56) studied the clonal composition of
meningococcal isolates cultured from the nasopharnyx of healthy carriers in
Norway and discovered that the frequency of recovery of clones (ET-5
complex and ET-37 complex) causing 80% of disease episodes were represented
by only 7% and 9%, respectively, of carrier isolates. This same study
demonstrated that the clones most commonly represented among carrier
isolates (19%) have never been recovered from patients with invasive
meningococcal infection. The data reinforce the concept that bacterial
clones vary dramatically in virulence potential.

Serogroup C Disease

An increase of invasive disease due to serogroup C N. meningitidis strains
has been reported in several countries in recent years (57-60). Study of
121 isolates recovered from patients in Greater Sao Paulo, Brazil, between
1976 and 1990 identified a striking increase in isolates assigned to ET 11
complex (58). The percentage of invasive disease episodes caused by complex
11 organisms increased from 8% in 1988 to 66% in 1990. Outbreaks of
serogroup C meningococci have also been recently reported from distinct
regions of the United States (59) and Canada (57, 60). Analysis of
organisms collected from 13 U.S. outbreaks identified five distinct
multilocus enzyme types, all very closely allied in overall chromosomal
relatedness (59). Moreover, strains causing 4 of these 13 outbreaks were
identical in multilocus enzyme type (designated ET-15) to organisms
responsible for outbreaks in eastern Canada (60). Canadian investigators
have reported (60) that ET-15 organisms had a significantly higher
case-fatality ratio than other invasive meningococcal disease isolates,
which may be due to a lower herd immunity to the newly emerged clone.

Serogroup A Disease

Unlike other serogroups of Neisseria meningitidis, which are usually
associated with endemic disease, isolates expressing serogroup A capsular
polysaccharide are unusual in that they may cause large epidemics. For
example, serogroup A organisms have been responsible for epidemics of
invasive disease in Africa, China, Iran, Greece, Finland, Brazil, and Nepal
(61). Major epidemics every 5-10 years in the Sahel region of sub-Saharan
Africa have led to the description of a "meningitis belt" and to detailed
studies by clonal analysis of the molecular epidemiology of serogroup A
organisms responsible for these and other outbreaks (62, 63).

A group led by M. Achtman assembled 423 serogroup A meningococcal isolates,
recovered primarily from invasive episodes, and representing organisms
responsible for 23 epidemics or outbreaks between 1915 and 1983.
Thirty-four distinctive clones were assigned to four complexes representing
groups of related clonal genotypes (61). Most epidemics were caused by a
single clone, and the same clone often was responsible for concurrent
epidemics in contiguous countries. For example, serogroup A clone I-1
caused a pandemic that began in North Africa and certain Mediterranean
countries in 1967 and spread throughout West Africa in the subsequent 2
years; clone III-1 has been responsible for disease outbreaks in Finland,
Brazil, Nepal, and China.

Recently Achtman's group has extensively characterized more than 300
serogroup A isolates from patients or carriers in one epidemic in The
Gambia in 1982-1983 and in 1984-1985 after an immunization program at the
end of 1983. Analysis of a representative subgroup of 64 isolates showed
that all were assigned to clone IV-1 (64, 65). Isolates of this clone were
examined for subclonal variation with SDS-PAGE profiling, LPS profiling,
and genomic restriction endonuclease profiling, and rare variants were
detected. Two cell-surface antigens (class 5 outer membrane protein and
pili) were unusually variable, and the hypothesis was formulated that
variation in the class 5 OMP occurs as a consequence of recombinational
events affecting the translational reading frame. The role, if any, of this
subclonal microheterogeneity in serogroup A meningococcal epidemics is
being assessed. Clonal analysis has provided a framework that is being
exploited to rationally select strains for further characterization by
molecular and serologic techniques that may provide insight into the forces
driving a bacterial epidemic (66-68).

Clonal analysis also has demonstrated that serogroup A isolates are a
restricted phylogenetic subpopulation of the species N. meningitidis (69).
This result may mean that the genotype bestowing the epidemic phenotype has
arisen a single time and that it has not been successfully transferred
horizontally to unrelated phylogenetic lineages of the species.

Moore et al. (70) employed clonal analysis to document the intercontinental
spread of an epidemic group A meningococcal clone complex by Muslim hajis
pilgrims in 1987. Apparently this clone was carried from South Asia (Nepal
and/or India) to Mecca, Saudi Arabia, where it was disseminated in epidemic
form to other hajis (pilgrims) and to indigenous Saudis. The report of
invasive serogroup A meningococcal disease in other Gulf nations and among
hajis returning to the United States, Europe (France and the United
Kingdom), and Africa (Ethiopia, Sudan, and Chad) and the recovery of
isolates of the same clone complex (designated ET III-1) from persons in
these diverse geographic localities strongly suggested that an unusually
virulent organism had been rapidly dispersed intercontinentally. Spread of
clone III-1 from Mecca to France by hajis was independently confirmed by
Riou et al. (71). This meningococcal clone also caused recent episodes of
invasive disease in Sweden (72) and Kenya (73).

Streptococcus pyogenes Invasive Disease

Severe invasive infections caused by S. pyogenes have been reported with
increased frequency in recent years in the United States (74, 75), Europe
(76-78), and elsewhere (79, 80). These include both soft tissue infections,
such as cellulitis, and deeper infections, including osteomyelitis,
necrotizing fasciitis, and sepsis, many of which have occurred in
previously healthy persons. The observation that many patients have
multiorgan failure and other signs and symptoms mimicking staphylococcal
toxic shock syndrome led to the characterization of a streptococcal
"toxic-shock-like syndrome" (TSLS) (81). Most S. pyogenes isolates
recovered from such patients produce one or more pyrogenic exotoxins with
significant amino acid sequence homology and functional similarity with
several enterotoxins synthesized by S. aureus (82).

To determine the genetic diversity and clonal relationships among S.
pyogenes isolates recovered from patients with TSLS or other invasive
diseases in the United States, 108 organisms were studied by multilocus
enzyme electrophoresis and analyzed for exotoxin A, B, and C synthesis
(82). The analysis showed that 33 distinctive clones were present among
isolates comprising the sample, but nearly half the disease episodes,
including more than two-thirds of the cases of TSLS, were caused by strains
of two related clones, designated ET-1 and ET-2 (82). The production of
pyrogenic exotoxin A (scarlet fever toxin, which is bacteriophage-encoded),
either alone or in combination with other pyrogenic exotoxins, was
associated with recovery in patients with TSLS. This association was
present with isolates of the same clone, as well as those of distantly
related phylogenetic lineages. The data were interpreted as strong
circumstantial evidence that scarlet fever toxin A itself, or, possibly,
the product of a gene tightly linked to it, is a factor in the pathogenesis
of TSLS.

Because an increase in disease caused by strains expressing the M1 serotype
protein had also been observed in England, Sweden, Norway, Germany, other
European countries, and elsewhere, we sought to determine if strains
recovered from these diverse localities were genetically allied.
Chromosomal diversity and relationships among 126 M1 strains from 13
countries on five continents were analyzed by multilocus enzyme
electrophoresis and restriction fragment profiling by pulsed-field gel
electrophoresis (83). All isolates were also examined for the speA gene by
PCR, and to increase the possibility of identifying interstrain variation,
strain subsets were examined by automated DNA sequencing for allelic
polymorphism in genes encoding M protein (emm), streptococcal pyrogenic
exotoxin A (speA), streptokinase (ska), pyrogenic exotoxin B (speB), and
C5a peptidase (scp). Seven distinct emm1 alleles were identified that would
express M proteins differing at one or more amino acids in the N-terminus
variable region. Although substantial levels of genetic diversity existed
among M1 organisms, most invasive episodes were caused by two subclones
marked by distinctive multilocus enzyme electrophoretic profile and PFGE
restriction fragment length polymorphism (RFLP) types. One of these
subclones (ET 1/RFLP pattern 1a) has the speA gene, and was recovered
worldwide. Identity of speA, emm1, speB, and ska alleles in virtually all
isolates of ET 1/RFLP type 1a means that these organisms have shared a
common ancestor, and that global dispersion of this M1 subclone has
occurred very recently. The occurrence of the same emm and ska allele in
strains that are well-differentiated in overall chromosomal character
demonstrated that horizontal transfer and recombination play a fundamental
role in diversifying natural populations of S. pyogenes.

The population genetic framework constructed for S. pyogenes has been
exploited to rationally choose strains for comparative molecular
characterization of the gene (speA) encoding scarlet fever toxin (84, 85).
An analysis by Nelson et al. (84) identified four alleles of speA in
natural populations, one of which (speA1) occurs in many distinct clonal
lineages and is, therefore, probably evolutionarily old. The presence of
identical exotoxin A structural genes in diverse phylogenetic lineages
means that the gene has been horizontally distributed among clones,
presumably by bacteriophage-mediated transfer. Two other alleles (speA2 and
speA3), characterized solely by single nucleotide changes resulting in
single amino acid substitutions, were each identified in single clones (ET
1 and ET 2) that together have caused most of TSLS episodes. The
restriction of speA2 and speA3 to single clonal lineages can be interpreted
as evidence that these two alleles are evolutionarily younger than speA1. A
fourth allele (speA4) also is present in a single phylogenetic lineage and
is 9% divergent from the other three toxin alleles. The absence of
synonymous (silent) nucleotide changes in speA2 and speA3 is unusual and
suggests that the allelic variation is not selectively neutral, which
implies that the toxins are not functionally equivalent. Moreover, the
mutations occur in a segment consisting of five amino acids that are highly
conserved in the aligned sequences of staphylococcal enterotoxin A (SEA),
staphylococcal enterotoxin B (SEB), SEC1, SEC3, SED, SEE, and streptococcal
pyrogenic exotoxin C (86). The segment of SPE A containing these variations
is immediately adjacent to a region containing cysteine residues involved
in the formation of a disulfide loop believed to be required for
mitogenicity of SPE A and other bacterial superantigens. Population genetic
analysis then suggests that there are functional correlates of the allelic
variation and that the alleles have been subject to natural selection.
Recent studies have shown that the ability of the SPEA2 and SPEA3 variants
to stimulate human peripheral blood mononuclear cells exceeds that of SPEA1
(87).

Mycobacterium tuberculosis

Perhaps no bacterial infection in recent years has generated as much
interest nationally as resurgent tuberculosis (TB) (88). Largely because of
the success of public health strategies, the incidence of TB declined
steadily in the United States since the early 1950s, and the disease was
thought to be eradicable by the end of the first decade of the 21st century
(89). However, the yearly decline in TB incidence ended in 1984, and after
several years of a plateau phase, resurged from 1988 through the present.
An estimated 63,000 excess cases occurred through 1993 (90). The HIV/AIDS
epidemic, immigration from countries with high TB prevalence, and outbreaks
in correctional institutions, nursing homes, shelters for the homeless, and
other congregative environments have contributed to the resurgence (88). On
a global scale, one-third of the world's population is infected with this
pathogen, and 8 million new TB cases occur each year. Moreover, nearly 3
million people die annually of TB, making it the leading cause of death due
to an infectious agent worldwide (88). Hence, there is a need to understand
the nature and extent of molecular variation in this pathogen.

Although the population genetics of M. tuberculosis has not been examined
by multilocus enzyme electrophoresis, a recent study (91) analyzed DNA
sequence diversity in eight loci (192,875 nucleotides) from unassociated
isolates recovered in North America and Europe. The data showed almost a
complete absence of coding sequence nucleotide variation. To rule out the
possibility that restricted geographic sampling biased the data set, 350-bp
fragments of genes encoding the beta subunit of RNA polymerase (rpoB), a
65-kilodalton heat shock protein (hsp65), the A subunit of DNA gyrase
(gyrA), an enzyme involved in aromatic amino acid biosynthesis (aroA), RecA
protein (recA), and a 1435-bp region of the gene (katG) encoding a
catalase-peroxidase enzyme important in isoniazid resistance (92-94) and
host-parasite interactions (95), were sequenced from one randomly selected
isolate from each of seven countries with well-differentiated human
populations (Switzerland, Turkey, Algeria, Somalia, Papua New Guinea,
Vietnam, and Tibet). A virtual lack of nucleotide variation was also found
in these seven isolates, and sequencing of several genes from many
additional TB isolates has reinforced the concept of extremely restricted
structural gene polymorphism. The paucity of sequence variation was
surprising for several reasons. First, paleopathologic evidence suggests
that humans got TB as early as 3700 BC in Egypt and 2500-1500 BC in Europe
and also pre-Columbian North and South America (96). Moreover, M.
tuberculosis DNA recovered from lung lesions in a 1000-year-old Peruvian
mummy confirmed that the disease existed in the pre-Columbian New World
(97). Second, as noted above, there is a very large global pool of infected
persons (88), and third, considerable chromosomal restriction fragment
length polymorphism has been identified by probing with mobile elements
such as IS6110 (98, 99). Based on a population genetic interpretation of
the data, it was posited that M. tuberculosis may be only 15,000 to 20,000
years old, an age that dates speciation and global dissemination to roughly
the same time as paleomigration into the New World. The time frame is also
consistent with speculation (100) that the agent of human TB arose from the
very closely related cattle pathogen M. bovis by host specialization
occurring since the domestication of this animal some 8,000 - 10,000 years
ago. Recent large-scale DNA sequencing results are also consistent with an
interpretation that M. tuberculosis and M. bovis have shared a recent
common ancestor.

These molecular population genetic findings have considerable implications
for M. tuberculosis pathobiology research. First, the virtual absence of
naturally occurring nucleotide substitutions greatly increases the
likelihood that missense mutations identified in genes associated with
resistance to antimicrobial agents actually confer resistance rather than
simply acting as convenient surrogate markers of resistance (93, 94, 101,
102). Second, restricted allelic diversity means that it is probable that
only nominal amino acid variation will occur in proteins of potential
immunoprophylaxis, diagnostic, or virulence interest.

Clones W and Son of W

Commensurate with the rise of TB cases in the United States was an increase
in the number of organisms resistant to one or more anti-TB medications
(103). This trend has been viewed with great concern by public health
authorities and clinicians, in part because no new first-line anti-TB
agents have been introduced in several decades. Certain communities have
contributed disproportionately to the documented increase is resistant
organisms, the most notable being New York City (104), which has accounted
for up to 60% of all drug-resistant M. tuberculosis reported nationally in
some surveys. Although strains with several antimicrobial agent
susceptibility patterns have been identified, approximately 300 organisms
are invariably resistant to isoniazid, streptomycin, rifampin, ethambutol,
and variably resistant to ethionamide, kanamycin, capreomycin, and
ciprofloxacin (105, 106). Early reports based on IS6110 restriction
fragment length polymorphism typing (99), other molecular techniques (98,
107), and classic epidemiologic investigations suggested that many of these
organisms were clonally related. More recent analysis using IS6110 typing,
several other molecular typing strategies, and automated DNA sequencing to
identify the exact nucleotide changes responsible for resistance to
isoniazid, rifampin, and streptomycin has unambiguously demonstrated the
existence of two abundant closely related subclones (arbitrarily named W
and W1, son of W) that have clearly shared a recent common origin (105).
Multidrug resistance in these strains is due to sequential accumulation of
amino acid substitutions conferring resistance to each drug alone, rather
than a single-step molecular event, such as acquisition of a
multidrug-resistance-conferring plasmid. Progeny of these two subclones
have now spread well beyond the New York City borders. The organisms have
been isolated from patients in other New York communities (108), Atlanta,
Miami, Denver, Las Vegas, and Paris, France (109). Thus far, all patients
documented to have infection caused by W or W1 organisms can readily be
epidemiologically connected with New York City, that is, secondary,
tertiary, or quaternary spread has not yet sufficiently obscured this
important epidemiologic thread. Dissemination of these difficult to treat W
and W1 organisms throughout New York City and other cities demonstrates the
devastating consequences of clonal origin and spread of a bacterial
pathogen. Because some persons now infected latently with W and W1 will
later experience reactivation disease, dissemination of W and W1 has
adverse implications for TB control in the 21st century.

Penicillin-Resistant Streptococcus pneumoniae

The Gram-positive bacterial pathogen S. pneumoniae is a major cause of
illness and death worldwide (110, 112). In the United States, the organism
is responsible for more than 500,000 cases of pneumonia, 55,000 episodes of
bacteremia, 6,000 cases of meningitis, and 40,000 deaths each year (113).
Until relatively recently, antibiotic resistance in S. pneumoniae was rare,
but it is now a global public health problem (114-116).

Resistance to penicillin in many organisms is due to the expression of
altered high molecular-weight penicillin-binding proteins (PBPs) that have
reduced antibiotic affinity (117). Among resistant strains, alterations in
four (1A, 2X, 2A, and 2B) of the five high molecular-weight PBPs expressed
by isolates of the species have been identified in resistant patient
isolates. Research has shown that two processes have contributed to the
rise of these organisms. First, many distinct susceptible strains are
independently evolving to the resistance phenotype. Acquisition of
penicillin binding protein gene segments from foreign donors, such as oral
streptococci, is apparently a primary driving force (118). At the molecular
level, the result is generation of mosaic genes, and thereby molecularly
remodelled PBP proteins with decreased affinity for penicillin (119-123).
Evidence shows that the Hex recombinational pathway (124) participates.
Once a distinct drug-resistant cell has been generated, progeny can be
transmitted locally and over intercontinental distances by person-to-person
spread (125-130). Multilocus enzyme electrophoresis has been applied to
analyze genetic relationships among penicillin-resistant strains of S.
pneumoniae from global sources and to infer patterns of epidemiologic
spread of these resistant organisms (125-131).

The importance of rapid local clonal spread of antibiotic-resistant S.
pneumoniae is illustrated by events in Iceland. Monitoring of antibiotic
resistance patterns of pneumococci in Iceland showed no detectable
penicillin-resistant organisms in 1983 to 1988. The first
penicillin-resistant strain was recovered in December 1988 (132). The
frequency of penicillin-resistant organisms rose sharply over the next 3
years from 2.3% to 17% of all isolates in the first quarter of 1992 (132).
Almost 70% of the resistant isolates expressed serogroup 6 capsule
polysaccharide and were also resistant to tetracycline, chloramphenicol,
erythromycin, and trimethoprim-sulfamethoxazole. To test the hypothesis
that these Icelandic isolates were clonally related, Soares et al. (128)
examined 57 organisms for serotype, PBP pattern, pulsed-field chromosomal
restriction endonuclease digestion pattern, and multilocus enzyme
electrophoretic genotype. All isolates were serotype 6B and had closely
similar or identical patterns for each of the molecular markers examined.
Surprisingly, the Icelandic organisms were indistinguishable from a
subgroup of multiresistant serotype 6B pneumococci that occurs with high
incidence in Spain. The authors concluded that the Spanish clone was
imported to Iceland and noted that in recent years a favored vacation
locality for Icelandic families with young children had been Spain. The
factors responsible for the precipitous spread of the clone in Iceland are
largely unknown. The frequency of use of beta-lactam antibiotics in Iceland
and Sweden (a country where resistant pneumococci are rare) in 1989 was
similar (133), and low compared to other industrialized countries (134).
However, Iceland has a very high use of antimicrobial agents such as
trimethoprim-sulfamethoxazole, metronidazol, and tetracycline (133, 134),
which means it is conceivable that selection for the multiresistant clone
occurred. A second possible factor contributing to the clonal spread of the
organisms is that 57% of Iceland's population of about 250,000 live in
Reykjavik and its suburbs, where most of these strains have been recovered.
Moreover, almost 80% of Icelandic children 2 to 6 years of age in Reykjavik
attend day-care centers. Together these factors may have provided a unique
set of circumstances for introduction and rapid spread of the
multiresistant clone.

Investigating the molecular population genetics of pneumococci has led to
the realization that horizontal transfer and recombinational processes are
also serving to generate variation in capsule type and immunoglobulin A1
(IgA1) protease gene alleles (135, 136). Coffey et al. (135) analyzed
European resistant strains expressing serotype 9 or 19 through a combined
approach employing clonal analysis and RFLP profiling of genes encoding
PBP1A, PBP2B, and PBP2X. Analysis of a resistant isolate synthesizing
serotype 19 capsule showed that it was identical in overall chromosomal
character to a clone of organisms resistant to multiple antibiotics which
expressed serotype 23F, a result that was interpreted as evidence that
horizontal transfer of capsular biosynthesis genes had occurred. More
recently, Lomholt (136) has shown that recombinational processes contribute
to allelic variation in the gene (iga) encoding IgA1 protease.

Methicillin-Resistant Staphylococcus aureus

Very soon after methicillin entered clinical use in the 1950s, strains of
S. aureus resistant to this antimicrobial agent were reported in the United
Kingdom (137). Within a few years, hospital outbreaks caused by
methicillin-resistant S. aureus (MRSA) occurred in Europe. MRSA were
recognized as an important hospital infection control problem in the United
States in the mid-1970s, and these organisms have now achieved global
distribution (138).

Intrinsic methicillin resistance is due to the expression of an altered
penicillin-binding protein (PBP) termed PBP 2a (139) that is encoded by the
chromosomal mec gene (140, 141). Evidence has been presented that mec
originated as a consequence of a recombinational event fusing about 300 bp
of a staphylococcal beta-lactamase gene and a segment of a gene encoding a
PBP from an unknown donor bacterium, perhaps E. coli (142).

Multilocus enzyme electrophoresis and other molecular population genetic
techniques were used to determine the extent of mec distribution among
phylogenetic lineages of the species and genetic relationships among MRSA
strains circulating in various geographic regions at different times (143,
144). The mec gene is harbored by many divergent phylogenetic lineages
representing a large portion of the breadth of chromosomal diversity in the
species S. aureus. On the basis of additional evidence, it was proposed
that multiple episodes of horizontal transfer and recombination have
contributed to the spread of the mec resistance determinant in natural
populations. The identification of a single multilocus enzyme genotype
among MRSA organisms recovered in the United Kingdom, Denmark, Switzerland,
Egypt, and Uganda, soon after the widespread introduction of methicillin
into clinical use in the 1960s, meant that MRSA isolates recovered from
those localities at that time were progeny of a single ancestral cell that
had probably acquired the mec determinant recently. The multilocus enzyme
electrophoretic data demonstrating association of mec with highly divergent
members of the S. aureus species effectively ruled out the idea (145) that
all extant MRSA are lineal descendants of a single clone, and that mec was
acquired just once by methicillin-sensitive clones of this pathogen.

Borrelia Species Associated with Lyme Disease

Molecular population genetic strategies have also been used to delineate
accurate phylogenetic relationships among emerging organisms. For example,
Boerlin et al. (146) studied 50 isolates classified as Borrelia burgdorferi
by multilocus enzyme electrophoresis and identified three distinct genetic
clusters that were well differentiated from one another in overall
chromosomal character. The investigators proposed that each cluster
represented a genospecies, and this idea was subsequently supported by
DNA-DNA reassociation studies (147), 16S rRNA gene sequencing (148),
genomic fingerprinting by arbitrarily primed polymerase chain reaction
(149), and other techniques (Valsangiacomo C, Balmelli T, Piffaretti J-C,
pers. comm.). Moreover, different genospecies of B. burgdorferi have been
associated with distinct clinical manifestations of Lyme borreliosis
(150-152).

Although population genetics has only been applied to the study of
pathogenic bacteria for approximately a decade, considerable insight has
been gained into the molecular mechanisms of temporal variation in disease
frequency and severity, host adaptation of clonal lineages, and the
relationship of disease severity and naturally occurring bacterial clones.
The work cited in this review represents only a small part of the
contribution of molecular population genetic investigations to an
understanding of temporal variation in disease frequency and severity,
microbial pathogenicity, and evolution of virulence genes. For example,
contributions have also been made from studies of Vibrio cholerae
(153-155), encapsulated H. influenzae (156), L. monocytogenes (157-158),
Salmonella spp. (159-161), and other pathogens (162).

Changes in human behavior, simple processes of microbial evolution, and
increasing resistance to antimicrobial agents will continue to supply
mankind with new infectious disease challenges and, therefore, motivation
for molecular population genetics studies. The genomes of two bacterial
pathogens have now been sequenced (163-164), and it is likely that the
genomes of most major human and veterinary viral and bacterial pathogens
will be sequenced in their entirety in the next decade. Hence, the trend
toward molecular dissection of microbial populations by large-scale DNA
sequencing will accelerate. DNA sequence-based and conventional molecular
population genetic studies are cost-effective and should be encouraged in
the fight to limit the detrimental impact of infectious agents on human,
animal, and plant health.

Acknowledgments

D. Meyer assisted in preparing the manuscript. Research in the author's
laboratory is supported by Public Health Service Grants AI-33119, AI-37004,
and DA-09238, and by awards from the Texas Advanced Technology Program and
the Moran Foundation. (The author is an Established Investigator of the
American Heart Association.)

Dr. Musser is a member of the Section of Molecular Pathobiology, Department
of Pathology, and associate professor of pathology, and microbiology and
immunology, Baylor College of Medicine, Houston, Texas. He is also
associate director, Clinical Microbiology Laboratory, and medical director,
Molecular Diagnostic Laboratory, The Methodist Hospital. He has had a
long-standing interest in elucidating the molecular forces responsible for
temporal variation in disease frequency and severity.

Address for correspondence:

James M. Musser, M.D., Ph.D.
Department of Pathology
Baylor College of Medicine
One Baylor Plaza
Houston, TX 77030, USA
Fax 713-798-4595

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SYNOPSES

(Pages 18-29)

Emergence of the Ehrlichioses as Human Health Problems

David H. Walker, M.D.
Department of Pathology, University of Texas Medical Branch at Galveston
J. Stephen Dumler, M.D.
Department of Pathology, The Johns Hopkins Medical Institutions

ABSTRACT

Ehrlichiae are small, gram-negative, obligately intracellular bacteria that
reside within a phagosome. The first human ehrlichial infection was
recognized in the United States in 1987. It was later shown to be caused by
a new species, Ehrlichia chaffeensis. In 1994, an ehrlichial pathogen
within neutrophils that is closely related to the known veterinary
pathogens E. equi and E. phagocytophila was found to infect humans.
Molecular methods were required to detect, characterize, and identify these
fastidious and uncultivated bacteria. Subsequently, E. chaffeensis
infection was documented in more than 400 patients in 30 states, Europe,
and Africa. Likewise, approximately 170 cases of human granulocytic
ehrlichiosis have been diagnosed, most since 1994, predominantly in the
upper midwestern and northeastern states, but also in northern California.
The disease caused by ehrlichiae is generally undifferentiated but is often
associated with leukopenia, thrombocytopenia, and elevated serum hepatic
transaminase levels in tick-exposed patients. Infection ranges from
subclinical to fatal; tetracycline appears to be an effective therapy. The
emergence of these two newly recognized tickborne infections as threats to
human health is probably due to increased clinical cognizance, but as in
other emerging tickborne infections, it is likely that the rapid increase
in identified cases signals a true emergence of disease associated with a
changing vector-host ecology.


