Table of Contents
EMERGING INFECTIOUS DISEASES
Volume 2, Number 3           July-September 1996

Table of Contents, Instructions to Authors, and General Information

Perspectives

   * Molecular Approaches to the Identification of Unculturable
     Infectious Agents, S-J. Gao and P.S. Moore 

   * DNA Vaccines for Emerging Infectious Diseases: What If?
     R.G. Whalen

Synopses

   * Conjugate Vaccines and the Carriage of Haemophilus influenzae 
     Type b, M.L. Barbour

   * Application of Molecular Techniques to the Diagnosis of
     Microsporidial Infection, D.P. Fedorko and Y.M. Hijazi 

   * Coccidioidomycosis: A Reemerging Infectious Disease,
     T.N. Kirkland and J. Fierer 

   * Antibody-Based Therapies for Emerging Infectious Diseases,
     A. Casadevall

Dispatches 

   * HIV-1 Group O Virus Identified for the First Time in the United
     States, M.A. Rayfield, P. Sullivan, C. I. Bandea, R. A. Otten, 
     C.P. Pau, D. Pieniazek, S. Subbarao, P. Simon, C. A. Schable, 
     A. Wright, J. Ward, and G. Schochetman 

   * Two Morbilliviruses Implicated in Bottlenose Dolphin Epizootics,
     J.K. Taubenberger, M. Tsai, A.E. Krafft, J.H. Lichy, A.H. Reid,
     F.Y. Schulman, and T.P. Lipscomb 

   * An Outbreak of Spotted Fever Rickettsiosis in U.S. Army Troops
     Deployed to Botswana, B.L. Smoak, J.B. McClain, J.F. Brundage,
     L. Broadhurst, D.J. Kelly, G.A. Dasch, and R.N. Miller 

   * A Highly Heterogeneous HIV-1 Epidemic in the Central African
     Republic, M. Massanga, J. Ndoyo, D.J. Hu, C-P. Pau, S. Lee-Thomas,
     R. Hawkins, D. Senekian, M.A. Rayfield, J.R. George, A. Zengais,
     N.N. Yatere, V. Yossangang, A Samori, G. Schochetman, and T. J.
     Dondero 

   * Legionella-Like Amebal PathogensPhylogenetic Status and Possible
     Role in Respiratory Disease, A. Adeleke, J. Pruckler, R. Benson,
     T. Rowbotham, M. Halablab, and B. Fields 

   * Role of Enterovirus 71 in Acute Flaccid Paralysis After the
     Eradication of Poliovirus in Brazil, E.E. da Silva, M.T. Winkler,
     F. Ferreira, and M. Pallansch 

   * Bancroftian Filariasis Distribution and Diurnal Temperature 
     Differences in the Southern Nile Delta, D.F. Thompson, J.B. Malone,
     M. Harb, R. Faris, O.K. Huh, A.A. Buck, and B.L. Cline Sentinel 

   * Surveillance for Yellow Fever in Kenya, 1993 to 1995, E.J. Sanders,
     P. Borus, G. Ademba, G. Kuria, P.M. Tukei, and J.W. LeDuc

   * Serologic Evidence for the Presence in Pteropus Bats of a 
     Paramyxovirus Related to Equine Morbillivirus, P.L. Young, 
     K. Halpin,  P.W. Selleck, H. Field, J.L. Gravel, M.A. Kelly, 
     and J.S. Mackenzie

Letters 

   * Acute Cervical Lymphadenopathy, M.B. Pasticci, F. Baldelli, 
     F. Bistoni, C. Piersimoni, G. Sbaraglia, G. Stagni, and S. Pauluzzi
   * AIDS: Dj Vu in Ancient Egypt? R.J. Ablin

News and Notes 

   * International Meeting on Research Advances and Rabies Control 
     in the Americas, C.E. Rupprecht 
   * Symposium Notice, Infect. Dis. Assoc. of California


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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, Bethesda, Maryland, USA

Dispatches Editor
Stephen Ostroff, M.D., National Center for Infectious Diseases,
CDC, Atlanta, Georgia, USA

Managing Editor
Polyxeni Potter, M.A., National Center for Infectious Diseases,
Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA


Liaison Representatives

Anthony I. Adams, M.D., Chief Medical Adviser,
Commonwealth Department of Human Services and Health,
Canberra, Australia

David Brandling-Bennett, M.D., Director,
Division of Communicable Diseases, Pan American Health Organization,
World Health Organization
Washington, D.C., USA

Gail Cassell, Ph.D.,
Liaison to American Society for Microbiology
University of Alabama at Birmingham
Birmingham, Alabama, USA

Richard A. Goodman, M.D., M.P.H., Editor, MMWR,
CDC, Atlanta, Georgia, USA

William Hueston, D.V.M., Ph.D, Acting Leader,
Center for Animal Health Monitoring, Centers for Epidemiology and Animal
Health Veterinary
Services, Animal and Plant Health Inspection Service, U.S. Dept. of
Agriculture
Fort Collins, Colorado, USA

James LeDuc, Ph.D., Advisor for Arboviral Diseases,
Division of Communicable Diseases, World Health Organization,
Geneva, Switzerland

Joseph Losos, M.D., Director General,
Laboratory Center for Disease Control
Ontario, Canada

Gerald L. Mandell, M.D., Liaison to Infectious Diseases Society of America,
University of Virginia Medical Center,
Charlottesville, Virginia, USA

Phillip P. Mortimer, M.D., Director, Virus Reference Division
Central Public Health Laboratory
London, United Kingdom

Robert Shope, M.D., Director, Yale Arbovirus Research Unit,
Yale University School of Medicine,
New Haven, Connecticut, USA

Natalya B. Sipachova, M.D., Ph.D.
Scientific Editor
Russian Republic Information & Analytic Centre
Moscow, Russia

Bonnie Smoak, M.D., Chief, Dept of Epidemiology, Division of Preventive
Medicine,
Walter Reed Army Institute of Research,
Washington, D.C., USA

Robert Swanepoel, B.V.Sc., Ph.D., Head, Special Pathogens Unit,
National Institute for Virology,
Sandrinham 2131, South Africa

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


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=========================================================================
Perspectives

Molecular Approaches to the Identification
of Unculturable Infectious Agents

Shou-Jiang Gao, Ph.D., and Patrick S. Moore, M.D., M.P.H., M.Phil.
School of Public Health, Columbia University,
New York, New York, USA
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          New molecular biologic techniques, particularly
          representational difference analysis, consensus
          sequencebased polymerase chain reaction, and
          complementary DNA library screening, have led to the
          identification of several previously unculturable
          infectious agents. New agents have been found in
          tissues from patients with Kaposi's sarcoma, non-A,
          non-B hepatitis, hantavirus pulmonary syndrome,
          bacillary angiomatosis, and Whipple's disease by using
          these techniques without direct culture. The new
          methods rely on identifying subgenomic fragments from
          the suspected agent. After a unique nucleic acid
          fragment belonging to an agent is isolated from
          diseased tissues, the fragment can be sequenced and
          used as a probe to identify additional infected tissues
          or obtain extended portions of the agent's genome. For
          agents that cannot be cultured by standard techniques,
          these approaches have proved invaluable for
          identification and characterization studies. Applying
          these techniques to other human diseases of suspected
          infectious etiology may rapidly elucidate novel
          candidate pathogens.

Identifying the causative agent of an infectious disease is the cornerstone
for its eventual control. In recent years, a great deal of progress has
been made in identifying new agents associated with both well-known and
newly emerging infectious diseases. A number of syndromes exist, however,
in which infectious etiology is likely, but the pathogen resists
cultivation with standard microbiologic techniques. For emerging diseases,
rapid identification and characterization of the responsible agent are
crucial first steps for epidemic control.

The rapid identification of a hantavirus responsible for an outbreak of
severe pulmonary distress syndrome in the southwestern United States (1, 2)
demonstrated that applying molecular biologic approaches can accelerate the
identification of an unknown agent. With extensive nucleic acid and protein
databases readily available, isolating and sequencing genomic fragments
from an unknown agent can provide important clues regarding its origin and
biologic behavior. Once a new agent's phylogenetic relationship to other
known organisms is established, appropriate culture conditions, serologic
tests, and perhaps even therapeutic strategies can be rapidly developed.

Although they have revolutionized our ability to identify new pathogens,
innovative molecular biologic techniques must be applied in conjunction
with traditional epidemiologic procedures. Beral, Jaffe and colleagues, for
example, showed that AIDS-related Kaposi's sarcoma (KS) is likely to be
caused by a transmissible agent other than human immunodeficiency virus
(HIV), before any likely causative agent was isolated (3, 4). These
findings focused the attention of investigators on AIDS-KS, which resulted
in the isolation of viral DNA from AIDS-KS lesions and the description of a
new human herpesvirus (5, 6). Spurious associations between infectious
agents and diseases are common, however, and only through careful
epidemiologic studies can a causal link between an organism and a disease
be established. Epidemiologic criteria for causality (7-9), superseding
Koch's postulates, have been used for 30 years, and a critical phase in the
process of new pathogen discovery involves the unambiguous establishment
that an agent is central to the disease process.

The various molecular biologic approaches to agent identification differ in
technical detail, but all rely on isolating nucleic acid fragments
belonging to the agent's genome from diseased tissue. The formidable tasks
of identifying and separating small unique nucleic acid fragments from
human genomic material have been approached by various means, each with its
own particular strengths and weaknesses. The appropriate technique depends
on the type of infectious agent involved (e.g., bacterial or viral),
whether the disease occurs in a normally sterile site, and whether it can
be passaged through animals.

Once a fragment from the agent's genome is isolated and sequenced, standard
genomic walking techniques are used to extend the known sequence, and
computer homology searches can be used to identify the likely phylogenetic
relationship of the agent to other known organisms. In this article, we
highlight recent successful situations when molecular approaches were used
to identify and characterize unknown agents of infectious diseases.

Representational Difference Analysis

Representational difference analysis is one of the more robust methods of
identifying new agents since it does not require prior knowledge of the
agent's class (10). The technique is based on polymerase chain reaction
(PCR) enrichment of DNA fragments present in diseased tissue but absent
from healthy tissues of the same patient (Figure) [Figure not available in 
ASCII). Representational difference analysis is also an important tool 
for identifying polymorphic DNA sequences associated with noninfectious 
diseases (11).

[Figure]
Figure. Schematic representation of different molecular approaches to the
identification of unculturable infectious agents.

Representational difference analysis depends first on digesting DNA from
both healthy and diseased tissues by using a restriction enzyme and then on
separately simplifying the resulting genomes to reduce their sequence
complexity. This is done by ligating PCR primers to both sets of DNA and
nonspecifically amplifying the mixtures. Since PCR most efficiently
amplifies fragments of 150 to1500 bp, restriction fragments in this size
range are enriched, and the fragments of the sequence represented outside
this size range (90%) are reduced.

Unique strands of DNA from diseased tissue representing restriction
fragments of an exogenous agent are isolated in a subtractive hybridization
process coupled to PCR amplification. First, the priming sequences ligated
on DNA restriction fragments of both normal and infected tissues are
removed. New primer sequences are ligated only to the diseased tissue DNA
fragments. These are then hybridized with an excess of the healthy tissue
representation. Human DNA fragments common to both diseased and healthy
tissues reanneal to each other and, since the healthy tissue fragments are
in excess, any given human fragment derived from the diseased tissue will
reanneal to a complementary strand from the healthy tissue representation.
Thus, common human sequences found in both representations will only have
one PCR priming sequence or none present on two complementary strands.
However, DNA fragments from the infectious agent will not find
complementary strands in the healthy tissue representation and will
reanneal with each other. Only hybrids with both strands derived from the
diseased tissue representation will have priming sites and be able to
undergo subsequent exponential PCR amplification. Several rounds of
representational difference analysis are performed, which successively
enrich the mixture for unique DNA sequences present only in the diseased
tissue representation.

KS-Associated Herpesvirus and KS

The power of representational difference analysis and the difficulties
encountered in establishing the etiology of disease are illustrated by its
application to KS (5), a vascular neoplasm that frequently occurrs in
homosexual men with AIDS (3). Geographic clustering (12, 13) and
association with specific sexual behavior (4, 14) suggest that the disease
is caused by a sexually transmitted agent. Several agents have been
investigated, including human cytomegalovirus (CMV), human papillomavirus,
human herpesvirus 6 (HHV-6), and HIV (for review, see [15]), but no
convincing etiologic link has been established.

Using representational difference analysis, Chang and colleagues isolated
two unique DNA sequences (KS330Bam and KS631Bam) from a KS lesion in an
AIDS patient (5). These DNA sequences are homologous to portions of minor
capsid and tegument protein genes from EpsteinBarr virus (EBV) and
herpesvirus saimiri (HVS), a New World monkey herpesvirus. Both EBV and HVS
are gamma herpesviruses associated with neoplastic disorders in humans
(EBV) (16) and nonhuman primates (HVS) (17). These results suggested that a
new human herpesvirus, now called KS-associated herpesvirus (KSHV or HHV8),
could be the KS agent.

The strong association between KS and KSHV was demonstrated by using
Southern hybridization and PCR to amplify a 233-bp DNA fragment (KS330 233)
internal to KS330Bam from all 25 intact and amplifiable DNA samples from KS
lesions that were examined (5). Ninety tissues from patients without AIDS
or KS were examined, and none showed evidence of KSHV infection.

Although controversy remains over the role of KSHV in KS (18-20),
epidemiologic and biologic studies strongly suggest that the agent is a
causal factor in KS (21). KSHV has now been identified in 211 (94.2%) of
224 KS lesions examined by a number of groups around the world, and the
virus is found in all forms of KS, AIDS-related and non-AIDS-related
(22-29). Two independent studies have shown that KSHV is present in
peripheral blood mononuclear cells of AIDS-KS patients before onset of KS
(30, 31), indicating that the virus is unlikely to be a passenger virus
in KS lesions. Further, the virus has been localized to KS tumor tissues by
semiquantitative PCR (25) and by PCR in-situ hybridization (26). The virus
has only been found in 8 (1.8%) of 449 solid tissues from control patients
without KS (5, 22, 25, 27, 28, 32, 38) and thus appears to be specifically
associated with KS. Although early reports suggested that the virus is
present in skin tumors from transplant patients without KS (19), subsequent
studies have not confirmed this finding (33, 34). One study has found
evidence of viral DNA by nested PCR, but not unnested PCR, in semen from
(23%) of healthy donors; these results suggest that the prevalence of
infection in North America may be higher than that indicated by tissue or
lymphocyte studies (35). This intriguing finding has not been reproduced by
other groups (23, 36); however, it should be explored in large rigorous
studies.

Lymphoproliferative disorders are common secondary malignancies in KS
patients with and without AIDS (37). KSHV has also been found in a rare
subset of AIDS-related, body cavity-based lymphomas, which are manifested
as primary effusions (5, 38). In these AIDS-related lymphomas, tumor cells
are coinfected with EBV (38, 39) but, unlike tumor cells in EBV-related
Burkitt's lymphomas, these cells do not exhibit c-myc gene rearrangement.
EBV-uninfected, KSHV-related body cavity-based lymphomas have also recently
been identified (40, 43). Another lymphoproliferative syndrome associated
with KS is Castleman's disease in both AIDS patients and HIV-seronegative
patients. KSHV has been found in AIDS-related multicentric Castleman's
disease lesions at a high copy number and less frequently in tissues from
Castleman's disease patients without AIDS (41).

Identifying KSHV DNA sequences by representational difference analysis also
led to the quick identification of an in vitro culture system for growing
the virus. KS330Bam and KS631Bam probes were used to identify a B-cell line
derived from a body cavity-based lymphoma that was stably infected with
both KSHV and EBV (42). Extended sequencing and transmission studies have
been performed with body cavity-base lymphoma cell lines, which clearly
define KSHV as a new human herpesvirus of the genus Rhadinovirus (6). This
has recently been confirmed by detecting herpesvirus particles in a body
cavity-based lymphoma cell line by electron microscopy (43).

Discovery of a continuously infected cell line has allowed the first
generation of serologic assays for KSHV antibodies to be developed (6).
Second-generation immunoblotting assays, which appear to be both sensitive
and specific for detecting KSHV antibodies, indicate that KS patients are
infected with the virus months to years before the disease develops and
that few North American blood donors are infected (S-J Gao, P.S. Moore,
unpublished observation). Thus, identifying a virus associated with KS by
using molecular approaches has rapidly led to new assays that use
traditional serologic techniques for detecting infection.

HHV-6 and Multiple Sclerosis

The difficulties of using representational difference analysis for new
pathogen identification are also illustrated by the search for the agent
that causes multiple sclerosis (MS) (44). An infectious cause for MS has
been proposed (45-47), and geographic and household clustering of cases
consistent with an infectious process have been shown (45, 48-50).
Challoner and colleagues used DNA from sclerotic plaques in brain tissues
from MS patients and performed representational difference analysis against
pooled DNA from peripheral blood leukocytes of healthy donors. Use of
pooled lymphocyte DNA in representational difference analysis opens the
possibility for examining rare banked tissues for which healthy control
tissues are not available. A 341-bp representational difference analysis
band was isolated from MS tissues with near sequence identity to a region
of the major DNA-binding protein gene of HHV-6 (44). Detecting exogenous
DNA in diseased tissue by this technique, however, does not exclude the
possibility that a commensal agent may be identified that is not the cause
of the disease being examined. HHV-6 is a neurotrophic virus present in
brain tissues from healthy control patients (51), and more than (70%) of
both MS patients and controls were positive for the representational
difference analysis sequence by PCR (44). Although case-patients and
controls cannot be differentiated by the presence or absence of these HHV-6
sequences, only MS tissues showed nuclear staining of oligodendrocytes
surrounding plaques when monoclonal antibodies against HHV-6 viral proteins
were used (44). There may be a subtle difference in tissue distribution for
the virus in MS patients not found in controls, or the HHV-6 variant B
group 2 infecting MS plaques may be particularly prone to generating the
autoimmune response seen in this disease.

GB Hepatitis Viruses

Since representational difference analysis relies on PCR amplification, the
technique is suited for detecting agents with a DNA-based genome; sequences
can be amplified by generating a cDNA intermediate, but detecting RNA
viruses is problematic since mRNA expression patterns differ between
tissues and are likely to give spurious representational difference
analysis bands. Simons and colleagues overcame this problem by passing an
agent associated with non-A, non-B (NANB) hepatitis through primates and
using cell-free extracts of primate plasma for representational difference
analysis and discovered two new human hepatitis viruses (52, 53).

A novel form of NANB hepatitis was first identified in a physician who
became ill with hepatitis, and the disease was transmitted to primates by
injecting serum from patients into the animals (54). Extensive studies
demonstrated that the virus, named the GB agent, was different from all
known human hepatitis viruses (hepatitis A [HAV], hepatitis B [HBV],
hepatitis C [HCV], and hepatitis E) (55-57). Total RNA from the samples was
reverse-transcribed to obtain cDNA by using cell-free preinfection and
acute-phase plasma from infected monkeys. After cDNA synthesis,
representational difference analysis was performed, and seven cDNA clones
were found to be specifically associated with this form of hepatitis.
Sequence analysis and comparison with other known hepatitis viruses
identified two unique flaviviruses, GBVA and GBVB, as the GB agents (52,
53, 58). A third virus, GBVC, was subsequently identified using the known
GBVA, GBVB, and HVC consensus sequences (52b and see below). GBVA and GBVC
are closely related phylogenetically. Cross-challenge experiments showed
that GBVC probably originated in human hepatitis patients, whereas GBVA and
B may be tamarin monkey viruses that were inadvertently passaged along with
the human virus (53, 58).

In addition to having difficulty in identifying RNA viruses,
representational difference analysis has several other limitations.
Polymorphic human DNA can be amplified through this technique (5), and not
all bands generated by it belong to the suspected agent. Representational
difference analysis may produce DNA fragments from an agent whose genome
has not been sequenced (e.g., most bacteria and fungi), and sequence
homology searches may be unable to distinguish the agent's genomic DNA
fragments from unsequenced human DNA. Only normally sterile site tissues
are appropriate for this technique because normal flora that differs
between tissue sites could result in spurious amplification. Finally, for
viruses with small genomes, multiple restriction digests may be required to
identify a unique restriction fragment of the appropriate size that can be
efficiently amplified. Although the technique has been successfully used by
several groups to identify polymorphic DNA, the procedure is complex and
not uniformly reproducible.

Consensus SequenceBased PCR

Consensus sequence PCR relies on the use of highly conserved DNA sequences,
such as ribosomal RNA (rRNA) gene sequences from known organisms, to
amplify DNA from related organisms not yet discovered (59). This technique
is simple and extremely successful in identifying new human pathogens.
Subunit rRNA genes evolve in a relatively slow and uniform manner, which
makes these sequences extremely useful for establishing phylogenetic
relationships (59). By using conserved DNA sequences from bacterial 16S
rRNA genes, at least two new bacteria associated with human diseases have
been identified (60, 61), and PCR amplification of conserved hantaviral
capsid DNA sequences resulted in the rapid identification of a new
hantavirus associated with an outbreak of severe pulmonary disease (1).

Unlike using representational difference analysis, using consensus
sequences to amplify DNA requires knowledge of the suspected agent's
phylogenetic relationship to other organisms. The technique generally
should be used on normally sterile site tissues if sequences from normal
flora are also likely to be amplified. Although the lack of broadly
amplifiable consensus primers limits use of this technique to prokaryotic
and eukaryotic pathogens, consensus sequences are to likely exist among
many of the classes of viruses that eventually could be used in screening
panels of diseased tissues when a viral cause is suspected.

Bartonella: Bacillary Angiomatosis and Cat-Scratch Disease

Phylogenetic studies of bacteria have relied on comparisons of highly
conserved rRNA gene sequences present in prokaryotes (62). rRNA genes are
present in all living cells and contain regions of highly conserved
sequences with intervening variable regions. Conserved sequences can be
used to amplify and sequence the intervening variable regions by PCR. This
technique was exploited by Relman and colleagues to identify the bacillus
associated with bacillary angiomatosis (60).

Bacillary angiomatosis is an inflammatory vascular proliferative process
that affects the skin, lymph nodes, and visceral organs of AIDS patients
(63). Although bacilli can be identified by Warthin-Starry staining of
lesions (64, 65), the suspected causal organism was resistant to
cultivation by standard techniques. Consensus oligonucleotide primers
complementary to the 16S rRNA genes of eubacteria were used to amplify 16S
rRNA gene fragments directly from tissue samples (60). Phylogenetic
analysis of the amplified DNA sequence showed that the organism belonged to
the genus Rochalimaea (now renamed Bartonella) (60). Bartonella organisms
were also cultured from bacillary angiomatosis lesions (66) and blood from
bacteremic patients (67, 68), and serologic analyses have associated the
organisms with cat-scratch disease (69). Current evidence suggests that
bacillary angiomatosis in HIV-seropositive patients results from infection
with either B. quitana or a newly described Bartonella species, B. henselae
(68, 70), whereas cat-scratch disease is primarily caused by infection with
B. henselae (71) (for review, see [72]).

Whipple's Disease

Consensus sequence PCR was also used to identify an organism associated
with Whipple's disease, one of the most persistent mysteries in
microbiology (61). Whipple's disease is a systemic illness, first described
in 1907, characterized by arthralgias, diarrhea, abdominal pain, and weight
loss (73). Rod-shaped bacilli were identified histologically in Whipple's
disease lesions in the early 1960s (74), but the suspected bacteria were
not culturable by standard techniques (73). The agent was found to be a
gram-positive actinomycete (Tropheryma whippelii gen.nov.sp.nov.),
unrelated to any characterized species when a bacterial 165 rRNA sequence
was amplified and sequenced directly from infected tissue (61, 75).

Hantavirus Pulmonary Syndrome

Consensus PCR primers have been successfully used to identify and classify
bacteria, and similar techniques can be used to diagnose new viral agents.
In May 1993, an outbreak of unexplained acute respiratory illness with a
high death rate occurred in the southwestern United States (76). In the
initial phases of the investigation, the cause of the syndrome was not
clearly known to be infectious. Serologic tests quickly detected
crossreactive antibodies to known hantaviral antigens in the serum of
patients; these results suggested that a previously unrecognized hantavirus
was the cause of the disease (77). PCR primers, based on consensus
sequences within the G2 protein coding region of the M segment of the
genomes of known hantaviruses, were designed and used in nested
reverse-transcription PCR to amplify a short segment of the viral genome
from diseased tissues (1).

Sequence analysis of the PCR products showed that the amplicon differed
from the other known hantaviral sequences by least 30%, and phylogenetic
analysis demonstrated that the new virus is most closely related to
Prospect Hill hantavirus (1), a zoonotic hantavirus endemic in North
America (78). Hantavirus antigens have been detected in endothelial cells
from patients (1, 79), and virus particles have been identified in infected
pulmonary endothelial cells and macrophages (80). Deer mice (Peromyscus
maniculatus) are the primary host for the virus (1), and the virus has been
passaged through laboratory-bred deer mice and cultured in Vero E6 cells
(2). This newly recognized virus was originally named Muerto Canyon virus
(2) but has since been renamed Sin Nombre virus in light of nomenclatural
disputes regarding the appropriateness of a descriptive name.

Complementary cDNA Library Screening

Identification of HCV and HGV

A third major approach to new organism identification relies on screening
cDNA libraries made from diseased tissues by using hyperimmune serum from
specimens. This method was successfully used to identify the cause of most
cases of NANB hepatitis, HCV. When serologic tests for HAV and HBV became
available, it became clear that most cases of transfusion-associated
hepatitis in the United States were not caused by either virus (81, 82).
Conventional techniques failed to identify the agent responsible for most
cases of NANB hepatitis (83), despite evidence that the disease was caused
by a bloodborne, small, enveloped virus readily transmissible to
chimpanzees (84, 85).

Since the virus was presumed to be an RNA virus, Choo and colleagues made a
cDNA expression library from RNA isolated from an infected chimpanzee's
plasma (86). While the GBVA, B, C were identified by direct detection of
viral cDNA through representational difference analysis, HCV cDNA was
identified by immunologic detection of cDNA that was encoding viral
protein. Viral antigens expressed from the cDNA library were identified by
immunoscreening with convalescent-phase human sera. A cDNA clone was
isolated encoding an antigen that could be used to screen
convalescent-phase sera from patients with NANB hepatitis. Identification
of the clone rapidly led to the production of a recombinant antigen used
for serologic screening to detect specific antibodies in infected
chimpanzees and patients with hepatitis after transfusion. Extended
sequence analysis demonstrated that the agent, now known as HCV, is closely
related to the family Flaviviridae and is the major cause of NANB hepatitis
throughout the world (86, 87). Recently, the same approach was successfully
used to identify HGV, which has been found to be almost identical to GBVC,
the human hepatitis virus identified by representational difference
analysis (88). The potential role of HGV and GBVC in human disease still
remains uncertain (88b).

Using convalescent-phase sera to screen cDNA libraries from diseased
tissues is a novel method for pathogen identification. It is a potentially
useful technique for diseases in which well-defined convalescent-phase sera
are available, and it requires tissues with a high titers of the agent. On
the other hand, constructing and screening DNA libraries are laborious, and
crossreactive antigens are likely to be detected, especially for diseases
in which autoantibodies are common.

Future Directions

Nucleic acid database information is rapidly expanding for all classes of
organisms, and a significant fraction of the human genome has already been
sequenced. Even small laboratories can exploit new and relatively
inexpensive molecular biologic technologies to search for new pathogens.
Once a small, unique nucleic acid fragment from a pathogen has been
identified, nucleic acid detection and serologic assays can often be
rapidly developed to establish an etiologic link with disease.

New pathogens are likely to be identified by some of these molecular
biologic approaches. A number of diseases have long been suspected to have
an infectious cause and are appropriate candidates for these techniques.
The eventual identification of infectious etiologic agents of diseases such
as sarcoidosis (89), Kawasaki's disease (90), and type I diabetes mellitus
is likely. New techniques, such as arbitrarily primed PCR and phage display
libraries (91), have not been used for pathogen discovery but show promise
for expanding the repertoire of techniques available for identifying
unculturable agents. Three years passed between the initial descriptions of
AIDS and the identification of HIV (92). Use of molecular biologic
approaches could lead to rapid identification and control of the next
pandemic caused by a newly emergent infectious disease.

Acknowledgments

We thank Drs. David Relman, Stanford University, and Yuan Chang, Columbia
University, for critical advice regarding new pathogen identification and
comments on the manuscript, and Francis Zappa for help in preparing the
manuscript.

Dr. Gao received his Ph.D. in virology from the University of Bordeaux,
Bordeaux, France. He is a research scientist in the School of Public
Health, Columbia University, New York, where his work focuses on the
serology and seroepidemiology of Kaposi's sarcomaassociated herpesvirus
(KSHV). He also works on the induction of lytic phase replication of KSHV
as well as genes that may be involved in the latent phase replication of
the virus.

Dr. Moore is an associate professor of clinical public health at Columbia
University. His current research is focused on delineating the relationship
between Kaposi's sarcoma and KSHV (HHV8) and understanding the tumorigenic
potential of this virus. Along with Dr. Yuan Chang, his laboratory
codirector and wife, he is working on both epidemiologic and basic
virologic studies of KSHV and other new potential pathogens.

Address for correspondence:  Shou-Jiang Gao, Ph.D., School of Public
Health, Columbia University, P&S 14-442, 630 West-168th St., New York,
NY 10032; fax: 212-305-4548; e-mail: sg192@columbia.edu.

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=========================================================================
Perspectives

DNA Vaccines for Emerging Infectious Diseases: What If?

Robert G. Whalen
Centre National de la Recherche Scientifique
Paris, France
---------------------------------------------------------------------------


        A novel and powerful method for vaccine research,
        colloquially known as DNA vaccines, involves the
        deliberate introduction into tissues of a DNA plasmid
        carrying an antigen-coding gene that transfects cells in
        vivo and results in an immune response. DNA vaccines have
        several distinct advantages, which include ease of
        manipulation, use of a generic technology, simplicity of
        manufacture, and chemical and biological stability. In
        addition, DNA vaccines are a great leveler among
        researchers around the world because they provide
        unprecedented ease of experimentation. To facilitate
        diffusion of information, an Internet site has been
        established called The DNA Vaccine Web
        (URL:http://www.genweb.com/Dnavax/dnavax.html). In this
        review, a brief survey is undertaken of the experimental
        models and preclinical work on DNA vaccines to contribute
        to a greater awareness of the possibilities for emerging
        infectious diseases.

But there is no single problem that is more pressing than our
fast-deteriorating relations with the microbial world wrote Barbara
Culliton (1), the editor-in-chief of Nature Medicine, at the end of 1995.
This stark statement concluded her comments on, among other threats, the
reemergence of cholera and plague, the growing number of Lyme disease
cases, and humanity's occasional but frenetic duels with Ebola virus. What
if one day an Ebola-infected traveler makes it to the boarding lounge and
embarks on an airplane? As pointed out by David Heymann, director of the
World Health Organization's new Division of Emerging Diseases, the virus
would spread to far corners of the world (2) with dramatic consequences.
This scenario has already been played out with human immunodeficiency virus
(HIV). Vaccines have traditionally been used as weapons against health
threats. In the case of HIV infection, the basis for one has not yet been
clearly delineated. For scientific, commercial, and practical reasons
vaccines cannot solve all the problems posed by emerging infectious
organisms. However, novel and powerful methods for vaccine research, and
possibly for vaccines themselves, hold some promise in our efforts to curb
emerging disease threats. These methods involve the deliberate introduction
of a DNA plasmid carrying a proteincoding gene that transfects cells in
vivo (albeit at a low efficiency) and expresses an antigen causing an
immune response (3). This procedure, known as a DNA vaccine, is perhaps
better described as DNA-mediated or DNA based immunization, with the
understanding that the objective is not to raise an immune response to the
DNA itself.

This method is conceptually sound and experimentally straightforward;
however, its most novel aspect is that it works at all! It was not expected
that pure plasmid DNA could be taken up by cells, after parenteral
introduction in a simple saline solution (4), to levels allowing expression
of enough protein to induce an immune response. A more radical method of
introducing DNA involves the bombardment of DNA-coated gold particles. When
applied to the skin, these particles produce good immune responses with
much less DNA than is required by other routes, such as intramuscular or
intradermal needle injection (5). More esoteric still is the application of
pure DNA solution (as nose drops) to the nasal membranes, which has been
reported to work (5) but is perhaps too inefficient for further
consideration.

DNA vaccines have distinct advantages: They can be manufactured far more
easily than vaccines composed of an inactivated pathogen, subcellular
fraction, or recombinant protein. Since almost all plasmids can be
manufactured in essentially the same way, substantial economies of scale
can be achieved. DNA is very stable and resists temperature extremes;
consequently, the storage, transport, and distribution of DNA-based
vaccines are more practical and less expensive. In addition to the
commercial, there are vaccine research and development considerations. It
is now possible to change the sequence of an ntigenic protein, or to add
heterologous epitopes, by simply introducing mutations to the plasmid DNA.
The immunogenicity of the modified protein can be directly assessed after
injecting the plasmid DNA. This simple method could increase considerably
our understanding of the immune response to antigens.

In addition, both in vaccine research and in actual use, DNA-mediated
immunization is the great leveler among researchers around the world. It is
easy to use because once the protein coding sequences are cloned into a
suitable expression vector, the direct introduction of the plasmid vector
(into mice for example) allows experimental assessment of the immune
response and its consequences, without further experimental steps such as
the preparation of a recombinant protein as antigen.

