----------------------------------------------------------------------------
Emerging Infectious Diseases  * Volume 2 * Number 4 * October-December, 1996
----------------------------------------------------------------------------

Perspectives

   * Guarding Against the Most Dangerous Emerging Pathogens: Insights from
     Evolutionary Biology, P.W. Ewald
   * Social Inequalities and Emerging Infectious Diseases, P. Farmer

Synopses

   * Molecular Mechanisms of Bacterial Virulence: Type III Secretion and
     Pathogenicity Islands, J. Mecsas and E.J. Strauss
   * New Vaccines for the Prevention of Pneumococcal Infections, H. Kyhty
     and J. Eskola
   * A Mathematical Model and CD4+ Lymphocyte Dynamics in HIV Infection, T.
     Hraba and J. Dolezal
   * Chlamydiae as Pathogens: New Species and New Issues, R.W. Peeling and
     R.C. Brunham

Dispatches

   * Experimental Inoculation of Plants and Animals with Ebola Virus, R.
     Swanepoel, P.A. Leman, F.J. Burt, N.A. Zachariades, L.E.O. Braack,
     T.G. Ksiazek, P.E. Rollin, S.R. Zaki, and C.J. Peters
   * Encephalitis Caused by a Lyssavirus in Fruit Bats in Australia, G.C.
     Fraser, P.T. Hooper, R.A. Lunt, A.R. Gould, L.J. Gleeson, A.D. Hyatt,
     G.M. Russell, and J.A. Kattenbelt
   * Creutzfeld-tJakob Disease in the United States, 1979-1994: Using
     National Mortality Data to Assess the Possible Occurrence of Variant
     Cases, R.C. Holman, A.S. Khan, E.D. Belay, and L.B. Schonberger
   * Rapid Increase of Resistance to Erythromycin and Clindamycin in
     Streptococcus pyogenes in Italy, 1993-1995 , G. Cornaglia, M. Ligozzi,
     A. Mazzariol, M.Valentini, G. Orefici, the Italian Surveillance Group
     for Antimicrobial Resistance, and R. Fontana
   * Assessing the Costs and Benefits of an Oral Vaccine for Raccoon
     Rabies: A Possible Model, M.I. Meltzer

Commentary

   * Traditional Healers and Global Surveillance Strategies for Emerging
     Diseases: Closing the Gap, N.E. Groce and M. E. Reeve

Letters

   * Widespread Foodborne Cyclosporiasis Outbreaks Present Major
     Challenges, D.G. Colley
   * Identification of Cyclospora in Poultry, H.L. Garca-Lpez, L.E.
     Rodrguez-Tovar, and C.E. Medina-De la Garza
   * PCR Confirmation of Infection with Cyclospora cayetanensis, N.J.
     Pieniazek, S.B. Slemenda, A.J. da Silva, E.M. Alfano, and M.J.
     Arrowood
   * Emerging Infectious Diseases and the Depopulation of French Polynesia
     in the 19th Century, P.M.V. Martin and C. Combes
   * Epidemic Zoster and AIDS, D.M. Morens, A.K. Agarwal, S. Sarkar, S.
     Panda, and R. Detels
   * Ancient Egypt and Today: Enough Scourges to Go Around, D.G. Colley
   * AIDS and [AAA] in Egypt? , R.J. Littman and D.M. Morens

Addendum

News and Notes

   * ABA Sponsors Program on Law and Emerging Infectious Diseases, D.P.
     Fidler
   * A Global Theme Issue: Bibliography of References, M.A. Winker
   * Conference on Emerging Foodborne Pathogens, D. Dalisera


---------------------------------------------------------------------------
ISSN 1080-6059
 

                                 About EID

Emerging Infectious Diseases is indexed in Index Medicus/Medline, Current
Contents, and several other electronic databases.

Emerging Infectious Diseases is part of CDC's plan for combatting emerging
infectious diseases; the plan is outlined in a recently published document,
Addressing Emerging Infectious Disease Threats--A Prevention Strategy for
the United States. One of the main goals of CDC's plan is to enhance
communication of public health information about emerging diseases so that
prevention measures can be implemented without delay. Emerging Infectious
Diseases is peer reviewed and will be providing information on emerging
infections in three broad categories: 1) Perspectives, a section addressing
factors that underlie disease emergence including microbial adaptation and
change, human demographics and behavior, technology and industry, economic
development and land use, international travel and commerce, and breakdown
of public health measures; 2) Synopses, concise, state-of-the-art summaries
of specific diseases or syndromes and related emerging infectious disease
issues; 3) Dispatches, brief laboratory or epidemiologic reports with an
international scope.
  ------------------------------------------------------------------------

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., Deputy 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

Thomas M. Gomez. D.V.M.,M.S., Staff Epidemiologist
U.S. Department of Agriculture Animal and Plant Health Inspection Service
Riverdale, Maryland, 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.
U.S. Army Medical Research Unit--Kenya
Unit 64109
Box 401
APO AE 09831-4109

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

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

Editorial and Computer Support

Editing and Production
Beatrice T. Divine, M.B.A.
Teresa M. Hood, M.S.

Editorial Assistance
Maria T. Brito

Electronic Distribution
Carol Y. Crawford
David L. Smith
  ------------------------------------------------------------------------

Emerging Infectious Diseases

Emerging Infectious Diseases is published four times a year by the National
Center for Infectious Diseases, Centers for Disease Control and Prevention
(CDC), 1600 Clifton Road, Mailstop C-12, Atlanta, GA 30333, USA. Telephone
404-639-3967, fax 404-639-3039, e-mail eideditor@cidod1.em.cdc.gov.

The opinions expressed by authors contributing to this journal do not
necessarily reflect the opinions of CDC or the institutions with which the
authors are affiliated.

All material published in Emerging Infectious Diseases is in the public
domain and may be used and reprinted without special permission; proper
citation, however, is appreciated.

Use of trade names is for identification only and does not imply
endorsement by the Public Health Service or by the U.S. Department of
Health and Human Services. Emerging Infectious Diseases is printed on acid
free paper that meets the requirements of ANSI/NISO 239.48-1992 (Permanence
of Paper).


---------------------------------------------------------------------------
                         Instructions to Authors

Editorial Policy and Call for Articles

Emerging Infectious Diseases (EID) is a peer-reviewed journal established
expressly to promote the recognition of new and reemerging infectious
diseases and to improve the understanding of factors involved in disease
emergence, prevention, and elimination.

Emerging infections are new or newly identified pathogens or syndromes that
have been recognized in the past two decades. Reemerging infections are
known pathogens or syndromes that are increasing in incidence, expanding
into new geographic areas, affecting new populations, or threatening to
increase in the near future.

EID has an international scope and is intended for professionals in
infectious diseases and related sciences. We welcome contributions from
infectious disease specialists in academia, industry, clinical practice,
and public health as well as from specialists in economics, demography,
sociology, and other disciplines. Inquiries about the suitability of
proposed articles may be directed to the editor at 404-639-3967
(telephone), 404-727-8737 (fax), or eideditor@cidod1.em.cdc.gov (e-mail).

EID is published in English and features three types of articles:
Perspectives, Synopses, and Dispatches. The purpose and requirements of
each type of article are described in detail below. A Spanish version of
the journal's first volume is available electronically from the Universidad
de la Plata, Argentina (ftp://ftp.unlp.edu.ar/pub/EID).
---------------------------------------------------------------------------

Instructions to Authors

Manuscripts should be prepared according to the "Uniform Requirements for
Manuscripts Submitted to Biomedical Journals" (JAMA 1993:269[17]: 2282-6).

Begin each of the following sections on a new page and in this order: title
page, abstract, text, acknowledgments, references, each table, figure
legends, and figures. On the title page, give complete information about
each author (full names and highest degree). Give current mailing address
for correspondence (include fax number and e-mail address). Follow Uniform
Requirements style for references. Consult List of Journals Indexed in
Index Medicus for accepted journal abbreviations. Tables and figures should
be numbered separately (each beginning with 1) in the order of mention in
the text. Double-space everything, including the title page, abstract,
references, tables, and figure legends. Italicize scientific names of
organisms from species name all the way up, except for vernacular names
(viruses that have not really been speciated, such as coxsackievirus and
hepatitis B; bacterial organisms, such as pseudomonads, salmonellae, and
brucellae).

All articles will be reviewed by independent reviewers. The Editor reserves
the right to edit articles for clarity and to modify the format to fit the
publication style of Emerging Infectious Diseases.

Documents sent in hardcopy should also be sent on diskette, or by e-mail.
Acceptable electronic formats for text are ASCII, WordPerfect, AmiPro,
DisplayWrite, MS Word, MultiMate, Office Writer, WordStar, or Xywrite. Send
graphics documents in Corel Draw, Harvard Graphics, Freelance, .TIF (TIFF),
.GIF (CompuServe), .WMF (Windows Metafile), .EPS (Encapsulated Postscript),
or .CGM (Computer Graphics Metafile). The preferred font for graphics files
is Helvetica. If possible, convert Macintosh files into one of the
suggested formats. Submit photographs in glossy, camera-ready photographic
prints.

Send all manuscripts and correspondence to the Editor, Emerging Infectious
Diseases, National Center for Infectious Diseases, Centers for Disease
Control and Prevention, 1600 Clifton Road, Mailstop C-12, Atlanta, GA
30333, USA, or by e-mail on the Internet to eideditor@cidod1.em.cdc.gov.

Perspectives: Contributions to the Perspectives section should provide
insightful analysis and commentary about new and reemerging infectious
diseases or related issues. Perspectives may also address factors known to
influence the emergence of infectious diseases, including microbial
adaption and change; human demographics and behavior; technology and
industry; economic development and land use; international travel and
commerce; and the breakdown of public health measures. Articles should be
approximately 3,500 words and should include references, not to exceed 40.
Use of additional subheadings in the main body of the text is recommended.
If detailed methods are included, a separate section on experimental
procedures should immediately follow the body of the text. Photographs and
illustrations are optional. Provide a short abstract (150 words) and a
brief biographical sketch.

Synopses: Submit concise reviews of infectious diseases or closely related
topics. Preference will be given to reviews of new and emerging diseases;
however, timely updates of other diseases or topics are also welcome.
Synopses should be approximately 3,500 words and should include references,
not to exceed 40. Use of additional subheadings in the main body of the
text is recommended. If detailed methods are included, a separate section
on experimental procedures should immediately follow the body of the text.
Photographs and illustrations are encouraged. Provide a short abstract (150
words) and a brief biographical sketch.

Dispatches: Provide brief updates on trends in infectious diseases or
infectious disease research. Dispatches (1,000 to 1,500 words of text)
should be in a letter to the editor format and should not be divided into
sections. Dispatches should begin with a brief introductory statement about
the relationship of the topic to the emergence of infectious diseases.
Provide references, not to exceed 10, and figures or illustrations, not to
exceed two.
---------------------------------------------------------------------------
---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
Perspectives

Guarding Against the Most Dangerous Emerging Pathogens:
Insights from Evolutionary Biology

Paul W. Ewald
Department of Biology, Amherst College,
Amherst, Massachusetts, USA
---------------------------------------------------------------------------


    Control of emerging infectious diseases will be difficult because
    of the large number of disease-causing organisms that are emerging
    or could emerge and the great diversity of geographic areas in
    which emergence can occur. The modern view of the evolution of
    pathogen virulencespecifically its focus on the tradeoff between
    costs and benefits to the pathogen from increased host
    exploitationallows control programs to identify and focus on the
    most dangerous pathogens (those that can be established with high
    virulence in human populations).

Studies of emerging diseases have focused chiefly on the spectrum of
different emerging pathogens, epidemiologic reasons for emergence, and
interventions to control emergence. The feasibility of disease control is
hampered by the potentially vast number of emerging and reemerging
pathogens, the diversity of geographic sources, the potential for rapid
global dissemination from these sources, and numerous ecologic and social
factors influencing emergence (1-4). Disease control could be made more
manageable if the most dangerous pathogens could be singled out for the
most intense study, surveillance, and control efforts. Experts who have
addressed this problem from an epidemiologic but not an evolutionary
perspective disagree about the feasibility of predicting and preventing the
emergence of the most damaging new pathogens (5-8). In this perspective, I
argue that improved understanding of the evolution of virulence (defined
broadly as the harmfulness of an infection) can make this goal more
feasible in two ways: 1) by facilitating identification and blocking of
pathogens that represent the greatest threat should they become established
in human populations (e.g., Yersinia pestis during the Middle Ages and
human immunodeficiency virus [HIV] during recent decades) and 2) by
providing methods for inhibiting the emergence of particularly virulent
variants of pathogens that are already established in human populations
(e.g., the pathogen that caused the 1918 influenza pandemic and virulent,
antibiotic-resistant strains of Staphylococcus aureus).

Modern understanding of the evolution of virulence focuses on a tradeoff to
which pathogens are subjected: the competitive benefits that pathogens
accrue through increased exploitation of hosts and the costs that result
from any effects of disease that reduce infectious contact between infected
and susceptible hosts. The traditional view presumed that natural selection
would favor evolution toward benign coexistence between host and parasite
(9-12). The modern view, however, stresses that such benign coexistence
will be unstable if pathogens that exploit hosts to a greater degree have
more overall success across transmission cycles than those that achieve
benign coexistence (13-17).

The primary assumption of this evolutionary argument is that increased
toxin virulence is correlated with increased pathogen propagation
(manifested as increases in pathogen reproduction within hosts and/or
pathogen shedding from infected hosts). This correlation need not be strong
across host/pathogen associations for the arguments to be valid;
differences in pathogenic mechanisms, for example, could make the
correlation virtually undetectable when extremely different kinds of
                             [end of page 245]

pathogens are compared. Rather, the tradeoff argument states that for a
given pathogen (with its particular tropisms and pathogenic mechanisms),
mutations that increase the level of host exploitation tend to increase
harmfulness. The association between virulence, exploitation, and pathogen
propagation is expected among "wild type" mutants, but not among novel
laboratory-generated virulent ones. Because there are many routes to
increased virulence and laboratory-generated variants are often not
selected on the basis of competitive superiority in vivo, the increased
virulence of variants generated in the laboratory may not be linked to
propagative superiority. In contrast, natural selection should eliminate
any variants for which increases in virulence are not linked to increases
in pathogen fitness.

The connection between virulence, host exploitation, and pathogen
propagation may be indirect or direct. If the pathogenic mechanism involves
toxin production, a positive association is expected between production and
pathogen propagation. In Vibrio cholerae, for example, high toxin
production is associated with increased densities of vibrios in the fecal
material, apparently as a result of the toxin's flushing of competing
organisms from the intestinal tract (17). In other organisms, the
association between virulence, host exploitation, and pathogen propagation
is more direct. The human plasmodia that reproduce more extensively often
cause more severe illness and are more life-threatening (16). Similarly,
more virulent strains of vector-borne dengue virus reproduce more
extensively in cell culture (18). Growth rates of Salmonella typhimurium
were reduced by eliminating one of its virulence plasmids and inhibiting
the plasmid's expression; introduction of an 8-kb region encoding the spv
genes restored increased growth rate (19). Comparison of Shigella species
suggests a similar association between virulence and pathogen reproduction
(20).

Sexually transmitted pathogens show analogous associations. For the best
studied pathogen, HIV, more rapidly replicative HIVs are associated with
greater cellular destruction in vitro, more rapid destruction of the immune
system, and more rapid onset of AIDS (21-35). Similarly, the more oncogenic
serotypes of human papilloma-viruses (HPV) generate greater numbers of
progeny by interfering with the cell's mechanisms for restricting cell
division (36). For both viruses, increased viral loads are associated with
increased probability of transmission to contacted persons (37-39), and
HIV-1, which propagates to higher densities than HIV-2, is more
transmissible per contact (40).

The association between virulence and viral propagation in pathogens
circulating naturally in human populations therefore supports the modern
emphasis on a tradeoff between the fitness benefits and the costs accrued
by pathogens as a function of changes in host exploitation.

Transmission Associated with High Virulence

Transmission from Immobile Hosts
Like the traditional view of host/parasite coevolution, the modern view
identifies host illness as a potential liability for the pathogen. When
pathogens rely on the mobility of their current host to reach susceptible
hosts, the illness caused by intense exploitation typically reduces the
potential for transmission. The modern perspective on host/parasite
coevolution differs from the traditional one, however, in its emphasis on
weighing these setbacks against the benefits of exploitation: high
virulence can contribute to evolutionary stability if the costs incurred by
parasites from exploitation-induced damage are particularly small and/or
the benefits obtained from exploitation are particularly big. Thus, if host
immobilization has little negative effect on transmission, pathogen
variants that exploit the host so intensely that it is immobilized will
reap the benefits of exploitation. Put more generally, when the costs
incurred from transmission associated with immobilization are small, the
costs of exploitation should outweigh the benefits at a higher level of
exploitationand hence virulencethan would occur if immobilization
severely impaired transmission (16).

Recognizing this version of the general tradeoff led to several
predictions: Because vector-borne parasites can be transmitted effectively
from immobilized hosts, they should evolve to a higher level of virulence
than
                             [end of page 246]

directly transmitted parasites (16). Similarly, aspects of human behavior
and culture can form "cultural vectors," which transmit pathogens from
immobile to susceptible hosts (41). For example, diarrheal pathogens that
are largely waterborne should evolve to relatively high levels of virulence
because effective transmission can occur even when infected hosts are
mobilized: persons carrying contaminated clothing and bedding, the water
used for washing bed sheets, and the movement of contaminated water into
drinking water together act like a swarm of mosquitoes, transmitting
pathogens from the immobilized host. Attendant-borne pathogens should also
become virulent. Attendant-borne transmission often occurs in hospitals,
when nurses and physicians transmit pathogens from one immobilized patient
to another. A reciprocal process occurs when parasites rely on the mobility
of susceptible persons rather than the mobility of the infected hosts to
reach the susceptible persons. Parasites that are durable in the external
environment should thus evolve toward a higher level of virulence than
nondurable pathogens because durable pathogens may remain viable in the
environment until the movement of susceptible individuals brings them into
contact with the pathogens.

Each of these hypotheses has been evaluated and in each case the expected
association occurred: virulence is positively associated with vector-borne
transmission, waterborne transmission, attendant-borne transmission, and
durability in the external environment (Table 1). This evolutionary
framework, therefore, explains the diversity of human parasites in a way
that contrasts starkly with the traditional view. Instead of being seen as
a sign of maladaption, the severity of diseases such as malaria,
tuberculosis, smallpox, cholera, and typhoid fever is seen as a consequence
of evolutionary adaptation because the causative parasites do not rely on
host mobility for transmission. The tradeoffs between the benefits and
costs of exploitation, therefore, favor evolution of relatively high levels
of exploitation for such pathogens and hence high degrees of harm to the
host.

Sexual Transmission
The evolutionary tradeoffs associated with virulence in sexually
transmitted diseases involve the requirements for sexual transmission
imposed on the pathogens by the sexual behavior of the host. Short
durations of infections would be ineffective for most sexually transmitted
pathogens. If people changed sex partners once per year, for example, a
pathogen that was rendered noninfectious by immunologic defenses or the
host's death within a few weeks would have little chance of being
transmitted. To survive, the pathogen must be transmissible for a period
that extends into the time of the next sexual partnership. To prosper, the
pathogen must be transmissible for periods that span more than one change
in sex partners; therefore, sexually transmitted pathogens may often need
cell and tissue tropisms that keep them from being eliminated by the immune
system for relatively long periods.

The evolutionary effects of changes in sexual behavior on virulence may be
strongly influenced by tropisms that were present before the behavior
change. Increased potential for sexual transmission should favor pathogen
variants that reproduce more extensively sooner after the onset of
infection. If the preexisting tropisms target nonessential cell types, this
selection for earlier reproduction will have relatively little effect on
virulence. If, for example, people changed sex partners every few days, the
sexually transmitted pathogen should evolve virulence levels much like
those of respiratory tract pathogens, which rely on host mobility for
transmission. Examples of such pathogens are sexually transmitted
unicellular pathogens such as Neisseria gonorrheae and Chlamydia
trachomatis, which tend to infect mucosal tissues and, therefore, have
relatively minor negative effects on the survival of adult hosts. If,
however, the tropisms involve critical cells, the damage associated with
increased levels of host exploitation should be more severe to the host.
HIV provides an example: HIV has a tropism for helper T cells, which are
critical regulators of immunologic responses. Although a high level of
replication in these cells can be tolerated over short periods, it
eventually leads (by mechanisms that are still being clarified) to the
decimation of this category of cells and the collapse of the immune system.

If these arguments about evolutionary forces and tissue tropisms are
applicable to
                             [end of page 247]

HIV, HIVs should be more virulent in areas where the potential for sexual
transmission is greater. In accordance with this prediction, HIV-2 tends to
be less virulent than HIV-1; moreover, evidence indicates that during the
early years of HIV infection in Africa, HIV-2 tended to be transmitted in
populations having a lower potential for sexual transmission (17,20). The
overall validity of this approach to HIV virulence, however, will be better
tested as different variants of HIV emerge in different geographic regions.
Information about the potential for sexual transmission can help predict
the evolution of HIV virulence in different geographic areas. On the basis
of the evolutionary tradeoffs mentioned above, for example, the type E
HIV-1s that are circulating in Thailand (where the potential for sexual
transmission has been great) are predicted to be particularly virulent
(17). Although this prediction needs to be evaluated rigorously, recently
gathered data support the prediction: the decline in CD4+ cell counts of
persons infected with HIV and the progression of illness in these patients
appear to be particularly rapid in Thailand (43-44).

    Table 1. Categories of pathogens that pose threats of being stably
    harmful in human populations because of reduced dependence on host
    mobility
    --------------------------------------------------------------------
    Characteristics         Association with lethality     Reference
    allowing
    transmission from
    immobile hosts
    --------------------------------------------------------------------
    arthropod-borne         lethality higher among         (16)
    transmission            arthopod-borne pathogens
                            than among directly
                            transmitted pathogens
    water-borne             lethality of diarrheal         (42)
    transmission            bacteria
                            correlated with tendencies
                            for
                            waterborne transmission
    attendant-borne         lethality of E. coli           (20, 41)
    transmission            correlated
                            with duration of
                            attendant-borne cycling
    durability in the       lethality of                   *
    external environment    respiratory-tract
                            pathogens correlated
                            with durability
    --------------------------------------------------------------------
    * B. A. Walther and P. W. Ewald, unpublished manuscript

The most important application of this evolutionary approach to HIV,
however, pertains to interventions that can be used to control the future
evolution of HIV. If the inherent virulences of HIVs depend evolutionarily
on the potential for sexual transmission, interventions that reduce this
potential should have a long-term evolutionary effect, as well as widely
recognized short-term epidemiologic effects in addition to reducing the
spread of HIV infection, such interventions should reduce the harmfulness
per infection. Follow-up of persons infected with HIV-1 for more than a
ecade without deterioration of the immune system indicates that the
mildness of the infections is sometimes attributable to inherently mild
viruses (45-47). The raw material for this evolutionary change, therefore,
appears to be already present in the HIV gene pool.

In Japan, which has a relatively low potential for sexual transmission
(48), type E HIV-1s have recently been introduced from Southeast Asia. If a
low potential for sexual transmission favors evolution toward mildness, the
Japanese type E viruses should become milder over the next few decades.

Assessing the Threat Posed by Pathogens

Assessment Goals
Focusing investigative and intervention efforts on the most significant
disease threats makes sense only if the threats can be reliably assessed.
The long-term threat depends on the evolutionary stability of high pathogen
virulence, and the most dangerous pathogens are those that threaten
widespread persistence with severely damaging manifestations. One of the
most important tasks in controlling emerging diseases is to identify and
block such pathogens during the early stages of emergence, or better yet,
before they emerge. If the most dangerous pathogensthe future analogs of
the causes of AIDS, malaria, smallpox, tuberculosis, and choleracould be
effectively blocked, the effort against emerging diseases would be
successful. If not, the effort may be looked on as a failure in spite of
successes against pathogens that are less able to effectively penetrate
human populations or relatively benign when they do establish
                             [end of page 248]

themselves. The emergence, spread, and persistence of pathogens with the
characteristics of rhinoviruses, for example, would not be looked on as a
great failure. The establishment of such pathogens would hardly be noticed
against the current backdrop of mild to moderately severe respiratory tract
pathogens.

To identify pathogens that must be studied and controlled most intensively,
each pathogen should be assessed for two characteristics that are
associated with high virulence: 1) an ability to spread well from human to
human (directly or indirectly through vectors) rather than infecting humans
as dead-end hosts, and 2) transmission features that select for high levels
of virulence.

The existing associations between virulence and transmission
characteristics (Table 1) can be used to make such identifications. Table 2
offers a checklist that could be applied to each emerging pathogen to
determine whether it makes the first cut in the process of identifying the
most dangerous candidates. Subsequent analyses of the pathogens would then
assess the nature of any barriers that limit the establishment of pathogens
in human populations (e.g., the absence of suitable arthropod vectors for
large proportions of the year).

        Table 2. First-level checklist for identifying the most
        dangerous emerging pathogens. If the answer to any of the
        questions is yes, the potential for continuous transmission
        between humans should be assessed. If this potential is
        high, the pathogen should be considered particularly
        dangerous.
        ------------------------------------------------------------
        Does it have a tendency for waterborne transmission?
        Is it vector-borne with the ability to use humans as part
        of the life cycle?
        If it is directly transmitted, is it durable in the
        external environment?
        Is it attendant-borne?
        Is it needle-borne?*
        If it is sexually transmitted, is it mutation-prone with a
        tropism for critical cell types or does it have invasive or
        oncogenic tendencies?
        ------------------------------------------------------------
        *The hypothesized importance of needleborne transmission
        has not yet been tested; it has been included in this
        listing on the basis of the harmfulness of needleborne
        pathogens and the hypothetical assocations between
        needleborne transmission and virulence (17).

Durability
Although durability in various external environments was quantified in
detail by microbiologists during the first half of this century (49),
modern studies have paid this attribute little attention. Evolutionary
considerations, however, indicate that it should be one of the first
variables quantified when a new pathogen is being studied. If a new,
directly transmitted pathogen can remain viable in the external environment
for many days to many weeks, it falls in the category of especially
dangerous pathogens. If, for example, Ebola virus were viable upon natural
desiccation for weeks instead of hours, its level of host exploitation and
potential for transmission from exploited hosts would not be so mismatched,
and it, like smallpox virus, would pose a much more serious threat.
Durability in the external environment depends largely on environmental
conditions (49), and thus assessments of viability should cover all
feasible environmental conditions.

Vector-borne Transmission
The most serious threat involved in vector-borne transmission comes from
pathogens that can be maintained by human/mosquito cycles but are absent
from suitable areas because of historical accidents or past eradication
campaigns. Dengue and malaria are members of this category; they have the
potential to spiral out of control immediately upon release into areas with
suitable vectors. Nonevolutionary analyses of emerging infections recognize
the threat posed by these pathogens because their damaging effects on human
populations are known.

Vector-borne pathogens that have not used humans as the primary vertebrate
host but may be capable of doing so represent less easily recognized
threats. Evolutionary considerations heighten concern because such
vector-borne pathogens are expected to become increasingly harmful as they
become adapted to human/vector cycles of transmission (16).

Rift Valley fever virus provides an example. For most of this century, this
virus was believed to infect humans only as dead-end hosts. Although it was
vector-borne in ungulates, humans were seen as acquiring the
                             [end of page 249]

infection either when involved in the slaughtering process or when bitten
by mosquitoes that had acquired infection from other vertebrates. Recent
outbreaks have spread to an extent consistent with substantial
human/mosquito cycling, but the existence of such cycling has not been
conclusively documented. If human/mosquito cycling is occurring, the door
is open for further adaptation to humans and for evolution of increased
virulence in humans, increased efficiency of human/vector transmission, and
increased spread through human populations. Rift Valley fever virus
viremias seem sufficient for human/mosquito cycling, and the lethality of
the largest outbreaks was particularly high, as one would expect if some
evolution toward increased virulence accompanied a temporary establishment
of human/mosquito cycles (50-51). To assess the long-term threat posed by
Rift Valley fever virus and to block this virus should it prove to be
particularly threatening, we need to emphasize the following research
priorities: 1) study the transmission of Rift Valley fever virus in
human/mosquito cycles, 2) assess the potential for such transmission over
extended periods, and 3) evaluate the effects of such transmission on virus
virulence.

All emerging vector-borne pathogens need not be viewed as equally
threatening. For example, Borrelia burgdorferi, the agent of Lyme disease
(an emerging vector-borne pathogen in human populations in North America),
does not need to be monitored to avoid its establishment as a human
pathogen because once emerged, it does not threaten to spiral out of
control; it is tick-borne, and ongoing human/tick cycles are not feasible
because of the limited exposure of infected humans to susceptible tick
populations of the appropriate instar. Tick- and mite-borne rickettsiae do
not present a great threat for similar reasons.

Sexual Transmission
The tradeoff concerning sexually transmitted pathogens may prove
particularly useful in identifying pathogens that are capable of sexual
transmission and have cell tropisms that would cause severe damage if host
exploitation increased but have not had high potential for sexual
transmission. Human T-cell lymphotropic virus (HTLV) is in this category,
even though by nonevolutionary criteria it could be dismissed because it
has been geographically widespread in humans for a long time (1). HTLV type
1 (HTLV-I) is less damaging than HIV; it kills or severely handicaps 5% to
10% of the people it infects, generally decades after infection. Although
HTLV-I and HIV infections share many characteristics, HTLV does not have
HIV's high mutation rate and hence does not have the potential for staying
ahead of immune responses and eventually decimating the immune system.
Instead, HTLV relies on modes of transmission that do not expose it to the
immune system: proviral replication through stimulation of host cell
proliferation and transmission through cell-to-cell contact. A concern with
HTLV is that a high potential for sexual transmission may favor increased
rates of viral replication leading to increased exposure to the immune
system and increased mutation rates (48).

A preliminary step toward evaluating the threat posed by the emergence of
particularly virulent HTLVs is assessing whether HTLVs exposed to different
levels of potential for sexual transmission vary in virulence. HTLV-I
infections tend to lead to leukemias and lymphomas at younger ages in
Jamaica, where the potential for sexual transmission is high, than in
Japan, where potential for sexual transmission is low (48). This difference
also occurs among North Americans of Japanese and Caribbean descent (52),
who presumably are infected predominantly (if not exclusively) by Japanese
and Caribbean HTLVs, respectively. The inherent virulence and
mutation-proneness of the Japanese and Caribbean HTLVs need to be assessed.
Similarly, HTLV virulence needs to be better studied in regions of Africa
where it has been long endemic to determine whether variations in HTLV
virulence are correlated with the potential for sexual transmission.

Although mutation-prone sexually transmitted viruses that infect critical
cell types are particularly threatening, sexually transmitted viruses in
general deserve special attention. Even if a sexually transmitted virus
invades only epithelial cells and replicates with low mutation rates, a
high potential for sexual
                             [end of page 250]

transmission may lead to evolution of increased lethality. Death caused by
HTLV-induced lymphomas and leukemias is one manifestation of the danger
posed by an RNA virus that replicates substantially in its DNA form and
hence is in a middle area within the spectrum of mutation-proneness. HPVs
illustrate dangers posed by sexually transmitted viruses that, because they
are DNA viruses, are even further away from HIV on the mutation-proneness
continuum. The mechanism by which HPV nudges infectious cells toward cancer
is associated with increased viral replication; moreover, high potential
for sexual transmission (as indicated by the number of lifetime sex
partners) is a strong risk factor for infection with the more oncogenic HPV
serotypes but not for the mild HPV serotypes (53). This association
supports the idea that reductions in the potential for sexual transmission
should cause evolution of reduced HPV virulence. Specifically, as the
potential for sexual transmission decreases, the risk for acquiring the
oncogenic serotypes (vs benign serotypes) should disproportionately
decrease. Similarly, if interventions prevent the potential for sexual
transmission from increasing, the emergence of oncogenic HPV serotypes
should be disproportionately suppressed.

Waterborne Transmission
Although such pathogens as Vibrio cholerae O139 and Shigella dysenteriae
type 1 threaten emergence in countries with inadequate water supplies, the
threat is much lower in countries with safe water supplies. Although such
pathogens continue to be brought into the countries with safe water
supplies by travelers and commerce, the pathogens show little potential for
emergence. For example, a major epidemic of S. dysenteriae type 1 spread
from Guatemala through Central America during the early 1970s. It entered
the United States in several places but dissipated without any great effort
at containment. Its transmission was studied in a Los Angeles neighborhood,
where each infection gave rise on average to about 0.4 new infections (54).
Without amplification by waterborne transmission, this outbreak, like other
introductions in the United States, was self-limited (54). The situation at
the other end of Central America was similar. The S. dysenteriae epidemic
dissipated as it moved into Costa Rica, where water supplies were
relatively pure (L. J. Mata, pers. comm.).

Attendant-borne Transmission
Emerging hospital-acquired pathogens may pose one of the greatest and most
controllable threats to people in countries like the United States, where
more than 5% of hospital admissions and about 14% of intensive care
patients acquire infections during their stay (55-57). According to some
estimates, nosocomial infections rank among the ten leading causes of death
in the United States (56), with dangerous bloodstream infections
approximately doubling during the 1980s (58).

Although high virulence has been documented in pathogens involved in
nosocomial outbreaks (59-63), the damage caused by nosocomial pathogens has
generally been attributed to the state of hospitalized patients, who may be
compromised by underlying disease, immunosuppressive drugs, and invasive
procedures. These factors, however, do not explain why nosocomial
pathogens, such as Staphylococcus aureus often cause symptomatic infections
in hospital staff (60) but rarely in persons in the outside community. They
also do not explain the association between the extent of nosocomial
transmission and the virulence of infection, or the differences in
symptomatic infections among otherwise healthy babies (17,20,41). In a New
York City hospital, for example, where attendant-borne transmission rates
were very low, only approximately one of 30 babies with S. aureus were
symptomatic (64). Among nosocomial outbreaks of endemic disease, the
analogous proportion may be 5- to 10-fold higher (65).

Without an evolutionary framework for understanding pathogen virulence,
researchers would have no reason for expecting to find particularly
virulent endemic pathogens in hospitals. The only serious attempts to
explain the apparently high-level of pathogen virulence in hospitals
involved the linking of virulence to another characteristic associated with
hospitals: antibiotic resistance. The emergence of antibiotic-resistant
organisms in hospitals in concert with the use of the antibiotics (66) led
researchers to conclude that high levels of antibiotic use caused the
emergence of resistant organisms and to speculate that antibiotic-resistant
organisms might be inherently
                             [end of page 252]

more virulent than their antibiotic-sensitive counterparts (67). Yet when
infections caused by resistant nosocomial organisms are compared with
sensitive (generally nosocomial) infections, the former are only sometimes
found to be associated with more severe infections. Even when they are
associated with more severe disease (62,63), any differences in inherent
virulence tend to be confounded with other factors, such as increased
severity due to lowered effectiveness of antibiotics. The increased
severity of disease, however, is sometimes associated with resistance to
antibiotics other than the one being used (61), suggesting that the
increased damage is not simply a result of ineffective antibiotics. The
presence of virulence-enhancing bacterial characteristics in damaging,
resistant nosocomial strains (63,68) also suggests a link between
nosocomial transmission, antibiotic resistance, and virulence:
antibiotic-resistant strains may have been particularly virulent because
they were nosocomial, but this virulence was not apparent in many of the
comparisons because the sensitive strains were also nosocomial.

Although the controversy regarding virulence and antibiotic resistance in
hospital-acquired infections can be explained by the hypothesized
connection between attendant-borne transmission and the evolution of both
virulence and antibiotic resistance, none of the investigations of the
topic made measurements that would allow assessment of the connection
between attendant-borne transmission and the emergence of variants with
increased virulence. The critical measure is the harmfulness per person
housing the organisms in question, and the critical comparison is between
nosocomial and community-acquired strains. Among persons that harbor
nosocomial strains of S. aureus, for example, the proportion that show
symptomatic infection could be compared with the analogous proportion of
matched persons who are harboring community strains. After
virulence-enhancing mechanisms are well understood, pathogens can be
assayed for their virulence directly. Thus Clostridium difficile pathogens
isolated from prolonged nosocomial outbreaks are predicted to be more
toxigenic than C. difficile isolated from the outside community. Similarly,
nosocomial Escherichia coli are predicted to have virulence-enhancing
characteristics (e.g., invasiveness, adherence) (69) more often than
community strains.

Further knowledge about virulence enhancing mechanisms and development of
techniques for rapid detection (e.g., [72-75]) should offer opportunities
for carefully controlled experiments to test whether reduction in
attendant-borne transmission causes a greater decline in the inherent
virulence of nosocomial pathogens in experimental hospitals than in control
hospitals in which interventions are not imposed. Long-term follow-up
should clarify the degree to which attendant-borne transmission may foster
the emergence of virulent variants among both established human pathogens
(e.g., S. aureus, E. coli) and new or newly recognized pathogens (e.g.,
Serratia spp., and Pseudomonas aeruginosa).

Harmful, often antibiotic-resistant, hospital-acquired pathogens can
readily emerge beyond a hospital's boundary, when patients are moved, or
attendants move between hospitals; the documentation is particularly strong
for dangerous variants of E. coli and S. aureus (62,74-78). The degree to
which emerging nosocomial pathogens spill over to generate outbreaks in the
outside community is not well understood, but evidence suggests that this
spillover represents a substantial threat when the organisms can infect
healthy people. When large-scale community-wide epidemics of pathogenic E.
coli have occurred, for example, transmission in hospitals often was
strongly implicated. During 1953 and 1954, an E. coli epidemic advanced up
the East Coast of the United States from the Carolinas through New England;
"As it spread, explosive outbreaks were limited to institutions, hospital
wards, and newborn nurseries" (59). A focal study of the U.S. Army Hospital
at Fort Belvoir, Virginia, indicated that the epidemic strain was brought
into the hospital by infected people in the community, with the proportion
of inpatient to outpatient cases reversing dramatically during the
hospital's 5-month outbreak (59). Similarly, during the winter of 1961, in
an outbreak in Chicago and adjacent communities in Indiana, about 5% of the
infants were affected, and nearly half of the affected infants had direct
or indirect contact with one of the 29 involved hospitals just before their
illnesses (75).

Studies of S. aureus have also shown that nosocomial and community
outbreaks are sometimes synchronous with transmission occurring in both
directions between the hospital and the outside community (79-80). The
long-term consequences of emergence of nosocomial strains for the outside
community, however, still need to be assessed. The possibility that
nosocomial pathogens may tend to be not only more resistant to antibiotics,
but also more inherently virulent lends some urgency to this need.

Almost no work has been done to determine the potential of pathogens
thought to be almost exclusively associated with nosocomial infection
(e.g., Enterococcus, C. difficile) to take hold in the outside community.
The high durability in the external environment of many nosocomial
pathogens heightens the need for additional information. Durable pathogens
that can infect uncompromised hosts (e.g., antibiotic-resistant S. aureus
and to a lesser extent C. difficile) possess the basic characteristics that
damaging organisms need to spread in the outside community. Durable
organisms unable to infect healthy people pose a relatively low threat, but
this inability is often presumed. Any transmission of durable nosocomial
organisms like P. aeruginosa from patients after discharge heightens the
threat to the outside community by providing an avenue for further
adaptation to humans. Molecular analyses that allow reconstruction of
epidemiologic patterns (e.g., molecular phylogenetics) could be used to
improve assessments of the degree to which nosocomial pathogens can emerge
in the outside community; such studies need to provide quantitative
assessments not only of the threats posed by nosocomial pathogens in their
current state, but also of their potential to breach by evolution the
barriers that have inhibited their broader spread in the past.

Conceptual Innovation, Explanatory Power, and Precision

Dangerous Emergences of the Past
Each of the organisms that caused devastating epidemics over the past 5
centuries, would have been identified as an extremely dangerous pathogen by
the criteria proposed here. Y. pestis, for example, is durable in the
external environment (49) and is vector-borne. Its threat is lower now than
centuries ago when fleas and rats were abundant domiciliary inhabitants,
but it still represents a threat where these hosts are present.

The periodic emergence of yellow fever in European and American cities
during the 18th and 19th centuries took a heavy toll; the 1878 epidemic,
for example, killed about a quarter of the population of Memphis, Tennessee
(81). If yellow fever virus were first encountered today, it would be
recognized as an important threat because it is vector-borne and can be
transmitted indefinitely through human/mosquito cycles.

With regard to the emergence of virulent variants from established
pathogens, the influenza viruses circulating at the Western Front during
World War I would be considered dangerous because barriers to transmission
from immobile hosts were removed by cultural practices and because
influenza virus is mutation prone (17,20). It is, therefore, not surprising
that the Western Front has been identified as the source of the highly
lethal variants of the 1918 influenza pandemic and that a pandemic of this
severity has never recurred (17). More importantly, evolutionary
considerations suggest that such a lethal pandemic will not recur unless
influenza viruses are again exposed to opportunities that allow
transmission from immobile hosts, as they are on poultry farms where highly
lethal influenza outbreaks periodically emerge (17).

Uncertainty about the Dangerous Epidemics of the Future
These arguments about the evolution of virulence provide only coarse
approximations of the selective processes in pathogen populations. To
determine whether the implications of these arguments need to be
substantially modified, we need empirical studies that evaluate these
arguments against alternative explanations. Considering the current state
of uncertainty, some might argue that it is dangerous to incorporate the
current coarse understanding of the evolution of virulence into policy
making. But failing to incorporate this understanding is dangerous.
                             [end of page 253]

If we do not adjust investments to take into account the evolutionary
arguments, and the arguments prove correct, the reduction in death and
illness per unit investment will be lower than it could have been. If we do
adjust investments on the basis of these evolutionary arguments, and the
arguments prove wrong, the nonevolutionary benefits of the investments
would still be obtained.

Although the precise mechanisms that increase virulence in pathogens in the
high-risk categories still need to be clarified, the associations (Table 1)
are strong. One could argue, for example, that durable or waterborne
pathogens are more harmful because hosts tend to pick up a greater
diversity of genotypes from the environment when pathogens are more durable
or are mixed in water; if the within-host genetic variability of such
pathogens is greater, they would have more potential for within-host
competition, which could favor the evolution of increased virulence. By
this argument, factors such as durability, vector-borne transmission, and
waterborne transmission would increase virulence indirectly by increasing
within-host genetic variation. With regard to the prevention of the
emergence of highly virulent disease, uncertainties about mechanisms are
not critical. Whether the effects of these factors are direct or indirect,
elimination of the factors should discourage the emergence of severe
disease and favor the decrease of highly virulent pathogens.

Decisions to invest in interventions without certainty about mechanisms is
not new to the health sciences. The hygienic interventions to control
hospital acquired diseases and the purification of water supplies to
control cholera were appropriately advocated on the basis of epidemiologic
data (from Ignaz Semmelweis and John Snow) a half century before the
causative agents of these or any other infectious diseases were first
identified. Jenner's smallpox vaccine program was accepted globally more
than a century before viruses were discovered or the mechanisms by which
vaccines provide protection were understood. Even now the mechanisms by
which the immune system provides protection encompass major areas of
uncertainty. This uncertainty is evidenced, for example, by the
controversies about the importance of the different legs of the immune
system (such as cytotoxic T cells, neutralizing antibody, and subsets of
helper T cells) in HIV pathogenesis.

If the evolutionary arguments are correct, the emergence of the most
harmful diseases can be countered not only for pathogens that are
recognized as threats but also for those posing threats that are not yet
recognized. Providing pure water supplies, reducing attendant-borne
transmission, and reducing vector-borne transmission preferentially from
ill people (e.g., by providing mosquito-proof houses [17]) should guard
against the emergence of virulent pathogens, whether the pathogens are
unidentified or are highly virulent variants of identified human pathogens.
An understanding of the evolutionary determinants of virulence may thus
make surveillance and prompt intervention much more manageable.

The emphasis thus is on suppression of the emergence of particularly
virulent variants rather than suppression of the emergence of new disease
organisms. The expectation is that the frequency of disease will drop even
though the frequency of individuals harboring organisms may decline little
if at all. The data on decentralization of nursery/maternity wards, for
example, indicate that the rates of nosocomial infection decline among
mothers and babies, even though the rates at which babies harbor pathogens
(colonization plus infection) do not decline (82). Indeed the disagreement
about the value of rooming-in as a mode of infection control (82) can be
attributed to a failure to distinguish the prevalence of disease organisms
from the prevalence of disease. Controversies about the value of waterborne
transmission can be traced to a similar failure (17).

The lead article of the first issue of this journal was entitled, "Emerging
infections: getting ahead of the curve" (4). I propose that integrating
evolutionary principles with epidemiology would enhance our ability to stay
ahead of the curve. Evolutionary insights should increase our ability to
distinguish emerging pathogens according to the long-term threat that they
pose and thereby adjust investments in accordance with the threat.
Knowledge of the evolution of virulence should also guide us to identify
for each pathogen the critical data that will allow us to make this
assessment. Finally, evolutionary considerations should allow
identification of
                             [end of page 254]

infrastructural investments that will guard against the most dangerous
pathogens, even if they are not blocked by surveillance and containment
efforts and even if they have not yet been identified or are never
identified as emerging pathogens.

    Dr. Ewald is a professor in the Department of Biology at Amherst
    College. Trained in ecology and evolutionary biology, he works at
    the interface of these areas with epidemiology, focusing on the
    evolution of virulence among infectious diseases of humans and
    insects.

Address for correspondence: Paul W. Ewald, Department of Biology, Amherst
College, Amherst, MA 01002-5000; fax: 413-542-7955; e-mail:
pwewald@amherst.edu.

References

  1. Morse SM. Emerging viruses: defining the rules for viral traffic.
     Perspect Biol Med 1991;34:387-409.
  2. Morse SM. Examining the origins of emerging viruses, In: Morse SM,
     editor. Emerging viruses. New York: Oxford, 1993;10-28.
  3. Morse SM. Factors in the emergence of infectious disease. Emerging
     Infectious Diseases 1995;1:7-15.
  4. Satcher D. Emerging infections: getting ahead of the curve. Emerging
     Infectious Diseases 1995;1:1-6.
  5. Henderson DA. Surveillance systems and intergovernmental cooperation.
     In: Morse SM, editor. Emerging viruses. New York: Oxford, 1993;283-9.
  6. Holland J. Replication error, quasispecies populations, and extreme
     evolution rates of RNA viruses. In: Morse SM, editor. Emerging
     viruses. New York, Oxford, 1993;203-18.
  7. Krause RM. Foreword. In: Morse SM, editor. Emerging viruses. New York:
     Oxford, 1993;xvii-xix.
  8. McNeill WH. Patterns of disease emergence in history. In: Morse SM,
     editor. Emerging viruses. New York: Oxford, 1993;29-36.
  9. Smith T. Parasitism and disease. Princeton: Princeton University
     Press, 1934.
 10. Dubos R. Man adapting. New Haven, Conn: Yale University Press, 1965.
 11. Burnet FM, White DO. Natural history of infectious disease, 4th ed.
     Cambridge: Cambridge University Press, 1972.
 12. Thomas L. Notes of a biology-watcher: Germs. N Engl J Med
     1972;247:553-5.
 13. Levin S, Pimentel D. Selection of intermediate rates of increase in
     parasite-host systems. American Naturalist 1981;117:308-15.
 14. Levin BR, Allison AC, Bremermann HJ, Cavalli-Sforza LL, Clarke BC,
     B\Frentzel-Beyme R, et al. Evolution of parasites and hosts. Group
     report, In: Anderson RM, May RM, editors. Population Biology of
     Infectious Diseases. Berlin: Springer-Verlag 1982;213-43.
 15. Anderson RM, May RM. Coevolution of hosts and parasites. Parasitology
     1982;85:411-26.
 16. Ewald PW. Host-parasite relations, vectors, and the evolution of
     disease severity. Annual Review of Ecology and Systematics
     1983;14:465-85.
 17. Ewald PW. Evolution of Infectious Disease. New York: Oxford University
     Press, 1994.
 18. Morens DM, Marchette NJ, Chu MC, Halstead SB. Growth of dengue type-2
     virus isolates in human peripheral blood leukocytes correlates with
     severe and mild dengue disease. Am J Trop Med Hyg 1991;45:644-51.
 19. Gulig PA, Doyle TJ. The Salmonella typhimurium virulence plasmid
     increases the growth rate of salmonellae in mice. Infect Immun
     1993;61:504-11.
 20. Ewald PW. Transmission modes and the evolution of virulence, with
     special reference to cholera, influenza and AIDS. Human Nature
     1991;2:1-30.
 21. sj B, Morfeldt-Mnson L, Albert J, Biberfeld G, Karlsson A, Lidman
     K, et al. Replicative capacity of human immunodeficiency virus from
     patients with varying severity of HIV infection. Lancet
     1986;334:660-2.
 22. ChengMayer C, Seto D, Tateno M, Levy JA. Biologic features of HIV-1
     that correlate with virulence in the host. Science 1988;240:80-2.
 23. Albert J, Bttiger B, Biberfeld G, Feny EM. Replicative and
     cytopathic characteristics of HIV-2 and severity of infection. Lancet
     1989;333:852-3.
 24. Feny EM, Albert J, sj B. Replicative capacity, cytopathic effects
     and cell tropism of HIV. AIDS 1989;3:S5-12.
 25. Levy JA. Human immunodeficiency viruses and the pathogenesis of AIDS.
     JAMA 1989;261:2997-3006.
 26. Tersmette M, Gruters RA, de Wolf F, de Goede REY, Lange JMA,
     Schellekens PTA, et al. Evidence for a role of virulent human
     immunodeficiency virus (HIV) variants in the pathogenesis of acquired
     immunodeficiency syndrome: Studies on sequential HIV isolates. J Virol
     1989;63:2118-25.
 27. Tersmette M, Lange JMA, de Goede REY, de Wolf F, Eefink Shaattenkerk
     JKM, Schellekens PTA, et al. Association beteween biological
     properties of human immunodeficiency virus variants and risk for AIDS
     and AIDS mortality. Lancet 1989;333:983-5.
 28. Ma X, Sakai K, Sinangil F, Golub E, Volsky DJ. Interaction of a
     noncytopathic human immunodeficiency virus type 1 (HIV-1) with target
     cells: Efficient virus entry followed by delayed expression of its RNA
     and protein. Virology 1990;176:184-94.
 29. Schneweis KE, Kleim J-P, Bailly E, Niese D, Wagner N, Brackmann HH.
     Graded cytopathogenicity of the human immunodeficiency virus (HIV) in
     the course of HIV infection. Med Microbiol Immunol 1990;179:193-203.
 30. Gruters RA, Terpstra FG, DeGoede REY, Mulder JW, DeWolf F, Schellekens
     PTA, et al. Immunological and virological markers in individuals
     progressing from seroconversion to AIDS. AIDS 1991;5:837-44.
                                [end of page 255]

 31. Schellekens PTA, Tersmette M, Roos MTL, Keet RP, Dewolf F, Coutinho
     RA, et al. Biphasic rate of CD4+ cell count decline during progression
     to AIDS correlates with HIV-1 phenotype. AIDS 1992;6:665-9.
 32. Hirsch I, Salaun D, Brichacek B, Chermann JR. HIV-1
     cytopathogenicity-genetic difference between direct cytotoxic and
     fusogenic effect. Virology 1992;186:647-54.
 33. Koot M, Keet IPM, Vos AHV, DeGoede REY, Roos MTL, Coutinho, RA, et al.
     Prognostic value of HIV-1 syncytium-inducing phenotype for rate of
     CD4+ cell depletion and progression to AIDS. Ann Intern Med
     1993;118:681-8.
 34. Connor RI, Mohri H, Cao YZ, Ho DD. Increased viral burden and
     cytopathicity correlate temporally with CD4+ T lymphocyte decline and
     clinical progression in human immunodeficiency virus type 1- infected
     individuals. J Virol 1993;67:1772-7.
 35. Connor RI, Ho DD. Human immunodeficiency virus type 1 variants with
     increased replicative capacity develop during the asymptomatic stage
     before disease progression. J Virol 1994;68:4400-8.
 36. Fisher SG. Epidemiology: a tool for the study of human
     papillomavirus-related carcinogenesis. Intervirology 1994;37:215-25.
 37. Puel J, Lheritier D, Guyader M, Izopet J, Briant L, Tricoire J, et al.
     Viral load and mother-to-infant HIV transmission. Lancet 1992;340:859.
 38. Weiser B, Nachman S, Tropper P, Viscosi KH, Grimson R, Baxter G, et
     al. Quantitation of human immunodeficiency virus type 1 during
     pregnancy: relationship of viral titer to mother-to-child transmission
     and stability of viral load. Proc Natl Acad Sci USA 1994;91:8037-41.
 39. Kaye JN, Cason J, Pakarian F, Jewers R, Kell B, Bible J, et al. Viral
     load as a determinant for transmission of human papillomavirus type 16
     from mother to child. J Med Virol 1994;44:415-21.
 40. Adjorloto-Johnson G, De Cock KM, Ekpini E, Vetter KM, Sibailly T,
     Brattegaard K, et al. Prospective comparison of mother-to-child
     transmission of HIV-1 and HIV-2 in Abidjan, Ivory Coast. JAMA
     1994;272:462-6.
 41. Ewald PW. Cultural vectors, virulence, and the emergence of
     evolutionary epidemiology. Oxford Surveys in Evolutionary Biology
     1988;5:215-45.
 42. Ewald PW. Waterborne transmission and the evolution of virulence among
     gastrointestinal bacteria. Epidemiol Infect 1991;106:83-119.
 43. Weniger BG, Tansuphaswadikul S, Young NL, Pau CP, Lohsomboon P,
     Yindeeyoungyeon W, et al. Differences in immune function among
     patients infected with distinct Thailand HIV-1 strains, In:
     Proceedings of the Tenth International Conference on AIDS. Yokohama,
     Japan, 7-12 August 1994; Abstract O12C.
 44. Kitayaporn D, Tansuphaswadikul S, Lohsomboon P, Pannachet K,
     Kaewkungwal J, Limpakarnjanarat K, et al. Survival of AIDS patients in
     the emerging epidemic in Bangkok, Thailand. J Acquir Immune Defic
     Syndr Hum Retrovirol 1996;11:77-82.
 45. Learmont J, Tindall B, Evans L, Cunningham A, Cunningham P, Wells J,
     et al. Long-term symptomless HIV-1 infection in recipients of blood
     products from a single donor. Lancet 1992;340:863-7.
 46. Cao YZ, Qin LM, Zhang LQ, Safrit J, Ho DD. Virologic and immunologic
     characterization of long-term survivors of human immunodeficiency
     virus type 1 infection. N Engl J Med 1995;332:201-8.
 47. Kirchhoff F, Greenough TC, Brettler DB, Sullivan JL, Desrosiers RC.
     Brief report: absence of intact nef sequences in a long-term survivor
     with nonprogressive HIV-1 infection. N Engl J Med 1995;332:228-32.
 48. Ewald PW. Evolution of mutation rate and virulence among human
     retroviruses. Phil Trans Roy Soc Lond, Ser B Biol Sci 1995;346:333-43.
 49. Mitscherlich E, Marth EH. Microbial Survival in the Environment.
     Berlin, Germany: Springer-Verlag, 1984.
 50. Meegan JM. Rift Valley fever in Egypt: an overview of the epizootics
     in 1977 and 1978. Contributions to Epidemiology and Biostatistics
     1981;3:100-13.
 51. House JA, Turell MJ, Mebus CA. Rift valley fever: present status and
     risk to the western hemisphere. Ann NY Acad Sci 1992;653:233-42.
 52. Levine PH, Manns A, Jaffe ES, Colclough G, Cavallaro A, Reddy G, et
     al. The effect of ethnic differences on the pattern of
     HTLV-I-associated T-cell leukemia/lymphoma (HATL) in the United
     States. Int J Cancer 1994;56:177-81.
 53. Franco EF, Villa LL, Ruiz A, Costa MC. Transmission of cervical human
     papillomavirus infection by sexual activity: differences between low
     and high oncogenic risk types. J Infect Dis 1995;172:756-63.
 54. Weissman JB, Murton KI, Lewis JN, Friedemann CHT, Gangarosa EJ. Impact
     in the U.S. of the Shiga dysentery pandemic of Central America and
     Mexico: A review of surveillance data through 1972. J Infect Dis
     1974;129:218-23.
 55. Dixon RE. Effect of infections on hospital care. Ann Intern Med
     1978;89:749-53.
 56. Haley RW, Culver DH, White JW, Morgan WM, Emori TG. The nationwide
     nosocomial infection rate: A new need for vital statistics. Am J
     Epidemiol 1985;121:159-67.
 57. Raju TNK, Kobler C. Improving handwashing habits in the newborn
     nurseries. Am J Med Sci 1991;302:355-8.
 58. Pittet D, Wenzel RP. Nosocomial bloodstream infections. Arch Intern
     Med 1995;155:1177-84.
 59. Belnap D, O'Donnell JJ. Epidemic gastroenteritis due to Escherichia
     coli 0:111. J Pediat 1955;47:178-93.
 60. Rountree P, Freeman BM. Infections caused by a particular phage type
     of Staphylococcus aureus. Med J Austr 1955;2:157-61.
 61. Jessen O, Rosendal K, Blow P, Faber V, Eriksen KR. Changing
     staphylococci and staphylococcal infections. A ten-year study of
     bacteria and cases of bacteremia. N Engl J Med 1969;281:627-35.
 62. Locksley RM, Cohen ML, Quinn TC, Tompkins LS, Coyle MB, Kirihara JM,
     et al. Multiply antibiotic-resistant Staphylococcus aureus:
     introduction, transmission, and evolution of nosocomial infection. Ann
     Intern Med 1982;97:317-24.
 63. Rello J, Torres A, Ricart M, Valles J, Gonzalez J, Artiga A, et al.
     Ventilator-associated pneumonia by Staphylococcus aureus. Am J Respir
     Crit Care Med 1994;150:1545-9.
 64. Holzman R, Florman A, Lyman M. Gentamicin resistant and sensitive
     strains of S. aureus. Factors affecting colonization and virulence for
     infants in a special care nursery. Am J Epidemiol 1980;112:352-61.
 65. Gezon HM, Rogers KD, Thompson DJ, Hatch TF. Some controversial aspects
     in the epidemiology of hospital nursery staphylococcal infections. Am
     J Public Health 1960;50:473-84.
 66. Gezon HM, Schaberg MJ, Klein JO. Concurrent epidemics of
     Staphylococcus aureus and group A streptococcus disease in a newborn
     nursery-control with penicillin G and hexachlorophene bathing.
     Pediatrics 1973;51:383-90.
 67. Craven DE, Reed C, Kollisch N, DeMaria A, Lichtenberg D, Shen K, et
     al. A large outbreak of infections caused by a strain of
     Staphylococcus aureus resistant to oxacillin and aminoglycosides. Am J
     Med 1981;71:53-8.
 68. Huebner J, Pier GB, Maslow JN, Muller E, Shiro H, Parent M, et al.
     Endemic nosocomial transmission of Staphylococcus epidermidis
     bacteremia isolates in a neonatal intensive care unit over 10 years. J
     Infect Dis 1994;169:526-31.
 69. Donnenberg MS, Kaper JB. Enteropathogenic Escherichia coli. Infect
     Immun 1992;60:3953-61.
 70. Donnenberg MS, Tacket CO, James SP, Losonsky G, Nataro JP, Wasserman
     SS, et al. Role of the eaeA gene in experimental enteropathogenic
     Escherichia coli infection. J Clin Invest 1993;92:1412-7.
 71. Franke J, Franke S, Schmidt H, Schwarzkopf A, Wieler LH, Baljer G, et
     al. Nucleotide sequence analysis of enteropathogenic Escherichia coli
     (EPEC) adherence factor probe and development of PCR for rapid
     detection of EPEC harboring virulence plasmids. J Clin Microbiol
     1994;32:2460-3.
 72. Schmidt H, Plaschke B, Franke S, Russman H, Schwarzkopf A, Heesemann
     J, et al. Differentiation in virulence patterns of Escherichia coli
     possessing eae genes. Med Microbiol Immunol 1994;183:23-31.
 73. Spitz J, Yuhan R, Koutsouris A, Blatt C, Alverdy J, Gecht G.
     Enteropathogenic Escherichia coli adherence to intestinal epithelial
     monolayers diminishes barrier function. Am J Physiol 1995;268:G374-9.
 74. Rogers KB, Koegler SJ. Inter-hospital cross-infection of epidemic
     infantile gastro-enteritis associated with type strains of Bacterium
     coli. Journal of Hygiene 1951;49:152-61.
 75. Marcy SM: Microorganisms responsible for neonatal diarrhea, in
     Remington JS, Klein JO, editors. Infectious diseases of the fetus and
     newborn infant. Philadelphia: WB Saunders, 1976:892-978.
 76. Saroglou G, Cromer M, Bisno AL. Methicillin-resistant Staphylococcus
     aureus:interstate spread of nosocomial infections with emergence of
     gentamicin-methicillin resistant strains. Infection Control
     1980;1:81-9.
 77. Pavillard R, Harvey K, Douglas D, Hewstone A, Andrew J, Collopy B, et
     al. Epidemic of hospital-acquired infection due to
     methicillin-resistant Staphylococcus aureus in major Victorian
     hospitals. Med J Aust 1982;1:451-4.
 78. Lyon BR, Iuorio JL, May JW, Skurray RA. Molecular epidemiology of
     multiresistant Staphylococcus aureus in Australian hospitals. J Med
     Entomol 1984;17:79-89.
 79. Gooch JJ, Britt EM. Staphylococcus aureus colonization and infection
     in newborn nursery patients. American Journal of Diseases of Children
     1978;132:893-6.
 80. Saravolatz LD, Markowitz N, Arking L, Pohlod D, Fisher E.
     Methicillin-resistant Staphylococcus aureus. Ann Intern Med
     1982;96:11-6.
 81. Keating JM. History of the yellow fever epidemic of 1878 in Memphis,
     Tennessee. Cincinnati, Ohio: Wrightson, 1879.
 82. Daschner F. Infectious hazards in rooming-in systems. J Perinat Med
     1984;12:3-6.