During the last decade, two previously unknown human diseases caused by
Ehrlichia species have emerged as public health problems in the United
States. Each of these infectious diseases is designated by the major target
cell: human monocytic ehrlichiosis is caused by Ehrlichia chaffeensis, and
human granulocytic ehrlichiosis by an E. equi-like organism. The taxonomic
positions of these bacteria have been determined with much greater
scientific clarity by the recent application of molecular methods, which
were critical for the identification of E. chaffeensis and essential for
that of human granulocytic ehrlichia (HGE) (1,2). Extreme difficulty in
developing methods for cultivation of these ehrlichiae and, until recently,
the lack of molecular approaches to the study of uncultivated organisms
partly explain why these far from rare diseases were not detected sooner
(3-5). However, it was the observation of Romanowsky-stained blood smears
by classic microscopy that actually opened the door to the discovery of
both diseases (6,7). This article reviews ehrlichiae and describes their
taxonomic and ecologic niches within the scheme of bacterial evolution and
the substantial knowledge, developed largely by the veterinary sciences,
that served as a foundation for the discovery and elucidation of the human
ehrlichioses.

Evolution of Ehrlichiae in Unusual Ecologic Niches

Among the obligately intracellular gram-negative bacteria, a genetically
related set is classified among the Protobacteria of the subgroup on the
basis of sequence analysis of the 16S rRNA gene (1,2,8,9). The overview of
the evolution of these organisms provided by this approach lacks many
details essential to the understanding of human disease; however, the
evolution of these organisms correlates well with the clonal divergence of
many species that do not have opportunities for genetic recombination
because of their intracellular isolation from other organisms. These
bacteria have evolved in close association with ticks, mites, chiggers,
fleas, other arthropods, and fish flukes into six genetically defined
clusters (1,2,8-11) (Table 1 and Figure 1 (Figure 1 cannot be viewed in 
ASCII). Future definition of the details determined by many other 
mportant genes may designate these six clusters as separate taxonomic 
genera. Currently, however, the presence of organisms named as 
Ehrlichia species within three of these clusters is
confusing, particularly since other genus names also fall into each of the
clusters (Tables 1 and 2).

  Table 1. Six related clusters of obligately intracellular gram-negative
                bacteria determined by 16S rRNA comparisons
 --------------------------------------------------------------------------
 Prototype species          Other genera within          Vector
 of the cluster             genetic confines             relationship
 --------------------------------------------------------------------------
   I. Ehrlichia canis       Cowdria                      Ticks
  II. E. phagocytophila     Anaplasma                    Ticks
 III. E. sennetsu           Neorickettsia                Fish flukes
                            Rhinocyllus, Nasonia
  IV. Wolbachia pipientis   incompatibility              Insects
                            intracellular symbionts

   V. Rickettsia rickettsii None                         Ticks, fleas,
                                                         mites, lice
  VI. Orientia
 tsutsuga-mushi* (R.        None                         Chiggers
 tsutsugamushii)
 --------------------------------------------------------------------------
 * Proposed new taxonomic designation (20).
---------------------------------------------------------------------------

Historical Foundations for Recognizing the Human Ehrlichioses in the United
States

The current, albeit incomplete, knowledge of the human ehrlichioses has a
long history. The emergence of ehrlichiae began in 1910, when Theiler
described Anaplasma marginale, the etiologic agent of an economically
important, severe worldwide disease of cattle. The contributions of
veterinary medical science to the understanding of ehrlichiae have
continued ever since; they include the description of Cowdria ruminantium
by Cowdry in 1925, of E. canis by Donatien and Lestoquard in 1935, and E.
phagocytophila by Gordon in 1940. The genus Ehrlichia was established in
1945 in honor of the German microbiologist Paul Ehrlich, and the general
scientific distinction of rickettsiae and ehrlichiae from viruses and
protozoa followed soon thereafter, mainly as a result of the advent of
antibiotics and electron microscopy. Molecular methods are now used to
identify various species. Indeed, E. sennetsu, the etiologic agent of a
human infectious mononucleosis-like illness described in Japan in 1954, was
considered a Rickettsia for many years. Numerous veterinary discoveries
preceded the recognition of human ehrlichial infections in the United
States: Neorickettsia helminthoeca in 1950, N. elokominica in 1964, E. equi
in 1969, E. ewingii in 1971, E. platys in 1978, and E. risticii in 1984
(12). Substantial emphasis on the study of ehrlichiae was stimulated by a
disastrous epizootic of canine ehrlichiosis that resulted in 200 to 300
deaths among military working dogs in Vietnam from 1968 until 1970. After
the discovery, in 1984, that the etiologic agent of Potomac horse fever was
a novel species designated as E. risticii, major advances in the scientific
investigation of ehrlichiae have been achieved, particularly in the
laboratory of Yasuko Rikihisa at the Ohio State University College of
Veterinary Medicine. Indeed, blinded to the potential relevance of
Ehrlichia ssp. to human medicine, we missed numerous opportunities during
the 1980s to investigate canine ehrlichiosis.
 --------------------------------------------------------------------------
 Table 2. Three related clusters of bacteria containing species designated
                               as Ehrlichia
 --------------------------------------------------------------------------
                     Vertebrate    Transmission      Major target cell
                     host
 --------------------------------------------------------------------------
 Genogroup I
  Ehrlichia canis    Canids        Rhipicephalus     Monocyte/macrophage
                                   sanguineus
                                   tick bite
  E. chaffeensis     Humans, deer  Amblyomma         Monocyte/macrophage
                                   americanum
                                   tick bite
  E. ewingii         Canids        A. americanum     Polymorphonuclear
                                   tick bite         leukocytes
  E. muris           Vole          Unknown           Monocyte/macrophage
  Cowdria            Cattle,       Amblyomma spp.    Endothelium
  ruminantium        sheep, goats, tick bite
                     antelope,
                     African
                     buffalo
 Genogroup II
  E. equi            Horses        Ixodes spp.       Neutrophils
                                   tick bite
  E. phagocytophila  Sheep,        Ixodes ricinus    Neutrophils
                     cattle,       tick bite
                     deer
  Human              Humans, deer, I. scapularis     Neutrophils
  granulocytic       rodents (?)   (dammini)
  ehrlichia                        tick bite
  E. platys          Canids        Unknown           Platelets
  Anaplasma          Cattle        Boophilus,        Erythrocytes
  marginale                        Rhipicephalus,
                                   and other
                                   tick bites
 Genogroup III
  E. sennetsu        Humans        Possibly          Monocyte/macrophage
                                   ingestion
                                   of raw fish
  E. risticii        Horses        Unknown           Monocytes,
                                                     enterocytes, mast
                                                     cells
  Neorickettsia      Canids        Salmon fluke      Macrophage
  helminthoeca                     ingestion
  N. elokominica     Canids        Salmon fluke      Macrophage
                                   ingestion
  Stellantchasmus    None known    Grey mullet       Unknown
  falcatus (SF)                    fluke
  agent
 --------------------------------------------------------------------------

Emergence of Human Monocytic Ehrlichiosis

A single, somewhat unusual case in 1986 led to the recognition of human
ehrlichial infections in the United States (6). A 51-year-old man who had
been bitten by a tick in Arkansas and had been sick for 5 days was admitted
to a hospital in Detroit. He was critically ill with fever, headache,
myalgia, confusion, azotemia, hypoxemia, thrombocytopenia and had
cytoplasmic inclusions in his peripheral blood leukocytes. Peripheral blood
smears and electron micrographs of the inclusion-bearing leukocytes
examined at the Centers for Disease Control (CDC) suggested that the
organisms were an Ehrlichia sp. Serologic evaluation showed a high titer of
antibodies reactive with E. canis that fell sharply during convalescence.
The patient had a prolonged hospitalization complicated by renal failure
which required hemodialysis; upper gastrointestinal hemorrhage; severe
central nervous system involvement; and systemic Candida infection.

State public health agencies and CDC demonstrated that many patients
originally suspected to have Rocky Mountain spotted fever or another
rickettsiosis (13) produced antibody to E. canis, a canine pathogen. In
Oklahoma, the state with the highest incidence of Rocky Mountain spotted
fever, serologic evidence suggested that human monocytic ehrlichiosis
occurred at the same incidence as Rocky Mountain spotted fever (14). In a
prospective active surveillance study of febrile hospitalized patients in
southeastern Georgia, ehrlichial disease was sixfold more prevalent than
Rocky Mountain spotted fever and was associated with history of tick bite,
anorexia, chills, weight loss, sweating, headache, nausea, myalgia,
thrombocytopenia, and elevated serum concentrations of hepatic
transaminases in 50% or more of the patients and by arthralgia, vomiting,
diarrhea, abdominal pain, cough, rash, and leukopenia in fewer than half of
the patients (15). Mild and asymptomatic seroconversion to ehrlichial
antigens has also been associated with tick exposure (16).

A practical method for the cultivation of E. canis (the suspected cause of
human ehrlichial infections), developed at CDC, used the cultivated
organisms to establish a reliable indirect immunofluorescence assay (IFA)
for the diagnostic demonstration of antibodies in serum (17). In
retrospect, it is apparent that E. canis was a relatively sensitive
surrogate antigen because it shares some, but by no means all, of the
antigens of the etiologic Ehrlichia. The method of ehrlichial cultivation
in a continuous canine histiocytoma cell line (DH82) was also successfully
applied to the isolation of an Ehrlichia species from the blood of a
febrile soldier at Fort Chaffee, Arkansas (3). Molecular methods were also
applied to establish, on the basis of the DNA sequence of the 16S rRNA
gene, that E. chaffeensis is a novel species (1).

Current Knowledge of E. chaffeensis and Human Monocytic Ehrlichiosis

Microbiology of the Etiologic Agent

Ultrastructural investigation of E. chaffeensis demonstrated that the inner
and outer leaflets of the cell wall were equal in thickness. Rickettsia
species have a thinner outer leaflet and thicker inner leaflet, and
Orientia (Rickettsia) tsutsugamushi has a thicker outer leaflet and thinner
inner leaflet (18-20) (Figures 2 and 3 cannot be viewed in ASCII). Two distinct morphologic forms,
larger reticulate cells with uniformly dispersed nucleoid filaments and
ribosomes and smaller cells with central condensation of nucleoid filaments
and ribosomes, were observed undergoing binary fission, evidence against a
developmental cycle (Figure 3). Many forms were observed, including
numerous intramorular vesicles and tubules originating from the ehrlichial
cell wall and fibrillar material consisting of ehrlichial antigen. Abnormal
forms included giant and multilobar ehrlichiae.

Western immunoblot technique has detected seven major proteins: 120, 66,
58, 44, 29, 28, and 22 kDa; the greatest number of antigens are shared with
E. canis (21). Monoclonal antibodies and monospecific polyclonal antibodies
demonstrated that the major, immunodominant 120-, 29-, 28-, and 22-kDa
proteins (as well as a minor 30-kDa protein) are surface-exposed and that
the 28- and 22-kDa proteins are related antigenically. Further human
isolations of ehrlichiae have shown antigenic and genetic diversity among
strains of E. chaffeensis (4).

DNA cloning has shown that 58- and 10-kDa proteins are genetically
homologous to the Escherichia coli GroEL and GroES heat shock proteins
(22). The 120-kDa protein includes a region of identical 80 amino acid
tandem repeat units, and preliminary evidence suggests that it mediates
adhesion to the host cell. Establishment and study of tick isolates and
identification of virulence factors are clearly important aims for future
research.


Clinical Manifestations

Most cases of human ehrlichiosis have been diagnosed after a
moderate-to-severe illness (23). Some patients have a life-threatening
illness resembling toxic shock syndrome (24). Deaths have occurred in
approximately 2% to 3% of patients, including previously healthy children
(13,23-28). Among 237 cases between 1985 and 1990 investigated by CDC, 62%
of patients were hospitalized (23). The median duration of illness,
including that for treated patients, was 23 days. The signs and symptoms
depict a systemic disease that has no clinically diagnostic features: fever
(97%), headache (81%), myalgia (68%), anorexia (66%), nausea (48%),
vomiting (37%), rash (6% at onset, 25% during the first week, and 36%
overall), cough (26%), pharyngitis (26%), diarrhea (25%), lymphadenopathy
(25%), abdominal pain (22%), and confusion (20%). Severe complications
include respiratory and renal insufficiency and serious neurologic
involvement. Of patients with chest radiographic examinations, nearly half
have pulmonary infiltrates (13). Clinical laboratory findings include
leukopenia (60%), thrombocytopenia (68%), and elevated hepatic
transaminases (86%). Hepatic involvement has been described as severe in
individual cases (29). Central nervous system involvement has been
documented by the occurrence of seizures, coma, and cerebral lesions at
autopsy as well as by cerebrospinal fluid (CSF) pleocytosis, increased CSF
protein concentration, and the presence of E. chaffeensis in CSF
demonstrated by immunocytology and polymerase chain reaction (PCR)
(24,30,31). Recognition of serious myocardial involvement further
emphasizes the potential gravity of this disease and the incompleteness of
our knowledge of its clinical manifestations (32). Seroconversion to E.
chaffeensis among 1.3% of 1,187 soldiers during training exercises with
tick exposures suggests that human risk for infection with it or an
antigenically related Ehrlichia is relatively high (16). However, the fact
that two-thirds of the seroconverters remained asymptomatic emphasizes that
the relationship between severity of illness and either host factors or
ehrlichial strain differences in virulence remains unknown.

Epidemiology of Human Monocytic Ehrlichiosis and Ecology of E. chaffeensis

More than 400 cases of serologically confirmed E. chaffeensis infection
have been documented at CDC, which has one of the few laboratories capable
of performing diagnostic ehrlichial serology. That these cases most likely
represent but the tip of the iceberg was confirmed when inquiry at MRL
Diagnostics, a commercial reference laboratory that offers IFA serology for
E. chaffeensis, reported 722 positive specimens between September 1992 and
June 15, 1995. In fact, there is no system, required or otherwise, for
notifying public health authorities of cases. Relatively few physicians
even know that these diseases exist and, thus, most of them are not making
the diagnosis. Even highly knowledgeable physicians find ehrlichiosis
virtually impossible to diagnose on the basis of clinical signs and
symptoms. Most patients (83%) report exposure to ticks or tick bite within
the 3 weeks of onset of illness (13,23). Cases are predominantly rural
(66%) and seasonal (68% during May-July). The median age of patients is 44
years, and three-quarters are male. Outbreaks of human monocytic
ehrlichiosis among groups of golfers and campers emphasize the risk for
infection during outdoor activities with tick exposure (16,33). Human
monocytic ehrlichiosis occurs not only as an acute illness of apparently
immunocompetent persons but also as an opportunistic infection of patients
with compromised host defenses, including acquired immunodeficiency
syndrome (AIDS) patients (24,26). The report of a fatal E. chaffeensis
infection in an AIDS patient, in whom a diagnostic antibody response to E.
chaffeensis never developed, suggests that such cases would not usually be
diagnosed correctly.


E. chaffeensis has been detected in two tick species, Amblyomma americanum
(the lone star tick) and Dermacentor variabilis (34,35) (the American dog
tick). Human monocytic ehrlichiosis has been confirmed in 30 states. Most
cases have occurred within the range of A. americanum, and a high
proportion of the remainder have occurred within the range of D.
variabilis. Ehrlichiae have been detected specifically by PCR in adult (but
not in nymphs) A. americanum ticks in Missouri, North Carolina, Kentucky,
and New Jersey; a single E. chaffeensis PCR-positive D. variabilis tick was
found in Arkansas. On the other hand, cases reported in Wyoming, Utah,
Washington, Europe, and Africa suggest the possibility of additional
vectors (23,36,37) or other antigenically related ehrlichial organisms.

White-tailed deer (Odocoileus virginianus) in Alabama, Arkansas, Florida,
Georgia, Illinois, Kentucky, Louisiana, Maryland, Mississippi, Missouri,
North Carolina, South Carolina, Tennessee, Texas, and Virginia have
antibodies reactive with E. chaffeensis. These deer are susceptible to
experimental infection with ehrlichiae that can circulate for weeks
(38,39). The close association among E. chaffeensis, the lone star tick,
and white-tailed deer was vividly illustrated by the sequential appearance
of A. americanum ticks and antibodies to E. chaffeensis in a population of
deer in Georgia (40). The prevalence of lone star ticks and seropositivity
both rose from 1983 to 1990 when 100% of deer examined were infested with
A. americanum and had serum antibodies to E. chaffeensis. The possibility
that the immature ticks acquired the ehrlichiae from another mammalian
host, such as a small rodent, before it was transmitted to deer cannot be
excluded. Experimentally infected dogs have ehrlichemia for at least 26
days and, thus, could also serve as a reservoir host (41). Related
ehrlichioses for which the ecology and transmission are known are
maintained in a cycle involving a mammalian host and a tick vector (12).
For example, Rhipicephalus sanguineus ticks acquire E. canis infection when
feeding on infected dogs as larvae or nymphs (42,43). Although the ticks
remain infected as they molt from stage to stage, transovarian transmission
does not occur. It seems likely that deer, dogs, or small rodents serve as
the reservoir hosts for E. chaffeensis, and that A.americanum ticks serve
as the major vector. Indeed, larval and nymphal A. americanum acquire E.
chaffeensis by feeding on infected deer, maintain the ehrlichiae
transstadially, and transmit E. chaffeensis while feeding as nymphs and
adults on naive deer (44).

Diagnosis and Treatment

The diagnosis of human monocytic ehrlichiosis is usually difficult to
establish during the acute stage of the infection, even in severe cases.
Ehrlichiosis should be considered in any febrile patient who has been
exposed to ticks during the previous 3 weeks, particularly if leukopenia
and thrombocytopenia are present.

Most cases have been confirmed by a humoral immune response generating
antibodies reactive with E. chaffeensis or the surrogate antigen, E. canis
(17,23). Use of E. chaffeensis antigens for IFA serology results in greater
diagnostic sensitivity than use of E. canis antigens (3). Diagnostic rises
in antibody titer usually occur by the third week after onset, and a
precipitous decline in antibody occurs in most patients during the
following year. A fourfold rise or fall in IFA titer with a peak titer of
64 or greater is considered diagnostic in a clinically compatible case.
Only two cases have been documented by ehrlichial isolation (3,4),
approximately 30 cases by E. chaffeensis-specific PCR (31,34,45), and even
fewer cases by immunohistology or immunocytology (25-28,30,46).
Furthermore, antibodies to E. chaffeensis did not develop in six
PCR-confirmed patients during convalescence, suggesting that serologic
testing may be less sensitive than generally assumed (31,45). The
sensitivity of PCR seems to be 80% to 87%; the specificity depends
critically upon avoiding contamination with ehrlichial DNA. Search for
morulae of E. chaffeensis in leukocytes is unrewarding. Even an exhaustive
search of buffy coat smears seldom yields a diagnostic result.

Retrospective analysis showed that treatment with tetracycline was
associated with reduced need for hospitalization of patients and with the
shortest median duration of treatment to effect defervescence for
hospitalized patients (2 days as compared with 3 days for chloramphenicol
and 7 days for all other antibiotics) (23). E. chaffeensis is killed in
cell culture in the presence of doxycycline or rifampin but is resistant to
chloramphenicol, ciprofloxacin, erythromycin, cotrimoxazole gentamicin, and
penicillin (47). E. chaffeensis can establish persistent infection even
after treatment with tetracycline and chloramphenicol (25). Persistent
ehrlichial infection, in some instances even after treatment, is a
well-documented aspect of the veterinary ehrlichioses, e.g., canine
monocytic ehrlichiosis (E. canis) and tick-borne fever (E. phagocytophila).
The long-term implications of persistent ehrlichiosis in humans are unclear
but might include subsequent reactivation of infection or altered host
defenses.

Pathology, Pathogenesis, and Immunity

E. chaffeensis is introduced into the dermis by the bite of an infected
tick and spreads hematogenously throughout the body. Intracellular
infection is established within phagosomes, most often in macrophages in
the spleen, liver, lymph nodes, bone marrow, lung, kidney, and
cerebrospinal fluid (25-28,30,46). Lesions potentially attributable to
ehrlichial infection include focal necroses of the liver, spleen, and lymph
nodes; multiorgan perivascular lymphohistocytic infiltrates;
hemophagocytosis in the spleen, liver, lymph nodes, and bone marrow;
interstitial pneumonitis; and pulmonary hemorrhage. In bone marrow
specimens from 12 patients, the most important findings related to the
hematopoietic response were myeloid hyperplasia (8 cases), myeloid
hypoplasia (1 case), pancellular hypoplasia (1 case), and megakaryocytosis
(7 cases) (46). The most striking discovery was the frequent occurrence of
granulomas (in 8 cases) and marrow histiocytosis (in 1 case) as
manifestations of the reaction of macrophages to this organism.

The pathogenic mechanisms of ehrlichial disease are poorly understood. E.
chaffeensis directly causes necrosis of heavily infected cells in vitro and
in immunocompromised patients (7,26); however; the role of host immune and
inflammatory responses as disease mechanisms has yet to be determined.
Observations of opportunistic fungal and viral infections in severe and
fatal cases suggest the possibility of an ehrlichial role in the
suppression or dysregulation of the immune response (6,25).

It is quite likely that E. chaffeensis is controlled by a combination of
cell-mediated and humoral immune mechanisms. Interferon-activated human
monocytes kill E. chaffeensis in vitro. The ehrlichicidal activity is
reversed by holotransferrin, suggesting that E. chaffeensis is inhibited by
intracellular iron depletion (48). The closely related E. canis organisms,
which grow in canine macrophages in the presence of normal canine serum and
cause macrophage necrosis, are killed by canine macrophages when the
ehrlichiae are opsonized by immune serum (49,50). Among patients who are
treated successfully with an anti-ehrlichial drug, the disease-associated
lymphocytopenia is corrected and within 2 to 3 days lymphocytosis develops
(51). The predominant (range, 41% to 97%) lymphocyte population comprises T
lymphocytes, cells that usually constitute only 3% to 8% of peripheral
lymphocytes at the institution where the patients were studied. The
function of these cells in this setting is unclear as are the consequences
of the low T-lymphocyte concentration.

Emergence of Human Granulocytic Ehrlichiosis

Human granulocytic ehrlichiosis was recognized originally in Duluth,
Minnesota, by Johan Bakken as a clinical syndrome of a potentially fatal
febrile illness in which the patient's neutrophils contained cytoplasmic
inclusions (7). Upon reading an update on human ehrlichioses at the time of
the index case, which had a dramatic fatal course, Bakken hypothesized that
the infection might have been ehrlichiosis. Collaboration with the authors,
who were working together at the University of Texas Medical Branch at
Galveston, resulted in the evaluation of a series of patients who were
suspected by Bakken to have the disease. Cytoplasmic inclusions were
observed in neutrophils, which differed from the findings in E. chaffeensis
infections, in which the monocyte/macrophage is the principal target cell
and the detection of circulating leukocytes with inclusions is a rare
event. Moreover, IFA tests for antibodies to E. chaffeensis performed in
the Texas laboratory and subsequently in Dumler's laboratory at the
University of Maryland were uniformly negative (4).

In retrospect, June 18, 1992, was a significant day in the history of human
granulocytic ehrlichiosis. It was Dumler's last day as a fellow in
Galveston before moving to Baltimore to become an assistant professor and
establish his own independent ehrlichial research laboratory, and it was
Sheng-min Chen's first day as a fellow. Momentously, the blood of a
78-year-old Wisconsin man was collected and sent to Galveston by Bakken on
the same day. Culture for ehrlichiae and acute- and convalescent-phase
serologic assays for chaffeensis, E. canis, E. sennetsu, and E. risticii
were all negative. PCR amplification of the 16S rDNA, the approach
developed by Wilson (52) and applied by Relman and co-workers to identify
the etiologic agent of bacillary angiomatosis (53), was performed
successfully on the specimen by Chen 3 months later (2). Sequencing the
gene was not a high priority compared with pursuit of the research aims of
funded projects during the season when the ticks were expected to be less
active. By April of 1993, DNA sequencing of half the 16S rDNA had been
accomplished, and it was recognized that the organism was most closely
related to E. phagocytophila and E. equi, closely related to E. platys,
less closely related to E. chaffeensis, and distantly related to E.
sennetsu. Completion and repeated confirmation of sequencing of both sense
and antisense strands enabled genogroup-specific primers to be designed for
nested PCR in July 1993.

What had progressed as a collaborative project became the major project in
Dumler's laboratory as Chen and Walker refocused their efforts on funded E.
chaffeensis research. The initial joint publications with Bakken documented
12 cases diagnosed by specific PCR and the identification of morulae in
circulating neutrophils (7). Two patients had died. Necropsy performed on
one patient showed ultrastructurally and immunohistologically identified
ehrlichiae in neutrophil phagosomes in the spleen. Convalescent-phase IFA
serology with surrogate antigens harvested from the blood of an E.
equi-infected horse and E. phagocytophila-infected sheep demonstrated
antibodies in 9 of the 10 survivors (7). The illness was characterized by
chills, fever, myalgias, and headache; some patients also had nausea,
confusion, cough, and arthralgias. Laboratory data included leukopenia in
50%, neutropenia in 17%, lymphopenia in 17%, anemia in 50%,
thrombocytopenia in 92%, and elevated aspartate aminotransferase in 91%.
Patients were predominantly older men (mean age, 68 years). Clinical
history showed strong association with tick bite preceding the onset of
illness.

Current Status of Human Granulocytic Ehrlichia and Human Granulocytic
Ehrlichiosis

Microbiology of Human Granulocytic Ehrlichia and E. equi

Evidence is accumulating to support the premise that a single Ehrlichia
species is the etiologic agent of a granulocytotropic ehrlichiosis of
humans, horses, and dogs. The DNA sequences of the 16S rDNA from the
peripheral blood of naturally infected horses and dogs in Sweden, dogs in
Minnesota and Wisconsin, and horses in Connecticut are identical with the
HGE and differ slightly from the published 16S rDNA sequence for E. equi
(2,54,55, and J. E. Madigan, J. E. Barlough, J. S. Dumler, N. S. Schankman,
E. DeRock, unpublished observations). Moreover, when infected human blood
from HGE patients is injected into horses, HGE develops, can be serially
transmitted to other horses, and induces protection against subsequent E.
equi challenge (56). Also, naturally occurring canine E. equi infections
have been transmitted to horses that had developed equine granulocytic
ehrlichiosis, and E. equi has been transmitted experimentally by injecting
equine blood into susceptible dogs (57,58). Reproducible, continuous in
vitro cultivation of all species of granulocytotropic ehrlichiae, including
E. ewingii and E. equi, is an achievable goal.