Because it is so straightforward and requires only simple molecular
biologic techniques, the method should be practical in many laboratories
around the world. It would be unrealistic to deny that certain diseases are
not of great interest to large pharmaceutical firms. Moreover, even when
triumphs over disease are achieved, as in the case of hepatitis B virus
(HBV) infection (6), marketing strategies can exclude the vaccine from
regions where it would do the most good. DNA-mediated immunization can be
used in countries that cannot implement more complicated and expensive
strategies.

What if DNA vaccine research could be carried out worldwide by a generic
technology, where human creativity adds substantial value to the work? This
short review outlines the possibilities offered by DNAmediated
immunization. (An Internet site, The DNA Vaccine Web (URL:
http://www.genweb.com/Dnavax/dnavax.html) provides journal references,
abstracts, experimental protocols, available plasmids, and other
information about DNA vaccines.) I will review experimental models and
preclinical work on DNA vaccines and discuss new developments that are
based on the idea that DNA delivery can be used to induce immune response
to proteins.

Animal Models of DNA Vaccines

Various experimental models of DNA vaccination have been reported (Table
1). Most of the pathogens studied have been viruses, which is consistent
with the method used: since the genes transferred by the plasmids require
the host cellular machinery to be expressed, DNA-based immunization most
resembles a virus infection. However, genes from other microorganisms have
also been used with success. The types of polypeptides expressed are often
the envelope proteins of viruses, but various proteins have been used.
Indeed, it is not obvious what aspects of a protein produce an effective
immune response by this unusual method of antigen delivery.

The immune responses obtained are clearly broad-based when they have been
well characterized. In several models, antibodies are reproducibly induced,
and the antibodies ultimately are of the immunoglobulin G (IgG) type,
indicating a T-dependent class switch. In the case of immunization for HBV
surface antigen, the fine specificity of the humoral response in mice
mimics, to a certain extent, that observed during infection in humans (12).
T-cell proliferation and cytokine secretion have been studied in several
models, and the cytokine profile indicates a Th1 type response,
characterized by the secretion of interleukin-2 and gamma-interferon. The
immune responses can be remarkably long lasting; however, the duration of
the immune response does not appear to have any deleterious effects on the
animals because they are protected against challenge long after imunization
(41). Also, in some cases, as in the DNA-mediated immunization against HBV
surface antigen, antibody levels reach a plateau at titers of about 10 to the 
4th power and remain stable for at least 18 months. If the mice are boosted 
with a second DNA injection at 7 months, a further 10-fold increase in titer 
can be obtained (14).

Cytotoxic T lymphocytes (CTL) are invariably induced to class I epitopes of
the proteins encoded by the transferred genes. These CTL responses can be
quite strong (10), and DNAmediated immunization can circumvent
haplotype-linked nonresponsiveness (13). Cytotoxic immune responses are
thought to be important in clearing viral infections because this type of
T-cell response allows the immune system to recognize virally infected
cells and destroy them, thus removing the virus.

What is surprising about the induction of CTL with DNA-mediated
immunization is that either professional antigen-presenting cells have been
successfully transfected, or the free protein can be processed more
effectively with CTL induction than with a classical immunization protocol.
A large body of immunologic evidence suggests that only professional
antigen-presenting cells can prime T lymph ocytes in the first stages of
the immune response. Thus, the transfection of muscle fibers, for example,
would not normally be expected to lead to such efficient immune responses.
Solution of this enigma should provide insight into some fundamental immune
processes. In the meantime, DNA-based immunization is an excellent and
simple tool to achieve CTL responses for which efficient methods have been
needed for years.

One of the first uses of DNA-mediated immunization was to induce such
cytotoxic cellular immunity to a conserved protein of influenza A virus to
determine if overcoming its seasonal variation was possible. Ulmer et al.
(26), obtained cytotoxic T lymphocytes directed against the conserved
influenza nucleoprotein, which protected mice against the disease, even
when they were challenged with a heterologous virus carrying a different
hemagglutinin but the same nucleoprotein sequence.

                  Table 1. Animal models of DNA vaccines
--------------------------------------------------------------------------
        Pathogen               Antigen             Animal      References
                                                  Species
--------------------------------------------------------------------------
 Bovine herpesvirus     Glycoprotein           Cattle, |     (7)
                                               mouse
 Hepatitis B virus      Capsid (core antigen)  Mouse         (48)
 Hepatitis B virus      Envelope protein       Mouse         (3, 8-14,
                        (surface antigen)      Rabbit        53)
                                               Rat
                                               Chimpanzee *

 Hepatitis C virus      Core/Nucleocapsid      Mouse         (12, 15)
 Herpes simplex virus   Glycoprotein B         Mouse | *     (16-18)
                        Glycoprotein D
                        ICP27
 Human immuno-          Envelope glycoprotein  Mouse         (19-22)
   deficiency virus-1   gp160                  Nonhuman
                        Noninfectious          primates
                        particles
 House dust mite        Allergen               Rat *         (52)

 Influenza virus        Hemagglutinin          Chicken *     (23-29)
                        Matrix protein         Ferrets *
                        Nucleoprotein
                                               Mouse *
                                               Nonhuman
                                               primates *

 Leishmania major       Major surface          Mouse *       (50)
                        glycoprotein
 Lymphocytic chorio-    Glycoprotein           Mouse | *@    (30-32)
   meningitis virus     Nucleoprotein
 Mycobacterium          M. leprae hsp65        Mouse |       (33)
    tuberculosis
 Mycoplasma pulmonis    M. pulmonis DNA        Mouse *       (34,35)
                        M. pulmonis DNA
                        expression library
 Papillomavirus         Major capsid protein   Rabbit *      (51)
                        L1
 Plasmodium yoelii      Circumsporozoite       Mouse |       (36-38)
                        protein
 Rabies virus           Glycoprotein           Mouse *       (39-41)

 Simian                 Env, gag               Monkeys       (49)
 immunodeficiency
 virus
 Schistosoma japonicum  Paramyosin (Sj97)      Mouse         (42)
--------------------------------------------------------------------------
 The symbols | and * refer to reports of partial or complete protection,
 respectively, to challenge by the infectious agent. In one case, enhanced
 immunopathology (@) after intracranial viral challenge has been
 reported. (32).

 Updated versions of this table can be found on The DNA Vaccine Web site
 (URL: http://www.genweb.com/Dnavax/Announce/table.html) where links are
 provided to the Medline abstracts of most references. See also a
 compendium of recent articles on DNA vaccines in a special issue of the
 Annals of the New York Academy of Sciences, Volume 772 (New York Academy
 of Sciences Conference: "DNA Vaccines: A Novel Approach to Vaccination,"
 Arlington, VA, April 7-9, 1995). The Medline abstracts of the articles in
 this issue can be found on The DNA Vaccine Web.
--------------------------------------------------------------------------

The crucial point, however, is the ability of DNA-based immunization to
protect animals from infection upon challenge, and this has been
demonstrated in several model systems, particularly with influenza and
rabies viruses, as well as Mycoplasma pulmonis and Plasmodium yoelii.
Donnelly et al. (29) have shown that a mixture of plasmids can be used to
induce antibody to the influenza hemagglutinin surface protein and
cytotoxic immunity to the viral nucleoprotein and matrix protein. This DNA
vaccine protected ferrets and African green monkeys against viral challenge
by using an antigenically distinct human influenza virus more effectively
than the contemporary commercial vaccine. The influenza model has now been
the subject of much preclinical work, and human trials are in progress.

One human trial uses plasmid vectors expressing HIV-1 genes delivered to
HIV-seropositive persons by intramuscular injection. This protocol uses
Marcaine to facilitate DNA uptake, although the mechanism of this effect
has not been clearly delineated (20). More clinical trials will likely be
initiated since plasmid DNA is now considered an innocuous substance
compared with other genetic vectors used in therapy.

Further Questions on Methods

Most studies listed in Table 1 have used intramuscular needle injection to
deliver DNA; however, intradermal particle bombardment works very well
(provided that one has access to biolistic apparatus). Intradermal particle
bombardment may ultimately work better than intramuscular injection in
primates, including humans, since extensive connective tissue in muscle may
impede DNA diffusion and transfection in larger mammals that do not lead
sedentary lives in cages.

In most of our own experimental work (8-12), we have used a protocol that
induces muscle regeneration and increases the number of transfected muscle
fibers (43), presumably because of improved diffusion of the DNA and better
transfection efficiency of immature fibers. The actual protocol is probably
not suitable for routine prophylactic vaccination in humans, although it
could be considered for therapeutic immunization. Nonetheless, this model
system shows that muscle fibers can take up DNA better under some
circumstances, and further understanding of this process may improve
formulations for DNA uptake in muscle. Such improvements will almost
certainly be a prerequisite for intramuscular DNA vaccination in humans.
The improved uptake of DNA in regenerating muscles composed of immature
fibers also suggests that newborn mammalian muscle may also take up DNA
more effectively than adult muscle. Since improving childhood vaccination
programs is a goal, this characteristic may be an important consideration
in the use of DNA vaccines, if they can be used for children.

The regeneration protocol is particularly useful for scientists who are
beginning research with DNA-mediated immunization. The use of available
control plasmids to inject DNA for validating the procedure is also an
important consideration. The various steps that can be taken to achieve
reproducible immunization with plasmid DNA transfer have been discussed
(44). Careful attention to the details of the intramuscular injection
protocol (43,45) can make a substantial difference in the outcome.

Vaccine Development and the Limits of the Protective Immune Response

A successful vaccine must confer protective response to the recipient, and
therefore, the limits of the immune response must be known. This knowledge
can be purely empirical, as in the case of the first polio vaccines for
which the precise protective epitopes were not known. Such knowledge is not
required when using a killed or attenuated viral preparation. In contrast,
for recombinant vaccines a single protein should induce an immune response
that will provide sterilizing immunity; this is also true for DNA vaccines
and represents a major conceptual limitation in the use of this approach
for vaccination. Although mixtures of individual proteins or their genes
can be envisioned, in the case of recombinant protein vaccines this would
be pro hibitively complicated and expensive. For DNA vaccines this is far
easier to imagine since the injected material is always DNA, and no matter
what genes are carried by the plasmid, the production process is the same.

The only human vaccine that uses a recombinant protein as its basis is that
against HBV, which has been used for nearly 10 years. Such a high-tech
vaccine was possible because empty viral particles, from the plasma of
persons chronically infected with HBV, could be purified and used to induce
humoral immunity against the so-called surface antigen that would protect
against infection. The plasma-derived particles are still used as a vaccine
in many countries, in part because of the cost of the recombinant product
(6). The envelope proteins of viruses are always good targets for inducing
protective immunity.

However, this rational clearly breaks down in the case of a virus, such as
HIV, which mutates so rapidly, apparently in response to immune pressure.
The fundamental knowledge required to determine what would comprise a
potential HIV vaccine is still lacking, and therefore, it is unlikely that
a vaccine can be developed until further basic research provides more
insight. Thus HBV and HIV illustrate two extremes in vaccine development:
with HBV a simple antibody response to a single antigen neutralizes the
virus, whereas with HIV some form of cytotoxic immune response is probably
necessary. DNA-mediated immunization has a role to play in further
research.

Expression Library Immunization

An attempt has been made to use DNA-mediated immunization to develop a
systematic method for producing a vaccine. Barry et al. (34) took advantage
of the very small amounts of DNA required with the particle bombardment
method (5,46). Since a single nanogram of DNA coated on the gold particles
can induce an immune response, one microgram can potentially introduce a
thousand different genes. On this basis, a library of gene fragments was
prepared from Mycoplasma pulmonis by cloning the genomic DNA into a plasmid
expression vector. Since this organism has a relatively small genome (about
10 to the 6th power base pairs), enough of the total DNA protein-coding 
sequences might be expressed to induce immunity to the pathogen. Since only 
a small part of the genetic complement of the organism is expressed and 
expression is mostly from only a fragment of genes and not entire functional 
proteins, pathogenic effects would be avoided, while all the advantages of
broad-based immunity produced by a DNA vaccine would be present.

Protection against M. pulmonis has been achieved after immunization with
different expression libraries (34). The next steps could consist of using
the method to screen for the gene or genes responsible for protective
immunity. Seen in this light, this approach solves the problems of having a
more general way to determine the limits of protection, which is vital in
establishing what the protein or DNA composition of a vaccine would be. The
search for a single gene, or a small number of genes, should be facilitated
by the use of the DNA-based immunization method. However, if many gene
products are required to confer protection, defining the correct mixture
may not be straightforward.

This discussion raises another consideration. From several points of view
(at least regulatory and manufacturing), a vaccine must contain defined
components. Therefore, the expression library immunization approach cannot
be used with simple mixtures of uncharacterized gene fragments. Although in
principle it is possible, would anyone be prepared to have a human vaccine
composed of an undefined mixture of HIV or Ebola virus gene fragments that
seemed to confer protection in animal models? Once again, the simple method
means that further creative research may rapidly provide insight into
vaccine design.

What If an Ebola Outbreak Threatens the World?

Let us take the most provocative scenario to illustrate what can perhaps be
done, in principle and in practice, in the face of a rampantly infectious
viral disease. When preventive measures against an agent like Ebola virus
are needed on an emergency basis, speed is imperative. The filoviruses
Ebola and Marburg are extremely pathogenic, causing a fulminating febrile
hemorrhagic disease; they grow fast, kill most cells, and the infected
person bleeds to death, usually within 48 hours of infection.

These viruses are enveloped filamentous particles with a nonsegmented
negative-strand RNA. The genes of both members of the filovirus genus, the
Marburg virus group and the Ebola group, have already been cloned and
sequenced. There does not, as yet, seem to be any great variation among the
proteins sequences for a given virus; however, Ebola and Marburg are not
highly related serologically. The filoviruses are similar to
paramyxoviruses, such as the respiratory syncytial virus.

This basic molecular information is enough for testing the ability of one
or more of the genes to induce an antibody and CTL response that might help
protect against disease. One would choose genes whose proteins are not
responsible for the pathology of the virus, but this should be relatively
easy, since the growth potential of the virus largely accounts for its
extreme toxicity. One option is the gene encoding the glycoprotein, a viral
membrane protein requiring glycosylation for its native structure. Such
glycosylation might be important for the antigenicity of the protein if it
is to mimic the filamentous envelope of the virus. Here, the fact that DNA
vaccination involves synthesis of the protein in the cells of the mammalian
recipient of the DNA vaccine means that the appropriate glycosylation
pattern can be produced.

If Ebola or Marburg, or a related filovirus, escaped a restricted ecologic
niche, the following scenario might unfold. If the emerged virus is found
(by serologic testing) to be one of the existing, characterized ones,
existing cloned genes could be used. Otherwise, a virus isolate would need
to be obtained, and the genes would need to be cloned. Cloning can be
accomplished easily since related genes are available for probes in
molecular cloning experiments.

Since one strategy for prophylactic vaccination is to induce a humoral
response to the outer coat of the virus and block entry to the cells,
cloning would allow an attempt to quickly counter the virus infection or at
least to slow down its spread in the organism or within the population. A
full-length envelope glycoprotein gene could be isolated from the cloned
virus genome (perhaps by polymerase chain reaction) and cloned directly
into a suitable DNA vaccine plasmid vector. A GMP production run could be
carried out, perhaps within a week, and DNA vaccine could begin to be
distributed to areas most critically at risk.

The above scenario assumes that DNA vaccines will be accepted for use in
humans, which seems likely for high-risk situations, and that the basic
method for preparing GMP-grade plasmid DNA is available, which is currently
the case. Since August 1995, a collaboration between Qiagen (Hilden,
Germany) and Pharos (Seraing, Belgium) has offered full-GMP production of
plasmid DNA to companies and the scientific community in general. The
molecular method needed to rapidly go from a virus isolate to the
vaccinating plasmid vector should represent only a relatively minor
bottleneck to the development of an urgently needed vaccine, at lest
compared with any other approach used before. It is not surprising that
Bernard Dixon, writing in Bio/Technology (47), has called DNA vaccines the
third vaccine revolution.

This review poses questions about DNA vaccines and suggests that the
answers lie in new methods of research and development. If DNA-mediated
immunization were used in all countries that have expertise in molecular
biology, novel vaccines would be developed. Ultimately, a major goal of the
DNA vaccine approach for public health might well be to bring vaccine
development within the reach of researchers working on infectious disease
problems in which there is no great commercial interest. If DNA vaccines
come into widespread use for public health applications, vaccines for many
diseases could be produced rapidly since, in the end, the product is simply
a DNA plasmid.

What if such a method were used for human vaccination? The cost of
production and delivery of vaccines would be reduced, thus allowing
vaccines to reach areas of the world somewhat deprived of preventive public
health measures, particularly the recent biotechnologic methods. If new
infectious diseases appear in the future, as they surely will, perhaps
these new tools will be used to combat them more effectively.

Today's research method can be tomorrow's vaccine. DNA vaccines will be
within the means of many more populations and countries since full GMP
production technology will be both simpler than technology for other
products and far more available to research scientists. A little more than
2 years stood between the first published description of a DNA vaccine and
the beginning of the first clinical trial, which indicates that the
necessary infrastructure for producing DNA vaccines was rapidly put in
place. But more importantly, this short time span bodes well for the
ability of public health agencies all over the world to bring scientific
research to bear on diseases relevant to their own situation and to disease
prevention.

Dr. Whalen is director of research, French National Center for Scientific
Research. His previous work at the Pasteur Institute in Paris, France,
concerned gene expression in muscle tissue, which serendipitously led to
the study of DNA-mediated immunization that used the hepatitis B surface
antigen as a model. Dr. Whalen collaborated closely with Drs. Heather
Davis, Loeb Institute for Medical Research and the University of Ottawa,
Ottawa, Canada, and Marie-Louise Michel and Maryline Mancini, both of the
Pasteur Institute.

Address for correspondence: Robert G. Whalen, Collge de France, 11, Place
Marcelin Berthelot, 75231 Paris cedex 5, France; fax: +33-1-44-27-13-09;
email: whalen@pasteur.fr; URL: http://www.genweb.com/Dnavax/dnavax.html.

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============================================================================
Synopses

Conjugate Vaccines and the Carriage of Haemophilus influenzae Type b

Marina L. Barbour, D.Phil., M.R.C.P.
John Radcliff Hospital, Oxford, England
---------------------------------------------------------------------------


        Pharyngeal carriage of Haemophilus influenzae type b (Hib)
        is important in the transmission of Hib organisms, the
        pathogenesis of Hib disease, and the development of
        immunity to the bacterium. The remarkable success of
        current vaccination programs against Hib has been due in
        part to the effect of conjugate Hib vaccines in decreasing
        carriage of Hib. This review explores evidence for this
        effect, and discusses the possible mechanisms of the
        mucosal influence of Hib conjugate vaccines.

Many junior doctors today have not had occasion to treat a child for
Haemophilus influenzae type b (Hib) disease. The remarkable absence of
cases of this disease is due to the use of conjugate vaccines. In countries
with established Hib vaccination programs the incidence of disease has
declined sharply (1). In fact, in some countries protection provided by Hib
conjugate vaccines appears to extend to unvacinated infants in the
population (2,3). This phenomenon has been attributed to the conjugate
vaccines' effect of decreasing Hib colonization; however, few controlled
studies have been conducted in this area and many aspects of the Hib
conjugate vaccines' influence on carriage remain speculative. This review
explores Hib carriage in the context of current efforts at elucidating the
effect of conjugate vaccines on Hib within human mucosae.

What Is Carriage?

A carrier is a person who harbors a specific infectious agent in the
absence of clinical illness with or without a detectable immune response
(4). The carrier state may reflect carriage of the organism in the
incubation period before clinical symptoms appear, during an illness, or
after recovery from illness. The carrier state may be short or long, and it
may be intermittent or continuous. Carriers may spread infection to others.
Latency should be distinguished from the carrier state, in that a latent
organism is not transmissible. Hib carriage can be synonymous with Hib
colonization and is defined as the presence of viable Hib organisms in the
human pharyngeal mucosa. This definition depends on the sensitivity and
specificity of the process used to identify viable bacteria in a healthy
host.

The Detection of Carriage

Because Hib carrier status is usually determined by culture techniques, the
efficiency of every step in the microbiologic investigation must be
maximized. Potential problems in the microbiologic detection of Hib include
consistency of swabbing technique, which is difficult to maintain; survival
of the organism during transport on the swab to the culture medium; and the
morphologic similarity of Haemophilus species on solid media, their
fastidious growth requirements, and the abundance of other bacteria in the
specimens.

The development of an antiserum agar culture method by Michaels et al. (5)
has overcome some of the problems in isolating Hib from a sample containing
mixed flora. This method has been successfully modified for use in large
scale studies (6). Pharyngeal swabs are plated onto a transparent solid
medium impregnated with high-titer antiserum for the serotype b capsular
polysaccharide. Isolated Hib colonies in a mixed culture are readily
recognized on this medium because they are surrounded by halos of
antigen-antibody precipitate.

Most surveys of Hib carriage use Michaels' antiserum agar method to
establish carrier status. Before this method became available, the
diversity of culture methods used to isolate Hib from pharyngeal swabs was
a major factor complicating the interpretation of data from different
studies. In most investigations, difficulties in isolating Hib may have
contributed to an underestimate of Hib carriage rates.

The Epidemiology of Hib Carriage

The most important factors contributing to the epidemiology of Hib carriage
are social and demographic. The probability of Hib carriage in a young
child seems closely related to the likelihood of exposure to the organism.

Most surveys agree that nasopharyngeal or throat cultures recover Hib in 3%
to 5% of young children (7,8), with age being a prominent determinant of
Hib carriage rates (9). Carriage rates are low in the first 6 months of
life, reach a maximum between the ages of 3 and 5 years, and gradually
decline in adulthood. Under circumstances of crowding or Hib disease within
a closed population, the carriage rate may be substantially higher
(7,10,11). Hib carriage rates among children increase with the number of
siblings in a family (9), and in the United States, especially after the
occurrence of Hib disease in a child care center, carriage rates of 50%
have been found (12). The influence of concurrent upper respiratory
symptoms on Hib carriage rates remains controversial, and studies differ in
their findings on the influence of season, sex, and race on carriage rates.
Most studies find no association of Hib carriage with these factors.
Antimicrobial therapy affects Hib carriage; in particular, if given
appropriately, rifampicin may eliminate throat carriage of Hib and reduce
the risk for secondary disease among contacts (13).

Longitudinal studies of children further characterize Hib carriage.
Although many children may be transient carriers of Hib (14), carriage
tends to persist for many weeks or months (10,12,13). It seems that close
contact and generous exchange of respiratory secretions is required for the
transmission of Hib between hosts. Even when the contact between a known
carrier and a susceptible child is intimate, spread of Hib occurs slowly
over weeks or months (7,8). Bijlmer has suggested that village population
dynamics and living conditions in the Gambia contribute to different
kinetics of colonization and transmission of Hib, as persistence of
carriage in Gambian children is short-lived (15).

Hib Carriage and the Transmission of Disease

Since most patients with Hib disease have not had contact with a person who
had invasive disease, and the organism has no known reservoir outside
humans, asymptomatic carriers have been recognized as the major source of
infection. The relationship between carriage rates and the risk for disease
is not simple. The spread of infection in the presence of low carriage
rates has been described (16), and no overt disease has been reported
despite high carriage rates (7,17). Furthermore, organisms isolated from
the pharynx appear to lack certain virulence attributes found in organisms
isolated from patients with invasive disease (18).

Like surveys of Hib carriage, epidemiologic surveys of Hib disease have
found that rapid dissemination of Hib strains has not been the rule,
although the temporal clustering of several episodes of systemic disease in
child care centers has occurred. The secondary attack rate for household
contacts is approximately 500 times higher than the endemicdisease risk for
the general population (1). The high frequency of Hib carriage in homes and
child care centers of patients who have Hib disease suggests either that a
high concentration of carriers precedes and increases the probability of
cases, or that affected children are potent sources of infection for others
in close contact.

Although encapsulation enhances the ability to survive the dehydrating
stress that occurs during transfer between hosts (20), a great deal remains
unknown about the transmission of Hib. What are the modes of transmission
between hosts? How long can organisms remain viable between hosts? Is there
a threshold colonizing population or dose of organisms to ensure successful
transmission? Is there any correlation between the size of the infective
dose or the number of carried organisms and the likelihood of invasive
disease?

Hib Carriage and the Pathogenesis of Disease

The accepted pathogenesis of invasive Hib disease begins with the pharynx
as the portal of entry. The infant rat model of H. influenzae meningitis
has been used to study the early pathogenic events of Hib disease. Infant
rats contracted meningitis after intranasal challenge with Hib (21). There
was an age-dependent susceptibility to meningeal invasion (22), and
bacteremia followed the nasopharyngeal inoculation of organisms. The
incidence of bacterial meningitis, irrespective of rat host age, was
directly related to the intensity of bacteremia (22). To reach the
bloodstream, this nonmotile bacterium must pass through or between
epithelial cells, penetrate the basement membrane and subepithelial tissue,
and enter the endothelium of a blood vessel. The manner by which this
occurs remains under investigation.

Untreated, Hib meningitis and epiglottitis are fatal in most cases. The
death of the host is disadvantageous to the infecting bacterium since death
terminates transmission and propagation of organisms. Vascular invasion by
Hib may be circumstantial or accidental rather than the result of
evolutionary advantage.

Hib Carriage and the Development of Immunity

The capsular polysaccharide of Hib is a linear polymer of ribose, ribitol,
and phosphate (23) and is called polyribosylribitol phosphate (PRP). PRP is
the principal determinant of virulence of Hib (24,25) and a target for
antibody-mediated immunity. Considerable evidence indicates that antibody
to PRP is a principal protective host factor (26).

At birth, maternal anti-PRP IgG confers protection; however, as the level
of maternal antibody in the infant declines, the risk for and incidence of
Hib disease rise. As children approach 2 years of age, their own antibody
to the capsular polysaccharide begins to appear. The antigenic stimulus for
this agedependent development of bactericidal activity may be through
mucosal exposure to Hib or to other crossreacting antigens (27).

High serum anti-capsular antibody concentrations are associated with the
Hib carrier state in children older than 18 months (17,28), but how
colonization increases serum anti-PRP antibody concentrations remains
unclear. Hib antigen may be absorbed across the mucosa, or the whole
organism may traverse this barrier during colonization, causing low grade
asymptomatic bacteremia and a systemic antibody respnse. It is possible
that Hib organisms are phagocytosed by lymphoid cells, in the pharyngeal
mucosa, that may act as antigen-presenting cells (29), leading to the
production of specific serum or mucosal antibodies, with or without direct
vascular invasion by the organism. Other mucosal surfaces, such as the
gastrointestinal mucosa, may play a role in anti-PRP immunogenesis. The
incidence of bacteremia and meningitis in infant rats was significantly
lower for rats fed with Escherichia coli that possessed K100 capsular
antigen (crossreactive with type b capsular antigen) than for rats fed with
E. coli K92 or saline (30).

Many have argued that the high prevalence of anti-PRP antibodies and the
low rates of Hib carriage in children point to sources other than Hib that
give rise to serum anti-PRP antibodies. However, the technical difficulties
in identifying Hib carriage may have led to an underestimate of carriage
rates. With more sensitive culture techniques, it now seems more plausible
that Hib carriage or infection could account for the acquisition of natural
immunity.

Vaccination and Hib Disease

The initial development of vaccines against Hib in the late 1960s was
prompted by three major concerns: the high case-fatality rate of Hib
disease, the high incidence of central nervous system sequelae in children
surviving Hib meningitis, and the gradual emergence of strains resistant to
preferred antibiotics. Because serum anti-PRP antibodies were known to be a
principal protective factor in the host, efforts were made to increase the
antibody concentration by active immunization with a vaccine composed of
purified capsular polysaccharide. While the PRP vaccine was effective in
protecting healthy children 18 months of age and older against invasive Hib
disease (with an estimated efficacy of 90%), it did not protect younger
children who have the highest incidence of Hib disease (31).

The explanation for the vaccine's inability to protect younger children
lies in the chemistry of the capsular material. PRP, a heteropolymer of
pentose sugars, does not elicit a T-cell--independent immune response and
is not an efficient immunogen, especially in young children. Carbohydrate
antigens, such as these capsular polysaccharides of encapsulated bacteria,
are characterized as T-cell--independent type 2 (TI-2) antigens. However,
for many antigens the classification T-cell--independent or
T-cell--independent refers more to the pattern of antibody response than to
the involvement of T-cells in eliciting that response. In the very young,
the antibody response to T-cell--independent antigens is low, consists of a
high proportion of IgM antibody, and there is no booster response to
repeated doses of antigen.

Converting polysaccharide into a T-cell--dependent antigen, to which
infants can respond, requires the covalent linkage to protein molecules,
thus producing a conjugate vaccine. Studies of the different conjugate
vaccines have confirmed that conjugation results in a T-cell--dependent
response (32). Even though the protein carriers and covalent linkages are
biochemically and structurally different, they appear to be more
immunogenic than pure PRP vaccines. Conjugate vaccines can confer
protection against Hib in infants under 6 months of age (33).

Four conjugate vaccines have undergone clinical evaluation: PRP-D
(Connaught Laboratories), PRP-T (Pasteur-Merieux), PRP-OMPC (Merck Sharp
and Dohme), and HbOC (Praxis Biologicals). Each of these vaccines is
distinguished by its carrier molecule, the size of the hapten saccharide,
the type of linkage between hapten and carrier, and the ratio of
polysaccharide to protein. These physicochemical differences between the
vaccines influence their immunogenicity. Additionally, dose variation, such
as the age at which the primary series of two or three immunizations is
given and the interval between each dose of vaccine, may affect the level
of antibody response and/or the protection provided.

The concentration of vaccine-induced serum antibody against Hib needed for
protection is not precisely known. The issue is complicated as similar
concentrations of antibodies may vary in functional activity (34). The
maintenance of a threshold concentration of serum anti-PRP antibody may not
be necessary if immunized children are primed for an effective booster
response on exposure to Hib.

Efficacy studies have shown that PRP-D, HbOC, PRP-OMP, and PRP-T can
prevent more than 90% of Hib disease (1,33,35,37). However, PRP-D proved to
be ineffective in preventing Hib disease in an efficacy trial among Alaskan
children (38). Thus, the choice between the conjugate vaccines may be
critical only in populations in which infection pressure is very high, and
the age of greatest incidence of disease is low. In these populations,
conjugate vaccines with high immunogenicity at the first dose may be the
best to use (39), so that the youngest children at the highest risk are
afforded some protection at the earliest opportunity. In most populations,
the choice of vaccine has to be based on other factors, the most important
of which is cost.

Conjugate Vaccination and Hib Carriage

After a 4-year national immunization program for infants in Finland, Takala
et al. (40) reported that Hib colonization was less prevalent among 327
children vaccinated with PRP-D vaccine than among 398, previously studied,
unvaccinated children (0% vs 3.5%). The children studied were healthy
3-year-olds, from whom throat swabs were taken during a well-child visit to
their local health center. However, the control cultures were obtained
before the nationwide vaccination study; thus temporal factors other than
vaccination may have influenced the results. Geographic factors
necessitated sending swabs by mail to the center of sample processing,
which may have limited the sensitivity of the microbiologic assay.
Additionally, possible exposure to the bacterium from contacts was not
measured.

In child care centers in Dallas, a prospective study was done to determine
the prevalence of Hib colonization between 1987 and 1989 (41). During this
period, conjugate vaccination was introduced in the United States, so both
vaccinated and unvaccinated children attended the centers. Of 283 children
studied, 59 had received unconjugated polysaccharide vaccine, and 89 had
received conjugate vaccine (of which 94% received PRP-D). The Hib
acquisition rates over the surveillance period were 21% and 9%,
respectively. Among children exposed to at least one child with a positive
culture result, the efficacy of conjugate vaccination to prevent Hib
colonization in an unmatched analysis was 64%. No effect on colonization
was found with PRP vaccination.

A clinic-based study in metropolitan Atlanta found a decreased Hib carriage
rate in a population of 2- to 5-year-old children, 75% of whom were
vaccinated with an Hib conjugate vaccine (42), and in Apache and Navajo
Indian children who had received the Hib-OMPC vaccine appropriate for their
age (43). These studies were performed after vaccination programs had
begun, when adequate control populations were not available, and logistic
factors often necessitated suboptimal microbiologic procedures.

The district by district implementation of a conjugate vaccine (PRP-T) in
the Oxford Region in England enabled examination of a conjugate vaccine's
effects over time in contemporary groups of vaccinated and unvaccinated
infants (44). The children were recruited at birth so as to minimize
selection bias concerning Hib carriage. Sampling error was limited, as one
person took all the throat swabs, and laboratory analysis was standardized
and took place in a single center. Other factors that could have influenced
exposure to Hib or susceptibility to carriage were subjected to statistical
control. Infants who had received Hib conjugate vaccine at the ages of 2,
3, and 4 months had significantly lower Hib acquisition rates than
controls. In accordance with this, the point prevalence of Hibcarriage was
consistently lower in vaccines than in controls at 6, 9, and 12 months of
age. The rates of acquisition and the period prevalence of H. influenzae
serotypes e and f did not differ between vaccines and controls.

How Does Conjugate Vaccine Against Hib Affect Carriage?

The unconjugated Hib polysaccharide vaccine raises the IgA antibody
concentration in nasal secretions and saliva of both adults and children
(45). However, it does not affect the oropharyngeal carriage of Hib (31).
The same lack of effect on pharyngeal carriage has been seen with other
parenterally administered polysaccharide vaccines: meningococcal group A
and C and several serotypes in the Streptococcus pneumoniae polysaccharide
vaccine (47,48).