                             [end of page 256]
---------------------------------------------------------------------------

---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
Perspectives

Social Inequalities and Emerging Infectious Diseases

Paul Farmer
Harvard Medical School and
Brigham and Women's Hospital
Boston, Massachusetts, USA
---------------------------------------------------------------------------


    Although many who study emerging infections subscribe to
    social-production-of-disease theories, few have examined the
    contribution of social inequalities to disease emergence. Yet such
    inequalities have powerfully sculpted not only the distribution of
    infectious diseases, but also the course of disease in those
    affected. Outbreaks of Ebola, AIDS, and tuberculosis suggest that
    models of disease emergence need to be dynamic, systemic, and
    critical. Such modelswhich strive to incorporate change and
    complexity, and are global yet alive to local variationare
    critical of facile claims of causality, particularly those that
    scant the pathogenic roles of social inequalities. Critical
    perspectives on emerging infections ask how large-scale social
    forces influence unequally positioned individuals in increasingly
    interconnected populations; a critical epistemology of emerging
    infectious diseases asks what features of disease emergence are
    obscured by dominant analytic frameworks. Research questions
    stemming from such a reexamination of disease emergence would
    demand close collaboration between basic scientists, clinicians,
    and the social scientists and epidemiologists who adopt such
    perspectives.

The past decade has been one of the most eventful in the long history of
infectious diseases. There are multiple indexes of these events and of the
rate at which our knowledge base has grown. The sheer number of relevant
publications indicates explosive growth; moreover, new means of monitoring
antimicrobial resistance patterns are being used along with the rapid
sharing of information (as well as speculation and misinformation) through
means that did not exist even 10 years ago. Then there are the microbes
themselves. One of the explosions in questionperhaps the most remarked
uponis that of "emerging infectious diseases." Among the diseases
considered "emerging," some are regarded as genuinely new; AIDS and
Brazilian purpuric fever are examples. Others have newly identified
etiologic agents or have again burst dramatically onto the scene. For
example, the syndromes caused by Hantaan virus have been known in Asia for
centuries but now seem to be spreading beyond Asia because of ecologic and
economic transformations that increase contact between humans and rodents.
Neuroborreliosis was studied long before the monikers Lyme disease and
Borrelia burgdorferi were coined, and before suburban reforestation and
golf courses complicated the equation by creating an environment agreeable
to both ticks and affluent humans. Hemorrhagic fevers, including Ebola,
were described long ago, and their etiologic agents were in many cases
identified in previous decades. Still other diseases grouped under the
"emerging" rubric are ancient and well-known foes that have somehow
changed, in pathogenicity or distribution. Multidrug-resistant tuberculosis
(TB) and invasive or necrotizing Group A streptococcal infection are cases
in point.

Like all new categories, "emerging infectious diseases" has benefits and
limitations. The former are well known: a sense of urgency, notoriously
difficult to arouse in large bureaucracies, has been marshaled, funds have
been channeled, conferences convened, articles written, and a journal
dedicated to the study of these diseases has
                             [end of page 259]

been founded. The research and action agendas elaborated in response to the
perceived emergence of new infections have been, by and large, sound. But
the concept, like some of the diseases associated with it, is complex. Its
complexity has, in some instances, hampered the learning process. A richly
textured understanding of emerging infections will be grounded in critical
and reflexive study of how learning occurs. Units of analysis and key terms
will be scrutinized and defined more than once. This process will include
regular rethinking not only of methods and study design, but also of the
validity of causal inference and reflection on the limits of human
knowledge. This study of the process, loosely known as epistemology, often
happens in retrospect, but many of the chief contributors to the growing
research in emerging infectious diseases have examined the epistemologic
issues surrounding their work and are familiar with the multifactorial
nature of disease emergence: "Responsible factors include ecological
changes, such as those due to agricultural or economic development or to
anomalies in the climate; human demographic changes and behavior; travel
and commerce; technology and industry; microbial adaptation and change; and
breakdown of public health measures" (1). A recent Institute of Medicine
report on emerging infections does not even categorize microbial threats by
type of agent, but rather according to factors held to be related to their
emergence (2).

In studying emerging infectious diseases, many thus make a distinction
between a host of phenomena directly related to human actionsfrom improved
laboratory techniques and scientific discovery to economic "development,"
global warming, and failures of public healthand another set of phenomena,
much less common and related to changes in the microbes themselves. Close
examination of microbial mutations often shows that, again, human actions
have played a large role in enhancing pathogenicity or increasing
resistance to antimicrobial agents. In one long list of emerging viral
infections, for example, only the emergence of Rift Valley fever is
attributed to a possible change in virulence or pathogenicity, and this
only after other, social factors for which there is better evidence (1). No
need, then, to call for a heightened awareness of the sociogenesis, or
"anthropogenesis," of emerging infections. Some bench scientists in the
field are more likely to refer to social factors and less likely to make
immodest claims of causality about them than are behavioral scientists who
study disease. Yet a critical epistemology of emerging infectious diseases
is still in its early stages of development; a key task of such a critical
approach would be to take existing conceptual frameworks, including that of
disease emergence, and ask, What is obscured in this way of conceptualizing
disease? What is brought into relief? A first step in understanding the
"epistemological dimension" of disease emergence, notes Eckardt, involves
developing "a certain sensitivity to the terms we are used to" (3).

A heightened sensitivity to other common rubrics and terms shows that
certain aspects of disease emergence are brought into relief while others
are obscured. When we think of "tropical diseases," malaria comes quickly
to mind. But not too long ago, malaria was an important problem in areas
far from the tropics. Although there is imperfect overlap between malaria
as currently defined and the malaria of the mid-19th century, some U.S.
medical historians agree with contemporary assessments: malaria "was the
most important disease in the country at the time." In the Ohio River
Valley, according to Daniel Drake's 1850 study, thousands died in seasonal
epidemics. During the second decade of the 20th century, when the
population of 12 southern states was approximately 25 million, an estimated
million cases of malaria occurred each year. Malaria's decline in this
country was "due only in small part to measures aimed directly against it,
but more to agricultural development and other factors some of which are
still not clear" (4). These factors include poverty and social
inequalities, which led, increasingly, to differential morbidity with the
development of improved housing, land drainage, mosquito repellents, nets,
and electric fansall well beyond the reach of those most at risk for
malaria. In fact, many "tropical" diseases predominantly affect the poor;
the groups at risk for these diseases are
                             [end of page 260]

often bounded more by socioeconomic status than by latitude.

Similarly, the concept of "health transitions" is influential in what some
have termed "the new public health" and in the international financial
institutions that so often direct development efforts (5). The model of
health transitions suggests that nation-states, as they develop, go through
predictable epidemiologic transformations. Death due to infectious causes
is supplanted by death due to malignancies and to complications of coronary
artery disease, which occur at a more advanced age, reflecting progress.
Although it describes broad patterns now found throughout the world, the
concept of national health transitions also masks other realities,
including intranational illness and death differentials that are more
tightly linked to local inequalities than to nationality. For example, how
do the variables of class and race fit into such paradigms? In Harlem,
where the age-specific death rate in several groups is higher than in
Bangladesh, leading causes of death are infectious diseases and violence
(6).

Units of analysis are similarly up for grabs. When David Satcher, director
of the Centers for Disease Control and Prevention (CDC), writing of
emerging infectious diseases, reminds us that "the health of the individual
is best ensured by maintaining or improving the health of the entire
community" (7), we should applaud his clearsightedness but go on to ask,
What constitutes "the entire community"? In the 1994 outbreak of
cryptosporidiosis in Milwaukee, for example, the answer might be "part of a
city" (8). In other instances, community means a village or the passengers
on an airplane. But the most common unit of analysis in public health, the
nation-state, is not all that relevant to organisms such as dengue virus,
Vibrio cholerae O139, human immunodeficiency virus (HIV),
penicillinase-producing Neisseria gonorrhoeae, and hepatitis B virus. Such
organisms have often ignored political boundaries, even though their
presence may cause a certain degree of turbulence at national borders. The
dynamics of emerging infections will not be captured in national analyses
any more than the diseases are contained by national boundaries, which are
themselves emerging entitiesmost of the world's nations are, after all,
20th-century creations.

Here I have discussed the limitations of three important ways of viewing
the health of populationstropical medicine, "the" epidemiologic
transition, and national health profilesbecause models and even
assumptions about infectious diseases need to be dynamic, systemic, and
critical. That is, models with explanatory power must be able to track
rapidly changing clinical, even molecular, phenomena and link them to the
large-scale (sometimes transnational) social forces that manifestly shape
the contours of disease emergence. I refer, here, to questions less on the
order of how pig-duck agriculture might be related to the antigenic shifts
central to influenza pandemics, and more on the order of the following: Are
World Bank policies related to the spread of HIV, as has recently been
claimed (9)? What is the relationship between international shipping
practices and the spread of cholera from Asia to South America and
elsewhere in the Western Hemisphere (10,11)? How is genocide in Rwanda
related to cholera in Zaire (12)?

The study of anything said to be emerging tends to be dynamic. But the very
notion of emergence in heterogeneous populations poses questions of
analysis that are rarely tackled, even in modern epidemiology, which, as
McMichael has recently noted, "assigns a primary importance to studying
interindividual variations in risk. By concentrating on these specific and
presumed free-range individual behaviors, we thereby pay less attention to
the underlying social-historical influences on behavioral choices,
patterns, and population health" (13). A critical (and self-critical)
approach would ask how existing frameworks might limit our ability to
discern trends that can be linked to the emergence of diseases. Not all
social-production-of-disease theories are equally alive to the importance
of how relative social and economic positioninginequalityaffects risk for
infection. In its report on
                             [end of page 261]

emerging infections, the Institute of Medicine lists neither poverty nor
inequality as "causes of emergence" (2).

A critical approach pushes the limits of existing academic politesse to ask
harder and rarely raised questions: What are the mechanisms by which
changes in agriculture have led to outbreaks of Argentine and Bolivian
hemorrhagic fever, and how might these mechanisms be related to
international trade agreements, such as the General Agreement on Tariffs
and Trade and the North American Free Trade Agreement? How might
institutional racism be related to urban crime and the outbreaks of
multidrug-resistant TB in New York prisons? Does the privatization of
health services buttress social inequalities, increasing risk for certain
infectionsand deathamong the poor of sub-Saharan Africa and Latin
America? How do the colonial histories of Belgium and Germany and the
neocolonial histories of France and the United States tie in to genocide
and a subsequent epidemic of cholera among Rwandan refugees? Similar
questions may be productively posed in regard to many diseases now held to
be emerging.

Emerging How and to What Extent? The Case of Ebola

Hemorrhagic fevers have been known in Africa since well before the
continent was dubbed "the white man's grave," an expression that, when
deployed in reference to a region with high rates of premature death,
speaks volumes about the differential valuation of human lives. Ebola
itself was isolated fully two decades ago (14). Its appearance in human
hosts has at times been insidious but more often takes the form of
explosive eruptions. In accounting for recent outbreaks, it is unnecessary
to postulate a change in filovirus virulence through mutation. The
Institute of Medicine lists a single "factor facilitating emergence" for
filoviruses: "virus-infected monkeys shipped from developing countries via
air" (2).

Other factors are easily identified. Like that of many infectious diseases,
the distribution of Ebola outbreaks is tied to regional trade networks and
other evolving social systems. And, like those of most infectious diseases,
Ebola explosions affect, researchers aside, certain groups (people living
in poverty, health care workers who serve the poor) but not others in close
physical proximity. Take, for example, the 1976 outbreak in Zaire, which
affected 318 persons. Although respiratory spread was speculated, it has
not been conclusively demonstrated as a cause of human cases. Most expert
observers thought that the cases could be traced to failure to follow
contact precautions, as well as to improper sterilization of syringes and
other paraphernalia, measures that in fact, once taken, terminated the
outbreak (15). On closer scrutiny, such an explanation suggests that Ebola
does not emerge randomly: in Mobutu's Zaire, one's likelihood of coming
into contact with unsterile syringes is inversely proportional to one's
social status. Local lites and sectors of the expatriate community with
access to high-quality biomedical services (viz., the European and American
communities and not the Rwandan refugees) are unlikely to contract such a
disease.

The changes involved in the disease's visibility are equally embedded in
social context. The emergence of Ebola has also been a question of our
consciousness. Modern communications, including print and broadcast media,
have been crucial in the construction of Ebolaa minor player,
statistically speaking, in Zaire's long list of fatal infectionsas an
emerging infectious disease (16). Through Cable News Network (CNN) and
other television stations, Kikwit became, however briefly, a household word
in parts of Europe and North America. Journalists and novelists wrote
best-selling books about small but horrific plagues, which in turn became
profitable cinema. Thus, symbolically and proverbially, Ebola spread like
wildfireas a danger potentially without limit. It emerged.

Emerging From Where? The Case of TB

TB is said to be another emerging disease, in which case, emerging is
synonymous with reemerging. Its recrudescence is often attributed to the
advent of HIVthe Institute of Medicine lists "an increase in
immunosuppressed populations" as the sole
                             [end of page 262]

factor facilitating the resurgence of TB (2)and the emergence of drug
resistance. A recent book on TB, subtitled "How the battle against
tuberculosis was wonand lost," argues that "Throughout the developed
world, with the successful application of triple therapy and the
enthusiastic promotion of prevention, the death rate from tuberculosis came
tumbling down" (17). But was this claim ever documented? Granted, the
discovery of effective anti-TB therapies has saved the lives of hundreds of
thousands of TB patients, many in industrialized countries. But TBonce the
leading cause of death among young adults in the industrialized worldwas
already declining there well before the 1943 discovery of streptomycin. In
the rest of the world, and in pockets of the United States, TB remains
undaunted by ostensibly effective drugs, which are used too late,
inappropriately, or not at all: "It is sufficiently shameful," notes one of
the world's leading authorities on TB, "that 30 years after recognition of
the capacity of triple-therapy . . . to elicit 95%+ cure rates,
tuberculosis prevalence rates for many nations remain unchanged" (18). Some
estimate that more than 1.7 billion persons are infected with quiescent,
but viable, Mycobacterium tuberculosis and, dramatic shifts in local
epidemiology aside, a global analysis does not suggest major decreases in
the importance of TB as a cause of death. TB has retreated in certain
populations, maintained a steady state in others, and surged forth in still
others, remaining, at this writing, the world's leading infectious cause of
adult deaths (19).

At mid-century, TB was still acknowledged as the "great white plague." What
explains the invisibility of this killer by the 1970s and 1980s? Again, one
must turn to the study of disease awareness, that is, of consciousness and
publicity, and their relation to power and wealth. "The neglect of
tuberculosis as a major public health priority over the past two decades is
simply extraordinary," wrote Murray in 1991. "Perhaps the most important
contributor to this state of ignorance was the greatly reduced clinical and
epidemiologic importance of tuberculosis in the wealthy nations" (20). Thus
TB has not really emerged so much as emerged from the ranks of the poor
(21,22). An implication, clearly, is that one place for diseases to hide is
among poor people, especially when the poor are socially and medically
segregated from those whose deaths might be considered more important.

When complex forces move more poor people into the United States, an
increase in TB incidence is inevitable. In a recent study of the disease
among foreign-born persons in the United States, immigration is essentially
credited with the increased incidence of TB-related disease (23). The
authors note that in some of the immigrants' countries of origin the annual
rate of infection is up to 200 times that registered in the United States;
moreover, many persons with TB in the United States live in homeless
shelters, correctional facilities, and camps for migrant workers. But there
is no discussion of poverty or inequality, even though these are, along
with war, leading reasons for both the high rates of TB and for immigration
to the United States. "The major determinants of risk in the foreign-born
population," conclude the authors, "were the region of the world from which
the person emigrated and the number of years in the United States."

Going Where? The Case of HIV

To understand the complexity of the issuesmedical, social, and
communicationalthat surround the emergence of a disease into public view,
consider AIDS. In the early 1980s, the public was informed by health
officials that AIDS had probably emerged from Haiti. In December 1982, for
example, a physician affiliated with the National Cancer Institute was
widely quoted in the popular press stating that "We suspect that this may
be an epidemic Haitian virus that was brought back to the homosexual
population in the United States" (24). This proved incorrect, but not
before damage to Haitian tourism had been done. Result: more poverty, a yet
steeper slope of inequality and vulnerability to disease, including AIDS.
The label "AIDS vector" was also damaging to the million or so Haitians
living elsewhere in the Americas and certainly hampered public health
efforts among them (25).

HIV disease has since become the most extensively studied infection in
                             [end of page 263]

human history. But some questions are much better studied than are others.
And error is worth studying, too. Careful investigation of the mechanisms
by which immodest claims are propagated (as regards Haiti and AIDS, these
mechanisms included "exoticization" of Haiti, racism, the existence of
influential folk models about Haitians and Africans, and the conflation of
poverty and cultural difference) is an important yet neglected part of a
critical epistemology of emerging infectious diseases. Also
underinvestigated are considerations of the pandemic's dynamic. HIV may not
have come from Haiti, but it was going to Haiti. Critical reexamination of
the Caribbean AIDS pandemic showed that the distribution of HIV does not
follow national borders, but rather the contours of a transnational
socioeconomic order. Furthermore, much of the spread of HIV in the 1970s
and 1980s moved along international "fault lines," tracking along steep
gradients of inequality, which are also paths of migrant labor and sexual
commerce (26).

In an important overview of the pandemic's first decade, Mann and
co-workers observe that its course "within and through global society is
not being affectedin any serious mannerby the actions taken at the
national or international level" (27). HIV has emerged but is going where?
Why? And how fast? The Institute of Medicine lists several factors
facilitating the emergence of HIV: "urbanization; changes in
lifestyles/mores; increased intravenous drug abuse; international travel;
medical technology" (2). Much more could be said. HIV has spread across the
globe, often wildly, but rarely randomly. Like TB, HIV infection is
entrenching itself in the ranks of the poor or otherwise disempowered.
Take, as an example, the rapid increase in AIDS incidence among women. In a
1992 report, the United Nations observed that "for most women, the major
risk factor for HIV infection is being married. Each day a further three
thousand women become infected, and five hundred infected women die" (28).
It is not marriage per se, however, that places young women at risk.
Throughout the world, most women with HIV infection, married or not, are
living in poverty. The means by which confluent social forces, such as
gender inequality and poverty, come to be embodied as risk for infection
with this emerging pathogen have been neglected in biomedical,
epidemiologic, and even social science studies on AIDS. As recently as
October 199415 years into an ever-emerging pandemica Lancet editorial
could comment, "We are not aware of other investigators who have considered
the influence of socioeconomic status on mortality in HIV-infected
individuals" (29). Thus, in AIDS, the general rule that the effects of
certain types of social forces on health are unlikely to be studied applies
in spite of widespread impressions to the contrary.

AIDS has always been a strikingly patterned pandemic. Regardless of the
message of public health slogans"AIDS is for Everyone"some are at high
risk for HIV infection, while others, clearly, are at lower risk.
Furthermore, although AIDS eventually causes death in almost all
HIV-infected patients, the course of HIV disease varies. Disparities in the
course of the disease have sparked the search for hundreds of cofactors,
from Mycoplasma and ulcerating genital lesions to voodoo rites and
psychological predisposition. However, not a single association has been
compellingly shown to explain disparities in distribution or outcome of HIV
disease. The only well-demonstrated cofactors are social inequalities,
which have structured not only the contours of the AIDS pandemic, but also
the course of the disease once a patient is infected (30-33). The advent of
more effective antiviral agents promises to heighten those disparities even
further: a three-drug regimen that includes a protease inhibitor will cost
$12,000 to $16,000 a year (34).

Questions for a Critical Epistemology of Emerging Infectious Diseases

Ebola, TB, and HIV infection are in no way unique in demanding
contextualization through social science approaches. These approaches
include the grounding of case histories and local epidemics in the larger
biosocial systems in which they take shape and demand exploration of social
inequalities. Why, for example, were there 10,000 cases of diphtheria in
Russia from 1990 to 1993? It is easy enough to argue that the excess cases
were due to a failure to
                             [end of page 264]

vaccinate (35). But only in linking this distal (and, in sum, technical)
cause to the much more complex socioeconomic transformations altering the
region's illness and death patterns will compelling explanations emerge
(36,37).

Standard epidemiology, narrowly focused on individual risk and short on
critical theory, will not reveal these deep socioeconomic transformations,
nor will it connect them to disease emergence. "Modern epidemiology,"
observes one of its leading contributors, is "oriented to explaining and
quantifying the bobbing of corks on the surface waters, while largely
disregarding the stronger undercurrents that determine where, on average,
the cluster of corks ends up along the shoreline of risk" (13). Neither
will standard journalistic approaches add much: "Amidst a flood of
information," notes the chief journalistic chronicler of disease emergence,
"analysis and context are evaporating . . . Outbreaks of flesh eating
bacteria may command headlines, but local failures to fully vaccinate
preschool children garner little attention unless there is an epidemic"
(38).

Research questions identified by various blue-ribbon panels are important
for the understanding and eventual control of emerging infectious diseases
(39,40). Yet both the diseases and popular and scientific commentary on
them pose a series of corollary questions, which, in turn, demand research
that is the exclusive province of neither social scientists nor bench
scientists, clinicians, or epidemiologists. Indeed, genuinely
transdisciplinary collaboration will be necessary to tackle the problems
posed by emerging infectious diseases. As prolegomena, four areas of
corollary research are easily identified. In each is heard the recurrent
leitmotiv of inequality:

Social Inequalities
Study of the reticulated links between social inequalities and emerging
disease would not construe the poor simply as "sentinel chickens," but
instead would ask, What are the precise mechanisms by which these diseases
come to have their effects in some bodies but not in others? What
propagative effects might social inequalities per se contribute (41)? Such
queries were once major research questions for epidemiol and social
medicine but have fallen out of favor, leaving a vacuum in which immodest
claims of causality are easily staked. "To date," note Krieger and
co-workers in a recent, magisterial review, "only a small fraction of
epidemiological research in the United States has investigated the effects
of racism on health" (42). They join others in noting a similar dearth of
attention to the effects of sexism and class differences; studies that
examine the conjoint influence of these social forces are virtually
nonexistent (43,44).

And yet social inequalities have sculpted not only the distribution of
emerging diseases, but also the course of disease in those affected by
them, a fact that is often downplayed: "Although there are many
similarities between our vulnerability to infectious diseases and that of
our ancestors, there is one distinct difference: we have the benefit of
extensive scientific knowledge" (7). True enough, but Who are "we"? Those
most at risk for emerging infectious diseases generally do not, in fact,
have the benefit of cutting-edge scientific knowledge. We live in a world
where infections pass easily across borderssocial and geographicwhile
resources, including cumulative scientific knowledge, are blocked at
customs.

Transnational Forces
"Travel is a potent force in disease emergence and spread," as Wilson has
reminded us, and the "current volume, speed, and reach of travel are
unprecedented" (45). Although the smallpox and measles epidemics following
the European colonization of the Americas were early, deadly reminders of
the need for systemic understandings of microbial traffic, there has been,
in recent decades, a certain reification of the notion of the "catchment
area." A useful means of delimiting a sphere of actiona district, a
county, a countryis erroneously elevated to the status of explanatory
principle whenever the geographic unit of analysis is other than that
defined by the disease itself. Almost all diseases held to be emergingfrom
the increasing number of drug-resistant diseases
                             [end of page 265]

to the great pandemics of HIV infection and cholerastand as modern rebukes
to the parochialism of this and other public health constructs (46). And
yet a critical sociology of liminalityboth the advancing, transnational
edges of pandemics and also the impress of human-made administrative and
political boundaries on disease emergencehas yet to be attempted.

The study of borders qua borders means, increasingly, the study of social
inequalities. Many political borders serve as semipermeable membranes,
often quite open to diseases and yet closed to the free movement of cures.
Thus may inequalities of access be created or buttressed at borders, even
when pathogens cannot be so contained. Research questions might include,
for example, What effects might the interface between two very different
types of health care systems have on the rate of advance of an emerging
disease? What turbulence is introduced when the border in question is
between a rich and a poor nation? Writing of health issues at the
U.S.-Mexican border, Warner notes that "It is unlikely that any other
binational border has such variety in health status, entitlements, and
utilization" (47). Among the infectious diseases registered at this border
are multidrug-resistant TB, rabies, dengue, and sexually transmitted
diseases including HIV infection (said to be due, in part, to "cross-border
use of `red-light' districts").

Methods and theories relevant to the study of borders and emerging
infections would come from disciplines ranging from the social sciences to
molecular biology: mapping the emergence of diseases is now more feasible
with the use of restriction fragment length polymorphism and other new
technologies (48). Again, such investigations will pose difficult questions
in a world where plasmids can move, but compassion is often grounded.

The Dynamics of Change
Can we elaborate lists of the differentially weighted factors that promote
or retard the emergence or reemergence of infectious diseases? It has been
argued that such analyses will perforce be historically deep and
geographically broad, and they will at the same time be processual,
incorporating concepts of change. Above all, they will seek to incorporate
complexity rather than to merely dissect it. As Levins has recently noted,
"effective analysis of emerging diseases must recognize the study of
complexity as perhaps the central general scientific problem of our time"
(49). Can integrated mathematical modeling be linked to new ways of
configuring systems, avoiding outmoded units of analyses, such as the
nation-state, in favor of the more fluid biosocial networks through which
most pathogens clearly move? Can our embrace of complexity also include
social complexity and the unequal positioning of groups within larger
populations? Such perspectives could be directed towards mapping the
progress of diseases from cholera to AIDS, and would permit us to take up
more unorthodox research subjectsfor example, the effects of World Bank
projects and policies on diseases from onchocerciasis to plague.

Critical Epistemology
Many have already asked, What qualifies as an emerging infectious disease?
More critical questions might include, Why do some persons constitute "risk
groups," while others are "individuals at risk"? These are not merely
nosologic questions; they are canonical ones. Why are some approaches and
subjects considered appropriate for publication in influential journals,
while others are dismissed out of hand? A critical epistemology would
explore the boundaries of polite and impolite discussion in science. A
trove of complex, affect-laden issuesattribution of blame to perceived
vectors of infection, identification of scapegoats and victims, the role of
stigmaare rarely discussed in academic medicine, although they are
manifestly part and parcel of many epidemics.

Finally, why are some epidemics visible to those who fund research and
services, while others are invisible? In its recent statements on TB and
emerging infections, for example, the World Health Organization uses the
threat of contagion to motivate wealthy nations to invest in disease
surveillance and control out of self-interestan age-old public health
approach acknowledged
                             [end of page 266]

in the Institute of Medicine's report on emerging infections: "Diseases
that appear not to threaten the United States directly rarely elicit the
political support necessary to maintain control efforts" (2). If related to
a study under consideration, questions of power and control over funds,
must be discussed. That they are not is more a marker of analytic failures
than of editorial standards.



Ten years ago, the sociologist of science Bruno Latour reviewed hundreds of
articles appearing in several Pasteur-era French scientific reviews to
constitute what he called an "anthropology of the sciences" (he objected to
the term epistemology). Latour cast his net widely. "There is no essential
difference between the human and social sciences and the exact or natural
sciences," he wrote, "because there is no more science than there is
society. I have spoken of the Pasteurians as they spoke of their microbes"
(50) (Here, perhaps, is another reason to engage in a "proactive" effort to
explore themes usually relegated to the margins of scientific inquiry:
those of us who describe the comings and goings of microbesfeints,
parries, emergences, retreatsmay one day be subjected to the scrutiny of
future students of the subject).

Microbes remain the world's leading causes of death (51). In "The conquest
of infectious diseases: who are we kidding?" the authors argue that
"clinicians, microbiologists, and public health professionals must work
together to prevent infectious diseases and to detect emerging diseases
quickly" (52). But past experience with epidemics suggests that other
voices and perspectives could productively complicate the discussion. In
every major retrospective study of infectious disease outbreaks, the
historical regard has shown us that what was not examined during an
epidemic is often as important as what was (53,54) and that social
inequalities were important in the contours of past disease emergence. The
facts have taught us that our approach must be dynamic, systemic, and
critical. In addition to historians, then, anthropologists and sociologists
accountable to history and political economy have much to add, as do the
critical epidemiologists mentioned above (55-58).

My intention, here, is ecumenical and complementary. A critical framework
would not aspire to supplant the methods of the many disciplines, from
virology to molecular epidemiology, which now concern themselves with
emerging diseases. "The key task for medicine," argued the pioneers
Eisenberg and Kleinman some 15 years ago, "is not to diminish the role of
the biomedical sciences in the theory and practice of medicine but to
supplement them with an equal application of the social sciences in order
to provide both a more comprehensive understanding of disease and better
care of the patient. The problem is not `too much science,' but too narrow
a view of the sciences relevant to medicine" (59).

A critical anthropology of emerging infections is young, but it is not
embryonic. At any rate, much remains to be done and the tasks themselves
are less clear perhaps than their inherent difficulties. The philosopher
Michel Serres once observed that the border between the natural and the
human sciences was not to be traced by clean, sharp lines. Instead, this
border recalled the Northwest Passage: long and perilously complicated, its
currents and inlets often leading nowhere, dotted with innumerable islands
and occasional floes (60). Serres' metaphor reminds us that a sea change is
occurring in the study of infectious disease even as it grows, responding,
often, to new challengesand sometimes to old challenges newly perceived.

Acknowledgments

The author acknowledges the editorial suggestions of Cassis Henry, Harvard
Medical School, and Haun Saussy, Stanford University.

    Dr. Farmer, an anthropologist/physician, is assistant professor of
    social medicine at the Harvard Medical School and divides his
    clinical practice between the Brigham and Women's Hospital and the
    Clinique Bon Sauveur in rural Haiti, where he directs the TB unit.
    His books
                             [end of page 267]

    include AIDS and Accusation and The Uses of Haiti; he is the editor
    of Women, Poverty and AIDS: Sex, Drugs, and Structural Violence.

Address for correspondence: Paul Farmer, Harvard Medical School, Department
of Social Medicine, 641 Huntington Avenue, Boston, MA 02115 USA; fax:
617-432-2565; e-mail: pefarmer@bics.bwh.harvard.edu.

References

  1. Morse S. Factors in the emergence of infectious diseases. Emerging
     Infectious Diseases 1995;1:7-15.
  2. Lederberg J, Shope RE, Oaks SC. Emerging infections: microbial threats
     to health in the United States. Washington, D.C.: National Academy
     Press, 1992.
  3. Eckardt I. Challenging complexity: conceptual issues in an approach to
     new disease. Ann New York Acad Sci 1994;740:408-17.
  4. Levine N. Editor's preface to selections from Drake D. Malaria in the
     interior valley of North America. Urbana: University of Illinois
     Press, 1964 (1850).
  5. Frenk J, Chacon F. Bases conceptuales de la nueva salud internacional.
     Salud Pblica Mx 1991;33;307-13.
  6. McCord C, Freeman H. Excess mortality in Harlem. N Engl J Med,
     1990;322:173-7.
  7. Satcher D. Emerging infections: getting ahead of the curve. Emerging
     Infectious Diseases 1995;1:1-6.
  8. MacKenzie W, Hoxie N, Proctor M, Gradus MS, Blair KA, Peterson DE, et
     al. A massive outbreak in Milwaukee of Cryptosporidium infection
     transmitted through the water supply. N Engl J Med 1994;331:161-7.
  9. Lurie P, Hintzen P, Lowe RA. Socioeconomic obstacles to HIV prevention
     and treatment in developing countries: the roles of the International
     Monetary Fund and the World Bank. AIDS 1995;9:539-46.
 10. World Health Organization. Cholera in the Americas. Weekly Epidemiol
     Rec. 1992;67:33-9.
 11. McCarthy S, McPhearson R, Guarino A. Toxigenic Vibrio cholera O1 and
     cargo ships entering the Gulf of Mexico. Lancet 1992;339:624.
 12. Goma Epidemiology Group. Public health impact of Rwandan refugee
     crisis: what happened in Goma, Zaire, in July, 1994? Lancet
     1995;345:339-44.
 13. McMichael A. The health of persons, populations, and planets:
     epidemiology comes full circle. Epidemiology 1995;6:633-6.
 14. Johnson KM, Webb PA, Lange JV, Murphy FA. Isolation and partial
     characterization of a new virus causing acute hemorrhagic fever in
     Zaire. Lancet 1977;1:569-71.
 15. World Health Organization. Ebola haemorrhagic fever in Zaire, 1976.
     Report of an international commission. Bull WHO 1978;56:271-93.
 16. Garrett L. The Coming Plague. New York: Farrar, Straus and Giroux,
     1995.
 17. Ryan F. The forgotten plague: how the battle against tuberculosis was
     wonand lost. Boston: Little, Brown, 1993.
 18. Iseman M. Tailoring a time-bomb. Am Rev Respir Dis 1985;132:735-6.
 19. Bloom B, Murray C. Tuberculosis: commentary on a resurgent killer.
     Science 1992;257:1055-63.
 20. Murray C. Social, economic and operational research on tuberculosis:
     recent studies and some priority questions. Bull Int Union Tuberc Lung
     Dis 1991;66:149-56.
 21. Farmer P, Robin S, Ramilus St-L, Kim J. Tuberculosis and "compliance":
     lessons from rural Haiti. Seminars in Respiratory Infections
     1991;6:373-9.
 22. Spence D, Hotchkiss J, Williams C, Davies P. Tuberculosis and poverty.
     British Medical Journal 1993;307:759-61.
 23. McKenna MT, McCray E, Onorato I. The epidemiology of tuberculosis
     among foreign-born persons in the United States, 1986 to 1993. N Engl
     J Med 1995;332:1071-6.
 24. Chabner B. Cited in the Miami News, December 2, 1982;8A.
 25. Farmer P. AIDS and Accusation: Haiti and the Geography of Blame.
     University of California Press, Berkeley, 1992.
 26. Farmer P. The exotic and the mundane: human immunodeficiency virus in
     the Caribbean. Human Nature 1990;1:415-45.
 27. Mann J, Tarantola D, Netter T. AIDS in the world. Cambridge, MA:
     Harvard University Press, 1992.
 28. United Nations Development Program. Young women: silence,
     susceptibility and the HIV epidemic. New York, UNDP, 1992.
 29. Sampson J, Neaton J. On being poor with HIV. Lancet 1994;344:1100-1.
 30. Chaisson RE, Keruly JC, Moore RD. Race, sex, drug use, and progression
     of human immunodeficiency virus disease. N Engl J Med 1995;333:751-6.
 31. Farmer P, Connors M, and Simmons J, eds. Women, poverty, and AIDS:
     sex, drugs, and structural violence. Monroe, ME: Common Courage Press,
     1996.
 32. Fife E, Mode C. AIDS incidence and income. JAIDS 1992;5:1105-10.
 33. Wallace R, Fullilove M, Fullilove R, Gould P, Wallace D. Will AIDS be
     contained within U.S. minority populations? Soc Sci Med
     1994;39:1051-62.
 34. Waldholz M. Precious pills: new AIDS treatment raises tough question
     of who will get it. Wall Street Journal, July 3, 1996, p. 1.
 35. Centers for Disease Control and Prevention. Diphtheria
     outbreak--Russian Federation, 1990-1993. MMWR 1993;42:840-1,847.
 36. Field M. The health crisis in the former Soviet Union: a report from
     the `post-war' zone. Soc Sci Med 1995;1469-78.
 37. Patz J, Epstein P, Burke T, Balbus J. Global climate change and
     emerging infectious diseases. JAMA 1996;275:217-33.
                                [end of page 268]

 38. Garrett L. Public health and the mass media. Current Issues in Public
     Health 1995;1:147-50.
 39. Centers for Disease Control and Prevention. Addressing emerging
     infectious disease threats: a prevention strategy for the United
     States. Atlanta, USA. Department of Health and Human Services, 1994.
 40. Roizman B., ed. Infectious diseases in an age of change: the impact of
     human ecology and behavior on disease transmission. Washington, D.C.:
     National Academy Press, 1995.
 41. Farmer P. On suffering and structural violence: a view from below.
     Daedalus 1996;125:261-83.
 42. Krieger N, Rowley D, Herman A, Avery B, Phillips M. Racism, sexism,
     and social class: implications for studies of health, disease, and
     well-being. Am J Prev Med 1993; (Supplement) 9:82-122.
 43. Navarro V. Race or class versus race and class: mortality
     differentials in the United States. Lancet 1990;336:1238-40.
 44. Marmot M. Social differentials in health within and between
     populations. Daedalus 1994;123:197-216.
 45. Wilson M. Travel and the emergence of infectious diseases. Emerging
     Infectious Diseases 1995;1:39-46.
 46. Haggett P. Geographical aspects of the emergence of infectious
     diseases. Geogr Ann 1994;76:91-104.
 47. Warner DC. Health issues at the US-Mexican border. JAMA
     1991;265:242-7.
 48. Small P, Moss A. Molecular epidemiology and the new tuberculosis.
     Infect Agents Dis 1993;2:132-8.
 49. Levins R. Preparing for Uncertainty. Ecosystem Health 1995;1:47-57.
 50. Latour B. The pasteurization of France. Sheridan A, Law J, trans.
     Cambridge, MA: Harvard University Press, 1988.
 51. Global Health Situation and Projections. Geneva: World Health
     Organization, 1992.
 52. Berkelman RL, Hughes JM. The conquest of infectious diseases: who are
     we kidding? Ann Int Med 1993;119:426-8.
 53. Epstein PR. Pestilence and poverty--historical transitions and the
     great pandemics. Am J Prev Med 1992;8:263-78.
 54. Packard R. White plague, black labor: tuberculosis and the political
     economy of health and disease in South Africa. Berkeley: University of
     California Press.
 55. Aach P, Carr-Hill R, Curtis S, Illsley R. Les ingalits sociales de
     sant en France et en Grande-Bretagne. Paris: INSERM, 1987.
 56. Fassin D. Exclusion, underclass, marginalidad. Revue Franaise de
     Sociologie 1996;37:37-75.
 57. Inhorn M, Brown P, eds. The anthropology of infectious diseases. New
     York: Gordon and Breach, 1996.
 58. Krieger N, Zierler S. What explains the public's health? A call for
     epidemiologic theory. Epidemiology 1996;7:107-9.
 59. Eisenberg L, Kleinman A. The relevance of social science to medicine.
     Dordrecht: Reidel, 1981.
 60. Serres M. Le passage du nord-ouest. Paris: Editions de Minuit, 1980.

                             [end of page 269]

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

---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
Synopses

Molecular Mechanisms of Bacterial Virulence: Type III Secretion and
Pathogenicity Islands

Joan Mecsas and Evelyn J. Strauss
Stanford University School of Medicine, Stanford, California
---------------------------------------------------------------------------

    Recently, two novel but widespread themes have emerged in the
    field of bacterial virulence: type III secretion systems and
    pathogenicity islands. Type III secretion systems, which are found
    in various gram-negative organisms, are specialized for the export
    of virulence factors delivered directly to host cells. These
    factors subvert normal host cell functions in ways that seem
    beneficial to invading bacteria. The genes encoding several type
    III secretion systems reside on pathogenicity islands, which are
    inserted DNA segments within the chromosome that confer upon the
    host bacterium a variety of virulence traits, such as the ability
    to acquire iron and to adhere to or enter host cells. Many of
    these segments of DNA appear to have been acquired in a single
    step from a foreign source. The ability to obtain complex
    virulence traits in one genetic event, rather than by undergoing
    natural selection for many generations, provides a mechanism for
    sudden radical changes in bacterial-host interactions. Type III
    secretion systems and pathogenicity islands must have played
    critical roles in the evolution of known pathogens and are likely
    to lead to the emergence of novel infectious diseases in the
    future.

Discovery of Two Traits Used by a Broad Range of Bacterial Pathogens
In the past decade, there has been an explosion of new information about
bacterial pathogens as researchers have begun to examine the molecular and
genetic bases of microbial pathogenicity. Because microbes invade many
niches in humans and cause a wide variety of syndromes, it initially
appeared that each disease might be created by a distinct molecular
mechanism. However, the spectrum of methods is not as broad as first
imagined; rather, bacteria exploit a number of common molecular tools to
achieve a range of goals (1). Among these tools are pathogenicity islands,
which enable bacteria to gain complex virulence traits in one step, and
type III secretion systems, which provide a means for bacteria to target
virulence factors directly at host cells. These factors then tamper with
host cell functions to the pathogens' benefit.

Early in the search for virulence genes, researchers discovered that many
of these genes resided on plasmids or phages; however, it was also clear
that these genes did not produce all of the physiologic changes induced in
host cells by various pathogens (2). Thus, researchers searched the
chromosome. Surprisingly, as when found on plasmids, virulence genes often
clustered in functionally related groups. Furthermore, these groups often
appeared to have been acquired from another organism, as features of their
DNA sequence differed from the bulk of the genome. These observations gave
rise to the concept of pathogenicity islandsdiscrete segments of DNA that
encode virulence traits and often appear to have a foreign origin (3,4).

Researchers found that a particular set of virulence genes appeared several
times on both plasmids and pathogenicity islands (5-8). These genes were
discovered in both plant and animal pathogens and were homologous
                             [end of page 271]

to genes encoded on a virulence plasmid of pathogenic Yersinia spp. (Table
1) (9-12). The Yersinia proteins are the components of a novel secretion
system (13), called type III (14). This machinery propels effector
molecules toward host cells where they alter host physiology (15,16). The
homology suggested that many divergent bacterial pathogens had acquired a
similar system from a common source. Pathogens use the type III system to
secrete different effector molecules that influence host cells in a variety
of ways (16-19).


[Figure 1] [Figures not available in ASCII]
Figure 1: Schematic diagram of type I, type II, and type III secretion systems.
All systems use the energy of ATP hydrolysis to drive secretion. Type I and type
III secrete proteins across both the inner membrane and the cell envelope (outer
membrane) in one step; secreted proteins do not make an intermediate stop in the
periplasm, as they do in type II secretion. Type I and type III systems are also
similar in that they do not remove any part of the secreted protein. In contrast,
the N-terminus of proteins secreted by the general secretory pathway is removed
upon transfer to the periplasm; the N-terminal signal sequence can be seen in the
periplasm, and the extracellular protein is clearly different from the
intracellular protein by virtue of its absence. Type I systems are composed of far
fewer components than type III systems; this is indicated by the number of
distinct proteins (indicated by shape and size) in the figure. Type II and type
III systems share a similar cell envelope component, as indicated by sequence
homology; this is reflected in the shape of a cell envelope component in the
figure.

Secretion Systems in Bacteria
Secreted or surface-exposed bacterial proteins have long been known to play
central roles in bacterial-host interactions. In gram-negative bacteria,
these proteins must pass through two membranes: the inner membrane, which
surrounds the cytoplasm, and the outer envelope, which encloses the
periplasm and acts as a barrier to the environment (Figure 1). The general
secretory pathway transports proteins to the periplasm. Before the Yersinia
secretion system was identified, two other specialized secretion systems,
type I and type II, were
                             [end of page 272]

known to transport molecules to the cell surface (14,20,21). Proteins
secreted by the Type I system cross directly from the cytoplasm to the cell
surface, bypassing the general secretory pathway completely (Figure 1).
Type II-secreted proteins use the general secretory pathway to reach the
periplasm and then traverse the outer membrane through distinct channel
proteins. Both type I and type II systems secrete proteins involved in
various functions, including pathogenesis. For example, [alpha]-hemolysin
of E. coli uses a type I system and bundle-forming pili of enteropathogenic
E. coli (EPEC) and enterotoxigenic E. coli (ETEC) use type II systems for
export.

In the past 5 years, the highly conserved, multicomponent type III
secretion system has been found in many gram-negative bacteria that cause
disease in animals and plants (8). This secretion system is responsible for
transporting effector molecules directly from the cytoplasm to the cell
surface, where they interact with mammalian cells and modify host cell
proteins (13). This one-step secretion process is reminiscent of the
mechanism used by type I systems (Figure 1). The genes that encode many
components of type III systems are homologous to those that encode
flagellar export machinery in both gram-negative and gram-positive bacteria
(Table 1) (22-24). Indeed, these two systems share many structural and
functional features. The differences reside at the outer membrane.
Flagellar components pass through an outer ring structure that is part of
the flagellum itself (23), whereas pathogenic effector molecules traverse
the outer membrane through a channel protein that is homologous to those
used in type II secretion systems (20,25). While our discussion of type III
secretion systems will focus primarily on those used by human pathogens,
many characteristics are common among systems found in plant pathogens and
bacteria that produce flagella.

Type III Systems Secrete Effector Proteins Upon Contacting Host Cells
In contrast to the secretion process in type I and type II systems, type
III secretion is triggered when a pathogen comes in close contact with host
cells (18,19,26,27), and hence, has been called contact-dependent secretion
(28). Temperature, growth phase, and salt conditions are environmental cues
known to induce synthesis of the secretion apparatus and effector molecules
in various pathogens (29-31). When the pathogen comes into close contact
with tissue culture cells, effector molecules move to the external surface
of the bacterium, sometimes forming appendages suggestive of flagellae
(18). In some cases, the bacterium binds to the host cells and these
molecules are delivered into the host cell (32). The effector molecules
cause changes in host cell function, which facilitate the pathogen's
ability to survive and replicate (15-17,33).

How Pathogens Use Type III Secretion Systems in the Host
The best studied bacterial pathogens that use type III secretion are
Yersinia pestis, which causes plague, and a number of enteropathogens.
Although these various enteropathogens (Yersinia spp., Salmonella spp.,
Shigella spp., and EPEC) cause diarrhea and, in some cases, systemic
disease, they produce distinct syndromes because their secreted proteins
target different host cells and molecules (Table 1) (34).

Yersinia spp. use their effector molecules to destroy key functions of
immune cells and render them innocuous (35). When these bacteria bind to
tissue culture cells, approximately 10 different effector molecules are
secreted (13) and at least three are injected into cells (27,36-38). Two of
these injected molecules, YopE and YopH, modify macrophage proteins and
destroy the cells' abilities to engulf and kill bacteria (16,39). During
the course of disease, immune cells are presumably neutralized by these
effector molecules, which enables Yersinia spp. to flourish in the
reticuloendothelial environment.

While effector molecules in Yersinia destroy normal cellular functions,
those from Shigella spp. and from one of the Salmonella spp. type III
secretion systems, encoded by genes located in SPI I (Table 2), stimulate
cells
                             [end of page 273]

Table 1. Function and location of components in Type III systems[a]
---------------------------------------------------------------------------------------------------
Yersinia      S.         S.     EPEC  Flagella Plant[d]     Possible          Possible     Reference
          typhimurium flexneri   (c)  [c]                   Function[d]        Cellular
  (p)[b]  SPI   SPI      (p)             (c)     (c)                         Location[f]
          I(c) II(c)
---------------------------------------------------------------------------------------------------
                                                                         cytoplasm,
YscN     SpaL  orf1   MxiB     SepB   FliI     HrpE   ATPase             associated       8, 43, 63
                                                                         with inner
                                                                         membrane (I.M.)
LcrD     InvA  orf9   MxiA     SepA   FlhA     HrpO   structural/        I.M.             28, 43,
                                                      regulatory                          63
YscR     SpaP  orf5   Spa24           FliP     HrpT   structural         I.M.             8, 43
YscS     SpaQ  orf6   Spa9            FliQ     HrpU                                     28, 43
YscT     SpaR  orf7   Spa40           FliR     HrpC                                     8, 43
YscU     SpaS  orf8   Spa40           FhlB     HrpN                                     8, 43, 63
YscC     InvG  orf11  MxiD     SepC            HrpA   structural/channel outer            28, 43,
                                                      forming            membrane         63
                                                                         (O.M.)
YscJ     PrgK  orf10  MxiJ     SepD   FliF     HrpI   lipoprotein/       O.M.             8, 28,
                                                      structural                          43, 63
VirG                                                                                    52
YscD                                                  essential, but     I.M.             48
                                                      unknown
YscE                                                                    unknown          51
YscF     PrgI         MxiH                            needed for YopB                    28, 51
                                                      and D secretion
YscG                                                  essential, but     equally distri-  48
                                                      unknown            buted between
                                                                         membrane
                                                                         and soluble
                                                                         fractions
YscI                                                                    unknown          51
YscK                                                                                    51
YscL                                           HrpF                                     8
YscO     SpaM  orf2   Spa13                           unknown                            12, 28,
                                                                                          43
YscP     SpaN  orf3   Spa32                                                             12, 28,
                                                                                          43
YscQ     SpaO  orf4   Spa33           FliN     HrpQ   SpaO needed for    Spa O: secreted  8, 28, 43
                                                      secretion
                                                      of Sips (see
                                                      below)
YopB     SipB         IpaB                            homology to pore   secreted/targeted26, 28,
                                                      forming toxins/    to host cell     55
                                                      delivery of
                                                      effector molecules
                                                      to host cells

                                         [end of page 274]

YopD                                                  delivery of        secreted         55
                                                      effector
                                                      molecules to host
                                                      cells
         SipD         IpaD                                              secreted         26
SycD     SicA         IpgC                            chaperone for      cytoplasm        56
                                                      YopB and
                                                      D/ IpaB
                                                      and IpaC
                                                      in Shigella
SycE                                                  chaperone for                      56
                                                      YopE
SycH                                                  chaperone for                      56
                                                      YopH
YopN     InvE         MxiC                            regulatory:        O.M./secreted    28
                                                      cell-contact
                                                      sensing pathway
LcrG                                                                    cytosol/I.M.     59, S.
                                                                                          Straley
                                                                                          pers.
                                                                                          comm.
LcrQ                                                  regulatory         cytoplasm/       54
                                                                         secreted
VirF     InvF         MxiE                     HrpB   regulatory:        cytoplasm        28
                                                      temperature
YopE                                                  effector molecule: host cell        27
                                                      depolymerizes
                                                      actin anti-
                                                      phagocytic
YopH                                                  effector molecule:                 36
                                                      tyrosine
                                                      phosphatase
                                                      anti-phagocytic
         SipC         IpaC                            effector molecule: secreted         17, 28
                                                      induces entry
                                                      into epithelial
                                                      cells
                               EspB                   effector molecule:                  33, 63
                               (EaeB)                 induces AE lesions
---------------------------------------------------------------------------------------------------
[a]Listed are names of proteins in type III secretion systems
[b]p-proteins are encoded by genes on a plasmid; c-proteins are encoded by genes on the chromosome.
[c]Protein names are those from the S. Typhimurium flagella; for list of flagellar protein names
from other bacteria, see ref. 23.
[d]Protein names are those from the plant pathogen Pseudomonas solanacearum; for a list of
components of type III secretion systems in other plant pathogens, see ref. 8.
[e]These are the possible functions for these factors in Yersinia, unless noted.
[f]These are the possible locations for these factors in Yersinia, unless noted.

                                         [end of page 275]

 Table 2. Characteristics of several pathogenicity islands
 ------------------------------------------------------------------------------------------------------------------------
     Organism             Name         Location    Borders    Stable Foreign origin          Functions         Size Ref.
                                                                ?    G+C:
                                                                     % island/
                                                                     % chromosome
 ------------------------------------------------------------------------------------------------------------------------
 Uropathogenic    Pathogenicity island selCa,   * 16 bp       no     * direct repeats  * [Image]-hemolysin I   70   3,
 E. coli 536      I, Pai I             82'      direct               * absent from                             kb   82,
                                                repeats,             normal fecal and                               84
                                                derived from         laboratory
                                                selC                 strains of E.
                                                * shared             coli
                                                motif with
                                                Pai II
                                                repeats
                  Pai II               leuXa,   * 18 bp       no     * direct repeats  * [Image]-hemolysin II  190  3,4,
                                       97'      direct               * absent from     * prf (fimbriae:        kb   67,
                                                repeats,             normal fecal and                               82,
                                                derived from         laboratory        adherence to host            84
                                                leuX                 strains of E.     cells)
                                                * shared             coli              * transcriptional
                                                motif with                             activators of
                                                Pai I repeats                          chromosomal genes
 Uropathogenic    Pai I                near                          * absent from     * [Image]-hemolysin I   >170 96
 E. coli J96                           pheVa,                        normal fecal and                          kb
                                                                                       * pap (fimbriae:
                                       64'                           laboratory        adherence to host
                                                                     strains of E.
                                                                     coli              cells)
                                                                                       * IS element sequences
                                                                                       * R plasmid sequences
                                                                                       * P4 phage sequences
                  Pai II               pheRa,   135 bp        no     * direct repeats  * [Image]-hemolysin II  106  4,
                                       94'      imperfect            * absent from     * prs (fimbriae:        kb   96,
                                                direct               normal fecal and                               97
                                                repeats              laboratory        adherence to host
                                                                     strains of E.     cells)
                                                                     coli              * cytotoxic necrotizing
                                                                                       factor type 1
                                                                                       * IS element sequences
                                                                                       * P4 phage sequences
                                                                                       * OmpR homolog
 Entero-pathogenicLocus of enterocyte  selCa,   no repeats or yesb   * G+C: 39%/51%    * mediates formation of 35   63,
 E. coli (EPEC)   effacement, LEE      82'      IS elements          * not present in  AE lesions              kb   83
                                                found                closely related,  * type III secretion
                                                                     non-AE-producing  system
                                                                     bacteria
 Salmonella       Salmonella           between  no repeats or yesb,c * G+C: 42%/52%    * invasion into         40   28,
 typhimurium      pathogenicity island fhl and  IS elements          * absent from E.  cultured epithelial     kb   68
                  1, SPI 1             mutS,    found in S.          coli              cells
                                       63'      typhimurium;                           * type III secretion
                                                IS3 on one                             system
                                                border in
                                                certain
                                                Salmonella
                                                serotypes

                                                    [end of page 276]

                  SPI 2                between                yesb   * G+C: 45%/52%    * type III secretion    40   43
                                       ydhE and                      * absent from E.  system                  kb
                                       pykF,                         coli; conserved
                                       31'                           among Salmonella
                  Salmonella induced   potB/    14 bp direct  yes    * G+C: 41%/52%    * required for          1.6  69d
                  filament gene A,     potC     repeats              * direct repeats  formation of structures kb
                  sifA                                               * absent in E.    associated with
                                                                     coli; conserved   Salmonella-associated
                                                                     among Salmonella  vacuoles within
                                                                                       epithelial cells
 Yersinia pestis  Ability to adsorb    phoE     2.2 kb direct no     * G+C: hemin      * hemin and congo red   102  70,
                  exogenous pigments,           repeats              storage region    binding                 kb   71
                  Pgm                           (=IS100)             47%/46-50%;       * pesticin sensitivity
                                                                     yersiniabactin    * iron acquisition
                                                                     receptor/         * growth at 37 C in
                                                                     iron-regulated    defined medium
                                                                     protein region
                                                                     56-60%/46-50%
                                                                     * direct repeats
 Helicobacter     Cytotoxin-associated glr      * 31 bp       see    * G+C: 35%/38-45% * induction of IL-8     40   e
 pylori           gene region, Cag              direct        text   * IS elements     secretion               kb
                                                repeats,             * not present in  * homologues to
                                                derived from         type II strains   membrane-associated
                                                glutamate                              proteins: environmental
                                                racemase gene                          sensors, translocases,
                                                * IS605 on                             permeases, pilus and
                                                one end                                flagella assembly
                                                                                       proteins
                                                                                       * IS elements
 Vibrio cholerae  otnA otnB            rfb      flanked by           * IS elements     * capsule and O antigen 35   93,
 O139                                           two different        * not present in  synthesis (by homology) kb   98
                                                IS elements          Vibrio cholerae
                                                                     O1 El Tor
 Listeria                              between  No IS         yesb   * not present in  * escape from vacuole   9.6  99
 monocytogenes                         prs and  elements             several           * intra-/inter-cellular kb
                                       ldh      found                nonpathogenic     spread
                                                                     species
 ------------------------------------------------------------------------------------------------------------------------
 atRNA gene
 bapparently
 cunstable in certain serotypes
 dadditional information received in personal communication with M. Stein
 eCensini S, et al., 1996, submitted for publication

                                                    [end of page 277]

to perform functions in addition to those in their usual repertoires.
Studies in epithelial tissue culture systems show that these bacteria
induce their own entry into normally nonphagocytic cells by using effector
molecules secreted by their type III systems (28,40). During the course of
disease, Shigella spp. enter and replicate in the mucosal epithelial cells
of the large intestine, while Salmonella spp. gain entry into the
peritoneal cavity by passing through the epitheloid-like M cells in the
small intestine (41). In the murine model for typhoid fever, S. typhimurium
that are defective in this secretion system are attenuated for infection
when administered orally, but not intraperitoneally (42). Presumably this
attenuation is a consequence of reduced entry into the M cells in the small
intestine, a barrier that is bypassed by intraperitoneal delivery.