E. equi antigens harvested from the blood of horses with high levels of
parasitemia have been examined for reactivity with convalescent-phase human
(HGE), equine (E. equi), canine (E. equi) and bovine (E. phagocytophila)
sera by Western immunoblotting (59). The antigens judged most specific for
E. equi were the 100-, 44-, 42-, and 25 kDa-bands. The most specific
antigen, the 44-kDa band, was strongly reactive with the convalescent-phase
human, equine, canine, and bovine sera, suggesting a specific antigenic
relationship among these organisms from different animals and geographic
origins. On the other hand, biologic studies of E. phagocytophila in
Finland have shown apparent strain differences in virulence and lack of
immunity to heterologous challenge (60). The molecular basis for these
differences and the actual taxonomic relationships of HGE, E. equi, and E.
phagocytophila remain to be determined.

Clinical Manifestations

A study of 41 cases of laboratory-confirmed human granulocytic ehrlichiosis
from Minnesota and Wisconsin showed male predominance (78%), a median age
of 59 years (range 6 to 91 years), median incubation period of 8 days,
median period of fever and other symptoms before initiation of effective
5-day treatment, and year-round occurrence with a peak in June and July
(61). Clinical manifestations included fever (100%), chills (98%), malaise
(98%), myalgias (98%), headaches (85%), nausea (39%), vomiting (34%), cough
(29%), confusion (17%), and rarely rash (2%). Four patients had pulmonary
infiltrates visible on roentgenograms. The severity of illness is reflected
in the rates of hospitalization (56%), admission to an intensive care unit
(7%), and death (5%). The course of illness in patients who were not
treated and yet survived included a 10-day febrile course in a child and 3-
to 11-week remittent febrile course in adults (61).

Diagnosis and Treatment

A clinical diagnosis of human granulocytic ehrlichiosis should be
considered in patients exposed to an Ixodes scapularis (dammini), I.
pacificus or I. ricinus tick-infested environment who have a flulike
febrile illness. Careful examination of a peripheral blood smear may show
neutrophils that contain cytoplasmic vacuoles filled with ehrlichiae. Two
different sets of PCR primers based upon the 16S rDNA sequence of HGE have
been designed to amplify, detect, and identify HGE in the patient's blood
during the acute stage of illness (2,62). Continued improvements in the
technology are expected to yield highly sensitive and specific results in
any well-managed clinical molecular diagnostics laboratory. Serologic
diagnosis by IFA employing E. equi-infected neutrophils harvested from the
blood of infected horses detects antibodies at a diagnostic titer of 80 or
greater in the convalescent-phase sera of 100% of patients, but antibodies
are usually not present in sera collected early in the illness.

Among 34 patients treated with doxycycline, 97% defervesced within 2 days.
One patient who had not been treated with doxycycline had E. equi
detectable by PCR in the blood 28 days after onset of symptoms.

Pathology, Pathogenesis, and Immunity

Following presumed injection of HGE into the patient's skin by the bite of
Ixodes ssp. ticks, virtually none of the subsequent events are known. It is
suspected that HGE infects a myeloid precursor in the bone marrow rather
than mature neutrophils. Bone marrow examinations demonstrated
hypercellularity in two patients and normocellular marrow in another
patient. Autopsies of three patients who died of HGE showed opportunistic
fungal pneumonia caused by a different agent (Aspergillus fumigatus,
Cryptococcus neoformans, and Candida albicans) in each patient, suggesting
altered host defenses (7,63). One of the patients who died had severe
herpes esophagitis. Co-infection with HGE and B. burgdorferi is suspected
to result in more severe disease. Examples from veterinary research
indicating that E. phagocytophila and E. equi suppress the host defenses
include opportunistic viral and fungal infections, decreased neutrophil
adherence, emigration, phagocytosis, and bacterial killing, decreased
production of antibodies, and decreased lymphocyte mitogenesis (64,65). The
mechanisms by which ehrlichiae impair the host phagocytic and immune
responses are not known.

Epidemiology and Ecology

Human granulocytic ehrlichiosis has been diagnosed in patients in
Minnesota, Wisconsin, Massachusetts, Connecticut, New York, Rhode Island,
Pennsylvania, Maryland, Florida, Arkansas, and California (7,63,66,67).
Serologic evidence suggests that HGE or an antigenically related organism
has also infected patients with Lyme borreliosis in Switzerland (68). An
organism apparently identical to HGE is present in Sweden (54). There is
compelling evidence that I. pacificus, the vector of Lyme borreliosis in
northern California, transmits E. equi to horses (69). Ten percent of I.
scapularis (dammini) ticks collected from vegetation in northwestern
Wisconsin in 1982 and 1991 were infected with HGE, including two specimens
containing both HGE and Borrelia burgdorferi. An engorged I. scapularis
(dammini) tick was removed from a patient with human granulocytic
ehrlichiosis in the same geographic area. PCR showed that the tick's
salivary glands contained DNA of HGE (62). Similarly, PCR-amplified HGE DNA
was detected in 50% of I. scapularis (dammini) ticks collected in
Connecticut; no E. chaffeensis was detected in these ticks (70). Although
the demonstration by PCR that blood from a high proportion of deer in a
study in Wisconsin contained HGE suggests that deer might be an important
reservoir, the possibility of a rodent reservoir should also be
investigated. Indeed, the illustrations in a 1938 Tyzzer article suggest
that Microtus pennsylvanicus and Peromyscus leucopus are naturally infected
with a granulocytotropic ehrlichia (71).

Acknowledgments

The authors express their gratitude to Dr. Vsevolod Popov for providing the
excellent electron micrographs and to Ms. Josie Ramirez and Ms. Kay
Kantowski for expert secretarial assistance in preparing the manuscript.
This research was supported by a grant from the National Institute of
Allergy and Infectious Diseases (AI31431).

Dr. Walker, professor and chair, Department of Pathology, and director,
Center for Tropical Diseases, University of Texas Medical Branch at
Galveston, has focused his biomedical research on rickettsial diseases
during the last 22 years. Particular efforts have been expended in his
laboratory since 1988 on the study of the agents and diseases of human
ehrlichial infections. His efforts have contributed to the characterization
of Rickettsia japonica, Ehrlichia chaffeensis, and R. felis as agents of
emerging infectious diseases. The agent causing human granulocytic
ehrlichiosis was first identified in his laboratory in Texas.

Dr. Dumler, assistant professor of pathology at the Johns Hopkins Medical
Institutions, is the key scientist and physician in the detection,
identification, and characterization of human granulocytic ehrlichiosis and
its etiologic agent. A major contributor to the understanding of Ehrlichia
chaffeensis, the human E. equi-like agent, and the diseases that they
cause, Dr. Dumler has authored or coauthored 25 articles on these topics.

Address for correspondence:

David H. Walker, M.D.
Department of Pathology
University of Texas Medical Branch
301 University Boulevard
Galveston, TX 77555-0609, USA
Fax: 409-772-2500

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(Pages 30-36)

Surveillance for Pneumonic Plague in the United States During an
International Emergency: A Model for Control of Imported Emerging Diseases

Curtis L. Fritz, D.V.M., Ph.D., David T. Dennis, M.D., M.P.H.,
Margaret A. Tipple, M.D., Grant L. Campbell, M.D., Ph.D.,
Charles R. McCance, B.A., and Duane J. Gubler, Sc.D.
Centers for Disease Control and Prevention,
Fort Collins, Colorado, and Atlanta, Georgia, USA

ABSTRACT

In September 1994, in response to a reported epidemic of plague in India,
the Centers for Disease Control and Prevention (CDC) enhanced surveillance
in the United States for imported pneumonic plague. Plague information
materials were rapidly developed and distributed to U.S. public health
officials by electronic mail, facsimile, and expedited publication.
Information was also provided to medical practitioners and the public by
recorded telephone messages and facsimile transmission. Existing quarantine
protocols were modified to effect active surveillance for imported plague
cases at U.S. airports. Private physicians and state and local health
departments were relied on in a passive surveillance system to identify
travelers with suspected plague not detected at airports. From September 27
to October 27, the surveillance system identified 13 persons with suspected
plague; no case was confirmed. This coordinated response to an
international health emergency may serve as a model for detecting other
emerging diseases and preventing their importation.


In the past 50 years, the speed of international travel, as well as the
number of travelers, has accelerated, providing a mechanism for the rapid
dissemination of disease agents from one country to another. For this
reason, vigilant surveillance is needed to prevent the importation and
spread of emergent infections. The United States needs a response plan that
involves international and domestic public health officials, physicians and
hospitals, and the public and can be implemented at the first indication of
an international health threat.

In 1994, in response to an epidemic of pneumonic plague in India, the
Centers for Disease Control and Prevention (CDC) developed and implemented
an enhanced surveillance system to supplement the existing regulations
concerning imported plague. The protocol described here may serve as a
model for detection and control of emerging diseases imported into the
United States or other countries with frequent and diverse international
traffic.

Background

In September 1994, India reported cases of plague for the first time in 28
years. Plague is caused by infection with the bacterium Yersinia pestis.
Bubonic plague is typically acquired by the bite of fleas from infected
rodents and is characterized by inguinal, axillary, and/or cervical
lymphadenitis. Pneumonic plague may occur as a secondary development to the
bubonic form or can be contracted by inhaling respiratory droplets from
humans or other animals with plague pneumonia(1).

Bubonic plague cases were first identified by Indian health officials in
the Beed District of Maharashtra State in late August. By September 24,
more than 300 unconfirmed cases of pneumonic plague and 36 deaths had been
reported from the city of Surat, Gujurat State, approximately 300 km west
of the Beed District (3). After these reports, hundreds of thousands of
Surat's two million residents fled, some to the major cities of Bombay,
Calcutta, and New Delhi (4). Unconfirmed pneumonic plague cases and
plague-related deaths were subsequently reported from several areas
throughout India (5,6).

With the reported epidemic of plague in India, the potential for spread of
the disease by infected travelers became a concern. Several countries
closed their borders to Indian travelers and cargo and discontinued all
flights of their air carriers to and from India (7). Because of its
epidemic potential, plague is listed as a Class 1 internationally
quarantinable disease in the International Health Regulations of the World
Health Organization (WHO) (2). These regulations authorize the detention
and inspection of any vehicle or passenger originating in an area where a
plague epidemic is in progress.

Response to the Epidemic

CDC's domestic response to the apparent plague epidemic in India involved
two simultaneous and complementary components: 1) information dissemination
and education, and 2) intensified active and passive surveillance to
identify and treat suspected plague patients and their contacts.

Information Dissemination

After the initial reports from India, information on plague and the
epidemic in India was urgently sought by the media, the public, medical
practitioners, and public health officials throughout the United States. To
meet this need, CDC circulated detailed and timely information to persons
concerned with the potential plague crisis. From September 26 to 29, CDC
produced six documents for distribution to public health officials and
agencies: 1) a general plague outbreak notice, 2) a plague alert notice for
international travelers from India, 3) a plague advisory for persons
traveling to India, 4) plague treatment and prophylaxis guidelines for
physicians, 5) guidelines for diagnosis and biosafety for persons handling
samples from patients with suspected plague, and 6) an article on the
Indian outbreak that appeared in CDC's widely circulated Morbidity and
Mortality Weekly Report (MMWR)(8).

CDC pursued several avenues to convey information to medical practitioners
and the public. Three articles on the epidemic were published in MMWR
(September 30, October 7, and October 21) (8-10). Information on plague in
general and the Indian epidemic in particular was made available on CDC's
Voice Information Service, Fax Information Service, and a special plague
hotline telephone number. A message intended for travelers to India
concerning the perceived risks and appropriate prophylactic measures was
added to the plague selections on the Voice Information Service menu.
Finally, all airline passengers disembarking in the United States from
India were given a plague alert notice that described the symptoms of
plague and advised them to seek medical attention and notify state and
federal public health authorities if they had any febrile illness within
the next 7 days. The standard Health Alert Notice (yellow card) of the
Division of Quarantine, CDC, was made available to all other international
arriving passengers and advised them of appropriate measures in the event
of illness.

Surveillance

The second component of CDC's response was to intensify active and passive
surveillance for persons entering the United States who potentially had
plague. Both the active (Figure 1 cannot be viewed in ASCII) and passive 
Figure 2 cannot be viewed in ASCII) surveillance systems identified not 
only persons suspected of having plague but also
those who might have been exposed to a patient with plague during the
contagious period.


Active Surveillance System

CDC's Division of Quarantine maintains staff at major international
airports in seven U.S. cities: Honolulu, Hawaii; Seattle, Washington; San
Francisco and Los Angeles, California; Chicago, Illinois; Miami, Florida;
and New York, New York. At airports where the division does not have staff,
officials of the Immigration and Naturalization Service (INS), Division of
Quarantine contract physicians, or both, serve as Quarantine Officers.

During the plague epidemic, crews on all commercial aircraft originating in
or continuing from India were reminded of the regulations requiring them to
notify the Quarantine Officer at the destination airport of any ill
passengers and were instructed to be especially alert for passengers with
fever, cough, or chills. When the aircraft landed, before passengers
disembarked, a Quarantine Officer and a Division of Quarantine contract
physician, in telephone consultation with the medical officer on call at
CDC's Division of Vector-Borne Infectious Diseases, examined any passenger
who reported illness and determined whether the suspicion of plague was
sufficient to warrant the passenger's hospitalization and further
evaluation. If deemed not likely to have plague, the passenger was placed
under the surveillance of the local health department and released with
instructions to consult a physician and to monitor his or her temperature
for the next 7 days, the maximal incubation period for pneumonic plague
after exposure (1). All other passengers were permitted to deplane and were
given a copy of the plague alert notice.

If plague had not beeen ruled out as a possible cause of the passenger's
illness, the passenger would have been considered a patient with suspected
plague and would have been placed in isolation at the airport until he or
she could be safely transported to a predetermined hospital. In the
hospital, the patient would have been placed under respiratory isolation
conditions, diagnostic specimens would have been obtained for testing in
the CDC plague laboratory, and appropriate antibiotic treatment for plague
would have been begun.

If the patient had been hospitalized, other passengers on the flight would
have been informed that they were under surveillance in accordance with
federal quarantine regulations. Locating information would have been
obtained from all passengers, who would have been instructed to monitor
their body temperature for 7 days and to report any illness to their county
or state health department. Because pneumonic plague is transmitted from
person to person through respiratory droplets (11) and air flow on
passenger airlines is directed toward the floor (12), only passengers
seated within 2 m of the patient (proximal passengers) and others with
close personal contact would have been considered at reasonable risk for
secondary transmission. Those proximal passengers would have been
identified and advised to begin antibiotic prophylaxis and to continue it
for 7 days. Had a suspected plague case been laboratory-confirmed, the
state health departments and state epidemiologists would have contacted all
proximal passengers and monitored completion of the antibiotic prophylaxis.
All other passengers would have also been contacted to ensure that they
continued to monitor themselves for febrile illness.


Passive Surveillance System

Private physicians, hospitals, and local public health officials were
relied on to identify international air travelers from India who became ill
within a short period (from hours to 7 days) after disembarkation and
report the illness to the appropriate state and federal public health
officials. The attending physician, in consultation with the CDC medical
officers on call, then determined on the basis of clinical and
epidemiologic evidence whether the ill person had a reasonable likelihood
of having plague. If so, the patient would have been placed under
respiratory isolation in a hospital, diagnostic specimens would have been
obtained, and antibiotic treatment would have been initiated. Close
contacts of the suspected plague patient during the putative contagious
period would have been identified and advised to begin antibiotic
prophylaxis.

A concerted effort would have been made to determine the time the suspected
plague patient became symptomatic, and thereby contagious (13), relative to
the person's arrival in the United States. If the patient had been
symptomatic at the time of the flight, a passenger list would have been
obtained from the airline and the U. S. Customs Service. State
epidemiologists in the states of residence of all passengers would have
been informed of the need to contact and maintain surveillance of
passengers within their jurisdiction who were possibly secondarily exposed.
If seating assignments for the flight could be obtained, passengers seated
within 2 m of the patient would have been advised to begin antibiotic
prophylaxis; all other passengers would have been instructed to monitor
their temperature for 7 days and to report any illness to state health
officials.

Results

On September 29, plague information documents were sent by electronic mail
or fax to four Executive Committee members and 50 members of the Council of
State and Territorial Epidemiologists, 60 members of the Association of
State and Territorial Public Health Laboratory Directors, 40 Executive
Board members and 50 state representatives of the National Association of
County and City Health Officials, 132 officers in CDC's Epidemic
Intelligence Service (EIS), 15 field supervisors of CDC's Field
Epidemiology Training Program, and one representative each in the U. S.
Department of State and the Quarantine Health Services in Canada. Although
an exact count is not available, more than 3,000 persons probably received
these documents directly from CDC or secondarily through other agencies.

From September 27 to October 31, the CDC Voice Information Service received
6,665 calls accessing information about plague; 2,692 of these calls were
received through the special plague hotline number. During this same
period, 5,589 documents about plague were requested and sent by the CDC Fax
Information Service.

On October 25, 1994, after an on-site investigation in India, a WHO team of
scientists that included four CDC staff members, determined that the plague
epidemic was of more limited scope than previously believed, and
recommended the lifting of travel restrictions. On October 27, 1994, CDC
authorized a stand-down of the heightened surveillance system at all ports
of entry and a return to normal operations. During the 30 days that the
surveillance system was in place, 13 airline travelers arriving in the
United States were evaluated. Six patients with suspected plague were
identified and evaluated in airports  JFK and La Guardia in New York City
(four), Dallas-Fort Worth (one), Chicago-O'Hare (one)  and seven by
private physicians in New York City (five), Albany, New York (one), and St
Louis, Missouri (one). All 13 had a history of recent travel in India. None
was found to have plague. Symptoms of illness included fever (eight), cough
(six), vomiting (four), and malaise (three). The final diagnoses of persons
evaluated were viral syndrome (four), malaria (two), concurrent malaria and
dengue (one), typhoid (one), end-stage liver failure (one), and no illness
(one) (14). The final diagnosis was unspecified in three patients.

Discussion

Plague pandemics have occurred throughout history (15). In the European
epidemic known as Black Death, from 1345 to 1360, an estimated quarter of
the world's known population of 24 million died. Originating in central
Asia and carried by ship to Sicily, the disease spread east to China, south
to Africa, north to Russia and Scandinavia, and west to Greenland in only a
few years. Plague in North America can be traced historically to infected
rats aboard ships from the Far East that docked in California during the
early 20th century (16). Today, air travel that can transport a person
anywhere in the world within 24 hours expands the opportunity for rapid
spread of a transmissible disease like pneumonic plague. The potential for
pneumonic plague to spread by air travel to the United States during the
recent Indian epidemic elicited considerable public concern (7).

Although rare, plague is enzootic in the United States, and 10 to 15 human
cases are reported each year; typically only one or two of these are
pneumonic plague cases (17). Thus, most public health officials and medical
practitioners in this country have limited experience with plague (18).
When the Indian epidemic began, detailed and reliable information from
India was sparse; therefore, CDC disseminated factual and comprehensive
information regarding pneumonic plague and the Indian epidemic to public
health officials, physicians, and private citizens. The development and
distribution of the e-mail, voice, fax, and printed documents were
coordinated through a single branch within CDC, which ensured the accuracy
and timeliness of the information conveyed. By serving as the central
clearing house for international and domestic reports, CDC was able to
gather and redistribute information rapidly and efficiently. Updates in
MMWR contained data obtained within hours of publication. Most public
health officials and agencies were accessible immediately by electronic
mail or fax, and group mailing codes were constructed to facilitate
simultaneous communication. These timely updates of information, which
included periodic results of the enhanced surveillance system, heightened
awareness of the public health threat and encouraged participation by
health practitioners in the passive component of the surveillance system.

Like the information network, the surveillance system was centrally
coordinated at CDC, but it relied on the contributions of many agencies and
individuals to function effectively. Federal, state, and local health
officials, the Immigration and Naturalization Service, The U.S. Customs
Service, commercial businesses (passenger airlines), medical practitioners,
hospital personnel, and the public played key roles in the successful
implementation of the system. Many state and local health departments made
additional efforts to alert the medical community to the potential for
imported plague cases, to reiterate the surveillance protocol, and to
emphasize the importance of obtaining a travel history from any patient
with unexplained fever (14). This distribution of responsibility through
the established public health network was essential to effective
surveillance.

In 1992, the Institute of Medicine's Committee on Emerging Microbial
Threats to Health recommended that surveillance of international infectious
diseases be implemented and coordinated by a single government agency,
ideally CDC (19); subsequently, CDC developed a comprehensive strategy for
preventing emerging infectious diseases in the United States (20). In its
response to the Indian plague epidemic, rather than constructing a new
system specific to this emergency, CDC used a surveillance protocol that
built on the existing quarantine framework to utilize trained staff in a
position to readily respond. Future responses to the threat of importation
of communicable diseases with epidemic potential will require a similar
network of individuals and agencies, with specific roles and
responsibilities but sufficiently flexible to adapt to the particular
epidemiologic circumstances. A system similar to the one described here was
put in place in response to the Ebola outbreak in Zaire in April and May
1995 (21).

A surveillance system must be effective without becoming overly burdensome
to either those conducting the surveillance or those under surveillance; it
must safeguard the public health without inhibiting commerce or interfering
with individual freedoms. In the 1370s, during the latter years of Black
Death, nautical travelers to the Republic of Ragusa, now part of Italy,
were detained for 40 days (from which the word "quarantine" [quaranti
giorni] derives) (15), a detention period inappropriately long in light of
the current knowledge of plague's incubation period of 2 to 7 days (1). In
the recent outbreak, closure of airports to all flights from India,
compulsory quarantine of all international travelers, and an embargo of
trade with India were extreme measures given the epidemiology of plague and
the risk of importing a case (18). Primary surveillance efforts were
focused at critical control points, i.e., international airports, where
personnel resources for identification and control of imported plague cases
are maximally efficient. The secondary system, utilizing private physicians
and state and local health departments, permitted continued surveillance
that was less intensive, but geographically expansive, without placing an
unnecessary burden on international air travelers.

If a case of plague had been confirmed in an airline passenger, tracing
passengers at risk would have been a substantial undertaking. Depending on
the interval between disembarkation and diagnosis, hundreds of persons
might have had to be located across the country. In addition to 39 of CDC's
Epidemic Intelligence Service (EIS) Officers stationed in state and local
health departments, 10 EIS Officers in CDC centers in Atlanta, Georgia,
Cincinnati, Ohio, Washington D.C., and Fort Collins, Colorado, were
recruited to assist state and local health departments in tracing contacts
if necessary. EIS Officers have often been called to assist in public
health crises in which a large complement of epidemiologists was required;
in 1993, 13 EIS Officers were among the scientists and public health
officials assembled during the outbreak of hantavirus pulmonary syndrome in
the southwestern United States (22). Because a rapid response to
importation of a disease with epidemic potential often requires a national
team of epidemiologists to assist local public health agencies, the
Institute of Medicine and others have recommended the expansion and
continued support of CDC's EIS program (19,20,23).

The surveillance system's first line of detection for plague cases depended
on airline personnel, Immigration and Naturalization Service and U.S.
Customs officials for the active component, and private physicians and
health care providers for the passive component. Since the former are not
trained medical personnel and may not detect an ill traveler in the absence
of obvious signs and symptoms, and the latter may not be sufficiently
alerted to the possibility of plague, diagnosis of some plague cases could
have been delayed and not been efficiently detected by the surveillance
system. It is unrealistic to expect any system to effectively screen all
travelers returning from areas of recognized disease outbreaks. It is
impossible to assess the sensitivity of the described surveillance system
since no cases of pneumonic plague were identified either within or outside
the system. In retrospect, the risk for an imported plague case was quite
small, since the epidemic in India was limited in time and space and had
far fewer cases than originally suspected (24). The WHO investigative team
found no evidence of transmission in metropolitan areas other than Surat.
Most of the patients with suspected plague in Surat came from poor
neighborhoods, residents of which would be unlikely to travel
internationally. In addition, the short incubation period and severe
symptoms of pneumonic plague and the rapid deterioration of the patient's
condition, substantially limited the contagious period and the opportunity
for secondary transmission.

Although the epidemic potential for plague makes it a good model for
developing emerging disease response capabilities, the direct applicability
of this program for other emerging diseases may not be straightforward. The
above protocol was developed in response to a regionally limited outbreak
that occurred during a relatively brief period, similar to the recent Ebola
outbreak in Zaire (21). To detect emerging diseases in the absence of a
recognized outbreak, surveillance would need to be maintained at some
baseline level for an indefinite period. Compliance with the enhanced
plague surveillance protocol during the short period it was in effect
appears to have been excellent, but how compliance might have waned over
weeks to months is unknown. In addition, the protocol was specific to
plague, a well-characterized disease with well-described pathogenesis and
clinical features. The severe manifestations of pneumonic plague, the short
incubation and contagion periods, and the availability of reliable
diagnostic tests allowed for a focused protocol that could confidently
identify cases. Other emerging diseases may be less well characterized, or
even entirely unknown, and may require surveillance protocols of lesser
specificity. Nevertheless, the plague surveillance system was broad enough
(and consistent with the Institute of Medicine's recommendation that a
global infectious disease surveillance system implement broad reporting
criteria for detection of emerging diseases [19]) to identify four persons
who had other potentially fatal notifiable infectious diseases.

Acknowledgments

The authors thank Dr. May C. Chu, Dr. Robert B. Craven, Mr. Thomas A.
DeMarcus, Ms. Rosamond R. Dewart, Dr. Kenneth L. Gage, Dr. Kathleen A.
Orloski, Mr. Tony D. Perez, Dr. Jack D. Poland, Dr. Martin E. Schriefer,
Mr. Thomas W. Skinner, Dr. Ofelia C. Tablan, and Dr. Theodore F. Tsai,
(CDC), and Dr. Brian Gushulak, (Health Canada), for expert consultation and
direct assistance; Ms. Mary Ellen Fernandez and Ms. Edwarda O. Lee, (CDC),
for administrative support; Ms. Kathy A. Bruce, Ms. Rebecca L. Deavours,
Ms. Anna M. Jimenez, and Ms. Karen A. Peterson (CDC), for secretarial
support; Mr. Jerome R. Cordts (Association of State and Territorial Public
Health Laboratory Directors), Mr. David E. Custer and Ms. Nancy Rawding,
(National Association of County and City Health Officials), Mr. Willis R.
Forrester and Ms. Kathy F. Getz (Council of State and Territorial
Epidemiologists), Dr. Martin Wolfe, (U.S. Department of State), and Ms.
Patsy R. Bellamy, Ms. Pamela K. Eberhardt, and Mr. Clyde S. Furney, Jr.
(CDC), for assistance with rapid dissemination of information; Mr. Richard
Heffernan, and Dr. Marcelle Layton, (New York City Department of Health),
and Dr. Rosalind J. Carter (New York City Department of Health and CDC),
for assistance in conducting surveillance.