Conjugate vaccines induce higher concentrations of serum antiPRP antibodies
in young children than polysaccharide vaccines. It has been suggested that
high serum concentrations might lead to passive transudation of IgG
antibodies to mucosal surfaces (49). In an infant rat model, the serum IgG
antibody concentration needed for an effect on Hib colonization was 7 g/mL
(50). More recent research has found that the presence and concentration of
IgG anti-PRP antibodies in saliva correlated with the concentration in
serum after conjugate vaccination (51). Secretory IgA anti-PRP antibody was
found in the saliva of children who had no similar, detectable serum IgA.
This suggests that the anti-PRP IgG in saliva was derived from serum,
whereas the IgA antibodies were locally produced. It seems that conjugate
vaccines can induce a mucosal IgA response as well as a systemic IgG
response, and while there is some evidence that high serum levels of IgG
are relevant to the prevention of acquisition of carriage, the role of IgA
remains unclear.

A decline in the serum, and thus perhaps mucosal, anti-PRP antibody
concentration after primary immunization offers a simple explanation for
the timedependent effect of conjugate vaccines on acquisition of Hib. The
geometric mean titer (GMT) of serum anti-PRP antibody in children in
Oxfordshire, at 5 months of age, soon after completing a course of Hib
immunization, was 5 g/mL (52). The GMT in these children at 12 months of
age was 0.83 g/mL (53). A concentration of 1 g/mL after immunization with
Hib polysaccharide vaccine has been accepted as the concentration
associated with long term protection against invasive Hib disease (54).
Acquisition of Hib and prolonged Hib carriage may occur only below a
threshold concentration of serum or mucosal antibody.

How specific antibody contributes to the demise, or inhibits the attachment
of Hib in the pharyngeal mucosa remains unresolved. Possible mechanisms
include antibody-mediated opsonophagocytosis, direct bactericidal activity,
or stereotactic inhibition. However, simple antibody-mediated bactericidal
mechanisms may not completely explain the modulation of colonization in
vaccinated persons. Carriage was not rapidly curtailed when conjugate
vaccine was administered to current Hib carriers (44). This result is not
easily explained, although the intracellular sequestration of Hib (55) may
render a source of organisms inaccessible to antibodies in secretions. It
is also possible that conjugate vaccine does not add significant antigenic
stimulus in a child carrying Hib, with Hib antigen present in the mucosa.

Vaccines against Hib intervene in the normal relationship between Hib and
host by increasing serum and/or mucosal anti-PRP antibody concentrations in
the host before the host has any exposure to the bacterium. One could
speculate that the primary role of vaccines in limiting Hib colonization is
to prevent the acquisition of organisms. Secondarily, a large boost in the
antibody concentrations, caused by mucosal contact with Hib in an immunized
host, could lead to the more rapid elimination of Hib from the mucosa.
There is also tenuous evidence of decreased Hib colony counts in cultures
from colonized, vaccinated children (28).

In ecologic terms, Hib may precariously occupy the pharynx. A small
influence that upsets the ecology in vaccinated persons may have a profound
effect on the population kinetics of Hib. A rise in the concentration of
serum and mucosal anti-PRP antibodies in infants could be enough to
dramatically affect the pattern of Hib transmission. It seems likely that
this effect would initially be greatest in places of close contact between
children: families and child care institutions. Subsequently, the effect
would become evident in a more widespread population.

The paucity of data on the ecologic behavior of Hib in the pharynx, the
influence of conjugate vaccines on this behavior, and the importance of
this influence, have prompted attempts at mathematical modeling to predict
the population kinetics of Hib carriage and disease after conjugate
vaccination. These models remain in their infancy but are awaited with
interest.

Because they differ in biochemical composition and immunogenicity, the
conjugate vaccines may vary in their long-term protective efficacy and
effect on Hib carriage and transmission. Additionally, differing
immunization regimens in populations may affect any generation of herd
immunity. For example, administering a booster dose of conjugate Hib
vaccine after the age of 12 months may prolong the presence of high
concentrations of anti-PRP antibody in children, thereby enhancing the
potential to limit transmission of Hib in the population.

In their relationship with the human host, Neisseria meninigitidis, S.
pneumoniae, and Hib have numerous and close parallels. With the use of
principles similar to those applied to Hib vaccines, conjugate vaccines
against these other encapsulated pathogenic bacteria are now being
developed and tested. The findings in studies of Hib may be a paradigm for
the effect of conjugate vaccines on colonization by N. meninigitidis and S.
pneumoniae. It will be important to examine this possibility closely as the
vaccines become available.

The effect of conjugate vaccines on Hib carriage has been established in
epidemiologic terms, but molecular knowledge about vaccination and mucosal
immunity is limited. Much remains to be learned about the interaction
between host and bacterium at the mucosal surface and about the
contribution of conjugate vaccines to this complex relationship.

Dr. Barbour, a Rhodes Scholar from Western Australia, received a D.Phil. at
Oxford University with work on the influence of conjugated Hib vaccine on
the oropharyngeal carriage of Hib. She is currently training in clinical
pediatrics at the John Radcliffe Hospital in Oxford.

Address for correspondence: Marina L. Barbour, D.Phil., M.R.C.P. Travis
Cottage, Whitebarn, Boars Hill, OX1 5HH, England; e-mail:
hughes@rmplc.co.uk.

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=============================================================================
Synopses

Application of Molecular Techniques
to the Diagnosis of Microsporidial Infection

Daniel P. Fedorko, Ph.D., and Yasmine M. Hijazi, M.D.
National Institutes of Health, Bethesda, Maryland, USA
---------------------------------------------------------------------------


          Microsporidia are now recognized as important pathogens
          of AIDS patients; the ability of these parasites to
          cause disease in immunocompetent persons is still being
          elucidated. Improved diagnostic tests for
          microsporidial infection are continually being sought
          for establishing diagnosis in order to avoid laborious
          electron microscopy studies that require invasively
          acquired biopsy specimens. Modified trichrome or
          chemofluorescent stains are useful for detecting
          microsporidia in bodily fluids and stool specimens, but
          they do not allow for speciation of microsporidia.
          Polymerase chain reaction with specific primers will
          allow the detection and speciation of microsporidia in
          biopsy tissue, bodily fluids, and stool specimens.

Microsporidiosis is truly an emerging infectious disease. Although
microsporidia were discovered more than 100 years ago (1), the first
well-documented case of human microsporidiosis was not reported until 1959
(2). Human microsporidiosis remained an uncommon infection until the human
immunodeficiency virus (HIV) pandemic; the first cases of microsporidial
infection in HIV-infected patients were reported in 1985 (3,4). Since then,
more than 400 HIV-associated microsporidial infections have been reported,
which indicates that microsporidia are common opportunistic pathogens in
patients with AIDS (5). Infections caused by three new species,
Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Encephalitozoon
hellem, have been described in patients with acquired immunodeficiency
syndrome (AIDS). Originally thought to be an opportunistic pathogen of AIDS
patients exclusively, E. bieneusi was reported in 1994 to be the cause of
acute, self-limited traveler's diarrhea in an immunocompetent person who
was not infected with HIV (6).

As microsporidia have been increasingly recognized as pathogens of both
immunosuppressed and immunocompetent persons, the need for rapid and
specific diagnosis of microsporidial infection has arisen. For instance,
albendazole therapy can cure E. intestinalis infections but is reported to
be of little benefit to patients infected with E. bieneusi (5); therefore,
identifying microsporidia to species level could have important
implications in the clinical management of patients. Current diagnostic
methods for microsporidia infection, though continually being improved,
have shortcomings that molecular diagnostic techniques may be able to
overcome.

Biologic Properties of Microsporidia

Microsporidia are obligate intracellular parasites that infect most
invertebrates and all classes of vertebrates. They belong to the phylum
Microspora in the subkingdom Protozoa. They are considered ancient
eukaryotes that multiply by binary fission and have a membrane bound
nucleus; they lack mitochondria and Golgi membranes (5). In addition to
causing disease in humans, these parasites cause disease in insects,
mammals, and fish; therefore, they may create economic headaches for
industries such as fisheries and silk production. They may also be
beneficial to humans as biologic control agents for such pests as
grasshoppers and locusts (5).

In host cells, microsporidia replicate either in a parasitophorous vacuole,
like the members of the genus Encephalitozoon, or directly in the cytoplasm
like E. bieneusi. Vegetative and spore stages of the organisms can be found
in the host cell as the parasite undergoes merogony and sporogony,
resulting in the production of the infective spore stage of the parasite
(7) (Figure 1) [Figures not available in ASCII]. The spores of microsporidia 
contain the uniquely characteristic coiled polar tubule (Figure 2). Under 
appropriate conditions within a suitable host, the polar tubule of the spore 
is extruded (Figures 3 and 4) [Figures not available in ASCII]. Contact of 
the end of the tubule with a host cell membrane allows the spore to transfer 
its contents (sporoplasm) to initiate infection within the new host cell. 
An influx of calcium into the spore coincides with the extrusion of the polar 
tubule, and this mechanism may provide a target for therapy for 
microsporidiosis since calcium channel blockers have
been shown to inhibit extrusion and the infection of host cells in vitro
(8).

The human microsporidial pathogens and their clinical manifestations are
shown in Table 1. The most common microsporidial disease is prolonged
diarrhea with wasting caused by E. bieneusi or E. intestinalis in AIDS
patients with CD4 T-cell counts below 50 cells/l. Microsporidia have been
reported in up to 39% of AIDS patients with diarrhea (9). Microsporidia may
disseminate to cause systemic infection (Table 1); these organisms have
been observed in urine, bile, and duodinal aspirates, as well as in ocular,
sinus, bronchial, renal, hepatic, and other tissue (5). E. hellem, which
primarily causes eye infections, has been exclusively found in AIDS
patients. Ingestion and inhalation of spores have been suggested as likely
modes of transmission for microsporidia (5,10). Biochemical, immunologic,
and molecular studies performed on E. cuniculi isolates from mice, rabbits,
and dogs indicate that the parasite can be classified into at least three
strains (11). An evaluation of the immunologic and molecular
characteristics of E. cuniculi isolated from humans and rabbits indicated
that the isolates were an identical strain, thus suggesting that this
microsporidion is a zoonotic parasite (12).

      Table 1. Named human microsporidial pathogens and their clinical
                                 syndromes
---------------------------------------------------------------------------
    Microsporidia genus and                 Clinical syndromes
            species
---------------------------------------------------------------------------
 Pleistophora species           Myositis
 Nosema connori                 Disseminated infection
 N. ocularum                    Keratitis
 Vittaforma corneae             Keratitis
 Encephalitozoon cuniculi       Peritonitis, fulminant hepatitis,
                                  seizures, rhinosinusitis
 E. hellem                      Conjunctivitis, keratoconjunctivitis,
                                  bronchiolitis, pneumonia,
                                rhinosinusitis,
                                  disseminated infection
 E. intestinalis                Diarrhea, disseminated infection
 Enterocytozoon bieneusi        Diarrhea, wasting syndrome,
                                  cholecystitis, cholangitis, bronchitis,
                                  pneumonia
 Source: ref. 5
--------------------------------------------------------------------------

Traditional Diagnostic Methods

Transmission Electron Microscopy

Definitive diagnosis of microsporidial infection relies on observating
microsporidia in biopsy tissue, bodily fluid specimens (e.g. urine, sinus
aspirates, bile, cerebral spinal fluid), or stool examined by transmission
electron microscopy (TEM). Microsporidia can be identified to genus or even
species level on the basis of morphologic characteristics. All stages of
the parasite's life cycle can be found in infected tissue, but only the
spore stage can be observed in bodily fluids and stool. The size and
ultrastructure of spores, particularly the configuration of the coiled
tubule, distinguish E. bieneusi from the Encephalitozoon species (Figures 5
and 6) [Figures not available in ASCII]. Limited success was reported when 
TEM was used to detect spores in
bodily fluids and stool specimens to avoid obtaining specimens by invasive
procedures (5). Tissue specimens are required for speciation within the
Encephalitozoon genus because the meronts and sporonts, nuclear
configuration, and location of replication in the host cell must be
carefully studied (7,10). Problems with the use of TEM include its lack of
sensitivity when performed on bodily fluids and stool, the requirement for
invasive procedures to acquire biopsy specimens, and the laborious
procedures for specimen preparation and examination.


Light and Fluorescence Microscopy

Histologic examination of biopsy specimens allows diagnosis of
microsporidial infection but not genus or species identification of the
parasites. Tissue stains used to detect microsporidia include hematoxylin
and eosin, Gram, Giemsa, Warthin-Starry, and chromotrope 2R modified
trichrome stains (13,14). The small size of the organisms and the lack of a
noticeable tissue inflammatory response make microsporidia detection
difficult (5). Once again, the invasive procedure required for obtaining
specimens and the length of time needed for processing them are major
drawbacks.

Diagnosing microsporidial infections by light microscopy examination of
noninvasively acquired specimens has been a challenge for laboratorians.
Microsporidial spores are not observed in traditional ova and parasite
examinations, and the parasites are generally overlooked in Gram-stained
preparations of stool samples because their size, shape, and staining
characteristics are similar to those of many enteric bacteria. Weber's
modification of the trichrome stain has allowed more definitive
identification of microsporidia by light microscopy (15). With this stain,
microsporidial spores appear bright pinkish-red, and most have a
distinctive diagonal or equatorial line that allows microsporidia and
bacteria to be easily differentiated (Figure 7) [Figures not available 
in ASCII]. The sensitivity and
specificity of the modified trichrome stain have not been well established.
When results of the modified trichrome stain performed on stool specimens
and a TEM examination of duodenal biopsy specimens from HIV-infected
patients were compared, E. bieneusi spores were detected in stool specimens
in 9 of 9 patients with moderate or abundant parasites in tissue and in 8
(57%) of 14 stool specimens from patients whose tissue contained few
parasites (16). This illustrates that microsporidial spores may not be
observed in the stool of some infected patients with the modified trichrome
stain.

Various chemofluorescent brighteners (such as calcofluor white and Uvitex
2B) bind to the endospore layer of microsporidia and allow spores to be
detected quickly and easily in smears of specimens examined with a
fluroescence microscope (5). Fluorescein-labeled polyclonal and monoclonal
antibodies are being developed for the detection and speciation of
microsporidia (5,17). Didier et al. (17) recently compared modified
trichrome, calcofluor white, and a fouorescent polyclonal antibody stain
and found that the polyclonal antibody stain was the least sensitive method
for detecting microsporidia in stool, urine, and duodenal lavage specimens.
They proposed screening specimens with calcofluor white and confirming
positive smears by using a modified trichrome stain. However, modified
trichrome and calcofluor white stains do not allow for speciation of
microsporidia; therefore, speciesspecific antibodies should be used to
provide definitive identifications. E. intestinalis-specific monoclonal
antibodies have been used in an immunofluorescence assay for detecting
spores in urine, stool, bronchial brush biopsy specimens, bronchoalvolar
lavage fluid, and samples obtained from nasal swabs (18). E.
bieneusi-specific antibodies may not be developed until the organism is
successfully cultivated in long-term culture.

Cell Culture

Microsporidia have been isolated from a variety of specimen types and in a
variety of cell lines (5). E. hellem, E. intestinalis, and Vittaforma
corneae have been isolated from human specimens and maintained in
continuous culture. Recently, E. bieneusi has been cultivated on a
short-term (6 months) basis in vitro (19). Detecting microsporidia in
infected cell cultures may take 3 to 10 weeks (19,20). Isolating
microsporidia in cell culture as a means of diagnosing infection is
laborious and lengthy and is prone to failure with specimens from
nonsterile sites. Therefore, cell culture is not recommended as a routine
laboratory technique for diagnosing microsporidiosis.

Serology

Serologic assays used to detect antibodies to microsporidia in human sera
include immunofluorescence, immunoperoxidase, enzyme-linked immunosorbent
assay (ELISA), and Western blot (21-23). The sensitivity and specificity of
these methods for detecting antimicrosporidial antibody are not known
because no comparative evaluations have been published. Studies have
demonstrated increased rates of seropositivity to E. cuniculi in persons
who live in tropical regions and have tropical diseases (21,24). Most
notable were E. cuniculi seropositivity rates of 4.7% in patients with
malaria and 9.1% in patients with schistosomiasis (24). A study of
homosexual men in Sweden found that 10 (33%) of 30 were seropositive for
antibodies to E. cuniculi, and all seropositive patients plus half of the
seronegative patients had sometimes visited tropical countries (25).
Studies to detect microsporidial antibodies in HIV-infected and
non-HIV-infected patients have demonstrated that AIDS patients can mount an
immune response to microsporidial infection; however, serologic methods are
not useful as diagnostic tools because these studies have found that at
least half of the serum specimens from persons without a history of
microsporidial infection had positive titers (22,23). Some of the problems
with serologic testing include the poor response to antigen challenge in
immunosuppressed persons (26), the probability that different (both
pathogenic and nonpathogenic) microsporidia contain common crossreactive
antigens (23,26), and the lack of species-specific reagents in part because
E. bieneusi cannot be grown in continuous culture.

Molecular Techniques

Characterization of the microsporidial genome has focused on the small
subunit ribosomal RNA (SSU-rRNA) gene. The sequence of the SSU-rRNA gene of
the microsporidium Vairimorpha necatrix was published by Vossbrinck et al.
in 1987 (27). The V. necatrix SSU-rRNA gene was far shorter than a typical
eukaryotic SSU-rRNA gene and lacked several universal (and
eukaryotic-specific) sequences. PCR amplification with primers
complementary with conserved sequences of the V. necatrix SSU-rRNA gene has
been used to generate sequence information from microsporidia that infect
humans. SSUrRNA gene sequences have been published for E. cuniculi
(28,29,30,31), E. hellem (30,31), E. intestinalis (31,32), and E. bieneusi
(31,33), and an unpublished sequence for V. corneae has been deposited into
GenBank National Center for Biotechnology Information, National Institutes
of Health, accession 0.5 U11046.

Taxonomic decisions concerning the microsporidia have been based
historically on morphologic characteristics as established by TEM. These
criteria are still valid for organisms with unique morphology and
ultrastructure and were used to justify the reclassification of Nosema
corneum to Vittaforma corneae (34). This reclassification was confirmed by
a study of microsporidial phylogeny based on evaluation of SSU-rRNA gene
sequences that indicated that N. corneum was more closely related to the
insect parasite Endoreticulatus schubergi than to the other Nosema species
(35) (Figure 8) [Figures not available in ASCII]. Phylogenetic analysis 
of the sequences of the SSU-rRNA
genes of microsporidia is often inconsistent with traditional
classifications that are based on morphologic characteristics observed by
TEM; this was demonstrated by the use of recent sequence data to determine
the correct taxonomic placement of E. intestinalis (32,35). This organism
was named Septata intestinalis because unique septations between spores
within the parasitophorous vacuole were observable by TEM (36). Although
the various stages of the microorganism throughout its life cycle are
indistinguishable from those of E. cuniculi, the observation of septations
allowed the organism to be identified. The E. intestinalis SSU-rRNA gene
contained sufficient unique sequences to establish this organism as an
independent species but shared about 90% sequence homology with the other
two characterized Encephalitozoon species, E. cuniculi and E. hellem.

Molecular techniques have confirmed the existence of different strains of
E. cuniculi that have been isolated from mice, rabbits, and dogs (11).
Three antigenically different strains were detected by sodium dodecyl
sulfate-polyacrylamide gel (SDS-PAGE) and Western blot. Double-stranded DNA
heteroduplex mobility, restriction fragment length polymorphism with the
restriction endonuclease Fok I, and DNA sequencing were performed on the
PCR products generated by using a set of primers to amplify the entire
microsporidial SSU-rRNA gene and a second set of primers specific for
Encephalitozoon species. These methods allowed three strains of E. cuniculi
to be clearly separated. The existence of different strains of E. bieneusi
was suggested after SSU-rRNA gene sequences of organisms from maxillary
sinus mucosa had been compared with those from intestinal enterocytes (37).
Sequencing errors in this study were minimized by analyzing multiple
recombinant DNA clones. SSU-rRNA sequences from E. bieneusi and
Encephalitozoon species derived from a variety of clinical specimens must
be compared to establish the existence of intraspecific genetic variation
in these organisms.

Molecular Diagnosis

Primer pairs that amplify the entire microsporidial SSU-rRNA gene sequence
produce amplicons of approximately 1,550 base pairs in length from
Encephalitozoon species and E. bieneusi (28,33). Although these primer
pairs have proven useful for sequencing and taxonomic studies, the targets
are too long to be efficiently amplified from clinical specimens in a
diagnostic assay. Targets for diagnostic PCR that can be amplified
efficiently usually range from 100 to 400 base pairs for formalin-fixed
tissue or up to 700 to 1,000 base pairs in fresh specimens (38). Several
primer pairs designed to amplify short regions (250 to 607 base pairs) of
the SSU-rRNA gene, and their application in the diagnosis of microsporidial
infection by PCR have been published (Table 2). Primers specific for E.
hellem and E. cuniculi-specific primers have been used to identify
microsporidia cultured from patient specimens (39,40), but only E. bieneusi
and E. intestinalis SSU-rRNA DNA have been amplified directly from patient
specimens (31,33,41,42). A pair of E. bieneusi-specific primers amplified
cloned E. bieneusi SSU-rRNA gene sequences but did not reliably amplify DNA
from infected tissue (33). Not unexpectedly, therefore, some primer sets
appear to be adequate for amplification from cultured organisms or cloned
sequences but may not reliably amplify microsporidial DNA in patient
specimens. The primer pair V1 and EB450 (Table 2) amplifies E. bieneusi
from TEM-confirmed infected tissue and E. hellem from cell culture (33).
This primer pair has not, however, been evaluated for its ability to
amplify E. hellem from patient specimens. Primer pair V1 and EB450 was
extensively tested by da Silva, et al. (43), who found that the primer pair
did amplify E. bieneusi DNA from some patient specimens that had evidence
of the parasite by electron microscopy. In addition, the primer pair did
not amplify DNA of E. bieneusi derived from a short-term culture. da Silva
et al. (43) described a pair of highly specific PCR primers for amplifing
E. bieneusi; these are called EBIEF1 and EBIER1 (Table 2), and are based on
SSU-rRNA sequences they generated. This primer pair amplified E. bieneusi
DNA from cultured organisms, cloned regions of the SSU-rRNA, and patient
specimens, but did not amplify SSU-rRNA-coding regions of 13 other genera
and species of microsporidia. The primer pair V1 and SI500 amplifies E.
intestinalis from intestinal biopsy material confirmed by TEM as infected,
but does not simplify E. bieneusi-infected tissue samples or E. cuniculi
from cell culture (31). This primer pair has not been evaluated for its
ability to amplify other microsporidia.

 Table 2. SSU-rRNA PCR primer pairs for the diagnosis of microsporidial infection
------------------------------------------------------------------------------------------
Primer pair (no. nucleotides)   Primer      Organisms       Amplicon Source     References
                                designation amplified       size     of Target
                                                            in base
                                                            pairs
------------------------------------------------------------------------------------------
5'-CACCAGGTTGATTCTGCCTGAC-3'(22) V1         Enterocytozoon  348      Biopsied   33,41
                                            bieneusi                 tissue     31
                                                                     Duodenal
                                                                     aspirates
5'-ACTCAGGTGTTATACTCACGTC-3'(22) EB450      Encephalitozoon          Cultured   33
                                            hellem                   organisms
5'-GAAACTTGTCCACTCCTTACG-3' (21) EBIEF1     E. bieneusi     607      Bile       43
5'-CCATGCACCACTCCTGCCATT-3' (21) EBIER1                              Duodenal
                                                                     aspirates
5'-TGAGAAGTAAGATGTTTAGCA-3' (21)            E. hellem       547      Cultured   39
                                                                     organisms
5'-GTAAAAACACTCTCACACTCA-3' (21)
5'-ATGAGAAGTGATGTGTGTGCG-3' (21)            Encephalitozoon 549      Cultured   39,40
                                            cuniculi                 organisms
5'-TGCCATGCACTCACAGGCATC-3' (21)
5'-CACCAGGTTGATTCTGCCTGAC-3'(22) V1         Encephalitozoon 370      Biopsied   31
                                            intestinalis             tissue
5'-CTCGCTCCTTTACACTCGAA-3'  (20) SI500
5'-CACCAGGTTGATTCTGCCTGAC-3'(22) PMP1*      E. bieneusi     250      Stool      42
5'-CCTCTCCGGAACCAAACCTG-3'  (21) PMP2       E. cuniculli    268      Cultured
                                                                     organisms
                                            E. intestinalis 270      Stool
                                            E. hellem       279      Cultured
                                                                     organisms
 *The nucleotide sequence of primer PMP1 is identical to that of primer V1.
------------------------------------------------------------------------------------

To avoid acquiring specimens by invasive procedures, PCR was used to detect
microsporidia in stool specimens (42). Primer pair PMP1 and PMP2 (Table 2)
allows E. bieneusi and the Encephalitozoon species (42) to be amplified and
will also amplify V. corneae from culture (D.P. Fedorko, unpublished data).
DNA is easily extracted from cultured organisms and biopsied tissue
specimens, but extracting DNA from spores requires harsh conditions
employing both mechanical and chemical disruption. A major problem with
performing PCR on stool specimens is the presence of PCR inhibitors.
Treating the stool specimens with sodium hypochlorite or 10% formalin
before DNA extraction inactivates microorganisms in the stool and has the
beneficial effect of inactivating Taq polymerase inhibitors.

Both DNA probes and restriction enzyme digestion have been used to confirm
the identity of PCR amplicons (33,41,42). An internal 30
meroligonucleotide5 EB150 (5'TGTTGCGGTATTTGGTCTCTGTGTGTAAA3'),
complementary to a region of the amplicon produced by primer pair V1 and
EB450, has been used to detect E. bieneusi by Southern blot (31,41). Probe
EB150 has been reported, however, to hybridize with E. hellem, amplified by
V1 and EB450, albeit at a lower intensity than E. bieneusi (31). PCR
products amplified from stool specimens by using primers PMP1 and PMP2 have
been digested with restriction endonucleases HaeIII and PstI to distinguish
between infection with E. bieneusi and E. intestinalis (42). These
restriction enzymes do not allow E. intestinalis to be differentiated from
E. cuniculi, thus limiting their use to diagnosing gastrointestinal
infection.

An efficient approach for the molecular detection of microsporidia in
patient specimens would involve using universal or pan-microsporidian
primers for amplification. The species of microsporidia detected could be
determined by using restriction endonuclease digestion or DNA probe assays
of the amplified DNA or repeat PCR with species-specific primers. Negative
specimens would require no further evaluation. Primers PMP1 and PMP2 appear
to be panmicrosporidian (42), but this primer pair needs to be evaluated
for its ability to amplify microsporidian DNA from a wide range of clinical
specimens. Didier et al. (44) have used pan-Encephalitozoon primers that
amplified a product approximately 1,000 base pairs in length, which
included a large portion of the SSU-rRNA gene and a small portion of the
large subunit rRNA gene. They successfully amplified E. hellem DNA from
urine and conjunctival specimens from a patient with AIDS. Southern
blotting and a species-specific probe were used to identify the organism to
species.

The application of molecular diagnostic techniques for microsporidiosis is
in its infancy. There have been no published reports of comparisons of PCR
to other methods to determine sensitivity and specificity. Careful
selection of primers and probes coupled with highly stringent conditions
will be required to detect and speciate microsporidia in patient specimens.
The potential for PCR to identify species of microsporidia from
noninvasively acquired specimens makes this technique an extremely
attractive diagnostic option. Although PCR can be used for detection and
speciation of microsporidia in patient specimens, screening for
microsporidia by using chemofluorescent stains and modified trichrome
stains followed by confirmation and speciation with PCR may become the
paradigm for the laboratory diagnosis of microsporidiosis.

Dr. Fedorko is a senior staff microbiologist in the Microbiology Service,
Clinical Pathology Department, Warren G. Magnuson Clinical Center, National
Institutes of Health, Bethesda, Maryland.

Dr. Hijazi is a senior staff pathologist in the Laboratory of Pathology,
National Cancer Institute, National Institutes of Health, Bethesda,
Maryland.

Address for correspondence: Daniel P. Fedorko, Ph.D., Clinical Pathology
Department, Warren G. Magnuson Clinical Center, National Institutes of
Health, Building 10, Room 2C-385, Bethesda, MD 20892-1508; fax:
301-402-1886; e-mail: dfedorko@nih.gov.

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Synopses

Coccidioidomycosis: A Reemerging Infectious Disease

Theo N. Kirkland, M.D., and Joshua Fierer, M.D.
Departments of Pathology and Medicine, University of California, San Diego
School of Medicine
Department of Veterans Affairs Medical Center, San Diego, California, USA
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          Coccidioides immitis, the primary pathogenic fungus
          that causes coccidioidomycosis, is most commonly found
          in the deserts of the southwestern United States and
          Central and South America. During the early 1990s, the
          incidence of coccidioidomycosis in California increased
          dramatically. Even though most infections are
          subclinical or self-limited, the outbreak is estimated
          to have cost more than $66 million in direct medical
          expenses and time lost from work in Kern County,
          California, alone. In addition to the financial loss,
          this pathogen causes serious and life-threatening
          disseminated infections, especially among the
          immunosuppressed, including AIDS patients. This article
          discusses factors that may be responsible for the
          increased incidence of coccidioidomycosis (e.g.,
          climatic and demographic changes and the clinical
          problems of coccidioidomycosis in the
          immunocompromised) and new approaches to therapy and
          prevention.

Emerging infectious diseases have been defined as infections that have
newly existed in a population or have existed but are rapidly increasing in
incidence or geographic range (1). In what sense is coccidioidomycosis an
emerging infectious disease? Coccidioidomycosis is not a new disease; it
was first recognized and reported slightly more than 100 years ago by a
medical student in Argentina (2). In fact, coccidioidomycosis has affected
inhabitants of the desert Southwest for thousands of years (3). However, in
the past several years, the number of cases of coccidioidomycosis has
increased dramatically, and the clinical symptoms of this illness have
changed in patients with acquired immunodefficiency syndrome (AIDS). In
this article, we explore some of the reasons for the increased incidence of
coccidioidomycosis, review the new clinical data, and discuss current
approaches to therapy and prevention.

Etiology

Coccidioidomycosis is caused by Coccidioides immitis, a dimorphic fungus
that grows as a mold in the soil. The mold forms arthroconidia within the
hypha, a type of conidia formation known as enteroarthric development
(Figure 1) [Figures not available in ASCII] (4). C. immitis is the only 
species within the primary
pathogenic fungi that has this type of conidia development. Alternate
conidia undergo autolysis, leaving empty spaces between viable
arthroconidia. The arthroconidia are released into the atmosphere when the
wind ruptures the hypha. C. immitis infects humans and animals almost
exclusively by the respiratory route (5). Once inhaled, the arthroconidia
cluster in the lungs and undergo a dramatic morphologic change. The round
cells, which develop into spherules, undergo repeated internal divisions
until they are filled with hundreds to thousands of offspring, termed
endospores. This process occurs over 48 to 72 hours (6). When the spherule
ruptures, each released endospore has the capacity to develop into a mature
spherule.

Epidemiology

C. immitis is primarily found in desert soil. It is present in highest
numbers in the San Joaquin Valley in California, southern Arizona, southern
New Mexico, west Texas, and the desert areas of northern Mexico (Figure 2)
 [Figures not available in ASCII].
The organism is also found in scattered foci in coastal southern
California, southern Nevada, and Utah (7) and is endemic in a few areas in
Central and South America, especially in Venezuela (7). C. immitis is
distributed unevenly in the soil and seems to be concentrated around animal
burrows and ancient Indian burial sites (8,9); it is usually found 4 to 12
inches below the surface of the soil (7).

Since C. immitis infects humans by the respiratory route, exposure to dust
is one critical factor determining the risk for infection (10).
Coccidioidomycosis is not spread from person to person, except in
extraordinary circumstances. Coccidioidomycosis probably had its most
profound effect on the population of the United States during World War II
when several training airfields were built in the San Joaquin Valley. The
rate of new infections in military personnel was 8% to 25% per year (10).
Coccidioidomycosis was the most common cause of hospitalization at many
airbases in the Southwest. Though the death rate was very low, many
soldiers were sick for weeks to months, and their training was completely
disrupted. At least in part because of efforts to minimize dust, the
infection rate declined as the war went on (10).

The incidence of coccidioidomycosis varies with the season; it is highest
in late summer and early fall when the soil is dry and the crops are
harvested (10). If it rains at this time of the year (which is unusual in
southern California), disease incidence declines as the amount of dust
decreases. Dust storms are frequently followed by outbreaks of
coccidioidomycosis. One particularly severe dust storm in 1977 carried dust
from the San Joaquin Valley up to the San Francisco Bay area and resulted
in hundreds of cases of nonendemic coccidioidomycosis in areas north of the
San Joaquin Valley (11). More recently, an earthquake centered in
Northridge, California, was associated with 170 cases of acute
coccidioidomycosis in Ventura County, which normally has a low incidence of
this disease. The airborne dust associated with landslides triggered by the
earthquake was implicated in the increase in the number of cases (12).

Occupational or recreational exposure to dust is also an important
consideration. Agricultural workers, construction workers, or others (such
as archeologists) who dig in the soil in the disease-endemic area are at
increased risk for the disease (13,14). During World War II, C. E. Smith,
one of the most perceptive and influential epidemiologists to study
coccidioidomycosis, recommended dust control as a primary measure to reduce
risk for exposure (10). However, because the desert is inherently dusty,
many cases of coccidioidomycosis are acquired just by driving through the
disease-endemic area.