Although neither its effector molecules nor target host cells have been
identified, a second type III secretion system, encoded by genes located in
SPI II (Table 2), has been described in S. typhimurium, on the basis of
sequence homology (43). The genes in SPI II, in contrast to those in SPI I,
are required for systemic disease regardless of the route of infection
(43,44). Presumably the factors encoded in SPI II act after the bacteria
have crossed the epithelial barrier of the small intestine.

Although the functions and sites of action of the secretion systems differ
among these enteric pathogens, effector molecules from one system can be
secreted by other systems, provided the appropriate chaperones are present.
Such heterologously expressed effector molecules can induce the same
cellular response as when expressed from their native systems. For
instance, an effector molecule from Yersinia that causes actin
depolymerization has the same effect on tissue culture cells when secreted
from Salmonella (45). Likewise, proteins of Shigella and Salmonella
involved in bacterial uptake into cultured epithelial cells are
functionally interchangeable (46).

The observation that Salmonella spp. have two contact-dependent systems
that function at distinct stages to cause disease raises several
interesting questions. Why do Salmonella spp. need different type III
secretion apparatuses when it is clear that effector molecules can be
secreted from heterologous systems? Could one suffice if the two sets of
effector molecules were expressed at appropriate times during the course of
infection? Alternatively, do these two sets of effector molecules need to
be delivered to different target cells in a specific manner, which is only
possible with distinctly customized machinery? Answers to these questions
will illuminate issues about both the course of salmonellosis and the basic
mechanics of the secretion apparatus.

The Nuts and Bolts of Type III Secretion
The type III secretion apparatus in Yersinia spp. has been the most
intensively investigated. However, this work has been done in three
Yersinia spp; thus several proteins have been shown to be essential for
effector molecule secretion in one species but have not yet been examined
in others. Our analysis of these studies assumes that proteins essential in
one will play a similar role in all (Table 1).

One essential feature of any secretion system is that energy must be
provided to move molecules through the membrane (14). Only one protein in
the system, YscN, has been shown to hydrolyze ATP and thus is a likely
candidate for generating energy to drive secretion (47). YscN is predicted
to be a cytoplasmic protein, closely associated with the inner membrane.

Several proteins essential to secretion including LcrD, YscD, R, S, T, and
U, are known or predicted to reside in the inner membrane (10,12,48-50). At
the outer membrane, only one protein, YscC (48), and two lipoproteins, YscJ
and VirG (51,52), appear essential for proper secretion. The roles and
subcellular locations are not known for several more essential proteins,
YscE, F, G, I, K, and L (9,48,51). How all of these proteins interact with
one another to form the secretion apparatus is not yet understood. It is
clear, however, that correct assembly of the apparatus is required not only
for secretion, but also for normal synthesis of effector molecules (47,48).
If one component of the export machinery is missing, production of the
effector molecules is altered. This
                             [end of page 278]

feedback regulation also occurs in systems that produce flagella (53). How
it works in Yersinia is under investigation; thus far, only one protein,
LcrQ, has been implicated (54).

Two proteins, YopB and YopD, are loosely associated with the outer membrane
(55) and are crucial for efficient delivery of effector molecules into
target cells. These two proteins use the type III secretion system to reach
the bacterial cell surface. Without YopB, which has homology to
pore-forming toxins (55), and YopD, effector molecules are secreted but not
efficiently internalized by host cells; thus, their activities on host
cells are severely abrogated (27,36). Presumably, YopB and YopD form a
mammalian cell through which effector molecules pass.

Several proteins, called chaperones, play critical roles in secretion by
binding to effector molecules in the bacterial cytoplasm. Chaperones have
several proposed functions (56-58). Chaperone binding may stabilize and
prevent proteins from folding into conformations that are impossible to
secrete. Alternatively, as has been shown for Shigella, they may prevent
effector molecules from improperly associating with one another before
secretion (58). Lastly, chaperones may deliver molecules to the secretion
apparatus.

In addition to the feedback regulation mentioned above, the synthesis of
and secretion from the Yersinia type III system is regulated by two
networks that respond to environmental cues (29,35). A temperature-sensing
network induces synthesis of the apparatus at 37C and includes VirF and
YmoA (35). A host cell-sensing network increases both synthesis of and
secretion from the type III system when Yersinia binds to target cells.
This regulatory system is called the low-calcium response networklow
calcium presumably mimics some signal generated by cell contactand
includes YopN, LcrG, and LcrQ (32,54,59,60). YopN localizes to the outer
membrane, where it senses cell contact and transduces this signal to the
cytoplasm by an unknown mechanism (60). The role of LcrG has not yet been
elucidated. LcrQ functions as a repressor of the Yops. When Yersinia comes
into close contact with host cells, LcrQ is secreted from the cytoplasm
through the type III secretion system. This lowers the intracellular
concentration of LcrQ and results in an increase in synthesis and secretion
of the Yops (32). Flagella synthesis is controlled in a similar manner
(61).

Many of the structural components of the Yersinia system have homologues in
Shigella, Salmonella, and EPEC (Table 1). A comparison of proteins found in
each system shows that certain core structural components are present in
all type III apparatuses, whereas others may exist in only one or a subset.
These differences may be due to particular functions of each system.

Several studies have examined whether structural components from different
bacteria are interchangable. In general, core constituents from Shigella
and SPI I of Salmonella, which both facilitate bacterial uptake by
epithelial cells, are interchangable with one another, but not with those
in the Yersinia system (7,62). These results may be due to the observation
that factors from Shigella and SPI I of Salmonella are predicted by
sequence homology to be more structurally similar to each other than to
those in Yersinia (7). Alternatively, some of the regulatory cues for
secretion and assembly may be different for Yersinia than for Salmonella
and Shigella.

Most proteins in the type III secretion systems, including effector
proteins, regulatory proteins, structural proteins, and chaperones (Table
1), are encoded by genes that belong to several large operons, which are
clustered together (7,9,12,63). These operons are on plasmids in some
species, and on the chromosome in others (Table 1). In some cases, such as
in Shigella and SPI I of Salmonella, the order of the genes within operons
and the arrangement of the operons with respect to each other are conserved
(7). These observations suggest that type III systems were inherited en
masse, and likewise, could be transmitted to other bacteria en masse. One
can speculate that the acquisition of a type III secretion system could
allow a bacterium to adapt to different environments or hosts. For
instance, a new pathogen could perhaps arise if a skin-commensal bacterium
were to acquire the means to penetrate and survive in the skin-associated
                             [end of page 279]

lymphoid tissue by obtaining a type III secretion system from an
enteropathogen.

Where Do Type III Secretion Systems Come From?
It seems plausible that the original type III secretion system for
virulence factors evolved from those for flagellar assembly (22,53). The
bacterial flagellum exists in a wide range of eubacteria and some
archaebacteria, which indicates that it probably emerged well before
gram-negative bacteria, the hosts of the type III virulence factor
secretion systems identified thus far.

Attempts to establish any of the known type III secretion systems as the
progenitor have been fruitless. On the basis of degrees of homology among
different type III systems and well-established evolutionary relationships
between the bacteria, each organism can be ruled out as the source (64).
For example, Shigella emerged from E. coli after Salmonella and E. coli
diverged from a common ancestor; thus, Shigella cannot have provided the
type III systems conserved in the Salmonella spp. Conversely, as Shigella
type III apparatus sequences have a G+C content well below that of
Salmonella and of the bulk of the Shigella chromosome, Salmonella could not
have been the source of the Shigella genes (Table 2). Thus, the ability to
secrete effector molecules by this mechanism seems to have been introduced
independently into each of these bacteria.

Examination of homologous genes in the epithelial cell invasion loci of
Salmonella and Shigella shows that some are highly conserved, while others
display much lower levels of homology (65). Li and colleagues have found a
relationship between evolutionary rate of change and subcellular location:
genes encoding several secreted proteins are hypervariable in relation to
genes encoding several proteins located in the bacterial inner membrane
(65). In principle, hypervariability could reflect antigenic variation or
adaptations to diverse host environments; however, neither of these
explanations appears to pertain to the particular proteins examined
(28,65).

Type III systems sometimes provide much of what distinguishes particular
organisms from closely related, often nonpathogenic, species. As described
above, many operons encoding type III secretion machinery are clustered.
DNA sequence analysis has shown that these loci are often distinguishable
from the bulk of the genomic DNA. The loci that are chromosomally located
represent "pathogenicity islands" (66).

History and Definition of "Pathogenicity Islands"
The phrase "pathogenicity island" was first used to describe two large,
unstable pieces of chromosomal DNA, unique to uropathogenic E. coli, that
encode a number of genes required for virulence (3,4). Since its
conception, the term has evolved to include regions of expression
chromosomal DNA essential for pathogenicity that do not appear to "belong"
(Table 2). Not all pathogenicity islands are genetically unstable, but each
one shows an indication of foreign origin. These pieces of DNA are often
missing in closely related, nonvirulent bacteria. Many pathogenicity
islands differ from the bulk of the genome in G+C content and codon usage,
and their borders are often marked by repeated sequences or insertion
elements, which suggests that some kind of recombination event delivered
them to the chromosome. Several encode multiple proteins that collaborate
to confer a single, complex virulence property to the bacterial host.

The definition of pathogenicity islands includes chromosomal location. As
such, the plasmid-borne type III gene clusters of Yersinia and Shigella do
not qualify (Table 1). This seems somewhat arbitrary. Indeed, phages and a
number of plasmids can easily insert into and excise from the chromosome.
Similarly, many transposable elements replicate and function equally as
well in the chromosome as on an extrachromosomal element. It seems to us
that a block of apparently foreign genes found uniquely in pathogenic
members of a genus and required for virulence is a more useful and relevent
defining feature of a pathogenicity island than location. Thus, it makes
sense to include the loci encoding type III secretion systems, regardless
of whether they reside on a plasmid or chromosome. In the
                             [end of page 280]

discussion below, however, we adhere to the established definition that
includes chromosomal location.

Pathogenicity Islands Contain Virulence Genes and Regulatory Elements
Pathogenicity islands also contain virulence genes other than those
encoding type III secretion systems; a common theme appears to be inclusion
of genes for secreted or cell surface-localized proteins such as
hemolysins, fimbriae, and hemin-binding factors (Table 2). In fact, the
similarities between pathogenicity islands extend further: examination of
the large ones shows that many also contain genes that encode a secretion
system and environmental sensors. They also can include proteins that
regulate expression of genes that lie outside the pathogenicity island. For
example, pathogenicity island II (Pai II) of uropathogenic E. coli contains
genes that encode transcriptional activators of S-fimbrial genes that
reside at a chromosomal locus remote from either of the known pathogenicity
islands in this species (67).

Pathogenicity Islands Can Exist in Various Structural Types and Numbers
Within a Bacterium
A single bacterial strain can harbor more than one pathogenicity island.
Salmonella contains at least five: the gene clusters encoding the two type
III secretion systems described above, sifA (see below), and two groups of
genes that are activated by the two-component regulator, PhoP/PhoQ. These
loci vary in size and complexity and reside at distinct chromosomal
locations (43,68,69, and S. Miller, pers. comm.).

Pathogenicity islands themselves can be composed of distinct segments. For
example, an unstable 102-kb region of DNA that encodes several traits
important for virulence of Y. pestis appears to consist of several regions
(70-72). One contains the hemin storage genes and has a G+C content similar
to that of the bulk of the chromosome (R. Perry, pers. comm.); the other
contains genes encoding the Yersiniabactin receptor and iron-regulated
proteins and has a significantly higher G+C content (R. Perry, pers. comm)
(73). Although the 102 kb region often deletes entirely, the two regions
can also act independently. In some strains the chromosomal region
containing the hemin storage genes spontaneously deletes from the
chromosome at a significant frequency, while the Yersiniabactin
receptor/iron-regulated protein region appears stable (72). Furthermore,
only the Yersiniabactin receptor/iron-regulated segment is present in Y.
enterocolitica (70).

Even more complex pathogenicity islands are harbored by strains of
Helicobacter pylori, the causative agent of gastritis and peptic ulcer
disease in humans. Strains of H. pylori have been divided into two classes:
type I strains express the cytotoxin-associated gene A (CagA) antigen and
induce secretion of the neutrophil attractant IL-8 by epithelial cells in
vitro, while type II strains lack both of these properties. Patients with
duodenitis, duodenal ulcers, and gastric tumors are most often infected by
type I strains. Likewise, type I strains are more likely than type II
strains to cause gastric injuries in murine model systems. Analysis of the
chromosomal region that contains the cagA gene has shown that it is a
pathogenicity island of approximately 40 kb of DNA, missing in type II
strains, and that mutations in this region abolish IL-8 induction in
gastric epithelial cell lines (Censini, S et al. A pathogenicity island of
Helicobacter pylori, encodes type I-specific and disease-associated
factors. 1996; submitted for publication).

Different type I strains display considerable heterogeneity in the cag
region (Censini, S et al. A pathogenicity island of Helicobacter pylori,
encodes type I-specific and disease-associated factors. 1996; submitted for
publication). In some isolates, the cag region is interrupted by one or
more insertion sequences. In a small number of strains, there is an
additional 20-kb sequence that is also present in type II strains. Partial
deletions of the cag region have been detected as well. Thus, the cag
pathogenicity island appears to be undergoing dynamic changes in natural
Helicobacter populations. Further study of the cag region may elucidate
details of pathogenicity island
                             [end of page 281]

acquisition and help correlate regions of the pathogenicity island with
disease symptoms in the murine model system.

Since its establishment, the definition of pathogenicity islands has
evolved to include genetic regions that are neither large nor complex;
single genes of apparently foreign origin can also be inserted into
chromosomal DNA. S. typhimurium has recently been shown to contain such a
gene, called sifA, which is required for formation of distinctive
structures associated with Salmonella-containing vacuoles within epithelial
cells and contributes to pathogenicity in the murine typhoid fever model
system (Table 2) (69).

Possible Origins of Pathogenicity Island DNA and Mechanisms of Transfer and
Insertion
While the sources of pathogenicity islands are unknown, their presence in a
wide variety of organisms (Table 2) indicates that bacteria can acquire DNA
despite multiple barriers to chromosomal gene transfer between species. The
existence of "foreign" genomic DNA is particularly intriguing as sequence
divergence is a major limitation to such transfers because it severely
limits the potential for homologous recombination (74).

Although the identity of the vectors that transport pathogenicity islands
from donor to recipient organisms is unknown, any number of mobile genetic
elements are candidates. Clear evidence showing an extrachromosomal stage
of a pathogenicity island is lacking; however, it is intriguing that the
G+C contents of the Helicobacter pathogenicity island and plasmid are
similar to each other and distinct from the chromosome (Censini, S et al. A
pathogenicity island of Helicobacter pylori, encodes type I-specific and
disease-associated factors. 1996; submitted for publication). Phages,
plasmids, transposons, integrons, and even free DNA carry genes from one
organism to another (74). Indeed, many phages and plasmids contain
virulence genes, and often these loci seem alien to the bacterial species
in which they reside (75). Furthermore, the animal host environment may be
particularly conducive to DNA transfer events. For example, the phage that
encodes cholera toxin infects V. cholerae more efficiently within the
gastrointestinal tract of a mammalian host than under laboratory conditions
(76).

Pathogenicity islands insert into the chromosome by an unknown mechanism;
however, the existence of insertion elements and repeated DNA motifs at the
boundaries of several pathogenicity islands suggest that recombination
events are involved. Recombination has recently been shown to be the major
factor governing the divergence of a group of E. coli strains and is a
significant driving force for evolution (74,77). Although the genetic
material comprising pathogenicity islands may be introduced into a new host
rganism in a single step, the events that generate known pathogenicity
islands are unlikely to be simple insertions, because DNA rearrangements
and alterations are common in the flanking chromosomal regions.

The identification of several tRNA genes as insertion sites for
pathogenicity islands is also notable, although the significance of this
remains obscure (Table 2). tRNA genes serve as integration sites for a
variety of prokaryotic genetic elements, including several phages and
transmissible plasmids (78-80). Perhaps the conserved portion of tRNA genes
is a useful landmark for mobile genetic elements that inhabit a variety of
prokaryotic hosts; in addition, the regions of dyad symmetry characteristic
of all tRNA genes could serve as binding sites for enzymes involved in
recombination.

Relative Advantages of Instability and Stability
Some pathogenicity islands can excise from the chromosome and are
apparently lost from the host bacterium (Table 2) (3,4,70). Such
instability may provide an adaptive advantage. Virulence properties may be
dispensable at certain stages of infection, and the coordinated loss of
these characteristics could be beneficial to the bacterium. Indeed,
expression of particular genes at inappropriate times can be detrimental to
bacterial pathogens (81). Natural selection of strains with deleted
                             [end of page 282]

virulence regions can occur in specific environments: diabetic patients are
more susceptible to uropathogenic E. coli strains not exhibiting virulence
phenotypes (4).

On the other hand, particular virulence traits could provide a continual
adaptive advantage, resulting in stable pathogenicity islands. The "foreign
nature" of pathogenicity islands may reflect this benefit; foreign DNA may
be actively maintained in the population because of its limited ability to
recombine with related organisms (75). It is not clear whether stable
islands exist because of a divergence of sequences at the borders (for
example, repeated elements that are no longer recognizable as such), an
integration mechanism completely different from that of the unstable
islands, or because of a lack of excision machinery.

Deletion of pathogenicity islands can affect gene expression by altering
the chromosomal site of insertion and by
removing the genes contained in the island. The locus of enterocyte
effacement (LEE) comprising the type III secretion apparatus in EPEC and
pathogenicity island I (Pai I) of uropathogenic E. coli both insert at the
selenocysteine tRNA (selC) gene (82,83). The presence of Pai I does not
interfere with selC expression. However, excision from the chromosome
appears to occur by a recombination event between the repeated sequences in
selC and the distal end of Pai I. This recombination event results in
deletion of part of the tRNA gene and inhibits anaerobic growth due to the
cell's inability to produce formate dehydrogenase, which contains
selenocysteine (84). Similarly, in Pai II deletion strains, the leuX tRNA
gene at the insertion site is disrupted, which interferes with its ability
to act as a global regulator of several virulence factors that lie outside
the pathogenicity island (84).

Bacteria may be able to have the best of both the stable and the unstable
worlds. The phenotypic loss of the enteroinvasive E. coli and Shigella
flexneri virulence plasmids is sometimes due to plasmid insertion into a
specific site on the chromosome (85,86). After integration, excision also
can be detected; strains containing precisely excised plasmids regain
virulence, while those with imprecisely excised plasmids remain
noninvasive. Several of the Y. pestis plasmids exhibit similar behavior
(87-89). Integration, which simultaneously maintains these plasmids in the
bacterial genome while downregulating their genes, may represent a
sophisticated adaptation to the requirements of different environments or
may represent? stages in the bacterial life cycle. Furthermore, integration
and excision remind us that strict definitions of "chromosomal" versus
"plasmid-borne" do not always reflect biological reality.

Foreign DNA Is a Significant Determinant in Recently Emerged Pathogens
Horizontal gene transfer has been invoked to explain the origin of
enterohemorrhagic E. coli (EHEC), which causes hemorrhagic colitis and
hemolytic uremic syndrome (90). Like EPEC, EHEC induces striking
morphologic changescalled attaching and effacing (AE) lesionsin host
cells of the small intestine; however, unlike EPEC, EHEC contains
Shiga-like toxins. After analyzing the genetic relationships between many
E. coli strains, Whittam and colleagues proposed that EHEC arose from an
EPEC-like progenitor strain, which then acquired the prophage-encoded
Shiga-like toxins, thus becoming a new pathogen that expresses both sets of
traits (90).

The new epidemic Vibrio cholerae O139 strain may have emerged after
acquisition of a pathogenicity island (91). Although it appears that V.
cholerae O139 arose from a strain of the same serotype (O1) that is causing
the ongoing cholera pandemic (O1 El Tor) (92), V. cholerae O139 contains an
additional piece of DNA that replaces part of the O antigen gene cluster of
O1 strains (93). The inserted DNA contains open reading frames homologous
to proteins involved in capsule and O antigen synthesis, two factors that
distinguish O139 and O1 El Tor, and are thought to mediate activities
important for pathogenesis and evasion of immunity.

Benefiting From Information About Type III Secretion and Pathogenicity
Islands
Pathogenic bacteria continue to exhibit impressive genetic flexibility and
exchange and use these abilities to adapt to varied
                             [end of page 283]

types of lifestyles within host organisms. It should be possible to use the
information from studies of pathogenicity islands and type III secretion
systems in the ongoing characterization of bacterial infections. When a
novel pathogen is isolated, it may be worthwhile to identify chromosomal
regions specific to it by comparing the gross genomic structure with that
of related organisms, which may provide a shortcut to the identification of
virulence genes. Likewise, simple molecular techniques can determine
whether bacteria contain type III secretion systems, because genes encoding
particular components are highly conserved; perhaps this procedure should
be part of our standard investigative arsenal as well.

Our knowledge of type III secretion systems may yield therapeutic benefits.
The contact-dependent systems appear to reside in pathogenic and not in
commensal bacteria. If this observation reflects a general truth,
antibiotics that target type III systems may specifically attack intruding
bacteria and spare the normal flora; therefore, these antibiotics might
produce minimal side effects. In addition, type III secretion systems will
provide new targets for therapeutic drugs that might not kill the bacterium
but would inhibit the disease process. We also may be able to exploit this
secretion system, by using appropriately attenuated bacteria, to prime
immunity. Chimeric proteinsfusions between effector and other proteinscan
be secreted in large quantities by the type III secretion machinery and be
internalized by host cells; furthermore, these proteins can elicit an
antibody response in mice (38,94).

The virulence traits of pathogenic microorganisms at the genetic and
molecular level remind us that bacterial pathogenicity does not arise by
slow adaptive evolution but by "quantum leaps" (95); therefore, microbes
can acquire complete systems that radically expand their capabilities to
exploit and flourish in different host environments. History teaches us
that infectious diseases may change in severity and form, but they will not
simply disappear. Microrganisms are, after all, survivors, and there is no
escaping our destinyto be consumed by them in one way or another. However,
the more we learn about the microbial tactics of survival, the longer we
forestall this destiny.

    Acknowledgments
    We thank A. Covacci, D. Frank, R. MacNab, S. Miller, R. Milkman,
    K. Nelson, R. Perry, K. Rudd, C. Stephens, S. Straley, M. Waldor,
    R. Welch, and T. Whittam for sharing unpublished data and
    manuscripts; R. Perry and R. Welch for patient explanations and
    helpful discussions; S. Fisher, D. Gunn, C. Lee, T. McDaniel, S.
    Mel, K. Ottemann, B. Raupach, J. Shea, S. Straley, and C. Stephens
    for thoughtful and incisive comments on the manuscript, and
    Stanley Falkow for his critical input on the manuscript, the
    fanciful and poignant concluding paragraph, a stimulating and
    exciting atmosphere in which to learn and think about bacterial
    pathogenesis, and his support and mentorship. We apologize to the
    many researchers whose work we did not cite.

Joan Mecsas received her Ph.D. at the University of Wisconsin-Madison. She
has been a post-doctoral fellow in Dr. Stanley Falkow's laboratory at
Stanford University for the past two-and-a-half years. Her work is
supported by a Damon Runyon-Walter Winchell Cancer Research Fund
postdoctoral fellowship (DRG#1277).

Evelyn J. Strauss received her Ph.D. at the University of California-San
Francisco. She has been a post-doctoral fellow in Dr. Stanley Falkow's
laboratory at Stanford University for the past 3 years. Her work is
supported by an American Cancer Society postdoctoral fellowship (Grant
#PF-4120).

Address for correspondence: Joan Mecsas, Department of Microbiology and
Immunology, Stanford University School of Medicine, Stanford, CA
94305-5402; fax: 415-723-1837; e-mail: mecsas@cmgm.stanford.edu.

References

  1. Finlay BB, Falkow S. Common themes in microbial pathogenicity.
     Microbiol Rev 1989;53:210-30.
  2. Hayes W. The Genetics of Bacteria and their Virues. In: 2nd ed. 1968,
     New York: John Wiley & Sons Inc.
  3. Knapp S, Hacker J, Jarchau T,Goebel W. Large, unstable inserts in the
     chromosome affect virulence properties of uropathogenic Escherichia
     coli O6 strain 536. J Bacteriol 1986;168:22-30.
  4. Hacker J, Bender L, Ott M, Wingender J, Lund B, Marre R, et al.
     Deletions of chromosomal regions coding for fimbriae and hemolysins
     occur in vitro and in vivo in various extraintestinal Escherichia coli
     isolates. Microb Pathog 1990;8:2213-25.
  5. Fenselau S, Balbo I,Bonus U. Determinants of pathogenicity in
     Xanthomonas campestris pv. vesicatoria are related to proteins
     involved in secretion in bacteria pathogens of animals. Mol Plant
     Microbe Interact 1992;5:390-6.
                                [end of page 284]

  6. Sasakawa C, Komatsu K, Tobe T, Fukuda I, Suzuki T, Yoshikawa M. Eight
     genes in region 5 that form an operon are essential for invasion of
     epithelial cells by Shigella flexneri. J Bacteriol 1993;175:2334-46.
  7. Groisman EA, Ochman H. Cognate gene clusters govern invasion of host
     epithelial cells by Salmonella typhimurium and Shigella flexneri. EMBO
     J 1993;12:3779-87.
  8. van Gijsegem F, Gough C, Zischek C, Niqueux E, Arlat M, Genin S, et
     al. The hrp gene locus of Pseudomonas solanacearum, which controls
     the production of type III secretion system, encodes eight proteins
     related to components of bacterial flagellar biogenesis complex. Mol
     Microbiol 1995;15:1095-114.
  9. Michiels T, Vanooteghem J-C, de Rouvroit C, China B, Gustin A, Boudry
     P, et al. Analysis of virC, an operon involved in secretion of Yop
     proteins by Yersinia enterocolitica. J Bacteriol 1991;173:4994-5009.
 10. Plano GV, Barve SS, Straley SC. LcrD, a membrane-bound regulator of
     the Yersinia pestis low-calcium response. J Bacteriol
     1991;173:7293-303.
 11. Haddix PL, Straley SC. Structure and regulation of the Yersinia pestis
     yscBCDEF operon. J Bacteriol 1992;174:4820-8.
 12. Bergman T, Erickson K, Galyov E, Persson C, Wolf-Watz H. The lcrB
     (yscN/U) gene cluster of Yersinia pseudotuberculosis is involved in
     Yop secretion and shows high homology to the spa gene clusters of
     Shigella flexeri and Salmonella typhimurium. J Bacteriol
     1994;176:2619-26.
 13. Michiels T, Wattiau P, Brasseur R, Ruysschaert J-M,Cornelis GR.
     Secretion of Yop proteins by Yersiniae. Infect Immun 1990;58:2840-9.
 14. Salmond GPC, Reeves PJ. Membrane traffic wardens and protein secretion
     in Gram-negative bacteria. Trends Biochem Sci 1993;18:7-12.
 15. Rosqvist R, Forsberg , Wolf-Watz H. Intracellular targetting of the
     Yersinia YopE cytotoxin in mammalian cells induces actin microfilament
     disruption. Infect Immun 1991;59:4562-9.
 16. Rosqvist R, Forsberg , Rimpilinen M, Bergman T, Wolf-Watz H. The
     cytotoxic protein YopE of Yersinia obstructs the primary host defense.
     Mol Microbiol 1990;4:657-67.
 17. Mnard R, Prvost M-CGP, Sansonetti P, Dehio C. The secreted Ipa
     complex of Shigella flexneri promotes entry into mammalian cells. Proc
     Natl Acad Sci USA 1996;93:1254-8.
 18. Ginocchio CC, Olmsted SB, Wells CL, Galn JE. Contact with epithelial
     cells induces the formation of surface appendages on Salmonella
     typhimurium. Cell 1994;76:717-24.
 19. Watarai M, Tobe T, Yoshikawa M, Sasakawa C. Contact of Shigella with
     host cells triggers release of Ipa invasins and is an essential
     function of invasiveness. EMBO J 1995;14:2461-70.
 20. Pugsley AP. The complete general secretory pathway. Microbiol Rev
     1993;57:50-108.
 21. Fath MJ, Kolter R. ABC transporters: bacterial exporters. Microbiol
     Rev 1993;57:997-1017.
 22. Stephens C, Shapiro L. Targetted protein secretion in bacterial
     pathogenesis. Current Biology 1996;6:927-30.
 23. Aizawa S-I. Flagellar assembly in Salmonella typhimurium. Mol
     Microbiol 1996;19:1-5.
 24. Harshey RM, Toguchi A. Spinning tails: homologies among bacterial
     flagellar systems. Trends Microbiol 1996;4:226-31.
 25. Kaniga K, Bossio JC, Galn JE. The Salmonella typhimurium invasion
     genes invF and invG encode homologues of the AraC and PulD family of
     proteins. Mol Microbiol 1994;13:555-68.
 26. Mnard R, Sansonetti PJ, Parsot C. The secretion of the Shigella
     flexneri Ipa invasins is induced by the epithelial cell and controlled
     by IpaB and IpaD. EMBO J 1994;13:5293-302.
 27. Rosqvist R, Magnusson K, Wolf-Watz H. Target cell contact triggers
     expression and polarized transfer of Yersinia YopE cytotoxin into
     mammalian cells. EMBO J 1994;13:964-72.
 28. Galn JE. Molecular genetic bases of Salmonella entry into host cells.
     Mol Microbiol 1996;20:263-71.
 29. Straley SC, Perry RD. Environmental modulation of the gene expression
     and pathogenesis in Yersinia. Trend Microbiol 1995;3:310-7.
 30. Lee C, Falkow S. The ability of Salmonella to enter mammalian cells is
     affected by bacterial growth states. Proc Natl Acad Sci USA
     1990;89:1847-51.
 31. Hromockyj AE, Tucker SC, Maurelli AT. Temperature regulation of
     Shigella virulence: identification of the repressor gene virR, an
     analogue of hns, and partial complementation by tyrosyl transfer RNA.
     Mol Microbiol 1992;6:2113-24.
 32. Pettersson J, Nordfelth R, Dubrinina E, Bergaman T, Gustfsson M,
     Magnusson KE, et al. Modulation of the virulence factor expression by
     pathogen target cell contact. Science 1996;273:1231-3.
 33. Kenny B, Lai L-C, Finlay BB, Donnenberg MS. EspA, a protein secreted
     by enteropathogenic Escherichia coli, is required to induce signals in
     epithelial cells. Mol Microbiol 1996;20:313-23.
 34. Salyers AA, Whitt DD. Bacterial Pathogenesis: A Molecular Approach,
     first ed. Washington D.C.: ASM Press, 1994.
                                [end of page 285]

 35. Cornelis GR. Yersinia Pathogenicity Factors. In: Hormaeche CE, Penn
     CW, Smyth CJ, editors. Molecular Biology of Bacterial Infection:
     Current Status and Future Perspectives. Cambridge: Cambridge
     University Press, 1992.
 36. Persson C, Nordfelth R, Holmstrm A, Hkansson S, Rosqvist R,
     Wolf-Watz H. Cell-surface-bound Yersinia translocate the protein
     tyrosine phosphatase YopH by a polarized mechanism into the target
     cell. Mol Microbiol 1995;18:135-50.
 37. Hkansson S, Galyov EE, Rosqvist R, Wolf-Watz H. The Yersinia YpkA
     Ser/Thr kinase is translocated and subsequently targeted to the inner
     surface of the HeLa cells plasma membrane. Mol Microbiol
     1996;20:593-603.
 38. Sory M-P, Cornelis GR. Translocation of a hybrid YopE-adenylate
     cyclase from Yersinia enterocolitica into HeLa cells. Mol Microbiol
     1994;14:583-94.
 39. Bliska JB, Guan K, Dixon JE, Falkow S. Tyrosine phosphatase hydrolysis
     of host proteins by an essential Yersinia virulence determinant. Proc
     Natl Acad Sci USA 1991;61:3914-21.
 40. Mnard R, Dehio C, Sansonetti PJ. Bacterial entry into epithelial
     cells: the paradigm of Shigella. Trends Microbiol 1996;4:220-6.
 41. Jones B, Pascopella L, Falkow S. Entry of microbes into the host:
     using M cells to break the mucosal barrier. Curr Opin Immunol
     1995;7:474-8.
 42. Galn JE, Curtiss IR., Cloning and molecular characterization of genes
     whose products allow Salmonella typhimurium to penetrate tissue
     culture cells. Proc Natl Acad Sci USA 1989;86:6386-7.
 43. Shea JE, Hensel M, Gleeson C, Holden DW. Identification of a virulence
     locus encoding a second type III secretion system in Salmonella
     typhimurium. Proc Natl Acad Sci 1996;93:2593-7.
 44. Hensel M, Shea JE, Gleeson C, Jones MD, Dalton E, Holden DW.
     Simultaneous identification of bacterial virulence genes by negative
     selection. Science 1995;269:400-3.
 45. Rosqvist R, Hkansson S, Forsberg , Wolf-Watz H. Functional
     conservation of the secretion and translocation machinery for
     virulence proteins of Yersiniae, Salmonellae and Shigellae. EMBO J
     1995;14:4187-95.
 46. Hermant D, Mnard R, Arricau N, Parsot C, Popoff MY. Functional
     conservation of the Salmonella and Shigella effectors of entry into
     epithelial cells. Mol Microbiol 1995;17:781-9.
 47. Woestyn S, Allaoui A, Wattiau P. YscN, the putative energizer of the
     Yersinia Yop secretion machinery. J Bacteriol 1994;176:1561-9.
 48. Plano GV, Straley SC. Mutations in yscC, yscD, and yscG prevent high
     level expression and secretion of V antigen and Yops in Yersinia
     pestis. J Bacteriol 1995;177:3843-54.
 49. Allaoui A, Woestyn S, Sluiters C, Cornelis GR. YscU, a Yersinia
     enterocolitica inner membrane protein involved in Yop secretion. J
     Bacteriol 1994;176:4534-42.
 50. Fields K, Plano GV, Straley SC. A low-Ca2+ response (LCR) secretion
     (ysc) locus lies within the lcrB region of the LCR plasmid in Yersinia
     pestis. J Bacteriol 1994;176:569-79.
 51. Allaoui A, Schulte R, Cornelis GR. Mutational analysis of Yersinia
     enterocolitica virC operon: characterization of yscE,F,G, I, J, K,
     required for Yop secretion and yscH encoding YopR. Mol Microbiol
     1995;18:343-55.
 52. Allaoui A, Scheen R, de Rouvroit CL, Cornelis GR. VirG, a Yersinia
     enterocolitica lipoprotein involved in Ca2+ dependency, is related to
     ExsB of Pseudomonas aeruginosa. J Bacteriol 1995;177:4230-7.
 53. Macnab RM. Flagella and Motility. In: Neidhardt FC et al., editors.
     Escherichia coli and Salmonella typhimurium Cellular and Molecular
     Biology. Washington, D. C.: ASM Press, 1996.
 54. Rimpilinen M, Forsberg , Wolf-Watz H. A novel protein, LcrQ,
     involved in the low-calcium response of Yersinia pseudotuberculosis
     shows extensive homology to YopH. J Bacteriol 1992;174:3355-63.
 55. Hkansson S, Bergman T, Vanooteghem J-C, Cornelis G, Wolf-Watz H. YopB
     and YopD constitute a novel class of Yersinia Yop proteins. Infect
     Immun 1993;61:71-80.
 56. Wattiau P, Woestyn S, Cornelis GR. Customized secretion chaperones in
     pathogenic bacteria. Mol Microbiol 1996;20:255-62.
 57. Frithz-Lindsten E, Rosqvist R, Johansson L, Forsberg . The
     chaperone-like protein YerA of Yersinia pseudotuberculosis stabilizes
     YopE in the cytoplasm but is dispensable for targeting to the
     secretion loci. Mol Microbiol 1995;16:635-47.
 58. Mnard R, Sansonetti P, Parsot C, Vasselon T. Extracellular
     association and cytoplasmic partitioning of the IpaB and IpaC invasins
     of S. flexneri. Cell 1994;79:515-25.
 59. Skrzypek E, Straley SC. LcrG, a secreted protein involved in negative
     regulation of the low-calcium response in Yersinia pestis. J Bacteriol
     1993;175:3520-8.
 60. Forsberg , Viitanen A-M, Skurnik M, Wolf-Watz H. The surface-located
     YopN protein is involved in calcium signal transduction in Yersinia
     pseudotuberculosis. Mol Microbiol 1991;5:977-86.
 61. Hughes KT, Gillen KL, Semon MJ, Karlinsey JE. Sensing structural
     intermediates in bacterial flagellar assembly by export of a negative
     regulator. Science 1993;262:1277-80.
 62. Ginocchio C, Galn JE. Functional conservation among members of the
     Salmonella typhimurium InvA family of proteins. Infect Immun
     1994;63:729-32.
                                [end of page 286]

 63. Jarvis KG, Girn JA, Jerse AE, McDaniel TK, Donnenberg MS, Kaper JB.
     Enteropathogenic Escherichia coli contains a putative type III
     secretion system necessary for the export of proteins involved in
     attaching and effacing lesion formation. Proc Natl Acad Sci USA
     1995;92:7996-8000.
 64. Ochman H, Groisman EA. The evolution of invasion by enteric bacteria.
     Can J Microbiol 1995;41:555-61.
 65. Li J, Ochman H, Groisman EA, Boyd EF, Solomon F, Nelson K, et al.
     Relationship between evolutionary rate and cellular location among the
     Inv/Spa invasion proteins of Salmonella enterica. Proc Natl Acad Sci
     USA 1995;92:7252-6.
 66. Lee CA. Pathogenicity islands and the evolution of bacterial
     pathogens. Infect Agents Dis 1996;5:1-7.
 67. Morschhuser J, Vetter V, Emdy L, Hacker J. Adhesin regulatory genes
     within large, unstable DNA regions of pathogenic Escherichia coli:
     cross-talk between different adhesin gene clusters. Mol Microbiol
     1994;11:555-66.
 68. Mills DM, Bajaj V, Lee CA. A 40 kb chromosomal fragment encoding
     Salmonella typhimurium invasion genes is absent from the corresponding
     region of the Escherichia coli K-12 chromosome. Mol Microbiol
     1995;15:749-59.
 69. Stein MA, Leung KY, Zwick M, Garcia-del Portillo F, Finlay BB.
     Identification of a Salmonella virulence gene required for formation
     of filamentous structures containing lysosomal membrane glycoproteins
     within epithelial cells. Mol Microbiol 1996;20:151-64.
 70. Fetherston JD, Schuetze P, Perry RD. Loss of the pigmentation
     phenotype in Yersinia pestis is due to the spontaneous deletion of 102
     kb of chromosomal DNA which is flanked by a repetitive element. Mol
     Microbiol 1992;6:2693-704.
 71. Fetherston JD, Perry RD. The pigmentation locus of Yersinia pestis
     KIM6+ is flanked by an insertion sequence and includes the structural
     genes for pesticin sensitivity and HMWP2. Mol Microbiol
     1994;13:697-708.
 72. Iteman I, Guiyoule A, de Almeida AMP, Guilvout I, Baranton G, Carniel
     E. Relationship between loss of pigmentation and deletion of the
     chromosomal iron-regulated irp2 gene in Yersinia pestis: evidence for
     separate but related events. Infect Immun 1993;61:2717-22.
 73. Rakin A, Urbitsch P, Heeseman J. Evidence for two evolutionary
     lineages of highly pathogenic Yersinia species. J Bacteriol
     1995;177:2292-8.
 74. Matic I, Taddei F, Radman M. Genetic barriers among bacteria. Trend
     Microbiol 1996;4:69-73.
 75. Falkow S. The evolution of pathogenicity in Escherichia, Shigella, and
     Salmonella. In: Neidhardt FC, et al. Escherichia coli and Salmonella
     Cellular and Molecular Biology. Washington, D. C.: ASM Press, 1996.
 76. Waldor MK, Mekalanos JJ. Cholera toxin is encoded by a filamentous
     bacteriophage that uses TCP pili as a receptor. Science
     1996;272:1910-4.
 77. Guttman DS, Dykhuizen DE. Clonal divergence in Escherichia coli as a
     result of recombination, not mutation. Science 1994;266:1380-3.
 78. Inouye S, Sunshine MG, Six EW, Inouye M. Retronphage R73: an E. coli
     phage that contains a retroelement and integrates into a tRNA gene.
     Science 1991;252:969-71.
 79. Reiter W, Palm P, Yeats S. Transfer RNA genes frequently serve as
     integration sites for prokaryotic genetic elements. Nucleic Acids Res
     1989;17:1907-14.
 80. Sun J, Inouye M, Inouye S. Association of a retroelement with a
     P4-like cryptic prophage (Retronphage R73) integrated into the
     selenocystyl tRNA gene of Escherichia coli. J Bacteriol
     1991;173:4171-81.
 81. Akerley BJ, Cotter PA, Miller JF. Ectopic expression of the flagellar
     regulon alters development of the Bordetella host interaction. Cell
     1995;80:611-20.
 82. Blum G, Ott M, Lischewski A, Ritter A, Imrich H, Tschape H, et al.
     Excision of large DNA regions termed pathogenicity islands from
     tRNA-specific loci in the chromosome of an Escherichia coli wild-type
     pathogen. Infect Immun 1994;62:606-14.
 83. McDaniel TK, Jarvis KG, Donnenberg MS, Kaper JB. A genetic locus of
     enterocyte effacement conserved among diverse enterobacterial
     pathogens. Proc Natl Acad Sci USA 1995;92:1664-8.
 84. Ritter A, Blum G, Emody L, Kerenyi M, Bock A, Neuhierl B, et al. tRNA
     genes and pathogenicity islands: influence on virulence and metabolic
     properties of uropathogenic Escherichia coli. Mol Microbiol
     1995;17:109-21.
 85. Zagaglia C, Casalino M, Colonna B, Conti C, Calconi A, Nicoletti M.
     Virulence plasmids of enteroinvasive Escherichia coli and Shigella
     flexneri integrate into a specific site on the host chromosome:
     integration greatly reduces expression of plasmid-carried virulence
     genes. Infect Immun 1991;59:792-9.
 86. Colonna B, Casalino M, Fradiani PA, Zagaglia C, Naitza S, Leoni L, et
     al. H-NS regulation of virulence gene expression in enteroinvasive
     Escherichia coli harboring the virulence plasmid integrated into the
     host chromosome. J Bacteriol 1995;177:4703-12.
 87. Zsigray RM, Hopper JB, Zukowski K, Chesbro WR. Integration of the Vwa
     plasmid into the chromosome of Yersinia pestis strains harboring F'
     plasmids of Escherichia coli. Infect Immun 1985;47:670-3.
 88. Zsigray RM, Lawton WD, Surgalla MJ. Repression of the virulence of
     Yersinia pestis by an F' plasmid. Infect Immun 1983;39:974-6.
 89. Protsenko OA, Filippov AA, Kutyrev VV. Integration of the plasmid
     encoding the synthesis of capsular antigen and murine toxin into
     Yersinia pestis chromosome. Microb Pathog 1991;11:123-8.
                                [end of page 287]

 90. Whittam TS, Wolfe ML, Wachsmuth IK, rskov F, rskov I, Wilson RA.
     Clonal relationships among Escherichia coli strains that cause
     hemorrhagic colitis and infantile diarrhea. Infect Immun
     1993;61:1619-29.
 91. Waldor MK, Mekalanos JJ. Vibrio cholerae O139 specific gene sequences.
     Lancet 1994;343:1366.
 92. Pajni S, Charu S, Bhasin N, Ghosh A, Ramamurthy T, Nair GB, et al.
     Studies on the genesis of Vibrio cholerae O139: identification of
     probable progenitor strains. Journal of Molecular Microbiology
     1995;42:20-5.
 93. Bik EM, Bunschoten AE, Gouw RD, Mooi FR. Genesis of the novel epidemic
     Vibrio cholerae 139 strain: evidence for horizontal transfer of genes
     involved in polysaccharide synthesis. EMBO J 1995;14:209-16.
 94. Sory M-P, Hermand P, Vaerman J-P, Cornelius GR. Oral immunization of
     mice with a live recombinant Yersinia enterocolitica O:9 strain that
     produces the cholera toxin B subunit. Infect Immun 1990;58:3830-6.
 95. Falkow S, Small P, Isberg R, Hayes SF, Corwin D. A molecular strategy
     for the study of bacterial invasion. Rev Infect Dis 1987;9:S450-5.
 96. Swenson DL, Bukanov NO, Berg DE, Welch RA. Two pathogenicity islands
     in uropathogenic Escherichia coli strain J96: cosmid cloning and
     sample sequencing. Infect Immun 1996;64:3736-43.
 97. Blum G, Falbo V, Caprioli A, Hacker J. Gene clusters encoding the
     cytotoxic necrotizing factor type 1, prs-fimbriae and -hemolysin form
     the pathogenicity island II of the uropathogenic Escherichia coli
     strain J96. Federation of European Microbiological Societies
     Microbiology Letters 1995;126:189-96.
 98. Comstock LE, Johnson JA, Michalski JM, Morris Jr JG, Kaper JB. Cloning
     and sequence of a region encoding a surface polysaccharide of Vibrio
     cholerae O139 and characterization of the insertion site in the
     chromosome of Vibrio cholerae 01. Mol Microbiol 1996:19:815-26.
 99. Gouin E, Mengaud J, Cossart P. The virulence gene cluster of Listeria
     monocytogenes is also present in Listeria ivanovii, an animal
     pathogen, and Listeria seeligeri, a nonpathogenic species. Infect
     Immun 1994;62:3550-3.

                             [end of page 288]

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

---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
Synopses

New Vaccines for the Prevention of Pneumococcal Infections

Helena Kyhty and Juhani Eskola
National Public Health Institute, Helsinki, Finland
---------------------------------------------------------------------------

    Streptococcus pneumoniae is a major cause of acute otitis media,
    pneumonia, bacteremia, and meningitis. Because in recent years
    antibiotic-resistant pneumococcal strains have been emerging
    throughout the world, vaccination against pneumococcal infections
    has become more urgent. The capsular polysaccharide vaccine that
    has been available is neither immunogenic nor protective in young
    children and other immunocompromised patients. Several
    pneumococcal proteins have been proposed as candidate vaccines,
    but no human studies associated with them have been reported.
    Clinical trials of first-generation pneumococcal conjugate
    vaccines have shown that covalent coupling of pneumococcal
    capsular polysaccharides to protein carriers improves the
    immunogenicity of the polysaccharides. The protective efficacy of
    the conjugate vaccines against carriage, acute otitis media, and
    invasive infections is being studied.

Streptococcus pneumoniae (pneumococcus [Pnc]) is a common bacterial agent
found in mild mucosal as well as severe systemic infections. Local
infections, such as acute otitis media, are rather common; every child has
at least one during the first 2 years of life (1), and Pnc is the causative
agent in approximately half of the bacterial culture-positive cases (2).
Pneumonia is another disease often caused by Pnc, both in industrialized
and developing countries. Pneumonia, which causes more than one million
deaths per year, is the most common cause of childhood death in the
developing world (3); pneumococcal pneumonia is a serious problem among the
elderly in industrialized countries. Pnc also causes frequent invasive
infections, especially among children. In Finland, the incidence of
bacteremic pneumococcal infections at 0 to 4 years of age has been 24.2 per
100,000 per year (4). The corresponding rate was 42 per 100,000 per year in
Israel (5) and 66 per 100,000 per year in the United States (6). In
addition to the young and the elderly, some of the other groups at
increased risk for Pnc infection are patients with chronic cardiac or
pulmonary diseases, immunocompromised patients, and especially persons with
functional or anatomic asplenia (7).

The treatment of recently emerged Pnc strains that are resistant to
penicillin and other antibiotics (8) is becoming a challenge. Because of
the high rates of illness and death associated with pneumococcal infections
and the rapidly increasing resistance of organisms that cause these
infections to antimicrobial drugs, development and use of effective
pneumococcal vaccines is of high priority. The progress has been rapid; in
addition to polysaccharide(PS)-protein conjugate vaccines, vaccines
containing pneumococcal proteins are also being developed.

Pneumococcal Capsular Polysaccharide Vaccine
Pnc can be divided into at least 90 serotypes according to the structure of
the PS in the capsule surrounding the bacterium. The capsule seems to be
the most important virulence factor; all strains isolated from infections
are encapsulated. The capsule helps the bacterium escape the host defense
mechanisms. However, only a small fraction of all capsular types are common
causes of pneumococcal infections. The list of the most common groups/types
(4, 6, 7, 9, 14, 18, 19, and 23) that cause
                             [end of page 289]

childhood infections is similar in most parts of the world. Types 1 and 5
are, however, more common in the developing world than in industrialized
countries (9).

Antibodies to capsular PSs protect from infection by opsonizing Pnc for
phagocytosis by neutrophils. A capsular PS vaccine containing 23 of the
most common serotypes/groups has proven protective in immunocompetent
adults and in some groups at risk (7,10,11); it has also been shown to have
an impact on death rates due to pneumonia in Papua New Guinea (12). Among
the immunocompromised and in preventing acute otitis media, (13) its
efficacy has been only marginal.

The reason for the vaccine's poor immunogenicity and its lack of efficacy
in children is thought to be the nature of the PS antigen. PS antigens are
type 2 T-cell independent (TI) antigens, which stimulate mature B cells
without the help of T cells. In humans, the B cells of newborns do not
respond to most of the PS antigens. Responsiveness develops only slowly
during the first years of life. Furthermore, the TI antigens do not induce
immunologic memory and the maturation of the immune response; anti-PS
antibodies have low avidity and the switch from one isotype to another does
not happen even after repeated immunizations. The TI antigens induce mainly
IgM responses, especially in mice. However, in humans the response also
contains the IgG and IgA components (14). Furthermore, the IgG response to
PS antigens contains a greater proportion of IgG2 (15,16) than found in a
response to protein antigens. The lack of memory has some important
implications for the vaccination. Because of the rapid decline of
antibodies, revaccination is often necessary (7).

Pneumococcal Protein Vaccine Candidates
Several ways have been and are being tried to solve the problem of poor
immunogenicity of pneumococcal PS vaccines in infancy. In addition to the
capsule, other pneumococcal virulence factors have been considered as
promising vaccine candidates or as carrier proteins in pneumococcal
conjugate vaccines (see above). The prime vaccine candidates are enzymes
and toxins that are excreted or released after the bacterium has autolyzed
or surface proteins whose exact functions are not known. Pneumococcal
proteins studied as potential vaccines include neuramididase, autolysin,
pneumolysin, pneumococcal surface protein A (PspA), and pneumococcal
surface adhesin A (PsaA) (17-19).

Pneumolysin is a cytolytic toxin produced by all types of Pnc. In mice,
immunization with inactivated pneumolysin or recombinant pneumolysin toxoid
offers at least partial protection or enhanced survival when challenged
with Pnc (20,21). PspA is a surface protein present in all clinically
relevant pneumococcal strains. PspAs from different pneumococcal strains
vary serologically. However, many PspA antibodies cross-react with PspAs
from unrelated strains. Furthermore, active immunization of mice with PspA
generates protective immune response against diverse pneumococcal strains
(22). Truncated PspAs, expressed as recombinant proteins, are also
immunogenic in mice and can elicit cross-protection (18).

Pneumococcal Conjugate Vaccines
Another approach to solving the poor immunogenicity of the capsular PS
antigens has already moved to the clinical phase-III trials. This approach
is based on the 1929 findings of Goebel and Avery (23), who showed that
covalent coupling of haptens to a protein carrier improves the
immunogenicity of the hapten. In this way, the anti-PS response gets T-cell
dependent characters: there is development of immunologic memory and
maturation of the immune response. This is seen as an increase in the
antibody concentrations and the antibody affinity and as a switch in the
isotype distribution after repeated immunizations. This approach has been
used successfully to prepare vaccines against Haemophilus influenzae type b
(Hib); the incidence of Hib infection has decreased drastically wherever
these conjugate vaccines have been used (24).

The PS antigen in a conjugate vaccine seems to benefit at least partly from
the immunologic characters of the carrier protein. The protein is presented
as peptides in association with the major histocompatibility complex class
II molecules
                             [end of page 290]

on the surface of the antigen-presenting cells. This stimulates the
T-helper cells, which then stimulate adjacent B cells for antibody
production and maturation into memory cells. Development of immunologic
memory means that the protection does not depend solely on the existing
antibody concentration. Instead, the vaccinated persons can respond with a
rapid, high, and effective antibody response to colonization or invasion by
the respective Pnc type. Studies in Finland suggest that this indeed
happens: the efficacy of an Hib conjugate vaccine, PRP-D, was more than 90%
in early infancy, even though a large proportion of the infants did not
have measurable antibody response after the primary course of immunization
(25). A study in the United Kingdom suggests that the carriage of Hib
indeed induces a high "booster type" immune response (26).

Conjugation of the PS to a protein carrier has repeatedly been shown to
work with Hib; vaccines based on the same principle would also decrease the
number of different infections caused by Pnc. Four vaccine manufacturers
have prepared pneumococcal conjugate vaccines with basically the same
approaches as the Hib conjugates (Table 1). PncOMPC vaccine contains PSs
from seven serotypes conjugated to the meningococcal outer membrane protein
complex (27). The PncCRM vaccine contains either oligosaccharides (OS) or
PSs coupled to a nontoxic mutant diphtheria toxin CRM197. The PS-containing
conjugate vaccine is at present heptavalent (28), but it is possible to add
types 1 and 5 to the product intended for use in developing countries. The
PncT vaccine contains eight PSs coupled to tetanus toxoid, and the PncD
product contains the same PSs coupled to diphtheria toxoid (29). Besides
these formulations, several other approaches have been tested in animals.
These include conjugates using pneumolysoid (30), pertussis toxin (31), and
salmonella protein (32) as a carrier. Recently, small peptides selected on
the basis of T-cell stimulating properties have also been coupled to
pneumococcal PS to form conjugate vaccines (33).

Preclinical Testing
Before human trials, these conjugates were immunogenic and protective in
animals, including mice, infant monkeys, and chinchillas (34-37). All these
studies indicate that conjugate vaccines have greater immunogenicity than
pneumococcal PS vaccines. Even though animal studies can tell if the
conjugate vaccine is immunogenic and evokes a T-cell dependent response,
the final proof of conjugate vaccines' superior immunogenicity and efficacy
over PS vaccines comes only from human studies. So far no animal model can
mimic human immunogenicity and efficacy studies.

Clinical Testing
Pneumococcal conjugates of all the manufacturers mentioned in Table 1 have
now been tested in phase-I and phase-II studies. The first human studies
were done in adults with mono- or bivalent conjugates and showed that the
conjugates were at least as immunogenic as the PS vaccine. Since then, up
to eight valent vaccines have been used in human studies, also among
infants.

          Table 1. Pneumococcal conjugate vaccines in phase-II
          and phase-III trials

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

           Vaccine   Serotype      Carrier        Manufacturer

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

          PncCRM   4, 6B, 9V,  CRM197          Wyeth-Lederle
                   14, 18C,                    Vaccines and
                   19F, 23F                    Pediatrics

          PncD     3, 4, 9V,   Diphtheria      Connaught
                   14, 18C,    toxoid          Laboratories
                   19F, 236B,

          PncT     3, 4, 6B,   Tetanus toxoid  Pasteur Merieux
                   9V, 14,                     Serums & Vaccins
                   18C, 19F,
                   23

          PncOMPC  4, 6B, 9V,  Meningococcal   Merck Research
                   14, 18C,    OMPC            Laboratories
                   19F, 23F

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

          CRM = CRM197, a nontoxic variant of diphtheria toxin;
          D = diphtheria toxoid; T = tetanus toxoid; OMPC =
          outer membrane protein complex

Adults and Toddlers
To show that they are safe and immunogenic, pneumococcal conjugates were
first given to small numbers of adults and toddlers. Most of the reported
studies have been conducted with mono- to tetravalent vaccines. The PncOMPC
studies in adults show that the conjugate vaccine was well tolerated but
not more immunogenic than the PS vaccine (38,39). One possible reason might
be the low dose (1g to 5g of each
                             [end of page 291]

conjugate) used in these studies. Different formulations of PncCRM
containing either PS or OS linked to CRM197 have been tested in adults. All
were well tolerated and evoked a comparable immune response (40). This was
confirmed in a study in which heptavalent OS conjugate was immunogenic
(28). Results of immunizing adults with PncT or PncD conjugates have been
reported in two studies; both vaccines were more immunogenic than the PS
vaccine (41,42). The Finnish study with tetravalent PncT and PncD showed
that these conjugates can also evoke a mucosal antibody response (42).

PncOMPC vaccine was given to 31 Finnish children at 24 months, and 10 of
them also received it at 26 months. The primary response was only slightly
higher than to the PS vaccine, but after the second dose a booster type
response was seen in most of the vaccinees (43). Studies conducted during
the second year of life showed that the heptavalent PncOMPC conjugate was
more immunogenic than the PS vaccine (44,45). Different formulations of
PncCRM have also been tested in toddlers (46). Conjugates were more
immunogenic than the PS vaccine; furthermore, the PS conjugate was more
immunogenic than the OS conjugate. One study showed a good booster response
to PS vaccine after primary immunization with pentavalent PS-based PncCRM
(47). The PncT and PncD conjugates have also proven immunogenic in
toddlers. A Finnish study compared 3-g and 10-g doses at 24 months, and a
U.S. study used 10-g doses of type 19F conjugates with PS vaccine booster
doses (48).

Infants
Keyserling et al. (49) have compared different dosages of type 14 PS
containing monovalent PncOMPC vaccine in infants and shown that 2.5g to
5g of type 14 PS in the conjugate gave better responses than the lower
doses. A Finnish study (50) showed that a primary series of three doses of
tetravalent PncOMPC at 2, 4, and 6 months was better than two doses at 4
and 6 months. Furthermore, a booster dose of PncOMPC given at 14 months
evoked a secondary response to all PS types. Concomitant administration of
PncOMPC with routine infant immunizations does not seem to have an effect
on either anti-Hib or anti-Pnc PS antibody responses (51). The heptavalent
PncOMPC formulation is as immunogenic as the previous formulations with
fewer serotypes (27).

hman et al. have shown that the pentavalent PncCRM vaccine containing OS
derived from pneumococcal capsule was immunogenic and tolerable in infants
(52). The same children developed a good antibody response when boostered
with PS vaccine at 24 months, suggesting that the immunologic priming had
been good even if the antibody response to the primary series had remained
rather low (53). The PS-based PncCRM has been shown to be more immunogenic
than OS conjugates also in infancy (54). A Gambian study evaluated the
pentavalent PncCRM conjugate (PS-based) in a developing country when given
at 2, 3, and 4 or at 2 and 4 months. The vaccine was immunogenic and well
tolerated; the schedule of three doses was better than the two-dose
schedule (55).

