---------------------------------------------------------------------------
 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
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                             Table of Contents

Perspective

   * Designing an International Policy and Legal Framework for the Control
     of Emerging Infectious Diseases: First Steps, B.J. Plotkin and A.M.
     Kimball

Synopses

   * Surface Antigens of the Syphilis Spirochete and Their Potential as
     Virulence Determinants, D.R. Blanco, J.N. Miller, and M.A. Lovett
   * Mycoplasmas: Sophisticated, Reemerging, and Burdened by Their
     Notoriety, J.B. Baseman and J.G. Tully
   * Fluoroquinolone Resistance in Neisseria gonorrhoeae, J.S. Knapp, K.K.
     Fox, D.L. Trees, and W.L. Whittington
   * Infectious Diseases and Immunity: Special Reference to Major
     Histocompatibility Complex, N. Singh, S. Agrawal, and A.K. Rastogi
   * Cryptosporidiosis: An Emerging, Highly Infectious Threat, R. L.
     Guerrant

Dispatches

   * Isolation and Phylogenetic Characterization of Ebola Viruses ausing
     Different Outbreaks in Gabon , M-C. Georges-Courbot, A. Sanchez, C-Y
     Lu, S. Baize, E. Leroy, J. Lansout-Soukate, C. Tvi-Bnissan,
     A.J. Georges, S.G. Trappier, S.R. Zaki, R. Swanepoel, P.A. Leman, P.E.
     Rollin, C.J. Peters, S.T. Nichol, and T.G. Ksiazek
   * The Epidemiology of Creutzfeldt-Jakob Disease in Canada: A Review of
     Mortality Data, E. Stratton, M.N. Ricketts, and P.R. Gully
   * Exudative Pharyngitis Possibly Due to Corynebacterium
     pseudodiptheriticum, a New Challenge in the Differential Diagnosis of
     Diphtheria, H.S. Izurieta, P.M. Strebel, T. Youngblood, D.G. Hollis,
     and T. Popovic
   * Mycoplasmal Conjunctivitis in Wild Songbirds: The Spread of a New
     Contagious Disease in a Mobile Host, J.R. Fischer, D.E. Stallknecht,
     M.P. Luttrell, A.A. Dhondt,and K.A. Converse
   * Knowledge-Based Patient Screening for Rare and Emerging
     Infectious/Parasitic Diseases: A Case Study of Brucellosis and Murine
     Typhus, C.N. Carter, N.C. Ronald, J.H. Steele, E. Young, J.P. Taylor,
     L.H. Russell, Jr., A.K. Eugster, and J.E. West

Letters

   * Emergence of Epidemic O'nyong-nyong Fever in Southwestern Uganda,
     After an Absence of 35 Years, E.B. Rwaguma, J.J. Lutwama, S.D.K.
     Sempala, N. Kiwanuka, J. Kamugisha, S. Okware, G. Bagambisa, R.
     Lanciotti, J.T. Roehrig, and D.J. Gubler
   * Prostatitis and Benign Prostatic Hyperplasia: Emerging Infectious
     Diseases? B. Hennenfent
   * Risk Factors for Severe Leptospirosis in the Parish of St. Andrew,
     Barbados, C.P. Douglin, C. Jordan, R. Rock, A. Hurley, and P.N. Levett
   * Electronic Communication and the Rapid Dissemination of Public Health
     Information, C.B. Dalton, P.M. Griffin, and L. Slutsker

News and Notes

   * Emerging Infectious Diseases Conference
   * "Emergent Illness and Public Scholarship" Fellowships
   * International Workshop on Molecular Epidemiology and Evolutionary
     Genetics

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                            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
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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

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

William J. Martone, M.D., Senior Executive Director
National Foundation for Infectious Diseases
Bethesda, Maryland, 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 UnitKenya
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
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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

Cover Design
Mindy Barringer
Martha F. Boyd
  ------------------------------------------------------------------------