References

  1. Benenson AS, editor. Control of Communicable Diseases in Man, 15th ed.
     Washington, DC: American Public Health Association, 1990.
  2. World Health Organization. International Health Regulations. 3rd ed.
     Geneva, Switzerland: WHO, 1983:26-9.
  3. Desai D, Samuel I. Surat flounders against medical crisis. The Indian
     Express 24 September 1994.
  4. Gupta S. 600,000 have fled Surat. The Statesman 25 September 1994.
  5. World Health Organization, Regional Office for Europe. Plague in
     India. Communicable Disease Report (CD News) Issue 4; 3 October 1994.
  6. Friese K, Mahurka U, Rattanani L, Kattyar A, Rai S. The plague peril.
     Are you at risk? India Today 15 October 1994.
  7. Post T, Clifton T, Mazumdar S, Cowley G, Raghavan S. The plague of
     panic. Newsweek 10 October 1994:40-1.<D%0>
  8. Centers for Disease Control and Prevention. Human plagueIndia, 1994.
     MMWR 1994;43:689-91.
  9. Centers for Disease Control and Prevention. Update: Human
     plagueIndia, 1994. MMWR 1994;43:722-3.
 10. Centers for Disease Control and Prevention. Update: Human
     plagueIndia, 1994. MMWR 1994;43:761-2.
 11. Craven RB. Plague. In: Hoeprich PD, Jorday MC, Ronald AR, editors.
     Infectious diseases: a treatise of infectious processes. 5th ed.
     Philadelphia: Lippincott, 1994:1302-12.
 12. National Research Council. The airliner cabin environment: air quality
     and safety. Washington, DC: National Academy Press; 1986.
 13. Poland JD, Barnes AM. Plague. In: Steele JH, editor. CRC handbook
     series in zoonoses: section A: bacterial, rickettsial, and mycotic
     diseases. Boca Raton, FL: CRC Press; 1979:515-58.
 14. Centers for Disease Control and Prevention. Detection of notifiable
     diseases through surveillance for imported plagueNew York,
     September-October 1994. MMWR 1994;44:805-7.
 15. Cartwright FF. Disease and history. New York: Dorset Press, 1972.
 16. Craven RB, Barnes AM. Plague and tularemia. Infect Dis Clin North Am
     1991;5:165-75.
 17. Centers for Disease Control and Prevention. Summary of notifiable
     diseases, United States. 1993. MMWR 1993;42:45.
 18. Campbell GL, Hughes JM. Plague in India: a new warning from an old
     nemesis. Ann Intern Med 1995;122:151-3.
 19. Lederberg J, Shope RE, Oaks SC, editors. Emerging infections:
     microbial threats to health in the United States. Washington, DC:
     National Academy Press, 1992.
 20. Centers for Disease Control and Prevention. Addressing emerging
     infectious disease threats: a prevention strategy for the United
     States. Atlanta, GA: U.S. Department of Health and Human Services,
     Public Health Service, 1994.
 21. Centers for Disease Control and Prevention. Outbreak of Ebola viral
     hemorrhagic feverZaire, 1995. MMWR 1995;44:381-2.
 22. Centers for Disease Control and Prevention. Update: outbreak of
     hantavirus infectionsouthwestern United States, 1993. MMWR
     1993;42:441-3.
 23. Henderson DA. Surveillance systems and intergovernmental cooperation.
     In: Morse SS, editor. Emerging viruses. New York: Oxford University
     Press, 1993:283-9.
 24. World Health Organization, South-East Asia Regional Office. Plague in
     India: World Health Organization team executive report. Geneva,
     Switzerland: WHO, December 1994.


(Pages 37-43)

Changing Patterns of Autochthonous Malaria Transmission in the United
States: A Review of Recent Outbreaks

Jane R. Zucker, M.D., M.Sc.
Centers for Disease Control and Prevention, Atlanta, Georgia, USA


ABSTRACT

Three recent outbreaks of locally acquired malaria in densely populated
areas of the United States demonstrate the continued risk for mosquitoborne
transmission of this disease. Increased global travel, immigration, and the
presence of competent anopheline vectors throughout the continental United
States contribute to the ongoing threat of malaria transmission. The
likelihood of mosquitoborne transmission in the United States is dependent
on the interactions between the human host, anopheline vector, malaria
parasite, and environmental conditions. Recent changes in the epidemiology
of locally acquired malaria and possible factors contributing to these
changes are discussed.


Malaria was endemic throughout much of the United States in the late 19th
and early 20th centuries (1). Interrupted human-vector contact, decreased
anopheline populations, and effective treatment contributed to a decline in
transmission and to subsequent eradication. However, environmental changes,
the spread of drug resistance, and increased air travel (2) could lead to
the reemergence of malaria as a serious public health problem. The
potential for the reintroduction of malaria into the United States has been
demonstrated by recent outbreaks of mosquitoborne transmission in densely
populated areas of New Jersey, New York, and Texas (3-5). A review of the
malaria life cycle and recent outbreaks illustrates key elements that
affect the risk for malaria transmission in the United States.

Life cycle and Entomologic Principles: Requirements for Transmission

The malaria parasites are protozoa of the genus Plasmodium. The four
species of Plasmodium that cause human malaria, P. falciparum, P. vivax, P.
ovale, and P. malariae, are transmitted by the bite of infective female
mosquitoes of the genus Anopheles. The immature stages of the vector's life
cycle (egg, larva, and pupa) are aquatic and develop in breeding sites,
whereas the aerial adult stage is terrestrial. Anopheline species capable
of transmitting malaria are found in all 48 states of the contiguous United
States (1). The most important vectors are An. quadrimaculatus and An.
freeborni, found east and west of the Rocky Mountains, respectively.
However, other anopheline species have been implicated in local
transmission, for example, An. hermsi in California (6).

Humans are the intermediate host and reservoir of the parasite, and the
mosquito is the definitive host and vector. Female anophelines become
infected only if they take a blood meal from a person whose blood contains
mature male and female stages (gametocytes) of the parasite. A complex
cycle of development and multiplication then begins with union of the male
and female stages in the stomach of the vector and ends with parasites,
called sporozoites, in its salivary glands, which are infective to humans
(Figure 1 not available in ASCII). The time required for the complete maturation of the parasite
(sporogonic cycle) in the mosquito varies and depends on the Plasmodium
species and external temperature. At 27 degrees C, approximately 8 to 13 days are
needed for the completion of this cycle for P. vivax and P. falciparum (7).
At lower temperatures, the time for the sporogonic cycle is considerably
longer: approximately 20 days at 20 degrees C and 30 days at 18C for P. vivax.
Similarly, for P. falciparum, the sporogonic cycle takes 30 days at 20C. At
a temperature below 16 degrees C or 18 degrees C, for these two species, respectively, the
cycle cannot be completed and transmission cannot occur. On the other hand,
33 degrees C is the upper limit for completion of the sporogonic cycle.

Only anophelines surviving longer than the sporogonic cycle can transmit
malaria, assuming they took an infective blood meal. Extrinsic factors that
affect the lifespan of the female anopheline, and thus the completion of
the sporogonic cycle, include ambient temperature, humidity, and rainfall.
The efficiency and potential for transmission have been mathematically
correlated to the survival of the mosquito population. Methods to determine
the age range of mosquito populations are imprecise. Thus, determining the
proportion of anophelines that have lived long enough to complete the
sporogonic cycle is difficult.

Anophelines feed at night; therefore, transmission occurs primarily between
dusk and dawn. When an infected mosquito takes a blood meal, it injects
sporozoites from its salivary glands into the bloodstream (Figure 1). The
sporozoites infect hepatocytes and begin a process of development and
multiplication. The life cycle is completed when an anopheline takes a
blood meal and ingests male and female gametocytes, allowing for sexual
reproduction.

P. vivax gametocytes develop within the first few days of infection, and so
a person may be infective early in the course of the illness. In contrast,
P. falciparum gametocytes do not appear for a minimum of 10 to 14 days, by
which time many people would have been symptomatic and received treatment.
In addition, both P. vivax and P. ovale may form dormant liver stages,
called hypnozoites, which may become active and cause a relapse of the
infection and gametocytemia months to years after a person has left a
malaria-endemic area. Hypnozoites are only formed at the time of the
initial sporozoite inoculation.

This review of the malaria life cycle identifies the three factors
essential for malaria transmission: adequate breeding sites and sufficient
abundance of anophelines, weather conditions that allow completion of the
sporogonic cycle, and gametocytemic persons. Historically, adequate
housing, water management, and mosquito-control activities acted to limit
anopheline populations and prevented anopheline-human contact. In addition,
conditions that promote mosquito survival and parasite development are not
usually sustained; hence, the balance of these factors does not favor
transmission. However, recent outbreaks demonstrate how tenuous the balance
among these factors is. Changes that effect human-vector contact and
increased density of gametocytemic persons during optimal weather
conditions may be all that is necessary for transmission.

Malaria Surveillance

Historical Background

It is believed that malaria was introduced into the continental United
States by European colonists (P. vivax and P. malariae) and African slaves
(P. falciparum) in the 16th and 17th centuries. It became endemic in many
areas of the country, paralleling the migration of people, with the
exception of northern New England and mountainous and desert areas (Figure
2 not available in ASCII). The incidence of malaria probably peaked in approximately 1875, and it
is estimated that more than 600,000 cases occurred in 1914 (1). Systematic
reporting of malaria cases began in 1933; in 1934 125,556 cases were
reported. The decline in transmission before the introduction of extensive
mosquito control measures was attributed to a population shift from rural
to urban areas, climatic conditions, increased drainage, improved housing
and nutrition, better socioeconomic conditions and standards of living,
greater access to medical services, and the availability of quinine for
treatment (1). Additional activities, conducted in the 1940s, that led to
the interruption of malaria transmission included larviciding, screening of
houses, house spraying (residual spray program with DDT), and use of DDT
(for residual spray and larviciding), which removed breeding sites,
decreased the density of anophelines, and interrupted anopheline-human
contact. Improved surveillance allowed treatment of parasitemic persons,
focused control activities geographically, and allowed accurate assessment
of the problem.

Surveillance was conducted by CDC to evaluate the progress toward malaria
eradication, and in the 1950s it was concluded that this goal had been
achieved. At that time, it was recognized that because of international
travel, presence of competent anopheline vectors, and environmental
conditions that could favor transmission, malaria could be reintroduced
into the United States. Surveillance activities have been maintained not
only to identify outbreaks of local malaria transmission, but also to
identify other cases acquired in the United States (for example,
transfusion-induced cases) and to monitor trends in imported cases that
guide CDC prevention recommendations.

Since 1957, nearly all cases of malaria diagnosed in the United States have
been imported, i.e., have been acquired by mosquito transmission
(autochthonous) in areas where malaria is known to occur (8). In general,
approximately half the cases occur among U.S. civilians and half among
foreign-born civilians. However, each year cases occur that are acquired
congenitally or are induced, i.e., acquired through artificial means, such
as blood transfusions. Rarely, cases occur that are classified as cryptic
(an isolated case of malaria determined after an epidemiologic
investigation not to be associated with secondary cases) or introduced (a
case documented to be acquired by mosquito transmission from an imported
case in an area where malaria does not normally occur) (8). In practice,
the distinction between a cryptic and an introduced case may be difficult
to ascertain. Frequently, epidemiologic investigations indicate that the
infection must have been acquired in the United States and circumstantial
evidence suggests it was mosquitoborne. Additional evidence to document
mosquitoborne transmission in the United States, such as the presence of
anopheline larvae or infective adults, or confirmation of secondary
transmission is rarely obtained. Therefore, all locally acquired cases
thought to be mosquitoborne will be included in the following discussion,
regardless of whether the final classification was cryptic or introduced.

Overview of Locally Acquired Cases

From 1957, when the current surveillance system began, through 1994, 76
cases of introduced and cryptic malaria were reported (9-27). Single cases
in Louisiana in 1983 and in Massachusetts in 1985 involved patients who had
recently received blood transfusions (28, 29). The infections were likely
induced by transfusion, although they were classified as cryptic because
serologic testing of available donors did not implicate a source person.
Apart from these two, 74 cases were reported from 21 states, including
three northern states (Oregon, north-central New York, and New Hampshire),
that were probably acquired by mosquitoborne transmission in the United
States (Figure 3 not available in ASCII). The most common species identified was P. vivax, which
accounted for 59 (80%) cases; P. malariae accounted for six (8%) cases, and
P. falciparum for five (7%); the species was not identified for the
remaining four (5%) cases. In 1992, P. vivax, P. falciparum, P. malariae,
and P. ovale were identified in 51%, 33%, 4%, and 3% of reported cases,
respectively (30). The species was not identified in the remaining 9% of
cases. The high proportion of locally acquired cases caused by P. vivax is
not surprising for several reasons: vivax malaria is diagnosed most often
among reported cases; the appearance of gametocytes early in the course of
infection may allow for transmission to mosquitoes before treatment is
received; relapse may occur months to years after leaving a malaria-endemic
area when hypnozoites are reactivated; and the temperatures required for
the completion of the sporogonic cycle are found in the United States.

The 74 cases represent 56 distinct episodes of probable transmission; 43
episodes involved one person without risk factors for malaria, nine
involved two persons without risk factors, and four involved three or more
persons. Before 1991, among cases with sufficient information, 41 (89%) of
46 outbreaks occurred in locations described as rural. Only three were in
areas described as suburban, and two were in army barracks. Since then, the
three episodes in New Jersey (1991), New York (1993), and Texas (1994) have
all occurred in densely populated suburban or urban areas.

California

From 1980 through 1990, 13 outbreaks of presumed mosquitoborne transmission
were reported from California. Most occurred in rural areas where medical
services were limited and sanitary facilities and housing were often
substandard, allowing for anopheline-human contact; they involved
undocumented migrant workers from - malaria-endemic areas who were
implicated as the gametocytemic source. During an outbreak in Carlsbad,
California, in 1986 (6), 28 cases (26 Mexican migrant workers and two
Carlsbad residents) of P. vivax were documented during a 3-month period.
The epidemic curve indicated secondary transmission, thus confirming
mosquitoborne transmission. The principal risk factor for malaria was
sleeping on a particular hillside outdoors during the evening. Adult female
anophelines (An. hermsi) were captured from a marsh area below the
hillside, and temperature and humidity were favorable for completion of the
sporogonic cycle.

New Jersey

In 1991, two separate episodes of locally acquired P. vivax malaria were
identified, occurring more than 70 miles apart (3); the first was
consistent with the expected epidemiologic pattern, but the second occurred
in a suburban and densely populated area. The index case-patient was an
8-year-old boy, without risk factors for malaria, (travel or exposure to
blood or blood products). Few undocumented agriculture workers were living
in this suburban area, but a large number of documented immigrants and
undocumented factory workers were identified. U.S. census data from 1990
indicated that the population of immigrants from the Indian subcontinent,
where malaria is endemic, increased by 230% compared with census data from
1980. The weather was hotter and more humid than usual, and higher
anopheline densities were reported from some regions of New Jersey. The
second case-patient had no clear exposure to mosquitoes but may have been
exposed during the early evening in a marshy area where he played ball.

New York City

In 1993, another outbreak of locally acquired malaria occurred in New York
City (4). The index patient had no travel history or other means of
acquiring malaria except local mosquitoborne transmission. The
investigation identified two other cases of malaria; one in a person who
had traveled internationally 2 years previously, and a third case which was
initially unreported. This outbreak was unusual, not only because urban
areas are poor habitats for anophelines, but also because the causative
parasite was P. falciparum. The area where the cases were identified in
northwest Queens had many immigrants; the 1990 census showed a 31% increase
in the number of foreign-born persons, which accounted for 48% of all
recent immigrants into Queens. Many of these immigrants were from
malaria-endemic areas, including parts of South and Central America and
Hispaniola (Dominican Republic and Haiti). In addition, more than 100 cases
of imported malaria were reported in New York City during 1993 (Malaria
Section/Division of Parasitic Diseases/CDC unpublished surveillance data).
As seen with the earlier outbreaks, the weather that summer was hotter and
more humid than usual. During the several weeks between the proposed dates
of transmission and the investigation, the weather had changed, interfering
with the identification of active anopheline breeding sites or adult
anophelines.

Houston, Texas

Three cases of locally acquired malaria were identified in Houston in 1994
(5). This investigation had features similar to those seen in previous
outbreaks in California. All three patients were homeless and lived in
substandard housing, which provided an opportunity for exposure to
anophelines at night. Two of the patients became ill, and malaria was
diagnosed in July; the duration of illness was 11 days to 3 weeks. The
third patient had symptoms in late July, but a diagnosis of malaria was not
made until December when he had a relapse of P. vivax, which could only
occur from mosquitoborne transmission. Results from an indirect
immunofluorescence assay for malaria antibodies conducted on serum
specimens obtained in August and December provided additional evidence that
his illness during the summer was malaria. The infected persons lived in
areas with large immigrant populations. Environmental investigation
identified possible breeding sites, and adult female An. quadrimaculatus
were captured in light traps. In addition, the average temperature and
humidity favored mosquito survival and development.

The three outbreaks that occurred in the early 1990s in densely populated
areas occurred in neighborhoods with many immigrants from countries with
malaria transmission and weather that was hot and humid and, therefore,
conducive to the completion of the sporogonic cycle and the survival of
adult female anophelines. The delay between mosquito inoculation,
diagnosis, and investigation often meant changes in weather and inability
to confirm the presence of adult anophelines and active breeding sites.

Discussion

Understanding the factors that contributed to these outbreaks and improving
case surveillance will facilitate detection of future outbreaks and
development of appropriate prevention and control measures.

Two necessary criteria must be met for malaria transmission: anopheline
vectors capable of transmitting malaria and gametocytemic persons. Both
exist throughout the United States. Under current conditions, the average
lifespan of anophelines in the United States is less than the duration of
the sporogonic cycle. A common feature of all recent outbreaks has been
weather that is hotter and more humid than usual, which may increase
anopheline survival and decrease the duration of the sporogonic cycle
enough to allow for the development of infective sporozoites. The possible
effect of weather on malaria transmission has been cited in recent articles
on the potential consequences of global environmental changes (31-33).

Detection of locally acquired cases depends on accurate diagnosis and
reporting of cases. Prompt reporting is not universal as suggested by the
Houston investigation (5). Delays in recognizing cases are caused by not
suspecting malaria in a person with a febrile illness who has not traveled
internationally, by laboratories inexperienced with blood smear diagnosis,
and by general lack of reporting of notifiable diseases. Prompt diagnosis,
treatment, and notification are essential for proper treatment and
evaluation of potentially gametocytemic persons.

Alternative hypotheses for explaining malaria infection acquired in areas
without ongoing transmission have included importation of infective
anophelines either on airplanes, ships, or in baggage (34-36). One recent
report of two persons who acquired P. falciparum in Germany indicates that
conditions supporting local mosquitoborne transmission were present in
Germany, although the authors concluded that infected mosquitoes must have
been imported in baggage (37). Like the United States, many parts of
Europe, including regions of Germany, have had endemic malaria transmission
and thus are at risk for introduced autochthonous transmission. These
alternative hypotheses have been addressed in the U.S. investigations, but
none of the episodes occurred close enough to international airports or
harbors to support these hypotheses. The possibility of "baggage malaria"
is intriguing but unlikely for reasons concerning mosquito survival during
transport and expected host-seeking behavior once the mosquitoes are
released from luggage.

Gametocytemic persons, both immigrants and native-born U.S. civilians, are
present in the United States and can serve as reservoirs of infection.
Water management, improved housing, and access to health care are critical
for preventing transmission. Diligent malaria surveillance can detect
outbreaks early and allow control measures to interrupt transmission.

Acknowledgments

I thank Dr. Monica Parise for her efforts in conducting the literature
review on locally acquired malaria in the United States, and Dr. Ray Beach
for his thoughtful comments and review of the manuscript.

Address for correspondence:

Jane R. Zucker
Centers for Disease Control and Prevention
1600 Clifton Rd., MS F22
Atlanta, GA 30333
Fax: 770-488-7761
e-mail: jxz2@ciddpd2.em.cdc.gov

References

  1. Report for registration of malaria eradication from United States of
     America. Washington, DC: Pan American Health Organization, December
     1969.
  2. Lederberg J, Shope RE, Oaks SC, Jr., editors. Emerging infections:
     microbial threats to health in the United States. Washington, DC:
     Institute of Medicine, National Academy Press, 1992.
  3. Brook JH, Genese CA, Bloland PB, Zucker JR, Spitalny KC. Malaria
     probably locally acquired in New Jersey. N Engl J Med 1994;331:22-3.
  4. 4. Layton M, Parise ME, Campbell CC, Advani R, Sexton JD, Bosler EM,
     Zucker JR. Malaria transmission in New York City, 1993. Lancet
     1995;346:729-31.
  5. Local transmission of Plasmodium vivax malaria -- Houston, Texas, 1994.
     MMWR 1995;44:295-303.
  6. Maldonado YA, Nahlen BL, Roberto RR, Ginsberg M, Orellana E, Mizrahi
     M, et al. Transmission of Plasmodium vivax malaria in San Diego
     County, California, 1986. Am J Trop Med Hyg 1990;42:3-9.
  7. Pampana E. A textbook of malaria eradication. London: Oxford
     University Press, 1963.
  8. World Health Organization. Terminology of malaria and of malaria
     eradication, 1963. Geneva, Switzerland: World Health Organization,
     1963:32.
  9. Dunn FL, Brody JA. Malaria surveillance in the United States,
     1956-1957. Am J Trop Med Hyg 1959;3:447-55.
 10. Shaw JD, Schrack WD, Jr. Malaria contracted in Pennsylvania. Public
     Health Rep 1966;81:413-8.
 11. Luby JP, Schultz MG, Nowosiwsky T, Kaiser RL. Introduced malaria at
     Fort Benning, Georgia, 1964-1965. Am J Trop Med Hyg 1967;16:146-53.
 12. Jacobs T. Cryptic malaria. Rocky Mountain Medical Journal
     1966;63:57-9.
 13. Steiner ML, Malaria in a Kentucky family: report of two cases in
     siblings. Clin Pediatr (Phila) 1968;7:493-4.
 14. Sartoriano GP, Rowden RM, Ginsburg DM. Malaria acquired in the United
     States: introduced and cryptic malaria. NY J Med 1971;71:1535-7.
 15. Hermos JA, Fisher GU, Schultz MG, Haughie GE. Case histories in 1968
     outbreak in Chambers. J Med Assoc Ala. 1969;39:57-66.
 16. Dover AS. A malaria outbreak in Texas, 1970. South Med J
     1972;65:215-8.
 17. Center for Disease Control. Introduced malaria in Texas. MMWR
     1970;19:407-8.
 18. Singal M, Shaw PK, Lindsay RC, Roberto RR. An outbreak of introduced
     malaria in California possibly involving secondary transmission. Am J
     Trop Med Hyg 1977;26:1-9.
 19. Malaria in California [letter]. West J Med 1981; 134:645-6.
 20. Centers for Disease Control. Introduced autochthonous vivax
     malaria -- California, 1980-1981. MMWR 1982;31:213-5.
 21. Centers for Disease Control. Transmission of Plasmodium vivax malaria
     San Diego County, California, 1986. MMWR 1986;35:679-81.
 22. Brillman J. Plasmodium vivax malaria from Mexicoa problem in the
     United States. West J Med 1987;147:469-73.
 23. Ginsberg MM. Transmission of malaria in San Diego County, California.
     West J Med 1991;154:465-6.
 24. State of California Health and Welfare Agency. Mosquito-transmitted
     malaria in California:1988-1989: part 1. California Morbidity,
     December 22, 1989.
 25. State of California Health and Welfare Agency. Mosquito-transmitted
     malaria in California:1988-1989: part 2. California Morbidity, January
     5, 1990.
 26. Centers for Disease Control. Transmission of Plasmodium vivax
     malaria -- San Diego County, California, 1988 and 1989. MMWR
     1990;39:91-4.
 27. Centers for Disease Control. Mosquito-transmitted malaria -- California
     and Florida, 1990. MMWR 1991;40:106-8.
 28. Centers for Disease Control. Malaria surveillance annual summary 1983.
     Atlanta, GA: Centers for Disease Control, October 1984.
 29. Centers for Disease Control. Malaria surveillance annual summary 1985.
     Atlanta, GA: Centers for Disease Control, September 1986.
 30. Zucker JR, Barber AM, Paxton LA, Schultz LJ, Lobel HO, Roberts JM, et
     al. Malaria surveillance -- United States, 1992. MMWR 1995;44(SS-5):1-17.
 31. Loevinsohn ME. Climatic warming and increased malaria incidence in
     Rwanda. Lancet 1994;343:714-7.
 32. Haines A, Epstein PR, McMichael AJ. Global health watch: monitoring
     impacts of environmental change. Lancet 1993;342:1464-9.
 33. Bouma MJ, Sondorp HE, van der Kaay HJ. Climate change and periodic
     epidemic malaria. Lancet 1994;343:1440.
 34. Isaacson M. Airport malaria: a review. Bull World Health Organ
     1989;67:737-43.
 35. Delmont J, Brouqui P, Poullin P, Bourgeade A. Harbour-acquired
     Plasmodium falciparum malaria. Lancet 1994;344:330-1.
 36. Castelli F, Caligaris S, Matteelli A, Chiodera A, Carosi G, Fausti G.
     Baggage malaria in Italy: cryptic malaria explained? Trans R Soc Trop
     Med Hyg 1993;87:394.
 37. Mantel CF, Klose C, Scheurer S, Vogel R, Wesirow AL, Bienzle U.
     Plasmodium falciparum malaria acquired in Berlin, Germany. Lancet
     1995;346:320-1.


DISPATCHES

(Pages 44-46)

Cluster of Lyme Disease Cases at a Summer Camp in Kent County, Maryland


Lyme disease is the second most prevalent emerging infectious disease in
the United States; more than 65,000 cases have been reported to the Centers
for Disease Control and Prevention since the disease was first described by
Steere and colleagues in 1977 (1).

In July 1994, a physician in Chestertown, Maryland, reported eight cases of
Lyme disease to the Kent County Health Department. Five were from a summer
camp 10 miles north of Rock Hall on the Chesapeake Bay. In one
case-patient, a 9-year-old camper from Pennsylvania, erythema migrans (EM)
rash and left facial nerve palsy developed the day after she arrived at the
camp.

To determine whether Lyme disease was present at the camp, we interviewed
the eight counselors who had EM or febrile illnesses during July and 43 of
the remaining 91 camp employees. Clusters of cases of Lyme disease with a
short and specific exposure period (i.e., 1012 weeks for the 100
counselors and 24 weeks for the 1,600 campers) had not been investigated
in recent years.

All 51 surveyed camp employees gave histories of tick exposure throughout
the summer. Four counselors had EM of 5 cm in diameter without other
symptoms or signs and were treated with amoxicillin by the camp physician.
Four other counselors had recurrent fever of 102F to 104F, severe
headaches, somnolence, malaise, fatigue, myalgia, and anorexia. All four
described extensive fatigue, drowsiness, and difficulty in getting out of
bed. Three described shaking chills, and one had watery diarrhea. The camp
physician admitted them all to the camp dispensary; Lyme disease was not
diagnosed in any of them; only the patient with diarrhea was given an
antimicrobial agent, trimethoprim/sulfamethoxazole. All patients improved
in 3 to 5 days.