A Finnish study compared three dosages of 1g to 10g of each PS in
tetravalent PncT and PncD conjugates when administered at 2, 4, and 6
months. These vaccines were immunogenic in infancy, and no difference could
be shown between PncT and PncD. The response after a primary series to
PncD, but not to PncT, was dose dependent (56). The children immunized with
PncD in infancy had a booster response after reimmunization with either
PncD or pneumococcal PS vaccine at 14 months (57). All who received PncT
were boostered with PS vaccine, and the response was dose dependent;
children that had received 10-g doses of PncT during the primary
immunization had the lowest mean booster responses (58). Another Finnish
study showed that octavalent (types 3, 4, 6B, 9V, 14, 18C, 19F, and 23F)
PncD (3g of each PS) and PncT (1g of each PS) induced immune responses
similar to the respective tetravalent formulations (29). An Icelandic study
showed that the octavalent vaccine was immunogenic in infants when given at
3, 4, and 6 months and that the IgG anti-PS concentrations correlated with
the opsonic activity (59).

Because no study has directly compared different pneumococcal conjugate
vaccines,
                             [end of page 292]

the comparison has to rely on data from separate studies. A Finnish group
has analyzed the antibody response in adults, toddlers, and infants to all
four types of Pnc conjugates. This comparison shows that there are vaccine-
and type-specific differences in the antibody responses (Table 2). However,
none of the vaccines used in these studies have the composition suggested
in the phase-III trials (29,50,52,56).

       Table 2. Antibody response of Finnish infants to pneumococcal
      conjugate vaccines administered at 2, 4, and 6 months of age*.
    -------------------------------------------------------------------
                       Geometric Mean of the Anti-PNC PS (g/ml)
                --------------------------------------------------------
                  Type 6B     Type 14     Type 19F    Type 23F
      Vaccine    Pre  Post   Pre   Post  Pre   Post  Pre   Post   Ref.
    -------------------------------------------------------------------
    PncOMPC     0.17  1.30  0.42  8.27   0.34  9.85  0.28  1.90  (50)
    PncCRM      0.25  0.50  0.30  2.49   0.46  1.13  0.18  0.83  (52)
    PncT01-4    0.25  0.89  0.24  2.84   0.36  3.73  0.18  0.82  (56)
    PncT01-8    0.20  1.28  0.30  2.56   0.56  4.23  0.22  1.03  (29)
    PncD03-4    0.26  0.88  0.44  2.20   0.43  5.29  0.21  0.67  (56)
    PncD03-8    0.17  1.44  0.31  4.62   0.37  4.94  0.24  1.07  (29)
    -------------------------------------------------------------------
    PncOMP = tetravalent conjugate vaccine with a meningococcal outer
    membrane protein complex as a carrier
    PncCRM = pentavalent oligosaccharide conjugate vaccine with CRM197
    protein as a carrier
    PncT01-4 = tetravalent conjugate vaccine with tetanus toxoid
    carrier; 1 g of each of four polysaccharides
    PncT01-8 = tetravalent conjugate vaccine with tetanus toxoid
    carrier; 1 g of each of four polysaccharides
    PncD03-4 = tetravalent conjugate vaccine with diphtheria toxoid
    carrier; 3g of each of four polysaccharides
    PncD03-8 = octavalent conjugate vaccine with diphtheria toxoid
    carrier; 3g of each of four polysaccharides
    *Serum samples are taken before immunization (pre) and at 7 months
    (post). The data have been gathered from separate studies done in
    the same population.

Comparing the data from different studies is difficult because there can be
interlaboratory variation in the enzyme-linked immunosorbent assay results.
The Centers for Disease Control and Prevention, Food and Drug
Administration, and World Health Organization are working on a standardized
anti-Pnc PS assay, which will, if not eliminate, at least reduce the impact
of this problem. A standard serum (60) to be used in all laboratories is
distributed by Center for Biologics Evaluation and Research/Food and Drug
Administration.

Pneumococcal Conjugates and the Carriage of Pnc
Experience with the Hib conjugates (24) suggests that Pnc conjugate
vaccines could also reduce the number of carriers of the vaccine types and
in this way decrease the spread of bacteria. The results from the only
reported study are encouraging. The PncOMPC vaccine decreased the carriage
rate among toddlers, while the pneumococcal PS vaccine did not (45).
Importantly, the carriage of antibiotic-resistant Pnc also decreased (61).

Efficacy Studies
Phase-III studies with the heptavalent formulations of PncOMPC and PncCRM
are ongoing or being started. These studies look at prevention of carriage,
acute otitis media, or invasive Pnc infection caused by Pnc of the vaccine
serotypes. Furthermore, there are several plans for studying the effect of
Pnc conjugates on Pnc invasive infection and pneumonia in developing
countries.

Questions to Be Answered in the Future
We do not know if conjugate vaccines can really prevent Pnc infections
better than the PS vaccine. We hope that the new vaccines can prevent
several types of infections, from symptomless Pnc carriage to serious
invasive infections with high death rates. It is quite probable that the
protective immune response needed is different for each type of infection.
We do not know if parenterally administered vaccine can prevent carriage or
mucosal infections such as acute otitis media. It is still unknown whether
a mucosal immune response is needed or whether transudation of antibodies
from the serum is enough for protection against local infection. Saliva
samples of infants immunized with Hib conjugate vaccines contain secretory
IgA but also IgG, which has most probably
                             [end of page 293]

transudated from serum (62). In an infant rat Hib colonization model, both
secretory IgA and serum derived IgG decreased colonization (63).
Furthermore, animal experiments suggest that immune response evoked by
parenteral administration of a conjugate vaccine would alone protect
against acute otitis media (64). In addition, passive immunization of
infants with hyperimmune serum pool containing antibodies to pneumococcal
PS-induced protection against pneumococcal acute otitis media suggests that
protection is offered when high enough serum antibody concentrations are
gained (65). At present, there are no data to show which antibody
concentrations are needed for protection. Deciding about the protective
concentration might be difficult because the development of immunologic
memory is an important factor; the protection does not solely depend on the
existing antibody concentration.

Most phase-II studies have used a schedule of two or three doses of Pnc
conjugate vaccine in infancy (usually at 2, 4, and 6 months) and a booster
dose of either conjugate or Pnc PS at the second year of life. The need for
a booster dose at the second year is not known; this information would be
important especially for planning the vaccination schedules for developing
countries, where administering a booster dose can be problematic. The
experience from the Hib conjugates suggests that a booster dose might not
be needed; the United Kingdom has successfully used a schedule of three
doses at 3, 4, and 5 months without a booster dose (66).

Reduction of pneumococcal infections among the elderly would probably
increase the quality of their lives. The immunogenicity of pneumococcal PS
vaccine in this age group has been satisfactory (67,68). An Hib conjugate
(PRP-D) has proven more immunogenic than the Hib PS vaccine in the elderly
(69). However, the immune response to PncCRM was not better than to the Pnc
PS vaccine, and no booster response was seen (70). Studies with other
pneumococcal conjugates in the elderly have not been reported.

Because pneumococcal infections of very young infants are a problem in
developing countries, several groups have studied the possibility of
maternal immunization with pneumococcal vaccines (71,72). So far only PS
vaccines have been used; even though these vaccines are immunogenic in
pregnant mothers, the immunity transferred to the neonate is not very long
lasting. If the conjugate vaccines induce higher antibody concentrations in
mothers, the concentration of passively acquired antibody in the baby would
stay high for a longer time. The Hib conjugate vaccines induce good
responses in mothers and, consequently, long-lasting protective
concentrations in infants born to these mothers (73,74). The effect of
simultaneous maternal tetanus immunization, especially if conjugates with a
tetanus toxoid carrier are used, and the effect of high maternal antibody
level on the antibody responses of the infants have to be determined.

By the year 2000, we may have pneumococcal conjugate vaccines to include in
routine childhood immunization programs. The price of the conjugate
vaccines has been so high that their use throughout the world has not been
possible. An important challenge in developing of pneumococcal conjugate
vaccines is to reduce the costs of manufacturing so that all children can
benefit from them.

Dr. Kyhty is a senior researcher at the National Public Health Institute,
Finland, working as the head of the Laboratory of Vaccine Immunology. Her
research focuses on systemic and mucosal immunity against encapsulated
bacteria, mainly Neisseria meningitidis, Streptococcus pneumoniae, and
Haemophilus influenzae type b.

Dr. Eskola is a research professor at the National Public Health Institute,
Finland, working as the director of the Division of Infectious Diseases,
and the head of the Department of Vaccines. A pediatric infectious disease
physician by training, he has focused his research interests on the
clinical evaluation of new vaccines. He is the principal investigator of
the Finnish efficacy trial of pneumococcal conjugates in prevention of
acute otitis media.

Address for correspondence: Helena Kyhty, Laboratory of Vaccine
Immunology, Department of Vaccines, National Public Health Institute,
Mannerheimintie 166, FIN-00300 Helsinki, Finland; fax: 358-9-4744238;
e-mail: helena.kayhty@ktl.fi.

References

  1. Alho OP. Acute otitis media in infancy: Occurrence and risk factors.
     Oulu, Finland: University of Oulu, 1990.
                                [end of page 294]

  2. Luotonen J, Herva E, Karma P, Timonen M, Leinonen M, Mkel PH. The
     bacteriology of acute otitis media in children with special reference
     to Streptococcus pneumoniae as studied by bacteriological and antigen
     detection methods. Scand J Infect Dis 1981;13:177-83.
  3. Stansfield SK. Acute respiratory infections in the developing world:
     strategies for prevention, treatment and control. Pediatr Infect Dis J
     1987;6:622-9.
  4. Eskola J, Takala AK, Kela E, Pekkanen E, Kalliokoski R, Leinonen M.
     Epidemiology of invasive pneumococcal infections in children in
     Finland; A five year prospective study with special implications for
     prevention. JAMA 1992;268:3323-7.
  5. Dagan R, Englehard D, Piccard E, Israeli Pediatric Bacteremia and
     Meningitis Group. Epidemiology of invasive childhood pneumococcal
     infections in Israel. JAMA 1992;268:3328-32.
  6. Bennett NM, Buffington J, LaForce FM. Pneumococcal bacteremia in
     Monroe County, New York. Am J Public Health 1992;82:1513-6.
  7. Advisory Committee on Immunization Practices. Prevention of
     pneumococcal disease. Recommendations of the Advisory Committee on
     Immunization Practices. MMWR (in press) 1996.
  8. Klugman KP. Pneumococcal resistance to antibiotics. Clin Microbiol Rev
     1990;3:171-96.
  9. Sniadack DH, Schwartz B, Lipman H, Bogaerts J, Butler JC, Dagan R, et
     al. Potential interventions for the prevention of childhood pneumonia:
     geographic and temporal differences in serotype and serogroup
     distribution of on sterile site pneumococcal isolates from children-
     implications for vaccine strategies. Pediatr Infect Dis J
     1995;14:503-10.
 10. Shapiro ED, Berg AT, Austrian R, Schroeder D, Parcells V, Margolis A,
     et al. The protective efficacy of polyvalent pneumococcal
     polysaccharide vaccine. N Engl J Med 1991;325:1453.
 11. Butler JC, Breiman RF, Campbell JF, Lipman HB, Broome CV, Facklam RR.
     Pneumococcal polysaccharide vaccine efficacy. JAMA 1993;270:1826-31.
 12. Riley ID, Lehmann D, Alpers MP, Marshall TF, Gratten H, Smith D, et
     al. Pneumococcal vaccine prevents death from acute lower respiratory
     tract infections in Papua New Guinean children. Lancet 1986;2:877-81.
 13. Mkel PH, Sibakov M, Herva E, Henricksen J. Pneumococcal vaccine and
     otitis media. Lancet 1980;2:547-51.
 14. Kyhty H, Eskola J, Peltola H, Stout M, Samuelson JS, Gordon LK.
     Immunogenicity in infants of a vaccine composed of Haemophilus
     influenzae type b capsular polysaccharide mixed with DPT or conjugated
     to diphtheria toxoid. J Infect Dis 1987;155:100-6.
 15. Sarvas H, Rautonen N, Sipinen S, Mkel O. IgG subclasses of
     pneumococcal antibodieseffect of allotype G2m(n). Scand J Immunol
     1989;29:229-37.
 16. Mkel O, Mattila P, Rautonen N, Seppl N, Seppl I, Eskola J, et
     al. Isotype concentrations of human antibodies to Haemophilus
     influenzae type b polysaccharide (Hib) in young adults immunized with
     the polysaccharide as such or conjugated to a protein (diphtheria
     toxoid). J Immunol 1987;139:1999-2004.
 17. Lock RA, Paton JC, Hansman D. Comparative efficacy of pneumococcal
     neuraminidase and pneumolysin as immunogens protective against
     Streptococcus pneumoniae. Microb Pathog 1988;5:461-7.
 18. Tart RC, McDaniel LS, Ralph BA, Briles DE. Truncated Streptococcus
     pneumoniae PspA molecules elicit cross-protective immunity against
     pneumococcal challenge in mice. J Infect Dis 1996;173:380-6.
 19. Sampson JS, O'Connor SP, Stinson AR, Tharpe JA, Russell H. Cloning and
     nucleotide sequence analysis of psaA, the Streptococcus pneumoniae
     gene encoding a 37-kilodalton protein homologous to previously
     reported Streptococcus sp. adhesins. Infect Immun 1994;62:319-24.
 20. Alexander JE, Lock RA, Peeters C, Poolman JT Andrew PW, Mitchell TJ,
     et al. Immunization of mice with pneumolysin toxoid confers a
     significant degree of protection against at least nine serotypes of
     Streptococcus pneumoniae. Infect Immun 1994;62:5683-8.
 21. Paton JC, Lock RA, Hansman DJ. Effect of immunization with pneumolysin
     on survival time of mice challenged with Streptococcus pneumoniae.
     Infect Immun 1983;40:548-52.
 22. McDaneiel LS, Sheffield JS, Delucchi P, Briles DE. PspA, a surface
     protein of Streptococcus pneumoniae, is capable of eliciting
     protection against pneumococci of more than one capsular type. Infect
     Immun 1991;59:222-8.
 23. Goebel W, Avery OT. Chemo-immunological studies conjugated
     carbohydrate proteins. I. The synthesis of p-aminophenol -glucoside,
     p-aminophenol -galactoside and their coupling with serum globulin. J
     Exp Med 1929;50:521-33.
 24. Mkel PH, Eskola J, Kyhty H, Takala A. Vaccines against Haemophilus
     influenzae type b. In: Ala-Aldeen D, Hormaeche C, editors. Molecular
     and Clinical Aspects of Bacterial Vaccine Development. Chichester:
     John Wiley & Sons, Ltd., 1995:41-91.
 25. Eskola J, Kyhty H, Takala AK, Peltola H, Rnnberg P-R, Kela E, et al.
     A randomized, prospective field trial of a conjugate vaccine in the
     protection of infants and young children against invasive Haemophilus
     influenzae type b disease. N Engl J Med 1990;323:1381-7.
 26. Barbour ML, Booy R, Crook DW, Griffiths H, Chapel HM, Moxon ER, et al.
     Haemophilus influenzae type b carriage and immunity four years after
     receiving the Haemophilus influenzae oligosaccharide-CRM197 (HbOC)
     conjugate vaccine. Pediatr Infect Dis J 1993;12:478-84.
                                [end of page 295]

 27. Anderson EL, Kennedy DJ, Geldmacher KM, Donnelly J, Mendelman PM.
     Immunogenicity of heptavalent pneumococcal conjugate vaccine in
     infants. Pediatrics 1996;128:649-53.
 28. Hogerman D, Kimura A, Malinoski F, Treanor J. Safety and
     immunogenicity of a heptavalent pneumococcal conjugate vaccine in
     healthy adult volunteers. Presented at the Infectious Diseases Society
     of America, Annual Meeting, San Francisco, CA, 1995; Abstract #389,
     page 114.
 29. hman H, Kyhty H, Leroy O, Froeschle J, Eskola J. Immunogenicity of
     octavalent pneumococcal (Pnc) conjugate vaccines (PncD, PncT) in
     Finnish infants. 36th Interscience Conference on Antimicrobial Agents
     and Chemotherapy (ICAAC), New Orleans, LA, 1996; Abstract G040, page
     150.
 30. Lee C-J, Lock RA, Andrew PW, Mitchell TJ, Hansman D, Paton JC.
     Protection of infant mice from challenge with Streptococcus pneumoniae
     type 19F by immunization with a type 19F polysaccharide-pneumolysoid
     conjugate. Vaccine 1994;12:785-878.
 31. Schneerson R, Levi L, Robbins JB, Bryla DM, Schiffman G, Lagergard T.
     Synthesis of a conjugate vaccine composed of pneumococcus type 14
     capsular polysaccharide bound to pertussis toxin. Infect Immun
     1992;60:3528-32.
 32. van de Wijgert JHHM, Verheul AFM, Snippe H, Check IJ, Hunter RL.
     Immunogenicity of Streptococcus pneumoniae type 14 capsular
     polysaccharide: influence of carriers and adjuvants on isotype
     distribution. Infect Immun 1991;59:2750-7.
 33. Alonso de Velasco E, Merkus D, Anderton S, Verheul AFM, Lizzio EF, van
     der Zee R, et al. Synthetic peptides representing T-cell epitopes act
     as carriers in pneumococcal polysaccharide conjugate vaccines. Infect
     Immun 1995;63:961-8.
 34. Schneerson R, Robbins JB, Parke JC, Bell C, Schlesselman JJ, Sutton A,
     et al. Quantitative and qualitative analyses of serum antibodies
     elicited in adults by Haemophilus influenzae type b and pneumococcus
     type 6A capsular polysaccharide-tetanus toxoid conjugates. Infect
     Immun 1986;52:519-28.
 35. Vella PP, Marburg S, Staub JM, Kniskern PJ, Miller W, Hagopian A, et
     al. Immunogenicity on conjugate vaccines consisting of pneumococcal
     capsular polysaccharide type 6B, 14, 19F, and 23F and meningococcal
     outer membrane protein complex. Infect Immun 1992;60:4977-83.
 36. Fattom A, Vann WF, Szu SC, Sutton A, Li X, Bryla D, et al. Synthesis
     and physicochemical and immunological characterization of pneumococcus
     type 12F polysaccharide-diphtheria toxoid conjugates. Infect Immun
     1988;56:2292-8.
 37. Giebink GS, Koskela M, Vella PP, Harris M, Le CT. Pneumococcal
     capsular polysaccharide-meningococcal outer membrane protein complex
     conjugate vaccines; immunogenicity and efficacy in experimental
     pneumococcal otitis media. J Infect Dis 1993;167:347-55.
 38. Kennedy EL, Anderson EL. Safety and immunogenicity of a heptavalent
     pneumococcal conjugate vaccine in adults and children. Presented at
     the 33rd Interscience Conference on Antimicrobial Agents and
     Chemotherapy (ICAAC), New Orleans, LA, 1993; Abstract #167, page 150.
 39. Nieminen T, Virolainen A, Kyhty H, Leinonen M, Eskola J. Immune
     response to tetravalent pneumococcal conjugate vaccine in adults.
     Presented at the 32nd Interscience Conference on Antimicrobial Agents
     and Chemotherapy (ICAAC), Anaheim, CA, 1992; Abstract #1283, page 324.
 40. Malinoski F, Hogerman D, Ginsberg H, Madore D. Safety and
     immunogenicity of pentavalent S. pneumoniae conjugate vaccines in
     healthy adults. Presented at the 33rd Interscience Conference on
     Antimicrobial Agents and Chemotherapy (ICAAC), New Orleans, LA, 1993;
     Abstract #168, page 150.
 41. Portier H, Choutet P, Duong M, Moreau M, Danve B. Serum antibody
     response to a tetravalent pneumococcal-tetanus toxoid conjugate
     vaccine in adult volunteers. Presented at the 34th Interscience
     Conference on Antimicrobial Agents and Chemotherapy (ICAAC), Orlando,
     FL, 1994; Abstract #G91, page 237.
 42. Nieminen T, Kyhty H, Eskola J. Mucosal and serum immune response to
     tetravalent pneumococcal conjugate vaccines in adults. Presented at
     the 34th Interscience Conference on Antimicrobial Agents and
     Chemotherapy (ICAAC), Orlando, FL, 1994; Abstract #G89, page 237.
 43. Kyhty H, Rnnberg P-R, Virolainen A, Eskola J. Immunogenicity of
     tetravalent pneumococcal capsular polysaccharide-meningococcal outer
     membrane protein conjugate vaccine in Finnish 2-year old children.
     Presented at the 33rd Interscience Conference on Antimicrobial Agents
     and Chemotherapy (ICAAC), New Orleans, LA, 1993; Abstract #172, page
     151.
 44. Zangwill K, Melamed R, Ward J, Marcy S, Patridge S, Greenberg D, et
     al. Safety and immunogenicity of a heptavalent pneumococcal conjugate
     vaccine among children 12-24 months of age. Presented at the Annual
     Meeting of the American Pediatric Society/Society for Pediatric
     Research, San Diego, CA, 1995; Abstract #1130, page 191A.
 45. Dagan R, Melamed R, Abramson O, Piglansky L, Greenberg D, Mendelman P,
     et al. Effect of heptavalent pneumococcal-OMPC conjugate vaccine on
     nasopharyngeal carriage when administered during the 2nd year of life.
     Presented at the Annual Meeting of the American Pediatric
     Society/Society for Pediatric Research, San Diego, CA, 1995; Abstract
     #1020, page 172A.
 46. Steinhoff D, Edward K, Keyseling H, Thoms ML, Johnson C, Madore D, et
     al. A randomized comparison of three bivalent Streptococcus pneumoniae
     glycoprotein conjugate vaccines in young children: effect of
     polysaccharide size and linkage characteristics. Pediatr Infect Dis J
     1994;13:368-72.
                                [end of page 296]

 47. Chiu SS, Grenberg DP, Partride S, et al. Safety and immunogenicity of
     a pentavalent pneumococcal conjugate vaccine in healthy toddlers.
     Presented at the 35th Interscience Conference on Antimicrobial Agents
     and Chemotherapy (ICAAC), San Francisco, CA, 1995; Abstract #G71, page
     171.
 48. Kennedy D, DeRousse C, Anderson E. Immunologic response of 12-18 month
     old children to licensed pneumococcal polysaccharide vaccine primed
     with Streptococcus pneumoniae 19F conjugate vaccine. Presented at the
     34th Interscience Conference on Antimicrobial Agents and Chemotherapy
     (ICAAC), Orlando, FL, 1994; Abstract #G88, page 236.
 49. Keyserling H, Bosley C, Starr S, Watson B, Laufer D, Anderson E, et
     al. Immunogenicity of pneumococcal type 14 conjugate vaccine in
     infants. Presented at the Annual Meeting of the American Pediatric
     Society/Society for Pediatric Research, Seattle, WA, 1994; Abstract
     #1087, page 184A.
 50. Kyhty H, hman H, Rnnberg P-R, Tillikainen R, Eskola J. Pneumococcal
     polysaccharide-meningococcal outer membrane protein complex conjugate
     vaccine is immunogenic in infants. J Infect Dis 1995;172:1273-8.
 51. Yogev R, Gupta S, Emanuel B, William K, Adams J. Safety, tolerability
     and immunogenicity of tetravalent (6B, 14, 19F, 23F) pneumococcal (Pn)
     conjugate vaccine in infants given concurrently with routine
     immunizations. Presented at the 33rd Interscience Conference on
     Antimicrobial Agents and Chemotherapy (ICAAC), New Orleans, LA, 1993;
     Abstract #170, page 150.
 52. hman H, Kyhty H, Tamminen P, Uistola A, Malinoski F, Eskola J.
     Pentavalent pneumococcal oligosaccharide conjugate vaccine PncCRM is
     well tolerated and able to induce an antibody response in infants.
     Pediatr Infect Dis J 1996;15:134-9.
 53. Kyhty H, hman H, Vuorela A, Malinkoski F, Eskola J. Response at 24
     months to a booster dose to pneumococcal (Pnc) polysaccharide (PS)
     vaccine in children immunized with pentavalent Pnc conjugate vaccine
     (PncCRM) in infancy. Presented at the 36th Interscience Conference on
     Antimicrobial Agents and Chemotherapy (ICAAC), New Orleans, LA, 1996;
     Abstract #G108, page 162.
 54. Daum RS, Steinhoff M, Rennels M, Rothstein E, Resinger K, Keyserling
     H, et al. Immunogenicity of S. pneumoniae oligo- and
     polysaccharide-CRM197 conjugate vaccines in healthy US infants.
     Presented at the 35th Interscience Conference on Antimicrobial Agents
     and Chemotherapy (ICAAC), San Francisco, CA, 1995; Abstract #GG5, page
     170.
 55. Leach A, Ceesay SJ, Banya WAS, Greenwood BM. Pilot trial of a
     pentavalent pneumococcal polysaccharide/protein conjugate vaccine in
     Gambian infants. Pediatr Infect Dis J 1996;15:333-9.
 56. hman H, Kyhty H, Leroy O, Froeschle J, Eskola J. Immunogenicity of
     tetravalent pneumococcal conjugate vaccines (PncD, PncT) in Finnish
     infants. Presented at the 35th Interscience Conference on
     Antimicrobial Agents and Chemotherapy (ICAAC), San Francisco, CA,
     1995; Abstract #G69, page 170.
 57. hman H, Kyhty H, Leroy O, Froeschle J, Eskola J. Booster response to
     polysaccharide and conjugate vaccine at 14 months after immunization
     with tetravalent pneumococcal (Pnc) conjugate vaccine PncD in infancy.
     Presented at the 36th Interscience Conference on Antimicrobial Agents
     and Chemotherapy (ICAAC), New Orleans, LA, 1996; Abstract G110, page
     163.
 58. hman H, Kyhty H, Leroy O, Eskola J. Booster response to
     polysaccharide vaccine at 14 months after immunization with
     tetravalent pneumococcal (Pnc) conjugate vaccine PncT in infancy is
     dose dependent. Presented at the 36th Interscience Conference on
     Antimicrobial Agents and Chemotherapy (ICAAC), New Orleans, LA, 1996;
     Abstract #G109, page 162.
 59. Jonsdottir I, Sigurdardottir STH, Vidarsson G, Ingolfsdottir G,
     Gudnason T, Dadidsdottir K, et al. Pneumococcal conjugate vaccines
     elicit functional antibodies in infants. Scand J Immunol 1996;43:710.
 60. Quataert SA, Kirch CS, Quackenbush Wiedl LJ, Phipps DC, Strohmeyer S,
     Cimino CO, et al. Assignment of weight-based antibody units to a human
     antipneumococcal standard reference serum, Lot 89-S. Clin Diagn Lab
     Immunol 1995;2:590-7.
 61. Dagan R, Melamed R, Muallem M, Piglansky L, Greenberg D, Abramson O,
     et al. Reduction of naso-pharyngeal carriage of penicillin-resistant
     pneumococci by pneumococcal-OMPC conjugate vaccine during second year
     of life. Presented at the 35th Interscience Conference on
     Antimicrobial Agents and Chemotherapy (ICAAC), San Francisco, CA,
     1995; Abstract #2, page 158.
 62. Kauppi M, Eskola J, Kyhty H. Anti-capsular polysaccharide antibody
     concentrations in saliva after immunization with Haemophilus
     influenzae type b conjugate vaccines. Pediatr Infect Dis J
     1995;14:286-94.
 63. Kauppi M, Saarinen L, Kyhty H. Anti-capsular polysaccharide
     antibodies reduce nasopharyngeal colonization by Haemophilus
     influenzae type b in infant rats. J Infect Dis 1993;167:365-71.
 64. Giebink GS, Meier JD, Quartey MK, Liebeler CL, Le CT. Immunogenicity
     and efficacy of Streptococcus pneumoniae polysaccharide-protein
     conjugate vaccines against homologous and heterologous serotypes in
     the chinchilla otitis media model. J Infect Dis 1996;173:119-27.
 65. Shurin PA, Rehmus JM, Johnson CE, Marchant CD, Carlin SA, Super DM, et
     al. Bacterial polysaccharide immune globulin for prophylaxis of acute
     otitis media in high-risk children. J Pediatr 1993;123:801-10.
 66. Booy R, Hodgson S, Carpenter L, Mayon-White RT, Slack MPE, Macfarlane
     JA, et al. Efficacy of Haemophilus influenzae type b conjugate vaccine
     PRP-T. Lancet 1994;344:362-6.
 67. Sankilampi U, Honkanen PO, Bloigu A, Herva E, Leinonen M. Antibody
     response to pneumococcal capsular polysaccharide vaccine in the
     elderly. J Infect Dis 1996;173:387-93.
                                [end of page 297]

 68. Musher DM, Groover JE, Graviss A, Baughn RE. The lack of association
     between aging and postvaccination levels of IgG antibody to capsular
     polysaccharide of Streptococcus pneumoniae. Clin Infect Dis
     1996;22:165-7.
 69. Powers D, Moore S, Mink CM. Vaccination of elderly adults with
     Haemophilus influenzae type b (Hib) polysaccharide or conjugate
     vaccine. Presented at the 35th Interscience Conference on
     Antimicrobial Agents and Chemotherapy (ICAAC), San Francisco, CA,
     1995; Abstract #976, page 171.
 70. Powers DC, Anderson EL, Lottenbach K, Mink CM. Reactogenicity and
     immunogenicity of a protein-conjugated pneumococcal oligosaccharide
     vaccine in older adults. J Infect Dis 1996;173:1014-8.
 71. Shahid NS, Steinhoff MC, Hoque SS, Begum T, Thompson C, Siber GR.
     Serum, breast milk, and infant antibody after maternal immunisation
     with pneumococcal vaccine. Lancet 1995;346:1252-7.
 72. O'Dempsey TJD, McArdle T, Ceesay S, Banya WAS, Demba E, Secka O, et
     al. Immunization with a pneumococcal capsular polysaccharide vaccine
     during pregnancy. Vaccine (in press).
 73. Englund JA, Glezen WP, Turner C, Harvey J, Thompson C, Siber GR.
     Transplacental antibody transfer following maternal immunization with
     polysaccharide and conjugate Haemophilus influenzae type b vaccines. J
     Infect Dis 1995;171:99-105.
 74. Mulholland K, Rahaman O, Suara R, Siber G, Roberton D, Jaffar S, et
     al. Maternal immunization with Haemophilus influenzae type b
     polysaccharide-tetanus protein conjugate vaccine in the Gambia. JAMA
     1996;275:1182-8.

                             [end of page 298]

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


---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
Synopses

A Mathematical Model and CD4+ Lymphocyte Dynamics in HIV Infection

Tom Hraba* and Jaroslav Dolezal
*Institute of Molecular Genetics, Prague, Czech Republic, Honeywell
Technology Center, Prague, and Institute of Information Theory and
Automation, Prague, Czech Republic
---------------------------------------------------------------------------

    The paper presents a model of CD4+ lymphocyte dynamics in
    HIV-infected persons. The model incorporates a feedback mechanism
    regulating the production of T lymphocytes and simulates the
    dynamics of CD8+ lymphocytes, whose production is assumed to be
    closely linked to that of CD4+ cells. Because CD4+ lymphocyte
    counts are a good prognostic indicator of HIV infection, the model
    was used to simulate such therapeutic interventions as
    chemotherapy and active and passive immunization. The model also
    simulated the therapeutic administration of anti-CD8 antibodies;
    this intervention was assumed to activate T-cell production by
    activating a feedback mechanism blocked by the high numbers of
    CD8+ lymphocytes present in HIV-infected persons. The character
    and implications of the model are discussed in the context of
    other mathematical models used in HIV infection.

The increased efficiency of modern computer techniques has expanded the
possibilities of mathematical modeling in an unprecedented way. However,
medical and biologic research has not taken full advantage of these
possibilities. Mathematical models are, in fact, working hypotheses that
require clear formulation and quantitative definition of factors and
relations included in the model. These requirements may discourage biologic
and medical research scientists from using mathematical models because
quantitative data are often not available to them, or are available only to
a limited extent. However, current computer techniques offer the
possibility of quickly testing different estimates of a realistic, probable
choice. The increased efforts required to construct mathematical models are
amply rewarded by the quantitative predictions generated by the models.

In the absence of adequate animal models, mathematical modeling of HIV
infection is especially important. Many adequate models of this infection
have been formulated (e.g., 1-3), but they have had a relatively small
impact on clinical and experimental research. Mathematical modeling,
however, was an integral and important part of evaluating recently obtained
data on HIV turnover in infected persons (4,5).

The Mathematical Model

We had simulated the dynamics of lymphocytes in immunologic tolerance (6)
for a decade when we became interested in modeling lymphocyte dynamics in
HIV-infected persons (7,8). The tolerance model simulates escape from
tolerance of a nonreplicating protein antigen and is based on the
assumption that lymphocytes specific to the tolerated antigen start to
appear when the concentration of the tolerated antigen drops below the
threshold level required for tolerance induction in differentiating new
lymphocytes. Our model of HIV infection concentrated on CD4+ lymphocytes
because the depletion of this T-cell subpopulation, and the parallel
decrease in the helper activity of T lymphocytes, seemed to be the major
immune system defect caused by HIV infection. When we started to construct
the model, the widely held view was that the decrease of this T-cell
subpopulation is not caused by the cytopathic effect of the virus
                             [end of page 299]

because in infected persons too few CD4+ cells expressed HIV. A direct or
indirect effect of HIV products on these cells was, therefore, considered
to cause this depletion (9).

In both immunologic tolerance and HIV infection, antigen seemed to
eliminate lymphocytes: in immunologic tolerance, lymphocytes carrying the
specific antigen receptor were affected, and in HIV infection, the entire
CD4+ lymphocyte population was the target of HIV products. The dynamics of
the affected lymphocyte pools were mutually inverse: in tolerance, the
number of specific lymphocytes increased with time because the antigen
concentration decreased and in HIV infection, the CD4+ lymphocyte count
decreased because of the rising level of HIV products as the infection
progressed.

             [Image] [Images not available in ASCII version]

             Figure 1.* Simulated CD4+ and CD8+ lymphocyte
             dynamics in HIV infection compared with observed
             mean T-cell values for CD4+ lymphocytes (circles)
             and CD8+ lymphocytes (squares).

             [Image] [Images not available in ASCII version] 
             
             Figure 2.* Comparison of only linear
             (curve 1, nu =1.0) T-helper activity decrease with
             non-linear (standard curve 2, nu = 1.6).
*CD4+ cell observed values are depicted as circles and those of CD8+
lymphocytes as squares. Both simulated and observed values are depicted as
a percentage of normal CD4+ lymphocyte numbers (the normal value of CD8+
lymphocytes is thus 66.7%).

In the model, CD4+ cell depletion was assumed to result, directly or
indirectly, from an effect of HIV products, where HIV proliferated at the
same rate during the whole course of the infection; therefore, once a
simulated substantial decline of CD4+ lymphocytes started, it progressed
rapidly to their total depletion. However, previous studies indicated that
an early decline in these cells occurs shortly after infection, is followed
by a period of slow decline, and then the decline accelerates again about
the time AIDS develops (10,11). However, if we assume that HIV
proliferation is limited by a helper T-cell-dependent immune reaction, our
model could simulate all the observed phases of CD4+ cell dynamics well
(8,12). In Figure 1, simulation curves of CD4+ and CD8+ lymphocytes
(Appendix)[Note: The Appendix contains detailed mathematical formulas
unable to be expressed in ASCII format. The complete Appendix is
available in the PDF, PostScript and HTML versions.] are compared with 
the mean observed values of these cells in HIV-infected persons (10,12).

The immune reaction limiting HIV proliferation, postulated in the model,
was assumed to be a specific cytotoxic activity of T cells that required
the cooperation of helper T cells. However, the model is not dependent on
this assumption, and any T helper-cell-dependent immune reaction can play
this role. Other mechanisms are possible candidates for this function.
Recently, interest was focused on cytokines produced by CD8+ lymphocytes
that inhibit HIV proliferation (13,14), an effect discovered much earlier
(15,16). As far as this activity depends on T-helper cells, this situation
can be simulated by our model. If T-helper cells did not play any
substantial role in this mechanism, our model would not be applicable.

In our model, CD4+ lymphocyte dynamics were successfully simulated only if
T-helper-cell activity did not decrease linearly with the decline of CD4+
lymphocytes but faster than these T cells. This relation is expressed by
the power coefficient [nu] in Equation 6
                             [end of page 300]

(Appendix)[Note: The Appendix contains detailed mathematical formulas
unable to be expressed in ASCII format. The complete Appendix is
available in the PDF, PostScript and HTML versions.], which must have a
value >1.0 (7,8). If the decrease of helper activity was assumed to be
directly proportional to the number of CD4+ lymphocytes ([nu] = 1.0), it
was possible to simulate only the initial phase of the CD4+ dynamicsthe
early drop of these cells. Then a permanent steady state of the CD4+ cell
level was established (Figure 2, curve 1). This finding could be of 
interest for elucidating mechanisms involved in the long-term, and 
possibly permanent, survival of some HIV-infected persons (17-19) whose
condition seems to be characterized by an equilibrium between HIV 
infection and a protective immune reaction. Although some of
these persons have CD4+ lymphocyte numbers in the normal range, their cases
do not necessarily contradict the model's prediction of a stabilized HIV
infection because with a small HIV load the steady-state CD4+ cell numbers
might be indistinguishable from normal ones, given the broad range of
normal values.

We are not able to ascribe a definite mechanism to the necessary assumption
that the activity of T-helper cells declines faster than the number of CD4+
lymphocytes. The faster decline could be caused by the disruption of the
lymphoid tissue structure by HIV infection (20). Another possible cause is
the increasing HIV variation as infection progresses (21), if this
variation led to decreased sensitivity, or even resistance, to the
protective immune reaction of at least a part of the virus population.

From our model of immunologic tolerance (6), two compartments of the
studied lymphocytes were retained in the model of HIV infection (7): mature
and immature CD4+ cells. We incorporated immature lymphocytes in the model
of immunologic tolerance because they were more sensitive to tolerance
induction. The assumption of immature CD4+ lymphocyte sensitivity to
elimination by HIV did not influence the simulation results substantially
(22); therefore, only mature CD4+ cells were considered to be eliminated by
the effect of HIV in most simulations we carried out, including those
described in this article.

Different mechanisms of CD4+ lymphocyte depletion caused by HIV infection
were simulated by various modifications of our model, which assumed either
a direct or indirect effect of HIV (22). Because no substantial difference
was observed in simulation results, for convenience reasons, most of the
work, including the examples presented in this article, assumed that
cytotoxic cells limiting HIV proliferation are also instrumental in
depleting CD4+ lymphocytes by eliminating those have HIV products. This
assumption seems to be supported by recent clinical findings (4,5).

Adleman suggested that the depletion of CD4+ lymphocytes might activate
some homeostatic mechanism that increases their production (24). He assumed
that this homeostatic mechanism increased production of both CD4+ and CD8+
lymphocytes and did not discriminate between the two T-cell subpopulations.

Adleman also suggested that the substantial and permanent depletion of CD4+
lymphocytes in HIV-infected persons might activate this mechanism. However,
because only CD4+ cells are destroyed in HIV infection, the newly produced
CD8+ lymphocytes would accumulate. An increase in the number of CD8+
lymphocytes was actually observed in HIV-infected persons, while the total
number of T cells remained in the normal range. The increase in the CD8+
cell count might switch off the homeostatic mechanism that increases T-cell
production, and as a consequence, cause or at least aggravate the CD4+
lymphocyte depletion (24,25). Because this view was supported by convincing
evidence, we incorporated this feedback mechanism in our model (22). When
we compared quantitatively the simulated CD8+ cell increase with the
observed values, they did not agree well, especially in later phases of the
infection when the simulated values continued to increase, while the
observed CD8+ lymphocyte counts started to decline. When it was assumed
that HIV infection constrained the influx of both CD4+ and CD8+
lymphocytes, satisfactory simulation results were obtained (26) (Figure 1).

Modeling Therapeutic Interventions

Because CD4+ lymphocyte counts are a good prognostic indicator of HIV
infection (27), our model is suitable for simulating different therapeutic
interventions.
                             [end of page 301]

The model has been used to simulate zidovudine (AZT) chemotherapy and
specific immunotherapy, both active and passive (28-30). The intensity of
most therapies we simulated did not completely eradicate the infection, a
situation common to the treatments now available. If a therapy that is
assumed not to eradicate HIV infection is simulated to be administered
permanently and to retain undiminished effectiveness, the observed overall
result (besides slight differences in the dynamics of the changes induced
by the various therapeutic measures) is the establishment of a new
steady-state level of CD4+ lymphocytes (Figure 3). The height of this level
reflects the effectiveness of the therapy: the nearer to normal values, the
more effective the interventions. This steady state is always lower than a
normal state. Even when the therapy is started at a later stage of HIV
infection, the obtained steady state is the same; its value depends only on
the effectiveness of the therapy, regardless of the onset of treatment
(Figure 3, curves 1 and 2).

             [Image] [Images not available in ASCII version]

             Figure 3.* Simulated effect of permanent AZT
             treatment ([zeta] = 0.005) started 2m 5m or 6
             years after the acquisition of HIV
             infectioncurves 1, 2, and 3, respectively.

             [Image] [Images not available in ASCII version] 

             Figure 4.* Simulated effect of temporary
             AZT treatment ([zeta] = 0.005) started 5 years
             after the acquisition of HIV infection and
             lasted 1, 2, or 3 yearscurves 1, 2, and 3,
             respectively.
*CD4+ cell observed values are depicted as circles and those of CD8+
lymphocytes as squares. Both simulated and observed values are depicted as
a percentage of normal CD4+ lymphocyte numbers (the normal value of CD8+
lymphocytes is thus 66.7%).

However, this result is valid up to a certain point only: when the CD4+
lymphocyte numbers are too low, therapy can no longer reverse the CD4+ cell
depletion; it can only slow the decrease and does not establish a steady
state (Figure 3, curve 3). The stage of infection in which depletion cannot
be reversed varies with different therapies and with different intensities
of the same therapy (e.g., AZT doses). It occurs earlier in the course of
the infection with less effective treatments than with more effective ones.
Actually, there is a point in the intensity of each treatment when it is
possible to stop further CD4+ cell decline and establish a steady state
with CD4+ cell numbers corresponding to their value at the onset of
therapy.

Another aspect of the therapies we simulated was their temporary
application. If the therapy eliminated the virus completely, CD4+
lymphocyte counts returned to normal for good. Our simulations concentrated
mainly on therapies leading only to limitation of the viral load, and in
consequence, to an increase in the numbers of CD4+ cells. When the
treatment was stopped, the decline of CD4+ cells started again and
essentially proceeded at the same rate as in untreated persons (Figure 4).
The maximal increase of CD4+ lymphocytes obtained by such intervention
corresponded to that induced by a permanent application of a treatment of
the same intensity. Of course, if the applied treatment did not last long
enough to allow the CD4+ cells to reach that level, only a lower maximal
value was obtained.

According to the results of our simulations temporary therapy only
prolonged survival, although extended survival could
                             [end of page 302]

exceed the length of treatment. This situation is relevant especially to
chemotherapy that loses effectiveness after a relatively short time,
probably because of drug resistance acquired by HIV. However, even
immunotherapy cannot be expected to retain its undiminished effectiveness
for a prolonged period.

Therapeutic Depletion of CD8+ Lymphocytes

It has been suggested that lowering the number of CD8+ lymphocytes in
HIV-infected persons by administering anti-CD8 antibodies could activate
the homeostatic mechanism, thus increasing production of both CD4+ and CD8+
T cells (24,25). However, this mechanism might be blocked by the high
numbers of CD8+ lymphocytes present in HIV infection, as discussed above.
Therefore, these authors assumed that a depletion of CD8+ lymphocytes
brought about by administering anti-CD8 antibodies might unblock this
feedback mechanism and that this could counteract the depletion of CD4+
cells caused by HIV infection.

When this regulatory mechanism was incorporated in our model, we were able
to simulate this situation (26,30). Reducing CD8+ lymphocyte values to
numbers not much below normal stops the further decline in CD4+ cells and
brings them to a steady state, which may be higher than their pretreatment
number (Figure 5). Lower doses of anti-CD8 antibodies also stopped further
decline of CD4+ cells, but the achieved steady-state level is lower than in
the illustrated case. On the contrary, the CD4+ lymphocyte increase is
larger with higher antibody doses. As in other cases of temporary
treatment, the decrease in CD4+ values starts again after anti-CD8 antibody
administration is discontinued.

             [Image] [Images not available in ASCII version]

             Figure 5.* Simulated CD4+ and CD8+ lymphocyte
             dynamics after permanent treatment with anti-CD8
             antibodies started 2 years after the acquisition
             of the HIV infection. Cells mediating the
             protective anti-HIV immune reaction are not
             affected by this treatment ([Rho] to the Rth
             power = 0.007, [Rho] to the Cth power = 0.0).

             [Image] [Images not available in ASCII version]

             Figure 6.* Simulated CD4+ and CD8+ lymphocyte
             dynamics after permanent treatment with anti-CD8
             antibodies started 2 years after the acquisition
             of the HIV infection. Cells mediating the
             protective anti-HIV immune reaction are also
             affected by this treatment ([Rho] to the Rth
             power = 0.007, Rho] to the Cth power = 0.007).

*CD4+ cell observed values are depicted as circles and those of CD8+
lymphocytes as squares. Both simulated and observed values are depicted as
a percentage of normal CD4+ lymphocyte numbers (the normal value of CD8+
lymphocytes is thus 66.7%).

However, such a therapeutic effect of CD8+ depletion is achieved only if
lymphoid cells eliminated by the administered antibody are assumed not to
participate in the anti-HIV immune reaction that limits virus
proliferation. This is not the case with cytotoxic T cells, or with CD8+
lymphocytes responsible for the production of cytokines that inhibit HIV
proliferation. This situation could also be simulated with our model. When
these cells assumed to cause a protective immune reaction were also
eliminated by the administered anti-CD8 antibody, the CD4+ lymphocyte
decrease did not stop; it even accelerated (Figure 6). All doses of
anti-CD8 antibody, even very small ones, increased the depletion of CD4+
lymphocytes under this assumption, although the deteriorating effect was
not so strong when small doses of antibody were used.
                             [end of page 303]

For the sake of brevity, we refrained from presenting examples from other
models, although in addition to providing some quantitative differences in
simulated results, these examples could be of interest in modeling other
aspects of HIV infection (31-33), and the comparison of other results with
those of our model would add another dimension to this article. Different
models, or some of their parts, can be combined, and in this way the actual
situation might be simulated even better; on the other hand, a particular
model might simulate a specific situation and help clarify some questions
posed by clinical or experimental studies.

    Dr. Hraba is affiliated with the Institute of Molecular Genetics
    of the Academy of Sciences of the Czech Republic. His early
    research activities involved primarily the problem of immunologic
    tolerance. The work of Dr. Hraba is supported in part by grant
    A50502604 from the Academy of Sciences of the Czech Republic.

    Dr. Dolezal is a manager of Honeywell Technology Center, Prague.
    His research interests include theoretical and applied aspects of
    optimal control and mathematical programming and applications of
    mathematical modeling methods to dynamic systems of both technical
    and biologic origin. He has also coordinated large applied
    projects with industry.

Address for correspondence: Tom Hraba, Institute of Molecular Genetics,
Flemingovo n. 2, 16637 Prague, Czech Republic; fax: 42-2-2431-0955; e-mail:
tomas.hraba@img.cas.cz.

References

  1. Nowak MA, May RM. Coexistence and competition in HIV infection. J
     Theor Biol 1992;159:329-42.
  2. Perelson AS, Kirschner DE, De Boer R. Dynamics of HIV infection of
     CD4+ T cells. Math Biosci 1993;114:81-125.
  3. McLean AR. The balance of power between HIV and the immune system.
     Trends Microbiol 1993;1:9-13.
  4. Ho DD, Neumann AU, Perelson AS, Chen W, Leonard JM, Markowitz M, et
     al. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1
     infection. Nature 1995;373:123-6.
  5. Wei X, Ghosh SK, Taylor ME, Johnson VA, Emini EA, Deutsch, P, et al.
     Viral dynamics in human immunodeficiency virus type 1 infection.
     Nature 1995;373:117-22.
  6. Klein P, Hraba T, Dolezal J. The use of immunological tolerance to
     investigate B lymphocyte replacement kinetics in chickens. J Math Biol
     1983;16:131-40.
  7. Hraba T, Dolezal J. Mathematical model of CD4+ lymphocyte depletion in
     HIV infection. Folia Biol (Praha) 1989;35:156-63.
  8. Hraba T, Dolezal J. Contribution of mathematical modelling to
     elucidation of AIDS pathogenesis. J Biol Systems 1993;1:349-62.
  9. Fauci AS. The human immunodeficiency virus: infectivity and mechanisms
     of pathogenesis. Science 1988;239:617-22.
 10. Lang W, Perkins H, Anderson RE, Royce R, Jewell N, Winkelstein W, et
     al. Patterns of T lymphocyte changes with human immunodeficiency virus
     infection: from seroconversion to the development of AIDS. J AIDS
     1989;2:63-9.
 11. Margolick JB, Donnenberg AD, Muos A, Park LP, Bauer KD, Giorgi JV, et
     al. Changes in T and non-T lymphocyte subsets following seroconversion
     to HIV-1: stable CD3+ and declining CD3- populations suggest
     regulatory responses linked to loss of CD4 lymphocytes. J AIDS
     1993;6:153-61.
 12. Dolezal J, Hraba T. Model-based analysis of CD4+ lymphocyte dynamics
     in HIV infected individuals. II. Evaluation of the model based on
     clinical observations. Immunobiology 1991;182:178-87.
 13. Baier M, Werner A, Bannert N, Metzner K, Kurth R. HIV suppression by
     interleukin-16. Nature 1995;378:563.
 14. Cocchi F, DeVico AL, Garzino-Demo A, Arya SK, Gallo RC, Lusso P.
     Identification of RANTES, MIP-1[alpha], and MIP-1[beta] as the major
     HIV-suppressive factors produced by CD8+ T cells. Science
     1995;270:1811-5.
 15. Walker CM, Moody DJ, Stites DP, Levy JA. CD8+lymphocytes can control
     HIV infection in vitro by suppressing virus replication. Science
     1986;234:1563-6.
 16. Mackewicz C, Levy JA. CD8+ cell anti-HIV activity: nonlytic
     suppression of virus replication. AIDS Res Hum Retrovir
     1992;8:1039-50.
 17. Muos A, Kirby AJ, He YD, Margolick JB, Visscher BR, Rinaldo CR, et
     al. Long-term survivors with HIV-1 infection: incubation period and
     longitudinal pattern of CD4+ lymphocytes. J AIDS 1995;8:496-505.
 18. Cao Y, Qin L, Zhang L, Safrit J, Ho DD. Virologic and immunologic
     characterization of long-term survivors of human immunodeficiency
     virus type 1 infection. N Engl J Med 1995;332:201-8.
 19. Pantaleo G, Menzo S, Vaccarezza M, Graziosi C, Cohen OJ, Demarest JR,
     et al. Studies in subjects with long-term nonprogressive human
     immunodeficiency virus infection. N Engl J Med 1995;332:209-16.
 20. Frost SDW, McLean AR. Germinal centre destruction as a major pathway
     of HIV pathogenesis. J AIDS 1994;7:236-44.
 21. Coffin JM. HIV population dynamics in vivo: implications for genetic
     variation, pathogenesis, and therapy. Science 1995;267:483-9
 22. Hraba T, Dolezal J, Celikovsk S. Model-based analysis of CD4+
     lymphocyte dynamics in HIV infected individuals. Immunobiology
     1990;181:108-18.
 23. Zinkernagel RM, Hengartner H. T-cell-mediated immunopathology versus
     direct cytolysis by virus: implications for HIV and AIDS. Immunol
     Today 1994;15:262-8.
 24. Adleman LM. On the maintenance of T cell populations. Technical
     Report. Los Angeles: Univ. of California: Dept. of Computer Science,
     1988.
                                [end of page 304]

 25. Adleman LM, Wofsy D. T-cell homeostasis: implications in HIV
     infection. J AIDS 1993;6:144-52.
 26. Hraba T, Dolezal J. A mathematical model of CD8+ lymphocyte dynamics in
     HIV infection. Folia Biol (Praha) 1995;41:304-18.
 27. Stein DS, Korvick JA, Vermund SH. CD4+ lymphocytes cell enumeration
     for prediction of clinical course of human immunodeficiency virus
     disease: a review. J Infect Dis 1992;165:352-63.
 28. Hraba T, Dolezal J. Mathematical modelling of chemotherapy in HIV
     infection. Folia Biol (Praha) 1994;40:103-11.
 29. Dolezal J, Hraba T. Mathematical modelling of immunotherapy in HIV
     infection. Folia Biol (Praha) 1994;40:193-9.
 30. Hraba T, Dolezal J. Mathematical modelling of HIV infection therapy.
     Int J Immunopharmacol 1995;17:523-6.
 31. De Boer JR, Boerlijst MC. Diversity and virulence thresholds in AIDS.
     Proc Natl Acad Sci USA 1994;94:544-8.
 32. Essunger P, Perelson AS. Modeling HIV infection of CD4+ T-cell
     subpopulations. J Theor Biol 1994;170:367-91.
 33. Nelson GW, Perelson AS. Modeling defective interfering virus therapy
     for AIDS: conditions for DIV survival. Math Biosci 1995;125:127-53.
 34. Dolezal J, Hraba T, Celikovsk S. Influence of the cell interaction
     parameters on the simulated CD4+ lymphocyte depletion in HIV
     infection. Folio Biol (Praha)1991:87:42-51.

Appendix

[Note: The Appendix contains detailed mathematical formulas
unable to be expressed in ASCII format. The complete Appendix is
available in the PDF, PostScript and HTML versions.]

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

---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
Synopses

Chlamydiae as Pathogens: New Species and New Issues

Rosanna W. Peeling* and Robert C. Brunham
*Laboratory Centre for Disease Control Health, Winnipeg, Manitoba, Canada
Department of Medical Microbiology, University of Manitoba, Winnipeg,
Canada
---------------------------------------------------------------------------

    The recognition of genital chlamydial infection as an important
    public health problem was made first by the recognition of its
    role in acute clinical syndromes, as well as in serious
    reproductive and ocular complications, and secondly by our
    awareness of its prevalence when diagnostic tests became widely
    accessible. The recent availability of effective single dose oral
    antimicrobial therapy and sensitive molecular amplification tests
    that allow the use of noninvasive specimens for diagnosis and
    screening is expected to have a major impact in reducing the
    prevalence of disease in the next decade. Clinical manifestations
    associated with Chlamydia pneumoniae infection continue to emerge
    beyond respiratory illness. In particular, its association with
    atherosclerosis deserves further investigation. Chlamydia pecorum,
    a pathogen of ruminants, was recently recognized as a new species.
    The continued application of molecular techniques will likely
    elucidate an expanding role for chlamydiae in human and animal
    diseases, delineate the phylogenetic relationships among
    chlamydial species and within the eubacteria domain, and provide
    tools for detection and control of chlamydial infections.

Chlamydiae are obligate intracellular bacteria that grow in eukaryotic
cells and cause a wide spectrum of human disease (Table). Species were
grouped according to their biologic and biochemical properties and a
greater than 95% homology in their 16s ribosomal RNA sequences (1).
Molecular analyses led to the reclassification of some Chlamydia psittaci
strains as Chlamydia pneumoniae, a human pathogen, and Chlamydia pecorum, a
pathogen of ruminants. Given the diverse host range of C. psittaci strains,
more reclassification within this species may be likely.

           Table. Spectrum of human diseases caused by Chlamydiae
---------------------------------------------------------------------------
Species        Acute Diseases          Sequelae/Chronic Diseases
---------------------------------------------------------------------------
C. trachomatis
  Serovars A-C conjunctivitis          trachoma
  Serovars D-K urethritis              proctitis, epididymo-orchitis,
                                       Reiter's Syndrome
               cervicitis              pelvic inflammatory disease, ectopic
                                       pregnancy, tubal infertility,
                                       Fitz-Hugh Curtis Syndrome
               ophthalmia neonatorum
               neonatal pneumonia
  LGV serovars lymphogranuloma
               venereum
C. pneumoniae  pharyngitis             ?cardiovascular disease
               sinusitis               ?asthma
               bronchitis
               community-acquired
               pneumonia

C. psittaci
  parrot       atypical pneumonia
  canaries     hepatic and renal
  pigeons      dysfunction
  turkeys      endocarditis
  ducks
  chickens
  cats         conjunctivitis
  ewes         abortion
---------------------------------------------------------------------------

The oldest reported disease associated with C. trachomatis infection is
trachoma, a sequela of ocular infection. This disease was described in
China and in the Ebers papyrus in Egypt thousands of years ago and
continues to be a major cause of preventable blindness, with an estimated
500 million cases of active trachoma worldwide (seven million include
blindness from conjunctival scarring and eyelid deformities [2]). In the
last two decades, genital chlamydial infection has been identified as a
major public health problem because of the recognition that chlamydial
infection is associated with disease syndromes such as nongonococcal
urethritis, mucopurulent cervicitis, pelvic inflammatory disease (PID),
ectopic pregnancy, and tubal infertility. The World Health Organization
estimated 89 million new cases of genital chlamydial infections worldwide
in 1995 (3). In the United States, each year an estimated four million new
cases occur and 50,000 women become infertile as a result of infection (4).

C. psittaci infection, acquired through respiratory droplet transmission of
chlamydiae from infected birds, has been considered for many years an
occupational hazard for employees of pet shops and poultry processing
plants (5). Sources of human C. psittaci infection other than infected
birds have been identified and may
                             [end of page 307]

be more common than currently recognized. Detection of C. psittaci in
household cats and breeding catteries illustrates the expanding number of
chlamydial diseases in animals that are transmissible to humans (6,7).

C. pneumoniae is a human pathogen recognized as an important cause of
respiratory illness (8). Approximately 40% to 60% of adult populations
around the world have antibodies to C. pneumoniae, which suggests that the
infection is extraordinarily prevalent, and reinfection is common. Current
interest centers on the emerging role of C. pneumoniae infection in the
pathogenesis of atherosclerosis and asthma.

Biology of Chlamydiae: An Update

Chlamydiae have a unique biphasic life cycle with dimorphic forms that are
functionally and morphologically distinct. An extracellular form, the
elementary body (EB), is infectious but metabolically inactive. Once
endocytosed, the EB differentiates into a larger pleomorphic form called
the reticulate body (RB), which replicates by binary fission. The precise
mechanism by which EBs attach and gain entry into the host cell is unknown.
Recent work suggests that chlamydiae employ a molecular mimic of heparan
sulfate to attach to glycosaminoglycan (GAG) receptors on eukaryotic cell
surfaces (9). GAG appears to form a trimolecular complex with the host cell
since (EB) infectivity is inhibited by the addition of heparan or heparan
sulfate to culture, and pretreatment of EBs with heparan sulfate lyase
abolishes EB infectivity. The mechanism of endocytic uptake remains
unclear. Once inside the host cell, chlamydiae reside in a membrane-bound
vacuole that can evade phagolysosomal fusion. The endosome is transported
to the distal region of the Golgi apparatus and incorporates host-derived
sphingolipids into the inclusion membrane (10,11). Thus it appears that
chlamydiae are able to intercept host vesicular traffic bound for the
plasma membrane to sequester lipids and possibly other host substances
synthesized in the Golgi. Subversion of host vesicular traffic may
represent a dual advantage for chlamydiae in obtaining materials from the
host for its metabolism as well as in modifying the inclusion membrane to
evade lysosomal fusion and immune detection.