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
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All material published in Emerging Infectious Diseases is in the public
domain and may be used and reprinted without special permission; proper
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Use of trade names is for identification only and does not imply
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                        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,
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proposed articles may be directed to the editor at 404-639-3967
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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://fcv.medvet.unlp.edu.ar).
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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
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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,
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Send all manuscripts and correspondence to the Editor, Emerging Infectious
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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.
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 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
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Perspective

Designing an International Policy and Legal Framework for the Control of
Emerging Infectious Diseases: First Steps

Bruce Jay Plotkin* and Ann Marie Kimball
*School of Law; School of Public Health and Community Medicine, University
of Washington, Seattle, Washington, USA
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      As the pace of emergence and reemergence of infectious
      diseases quickens, the International Health Regulations, which
      have served as the legal and policy framework of epidemic
      control for 45 years, are being revised by the World Health
      Organization (WHO). In this article, we review the recent
      history, legal construction, and application of these
      regulations and related international treaty-based sanitary
      measures, especially the General Agreement on Tariffs and
      Trade and the Agreement on the Application of Sanitary and
      Phytosanitary Measures, and the history of applying the
      regulations in the maritime and aviation industries. This
      review indicates that revision efforts should address 1) the
      limited scope of disease syndromes (and reporters of these
      syndromes) now in the regulations and 2) the mismatch between
      multisectoral factors causing disease emergence and the single
      agency (WHO) administering the regulations. The revised
      regulations should expand the scope of reporting and
      simultaneously broaden international agency coordination.

The Division of Emerging and Other Communicable Disease Surveillance and
Control, World Health Organization (WHO), is revising the often criticized
International Health Regulations (referred to as regulations hereon), the
central legal framework for addressing the international spread and control
of infectious disease (1,2). This process is under way as emerging and
reemerging infectious diseases are the objects of broadening efforts and
analysis by the national (3) and international public health communities
(4). At least 30 new diseases have emerged in the last 20 years, and still
others, including tuberculosis, malaria, cholera, dysentery, and pneumonia,
have developed varying degrees of resistance to antimicrobial drugs (4).

The Intersectoral Nature of the Factors Involved in Disease Emergence

Infectious diseases are emerging in the increasingly global context of
commercial and demographic activities. The journey of microbial agents from
one country to another, often shorter than the incubation period of the
disease, is rendering border controls futile. The response to emerging
diseases is increasingly global as well: national agencies, international
organizations, and other groups coordinate efforts to monitor, prevent, and
control the spread of these diseases (5). While no substitute for adequate
national health services and infrastructure, international efforts against
emerging diseases have increased in importance as national programs have
been compromised by complacency, economic recession, international debt,
civil turmoil, or natural disasters (3,5,6). At the same time, detecting
new agents often requires up-to-date diagnostic facilities unavailable in
many parts of the world. International agencies have improved access to
such facilities to help define the causes of disease outbreaks.

The Current Legal Framework: International Health Regulations

The regulations, which play a central, albeit limited, role in addressing
global disease outbreaks (Table 1), have served as the primary legally
binding framework for preventing the international spread of infectious
disease (Table 2). In addition to its other legal and policy options for
promoting international public health, WHO's World Health Assembly is
specifically authorized to adopt regulations concerning "sanitary and
quarantine requirements and other procedures designed to prevent the
international spread of disease" (7, art. 21[a]). The twin objectives of
the regulations balance "maximum security against the international spread
of diseases with a minimum interference with world traffic" (1, Foreword).

          Table 1. Selected international health regulatory
          provisions pertinent to emergent infectious disease
          -------------------------------------------------------
                     Provision              Source/Lead Agency
          -------------------------------------------------------
          International Health            World Health
          Regulations                     Organization
          Agreement on the Application
          of Sanitary and Phytosanitary   World Trade
          Measures                        Organization

          Codex Alimentarius              FAO/WHO Codex
                                          Alimentarius Commission
          Annex 9: Facilitation to the
          Convention on International     International Civil
          Civil Aviation                  Aviation Organization
          -------------------------------------------------------

          Table 2. Development of the International Health
          Regulations
          --------------------------------------------------------
          Year(s)                     Action
          --------------------------------------------------------