Sera were obtained in mid-August from the 51 employees; for the eight
patients described above, this was 4 to 7 weeks after the onset of illness.
All sera were nonreactive in indirect fluorescence antibody (IFA) tests
against antigens for Rickettsia rickettsii and Ehrlichia equi (used to
screen for human granulocytic ehrlichiosis). One patient, who had an
EM-like rash but no other symptoms, had an IFA titer of 512 for E.
chaffeensis (used to screen for human monocytic ehrlichiosis). Serologic
testing for Borrelia burgdorferi by enzyme immunoassay (EIA) (Lyme Stat,
BioWhittker, Walkersville, Maryland) identified patients with positive or
borderline results (Table 1).

Hard ticks were collected by dragging felt material at several sites within
the camp on three occasions during August. Collected adult Ixodes
scapularis were tested by an antigen capture EIA for outer surface protein
A (2). Ten (16.9%) of 59 male ticks were positive for B. burgdorferi.
Although the infection rate was higher in female ticks collected from the
camp, the results cannot be interpreted because the female ticks were
co-fed on rabbits; it is not certain whether this could cross-infect ticks
feeding on the same animals.

We considered exposure to B. burgdorferi in this camp to be high (suspected
acute Lyme disease-like illness incidence of 6% to 8%). The incidence rate
depends on whether patients 6 and 7, who had flulike illnesses and positive
EIAs and negative Western blot results (Marblot Strip Test System, Mardex
Diagnostics, Carlsbad, California) are considered to have had Lyme disease,
and on assuming that the 49 unexamined counselors did not have Lyme
disease. Also, Kent County has one of the highest incidences of Lyme
disease in the state (3), many deer were present in the woods and fields in
and around the camp, and all counselors reported frequent exposure to
ticks.

-------------------------------------------------------------------
Table.  Results of WB antibody tests for Borrelia burgdorferi in
summer camp residents with positive (titer >= 1.00) and borderline 
(tite = 0.80-0.99) EIA results
____________________________________________________________________
                                       Serology
                             ____________________________                         
                                               WB 
                                         ________________
Subject      Syndrome         EIA        IgM          IgG
________________________________________________________________
1           EM                1.82       Pos          Pos
2           EM                1.40       Neg          Pos
3           EM                1.21       Neg          Neg
4           EM                3.21       Pos          Pos
5           Flulike           1.00       Neg          Pos
6           Flulike           1.04       Neg          Neg
7           Flulike           1.80       Neg          Neg
8           Flulike           2.46       Neg          Pos
9           None              0.96       Neg          Neg
10          None              0.96       ND           Pos
11          None              1.82       Neg          Pos
12          None              2.21       Neg          Neg
13          Sinusitis         1.11       Pos          Neg
14          Sinusitis         0.93       Neg          Neg
15          None              1.00       Neg          Neg
16          Rocky Mountain    1.14       Neg          Neg
             spotted fever,
             1991
_____________________________________________________________________
EIA=enzyme immunoassay;EM=erythema migrans;ND=no data;WB=Western blot.


The four patients who had an acute febrile illness without cutaneous
lesions were not initially suspected to have acute Lyme disease. We believe
that flulike illness without EM is a more common manifestation of acute
Lyme disease than is generally appreciated since, as in patients 5 through
8 (Table), Lyme disease is often not considered in the differential
diagnosis (4). Acutely febrile patients, who have been bitten by a tick in
Lyme disease-endemic areas also should be considered for early antibiotic
therapy. Doxycycline or another tetracycline is effective for Lyme disease
as well as for infections with E. chaffeensis and R. rickettsii, which are
also transmitted by ticks and may have a similar clinical syndrome (5).
Serologic testing, although it can confirm the diagnosis during the
convalescence phase, may not establish an early diagnosis in either case,
since antibody responses to all three infections are usually delayed until
2 to 4 weeks after the onset of symptoms and may not occur in patients
treated with antibiotics (5-8).

We interpreted the Western blots according to criteria proposed at the
Second National Meeting on Serological Diagnosis of Lyme disease (6). Of
the four patients with EM and with EIAs positive for B. burgdorferi,
patient 3, who had antibodies to E. chaffeensis, did not have IgG and/or
IgM evidence of B. burgdorferi infection by Western blot. He also had no
symptoms compatible with monocytic ehrlichiosis. Two (patients 6 and 7) of
the four with flulike illnesses and with EIAs positive for B. burgdorferi
did not have B. burgdorferi infection confirmed by Western blot. Positive
or borderline serologic results for B. burgdorferi infection in patients 9
through 16 (Table) who did not have a clinical history compatible with Lyme
disease could have been caused by asymptomatic infection, antibody
responses from prior infections, cross-reactions from other infections, or
false-positive reactions (8). Many of the counselors had been at the camp
during previous summers and could have had prior mild, nondiagnosed
infections with B. burgdorferi. Another possibility is that the EIA titers
in some of the patients were high normal values, which may have been the
case for patients 9, 14, 15, and 16. Patient 13 who had IgM evidence of
recent infection on Western blot may have had a mild infection with B.
burgdorferi during the previous month. However, this is impossible to
confirm without acute-phase and convalescent-phase (or preexposure and
post-exposure) serum samples. This is also pertinent to those with flulike
symptoms and negative Western blot results (patients 6 and 7).

The usefulness of using EIA screening and Western blot confirmation in
seroepidemiologic studies for Lyme disease has not been established. The
positive predictive value of a diagnostic test is highly dependent on the
prevalence of the disease being studied. If the prevalence of Lyme disease
in the population screened is very low, the positive predictive value of
testing may be too low to be diagnostically useful.

G. Thomas Strickland, M.D., Ph.D.,* Leena Trivedi, Ph.D., Stanley Watkins,
B.S.,* Margaret Clothier, R.N., John Grant, M.D., M.P.H., John Morgan, M.
D., Edward Schmidtman, Ph.D., Thomas Burkot, Ph.D.#

*University of Maryland School of Medicine, Baltimore, Maryland, USA;
Maryland Department of Health and Mental Hygiene, Baltimore, Maryland,
USA; Kent County Health Department, Chestertown, Maryland, USA; Private
practice of medicine, Chestertown, Maryland, USA; U.S. Department of
Agriculture Animal Research Station, Laramie, Wyoming, USA; #Division of
Vector-Borne Infectious Diseases, National Center for Infectious Diseases,
Centers for Disease Control and Prevention, Fort Collins, Colorado, USA.

Acknowledgements

Dr. James Olson, Viral and Rickettsial Diseases Branch, National Center for
Infectious Diseases, Centers for Disease Control and Prevention, performed
IFA testing for R. rickettsii and E. chaffeensis antibodies. Dr. J. Stephen
Dumler, Pathology Department, University of Maryland School of Medicine,
performed the IFA testing for E. equi antibodies. We also thank Mr. Charles
Hayword and his camp staff, including Ms. Carol M. Brown, for assisting in
this investigation and Dr. Ebenezer Israel, Maryland Department of Health
and Mental Hygiene, for advice and assistance. This project was supported
by the Epidemiology and Disease Control Program, Maryland Department of
Health and Mental Hygiene, and by the Agency for Health Care Policy and
Research Grant 5 RO1 HSO7813.

References

  1. Steere AC, Maliwista SE, Snydman DR, Shope RE, Andiman WA, Ross MR,
     Steele FM. Lyme arthritis: an epidemic of oligoarticular arthritis in
     children and adults in three Connecticut communities. Arthritis Rheum
     1977; 20:7-17.
  2. Burkot TR, Patrican L, Piesman J. Field trial of an outer surface
     protein A (OspA) antigen-capture enzyme-linked immunosorbent assay
     (ELISA) to detect Borrelia burgdorferi in Ixodes scapularis. Am J Trop
     Med Hyg 1994; 50:354-8.
  3. Steinberg SH, Strickland GT, Pena C, Israel E. Lyme disease
     surveillance in Maryland, 1992. Ann Epidemiol 1996; 6: In press.
  4. Feder HM Jr., Gerber MA, Krause PJ, Ryan R, Shapiro Ed. Early Lyme
     disease: a flu-like illness without erythema migrans. Pediatrics 1993;
     91:456-9.
  5. Dumler JS, Bakken JS. Ehrlichial diseases of humans: emerging
     tick-borne infections. Clin Infect Dis 1995; 20:1102-10.
  6. Recommendations for test performance and interpretation from the
     Second National Conference on Serologic Diagnosis of Lyme Disease.
     MMWR 1995; 44:590-1.
  7. Engstrom SM, Shoop E, Johnson RC. Immunoblot interpretation criteria
     for serodiagnosis of early Lyme disease. J Clin Microbiol 1995;
     33:419-27.
  8. Magnarelli LA. Current status of laboratory diagnosis for Lyme
     disease. Am J Med 1995; 98:10S-14S.


(Pages 47-54)

Unexplained Deaths Due to Possibly Infectious Causes in the United States:
Defining the Problem and Designing Surveillance and Laboratory Approaches

Many new infectious diseases have been identified in the United States
during the last several decades (1). Among these are AIDS, Legionnaires'
disease, toxic-shock syndrome, hepatitis C, and most recently, hantavirus
pulmonary syndrome; all caused serious illness and death. In each instance,
the disease was recognized through investigation of illness for which no
cause had been identified. Retrospective studies of these and other newly
recognized infectious diseases often identified cases that occurred before
the recognition of the new agent; therefore, a more sensitive detection
system may make the earlier recognition of new infectious agents possible.

Delays in recognizing new infectious agents have often been substantial.
For instance, Legionella pneumophila was established as the cause of
Legionnaires' disease in 1976 after an epidemic in Philadelphia, but
sporadic cases in 1947 and an outbreak in 1957 were retrospectively
identified (2, 3). Similarly, toxic shock syndrome was recognized in late
1979 and early 1980, but retrospective reporting and chart reviews
documented cases as early as 1960 (4). HIV was identified in 1983 (5) yet
retrospective investigations documented AIDS cases in the late 1970s and
possibly as early as 1968 in the United States (6, 7).

The difficulty of identifying unknown etiologic agents is part of the
reason for delays between the occurrence and recognition of new infectious
diseases. Until recently, to identify new infectious agents we relied
primarily on culture techniques. For fastidious bacteria such as Legionella
sp., and new viruses, such as HIV, which have very specific growth
requirements, successful isolation usually required numerous attempts with
various culture systems, often extending over years. Advances in molecular
techniques, including polymerase chain reaction (PCR) amplification and
other DNA- (and RNA-) based techniques (e.g., representational difference
analysis), allow identification and classification of unknown etiologic
agents without having to culture them (8-10) and provide clues concerning
appropriate conditions for subsequent isolation of the agent in culture
(11,12).

A more systematic public health approach for the early detection of unknown
infectious agents is needed. This need was acknowledged in Addressing
Emerging Infectious Diseases Threats: A Prevention Strategy for the United
States, a CDC publication about emerging infections (13). CDC has
established an emerging infections program (EIP) network to conduct special
population-based surveillance projects, develop surveillance methods, pilot
and evaluate prevention strategies, and conduct other epidemiologic and
laboratory studies. In late 1994, CDC funded four programs based at state
health departments and academic institutions in California (Alameda, Contra
Costa, Kern, and San Francisco counties), Connecticut, Minnesota, and
Oregon. Some projects are conducted at all program sites and others,
depending on local interest and expertise, at only one or two sites.

Surveillance for unexplained deaths due to possibly infectious causes
(UDPIC) for early detection of new infectious diseases is one of the core
activities being conducted at all sites. This paper estimates the number of
UDPIC at the EIP programs and summarizes the surveillance and laboratory
approaches that will be used to identify their cause. This is the first
attempt to conduct surveillance for early detection of new infectious
diseases in a large U.S. population.

To estimate the number of deaths that might be identified in surveillance
for UDPIC, we used multiple cause-of-death data for the United States for
1992 from the National Center for Health Statistics (14). The year 1992 was
the most recent for which national data were available at the time of this
study. The analyses of death records were restricted to the EIP program
populations and age group (1-49 years of age) in which surveillance for
UDPIC was planned. Multiple cause-of-death data listed on the National
Center for Health Statistics death record allow for analysis of mortality
data based on the different causes (15). The International Classification
of Diseases, 9th Revision (ICD-9) was used to define UDPIC (16). We
selected 77 codes likely to represent UDPIC when listed on the death record
(Table 1) (17).
 ----------------------------------------------------------------------------------------------------------
 Table 1. Selected codes from Intemational Classification of Diseases, 9th 
revision 
  (ICD-9) used to identify unexplained deaths due to possibly 
infectious causes (UDPIC)
 ----------------------------------------------------------------------------------------------------------
 007.9 unspecified                420.9 other and                      785.6 enlargement of
           protozoal                             unspecified                             lymph nodes
           intestinal disease                 acute pericarditis
 008.5 bacterial                    421.0 acute and subacute        786.0 dyspnea and
          enteritis,                               bacterial                                   respiratory
          unspecified                          endocarditis                              abnormalities
 008.8 intestinal                    421.9 acute endocarditis,         792   nonspecific
           infectious                             unspecified                            abnormal
           due to other                                                                       findings in 
other
           organisms:                                                                         
body substances
           other organism,                                     
           not
 classified
           elsewhere
 009.0 infectious                   422.9 other and                       792.0 cerebrospinal
           colitis,                                  unspecified                              fluid
           enteritis, and                        myocarditis
           gastroenteritis
 009.1 colitis,                        424.9 endocarditis,                  792.1 stool contents
          enteritis, and                        valve unspecified
          
gastroenteritis
          of presumed
          infectious
 origin
 009.2 infectious                   425.4 other primary                  792.2 semen
          diarrhea                               cardiomyopathies
 009.3 diarrhea of                 425.9 secondary                      792.3 amniotic fluid
          presumed                            cardiomyopathy,
          infectious origin                   unspecified
 027.9 unspecified                446.6 thrombotic                       792.4 saliva
          zoonotic                              microangiopathy
          bacterial disease
 038.9 unspecified                465.0 acute                              792.9 other 
nonspecific
          septicemia                           laryngopharyngitis                    
abnormal findings
                                                                 
                                  in body
                                                           
                                                                                                   substances
 041.9 bacterial                     465.8 acute upper                    795   nonspecific
          infection                                respiratory                              abnormal
          in conditions                          infections of                            histologic and
          classified                               multiple or                               immunologic
          elsewhere and of                    unspecified sites:                    findings
          unspecified site:                     other multiple sites
          bacterial
          infection,
          unspecified
 046.9 unspecified slow          465.9 acute upper                    795.3 nonspecific
          virus                                     respiratory                               positive
 culture
          infection of the                      infections of                             
findings
          central                                  multiple or                
          nervous system                     unspecified sites:
                                                      unspecified site
 047.9 unspecified viral           466.0 acute bronchitis              795.4 other
          meningitis                                                                            nonspecific
                                                                                                     abnormal
                                                                                                     histologic
                                                                                                     findings
 049.9 unspecified                  466.1 acute bronchiolitis           795.7 other
          non-arthropodborne                                                              nonspecific
          viral diseases of                                                                    immunologic
          central nervous                                                                     findings
          system
 057.9 viral exanthem,             480.9 viral pneumoma,              796.4 other
          unspecified                            unspecified                               nonspecific
                                                                                                      abnormal
                                                                                                      findings:
                                                                                                      other abnormal
                                                                                                      clinical
 findings
 079.9 viral infection in            482.9 bacterial pneumonia,       798   sudden death,
          conditions classified               unspecified                             cause unknown
          elsewhere and of
          unspecified site:
          unspecified viral
          and
 chlamydial
          infection
 099.0 venereal disease,          485 bronchopneumonia,           798.1 instantaneous
          unspecified                         organism unspecified                  death
 136.9 other and                      486 pneumonia, organism        798.2 death occurring
          unspecified                          unspecified                                in less than 24
          infectious and                                                                       hours from the
          parasitic                                                                               onset of
          diseases:                                                                              symptoms, not
          unspecified                                                                           otherwise
          infectious and                                                                        explained
          parasitic diseases
 283.1 non-autoimmune            511.9 unspecified pleural         798.9 unattended
          hemolytic anemias                   effusion                                  death
 284.8 other specified               518.4 acute edema of lung,      799    other 
ill-defined
          aplastic anemias                      unspecified                              and
 unknown 
                                                                                                      causes
 of morbidity
                                                                                                      
and mortality
 286.6 defibrination                   518.8 other diseases of            799.0 asphyxia
          syndrome                                lung
 287.3 primary                          519.9 unspecified disease        799.1 respiratory
          thrombocytopenia                    of respiratory system               failure

 287.5 thrombocytopenia,          558   other and                       799.3 debility,
          unspecified                              unspecified                             unspecified
                                                         noninfectious
                                                         gastroenteritis
                                                         and colitis
 320.9 meningitis due to             780.6 pyrexia of unknown         799.4 cachexia
          unspecified                              origin
          bacterium
 322.9 meningitis,                      782.1 rash and other                799.8 other
          unspecified                              nonspecific skin                        ill-defined
                                                         eruption                                    conditions
 323.9 unspecified cause           782.7 spontaneous                   799.9 other unknown
          of encephalitis                          ecchymoses                            and 
unspecified
                                                                                                        
cause
 357.0 acute infective                 785.5 shock without
          polyneuritis                               mention of trauma
 ----------------------------------------------------------------------------------------------------------

Analyses for UDPIC were restricted to previously healthy persons 1 to 49
years of age by excluding persons outside this age-group and those who had
any of the following ICD-9 codes as an underlying cause of death: 140 to
239.9, neoplasms; 250.0 to 250.9, diabetes mellitus; 279.0 to 279.9,
disorders involving the immune mechanism; 295.5, other disease of spleen;
800 to 999.9, injury and poisoning; E800 to E998, supplementary
classification of external causes of injury and poisoning. Patients with
HIV disease listed anywhere on the death record were also excluded (codes
042, 042.0, 042.1, 042.2, 042.9, 043, 043.0, 043.1, 043.2, 043.3, 043.9,
044, 044.0, 044.9, and 795.8) (18).

Deaths meeting the study criteria were identified along with patient age,
gender, race (black, white, and other), and autopsy status for the four
EIPs (aggregate and by EIP program). To determine rates of UDPIC, we used
1992 census estimates for the four EIP programs (19).

In 1992, 744 UDPIC were identified among previously healthy persons 1 to 49
years of age in the four EIP sites. These deaths accounted for 14% of all
deaths (n = 5,304) among persons 1 to 49 years of age in hospitals and
emergency rooms. Most of the 744 UDPIC occurred among male patients (60%)
and whites (72%) (Table 2). Overall rates among blacks were almost four
times as high as those among whites (29.5 vs. 7.7 per 100,000). By site,
overall rates ranged from 5.6 (in Minnesota) to 14.5 (in California) per
100,000 population. These geographic differences could be accounted for
only in part by differences in the proportions of blacks by site. In
Minnesota and Oregon the proportions of blacks were 2.8% and 1.9%,
respectively, whereas in California and Connecticut the proportions were
14.7% and 12.4%, respectively.

Figure 1 shows the age-specific rates of UDPIC for persons 1 to 49 years of
age. Persons 1 to 24 years of age accounted for only 19% of deaths, while
persons 40 to 49 years of age accounted for 50%.

[Image]
Figure 1. Age-specific rates of unexplained deaths due to possibly
infectious causes (UDPIC) among previously health persons 1 to 49 years of
age in the four emerging infections program sites, 1992.

Of selected ICD-9 codes (Table 1), the six disease classifications (and
codes) accounting for the most of the UDPIC are shown in Table 3. A
selected ICD-9 code was listed as the underlying cause of death in 253
(34%) of 744 UDPIC. Autopsies were performed in 293 (39%) of the 744 UDPIC.

Two approaches for surveillance were proposed as a basis for the EIP
project. In the first, clinicians will be asked to report unexplained
deaths and serious illnesses from possibly infectious causes. In the
second, death certificate databases will be used to select patients with
ICD-9 codes likely to represent UDPIC. The first approach allows
prospective collection of data and specimens for deaths and serious
illnesses. In the second approach, UDPIC will be identified retrospectively
through information on death certificates.
 ----------------------------------------------------------------------------------------------------------

   Table 2. Unexplained deaths due to possibly infectious causes (UDPIC)
   among previously healthy persons by emerging infection program (EIP)
                                site, 1992
 -----------------------------------------------------------------------------------------------------------
                                  Rate (per 100,000 population aged 1-49 years)
                          -------------------------------------------------
--------------------------------
                                                      Gender               Race
                                    ---------------------------------------
--------------------------------
                        No. of
 EIP site           UDPIC   Overall   Female  Male   Black  White    Other
 --------------------------------------------------------------------------
--------------------------------
 California*          316       14.5      10.8    18.5     34.0    12.2       8.9
 Connecticut        83       14.2      10.5    18.5     37.9    11.4        --
 Minnesota           189        5.6        4.8      6.6     11.0     5.4        9.5
 Oregon               156        7.2        6.9      7.7     21.8     7.0        7.8
 Total                   744        8.9        7.4    10.9     29.5     7.7        8.7
 --------------------------------------------------------------------------
--------------------------------
 *Alameda, Contra Costa, and San Francisco counties.
 New Haven County.
---------------------------------------------------------------------------
--------------------------------
--------------------------------------------------------------------------
--------------------------------
-
 Table 3. Of selected ICD-9 codes, disease classifications accounting for
 most unexplained deaths due to possibly infectious causes (UDPIC) in the
                          four study sites, 1992
--------------------------------------------------------------------------
--------------------------------

                                                            UDPIC with ICD-9 code included on death
                                                                          record by age group (%)
                                                            ----------------------------------------
--------------

 Disease classification (ICD-9)*             1-49 yr;      1-14 yr;    15-39 yr;     40-49 yr;
                                                            n = 744      n = 75      n = 295       n = 374
 --------------------------------------------------------------------------
--------------------------------
 Respiratory failure (799.1)               205 (28)     14 (19)     91 (31)      100 (27)
 Unspecified septicemia (038.9)        108 (14)       8 (11)     42 (14)        58 (16)
 Pneumonia, organism unspecified
 
(486)                                              101 (14)       7 (9)       33 (11)        61 (16)
 Other primary cardiomyopathy
 
(425.4)                                             84 (11)       5 (7)        26 (9)         53 (14)
 Shock without mention of trauma
 (785.5)                                             83 (11)     10 (13)       29 (10)       44 (12)
 Other unknown or unspecified
 
(799.9)                                             75 (10)       9 (12)       35 (12)       31 (8)
 Totals                                            505 (68)     39 (52)     193 (65)      273 (73)
 -------------------------------------------------------------------------
--------------------------------
 *More than one of these disease classifications (ICD-9 code) may be
 listed on a death record.
 UDPIC with at least one of the six disease classifications included on
 the death record.
 --------------------------------------------------------------------------

Clinicians in the EIP areas have been asked to report by telephone to EIP
program surveillance personnel all previously healthy persons 1 to 49 years
of age who are hospitalized (or admitted to an emergency room) with a
life-threatening illness with hallmarks of an infectious disease for which
no cause is identified. Inclusion and exclusion criteria are shown below.

Inclusion criteria

  1. 1 to 49 years of age
  2. Admitted to a hospital or emergency room with life-threatening illness
     of potentially infectious etiology
  3. No cause for illness identified by preliminary testing

Exclusion criteria

  1. Preexisting chronic medical condition: malignancy; HIV infection;
     chronic cardiac, pulmonary, renal, hepatic or rheumatologic disease;
     or other known underlying chronic illness (e.g., diabetes mellitus)
  2. Immunosuppressive therapy
  3. Trauma
  4. Toxic ingestion or exposure
  5. Nosocomial infection

Clinicians and pathologists in the four EIP programs were informed of the
surveillance system through a combination of mailings, oral presentations,
and posters.

Classifying patients as having one or more infectious disease-related
syndrome(s) as listed below should help identify groups of patients with
similar illnesses for laboratory testing.

  1. Acute abdominal symptoms (e.g, diarrhea, pain, nausea/vomiting) and
     history of (h/o) fever
  2. Arthritis or osteomyelitis and h/o fever
  3. Blood cell dyscrasia or coagulopathy and h/o fever
  4. Conjunctivitis, keratitis, endophthalmitis, or periocular infection
     and h/o fever
  5. Endocarditis, myocarditis, pericarditis and h/o fever
  6. Hepatitis or hepatic insufficiency/failure and h/o fever
  7. Meningitis, encephalitis, encephalopathy, dementia, or other
     neurologic syndrome with or without a h/o of fever
  8. Rash, skin or mucosal membrane lesions, cellulitis, myositis,
     lymphadenitis, or lymphangitis and h/o of fever
  9. Renal insufficiency/failure and h/o of fever
 10. Respiratory failure, pulmonary infiltrates, or other pleuropulmonary
     manifestation and h/o of fever
 11. Shock or sepsis and h/o of fever or hypothermia
 12. Other

Information about exposures (e.g., travel or contact with animals or
insects) resulting in infectious diseases will be collected. For patients
who are still alive or have died recently, clinical and pathology
laboratories will be asked to save clinical specimens (including biopsied
tissues) obtained during clinical care and diagnostic evaluation. Range of
specimens will vary but be appropriate for the given illness and organ
systems affected. These specimens will be collected, divided into aliquots,
and stored. Autopsies will be encouraged. With the exception of pathology
specimens, specimens will be initially banked at the EIP sites. Fixed or
frozen tissue specimens (premortem and postmortem) will be sent directly to
CDC for examination. A CDC pathologist will be available to consult with
the local pathologist and to discuss preparation and transport of tissues.
Pathology results are expected to guide further laboratory testing on
specimens.

Clinical and epidemiologic data will be periodically reviewed locally at
each EIP and at CDC in aggregate. Each EIP will identify UDPIC not reported
through the clinician-based system by using state-based (rather than
national) electronic data systems to reduce delays in relaying information.
When deaths not reported through the clinician-based system are identified,
the medical chart will be reviewed, the patient's illness will be
classified by syndrome and information available in the medical record
concerning exposures will be collected. Samples of specimens will be
obtained at autopsy. Deaths will be handled as in the clinician-based
system with regard to periodic review and laboratory testing, although it
is expected that fewer clinical specimens will be available from patients
whose deaths were not reported through the clinician-based system.

Additional reference level laboratory tests for known pathogens will be
done in state health laboratories and CDC. CDC will test for previously
unrecognized infectious agents.