Chlamydiae are considered energy parasites because they lack the enzymes of
the electron transport chain and thus require adenosine triphosphate (ATP)
and nutrient
                             [end of page 308]

resources from the host to fuel their metabolism and replication.
Chlamydiae are incapable of de novo nucleotide biosynthesis and are
dependent on host nucleotide pools (12). In spite of the successful
selection of various metabolic mutants of C. trachomatis, progress in
elucidating the host-parasite metabolic relationship has been hampered by
multiple salvage metabolic pathways in the host and the lack of a genetic
shuttle system for chlamydiae.

C. trachomatis

Epidemiology
Genital infections due to C. trachomatis are the most common sexually
transmitted diseases in many industrialized countries (3). Each year, an
estimated four million new cases occur in the United States and three
million in Europe. These infections present unique problems for public
health control programs because 50% to 70% of infections in women (and
perhaps men) are clinically silent. Unrecognized and untreated, the
bacteria may remain infectious in the host for months and be readily
transmitted to sex partners. Furthermore, most reported infections occur in
the 15- to 24-year-old age group. Young women with cervical chlamydial
infections are at risk for pelvic inflammatory disease, which can lead to
long-term reproductive sequelae such as chronic pelvic pain, ectopic
pregnancy, and tubal infertility. Babies born to infected mothers are also
at risk for conjunctivitis and pneumonia. The annual direct and indirect
costs of genital chlamydial infections in the United States are estimated
at $2.4 billion (4).

Control programs emphasizing early diagnosis, targeted screening, partner
notification, and effective treatment have led to a slow decline in the
incidence of genital chlamydial infection in countries where these programs
have been implemented (13). The true rate of decline may be higher than the
reported rate because of increased sensitivity of laboratory testing and
more widespread screening. In women, screening of chlamydial infection at
the time of Papanicolaou tests, prenatal visits, or attendance at family
planning or pregnancy counseling clinics have been effective. In
asymptomatic men, who are less likely to access care, asymptomatic
infection is not adequately addressed by current public health programs.

In contrast to genital chlamydial infection, trachoma is a household
disease that has disappeared in many parts of the world because of improved
living conditions and hygiene. In trachoma-endemic areas, severe disease
leading to scarring and blindness may be the result of frequent reinfection
or persistent infection in those whose immune system does not mount an
adequate response to clear the infection. For both ocular and genital
chlamydial infections, recent advances in diagnostic and screening
technology and single dose antimicrobial therapy will likely have a
significant impact on the efficacy of disease control programs and the
opportunity for eventual disease eradication.

Laboratory Diagnosis
Since curative antibiotic therapy for chlamydial infections is readily
available and inexpensive, early diagnosis is an essential component of
public health programs to control these infections. The goals of early
identification are to interrupt the chain of transmission in the community
and to prevent long-term sequelae. Isolation of the organism in cell
culture had been the traditional method for laboratory diagnosis and has
remained the method of choice for medicolegal specimens because of its
specificity. However, culture requires expensive equipment, technical
expertise, and stringent transport conditions to preserve specimen
viability; it also has a turnaround time of 2 to 3 days. Hence, in many
settings, culture has been replaced by antigen-detection methods, such as
enzyme immunoassays (EIA) and direct fluorescence assays (DFA), which have
less demanding transport requirements and can provide results on the same
day. EIAs are suitable for public health laboratories serving large
geographic areas because specimens are stable in transport under ambient
conditions and are inexpensive because they allow specimens to be processed
in batches by automated equipment. Assays are typically based on the
capture of the
                             [end of page 309]

chlamydial lipopolysaccharide (LPS) using monoclonal or polyclonal
antibodies linked to a solid-phase support. Early problems with low
specificity because of cross-reactivity between the chlamydial LPS and that
of other gram-negative bacteria have been largely overcome by confirmation
with DFA or a blocking antibody assay. With a lower detection limit of
10,000 elementary bodies, EIA lacks sensitivity as a screening assay,
especially for asymptomatic men (14,15). Nucleic acid-based hybridization
probe tests offer higher specificity but no substantial improvement on
sensitivity (15). Nucleic acid amplification tests based on polymerase
chain reaction (PCR), ligase chain reaction (LCR), and
transcription-mediated amplification technology are now commercially
available. The precision of nucleic acid hybridization and the rapid
amplification of a single gene target facilitated the design of diagnostic
tests with specificities in excess of 99% and lower detection limits of
1-10 EBs. In addition, these tests offer all the advantages of nonculture
tests in terms of ambient specimen transport, batching, automation, and
rapid processing time of 4 hours. Duplex testing for the simultaneous
detection of chlamydial and gonococcal DNA from a single specimen is also
commercially available in some countries.

A major advantage of the increased sensitivity of these molecular
amplification tests is that noninvasive specimens, such as urine, can be
used for testing. The ease of collection and the lack of sampling bias of
urine specimens make screening feasible in settings outside physicians'
offices. PCR assays on urethral or cervical swabs for the laboratory
diagnosis of genital chlamydial infection in symptomatic men and women show
sensitivities of 89% to 100% and specificities of 99% to 100% compared with
the traditional culture or PCR test, confirmed by a second PCR reaction
targeting a different gene (16-18). For urine specimens, PCR assays show
sensitivities of 87% to 100% for men and 92% for women and specificities of
96% to 100% for men and 95% for women (18-20). In a study of 447 women with
a prevalence of infection of 6%, the sensitivity of urine LCR was 96%
compared with 56% for cervical swab culture, 78% for cervical swab EIA, and
37% for urine EIA (21). For men in the same study, the sensitivity of urine
LCR was 96% compared with 68% for urine EIA, and 38% for urethral swab
culture. In a multicenter study of 2,132 women, cervical swab LCR showed a
sensitivity of 87% to 98% compared with a sensitivity of 52% to 92% for
culture (22). In LCR studies, a true positive was defined as culture
positive or LCR positive confirmed with DFA or another LCR assay with a
different DNA target. Thus it appears that molecular amplification
techniques for the detection of C. trachomatis in urine specimens from both
men and women are a substantial improvement over conventional diagnostic
and screening methods and will provide an important tool for decreasing the
reservoir of infection, especially in asymptomatic men.

In the diagnostic laboratory, molecular techniques present different
problems for specimen handling and interpretation of results than cell
culture or antigen detection (15). Inherent in the increased sensitivity of
these molecular techniques is the potential for false-positive results due
to cross contamination between specimens, and run-to-run contamination from
equipment, reagents, and supplies. These problems can be overcome by
observing stringent rules for specimen preparation (e.g., dedicated
equipment) and separating specimen processing and reagent preparation areas
to prevent contamination. Enzymatic or photochemical sterilization can be
used to eliminate run-to-run contamination. False-negative results may be
due to substances in specimens inhibitory to enzymes used for
amplification. Known inhibitors include phosphate ions, heparin, heme,
crystals in the urine specimens, and detergents used in specimen
processing. Internal controls are now commercially available to detect
false negatives.

Although molecular tests are more expensive than EIA, cost-effectiveness
studies should take into consideration the benefits of averting the
enormous costs of long-term reproductive sequelae in women with undetected
infections, adverse pregnancy outcomes, and HIV infection. Targeted
screening of women to detect cervical chlamydial infection decreases the
incidence
                             [end of page 310]

of symptomatic PID (23). Patients with genital gonococcal or chlamydial
infections are also at increased risk for human immunodeficiency virus
(HIV) (24). Although the risk for HIV may be lower in patients with
chlamydial infection than in those with genital ulcer disease, the higher
prevalence of chlamydial infection in some populations means that the
population attributable risk for HIV may be substantially higher for
chlamydia. Shortening the duration of infectiousness by early diagnosis and
treatment could have a major impact on risk reduction for HIV infection. A
recent study showed that strengthening sexually transmitted disease control
through education, access to diagnosis, and treatment reduced the incidence
of HIV by 42% in study communities in Tanzania over 2 years (25).

Treatment
Azithromycin prescribed as a single oral 1-g dose is equivalent to the
traditional 7-day regimen of doxycycline for treating ocular and
uncomplicated genital chlamydial infections (26-28). Compared with
conventional therapy, azithromycin has excellent pharmacokinetic
characteristics, such as increased bioavailability; lower incidence of
gastrointestinal tract side effects; and increased concentration in mucus,
macrophages, and tissues with a half life of 5 to 7 days (29). These
characteristics allow for single dosing, which alleviates the problem of
patient noncompliance with multiday regimens. With single-dose therapy, the
potential for reinfection due to earlier resumption of sexual activity is a
concern. At present, there are limited data on the use of single-dose
therapy in adolescents, during pregnancy, and for syndromes such as PID,
cervicitis, and nongonococcal urethritis (30-33). Studies are needed to
determine if these regimens achieve clinical and microbiologic cure while
preserving fertility and preventing further tissue damage to the upper
genital tract.

Although the higher cost of azithromycin may be prohibitive for its use in
resource-limited settings, selective use in persons at high risk or in
those with a history of noncompliance may prove cost-effective. The cost of
retreatment as a result of noncompliance and the additional cost of contact
tracing can make single dose azithromycin more cost-effective than
doxycycline (34).

Pathogenesis
Interesting findings in three areas of C. trachomatis pathogenesis further
delineate the complex bacteria-host relationship in disease and may have
implications for vaccine design. These new observations include the
extensive but unexpected polymorphism of the major outer membrane protein
(MOMP), the evidence for genetic susceptibility to disease, and the
association of antibody response to the 60 kDa heat shock protein (CHSP60)
with the development of adverse sequelae following ocular and genital
infections.

Polymorphism of MOMP
The ecologic success of a pathogen is determined in part by its ability to
evade host defenses. With C. trachomatis, MOMP is a major target for
protective host immune responses, such as neutralizing antibodies and
possibly, protective T-cell responses (35,36). The basis for MOMP antigenic
variation is allelic polymorphism at the omp-1 locus, and immune selection
appears to be occurring in host populations frequently exposed to C.
trachomatis (37). Each variant apparently only infects hosts lacking
serovar-specific immunity to that variant, and the ecologic success of
chlamydiae may be due to their ability, under immune selection pressure, to
generate successive allelic variants (36). DNA sequence analyses of
isolates from different populations show that most MOMP variants are
results of single amino acid substitutions (37-39). Recombination of
sequences from MOMP during mixed infections may also have occurred.
Recombinant variants with mosaic sequences of MOMP from different strains
were especially frequent in persons with high rates of infection. MOMP
variants were also more frequently found in women with PID than in those
with lower genital tract infections, which suggests a relationship between
sequence variation in MOMP and more invasive disease (39). Clearly, the
extensive polymorphism of MOMP, the tempo for variation, and the mechanism
of immune selection have
                             [end of page 311]

important implications for vaccine design (35).

Genetic Susceptibility to Disease
HLA B27 has been associated with Reiter's syndrome following genital
chlamydial infection (40). Only a subset of infected persons appear to have
long-term complications after acute or repeated chlamydial infections. In a
study of 306 persons from trachoma-endemic communities in the Gambia, the
HLA class I antigen HLA-A28 was significantly more common in case-patients
than in age-, sex-, and location-matched controls (41). In particular, the
A*6802 allele was overrepresented among case-patients. It may be that
immunopathology is associated with HLA-A*6802 restricted cytotoxic
T-lymphocyte responses. The frequency of HLA class II alleles was similar
among cases and controls suggesting that, if class II restricted T-cell
responses are important in immunopathology, they were not targeted at
single epitopes. No individual HLA type was associated with protection from
scarring, which suggests that multiple or complex T-cell responses may be
involved in protective immunity. Susceptibility to chlamydial PID in a
study of sex workers in Nairobi, Kenya, has been associated with a HLA
class I allele, HLA A-31 (42). Studies are needed to determine whether
susceptibility to silent PID, ectopic pregnancy, and progression to tubal
factor infertility are associated with HLA class I restricted immune
responses.

Role of CHSP60 in Immunopathology
Antibody response to a 57 kDa chlamydial protein was initially observed
more frequently in women with tubal infertility than in controls (43). This
protein was subsequently identified as a heat shock protein of the GroEL
family of stress proteins. The association between antibody response to
CHSP60 and PID, ectopic pregnancy, tubal infertility, and trachoma (44-48)
has been documented. The risk factors associated with CHSP60 antibody
response are similar to those for chlamydial PID and include older age and
chronic or repeated infections. There appears to be genetic restriction for
the CHSP60 antibody response. In a study of trachoma in the Gambia, HLA
DRB1*0701 was positively correlated with CHSP60 response, while DRB1*0301
and DQB1*0501 were negatively associated (48). However, these alleles were
not associated with trachoma and may reflect linkage disequilibrium between
HLA class II alleles and polymorphic markers for other immune response
genes.

At present, it remains unclear whether antibody to CHSP60 is causally
involved in chlamydial immunopathogenesis or is merely a marker of
persistent chlamydial infection (35). Both may be true. In cells
persistently infected with C. trachomatis, the expression of CHSP60 is
normal, while other antigens, such as MOMP, are downregulated, thus
providing continued antigenic stimulation for the CHSP60 antibody response
observed in persons with long-term sequelae (49). T-cell responses to
chlamydial antigens, including CHSP60, were more depressed in persons with
trachoma than in those who recovered from infection without sequelae (50).
Persons with trachoma or reproductive sequelae have high levels of serum
antibody response to C. trachomatis. In guinea pigs and in gene knock-out
mice, both B- and T- cell responses have been important in immunity and
resolution of infection (51,52). Therefore, persons with long-term sequelae
may have predominantly Th2 responses, characterized by high levels of
B-cell response and inadequate T-cell responses that may not clear the
infection thus leading to chronic inflammation. Immunopathology may also be
the result of a hit-and-run mechanism in which immune response to CHSP60
breaks self-tolerance to the human HSP60 and leads to an autoimmune
reaction that results in tissue damage (35).

C. psittaci

Epidemiology
Human infections with C. psittaci are caused by occupational exposure to
infected birds or household handling of nasal discharge or fecal material
from pet birds. Birds can be healthy carriers of C. psittaci. Increased
shedding and susceptibility to disease occur under conditions of stress
such
                             [end of page 312]

as shipping, crowding, starvation, or egg laying. Person-to-person
transmission is rare but has been observed in outbreaks. In the C. psittaci
pandemic of 1929-30, infected birds from Argentina were shipped to
different parts of the world causing outbreaks of infection worldwide with
death rates of up to 40% (5). Since then C. psittaci has been isolated from
more than 130 species of birds. Thus, all avian species, including wild
birds, should be regarded as potential sources of zoonosis.

Reports of outbreaks of psittacosis in duck and turkey processing plants
show that, in spite of availability of medicated feed, diagnostic testing,
and screening of poultry, C. psittaci infections continue to be a public
health concern (53,54). High rates of chlamydial infection in household
cats and asymptomatic carriage of C. psittaci in cats from breeding
catteries raise the possibility that human C. psittaci infection from pets
other than birds may be underdiagnosed (6,7,55,56). Studies of animal and
cellular tropism of various strains within the species may give important
clues to the pathogenesis of C. psittaci infections.

Clinical Manifestations
Human infection caused by exposure to infected birds or poultry is
manifested as a flulike illness characterized by fever, chills, headache,
and less frequently, cough, myalgias, rash, arthralgia and joint swelling,
and atypical pneumonia in more severe cases. The incubation period is 6 to
19 days. Infections transmitted from ruminants are rare, but placentitis,
disseminated intravascular coagulation, and spontaneous abortion in women
exposed to infected sheep during lambing have been reported (56). Zoonoses
associated with exposure to ruminants are characterized by multiorgan
involvement often resulting in hepatic and renal dysfunction and
endocarditis. Human conjunctivitis, glomerulonephritis, and endocarditis
caused by C. psittaci from infected cats and pigeons have been reported
(55).

Diagnosis and Treatment
Serodiagnosis has been the method of choice for human C. psittaci
infections because culture is technically demanding and represents an
important biohazard. The complement fixation assay is genus specific. Its
interpretations should depend on clinical symptoms and patient history. The
microimmunofluorescence (MIF) assay can detect species-specific IgM or IgG
antibodies. Antigen detection methods, such as EIA, have been used, but
they are based on the capture of the genus-specific LPS. PCR assays are not
yet commercially available but can offer lower detection limits of 10 EBs
or less (57,58). Molecular techniques not only provide more sensitive and
rapid diagnosis than serology, but they also provide the opportunity for
fingerprinting strains. This is particularly useful in outbreak
investigations and for the confirmation of zoonotic transmission from
infected birds or animals.

The recommended treatment for C. psittaci infection is 250 mg of
tetracycline 4 times daily for 21 days. Although the death rate is low,
prolonged hospitalization may be required. Protracted recovery and high
incidence of relapse have also been noted.

C. pneumoniae

Epidemiology
C. pneumoniae is a common cause of acute respiratory tract infections and
accounts for 6% to 10% of community-acquired pneumonia (8). Infection is
usually mild or asymptomatic but can be severe, especially in the elderly,
probably as a result of underlying illness, impaired mucociliary clearance,
and immune senescence. Unlike C. psittaci, C. pneumoniae is spread by
person-to-person transmission by respiratory droplet and has an incubation
period of 7 to 21 days. Outbreaks of infection have been reported in
families, schools, military barracks, and nursing homes. Coinfection with
viruses (e.g., influenza and respiratory syncytial virus) and with bacteria
has been reported frequently. Seroepidemiologic studies show that most
primary infections occur during school age and the early teenage years;
among adults seroprevalence is 40% to 70%. Reinfections are common, and
serum antibodies do not appear to be protective.
                             [end of page 313]

Laboratory Diagnosis
Accurate and rapid laboratory diagnostic methods leading to improved
patient care, appropriate use of antimicrobial therapy, and better
understanding of the epidemiology of this emerging pathogen (59,60) are
needed. Culture is highly specific but is technically demanding often
requiring multiple passages over a period of weeks to show a positive
result. C. pneumoniae has been isolated from the nasopharynx of healthy
persons, but the rate of asymptomatic carriage in a normal population is
unknown (61).

Antigen detection tests, such as EIA and DFA, and molecular detection
methods, such as PCR assays, provide a rapid diagnosis without stringent
transport requirements. Monoclonal antibodies specific for C. pneumoniae
are now commercially available for DFA and for culture confirmation (62).
PCR assays have lower detection limits of 10 to 100 EBs (57,58,63-65). The
protocol developed by Tong and Sillis amplifies a target sequence conserved
between C. pneumoniae and C. psittaci and hence can detect DNA from either
pathogen in a single assay (57). A nested PCR procedure is used to
differentiate between the C. pneumoniae and C. psittaci amplicons. The
protocol of Rasmussen et al. amplifies a genus-specific target, followed by
species differentiation using restriction enzyme digestion (58). The
development of multiplex PCR assays containing primers specific for a panel
of respiratory pathogens will be useful.

The MIF assay is the standard method used for chlamydia serology today.
Ekman compared the performance of the complement fixation (CF), LPS-based
EIA, and MIF tests for the serodiagnosis of C. pneumoniae and C. psittaci
infections in an elderly population and found that the CF test has a
sensitivity of 10% compared with 88% and 72% for MIF and EIA, respectively
(66). IgM antibodies were only detected in 11% of the cases. IgM antibodies
are rarely produced in reinfections with C. pneumoniae. CF tests may be
useful in early initial infections as LPS antibodies are produced early in
infection. Serodiagnosis may be made by demonstrating a fourfold rise in CF
or EIA titer in paired sera taken a week apart, compared with the 3 weeks
or more that it takes by MIF to demonstrate seroconversion. Because
reinfections are common and LPS-based serologic tests are not useful in
reinfection, the MIF assay remains the most useful and specific tool for
the serodiagnosis of respiratory infections due to C. pneumoniae.

Treatment
The newer macrolides, clarithromycin and azithromycin, with longer tissue
half-life and concentration in mucus and macrophages and improved
bioavailability can potentially provide shorter and better-tolerated
regimens for the treatment of respiratory infections due to C. pneumoniae
than doxycycline or erythromycin, which have to be given for 2 to 3 weeks
to avoid relapse. They may also be preferred for empiric therapy as they
provide broader coverage than erythromycin against etiologic agents in
community-acquired pneumonia. The optimal duration of treatment for
respiratory infections due to C. pneumoniae needs to be determined since
studies with documented microbiologic cure are limited, and recurrence of
infection is common (67).

Association with Atherosclerosis
The association of C. pneumoniae infection with coronary heart disease and
acute myocardial infarction was first made on the basis of elevated IgG and
IgA antibodies and LPS containing immune complexes in 50% to 60% in
patients with coronary heart disease or acute myocardial infarction
compared with 7% to 12% in the controls. This study did not take into
account risk factors for heart disease such as smoking, hypertension, or
serum lipid levels. Subsequently, several cross-sectional studies involving
46 to 461 study participants have shown that a similar association of IgG
antibodies against C. pneumoniae with coronary artery disease and carotid
disease with adjusted odds ratios of 1.6 to 2.6 after controlling for known
risk factors (68-72). Electron microscopy, PCR, and immunochemical evidence
of C. pneumoniae in coronary arterial fatty streaks and atheromatous
plaques have also been described (72,73).
                             [end of page 314]

Two more recent studies reported equivocal findings. In one, C. pneumoniae
was detected in 79% of 90 coronary atherectomy specimens from symptomatic
patients by direct immunofluorescence and was confirmed by electron
microscopy. Only 4% of 24 control nonatherosclerotic coronary specimens
were positive for C. pneumoniae (74). The 24 control samples included 12
from heart transplant patients whose arteries were damaged, but not by
atherosclerosis. The absence of C. pneumoniae in these tissue samples
argues against its role as a passenger recruited to the site of injury in
macrophages. In the other study, C. pneumoniae was not detected in 58
coronary atheroma specimens by culture, PCR, or electron microscopy (75).
The seroprevalence of C. pneumoniae in 65 case-patients was not different
from that in 28 asymptomatic controls. In fact, IgG titers were higher in
controls than in case-patients. Nonetheless, data suggest that the
association of C. pneumoniae with atherosclerosis is consistent and
biologically plausible. Whether C. pneumoniae is causally involved or is a
bystander trapped in the atherogenic process is unclear.

The sustained IgA and IgG antibody levels against C. pneumoniae in persons
with atherosclerosis suggest that chronic infection may be frequent after
infection. The site of colonization for a chronic C. pneumoniae infection
may be in the alveolar macrophages of the lung. Thus the initial event in
atherogenesis may be the formation of the fatty streak. Fatty streaks
consist of lipid-laden macrophages derived from blood monocytes and T
lymphocytes attracted to the arterial subintima. Conversion of the fatty
streak to atheroma depends on many factors, e.g., the proliferation and
differentiation of smooth muscle cells and fibroblasts. Chronic infection
with C. pneumoniae may result from organisms harbored in macrophages
trapped in the arterial wall. Growth of C. pneumoniae in endothelial,
smooth muscle cells, and macrophages from peripheral blood monocytes has
been reported (76). Injured blood vessels initiate events that promote
thrombosis and platelet adhesion at the site of injury. These events in
turn promote atherosclerosis. Tissue injury through C. pneumoniae-specific
circulating immune complexes in patients with chronic heart disease may be
an alternate mechanism or compounding atherogenesis. The idea that an
infectious agent is involved in the atherogenic process is not new, but the
role of C. pneumoniae in this process needs to be defined.

Association with Asthma
The prevalence of asthma, an important chronic respiratory disorder, has
been steadily increasing. Viral and Mycoplasma pneumoniae infections have
been implicated in exacerbating the disease. The first observations on the
association of C. pneumoniae infection with the exacerbation of asthma were
made in 1986 when wheezing was associated with acute bronchitis due to
pneumoniae infection (8,77). Subsequent studies showed that exacerbation of
asthma due to C. pneumoniae infection may occur in 1% to 11% of respiratory
infections in adults as well as children. The mechanism underlying the
association is unclear. Preliminary results in animal models suggest that
C. pneumoniae can produce persistent infection and cause pulmonary
inflammation, and production of chlamydia-specific IgE antibodies in
children with reactive airway disease has been demonstrated (78). A
possible scenario for this association is an antigen-specific allergic
reaction with the release of pulmonary inflammatory mediators and
recruitment of inflammatory cells to the airways, causing airway epithelial
damage. Activated T lymphocytes and cytokines appear to play a critical
role as mediators of persistent inflammation in asthma. IL-4 is essential
for B lymphocytes class switching from IgG to IgE. In vitro human IgE
synthesis is reciprocally regulated by IL-4 and interferon-gamma. Thus
cytokines from a Th2 response to infection would facilitate and promote IgE
production. Immunotherapy or glucocorticoid therapy targeting CD4+ T cells
may decrease the proinflammatory role of these cells and alleviate symptoms
of asthma. The role of persistent infection in the pathogenesis of asthma
merits further study because, unlike
                             [end of page 315]

viral infections, C. pneumoniae infections can be eradicated through
appropriate antimicrobial therapy.

The hallmark of chlamydial infection is that most persons infected have
mild to no apparent clinical disease and some have severe disease.
Asymptomatic infection not only creates a problem in detecting cases for
disease control programs but also contributes to the development of
long-term adverse sequelae, such as scarring trachoma from ocular C.
trachomatis infection, pelvic inflammatory disease, ectopic pregnancy, and
tubal factor infertility from genital C. trachomatis infection. The recent
availability of effective single dose oral antimicrobial therapy and
sensitive molecular amplification tests that allow the use of noninvasive
specimens for diagnosis and screening is expected to have a major impact in
reducing the prevalence of disease in the next decade. New information from
cell biology as well as data from observing the interaction of chlamydiae
with the host in terms of metabolic requirements and immune evasion
strategies offer clues about the pathogenesis of chlamydia infections and
may eventually lead to an effective vaccine. Sporadic outbreaks of
psittacosis continue to be reported despite the use of medicated feed and
the screening of poultry. Recent reports of C. psittaci in cats from
breeding catteries illustrate the potential of zoonotic diseases
transmissible to humans from pets other than birds. Two new species of
chlamydiae, C. pneumoniae and C. pecorum, were desig-nated in 1989 and
1992, respectively. Clinical manifestations associated with C. pneumoniae
infection continue to emerge. Possible links to chronic conditions, such as
atherosclerosis and asthma remain to be elucidated. With the recent
discovery of the involvement of infectious agents in other chronic
conditions, it seems reasonable to apply molecular tools for chlamydial
detection to identify their potential involvement in other etiologically
undefined chronic inflammatory conditions such as inflammatory bowel
disease and rheumatoid arthritis.

    Dr. Peeling is a research scientist and chief of the Division of
    Chlamydial and Mycoplasma Diseases at the Laboratory Centre for
    Disease Control, Health Canada. She is interested in the diagnosis
    and pathogenesis of chlamydial infections with particular emphasis
    on the development, risk assessment, and possible prevention of
    adverse ocular and reproductive sequelae in human chlamydial
    infections.

    Dr. Brunham is professor and head of the Department of Medical
    Microbiology at the University of Manitoba. He has a long standing
    interest in the immunology of chlamydial infections, and his
    current research focus is on vaccine development.

Address for correspondence: Rosanna W. Peeling, LCDC Chlamydia Laboratory,
Health Sciences Centre MS 673C, 820 Sherbrook St., Winnipeg, Manitoba,
Canada R3A 1R9; fax: 204-787-4699; e-mail: rosanna_peeling@isdtcp3.hwc.ca.

References

  1. Weisburg WG, Hatch TT, Woese CR. Eubacterial origin of chlamydiae. J
     Bacteriol 1986;167:570-4.
  2. Dawson CR, Jones BR, Tarizzo ML. Guide to trachoma control in
     programmes for the prevention of blindness. Geneva: World Health
     Organization, 1981.
  3. Sexually Transmitted Diseases. World Health Organization Press Release
     WHO/64, 25 August 1995.
  4. Washington A, Johnson RE, Sanders L Jr. Chlamydia trachomatis
     infection in the United States: what are they costing us? JAMA
     1987;257:2070-2.
  5. Schachter J, Dawson CR. Psittacosis. Human Chlamydial Infections.
     Littleton, MA: PSG Publishing Co., 1978.
  6. Nasisse MP, Guy JS, Stevens JB, English RV, Davidson MG. Clinical and
     laboratory findings in chronic conjunctivitis in cats: 91 cases
     (1983-1991). J Am Vet Med Assoc 1993;203:834-7.
  7. Pointon AM, Nicholls JM, Neville S, Allanson M, Coles C, Lawrence D.
     Chlamydia infection among breeding catteries in south Australia.
     Australian Veterinary Practitioner 1991;21:58-63.
  8. Grayston JT. Infections caused by Chlamydia pneumoniae strain TWAR.
     Clin Infect Dis 1992;15:757-63.
  9. Stephens RS. Molecular mimicry and Chlamydia trachomatis infection of
     eucaryotic cells. Trends Microbiol 1994;2:99-101.
 10. Hackstadt T, Scidmore MA, Rocky DD. Lipid metabolism in Chlamydia
     trachomatis-infected cells: directed trafficking of Golgi-derived
     sphingolipids to the chlamydial inclusion. Proc Natl Acad Sci U S A
     1995;92:4877-81.
 11. Hackstadt T, Rocky DD, Heinzen RA, Sidmore MA. Chlamydia trachomatis
     interrupts an exocytic pathway to acquire endogenously synthesized
     sphingomyelin in transit from the Golgi apparatus to the plasma
     membrane. EMBO J 1996;15:964-77.
 12. McClarty G. Chlamydiae and the biochemistry of intracellular
     parasitism. Trends Microbiol 1994;2:157-64.
                                [end of page 316]

 13. Peeling RW. Chlamydia trachomatis and Neisseria gonorrhoeae: Pathogens
     in retreat? Current Opinion in Infectious Diseases 1995;8:26-34.
 14. Lin JSL, Jones WE, Yan L, Wirthwein KA, Flaherty EE, Haivanis RM, et
     al. Underdiagnosis of Chlamydia trachomatis infection: diagnostic
     limitations in patients with low-level infection. Sex Transm Dis
     1992;19:259-65.
 15. Peeling RW, Brunham RC. Molecular techniques for the laboratory
     identification of Chlamydia trachomatis. Journal of the International
     Federation of Clinical Chemistry 1994;6:78-82.
 16. Bauwens JE, Clark AM, Stamm WE. Diagnosis of Chlamydia trachomatis
     endocervical infections by a commercial polymerase chain reaction
     assay. J Clin Microbiol 1993;31:3023-7.
 17. de Barbeyrac B, Pellet I. Dutilh B, Bebear C, Dumon B, Geniaux M, et
     al. Evaluation of the Ampicor Chlamydia trachomatis test versus
     culture in genital samples in various prevalence populations.
     Genitorurin Med 1994;70:162-6.
 18. Wisenfeld HC, Uhrin M, Dixon BW, Sweet RL. Diagnosis of male Chlamydia
     trachomatis urethritis by polymerase chain reaction. Sex Transm Dis
     1994;21:268-71.
 19. Jaschek G, Gaydos CA, Welsh L, Quinn TC. Direct detection of Chlamydia
     trachomatis in urine specimens from symptomatic and asymptomatic men
     by using a rapid polymerase chain reaction assay. J Clin Microbiol
     1993;31:1209-12.
 20. Toye B, Peeling RW, Jessamine P, Claman P, Gemmill I. Diagnosis of
     Chlamydia trachomatis infections in asymptomatic men and women by PCR
     assay. J Clin Microbiol 1996;34:1396-400.
 21. Chernesky MA, Jang D, Lee H, Hu H, Sellors J, Tomazic-Allen SJ, et al.
     Diagnosis of Chlamydia trachomatis infections in men and women by
     testing first-void urine by ligase chain reaction. J Clin Microbiol
     1994;32:2682-5.
 22. Schachter J, Stamm WE, Quinn TC, Andrews WW, Burczak JD, Lee H. Ligase
     chain reaction to detect Chlamydia trachomatis infection of the
     cervix. J Clin Microbiol 1994;32:2540-3.
 23. Scholes D, Stergachis A, Heidrich FE, Andrilla HA, Holmes KK, Stamm
     WE. Selective screening for chlamydia reduces the incidence of pelvic
     inflammatory disease: results from a randomised intervention trial. J
     Infect Dis 1996 (in press).
 24. Cameron DW, Simonsen JN, D'Costa LJ, Ronald AR, Maitha GM, Gakinya MN,
     et al. Female to male transmission of human immunodeficiency virus
     type 1: risk factors for seroconversion in men. Lancet 1989;ii:403-7.
 25. Grosskurth H, Mosha F, Todd J, Mwijarubi E, Klokke A, Senkoro K, et
     al. Impact of improved treatment of sexually transmitted diseases on
     HIV infection in rural Tanzania: randomised controlled trial. Lancet
     1995:346:530-6.
 26. Martin DH, Mroczkowski TF, Dalu ZA, McCarty J, Jones RB, Hopkins SJ,
     et al. A controlled trial of a single dose of azithromycin for the
     treatment of chlamydial urethritis and cervicitis. N Engl J Med
     1992;327:921-5.
 27. Bailey RL, Arullendran P, Whittle HC, Mabey DCW. Randomised controlled
     trial of single-dose azithromycin in the treatment of trachoma. Lancet
     1993;342:453-6.
 28. Ossewaarde JM, Plantema FHF, Rieffe M, Nawrocki RP, de Vries A, van
     Loon AM. Efficacy of single-dose azithromycin versus doxycycline in
     the treatment of cervical infections caused by Chlamydia trachomatis.
     Eur J Clin Microbiol Infect Dis 1992;11:693-7.
 29. Worm A-M, Osterlind A. Azithromycin levels in cervical mucus and
     plasma after a single 1.0 g oral dose for chlamydia cervicitis.
     Genitourin Med 1995;71:244-6.
 30. Hammerschlag MR, Golden NH, Oh MK, Gelling M, Sturdevant M, Pernell
     PR, et al. Single dose of azithromycin for the treatment of genital
     chlamydial infections in adolescents. J Pediatr 1993;122:961-5.
 31. Bush MR, Rosa C. Azithromycin and erythromycin in the treatment of
     cervical chlamydial infection during pregnancy. Obstet Gynecol
     1994;84:61-3.
 32. Lauharanta J, Saarinen K, Mustonen M-T, Happonen H-P. Single-dose oral
     azithromycin versus seven-day doxycycline in the treatment of
     non-gonococcal urethritis in males. J Antimicrob Chemother
     1993;31:177-83.
 33. Lister PJ, Balechandran T, Ridgway GL, Robinson JA. Comparison of
     azithromycin and doxycycline in the treatment of non-gonococcal
     urethritis in men. J Antimicrob Chemother 1993;31:185-92.
 34. Magid D, Douglas JM, Schwartz JS. Doxycycline compared with
     azithromycin for treating women with genital Chlamydia trachomatis
     infections: an incremental cost-effectiveness analysis. Ann Intern
     Med 1996;124:389-99.
 35. Brunham RC, Peeling RW. Chlamydia trachomatis antigens: role in
     immunity and pathogenesis. Infect Agents Dis 1994;3:218-33.
 36. Brunham RC, Plummer F, Stephens RS. Bacterial antigenic variation,
     host immune response and pathogen-host co-evolution. Infect Immun
     1993;61:2273-6.
 37. Brunham R, Yang C, Maclean I, Kimani J, Maitha G, Plummer F. Chlamydia
     trachomatis from individuals in a sexually transmitted disease core
     group exhibit frequence sequence variation in the major outer membrane
     protein (omp1) gene. J Clin Invest 1994;94:458-63.
 38. Hayes LJ, Bailey RL, Mabey DCW, Clarke IN, Pickett MA, Watt PJ, et al.
     Genotyping of Chlamydia trachomatis from a trachoma endemic village in
     the Gambia by a nested polymerase chain reaction: identification of
     strain variants. J Infect Dis 1992;166:1173-7.
                                [end of page 317]

 39. Dean D, Schachter J, Dawson CR, Stephens RS. Comparison of the major
     outer membrane protein variant sequence regions of B/Ba isolates: a
     molecular epidemiologic approach to Chlamydia trachomatis infections.
     J Infect Dis 1992;166:383-92.
 40. Schachter J, Dawson CR. Reiter's Syndrome. Human Chlamydial
     Infections. Littleton, MA: PSG Publishing Co., 1978.
 41. Conway DJ, Holland MJ, Campbell AE, Bailey RL, Krausa P, Peeling RW,
     et al. HLA class I and class II polymorphism and trachomatous scarring
     in a chlamydia trachomatis-endemic population. J Infect Dis
     1996;174:643-6.
 42. Kimani J, Maclean IW, Bwayo JJ, MacDonald K, Oyugi J, Maitha GM, et
     al. Risk factors for Chlamydia trachomatis pelvic inflammatory disease
     among sex workers in Nairobi, Kenya. J Infect Dis 1996;173:1437-44.
 43. Brunham RC, Maclean IW, Binns B, Peeling RW. Chlamydia trachomatis:
     its role in tubal infertility. J Infect Dis 1985;152:1275-82.
 44. Wagar EA, Schachter J, Bavoil P, Stephens RS. Differential human
     serologic response to two 60,000 molecular weight Chlamydia
     trachomatis antigens. J Infect Dis 1990;162:922-7.
 45. Brunham RC, Peeling R, Maclean I, Kosseim ML, Paraskevas M. Chlamydia
     trachomatis-associated ectopic pregnancy:serologic and histologic
     correlates. J Infect Dis 1992;165:1076-81.
 46. Toye B, Laferriere C, Claman P, Jessamine P, Peeling R. Association
     between antibody to the chlamydial heat shock protein and tubal
     infertility. J Infect Dis 1993;168:1236-40.
 47. Arno JN, Yuan Y, Cleary RE, Morrison RP. Serologic responses of
     infertile women to the 60-kd chlamydial heat shock protein. Fertil
     Steril 1995;64:730-5.
 48. Peeling RW, Bailey RL, Conway D, Holland MJ, Dillon E, Mabey DCW.
     Antibody response to the chlamydial heat shock protein 60 is
     associated with scarring trachoma. 96th American Society for
     Microbiology meeting, New Orleans, May 1996. Abstract #2871.
 49. Beatty WL, Byrne GI, Morrison RP. Morphologic and antigenic
     characterization of interferon gamma mediated persistent Chlamydia
     trachomatis infection in vitro. Proc Natl Acad Sci U S A
     1993;90:3998-4002.
 50. Holland MJ, Bailey RL, Hayes LJ, Whittle HC, Mabey DCW. Conjunctival
     scarring in trachoma is associated with depressed cell-mediated immune
     responses to chlamydial antigens. J Infect Dis 1993;168:1528-31.
 51. Morrison RP, Feilzer K, Tumas DB. Gene knock-out mice establish a
     primary role for major histocompatibility complex class II-restricted
     responses in Chlamydia trachomatis genital tract infection. Infect
     Immun 1995;63:4661-8.
 52. Igietseme JU, Magee DM, Williams DM, Rank RG. Role for CD8+ T cells in
     antichlamydial immunity defined by chlamydia-specific T-lymhocyte
     clones. Infect Immun 1994;62:5195-7.
 53. Hinton DG, Shipley A, Galvim JW, Harkin JT, Brunton RA. Chlamydiosis
     in workers at a duck farm and processing plant. Aust Vet J
     1993;70:174-6.
 54. Hedberg K, White KE, Forfang JC, Korlath JA, Friendshuh, Hedberg CW,
     et al. An outbreak of psittacosis in Minnesota turkey industry
     workers: implications for modes of transmission and control. Am J
     Epidemiol 1989;130:569-77.
 55. Hadley KM, Carrington D, Frew CE, Gibson AAM, Hislop WS. Ovine
     chlamydiosis in an abattoir worker. J Infect Dis 1992;25:105-9.
 56. Regan RJ, Dathan JRE, Treharne JD. Infective endocarditis with
     glomerulonephritis associated with cat chlamydia (C. psittaci)
     infection. British Heart Journal 1979;42:349-52.
 57. Tong CYW, Sillis M. Detection of Chlamydia pneumoniae and Chlamydia
     psittaci in sputum samples by PCR. J Clin Pathol 1993;46:313-7.
 58. Rasmussen SJ, Douglas FP, Timms P. PCR detection and differentiation
     of Chlamydia pneumoniae, Chlamydia psittaci and Chlamydia trachomatis.
     Mol Cell Probes 1992;6:389-94.
 59. Saikku P. Diagnosis of acute and chronic Chlamydia pneumoniae
     infections. In: Orfila J, et al., editors. Chlamydial Infections.
     Bologna: Societa Editrice Esculapio, 1994.
 60. Peeling RW. Laboratory diagnosis of Chlamydia pneumoniae infections.
     Canadian Journal of Infectious Diseases 1995;6:198-203.
 61. Gnarpe J, Gnarpe H, Sundelof B. Endemic prevalence of Chlamydia
     pneumoniae in subjectively healthy persons. Scand J Infect Dis
     1991;23:387-8.
 62. Montalban GS, Roblin PM, Hammerschlag MR. Performance of three
     commercially available monoclonal reagents for confirmation of
     Chlamydia pneumoniae in cell culture. J Clin Microbiol 1994;32:1406-7.
 63. Campbell LA, Melgosa MP, Hamilton DJ, Kuo C-C, Grayston JT. Detection
     of Chlamydia pneumoniae by polymerase chain reaction. J Clin Microbiol
     1992;30:434-9.
 64. Gaydos CA, Roblin PM, Hammerschlag MR, Hyman CL, Eiden JJ, Schacter J,
     et al. Diagnostic utility of PCR-Enzyme Immunoassay, culture, and
     serology for the detection of Chlamydia pneumoniae in symptomatic and
     asymptomatic patients. J Clin Microbiol 1994;32:903-5.
 65. Black CM, Fields PI, Messmer TO, Berdal BP. Detection of Chlamydia
     pneumoniae in clinical specimens by polymerase chain reaction using
     nested primers. Eur J Clin Microbiol Infect Dis 1994;13:752-6.
 66. Ekman MR, Leinonen M, Syrjala H, Linnanmaki E, Kujala P, Saikku P.
     Evaluation of serological methods in the diagnosis of Chlamydia
     pneumoniae during an epidemic in Finland. Eur J Clin Microbiol Infect
     Dis 1993;12:756-60.
                                [end of page 318]

 67. Roblin P, Montalban G, Hammerschlag MR. Susceptibilities to
     clarithromycin and erythromycin of isolates of Chlamydia pneumoniae
     from children with pneumonia. Antimicrob Agents Chemother
     1994;38:1588-9.
 68. Grayston JT, Thom DH, Kuo C-C, Campbell LA, Wang S-P. Chlamydia
     pneumoniae (TWAR) and atherosclerosis. In: Orfila J, Byrne G,
     Chernesky MA, et al, editors. Chlamydial Infections. Bologna: Societa
     Editrice Esculapio, 1994.
 69. Saikku P, Leinonen M, Tenkanen L, Linnanmaki E, Ekman M-R, Manninen V,
     et al. Chronic Chlamydia pneumoniae Infection as a Risk Factor for
     Coronary Heart Disease in the Helsinki Heart Study. Ann Intern Med
     1992;116:273-8.
 70. Thom D, Grayston JT, Siscovick D, Wang S-P, Weiss N, Daling J.
     Association of Prior Infection With Chlamydia pneumoniae and
     Angiographically Demonstrated Coronary Artery Disease. JAMA
     1992;268:68-72.
 71. Melnick S, Shahar E, Folsom A, Grayston JT, Sorlie P, Wang S-P, et al.
     Past Infection by Chlamydia pneumoniae Strain TWAR and Asymptomatic
     Carotid Atherosclerosis. Am J Med 1993;95:499-504.
 72. Shor A, Kuo CC, Patton D. Detection of Chlamydia pneumoniae in
     coronary arterial fatty streaks and atheromatous plaques. S Afr Med J
     1992;82:158-61.
 73. Kuo CC, Shor A, Campbell L, Fukushi H, Patton D, Grayston JT.
     Demonstration of Chlamydia pneumoniae in Atherosclerotic Lesions of
     Coronary Arteries. J Infect Dis 1993;167:841-9.
 74. Muhlestein JB, Hammond E, Carlquist JF, Radicke E, Thomson MJ,
     Karagounis LA, et al. Increased incidence of Chlamydia species within
     the coronoary arteries of patients with symptomatic atherosclerotic
     versus other forms of cardiovascular disease. J Am Coll Cardiol
     1996;27:1555-61.
 75. Weiss SM, Roblin PM, Gaydos C, Cummings P, Patton D, Schulhoff N, et
     al. Failure to detect Chlamydia pneumoniae in coronary atheromas of
     patients undergoing atherectomy. J Infect Dis 1996;173:957-62.
 76. Godzik K, O'Brien E, Wang S-K, Kuo CC. In Vitro Susceptibility of
     Human Vascular Wall Cells to Infection with Chlamydia pneumoniae. J
     Clin Microbiol 1995;33:2411-4.
 77. Hahn DL, Dodge RW, Golubjatnikov R. Association of Chlamydia
     pneumoniae (TWAR) infection with wheezing, asthmatic bronchitis and
     adult-onset asthma. JAMA 1991,266:225-30.
 78. Emre U, Sokolovskaya N, Roblin PM, Schachter J, Hammerschlag M.
     Detection of anti-Chlamydia pneumoniae IgE in children with reactive
     airway disease. J Infect Dis 1995;148:727-32.

                             [end of page 319]

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

---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
Dispatches

Experimental Inoculation of Plants and Animals with Ebola Virus

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


    Thirty-three varieties of 24 species of plants and 19 species of
    vertebrates and invertebrates were experimentally inoculated with
    Ebola Zaire virus. Fruit and insectivorous bats supported
    replication and circulation of high titers of virus without
    necessarily becoming ill; deaths occurred only among bats that had
    not adapted to the diet fed in the laboratory.

The taxonomy of the Filoviridae is in a state of flux; the family includes
viruses currently designated Marburg, Ebola Zaire, Ebola Sudan, and Ebola
Ivory Coast, which are believed to be endemic to Africa, and Ebola Reston,
which putatively originates in the Philippines (1,2). The viruses are known
particularly for their propensity to cause fatal hemorrhagic disease of
humans with person-to-person spread, but their pathogenicity varies from
asymptomatic infection (Ebola Reston) to epidemics with death rates of 77%
to 88% (Ebola Zaire) (1,3). All of the viruses appear to be highly
pathogenic for nonhuman primates. Outbreaks of disease have occurred in
Europe and North America in monkeys imported from Africa and the
Philippines, sometimes with spread of infection to humans (1). Contact with
the tissues of dead nonhuman primates was a source of infection for humans
on at least two occasions in Africa (2,4). Nevertheless, the lethality of
the viruses for nonhuman primates suggests that, like humans, nonhuman
primates are incidental victims of infection and are not true reservoir
hosts (1). The source of filoviruses in nature remains unknown, but in some
instances, bats roosted in buildings or a cave visited or frequented by
people who subsequently were found to have primary cases of infection in
outbreaks of disease in Africa; one patient was bitten or stung by what is
presumed to have been an arthropod 7 days before coming down with Marburg
disease (1). Informal speculation has included the suggestion that
filoviruses may be plant viruses, perhaps even involving transmission by
arthropod vectors.

The search for the source of the viruses in nature has been hampered by the
erratic recognition of outbreaks of filovirus infection in widely separated
geographic locations at unpredictable intervals; therefore, comparatively
few field investigations have been reported (1). After the 1995 epidemic of
Ebola fever in Kikwit, Zaire, teams of scientists coordinated by the
Centers for Disease Control and Prevention, Atlanta, and the U.S. Army
Medical Research Institute of Infectious Diseases, Fort Detrick, collected
large numbers of vertebrate and arthropod specimens during June, July, and
August, 1995. Because evidence indicated that the outbreak had actually
started in January 1995, it was possible that the virus was no longer
circulating in its natural hosts in the vicinity of Kikwit by the time
ecologic studies were undertaken. To allow for the possibility that the
filoviruses manifest seasonal activity in their natural hosts, a team from
the National Institute for Virology (NIV) in South Africa visited Kikwit in
January 1996, to trap wild vertebrates and arthropods in the sites
investigated by the other teams in mid-1995. Testing of the field material
is a lengthy process, and no filoviruses have been detected so far. We
decided to narrow the search by performing pathogenicity studies with Ebola
virus in representatives of different classes and orders of living things,
including vertebrates, invertebrates, and even plants. The underlying
assumptions were that if a group of species is either refractory or
hypersusceptible to the virus, members of the taxon are unlikely reservoir
hosts of the virus, whereas members of taxa capable of circulating virus
for prolonged periods without becoming ill are suspected reservoirs.

The strain of Ebola Zaire virus used in the experiments, Zaire-95, had been
isolated from the blood of a patient in the 1995
                             [end of page 321]

epidemic in Kikwit and designated as the prototype strain of the outbreak.
Stocks were prepared from virus at pass level 4 in Vero V76 cell cultures
by freeze-thawing infected cultures, clarifying the supernatant culture
fluid at 3,000g, and storing it in small volumes at -70C. Virus titers
were determined by fluorescent focus assay in 8-chamber slide cultures as
described for rabies (5), except that Vero cells were used, and cultures
were stained with immune mouse ascitic fluid followed by
fluorescein-labeled anti-mouse immunoglobulin and read on day 3 to 5
postinoculation to detect infected foci. Inoculum volumes of 10l of
10-fold serial dilutions of stock virus or tissue suspensions were adsorbed
to cultures, and titers were expressed as fluorescent focus-forming units
(FFU) per ml.

One-month-old potted seedlings of 33 varieties of 24 species of weeds and
crop plants used in plant virology (Table 1), plus colonized leafhoppers,
were obtained from Dr. G. Pietersen of the Plant Protection Research
Institute (PPRI) at Rietondale, Pretoria, South Africa. The plants were
selected because collectively they could provide culture substrates for a
broad spectrum of the known viruses of economically important plants. They
were kept under suitable lighting for 10 hours each day and watered as
necessary to sustain growth in the laboratory. Pigeons were obtained from
the South African Institute for Medical Research in Johannesburg; 1- to
3-month-old hatchling snakes from the Transvaal Snake Park, Midrand;
cockroaches from a colony at the Bureau of Standards in Pretoria; and
Mastomys natalensis and NIH mice from colonies at NIV. All other animals
used in the studies (Table 2) were collected in the Kruger National Park,
South Africa, with the permission of the National Parks Board as part of a
long-standing research project on hemorrhagic fever viruses. The animals
were translocated from the park under permit from the Department of
Veterinary Services, and the experiments were conducted in a biosafety
level 4 containment laboratory at NIV with clearance from the Department of
Agriculture, Conservation and Environment of Gauteng Province, and the
Animal Ethics Committee of NIV. All animals were fed a diet similar to
their natural diets and were provided with fresh drinking water daily.

         Table 1. Plants experimentally inoculated with Ebola virus
         ----------------------------------------------------------
         Scientific name                     Common name
         ----------------------------------------------------------
         Arachis hypogaea                    Groundnut
         Beta vulgaris                       Beetroot
         Chenopodium amaranticolor           Goosefoot weed
         Chenopodium quinoa                  Goosefoot weed
         Cucumis sativus                     Cucumber
         Cucurbita pepo                      Pumpkin
         Glycine max(a))                     Soybean

         Gomphrena globosa                   Weed
         Gossypium hirsutum                  Cotton
         Lupinus albus                       Lupin
         Lycopersicon esculentum             Tomato
         Macroptilium atropureum             Siratro bean
         Nicotiana benthamiana               Wild tobacco
         Nicotiana clevelandii               Wild tobacco
         Nicotiana glutinosa                 Wild tobacco
         Nicotiana langsdorfi                Wild tobacco
         Nicotiana rustica                   Wild tobacco
         Nicotiana tabacum                   Tobacco
         Phaseolus vulgaris(a)               French bean

         Pisum sativum                       Green pea
         Triticum aestivum                   Wheat
         Vicia faba                          Broadbean
         Vigna unguiculata(b)                Cowpea

         Zea mays                            Maize
         ----------------------------------------------------------
         (a)Five varieties inoculated
         (b)Two varieties inoculated

Vertebrates were inoculated subcutaneously with 0.1 ml of stock virus
diluted 1:10 in cell culture medium, and back titration of the inoculum
indicated that each animal received a dose of 40,000 or 10 to the 4.6th power FFU virus.
Invertebrates were inoculated with undiluted stock virus and received
approximately 1.0l containing 4,000 or 10 to the 3.6th power FFU of virus,
except for leafhoppers, which received about 0.3l inoculum. The arthropods
were inoculated intrathoracically (6), except for ants and millipedes, 
which were inoculated into the hemocoel through the membranous integument
between tergites. To simulate mechanical transmission, undiluted stock
virus mixed with Carborundum powder was rubbed gently with cotton buds 
onto two leaves on each of the plant varieties on experiment; to 
simulate vector-borne transmission, a second plant of each type was
 inoculated with 1.0l virus suspension into the phloem of the stem, 
using the same apparatus as for arthropods. A third plant
                             [end of page 322]

of each type served as control. Plants were observed daily and those that
wilted or developed apparent lesions were harvested; some material was
fixed in 2.5% glutaraldehyde in buffer for examination by electron
microscopy, and the rest was stored at -70C for virus assay. The process
of embedding, sectioning, and examining the plant tissues by electron
microscopy was performed by H.J. van Tonder of PPRI. Animals were
sacrificed and assayed for virus content either in pools or individually,
before inoculation and at intervals postinoculation (Table 2). Serum and
pooled visceral organs were tested separately, and individual organ samples
were preserved at -70C and in Formalin fixative for more detailed study
later. Urine and feces samples were tested on some occasions. Materials for
virus assay were prepared as 10% suspensions in culture medium, and in
parallel with attempts to titrate infectivity, 0.1 ml volumes were injected
into Vero cell monolayers in 25 cm-square flasks, which were subcultured
thrice at weekly intervals before specimens were recorded as negative.


                      Table 2. Results of experimental infection of various animals with Ebola virus
NOTE: Wide table, print with condensed font or landscape mode.
-----------------------------------------------------------------------------------------------------------
                                                                   Day postinfection
Species                              Pool
     Common name                     size  1    2   3   4   5   6   7   8   9   10  11  12  13  14  21  28
-----------------------------------------------------------------------------------------------------------
Columba livia             
     Domestic pigeon                 1     0/1* 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/2 0/2
Hyperolius viridiflavus   
     Painted reed frog               2     0/1  0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/2
Bufo regularis            
     Common toad                     1                  0/1     0/1                     0/1         0/1
Chiromantis xerampelina   
     Grey tree frog                  1                      0/1     0/1                         0/1 0/1
Hemidactylus mabouia      
     Tropical house gecko            1     0/1  0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1     0/1
Lamprophis fuliginosus    
     Brown house snake               1     0/1  0/1 1/1 0/1 0/1 0/1 0/1 1/1 0/1 0/1 1/1 0/1 0/1 0/1 0/2 0/2
Geochelone pardalis       
     Leopard tortoise                1                          0/1     0/1     0/1                     0/1
Kinixys belliana          
     Hinged-back tortoise            1                          0/2     0/2     0/2                     0/2
Tadarida condylura          
     Angola free-tailed bat          1                  1/2 1/2         2/2 1/1         1/1 
Tadarida pumila           
     Little free-tailed bat          1     1/1  1/1 1/1     0/1 1/2 0/2 0/1     1/1 1/1 1/1 0/1 1/1 0/4 0/5
Epomophorus wahlbergi     
     Wahlbergs epauletted fruit bat 1          0/1     1/1     1/1 1/1 1/1     1/1 1/1 1/1         1/1 0/1
Mastomys natalensis ss    
     Multimammate mouse              1     0/3  0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3
Mus musculus              
     NIH mouse                       1          1/1 0/1 0/1         1/1 0/1 1/1 0/1 0/1         0/1 0/2
Periplaneta americana     
     American cockroach              2     0/1  0/1 0/1 0/1 0/1 0/1 0/1 0/1 
Austria agallia           
     Leafhopper                      50                                                                 0/1
Messor barbarus capensis   
     Myrmicine ant                   5              0/1     0/1     0/1  
Stegodyphus dumicola      
     Social spider                   2     0/1  1/1 0/1 0/1 0/1 0/1 0/1 0/1                     1/1
Alloporus sp.             
     Millipede                       1     0/1  0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 
Achatina sp.              
     African landsnail               1              0/1     0/1                                         0/1
------------------------------------------------------------------------------------------------------------
*Virus isolations/pools tested

Thirteen plants either wilted or developed lesions on the leaves ascribed
to mechanical injury during the inoculation process, but no infectivity
could be recovered from the tissues, and no evidence of virus infection was
observed by electron microscopy.
                             [end of page 323]

All animal experiments (Table 2) were performed in parallel on a single
occasion except for a second experiment with insectivorous bats (Tadarida
spp.). Insectivorous bats had difficulty in adapting to a laboratory diet,
and consequently 10 of 18 died in the first experiment. Although virus was
recovered from the blood and organs of some, no histopathologic lesions
were observed, and no evidence of widespread infection was detected by
immunohistochemical technique (7). All of the bats that died had not been
eating well, and hence more insectivorous bats (T. pumila) were collected
from the same colony as before and adapted to a diet of mealworms over a
period of 3 weeks before inoculation. The bats in the second experiment
received the same dose of virus as the previous group, and no deaths
occurred before the 12 animals in the study, including nine that were kept
21 to 28 days, were sacrificed (Table 2). None of the other vertebrates
died, although some of the ants, cockroaches, and spiders died, possibly
from desiccation as a result of injury during inoculation.

The virus replicated in bats: titers of 10 to the 4.6th power - 10 to
the 7.0th power FFU/ml were recorded in sera and titers of 10 to the
2.0th power - 10 to the 6.5th power FFU/ml in pooled viscera of fruit bats.
In addition, virus was recovered from the feces of a fruit bat on day 21
postinoculation. Virus was also recovered on a few occasions from snakes,
and NIH mice and spiders (Table 2), but this was at a minimal titer of
10 to the 1.0st power FFU/ml and could represent residual infectivity 
from inoculation. Histopathologic and immunohistochemical investigations
have thus far been limited to some sets of the bat organs, and the only
virus antigen detected was present in the endothelial cells of lung tissue
of a bat sacrificed on day 8 postinoculation (Figure). Four insectivorous
bat sera collected on day 28 postinoculation and four samples from 
noninfected bats were tested for Ebola virus antigen or antibody by 
enzyme-linked immunoassay (8) using conjugated chicken anti-rodent 
immunoglobulin; antibody was found in only one of the infected bats.

             [Image] [Figures not available in ASCII version]

             Figure 1. Ebola virus antigen-positive cells
             (red) in lung of an insectivorous bat as
             determined by immunohistochemistry. Note
             prominent endothelial immunostaining. (Rabbit
             anti-Ebola virus serum, napthol/fast red with
             hematoxylin counterstain, original magnification
             x 250).