Clinical Illness

C. immitis is transmitted by the respiratory route. Smith et al., in a
prospective study of cases of coccidioidomycosis acquired during World War
II by soldiers at three San Joaquin Valley airbases, skin-tested the airmen
periodically and questioned them about illnesses in the interval. They
found that most infections (60%) were asymptomatic and resolved
spontaneously; 15% were not severe enough to require medical care, and 25%
were clinically important and required a substantial amount of time off
work (15). In symptomatic patients, the pulmonary illness ranges from a
self-limited flulike illness to pneumonia (16). Approximately 5% of primary
infections result in erythema nodosum or erythema marginatum with
associated noninfectious arthritis; most of those patients have a
self-limited infection (17). Particularly in persons with diabetes,
multiple thin-walled chronic cavities tend to develop as a residual effect
of pulmonary coccidioidomycosis (18). Unlike in tuberculosis, in
coccidioidomycosis, dissemination almost always becomes evident within a
few weeks of the primary pneumonia, although in cases of limited
dissemination it may not become clinically evident until months later
(15,19). Cocidioidomycosis can disseminate and cause miliary disease, bone
and joint infection, skin disease, soft tissue abscesses, and meningitis
(15,16). These extrapulmonary complications are uncommon (<5% of
infections).

The risk for disseminated coccidioidomycosis is much higher among some
ethnic groups, particularly African-Americans and Filipinos. In these
ethnic groups, the risk for disseminated coccidioidomycosis is tenfold that
of the general population (5,20). Presumably, a gene (or genes) that
increases susceptibility to infection is more prevalent in these ethnic
groups than in the general population. Such a resistance gene has been
identified in mice (21-23), but not yet in humans. The mechanism by which
the resistance genes affect the course of the disease in mice is not clear.
Pregnant women and the immunosuppressed are also at high risk for
developing disseminated disease (Figure 3) [Figures not available in 
ASCII] (24). One study demonstrated
that the growth rate of spherules was influenced by human sex hormones,
which may partially account for the increased risk of disseminated disease
in pregnancy (25). Pregnancy also redirects the immune response toward
humeral (TH2) immunity and away from delayed hypersensitivity (TH1) (26),
which may influence resolution of coccidioidomycosis. Generalized
suppression of cell mediated immunity also increases the risk of
disseminated disease (27). Coccidioidomycosis is particularly severe in
patients with organ transplants or AIDS.

Though disseminated coccidioidomycosis is uncommon, and symptomatic
coccidioidal pneumonia usually resolves without therapy, many of these
patients are very ill for weeks to months. Galgiani reported that a group
of college students in Tucson who had coccidioidomycosis required an
average of six clinic visits before the disease resolved (16). Therefore,
this can be an expensive illness in terms of medical costs and time lost
from work or school, even when the infection resolves spontaneously.

Coccidioidomycosis Epidemic in California

Kern County, in the San Joaquin Valley, California, is one of the most
highly coccidioidomycosis-endemic regions. The number of new cases of
coccidioidomycosis in the area has varied widely from year to year; a low
incidence of coccidioidomycosis from 1987 to 1990 (<500 reported cases a
year in Kern County), was followed by a high incidence from 1991 to 1994
(28-30). The number of reported cases, which were identified by serologic
testing at the Kern County Health Department (the reference serology
laboratory for the county), probably represent approximately 10% of the
total number of infected persons in that county (Figure 4) [Figures not 
available in ASCII] (28). The
medical costs for infected persons in Kern County are estimated at $66
million (29). In 1992, 4,500 new cases were reported to the California
State Department of Health Services (30), most from Kern County; the number
of new cases also increased in almost all counties in central and southern
California (30). The increase in reported cases in California in 199192 was
dramatic but certainly an underestimate of the magnitude of the problem
(31).

The epidemic seems to be waning, for reasons that are not clear, but the
marked increase in incidence from the 1980s to 1991 through 1993 is
indisputable. What factors may account for this increase? One major
consideration is the weather. C. E. Smith observed years ago that the
number of cases of coccidioidomycosis was higher in the summer after a
rainy winter than after a dry winter (10). In March 1991, a 5-year drought
in California ended with a heavy rainfall. Rainfall was also heavy in the
winters of 1992 and 1993. Though the relationship between the weather and
the density of C. immitis in the soil may never be understood in detail,
the following scenario seems plausible. During drought years, the number of
organisms competing with C. immitis decreases. C. immitis does not thrive,
but it remains viable though dormant. After heavy rain, the arthrocondia
germinate and multiply to a higher density than usual because of the lack
of competing organisms. Once the soil dries in the late summer and fall,
the arthroconidia become airborne and potentially infectious (29).

Another reason for the sudden increase in disease incidence might have been
the number of susceptible persons in the disease-endemic area. The number
may have been the result of both increased migration of susceptible persons
and decreased immunity in the indigenous population. Immunity comes from
prior infection and is manifest as a positive coccidioidin skin test. In
almost all cases, coccidioidomycosis confers lifelong immunity. As a result
of years of low incidence, the number of nonimmune persons may have
increased, as evidenced by the decrease in prevalence of positive
coccidioidin skin tests among local high school students. In 1939, 50% to
60% of high school students in the San Joaquin Valley had positive skin
tests (17), but in the 1980s only 3% to 5% of high school students had
positive skin tests (T. Larwood, pers. comm.). Given the historical data,
this estimate seems low, but another study also found a low prevalence. In
1985, workers in Tucson estimated that 30% of a random sample of persons in
a Hispanic neighborhood had positive skin tests (32). In addition to the
drought, irrigation of fields, the increasing amount of land under
cultivation, and a decrease in indoor dust due to the widespread use of air
conditioning may also have played a role in the relatively low incidence of
infections in the 1980s.

Coccidioidomycosis in the Immunosuppressed

C. immitis is a primary pathogen that can cause disease in immunologically
healthy persons. In the population as a whole, fewer than 5% of infected
persons have persistent pulmonary infection or extrapulmonary dissemination
of the disease (16). The incidence of clinically significant disease in
immunosuppressed patients is much higher. In one study symptomatic
coccidioidomycosis developed in 18 (7%) of 260 renal transplant patients in
Arizona over a 10-year period, primarily in the first year after
transplantation (33). This rate was substantially higher than the rate of
infection in patients who were undergoing hemodialysis. Approximately 12
(67%) of infections in the patients with renal transplants were
disseminated; the remainder were confined to the lung. Of patients with
disseminated disease, 10 (83%) died, despite intensive therapy with
amphotericin B. In another study from Tucson, all confirmed cases of
coccidioidomycosis during a 4-year period were reviewed. The dissemination
rate was 8 (73%) of 11 of patients who were receiving immunosuppressive
therapies, compared with only 15 (14%) of 110 healthy controls (34). As
more patients in the disease-endemic area receive liver, lung, and heart
transplants, this problem will increase.

Pregnant women, especially those in the third trimester, are at high risk
for developing disseminated coccidioidomycosis if they become infected
(24). In the first and second trimesters, the risk is much lower. The
reason is not entirely clear, but two factors may play a role: 1) the high
sex hormone levels found in late pregnancy enhance the growth of C. immitis
in vitro (25), and 2) the shift in the T-cell immune response late in
pregnancy toward TH2 cytokines (26) interferes with resolving the
infection. In experimental animals, pregnancy increases the severity of
leishmaniasis, another infection hat is controlled by a TH1 T-cell response
(35).

Coccidioidomycosis in AIDS patients is also very likely to be
life-threatening. The first cases of coccidioidomycosis described in AIDS
patients were atypical, with a reticulonodular chest x-ray pattern,
positive blood cultures, and infection of multiple organs (36). As we have
gained more experience with coccidioidomycosis in HIV-infected persons, we
have learned that the clinical spectrum is broader than originally
reported. Fish and his colleagues collected data from 77 AIDS patients with
coccidioidomycosis who were treated by physicians in Arizona and California
(37). They grouped the patients according to their clinical symptoms
(Figure 5) [Figures not available in ASCII].

Although the largest group of patients had diffuse pulmonary infiltrates, a
significant fraction had focal pulmonary disease, meningitis, or other
extrapulmonary disease. Six patients had only a positive serologic test
with no other evidence of infection. Excluding the patients who had only a
positive serologic test, 81% of the patients in this series had a positive
serologic test for coccidioidomycosis, either for IgM or IgG antibodies.
However, only 69% of patients with diffuse pulmonary disease had a positive
serologic test, and the death rate in this group was also the highest
(70%). In all clinical groups, death was correlated with the number of
circulating CD4 T-cells at the time of diagnosis. For clinicians, however,
the most important message from this study is that coccidioidomycosis is
not a uniformly fatal complication in patients with AIDS, and that many
forms of this disease, including meningitis, respond to therapy. Patients
with <200 CD4 T-cells/l are more likely to have severe, disseminated
infections.

A more recent prospective study of 170 HIV-infected persons in an area of
Arizona where coccidioidomycosis is endemic showed a cumulative incidence
of coccidioidomycosis of 25% over 41 months (38). The most important risk
factors were the level of CD4 T-cells and the diagnosis of AIDS (as opposed
to HIV infection). HIV-infected patients with AIDS or <250 CD4 T-cells/l
were 8 to 35 times more likely to get coccidioidomycosis. History of
coccidioidomycosis, a history of a positive skin test for
coccidioidomycosis, or a prolonged stay in the disease-endemic area were
not associated with an increased risk for infection. These data suggest
that most cases were primary infections in severely immunosuppressed
patients. Since patients with AIDS were not more likely to be exposed to
the spores of C. immitis and all patients were seen prospectively at
4-month intervals and tested for antibody to C. immitis, severe
immunosuppression appeared to increase their risk for infection, as well as
disease. As in the retrospective study reviewed above, the clinical
symptoms varied widely, ranging from mild to extremely severe. Only one
patient had antibody titers to C. immitis by complement fixation test
without any other evidence of disease.

Treatment

Various drugs are now available for treating coccidioidomycosis. In
addition to amphotericin B, which must be given intravenously and is
considerably toxic, triazole compounds have been found to be active agents
for treating most manifestations of coccidioidomycosis. Fluconazole, in an
uncontrolled study, was reported to be effective primary therapy for
coccidioidal meningitis; since untreated coccidioidal meningitis is
uniformly fatal, robust conclusions could be drawn from this trial (39).
Forty-seven consecutive patients were treated with 400 mg/day of
fluconazole; during the first 6 months of therapy, 33 (70%) of the patients
responded to therapy. (A response was defined as a 40% reduction in a
score, on the basis of clinical measurements and cerebrospinal fluid
findings.) Two patients who did not respond to therapy died of
coccidioidomycosis; both were HIV-positive. Because of previous experience
with high relapse rates when azole therapy is stopped, the authors
recommended lifelong treatment with fluconazole. In a small study, four of
five patients treated for meningitis with itraconazole as sole therapy
responded favorably (40). A recent article emphasized the high relapse rate
after azole therapy is stopped (41). The alternative treatment to the
azoles is amphotericin B. If amphotericin B is used to treat meningitis,
however, it must be given intrathecally as well as intravenously, and this
greatly increases the risk for a toxic reaction to that drug.

Clearly, fluconazole and itraconazole can be used to treat patients with
nonmeningeal coccidioidomycosis (42-44). Whether one of these drugs is
superior to the other, or how either one compares to amphotericin B is not
known. It seems prudent to treat extremely ill patients with amphotericin
B, at least until their clinical situation stabilizes, although no
published studies support that point of view. However, few (if any)
patients with the acute miliary form of coccidioidomycosis have been
included in any of the reported studies of any of the azole drugs. New
agents that are more active against coccidioidomycosis are still sorely
needed.

Prevention

Simple environmental measures, such as planting grass or paving roads in
highly populated areas, decrease the amount of airborne dust and lower the
risk for coccidioidomycosis (10). These measures do not necessarily
eradicate C. immitis from the soil but lower the risk for airborne
dispersion of the organism. At present, no practical method exists for
eliminating C. immitis from the soil.

Vaccine Development

An alternative approach is to vaccinate persons at risk. A vaccine is
feasible because natural infection almost always confers lifelong immunity
from reinfection. Furthermore, good animal models exist to test vaccine
candidates (21). Finally, genetically susceptible mice can be successfully
immunized, which suggests that the genetically susceptible human population
would also benefit from vaccination (21).

One vaccine that has been tested is a killed spherule vaccine developed by
Pappagianis and Levine. It protected mice and other animals from
experimental infection with C. immitis (45). Between 1980 and 1985, a
double-blinded human study compared results of a formalin-killed spherule
vaccine with results obtained from a placebo. In this study, which involved
almost 3,000 people, only a minority of the vaccinated persons had positive
skin test results to C. immitis. Although the incidence of
coccidioidomycosis was low while this study was conducted, no difference
was found in the number of cases of coccidioidomycosis or the severity of
the disease in the vaccinated group compared with that for the
placebo-receiving control group (46). One explanation for the
ineffectiveness of this vaccine may be that relatively small numbers of
killed organisms could be injected into human without unacceptable local
side effects of pain and swelling. Nevertheless, the vaccine trial made it
clear that immunization with tolerable numbers of whole killed-spherules
does not provide immunoprotection against coccidioidomycosis in humans.

Since the cell wall of C. immitis is made up primarily of nonprotein
macromolecules, it contains a large amount of material presumably
nonantigenic for T lymphocytes. Therefore, the whole organism is not the
ideal vaccine candidate. Ideally, one would like to vaccinate patients
selectively only with antigens that stimulate a protective T-cell-mediated
immune response. These antigens have been difficult to identify, and a
consensus on what they are does not exist. Various approaches have been
used to obtain antigenic proteins. In one, a lysate of arthroconidia
(coccidioidin) or spherules (spherulin) was made (47). Alkali treatment has
also been used to extract antigens from arthroconidia and spherules (48).
Another approach has been to use C. immitis antigens obtained without
extraction or autolysis. The advantage of this method is that one should
obtain reproducible preparations of intact proteins. Cole and co-workers
(49) found that when the outer conidial wall was removed from
arthroconidia, the organism released various proteins (called the soluble
conidial wall fraction). This mixture of proteins was extraordinarily
effective in stimulating the proliferation of C. immitis-immune T cells in
mice. Another antigenic mixture is a membranous material consisting
primarily of proteins and lipids that the spherule phase of the organism
spontaneously releases (the spherule outer wall). This spherule wall
fraction has been shown to be an active antigen in T-cell-mediated immune
responses in mice (50).

All of these mixtures are heterogeneous and difficult to fractionate
biochemically. This is probably due, at least in part, to differences in
glycosylation, which makes physically separating the proteins difficult. To
resolve this problem, Galgiani and his colleagues deglycosylated the
proteins from a toluene spherule lysate by using hydrogen fluoride (51,52).
Although this treatment does remove all sugars, it is extraordinarily harsh
and yields less than 10% of the initial protein, with most of the protein
forming an insoluble precipitate. Nevertheless, the resulting product
reacts with reference antiserum to C. immitis in immunoelectrophoresis.
This antigen also stimulated a proliferative T-cell response in patient
lymphocytes but not in those of the control group (noninfected donors).

Another way to attack the problem of generating pure C. immitis antigens is
to use molecular biologic techniques. The advantage to this approach is
that once antigens are molecularly cloned, and the protein is expressed, an
essentially unlimited source of completely defined antigen is available.
Therefore, one would not have to repeatedly grow C. immitis, extract the
antigen, and purify it from a complex mixture. In addition, with the
molecular approach, antigens could be delivered as part of a living vaccine
system, should that be required to effectively immunize people against
coccidioidomycosis. We believe that systematically identifying and
evaluating C. immitis T-cell reactive antigens in experimental animals is a
rational approach to the ultimate development of a vaccine. Our
laboratories, in collaboration with Garry Cole, have used a murine T-cell
line that is specific for soluble conidial wall fraction antigens to
identify one cloned fragment of a C. immitis protein (53). Recently,
genomic DNA clones coding for this protein have been identified and
sequenced. Significant homology exists between this C. immitis antigen and
the human enzyme 4 hydroxyphenylpyruvate dioxygenase (54). This protein has
been expressed in bacteria and was found to elicit T-lymphocyte
proliferative responses in mice immune to C. immitis. We are testing its
efficacy as an experimental vaccine.

With the exception of alkali extracted spherules (55) and whole killed
spherules (45), none of the T-cell reactive antigens have been shown to be
immunoprotective in experimental models. However, it is reasonable to
expect that some antigen, or mixture of antigens, will be found that can
confer protective immunity in experimental animals. Molecular strategies
are available to accomplish this task and are an important area of future
research. Once a vaccine has been successfully tested in animals, another
human vaccine trial would be feasible.

Dr. Kirkland is associate professor of pathology and medicine; Dr. Fierer
is professor of medicine and pathology and head of the Division of
Infectious Diseases, University of California, San Diego School of
Medicine. Drs. Kirkland and Fierer have worked together for the past 15
years. Currently, they are focusing on the genetic determinants for
resistance to infection and on identifying candidates for a
coccidioidomycosis vaccine.

Acknowledgments

We are grateful to Dr. Ron Talbot, Director of the Kern County Public
Health Laboratory, and Dr. Tom Larwood for sharing unpublished data with
us. The experimental work done in our laboratories has been supported by
National Institutes for Health grants AI19149 and AI37232 and by the
Research Service of the Department of Veterans Affairs.

Address for correspondence: Theo N. Kirkland, M.D., Department of Pathology
and Medicine, University of California, San Diego School of Medicine,
Department of Veterans Affairs Medical Center (111-F) 3350 La Jolla Village
Dr., San Diego, CA 92161, USA; fax: 619-552-4398; e-mail:
tkirkland@ucsd.edu.

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     1983;39:473-5.


===========================================================================
Synopses

Antibody-Based Therapies for
Emerging Infectious Diseases

Arturo Casadevall, M.D., Ph.D.
Albert Einstein College of Medicine, Bronx, New York, USA
---------------------------------------------------------------------------


          In the 19th century, it was discovered that immune sera
          were useful in treating infectious diseases. Serum
          therapy was largely abandoned in the 1940s because of
          the toxicity associated with the administration of
          heterologous sera and the introduction of effective
          antimicrobial chemotherapy. Recent advances in the
          technology of monoclonal antibody production provide
          the means to generate human antibody reagents and
          reintroduce antibody therapies, while avoiding the
          toxicities associated with serum therapy. Because of
          the versatility of antibodies, antibody-based therapies
          could, in theory, be developed against any existing
          pathogen. The advantages of antibody-based therapies
          include versatility, low toxicity, pathogen
          specificity, enhancement of immune function, and
          favorable pharmacokinetics; the disadvantages include
          high cost, limited usefulness against mixed infections,
          and the need for early and precise microbiologic
          diagnosis. The potential of antibodies as
          anti-infective agents has not been fully tapped.
          Antibody-based therapies constitute a potentially
          useful option against newly emergent pathogens.

In the mid-1990s, successful implementation of anti-infective therapy has
become increasingly difficult because of widespread antimicrobial
resistance, the emergence of new pathogens, and the occurrence of many
infections in immunocompromised patients in whom antimicrobial drugs are
less effective. Infections caused by some new pathogens (e.g., human
immunodefficiency virus [HIV] and Cryptosporidium parvum) cannot be cured
with existing antimicrobial drugs. Regaining the upper hand in the struggle
against microbes requires multidisciplinary efforts which include
developing new antimicrobial agents (1), improving surveillance for
emerging microbial threats (2), teaching the correct use of antimicrobial
therapy (3), expanding the use of vaccines to prevent infection (4),
developing adjunctive immunotherapies (5), and conducting new basic
research on the mechanisms of pathogenesis and drug resistance. In this
article, the potential of antibody therapy in confronting the threat of
emergent infections will be explored.

Antibody-Based Therapies: Then and Now

Antibody-based (serum) therapies were first used to treat human infections
in the 1890s (6,7). In the early 20th century, serum therapy was used to
treat a variety of bacterial infections, including those cased by
Corynebacterium diphtheriae, Streptococcus pneumoniae, Neisseria
meningitides, Haemophilus influenzae, group A streptococcus, and
Clostridium tetani (6,7). By the 1930s, serum therapy was standard
treatment for lobar pneumonia (Figure 1)  [Figures not available in ASCII]. 
Several large controlled trials
showed that administering type-specific serum reduced the death rate in
patients with pneumococcal pneumonia by approximately 50% (6). However,
when antimicrobial chemotherapy was discovered in the mid1930s, serum
therapy for bacterial infections was rapidly abandoned. Antimicrobial
chemotherapy had important advantages over serum therapy: it was more
effective and less toxic. The immediate side effects of serum therapy
included fevers, chills, and allergic reactions (8,9). A delayed toxic
reaction of serum therapy was serum sickness, a syndrome characterized by
rash, proteinuria, and arthralgia; this occurred in 10% to 50% of patients
who received heterologous serum and was probably caused by immune
complexes. Other disadvantages of serum therapy included the need to
establish a precise diagnosis before selecting serum, lot-to-lot variation
of serum, and the need for considerable physician expertise (Table 1).
Serum therapy could fail because of inadequate dosage, delayed treatment,
mislabeling of serum, and because the infection was mixed or complicated
(i.e., empyema) (10). Producing therapeutic sera was very expensive because
of the costs of animal husbandry, antibody purification, refrigeration, and
standardization by the mouse protection tests. When antimicrobial
chemotherapy was first introduced, enthusiasm was expressed for combining
serum therapy and antimicrobial chemotherapy. Support for combined therapy
came from animal studies, which suggested that combination therapy was more
effective than either therapy alone against several pathogens, including
group A streptococcus (11), pneumococcus (12), and meningococcus (13), and
some authorities recommended combined therapy for serious infections
(14,15). However, several studies showed that combined therapy was not more
effective than antimicrobial chemotherapy alone and that it caused
significantly more side effects (16-18). Therefore, serum therapy was
abandoned because it offered no measurable advantage in efficacy over
chemotherapy and had substantial disadvantages in implementation, cost, and
toxicity.


         Table 1. Serum therapy, human MAbs, and antimicrobial chemotherapy
----------------------------------------------------------------------------------
                        Antibody therapy         Chemotherapy       Comment
                   Immune serum     Human MAb
----------------------------------------------------------------------------------
 Specificity     Narrow          Narrow          Broad         Narrow specificity
                                                               avoids selection of
                                                               resistant organims
                                                               among nontargeted
                                                               microbes.
                                                               Narrow specificity
                                                               requires a precise
                                                               diagnosis before
                                                               use.
 Source              Animals      Tissue culture  Fermentation Before antibiotics,
                      Humans        Bioreactor      Chemical   most serum
                                   Fermentation    synthesis   preparations were
                                                               from horses and
                                                               rabbits. MAbs are
                                                               produced by tissue
                                                               culture techniques.
                                                               Industrial
                                                               production of MAbs
                                                               may utilize
                                                               immunoglobulin
                                                               synthesis in yeast,
                                                               bacteria, or plants.
 Toxicity        High            Low             Low           Toxicity of serum
                                                               was due to allergic
                                                               reactions to animals
                                                               protein. Human
                                                               immunoglobulin
                                                               preparations are
                                                               well tolerated (42).
                                                               Antiidiotypic
                                                               responses remain a
                                                               problem for
                                                               humanized MAb
                                                               therapy.
 Cost            High            High            Low           Serum therapy for
                                                               pneumococcal
                                                               pneumonia in the
                                                               1930s was costly(6).
                                                               Immunoglobulin
                                                               therapy remains very
                                                               expensive.
 Administration  Difficult       Easy            Easy          Serum therapy
                                                               required
                                                               considerable
                                                               expertise, and
                                                               because of
                                                               life-threatening
                                                               allergic reactions,
                                                               dosage was often
                                                               based on clinical
                                                               experience.
 Pharmaco         Variable       Consistent      Consistent    Pharmacokinetics of
 kinetics                                                      hererologous  
                                                               polyclonal antibody
                                                               depends on multiple
                                                               variables, e.g.,
                                                               animal source,
                                                               isotype composition,
                                                               and immune status of
                                                               the recipient.
                                                               Human MAbs are
                                                               homogeneous reagents
                                                               and can be expected
                                                               to have more
                                                               consistent
                                                               pharmacokinetics.
 Mechanism        Antimicrobial   Antimicrobial  Antimicrobial Conventional
 of action            Immune          Immune                   antimicrobial
                   enhancement     enhancement                 chemotherapy kills
                      Toxin           Toxin                    or inhibits the
                  neutralization  neutralization               replication of
                                                               microorganisms.
                                                               Antibodies function
                                                               through a variety of
                                                               mechanisms, e.g.,
                                                               promoting
                                                               complement-mediated
                                                               lysis, enhancing
                                                               antimicrobial
                                                               efficacy of host
                                                               effector cells,
                                                               promoting
                                                               phagocytosis,
                                                               preventing
                                                               attachment, and
                                                               neutralizing toxins.
----------------------------------------------------------------------------------

Today antibody therapy is indicated in infectious diseases in relatively
few situations, including replacement therapy in immunoglobulin-deficient
patients, post-exposure prophylaxis against several viruses (e.g., rabies,
measles, hepatitis A and B, varicella), and toxin neutralization
(diphtheria, tetanus, and botulism). Ironically, the general abandonment of
antibodies as antimicrobial agents was followed by major advances in the
technology of antibody production. In 1975, hybridoma technology provided
the means to generate unlimited amounts of monoclonal antibodies (MAbs)
(19). In recent years, major advances have been made in the techniques used
to generate human antibodies and humanize murine MAbs (20).

The juxtaposition of three recent developments makes the reintroduction of
antibody-based therapies an option for serious consideration. First,
because of advances in technology, human antibody reagents can be
synthesized; thus the toxicities traditionally associated with serum
therapy can be avoided. Second, the emergence of new pathogens, the
reemergence of old pathogens, and the increased prevalence of
drug-resistant microorganisms have caused the effectiveness of existing
therapeutic options to decline. Third, the difficulties involved in
treating infections in immunocompromised patients suggest the need for
adjunctive immunotherapy.

Polyclonal Sera Versus Monoclonal Antibodies for Therapy

Immune sera contain antibodies of multiple specificities and isotypes.
Problems with immune sera include lot-to-lot variation (21), low content of
specific antibodies (22), and some hazards in the transmission of
infectious diseases (23). Commercially available intravenous immunoglobulin
preparations obtained from human donors differ in their opsonic activity
for common pathogens such as Staphylococcus epidermidis, H. influenzae type
b, S. pneumoniae, group B streptococcus, and Escherichia coli, reflecting
the characteristics of the donor pool (22). In contrast, MAbs are generated
in vitro by either hybridoma technology or recombinant DNA techniques. MAbs
are homogenous immunoglobulins that, by definition, recognize one epitope
and have markedly higher specific activity than polyclonal preparations.
For example, 0.7 mg of two human MAbs to tetanus toxin have the same
activity as 100 to 170 mg of immune globulin (24). The higher specific
activity of MAbs may also translate into greater therapeutic efficacy. MAbs
formulations are superior to polyclonal sera in homogeneity, constancy,
specific activity, and (possibly) safety. For some infections,polyclonal
preparations may be superior to MAbs because MAbs contain antibodies to
multiple epitopes (i.e., they are polyvalent). However, different
therapeutic MAbs can be combined to generate polyvalent preparations
composed of antibodies with multiple specificities and isotypes. Given the
advantages of MAb preparations over immunesera, antibody-based therapies
for emergent infections, if used, will likely rely primarily on MAb
technology.

Advantages of Antibody-Based Therapies

Humans can produce antibodies to practically all existing pathogens.
Antibody molecules are assembled from combinations of variable gene
elements, and the possibilities resulting from combining the many variable
gene elements in the germline enable the host to synthesize antibodies to
an extraordinarily large number of antigens. During the generation of the
antibody response, somatic mutations are introduced into immunoglobulin
genes, which result in higher affinity antibodies and more diversity in
specificity (25). Thus, antibodies are, as a class, broad-spectrum
antimicrobial agents with activity against all classes of pathogens.
However, individual antibodies are usually pathogen-specific.
Pathogen-specific antimicrobial agents have the theoretical advantage that
they do not select for resistant organisms among nontargeted microbes and
are unlikely to produce great disturbances in the normal host flora.

Antibody-based therapies could, in theory, be developed against any
pathogen. Although the level of antibody immunity differs among pathogens,
it may be possible to develop useful antibody therapies even if natural
antibody immunity plays little or no role in protection. Two fungi, Candida
albicans and Cryptococcus neoformans, are pathogens for which protective
antibodies can be generated despite uncertainty about the role of natural
antibody immunity (26). The MAbs to C. neoformans enhance the therapeutic
efficacy of amphotericin b (27,28), fluconazole (29), and 5-flucytosine
(30) in murine models of cryptococcosis. Therefore, uncertainty regarding
the role of natural antibody immunity in protecting against a given
pathogen does not rule out the existence of antibodies that may be useful
in therapy.

Microbial targets for therapeutic antibody development are not necessarily
limited to extracellular pathogens. Although intracellular pathogens are
commonly believed to be outside the reach of antibody immunity, several
reports have suggested that some MAbs are active against some intracellular
microorganisms. Some IgA MAbs can neutralize intracellular viruses (31),
and an MAb to Toxoplasma gondii has been reported to interfere with
intracellular replication of the parasite (32). It has been proposed that
intracellular virus neutralization by IgA occurs by antibodies binding to
viral proteins and interfering with viral assembly (31). Additional
evidence for intracellular antibody activity comes from the observation
that IgG anti-DNA autoantibodies can enter the cytoplasm and nucleus of
living cells (33).

Antibodies mediate antimicrobial function through a variety of mechanisms,
including inhibition of microbial attachment, agglutination, viral
neutralization, toxin neutralization, antibody-directed cellular
cytotoxicity, complement activation, and opsonization (34). Antibodies are
extremely versatile antimicrobial molecules: some are active directly
against the pathogen, some neutralize the toxic products of infection, and
others enhance the efficacy of host effector cells. Some MAbs to poliovirus
are neutralizing only at fever temperatures (35), which demonstrates their
ability to function at physiologic extremes. The versatility of
antibody-based therapies is illustrated by the ability of digoxin-binding
antibodies to reverse digoxin toxicity (36) and recent attempts to treat
septic shock by employing MAbs that bind cytokines (37).

Human IgG has favorable pharmacokinetics for use as an antimicrobial agent,
including good tissue penetration (38) and a half-life of about 20 days
(39). Murine MAbs have much shorter half lives in humans, and these usually
elicit human antibody responses (40). Human-mouse chimeric antibodies and
humanized MAbs are synthetic molecules composed primarily of human antibody
protein sequences that retain the antigen-binding site of the heterologous
antibody (20). Human-mouse chimeric antibodies and humanized MAbs have
longer half-lives than the murine precursor, but their half lives are still
much shorter than that of native human IgG (41). This area is being
intensively investigated, and genetic engineering of antibody molecules may
be used to synthesize MAbs with longer half-lives.

Immunoglobulin therapy with human reagents is generally well tolerated
(42). Serious adverse reactions, including renal failure (43), aseptic
meningitis (44), and thromboembolic events (45) can occur with high-dose
(0.5 to 2 g/kg) antibody therapy. However, anti-infective immunoglobulin
therapy with MAb preparations is unlikely to require the high doses of
immunoglobulin used to treat rheumatic disorders and other conditions. For
example, the heterologous immune sera used in the pre-antibiotic era were
effective, although they contained small amounts of specific antibody. The
higher activity of MAb preparations should permit a smaller amount of
immunoglobulin proteins to be used and thus avoid the occasional toxicity
reported with high-dose antibody therapy.

Problems with Antibody-Based Therapies

Most antibody therapies are pathogen-specific. This is a disadvantage in
dealing with mixed infections. Mixed infection with multiple S. pneumoniae
serotypes was recognized as a cause for the failure of serum therapy (46).
For pathogens that are antigenically variable, one solution is to use
antibody cocktails of agents active against the most common antigenic
types. Antibody cocktails may also be designed to include antibodies of
different isotypes to enhance antibody effector function. The successful
implementation of antibody-based therapies would also require improvements
in diagnostic microbiology. In the pre-antibiotic era, for lobar pneumonia,
rapid protocols were developed for the isolation and typing of pneumococci
from sputum (47). Recent advances in diagnostic microbiology, including
polymerase chain reaction and nucleic acid hybridization, could
substantially shorten the time required to establish a microbiologic
diagnosis. The narrow spectrum of antimicrobial activity that characterizes
antibody-based therapies is a drawback for commercial development, however.
Pathogen-specific drugs, have smaller potential markets than broad-spectrum
antimicrobial agents, and this makes them less attractive to the
pharmaceutical industry. Conversely, the emergence of multidrug-resistant
microorganisms and new pathogens for which no drugs exist could make
pathogen-specific drugs attractive for commercial development.

Widespread use of antibody-based therapies could produce selective pressure
on microbial populations for the emergence of antibody-resistant variants.
Antibody-resistant mutants of Borrelia burdorferi have been produced in the
laboratory (48), and may be selected in patients who undergo antibody-based
therapies. Microorganisms may become resistant to antibodies by acquiring
mutations that change the antigenic site recognized by the antibodies or by
producing proteases that destroy immunoglobulins. The IgA protease genes of
N. gonorrhoeae can be transferred between strains, and the widespread use
of antibody therapies may select for protease-producing strains (49).
However, the versatility of antibody technologies provides alternatives for
countering antibody-resistant strains. For instance, new antibodies
directed toward the mutated epitope could be developed, or antibodies that
bind other antigenic targets could be introduced. Protease-producing
strains could be countered with protease-resistant immunoglobulin molecules
generated by introducing amino acid changes at protease cleavage sites.
Alternatively, MAbs that neutralize proteases could be incorporated into
therapeutic antibody cocktails in a manner analogous to the present
practice of using beta lactamase inhibitors to increase the effectiveness
of beta lactam antibiotics. Recognizing that the introduction of new agents
has been inevitably followed by the emergence of resistance, researchers
could attempt to minimize the emergence of antibody-resistant organisms
from the outset by using cocktails of MAbs directed at multiple antigenic
targets.Combining antibody therapy with chemotherapy also could reduce the
probability of selecting for organisms resistant to either therapeutic
modality.