Initial identification of unrecognized etiologic agents at CDC will
primarily rely on serology, immunohistochemistry, and nucleic acid probes.
When a sufficient number of patients with similar illnesses are identified,
a customized strategy for laboratory testing will be designed. Serology and
immunohistochemistry will be used to narrow the scope of possible
etiologies. Nucleic acid probes will be used with PCR to amplify from
clinical specimens specific fragments of genetic material that can be
sequenced and used for phylogenetic comparisons to known infectious agents.
Clinicians who reported cases will be informed of laboratory results, but
information will usually not be available in time to affect treatment of
individual patients.

Until now, unexplained deaths and serious illnesses due to possibly
infectious causes have not been addressed as a specific public health
problem. The data obtained in the first phase of this project suggest that
UDPIC in previously healthy persons account for 13% of hospitalized deaths
among persons 1 to 49 years old in the EIP sites. Experience in recent
years with new infectious diseases suggests that systematic study of UDPIC
and similarly unexplained serious illnesses may allow earlier detection of
emerging infections. This has been made more feasible by newly developed
nucleic acid-based methods for identification of unknown etiologic agents.

Use of the 1992 National Center for Health Statistics multiple
cause-of-death data to estimate the number of UDPIC has its limitations.
The most important is in the selection of ICD-9 codes to identify these
deaths. Even with codes such as 038.9 ("unspecified septicemia"), which
seem relevant, without reviewing the medical record it is impossible to
know if the cause of the septicemia was known by the clinician but not
specified or was nosocomial. Codes representing potentially infectious
deaths (e.g., 799 for "other ill-defined and unknown causes of morbidity
and mortality") might also be assigned to noninfectious deaths. Another
critical limitation is failure to identify deaths that are, in fact,
unexplained but have been given an incorrect diagnosis.

For several reasons, our surveillance is limited to persons 1 to 49 years
of age who have been healthy. The 1-year lower age limit was selected to
avoid confusion with congenital problems in infants but include most
children in day-care, where infectious diseases are common and a new
infectious disease might spread rapidly. The upper age limit was set to
exclude an expected increased proportion of unexplained deaths from
noninfectious causes in persons 50 years and older. Many of the recently
recognized life-threatening infectious diseases would have been detected
among previously healthy persons in this age-group. Previously healthy
persons might also be considered better sentinels for new infectious
diseases because of their generally more vigorous interaction with people
and higher likelihood of exposure to infections (e.g., travel or contact
with animals or insects). However, restricting surveillance to previously
healthy persons is likely to decrease the sensitivity of our system.

Patients who are immunocompromised whether from HIV infection, malignancy,
or immunosuppressive therapy and many patients with other chronic
illnesses, are more susceptible to known and unknown infectious diseases.
New infectious diseases first identified in persons who are
immunocompromised or have chronic illnesses have subsequently been found to
also cause infection in persons with normal immune systems (20, 21).
Although sensitivity could be improved by including these populations in
surveillance, available resources and a concern that laboratory evaluation
would be complicated by the broader range of infectious possibilities
compelled us to focus on previously healthy persons.

Clinician-based and death certificate based systems for surveillance and
laboratory evaluation are being used in combination because of their
complementary strengths and weaknesses. The notable strengths of the
clinician-based system are the contribution of clinicians and the
timeliness of reporting. Because of their training and their relationship
with patients, clinicians can recognize unusual and potentially new
infections. This system also offers opportunities to collect and store
clinical specimens (pre-mortem and postmortem) that would not normally be
saved, in addition to providing systematic and timely collection of
exposure information that might not be available in the medical record.
This system might also increase the likelihood of an autopsy. However,
reporting is time-consuming and is not likely to affect the patient's care,
which may lower the sensitivity of this approach.

The primary strengths of the death certificate based system are its
completeness and relative ease, once the data are electronically available.
The completeness may make it sensitive for detection of new infections
resulting in death (but assumes that the correct ICD-9 codes are selected
and that they are coded accurately). Sensitivity is important because, to
be effective, the combined approaches should detect relatively rare
illnesses (e.g., in the range of one case per 100,000 to 1,000,000
population per year). The main disadvantages of this system are the
vagaries of ICD-9 classification: codes are not designed to identify new
infectious diseases and are assigned by persons not directly familiar with
the case. The list of ICD-9 codes used to identify UDPIC is likely to be
modified on the basis of information collected in this system and in the
clinician-based system. Another problem is the delay in getting information
on the death certificate into the database for review, which makes this
system relatively slow. Further, the only clinical specimens likely to be
available for laboratory evaluation are those collected at autopsy.

The goal of our project is early detection of new life-threatening
infectious diseases. However, it is likely that in the process, we will
identify cases in which known, but poorly recognized, infectious diseases
are responsible, either because the diagnostic tests being used clinically
are of poor sensitivity or because the diagnosis was unexpected by
clinicians. Findings concerning such cases may be useful in identifying
areas in which better diagnostic capabilities are needed and in improving
estimates of infectious disease prevalence (22). A population-based bank of
clinical specimens will be invaluable in current and future testing for
newly recognized etiologic agents and for developing diagnostic tests. This
project will better clarify surveillance strategies and help standardize
nucleic acid-based techniques for identification of previously unknown
etiologic agents. Through it, we expect to build U.S. capacity for
detecting and responding to newly recognized infectious diseases not only
at the EIP sites but elsewhere, nationally and internationally.

Bradley A. Perkins,* Jennifer M. Flood, Richard Danila, Robert C.
Holman,* Arthur L. Reingold, Laura A. Klug,* Michael Virata, Paul R.
Cieslak, Sherif R. Zaki,* Robert W. Pinner,* Rima F. Khabbaz,* and the
Unexplained Deaths Working Group#

*National Center for Infectious Diseases, Centers for Disease Control and
Prevention, USA, Atlanta, Georgia; School of Public Health, University of
California at Berkeley, California, USA;

Minnesota Department of Health, Minneapolis, Minnesota, USA; Yale
University School of Medicine, New Haven, Connecticut, USA; Oregon
Department of Human Resources, Portland, Oregon, USA

#The Unexplained Deaths Working Group: Grechen Rothrock, University of
California at Berkeley; Duc Vugia, California Department of Health
Services; James Hadler, Matt Cartter, Connecticut Department of Public
Health and Addiction Services; James Meek, Robin Ryder, Mark Wilson, Yale
University School of Medicine; Michael Osterholm, Kristine L. MacDonald,
Jean Rainbow, Norman Crouch, Kathy LeDell, Minnesota Department of Health;
David Fleming, Katrina Hedberg, Oregon Health Division; Don Brenner, Mark
Eberhard, James Olson, Pierre Rollin, R. Gibson Parrish, CDC.

References

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  2. McDade JE, Brenner DJ, Bozeman FM. Legionnaires' disease bacterium
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  3. Osterholm MT, Chin TDY, Osborne DO, et al. A 1957 outbreak of
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  4. Osterholm MT, Forfang JC. Toxic-shock syndrome in Minnesota: results
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  5. Barre-Sinoussi F, Chermann JC, Rey F, et al. Isolation of a
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  6. CDC Task Force on Kaposi's Sarcoma and Opportunistic Infections.
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  7. Garry RF, Witte MH, Gottlieb A, et al. Documentation of an AIDS virus
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  8. Relman DA, Loutit JS, Schmidt TM, Falkow S, Tompkins LS. The agent of
     bacillary angiomatosis: an approach to the identification of
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  9. Nichol ST, Spiropoulou CF, Morzunov S, et al. Genetic identification
     of a hantavirus associated with an outbreak of acute respiratory
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 10. Chang Y, Cesarman E, Pessin MS, et al. Identification of
     herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma.
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     angiomatosis. N Engl J Med 1992;327:1625-31.
 12. Elliot LH, Ksiazek TG, Rollin PE, et al. Isolation of the causative
     agent of hantavirus pulmonary syndrome. Am J Trop Med Hyg
     1994;51:102-8.
 13. Centers for Disease Control and Prevention. Addressing emerging
     infectious disease threats: a prevention strategy for the United
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     documentation. Multiple cause of death for ICD-9 1992 data.
     Hyattsville, MD: U.S. Department of Health and Human Services, 1994.
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     Milwaukee of Cryptosporidium infection transmitted through the public
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     bacillary splenitis in immunocompetent adults. Ann Intern Med
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     mortality in the United States. JAMA 1996;275:189-93.



(Pages 54-56)

Trends in Bacteremic Infection Due to Streptococcus pyogenes 
(Group A Streptococcus), 1986-1995

During the past 7 years, severe invasive infections caused 
by Streptococcus pyogenes (group A streptococcus [GAS]) have 
been reported with increasing frequency (1). It is not certain 
whether these reports reflect actual increase in the incidence 
of disease caused by this pathogenic bacterium or merely enhanced 
awareness and interest on the part of the medical community. 
This difficulty prevails whether the apparent resurgence of
life-threatening infection is described within a region (2), 
a province (3), or in discrete outbreaks (4,5) and is heightened 
by the fact that streptococcal infection has not been a 
reportable disease in most locales.

An outbreak of infection may awaken interest in streptococcal 
disease, leading to publication bias (6,7). Continued 
study may show that what appears to be a general increase 
in the incidence of GAS infection may actually represent an 
outbreak, as has been demonstrated in Colorado (8,9), Sweden 
(10), and the United Kingdom (11). In a laboratory-based study,
Burkert and Watanakunakorn (12) found that the frequency and 
apparent severity of bacteremic infection due to S. pyogenes 
did not change from 1980 to 1989. A meticulous, population-
based study in Pima County, Arizona (13), found no change 
in the incidence of invasive GAS disease between 1985
and 1990 but did suggest the emergence of streptococcal 
toxic shock syndrome in the late 1980s, on the basis of 
the appearance of six such cases in 1987-1990 compared to 
zero cases in 1985-1987. In contrast, a preliminary, 
population-based report from Israel (14) found no such
increase.

The Houston Veterans Affairs Medical Center (HVAMC) serves 
about 50,000 eligible veterans, most in Harris County, 
Texas, and surrounding counties, although a small number 
of these veterans live elsewhere in Texas or in bordering 
states. Most patients are middle-aged or elderly men, although
approximately 20% are under the age of 40. Within the past
decade, there have been broader medical options for some 
of our patients, especially those who are indigent and/or 
elderly, and narrower options for others, especially 
those whose medical insurance was initially marginal.
Nevertheless, most persons who select the HVAMC for care 
of any problem tend to identify themselves with this 
medical center and look to it to provide complete 
medical care (15).

In contrast to that reported by some medical centers in 
the United States, our clinical experience did not 
suggest that the number or severity of infections due to 
S. pyogenes had increased during the past decade. Because
our population was relatively stable and data were 
available in our Microbiology Laboratory for 1986 
through 1995, we studied trends in bacteremic infection 
at the medical center during this period.

In the 9 years under study, the number of yearly inpatient 
admissions to the HVAMC decreased by 4.1%, while the 
number of outpatient visits increased by 33.9%. The 
number of patients who, in a given year, sought
medical attention at the center (determined by social 
security numbers), increased by 18.7%, with a 5.2% 
decrease in those persons admitted to the hospital and 
a 19.4% increase in those who sought outpatient care. 
During this period, the number of blood specimens 
submitted for cultures each year remained essentially 
unchanged. We have noted no change in the clinical 
findings that prompt interns and residents to request 
blood cultures, although the possibility of unrecognized, 
subtle changes cannot be excluded.

The rate of isolation of GAS from blood cultures 
(Figure 1 is not available in ASCII) or from all sterile 
sites (data not shown) remained unchanged between 1986 
and 1995. The number of positive blood cultures tended to 
be lower in the summer and early fall and to rise in 
midwinter, but the average number has remained at 
approximately five to eight per year during the entire 
decade. The frequency with which GAS were isolated from 
all specimens declined significantly (p = 0.003) during 
the decade under observation.

These data suggest that the frequency of bacteremic 
infection due to S. pyogenes has not increased in a 
population of veterans residing in and around Houston, 
Texas. The number of isolates of S. pyogenes from blood 
and from all sterile sites has remained unchanged, 
whereas the number of persons served in inpatient and 
outpatient encounters actually increased. The slight, 
but significant decrease in the total number of isolates 
from all sources might suggest that an unchanging number 
of blood isolates represents an increased prevalence 
of virulent strains, but the large number of variables 
involved in collecting all specimens makes these data
difficult to interpret.

By showing that the incidence of S. pyogenes bacteremia 
has not changed, these results appear to support our 
anecdotal observation that the incidence of life-
threatening GAS infection has not increased. Without
reviewing every single case, which is best done 
prospectively rather than retrospectively, we cannot 
exclude the possibility that the number of cases
associated with streptococcal shock syndrome has 
increased, as was suggested in Pima County (13). 
Even that study, however, showed only six cases of 
shock (two per year) in 1987 through 1990 compared to 
none in 1985 through 1987; one wonders whether bias 
in recording data might have been partially responsible, 
and whether this increase will persist. We also do
not know what proportion of our cases is associated 
with necrotizing fasciitis, although it must be noted 
that Hoge, et al. (13) specifically did not document 
the association between this clinical syndrome and
streptococcal toxic shock. Our findings cannot exclude 
the possibility that serious GAS has increased in younger 
adults, as has been reported by others (1,13), since our 
population tends to be (but is not exclusively) middle-aged 
or older. Also, the effect of increases may not have been
observed in Houston. In any case, reporting bias is 
difficult to avoid, and prospective population-based 
studies must be carried out over an adequate
number of years before they can provide valid conclusions.

If the frequency of severe GAS infection has, in fact, 
increased, a virulent clone or the mutation of a clone 
that has antigens to which most adults lack antibody 
could be responsible. Serotype M1 has predominated in
reports of streptococcal toxic shock syndrome (16-18). 
Some investigators (19) have proposed that allelic 
variation in the streptococcal pyrogenic exotoxin A 
gene (speA) may contribute to disease severity; however, 
others have not found a high prevalence of isolates that 
produce pyrogenic exotoxin A among organisms that cause 
streptococcal toxic shock syndrome. Although the gene 
that encodes pyrogenic exotoxin B, a cysteine protease,
has been universally present in virulent isolates, this 
gene is also present in nearly all other GAS isolates; 
therefore, its importance in toxic shock-producing 
strains remains unclear. Finally, changes in herd 
immunity to certain exotoxin B variants and/or M protein 
serotypes may result in an increased susceptibility to 
newly emerging mutant strains (20).

Acknowledgment

The authors are grateful to Nancy J. Petersen, M.S., for 
help in obtaining VA Medical Center utilization data.

Daniel M. Musher, Richard J. Hamill, Charles E. Wright, 
Jill E. Clarridge, Carol M. Ashton
Veterans Affairs Medical Center, Houston Veterans 
Affairs Health Services Research and Development 
Field Program, and Baylor College of Medicine,
Houston, Texas

References

  1. Stevens DL. Streptococcal toxic shock syndrome: spectrum 
  of disease, pathogenesis, and new concepts in treatment. 
  Emerging Infectious Diseases 1995; 1:69-78.
  2.  Stevens DL, Tanner MH, Winship J, et. al. Reappearance 
  of scarlet fever toxin A among streptococci in the Rocky 
  Mountain West: severe group A streptococcal infections 
  associated with a toxic shock-like syndrome. N Engl J 
  Med 1989; 321:1-7.
  3.  McGeer K, Green R, Cann D, et. al. Changing 
  epidemiology of invasive group A streptococcal 
  infectionpopulation based surveillance, Ontario Canada, 
  1992-1995. Presented at the 35th Interscience Conference on
  Antimicrobial Agents and Chemotherapy, 1995; Abstract K135,
  4.  Cockerill F III, Thompson R, Roberson F, et. al. 
  Outbreak of invasive group A streptococcal infections 
  (ISI) in residents of southeastern Minnesota. 
  Presented at the 35th Interscience Conference on
  Antimicrobial Agents and Chemotherapy, 1995; Abstract K137.
  5.  Levine OS, Turf E, Ginsberg R, et. al. An outbreak 
  of invasive group A streptococcal disease in the 
  Shenandoah Valley of Virginia. Presented at the 35th 
  Interscience Conference on Antimicrobial Agents
  and Chemotherapy, 1995; Abstract K134.
  6.  Braunstein H. Characteristics of group A 
  streptococcal bacteremia in patients at the San 
  Bernardino County Medical Center. Rev Infect Dis
  1991;13:8-11.
  7.  Demers B, Simor AE, Vellend H, et al. Severe 
  invasive group A streptococcal infections in Ontario, 
  Canada: 19871991. Clin Infect Dis 1993;16:792-800.
  8.  Centers for Disease Control. Group A beta-
  hemolytic streptococcal bacteremiaColorado, 1989. 
  MMWR 1990;39:3-11.
  9.  Colorado Department of Health. Surveillance for 
  group A streptococcal bacteremia in metropolitan 
  Denver. Colo Dis Bull 1991;2:18.
  10.  Stromberg A, Romanus V, Burman LG. Outbreak of 
  group A streptococcal bacteremia in Sweden: an 
  epidemiologic and clinical study. J Infect Dis 
  1991;164:595-8.
  11.  Centers for Disease Control and Prevention. 
  Invasive group A streptococcal infections United 
  Kingdom, 1994. MMWR 1994;43:401-2.
  12.  Burkert T, Watanakunakorn C. Group A 
  streptococcal bacteremia in a community teaching 
  hospital--1980-1989. Clin Infect Dis 1992;14:29-37.
  13.  Hoge CW, Schwartz B, Talkington DF, et al. The 
  changing epidemiology of invasive group A streptococcal 
  infections and the emergence of streptococcal toxic 
  shock-like syndrome. JAMA 1993;269:384-9.
  14.  Ashkenazi S, Livni G, Leibovici L, et. al. 
  Trends in incidence and severity of group A 
  streptococcal (GAS) bacteremia. Presented at the
  35th Interscience Conference on Antimicrobial Agents 
  and Chemotherapy,1995; Abstract K138.
  15.  Fleming C, Fisher E, Chang C, et. al. Studying 
  outcomes and hospital utilization in the elderly. 
  Med Care 1992; 30:377-91.
  16.  Holm SE, Norrby A, Bergholm A-M, Norgren M. 
  Aspects of pathogenesis of serious group A 
  streptococcal infections in Sweden, 19881989. J
  Infect Dis 1992;166:31-7.
  17.  Cleary PP, Kaplan EL, Handley JP, et al. 
  Clonal basis for resurgence of serious Streptococcus 
  pyogenes disease in the 1980s. Lancet 1992;339:518-21.
  18.  Talkington DF, Schwartz B, Black CM, et al. 
  Association of phenotypic and genotypic 
  characteristics of invasive Streptococcus pyogenes
  isolates with clinical components of streptococcal 
  toxic shock syndrome. Infect Immun 1993;61:3369-74.
  19.  Musser JM, Kapur V, Kanjilal S, et al. Geographic 
  and temporal distribution and molecular characterization 
  of two highly pathogenic clones of Streptococcus 
  pyogenes expression allelic variants of pyrogenic 
  exotoxin A (scarlet fever toxin). J Infect Dis
  1993;167:337-46.
  20.  Musser JM, Nelson K, Selander RK, et al. Temporal 
  variation in bacterial disease frequency: molecular 
  population genetic analysis of scarlet fever epidemics 
  in Ottawa and in Eastern Germany. J Infect Dis 
  1993;167:759-62.


LETTER

(Pages 57-58)


PHLS Surveillance of Antibiotic Resistance, England and Wales:
Emerging Resistance in Streptococcus pneumoniae

To the Editor: The commentary, by Dr. M. S. Cetron and
colleagues, on the action plan for drug-resistant Streptococcus
pneumoniae (1) prompts us to describe the main system for
surveillance of antibiotic resistance in use by the Public Health
Laboratory Service (PHLS) in England and Wales and our recent
results for resistance in S. pneumoniae.

Since 1974, the diagnostic laboratories in the PHLS network (53
in 1993-94) and increasing numbers of National Health Service and
private laboratories have reported, on a voluntary basis, all
bacterial isolations from blood or cerebrospinal fluid to the
PHLS Communicable Disease Surveillance Centre. Since 1989, they
have been asked to include their antimicrobial susceptibility
test results on all isolates. In 1993, for example, antimicrobial
susceptibility test results were received from 195 laboratories,
on >28,000 nonduplicate isolates. Some data are sent as written
entries on the report forms, but increasingly they are being
transmitted electronically. Results may be analyzed according to
region or reporting laboratory or by patient characteristics,
such as age.

All laboratories do not test the same antimicrobial agents, but a
nucleus set is tested by most laboratories for each species.
Results are reported as "susceptible" (S), "resistant" (R) or
"intermediate" (I). Although the methods are not standardized,
external quality assurance is provided by the UK National
External Quality Assessment Scheme (2), to which almost all
laboratories subscribe. In addition, many laboratories refer
isolates that show particularly critical resistance traits (such
as -lactam resistance in S. pneumoniae, or glycopeptide
resistance in Enterococcus species) to the PHLS Antibiotic
Reference Unit (ARU) for determination of minimum inhibitory
concentrations (MICs); these can often be matched against the
submitted results. Occasional prevalence surveys in the PHLS
network, with testing of isolates in the ARU, act as a further
monitoring measure.

The application of the system can be seen in the recently
published results of 6 years' surveillance of resistance amongst
isolates of S. pneumoniae causing bacteremia or meningitis (3,4).
There has been a statistically significant trend to increased
resistance to penicillin (from 0.3% in 1989 to 2.5% in 1994),
although these are low percentages in comparison with those seen
in many other countries (5). Moreover, a considerable increase
has been observed in resistance to erythromycin (from 3.3% in
1989 to 11.2% in 1994). These figures are based on susceptibility
testing of more than 2,500 isolates in each of the 6 years. In
1993, resistance to penicillin and erythromycin was significantly
more common amongst pneumococci from bacteremia and meningitis in
the younger age groups (9 years). A significant rise during the
6 years was also seen in trimethoprim resistance, but no
significant change was observed in resistance to tetracycline or
chloramphenicol.

The resistance totals include isolates reported as resistant (R),
and as intermediate (I), as we cannot be sure of the basis for
this discrimination in the diagnostic laboratories. The
proportion of isolates reported as I is very small, with the
exception of antimicrobial agents for which many strains have
MICs near the breakpoint defined to separate sensitive and
resistant strains. In the case of S. pneumoniae, this applied to
trimethoprim (6.3% of isolates reported as I in 1993); <0.5% of
isolates were reported as I to other antimicrobial agents.

The results of the National External Quality Assessment Scheme
exercises have shown acceptable proficiency. For example, in the
detection of penicillin resistance, in five distributions of
pneumococcal strains that require an MIC of 0.25 mg/l, 74% to 90%
of laboratories obtained correct results; in six distributions of
strains that require an MIC of 1.0 mg/l. 95% to 99% of
laboratories did so (J.J.S. Snell, pers. comm.). The isolates
included in the analysis that were also tested in the ARU gave
closely similar results: for example, of 86 pneumococcal isolates
tested for susceptibility to penicillin in early 1995, 82 (95%)
gave the same result in the sender's laboratory and in the ARU. A
survey of resistance in pneumococci (from all sites) conducted in
March l995 with MIC determination by the ARU (unpublished) showed
similar proportions of resistant strains.

These observations demonstrate that the results of susceptibility
tests undertaken for the management of individual patients may be
compiled and analyzed for surveillance purposes. Duplicate
isolates from the same infection episode should not be included,
and satisfactory quality assurance should be undertaken.
Increasing use of computers and networking among the clinical
laboratories should facilitate the process of data collection.

David C.E. Speller, Alan P. Johnson, Barry D. Cookson, Pauline
Waight,* Robert C. George
Central Public Health Laboratory and *Communicable Disease
Surveillance Centre, 61 Colindale Avenue, London NW9 5HT, UK

References
1.  Cetron MS, Jernigan DB, Breiman RF, DSRP Working Group.
Action plan for drug-resistant Streptococcus pneumoniae. Emerging
Infectious Diseases 1995;1:64-5.
2.  Snell JJS, de Mello JV, Gardner PS. The United Kingdom
national microbiological quality assessment scheme. J Clin Pathol
1982;35:82-93.
3.  Aszkenazy OM, George RC, Begg NT. Pneumococcal bacteraemia
and meningitis in England and Wales 1982 to 1992. CDR Review
1995;5:R45-R50.
4.  Antibiotic resistance in Streptococcus pneumonia 1993 and
1994. Commun Dis Rep 1995;5:187-8.
5.  Friedland IR, McCracken GH. Management of infections caused
by antibiotic-resistant Streptococcus pneumoniae. N Engl J Med
1994;331;377-82.



COMMENTARY

(Page 59-61)

Infectious Diseases in Latin America and the Caribbean: Are They Really
Emerging and Increasing?


During 1995, infectious disease epidemics in Latin America and the
Caribbean received wide publicity: dengue throughout the region, Venezuelan
equine encephalitis (VEE) in Venezuela and Colombia, and hemorrhagic fever
in Nicaragua. Increased awareness of these diseases followed extensive
reports in the scientific community about the threat of emerging infections
(1,2). Are infectious diseases increasing in the region or are we simply
seeing the results of better reporting of persistent problems? Analysis
suggests that both factors are at work.

Dengue and Dengue Hemorrhagic Fever

During the 1950s and 1960s, under the leadership of the Pan American Health
Organization (PAHO), most countries in the Americas successfully reduced or
eliminated infestation with Aedes aegypti, the principal vector of dengue
and urban yellow fever. As a result, much of the Americas became free of
dengue. Dengue transmission, however, persisted in many Caribbean islands
and in some countries of northern South America that failed to control the
vector; therefore, several outbreaks occurred during the 1960s and in
subsequent decades (3).

Ae. aegypti eradication programs, however, were not sustained and the
mosquito reinfested all Latin American countries except Chile and Uruguay.
As a consequence, dengue spread throughout the region, causing severe
epidemics or even pandemics during the 1970s and 1980s. Currently, dengue
is endemic in virtually all countries with Ae. aegypti, and epidemics occur
periodically.

Between 1968 and 1980, only 60 suspected or confirmed cases of dengue
hemorrhagic fever (DHF) were reported, all by five countries in and around
the Caribbean. After the 1981 DHF outbreak in Cuba, reports of DHF in the
Americas markedly increased. The Cuban epidemic was the most notable event
in the history of dengue in the Americas: almost 400,000 cases of dengue,
over 10,000 cases of DHF, and 158 deaths were reported. The Cuban
authorities implemented a successful vector control program and the country
is still virtually free of Ae. aegypti. After this outbreak, cases of DHF
continued to occur in the Americas, although at relatively low levels,
until 1989 when another large epidemic with 2,500 cases of DHF occurred in
Venezuela. Since then, Venezuela has reported large numbers of DHF cases
every year, and in 1995 the country reported the largest outbreak of
dengue/DHF in its history: almost 30,000 dengue cases and 5,000 DHF cases.
Since 1968, 25 countries of the Americas have reported more than 35,000
confirmed or suspected DHF cases and approximately 500 deaths.