          1951   WHO Int'l Sanitary Regulations and additional
                 regulations in 1955, 1956, 1960, 1963, 1965)

          1969   Revised and renamed Int'l Health Regulations
                 (regulations)
          1973   Regulations revised
          1981   Regulations revised (current version)
          1995   WHO resolves to revise regulations
          --------------------------------------------------------

The regulations evolved from efforts to deal with epidemics. The use of
quarantine dates back at least to the Middle Ages (3). Steps to regulate
countries' actions to protect themselves against introduction of
communicable diseases and to report disease outbreaks developed before the
International Sanitary Regulations were adopted in 1951 (8). Inadequacies
of the current regulations have been associated with outdated quarantine
and frontier-based practices.

The current regulations outline procedures for limiting disease
transmission in international traffic, including the disinsectization,
disinfection, and deratting of ships and aircraft and the provision of
sanitary conditions and health facilities at sea and airports. In addition,
the regulations focus on two core obligations of member states relating to
disease incidence reporting and response; these obligations apply primarily
to the three diseases subject to the regulations: cholera, plague, and
yellow fever (1, art. 1). First, states must report to WHO, within specific
periods, cases of these three diseases within their territories (1, arts.
3-7,9,11-12). Second, to facilitate reporting and deter unnecessary
interference with international travel and trade, members must limit their
responsive health measures (applied to international traffic for the
protection of their territories against these diseases) to maximum measures
permitted by these regulations (1, art. 23). States must also report to WHO
any health measures they have applied to international traffic (1, art. 8).
Most of such permitted health measures (and related provisions in the
regulations limiting or prohibiting health measures) focus on cholera,
plague, and yellow fever; some more general provisions, however, limit
measures directed at other diseases (1, arts. 28, 29, 31, 81; 9).

Like most international agreements, the regulations are to be implemented
through national laws and policies that incorporate or otherwise
accommodate the regulations' various provisions, minimum requirements, and
limitations. The regulations have served as international reference
standards for some states creating their own national quarantine
provisions; this relationship magnifies the effect of outdated and
problematic aspects of the regulations (and increases the urgency of their
timely revision).

Even though the regulations pose a legal obligation for WHO members who
have not officially "opted out" from participating, lack of compliance has
been an ongoing problem (8,9). The regulations have weaknesses: 1) the
limited scope of reported information vis-a-vis the burgeoning scope of new
infections, and 2) the mismatch between the narrow institutional,
political, and legal bases of the regulations issued under the
circumscribed authority of a single specialized United Nations (U.N.)
agency, WHO, and the varied factors affecting the international emergence
and control of infectious disease. The factors include international trade
and travel, economic development and land use, changes in human
demographics and behavior, and the breakdown of public health
infrastructure (3). WHO's broad mandate within the U.N. system (10, art.
57) and under the WHO Constitution (7, preamble, arts. 1-2), to address
these factors is ultimately rooted in the one agency. While WHO's natural
institutional allies tend to be national health ministries, the factors
affecting disease emergence are multisectoral and can be more directly
addressed by a constituency of international organizations and agencies.
WHO members' violations of regulations during epidemics (e.g., the
implementation of unjustified and illegal health-based trade barriers) are
critical symptoms of the institutional mismatch. Such barriers are rarely
the product of sole actions by the Ministry of Health, and WHO has little
influence over other national agencies such as Ministries of Trade,
Commerce, or Planning.

These core weaknesses are exacerbated by the administration of regulations.
In spite of the regulations' strong legal basis, WHO has frequently
preferred nonmandatory urging or mediation to a legally binding approach to
members' obligations (8,11). In the area of trade, for example, a Pan
American Health Organization epidemiologic bulletin on the 1991 cholera
outbreak in Latin America referred to unjustified trade restrictions
imposed on Peruvian marine exports as "not in accordance with the
recommendations of WHO" (12). More substantive is the ambiguous,
indeterminate, or otherwise vague nature of many of the regulations'
articles, which create further difficulties in application (1, arts. 46[1]
and 36 [3]). WHO is unlikely to use formal mandatory enforcement such as
sanctions against member states who do not comply with the regulations
(13). Under the WHO Constitution, typical of such treaties (13), there are
no formal punitive sanctions. While under article 7 of the Constitution,
the World Health Assembly is authorized in "exceptional circumstances" to
withdraw membership privileges, under treaties, such provisions are rarely
invoked and then usually on political grounds against otherwise
marginalized states (13). These sanctions would also hinder the WHO
objectives of tracking, controlling, and preventing incidence and
transmission of disease.