Antibodies are more effective in preventing infection than in treating
established infection. Antibody-based therapies have been most useful when
administered early in the course of disease: serum therapy for pneumococcal
pneumonia was most effective if serum was administered within 3 days of the
onset of clinical symptoms (6). Because antibodies are proteins, therapy
for invasive infections is likely to require systemic administration. This
is a serious disadvantage in developing countries where access to medical
care is limited. For enteric pathogens, oral antibody administration may be
most effective (50,51). Efforts to develop MAbs for cancer therapy have run
into unexpected pharmacologic problems (38,40). The finding that MAb uptake
by tumors is partially dependent on antigen expression in the tumor (40)
suggests that MAb uptake by infected tissues could depend on microbial
antigen expression at the site. Th same problems may be found when antibody
penetrates tumors.

Antibodies can, in principle, elicit neutralizing antibody, allergic
responses, or both. Administering rodent MAbs to patients elicits human
antibodies to the rodent MAb (52). Antibody therapies against emergent
pathogens, if attempted, are likely to use human, human-animal chimeric, or
humanized antibodies. Mouse-human chimerics and humanized antibodies are
less immunogenic than heterologous antibodies (41,53,54); therefore, the
likelihood that the patient will mount a neutralizing antibody response to
the therapeutic antibody may be reduced. Nevertheless, anti-idiotypic
responses have been observed in patients receiving humanized antibody
therapy (54). The clinical importance of such anti-idiotypic responses is
uncertain. Many infections are single life-threatening episodes in the life
of a person, and the occurrence of anti-idiotypic antibodies following
therapy may require repeated or long-term antibody administration.

Antibodies are largely excluded from the central nervous system by the
blood-brain barrier. Nevertheless, antibody treatment of brain infections
is feasible. In some brain infections, the blood-brain barrier is more
permeable to serum components because of inflammation, and systemic
antibody therapy was used successfully to meningococcal meningitis (55). If
antibody penetration to brain tissue is a problem, two alternatives exist.
First, antibodies can be administered directly into the sub-arachnoid space
(as was done for the treatment of meningococcal meningitis in the
pre-antibiotic era) (15,56). Second, antibody molecules can be engineered
for enhanced brain penetration by altering their charge (57) or by linking
them to proteins that cross the blood-brain barrier (58).

Antibody therapies are also costly to develop and expensive for the
patient. For example, antibody prophylaxis for cytomegalovirus infections
can cost several thousand dollars per patient (59). To be cost-effective,
antibody-based therapies would have to provide a clear benefit over
existing therapy. For emerging pathogens for which no therapy is available,
the cost of antibody therapies may be justifiable, depending on the
potential for death, illness, and long-term consequences of the infection.
In the long run, advances in antibody production and improvements in
technology may greatly lower costs and make antibody-based therapy more
competitive with antimicrobial chemotherapy.

Historical Perspective on the Development of Antibody Therapies

In recent years, considerable interest has been expressed in using
antibody-based therapies to treat septic shock (60). Unfortunately, MAbs to
endotoxin have not been as effective in clinical trials as anticipated
(61,62), and this has dampened some of the enthusiasm for MAb therapies.
The development of serum therapy for S. pneumoniae also encountered
considerable difficulties in the pre-antibiotic era.

The Klemperers demonstrated that immune serum protected rabbits in 1891
(63), but reliable antibody therapies for pneumococcal pneumonia were not
available until the 1920s (Figure 2) [Figures not available in ASCII]. 
Translating the laboratory finding
that immune sera protected rabbits against experimental pneumococcal
infection to the successful use of serum therapy for lobar pneumonia in
humans required extensive basic and clinical research. At the laboratory
bench, developing antibody therapy for pneumococcal pneumonia required the
discovery that antigenic variation existed among pneumococcal strains, that
only type-specific sera provided protection, that certain vaccination
schedules were necessary to elicit good antibody responses, the ability to
standardize the serum potency (by the mouse protection test), and improved
antibody purification techniques (6). At the bedside, implementing
successful serum therapy required learning when and how to give serum,
managing the side effects of serum therapy, and developing rapid protocols
for recovering pneumococci from sputum for serum typing (64). The mouse
protection test reduced but did not eliminate the problems of lot-to-lot
variation in serum efficacy (21). The development and perfection of
antibody therapies for pneumococcus contributed important research
findings, which led to major discoveries in immunology (65). The high death
rate for meningococcal meningitis also led to the rapid development of
serum therapy (6). In the early 1900s, serum therapy markedly reduced the
death rate of meningococcal epidemics, possibly because of antigenic
changes in the pathogen (6). The lengthy time required for the development
of serum therapy for pneumococcus, the variable efficacy of
anti-meningococcal sera (depending on the epidemic), and the more recent
difficulties encountered in developing MAb therapy for septic shock suggest
that developing antibody-based therapies for emergent pathogens will
require extensive preclinical and clinical testing.

Other Antibody-Based Strategies Against Emerging Pathogens

Vaccines that elicit protective antibody immunity could be used to protect
against emergent pathogens. A polysaccharide-protein conjugate vaccine is
being developed (66) against C. neoformans. This vaccine elicits protective
antibodies in mice (67), and it is hoped that vaccination will result in
the production of effective anti-cryptococcal antibodies to prevent disease
in patients at risk. The conjugate vaccine against C. neoformans is
intended to elicit protective antibody immunity, even though the role of
natural antibody immunity in protection against cryptococcosis is uncertain
(26). Newer vaccines against common pathogens could help limit the spread
of drug-resistant microorganisms. Dissemination of penicillin-resistant
pneumococci has been associated with infection and carriage by young
children among whom the current 23-valent pneumococcal polysaccharide
vaccine is ineffective in inducing protective immunity (4). However, the
effectiveness of polysaccharide-protein conjugate vaccine to H. influenzae
type b suggests that a similar conjugate vaccine to S. pneumoniae, if
available, could effectively abort childhood infection with
antibiotic-resistant pneumococci and thereby limit the spread of these
strains (4).

Future Directions

Immunoglobulins are an extremely versatile class of antimicrobial proteins
that can be used to prevent and treat emerging infectious diseases.
Antibody therapy has been effective against a variety of diverse
microorganisms. The historical record clearly documents both the usefulness
and the difficulties in developing and implementing passive antibody
therapies. The experience with serum therapy for pneumococcal pneumonia and
meningococcal meningitis suggests that extensive basic and clinical
research is essential for the successful implementation of antibody
therapy. Given the multitude of pathogens, the pathogen-specific nature of
antibody therapies, and the costs of developing and using antibody
therapies, the development of such therapies for most pathogens at present,
would be impractical. However, for selected pathogens, antibody-based
therapies could provide new therapeutic options. Opportunities for the
development of antibody-based strategies include 1) pathogens for which
there is no available antimicrobial therapy (e.g., C. parvum and
vancomycin-resistant enterococcus); 2) pathogens that affect primarily
immunocompromised patients in whom antimicrobial therapy is not very
effective (e.g., invasive fungal infections); 3) pathogens for which
drug-resistant variants are rapidly spreading (e.g., Pseudomonas aeruginosa
[68]); and 4) highly virulent pathogens for which few effective
antimicrobial agents are available (e.g., methicillin-resistant S. aureus).

Acknowledgments

The author thanks Drs. L. Pirofski, M.D. Scharff, and R. Soeiro for many
useful discussions over the years. This research was supported in part by
NIH grants R01AI33774 and R01AI13342.

Dr. Casadevall is assistant professor of medicine and microbiology and
immunology at the Einstein College of Medicine. His research interests
include antibody immunity, fungal pathogenesis, and molecular epidemiology.

Address for correspondence: Arturo Casadevall, M.D., Ph.D., Department of
Medicine, Albert Einstein College of Medicine, 1300 Morris Park Ave.,
Bronx, New York 10461, USA; fax: 718-340-8968; e-mail:
casadeva@aecom.yu.edu.

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============================================================================
Dispatches

HIV-1 Group O Virus Identified for the First Time in the United States

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


In 1994, seven unusual cases of HIV infection were identified in France
(1). These cases occurred in foreign nationals whose specimens had atypical
Western blot patterns and weak or no seroreactivity in HIV-1 peptide or
recombinant-based enzyme immunoassays (EIA). In previous cases, such
isolates had been loosely grouped as phylogenetic outliers, termed Group O
strains, highly divergent from the Group M strains that gave rise to the
HIV-1 pandemic (2). The 1994 cases were soon recognized as representing the
global emergence of genetically distinct viruses known to be circulating in
Cameroon and Gabon. This recognition prompted an immediate reassessment of
HIV screening in Europe and North America. Subtype B strains of HIV-1
predominate in Europe and North America, and there has been increasing
evidence for the entry of non-B strains into these populations through
persons who were infected while abroad. However, there was no evidence of
indigenous spread of the strains in North America (3,4).

We report here the laboratory findings related to the first documented
HIV-1 Group O infection in the United States. The case was identified in
April of this year through the CDC sentinel surveillance program for
unusual HIV variants and involves a West-Central African woman (residing in
the United States), with a 2-year clinical history of lymphadenopathy,
thrombocytopenia, and progressive CD4 lymphocyte loss. In February 1995,
the patient was tested for HIV-1-specific antibodies and was determined to
be seronegative (Abbott HIV-1/2; signal/cutoff value = 0.7) by recombinant
antigen-based EIA. The patient's serostatus was reevaluated the following
October and November and again in April of 1996 with inconclusive results
(Table 1). On these occasions, whole virus lysate-based EIAs yielded
negative or weakly positive results (signal/cutoff ratios were 0.68 to
4.20); however, confirmatory Western blot assays remained indeterminate.
The November 1995 sample was negative in both Genetic Systems formats;
however, a signal/cutoff ratio of 0.89 was noted. The Western blot pattern
remained remarkably stable through the year, and the patient's sera
remained HIV-1 antigen-negative on each sampling occasion. Attempts by
commercial reference laboratories to amplify proviral sequences from the
patient's peripheral blood mononuclear cells by standard Group M
gag-specific primers for polymerase chain reaction (PCR) or reverse
transcriptase (RT)-PCR did not show HIV sequences.

       Table 1. Results of enzyme immunoassay for antibodies to HIV
-------------------------------------------------------------------------
                                Genetic
                    Genetic     Systems      OTC     Abbott      Abbott
                    Systems     HIV-1/2     HIV-1     HIV-1     HIV-1/2
 Sample Date      HIV-1 EIAa      EIAa      EIAa      EIAa        EIAa
-------------------------------------------------------------------------
 February 1995        ND           ND        ND        ND         0.70
 October 1995 a      0.51         1.35      1.60       ND          ND

 November 1995 b     0.41         0.89      2.10       ND          ND

 April 1996 c        4.20         3.40      0.68      3.10        1.15

 a HIV-1 WB = indeterminate; detected p17, p24, p31, p50, p66, and
 equivocal gp41; HIV-1
    antigen = negative (0.40).
 b HIV-1 WB = same as above; HIV-1 antigen = negative (0.34).
 c HIV-1 WB = same as above; HIV-1 IFA = positive (2.5+).
 ND= not done.

To reconcile the serologic findings, a panel of HIV subtype--specific,
peptide-based EIAs was performed at CDC (6) (Table 2). As points of
reference, examples of results with sera representing the Group M subtype
A, B, C, D, E, and F are shown. A high degree of cross-reactivity exists
among the Group M sera, with the strongest response usually directed
against the V3 peptide from the subtype that infected the source patient.
The exception to this (Table 2) is seen in the Brazilian subtype F sera,
which react strongly with the subtype B peptide but not with the subtype F
peptide, which was derived against subtype F isolates from Romania. The
distinction between Brazilian and Romanian sera is reflected in the genetic
diversity between isolates from the two regions (7). This broad
seroreactivity among group M sera is also reflected in their response to
group-specific peptides from the gp41. However, the patient's sera were
only reactive to the Group O-specific V3 and gp41 peptides (OD = 1.704 and
2.467, respectively). This provided the first direct evidence that this
patient was infected with a Group O variant.

               Table 2. Typical peptide reactivity patterns.
-------------------------------------------------------------------------
Subtype   V3-A   V3-B   V3-C  V3-D   V3-E   V3-F   V3-O   GP41-M  GP41-O
-------------------------------------------------------------------------
A         2.742  1.144  1.628 0.065  0.083  0.724  0.055  2.683   2.627
B         0.071  1.271  0.051 0.030  0.033  0.038  0.028  2.570   2.414
C         2.320  0.084  2.515 0.079  1.647  1.637  0.061  2.746   2.858
D         0.210  0.079  0.056 1.203  0.048  0.051  0.040  1.380   0.279
E         0.055  0.043  0.385 0.042  1.291  0.038  0.025  0.619   0.340
F         0.062  0.857  0.041 0.037  0.039  0.033  0.037  2.615   2.670
O         0.089  0.088  0.089 0.188  0.088  0.081  1.704  0.070   2.457
GP41-M and GP41-O refer to peptides derived from the consensus gp41
sequences for Group M and Group O variants.
-------------------------------------------------------------------------

The virus CDC7755 was isolated by cocultivating the patient's peripheral
blood mononuclear cells with phytohemagglutinin A, which stimulated normal
human peripheral blood mononuclear cells. Viral replication was monitored
by antigen capture (Coulter SIV Core Antigen Assay, Hialeah, FL) and PCR
amplification of the viral protease gene (8). Virus growth was rapid,
highly cytolytic, and characterized by punctate syncytial formation
throughout the primary culture. All PCR amplifications were performed with
hotstart technology and nested primer sets to facilitate direct sequencing.
A consensus set of nested primers, which is generic for Group M viruses,
was first used to attempt amplification and detection of the viral
protease. This set did not detect proviral sequences during direct
evaluation of the patient's peripheral blood mononuclear cells; however, a
comparably derived consensus primer set for the Group O proteases readily
amplified proviral sequences from the patient's cells and cocultures.

For sequence comparison and phylogenetic analysis, proviral sequences
spanning the viral protease gene and portions of the p24 and gp120 gene
regions were amplified from the patient's peripheral blood mononuclear
cells and cocultures.1 Amplification of a 738-bp fragment spanning the
junction of the p24 and p17 genes of the gag region was accomplished
through a nested set of generic primers (9). Group O consensus primer sets
were derived to amplify the 297-bp viral protease gene and a 381-bp
fragment spanning the C2V3 portion of gp120 within the env gene. Sequence
identity was demonstrated between repeated patient samplings and successive
days in cocultivation for each of the gag, pol, and env elements, thus
confirming a common but unique source for the isolate. For phylogenetic
analysis, these sequences were further edited to obtain the relevant
portions of the protease gene (254 bp), p24 (419 bp), and C2V3 region (233
bp) of gp120. Phylogenetic analysis was performed by neighbor-joining and
maximum-likelihood methods (10,11).

1 Primers: gag/outer/forward - AGTACATGTTAAAACATGTAGTATGGGC; gag/outer/
reversed - CCTACTCCCTGACAGGCCGT CAGCATTTCTTC; gag/inner/forward -
AGTACATGTTAAAACATGTAGTATGGGC; gag/inner/reverse - CCTTAAGCTTTTGT
AGAATCTATCTACATA; protease/outer/forward - TTTGCCTCCCTCAAATC;
protease/outer/reverse - TTACTGGCACTG GGGCTATGG; protease/inner/ forward -
CCTCAAATCCCTCTTTG; protease/inner/reverse - TATAGGGAAGTTTAGTGTACA;
env/outer/forward - CACAGAATTTAATGGAACAGGC; env/outer/reverse - TGTGTT
ACAATAAAAGAATTCTCC; env/inner/forward - GTTACTTGTACACATGGCAT; and
env/inner/reverse - AGAATTCTCCATGACAGTTAAA.

In the phylogenetic analysis of the protease gene, we included the two
available Group O prototypes (ANT70 and MVP5180), reference strains for the
HIV-1 subtypes representing Group M variants, and HIV-2 strains (Figure
1A) [Figures not available in ASCII]. The fidelity of the phylogenetic 
results was verified by bootstrap
analysis (100 data sets) and pruning, i.e., the sequential removal of
representative strains followed by reanalysis. The segregation of CDC7755
and Group O strains was absolute and is consistent with a divergence
independent of the rooting of the Group M clades (Figure 1A) [Figures not 
available in ASCII]. For each gene
region, all resulting trees were consistent with the exclusion of the
CDC7755 isolate from the Group M family and its strong inclusion with the
Group O strains.

To better understand the evolutionary relationship between the patient s
strain and other Group O isolates, we analyzed the C2V3 region of gp120
from multiple Group O strains. This gene region exhibits a high degree of
divergence and has been sequenced for the largest number of Group O
variants (Figure 1B) [Figures not available in ASCII]. The chimpanzee 
isolate SIV-CPZ was used as an
outgroup. We took this approach to avoid possible errors introduced through
an alignment of very distant HIV-1 and HIV-2 strains. Our findings indicate
that srain CDC7755 retains a distant evolutionary relationship to the
MVP5180 isolate (Figure 1B, MVP51) [Figures not available in ASCII] 
yet represents a distinct evolutionary
lineage within Group O. The nucleotide sequence divergence among the
represented strains is 19% to 26%, values characteristic of Group O viruses
and typical of intersubtype rather than intrasubtype genetic distances
among the Group M variants. This level of diversity among the Group O
members emphasizes the potential that the limited number of O variants
yet recognized may represent a set of genetic lineages as distinct as the
Group M subtypes.

An alignment of the deduced C2V3 amino acid sequence of strain CDC7755 with
those of three Cameroonian Group O reference strains is presented in Figure
2 [Figures not available in ASCII]; the V3 loop is bound by the two cysteine 
residues (residues 45 and 83).
Unlike Group M viruses, which show greater heterogeneity in the regions
flanking the V3 loop, the Group O viruses, including the CDC7755 strain,
show substantial divergence within the loop itself. The pattern of
potential N-glycosylation sites over the C2V3 region of the CDC7755 strain
is consistent with that found among the Group O reference strains. Also of
interest is the increased number of amino acid residues in the V3 loop of
the CDC7755 strain (37 amino acids)--a feature common to other Group O
viruses but two to three residues more than most Group M viruses; however,
the significance of these differences is not clear.

CONS-O  KP TV STQ LIL N GTL SK GKI R IMG KNI SE SG KN I?V TLN ?T IN MT CER PG?
ANT70   R- -- --- --- - --- -- --- - M-A -D- L- G- -- -I- --- S- L- -- --- -QI
MVP5180 -- -- --- --- - --- -R E-- - --- --- T- -A -- -I- --- TP -- -- -I- E-I
VAU     -- -- --- --- - --- -- -N- T --- --- -D -- E- -LI --- TN -T IA --- --N
CDC7755 -- -- --H --F - --I -- --- - --V --- -S NS D- -L- --- S- -- -- -V- --N
CONS-O  ?? ?Q E?R IGP M AWY SM GLG R T?G ??? ?S RA AY CTY NAT DW ?K AL ?Q? AER
ANT70   D* I- -M- --- - --- -- -I- G -A- N** S- -- -- -K- --- -- G- I- K-T ---
MVP5180 AE V- DIY T-- - R-R -- T-K - SNN TSP R- -V -- --- -K- V- EN -- Q-T -I-
VAU     QT I- KIM A-- - --- -- A-S N -K- D** *T -- -- -N- S-- -- N- -- KNI T--
CDC7755 NS V- -M- --- L --- -- --- - gYA S** K- -I -F --- --- E- KE T- QGI ---
CONS-O  YL EL VN? NTE N VT? TF ??S S GGD AEV TH LH .. ... ... .. .. .. ... ...
ANT70   -- -- --* --G S INM -- NH- - --- L-- -- -- .. ... ... .. .. .. ... ...
MVP5180 -- N- --* Q-- - --I I- SRT - --- --- S- -- .. ... ... .. .. .. ... ...
VAU     -- -- -EY -QT D --M K- G-H - -E- --- -N FF .. ... ... .. .. .. ... ...
CDC7755 -- -- --* H-- - S-I -- KN- T --- I-- -R -- .. ... ... .. .. .. ... ...

Figure 2. Alignment of deduced amino acid sequences of the env C2V3 region
of the CDC7755 strain with those of three representative Group O
Cameroonian strains (ANT70C, MVP5180 and FR.VAU). The CONS-O represents the
consensus amino acid sequence derived from the four strains presented in
this alignment. (?) represents positions where a consensus could not be
derived. (-) indicates identical amino acids with the CONS-O and (*)
indicates gaps (insertion\deletions) that were introduced to align the
sequences.
NOTE: This table may not fit on page unless printed in landscape mode.

To date, nearly all of the Group O infections reported worldwide (fewer
than 100), have been detected in persons from West-Central Africa. This is
the first Group O case reported in the United States, and the presence of
these infections here is believed to be exceedingly low. However, the
discovery of any divergent HIV strains should be considered a sentinel
event and should prompt the reappraisal of HIV antibody tests used to
protect of the blood supply. In the United States guidelines for preventing
HIV transmission by blood and blood products include testing for HIV-1 and
-2; the refinements necessary to improve the sensitivity of these
diagnostic tests for the detection of Group O variants are known. Other
criteria for donor deferral (e.g., self-deferral for donors at high risk
for HIV infection and temporary exclusion of donors from malaria-endemic
regions) that may confer a measure of protection against divergent HIV
viruses present in equatorial Africa are in place. The identification of
Group O infections underscores the potential for the emergence of other
highly divergent HIV strains and highlights the importance of maintaining
active worldwide surveillance for these variants.

M. A. Rayfield,* P. Sullivan, C. I. Bandea,* L. Britvan, R. A. Otten,* C.
P. Pau,* D. Pieniazek,* S. Subbarao,* P. Simon, C. A. Schable,* A. C.
Wright,* J. Ward, and G. Schochetman*

*National Center for Infectious Diseases, National Center for HIV, STD and
TB Prevention, Centers for Disease Control and Prevention, Atlanta,
Georgia, USA; Kaiser Permanente Medical Group, Los Angeles, California,
USA

References

  1. Loussert-Ajaka I, Chaix M, Korber B, et al. Variablility of human
     immundeficiency virus type 1 roup O strains isolated from Cameroonian
     patients living in France. J Virol 1995;69:5640-9.
  2. Myers G, Korber B, Wain-Hobson S, Smith R, Pavlakis G., Eds. Human
     retroviruses and AIDS 1994: a compilation and analysis of nucleic acid
     and amino acid sequences. Los Alamos, NM: Los Alamos National
     Laboratory, 1993.
  3. Brodine SK, Mascola JR, Weiss PJ, et al. Detection of diverse HIV-1
     genetic subtypes in the USA. Lancet 1995;346:1198-9.
  4. Schable C, Zekeng L, Pau CP, et al. Sensitivity of United States HIV
     antibody tests for detection of HIV-1 Group O infections. Lancet
     1994;344:1333-4.
  5. Identification of HIV-1 Group O Infection  Los Angeles County,
     California, 1996. MMWR 1996;45:561-5.
  6. Pau CP, Hu DJ, Spruill C, et al. Surveillance for human
     immunodeficiency virus type 1 group O infections in the United States.
     Transfusion. 1996;36:398-400.
  7. Bandea C, Ramos A, Pieniazek D, et al. Epidemiologic and evolutionary
     relationships between Romanian and Brazilian HIV-1 subtype F strains.
     Emerging Infectious Diseases 1995;1:91-3.
  8. Pieniazek D, Janini LM, Ramos A, et al. HIV-1 patients may harbor
     viruses of different phylogentic subtypes: implications for the
     evolution of the HIV/AIDS pandemic. Emerging Infectious Diseases
     1995;1:86-8.
  9. Gao F, Yue L, Robertson DL, et al. Genetic diversity of human
     immunodeficiency virus type 2: evidence for distinct sequence subtypes
     with differences in virus biology. J Virol 1994;68:7433-47.
 10. Larsen N, Olsen GJ, Maidak BN, et al. The ribosomal database project.
     Nucleic Acids Res 1993;21:3021-3.
 11. Felsenstein J. PHYLIP-phylogeny inference package (version 3.2).
     Cladistics 1989;5:164-6.

===========================================================================
Dispatches

Two Morbilliviruses Implicated in Bottlenose Dolphin Epizootics

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


Sequence analysis was performed on viral RNA isolated from bottlenose
dolphins (Tursiops truncatus) that died during two chronologically and
geographically separate epizootics in North America. Both dolphin
morbillivirus (DMV) and porpoise morbillivirus (PMV) were detected in
bottlenose dolphins that died during the 1987 U.S. Atlantic coast
epizootic. Our results indicate not only that these viruses are not species
specific, but also that both viruses were present in North America before
outbreaks in the Mediterranean and Irish Seas. Samples taken along the
Atlantic coast showed a statistically significant trend with DMV in the
north and an increasing incidence of PMV in samples isolated farther south.
In the 1993 Gulf of Mexico epizootic, only PMV was detected in bottlenose
dolphins that died. Thus, DMV and PMV are implicated as the causes of the
earliest known aquatic mammal morbilliviral outbreak, the U.S. Atlantic
coast epidemic; PMV is implicated in the Gulf of Mexico epidemic. The
presence of two pathogenic morbilliviruses that may circulate together or
separately complicates the epidemiology of cetacean morbilliviral diseases.

The only morbilliviruses known before 1989 were human measles virus, canine
distemper virus, rinderpest, and peste-des-petits-ruminants virus (1).
Recently, newly characterized morbilliviruses have been shown to be
epizootic-associated pathogens in pinnipeds and cetaceans. Phocine
distemper virus (2) was associated with a massive epizootic of harbor seals
(Phoca vitulina) in northwestern Europe (3) in 1988. During the harbor seal
phocine distemper virus outbreak, PMV was isolated from harbor porpoises
(Phocoena phocoena) that died along the Irish coast (4). DMV (5), was
isolated during an epizootic of striped dolphins (Stenella coeruleoalba) in
the Mediterranean Sea (6,7) in 1990-92.

Between June of 1987 and May of 1988, a morbillivirus epizootic caused a
tenfold increase in bottlenose dolphin stranding along the U.S. Atlantic
coast from New Jersey to Florida (8,9). More than half of the inshore
population of bottlenose dolphins in this area may have died.
Morbillivirus-related strandings of bottlenose dolphins along the Gulf of
Mexico coasts of Alabama, Mississippi, and Texas were also observed from
October 1993 through April 1994 (10,11).

Using reverse transcriptasepolymerase chain reaction (RT-PCR), we examined
tissue lysates made from formalin-fixed paraffin-embedded lung tissue from
stranded dolphins from the Atlantic epizootic and frozen unfixed lung
tissue from the Gulf of Mexico epizootic (11,12). RT-PCR-positive cases
were subsequently characterized by sequence analysis of segments of the
morbillivirus P gene in regions with the greatest diversity between DMV and
PMV.

We sequenced segments of the morbillivirus P gene from 29 dolphin specimens
(1 striped and 28 bottlenose dolphins) from the 1987-88 Atlantic epizootic;
sequence sufficient for viral identification was obtained in 25 (86%) of 29
cases. Sequence was also obtained from 7 (37%) of 19 bottlenose dolphin
specimens stranded in Texas during the Gulf of Mexico epizootic. Because of
the advanced state of postmortem decomposition of most of these specimens,
histologic and immunophenotypic analysis was not possible (13). Data
(stranding date, location, species, and morbillivirus sequence analysis
results) for these 32 cases are shown in Table 1. In addition, P gene
sequence was obtained from formalin-fixed paraffin-embedded lung tissue
from four of four striped dolphins from the 1990 Mediterranean Sea
epizootic and from one harbor porpoise recovered off the coast of Northern
Ireland in 1988 during the harbor seal epizootic (Table 1).

Morbillivirus P gene sequencing allowed viral identification of 37 stranded
cetaceans from four geographically and chronologically separate epizootics.
In the U.S. Atlantic Coast 1987-88 epizootic, mixed infection was present
involving both recognized cetacean morbilliviruses, DMV and PMV (Table 1).
Twelve animals (including the striped dolphin) were infected with DMV, nine
with PMV, and four with both. From the later Gulf of Mexico
morbillivirus-associated epizootic, only PMV was identified. The
morbillivirus infecting striped dolphins in the Mediterranean Sea epizootic
was identified as DMV (6). All four striped dolphin specimens examined in
this study were typed as DMV infected. A harbor porpoise stranded on the
coast of Northern Ireland during the harbor seal epizootic in northwestern
Europe was typed as PMV-infected, consistent with the published analysis of
these animals (14).

                          Table 1. Case summary
-------------------------------------------------------------------------
  Date found         Stranding               Dolphin       Morbillivirus
                     by state                species         sequence(a)
-------------------------------------------------------------------------
 4 Aug. 1987 New Jersey               Tursiops truncatus   DMV
 5 Aug. 1987 New Jersey               T. truncatus         DMV
 9 Aug. 1987 New Jersey               T. truncatus         DMV
 11 Aug. 1987New Jersey               T. truncatus         DMV
 23 Aug. 1987New Jersey               T. truncatus         DMV + PMV
 3 Sep. 1987 New Jersey               Stenella coeruloalba DMV
 5 Sep. 1987 New Jersey               T. truncatus         DMV + PMV
 6 Sep. 1987 New Jersey               T. truncatus         PMV
 14 Aug. 1987Virginia                 T. truncatus         PMV
 14 Aug. 1987Virginia                 T. truncatus         DMV + PMV
 15 Aug. 1987Virginia                 T. truncatus         PMV
 26 Aug. 1987Virginia                 T. truncatus         DMV + PMV
 29 Aug. 1987Virginia                 T. truncatus         PMV
 29 Aug. 1987Virginia                 T. truncatus         DMV
 4 Sep. 1987 Virginia                 T. truncatus         DMV
 5 Oct. 1987 Virginia                 T. truncatus         DMV
 6 Oct. 1987 Virginia                 T. truncatus         DMV
 7 Oct. 1987 Virginia                 T. truncatus         DMV
 21 Dec. 1987Florida (Atlantic Coast) T. truncatus         PMV
 21 Dec. 1987Florida (Atlantic Coast) T. truncatus         PMV
 1 Jan. 1988 Florida (Atlantic Coast) T. truncatus         PMV
 18 Jan. 1988Florida (Atlantic Coast) T. truncatus         PMV
 9 Feb. 1988 Florida (Atlantic Coast) T. truncatus         DMV
 9 Feb. 1988 Florida (Atlantic Coast) T. truncatus         PMV
 10 Feb. 1988Florida (Atlantic Coast) T. truncatus         DMV
 4 Apr. 1993 Texas                    T. truncatus         PMV
 4 Apr. 1993 Texas                    T. truncatus         PMV
 4 Apr. 1993 Texas                    T. truncatus         PMV
 11 Apr. 1993Texas                    T. truncatus         PMV
 15 Apr. 1993Texas                    T. truncatus         PMV
 19 Apr. 1993Texas                    T. truncatus         PMV
 21 Apr. 1993Texas                    T. truncatus         PMV
 1991        Mediterranean Sea        S. coeruloalba       DMV
 1991        Mediterranean Sea        S. coeruloalba       DMV
 1991        Mediterranean Sea        S. coeruloalba       DMV
 1991        Mediterranean Sea        S. coeruloalba       DMV
 1988        Northern Ireland         Phocoena phocoena    PMV

 a RNA isolation, RT-PCR, and cycle sequencing were performed (11,20).
--------------------------------------------------------------------------

Partial P gene nucleotide sequence data are presented with published DMV
and PMV P gene sequences (Figure). Sequence data from the PMV-infected
cases (Atlantic epizootic cases, Gulf of Mexico epizootic cases, Irish
harbor porpoise) showed no within-group variation. Data from all cases
showed two single nucleotide changes from the published PMV sequence (12),
at positions 206 and 209 (aligned with the measles virus P gene sense
mRNA). Likewise, no sequence variation was noted for the DMV-infected
animals (Atlantic and Mediterranean epizootics). However, a two-nucleotide
inversion, at coding positions 206-207, was consistently noted when
compared to the published DMV sequence (12) (Figure). In four cases from
the Atlantic epizootic cycle sequencing indicated simultaneous infection
with DMV and PMV. To confirm that the two different sequences were present
simultaneously, PCR product was cloned, and plasmid DNA from individual
colonies was sequenced. In each of the four doubly infected cases,
approximately half the clones contained the DMV sequence, and half
contained the PMV sequence. No clones containing hybrid sequences were
found. Since PMV and DMV cause non-species-specific infections, more
appropriate designations for these viruses might be cetacean morbillivirus
1 for PMV (the first detected cetacean morbilivirus [4]) and cetacean
morbillivirus 2 for DMV.