Although they do not provide conclusive evidence that bats are potential
reservoir hosts of filoviruses or that the other animals are not, the
findings demonstrate the validity of the experimental approach to the
search for the source of the viruses in nature. If it can be shown, for
instance, that a further two to three species of birds of widely divergent
orders or families are refractory to the virus, birds can be accorded low
priority in field studies, and efforts can be concentrated on animals
capable of circulating virus. Even if evidence is obtained that certain
animals become infected in the field, it would remain desirable to study
the nature of the infection they undergo in the laboratory to determine
whether they can harbor virus for prolonged periods and transmit it to
other animals. Additionally, materials derived from the experiments can be
used to develop, test, and perfect methods for detecting infectious virus,
viral antigen, nucleic acid, or antibodies in different species, and to
establish whether or not demonstrable immune response develops in
ostensibly refractory animals. There are no accepted methods for
demonstrating antibody in many wild vertebrates; anti-bat immunoglobulin is
being produced as part of the present project.

The two tadarids studied here, and many other bats, have a distribution
that overlaps the sites of known filovirus outbreaks in Africa, and the
migratory habits of some species would facilitate dissemination of
                             [end of page 324]

virus (9). The presence of virus in lung tissue implies that respiratory or
oral spread of infection could occur in the confined spaces where bats
roost, and isolation of virus from feces suggests the existence of
mechanisms for transmission of infection to other animals. However, much
remains to be learned about the nature of the infection in bats, including
the sites of virus replication, persistence, and the behavior of the virus
in further genera and species.

Robert Swanepoel,* Patricia A. Leman,* Felicity J. Burt,* Nicholas A.
Zachariades,* Lawrence E.O. Braack, Thomas G. Ksiazek, Pierre E. Rollin,
Sherif R. Zaki, and Clarence J. Peters
*National Institute for Virology, Sandringham, South Africa; National
Parks Board, Skukuza, South Africa; Centers for Disease Control and
Prevention, Atlanta, Georgia, USA

References

  1. Peters CJ, Sanchez A, Rollin PE, Ksiazek TG, Murphy FA. Filoviridae:
     Marburg and Ebola viruses. In: Field's Virology, Philadelphia:
     Lippincott-Raven. 1996;1161-76.
  2. Le Guenno B, Formenty P, Wyers M, Gounon P, Walker F, Boesch C.
     Isolation and partial characterization of a new strain of Ebola virus.
     Lancet 1995;345:1271-4.
  3. Muyembe T, Kipasa M. Ebola haemorrhagic fever in Kikwit, Zaire. Lancet
     1995;345:1448.
  4. World Health Organization. Outbreak of Ebola haemorrhagic fever in
     Gabon officially declared over. Wkly Epidemiol Rec 1996;71:125-6.
  5. Smith JS, Yager PA, Baer GM. A rapid tissue culture test for
     determining rabies neutralizing antibody. In: Laboratory techniques in
     rabies, Geneva: World Health Organization. 1973;354-7.
  6. Rosen L, Gubler D. The use of mosquitoes to detect and propagate
     dengue viruses. Am J Trop Med Hyg 1974;23:1153-60.
  7. Zaki SR, Greer PW, Goldsmith CS, Coffield LM, Rollin PE, Callain P, et
     al. Ebola virus hemorrhagic fever: pathologic, immunopathologic and
     ultrastructural studies. Lab Invest 1996;74.
  8. Ksiazek TG, Rollin PE, Jahrling PB, Johnson E, Dalgard DW, Peters CJ.
     Enzyme immunosorbent assay for Ebola virus antigens in tissues of
     infected primates. J Clin Microbiol 1992;30:947-50.
  9. Smithers RHN. Chiroptera. In: The mammals of the southern African
     region, Pretoria: University of Pretoria. 1983;51-137.

                             [end of page 325]

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


---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
Dispatches

Encephalitis Caused by a Lyssavirus in Fruit Bats in Australia

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

    This report describes the first pathologic and immunohistochemical
    recognition in Australia of a rabies-like disease in a native
    mammal, a fruit bat, the black flying fox (Pteropus alecto). A
    virus with close serologic and genetic relationships to members of
    the Lyssavirus genus of the family Rhabdoviridae was isolated in
    mice from the tissue homogenates of a sick juvenile animal.

The Lyssavirus genus of the family Rhabdoviridae consists of five
serotypes: classical rabies virus (serotype 1), Lagos bat virus (LBV)
(serotype 2), Mokola virus (serotype 3), Duvenhage virus (DUVV) (serotype
4), and European bat virus (EBV) (serotype 5). The viruses within the genus
share serologic relationships, but the serotypes and stable
species-associated variants within serotypes can be distinguished by the
reactivity profiles of monoclonal antibodies (Mab) directed against
nucleoprotein and glycoprotein antigens. Analysis of the nucleotide
sequence of the nucleoprotein gene has also shown genetic clusters along
the same lines as serologic analysis, except that serotype 5, EBV, has been
separated into two genotypes, EBV1 and EBV2 (1). Lyssaviruses have not been
isolated in Australia before, although rhabdoviruses in the genus
Ephemerovirus are present, and viruses with some serologic relationship to
the Lyssavirus genus, for example Adelaide River virus (2), have been
identified but not characterized. All members of the Lyssavirus genus can
cause rabies or rabies-like diseases in infected animals.

Rabies-like disease has been recorded in bats on all continents except
Australia. Classical rabies virus infections are common in insectivorous
and hematophagous bats and less common in frugivorous bats in the Americas,
while rabies-related viruses (EBV 1 and 2) are found in insectivorous bats
in Europe. Two other rabies-related viruses, LBV and DUVV, are found in
frugivorous bats and insectivorous bats, respectively, on the African
continent. Rabies has been described in a flying fox (Pteropus
poliocephalus) in India, although the virus causing the disease was not
characterized (3). An outbreak of rabies involving several dogs occurred in
the Australian island state of Tasmania in 1867 but was quickly eradicated
(4). Two cases of rabies in children were reported in Australia (in 1987
and 1990). Both cases were caused by classical rabies virus and were
contracted in endemic-disease countries (5). We report for the first time
apparent endemiclyssavirus-induced disease in Australia.

The four largest species of frugivorous bats in Australia are called flying
foxes and belong to the genus Pteropus (Order Chiroptera, Suborder
Megachiroptera, Family Pteropodidae). The Australian range of the flying
foxes extends from temperate eastern and coastal Australia into the eastern
tropics, around the tropical northern coastline, and down as far as the
subtropical west coast. The gray-headed flying fox (Pteropus poliocephalus)
range is the temperate and subtropical east coast, the black flying fox (P.
alecto) inhabits primarily the subtropical and tropical range, and the
little red flying fox (P. scapulatus) occupies the entire range except the
coolest southern areas. The fourth species, the spectacled flying fox (P.
conspicillatus) occupies a smaller range in tropical northeast Queensland.
Large flying fox "camps," with possibly tens of thousands of foxes, often
contain more than one species. Analysis of population genetic markers shows
a considerable movement of both P. alecto and P. poliocephalus across their
geographic ranges within Australia (6). The range of P. alecto extends to
the north of Australia into Papua New Guinea and the eastern islands of
Indonesia (7,8). Regular patterns of movement suggest that flying foxes
move between northern Australia and Papua New Guinea (L. Hall, pers.
comm.). It is, therefore, possible that the virus described in this paper
also extends across the
                             [end of page 327]

range of these mammals outside Australia.

The flying foxes (P. alecto) described in this paper were wild native
Australian animals collected near Ballina, in northern New South Wales,
Australia. The first case, in 1996, was in a 5-month-old female black
flying fox found under a fig tree, unable to fly. It was euthanized by
intravenous sodium pentobarbitone injection. Fresh blood, lung, kidney, and
spleen were submitted for equine morbillivirus (EMV) isolation; antibody to
EMV has been detected in P. alecto (9), and it is conjectured that this
species may be the reservoir for EMV. Paraffin-embedded formalin-fixed
samples, processed by standard techniques, showed a severe nonsuppurative
encephalitis. The second case, in 1995, was identified after a
retrospective examination of archived paraffin-embedded tissues. The
affected animal, a juvenile female of the same species, was reported to be
more aggressive than usual, and was euthanized and necropsied in a similar
manner to the first. Histologically, although encephalitis was very mild,
many eosinophilic, cytoplasmic inclusion bodies were present in various
parts of the brain. All tests for EMV were negative.

An indirect immunoperoxidase test for rabies was carried out on tissues
from paraffin blocks (10) by using an antirabies Mab (HAM) (Clone `HAM',
c/o Drs. R. Zanoni and E . Peterhans, Institut fr Veterinr-Virologie,
Lnggasstr. 122, CH-3012, Bern Switzerland) that gave good reactions
without background staining when used at 1:100. The 1996 bat had positive
results over wide areas of the brain, particularly in parts of the
hippocampus, the mesenchymal cells of the trigeminal nucleus, and larger
motor neurons of the medulla oblongata. The brain of the 1995 bat reacted
strongly over all areas. The reactions were either granular, or
characteristically, had ring formations in large neurons. In addition,
similar reactions were seen in neuronal cytoplasms in nerve plexuses of the
gastrointestinal tract from both bats. Electron microscopy examination of
ultrathin sections of hippocampus from the 1996 bat showed aggregates of
viral nucleocapsids within the cytoplasm of cell bodies. These inclusion
bodies were specifically labeled with anti-rabies HAM Mab and gold-labeled
rabbitanti-mouse.

The only fresh samples available were blood, lung, kidney, and spleen from
the 1996 bat. The blood was examined for neutralizing antibody to rabies
virus (CVS-11) by the rapid fluorescent focus inhibition method (11). No
neutralizing antibody was detected. Tissue homogenates (lung, kidney, and
spleen) were injected into mouse neuroblastoma cells, individually injected
intracerebrally into 3-week-old mice (five mice per sample), and, as a pool
of the three tissues, injected into day-old suckling mice (two litters, 14
mice). No virus was isolated from cell culture after two serial passages of
4 days. One weanling mouse injected with kidney homogenate showed hind leg
paraplegia 16 days postinoculation. All other mice remained normal until
termination (suckling mice at 21 days and weanling mice at 28 days
postinoculation). The affected mouse was euthanized, and acetone-fixed
smears of brain material were positive for a lyssavirus when tested by the
Centocor fluorescein-labeled Mab (Centocor Inc., 244 Great Valley Park,
Malvern, PA 19355, USA). Formalin-fixed brain material showed
nonsuppurative encephalitis and was positive to the indirect
immunoperoxidase test for rabies virus by the HAM Mab.

Polymerase chain reaction (PCR) tests were done on nucleic acids extracted
from the brain, lung, kidney, and spleen of the 1996 bat and on
paraffin-embedded formalin-fixed brain tissues from the 1995 and 1996 bats
by using oligomers designed for the amplification of lyssavirus N protein
(12) or for nested PCR amplification of the nucleocapsid protein (5).
Results from these primers were consistently negative, presumably because
of formalin-fixation and/or sequence heterogeneity. Therefore, another
nested PCR system was devised for the amplification of N protein. Nucleic
acids were extracted (5) and transcribed into cDNA by using a degenerate
oligomer NP1087 (5' GAGAAAGAG[A/C]T[G/T]CAAGA[A/C/T]TA. Primary PCR was
done with primers NP1087 and NP1279 (5' CAG
AGACATATCT[G/C]C[G/T][G/T]ATGTG) with amplification conditions of 94C for
1 min, 37C for 2 min, and 72C for 2 min for 35
                             [end of page 328]

cycles. Nested PCR was done by using primers NP1087 and NP1227 (5' CTTCA
[C/T]C[G/T]ACC[A/T][C/T][C/T]GTTCATCAT) as above except that the number of
cycles was reduced to 25. PCR products were excised and sequenced. Positive
PCR amplification signals were derived from the tissue culture virus and
paraffin-embedded formalin-fixed brain tissues by using primers NP1087 and
NP1227. Sequence analysis of these products showed that they were
identifical. Sequence comparisons were done by using the nucleocapsid
proteins of known lyssaviruses and the virus reported in this paper,
designated pteropid lyssavirus (PLV) (Table 1). Nucleotide sequence
comparisons showed that PLV had a 75% homology with LBV, 75% homology with
EBV-2, and 79% with Pasteur vaccine rabies virus; at the amino acid level,
the virus was 85% homologous with both EBV-2 and LBV (but 92% homologous
with the rabies virus), 89% with DUVV, and 93% homologous with EBV-1
viruses. Phylogenetic analysis of both the nucleotide and amino acid
sequences (not shown) showed that the virus is closely related to the EBV
as well as the classic street rabies strains (12).

 Table 1. Amino acid sequences of the virus designated Pteropid lyssavirus
            (PLV) and those of rabies and rabies-like viruses(a,b)

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

         PLV(c) PV   4FRA   POL   8FRA   AS   FIN  HOL   NGA    Genotype

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

 PLV      -

 PV       92    -                                                  1

 4FRA     93    99    -                                            1

 POL      93    94   94     -                                      5

 8FRA     93    94   94    100     -                               5

 AS       89    90   91     95     95    -                         4

 FIN      85    87   88     87     87   84    -                    6

 HOL      85    88   89     87     87   83    98    -              6

 NGA      82    82   83     85     85   85    76   77    -         2

 MOK      76    75   76     79     79   80    72   71    86        3

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

 (a) Described in Ref. 13.
 (b) Comparisons were made of cognate regions of the N protein (amino acids
 298 to 426 inclusive). Genebank accession numbers are given in brackets.
 PV, PV rabies virus (X03673); 4FRA, fox rabies virus (U22844); POL,
 European bat virus (U22844, 8615POL, EBV1); 8FRA, European bat virus
 (U22845, 8918FRA, EBV1); AS, Duvenhage virus (U22848); FIN, European bat
 virus (U22846, 9007FIN, EBV2); HOL, European bat virus (U22847, 9018HOL,
 EBV2); NGA, Lagos bat virus (U22842), Mokola virus (U22843).
 (c)PLV, the virus now reported, provisionally designated Pteropid
 lyssavirus; PV, PV rabies virus (X03673); 4FRA, fox rabies virus
 (U22844); POL, European bat virus (U22844, 8615POL, ELB1); 8FRA, European
 bat virus (U22845, 8918FRA, EBV1); AS, Duvenhage virus (U22848); Fin,
 European bat virus (U22846, 9007FIN, EBV2); HOL, European bat virus
 (U22847, 9018HOL, EBV2); NGA, Lagos bat virus (U22842), Mokola virus
 (U22843).


Brain material from the affected mouse was repassaged by intracerebral
inoculation into 3-week-old mice, in which neurologic signs developed 8 to
11 days postinoculation. Examination of brain homogenate from these mice by
negative-contrast electron microscopy showed classical bullet-shaped
rhabdoviruses. The isolate was also passaged to mouse neuroblastoma cells,
which were acetone-fixed and tested by indirect immunofluorescence using a
panel of Mabs against various rabies and rabies-like viruses. The CVS-11
strain of rabies was also tested for comparison. The results (Table 2)
confirm that the isolate is a lyssavirus but is different from previously
described isolates. Additional nucleocapsid Mab reaction patterns (results
not shown) indicated a unique profile that shared the greatest number of
positive reactions with serotype 1 rabies (CVS-11) compared with published
profile data on other viruses (15). Preliminary testing of the isolate in a
modified (incubated 3 days) rapid fluorescent focus inhibition
neutralization assay indicated that the virus was neutralized by antisera
to rabies virus (mouse anti-Evelyn-Rokitnicki-Abelseth [ERA] virus). The
titer of the immune mouse serum against CVS rabies virus was 1,194, and
against the bat virus, 1,640.

This is the first evidence of an endemic lyssavirus in Australia. The
isolate described has been provisionally called pteropid lyssavirus. The
natural history of this virus in bats in Australia needs to be
investigated. Further genetic and antigenic analyses are
                             [end of page 329]

  Table 2. Reactivity patterns of nucleocapsid monoclonal antibodies (Mab)
             with rabies (CVS-11) and Pteropid lyssavirus (PLV)
---------------------------------------------------------------------------
                                                          IFAT(a)
 MAb(b)                Specificity(a)             CVS-11          PLV
---------------------------------------------------------------------------
W502-2(c)           lyssavirus                       +             +
HAM(d)              lyssavirus                       +             +
C15-2(e)            rabies                           +             -
62-143-1(f)         rabies                           +             +
62-3-1(f)           rabies +, EBV +                  +             -
62-146-3(f)         rabies +, DUVV -                 +             +
W422(c)             Mokola +, LBV +                  -             -
---------------------------------------------------------------------------
(a) + indicates a positive reaction; - indicates a negative reaction
(b) Monoclonal antibody were specificities indicated by the following
sources:
(c) (14,15); (d) (10); (e) (J Smith, pers. comm.); (f) (15,16)

also needed to fully determine the relationship of the virus to existing
Lyssavirus serogroups and genogroups and to confirm its separate identity
from other as yet uncharacterized rhabdoviruses isolated in Australia. The
virus has been submitted to the Rabies Laboratory at the Centers for
Disease Control and Prevention, Atlanta, for further Mab profile analysis
and crossprotection studies with classical rabies vaccines. Findings will
result in a better understanding of the public health implications of this
newly emerged lyssavirus.

    Acknowledgments

    We thank Peter Young, Queensland Department of Primary Industries,
    for conducting EMV exclusion tests and forwarding the fresh
    tissues to the Australian Commonwealth Scientific and Industrial
    Research Organization laboratory for further testing for rabies;
    Megan Braun and Stuart Blacksell for assisting with many of the
    tests; Harvey Westbury and David Boyle for professional advice;
    Keith Murray for assisting in the preparation of this paper; Jean
    Smith, Rabies Laboratory, CDC, Atlanta, Georgia, for her advice
    and the reagents; and Roger Kelly, University of Queensland, for
    his recommendation for the `HAM' anti-rabies monoclonal antibody,
    which gave such good results on formalin-fixed paraffin-embedded
    tissues.

Graeme C. Fraser,* Peter T. Hooper, Ross A. Lunt, Allan R. Gould,
Laurence J. Gleeson, Alex D. Hyatt, Gail M. Russell, and Jaqueline A.
Kattenbelt
*NSW Department of Agriculture, Wollongbar, Australia; CSIRO Division of
Animal Health, Geelong, Australia

References

  1. Bourhy H, Kissi B, Lafon M, Sacramento D, Tordo N. Antigenic and
     molecular characterisation of bat rabies virus in Europe. J Clin
     Microbiol 1992;30:2419-26.
  2. Calisher CH, Karabatsos N, Zeller H, Digoutte J-P, Tesh RB, Shope RE,
     et al. Antigenic relationship among rhabdoviruses from vertebrates and
     hematophagous arthropods. Intervirology 1989;30:241-57.
  3. Brass DA. Rabies in bats: Natural history and public health
     implications. Ridgerfield, Connecticut: Livia Press, 1994.
  4. Geering WA, Forman AJ, Nunn MJ. Exotic diseases of animals: a field
     guide for veterinarians. Canberra: Australian Government Printing
     Service, 1995.
  5. McColl KA, Gould AR, Selleck PW, Hooper PT, Westbury HA, Smith JS.
     Polymerase chain reaction and other laboratory techniques in the
     diagnosis of long incubation rabies in Australia. Aust Vet J
     1993;70:84-9.
  6. Webb NJ, Tidemann CR. Mobility of Australian flying-foxes, Pteropus
     spp. (Megachiroptera): evidence from genetic variation. Proc R Soc
     Lond Biol Sci 1996;263:497-502.
  7. Ride WDL. A guide to the native mammals of Australia. Melbourne:
     Oxford University Press, 1970.
  8. Richards GC, Hall LS. Placental Mammals, Bats, Order: Chiroptera. In:
     Strahan R, editor. The Australian museum complete book of Australian
     mammals. Sydney: Angus and Robertson, 1983.
  9. Young PL, Halpin K, Selleck PW, Field H, Gravel JL, Kelly MA, et al.
     Serologic evidence for the presence in pteropus bats of a
     paramyxovirus related to equine morbillivirus. Emerging Infectious
     Diseases 1996;2:239-40.
 10. Feiden W, Kaiser E, Gerhard L, Dahme E, Gylstorff B, Wandeler A, et
     al. Immunohistochemical staining of rabies virus antigen with
     monoclonal and polyclonal antibodies in paraffin tissue sections.
     Zentralbl Veterinrmed 1988;35:247-55.
 11. Smith JS, Yager PA, Baer GM. A rapid tissue culture test for
     determining rabies neutralising antibody. In: Kaplan MM and Koprowski
     H, editors. Laboratory Techniques in Rabies, 3rd ed. Geneva,
     Switzerland: World Health Organization, 1973.
 12. Bouhry H, Kissi B, Tordo N. Molecular diversity of the Lyssavirus
     genus. Virology 1993;194:70-81.
 13. Kissi B, Tordo M, Bourhy H. Genetic polymorphism in the rabies virus
     nucleoprotein gene. Virology 1995;209:526-37.
 14. Wiktor TJ, Koprowski H. Antigenic variants of rabies virus. J Exp Med
     1980;152:99-111.15
 15. Smith JS. Rabies virus epitopic variation: use in ecologic studies.
     Adv Virus Res 1989;36:215-53.
 16. Fekadu M, Shaddock JH, Sanderlin DW, Smith JS. Efficacy of rabies
     vaccines against Duvenhage virus isolated from European house bats
     (Eptesicus serotinus), classic rabies and rabies-related viruses.
     Vaccine 1988;6:533-9.

                             [end of page 330]

    Addendum

    Since this report was submitted in September, 1996, the host and
    geographic range of the virus have been extended. The virus has
    been recognized by immunohistochemical techniques in five bats in
    three different virus isolations. Some of these bats were from
    another species, (the little red flying fox [P. scapulatus]), and
    from locations as far apart as 1,700 km along the Australian east
    coast.

                             [end of page 331]

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

---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
Dispatches

Creutzfeldt-Jakob Disease in the United States, 1979-1994: Using National
Mortality Data to Assess the Possible Occurrence of Variant Cases

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


        After a cluster of Creutzfeldt-Jakob disease (CJD) cases
        among unusually young patients was reported recently from
        the United Kingdom, we examined trends and the current
        incidence of CJD in the United States. We found that the
        age-adjusted CJD death rate in the United States is similar
        to published estimates of the crude incidence of CJD
        worldwide and has continued to be stable from 1979 through
        1994. The number of CJD deaths in persons <45 years of age
        remained stable during this period. We found no evidence of
        the variant form of CJD.

Transmissible spongiform encephalopathies (TSE) are rare forms of
progressive neurodegenerative disorders that affect both humans and animals
(1). They are distinguished by long incubation periods, characteristic
spongiform changes associated with neuronal loss, and a failure to induce
inflammatory response (2). The prototype TSE in humans, Creutzfeldt-Jakob
disease (CJD), occurs sporadically (approximately 90% of cases), through
iatrogenic transmission of the infective agent (<1% of cases), or as an
autosomal dominant inheritance (approximately 10% of cases) (3,4). CJD is a
rapidly fatal dementing illness that occurs worldwide, with an estimated
incidence of approximately one case per million persons. Unconventional
agents termed prion proteins (PrPs), which are encoded by genes on
chromosome 20, are considered the etiologic agent of CJD. The pathologic
properties of these proteins lie in their three-dimensional configuration
and their ability to recruit and influence normal PrPs to undergo similar
conformational changes. The transmissibility of CJD has been verified with
reports of iatrogenic transmission from a corneal transplant,
electroencephalographic depth electrodes, neurosurgical procedures,
cadaveric dura mater grafts, and pituitary hormone administration (5-11).

A cluster of CJD cases with a unique neuropathologic picture among
unusually young patients in the United Kingdom (12), which was reported
during a widespread epizootic of bovine spongiform encephalopathy (BSE),
has alerted many countries, including the United States, to update their
surveillance for CJD and look for similar cases (13). This increased
attention, and the fact that CJD is rapidly and invariably fatal (14),
prompted us to analyze the most current CJD deaths and update our analysis
of national trends of CJD mortality in the United States. Because of
concerns about the theoretical risk for transmission of CJD by blood
transfusion, we also examined the mortality records for evidence of
diseases associated with increased exposure to blood or blood products.

Multiple cause-of-death data for the United States from 1979 through 1994
were obtained from the National Center for Health Statistics, Centers for
Disease Control and Prevention (CDC) (15), with 1994 data reported as
provisional. Cause-of-death classifications were based on the Ninth
Revision of the International Classification of Diseases (ICD-9) (16-18).
CJD deaths were defined as those for which ICD-9 code 046.1 appeared as an
entity-axis code. The alternative CJD ICD-9 code 331.5 is not valid in the
United States (17,19,20). The case of a 3-year-old child with spongiform
degeneration of infancy (ICD-9 code 046.1) was included in the study. We
excluded the case of a 5-year-old child with Kawasaki disease (ICD-9 code
446.1) because CJD was not listed on the death certificate and the case of
a 17-year-old patient who had had diffuse T-cell proliferative disease.
                             [end of page 333]

Annual CJD death rates were calculated as the number of CJD deaths per
million persons, on the basis of U.S. resident population estimates, and
standardized by the direct method, using the 1990 census population (21).
Age-adjusted annual death rates were calculated by sex and race and for the
United States overall; age-, sex-, and race-adjusted annual death rates
were calculated by standard region (22). Risk ratios (RRs) with 95%
confidence intervals (CIs) were calculated by Poisson regression analysis
(23). Deaths were examined according to whether autopsy was indicated on
the death certificate, as available from 1979 through 1993. We also
examined other causes of death listed and searched the records for several
diseases associated with increased exposure to blood or blood products,
specifically hemophilia A (ICD-9 code 286.0), hemophilia B (ICD-9 code
286.1), thalassemia (ICD-9 code 282.4), and sickle cell disease (ICD-9 code
282.6).

From 1979 through 1994, CJD was recorded as a cause of 3,642 deaths in the
United States; 83.4% of these deaths had CJD recorded as the underlying
cause. The average annual age-adjusted death rate during the study period
was 0.95 deaths per million persons, from 0.78 in 1980 to 1.11 in 1987
(Figure 1). The mean age of death was 67 years (median age = 68 years).
Approximately 98% of the deaths were among persons
[greater than or equal to] 45 years of age. The age-specific death rates
were highest for persons in their 70s (Figure 2).

        [Figure 1] [Figures not available in ASCII version]

        Figure 1. Creutzfeldt-Jakob disease age-adjusted and
        age-specific death rates, United States, 1979 through 1994.

        [Figure 2] [Figures not available in ASCII version]

        Figure 2. Creutzfeldt-Jakob disease deaths and death 
        rates by age group, United States, 1979 through 1994.

Although 52.9% of the deaths were in female patients, the age-adjusted
death rate of male patients was slightly higher than that of female
patients (RR = 1.16, 95% CI = 1.09-1.24; Table 1). In contrast to the death
rate in age groups <60 years, the CJD death rate in age groups
[greater than or equal to] 60 years was higher in men than in women (Table
2). Most (95.2%) deaths were among whites. The age-adjusted death rate of
whites was higher than that of blacks (RR = 2.66, 95% CI = 2.22-3.18)
(Table 1). Other races accounted for only 1.4% of the CJD deaths, with an
age-adjusted rate intermediate to that of blacks and whites. The death rate
of blacks was lower than that of whites in each age group. Geographically,
the age-sex-race-adjusted rate of the South was lower than that of each of
the other regions. The previously noted differences by sex and race
remained consistent for each region.

Approximately 80% of the CJD deaths were among persons
[greater than or equal to] 60 years of age. The average annual death rate
during the study period of this age group was 4.58 per million persons
(3.71 per million in 1983 to 5.52 per million persons in 1987) (Figure 1).
Among persons 45 through 59 years of age, the
                             [end of page 334]

average annual death rate was 1.15 per million persons (0.87 to 1.45 per
million). Among persons <45 years of age, the annual death rate was
consistently at or below 0.05 per million persons; the number of deaths
among such young persons was zero in 1984 and eight in 1981 and 1993. Only
five persons who died of CJD during the study period were younger than 30
years of age, and in any single year, there was no more than one such
death. In the most recent 5-year period, 1990-1994, the only person with
CJD in this young age group had received pituitary-derived human growth
hormone.

Table 1: Creutzfeldt-Jakob disease deaths and death rates in the United
States, 1979-1994
---------------------------------------------------------------------------
           Characteristics                  Number of           Death
                                             Deaths              Rate
---------------------------------------------------------------------------
Sex(a)     Male                               1714              1.04
           Female                             1928              0.89
Race(a)
           White                              3466               1.01
           Black                               125              0.37
           Other                               51                0.67
Age group, years
           0-4                                  1               <0.01
           5-9                                  0                 0
           10-14                                0                 0
           15-19                                0                 0
           20-24                                1               <0.01
           25-29                                3               <0.01
           30-34                               13               0.04
           35-39                               23               0.08
           40-44                               39               0.16
           45-49                               91                0.45
           50-54                               183              0.99
           55-59                               378              2.14
           60-64                               598              3.55
           65-69                               765              5.03
           70-74                               706              5.75
           75-79                               508               5.60
           80-84                               225              3.94
           85+                                 108              2.42
Region(b)
           Northeast                           926               1.06
           Midwest                            1008              1.04
           South                              1068              0.83
           West                                640              0.89
---------------------------------------------------------------------------
U.S.(a)                                       3642               0.95
---------------------------------------------------------------------------
(a)Age-adjusted death rates per million persons.
(b)Age-sex-race-adjusted death rates per million persons

None of the persons who died of CJD had hemophilia A, hemophilia B,
thalassemia, or sickle cell disease mentioned on the death record. During
1979 through 1993, autopsies were performed on 907 persons who died of
CJD-related causes (29.7% of those whose autopsy status was known). The
proportion of autopsies ranged from 20.7% in 1993 to 37.8% in 1983.
Autopsies of persons <45 years of age were indicated in 51.5% of the known
deaths, while 30.2% and 29.0% were indicated in the 45- to 59-year-old and
[greater than or equal to] 60-year-old age groups, respectively.

During 1979 through 1994, the average annual age-adjusted death rate was
0.95 per million persons, which is consistent with published estimates of
the crude incidence worldwide of one case per million persons (3,11). The
addition of the 4 recent years of data to our previous report did not alter
our initial findings (24). The previously reported racial differences in
the U.S. CJD death rates were found again and should be further studied. In
addition, no CJD-related deaths were found among persons with hemophilia A,
hemophilia B, thalassemia, or sickle cell disease.

In 1994 and 1995, a cluster of 10 unusually young (median age at onset 28
years, range 16 to 39 years) CJD patients in the United Kingdom were
reported with atypical clinical features (12). These features included
behavioral change and dysesthesia when patients sought treatment, followed
within weeks or months by a cerebellar syndrome, dementia, and myoclonus in
the late stages, a duration of illness of at least 6 months, and
electroencephalogram changes that were not diagnostic of CJD. These
patients had a characteristic neuropathologic profile that consisted of
severe spongiform change, neuronal loss, and astrocytosis in the basal
ganglia and thalamus, with abundant kuru-type amyloid plaques surrounded by
vacuoles in the cerebrum and cerebellum and PrP accumulation in high
density shown by immunocytochemistry. These findings, coupled with the
unusually young age of the patients, led to the conclusion that the
clinicopathologic features constituted a new variant of CJD. A causal
association with the BSE epizootic in British cattle was also hypothesized
and recently supported by experimental evidence involving intracerebral
inoculation of cynomolgus
                             [end of page 335]

macaques with brain tissue obtained from cattle with BSE (25).

 Table 2: Creutzfeldt-Jakob disease deaths and age-specific death rates (per million
 persons) by sex and by race, United States, 1979-1994

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

   Age        U.S.               Sex                              Race
  group
 (years)
                            male         female       white        black        other


          deaths rate  deaths rate  deaths  rate deaths  rate  deaths rate  deaths  rate

 0-44        80  0.03      44  0.03    36   0.03    72   0.03       6 0.02      2   0.02

 45-59       652 1.15     303  1.11   349   1.19   605   1.23      31 0.54     16   0.98

 60-69      1363 4.25     678  4.63   685   3.93  1307   4.58      39 1.38      17  2.58

 70-79      1214 5.68     548  6.26   666   5.28  1167   6.05      37 2.15      10  2.87

 80+         333 3.27     141  4.33   192   2.77    315  3.38      12 1.62      6   4.79

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

To help assess the purported uniqueness and distribution of the newly
described CJD variant, CDC intensified CJD surveillance activities to seek
evidence for the presence of this variant in the United States (13). In
analyzing the U.S. multiple cause-of-death data, we did not find any
increase in the number of CJD deaths among persons <45 years of age; also,
there were no recent CJD deaths in persons <30 years of age, except for the
death of the recipient of pituitary-derived human growth hormone (in
contrast, five of the eight patients originally reported in the United
Kingdom died before the age of 30). To supplement these findings in the
United States, CDC also conducted active surveillance in its four Emerging
Infections Program sites and in the Metropolitan Atlanta Active
Surveillance Program in Georgia (total 1993 population 16.3 million)
(13,26); 92% to 100% of neuropathologists and neurologists were contacted,
and information was obtained on their patients who died of CJD from 1991
through 1995. Medical records and neuropathologic reports of decedents <55
years of age were also sought for review. Consistent with national
mortality data, nine (10%) of the case patients were <55 years of age, only
one case patient was <45 years of age, and no case patient was <30 years of
age. Review of clinical and neuropathologic records of the nine patients
<55 years of age did not show any with the variant form of CJD.

The validity of multiple cause-of-death data is potentially a problem
because of possible coding and reporting discrepancies, including
misdiagnoses (24). However, past reports have indicated that approximately
80% of histologically confirmed CJD patients were identified through death
certificate searches, using national death registries (27). Furthermore,
the consistency of data from the active surveillance sites with the
national multiple cause-of-death data supports the conclusion that annual
review of multiple cause-of-death data provides an efficient and
cost-effective method to monitor CJD incidence (24,27). Active follow-up of
reported CJD deaths in persons <55 years of age has been initiated in the
United States to improve the national surveillance of potential cases of
the newly described CJD variant. Access to more current national mortality
data makes this data source even more beneficial in monitoring CJD deaths
in the United States. Although currently available CJD surveillance data do
not provide evidence for the variant CJD in the United States, ongoing CJD
surveillance in this country and elsewhere, especially in the United
Kingdom, will be critical for ultimately determining the geographic
distribution of this illness and its possible relationship to BSE.

        Acknowledgments
        We thank Thomas J. Trk, MD, for his support of the CJD
        surveillance efforts; Rima F. Khabbaz, MD, and Harry M.
        Rosenberg, PhD, for their critical review; Ken D. Kochanek,
        MA, Mary Ann Freedman, Deborah D. Ingram, PhD, and Charles
        E. Royer for technical assistance; Marilyn Velez, MPH, for
        manuscript preparation assistance; and John O'Connor, MS,
        for editorial assistance.

Robert C. Holman, Ali S. Khan, Ermias D. Belay, and Lawrence B. Schonberger

Centers for Disease Control and Prevention, Atlanta, Georgia USA

References

  1. DeArmond SJ, Prusiner SB. Etiology and pathogenesis of prion diseases.
     Am J Pathol 1995;146:785-811.
                                [end of page 336]

  2. Budka H, Aguzzi A, Brown P, Brucher J, Bugiani O, Gullotta F, et al.
     Neuropathalogical diagnostic criteria for Creutzfeldt-Jakob disease
     and other human spongiform encephalopathies [Prion diseases]. Brain
     Pathol 1995;5:459-66.
  3. Masters CL, Harris JO, Gajdusek DC, Gibbs CJJ, Bernoulli C, Asher DM.
     Creutzfeldt-Jakob disease: patterns of worldwide occurrence and the
     significance of familial and sporadic clustering. Ann Neurol
     1979;5:177-88.
  4. Will RG. Epidemiology of Creutzfeldt-Jakob disease. Br Med Bull
     1993;49:960-70.
  5. Gibbs Jr CJ, Gajdusek DC, Asher DM, Alpers MP, Beck E, Daniel PM, et
     al. Creutzfeldt-Jakob disease (spongiform encephalopathy):
     transmission to the chimpanzee. Science 1968;161:388-9.
  6. Duffy P, Wolf J, Collins G, DeVoe AG, Streeten B, Cowen D. Possible
     person-to-person transmission of Creutzfeldt-Jakob disease. N Engl J
     Med 1974;290:692-3.
  7. Bernoulli C, Siegfried J, Baumgartner G, Regli F, Rabinowicz T,
     Gajdusek DC, et al. Danger of accidental person-to-person transmission
     of Creutzfeldt-Jakob disease by surgery. Lancet 1977;1:478-9.
  8. Will RG, Matthews WB. Evidence for case-to-case transmission of
     Creutzfeldt-Jakob disease. J Neurol Neurosurg Psychiatry
     1982;45:235-8.
  9. Centers for Disease Control. Rapidly progressive dementia in a patient
     who received a cadaveric dura mater graft. MMWR 1987;36:49-50,55.
 10. Fradkin JE, Schonberger LB, Mills JL, Gunn WJ, Piper JM, Wysowski DK,
     et al. Creutzfeldt-Jakob disease in pituitary growth hormone
     recipients in the United States. JAMA 1991;265:880-4.
 11. Brown P, Preece MA, Will RG. "Friendly fire" in medicine: hormones,
     homografts, and Creutzfeldt-Jakob disease. Lancet 1992;340:24-7.
 12. Will RG, Ironside JW, Zeidler M, Cousens SN, Estibeiro K, Alperovitch
     A, et al. A new variant of Creutzfeldt-Jakob disease in the UK. Lancet
     1996;347:921-5.
 13. Centers for Disease Control and Prevention. World Health Organization
     consultation on public health issues related to bovine spongiform
     encephalopathy and the emergence of a new variant of Creutzfeldt-Jakob
     disease. MMWR 1996;45:295-6,303.
 14. Brown P, Cathala F. Creutzfeldt-Jacob disease in France. In: Hadlow
     WJ, Prusiner SB, eds. Slow transmissible diseases of the nervous
     system. New York: Academic Press, 1979;vol 1: 213-27.
 15. U.S. Department of Health and Human Services. Vital statistics
     mortality data, multiple cause detail, 1979-1993. Public use data tape
     contents and documentation package. Hyattsville, MD: Centers for
     Disease Control and Prevention, National Center for Health Statistics,
     1996.
 16. U.S. Department of Health and Human Services. Vital statistics of the
     United States, 1988, vol II, mortality, Part A. Washington, D.C.:
     Public Health Service, Centers for Disease Control, National Center
     for Health Statistics; DHHS pub. no. (PHS) 91-1101; 1991.
 17. World Health Organization. Manual of the international statistical
     classification of diseases, injuries, and causes of death. Based on
     recommendations of the 9th revision conference, 1975, and adopted by
     the 29th World Health Assembly. Geneva: World Health Organization,
     1977.
 18. Israel RA, Rosenberg HM, Curtin LR. Analytical potential for multiple
     cause-of-death data. Am J Epidemiol 1986;124:161-79.
 19. National Center for Health Statistics. Instruction manual, part 9.
     ICD-9 underlying cause of death lists for tabulating mortality
     statistics. Hyattsville, MD: Public Health Service, 1979.
 20. National Center for Health Statistics. Instruction manual: part 11.
     Computer edits for mortality data. Hyattsville, MD: Public Health
     Service, 1989.
 21. Bureau of Census. Intercensal estimates of the population by age, sex,
     and race: 1970-1993. Washington, DC: Bureau of Census, 1996.
 22. Armitage P, Berry G. Statistical methods in medical research. Oxford:
     Blackwell Scientific Publications, 1987.
 23. Kleinbaum DG, Kupper LL, Muller KE, editors. Applied regression
     analysis and other multivariable methods. Boston: PWS-KENT Publishing
     Company, 1988.
 24. Holman RC, Khan AS, Kent J, Strine TW, Schonberger LB. Epidemiology of
     Creutzfeldt-Jakob disease in the United States, 1979-1990: Analysis of
     national mortality data. Neuroepidemiology 1995;14:174-81.
 25. Lasmezas CI, Deslys JP, Demaimay R, Adjou KT, Lamoury F, Dormont D.
     BSE transmission to macaques. Nature 1996;381:743-4.
 26. Centers for Disease Control and Prevention. Surveillance for
     Creutzfeldt-Jakob Disease--United States. MMWR 1996;45:665-8.
 27. Davanipour Z, Smoak C, Bohr T, Sobel E, Liwnicz B, Chang S. Death
     certificates: an efficient source for ascertainment of
     Creutzfeldt-Jakob disease cases. Neuroepidemiology 1995;14: 1-6.

                             [end of page 337]

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

---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
Dispatches

Rapid Increase of Resistance to Erythromycin and Clindamycin in
Streptococcus pyogenes in Italy, 1993-1995

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


        A survey of antibiotic resistance in Streptococcus pyogenes
        in Italy showed a sharp increase in erythromycin
        resistance. In 1993, the incidence of
        erythromycin-resistant strains was on average 5.1%, with
        marked variations by geographic area. Two years later, the
        incidence of these strains had registered a 1.5- to roughly
        20-fold increase, with a mean value of 25.9%, exceeding 40%
        in three centers out of 13 and 30% in another four. For all
        the strains studied, normal levels of susceptibility to
        penicillin were reported.

Over the past few years, the increased frequency of infections caused by
Streptococcus pyogenes (group A streptococcus [GAS]) and their sequelae has
been reported in several parts of the world (1,2). Even though these
reports may reflect an enhanced awareness of and interest in these possibly
life-threatening infections on the part of the medical community (3), in at
least some areas, an increase in severe infections over time has been
documented (4,5).

Meanwhile, the increased clinical use of erythromycin and its derivatives,
mostly in upper respiratory tract infections, has been related to an
increased resistance of GAS to this antibiotic. Even though fewer than 5%
of GAS isolates are reported as resistant to macrolide, lincosamide, and
streptogramin (MLS) antibiotics (2,6), local exceptions have been reported,
and widespread GAS resistance to erythromycin has so far been reported in
Australia (17.6%) (7), Finland (20%) (8), the United Kingdom (22.8%) (9),
Japan (60%) (10), and Taiwan (percentage not specified) (11).

Awareness of GAS resistance to erythromycin seems limited. Clinical
microbiology laboratories rarely determine erythromycin susceptibility on a
routine basis, and only recently have erythromycin breakpoints for
streptococci other than S. pneumoniae been added in the latest National
Committee for Clinical Laboratory Standards (NCCLS) document (12). Since
the late 1980s, appreciable incidences of macrolide resistance in cases of
pharyngo-tonsillitis and scarlet fever have also been reported from Italy
(13-15).

Temporal trends in GAS resistance to erythromycin and clindamycin were
systematically appraised on the basis of data collected over the last 3
years from 15 laboratories that participated in the Italian Surveillance
Group for Antimicrobial Resistance (ISGAR). All the strains were isolated
from throat swabs collected from symptomatic patients (mostly schoolage
outpatients) from 1993 through 1995. The number of isolates tested per year
and the percentage of resistant ones are represented in the Figure.

GAS were identified by beta-hemolysis production on sheep or horse blood
agar plates and by the presence of Lancefield group A antigen tested by
commercial latex agglutination techniques (Streptex, Murex Diagnostics
Ltd., Dartford, England; Phadebact, Boule Diagnostics AB, Huddinge,
Sweden). The susceptibility tests used either the disk diffusion method
(according to NCCLS performance standards [16,17]) or semiautomated
microdilution tests (ATB, bioMrieux S.A., Marcy-l'Etoile, France; Sceptor,
Becton Dickinson Diagnostic Instrument Systems, Sparks, Maryland), which
were carried out as recommended by the respective manufacturers. The disk
diffusion tests were read by manual measurement of the inhibition diameters
or by a semiautomated system equipped with a video camera and image
processing software that records the inhibition diameters (Bio-Videobact,
Biokit S.A., Barcelona, Spain). The data came from each automated reader
device through data acquisition interfaces (created for that purpose by the
respective manufacturers) and were subsequently translated through the
MyMic software package (18) from individual proprietary formats into a
common file format (Xbase) and transmitted to the reference center (Verona)
on floppy disks or by electronic mail.

Test results were originally attributed to the different interpretive
categories according to the NCCLS documents in force up until late 1995
(16,17,19). After the data arrived in the reference center, they were
reinterpreted on the basis of the new criteria for testing streptococcal
species in the latest NCCLS document (12). The zone diameter criteria for
resistant and susceptible isolates were <= 15 and >= 21, respectively, for
erythromycin, and <= 15 and >= 19, respectively, for clindamycin. The
equivalent minimum inhibitory concentration breakpoints (g/ml) for
resistant and susceptible isolates were >= 4 and <= 0.5, respectively, for
erythromycin, and >= 1 and <= 0.25, respectively, for clindamycin.

The survey showed a dramatic increase in the isolation of
erythromycin-resistant strains of GAS (Figure). Both the rapid increase in
the resistance rate in the areas involved and the subsequent involvement of
other geographic sites caused considerable immediate concern since
erythromycin had hitherto been effective against most isolates of this
species and had been the drug of choice for treating streptococcal
infections in patients allergic to penicillin.

             [Figure]
             Figure. Number of GAS isolates tested per year
             and percentage of resistance to erythromycin and
             clindamycin, Italy, 1993-1995. Dark gray bars
             represent erythromycin resistance and light gray
             bars, clindamycin resistance.

In 1993, the first year surveyed, the incidence of erythromycin-resistant
strains was on average 5.1%, with marked variations according to geographic
area, from 0% (all 19 strains from Pistoia) to 19.1% (Sassari, 18 strains
out of 94). Two years later, in 1995, the incidence of resistant strains
had registered a 1.5- to roughly 20-fold increase, with a mean value of
26.8%, from 13% (Palermo area, 3 strains out of 23) to 62% (Venice area, 31
strains out of 50). This incidence again showed geographic variations, but
exceeded 40% in three centers out of 13 and 30% in another four. The
Palermo area yielded the lowest rate of resistant strains, but its
incidence of intermediate strains was exceptionally high (39.1%, 9 strains
out of 23, versus a 5% to 10% rate in all other centers).

Resistance to clindamycin was more difficult to evaluate since this
antibiotic was tested in only a few centers and on limited numbers of
isolates. An increase in clindamycin resistance was recorded in five out of
the seven centers that made this kind of data available. The highest rate
of clindamycin resistance in 1995 was recorded in Verona (28.9%, 39 strains
out of 135), while the lowest was recorded in Sassari (2.2%, one single
strain out of 45). For all the strains studied, normal levels of
susceptibility to penicillin and ampicillin were reported.

Molecular typing of erythromycin-resistant isolates was performed on
strains isolated in the area of Verona in the first 2 months of 1995
(Table). Nine strains out of 14 were resistant to erythromycin, the
16-membered macrolide miokamycin, and the lincosamide clindamycin (the
so-called MLSB phenotype, which has reduced binding of MLS antibiotics to
their shared 50S rRNA target site [9,20]); the other five strains were
resistant to erythromycin but not to miokamycin or clindamycin (the
so-called M phenotype, in which resistance is attributed to an efflux
system [21]). All strains of the MLSB phenotype carried the ermAM gene,
which determines resistance to all MLSB antibiotics, as investigated by
polymerase chain reaction (PCR) performed on total DNA (22), by using the
following oligonucleotide primers (sequence 5' to 3') derived from the
published sequence of the gene (23):

     MLS1: AGAAACCGATACCGTTTACGA
     MLS2: GGTCAATCGAGAATATCGTCA

The PCR studies used the control strain Streptococcus sanguis V736, which
carries the ermAM gene in plasmid pVA736 (24). In contrast, all strains of
the M phenotype were negative to the PCR analysis.

Five different DNA restriction profiles (Table) were found by pulse-field
gel electrophoresis (PFGE) of genomic DNA fragments digested with SmaI
(Boehringer, Mannheim, Germany), with C predominant. Three profiles were
found among MLSB strains, and two among M strains; no profile was common to
both MLSB and M strains.

Serologic analysis with T-protein-specific antisera (Institute of Sera and
Vaccines, Prague, Czech Republic) showed seven T-types. Within each PFGE
type, similar but not identical T-types were identified. Again, no T-type
was common to both MLSB and M strains (Table).

         Table. Antibiotic susceptibilities,* presence of the ermAM
          gene, pulse-field gel electrophoresis type, and T-protein
           pattern of Streptococcus pyogenes strains isolated in
                                   Verona
         ----------------------------------------------------------
         Strain ERY  MIO CLI  ermAM PFGE type  T-protein pattern
         ----------------------------------------------------------
          VR1     R    S     S     -       A           8, 25
          VR2     R    S     S     -       A           8, 25
          VR3     R    S     S     -       A             25
          VR4     R    S     S     -       A           8, 25
          VR5     R    R     R     +       B           2, 28
          VR6     R    R     R     +       C         5, 12, 27
          VR7     R    S     S     -       D             4
          VR8     R    R     R     +       C           12, 27
          VR9     R    R     R     +       E         5, 12, 27
          VR10    R    R     R     +       B             2
          VR11    R    R     R     +       C           12, 27
          VR13    R    R     R     +       C         5, 12, 27
          VR14    R    R     R     +       C           12, 27
          VR15    R    R     R     +       C          5, 12, 27
         ----------------------------------------------------------
         *Resistant, susceptible
         Erythromycin
         Miokamycin
         Clindamycin

Molecular typing results showed a great heterogeneity of
erythromycin-resistant isolates in Verona: by combining the PFGE-type and
the serotype, at least eight different clones could be identified.

The polyclonality of Verona isolates and the largely different rates of
erythromycin- and clindamycin-resistance in most centers seem to confirm
that the M phenotype of resistance has become fairly frequent (21,25). The
diffusion of GAS strains resistant to erythromycin and susceptible to
miokamycin and clindamycin implies that testing of erythromycin alone is no
longer sufficient to assess the susceptibility of GAS to all MLS
antibiotics, contrary to the claims made by Leclerq and Courvalin (26).

Giuseppe Cornaglia,* Marco Ligozzi,* Annarita Mazzariol,* Myriam
Valentini,* Graziella Orefici, the Italian Surveillance Group for
Antimicrobial Resistance, and Roberta Fontana*
*Institute of Microbiology, University of Verona, Italy; National Health
Institute, Rome, Italy.
The Italian Surveillance Group for Antimicrobial Resistance: Antonio
Goglio, Cristiana Passerini Tosi, Ospedali Riuniti di Bergamo; Massimo
Gallina, Panaiota Troupioti, Azienda Ospedaliera 'Morelli, Bormio; Bruno
Maranini, Riccardo Cioni, Ospedale 'San Giuseppe Empoli; Pierluigi
Nicoletti, Laura Martelli, Azienda Ospedaliera 'Careggi', Florence, Sergio
Frugoni, Amelia Berlusconi, Pio Albergo Trivulzio, Milan; Roberto
Rescaldani, Simone Bramati, Ospedale 'San Gerardo', Monza; Alfredo
Chiarini, Anna Giammanco, Dipartimento di Igiene e Microbiologia,
Universit di Palermo; Roberto Rossetti, Spedali Riuniti, Pistoia;
Gianfranco Santini, Annapaola Callegaro, Ospedale Civile, Pordenone;
Giovanni Fadda, Teresa Spanu, Istituto di Microbiologia, Universit
Cattolica del Sacro Cuore, Rome; Alessandro Maida, Elena Muresu, Bianca
Maria Are, Istituto di Igiene e Medicina Preventiva, Universit di Sassari;
Piero Cappuccinelli, Silvana Sanna, Istituto di Microbiologia, Universit
di Sassari; Alessandro De Toffoli, Margherita Bergamasco, Ospedali Civili
Riuniti, Venice; Mariuccia Scagnelli, Patrizia Reatto, Ospedale 'San
Bortolo', Vicenza. Data manager: Giuseppe Cornaglia.

        Acknowledgments

        We thank Becton Dickinson Italia S.p.A., biokit Italia
        s.r.l., and bioMrieux Italia S.p.A. for interfacing their
        instruments with the MyMic software; Andrea Di Clemente and
        Maurizio Trombini for their excellent technical assistance;
        and Anthony Steele for his help with the English language
        version of this paper. We dedicate this article to 
        Giuseppe Satta, long-time promoter of the Italian 
        Surveillance Group for Antimicrobial Resistance, who 
        died 9 October 1994 at age 52.

References

  1. Stevens DL. Streptococcal toxic shock syndrome: spectrum of disease,
     pathogenesis, and new concepts in treatment. Emerging Infectious
     Diseases 1995;1:69-78.
  2. Kaplan EL. The resurgence of group A streptococcal infections and
     their sequelae. Eur J Clin Microbiol Infect Dis 1991;10:55-7.
  3. Musher DM, Hamill RJ, Wright CE, Clarridge JE, Ashton CM. Trends in
     bacteremic infection due to Streptococcus pyogenes (group A
     streptococcus), 1986-1995. Emerging Infectious Diseases 1996;2:54-6.
  4. Strmberg A, Romanus V, Burman LG. Outbreak of Group A streptococcal
     bacteremia in Sweden: an epidemiologic and clinical study. J Infect
     Dis 1991;164:595-8.
  5. Martin PR, Hiby EA. Streptococcal serogroup A epidemic in Norway
     19771988. Scand J Infect Dis 1990;22:421-9.
  6. Duval J. Evolution and epidemiology of MLS resistance. J Antimicrob
     Chemother 1985;16:137-49.
  7. Stingemore N, Francis GR, Toohey M, McGeechie DB. The emergence of
     erythromycin resistance in Streptococcus pyogenes in Fremantle,
     Western Australia. Med J Aust 1989;150:626-31.
  8. Seppl H, Nissinen A, Jrvinen H, Huovinen S, Henriksson T, Herva E,
     et al. Resistance to erythromycin in Group A streptococci. N Engl J
     Med 1992;326:292-7.
  9. Phillips G, Parratt D, Orange GV, Harper I, McEwan H, Young N.
     Erythromycin-resistant Streptococcus pyogenes. J Antimicrob Chemother
     1990;25:723-4.
 10. Maruyama SH, Yoshioka H, Fujiita K, Takimoto M, Satake Y. Sensitivity
     of group A streptococci to antibiotics: prevalence of resistance to
     erythromycin in Japan. Am J Dis Child 1979;133:1143-5.
 11. Hsueh P-R, Chen H-M, Huang A-Y, Wu J-J. Decreased activity of
     erythromycin against Streptococcus pyogenes in Taiwan. Antimicrob
     Agents Chemother 1995;39:2239-42.
 12. National Committee for Clinical Laboratory Standards. Performance
     standards for antimicrobial susceptibility testing: sixth
     informational supplement. National Committee for Clinical Laboratory
     Standards document M100-S6. National Committee for Clinical Laboratory
     Standards, Villanova, Pa. 1995.
 13. Borzani M, Varotto F, Garlaschi L, Conio F, Dell'Olio M, Careddu P.
     Clinical and microbiological evaluation of miocamycin activity against
     group A in pediatric patients: three years' incidence of erythromycin
     resistant group A streptococci. J Chemother 1989;1:35-8.
 14. Scopetti F, Pataracchia M, Pacifico L, Ranucci A, Morini C, Cherchi G,
     et al. A multicenter study on antimicrobial susceptibility and typing
     of group A streptococci from cases of pharyngo-tonsillitis and scarlet
     fever. Microecology and Therapy 1995;25:348-55.
 15. Cellesi C, Chigiotti S, Zanchi A, Mencarelli M, Corbisiero R,
     Rossolini GM. Susceptibility to macrolide and beta-lactam antibiotics
     of Streptococcus pyogenes strains isolated over a four-year period in
     central Italy. J Chemother 1996;8:188-92.
 16. National Committee for Clinical Laboratory Standards. Methods for
     dilution antimicrobial susceptibility tests for bacteria that grow
     aerobically, M7-A3. National Committee for Clinical Laboratory
     Standards, Villanova, Pa. 1993.
 17. National Committee for Clinical Laboratory Standards. Performance
     standards for antimicrobial disk susceptibility tests, M2-A5. National
     Committee for Clinical Laboratory Standards, Villanova, Pa. 1993.
 18. Cornaglia G, Satta G. MyMic: computerised listing of antimicrobials
     with optimal activity and pharmacokinetic properties for individual
     infections. Binary 1993;5:159-64.
 19. National Committee for Clinical Laboratory Standards. Performance
     standards for antimicrobial susceptibility testing: fifth
     informational supplement. National Committee for Clinical Laboratory
     Standards document M100-S5. National Committee for Clinical Laboratory
     Standards, Villanova, Pa. 1994.
 20. Weisblum B. Erythromycin resistance by ribosome modification.
     Antimicrob Agents Chemother 1995;39:577-85.
 21. Sutcliffe J, Tait-Kamradt A, Wondrack L. Streptococcus pneumoniae and
     Streptococcus pyogenes resistant to macrolides but sensitive to
     clindamycin: a common resistance pattern mediated by an efflux system.
     Antimicrob Agents Chemother 1996;40:1817-24.
 22. Arthur M, Molinas C, Mabilat C, Courvalin P. Detection of erythromycin
     resistance by the polymerase chain reaction using primers in conserved
     regions of erm rRNA methylase genes. Antimicrob Agents Chemother
     1990;34:2024-6.
 23. Arthur M, Brisson-Nol A, Courvalin P. Origin and evolution of genes
     specifying resistance to macrolide, lincosamide and streptogramin
     antibiotics: data and hypothesis. J Antimicrob Chemother
     1987;20:783-802.
 24. Macrina FL, Jones KR, Wood PH. Chimeric plasmids and their use as
     molecular cloning vehicles in Streptococcus sanguis (Challis). J
     Bacteriol 1980;143:1425-35.
 25. Seppl H, Nissinen A, Yu Q, Huovinen P. Three different phenotypes of
     erythromycin-resistant Streptococcus pyogenes in Finland. J Antimicrob
     Chemother 1993;32:885-91.
 26. Leclerq R, Courvalin P. Bacterial resistance to macrolide, lincosamide
     and streptogramin antibiotics by target modification. Antimicrob
     Agents Chemother 1991;35:1267-72.

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

---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
Dispatches

Assessing the Costs and Benefits of an Oral Vaccine for Raccoon Rabies: A
Possible Model

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

    Any cost-benefit analysis of the use of an oral vaccine to control
    raccoon rabies should include calculating both costs and benefits
    in terms of $/unit area. Further, cost savings must be adjusted to
    match the stages of an epizootic: pre-epizootic, epizootic, and
    post-epizootic. A generic model, which can be adapted to different
    sites, illustrates the use of threshold analysis to link
    distribution costs, cost savings, bait density, and vaccine price.
    Initial results indicate the need to lower the cost of the
    vaccine, continue research to determine optimal bait densities,
    and examine distribution plans that do not require continued
    protection of areas in which raccoon rabies was eliminated through
    previous vaccination programs.