In 1995, dengue and DHF activity in the region was higher than in any year
except 1981. As of November, countries in the Americas had reported more
than 200,000 dengue cases and 6,000 DHF cases, and approximately 90 deaths.
Brazil has had the largest number of dengue cases, but more than 80% of the
DHF cases occurred in Venezuela. The reinvasion of the Americas by dengue
virus type 3, which had been absent for 16 years, has increased the threat
of large epidemics and consequent risk for DHF (4). This serotype was
isolated in Panama and Nicaragua at the end of 1994, and in 1995 it spread
to other Central American countries (except Belize) and Mexico, causing
severe outbreaks. High levels of infestation with Ae. aegypti are common
from the United States to Argentina, making it likely that dengue epidemics
will increase in frequency and severity.

Cholera

Another disease reemerging in the Americas is epidemic cholera, which had
been absent from this hemisphere for approximately 90 years before it was
introduced into Peru in January 1991 (5). Since then more than 1 million
cases of cholera have been reported in 20 countries in the region. Only
Uruguay and the islands of the Caribbean have been spared. Though the
annual total of reported cases has decreased since 1991, the disease is
persistent and problematic in several Latin American countries.

Venezuelan Equine Encephalitis

An outbreak of human infection with VEE virus associated with a large
number of equine cases and deaths was detected in northwestern Venezuela in
April 1995. The disease spread to the adjacent Colombian state of La
Guajira in September (6). Unusually heavy rains during 1994 and 1995
contributed to the epidemic by increasing breeding sites for the mosquito
vectors Ae. taeniorrhynchus and Psorophora confinnis. Viral strains with
epizootic and epidemic potential appear to have emerged from enzootic
strains maintained in enzootic rodent-mosquito cycles (7). In addition,
failure to immunize wild and domestic equine populations allowed the virus
to amplify and spread. By mid-October 1995, reported human cases totaled
26,500 in Venezuela and 22,300 in Colombia, with 24 deaths in the latter.
Attack rates in severely affected communities were 18% to 57%.

This epidemic of VEE is the largest since that of 1962-1971, when the
disease extended from northern South America through Central America and
Mexico to the United States (8). Intensified vector control (including
application of adulticides and larvicides), equine vaccination, and
restriction of equine movement appear to have at least temporarily
controlled the epidemic.

Leptospirosis

In late October 1995, Nicaragua reported several hundred cases of a
hemorrhagic febrile illness in and near the community of Achuapa,
approximately 110 km northwest of Managua; eight patients died. The
affected communities had experienced unusually heavy rains and flooding
during the 2 weeks before the cases were noted. Although dengue was
occurring elsewhere in Nicaragua at the same time, that diagnosis was
excluded by negative laboratory tests and the absence of Ae. aegypti in the
local area. In addition, some of the patients had frank pulmonary
hemorrhage [not typical of dengue]. By the end of October, the Centers for
Disease Control and Prevention had ruled out dengue, other arboviruses, and
hemorrhagic fever viruses as causes but had identified leptospira by
immunohistochemistry in tissues from four patients with fatal cases. By
mid-November, the Ministry of Health reported that 2,480 persons had been
ill, 750 were hospitalized, and at least 16 died. (Investigation to define
the extent of illness and the responsible serovars was still in progress at
this writing.)

Of the examples discussed here, dengue and DHF are certainly reemerging.
Dengue has been signaling its return for more than 15 years and DHF since
1989; these diseases will likely persist as epidemic problems unless
drastic changes in vector control are achieved. Enzootic VEE has persisted
in northern Venezuela and Colombia since the previous major epidemic of
1962-1971. PAHO had urged the countries to increase vaccination coverage of
equines because of increased viral activity in 1993 and 1994. The
appearance in 1995 of strains similar to these of the 1962-1971 epidemics,
with locally intense transmission, raised the possibility that VEE would
reemerge as a major epidemic disease. Whether vigorous vector control
measures and immunization programs have contained that threat is not yet
known, but we must continue to regard the threat as real. Leptospirosis is
a persistent, often under-recognized, problem to which the international
community has paid relatively little attention. In Nicaragua, public health
interest was sparked by concern that the epidemic of a new disease would
pose a threat to other communities and countries, but attention waned as
that threat diminished. Yellow fever, which is usually present in
relatively low numbers in remote areas of South America, reemerged with
force in Peru during 1995. At least seven departments of that country have
been affected (470 cases and a 40% case-fatality rate by September 1995).

Several factors have contributed to the reemergence of infectious diseases
in the Americas. Investments in public health have been decreasing because
of economic recession and a shrinking public sector or have been diverted
from infectious disease programs to other pressing problems (9). Human
populations throughout the region have grown and become increasingly urban,
with many living in inadequate housing without sanitation or potable water.
At the same time, population and commercial pressures have led to the
invasion of forests, exposing people to exotic agents and enzootic
diseases, including yellow fever, rabies transmitted by vampire bats,
arenaviruses, and others. Human behavior has contributed to epidemic plague
in Peru and the rapid spread of diseases such as cholera. To this list can
be added the effects of deforestation and habitat and climate change.
Unusually heavy rainfall contributed to at least three of the epidemics
considered in this commentary (10).

Infectious diseases (whether new or reemerging) are a real and serious
problem in Latin America and the Caribbean. To combat the threat of these
diseases, PAHO, with the participation of other institutions in the region,
has prepared a regional plan to improve surveillance for emerging disease
and enhance countries' ability to respond effectively by strengthening
laboratory capacity, training, and research and by implementing prevention
and control strategies. Ministers of health from countries throughout the
region discussed and endorsed the plan at a meeting of PAHO's Directing
Council in September 1995. Successful implementation of the plan will
require committed action by public health authorities and collaboration and
cooperation by many institutions and experts throughout the region.

A. David Brandling-Bennett, Francisco Pinheiro
Pan American Health Organization, Washington, D.C., USA

References

  1. Institute of Medicine. Emerging infections: microbial threats to
     health in the United States. Washington, DC: National Academy Press,
     1992.
  2. Centers for Disease Control and Prevention. Addressing emerging
     infectious disease threats: a prevention strategy for the United
     States. Atlanta, GA: Centers for Disease Control and Prevention, 1994.
  3. Pan American Health Organization. Dengue and dengue hemorrhagic fever
     in the Americas: guidelines for prevention and control. Washington,
     DC: Pan American Health Organization, 1995.
  4. Centers for Disease Control and Prevention. Dengue type 3
     infectionNicaragua and Panama, October-November 1994. MMWR
     1995;44:21-4.
  5. Tauxe RV, Mintz ED, Quick RE. Epidemic cholera in the New World:
     translating epidemiology into new prevention strategies. Emerging
     Infectious Diseases 1995;1:141-6.
  6. Centers for Disease Control and Prevention. Venezuelan equine
     encephalitisColombia, 1995. MMWR 1995;44:721-4.
  7. Centers for Disease Control and Prevention. Update: Venezuelan equine
     encephalitisColombia, 1995. MMWR 1995;44:775-7.
  8. Walton TE, Grayson MA. Venezuelan equine encephalomyelitis. In: Monath
     TP, ed. The arboviruses: epidemiology and ecology. Boca Raton, FL: CRC
     Press, 1989;203-32.
  9. Pan American Health Organization. Social response to health problems.
     In: Health Conditions in the Americas. Washington, DC: Pan American
     Health Organization, 1994;303-420.
 10. Epstein PR, Pena OC, Racedo JB. Climate and disease in Colombia.
     Lancet 1995:346;1243-4.


(Page 62-63)

Microbial Threats and the Global Society

The public health threat of microbial organisms living in what
can be regarded as one large global society was the subject of a
recent interactive workshop sponsored by Tufts University's
Education for Public Inquiry and International Citizenship 10th
anniversary celebration, held at Tufts University in Medford,
Massachusetts. The participants* discussed microbes as the
harbingers of disease and society as both potential victim and
guardian of health. Microbial threats were identified as new,
reemerging, and not yet known.

The forum examined the many unanswered questions regarding the
origin and causes of infectious disease agents. The failure of
traditional treatments due to antibiotic resistance and the
ineffective control and continued spread of infectious agents
were also discussed. Participants addressed environmental and
behavior factors that foster the "amplification" and "spread" of
disease organisms: bathhouses conducive to the spread of HIV
infection, homelessness and crowded living promoting the spread
of tuberculosis, day-care centers that are ideal environments for
the spread of drug-resistant pneumococcus.

In the context of the workshop at Tufts, analyses of emerging
infectious disease issues generated insights about the political
and social framework within which to address these threats to
health: A minority group may be particularly affected by new or
reemerging disease, as was the case, for example, of AIDS in the
gay population or tuberculosis in the immigrant and homeless
population. These groups become valuable resources for
understanding the factors leading to the emergence or reemergence
of the disease and should be the focus of public health efforts
for curtailing its spread. However, as the history of AIDS
demonstrates, because of political concerns, investigative
efforts are often delayed or inadequate to stop the spread of the
disease.

An emerging or reemerging organism, however, grows unhindered by
the social concerns of its potentially infectable host. To
microorganisms, the world is a single entity without borders.
Microorganisms have more freedom than we do and also more genetic
flexibility. Thus, in the contest between humans and microbes, we
are at a disadvantage. We can neither easily acquire resistance
mechanisms against the organisms, nor rapidly respond to an
infectious disease problem in another country. The recent
difficulties in dealing with a possible plague epidemic in India
are just one example. Moreover, antibiotics which have been a
front-line weapon against diseases are becoming increasingly
ineffective, and new antibiotics to treat and contain
drug-resistant bacterial strains are not available.

Inadequate microbiologic diagnostic capability also the result of
the national and international political climate works to the
advantage of emerging microbes. During the plague outbreak in
India, laboratory facilities that could confirm the diagnosis
were lacking. In the United States, similar inadequacies in
laboratory diagnostic capacity interfere with rapid reporting of
common community-acquired infections and their susceptibility to
antibiotics. If physicians promptly knew what they were treating,
the need for use of an antibiotic as well as the proper kind of
antibiotic would be based on data, not guesswork.

The emergence of antibiotic resistance was not factored into
strategic planning by public health authorities. If it had been,
perhaps conditions could have been in place to handle it, as well
as AIDS, tuberculosis, and other emerging pathogens. Insurance
against devastating happenings in infectious disease has never
been given the attention it deserves. Such insurance would have
been helpful, not just in money, but also in expertise to forfend
and then cope with the calamity, like insurance for earthquake
damage to structure and other unexpected disasters. Should we not
consider insuring our future by putting more money and expertise
into basic research, into systems for surveillance, and into ways
to curtail the spread of a disease once it has emerged?

To meet the demands of increased public health activity and to
implement an "insurance policy" for the future, we need to be
able to communicate the problem to a broad audience that
sometimes has little understanding of the science. To some public
health officials, recognition that an infectious disease problem
exists is sufficient to address the problem. However, to those
not trained in the field who may be making important policy
decisions, the "public safety" aspect of the problem can be
emphasized. Health, like crime and traffic, should become once
again a major society issue.

Requests for increasing support for surveillance, education, and
research must take into account current political and social
priorities and emphasize direct benefits to the U.S. population;
international efforts should involve the collaboration of other
countries.

Nongovernment agencies need to be enlisted in this public health
effort; thus a larger portion of society will be involved in the
fight against the ever-increasing threat of infectious diseases.

Stuart B. Levy
Center for Adaptation Genetics and Drug Resistance, Tufts
University School of Medicine, Boston Massachusetts, USA
*Participants: Stuart B. Levy, Tufts University School of
Medicine, and Ruth L. Berkelman, Centers for Disease Control and
Prevention (co-conveners); Christopher Foreman, The Brookings
Institute; Laurie Garrett, Newsday; Margaret Hamberg, New York
City Department of Health; Joshua Lederberg, Rockefeller
University; Jonathan Mann, Christopher Murray, Harvard School of
Public Health.


(Pages 64-70)

Xenotransplantation: Risks, Clinical Potential, and Future Prospects


The reemergence of xenotransplantation as a therapeutic option for the
hundreds of thousands of people dying each year of heart, kidney, lung, and
liver failure has raised ethical, social, and scientific questions.
End-stage organ failure is one of the most important public health problems
facing Americans today. Heart failure, for example, kills four times as
many people as does HIV infection and three times as many people as does
breast cancer; it is a disease with an increasing incidence, and the cost
of taking care of affected patients is 8 to 35 billion dollars each year.
The single most effective therapy for it is transplantation. Preventive
therapies have had little impact on diseases due to end-stage organ failure
and are unlikely to have an impact at least in the next decade. In the
meantime, demand for organs, which far outstrips the supply, continues to
grow. It has been estimated that approximately 45,000 Americans under age
65 could benefit each year from heart transplantation, yet only 2,000 human
hearts are available annually. Patients are more likely to die waiting for
a human donor heart than in the first 2 years after transplantation.

Although clearly an experimental procedure, xenotransplantation between
closely related species, such as baboons and humans, offers an alternative
to allotransplantation as a source of human organ replacement. Alternatives
to allograft donors, such as baboon or pig xenografts, require serious
investigation if clinical transplantation is ever to meet the current
demand and continue the explosive growth pattern it has established over
the past quarter century.

Biologic cardiac replacement poses the immunologic problems of rejection
and infection associated with transplantation. Increasing clinical
experience worldwide has shown that rejection and infection can be managed
successfully in most patients who receive human cardiac allografts.
Further, the introduction of cyclosporine as the primary immunosuppressive
agent for cardiac transplant recipients has resulted in excellent survival
rates (85% 1-year survival at most centers) and has decreased illness
associated with infection and rejection. Although considerable advances
have been made in the field of cardiac xenotransplantation since its first
clinical application by Hardy in 1964 (1), it remains uncertain whether
xenotransplantation as destination therapy can be successfully applied to
humans. However, heart, kidney, and liver xenografts have been able to
support human life for an extended period. It is this fact that
investigators wish to exploit in clinical bridging studies. By providing
temporary heart, kidney, or liver support as a bridge-to-transplantation,
these biological devices may allow patients to recover end-organ function
and await allograft transplantation in a more stable clinical state, thus
improving their chances of survival. Bridging strategies cannot alleviate
the human organ donor shortage. However, if one views bridging strategies
as a first feasibility test, then cross-species transplantation does offer
the possibility of eventual long-term organ replacement. Success in this
more ambitious goal would help alleviate the human organ donor shortage.

Nonhuman primate organ donors have been favored by those wishing to
minimize the genetic disparity between donors and human recipients.
Chimpanzees, although most compatible with standard selection criteria
(e.g., compatibility of size and blood types), are unavailable as an
acceptable source of clinical xenotransplantation. Another choice is the
baboon, which is not endangered, has an anatomy and physiology similar to
those of humans, and grows to a weight of approximately 70 pounds. Baboon
size would limit the clinical application of xenotransplantation with
baboon organs to pediatric patients and small adults. Small body size, the
infrequency of blood group O (universal donor) animals, and the limited
number of colony-bred animals are distinct disadvantages to the baboon as a
donor.

Extended graft survival is possible, but ABO blood group compatibility is
mandatory before xenotransplantation (2). The distribution of ABO blood
groups found in baboons indicates that approximately one third are group A,
one-third group B, and one-third group AB. Universal donor group O,
however, is exceedingly rare. In Americans of Western European descent, the
relative frequency of blood types is approximately 45% group A, 8% group B,
4% group AB, and 43% group O (2).

Although available in large numbers, wild baboons are not suitable from an
infectious disease perspective. Most experts have suggested that
colony-bred animals represent a more suitable donor pool. However, these
animals number only in the hundreds and are, therefore, only likely to
partially meet the epidemiologic demands of the pediatric population with
end-stage organ failure.

Xenotransplantation between baboons and humans raises the issue of
xenozoonoses (3,4). The organisms of greatest concern are the herpesviruses
and retroviruses, which can be screened for and eliminated from the donor
pool. Others include Toxoplasma gondii, Mycobacterium tuberculosis, and
encephalomyocarditis virus. Less likely to be found in animals raised in
captivity in the United States are the filoviruses (Marburg and Ebola),
monkeypox, and Simian hemorrhagic fever virus. Organisms that are unlikely
to be transmitted with an organ transplant (but should be screened for)
include lymphocytic choriomeningitis virus, gastrointestinal parasites, and
GI bacterial pathogens.

The risk for xenozoonoses is likely to be restricted to the xenogeneic
tissue recipient. Nevertheless, one must consider and anticipate the
potential for xenozoonotic transmission through the human population,
constituting a public health concern. The risk for recognized zoonotic
pathogens can be reduced, if not eliminated, by controlling the donor
animal vendor source and the individual donor animal by employing described
screening tests and strict sterile procedures during organ harvesting and
donor autopsy for tissue and blood. The risk for unrecognized pathogens is
present but ill defined.

Surveillance for the transmission of known or unknown pathogens among
health care workers must be conducted by monitoring for unexpected or
unexplained adverse health events. It is difficult to monitor for the
unknown; therefore, surveillance should include notifying the principal
investigator's office of any unexplained illness in exposed health care
workers, as well as telephone interviews of these personnel every 6 months
by the principal investigator's office.

Concurrent with scientific advances in xenotransplantation have been the
necessary ethical debates concerning the appropriateness of this endeavor
(5). Disputes regarding animal experimentation notwithstanding, the ethical
issues raised by many of these debates are strikingly similar to those put
forth 25 years ago in reference to the (then new) field of human heart
transplantation. Indeed, the timeless nature of these queries itself
attests to their essence, for such ethical concerns are appropriate in the
appraisal of any new therapeutic procedure in medicine.

Can one ever hope to determine if or when the clinical application of
xenotransplantation is justified? The assessment of any experimental
therapy, as Fox and Swazey (6) have suggested, should encourage the
investigator to address three critical questions: 1) in the laboratory,
what defines "success" sufficient to warrant advancement to the clinical
arena? 2) under what clinical conditions should this advancement proceed?
and 3) in the clinical arena, what defines "success" sufficient to warrant
further evaluation (6)? Providing answers to this threefold inquiry
requires a reliance upon defined "success," itself an appraisal of judgment
that can only confidently be made in retrospect.

Because human heart transplantation is now considered by most justifiable
for the treatment of end-stage heart disease, I would first like to review
the history of cardiac allotransplantation in light of its ability to
address the above threefold inquiry. I will also discuss the history of
cardiac xenotransplantation with reference to scientific advances made in
the field throughout the past quarter century. Finally, in light of these
analyses, I hope to illustrate the role of baboon heart xenotransplantation
as an alternative to allotransplantation for permanent cardiac replacement
in the treatment of end-stage heart disease.

After the first human cardiac allograft procedure performed by Barnard in
1967 (7) the field of cardiac transplantation witnessed a surge in both
enthusiasm and attempted trials, which was followed by a marked drop in
procedures throughout the 1970s because of poor survival rates. During the
initial peak, 21 human heart allotransplants were performed in the 6-month
interval between December 1967 and June 1968 (with a cumulative 1-year
survival of 22%), and 105 cardiac allotransplantations were performed in
1968 alone (8-10). However, these early clinical trials were marred by
numerous failures, as 65% of persons undergoing the procedure before June
1970 died within 3 months of transplantation (6).

Few centers continued animal research and human procedures during the
so-called black years of cardiac transplantation. The initial explosion in
clinical trials accordingly elicited numerous responses suggesting that too
much was being attempted too soon.

Some would propose that this was the price of eventual "success," and that
further experimental studies at the time could not have avoided early
losses. And yet, there has been, and may always be, a tacit recognition by
medical innovators that the ultimate experiment must be performed in
humans, for no animal model can truly reflect the human condition.
Proponents of allotransplantation at the time of the first heart
transplantation cited the more than 60-year history of experimental cardiac
transplantation, beginning with Carrel's original work in 1905. Although
most of this work began in the 1930s, subsequent investigations regarding
the experimental transplantation of mammalian hearts showed that cardiac
transplantation was technically feasible and suggested the possibility of
clinically relevant survival rates. During the decade before Barnard's
first clinical application, cardiac allograft survival had been shown to
exceed 250 days (mean 103 days) in adult dogs treated with an
immunosuppressive regimen that included azathioprine and methylprednisone
used intermittently. The mean survival in untreated dogs used as controls
was 7 days (11).

Since that time, with further expansion of knowledge in virtually all areas
of clinical cardiac transplantation, 1-year survival has increased from 67%
in 1976, to approximately 85% reported currently at most hospital centers
(3). Human recipients have survived for as long as 20 years after
transplantation, and the 10-year posttransplant survival rate is now
approximately 45% (12). While these figures depict a clear improvement in
raw survival, cardiac transplantation is still not a cure for end-stage
heart disease. Recipients must take immunosuppressive medication for life
and be monitored for infection, rejection, and graft arteriopathy. However,
these results are impressive considering that the recipient population
today is considerably sicker than earlier allograft candidates. In light of
these findings, few would deny cardiac allotransplantation its present
claim to "success." To further understand the evolution of this
achievement, however, we may now look back upon the early years of cardiac
allotransplantation and try to address the proposed threefold inquiry.

First, for Barnard and co-workers what can we presume as "success"
warranting advancement to the clinical arena? They performed the first
human adult cardiac allotransplantation when the maximum survival in
immunosuppressed adult dogs had been 250 days (average survival 103 days)
(11) and suggested that "against the background of this research . . . the
time arrived when a cardiac transplant could be contemplated with hope of
success" (7). Indeed, in their report of this case, they further described
the scientific basis of their clinical advancement by explaining that "this
achievement did not come as a surprise to the medical world. Steady
progress toward this goal had been made by immunologists, biochemists,
surgeons, and specialists in other branches of medical science all over the
world during the past decades to ensure that this, the ultimate in cardiac
surgery, would be a success" (7). Although we may, in retrospect, consider
them justified in their declaration, in fact, at that time the endeavor was
highly controversial and came as a surprise to much of the medical world.

Second, under what conditions did they proceed with this clinical trial?
Given the "hope of success," Barnard and colleagues selected a patient
"considered to have heart disease of such severity that no method of
therapy short of cardiac transplantation could succeed" (7). The patient, a
54-year-old man, had remained in intractable congestive heart failure
(following multiple myocardial infarctions) despite all medical management
(13).

Finally, in this clinical arena, what defined for Barnard and colleagues
"success" warranting further investigation? A concurrent editorial in the
South African Medical Journal may provide some insight into their thinking:
"The claim `successful' can be used even at this early stage because
to-date it is a feat which makes medical history, no matter how short the
further survival of the patient might be (4). "Success," by such an
analysis, was thus not targeted posttransplant survival time, but rather
any posttransplant survival time (given the ground-breaking nature of the
endeavor). Further editorials regarding the ethics of cardiac
transplantation viewed the procedure as a legitimate experiment but not a
treatment (15), while in 1968, the American College of Cardiology suggested
(with regard to the "success" of allotransplantation) that results varied:
". . . the spectrum of success ranges from short-term restoration of
circulation to complete physical recovery" (16).

Indeed, "success" did vary along a spectrum of results. Barnard and
colleagues' first allotransplant recipient lived for 18 days and ultimately
died of pneumonia. However, their second recipient, 1 month later, survived
more than 19 months before dying of chronic rejection (17). Their third
patient also lived more than 20 months after allotransplantation and
ultimately died of carcinoma of the stomach without signs of acute or
chronic rejection (18). One can only speculate how different the world
reception to allotransplantation would have been had the latter two
patients represented the first and second recipients of cardiac
allotransplants. Would these survival data be considered "success," or
would they still pale in comparison with the theoretical goal of obtaining
a graft that could function normally indefinitely?

Clinical cross-species transplantation dates to the early twentieth
century, with kidney xenografts from rabbit, pig, goat, non-human primate
and lamb donors (19). After these early failures, the scientific literature
was largely devoid of reports of clinical xenotransplantation for nearly 40
years. In 1963, Reemtsma and colleagues described six human recipients of
chimpanzee kidneys, the longest survivor of whom died of causes unrelated
to rejection 9 months after xenotransplantation (20).

The first cardiac xenotransplantation, performed by Hardy in 1964, also
represented the first attempt at cardiac transplantation in humans,
predating Barnard's report by nearly 4 years (1). Since 1964, when Hardy
and colleagues at the University of Mississippi performed the world's first
heart xenotransplant using a chimpanzee as a donor, there have been eight
documented attempts at clinical heart xeno-transplantation. Five of these
donors were non-human primates (2 baboons, 3 chimpanzees) and three were
domesticated farm animals (1 sheep, 2 pigs) (21-25). The longest survivor
was a newborn infant with hypoplastic left heart syndrome. "Baby Fae" was
the recipient of an ABO-blood group mismatched baboon heart that functioned
for 20 days (26). However, by the time the first human neonatal cardiac
xenotransplantation was performed by Bailey in 1984 (the so-called "Baby
Fae" case), there had been only limited experimental experience with
prolonged graft survival in the newborn xenotransplant recipient. Studies
presented by Bailey and co-workers shortly before the Baby Fae case
described a mean survival time of 72 days in newborn lamb-to-goat
xenotransplants, with one survivor living to 165 days (27).

This advancement of xenotransplantation into the clinical forum was met
with resistance in the medical community because of a perception that
research with acceptable survival "success" had not been achieved
experimentally. As Losman in an editorial regarding the Baby Fae experience
stated, "It appears that this baboon-to-infant transplantation did not rest
on such a [scientific] basis [as did Barnard's earlier operation in 1967]
"(28).

During the past 3 years, investigators at the University of Pittsburgh
reported two cases in which they transplanted a baboon liver into a human
recipient, obtaining a 70-day survival in their first reported case, and a
26-day survival in the second (29; J.J. Fung, pers. comm.) The
investigators' overwhelming effort to prevent rejection led them to use a
harsh immunosuppressive regimen that permitted multiple life-threatening
infections. Rejection was not the major clinical obstacle they encountered;
therefore, they recommended a more directed and less arduous
immunosuppressive regimen for future patients.

More alarming have been the attempts to apply xenotransplantation of
distantly related species to the clinical arena. In 1968, both Cooley and
Ross transplanted sheep and pig hearts, respectively, into dying human
recipients (30,31). Both grafts failed upon reperfusion, presumably because
of hyperacute rejection.

More recently, Czaplicki and co-workers in 1992 described a case in which
they attempted the xenotransplantation of a pig heart into a human
recipient with Marfan's syndrome (32). By their report, no evidence of
hyperacute rejection was present at the time of death nearly 24 hours after
xenotransplantation. Their protocol used an unusual immunosuppressive
regimen in which both donor and recipient received, in addition to
conventional immunosuppression, both thymic tissue extracts and fetal calf
sera. This regimen also included the extracorporeal perfusion of two pig
hearts with the recipient's blood in an attempt to remove human anti-pig
antibodies before the orthotopic transplantation of the functional pig
heart (33). As astonishing as this case may be in its extension to the
clinical arena of a technique not yet shown to be effective in the
experimental laboratory, it is not unique. Also in 1992, Makowka and
colleagues transplanted a pig liver into a 26-year-old woman dying of acute
liver failure from autoimmune hepatitis (pers. comm.). Despite the fact
that, at present, it appears unlikely that sufficient "success" has been
achieved in the laboratory regarding xenotransplantation between distantly
related species to warrant advancement to the clinical arena, these
investigators were able to obtain approval from their hospital's ethics
committee and institutional review board to proceed with the clinical
trial. Most experts in the field of xenotransplantation share the opinion
that pig-to-human organ transplantation remains at least 3 to 5 years from
clinical trials.