                          176                                        215
Published DMV             ATCTGCTCCC  AGGATTAAGG  TCGAGAGATC  TGCTGACGTT
Atlantic/Mediter. DMV     ----------  ----------  ----------  GT--------
Atlantic/Gulf/Irish PMV   ----------  ----------  ----------  AT-C--T---
Published PMV             ----------  ----------  ----------  -T----T---

                          216                                      255
Published DMV             GAGACTATAA  GCAGTGAAGA  GCTACAAGGA  CTGATTAGAT
Atlantic/Mediter. DMV     ----------  ----------  ----------  ----------
Atlantic/Gulf/Irish PMV   ----------  -------G--  A--T------  -----C----
Published PMV             ----------  -------G--  A--T------  -----C----

                          256                                        295
Published DMV             CTCAGAGTCA  AAAACATAAT  GGATTTGGAG  TAGACAGATT
Atlantic/Mediter. DMV     ----------  ----------  ----------  ----------
Atlantic/Gulf/Irish PMV   ------AC--  C--G--G---  -----C----  G-----A--C
Published PMV             ------AC--  C--G--G---  -----C----  G-----A--C

                          296         313
Published DMV             CCTAAAGGTC  CCACCAAT
Atlantic/Mediter. DMV     ----------  --------
Atlantic/Gulf/Irish PMV   TT--------  --------
Published PMV             TT--------  --------

Figure. Partial nucleotide sequence of DMV and PMV morbillivirus P gene
compared with published DMV and PMV sequences (12). Primer sequences used
were 5'-ATC TGC TCC CAG GAT TAA GGT CGA-3' (forward) and 5'-CGG GAT TGG TGG
GAC CTT TA-3' (reverse). RT- PCR was performed (11). PCR products were
cycle sequenced (20) or cloned into PDK101 (21) and sequenced by using T7
Sequenase (Amersham Corporation, Arlington Heights, IL) according to the
manufacturers instructions.

Bottlenose dolphins of the U.S. Atlantic coast migrate seasonally (15). The
1987-88 epizootic moved south from New Jersey to the east coast of Florida
with the southerly fall migration. In-shore bottlenose dolphins rarely
travel around the southern tip of the Florida peninsula (Randall Wells,
pers. comm.), which explains the end of the 1987 epizootic on the east
coast of Florida in May of 1988. Sequences were obtained from dolphins
stranded during this epizootic in three U.S. states, New Jersey, Virginia,
and Florida. From the Gulf of Mexico epizootic, which occurred 5 years
later, sequence data from seven dolphins stranded in Texas were obtained. A
significant trend (chi square for trends [16], p = 0.00023) was noted among
the specimens analyzed from the New Jersey, Virginia, and Florida Atlantic
coasts in which the percentage of animals infected with DMV decreased,
while PMV infection increased (Table 2). In the 1993 die-off, 100% of the
animals from which sequence information was obtained were PMV-infected. The
epidemiologic significance of this finding is unknown. It is possible that
dolphins of the southern Atlantic coast and those in the Gulf of Mexico
were more susceptible to infection with (or had an increased death rate
from) PMV than with DMV.

 Table 2. Percentage of DM-V and PMV-infected animals by location
------------------------------------------------------------------
    Stranding location       No.        No. DMV        No. PMV
                           dolphins  infected (%)   infected (%)
------------------------------------------------------------------
 Atlantic, New Jersey         8         7 (70)a        3 (30)a

 Atlantic, Virginia           10        7 (58)a        5 (42)a

 Atlantic, Florida            7         2 (29)         5 (71)
 Gulf of Mexico, Texas        7            0           7 (100)

 a Including the four cases of simultaneous DMV and PMV infection.
------------------------------------------------------------------

To test whether enzootic morbillivirus infection could be detected in
bottlenose dolphins, RT-PCR was performed on formalin-fixed
paraffin-embedded tissue samples from 11 dolphins stranded along the U.S.
Atlantic coast from 1974 to 1985 (before the 1987 epizootic). None of the
dolphins had histologic or immunohistochemical evidence of morbillivirus
infection (9). Amplifiable RNA was obtained from samples from eight
animals, and all were negative for morbillivirus. Tissue specimens from
seven bottlenose dolphins stranded on the Florida coast of the Gulf of
Mexico in the interval between the Atlantic and Gulf of Mexico epizootics
were also examined by RT-PCR for morbillivirus. None of these animals had
histologic or immunohistochemical evidence of morbillivirus infection (data
not shown). However, one of these samples was weakly positive for the
morbillivirus P gene 78-bp product (11). Sequence information, however,
could not be obtained from this sample. This result suggests the
possibility of an enzootic infection in dolphins along the U.S. coast after
the initial 1987 morbillivirus epizootic.

Serum antibodies to canine distemper virus were detected in specimens of 6
of 13 free-ranging bottlenose dolphins during the U.S. Atlantic epizootic
in 1987 (17), indicating exposure to morbillivirus. A recent study by
Duignan, et al. (18) presented serologic evidence of morbillivirus
infection in 11 of 15 cetacean species in the western Atlantic since 1986.
Virus neutralizing titers were higher against DMV and PMV than against
peste-des-petits ruminants virus, phocine distemper virus, or canine
distemper virus. Enzootic morbillivirus infection of two species of pilot
whale (Globicephala melas and G. macrorhynchus) was recently demonstrated
in animals in the western Atlantic (19), with the earliest titer from a
pilot whale stranded in 1982. Neutralizing antibody titers in these species
were also higher against DMV and PMV, and were observed in 108 of 125
animals tested. However, confirmation of active infection by viral sequence
analysis was not performed in either of these studies. It is, therefore,
possible that the pilot whale mass strandings reported in 1982 and 1986
were morbillivirus induced, but confirmation of this hypothesis would
require histologic and/or viral sequence confirmation.

Geographically and temporally distinct morbillivirus die-offs, as well as
the evidence presented in this study that the two previously described
cetacean morbilliviruses are not species-specific, raise many questions
about the epidemiology of this family of viruses. The viruses implicated in
the 1988 European porpoise deaths and 1990 Mediterranean dolphin epizootic
were present in bottlenose dolphins of the 1987 U.S. die-off. Duignan, et
al. hypothesized that pilot whales enzootically infected with
morbilliviruses could act as long-distance vectors between America and
Europe (19). Confirmation would require sequence analysis of pilot whale
samples collected in both regions. The distribution of DMV and PMV
infection in the U.S. epizootics suggests that the later epizootic may have
been initiated by rare contact between PMV-infected Atlantic dolphins and
immunologically naive Gulf dolphins. Further investigation of serum
antibody titers, PCR evidence of enzootic infection, and sequence
identification of morbillivirus species are required for the better
understanding of these epizootics.

Acknowledgments

This study was partially supported by a grant from the Center for Marine
and Estuarine Disease Research, U.S. Environmental Protection Agency. We
thank S. Kennedy, M. Domingo, the U.S. National Marine Fisheries Service,
and the U.S. Marine Mammal Stranding Networks for providing samples.

Jeffery K. Taubenberger, M.D., Ph.D.,* Mark Tsai, M.S.,* Amy E. Krafft,
Ph.D.,* Jack H. Lichy, M.D., Ph.D.,* Ann H. Reid, M.A.,* F. Yvonne
Schulman, D.V.M., and Thomas P. Lipscomb, D.V.M.

*Division of Molecular Pathology, Department of Cellular Pathology, and
Department of Veterinary Pathology, Armed Forces Institute of
Pathology,Washington, DC 20306-6000 USA

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     bottlenose dolphins along the US central and south Atlantic coast:
     final report to National Marine Fisheries Service and U.S. Navy.
     Guelph, Canada: Office of Naval Research, and Marine Mammal
     Commission; 1989.
 18. Duignan PJ, House C, Geraci JR, Duffy N, Rima BK, Walsh MT, et al.
     Morbillivirus infections in cetaceans of the western Atlantic. Vet
     Microbiol 1995;44:241-9.
 19. Duignan PJ, House C, Geraci JR, Early G, Copland H, Walsh MT, et al.
     Morbillivirus infection in two species of pilot whales (Globicephala
     sp.) from the western Atlantic. Mar Mam Sci 1995;11:150-62.
 20. Reid AH, Tsai MM, Venzon DJ, Wright CF, Lack EE, O'Leary TJ. MDM2
     amplification, P53 mutation, and accumulation of the P53 gene product
     in malignant fibrous histiocytoma. Diagn Mol Pathol 1996;5:65-73.
 21. Kovalic D, Kwak JH, Weisblum B. General method for direct cloning of
     DNA fragments generated by the polymerase chain reaction. Nucleic
     Acids Res 1991;19:4560.


=============================================================================
Dispatches

An Outbreak of Spotted Fever Rickettsiosis in U.S. Army Troops Deployed to
Botswana

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


Novel infectious diseases are recognized when nonimmune persons move into
the ecologic niche of a pathogen and become inadvertent hosts. One
population at frequent risk to such emerging pathogens is the military.
Historical examples of this phenomenon include outbreaks of scrub typhus
during World War II and Korean hemorrhagic fever during the Korean War (1).
In this report, we describe a modern example as we document an unusually
high attack rate of a rickettsial disease among U.S. troops camping for a
brief period in Africa.

In January 1992, U.S. soldiers participated in a 10-day training exercise
with the Botswana Defense Force near Shoshong, Botswana. Activities
included individual weapons training and a series of mock battles. The
terrain was generally flat with scattered bushes and small trees. The
climate was semiarid, and temperatures reached 120F during the day. During
the field exercise, one soldier sought medical assistance to remove small
insects crawling rapidly on his body. No specimens were reliably
identified in the field or preserved for subsequent examination. No unusual
illnesses were reported to medical personnel during the training exercise.
After the field-training exercises, the soldiers spent 2 days in Gaborone,
the capital of Botswana. Within 2 days of their return to their home
station, approximately 30% of the deployed soldiers sought medical
attention with symptoms of fever, headache, and regional lymphadenitis.
Several soldiers reported insect bites. An epidemiologic team from Walter
Reed Army Institute of Research was sent to Botswana to assist with the
outbreak investigation.

From airplane manifests, 169 soldiers were identified as having been
deployed to Botswana. One week after the soldiers returned to their home
station, a questionnaire requesting information on symptoms, personal
protective measures taken against arthropod vectors, reservoir exposures,
and other potential risk factors for infection was administered to all
available soldiers (n = 140); 132 soldiers underwent directed physical
examinations, in which skin lesions, lymphadenopathy, and right upper
quadrant tenderness were noted. Clothing and equipment used in the field
that had not been cleaned since the exercise were visually examined in
detail by a team of entomologists. Blood samples from 126 soldiers were
obtained at 1 week and 10 weeks postdeployment. Serum specimens were
divided into aliquots and frozen at -20C for laboratory analyses.

Sera were tested for antibodies to rickettsiae by using an indirect
fluorescence antibody technique (IFA) (2). Sera drawn at 10 weeks
postdeployment were initially screened for whole immunoglobulin reactivity
against Rickettsia conorii Moroccan strain, a member of the spotted fever
group of rickettsiae that cause boutonneuse fever. Subsequently, sera
showing titers >=1:64 were paired with their respective 1-week samples and
titered to the endpoint, defined as the highest dilution showing
discernible fluorescing organisms. The rickettsial species used as antigens
for antibody titration included cell culturepropagated R. conorii
(Moroccan, ATCC VR141), Rickettsia akari Hartford, and Rickettsia typhi
(Wilmington, ATCC VR144). The latter two microorganisms are the etiologic
agents of rickettsial pox and murine typhus, respectively. Slides were read
by one technician who was blinded to the clinical history corresponding to
the sample.

A case-patient was defined as a soldier who had either a fourfold or
greater rise in immunoglobulin G (IgG) titer to R. conorii within 9 weeks
to a titer >=1:128 by IFA, or clinically, by the presence of a tache noire
(eschar) and at least two of the following symptoms: regional
lymphadenitis, fever, chills, severe headache, and muscle or joint pains.
By this case definition, the overall attack rate was 23% (39 of 169
soldiers). Of 36 case-patients who provided paired serum samples, 24 (67%)
seroconverted during the postdeployment period. All 24 case-patients who
seroconverted were symptomatic, but six did not have an eschar. Of the 15
case-patients without documented fourfold rises in titer, all had at least
one eschar and regional lymphadenitis observed by a physician. Three of the
soldiers who did not seroconvert, but were identified as case-patients by
the clinical definition, had high standing titers, >=1:256.

One week after returning from Botswana, the case-patients (n = 36) had a
geometric mean reciprocal (GMT) IgG titer to R. conorii of 43, which
increased to 185  9 weeks later (Student's t test paired, p < 0.0001). Over
the same period, the GMT of non-case-patients (n = 38) who had positive
titers on the initial whole immunoglobulin screening remained the same at
38. To determine whether early treatment impaired antibody production, the
case-patients were divided into two groups: those who received treatment
within 2 days of the start of their symptoms (n = 12) and those who
received treatment 3 or more days later (n = 21). The group that was
treated early had a lower mean GMT at follow-up, but the difference was not
statistically significant (early 136, 95% confidence interval [C.I.]: 77,
239: late 197, 95% C.I.: 114, 338).

A Western blot immunoassay was used (3). Rickettsial antigens (R. typhi
Wilmington, R. akari, Rickettsia africae F (4,5), R. conorii Malish (ATCC
VR613), and Israeli tick typhus rickettsiae (T487)) used in the
immunoblots, either boiled or solubilized at room temperature, were
electrophoresed on an 8% to 16% gradient PAGE gel. Renografin density
gradient purified antigen was applied at 20 g protein per lane. R. typhi
was yolk sacpropagated, whereas spotted fever group antigens were grown in
irradiated L cells. Patients' serum specimens were reacted with the
electrophoresed antigens at 1:250 dilution. Horseradish peroxidaselabeled
oat antibodies against human IgG (gamma-chain-specific) and IgM (
chain-specific) (Calbiochem, San Diego, CA) were used at 1:1000 dilution,
and bound antibody was detected with 4-Cl-1-naphthol-H2O2 reagent.
Prestained SDS-PAGE molecular weight standards (Bio-Rad, Melville, NY) were
used to estimate antigen sizes

Western blots were used to characterize the serologic reactivities of 38
case-patients and 37 noncase-patients. Serum specimens were considered
positive for reactivity with spotted fever rickettsial antigens if a
characteristic broad washboard of spotted fever group lipopolysaccharide
reactivity (SFG LPS) was observed in the 20- to 40-kDa region with anti-IgG
conjugate (Figure 1) [Figures not available in ASCII]. 
Most of these serum specimens also exhibited
pronounced IgM reactivity, which sometimes included R. typhi LPS. Serologic
specificity toward the individual spotted fever species was determined by
the relative IgG reactivity to species-specific antigens (SPAs) solubilized
at room temperature found at 125 to 145 kDa. Thirty-two of the 38
case-patient specimens tested by Western blot were positive for spotted
fever group IgG LPS.

On Western blot, the sera of 23 case-patients reacted most strongly with R.
africae SPA (not shown), one with R. conorii SPA, and eight showed no
specificity (Figure 1)[Figures not available in ASCII]. Nine of 37 soldiers 
who did not meet our case
definition were positive for spotted fever group IgG LPS. The specimens of
two reacted primarily with R. africae SPA, and specimens from the remaining
seven had nonspecific patterns. Five of these nine soldiers reported no
symptoms. Two soldiers reported a runny nose, another reported a rash on
the dorsal portion of the right upper arm, and another had a tender raised
papule. By Western blot criteria, these nine soldiers would be considered
infected and may represent asymptomatic cases. However, they did not meet
our case definition and were not counted as case-patients in our analyses.

Patients' sera showed expected broad cross-reactivity with other spotted
fever group rickettsiae by IFA. In sera collected at 10 weeks, 17 (51%) of
the case-patients had identical R. akari and R. conorii titers; a
difference of one dilution in titers was observed in 12 (36%) of the
case-patients. The remaining cases had R. akari titers that were fourfold
less than their R. conorii titer. However, no Western blots were positive
for R. akari. All but two 10-week serum samples from the case-patients were
nonreactive at a titer of <1:32 with R. typhi antigen by IFA, and none were
positive by Western blot. Serum from one case-patient had titers of 1:128
to R. akari, R. typhi, and R. conorii. Serum from the second case-patient
had titers of 1:512 to R. conorii and 1:64 to R. typhi.

Median ages of the case-patients and noncase-patients were 23.5 and 22
years, respectively. Although a larger proportion of cases was found among
the older age groups, this trend was not significant (chi-squared trend, p =
0.28). Most of the soldiers (77%) were non-Hispanic white. There were too
few soldiers in other race-ethnic groups to make separate comparisons, but
when those from other ethnic backgrounds were combined into one group, the
group had an attack rate (22%) similar to that of the non-Hispanic white
soldiers (24%).

The illness observed in the soldiers was mild to moderately severe.
Although all cases were symptomatic, some came to medical attention only
through active case finding. Many case-patients had systemic complaints,
commonly described as flu-like. Each of the classic symptoms associated
with rickettsial diseases (i.e., fever, chills, headache, myalgia, and
arthralgia) was reported by at least 59% of the case-patients but by fewer
than 10% of the noncase-patients (Table 1). Gastrointestinal complaints of
stomach pain, anorexia, nausea, or vomiting were also reported more
frequently by the case-patients than by the noncase-patients. In contrast,
diarrhea and upper respiratory symptoms were reported at similar rates
among case-patients and noncase-patients. On physical examination,
lympadenitis was observed in 36 (92%) and right upper quadrant tenderness
in 17 (44%) of the case-patients. The skin lesions began as a tender
erythematous papule, which progressed over 24 to 36 hours to form one to
three clear vesicles. The fragile vesicles ruptured, exposing an
erythematous base, which darkened into a small eschar. The total duration
of the evolution was 72 to 96 hours; 34 (87%) of case-patients had at least
one eschar, and 10 of the case-patients had more than one. Of all eschars
reported during physical examinations, 50% were located on the trunk;
others were located on the head, neck, extremities, penis, and scrotum. No
soldiers were hospitalized, and all case-patients responded rapidly to
antibiotic treatment.

   Table 1. Symptoms of case-patients and noncase-patients during a
                        spotted fever outbreak.
----------------------------------------------------------------------
                 Casepatients; Noncase-patients; Risk 95% Confidence
 Symptoms            n=32(%)         n=107(%)     ratio    interval
----------------------------------------------------------------------
 Fever               19 (59)          8 (7)        6.06   3.44,10.68
 Chills              25 (78)          6 (6)       12.44   5.96,26.00
 Headache            23 (72)          8 (7)        8.90   4.61,17.21
 Muscle ache         26 (81)          10 (9)      12.40   5.56,27.66
 Joint pain          21 (66)          9 (8)        6.94   3.78,12.73
 Fatigue             27 (84)          6 (6)       17.35   7.26,41.43
 Lymphadenitis       27 (84)          3 (3)       19.62   8.27,46.57
 Abdominal pain      9 (28)           4 (4)        3.79    2.26,6.36
 Anorexia            11 (34)          3 (3)        4.68    2.90,7.53
 Nausea/vomiting     13 (41)          3 (3)        5.26    3.27,8.46
 Diarrhea            7 (22)          21 (20)       1.11    0.54,2.30
 Cough               4 (12)           9 (8)        1.38    0.58,3.33
 Nasal congestion    7 (22)          17 (16)       1.34    0.66,2.74
 Sore throat         6 (19)          16 (15)       1.23    0.57,2.63
----------------------------------------------------------------------

An incubation period of up to 6 days was estimated from the histories of
the first two case-patients (Figure 2) [Figures not available in ASCII]. 
The first case-patient reported
onset of symptoms on January 20, 6 days after he arrived in Botswana as a
member of an advance team. The second case-patient arrived in Botswana on
January 18 and had onset of symptoms 6 days later. Thus, assuming a
Botswana exposure, the time from infection to symptoms in these two
case-patients could not have exceeded 6 days, an incubation time consistent
with the 5- to 7-day period commonly reported for African spotted fever
rickettsioses. Finally, since most case-patients reported symptoms during
the 3 days before or the 4 days after leaving Botswana, we concluded that
the period of high risk exposures occurred during field training in
Shoshong and not in Gaborone.

No significant differences were found between case-patients and others in
reported exposure to vectors, such as mosquitoes (68% vs. 62%; chi-squared, p =
0.53) and chiggers (19% vs 10%; chi-squared, p = 0.14). However, case-patients
(68%) were more likely than noncase-patients (40%) to report tick bites (
chi-squared, p = 0.007). Only one soldier reported finding an engorged tick on
his body. Fewer than 50% of all deployed soldiers reported using a
DEET-containing insect repellent at any time during the exercise, and fewer
than 10% of the soldiers reported using permethrin (anacaricide and
repellent applied to clothing). No statistical differences were found
between case-patients and noncase patients in reported uses of these
personal protective measures. Self-reported exposures of case-patients (n =
31) vs non-case-patients (n = 101) to potential reservoirs of R. conorii
(dogs: 52% vs 56%; rodents: 10% vs 35%; antelopes: 3% vs 7%; cattle: 81% vs
87%; horses:16% vs 25%; sheep: 42% vs 38%; and goats: 90% vs 87%) were not
statistically different.

More than 85% (n = 117) of the soldiers slept on sleeping pads placed
directly on the ground. Few (n = 18) soldiers reported sleeping on cots,
litters, hammocks, or pallets. The attack rate was approximately twice as
high among men sleeping on the ground (25%) as among those who did not
(11%). Because of the small number who slept off the ground, this
difference, was not statistically significant (risk ratio = 2.23; 95% C.I.:
0.58, 8.56). The attack rate among soldiers who maneuvered in the field
during daily training exercises was lower than the rate among soldiers who
remained at base camp (24% vs. 33%, respectively), but the difference was
also not statistically significant (odds ratio = 1.40, 95% C.I.: 0.77,
2.56).

This report documents the occurrence of a large focal outbreak of a spotted
fever group rickettsiosis among American soldiers participating in a short
field-training exercise in Botswana. Little is known about the epidemiology
of rickettsial diseases in Botswana. However, during the civil unrest that
occurred in neighboring Rhodesia (now Zimbabwe) in the late 1970s, several
thousand cases of tick typhus were reported among military personnel. In
Zimbabwe, ticks removed from 100 soldiers were all larvae of Amblyomma
hebreum (6). In another study, 33% to 75% of A. hebraeum ticks, collected
from animals or vegetation in four regions of Zimbabwe, were heavily
infested with rickettsia-like organisms (7). Only the recently
characterized new rickettsia R. africae, but not R. conorii, the etiologic
agent of boutonneuse fever, was detected in these ticks (4,7). In contrast,
the dog ticks Rhipicephalus simus and Haemaphysalis leachi were less
heavily infected (0% to 21%) and were only infected with R. conorii. R.
africae has also been isolated from a hospitalized patient with fever,
severe headache, and regional lymphadenopathy but no rash other than an
erythematous tick bite site (8).

On the other hand, in his original 1934 clinical description of African
tick bite fever, Pijper (9) described a patient with a rash and eschar,
whose condition he carefully distinguished from boutonneuse fever. He
clearly associated this milder disease with the hardly visible swarming
larval veld ticks (little pests) and not dog ticks. He also noted that
even if the disease was not severe, it needed to be recognized by
physicians and not confused with other potentially more devastating
illnesses requiring more aggressive therapy.

Recent surveys of human antibodies in eight African countries have
suggested a seroprevalence of antibodies to spotted fever rickettsiae of 0%
to 52%, which parallels the distribution of Amblyomma ticks (10,11). The
etiologic agent appeared to be R. africae, based on Western blot analysis
(10). IFA analysis has been found inadequate in distinguishing human
infections with R. africae and R. conorii (11).

The collective observations parallel our clinical, epidemiologic, and
serologic findings on the Botswana outbreak and are most consistent with
disease caused by R. africae. R. africae is closely related to R. conorii
but has been proposed as a new species (5). Whether we consider this a
newly emerging rickettsiosis or, in deference to the pioneering work of
Pijper, a disease reemerging into our consciousness, African tick bite
fever caused by R. africae may be epidemiologically distinct from urban R.
conorii infections.

We have established the potential for high attack rates of this spotted
fever rickettsiosis, but much more needs to be understood about the
clinical severity of this disease and its potential for asymptomatic
infections. Physically fit, healthy, young soldiers may not represent a
particularly good model for assessing the severity of disease in
populations residing in areas where the disease is endemic.

Bonnie L. Smoak, M.D., Ph.D.*; J. Bruce McClain, M.D.; John F. Brundage,
M.D.*; Laurel Broadhurst, M.D.*; Daryl J. Kelly, Ph.D.; Gregory A. Dasch,
Ph.D.; and Richard N. Miller, M.D.*

*Divisions of Preventive Medicine and Communicable Diseases and
Immunology, Walter Reed Army Institute of Research, Washington, D.C., USA;
U.S. Army MEDDAC, Vicenza, Italy; Viral and Rickettsial Diseases Program,
U.S. Naval Medical Research Institute, Bethesda, Maryland, USA

Acknowledgment

This investigation was supported in part by the Naval Medical Research and
Development Command, Research Task No. 61102A.010BJX.1293.

References

  1. Institute of Medicine. Emerging infections: microbial threats to
     health in the United States. Washington, DC: National Academy Press,
     1992:110-2.
  2. Philip RN, Casper EA, Burgdorfer W, et al.. Serologic typing of
     rickettsiae of the spotted fever group by microimmunofluorescence. J
     Clin Microbiol 1978;3:51-61.
  3. Raoult D, Dasch GA. Line and Western blot immunoassays for diagnosis
     of Mediterranean spotted fever. J Clin Microbiol 1989;27:2073-9.
  4. Kelly PJ, Beati L, Matthewman LA, Mason PR, Dasch GA, Raoult D. A new
     pathogenic spotted fever group rickettsia from Africa. J Trop Med Hyg
     1994;97:129-37.
  5. Kelly PJ, Beati L, Mason PR, Matthewman LA, Roux V, Raoult D.
     Rickettsia africae sp. nov., the etiological agent of African tick
     bite fever. Int J Syst Bacteriol 1996;46:611-4.
  6. Yunker CE, Norval RAI. Observations on African tick typhus (tick-bite
     fever) in Zimbabwe. In: Fivaz B, Petney T, Horak I, editors. Tick
     vector biology: medical and veterinary aspects. Berlin: Springer
     Verlag 1989:143-7.
  7. Beati L, Kelly PJ, Mattewman LA, Mason PR, Raoult D. Prevalence of
     rickettsia-like organisms and spotted fever group rickettsiae in ticks
     (Acari: Ixodidae) form Zimbabwe. J Med Entomol 1995;32:787-92.
  8. Kelly P, Mattewman L, Beati L, Raoult D, Mason P, Dreary M, et al.
     African tickbite fever: a new spotted fever group rickettsiosis under
     an old name. Lancet 1992;340:982-3.
  9. Pijper A. Tickbite fever: a clinical lecture. SA Med J 1934;8:551-6.
 10. Dupont HT, Brouqui P, Faugere B, Raoult D. Prevalence of antibodies to
     Coxiella burnetii, Rickettsia conorii, and Rickettsia typhi in seven
     African countries. Clin Infect Dis 1995;21:1126-33.
 11. Kelly PJ, Mason PR, Matthewman LA, Raoult D. Seroepidemiology of
     spotted fever group rickettsial infections in Zimbabwe. J Trop Med Hyg
     1991;94:304-9.


============================================================================
Dispatches

A Highly Heterogeneous HIV-1 Epidemic in the Central African Republic

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


The Central African Republic has been strongly affected by the human
immunodeficiency virus (HIV) epidemic. Although itself sparsely populated,
the republic borders five other African countries and is crossed by the
trans-African highway, which connects West and Central Africa with East
Africa (Figure 1). Wide genetic variation reported among HIV-1 subtypes (on
the basis of small numbers of samples) suggests multiple introductions of
HIV into the republic (1). However, except in small studies and unpublished
reports (2), the distribution and serologic reactivities of HIV within the
Central African Republic have not been characterized.

The need to conduct HIV surveillance has also been highlighted by reports
of highly divergent strains of HIV-1 group O from neighboring Cameroon
(3,4) and by recent findings that some of these strains were not reliably
detected by current antibody screening tests (5-7). To assess the
prevalence and distribution of HIV in the Central African Republic, the
local Ministry of Health AIDS Program, assisted by the Centers for Disease
Control and Prevention (CDC), initiated a nationwide sentinel surveillance
survey in late 1994. Sentinel surveillance has been useful for obtaining
information on HIV infection (8). In particular, serial sentinel studies
have permitted the spread of HIV to be monitored and trends to be followed
over time. In this report, we present findings on the distribution of HIV-1
infections and serologic reactivities of HIV in the Central African
Republic.

From October through December 1994, a nationwide sentinel surveillance
survey was conducted in 10 cities and towns: Bambari, Bangassou, Bangui,
Berberati, Bossangoa, Bozoum, Bria, Gamboula, MBaiki, and Mobaye (Figure
1) [Figures not available in ASCII]. The two primary population groups 
included were women attending
prenatal clinics and male and female attendees at sexually transmitted
disease (STD) clinics. Two additional sites in the capital city, Bangui,
were included to perform testing for laborers and university students.

Serum samples, anonymous and without any personal identifiers, were
collected and transported to the National Public Health Laboratory in
Bangui. Testing for this study was supported by CDC, which used U.S. Public
Health Service guidelines for confirmation (9). All sera were tested with
the Genetic Systems HIV-1/2-enzyme immunoassay (EIA) kit and all repeat
positive sera were confirmed by HIV-1 Western blot (Cambridge Biotech).
Specimens positive by initial EIA but negative for HIV-1 by Western blot or
indeterminate were further tested for HIV-2 antibodies by an HIV-2 Western
blot (Cambridge Biotech). In addition, samples from 11 sites (with
sufficient quantity) that were positive by initial EIA were serotyped by
using peptides representing the known HIV-1 subtypes A-F and the divergent
HIV-1 group O (10,11).

A total of 2,259 persons were tested from 17 sites from 10 cities and
towns. Between 2.7% and 30.7%, by site, were positive for HIV-1 by repeat
EIA and Western blot confirmation (Table). A higher HIV-1 prevalence (25.3%
to 30.7%) was observed among STD clinic attendees, whereas the prevalence
among women at prenatal care clinics was generally >5% and as high as 16.7%
(the exception was the lower rate in women from the prenatal care clinic in
Gamboula. No HIV-2 infection was detected among the 175 persons whose serum
was initially positive by EIA but negative for HIV-1 by Western blot or
indeterminate by Western blot for HIV-2.

Among 247 samples serotyped by peptide EIA, 173 were HIV-1 positive by
Western blot, 60 were indeterminate, and 14 were HIV-1 negative (Figure 2)
[Figures not available in ASCII].
No divergent HIV-1 group O infection was found among these persons.
Serotype C (24%) was most prevalent among the monoreactive specimens. Many
of the specimens were reactive to multiple peptides; 16 (9%) did not
recognize any of the peptides and may represent more divergent HIV-1
strains.

The high seroprevalences (Table) indicate that HIV-1 infection is a serious
public health problem in a number of groups in the Central African
Republic. The wide variation of HIV-1 prevalence even within similar groups
in a region illustrates the heterogeneous nature of this epidemic. The
HIV-1 prevalence was especially high among STD clinic attendees. Although
the number of specimens from STD clinic attendess was relatively small, the
high proportion of HIV-infected persons at all four STD clinics is a cause
for concern. Because Bangui and Bambari are major crossroads for transport
in the Central African Republic as well as on the east-west highway to
other countries, the potential for further HIV spread is great.

       Table. Prevalence of HIV-1 by sentinel
                  site/population
-----------------------------------------------------
                              Positive by
 Sentinel site/Population  Western blot (%)  Total
-----------------------------------------------------
 Bambari

  Prenatal clinic women        20 (13.3)       150

  STD clinic attendees         23 (26.7)        86

 Bangassou

  Prenatal clinic women        16 (10.0)      160

 Bangui

  Prenatal clinic women A      20 (13.3)       150

  Prenatal clinic women B      8 ( 5.3)        150

  STD clinic attendees         20 (25.3)        79

  University students          14 ( 9.3)       150

  Workers                      23 (15.4)       149

 Berberati

  Prenatal clinic women        16 (10.7)       150

 Bossangoa

  Prenatal clinic women        9 ( 6.0)        148

 Bozoum

  Prenatal clinic women        20 (13.2)       151

  STD attendees                23 (30.7)        75

 Bria

  Prenatal clinic women        22 (16.7)       132

  STD attendees                24 (30.4)        79

 Gamboula

  Prenatal clinic women        4 ( 2.7)        150

 MBaiki

  Prenatal clinic women        8 ( 5.3)        150

 Mobaye

  Prenatal clinic women        9 ( 6.0)        150

 Total number tested                         2,259
-------------------------------------------------------

Since STDs are associated with (and may facilitate) HIV transmission (12),
STD diagnosis and control are integral components of HIV prevention. CDC,
with financial support from the U.S. Agency for International Development,
recently helped establish two pilot STD treatment sites in Bambari and
Bria.

Although HIV-2, which is endemic to West Africa, has not been reported from
the Central African Republic, reports of HIV-1 Group O from neighboring
Cameroon highlighted the importance of monitoring these strains. Although
no HIV-2 or divergent HIV-1 group O infections were found in this study,
the extremely wide pattern of serologic reactivity among HIV-1-infected
persons suggests a very heterogenous distribution of HIV-1 strains in the
republic. This is confirmed by the wide variety of genotypes (subtypes)
found in the republic in the past (1), and suggests that in certain
populations, at least, multiple subtypes of HIV-1 have been introduced over
time (7). This finding may be important to the development of effective
future HIV vaccines for use in this region.

Since the quantity of specimens collected in this large-scale survey was
not sufficient for genetic analysis, further studies are needed to
characterize the genetic diversity of HIV in the Central African Republic.
Given the limited search for HIV variants and the diversity of HIV subtypes
recognized thus far, both a wider distribution of the HIV strains and the
existence of yet more divergent variants seem likely (7). For these
reasons, CDC plans to assist the Republics National AIDS Control Program
with further characterization of virus for divergent subtypes; these
findings indicate the need for more resources to help the program maintain
and expand HIV and STD prevention.