Over the past 30 years, reported cases of animal rabies in the United
States have increased, from fewer than 5,000 per year in the early 1960s to
almost 10,000 per year in the mid-1990s (1,2). Most of the increase is
attributable to the spread of raccoon rabies from Florida to the
northeastern states (1). The impact of this epizootic has been
considerable. For example, hospitals in Connecticut reported a 2,000%
increase in the annual number of postexposure rabies prophylaxis treatments
after raccoon rabies was first detected in the state in 1991 (3).
Postexposure prophylaxis costs $2,000 to $3,000, or more, per person (3,4).
In addition, a raccoon rabies epizootic increases other costs, including
those associated with animal control and laboratory diagnosis (5; Tysmans,
J., Costs of rabies in Cumberland County, NC: 1993 and 7/1/94-6/30/95 [MPH
thesis], Chapel Hill (NC): Dept. of Health Policy and Admin., Univ. of
North Carolina, Chapel Hill, 1996).

Oral vaccines offer a potential solution to rabies in wildlife populations
(6,7). The spread of rabies in foxes in an area of Switzerland was halted
when an oral vaccine was used to vaccinate 60% of the fox population (8).
An oral vaccine to control raccoon rabies (9) is undergoing evaluation in
New York and Massachusetts (10,11). Also, large-scale programs are using an
oral vaccine against rabies in coyote and gray fox populations in Texas
(12).

Uhaa et al. (5) examined the economics of using an oral vaccine in
Hunterdon and Warren Counties, New Jersey. However, their study was limited
to a 5-year period and only to costs borne by the two counties. To aid the
collection of data needed to conduct a cost-benefit analysis of the use of
an oral vaccine in large areas (e.g., an entire state) and over prolonged
periods (e.g., 30 years), this article outlines some basic premises
required for such an analysis and a generic cost-benefit model, which can
be modified to fit specific geographic areas. Since many of the necessary
data for the model are still being collected, the data used in the model
are illustrative rather than definitive.

Any evaluation of the costs and benefits of using an oral vaccine to
protect wild raccoon populations must be based on three premises: 1) Oral
vaccine is distributed on a per unit area basis, calculated by using
estimates of raccoon population density (i.e., a predetermined number of
baits per raccoon times the number of raccoons per unit area). Costs of an
oral vaccine program, therefore, are expressed in $/unit area. 2) To
compare costs and benefits, all benefits that might accrue because of oral
vaccine use must also be converted into $/unit area. 3) The cost savings
(benefits) must be adjusted to match three broad, time-based categories
describing different stages of an epizootic: pre-epizootic, epizootic, and
post-epizootic (rabies still present in the population). The greatest cost
savings is likely to occur during the epizootic since this stage is
typically associated with the highest cost of raccoon rabies control (5;
Tysmans, J., MPH thesis, 1996). In New Hampshire, for example, the annual
number of requests for Animal Damage Control services involving raccoons
                             [end of page 343]

went from 250 per year in pre-epizootic years (1988-1991), to 667 during an
epizootic year (1993), and then declined to 165 in the post-epizootic years
(13).

The Model
A cost-benefit model with a societal perspective was constructed (on Excel
5.0, Microsoft, Inc.) by using a discount rate of 3% (14) over 30 years to
provide a Net Present Value (NPV).1 The costs of a raccoon rabies
vaccination program include purchasing and distributing the oral vaccine in
bait form. The benefits of such a program are the direct and indirect cost
savings obtained by successfully halting or preventing a raccoon rabies
epizootic and subsequent enzooticity. Since the vaccine technology is new,
and no data exist on how the technology might change over the 30 years, it
is assumed that the real costs and benefits are constant over the 30 years.
Parameters and values used in the model are presented in Table 1.

       Table 1: Illustrative data used to examine the costs and
       benefits of using an oral vaccine to control raccoon rabies
       --------------------------------------------------------------

               Item            Baseline case      Values used for
                                               sensitivity analyses
       --------------------------------------------------------------
       Discount rate         3%                0%, 5%
       Timeline              30 years          -
       Oral vaccine density  250 units/sq. mi  100-300 units/sq. mi
       Raccoon density       50/sq. mi         threshold analysis
       Baits required/raccoon5                 threshold analysis
       Cost of oral vaccine
       bait                  $1.50/unit        threshold analysis
       Cost of distribution  $100/sq. mi       $260(a)
       Benefits: Cost savings
         During epizootic
       years(b)              $1.52/person/yr   $2.61/person/year(b)
         During
       post-epizootic years(b)$0.30/person/yr   -
         Density human
       population            103/sq. mi        -
       Distribution
       costs:cost savings
         Ratio of max. $/sq.
       mi                    1:1.57            1:1.03
       Sensitivity analysis
         Cost of pet
       vaccination           -                 $16/pet
         Extra vaccinations:
       epizootic             -                 11/sq. mi
         Extra vaccinations:
       post-epizootic        -                 2.75/sq. mi
       --------------------------------------------------------------
       (a) Source: New Jersey data (1).
       (b) On the basis of the New Jersey data (1), four categories
       of costs contribute to the cost-savings: animal control,
       laboratory diagnoses, educational activities, and human pre-
       and post-exposure treatments.

        1 A cost-benefit analysis uses the following formula (15):
        Threshold costs were calculated by altering vaccine cost so
        that benefits minus cost, when discounted and summed over
        time, had an NPV of $0.

                                      t=n  (Benefits-Costs) to the tth power
          Net Present Value (NPV) =  Sigma   
                                      t=0        (1+r) to the tth power

        where:
             t = year, from t = 0, . . . , n
             r = discount rate.

Two Distribution Scenarios
Since what constitutes an "ideal" plan for large-scale oral vaccine bait
distribution is not known, two scenarios were constructed. The area covered
and the rate of barrier movement were chosen merely for illustration. Once
actual proposals are made, the area covered and the time line may be
modified to suit specific geographic areas. The first scenario assumes that
bait is distributed in ever-expanding circles, starting with an area
covered by a radius of 20 mi (area: 1,257 sq. mi). This area would be
baited for 2 years. In the third year, the radius of the circle would be
increased to 25 mi. The area between the 20-mi and 25-mi radius would then
be baited for 2 years, at which time the radius defining the outer boundary
would be increased by another 5 mi. This process would continue for 20
years, at the end of which the radius of the circle would be 65 mi,
encompassing 13,273 sq. mi, which is approximately equal to one-third the
area of New York State. At the end of the 20 years, a 10-year period would
follow during which a 5-mi wide barrier zone would be baited every year.
The yearly and cumulative total areas baited are shown in Figure 1. The
barrier zone is necessary because the described plan cannot guarantee
elimination, and the area covered by a vaccine program is vulnerable to the
reintroduction of rabid animals. The second scenario considers the entire
13,273 sq. mi baited during the first 2 years, followed by 28 years of
baiting a 5-mi-wide barrier zone.

Costs: Bait
In an economic analysis, opportunity costs should be used (15). However,
                             [end of page 344]

the vaccine is still experimental, and data concerning discount prices
could not be obtained. Therefore, the cost of oral vaccine in bait form was
calculated at $1.50 per unit. This is the cost reported for trials of the
oral vaccine in New York State (C. Hanlon, unpublished report, New York
State Department of Health, 1995). In lieu of accurate opportunity costs, a
sensitivity analysis evaluates the threshold price (i.e., NPV = $0) of the
vaccine. This threshold value can then be compared with actual opportunity
costs when more data become available.
      [Figure 1]
      Figure 1: Annual and cumulative total areas baited with oral
      vaccine using an expanding circles scenario
      Notes: Initial circle has a radius of 20 mi, expanded out by 5
      mi every 2 years. After 20 years, radius is 65 mi. Thereafter,
      a 5-mi wide boundary is maintained around the circle for the
      next 10 years.

Costs: Bait Density
The bait density required to successfully halt or stop raccoon rabies has
not been definitively determined (10,11,16,17). Raccoon density can vary
enormously with local ecology and weather (16,18-20). On the basis of
densities currently under trial, for each of the two scenarios it is
assumed that bait is distributed at 250 units per sq. mi, with an assumed
raccoon density of approximately 50 per sq. mi (i.e., five baits per
raccoon). Bait density is also the subject of sensitivity analysis.

Costs: Distribution
Distribution costs for oral rabies vaccine have yet to be comprehensively
documented. In current trials in New York and Massachusetts, bait was
dropped by people walking and from aircraft, helicopters, and cars,
depending on the terrain and available resources. The use of some vehicles
has been donated, as have large amounts of personnel time; therefore, their
cost is difficult to determine (an economic analysis must contain such
costs). To illustrate the generic model, total distribution costs were
assumed to be $100 per sq. mi. This cost is also the subject of a
sensitivity analysis.

Benefits: Cost-savings
In the model described here, the benefits from using the oral vaccine are
assumed to be the cost savings derived directly from preventing a
raccoon-related rabies epizootic and post-epizootic. These cost savings
are, therefore, the difference between the pre-epizootic costs of
controlling rabies and the costs incurred during the epizootic and
post-epizootic periods. It was assumed that a rabies epizootic lasts 2
years (1) and that the years following were defined as post-epizootic. The
direct costs associated with the control of animal rabies have been listed
(5) and can be broken down into four categories. These categories, with the
estimated proportional contribution to the entire cost savings, are animal
control costs (41%); laboratory diagnosis costs (13%); education and
administration costs (9%); and human costs, such as pre- and post-exposure
prophylaxis treatments (37%). Just as opportunity costs should be used for
input costs, so potential cost savings should be valued at costs and not
charges (15). For example, the average operating cost-to-charge ratio for
urban hospitals in New York is 0.635:1 (15).

To illustrate the generic model, data from New York (Harris-Valente et al.,
unpublished
                             [end of page 345]

report, 1995) were used to provide a preliminary estimate of cost savings
($1.52 per person), which will occur during the epizootic phase. The same
data source yielded a figure of $0.30 per person during the post-epizootic
phase. These savings per person are converted to savings per unit area as
follows: On the basis of population estimates (21) from 53 counties in New
York State, the median population density was estimated at 103 persons per
sq. mi (25th percentile = 67; 75th = 204). Thus, for the areas baited, the
savings were calculated at $156.56 per sq. mi for the first 2 epizootic
years ($1.52 per person x 103 persons per sq. mi), and $30.90 per sq. mi
for the post-epizootic years ($0.30 per person x 103 persons per sq. mi).
Cost-savings data from New Jersey (5) are used in the sensitivity analysis.

Distribution Costs: Cost-savings Ratio
The relative importance of distribution costs (excluding costs of vaccine)
with respect to the amount of cost savings can be examined by constructing
a distribution costs:cost savings ratio on a per-unit-area basis. Thus, the
baseline case of $100 per sq. mi distribution costs and maximum cost
savings during epizootic years of $156.56 per sq. mi ($1.52 per person x
103 persons per sq. mi) gives a ratio of 1:1.57. This ratio can be altered
for sensitivity analyses.

Sensitivity Analyses: Animal Vaccinations
The proportion of household pets and domesticated animals vaccinated before
an epizootic can be quite low. For example, in Cumberland County, North
Carolina, it was estimated that only 20% of dogs and cats were vaccinated
against rabies before the epizootic (Tysmans, J., MPH thesis, 1996). During
an epizootic, public health officials often encourage or enforce pet
vaccinations, and vaccination rates often increase (5; Tysmans, J., MPH
thesis, 1996). Successful elimination of raccoon rabies from an area could
be considered a good reason for allowing vaccination rates to remain at
lower levels, thus avoiding the costs associated with increased
vaccinations. However debatable this proposition is, the economic impact of
considering reduced pet vaccinations as a benefit is demonstrated by
rerunning the two distribution scenarios and including the potential cost
savings associated with reduced pet vaccination levels.

An example of the savings estimate follows: In 1991, there were 52.5
million dogs and 57 million cats in the United States (22), an average of
0.41 pets per person. During pre-epizootic periods, 0.14 pets per person
(32% of average pets per person) are vaccinated each year against rabies
(5). In the epizootic year this rate increased to 0.24 pets per person (55%
of average pets per person) (5). Thus, during an epizootic period, there is
an increase of approximately 11 extra pet vaccinations per sq. mi (0.24 -
0.14 pets per person x 103 persons per sq. mi). Pet vaccinations are costed
at $16 per pet (Tysmans, J., MPH thesis, 1996). When baseline figures are
used for distribution costs and cost savings (Table 1), a total
distribution cost:cost savings ratio of 1:3.33 is calculated. No data are
available concerning post-epizootic pet vaccination rates, and it is
arbitrarily assumed that there are 2.75 extra pet vaccinations in
post-epizootic stages (25% of the epizootic increase).

Additional Sensitivity Analyses
The impact of two alternate discount rates (0% and 5%) was evaluated, and
the impact of higher distribution costs was assessed. In New Jersey (5),
the costs of distribution were calculated at approximately $260 per sq. mi,
while cost savings during the epizootic period were calculated at $2.60 per
person (without reduced pet vaccinations considered as a benefit). This
gives a distribution costs:cost savings ratio of 1:1.03 (compared with
1:1.57 in the baseline scenario) increasing the costs of distribution
relative to cost-savings. The impact of removing the cost of having to
continuously bait a barrier zone around areas covered by previous
vaccination programs was then considered. In the first distribution
scenario, costs associated with baiting a barrier for years 21 through 30
were removed from the model. In the second distribution scenario, it was
assumed that the barrier need only be maintained for 2 years after the
first 2 years of baiting (i.e., no baiting for years 5 through 30). The
final
                             [end of page 346]

sensitivity analysis used the first distribution scenario to examine the
threshold price of oral vaccine for levels of bait density ranging from 100
sq. mi to 300 sq. mi, and using three different distribution costs:cost
savings ratios. Threshold price was determined by altering the price of the
vaccine until the NPV for a given bait density equaled $0 (see footnote)
The first two price ratios were 1:1.57 (baseline, Table 1) and 1:1.96. The
latter represents an arbitrary increase of +25% in cost-savings relative to
distribution costs. The last price ratio used for the threshold analyses
was constructed by assuming that reduced rates of pet vaccination resulted
in additional cost-savings of $176 per sq. mi ($16 per pet x 11 extra
vaccinations per sq. mi), giving a ratio of 1:3.33.

Results: Baseline Case
The first distribution scenario (expanding circles) provides smaller losses
than the second distribution scenario (baiting the entire area in 2 years,
followed by 28 years of baiting a 5-mi boundary) (Table 2). Considering the
avoidance of increased pet rabies vaccinations as a benefit improves the
NPVs of both scenarios and changes the NPV of the expanding circles
distribution scenario from negative to positive. For the baseline case,
using different discount rates changes the absolute values but not the
signs.

      Table 2: Net present values (NPV) of different distribution
      scenarios for using an oral vaccine to control raccoon rabies
      ---------------------------------------------------------------
                                 NPV without pet     NPV with pet
                                   vaccination      vaccinations(a)
      Baiting scenario             ($ millions)      ($ millions)
                                  Discount rate      Discount rate
                                 0%    3%     5%    0%    3%    5%
      ---------------------------------------------------------------
        Expanding circles(b)    -10.2 -6.2   -4.7  +5.5  +3.1  +2.2
        Entire area at once(b)  -21.3 -15.7  -13.3 -0.5  -1.1  -1.3

      Sensitivity analyses
      Reduced
      distribution:savings(c)
        Expanding circles       -14.3 -8.5   -6.3  +1.4  +0.9  +0.6
        Entire area done at
      once                      -30.8 -21.9  -18.2 -12.5 -8.9  -7.3
      No baiting of boundary
        Expanding circles       -0.7  -1.8   -2.1  +14.1 +7.9  +4.5
        Entire area at once     +2.9  -0.9   -2.3  +23.7 +13.7 +9.7
      ---------------------------------------------------------------
      (a) Cost savings (benefits) of 11 pets/sq. mi during an
      epizootic period, and 2.75 pets/sq. mi during the
      post-epizootic period, both at $16/pet.
      (b) Expanding circles assumes start with 20-mi radius, expanding
      by 5 mi every 2 years for 20 years, and then baiting a 5-mi
      wide boundary for next 10 years. Entire area assumes baiting
      65-mi radius for 2 years, followed by 28 years of baiting 5-mi
      wide radius.
      (c) Ratio of distribution costs:cost savings is 1:1.03,based on
      New Jersey data (1). In baseline scenario, ratio is 1:1.57.

      [Figure 2] [Figures not available in ASCII version]
      Figure 2: Threshold price for the oral vaccine at different
      levels of bait density and three distribution
      costs:cost-savings ratios for the expanding circles scenario

      Notes: Threshold price occurs at the point that the net present
      value of a vaccination program = $0.
      The distribution costs:cost-savings ratios are calculated on a
      per unit area basis, using the maximum cost-savings figures
      (i.e., savings resulting from avoiding an epizootic).

Results: Sensitivity Analysis
The smaller distribution costs:cost saving ratio of 1:1.03 increased the
losses (larger negative NPVs) and reduced the sole positive NPV to +$0.9
million (3% discount rate). Eliminating the need for baiting a protective
barrier saves $4.8 million in the expanding circles scenario ($7.9-$3.1
million, 3% discount rate) and $14.8 million in the entire area scenario
($13.7 million + $1.1 million, 3% discount rate) (Table 2). Figure 2 shows
the threshold prices for the oral
                             [end of page 347]

vaccine in the expanding circles scenario, given different levels of bait
density used and three different distribution costs:cost-savings ratios. At
the baseline distribution costs:cost-savings ratio of 1:1.57, and at bait
density of 250 baits per sq. mi, the threshold price for the oral vaccine
is $0.63 per unit. When the distribution costs:cost-savings ratio is
increased to 1:1.96, the threshold price for 250 baits per sq. mi is $0.91
per unit (+44% from baseline threshold). When reduced pet vaccinations are
considered a benefit and the distribution costs:cost-savings ratio
increases to 1:3.33, the threshold prices are always greater than the $1.50
cost of the vaccine (Figure 2).

Many of the data used in the generic model are assumed for illustrative
purposes. The results presented here, therefore, should not be used to
decide if a large-scale oral vaccine program is economically worthwhile.
These results identify at least two priorities: the need to continue
research that will determine the optimal level of bait density and the
importance of better defining the distribution costs:cost-savings ratio.
Both of these priorities are the focus of research efforts in New York and
Massachusetts.

The results of the generic model (Table 2) clearly identify the importance
of pet vaccinations when considering the costs and benefits of a raccoon
vaccination program. It can be argued that avoidance of increased pet
vaccinations is a benefit only if the laws requiring small animal
vaccinations were passed solely as a result of the raccoon epizootic. It
could be counterargued that, because past rabies control programs were so
successful, the current laws are unnecessary and an economic burden to
society. However, this would suggest that public health officials would
accept a repeal of mandatory vaccination requirements except under
extenuating circumstances (e.g., a rabies epizootic). Further, there are
other sources of rabies exposure (e.g., bats), and the risk for pet
exposure from these will most likely not be altered by a raccoon rabies
vaccination program.

In evaluating an actual proposed rabies vaccine program, the use of
expanding circles, although more economical than baiting entire areas at
once (Table 2), may be unrealistic because of natural terrain or political
boundaries. The sensitivity analyses demonstrated the costs of having to
maintain a barrier (Table 2). The only way to avoid having to maintain a
barrier over a long time may be to conduct a large-scale program, involving
many contiguous states. However, such a program would pose a financial
burden on governments and, at its conclusion, would still not stop rabies
from being reintroduced in a manner similar to that which caused the
current epizootic.

In addition to the issues related to distribution, two important implicit
assumptions in the model affect costs: 1) the vaccine is 100% effective in
stopping rabies in baited areas in 2 years; and, 2) rabies is not
reintroduced. Removal of these assumptions would effectively increase the
cost of the program by requiring increased bait densities, longer baiting
periods, or both. Any of these situations would move the optimal bait
density in Figure 2 (X-axis) to the right, further reducing the threshold
value of the vaccine. Obviously, increased costs would merely increase the
negative value of the NPVs presented in Table 2. The threshold analysis
used here (Figure 2) provides a basis, even when some data are uncertain,
for discussing discount pricing of the vaccine with the manufacturers when
bulk purchases for large-scale programs are being planned.

Martin I. Meltzer
Centers for Disease Control and Prevention, Atlanta, Georgia, USA

References

  1. Rupprecht CE, Smith JS. Raccoon rabiesthe re-emergence of an
     epizootic in a densely populated area. Seminars in Virology
     1994;5:155-64.
  2. Krebs JW, Strine TW, Smith JS, Rupprecht CE, Childs JE. Rabies
     surveillance in the United States during 1993. J Am Vet Med Assoc
     1994;205:1695-709.
  3. Centers for Disease Control and Prevention. Rabies postexposure
     prophylaxisConnecticut, 1990-1994. MMWR Morb Mortal Wkly Rep
     1996;45:232-4.
  4. Centers for Disease Control and Prevention. Animal rabiesSouth
     Dakota, 1995. MMWR Morb Mortal Wkly Rep 1996;45:164-6.
  5. Uhaa IJ, Dato VM, Sorhage FE, Beckly JW, Roscoe DE, Gorsky RD, et al.
     Benefits and costs of using an orally absorbed vaccine to control
     rabies in raccoons. J Am Vet Med Assoc 1992;201:1873-82.
                                [end of page 348]

  6. Winkler WG, Bogel K. Control of rabies in wildlife. Scientific
     American June 1992:86-92.
  7. Rupprecht CE, Smith JS, Fekadu M, Childs JE. The ascension of wildlife
     rabies: A cause for public health concern or intervention? Emerging
     Infectious Diseases 1995;1:107-14.
  8. Steck F, Wandler A, Bischel P, Capt S, Hafliger U, Schneider L. Oral
     immunization of foxes against rabies. Laboratory and field studies.
     Comp Immunol Microbiol Infect Dis 1982;5:165-79.
  9. Rupprecht CE, Wiktor TJ, Johnson DH, Hamir AN, Dietzschold B, Wunner
     Wh, et al. Oral immunization and protection of raccoons (Procyon
     lotor) with a vaccinia-rabies glycoprotein recombinant virus vaccine.
     Proc Natl Acad Sci U S A 1986;83:7947-50.
 10. Robbins AH, Niezgoda M, Levine S, Windmiller BS, McGuill MW, Rowell
     SL, et al. Oral rabies vaccination of raccoons (Procyon lotor) on the
     Cape Cod isthmus, Massachusetts. Presented at the 5th Annual
     International Meeting of Rabies in the Americas, Niagara Falls,
     Ontario, Canada, 1994; Conference Abstracts, p.29.
 11. Hanlon CA, Trimarchi CE, Rupprecht CE, Debbie JG. Oral wildlife rabies
     vaccination in New York state: First field evaluation in an enzootic
     raccoon rabies area. Presented at the 6th Annual International Meeting
     on Research Advances and Rabies Control in the Americas, Merida,
     Mexico, 1995; Program and Abstracts, p.29.
 12. Manning, A. Texas officials take battle against rabies to the air. USA
     Today January 11, 1996:27.
 13. Slate D, Calvert RT, Chipman RB, Barden ME. Raccoon pest management
     patterns before, during and after a rabies epizootic in northern New
     England: What do they mean? Presented at the 6th Annual International
     Meeting on Research Advances and Rabies Control in the Americas,
     Merida, Mexico; 1995; Program and Abstracts, p.20.
 14. Gold MR, Siegel JE, Russell LB, Weinstein MC, editors. Cost
     effectiveness in health and medicine. New York: Oxford University
     Press, 1996.
 15. Haddix AC, Teutsch SM, Shaffer PA, Dunet DO, editors. Prevention
     effectiveness: A guide to decision analysis and economic evaluation.
     New York: Oxford University Press, 1996.
 16. Perry BD, Garner N, Jenkins SR, McCloskey K, Johnston DH. A study of
     techniques for the distribution of oral rabies vaccine to wild raccoon
     populations. J Wildl Dis 1989;25:206-17.
 17. Linhart SB, Blom FS, Engeman RM, Hill HL, Hon T, Hall DI, et al. A
     field evaluation of baits for delivering oral rabies vaccines to
     raccoons (Procyon lotor). J Wildl Dis 1994;30:185-94.
 18. Hanlon CL, Hayes DE, Hamir AN, Snyder DE, Jenkins S, Hable CP, et al.
     Proposed field evaluation of a rabies recombinant vaccine for raccoons
     (Procyon lotor): sites election, target species characteristics, and
     placebo baiting trials. J Wildl Dis 1989;25:555-67.
 19. Hoffmann CO, Gottschang JL. Numbers, distribution, and movements of a
     raccoon population in a suburban residential community. Journal of
     Mammalogy 1977;58:623-36.
 20. Hudson EM. The raccoon (Procyon lotor) on St. Catherines Island,
     Georgia: Part 2: Relative abundance in different forest types as a
     function of population density. American Musgum Novitates 1978;1-16.
 21. Population Estimates and Population Distribution Branches, Division of
     Population, U.S. Bureau of the Census. Estimates of the resident
     population of states and counties, and percent change 4/1/90 to
     7/1/94. Posted on the Internet: 1995 Jan 18.
 22. American Veterinary Medical Association. US pet ownership and
     demographics source book. Schaumburg, IL: American Veterinary Medical
     Association, 1995.

                             [end of page 349]

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

---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
Commentary

Traditional Healers and Global Surveillance Strategies for Emerging
Diseases: Closing the Gap

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


A recent position paper by the Centers for Disease Control and Prevention
(CDC) stresses that surveillance is critical to an effective defense
against new and reemerging infectious diseases and indicates that current
international monitoring of such diseases is fragmentary and inadequate
(1). Other major studies have also recorded the weaknesses in the present
disease reporting system (2-4).

The concept of "global surveillance" implies the coordination of existing
networks as well as the addition of state-of-the-art electronic networks to
ensure close monitoring of and rapid response to outbreaks, even in the
most remote locations (1,2,5-8). Plans for strengthening current
surveillance efforts include a global consortium with specialists in
epidemiology and infectious diseases working in close collaboration with
international agencies, ministries of health, universities, and research
laboratories (1,2,6,9-11). Existing programs at the World Health
Organization, CDC, the Pan American Health Organization, and elsewhere will
be reconfigured to work as a more cohesive system (1). Secure networks will
be developed for 1) the transmission of sensitive information; 2) automatic
reporting from physicians' offices, hospitals, and laboratories; and 3) the
integration of existing and planned information systems. The field
application of computer technology, satellite imagery that allows
geographically oriented information to be visually and analytically linked
to images of the environment, and the development of new statistical and
mathematical modeling methods are under discussion (1,3,12).

As medical anthropologists, we note the absence in current plans for global
reporting systems of "traditional" or non-Western health care providers,
who in communities worldwide are usually the first, and often the only,
health specialists to see patients with new or reemerging diseases. These
local health specialists, called traditional healers, may have a role to
play in the early identification of new or reemerging diseases and could
assist in coordinating responses to outbreaks and providing public health
education at the local or regional levels.

Most people around the world have little access to modern medical systems
(13-15). Even though immunizations and antibiotics increasingly find their
way into indigenous systems, healers, midwives, bone setters, herbalists,
and other traditional health experts provide most or all medical care. The
more remote, indigent, or traditional the population, the greater the
likelihood that it will have little access to modern medical care (13,16).
If such care is sought, it will be only as a last resort, should
traditional healers prove unable to address the illnesses (16). In many
communities, modern medicine is not perceived as better than traditional
healing, and it is often more costly. Distance from modern medical
resources is another barrier. Medical care that is not sensitive to
cultural differences as well as the belief that some types of diseases are
not treatable by modern medicine are also prevalent. These beliefs are
particularly common in developing countries; however, traditional healers
also practice in many ethnic and minority communities in industrialized
societies throughout North America, Europe, and Australia (17-19).

A primary dependence on traditional healers continues in areas that, until
recently, were considered largely untouched by modern development. It is in
just such areas that much of the recent economic development has triggered
rapid ecologic change. These once sparsely populated areas, now being
pulled into the global economic sphere through logging, mining, and
agriculture, are precisely the areas where it is anticipated that many new
infectious diseases will originate, as increasing populations come in
contact with previously undisturbed vectors of infectious diseases. In such
areas, traditional healers are often in a unique position to identify new
and reemerging diseases. Whatever their specialty, traditional healers are
1) familiar with diseases commonly found locally; 2) aware of an increase
or decrease in the incidence of such diseases in their patient population;
3) among the very first to see
                             [end of page 351]

cases of new diseases; and 4) cognizant of the recurrence of a disease they
have not seen in some time. If traditional healers are not tied into the
global reporting network in a systematic and effective manner, their
knowledge of new or reemerging disease information may reach the outside
world late or in many cases, not at all. Traditional healers differ not
only from country to country, but often from region to region and from one
ethnic or minority group to the next. An adequate surveillance system must
ensure that in each instance the most appropriate traditional healers are
included in some type of timely warning system.

Including traditional healers into a global system does not mean that
scientists and clinicians must agree with indigenous explanations of the
causes or treatments of infectious diseases. Nor does it require that
traditional healers accept modern assumptions about the causes, presence,
or treatment of such diseases. However, a complete surveillance system does
require that participants cooperate and maintain professional respect and
courtesy. The goal is a surveillance system that is sensitive to cultural
differences and in which new or unusual medical events can be reported
quickly and accurately from the traditional healer to the local medical
officials in the hospital or laboratory linked to the global surveillance
system.

In recommending the inclusion of traditional healers in a global
surveillance network, we do not seek to minimize the differences, or the
animosity, between these healers and modern medical practitioners (14).
Moreover, the relevant strengths and weakness of traditional healing are
not the issue here. Critical time, however, may be lost unless all
resources are tied into a disease reporting system.

Lines of communication must be established between traditional healers and
local health care systems that serve as the "up-links" to the regional,
national, and international early warning systems. A system in which
traditional healers know whom to contact and how to establish contact
quickly is essential. Traditional healers must be taught why, what, when,
and how to report unusual symptoms in their patients to local officials.
Training for traditional healers must include explaining, (in terms that
are culturally relevant to their understanding of illness and health) why
scientists outside their communities need timely medical information from
their local practices. What to report is of equal concern. Healers must be
briefed in what is reportable. A checklist of specific symptoms, such as
new or unusual fevers, rashes, or lesions could be developed for reference.
Such a checklist could also include questions on the apparent mode of
transmission of the disease, (whether it is appearing in members of the
same household; in specific parts of a local area, such as households that
share a common water source or are located near a forested area; or in sex
partners). The development and circulation of a pictorial reference guide
of diseases found in an area might facilitate communication between healers
and local officials. Specific guidelines should ensure that reporting is
done quickly. Finally, a clear and workable reporting system, with specific
information about whom to contact at the local level should be established.

An effective surveillance program must include a systematic educational
component for local health officials, with specific discussion about the
need to include traditional healers, what information these healers are
asked to provide, and how this information, once conveyed to local health
officials, must be transmitted to the regional hospital, universities, and
ministries of health quickly and effectively. Because many local health
officials have heavy demands placed on their time, the more straightforward
this transmission link is made, the better for all concerned. The local
health official is the key "up-link" between the remote field and the
regional or national surveillance centers where a more careful and
systematic evaluation of the new or reemerging infectious disease should
begin.

Finally, training for both healers and those to whom they report must be
comprehensive, and its effectiveness must be evaluated often. A
communications bridge must be established and maintained if global warning
is to be truly effective.
                             [end of page 352]

Acknowledgment
We thank Mark L. Wilson, Infectious Disease Division, Yale School of Public
Health, for his helpful comments on this manuscript.

Nora Ellen Groce* and Mary Elizabeth Reeve
*Division of Health Policy, Yale School of Public Health and Division of
International Health, Yale School of Public Health

References

  1. Centers for Disease Control and Prevention. Addressing emerging
     infectious disease threats: a prevention strategy for the United
     States. Atlanta, GA: U.S. Department of Health and Human Services,
     Public Health Service, 1994.
  2. Wilson ME, Levins R, Spielman A., editors. Detection, surveillance,
     and response to emerging diseases. In Disease in evolution: global
     changes and emergence of infectious diseases. New York Academy of
     Sciences 1994;70:336-8.
  3. World Health Organization. Emerging infectious diseases: Memorandum
     from a WHO meeting. Bull World Health Organ 1994;72:845-50.
  4. Hughes J. Conference on "Emerging infectious diseases: meeting the
     challenge." Emerging Infectious Diseases 1995;1:101.
  5. Bartlett C, Gill N. International surveillance of disease. Lancet
     1993;341:1003-6.
  6. Henderson D. Surveillance systems and intergovernmental cooperation.
     In: More, SS, editor. Emerging Viruses. New York: Oxford University
     Press, 1993.
  7. Marwick C. Effective response to emerging diseases called an essential
     priority worldwide. JAMA 1995;273:189-90.
  8. Institute of Medicine. Emerging infections, microbial threats to
     health in the United States. Washington, DC: National Academy Press,
     1992.
  9. Pan American Health Organization. Combating emerging infectious
     diseases. Washington, DC: PAHO, June 1995.
 10. LeDuc J, Tikhomirov E. Global surveillance for recognition and
     response to emerging diseases. New York Academy of Sciences.
     1994;740:341-5.
 11. O'Brien T, Stelling J. WHONET: An information system for monitoring
     antimicrobial resistance. Emerging Infectious Diseases 1995;1:66.
 12. Vacalis T, Bartlett C, Shapiro C. Electronic communication and the
     future of international public health surveillance. Emerging
     Infectious Diseases 1995;1:34-5.
 13. Foster G, Anderson B. Medical Anthropology. New York: Knopf, 1978.
 14. Velimirovic B. Is integration of traditional and western medicine
     really possible? In: Coriel J, Mull JD, editors. Anthropology and
     Primary Health Care. Boulder, CO: Westview Press 1990.
 15. Gumede MV. Traditional healers: a medical practitioner's perspective.
     Braamfontein: Skotaville Publishers, 1990.
 16. Pillsbury B. Policy and evaluation perspectives on traditional health
     practitioners in national health care systems. Social Science and
     Medicine 1982;16:1825-34.
 17. Galanti GA. Caring for Patients from Different Cultures Philadelphia:
     University of Pennsylvania Press, 1991.
 18. Lynch E, Hanson M. Developing cross-cultural competence. Baltimore:
     Paul Brooks 1992.
 19. Brown K. Mama Lola: A Vodou Priestess in Brooklyn. Berkeley:
     University of California Press, 1991.

                             [end of page 353]

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


---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
Letters

Widespread Foodborne Cyclosporiasis Outbreaks Present Major Challenges

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


To the Editor: The organism now named Cyclospora cayetanensis was first
recognized as a cause of human illness in 1977. For several years, as its
taxonomy was deliberated, it was referred to as "cyanobacterium-, or
coccidia-like bodies" (CLBs), or considered to be blue-green algae. In
1993, C. cayetanensis was reported to be a protozoan parasite, a coccidian
member of the family Eimeriidae. To be infectious, the spherical,
chlorine-resistant oocyst (8mm to 10mm) found in the feces of infected
persons must sporulate in the environment, a process that, depending on
conditions, takes at least several days. Upon examination by ultraviolet
microscopy, Cyclospora oocysts autofluoresce and upon staining, they are
variably acid-fast. The incubation period between infection and onset of
symptoms averages approximately 1 week. Cyclospora infects the small
intestine and usually causes watery diarrhea, with frequent stools. It can
also cause loss of appetite, weight loss, stomach cramps, nausea, vomiting,
fatigue, increased flatus, and low-grade fever. The duration of symptoms is
often several weeks, and remitting courses spanning 1 to 2 months, with
several relapses, have been reported. Cyclosporiasis is effectively treated
with trimethoprim/sulfamethoxazole; however, therapy for patients who are
sulfa-intolerant has not been identified.

Before 1996, only three outbreaks of Cyclospora infection had been reported
in the United States. However, between May 1 and mid-July 1996 almost 1,000
laboratory-confirmed cases were reported to the Centers for Disease Control
and Prevention (CDC). A few hospitalizations (<20) were reported, but no
Cyclospora-related deaths were confirmed. These infections occurred in at
least 15 states and Canadian provinces and the District of Columbia.
Investigations of approximately 50 event-related outbreaks of diarrheal
illness due to C. cayetanensis, as well as case-control studies of
sporadic, laboratory-confirmed cases by several states, now clearly
implicate consumption of fresh raspberries. Complete, high confidence level
trace-backs of raspberry shipments related to more than 25 of the events
have indicated that the raspberries responsible were imported from
Guatemala between early May and mid-June 1996.

On June 17, 1996, CDC began hosting thrice-weekly conference calls to
ensure close coordination among CDC, the U.S. Food and Drug Administration
(FDA), and the many state and local health agencies investigating these
widespread outbreaks and cases. The conference calls provided coordination
in tracking and discussing this multifocal problem. In addition, on July
17, 1996, in Atlanta, CDC and FDA held a 1-day work-shop entitled
"cyclospora - 1996," which was attended by more than 80 persons
representing CDC, FDA, the U.S. Department of Agriculture, 16 states, one
province, five cities, five universities, the Council of State and
Territorial Epidemiologists, the Association of State and Territorial
Public Health Laboratory Directors, the Pan American Health Organization,
and the government of Canada. The participants in the investigations of
Cyclospora shared the knowledge gained through their individual
investigations of this multistate, multicountry outbreak. The goals of the
workshop were to begin to formulate effective prevention strategies for
Cyclospora infection, to discuss the strength of the evidence implicating
Guatemalan raspberries, and to formulate research needs. The workshop
allowed for discussions about the epidemiologic and trace-back studies
conducted and speculation about where and how the raspberries became
contaminated. Representatives from Texas, South Carolina, New York City,
Florida, and New Jersey presented data from their respective case-control
and cohort studies; CDC representatives provided an overview of the
outbreaks and focused on multiple, specific trace-backs from more than 20
of the event-related outbreaks. FDA representatives discussed their roles
and regulatory authority in foodborne investigations.

The workshop also addressed the array of scientific challenges concerning
C. cayetanensis, such as clinical diagnostic techniques, protocols for
detection of the organism on produce, and the basic biology of
                             [end of page 354]

this protozoon. We do not know the infectious dose, the proportion of the
infected persons who have diarrhea, the proportion of diarrheal illness
caused in various different environmental conditions. It can be transmitted
by water and food, and its transmission is seasonal (late spring/early
summer), at least where it has been studied (primarily temperate, seasonal
climates).

The poor sensitivity and specificity of current methods for diagnosis and
detection of Cyclospora were discussed. A photomicrographic demonstration
convinced the participants that currently the foremost requirement for
accurate clinical diagnosis is a skilled microscopist. The status of
polymerase chain reaction technologies for detection and diagnosis of
Cyclospora was presented and discussed, including the inhibitory aspects of
berry juices and the difficulty in oocyst recoveries from spiked berry
samples. Participants stated the need for a bank of Cyclospora organisms
and their DNA (molecular libraries) from different locations and outbreaks.
Currently, we may not be able to take full advantage of such
epidemiologically well-documented specimens; however, the technologies and
tools will continue to advance, and these specimens need to be centrally
banked now, to be made available when the tools are up to the task. An
animal model needs to be developed, or at least explored. The uses for such
a model include providing material (oocysts and other life-cycle stages)
for reagent development (monoclonal antibodies) to allow studies of the
organisms, the disease, immune responses, and potential environmental
transmission. Such a model will facilitate the development of prevention
and treatment strategies.

Ongoing investigations into how the raspberries were contaminated were
discussed. The lack of sensitive and reproducible detection assays for
Cyclospora, which does not replicate outside the human host, remains the
major stumbling block in providing proof of contamination of suspected
transmission vehicles. Studies were too preliminary for conclusions. Both
the government of Guatemala and the producer/exporter associations were
most helpful in investigations and need to remain involved if we are to
better understand what occurred in May and June of this year.

Throughout the workshop, a wider issue than the current situation with
Cyclospora was discussed: the management of the emerging problem of
widespread multistate and international foodborne outbreaks of both
infectious and toxic nature. Such outbreaks are increasing and can be
expected to worsen as the world moves toward a global food economy. What
contaminates a particular food item on a farm, in a herd or crop, at a
processing shed, or from a handler, can now cause widely distributed
outbreaks, continents away, in a day. More coordination is needed on
several fronts in the management of such outbreaks: 1) the development of a
structured process for integration and coordination of epidemiologic
studies; 2) more aggressive laboratory diagnostic training related to
poorly recognized or understood emerging infections; 3) better coordination
of press releases related to multistate outbreaks; 4) better understanding
and clarification of the legal roles and responsibilities of federal,
state, and local agencies; 5) and earlier involvement of industrial
partners at all levels, including growing/processing, exporting/importing,
transporting, and wholesale/retail sales. Because these types of outbreaks
are likely to become international this aspect must be addressed in
considering appropriate approaches.

The Cyclospora outbreaks of May and June 1996 underlined that without the
ability to culture and grow the organisms, without a supply of the organism
to develop expedient assays, without an established coordinating body to
expedite agreed-upon means for dissemination of information, we, as public
health officials, are called upon to provide guidance without the benefit
of all the appropriate knowledge. The workshop engendered interchange and
discussion on critical issues concerning what is known and unknown about
Cyclospora and the outbreaks of cyclosporiasis during May and June 1996.
The workshop also provided a forum in which it became apparent that public
health officials must launch a committed effort to develop an established,
                             [end of page 355]

coordinating system among agencies at all levels and deal with the threat
of widespread, multistate/international foodborne outbreaks caused by
infectious or toxic agents.

Daniel G. Colley
Centers for Disease Control and Prevention, Atlanta, Georgia, USA

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


---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
Letters

Identification of Cyclospora in Poultry

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


To the Editor: Human infection with the parasitic protozoa, Cyclospora, was
first described in 1979 (1), and the organism was only recently categorized
as an important gastrointestinal parasite. A single species, Cyclospora
cayetanensis, has been described in humans (2), while most species in the
genus Cyclospora have been described only in reptiles and rodents (3). The
consumption of undercooked meat and exposure to contaminated water have
been considered possible sources of human infection with C. cayetanensis
(1,4). Coccidia were detected in drinking water in Nepal (5), and the
parasite was identified in an animal species (one duck in Peru, by Zerpa et
al. [6]) different from those in which it was described earlier. To
determine whether a domestic animal is either a host or a reservoir for C.
cayetanensis, we first examined feces from cats, which are hosts and
reservoirs of Toxoplasma gondii, a coccidia causing human illness, but got
negative results. Because Cyclospora were recently phylogenetically linked
to Eimeria mitis and E. tenella (7), coccidial parasites of chickens, we
investigated the presence of Cyclospora in poultry.

We pooled feces from approximately 600 4- to 6-week-old chickens from a
poultry farm near Monterrey, Mexico, and extracted feces from the caecum of
50 6- to 8-week-old chickens from a poultry market at that location. By
Percoll discontinuous-gradient centrifugation (Medina-De la Garza et al.,
submitted), both fecal pools were positive for coccidia, mainly Eimeria
species and what we regarded as C. cayetanensis oocysts. Presence of
Cyclospora was confirmed by 1) characteristic morphology and size (8mm to
10mm), 2) positive staining with Kinyoun's acid-fast stain, 3) positive
autofluorescence under ultraviolet light, and 4) sporulation of oocysts
with formation of sporocysts after a 10-day incubation. All these are
diagnostic features of C. cayetanensis (8) and to our knowledge are not
described for any known poultry coccidia.

On the basis of these findings, we suggest that poultry may serve as a
possible source for human infection with Cyclospora. Consumption of chicken
has been reported in one infected patient in the original description by
Ashford (1) and in a patient reported recently by Connor and Shlim (9).
Moreover, the only existing report of C. cayetanensis found in feces from a
domestic farm animal concerned a farm duck (6). Zerpa et al. suggest that
besides consumption of contaminated water, other modes of transmission
involving contact with domestic animals must be considered. So far,
however, a possible infection route involving poultry, whether it may be
direct consumption of undercooked chicken meat, contamination of food and
water sources with chicken feces, or both, remains to be determined. It
should be noted that sanitary standards in poultry-breeding facilities in
developing countries may not be adequate. This would account for the fact
that reports implicating chickens in the transmission of Cyclospora (1,9)
have occurred in, or in relation to, developing countries. The Cyclospora
found in the chickens in our study have the diagnostic features of C.
cayetanensis. Nevertheless, the existence of another, not yet described,
Cyclospora species infecting poultry, which has similar features but is
different from C. cayetanensis, cannot be excluded at this stage. In
addition, the number of oocysts recovered was not large and because feces
were pooled, we could not calculate the number of oocysts passed by each
bird. The possibility that oocysts were acquired as a contaminant from food
or water sources and were only passing though the gut of the chickens
(making the chickens a paratonic host) cannot be ruled out.

The increased recognition of Cyclospora as an important cause of diarrhea
in both immunocompromised and immunocompetent
                             [end of page 356]

persons and the public health relevance of this emerging pathogen as a
potential cause of diarrheal outbreaks (3,4) make prompt disclosure of the
epidemiologic features and behavior of the parasite necessary. As we
propose the possible participation of poultry in the epidemiologic cycle of
the coccidia, we invite other Cyclospora working groups worldwide to
confirm the so far putative reservoir described in this communication and
to further study other possible hosts or reservoirs.

H. Leslie Garca-Lpez, Lus E. Rodrguez-Tovar, and Carlos E. Medina-De la
Garza*
Facultad de Medicina y Hospital Universitario "Dr. J.E. Gonzlez,"
Universidad Autnoma de Nuevo Len, Monterrey, Mexico

References

  1. Ashford RW. Ocurrence of an undescribed coccidian in man in Papua New
     Guinea. Ann Trop Med Parasitol 1979;73:497-500.
  2. Ortega YR, Gilman RH, Sterling CR. A new coccidian parasite
     (Apicomplexa: Eimeriidae) from humans. J Parasitol 1994;80:625-9.
  3. Soave R, Johnson WD. Cyclospora: conquest of an emerging pathogen
     (commentary). Lancet 1995;345:667-8.
  4. Huang P, Weber JT, Sosin DM, Griffin PM, Long EG, Murphy JJ, et al.
     The first reported outbreak of diarrheal illness associated with
     Cyclospora in the United States. Ann Intern Med 1995;123:409-14.
  5. Rabold JG, Hoge CW, Shlim DR, Kefford C, Rajah R, Echeverra P.
     Cyclospora outbreak associated with chlorinated drinking water. Lancet
     1994;344:1360.
  6. Zerpa R, Uchima N, Huicho L. Cyclospora cayetanensis associated with
     watery diarrhoea in Peruvian patients. J Trop Med Hyg 1995;98:325-9.
  7. Relman DA, Schmidt TM, Gajadhar A, Sogin M, Cross J, Yoder K, et al.
     Molecular phylogenetic analysis of Cyclospora, the human intestinal
     pathogen, suggests that is closely related to Eimeria species. J
     Infect Dis 1996;173:440-5.
  8. Chiodini PL. A "new" parasite: human infection with Cyclospora
     cayetanensis. Trans Roy Soc Trop Med Hyg 1994;88:369-71.
  9. Connor BA, Shlim DR. Foodborne transmission of cyclospora . Lancet
     1995;346:1634.

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


---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
Letters

PCR Confirmation of Infection with Cyclospora cayetanensis

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


To the Editor: Cyclospora cayetanensis, formerly known as
cyanobacterium-like body, is a variably acid-fast microorganism. Recently,
it was classified as a coccidian parasite (1) closely related to the genus
Eimeria (2). Humans infected with C. cayetanensis typically have diarrheal
illness with a variable number of stools per day and sometimes have nausea
and vomiting (3,4). Cyclospora infection has been reported in many parts of
the world as clustered or sporadic cases (1,3-5).

Variable success in diagnosing infection with this parasite underscores the
need for using (as quality control) molecular methods, which do not rely on
the level of expertise of laboratory personnel in microscopy. The key
features for diagnosis by light microscopy are size (8mm to 10mm in
diameter), internal features of stained and unstained oocysts, and
autofluorescence of oocysts (1,6). The definitive diagnosis is understood
as visualization of characteristic sporulated oocysts, which contain two
sporocysts. However, sporulation typically requires incubating oocysts for
up to 2 weeks, and this approach cannot be applied to Formalin or
polyvinylalcohol-preserved stool smears.

Sporadic and clustered cases of Cyclospora infections were reported in the
United States and Canada during May and June 1996 (5,7). From these
outbreaks, more than 900 cases were diagnosed by examining stool specimens
under light microscopy (Barbara Herwaldt, pers. comm.). Epidemiologic
studies indicated risk for Cyclospora infection from consuming raspberries
imported from Guatemala (7). Forty-two stool specimens supplied in 2.5%
potassium dichromate from patients with intestinal symptoms were forwarded
to the Centers for Disease Control and Prevention to be evaluated by
microscopy and by polymerase chain reaction (PCR) amplification. In
addition, one well-characterized positive stool specimen from Nepal was
provided by John Cross, Armed Forces Research Institute of Medical
Sciences,
                             [end of page 357]

Bangkok, Thailand, to use as the positive control.

Using techniques we developed for diagnosis of other protozoan parasites in
stools, we extracted DNA from all stools. The techniques we used employ
glass-bead disruption of oocysts in a buffer containing Laureth-12,
purification with the RapidPrep Micro Genomic DNA Isolation Kit for Cells
and Tissue (Pharmacia Biotech Inc., Piscataway, N.J.), followed by a final
purification step employing the QIAquick PCR purification kit protocol
(Qiagen, Inc., Chatsworth, Calif.) (8). The glass-bead disruption of
oocysts was far more effective than sonication (2) or freeze-thawing
techniques (9). We performed nested PCR in all stool specimens by using
Relman et al. (2) primers CYCF1E and CYCR2B for the first step of nested
amplification and primers CYCF3E and CYCR4B for the second (nested) step of
the PCR. These are the only primers described for amplification of
Cyclospora DNA. We found optimal conditions for the first step PCR to be
denaturation at 94C for 30 s, annealing at 55C for 30 s, and extension at
72C for 90 s, 45 cycles. The same conditions were used for the second step
of the nested PCR, but the annealing temperature was 60C.

By using this approach, we amplified the Cyclospora-specific DNA fragment
in 16 (38%) of the 26 (62%) specimens reconfirmed as positive by light
microscopy. The 10 specimens negative by PCR but positive by microscopy
showed either few or moderate numbers of Cyclospora oocysts. None of the 16
(38%) specimens negative by microscopy generated positive results in the
PCR Cyclospora test. Upon further examination by the PCR technique we
developed (9), three of these samples were positive for another enteric
coccidian, Cryptosporidium parvum.

Preliminary evaluation indicates that the sensitivity of PCR is 62%, and
the specificity is 100%. Although the sensitivity of the technique should
be evaluated further, these results indicate that PCR can be used to detect
Cyclospora. We assessed the sensitivity of this PCR again by using the
Nepalese specimen described above. This specimen, which was used as
positive control in all reactions, was amplified even when the extracted
DNA was diluted at 10 to the -5th power.

Lastly, a note of caution. As noted by Relman et al. (2) and confirmed by
us through GenBank searches, the nested PCR Cyclospora primers
cross-amplify other coccidians, especially those belonging to the genus
Eimeria (because no molecular data exist for another human coccidian
enteric parasite, Isospora belli, potential cross-amplification remains to
be determined). This cross-amplification with Eimeria should not present a
problem in diagnosing Cyclospora in human stool, as no human infections by
Eimeria are known. However, when analyzing food or environmental specimens,
this cross-amplification may complicate precise detection of Cyclospora.

Norman J. Pieniazek, Susan B. Slemenda, Alexandre J. da Silva, Edith M.
Alfano, and Michael J. Arrowood
Centers for Disease Control and Prevention, Atlanta, Georgia, USA

References

  1. Ortega YR, Sterling CR, Gilman RH, Cama VA, Diaz F. Cyclospora
     cayetanensis: a new protozoan pathogen of humans. N Engl J Med
     1993;328:1308-12.
  2. Relman DA, Schmidt TM, Gajadhar A, Sogin M, Cross J, Yoder K, et al.
     Molecular phylogenetic analysis of Cyclospora, the human intestinal
     pathogen, suggests that it is closely related to Eimeria species. J
     Infect Dis 1996;173:440-5
  3. Soave R, Dubey JP, Ramos LJ, Tummings M. A new intestinal pathogen?
     Clin Res 1986;34:533A.
  4. Long EG, Ebrahimzadeh A, White EH, Swisher B, Callaway CS. Alga
     associated with diarrhea in patients with aquired immunodeficiency
     syndrome and in travelers. J Clin Microbiol 1990;28:1101-4.
  5. Centers for Disease Control and Prevention (CDC). Outbreaks of
     Cyclospora cayetanensis infection - United States, 1996. MMWR Morb
     Mortal Wkly Rep 1996;45:549-51.
  6. Garcia LS, Bruckner DA. Intestinal protozoa: Coccidia and
     microsporidia. In: Garcia LS, Bruckner DA, editors. Diagnostic medical
     parasitology, 2nd ed. Washington: American Society for Microbiology,
     1993;49-74.
  7. Centers for Disease Control and Prevention (CDC). Update: Outbreaks of
     Cyclospora cayetanensis infection - United States and Canada, 1996.
     MMWR Morb Mortal Wkly Rep 1996;45:611-2.
  8. da Silva AJ, Schwartz DA, Visvesvara GS, de Moura H, Slemenda SB,
     Pieniazek NJ. Sensitive PCR diagnosis of infections by Enterocytozoon
     bieneusi (microsporidia) using primers based on the region coding for
     small-subunit rRNA. J Clin Microbiol 1996;34:986-7.
                                [end of page 358]

  9. Johnson DW, Pieniazek NJ, Griffin DW, Misener L, Rose JB. Development
     of a PCR protocol for sensitive detection of Cryptosporidium oocysts
     in water specimens. Appl Env Microbiol 1995;61:3849-55.

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


---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
Letters

Emerging Infectious Diseases and the Depopulation of French Polynesia in
the 19th Century

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


To the Editor: The same dynamics now considered factors in the emergence of
infectious diseases may have been involved in the dramatic depopulation of
French Polynesia in the 19th century. Temporal and geographic variation in
the frequency and severity of infectious diseases are the result of the
encounter and interaction of a population of parasites and a population of
hosts. J. Musser reviewed the "bacterial side of the equation" (1). On the
host side, there are two historical models that describe the influence of
parasitism on human populations (2-4): 1) the South American model, in
which new pathogens were introduced into native populations by the European
conquistadores, causing the death of 50 million people; and 2) the African
model, in which infectious diseases present in native populations protected
them from the effects of colonization until modern times when the discovery
of quinine and other efficient antipathogenic drugs provided added
protection. The second model is well illustrated by the attempted
colonization of Madagascar, where the French lost five men to war and 5,000
to malaria (2). This letter intends to illustrate the first model. We
suggest that during their first contacts with European navigators in the
very late 18th century and the 19th century, Polynesian islanders, much
like populations in the South American model, were decimated by newly
introduced infectious diseases.

It is difficult to know precisely which infectious diseases were present in
Tahiti and the other French Polynesian islands before the arrival of the
first Europeans. However, a study of Polynesian languages indicates that
Bancroftian filariasis and leprosy were already present, while syphilis and
other venereal diseases, influenza, and tuberculosis (TB) were probably
unknown. Epidemic diarrhea and dysenteriae could have existed, although
first reports mentioned that the oldest Polynesians "never heard of
dysenteriae before" (5). In the Marquesian language, names exist for
leprosy, bronchitis, abscesses, and impetigo.

The number of inhabitants in Tahiti, as well as in the Marquesas and the
Austral Archipelago, was at first only estimated by European explorers.
However, a precise census was performed as soon as missionaries and French
authorities noted the high death rates in most of the islands (5,7,15,16).
Tahiti was annexed by France in 1843; the first census was performed in
1848, and the population size was assessed approximately every 5 years
until 1911.

Four major epidemic diseases (TB, typhoid, influenza, and smallpox)
devastated the Marquesas from 1791 to 1863/64; approximately 80% of the
population died. During that period, exchange of populations between the
Marquesas Islands also increased, as a consequence of colonization. Thus,
leprosy increased dramatically during the second half of the 19th century,
to a prevalence of 4.11% in 1884 (6).

In Rapa, the remote, southern island of the Austral Archipelago, at least
three epidemics were reported, resulting in the loss of more than 90% of
the population. Although the cause of the first epidemic remained unknown,
dysenteriae and smallpox were identified as causes of the second and third
epidemics, respectively.

From Rapa, a missionary went to Mangareva in 1831 or 1832, and his visit
there was followed by an epidemic that the natives attributed "to his god."
He had to flee back to Rapa. The second recorded epidemic disease was
"Chinese scabies" in 1865, which decimated the child population. Then, the
warship "La Zle" brought an epidemic of influenza in 1908. In 1910, TB
and leprosy were reported "to spread rapidly" (7), and in 1911, the ship
"La Gauloise" brought whooping cough to Mangareva.

In Tahiti and the Society Islands, the number and diversity of
international and interisland exchanges, involving numerous commercial
ships and whalers, make the origin of epidemics more difficult to trace.
                             [end of page 359]

However, at least five were reported successively in the Leeward Islands in
1843, 1848, 1854, and 1864 (7), and at least 11 in Tahiti: influenza (1772
to 1774), pulmonary TB (1775), dysentery following the passage of the ship
of Vancouver (1790), dysentery after the passage of the whaler "Britania"
(1807), disastrous influenza in 1820, whooping cough in 1840, smallpox in
1841, dysentery again in 1843, scarlet fever in 1847, measles in 1852-1854
(800 deaths were recorded) and typhoid fever after the passage of "La
Magicienne" in 1877 (8).

Almost without exception, authors attributed the dramatic depopulation of
French Polynesia during the 19th century to infectious diseases. Other
causes, such as alcohol, opium, local wars, infanticides, and even
orgiastic behavior were also mentioned as possible causes. Depopulation
occurred to a similar extent in other South Pacific countries (9), e.g.,
the Cook Islands, Hawaii, Tonga, Samoa, and particularly Fiji, where 50% of
the population died. Thus, after limited initial contact with persons
exposed to infectious diseases, most of the Polynesian populations died.
Why did it happen? Why were epidemics so intense and so severe? It is
unlikely that clones of bacteria, viruses, fungi, or parasites with
particularly high virulence were introduced into native populations since
the long crossing by sailing boats would have selected clones with lower
virulence. Moreover, epidemics are also intense and severe in animal
populations when new infectious agents are introduced. In Hawaii, the
introduction of Plasmodium from birds had catastrophic consequences for the
local fauna (10).

Host population factors that may influence the spread of an infectious
agent (i.e., the intensity of an epidemic) are diverse: 1) social
disruption was certainly a major cause for the increase of leprosy and TB
in the Marquesas during the 19th century: pacification of the archipelago
by Dupetit-Thouars changed traditional behavior and destroyed tribal
barriers against leprosy by permitting the development of interisland
exchanges, thus contributing to the spread of both leprosy (within the
Marquesas) and TB (from Tahiti to one Marquesas island, then between the
Marquesas) (11); 2) the absence of most infectious diseases in Polynesia
before the 18th century probably slowed the selection of behavioral methods
of prevention and the development of traditional medicine; 3) a small
population without exposure to infectious diseases would not have selected
resistance genes against nonexistent infectious agents; and 4) the lack of
population immunity probably had a major role in the spread of new
infectious agents.