Considerable advances in the field of cardiac xenotransplantation have
subsequently emerged worldwide since Hardy's first clinical attempt in
1964, with a better understanding of the xenorejection process and a more
sophisticated insight into mechanisms for its control. Extended graft
survival has been achieved in a number of different experimental models,
including a greater than tenfold graft survival in non-human primates
treated with conventional cyclosporine-based immunosuppression (34,35) a
more than thirtyfold increase in survival over controls described by Celli
and colleagues in a rodent model (36), and survival beyond 1 year reported
by Kawauchi and colleagues in a non-human primate model (37). These
findings support the potential for achieving clinically relevant graft
survival in humans.

The question is whether we have reached a stage in laboratory
experimentation to justify further attempts at advancing cardiac
xenotransplantation to the clinical arena. If we view the current status of
experimental accomplishments in xenotransplantation with the same scrutiny
as that of allotransplantation at the time of Barnard's endeavor, we are
left with similar conclusions; first, comparable graft survival time has
been achieved in animal models of xenotransplantation as was evident for
allotransplantation before 1967. Second, with our current understanding of
cardiac allotransplantation has also come a greater awareness of its
limitations. Thus, the conditions for the advancement of
xenotransplantation arguably could be fulfilled by a patient with end-stage
heart disease who is a candidate for allotransplantation, but for whom a
donor cannot be identified in time. Finally, the clinical "success" of
xenotransplantation might also be considered (as was the case for
allotransplantation) any graft survival, and the goal of
xenotransplantation to strive for extended graft survival.

However, political and scientific sensibilities today clearly differ from
those of the 1960s, and so the critical assessment of xenotransplantation
must be more rigorous than our previous discussion. Indeed, the above
comparison was put forth largely to underscore the more humble origins of
the (now) successful therapy (allotransplantation) to which
xenotransplantation is currently compared.

What then defines "success" in the laboratory warranting advancement from
the laboratory to the operating room? Having demonstrated dramatic
prolongation of cardiac xenograft survival through experiments in rodent
and non-human primate models (27,34-37), which model most closely
approximates the human condition (and thus which therapy will be most
successful in avoiding clinical rejection) remains to be established.
Therefore, it is reasonable to suggest both that we have reached a
formidable limitation for precisely predicting the applicability of
experimental laboratory evidence and that answers may only be sought from
experiment in humans. This concept was realized by the American Medical
Association with regard to allotransplantation, in reference to which it
released an official statement acknowledging this notion in 1969 (38).

Concerns most commonly voiced with respect to the clinical application of
xenotransplantation, however, pertain to a larger ethical controversy
regarding human experimentation. Reemtsma, in a related comment concerning
the Baby Fae case, suggested the following: "There is a widespread
misperception that medical treatments and surgical procedures are easily
classified as either experimental or accepted. In fact, all treatments have
an element of experimentation, and new surgical procedures are based on
extrapolations from prior work. . . . When does a surgeon decide to apply a
new operation to a patient? . . . the decision is based on balancing, on
the one hand, the experimental evidence suggesting that the procedure may
succeed, and, on the other, the clinical urgency. . . (39).

Under what conditions will the clinical advancement of xenotransplantation
proceed? For those initial patients in whom clinical xenotransplantation
will first be applied, clinical urgency, in the complete absence of other
suitable alternatives, undoubtedly will represent the motivating factor to
proceed. Who will comprise this initial cohort? As Caplan has pointed out:
"There would appear to exist a pool of terminally ill persons, both
children and adults for whom no therapeutic alternatives exist or are
likely to exist in the near future. . . . It would [thus] appear ethically
defensible to allow research involving xenografting in human subjects to
proceed in those areas where no reasonable alternative to therapy exists
(40). In this context, innumerable reservations have been voiced regarding
the ethics of proposing alternative experimental therapies to such patients
for whom therapy has either failed or is non-existent. However, with regard
to clinical experimentation under these circumstances, one must also recall
(as Shimkin has suggested): "To do nothing, or to prevent others from doing
anything, is itself a type of experiment, for the prevention of
experimentation is tantamount to the assumption of responsibility for an
experiment different from the one proposed" (41).

What is the goal of the clinical application of xenotransplantation? The
need for donor organs irrefutably outweighs the resources available, and
mechanical devices and xenotransplantation have emerged as the two most
promising alternatives to allograft cardiac replacement. Mechanical left
ventricular assist devices (LVADs) have witnessed relative success as
"bridges" in carefully selected patients with heart failure. (A "bridge" is
a temporary method of life support designed to carry a patient indefinitely
until a human heart can be found and transplanted. It is not a
"destination" therapy.)

Criteria for LVADs exclude patients with biventricular failure, and
(because of the relatively large size of the device) patients with a total
body surface area less than 1.5 square meters (~120 lb). Thus, many women
and virtually all children are not candidates for mechanical left
ventricular assistance. As has been the case for Food and Drug
Administration protocols using LVADs, proposed investigations involving
biologic assist devices (xenografts), have sought to evaluate a short-term
alternative to allotransplantation in patients for whom a donor heart is
not immediately available, and death is imminent. Only candidates who meet
criteria for heart transplantation, but do not meet criteria for LVAD
insertion, would be considered for a heart xenobridge. Similar clinical
scenarios have been proposed for other solid organ transplants. Since they
were first introduced by Cooley in 1969, temporary mechanical circulatory
support devices have become critically useful tools in the therapeutic
armamentarium available to patients awaiting transplantation (42).
Nevertheless, at present, the widespread application of mechanical
circulatory support is limited both by patient selection criteria and by
the temporary nature of the device. For excluded patients, as well as many
adult male candidates. For excluded patients, cardiac xenotransplantation
may be the only reasonable alternative to cardiac allograft replacement.

Investigations in clinical xenotransplantation have been accused of using
"the guise of [being a] bridge-to-transplantation" to appear acceptable to
Institutional Research/Ethical Boards (5). However, the use of xenografts
(or mechanical devices) solely as bridges to allotransplantation will not
increase the donor pool, and, therefore, successful permanent
xenotransplantation must itself be seen as the target for future clinical
investigations. The goal of these studies is thus not to engage, as
Hastillo and Hess (5) would suggest, in the "premature use of unproven
procedures in fellow humans," but rather to impact positively on the
current shortage of human donor organs (6). In 1996, the clinical picture
is no less bleak and the conclusions no less valid. The question that
remains is not how but rather when xenotransplantation should advance to
the clinical arena. Most of the uncertainties surrounding its advancement
will only be answered by its undertaking.

In the foreseeable future, clinical xenotransplantation may achieve its
targeted goal of extended graft survival. As was the case during the early
years of allotransplantation, clinical xenotransplantation must persevere
under the consideration of and often in spite of scrutiny by its most
demanding critics, for while "success has a hundred fathers, failure is an
orphan" (43).

Robert E. Michler
Director of Heart Transplant Service
Division of Cardiothoracic Surgery
Columbia-Presbyterian Medical
Center, New York, NY, USA

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NEWS AND NOTES

(Pages 71-72)

Another Human Case of Equine Morbillivirus Disease in Australia

Another human case of equine morbillivirus (EMV) disease has
occurred in Australia. The patient was a 35-year-old farmer, who
lived near Mackay, in northern Queensland. He died in the Royal
Brisbane Hospital on October 21, 1995 (1). The patient was
probably infected with the novel virus 12 months before his
death approximately the time of the first reported outbreak of
EMV.

A hitherto unknown infectious disease, EMV was first observed in
Brisbane, Queensland, in September 1994, when an outbreak of
acute respiratory disease in horses at three stables in Hendra, a
Brisbane suburb, was reported (2). In a 2-week period, 14
racehorses and two persons at the stable contracted the disease.
One of the human cases and some of the equine cases were fatal. A
total of 21 horses were infected with the virus. Fourteen horses
died as a result of clinical illness (they either died from the
infection or were euthanized). The remaining horses had either
symptomatic or asymptomatic infection and were euthanized.

The cause of infection in the recent case has been confirmed as
EMV at the Australian Animal Health Laboratory in Geelong through
the testing of samples taken from the patient before he died  (I.
Douglas, Australian Communicable Disease Service; PROMED).

The Mackay patient was married to a veterinary surgeon. The
couple bred horses and grew sugar cane. In August 1994 (a month
before the outbreak of EMV in southern Queensland), two horses
died on the couple's property. The veterinary surgeon, assisted
by her husband, performed autopsies on the two animals. The
diagnoses, based on these autopsies, were "avocado poisoning" and
"brown snake bite," respectively (1).

In August-September 1994, soon after the death of the horses, the
husband became ill with a mild meningoencephalitis, which
improved with antibiotics. Cerebrospinal fluid examination showed
a neutrophilic pleocytosis suggestive of a viral infection (1).
Serum collected at the time of the examination and stored was
found to contain a low but significant titer of antibody to EMV.

The patient appeared to have recovered; however, he was admitted
to the hospital 5 weeks before his death with signs of
encephalitis. Evidence of EMV infection included a high serum
neutralizing antibody titer against the virus and a positive
polymerase chain reaction (PCR) test of cerebrospinal fluid
collected before his death. Tests of autopsy specimens confirmed
the infection.

Direct fluorescence antibody and PCR tests of fixed tissue blocks
from one of the horses at the Australian Animal Health Laboratory
have confirmed that it was infected with EMV (I. Douglas,
PROMED). However, it is likely that both horses were infected.
The Mackay patient's symptoms were predominantly neurologic. He
displayed no respiratory symptoms until aspiration pneumonia 
developed. By contrast, the major clinical symptoms of EMV in
Hendra were respiratory.

No recent outbreaks of clinical illness have been reported in the
horses on the Mackay property or elsewhere in Queensland since
the 1994 outbreak. Also, investigation has not shown a link
between the horses on the Mackay property and those in the Hendra
stables. A serologic survey of over 2,000 horses, undertaken in
1994 after the Hendra outbreak, yielded negative results. That
survey included more than 200 horses in the
Mackay/Rockhampton/Townsville areas. Similarly, the Queensland
veterinary authorities  have obtained samples from more than
3,000 animals from 294 populations (including farms, race
meetings, and horse events) since October 23, 1995; 2,349 of the
samples have been tested, all with negative results. Moreover,
blood samples recently taken from all the domestic animals on the
Mackay property, including approximately 90 horses, have been
tested for virus by the Animal Health Bureau, Queensland
Department of Primary Industry and Energy. All results were
negative.

An extensive epidemiologic investigation is being conducted by
the Queensland Department of Health and the Department of Primary
Industry and Energy. All persons who may have had exposure to the
virus in either episode have been tested for EMV infection and
had negative results (1). No serologic evidence of further human
infection has been found.

No human-to-human transmission of EMV has been reported. It is
believed that the disease is spread through contact with the body
fluids of infected sick or dying animals.

References
l. Allworth T, O'Sullivan J, Selvey L, Sheridan J. Equine
morbillivirus in Queensland. Communicable Diseases Intelligence
1995;19:575.
2. Murray K, Rogers R, Selvey L, Selleck P, Hyatt A, Gould A, et
al. A novel morbillivirus pneumonia of horses and its
transmission to humans. Emerging Infectious Diseases 1995;1:31-3.



(Page 72)

Social Science and the Study of Emerging Infectious Diseases

Topics related to emerging and reemerging infectious diseases
attracted a considerable audience at the annual meeting of the
American Anthropological Association, November 15 19, 1995, in
Washington, D.C. The meeting had a separate session entitled
"Emerging and Reemerging Infectious Diseases: Biocultural and
Sociocultural Approaches."

The session brought together anthropologists interested in and
working on emerging infectious diseases from various
subdisciplinary perspectives. Presentations were made on the
following subjects: outline of a research agenda, deforestation
and the emergence of infectious diseases in the rain forests of
Papua-New Guinea, the cholera epidemic in Latin America,
evolutionary aspects of emergent infections, societal impacts of
the test for acquired immunodeficiency syndrome, compliance and
iatrogenesis in tuberculosis treatment in the United States,
patchwork policies that affect long-term treatment of
tuberculosis in Nepal and Uganda, the reemergence of
schistosomiasis in Egypt, dengue control in Latin America,
cultural and political ecologic models of emergent infections,
and the politics of leprosy eradication. Abstracts are available
from the conference organizers, listed below.

Anthropologists interested in international health and the social
science aspects of infectious diseases are organized in a working
group called the International Health and Infectious Disease
Study Group of the Society of Medical Anthropology (American
Anthropological Association). Requests to subscribe to this
group's newsletter can be sent to

Johannes Sommerfeld
Harvard Institute for International Development
Cambridge, Massachusetts, USA
Phone: 617-495-9791
E-mail: jsommerf@hiid.harvard.edu

Sandra Lane
Case Western Reserve University
Cleveland, Ohio, USA
E-mail: sxl45@po.cwru.edu



(Pages 72-73)

WHO Establishes New Rapid-Response Unit for Emerging Infectious
Diseases

The World Health Organization (WHO) has established a new
rapid-response unit to control and prevent the growing incidence
of new and reemerging diseases worldwide. The unit's focus will
be improved containment of disease outbreaks, such as that caused
by the deadly Ebola virus, which struck Zaire in 1995.

The WHO unit will be called the Division of Emerging Viral and
Bacterial Diseases Surveillance and Control (EMC). It will be
capable of mobilizing staff from WHO headquarters in Geneva and
from the organization's regional offices.

In addition to mobilizing WHO's own technical staff and
expertise, EMC will coordinate the activities of the agency's
traditional partners, for example, its international network of
collaborating centers, bilateral donors, expert advisers, and
nongovernmental organizations.

Teams equipped to implement epidemic control measures will be
placed on-site within 24 hours' notification of an outbreak. This
strategy, when implemented in Zaire, not only rapidly contained
the recent Ebola outbreak but also prevented its spread to
Kinshasa, the capital city of 2 million.

Among EMC's goals are 1) to strengthen local surveillance and
disease control so that countries can develop the early warning
systems needed to detect emerging or reemerging diseases through
innovative field epidemiology and public health laboratory
training programs and 2) to continue WHO's activities in
developing a network of public health laboratories to strengthen
regional and international collaboration in outbreak detection
and control.

EMC will continue to expand WHO's network termed WHONET that
detects and monitors antibiotic resistance worldwide. WHO will
use the information collected to continue to advocate research
and development of new antibiotics to replace those that are no
longer effective.

For further information on WHO's rapid-response unit, contact

Philippe Stroot
Health Communications and Public Relations
World Health Organization
Geneva, Switzerland
Phone: 41-22-791-2535
Fax: 41-22-791-4858


(Page 73)

Rotavirus Vaccine Workshop Held

More than 125 participants from at least 15 countries attended
the Fifth Rotavirus Vaccine Workshop at the Centers for Disease
Control and Prevention (CDC) in Atlanta, Georgia, October 16-17,
1995.

Rotavirus has emerged as the most important cause of severe
diarrhea in children worldwide. It is a problem not only in
developing countries, where it kills an estimated 870,000
children each year, but also in the United States, where it
remains the most important single cause of hospitalization or
clinic visits for childhood diarrhea.

Moreover, although studies from many countries indicate that only
four serotypes are predominant worldwide, some strains at every
site studied cannot be serotyped. In some countries such as
India, the diversity of strains is extensive. Further studies are
needed to define the extent of cross-protection against these
strains that is induced by the vaccine to determine whether
additional antigens need to be included in vaccines for such
areas.

This workshop included sessions on epidemiology, virology,
pathogenesis and immunity, and vaccines currently being tested.
Each session had numerous presentations by leaders in the field
of rotavirus research. Researchers reported that several live
oral rotavirus vaccines, based on animal strains of rotavirus
combined with reassortant strains, have been tested in field
trials in children. These appear to protect American children
against rotavirus and are more efficacious against severe
disease. These vaccines like natural protection, are not 100%
protective so many investigators are exploring alternative
approaches to vaccines such as the use of virus-like particles,
native DNA, and microencapsulation of antigens.

No published volume of proceedings from the workshop is planned,
but a supplemental issue of the Journal of Infectious Diseases
scheduled for early 1996 will contain papers from the meeting.

The workshop was held under the auspices of the National
Institutes of Health, Emory University School of Medicine, and
the World Health Organization.

For further information, contact

Roger I. Glass
Global Program for Vaccines and Immunizations
World Health Organization
Geneva, Switzerland
Phone: 41-22-791-2698/2681
Fax: 41-22-791-4860



(Pages 73-74)

International Conference Addresses Preparedness for Emerging
Strains of Pandemic Influenza

An international meeting on pertinent issues related to
recognizing, identifying, and controlling newly emerging strains
of pandemic influenza was held in Bethesda, Maryland, December
11 13, 1995. The conference, "Pandemic Influenza: Confronting a
Reemergent Threat," was sponsored by the National Institutes of
Health, the University of Michigan, the Centers for Disease
Control and Prevention, the Food and Drug Administration, the
U.S.-Japan Cooperative Medical Science Program, and the World
Health Organization.

Epidemic strains of influenza cause infections almost every year
throughout the world because of continuous minor genetic changes
in the virus. However, periodically a major change occurs, such
as reassortment between mammalian and avian strains of the virus.
These pandemic strains are novel to the human immune system and,
therefore, can cause substantial disease worldwide. The
conference concentrated on issues that would be crucial to
controlling an influenza pandemic.

Plenary and workshop sessions examined the following topics: Can
pandemics be predicted? What are the specific approaches for
pandemic control? What are the advantages and limitations of
vaccines and antiviral agents? The workshops also focused on
factors contributing to the emergence of pandemic strains and
various aspects of surveillance, such as the adequacy of current
global surveillance structure for early identification of a
pandemic strain, the use of virologic and epidemiologic
surveillance once a strain is identified, and the rapid exchange
of information globally. The following immunologic and molecular
questions were addressed as well: What basic research advances
would allow us to respond more rapidly after the next human -
pandemic strain is detected? Is the presence of novel influenza A
virus in pigs a predictor of the next influenza pandemic? Is an
H2 influenza virus the next human pandemic subtype or are H7
viruses equally possible? Also discussed were the practical
issues of vaccine needs, production, and distribution.

Conference participants then reviewed international pandemic
plans and the U.S. pandemic plan being prepared by the Federal
Interagency Group on Pandemic Preparedness.

Dominick A. Iacuzio
Division of Microbiology and Infectious Diseases
National Institutes of Health
Bethesda, Maryland, USA



(Page 74)

Course Offered on Clinical and Pathologic Features of Emerging
Infections

The Armed Forces Institute of Pathology (AFIP), Emory University,
and the Centers for Disease Control and Prevention (CDC) are
cosponsoring a course on emerging and reemerging pathogens. The
course will be taught in Atlanta, Georgia, from April 27 to May 1
and will discuss the epidemiology, clinical features, pathology,
and pathogenesis of such diseases as plague, Lyme disease, Kaposi
sarcoma, microsporidiosis, Buruli ulcer, ehrlichiosis, hantavirus
pulmonary syndrome, and Ebola virus infection. Emerging drug
resistance in pneumococci and other streptococcal infections will
also be discussed.

The course is designed for pathologists, epidemiologists,
infectious disease physicians, veterinarians, microbiologists,
parasitologists, and others interested in the pathology as well
as the emergence of infectious diseases. The course, to be held
at the Emory Conference Center Hotel, will provide 38 hours of
Category I CME credit and will consist of 32 hours of lectures
with open discussion periods, 6 hours of glass and color slide
review, and a visit to CDC laboratories. For more information,
contact the course director, Center for Advanced Medical
Education, AFIP, Washington, D.C. 20306-6000 (phone: 800-577-3749
or 301-295-7921; fax: 301-427-5001).

C. Robert Horsburgh, Jr.
Emory School of Medicine
Atlanta, Georgia, USA


(Page 74)

NASA Sponsors Symposium on Remote Sensing and Control of
Insect-Transmitted Diseases 

Health officials and disease control experts met November 28-30
in Baltimore, Maryland, for a symposium on the use of satellites
to monitor and control insect-transmitted diseases.

Sponsored by the National Aeronautics and Space Administration
(NASA) and the Third World Foundation of North America, the 
symposium was held to inform government officials from various
countries of NASA's scientific and technologic capabilities for
detecting, monitoring, and improving the control of diseases.
Health ministers and medical directors from more than 20
countries, including Bangladesh, Belize, China, Ghana, Indonesia,
Kenya, Malaysia, Nigeria, Peru, and Rwanda, attended.

The symposium featured discussions on the economics of disease
surveillance, deforestation, and urbanization. The keynote
address, "The resurgence of vector-borne infectious diseases as
major public health problems in the 1990s," was given by Duane
Gubler, director, Division of Vector-Borne Infectious Diseases,
National Center for Infectious Diseases, Centers for Disease
Control and Prevention (CDC). Participants also discussed
possible joint activities between NASA and interested countries.
Further information can be obtained from NASA's Office of Life
and Microgravity Sciences, Washington, D.C., which manages the
agency's global monitoring and human health research program in
conjunction with the National Institute of Allergy and Infectious
Diseases, National Institutes of Health, and CDC. 

Michael Braukus
National Aeronautics and Space Administration
Washington, D.C., USA

Raj Khanna 
Third World Foundation of North America
College Park, Maryland, USA



(Page 75)

CDC Convenes Meeting to Discuss Strategies for Preventing
Invasive Group A Streptococcal Infections

Since the mid-1980s, the epidemiology of invasive group A
streptococcal (GAS) infections in the United States and worldwide
has changed, and the incidence of invasive infections,
streptococcal toxic shock syndrome (strep TSS), and necrotizing
fasciitis has increased. These changes may be the result of a
shift in GAS M-types and a corresponding increase in strains that
produce certain pyrogenic exotoxins. Recognizing the importance
of monitoring changes in the occurrence of severe group A
streptococcal disease, the Council of State and Territorial
Epidemiologists recommended in April 1995 that invasive GAS
infections and strep TSS be added to the National Public Health
Surveillance System.

Most invasive GAS infections occur sporadically and are acquired
in the community. For these cases, preventing illness and death
depends on improving recognition and treatment. Primary
prevention of invasive GAS disease may be more feasible for
infections that are acquired in institutions (such as hospitals
and nursing homes) and for secondary cases that occur among
contacts of persons with invasive disease. Most nosocomial
infections (for example, wound infections, postpartum
endometritis, and sepsis) occur in surgical or obstetric
settings, or are associated with intravenous catheters. Secondary
invasive disease in the community is uncommon, although studies
of household contacts of those with GAS infectionshave found a
substantially increased risk for infection in this group. GAS
infections spread easily from person to person after contact with
respiratory secretions of an infected person and have
traditionally caused epidemics of pharyngitis, scarlet fever, and
rheumatic fever. Recently, clusters of invasive infections have
been reported in families, hospitals, and nursing homes;
community-wide outbreaks have also been reported.

As state health departments initiate surveillance for invasive
GAS disease and strep TSS, guidelines for prevention will help in
interpreting these data and in formulating a public health
response. CDC convened a meeting of experts from academia and
public health (October 10-11, 1995), to discuss existing data and
strategies for preventing invasive GAS disease in institutions
and the community. Discussions centered on the magnitude of risk
for secondary disease among close contacts of persons with
invasive infection and the potential for preventing disease by
chemoprophylaxis, and on approaches for investigating and
preventing infections in institutions. Recommendations are being
developed, and the conclusions of the participants will be
presented at a later date.

The Working Group on Prevention of Severe Group A Streptococcal
Infections*
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, Georgia, USA

*The members of the Working Group on Prevention of Severe Group A
Streptococcal Infections are Gus Birkhead, New York State
Department of Health; John Brundage, U.S. Army; Matt Cartter,
Connecticut State Department of Health; Mike Gerber, University
of Connecticut; Walter Hierholzer, Yale University; Ed Kaplan,
University of Minnesota; Kris MacDonald, Minnesota Department of
Health; Dennis Stevens, V.A. Medical Center, Boise, Idaho; Karen
Green and Allison McGeer, Princess Margaret Hospital, Toronto,
Ontario, Canada; Stan Shulman and Ram Yogev, Children's Memorial
Hospital, Chicago, Illinois; Richard Facklam, Julia Garner,
William Jarvis, Orin Levine, Benjamin Schwartz and Jay Wenger,
CDC.


(Pages 75-76)

Regional Conference on Emerging Infectious Diseases Sparks Plan
for Increased Collaboration

The World Health Organization (WHO), the Naval Medical Research
Unit Three (NAMRU-3), and the Centers for Disease Control and
Prevention (CDC) jointly sponsored the first conference in the
region on issues of emerging and reemerging infectious diseases
for members of WHO's Eastern Mediterranean Regional Office
(EMRO). The meeting was held in Cairo, Egypt, November 26-29,
1995. Delegates from the WHO South-East Asia Regional Office and
African Regional Office also participated.

The meeting brought together persons representing key resources
that have begun working together to organize a regional program
of laboratory assistance and enhanced surveillance communications
for infectious diseases. Participants included WHO infectious
disease program officers and key personnel from WHO collaborating
centers, national reference laboratories, national infectious
disease programs, ministries of health, and university public
health programs.  Thirty countries were represented by more than
200 participants. On the final day of the conference, the
participants adopted a regional plan of action for strengthening
surveillance, laboratory capabilities, and communications.

To begin this cooperative effort, a 1-day meeting of WHO-EMRO
collaborating centers was convened on November 30.
Representatives from six EMRO collaborating centers attended as
well as representatives from WHO-EMRO, WHO headquarters, CDC, the
Naval Medical Research Institute in Washington, D.C., and the
Battelle Foundation.

Participants noted that WHO collaborating centers are an
excellent resource for the implementation of programs to address
emerging infections because they represent some of the most
competent and experienced diagnostic and reference capacities in
the region.  However, the representatives agreed that the
relationships among the collaborating centers themselves are not
well developed.  Many were unaware of the existence or
capabilities of most other regional collaborating centers and
most had no regular communications or interactions with other
collaborating centers.  All welcomed closer ties with an increase
in communication, collaboration, and interaction.

The participants unanimously approved a recommendation to
establish an Association of EMRO collaborating centers composed
of representatives from each WHO collaborating center within the
region. Member organizations of the association will work
together, in coordination with EMRO, to address the complex needs
of surveillance, laboratory diagnostics, and disease reporting of
emerging infections at the local and regional level.

Bradford A. Kay
Naval Medical Research Unit 3
PSC 452, Box 112
FPO AE 09835