Acknowledgments

We thank Jacob Ngaba, Harold Jaffe, Grard Grezenguet, Abdoulaye Kozemaka,
David Gittelman, Lucienne Gaba, Franoise Jabot, David Espey, Benoit Soro,
Robert Gribbin, Samuel Laeuchli, and the staff of the American Embassy,
Bangui, Central African Republic.

Marcel Massanga,* Justin Ndoyo,* Dale J. Hu, Chou-Pong Pau, Stephanie
Lee-Thomas, Reginald Hawkins, Dominique Senekian,* Mark A. Rayfield, J.
Richard George, Amde Zengais,* Noel Ngalla Yatere,* Victor Yossangang,*
Aliou Samori,* Gerald Schochetman, and Timothy J. Dondero

*Ministre de la Sant Publique et de la Population, Central African
Republic; Centers for Disease Control and Prevention, Atlanta, Georgia,
USA

References

  1. Murphy E, Korber B, Georges-Courbot MC, You B, Pinter A, Cook D, et
     al. Diversity of V3 region sequences of human immunodeficiency viruses
     type 1 from the Central African Republic. AIDS Res Hum Retroviruses
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=============================================================================

Dispatches

Legionella-Like Amebal PathogensPhylogenetic Status
and Possible Role in Respiratory Disease

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


Legionella-like amebal pathogens (LLAPs) are nonculturable, protozoonotic,
gram-negative bacilli. They were named LLAPs because of their ability to
infect and multiply intracellularly within amebae (Figure 1) in the same
way that legionellae infect and multiply in protozoa and human alveolar
macrophages (1-3). However, unlike other known legionellae, LLAPs do not
grow routinely on buffered charcoal yeast extract agar (BCYE), or on any
other known bacteriologic media.

[Figure 1]
Figure 1. Transmission electron micrograph of Sarcobium lyticum within
Acanthamoeba castellanii. Bar = 1 m. (Reproduced with permission from
reference 7.)

The first known LLAP was isolated from Polish soil by Drozanski in 1954
(4). Until 1991 when it was described and given the name Sarcobium lyticum
(5), it was simply referred to as an obligate intracellular bacterial
parasite of free-living amebae (6, 7). The next isolation of an LLAP was in
England more than 20 years later. Since then, LLAPs have been commonly
isolated from various sources and found to infect a variety of amebae
(Table 1). Because one LLAP cannot be differentiated from another, each
strain is given the name LLAP suffixed by a designated number. Except for
S. lyticum, LLAP-1, LLAP-5, and LLAP-3, which was a clinical isolate, all
LLAPs were originally isolated from sources associated with confirmed cases
or outbreaks of Legionnaires' disease (Table 1).

         Table 1. Source of Legionella-like amebal pathogen (LLAP)
     Strain      Host     Temp(C)          Original source          Year
 S. lyticum    AP**     35          Soil isolatea                   1954
 LLAP-1        AP       30N35       Tank of portable water well     1981
 LLAP-2        AP**     35          Garage steam cleaning pit       1986
 LLAP-3        AP1      35          Sputum from pneumonia patient   1986
 LLAP-4        AP       30N35       Hospital whirlpool bath         1986
 LLAP-5LST     AP       30          Nursing home plant spray        1988
 LLAP-6        AP**     35          Factory liquefier tower         1988
 LLAP-7        AP1**    35          Hotel whirlpool spa             1991
 LLAP-8        HVNAP    35          Hospital shower                 1990
 LLAP-9        AP1**    35          Factory cooling water           1992
 LLAP-10       AP1      35          Ship air-conditioning system    1994
 LLAP-11       AP       30          Furnace cooling system          1993
 LLAP-12       AP       30          Furnace cooling system          1994
 a = first LLAP isolate. AP = infected Acanthamoeba polyphaga isolated from
 original material. AP1 = isolated after coculitivation of sample with A.
 polyphaga. HV = Hartmannella vermiformis isolated from original material.
 ** = also multiplies in H. vermiformis. NAP = does not infect A.
 polyphaga. LST = lost strain. N35 = does not multiply at 35C.

Amebae are natural hosts for legionellae in the environment (2) and have
been used to isolate legionellae from clinical samples (8). The
relationship between these organisms is unique in that amebae, which
generally use other bacteria as food, are parasitized by legionellae. The
mechanism by which legionellae multiply and evade host defenses in amebae
simulates the mechanism of infection in humans (9,10). The ability of
Legionella pneumophila to infect epithelial cells in vitro is also enhanced
by prior cultivation of the bacteria in amebae (11). This suggests that
there may be a connection between the pathogenicity of legionellae for
mammalian cells and animal models and their pathogenicity for amebae.

Whereas members of the genus Legionella, particularly L. pneumophila, are
recognized as important etiologic agents of pneumonia (12), little is known
about the clinical relevance of LLAPs; nevertheless, some evidence suggests
that they may be an unrecognized and possibly significant cause of
respiratory disease. LLAPs inability to grow or multiply in the absence of
amebae makes it difficult to isolate or identify them in clinical samples
by conventional techniques. No serologic reagents are currently available
for the detection or identification of LLAPs in clinical specimens. It is
particularly noteworthy that one LLAP strain was originally isolated by
amebal enrichment of a sputum specimen from a patient with pneumonia (13).
Clinical samples from the patient were culture and serologically negative
for L. pneumophila serogroups 1 through 6. However, incubation of the
sputum sample with Acanthamoeba polyphaga resulted in numerous
bacteria-infected amebae. The patient demonstrated a fourfold rise in
antibody titer to the bacteria from the infected amebae. Even though the
bacteria, now known as LLAP-3, could not be recovered on BCYE, the patient
was treated for Legionella infection and recovered (13). Attempts to
culture LLAP-3 on BCYE or on any other artificial media have been
unsuccessful.

Since the discovery of LLAP-3, there has been further serologic evidence
for the involvement of LLAPs in pneumonia. Between 1989 and 1993, the
Public Health Laboratory (Leeds, UK) routinely screened sera from patients
with suspected Legionnaires' disease for antibodies to LLAP-3 and found
more than 10 cases with appropriately timed rising titers and several
convalescent-phase sera. A few cases of respiratory disease with rising
titers to LLAP-1 and little activity to LLAP-3 were also found (13).

In a study to assess the role of LLAPs in community-acquired pneumonia, 19%
of 500 hospitalized patients with pneumonia of unidentified origin
demonstrated a >= fourfold rise in antibody titer to 1:128 to at least one
of nine LLAPs tested (14). These patients had no evidence of infection with
known Legionella species (15). Cross-reactivity or nonspecific antibody
rises may have accounted for some of the cases. Nevertheless, these
findings are particularly important since no etiologic agent is identified
for an estimated 50% of the 500,000 adult pneumonia cases occurring
annually in the United States (16-18).

With the exception of S. lyticum, LLAPs have not been named or classified
because the inability to cultivate them on laboratory media has limited
their genetic and biochemical characterization. In this study, LLAPs were
cocultured with Hartmannella vermiformis and Acanthamoeba polyphaga,
depending on the most suitable host for each LLAP. Samples were cultured on
blood agar and BCYE without cysteine to rule out bacterial contaminants.
Bacteria were separated from amebae by differential centrifugation of
cocultures before isolation of genomic DNA for ribosomal DNA sequencing.

The genes that code for rRNA in bacteria are highly conserved and can be
used to establish the phylogenetic relationship between organisms (19).
Previous 16S rDNA analysis of S. lyticum (20) and LLAP-3 (21), showed that
they are closely related (91.6% to 95.8% rRNA similarity) to members of the
genus Legionella. It was, therefore, suggested that LLAPs may have been
responsible for the Gen-Probe positive specimens from patients with
pneumonic illness, for whom conventional culture and serologic tests for
legionellae were negative (13,22,23). The Gen-Probe system is a rapid
diagnostic test for legionellae that uses a DNA probe complementary to the
rRNAs of Legionella species (23,24).

To establish the degree of relatedness of all known LLAPs to each other and
to other members of the family Legionellaceae, we recently amplified the
16S rRNA gene from five LLAP strains (LLAP-1, LLAP-2, LLAP-7, LLAP-8, and
LLAP-9), using primers specific for Eubacteria. The amplified DNA was
sequenced and compared with other bacterial sequences (including those of
all Legionella species and LLAPs) held by GenBank/EMBL. Analysis of the 16S
rRNA gene sequences showed that all 12 LLAPs in this study are closely
related (91.2% to 97.0% similarity over 1303 bases of 16S rRNA sequence) to
members of the genus Legionella. This is consistent with the 16S rRNA
sequence identity (90.2% to 99.1% over 1303 bases) that exists between the
39 validly described members of the genus1

1Genomic DNA was isolated by standard methods and extracted with
phenol/chloroform. A 1400-bp 16S rDNA segment was amplified by using
primers specific for Eubacteria and based on a consensus of Legionella 16S
rRNA sequences. Each strain was sequenced at least 3 times to ascertain
accuracy of obtained results. 16S rRNA sequences for all Legionella
species, those of some previously sequenced LLAPs and Coxiella burnetii
were obtained from GenBank/EMBL and aligned with sequences generated in
this study, using the GCG sequence analysis package (version 8.0).
Comparisons were performed at NCBI using the BLAST network service (32).
Ambiguous and hypervariable regions were removed before phylogenetic
analysis, which was carried out on 53 strains for 1303 nucleotides by the
neighbor-joining method from the Phylogeny Inference Package (Phylip) (33),
version 3.5. Multiple datasets (X100) were analyzed, and different distance
models were compared to ensure reliability. Some of the 16S rRNA sequences
included in the data analysis are of unpublished and undescribed stains.

Accession numbers for sequences included in this study are as follows:
LLAP-1 U64034, LLAP-2 U44909, LLAP-3 X60080, LLAP-4 X97357, LLAP-6 X97357,
LLAP-7 U44910, LLAP-8 U64035, LLAP- 9 U44911, LLAP-10 X97363, LLAP-11
X97362, LLAP-12 X97366. L. adelaidensis Z49716, L.anisa X733394, L.
birminghamensis Z49717, L. brunensis X73403, L. cherrii X73404, L.
cincinnatiensis X73407, Coxiella burnetii M21291, L. donaldsonii Z49724,
L.dumoffii X73405, L.erythra Z32638, L. fairfieldensis Z49722, L. feelii
X73395, L. geestiana Z49723, L. gormanii Z32639, L. gratiana Z49725, L.
hackeliae M36028, L. israelensis X73408, L. jamestowniensis X73409, L.
jordanis X73396, L. lansingensis Z49727, L. londiniensis Z49728, L.
longbeachae M36029, L. maceachernii X60081, L. micdadei M36032, L. moravica
Z49729, L. nautarum Z49730, L. oakridgensis X73397, L. parisiensis Z49731,
L. pneumophila M59157, L. quateirensis Z49732, L. worsliensis Z49739, L.
quinlivanii Z49733, L. rubrilucens X73398, L. santicrucis Z49735, Sarcobium
lyticum X66835, L. shakespearei Z49736, L. spiritensis M36030, L.
steigerwaltii X73400, L. sainthelensi Z49734, L. tusconensis Z32644, L.
wadsworthii X73401.

Phylogenetic analysis showed that all the LLAPs in this study form a
coherent cluster with other members of the family Legionellaceae (Figure
2). LLAP-1, LLAP-8, and LLAP-10 were grouped in distinctly different
clades, whereas LLAP-2, LLAP-3, LLAP-6, LLAP-7, LLAP-9, and S. lyticum
formed a single well-defined clade, most closely related (>96.9%
similarity) to another clade consisting of LLAP-4, LLAP-11, and LLAP-12
(Figure 2). LLAP-2, LLAP-3, LLAP-6, LLAP-7, LLAP-9, and S. lyticum shared a
16S rRNA sequence similarity of >98.9%. LLAP-11 and LLAP-12 had 16S rRNA
sequence homology of 100%. Overall 16S rRNA sequence similarity values from
some Legionella species, all the LLAPs in this study, and Coxiella burnetii
are shown in Table 2. These results suggest that these 12 LLAP strains may
represent up to five new species of Legionella and their various subtypes
on the basis of a minimum sequence identity of 97.0% between strains of the
same species (Table 3). However, they may include new subtypes of
previously described species. It is also possible that these strains
represent more than five species because, even though 16S rRNA sequence
identity can be used routinely to distinguish and establish relationships
between genera and well-resolved species, it may not be sufficient to
guarantee species identity (25,26). For example, in this study, several
Legionella species had sequence similarities exceeding 98.0%. L. cherrii
and L. wadsworthii shared a sequence similarity of 98.7%. L. rubrilucens
and L. erythra had a sequence identity of 99.1%. L. sainthelensi and L.
cincinnatiensis were likewise identical by 99.1%. Furthermore, bacterial
strains exhibiting more than 99.5% rRNA sequence identity may belong to
different species by current standards for DNA-DNA hybridization (70% DNA
reassociation) (25). DNA hybridization studies are, therefore, needed to
determine species identity.

   Table 2. 16S rRNA similarities (%) between members of the genus Legionella and different LLAP
                                             strains.
 L1  L2  L3   L4   SL  L6   L7  L8   L9  L10  L11  L12  Lpn  Lfl Lgr  Lts  Lpr Lst  Ldm Lwd   Cox
 L1 94.1 93.693.9 93.7 94.093.7 94.1 93.794.4 94.194.1  93.8 96.693.7 94.0 93.994.3 94.194.0 85.4
 L2      98.996.9 99.2 99.899.4 95.4 99.396.6 97.497.4  95.8 94.596.2 96.3 96.396.7 96.896.3 85.6
 L3          96.9 99.4 99.099.5 94.9 99.596.3 97.097.0  95.8 93.995.7 96.2 96.196.5 96.496.0 85.6
 L4               97.0 97.097.2 95.1 97.196.0 98.598.5  95.5 93.495.5 95.9 95.996.0 95.996.0 85.2
 SL                    99.199.9 94.9 99.896.3 97.297.2  95.8 94.195.5 96.1 95.996.4 96.396.0 85.6
 L6                        99.3 95.1 99.296.8 97.397.3  95.6 94.496.1 96.4 96.396.5 96.596.1 85.5
 L7                             95.0 99.996.5 97.497.4  95.9 94.195.7 96.3 96.096.6 96.596.1 85.8
 L8                                  94.994.9 95.795.7  96.1 95.195.9 95.9 95.995.8 96.195.4 85.5
 L9                                      96.4 97.397.3  95.8 94.095.6 96.2 95.996.5 96.496.0 85.8
 L10                                          96.396.3  96.7 94.496.3 95.9 97.096.3 96.496.6 85.9
 L11                                              100.0 96.3 94.096.3 97.0 96.796.9 96.596.5 85.0
 L12                                                    96.3 94.096.3 97.0 96.796.9 96.596.5 85.0
 Lpn                                                         94.396.8 96.9 97.397.5 97.096.8 85.5
 Lfl                                                             94.4 94.9 95.095.2 95.495.1 86.2
 Lgr                                                                  97.1 97.297.4 97.696.4 85.5
 Lts                                                                       97.898.1 97.396.8 85.8
 Lpr                                                                           98.1 98.498.0 86.3
 Lst                                                                                98.497.6 85.7
 Ldm                                                                                    98.0 86.1
 Lwd                                                                                         85.8
 L. LLAP, Lpn. L. pneumophila, Lfl. L. feeleii, Lgr. L. gratiana, Lts. L. tusconensis, Lpr. L.
 parisiensis, Lst. L. steigerwaltii, Ldm. L. dumoffii, Lwd. L. wadsworthii, Cox. Coxiella
 burnetii.

        Table 3. Possible number of species represented by
              Legionella-like amebal pathogens (LLAP)
  Species 1    Species 2    Species 3     Species 4    Species 5
 LLAP-2       LLAP-1       LLAP-8       LLAP-10       LLAP-4
 LLAP-3                                               LLAP-11
 LLAP-6                                               LLAP-12
 LLAP-7
 LLAP-9
 S. lyticum
 Note. The table above includes the minimum number of species that
 may be represented by the 12 LLAPs in this study.

Preliminary fatty acid analysis showed that these LLAPs have branched fatty
acids, characteristic of members of the genus Legionella. The gas-liquid
chromatography fatty acid profiles obtained, however, appeared to be
distinct from those of other known legionellae (data not shown).

LLAP-2, LLAP-3, LLAP-7, and LLAP-9 reacted positively with antiserum
prepared against S. lyticum. Fluorescent antibody tests also showed some
LLAPs to react positively with the polyvalent Legionella antiserum
contained in the Remel Legionella Poly-ID Test Kit.2

2 The Remel Poly-ID kit used in this study is designed to identify 22
species and 31 serogroups of Legionella (34,35).

Several attempts have been made to culture LLAPs on conventional Legionella
media, BCYE. S. lyticum could occasionally be recovered from a coculture
with amebae on alanine supplemented BCYE (27). We attempted to grow 12 LLAP
strains on regular BCYE and BCYE supplemented with various amino acids,
including alanine, and have recently succeeded in cultivating LLAP-6,
LLAP-7, LLAP-9, and LLAP-10 on bacteriologic media for the first time. All
colonies had the characteristic cut-glass appearance associated with
legionellae. LLAP-1 had previously been cultured on BCYE supplemented with
sodium selenate (13,28). In this study, LLAP-1 was successfully cultured on
regular BCYE and BCYE supplemented with alanine in the absence of sodium
selenate. S. lyticum, LLAP-1, LLAP-6, LLAP-7, LLAP-9, and LLAP-10 had a
cysteine requirement for growth. Other factors influencing growth are yet
to be determined, but sodium selenate and alanine may not necessarily be
required for growth. Cultivation of LLAPs on bacteriologic media still
cannot be considered a routine process because recovery is not always
possible, and extremely low numbers of the plated bacteria produce
colonies.

Some species of Legionella are known to produce colonies that autofluoresce
on exposure to long-wave ultraviolet (UV) light. This facet of their
colonial morphology often assists in identifying and speciating isolates.
LLAP-2, LLAP-6, LLAP-7, LLAP-9, and S. lyticum exhibited blue-white
autofluorescence when exposed to long-wave UV light while LLAP-1 and
LLAP-10 did not autofluoresce. This is consistent with the clustering
pattern (Figure 2) where the blue-white fluorescent LLAPs formed a
well-defined clade.

[Figure 2]
Figure 2. Unrooted consensus tree showing the relationship between LLAPs,
members of the genus Legionella and the outgroup Coxiella burnetti. Bar
represents 5% nucleotide difference. *Species shown to exhibit blue-white
autofluorescence.

On the basis of these results, we propose that all 12 Legionella-like
amebal pathogens be included in the genus Legionella as Legionella species,
with strain specifications. A proposal was recently made for the transfer
of the species S. lyticum to the genus Legionella as L. lytica comb. nov.
(29). It appeared that L. lytica was being proposed for LLAPs. However, our
study suggests that LLAPs may represent a minimum of five species.
Consequently, the species name L. lytica, though appropriate for the strain
formerly classified as S. lyticum, cannot be applied to all LLAPs.

The genus Legionella is defined in Bergey's Manual of Determinative
Bacteriology as follows:

     Rods measuring 0.3-0.9 x 2.0-20.0 m or more. Do not form endospores
     or microcysts and are not encapsulated. Not acid fast. Cells stain
     gram negative. Motile by one, two, or more straight or curved polar or
     lateral flagella; non-motile strains are occasionally seen. Aerobic.
     L-Cysteine hydrochloride and iron salts are required for growth.
     Oxidase test is negative or weakly positive. Nitrates are not reduced.
     Urease negative. Gelatin is liquified. Branched chain fatty acids
     predominate cell wall. Chemoorganotrophic, using amino acids as carbon
     and energy sources. Carbohydrates are neither fermented nor oxidized.
     Isolated from surface water, mud, and from thermally polluted lakes
     and streams. There is no known soil or animal source. pathogenic for
     humans, causing pneumonia (Legionnaires' disease) or a mild, febrile
     disease (Pontiac fever) (30,31).

The description in Bergey's Manual should be amended to include the
following statement: Some legionellae appear to be primarily obligate
intracellular parasites of amebae that exhibit little or no growth on
current laboratory media.

Further characterization is needed to classify each LLAP strain to species
level and assess the need for additional amendments to the present
description of the genus Legionella. Appropriate diagnostic techniques are
needed to determine the clinical relevance of LLAPs, and particularly their
possible role in respiratory disease.

Acknowledgements

We are very grateful to Drs. Anne Whitney, Chi-Cheng Luo, and Catherine
Bender for valuable assistance with analysis of sequence data. The picture
of S. lyticum within A. castellanii was reprinted with permission from
Prof. W. Drozanski of the Institute of Microbiology, Maria Curie-Sklodowska
University, Poland.

Some of these data were presented at the 96th General Meeting of the
American Society for Microbiology in May 1996.

Adenike Adeleke, M.S.,* Janet Pruckler, Robert Benson, M.S., Timothy
Rowbotham, Ph.D., Mahmoud Halablab, Ph.D.,* Barry Fields, Ph.D.

*Kings College, London, UK; Centers for Disease Control and Prevention,
Atlanta, Georgia, USA; Public Health Laboratory, Leeds, UK

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     Prospective evaluation of the Gen-Probe assay for detection of
     legionellae in respiratory specimens. Eur J Clin Microbiol Infect Dis
     1988;7:748-52.
 24.  Edelstein PH, Bryan RN, Enns RK, Kohne DE, Kacian DL. Retrospective
     study of Gen-Probe Rapid Diagnostic System for detection of
     legionellae in frozen clinical respiratory tract samples. J Clin
     Microbiol 1987;25:1022-6.
 25.  Fox GE, Wisotzkey JD, Jurtshuk P JR. How close is close; 16S rRNA
     sequence identity may not be sufficient to guarantee species identity.
     Int J Syst Bacteriol 1992;42:166-70.
 26.  Stackebrandt E, Goebel BM. Taxonomic note: a place for DNA-DNA
     reassociation and 16S rRNA sequence analysis in the present species
     definition in bacteriology. Int J Syst Bacteriol 1994;44:846-9.
 27.  Giles DL, Fields BS, Newsome AL, Drozanski WJ. Cultivation of
     Sarcobium lyticum on artificial medium. Proceedings of the 96th Annual
     General Meeting of the American Society for Microbiology, Washington
     DC, 1995: Abstract Q-447.
 28.  Smalley DL, Jaquess PA, Layne JS. Selenium-enriched medium for
     Legionella pneumophila. J Clin Microbiol 1980;12:32-4.
 29.  Hookey JV, Saunders NA, Fry NK, Birtles RJ, Harrison TJ. Phylogeny of
     Legionellaceae based on small-subunit ribosomal DNA sequences and
     proposal of Legionella lytica comb. nov. for Legionella-like amoebal
     pathogens. Int J Syst Bacteriol 1996;46:526-31.
 30.  Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST. Bergey's
     manual of determinative bacteriology, Baltimore: Williams & Wilkins,
     1994:86.
 31.  Brenner DJ, Feeley JC, Weaver RE. Family VII. Genus I. Legionella
     Brenner, Steigerwalt and McDade 1979. 658AL. In: Krieg NR, Holt JG
     editors: Bergey's manual of systematic bacteriology, 1. Baltimore:
     Williams & Wilkins, 1984:279-88.
 32.  Atschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local
     alignment search tool. J Mol Biol 1990;215:403-10.
 33.  Felsenstein J. PHYLIP-Phylogeny inference package. Cladistics
     1989;5:164-6.
 34.  Thacker WL, Plikaytis BB, Wilkinson HW. Identification of 22
     Legionella species and 33 serogroups with the slide agglutination
     test. J Clin Microbiol 1985;21:779-82.
 35.  Brown SL, Bibb WF, McKinney RM. Retrospective examination of lung
     tissue specimens for the presence of Legionella organisms: comparison
     of an indirect fluorescent antibody system with direct fluorescent
     antibody testing. J Clin Microbiol 1984;19:468-72.

=============================================================================
Dispatches

Role of Enterovirus 71 in Acute Flaccid Paralysis After the
Eradication of Poliovirus in Brazil

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


As a result of the successful initiative to eradicate poliomyelitis in the
Americas, Brazil is now free of circulating wild poliovirus. The last cases
of acute flaccid paralysis (AFP) with confirmed wild poliovirus isolation
occurred in March 1989 (1). Since the elimination of wild
poliovirus-associated poliomyelitis in Brazil and the American region by
intensive mass vaccination campaigns, acute paralytic illnesses from other
causes constitute a greater proportion of suspected cases. Enterovirus 71
(EV71) can cause paralytic disease with persistent flaccid paralysis (which
may be confused with paralysis caused by wild polioviruses); therefore, the
presence of this enterovirus in a community may complicate the evaluation
of poliomyelitis control progress. Serologic evidence of EV71 infections
(demonstrated by neutralization assays) has been observed in AFP patients
in Brazil (9). In addition, EV71 isolates have been obtained from patients
with suspected cases of poliomyelitis and their healthy contacts from
different areas in Brazil and from AFP patients in Peru and Bolivia (3,
Ferreira et al., in preparation).

EV71, the most recently recognized human enterovirus in the family
Picornaviridae, has been associated with outbreaks of hand-foot-and-mouth
disease and central nervous system diseases (e.g., aseptic meningitis,
encephalitis, and poliomyelitis-like paralytic diseases) with persistent or
transient paralysis (2,4,5). Despite some understanding of the variability
in clinical manifestations and epidemiologic pattern, little is known about
the contribution of EV71 to overall AFP in different countries.

Immunoglobulin M (IgM) antibody to EV71 was measured by using an -capture
enzyme immunoassay (EIA) (6). The antigen was prepared from the BrCr strain
of EV71, and biotinylated anti-EV71 monoclonal antibody as detector was
also prepared (6). The results were expressed as the difference in mean
optical density values measured in triplicate wells of positive antigen (P)
and negative controls (N). A specimen was considered positive if the
observed P-N was >= 2 standard deviations (SD) above the mean of the
optical densities for the negative control serum (P-N >= 0.25).

Serum samples were obtained from 92 infants from different regions of
Brazil who had suspected poliomyelitis and symptoms of AFP during 1989 and
1990. The 138 samples included paired serum samples (S1 and S2) from 46
patients (92 sera) and 46 serum samples (S1 only) from 46 patients.
Acute-phase serum samples (S1) were obtained 1 to 15 days after the onset
of symptoms, and a second group of samples were obtained (S2) 15 to 45 days
later; all were from children whose stool specimens were negative for wild
poliovirus.

To check the assay reagent cross-reactivity with polioviruses, all 138
serum specimens were tested in parallel by the same described method, but
by using poliovirus serotypes 1, 2, and 3 Sabin strains instead of EV71 as
the antigen. The assay had also been checked in a limited fashion for
heterotypic response due to poliovirus infection by testing sera from a
limited number of known vaccine-associated paralytic poliomyelitis cases
(4). These vaccine associated case sera showed no evidence of IgM response
to EV71 antigen from poliovirus infection .

Specimens from 20 of 92 patients with suspected cases of poliomyelitis had
positive IgM responses to EV71 (Table). Three patients (2287, 3805, and
3489) whose specimens were positive had only a single serum specimen (S1)
available. Four serum specimens (1819, 2018, 1918, and 1906) only had
positive IgM titers for the first serum (S1), and five specimens from Bahia
State (429, 784, 1130, 1325, and 780) had an IgM response only for the
convalescent-phase sera (S2). The IgM-positive AFP specimens were from
residents of eight widely dispersed regions of Brazil with eight specimens
collected in 1989 and 12 collected in 1990. Six of 19 AFP patients with IgM
positive results had residual paralysis after 60 days, which is considered
to be clinically characteristic of poliomyelitis due to poliovirus. The
remainder of the AFP patients only had a transient paralysis from which
they recovered.

        Table. Data from patients positive for enterovirus 71(EV71)
--------------------------------------------------------------------------
                                    hEV71 IgMh            Sequelae after
  Case  Initial       Brazilian     ----------   Year
        symptom        state        S1    S2                60 days

    429 AFP          BA              -    +      1989    Yes

    784 AFP          BA              -    +      1989    No

   1130 AFP          BA              -    +      1989    No

   1325 AFP          BA              -    +      1989    Yes

    780 AFP          BA              -    +      1989    NF

   2287 AFP          BA              +    NAa    1990    No

    907 AFP          DF              +    +      1989    No

   1014 AFP          DF              +    +      1989    No

   1394 AFP          RS              +    +      1989    Yes

   2593 AFP          RS              +    +      1990    Yes

   1826 AFP          SC              +    +      1990    No

   1819 AFP          SC              +    -      1990    Yes

   1815 AFP          SC              +    +      1990    Yes

   1809 AFP          SC              +    +      1990    No

   1940 AFP          PE              +    +      1990    No

   2018 AFP          RN              +    -      1990    No

   1918 AFP          PB              +    -      1990    No

   1906 AFP          RN              +    -      1990    No

   3805 AFP          RJ              +    NA     1990    No

   3849 AFP          RJ              +    NA     1990    No

 BA = Bahia State; DF = Distrito Federal; RS = Rio Grande do Sul; SC =
 Santa Catarina;
 PE = Pernambuco; RJ = Rio de Janeiro; RN = Rio Grande do Sul; PB =
 Paraiba.
 aNA = not available; NF = no follow-up.
----------------------------------------------------------------------

The presence of residual paralysis and EV71 IgM antibodies in 20 (21.7%) of
the 92 AFP patients from eight geographic regions indicates that EV71 may
be causing AFP-like poliomyelitis throughout Brazil. The incidence of EV71
infection associated with AFP in Brazil is high when compared with its
incidence in the United States, where EV71 infections leading to AFP are
very uncommon but have rates not much lower than those of
vaccine-associated paralytic poliomyelitis (5,8). Poliomyelitis-like
paralytic illnesses in Brazil in 1989 and 1990 are not likely attributable
to polioviruses; most of these cases occurred after the last confirmed case
of wild poliovirus paralysis, and poliovirus was not isolated from any
patient's stool specimens. EV71 causes persistent flaccid paralysis; our
results link EV71 with some of these AFP cases in Brazil. Subsequent
testing on specimens from some of these patients resulted in the isolation
of EV71; neutralizing antibodies were found with seroconversion (3, 9,
Ferreira et al., in preparation). Before the eradication of wild
polioviruses in Brazil, all AFP patients who had residual paralysis as
described in this study would have generated concern about undetected wild
poliovirus circulation because their cases resembled poliovirus-caused
paralysis.

The pattern of results obtained with the acute-phase and convalescent-phase
sera in this study is not uncommon for serologic tests of enterovirus
infections. Only seven patients with AFP had two positive serum specimens;
for nine AFP patients, only one of the two specimens was positive. Some S1
samples were collected during the early stages of the disease when the IgM
titers may not have been detectable. Our results are consistent with
seroconversion data as has been observed with enterovirus 70 infections and
a similar assay (7); however, neutralizing antibodies were detected in this
study's convalescent-phase sera (9). Additionaly, late specimen collection
or inadequate specimen storage or handling before receipt in the laboratory
might have affected IgM titers adversely because these conditions were not
carefully controlled. Although cross-reactivity of antibody from other
nonpolio enteroviruses cannot be excluded, the possibility can only be
addressed directly by additional testing with other serologic assays.
However, when the same assay was used to examine specimens from known cases
of vaccine-associated paralytic poliomyelitis from the United States, no
heterotypic response was observed (4).

Since 1990, for technical and operational reasons, serologic methods have
not been used to confirm cases of AFP as caused by wild poliovirus;
therefore, serum samples are no longer available for routine poliovirus
diagnosis. Our recent efforts have been directed towards isolating and
characterizing nonpolio enteroviruses (with emphasis on EV71) from patients
with suspected poliomyelitis and towards examining the geographic and
temporal relationship between these AFP cases.

Public health personnel and pediatricians should be alerted to the possible
role of nonpolio enterovirus infections in the differential diagnosis of
AFP or other severe central nervous system diseases, particularly in areas
where the circulation of wild poliovirus has been interrupted. The
laboratory diagnosis of all AFP cases should routinely include tests
capable of detecting EV71 as well as other enteroviruses once the primary
objective of poliovirus eradication has been achieved.

Edson E. da Silva,* Maria T. Winkler,* and Mark A. Pallansch

*Instituto Oswaldo Cruz, Rio de Janeiro, Brazil; Centers for Disease
Control and Prevention, Atlanta, Georgia, USA

References

  1. de Quadros CA, Andrus JK, Oliv JM, da Silveira CM, Eikhof RM,
     Carrasco P, et al. Eradication of poliomyelitis: progress in the
     Americas. Pediatr Infect Dis J 1991;10:222-9.
  2. Melnick JL. Enterovirus type 71 infections: a varied clinical pattern
     sometimes mimicking paralytic poliomyelitis. Rev Infect Dis 1984;
     6(S2):S387-S90.
  3. da Silva EE, Fillipis AM, Cortes LC, Schatzmayr HG. Isolation of
     enterovirus 71 from suspected poliomyelitis cases and their healthy
     contacts in Brazil. Abstracts of the VIIIth International Congress of
     Virology 1990; P22-067.
  4. Nagy G, Taktsy S, Kurn E, Mihly I, Dmk I. Virological diagnosis
     of enterovirus type 71 infections: experiences gained during an
     epidemic of acute CNS diseases in Hungary in 1978. Arch Virol
     1982;71:217-27.
  5. Hayward JC, Gillespie SM, Kaplan KM, Packer R, Pallansch MA, Plotkin
     S, et al. Outbreak of poliomyelitis-like paralysis associated with
     enterovirus 71. Pediatr Infect Dis J 1989;8:611-6.
  6. Helfand RF, Khan AS, Pallansch MA, Alexander JP, Meyers HB, DeSantis
     RA, et al. Echovirus 30 infection and aseptic meningitis in parents of
     children attending a child care center. J Infect Dis 1994;169:1133-7.
  7. Bern C, Pallansch MA, Gary HE, Alexander JP, Trk TJ, Glass RI, et
     al. Acute hemorrhagic conjunctivitis due to enterovirus 70 in American
     Samoa: serum- neutralizing antibodies and sex-specific protection. Am
     J Epidemiol 1992;136:1502-6.
  8. Alexander JP, Baden L, Pallansch MA, Anderson LJ. Enterovirus 71
     (EV71) infections and neurologic diseaseUnited States, 1985-1989. J
     Infect Dis 1994; 169:905-8.
  9. da Silva EE, Fillipis AM, Schatzmayr HG, Cortes LC. Evidence of
     enterovirus 71 infections in Brazil. Mem Inst Oswaldo Cruz
     1990;85:131-2.