Host population factors that can influence the virulence of parasites
(i.e., the severity of an epidemic) are less frequent. Successive epidemics
of closely related viruses or bacteria can enhance the severity of the
disease, as in dengue fever (12), or can inversely provide
cross-protection, as was suggested between yaws and syphilis (13), whose
causative organisms are almost indistinguishable. Reduced genetic
polymorphism of 19th century Polynesians who had no immunity to infectious
diseases could have contributed to the severity of epidemics in the South
Pacific, as it was speculated for South America (4,14).

Paul M. V. Martin* and Claude Combes
*Institut Territorial de Recherches Mdicales Louis Malard, Papeete,
French Polynesia; and Centre de Biologies et d'Ecologie Tropicale et
Mditerranenne, Universit de Perpignan, Perpignan, France

References

  1. Musser JM. Molecular population genetic analysis of emerged bacterial
     pathogens: selected insights. Emerging Infectious Diseases
     1996;2:1-17.
  2. Combes C. Interactions durables: cologie et volution du parasitisme.
     Paris: Masson, 1995.
  3. Haldane JBS. Disease and evolution. Ricerca Scientifica Supplement
     1949;19:68-76.
  4. Black FL. Why did they die? Science 1992;258:1739-40.
  5. De Bovis E. Etat de la socit tahitienne  l'arrive des europens.
     Papeete: Socit des Etudes Ocaniennes, 1978.
  6. Clavel CL. Les Marquisiens. Arch Med Nav 1884;42:194-212.
  7. Toullelan PY. Tahiti colonial. Paris: Publication de la Sorbonne,
     1987.
  8. Vigneron E. Hommes et sant en Polynsie Franaise. Montpellier, 1991.
  9. Mac-Arthur N. Islands population of the Pacific. Canberra: Australian
     National University Press, 1968.
                                [end of page 360]

 10. van Ripper III C, Van Ripper SG, Lee Goff M, Laird M. The
     epizootiology and ecological significance of malaria in Hawaiian land
     birds. Ecological Monographs 1986;56:127-44.
 11. Buisson GPE. Les les Marquises et les Marquisiens. Annales d'Hygine
     et de Mdecine Coloniales 1903;6:535-59.
 12. Halstead SB. Pathogenesis of Dengue: challenge to molecular biology.
     Science 1988;239:476-81.
 13. Turner TB. Studies on the relationship between yaws and syphilis.
     American Journal of Hygiene 1937;25:477-506.
 14. Garenne M, Aaby P. J Infect Dis 1990;161:1088.
 15. Rollin L. Moeurs et coutumes des anciens Maoris des les Marquises.
     Papeete: Stepolde, 1974.
 16. Hanson A. Rapa, une le polynsienne hier et aujourd'hui. Paris:
     Publications de la Socit des Ocanistes, 1973.
 17. Vallaux F. Mangareva et les Gambier. Papeete: Etablissement
     Territorial d'Achats Groups, 1994.

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


---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
Letters

Epidemic Zoster and AIDS

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


To the Editor: Zoster (exogenously reactivated varicella-zoster virus
infection) may seem an unlikely candidate for emergence and epidemicity. A
recent report, however, describes a zoster outbreak associated with
epidemic HIV in injecting drug users in Manipur State, India (1). In
addition to underscoring the variety of ways in which "old" diseases may
reemerge under complex bio-ecologic conditions, this outbreak may also have
implications for anticipating and diagnosing HIV infections and AIDS in
developing countries. The Manipur outbreak was associated with a doubling
of zoster frequency above background levels, with increased occurrence most
notable in males 12-44 years old, who also had the highest HIV prevalence.
In a separately studied group of 120 injecting drug users, 20 developed
zoster and all were found to be HIV positive (1), a correlation
substantially greater than for such other clinical predicters of HIV
infection as persistent lymphadenopathy, weight loss, or recurrent
dermatoses. Increased zoster occurrence associated with HIV transmission
has also been seen in Ho Chi Minh City, Vietnam, and in other Southeast
Asian countries, particularly in injecting drug using populations
(unpublished). Zoster as a sentinel indicator of community HIV transmission
is also suggested by reports from Africa (2).

For over 150 years, it was believed that zoster occurred in local epidemics
(3,4). By the 1950s, however, it was generally agreed that zoster
represented reactivation of latent gangliar varicella virus either
sporadically, or in response to immunosuppression or trauma. Epidemics of
"endogenous" immunosuppression, such as those associated with epidemic HIV
infection, might thus be expected to produce outbreaks of zoster, as seems
to have occurred in Manipur and Vietnam. In the Indian outbreak traumatic
zoster seemed unlikely: truncal and facial dermatomes predominated, rather
than dermatomes corresponding to drug injection sites (usually the hands or
legs). Recognition of zoster outbreaks may be important in developing
countries where HIV diagnosis is limited, CD4 cell counts are unavailable,
and diagnosis of AIDS is delayed. Zoster is not currently accepted as an
AIDS-defining condition (5), and the extent to which it may reflect immune
collapse or predict HIV disease progression is uncertain. Nevertheless,
greater awareness of zoster as a sentinel indicator of community HIV
transmission may be of help not only in clinical diagnosis, but also in
public health efforts to recognize epidemic HIV occurrence.

D. M. Morens,* A.K. Agarwal, S. Sarkar, S. Panda, and R. Detels

*University of Hawaii School of Medicine, Honolulu, Hawaii; ICMR Unit for
Research on AIDS in Northeastern States of India, Calcutta, India; and
University of California at Los Angeles, Los Angeles, California

References

  1. Panda S, Sarkar S, Mandal BK, Singh TBK, Lokendra Singh K, Mitra DK,
     et al. Epidemic of herpes zoster following HIV epidemic in Manipur,
     India. J Infect 1994;28:167-73.
  2. Tyndall MW, Nasio J, Agoki E, Malisa W, Ronald AR, Ndinya-Achola JO,
     Plummer A. Herpes zoster as the initial presentation of human
     immunodeficiency virus type 1 infection in Kenya. Clin Infect Dis
     1995;21:1035-7.
  3. Simon L. Questions sur diverses branches des sciences mdicales. Thse
     202. Paris: Rignoux, 1840.
  4. Kaposi M. Bemerkungen ber die jngste Zoster-Epidemie und zur
     Aetiologie des Zoster. Wien Med Wochenschr 1889;25:961-4, 26:1001-4.
                                [end of page 361]

  5. World Health Organization. Interim proposal for a WHO staging system
     for HIV infection and diseases. Wkly Epidemiol Rec 1990;65:221-4.

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

---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
Letters

Epidemic Zoster and AIDS

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


To the Editor: Zoster (exogenously reactivated varicella-zoster virus
infection) may seem an unlikely candidate for emergence and epidemicity. A
recent report, however, describes a zoster outbreak associated with
epidemic HIV in injecting drug users in Manipur State, India (1). In
addition to underscoring the variety of ways in which "old" diseases may
reemerge under complex bio-ecologic conditions, this outbreak may also have
implications for anticipating and diagnosing HIV infections and AIDS in
developing countries. The Manipur outbreak was associated with a doubling
of zoster frequency above background levels, with increased occurrence most
notable in males 12-44 years old, who also had the highest HIV prevalence.
In a separately studied group of 120 injecting drug users, 20 developed
zoster and all were found to be HIV positive (1), a correlation
substantially greater than for such other clinical predicters of HIV
infection as persistent lymphadenopathy, weight loss, or recurrent
dermatoses. Increased zoster occurrence associated with HIV transmission
has also been seen in Ho Chi Minh City, Vietnam, and in other Southeast
Asian countries, particularly in injecting drug using populations
(unpublished). Zoster as a sentinel indicator of community HIV transmission
is also suggested by reports from Africa (2).

For over 150 years, it was believed that zoster occurred in local epidemics
(3,4). By the 1950s, however, it was generally agreed that zoster
represented reactivation of latent gangliar varicella virus either
sporadically, or in response to immunosuppression or trauma. Epidemics of
"endogenous" immunosuppression, such as those associated with epidemic HIV
infection, might thus be expected to produce outbreaks of zoster, as seems
to have occurred in Manipur and Vietnam. In the Indian outbreak traumatic
zoster seemed unlikely: truncal and facial dermatomes predominated, rather
than dermatomes corresponding to drug injection sites (usually the hands or
legs). Recognition of zoster outbreaks may be important in developing
countries where HIV diagnosis is limited, CD4 cell counts are unavailable,
and diagnosis of AIDS is delayed. Zoster is not currently accepted as an
AIDS-defining condition (5), and the extent to which it may reflect immune
collapse or predict HIV disease progression is uncertain. Nevertheless,
greater awareness of zoster as a sentinel indicator of community HIV
transmission may be of help not only in clinical diagnosis, but also in
public health efforts to recognize epidemic HIV occurrence.

D. M. Morens,* A.K. Agarwal, S. Sarkar, S. Panda, and R. Detels

*University of Hawaii School of Medicine, Honolulu, Hawaii; ICMR Unit for
Research on AIDS in Northeastern States of India, Calcutta, India; and
University of California at Los Angeles, Los Angeles, California

References

  1. Panda S, Sarkar S, Mandal BK, Singh TBK, Lokendra Singh K, Mitra DK,
     et al. Epidemic of herpes zoster following HIV epidemic in Manipur,
     India. J Infect 1994;28:167-73.
  2. Tyndall MW, Nasio J, Agoki E, Malisa W, Ronald AR, Ndinya-Achola JO,
     Plummer A. Herpes zoster as the initial presentation of human
     immunodeficiency virus type 1 infection in Kenya. Clin Infect Dis
     1995;21:1035-7.
  3. Simon L. Questions sur diverses branches des sciences mdicales. Thse
     202. Paris: Rignoux, 1840.
  4. Kaposi M. Bemerkungen ber die jngste Zoster-Epidemie und zur
     Aetiologie des Zoster. Wien Med Wochenschr 1889;25:961-4, 26:1001-4.
                                [end of page 361]

  5. World Health Organization. Interim proposal for a WHO staging system
     for HIV infection and diseases. Wkly Epidemiol Rec 1990;65:221-4.

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


---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
Letters

AIDS and [AAA] in Egypt?

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


To the Editor: A recent letter concerning Egyptian hieroglyphs on the
disease [AAA] asks if this disease could be AIDS or an HIV-associated
condition prevalent in Egypt during the time of the pharaohs (1). We
believe this possibility is highly unlikely. Aside from conflicts with
current thought on the origin and evolution of lentiviruses, there is a
problem of linguistic interpretation. The initial hieroglyph in the series
of hieroglyphs comprising the word [AAA], a picture of a discharging
phallus, is a "determinative," indicating the class or category to which
the word belongs. Although scholars once took this determinative to
indicate a phallic connection with disease, even suggesting that [AAA]
meant hematuria, consistent with schistosomiasis (2,3), it was later
proposed that the determinative meant semen or poison, reflecting the
Egyptian concept that diseases may be transmitted by an evil spirit in the
form of an incubus, impregnating a victim with poisonous semen. This
interpretation is now generally accepted (4,5). The phallus-with-discharge
thus came to indicate a deadly disease, and [AAA] a poisonous
disease-causing substance introduced into the body by magic. The word [AAA]
is used elsewhere in the Egyptian medical papyri in other contexts, such as
"[AAA] of the heart" and "[AAA] of the belly and heart," and is not known
to have been used in connection with the bladder or genitalia. While the
determinative meaning may not be absolutely established, it is clear from
its usage in other contexts that the phallus-with-discharge determinative
can indicate fatal or serious illness. The notion that the
phallus-with-discharge determinative refers to sexually transmitted disease
is not consistent with its usage. To further argue that [AAA] represents
AIDS or HIV disease is not justified by the linguistic evidence. Without
further archaeologic or inscriptional evidence, we would doubt that HIV
circulated in ancient Egypt.

Robert J. Littman and David M. Morens
University of Hawaii, Honolulu, Hawaii

References

  1. Ablin RJ. Dj vu in ancient Egypt? Emerging Infectious Diseases
     1996;2:242.
  2. Ebbell B. The Papyrus Ebers. London: Oxford University Press, 1937.
  3. Jonckheere F. Une Maladie gyptienne, l'Hmaturie Parasitaire.
     Bruxelles: Fondation gyptologique Reine lizabeth, 1944.
  4. von Deines H, Westendorf W. Wrterbuch der medizinischen Texte. Erste
     Hlfte. Berlin: Akademie-Verlag, 1961;7(Part 1):129.
  5. Nunn JF. Chapter 3. Concepts of anatomy, physiology and pathology.
     Chapter 4. The pattern of disease. Chapter 5. Magic and religion in
     medicine. In: Nunn JF: Ancient Egyptian Medicine. London: British
     Museum Press, 1966:42-63,64-95,96-112.

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

---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
News and Notes

ABA Sponsors Program on Law and Emerging Infectious Diseases

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


In August 1996, the International Health Law Committee, International Law
and Practice Section, American Bar Association, sponsored a program
entitled "Law and Emerging and Re-Emerging Infectious Diseases" to examine
how the emergence and reemergence of infectious diseases affects
international and U.S. federal, state, and local law. The issues addressed
included international legal rules on infectious disease control and the
need for their revision in light of emerging and reemerging infectious
diseases; emerging infectious diseases and U.S. federal law, especially as
it affects the mission of public health agencies; and the importance of
state and local law in dealing with emerging infectious diseases.

Common themes included the challenge posed by emerging infectious diseases
as law at every level (international, national, and local) is involved and
law in various forms (treaties, constitutions, statutes, and regulations)
is affected; the need for legal reform at the international and U.S.
federal, state, and local levels; the challenges posed by a complex
jurisdictional environment (e.g., World Health Organization's relationship
to independent states and U.S. public health agencies' relationships to
state governments); in considering legal reform, the need to balance
competing policy objectives, such as the control of infectious diseases
versus the freedom of global trade and travel or protecting the community
versus privacy rights; the interdependence of legal reform efforts in that
local and national implementation of revised international rules will be
critical to any global strategy; the need to integrate the efforts of
lawyers and public health officials to effectively promote epidemiologic
principles and objectives; and the massive scope of the emerging diseases
threat stemming from not only its global reach but also the long list of
causes behind the emergence and reemergence of infectious diseases (e.g.,
political and medical complacency, international trade, global travel, war,
human behavior, environmental degradation, urbanization, poverty, and
inadequate public health infrastructures).

The International Health Law Committee program marks a first step in
raising awareness in the legal and public health communities of the many
and complex legal issues involved in addressing emerging infectious
diseases.

For copies of program presentations and other information, contact David P.
Fidler by e-mail at davidfidler@law.indiana.edu.

David P. Fidler
Indiana University School of Law
Bloomington, Indiana, USA
[page 365]
---------------------------------------------------------------------------

---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------

ADDENDUM

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


Adeleke A, Pruckler J, Benson R, Rowbotham T, Halablab M, Fields B.
Legionella-like amoebal pathogensphylogenetic status and possible role in
respiratory disease. Emerging Infectious Diseases 1996;3:225-30.

Since the publication of the above article, accession numbers have been
allocated to the following sequences:
     LLAP -1 U64034
     LLAP -8 U64035

Information about the phylogeny of S. lyticum and LLAP-3 can be found in
references 20 and 21, respectively. The derivation of sequence data
(accession numbers: LLAP-4x97357, LLAP-6x97359, LLAP-10x97363,
LLAP-11x97362, LLAP-12x97366J) referred to in our publication, can be found
in Birtles RJ, Rowbotham TJ, Raoult D, Harrison TG. Phylogenetic diversity
of intra-amoebal legionellae as revealed by 16S rRNA gene sequencing.
Microbiology 1996; 142:3525-30. In this study, which was carried out
concurrently with the study described in the article by Adeleke et al. 16S
rRNA sequence data were also obtained for LLAP1 (accession number x97355),
LLAP2 (x97356), LLAP7 (x97365), LLAP8 (x97361), LLAP9 (x97360), and the L.
lytica strains L2 (x97364) and LLAP3 (x97358).
                             [end of page 364]

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




---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
News and Notes

A Global Theme Issue: Bibliography of References

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


    Emerging and reemerging infections respect no national boundaries;
    therefore, they were an appropriate topic for the first "global
    medical theme issue," introduced in January 1996. This global
    theme issue was conceived by three editors, Linda Hawes Clever,
    The Western Journal of Medicine, Magne Nylenna, Journal of the
    Norwegian Medical Association, and George D. Lundberg, Journal of
    the American Medical Association, who in 1995 invited the editors
    of 78 journals worldwide to participate. A year later, 36 journals
    in 21 countries on six continents published more than 200 articles
    pertaining to emerging and reemerging global microbial threats
    (1). The articles addressed topics ranging from factors
    contributing to increasing antimicrobial resistance to the impact
    of global warming on infectious disease. While some solutions were
    suggested, the global issue primarily served as a call to medical
    communities and people worldwide to identify contributing factors
    and begin to develop strategies to control emergent infections.

    Following this note is a bibliography of articles published by the
    36 journals participating in the global theme issue.

Margaret A. Winker
Journal of the American Medical Association, Chicago, Illinois, USA
                             [end of page 365]

Bibliography of References

  1. Addy PAK, Esani RK, Atusheno SKN. Possible contributing factors to the
     paucity of yellow fever epidemic in the Ashanti Region of Ghana, West
     Africa. East Afr Med J 1996;73:3-9.
  2. Adlestein S, McKnight I, Pethebridge AM. HIV and intermediate immune
     deficiency. Med J Aust 1996;164:109-10.
  3. Aita J, Barrera L, Reniero A, et al. Hospital transmission of
     multidrug resistant Mycobacterium tuberculosis in Rosario Argentina.
     Medicina 1996;56:48-50.
  4. Albrecht H, Sobottka I, Emminger C, et al. Visceral leishmaniasis
     emerging as an important opportunistic infection in HIV-infected
     persons living in areas non-endemic for Leishmania donovani. Arch
     Pathol Lab Med 1996;120:189-98.
  5. Altekruse SF, Swerdlow DL. The changing epidemiology of foodborne
     diseases. Am J Med Sci 1996;311:23-9.
  6. Aseffa A, Yohannes G. Antibiotic sensitivity pattern of prevalent
     bacterial pathogens in Gondar, Ethiopia. East Afr Med J 1996;73:67-71.
  7. Bakken JS, Krueth MT, Wilson-Nordskog C, Tilden RL, Asanovich K,
     Dumler JS. Clinical and laboratory characteristics of human
     granulocytic ehrlichiosis (HGE). JAMA 1996;275:199-205.
  8. Bn E. Changes in the role of gram-positive bacteria in the infections
     of immunocompromised patients. LAM 1996;6:pages not available.
  9. Banerjee G, Ayyagari A, Prasad KN, Dhole TN, Singh SK. Nosocomial
     infection due to Enterobacter cloacae in a tertiary care hospital in
     Northern India. Indian J Med Res 1996;103:62-3.
 10. Bnhegyi D. Current situation of HIV/AIDS in Hungary. LAM 1996;6:pages
     not available.
 11. Barbour AG. Does Lyme disease occur in the South? Am J Med Sci
     1996;311:34-40.
 12. Barnes PF, El-Hajj H, Preston-Martin S, et al. Transmission of
     tuberculosis among the urban homeless. JAMA 1996;275:305-7.
 13. Barton LL, Jeck DT, Vaidya VU. Necrotizing fascitis in children:
     report of two cases and review of the literature. Arch Pediatr Adolesc
     Med 1996;150:105-8.
 14. Berkelman RL, Pinner RW, Hughes JM. Addressing emerging microbial
     threats in the United States. JAMA 1996;275:315-7.
 15. Beyers AD, Donald PR, van Helden PD, Ehlers MRW, Steyn L, Mizrahi V.
     Tuberculosis research - the way forward. S Afr Med J 1996;86:30-3.
 16. Beyers N, Gie RP, Zietsman HL, et al. The use of a geographical
     information system (GIS) to evaluate the distribution of tuberculosis
     in a high-incidence community. S Afr Med J 1996;86:40-4.
 17. Bhatt KM, Bhatt SM, Mirza NB. Meningococcal meningitis. East Afr Med J
     1996;73:35-9.
 18. Bifani P, Plikaytis BB, Kapur V, et al. Origin and interstate spread
     of a New York City multidrug-resistant Mycobacterium tuberculosis
     clone family: adverse implications for tuberculosis control in the
     21st century. JAMA 1996;275:452-7.
 19. Birrie H, Balcha F, Bisuneh A, Bero G. Susceptibility of Ethiopian
     Bulinid snails to Schistosoma haematobium from Somalia. East Afr Med J
     1996;73:76-7.
 20. Bloch AB, Simone PM, McCray E, Castro KG. Preventing
     multidrug-resistant tuberculosis. JAMA 1996;275:487-9.
 21. Borus P. Rabies: the emergence of a microbial threat. East Afr Med J
     1996;73:32-4.
 22. Bowden DS, Moaven LD, Locarnini SA. New hepatitis viruses: are there
     enough letters in the alphabet? Med J Aust 1996;164:87-9.
 23. Briem H, Thorsteinsson SB, Gudmundsson S, Erlendsson K, Lve A. The
     epidemiology of AIDS in Iceland. Laeknabladid 1996;82:21-31.
 24. Bronze MS, Dale JB. The reemergence of serious group A streptococcal
     infections and acute rheumatic fever. Am J Med Sci 1996;311:41-54.
 25. Budai J. The present and the future of vaccination in Hungary. LAM
     1996;6:pages not available.
 26. Butler JC, Peters CJ. Lessons learned from the hantavirus and other
     hemorrhagic fever viruses. Am J Med Sci 1996;311:55-9.
 27. Cabreros LJ, Rajendran R, Drimoussis A, Brandstetter RD. Radiographic
     mimics of pneumonia: pulmonary disorders to consider in differential
     diagnosis. Postgrad Med 1996;99:139-42, 145, 146.
 28. Cairney R. Global spread of tuberculosis: worrying trend for Canadian
     physicians. Can Med Assoc J 1996;154:236-8.
 29. Campbell CM, Williams BG. Academic research and HIV/AIDS in South
     Africa. S Afr Med J 1996;86:55-60.
 30. Carballal G. Emerging or reemerging virus. [in Spanish] Medicina
     1996;56:97-101.
 31. Carr A, Boyle MJ. Primary HIV infection. Med J Aust 1996;164:105-6.
 32. Cassiere H, Rodrigues JC, Fein AM. Delayed resolution of pneumonia:
     when is slow healing too slow? Postgrad Med 1996;99:151-4, 157, 158.
 33. Celentano DD, Nelson KE, Suprasert S, et al. Risk factors for HIV-1
     seroconversion infection among young men in northern Thailand. JAMA
     1996;275:122-7.
 34. Chen B, Tao S. Arbovirus survey in China in recent ten years. Chin Med
     J 1996;109:13-5.
 35. Chen D-S. Viral hepatitis in East Asia. J Formos Med Assoc
     1996;95:6-12.
 36. Chopra I, Hodgson J, Metcalf B, Poste G. New approaches for the
     control of infections caused by antibiotic-resistant bacteria: an
     industrial perspective. JAMA 1996;275:401-3.
                                [end of page 366]

 37. Chukwuma C Sr. Microsporidium in AIDS and HIV-infected patients: a
     perspective. East Afr Med J 1996;73:72-5.
 38. Claus JJ, Kuijper EJ, Gans J de. Listeria-meningitis. [Listeria
     meningitis.] Ned Tijdschr Geneeskd 1996;140:113-116.
 39. Clemens J, Brenner R, Rao M, Tafari N, Lowe C. Evaluating new vaccines
     for developing countries: efficacy or effectiveness? JAMA
     1996;275:390-7.
 40. Clever LH. Infectious diseasesgetting out of a deep hole. West J Med
     1996;164:17.
 41. Collignon PJ, Bell JM, and the AGAR. Drug resistant Streptococcus
     pneumoniae: the beginning of the end for many antibiotics? Med J Aust
     1996;164:64-7.
 42. Colquhoun SD. Hepatitis C: a clinical update. Arch of Surg
     1996;130:18-23.
 43. Cunha BA. Community-acquired pneumonia: cost-effective antimicrobial
     therapy. Postgrad Med 1996;99:109, 110, 113, 114, 117-9, 122.
 44. Cunha BA, Ortega AM. Atypical pneumonia: extrapulmonary clues guide
     the way to diagnosis. Postgrad Med 1996;99:123-8, 131, 132.
 45. Davis KJ, Fritz DL, Pitt ML, Welkos SL, Worsham PL, Friedlander AM.
     Pathology of experimental pneumonic plague produced by fraction
     1-positive and fraction 1-negative Yersinia pestis in African Green
     Monkeys (Cercopithecus aethiops). Arch Pathol Lab Med
     1996;120(February):pages not yet available.
 46. Dawson JE, Warner Ck, Standaert S, Olson JG. The interface between
     research and the diagnosis of an emerging tick-borne disease, human
     ehrlichiosis due to Ehrlichia chaffeensis. Arch Intern Med
     1996;156:137-42.
 47. de Quadros CA, Oliv JM, Hersh BS, et al. Measles elimination in the
     Americas: evolving strategies. JAMA 1996;275:224-9.
 48. de Kantor IN, Ritacco V, Barrera L. Is multidrug resistant
     tuberculosis an emerging infection in Buenos Aires? [in Spanish].
     Medicina 1996;56:102-4.
 49. Dhanda V, Das PK, Lal R, Srinivasan R, Ramaiah KD. Spread of lymphatic
     filariasis and reemergence of leishmaniasis in India. Indian J Med Res
     1996;103:46-54.
 50. Doherty RR. HTLV-1 in Australia and Oceania: long term resident or
     recent immigrant? Med J Aust 1996;164:84-6.
 51. Duodenal ulcer: the helicobacterization of a psychosomatic disease?
     Isr J Med Sc 1996;(March):pages not yet available.
 52. Dwyer B. What we should be doing about tuberculosis in Australia. Med
     J Aust 1996;164:62-3.
 53. Eskild A. The AIDS epidemic in Norway - where did it go? Tidsskr Nor
     Laegeforen 1996;116:(January): pages not yet available.
 54. Espersen F. Kampen mod mikroberne - et internationalt anliggende (The
     fight against the microbial threat - an international issue). Ugeskr
     Laeger 1996;158:251-2.
 55. Espinol CH. On the trail of color. JAMA 1996;275:168.
 56. Fadl Alla AI, Fahal AH, Ahmed ME, Hasson MA. Pattern of postoperative
     pyrexia in Khartoum. East Afr Med J 1996;73:78-80.
 57. Fattom AI, Naso R. Staphylococcal vaccines: a realistic dream. Ann Med
     1996;28:43-6.
 58. Feingold DS, Weinberg AN. Group A streptococcal (gas) infections: and
     old adversary reemerging with new tricks. Arch Dermatol
     1996;132:67-70.
 59. Feldman C, Klugman K. Antibiotic-resistant pneumococcal pneumonia. S
     Afr Med J 1996;86:28-30.
 60. File TM, Tan JS, Plouffe JF. Community-acquired pneumonia: what's
     needed for accurate diagnosis. Postgrad Med 1996;99:95-8, 100, 102,
     105-7.
 61. Fine MJ, Smith MA, Carson CA, et al. Prognosis and outcomes of
     patients with community-acquired pneumonia: a meta-analysis. JAMA
     1996;275:134-41.
 62. Fodor T. Pataki G. Bacteriology to the diagnosis of tuberculosis. LAM
     1996;6:pages not available.
 63. Fonn S. A blood-result turn-around time survey to improve congenital
     syphilis prevention in a rural area. S Afr Med J 1996;86:67-71.
 64. Frost GW, Johns M. Vaccine-preventable childhood diseases in
     Australia. Med J Aust 1996;164:61.
 65. Fry DE. The reemergence of mycobacterial infections. Arch of Surg
     1996;130:14-7.
 66. Galama JMD. Humane herpesvirussen type 6 en 7, verwekkers van onder
     meer exanthema subitum. [Human herpesviruses type 6 and 7, causative
     agents of e.g. exanthem subitum.] Ned Tijdschr Geneeskd
     1996;140:124-8.
 67. Gaston DA, Zurowski SM. Arcanobacterium haemolyticum pharyngitis and
     exanthem: three case reports and literature review. Arch Dermatol
     1996;132:61-4.
 68. George V, Jesudason MV, John TJ. CAMP test for the identification of
     Vibrio cholerae 0139. Indian J Med Res 1996;103:55-7.
 69. Gilbert GL. Multidrug-resistant tuberculosis: implications for the
     future; lesions from the past. Med J Aust 1996;164:60.
 70. Gilbert GL. Multidrug-resistant tuberculosis: prevention is better
     than cure. Med J Aust 1996;164:120-3.
 71. Githure JI, Ngumbi PM, Anjili CO, et al. Animal reservoirs of
     Leishmaniasis in Marigat, Baringo District, Kenya. East Afr Med J
     1996;73:44-7.
 72. Glanz K, Yang H. Communicating about risk of infectious diseases. JAMA
     1996;275:253-6.
 73. Gold SM, Shott SR, Myer CM III. Membranous laryngotracheobronchitis.
     Arch Pediatr Adolesc Med 1996;150:97-8.
 74. Goldman DA, Weinstein RA, Wenzel RP, et al for the Workshop to Prevent
     and Control the Emergence and Spread of Antimicrobial-Resistant
     Microorganisms in Hospitals. Strategies to prevent and control the
     emergence and spread of antimicrobial-resistant microorganisms in
     hospitals: a challenge to hospital leadership. JAMA 1996;275:234-40.
                                [end of page 367]

 75. Golledge CL, Riley TV. Natural therapy for infectious diseases. Med J
     Aust 1996;164:94-6.
 76. Gool T van, Dankert J. Drie opkomende protozaire infectieziekten in
     Nederland: Cyclospora-, Dientamoeba- en Microspora-infecties. [Three
     emerging protozoal infections in the Netherlands: cyclospora,
     dientamoeba, and microspora infections.] Ned Tijdschr Geneeskd
     1996;140:
 77. Grant CC, Duggan AK, Santosham M, DeAngelis C. Oral prednisone as a
     risk factor for infections in children with asthma. Arch Pediatr
     Adolesc Med 1996;150:58-63.
 78. Gudjnsson Ath, Kristinsson KG, Gudmundsson S. Antibiotic use and
     misuse at the National University Hospital, Iceland. Laeknabladid
     1996;82: 39-45.
 79. Gumundsdttir BK, Gumundsdttir S, Mangnadttir B, Helgason S.
     Research in bacterial diseases of salmonid fish. Laeknabladid 1996;82:
     72-7.
 80. Haemophilus influenza infection in Singapore: antimicrobial
     resistance, serotypes and clinical spectrum. Ann Acad Med Singapore
     1996;25(March):pages not yet available.
 81. Hattum J van. Hepatitis C, een ontwakende reus. [Hepatitis C, an
     awakening giant.] Ned Tijdschr Geneeskd 1996;140:119-24.
 82. Haug C. Emerging and reemerging serious infections: doctors around the
     world, unite! Tidsskr Nor Laegeforen 1996;116:(January):pages not yet
     available.
 83. Hayden FG, Treanor JJ, Betts RF, Lobo M, Esinhart JD, Hussey EK.
     Safety and efficacy of the neuroaminidase inhibitor GG167 in
     experimental human influenza. JAMA 1996;275:295-9.
 84. Haynes BF, Putman SB, Winberg JB. Update on the issues of HIV vaccine
     development. Ann Med 1996;28:39-41.
 85. Heath CH, Blackmore TK, Gordon DL. Emerging resistance in Enterococcus
     species. Med J Aust 1996;164:116-9.
 86. Heikkinen T, Ruuskanen O. New prospects in the prevention of otitis
     media. Ann Med 1996;28:23-30.
 87. Herndon RM. Evasion of immulogic defenses and emerging viral threats.
     Arch Neurol 1996;53:23-7.
 88. Hilleman MR. Cooperation between government and industry in combating
     a perceived emerging pandemic through vaccination: the 1976 swine
     influenza example. JAMA 1996;275:241-3.
 89. Hoge CW, Shlim DR, Echeverria P, Rajah R, Herrmann JE, Cross JH.
     Epidemiology of diarrhea among expatriate residents living in a highly
     endemic environment. JAMA 1996;275:533-8.
 90. Hogg GG, Strachan JE, Huayi L, Beaton SA, Robinson PM, Taylor K.
     Non-toxigenic Corynebacterium diphtheriae biovar gravis: evidence for
     an invasive clone in a south-eastern Australian community. Med J Aust
     1996;164:72-5.
 91. Hollister JM, Laing P, Mednick SA. Rhesus incompatibility as a risk
     factor for schizophrenia in male adults. Arch Gen Psychiatry
     1996;53:19-24.
 92. Honan GM, White GW, Eisenberg GM. Spontaneous infectious discitis in
     adults. Am J Med 1996;100:85-9.
 93. Hong T. Human group B rotavirus: adult diarrhea rotavirus. Chin Med J
     1996;109:11-2.
 94. Hoogkamp-Korstanje JAA. Yersiniosis. [Yersiniosis.] Ned Tijdschr
     Geneeskd 1996;140:128-30.
 95. Hopewell PC. Mycobacterium tuberculosisan emerging pathogen? West J
     Med 1996;164:33-5.
 96. Horton R. The infected metropolis. Lancet 1996;347:pages not yet
     available.
 97. Hsueh P-R, Chen H-M, Lu Y-C, Wu J-J. Antimicrobial resistance and
     serotype distribution of Streptococcus pneumoniae strains isolated in
     Southern Taiwan. J Formos Med Assoc 1996;95:29-36.
 98. Hu DJ, Dondero TJ, Rayfield MA, et al. The emerging genetic diversity
     of HIV: the importance of global surveillance for diagnostics,
     research, and prevention. JAMA 1996;275:210-6.
 99. Huang G, Lin T. Bacterial L-forms research in China. Chin Med J
     1996;109:18-20.
100. Hughes JM. Emerging pathogensan epidemiologist's perspective on the
     problem and priorities for the future. West J Med 1996;164:21-2.
101. Hung C-C, Chen Y-C, Chang S-C, Luh KT, Hsieh W-C. Clinical study of
     nosocomial candidemia in a university hospital in Taiwan. J Formos Med
     Assoc 1996;95:19-28.
102. Indriason S, Kristinsson KG, smundsson P, Bvarsson M. The
     incidence of infection associated with peritoneal dialysis.
     Laeknabladid 1996;82:53-9.
103. Infectious diseases - the old and the new. Ann Acad Med Singapore
     1996;25(March):pages not yet available.
104. Jaax NK, Davis KJ, Geisbert TJ, et al. Lethal experimental infections
     of rhesus monkeys with Ebola-Zaire (Mayinga) virus by the oral and
     conjunctival route of exposure. Arch Pathol Lab Med 1996;120:140-55.
105. Jacob John T. Emerging and reemerging global microbial threats. Natl
     Med J India 1996;9:1-2.
106. Jankovics I. The influenza viruses: past, present and future. LAM
     1996;6:pages not available.
107. Jernigan DB, Cetron MS, Breiman RF. minimizing the impact of
     drug-resistant Streptococcus pneumoniae (DRSP): a strategy from the
     DRSP Working Group. JAMA 1996;275:206-9.
108. John TJ. Emerging and reemerging bacterial pathogens in India. Indian
     J Med Res 1996;103:4-18.
109. John TJ, Jesudason MV, Lalitha MK, et al. Melioidosis in India: tip of
     the iceberg. Indian J Med Res 1996;103:64-5.
110. Johnson RT. Emerging viral infections. Arch Neurol 1996;53:18-22.
111. Johnson PDR, Veitch MGK, Leslie DE, Flood PE, Hayman JA. The emergence
     of Mycobacterium ulcerans infection near Melbourne. Med J Aust
     1996;164:76-8.
112. Jones D. Emergence of antibiotic resistance: role of ophthalmology.
     Arch Ophthalmol 1996;114:91-2.
                                [end of page 368]

113. Jnsdttir KE, Hansen H, Arnrsson VH, Laxdal Th, Stefnsson M.
     Immunization against Haemophilus influenzae type b in Iceland: results
     after six years use of PRP-D (ProHIBiT). Laeknabladid 1996;82:32-8.
114. Joyce T, McGuigan KG, Elmore-Meegan M, Conroy RM. Prevalence of
     entropathogens in stools of rural Maasai children under five years of
     age in the Maasailand region of the Kenyan Rift Valley. East Afr Med J
     1996;73:59-62.
115. Joynt RT. Microbial threats. Arch Neurol 1996;53:17.
116. Jupp PG, A Kemp. What is the potential for future outbreaks of
     chikungunya, dengue and yellow fever in southern Africa. S Afr Med J
     1996;86:35-7.
117. Kager PA, Wetsteyn JCFM. Malaria en geneesmiddelenresistentie.
     [Malaria and drug resistance.]. Ned Tijdschr Geneeskd 1996;140:151-5.
118. Kar NCAJ van de, Heuvelink AE, Boer E de, Monnens LAH. Infecties met
     verocytotoxine-producerende Escherichia coli en het
     hemolytisch-uremisch syndroom. [Infections with
     verotoxinogeneic-producing Escherichia coli and the hemolytic-uremic
     syndrome.] Ned Tijdschr Geneeskd 1996;140:134-7.
119. Kelleher AD, Orth D. Early HIV-induced immune deficiency. Med J Aust
     1996;164:107-8.
120. Kent C. Microbial menaces: long neglected system strains to respond to
     a rising threat. AMNews 1996;39:9-12.
121. Kent C. Microbial menaces: new era feared, when common bacteria will
     kill again. AMNews 1996;39:9-12.
122. Khan AS. Hantavirus pulmonary syndrome in Florida: association with
     the newly identified Black Creek Canal Virus. Am J Med 1996;100:46-8.
123. Kilstein J, Sala R, Greca A, Battagliotti C. Oxygen radicals and HIV
     infection: can antioxidants be of any use? [in Spanish]. Medicina
     1996;56:105-7.
124. King DE. Bronchodilators and antibiotics in the treatment of acute
     bronchitis. Arch Fam Med 1996;5
125. Kolmos HJ. Antibiotika i almen praksis (Antibiotics in general
     practice). Ugeskr Laeger 1996;158:258-60.
126. Kolmos HJ. Rationel anvendelse af antibiotika p hospital (The
     rationale for the use of antibiotics in hospitals). Ugeskr Laeger
     1996;158:255-7.
127. Koo D, Maloney K, Tauxe R. Epidemiology of diarrheal disease outbreaks
     on cruise ships: 1989 - 1993. JAMA 1996;275:545-7.
128. Koziel MJ. Immunology of viral hepatitis. Am J Med 1996;100:98-109.
129. Kristinsson KG. Epidemiology of penicillin resistant pneumococci.
     Laeknabladid 1996;82:9-19.
130. Kullberg BJ, Voss A. Het veranderend patroon van Candida-infecties:
     andere soorten en meer resistentie. [The changing pattern of Candida
     infections.] Ned Tijdschr Geneeskd 1996;140:148-151.
131. Lawson KA. Australian contributions to the study of infectious
     disease. Med J Aust 1996;164:113-5.
132. Lederberg J. Infection emergent. JAMA 1996;275:243-5.
133. LeDuc JW. World Health Organization: strategy for emerging infectious
     diseases. JAMA 1996;275:318-20.
134. Lee C-M, NG S-H, Wan Y-L, Tsai C-H. Gastric actinomycosis. J Formos
     Med Assoc 1996;95:66-8.
135. Lee T, Esterhuyse T, Steinberg M, Schneider H. Demographic modelling
     of the HIV/AIDS epidemic on the Soweto population- results and health
     policy implications. S Afr Med J 1996;86:60-3.
136. Lore W. Emerging and reemerging global microbial threats. East Afr Med
     J 1996;73:1-2.
137. Ludwig E. Changes in antibiotic usage. LAM 1996;6:pages not available.
138. Mackenzie JS, Smith DW. Mosquito-borne viruses and epidemic
     polyarthritis. Med J Aust 1996;164:90-3.
139. MacLean JD, Arthur JR, Ward BJ, Gyorkos TW, Curtis MA, Kokaskin E. The
     North American liver fluke: Metorchis conjunctus. Lancet
     1996;347:154-8.
140. Magnason S, Gudmundsson S, Stefnsson TSv, Erlendsdttir H,
     Baldursdttir L, Kristinsson KG. Nosocomial infections in the ICU at
     Landspitalinn: an interim report. Laeknabladid 1996;82:60-5.
141. Mainous AG III, Zoorob RJ, Hueston WJ. Current management of acute
     bronchitis in ambulatory care: the use of antibiotics and
     bronchodilators. Arch Fam Med 1996;5
142. Manders SM, Heymann WR, Atillasoy E, Kleeman J, Schlievert PM.
     Recurrent toxin-mediated perineal eyrhtema. Arch Dermatol
     1996;132:57-60.
143. Marriott D, McMurchie M. HIV and advanced immune deficiency. Med J
     Aust 1996;164:111-2.
144. Martnez-Vzquez JM, Cabarcos A. Present and future of the retroviral
     therapy. An Med Intern 1995;13:1-3.
145. McClure HM, Novembre FJ. Simian immunodeficiency virus variants:
     threat of new retroviruses. Am J Med Sci 1996;311:30-3.
146. McIntyre J. HIV/AIDS in South Africa - a relentless progression? S Afr
     Med J 1996;86:27-8.
147. Meer JWM van der, Hoogkamp-Korstanje JAA, Kager PA, Overbeke AJPM.
     Opdujkende pathogenen. [Emerging pathogens.] Ned Tijdschr Geneeskd
     1996;140:116-8.
148. Meis JFGM, Neeleman C. Resistentieproblemen bij Streptococcus
     pneumoniae. [Resistance problems with Streptococcus pneumoniae.] Ned
     Tijdschr Geneeskd 1996;140:141-4.
149. Mekasha A, Meharie S. Outbreak of louse-borne relapsing fever in
     Jimma, south western Ethiopia. East Afr Med J 1996;73:54-8.
150. Mhalu FS, Lyamuya E. Human immunodeficiency virus infection and AIDS
     in East Africa: challenges and possibilities for prevention and
     control. East Afr Med J 1996;73:13-9.
151. Mller AD, Gumundsson S, Gunnarsdttir K, Jnsson Th. Infections in
     the intensive care unit in Reykjavik City Hospital. Laeknabladid
     1996;82:46-52.
                                [end of page 369]

152. Morcillo N, Alito A, Romano MI, et al. A multidrug resistant
     tuberculosis outbreak in Buenos Aires: DNA fingerprinting analysis.
     Medicina 1996;56:45-7.
153. Mqoqi NP, Appleton CC, Dye AH. Prevalence and intensity of Schistosoma
     haematobium urinary schistosomiasis in the Port St Johns district. S
     Afr Med J 1996;86:76-80.
154. Mugerwa RD, Marum LH, Serwadda D. Human immunodeficiency virus/AIDS in
     Uganda. East Afr Med J 1996;73:20-26.
155. Mulhausen PL, Harrel LJ, Weinberger M, et al. Contrasting
     methicillin-resistant Staphylococcus aureus colonization in Veterans
     Administration and community nursing homes. Am J Med 1996;100:24-31.
156. Mutinga MJ. Phlebotomine sandflies (Diptera Psychodidae) their
     importance and some aspects of control. East Afr Med J 1996;73:48-9.
157. Nelson AM, Sledzik PS, Mullick FG. The Army Medical Museum/Armed
     Forces Institute of Pathology and emerging infections: from camp
     fevers and diarrhea during the American Civil War in the 1860s to
     global molecular epidemiology and pathology in the 1990s. Arch Pathol
     Lab Med 1996;120:129-33.
158. Nelson RP, Price LJ, Halsey AB, et al. Diagnosis of pediatric human
     immunodeficiency virus infection by means of a commercially available
     polymerase chain reaction gene amplification. Arch Pediatr Adolesc Med
     1996;150:40-5.
159. Nolte KB, Simpson GL, Parrish RG. Emerging infectious agents and the
     forensic pathologist: the New Mexico model. Arch Pathol Lab Med
     1996;120:125-8.
160. Olafsson M, Sigurdsson JA. Increased antimicrobial resistance in
     urinary tract infections. Laeknabladid 1996;82:66-70.
161. Olliaro P, Cattani J, Wirth D. Malaria: the submerged disease. JAMA
     1996;275:230-3.
162. Oloo AJ, Vulule JM, Koech DK. Some emerging issues on the malaria
     problem in Kenya. East Afr Med J 1996;73:50-3.
163. Osborn JE. Reemerging pathogens. West J Med 1996;164:23-4.
164. Parisi MN, Enra D, Pini N, Sabattini M. Retrospective detection of
     clinical infection caused by hantavirus. Medicina 1996;56:1-13.
165. Patz JA, Epstein PR, Burke TA, Balbus JM. Global climate change and
     emerging infectious diseases. JAMA 1996;275:217-23.
166. Peters CJ. Emerging infectionsebola and other filoviruses. West J Med
     1996;164:36-8.
167. Petersen EA. Emerging infectious diseases. Arch Intern Med
     1996;156:124.
168. Pilote L, Tulsky JP, Zolopa AR, Hahn JA, Schecter GF, Moss AR.
     Tuberculosis prophylaxis in the homeless: a trial to improve adherence
     to referral. Arch Intern Med 1996;156:161-5.
169. Pinner RW. Addressing the challenges of emerging infectious diseases.
     Am J Med Sci 1996;311:3-8.
170. Pinner RW, Teutsch SM, Simonsen L, et al. Trends in infectious
     diseases mortality in the United States. JAMA 1996;275:189-93.
171. Plouffe JF, Breiman RF, Facklam RR, for Franklin County Pneumonia
     Study Group. Bacteremia with Streptococcus pneumoniae: implications
     for therapy and prevention. JAMA 1996;275:194-8.
172. Povinelli M, Remafedi G, Tao G. Trends and predictors of human
     immunodeficiency virus antibody testing by homosexual and bisexual
     adolescent males, 1980-1994. Arch Pediatr Adolesc Med 1996;150:33-8.
173. Prabhakar R. Tuberculosis--the continuing scourge of India. Indian J
     Med Res 1996;103:19-25.
174. Prado V. Emerging infections: a new problem? Rev Med Chil
     1996;124:1-4.
175. Pretorius GS, Sirgel FA, Schaaf HS, van Helden PD, Victor TC.
     Rifampicin resistance in Mycobacterium tuberculosis - rapid detection
     and implications in chemotherapy. S Afr Med J 1996;86:50-5.
176. Quinlan KP, Hayani KC. Vitamin A and respiratory syncytial virus
     infection: serum levels and supplementation trial. Arch Pediatr
     Adolesc Med 1996;150:25-30.
177. Rathinamisivakumar, Ratnam S, Sureshbabu L, Natarajaseenivasan K.
     Leptospiral antibodies in patients with recurrent ophthalmic
     involvement. Indian J Med Res 1996;103:66-8.
178. Reichman L. How to ensure the continual resurgence of tuberculosis.
     Lancet 1996;347:pages not yet available.
179. Samore MH, Venkatarami L, DeGirol PC, et al. Clinical and molecular
     epidemiology of sporadic and clustered cases of nosocomial Clostridium
     difficile diarrhea. Am J Med 1996;100:32-40.
180. Sande M. The public health risk of emerging and reemerging infections.
     West J Med 1996;164:18-20.
181. Sanders EJ, Tukei PM. Yellow fever an emerging threat for Kenya and
     other East African countries. East Afr Med J.1996;73:10-2.
182. Satyanarayana K, Medappa N. Emerging and reemerging infections. Indian
     J Med Res 1996;103:1-3.
183. Schellekens JFP. Kattekrabziekte en andere infecties met
     Bartonella-species. [Cat-scratch disease and other infections of the
     Bartonella species.] Ned Tijdschr Geneeskd 1996;140:144-7.
184. Schmitt ED, Camozzi S, Vigo G, Tadini G. Tinea corporis in an HIV-1
     infected patient resembled dermatophyte colonies on Sabouraud's agar.
     Arch Dermatol 1996;132:233-4.
185. Schmitt H-J, von Knig CHW, Neiss A, et al. Efficacy of an acellular
     pertussis vaccine in infants after household exposure to pertussis.
     JAMA 1996;275:37-41.
186. Schwartz DA, Bryan RT. Infectious disease pathology and emerging
     infections: are we prepared? Arch Pathol Lab Med 1996;120:117-24.
                                [end of page 370]

187. Schwartz DA, Sobottka I, Leitch GJ, Cali A, Visvesvara GS. Pathology
     of microsporidiosis: emerging parasitic infections in patients with
     AIDS. Arch Pathol Lab Med 1996;120:173-88.
188. Shah K, Davis C, Wilson J, Parekh B. Chimeric synthetic peptides as
     antigens for detection of the antibodies to HIV-1 and HIV-2. East Afr
     Med J 1996;73:63-6.
189. Sharma VP. Reemergence of malaria in India. Indian J Med Res
     1996;103:26-45.
190. Sharp B, le Sueur D. Malaria control in South Africa - the past, the
     present and selected implications for the future. S Afr Med J
     1996;86:83-9.
191. Somekh E, Abishai V, Hanani M, Gutman R, Mintz M. The clinical
     significance of Pseudomonas aeruginosa isolation from stool of
     neonates. Arch Pediatr Adolesc Med 1996;150:108-9.
192. Soni PN, Tait DR, Gopaul W, Sathar MA, Simjee AE. Hepatitis C virus
     infection in chronic liver disease in Natal. S Afr Med J 1996;86:80-3.
193. Speed BR, Gerrard MP, Kennett ML, Carron MG, Harvey BM. Viral
     haemorrhagic fevers - current status, future threats. Med J Aust
     1996;164:79-83.
194. St. Geme JW III. Progress towards a vaccine for nontypable Haemophilus
     influenzae. Ann Med 1996;28:31-7.
195. Stanton BF, Li X, Galbraith J, Feigelman S, Kaljee L. Sexually
     transmitted diseases, human immunodeficiency virus, and pregnancy
     prevention: combined contraceptive practices among urban
     African-American early adolescents. Arch Pediatr Adolesc Med
     1996;150:17-24.
196. SAtephens DS, Farley MM. Emerging and reemerging microbial threats. Am
     J Med Sci 1996;311:1-2.
197. Stevens DL. Group A streptococcusfrom basic science to clinical
     disease. West J Med 1996;164:25-7.
198. Stewart GJ. HIV medicine in the mainstream. Med J Aust 1996;164:97-8.
199. Stewart GJ, Irvine SS, Scott M, et al. Strategies of care in managing
     HIV. Med J Aust 1996;164:99-104.
200. Strange P, Skov L, Lisby S, Lovgreen P. Stahpyloccocal enterotoxin B
     applied on intact normal and intact atopic skin induces dermatitis.
     Arch Dermatol 1996;132:27-33.
201. Straub I. Epidemiological situation in Hungary: past, present and
     future. LAM 1996;6:pages not available.
202. Su I-J, Huang L-M, Wu S-J, et al. Detection and sequence analysis of a
     new herpesvirus-like agent (KSHV) in AIDS and non-AIDS Kaposi's
     sarcoma in Taiwan: a comparison with related herpesviruses. J Formos
     Med Assoc 1996;95:13-8.
203. Subtype B isolates of the human immunodeficiency virus type 1 detected
     in Australia. Ann Acad Med Singapore 1996;25(March):pages not yet
     available.
204. Susser E, Neugebauer R, Hoek HW, et al. Schizophrenia after prenatal
     famine: further evidence. Arch Gen Psychiatry 1996;53:25-31.
205. Szalka A, Printz G. Hanta viruses and the pulmonal syndrome caused by
     hanta virus. LAM 1996;6:pages not available.
206. Taller AM, Asher DM, Pomeroy KL, et al. Search for viral nucleic acid
     sequences in brain tissues of patients with schizophrenia using nested
     polymerase chain reaction. Arch Gen Psychiatry 1996;53:32-40.
207. Tamssy K, Microbiological diagnosis and complex treatment of
     gastrobulbar alterations: the role of Helicobacter pylori. LAM
     1996;6:pages not available.
208. Tambi A, Tambi T, Kuiec-Tepe N. Prevalence and antibiotic
     sensitivity pattern variations of bacterial isolates in different
     settings and different periods of time. Acta Med Croatica 1996;pages
     not available.
209. Tappero JW, Khan AS, Pinner RW, et al. Hantavirus pulmonary syndrome
     1993: utility of an emergency, telephone-based national surveillance
     system. JAMA 1996;275:398-400.
210. Tenover FC, McGowan JE. Reasons for the emergence of antibiotic
     resistance. Am J Med Sci 1996;311:9-16.
211. Tenover FC, Hughes JM. The challenges of emerging infectious diseases:
     development and spread of multiply resistant bacterial pathogens. JAMA
     1996;275:300-4.
212. Thomas DL, Gruninger SE, Siew C, et al. Occupational risk of hepatitis
     C infections among general dentists and oral surgeons in North
     America. Am J Med 1996;100:41-5.
213. Thompson J. Enterokokken: toename van het aantal infecties en van
     antibioticumresistentie. [Enterococci: increase of the number of
     infections and of antimicrobial resistance.] Ned Tijdschr Geneeskd
     1996;140:130-3.
214. Thornsberry C. Emerging resistance in clinically important gram
     positive cocci. West J Med 1996;164:28-32.
215. Thorsteinsson SB. Infectious diseases in Iceland: current issues and
     future challenges. Laeknabladid 1996;82:6-7.
216. Tompkins LS. Bartonella species infections, including cat-scratch
     disease, trench fever, and bacillary angiomatosiswhat molecular
     techniques have revealed. West J Med 1996;164:39-41.
217. Tswana S, Chetsanga C, Nystrm L, Moyo S, Nzara M, Chieza L. A
     sero-epidemiological cross-sectional study of hepatitis B virus in
     Zimbabwe. S Afr Med J 1996;86:72-5.
218. Tukei PM. The threat of Marburg and Ebola viral haemorrhagic fever in
     Africa. East Afr Med J 1996;73:27-31.
219. Turnidge JD, Nimmo GR, Francis G, and the AGAR. Evolution of
     resistance in Staphylococcus areus in Australian teaching hospitals.
     Med J Aust 1996;164:68-71.
220. Ujhelyi E. The update of the croup-syndrome. LAM 1996;6:pages not
     available.
221. Vandenbroucke-Grauls CMJE. Meticilline-resistente Staphylococcus
     epidermidis. [Meticillin-resistant Staphylococcus epidermidis.] Ned
     Tijdschr Geneeskd 1996;140:138-41.
                                [end of page 371]

222. Vogel P, Abplanalp D, Kell W, et al. Venezuelan equine encephalitis in
     BALB/c mice: kinetic analysis of central nervous system infection
     following aerosol or subcutaneous inoculation. Arch Pathol Lab Med
     1996;120:164-72.
223. Waiyaki PG. Cholera: its story in Africa with special reference to
     Kenya and other east African countries. East Afr Med J 1996;73:40-3.
224. Walker DH, Barbour AG, Oliver JH, et al. Emerging bacterial zoonotic
     and vector-borne diseases: ecologic and epidemiologic factors. JAMA
     1996;275:463-9.
225. Wallace MR, Sharp TW, Smoak B, et al. Malaria among US troops in
     Somalia. Am J Med 1996;100:49-55.
226. Wann S-R, Liu Y-C, Yen M-Y, Wang J-S, Chen Y-S, Wang J-H, Cheng D-L.
     Endogenous Escherichia coli endophthalmitis. J Formos Med Assoc
     1996;95:56-60.
227. Warren R, Hauman J, Beyers N, et al. Unexpectedly high strain
     diversity of M tuberculosis in a high incidence community. S Afr Med J
     1996;86:45-9.
228. Weissenbacher M, Cura E, Segura E, et al. Serological evidence of
     human hantavirus infection in Argentina, Bolivia and Uruguay. Medicina
     1996;56:17-22.
229. Whitcup SM. Ocular manifestations of AIDS. JAMA 1996;275:142-4.
230. Wilkinson D, de Cock KM. Tuberculosis control in South Africa - time
     for a new paradigm? S Afr Med J 1996;86:33-5.
231. Wilkinson D, Moore DAJ. HIV-related tuberculosis in South Africa -
     clinical features and outcome. S Afr Med J. 1996;86:64-7.
232. Williamson H. Treatment of acute bronchitis: there's much work to be
     done. Arch Fam Med 1996;5
233. Winker MA, Flanagin A. Infectious diseases: a global approach to a
     global problem. JAMA 1996;275:245-6.
234. Woelffer GB, Bradford WZ, Paz A, Small PM. A computer assisted
     molecular epidemiologic approach for confronting the reemergences of
     tuberculosis. Am J Med Sci 1996;311:17-22.
235. Xu J, Cheng B, Wu Y, et al. A new diarrhea pathogen:
     entero-adherent-invasive-toxigenic Escherichia coli. Chin Med J
     1996;109:16-7.
236. Yahav J, Oderda G, Diver-Haber A, et al. Serum pepsinogen I in
     childhood Helicobacter pylori gastritis: its relation to mucosal
     peptic activity. Isr J Med Sc 1996;32:56-9.
237. Yao C, Wang W-W, Chung Y-M, Su Y-L, Liu C-Y, Chen Y-M A.
     Transfusion-transmitted AIDS: report of the first case in Taiwan. J
     Formos Med Assoc 1996;95:51-5.
238. Young LHY. Therapy for cytomegalovirus retinitis: still no silver
     lining. JAMA 1996;275:149-50.
239. Ytterdahl T. Tropical diseases: an interview with David Stevenson, MD.
     Tidsskr Nor Laegeforen 1996;116:(January):pages not yet available.
240. Zaki SR, Khan AS, Goodman, et al. Retrospective diagnosis of
     hantavirus pulmonary syndrome, 1978-1993: implications for emerging
     infectious diseases. Arch Pathol Lab Med 1996;120:134-9.
241. Zhu CM. Comments on some new viruses associated with old diseases in
     China. Chin Med J 1996;109:5-10.

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


---------------------------------------------------------------------------
Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
---------------------------------------------------------------------------
News and Notes

Conference on Emerging Foodborne Pathogens

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


The conference on "Emerging Foodborne Pathogens: Implications and Control,"
March 24-26, 1997, Radisson Plaza Hotel at Mark Center, Alexandria,
Virginia, USA, is organized by the International Life Sciences Institute
(ILSI), ILSI North America Technical Committee on Food Microbiology, the
U.S. Centers for Disease Control and Prevention, Department of Agriculture,
and Food and Drug Administration, in cooperation with the Food and
Agriculture Organization of the United Nations, and the Pan American Health
Organization/World Health Organization. The conference will review the
lessons learned and knowledge gained concerning the emergence/reemergence
and dissemination of food-related microbial threats to health; identify
factors that foster emergence/reemergence and dissemination of these
hazards; identify scientific and food safety strategies to address emerging
foodborne microbial hazards; and identify future research needs. The
conference will be of special interest to food protection and public health
professionals, including microbiologists, epidemiologists, physicians, and
health policy makers; industry, academic, and government researchers; and
others interested in microbial food safety hazards.

For program and registration information, contact

Ms. Diane Dalisera
Emerging Foodborne Pathogens Conference International Life Sciences
Institute (ILSI)
1126 Sixteenth Street, NW
Washington, DC 20036-4810, USA
Telephone: 202-659-0074
Fax: 202-659-3859
E-mail: meetings@dc.ilsi.org
                             [end of page 372]

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