===========================================================================
Dispatches

Bancroftian Filariasis Distribution and Diurnal Temperature
Differences in the Southern Nile Delta

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


In recent years the prevalence of Bancroftian filariasis has increased
dramatically in the Nile delta (1). The resurgence is believed to be
associated with an increasing accumulation, in and near villages, of
surface and subsurface water that serves as breeding sites for Culex
pipiens mosquitoes. This water increase could be due to the growing number
of irrigation channels and to more reliable water supply and distribution
since the Aswan High Dam was completed in 1971.

A correlation between remotely characterized environmental features and the
distribution of some focal diseases (2) has been suspected; however, no
direct association has been reported. Intermediate factors affecting
disease distribution, such as vector abundance (3,4) and risk for disease
transmission (5), have been described. Recently, we examined diurnal
temperature difference (dT) maps derived from the advanced very high
resolution radiometer of the polar-orbiting National Oceanographic and
Atmospheric Administration environmental satellite in relationship to the
distribution of schistosomiasis in the Nile delta (6). dTs are derived from
thermal scanning radiometry, and they indicate surface and subsurface
moisture contained in the soil and plant canopy. The distribution of
schistosomiasis has been stable for decades, which suggests that infection
prevalence relates to intermediate host snail abundance which in turn is
influenced by environmental moisture factors. Because dT values are
associated with surface moisture and standing water, we hypothesized that
those values should correlate well with the distribution of Bancroftian
filariasis cases. We report here the initial test of this hypothesis.

First, we determined the prevalence of Bancroftian filariasis between 1985
and 1993, by surveying 297 villages in the southern Nile delta (1).
Prevalence was determined on the basis of microfilaremia diagnosis and on
the presence of filarial antigenemia and clinical evidence of prior
infection, or both. We grouped the prevalence data into four categories (0%
to 1%, 2% to 9%, 10% to 19%, and 20+%), and used approximate village
coordinates to plot the results on a map. Next, a more accurate version of
this map (provided by one of the authors) was digitized to create a file of
village latitude and longitude coordinates; this file was overlaid onto the
dT map with TeraScan software (SeaSpace Corporation, San Diego, CA)
(Figure) [Figures not available in ASCII].

Satellite image data from the advanced very high resolution radiometer from
August 16, 1990, were analyzed by using TeraScan to determine dTs for the
southern Nile delta. The time of data collection was approximately 01:00
and 13:00 hours, local time. Point dT values were averaged for 3 x 3 pixel
areas (10 km squared), centered on the latitude and longitude of each study site
to reduce false precision of individual pixels. Digitized filariasis
prevalence data were superimposed and assigned mid-point values for each of
the four prevalence categories (0.5%, 5%, 15%, and 25%, respectively). The
number of villages assigned to each of these categories, respectively, was
169, 74, 37, and 13. Four villages in the 0% to 1% prevalence category fell
beyond the southern border of the computerized dT map.

Single factor analysis of variance between the four categories showed that
for the four groups, values were significantly different (F = 5.42, p =
.0012). Stepwise polychotomous logistic regression was used to model
prevalence by using the dT value as the only predictor. The model that
predicts prevalence showed significant improvement for each of the three
prevalence levels over the (negative) reference category of 0.5%
(improvement, chi-squared = 17.11, p = .002).

The high degree of correlation between the remotely sensed dT values and
the ground truth filariasis prevalence data was unexpected, considering
the low resolution of the data and the relative imprecision of the village
coordinates. We are now gathering data on the precise numerical prevalence
level for each village and are using a hand-held global position system
receiver in the villages to obtain exact coordinates. Because the effect of
random error due to the imprecision of the measurements can only diminish
correlations, we anticipate that more precise data will result in even
closer associations.

Our efforts are directed towards prospectively determining the predictive
power of dT with respect to the village-specific prevalence of Bancroftian
filariasis from adjacent parts of the Nile delta for which prevalence data
are not yet available. Ultimately, we hope to determine the degree to which
dT values, which are available for much of the earth's surface, can be used
to signal the presence of Bancroftian filariasis in the Nile delta and
perhaps in other disease-endemic regions.

Acknowledgments

We thank Melissa Seymour and Michael Lavestere of the Louisiana State
University Earth Scan Laboratory, Coastal Studies Institute, for satellite
data processing support, and Stephanie Posner, M.P.H., for statistical
assistance.

Donald F. Thompson, M.D.,* John B. Malone, D.V.M., Ph.D., Mustafa Harb,
M.D., Rifki Faris, M.D., Oscar K. Huh, Ph.D., Alfred A. Buck, M.D.,**
and Barnett L. Cline, M.D., Ph.D.

*Tulane University Medical Center, New Orleans, Louisiana, USA; Louisiana
State University, Baton Rouge, Louisiana, USA;  Ministry of Health, Cairo,
Egypt; Ain Shams University, Cairo, Egypt; **Johns Hopkins University,
Baltimore, Maryland, USA; Tulane University School of Public Health and
Tropical Medicine, New Orleans, Louisiana, USA

References

  1. Harb M, Faris R, Gad AM, Hafez ON, Ramzy R, Buck AA. The resurgence of
     lymphatic filariasis in the Nile delta. Bull WHO 1993;71:49-54.
  2. Cline BL. New eyes for epidemiologists: aerial photography and other
     remote sensing techniques. Am J Epidemiol 1970;92:85-9.
  3. Wood BL, Washino RK, Beck LR, Hibbard K, Pitcairn M, Roberts D, et al.
     Distinguishing high and low anopheline-producing rice fields using
     remote sensing and GIS technologies. Prev Vet Med 1992;11:277-88.
  4. Rogers DJ, Randolph SE. Mortality rates and population density of
     tsetse flies correlated with satellite imagery. Nature
     1991;351:739-41.
  5. Beck LR, Rodriguez MH, Dister SW, Rodriguez AD, Rejmankova E, Ulloa A,
     et al. Remote sensing as a landscape epidemiologic tool to identify
     villages at high risk for malaria transmission. Am J Trop Med Hyg
     1994;51:271-80.
  6. Malone JB, Huh OK, Fehler DP, Wilson PA, Wilensky DE, Holmes RA, et
     al. Temperature data from satellite imagery and the distribution of
     schistosomiasis in Egypt. Am J Trop Med Hyg 1994;50:714-22.


============================================================================
Dispatches

Sentinel Surveillance for Yellow Fever
in Kenya, 1993 to 1995

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


Yellow fever (YF) is an acute mosquito-transmitted viral infection, endemic
and occasionally epidemic in many countries of Africa and South America.
Periodic outbreaks of yellow fever in East Africa have been reported since
1940 (1,2) (Figure 1) [Figures not available in ASCII]. The largest 
outbreak, an estimated 300,000 cases,
occurred from 1960 to 1962 in Ethiopia (3). In 1966, YF reappeared in
Arba-Minch, Ethiopia, east of Lake Abaya, in an area not affected by the
outbreak of 1960 to 1962. During the 1966 outbreak, 2,200 cases (450
deaths) were reported (4). Despite the prevalence of anti-YF antibody in
northern Kenya (up to 14%) (5,6), no outbreaks of YF were reported in Kenya
until 1992 when epidemic YF emerged in Kenya for the first time, with at
least 54 cases and 29 deaths (7). Cases of hemorrhagic fever were first
reported in September 1992, from the southern parts of Keiyo (Kerio
Valley), Baringo, and the Koibatek Districts (former Elgeyo-Marakwet and
Baringo districts) in the Rift Valley Province, northwest Kenya (Figure 2).
Of the 54 patients with hemorrhagic fever, 35 (67%) were male. Of the 48
patients whose definite age was known, 41 (85%) were 10 to 39 years old.
Twenty-one (39%) of the 54 patients had serologic evidence of recent YF
infection, and three of the 21 had cases confirmed by isolation of YF
virus. The outbreak ended when the entire population of these districts was
vaccinated. A surveillance system involving 13 sentinel sites was
established 6 months after the epidemic was over. Six health facilities
were located within the outbreak area of Keiyo, Koibatek, and Baringo
districts. Seven facilities were situated in the surrounding areas of
Nakuru, Nyandarua, Kericho, and the Uasin Gishu districts. In 1994, the
number of sentinel sites was increased to 18. Sentinel facilities were
provided with case investigation forms and equipment for blood collection
and storage. Surveillance activities were ongoing, and supervisory visits
were conducted once every 6 weeks.

In Kenya, there are three levels of health care delivery: hospitals, health
centers, and dispensaries. In addition, missionary hospitals provide
independent health services. Surveillance training candidates were selected
from district hospitals, health centers, and mission hospitals. Training
focused on case recognition, record keeping, use of a YF case investigation
form, handling of serum samples, and obtaining exposure information. A
field guide for YF surveillance was developed in collaboration with the
World Health Organization (WHO) and the Centers for Disease Control and
Prevention (CDC). A 1-day training workshop was followed by a visit to the
collaborating surveillance centers. Medical staff at the health facility
who could not attend the workshop received on-the-job training. A
surveillance team consisted of a medical officer (or scientist from the
Virus Research Centre), a laboratory technologist, the provincial public
health officer from Rift Valley Province, and a driver. In October 1994, a
second 1-day training workshop was offered for five additional sentinel
health facilities. For surveillance purposes, a patient was considered to
have a case of YF if two of the following symptom complexes were present:
jaundice; hemorrhage; signs of encephalitis or renal involvement; a
temperature >= 38C.

Serum was separated from blood samples on the day of collection and stored
frozen, until the serum specimens could be transported by the surveillance
team. Vaccine carriers with ice packs were used to transport samples (along
the surveillance route) to the Kenya Medical Research Institute (KEMRI). In
1994, a liquid nitrogen container was added for transporting samples.
Samples were tested for YF antibodies by the IgM-capture enzyme-linked
immunosorbent assay (MAC ELISA) (8) at the Virus Research Centre. Selected
acute-phase surveillance samples were also subjected to virus isolation
attempts, in which newborn mice and tissue cultures were used. All
serologic results were confirmed by either CDC's laboratory in Fort
Collins, Colorado, or the Institute Pasteur, Paris. Persons whose illness
met the surveillance case definition were considered positive for recent YF
infection if YF-specific IgM antibody was present in acute-phase serum
samples by MAC ELISA. Samples that were borderline positive by IgM MAC
ELISA were tested for neutralizing antibody by the plaque reduction
neutralization test with YF virus. Results of this sentinel surveillance
from October 7, 1993, to December 31, 1995, are reported here.

One hundred-fifty persons, of whom 131 (87%) had jaundice, met the
surveillance case definition. These were identified by 17 health
facilities, during a 27-month period (average, 5.5 per month; range, 0-12);
85 (57%) were male. Of the 138 persons whose age was known, 117 (85%) were
under 40 years of age (average age, 21.4 years; range, 1 to 70 years).

Ten serologically confirmed cases were identified by four health
facilities. All attempts to isolate the virus were unsuccessful. The onset
of the first confirmed case was March 1, 1994. The onset of the latest case
was May 15, 1995. Seven confirmed cases were from an area not known to be
affected during the 1992 to 1993 epidemic. The homes of six patients border
the districts where no YF vaccinations are given (Figure 2) [Figures not 
available in ASCII]. One patient
(1994, Songhor, Nandi District) lived far outside this area and had no
history of travel to the known YF-endemic area. Five patients with
confirmed cases were male: eight (80%) were under 40 years of age (average
age, 26.5 years; range, 8 to 48 years). All patients with confirmed cases
had jaundice, and five (50%) hemorrhaged from the nose or gums. Three
patients died (two were female). One was not vaccinated for YF, and the
vaccine status of two was unknown. The case-fatality rate for patients with
confirmed YF was 30%. All surviving patients had been vaccinated (Table).

     Table. Characterisitics of patients with
 confirmed YF cases, October 7, 1993 to December
                     31, 1995
--------------------------------------------------
      Characteristics      Number  Percentage (%)
--------------------------------------------------
 Sex, male                      5              50

     female                     5              50

 Age, < 40 years                8              80

 Jaundice                      10             100

 Hemorrhage                     5              50

 Deaths                         3              30

 History of YF vaccination     7a              70

 a Only non-fatal cases.
--------------------------------------------------


Timely reporting of disease emergencies is not feasible through the
existing health information system in Kenya. When YF emerged there in
September 1992, disease investigation was delayed, and the diagnosis was
not confirmed until January 1993. In the case of a limited health
information system, sentinel surveillance for YF, with active follow-up by
collaborating centers, seems appropriate for monitoring disease after an
epidemic.

Most patients surveyed had fever and jaundice. All patients with confirmed
YF cases had jaundice, and 50% hemorrhaged from the nose and gums. Mild,
nonspecific, and nonfatal clinical forms of yellow fever may have gone
unnoticed as they did during the 1992 to 1993 epidemic when YF virus was
isolated from a 12-year-old girl, who had fever, headache and joint pains
but no jaundice or signs of hemorrhagic illness. (Outbreak of YF in Kenya,
1992-1993 Epidemiological Investigations, Sanders et al., in preparation).

The continued low-level of transmission of YF virus would likely have gone
unnoticed if the enhanced surveillance and diagnostic capacity instituted
by the government of Kenya after the 1992-93 epidemic was not in place.
When cases of YF were confirmed from an area not known to have been
affected during the outbreak, the Kenya Ministry of Health quickly
recognized the problem and resumed vaccinations (9,10).

Operational costs of the field visits to 18 sentinel sites are relatively
high. Since nine (90%) of the confirmed cases of YF were recognized by
three hospitals, which had cared for YF patients during the previous
epidemic, limiting the number of sentinel sites may be considered.
Distributing materials to obtain blood from persons with suspected cases
does not seem feasible countrywide.

The WHO initiative to create a global surveillance program for recognition
of and response to emerging diseases emphasizes that a critical element of
surveillance includes a network of reference centers that assist national
services in disease diagnosis and investigation (11). In Kenya, the first
steps have been taken to establish such a reference center at KEMRI in
Nairobi. In 1994, two technologists were trained for an extensive period at
CDC's laboratory in Fort Collins, Colorado, in the diagnosis of YF and 
other arboviral diseases. An epidemiologic support unit
was established at the KEMRI laboratory to provide the link between
possible disease occurrences in the field and the diagnostic facilities at
the distant laboratory.

Will this effort ensure that future epidemics will be recognized? It is
often argued that the critical element of surveillance is disease
recognition by front-line health-care providers, subsequent investigation,
sample collection, and the forwarding of samples to available diagnostic
facilities. In Kenya there is no focus on what diseases should be reported
immediately (monthly disease reporting at district level requires health
care workers to compile information on 37 diseases). Communication between
the field and laboratory is often difficult. Once a specimen is forwarded,
results often do not reach the clinician who submitted the sample in a
timely fashion. Separate disease monitoring systems have been initiated by
different health programs, but they rely on the same district officers to
fill out the forms. Admittedly, sentinel surveillance for YF has not been
integrated into one of the existing disease control programs, and its
sustainability as anything more than a research program is questionable.

Hemorrhagic fever surveillance can be improved by 1) arranging sentinel
sites according to the presumed southward extension of the YF-endemic area,
2) managing supervision of the sentinel sites at provincial level, 3)
integrating disease reporting for YF in the surveillance system for EPI
target diseases, 4) clearly defining which diseases need to be actively
monitored by health care providers, 5) instituting improved diagnostic
capabilities, and 6) forming a disease outbreak team.

Transmission of YF poses a potential threat for the largely unvaccinated
population of Kenya and other East African countries, especially since the
YF-endemic area appears to be expanding (12). YF vaccination appears to be
the only practical public health intervention. Vaccination of children 6
months of age and older in the YF-endemic districts (Keiyo, Marakwet,
Baringo, and Koibatek) should be continued. Districts adjacent to the
disease-endemic area (Uasin Gishu, Kericho, Nandi, and Nakuru) should be
included in the vaccination program.

Acknowledgments

We thank our colleagues at the Centers for Disease Control and Prevention
(CDC) and at the Institute Pasteur for confirming our preliminary serologic
results; the medical staff at the sentinel surveillance facilities, the
Kenya Ministry of Health, and the Kenya Medical Research Institute (KEMRI)
for their continued support for this program. Special thanks go to Dr.
Duane Gubler and his staff at the CDC laboratory in Fort Collins, Colorado,
for training two of KEMRI's senior technologists during a 4-month period in
1994.

Eduard J. Sanders, M.D., M.P.H.,* Peter Borus, D.V.M.,* George Ademba,*
Grace Kuria,* Peter M. Tukei, M.D., M.Sc.,* and James W. LeDuc, Ph.D.

*Virus Research Centre, Kenya Medical Research Institute, Nairobi, Kenya;
Division of Emerging and Other Communicable Diseases Surveillance and
Control, World Health Organization, Geneva, Switzerland

References

  1. Kirk R. Epidemic of yellow fever in the Nuba Mountains, Anglo-Egyptian
     Sudan. Ann Trop Med 1941;35:67-112.
  2. Satti MH, Haseeb MA. An outbreak of yellow fever in the southern Fung
     and Upper Nile Province, Republic of the Sudan. J Trop Med Hyg
     1966;69:36-44.
  3. Berdonneau R, Sri C, Panthier R, Hannoun C, Papaioannou SG,
     Georgieff P. Sur L'pidemie de fivre jaune de l'anne 1959 en
     Ethiopie. Bull Soc Path Exot 1961;54:276-86.
  4. Ardoin P, Rodhain F, Hannoun C. Epidemiologic study of arboviruses in
     the Arba-Minch District of Ethiopia. Trop Geogr Med 1976;18:309-15.
  5. Henderson BE, Metselaar D, Cahill K, Timms GL, Tukei PM, Williams MC.
     Yellow Fever Immunity Surveys in northern Uganda and Kenya and eastern
     Somalia, 1966-67. Bull WHO 1968;38:229-37.
  6. Henderson BE, Metselaar D, Kirya GB, Timms GL. Investigations into
     yellow fever virus and other arboviruses in the northern regions of
     Kenya. Bull WHO 1970;42:787-95.
  7. WHO Weekly Epid Rec 1993; No. 22:159-60. Yellow fever, Kenya.
  8. Monath TP, Nystrom RR, Bailey RE, Calisher CH, Muth DJ. Immunoglobulin
     M antibody capture enzyme-linked immunosorbent assay for diagnostics
     of St. Louis encephalitis. J Clin Microbiol 1984;20:784-90.
  9. WHO Weekly Epid Rec 1995; No. 24:175-6. Yellow fever, Kenya.
 10. WHO Weekly Epid Rec 1996; No. 13:103. Yellow fever, Kenya.
 11. LeDuc JW, Tikhomirov E. Global surveillance for recognition and
     response to emerging diseases. Ann NY Acad Sci 1994;740:341-5.
 12. Sanders EJ, Tukei PM. Yellow fever: an emerging threat for Kenya and
     other East African countries. E Afr Med J 1996;73:10-2.


============================================================================
Dispatches

Serologic Evidence for the Presence in Pteropus Bats of a Paramyxovirus
Related to Equine Morbillivirus

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


Two outbreaks of a previously unknown disease in horses and humans occurred
in Queensland in 1994. The outbreaks occurred within 1 month of each other
in Brisbane and Mackay, which are approximately 1000 km apart. In the
Brisbane incident, 21 horses were infected of which 14 died or were
euthanized after severe clinical signs of an acute respiratory disease. Two
human cases were in patients with less well defined clinical signs; one
patient died (1,2). In the Mackay incident, two horses became seriously ill
and died, and one person also died (3). Although it is now known that the
two outbreaks occurred in August and September 1994, knowledge of the
Mackay outbreak did not occur until late 1995 when the infected person died
of a relapsing encephalitis. The name equine morbillivirus (EMV) has been
proposed for a paramyxovirus isolated from four of the Brisbane horses and
the first patient who died (2).

In both locations, the index case appears to have been in a mare in late
pregnancy, on pasture. The mode of transmission to other horses and to
humans is unknown. In spite of intensive investigations, no connection has
been established between the two incidents. At both locations, initial
serologic studies of in-contact horses and humans failed to show evidence
of neutralizing antibody, and it was concluded that infection by contact is
uncommon. A subsequent statewide serologic survey of 2,411 horses has also
shown no evidence of infection (4). At present, only seven horses have had
antibody, and all were involved in the Brisbane outbreak.

To evaluate the theory that EMV originated from a wildlife source, a
trapping program was initiated, focusing on the location of the index
cases, first in Brisbane and later in Mackay when the details of that
incident became known. A total of 5,264 sera from 46 species were tested,
none of which showed any evidence of antibody.

Further information from the Mackay cases enabled us to think more
logically about a possible reservoir host. Although virus was not isolated
from the two horses, it was possible to amplify part of the EMV matrix
protein gene by PCR. A comparison of the sequence of this PCR product with
PCR product from the Brisbane cases showed that they were identical,
indicating a common source of origin (5). In our consideration of possible
reservoir hosts, the following criteria were applied to prioritize species
for investigation: 1) The species should be present in both the Brisbane
and Mackay areas; 2) the species should be capable of migrating between
these areas, and 3) contact with horses should be possible. The two groups
of animals which readily fit this description were birds (especially
migratory waders) and flying foxes. Because EMV is a mammalian virus and
transmission of paramyxoviruses from birds to mammals is uncommon, flying
foxes were given a higher priority than birds.

In Australia four species of bats belong to the Suborder Megachiroptera
(fruit bats or flying foxes). The spectacled fruit bat (Pteropus
conspicillatus) occurs in the northern and eastern parts of Queensland
while the black fruit bat (P. alecto) has a much wider distribution across
the northern part of Australia. The little red (P. scapulatus) is found
across northern and eastern Australia while the grey-headed (P.
poliocephalus) occurs in eastern and southern areas of the country.

Serologic sampling of these species soon showed the presence of antibody to
EMV. At present, 224 samples have been tested of which 20 have neutralizing
antibody to EMV (prevalence rate of approximately 9%). Positives have been
detected in all species but insufficient samples are available to provide
any meaningful estimate of comparative prevalence. Animals with positive
results have been recorded along the whole of the eastern coast of
Queensland, from Cairns to Brisbane. Again, insufficient data are available
to compare the seroprevalence in different locations. Titers vary from 1/5,
the lowest dilution tested, to 1/640. For comparison, the neutralization
titer of the control positive equine sera is 1/160.

In relation to the origin of EMV, the significance of finding antibody in
fruit bats remains to be determined. Our interpretation of the
neutralization test results is that a virus closely related to EMV is
circulating in fruit bats. We propose referring to this virus as bat
paramyxovirus to emphasize that its relationship to EMV is uncertain.

A serologic survey of persons who have had prolonged and close contact with
fruit bats is under way. To our knowledge there have been no unexplained
occurrences of severe infectious disease in fruit bats, nor have there been
any recorded cases of severe disease in persons who have had extensive
exposure to these animals.

We will continue sampling birds and other animals because fruit bats may
not be the only reservoir of this virus. Also transmission from bats to
another species may be required before spillover occurs in horses. A high
priority will be to isolate the bat paramyxovirus and compare it to EMV.
The natural history of bat paramyxovirus infection should be investigated
so that natural routes of infection can be established. This information
should then lead to testable hypotheses about how infection of other
species occurs.

Peter L. Young,* Kim Halpin,* Paul W. Selleck, Hume Field,* Jenny L.
Gravel,* Mark A. Kelly,* and John S. Mackenzie

*DPI Animal Research Institute, Brisbane, Australia; CSIRO Australian
Animal Health Laboratory, Geelong, Australia; Department of Microbiology,
University of Queensland, Brisbane, Australia

References

  1. Selvey L, Wells RM, McCormack JG, Ansford AJ, Murray PK, Rogers RJ, et
     al. Outbreak of severe respiratory disease in humans and horses due to
     a previously unrecognised paramyxovirus. Med J Aust 1995;162:642-5.
  2. Murray P, Selleck P, Hooper P, Hyatt A, Gould A, Gleeson L, et al. A
     morbillivirus that caused fatal disease in horses and humans. Science
     1995;268:94-7.
  3. Rogers RJ, Douglas IC, Baldock FC, Glanville RJ, Seppanen KT, Gleeson
     LJ, et al. Investigation of a second focus of equine morbillivirus
     infection in coastal Queensland. Aust Vet J 1996 (in press).
  4. Ward MP, Black PF, Childs AJ, Baldock FC, Webster WR, Rodwell BJ, et
     al. Negative findings from serological studies of equine morbillivirus
     in the Queensland horse population. Aust Vet J 1996 (in press).
  5. Hooper PT, Gould A, Mitchell G, Russell GW, Kattenbelt J. The
     retrospective diagnosis of a second outbreak of equine morbillivirus
     infection. Aust Vet J 1996 (in press).


============================================================================
Letters

Acute Cervical Lymphadenopathy

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


To the Editor: Acute cervical lymphadenopathy has not been reported as a
symptom of Mycobacterium genavense infection. In June 1994, a 32-year-old
injecting drug user, who had been monitored since 1987 for human
immunodeficiency virus (HIV) infection at the outpatient clinic of the
Infectious Disease Institute, Perugia, Italy, was admitted to a hospital
with fever (39C) and progressive swelling over the submandibular region
and neck. In addition to being febrile, upon physical examination the
patient had tender left submandibular and cervical lymphadenopathy
approximately 3 cm in diameter, with redness and edema of the overlying
skin. The CD4+ lymphocyte count was 0.01 x 10 to the 9th power/L. A 
specimen obtained by needle aspiration of the submandibular lymph node 
contained numerous acidfast bacilli, and the patient was treated with 
isoniazid, rifampin, ethambutol, and amikacin for presumed Mycobacterium 
tuberculosis with a good response; however, 10 days later, the patient's 
submandibular pain recurred along with abdominal pain and bowel 
irregularities. Gastroscopy showed superficial duodenal erosions, and 
acidfast bacilli were visualized by microscopy. Shortly thereafter, pain 
and swelling of the patient's right ankle developed, and small lesions 
were noted on the dorsum of the right foot. Clarithromycin was substituted 
for the amikacin for suspected without a clear response, and a course of 
steroids was initiated with clinical improvement. Symptoms recurred when 
the steroids wre tapered. Ciprofloxacin was substituted for isoniazid, 
and amikacin was readministered. Material from a repeat needle aspiration 
of the submandibular node 1 month later also showed acidfast bacilli by 
microscopy.

Cultures of the initial submandibular aspirate demonstrated poor growth in
Bactec 13A broth and did not grow on solid media. The specimen was sent to
a reference laboratory where acidfast bacilli were successfully isolated 10
weeks later in Middlebrook agar containing mycobactin J. These acidfast
bacilli were subsequently identified as M. genavense by highpressure liquid
chromatography and nucleic acid sequencing of the 16S rRNA. By this point,
the patient had improved on a regimen of isoniazid, pyrazinamide,
clofazimine, and amikacin for presumed M. genavense infection, and this
regimen was continued. The patient died 19 months later; cultures for
mycobacteria were persistently negative even when antimycobacterial drugs
were discontinued 16 months after the initial episode.

M. genavense is a novel mycobacterial species that causes serious
disseminated infections with massive involvement of the small intestine,
spleen, liver, and abdominal lymph nodes in profoundly immunocompromised
persons. Cultures with Bactec 13A vials containing radiometric liquid
medium are generally positive but subcultures on solid media are
unsuccessful (1). Lowering the pH of medium to six enhances its growth (1),
while adding mycobactin J to Middlebrook 7H11 (2) solid media can help in
the isolation. The suppression of growth of M. genavense by NAP can cause
confusion with the M. tuberculosis complex; however, M. genavense can be
easily distinguished by its slow growth and its dysgonic nature. At
present, the way to identify M. genavense is by 16S rRNA sequencing (3).
Highpressure liquid chromatography can be used (4).

Maria Bruna Pasticci, M.D.,* F. Baldelli,* F. Bistoni, C. Piersimoni, G.
Sbaraglia, G. Stagni, and S. Pauluzzi

*Infectious Disease Institute, Perugia University, Policlinico Monteluce,
Perugia, Italy; Department of Experimental Medicine and Biochemical
Sciences, Microbiology Section, Perugia University, Perugia, Italy; and
Department of Clinical Microbiology, Umberto I Torrette Hospital, Ancona,
Italy

References

  1. Tortoli E, Simonetti MT, Dionisio D, Meli M. Cultural studies on two
     isolates of Mycobacterium genavense from patients with acquired
     immunodeficiency syndrome. Diagn Microbiol Infect Dis 1994; 18:7-12.
  2. Kiehn TE. The diagnostic mycobacteriology laboratory of the 1990s.
     Clin Infect Dis 1993; 17(Suppl 2):S447-454.
  3. Kirshner P, Springer B, Meier A, Wrede A, Kiekenbeck M, Bange FC.
     Genotypic identification of mycobacteria by nucleic aid sequence
     determinationreport of a 2-year experience in a clinical laboratory
     1993; J Clin Microbiol; 31:2882-9.
  4. Toroli E, Bartoloni A, Burrini C, Mantella A, Simonetti MT. Utility of
     high-performance liquid chromatography for identification of
     mycobacterial species rarely encountered in clinical laboratories. Eur
     J Clin Microbiol Infect Dis 1995; 14:240-3.

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AIDS: Dj Vu in Ancient Egypt?

To the Editor: The recent letter by Olson et al. (1) on the plague of
Athens and the reemergence (?) of Ebola virus prompts brief reiteration
of an earlier communication (2) placing human immunodeficiency virus (HIV)
back in history, possibly in the time of the pharaohs.

Translations of the Papyrus Ebers from ancient Egyptian literature
repeatedly make reference to difficulties in the diagnosis of conditions
under the names AAA disease, uxedu-disease, and uha-disease (3). The
interpretation of these diseases has been inconclusive among Egyptologists.
However, the many remedies proposed for the ravages of AAA, whether by
itself or complicated by uxedu- or uha-disease, brand it as a scourge of
first magnitude. AAA is mentioned 50 times in four papyri (28 times in
Papyrus Ebers, 12 times in Papyrus Berlin, 9 times in Papyrus Hearst, and
once in the London Papyrus [4]). The hieroglyphic symbol for AAA (5) is
shown below [Figure not available in ASCII].

The hieroglyphic symbol for AAA disease, translated as "semen" or
"poison" (From: Von Dienes and Westeneorf [5].)
Could this be documentation of HIV, or more accurately its prototype,
occurring in Egypt during the time of the pharaohs?

Richard J. Ablin, Ph.D.
Innapharma, Inc.
Suffern, New York, USA

References

  1. Olson PE, Hames CS, Benenson AS, Genovese EN. The Thucydides syndrome:
     Ebola dj vu? (or Ebola reemergent?). Emerging Infectious Diseases
     1996;2:1556.
  2. Ablin RJ, Immerman RS, Gonder MJ. AIDS: a disease of ancient Egypt? NY
     State J Med 1985;85:2001.
  3. Bryan CP, Smith GE. The AAA In: Ancient Egyptian medicine: the Papyrus
     Ebers. Chicago: Ares Publishers, 1924:11923.
  4. Ghalioungui P. Magic and medical science in ancient Egypt. London, UK:
     Hodder and Stoughton, 1963.
  5. Von Deines H, Westeneorf W. Wterbuch der medizimischen texte. Berlin,
     Germany: AkademieVerlag, 1961;7,Part 1:129.


=============================================================================
News and Notes

International Meeting on Research Advances and Rabies Control in the
Americas

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


The "VIIth Annual International Meeting on Research Advances and Rabies
Control in the Americas" will be convened at the Centers for Disease
Control and Prevention in Atlanta during 913 December 1996. The conference
will focus on both basic scientific contributions and applied research in
the understanding and control of this fatal viral disease. Conference
sessions are designed for a diverse public health and biomedical audience,
particularly professionals in Canada, the United States, and Latin America;
the sessions will explore research advances, diagnostic issues, viral
pathobiology, oral immunization, descriptive and molecular epizootiology,
host ecology, and human preexposure and postexposure vaccination. Workshops
detailing techniques used in the diagnosis and surveillance of rabies by
conventional and molecular methods will also be held.

For further information, contact

Dr. Charles E. Rupprecht
Chair, Organizing Committee, Centers for Disease Control and Prevention,
1600 Clifton Road, MS G33, Atlanta, GA 30333, USA; fax: 404-639-1058
email: cyr5@ciddvd1.em.ced.gov.
---------------------------------------------------------------------------

Symposium Notice

The Infectious Disease Association of California (IDAC) will hold its fall
symposium on Saturday, October 19, 1996, at the Hyatt Regency in Irvine,
California.

Registration, including lunch, is free to IDAC members; $50 for nonmembers.

For information, call
(310) 398-5931 or email: IDAC@aol.com.


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