The regulations contain a dispute resolution provision that authorizes
member states to refer "any question or dispute" concerning the regulations
to the Director General or a WHO committee to "settle," rather than to
enforce (1, art. 93[1]). If this referral process fails, a member state is
authorized to bring the dispute to the International Court of Justice in
the Hague for decision (1, art. 93[3]). The International Court of Justice
has, however, never determined a case under the regulations and is a
relatively rare choice for disposition of international disputes (13).
WHO's right to request advisory opinions from the International Court of
Justice has yielded court rulings in only two cases. Thus, disputes appear
to be usually handled informally through the WHO bureaucracy (8).

In this article, we propose two steps for improving the effectiveness of
the regulations. Some of the following issues are addressed in the December
1995 Report of WHO Informal Consultation of Experts on Revision of the
Regulations (14,15).

Step One: Expand the Information Base

The Limited Scope of Reporting Under the Regulations
The current narrow scope of disease reporting undercuts the relevance of
the regulations and has been criticized (16). In contrast to the long list
of known emerging and other infectious diseases that threaten world
communities and the threats posed by as yet unknown or unrecognized
diseases or syndromes, only three diseases (cholera, plague, and yellow
fever) are expressly covered by the regulations' reporting requirements.

Additional obstacles to effectiveness and compliance regarding both
outbreak reporting and minimizing of health restrictions arise from
limiting the range of information sources that can report on these issues
to WHO. WHO has been criticized for relying solely on information reported
officially by member states regarding outbreaks within their borders or
health measures applied to traffic from the country involved in an outbreak
(9). To avoid self-incrimination, members do not report on a timely basis,
if at all, as required (8,9,16). A related problem arises from the right of
the affected country's health administration to determine the "infected
area" of an outbreak (1, art. 1), although that decision can influence
health measures other countries may apply to persons, vehicles, and cargo
from the outbreak area (1, arts. 46[1], 59, 64[2], and 66). WHO appears to
limit its sources for listing infected areas in the Weekly Epidemiological
Review "only [to] official governmental information" (17).

Consequences of an Inadequate Information Base
Driven by political and economic pressures and other concerns (8,18),
neighbors or trading partners of countries affected by epidemics often
overreact by setting up border or other restrictions in excess of those
permitted under the regulations. From the beginning of the seventh cholera
pandemic into Latin America in 1991, for example, trading partners of
affected countriesparticularly Peruat times rejected food imports (19) or
even non-food manufactured goods (20), restricted travel (19), or closed
their borders. Peru lost an estimated $770 million because of the epidemic
(4). In response to the 1994 plague outbreak in India, some countries
severed air and shipping links with India; the country sustained a reported
$1.3 billion in export losses in 2 months (21). Concerns about potential
trade and travel restrictions have caused countries not to report outbreaks
within their borders (8,12,22).

Proposed Solutions to Information Weaknesses in Regulations
The regulations' disease coverage, reporting of outbreaks, and disease
incidence (and permitted responsive health measures) should be based upon
an up-to-date schedule of the most relevant clinical syndromes in
conjunction with a broadened list of relevant serious diseases. Syndromes
should be reportable until the underlying disease is identified.
Descriptions of syndromes and appropriate and inappropriate health
responses should be stated as clearly and specifically as possible in the
regulations. This approach would align the coverage of the binding
regulations with the most appropriate and dangerous diseases; it would also
facilitate more direct and coherent risk evaluation and present a framework
for addressing unknown or unrecognized diseases and syndromes. It is also
likely to expedite reporting as there would be no need for disease
identification. It may also stimulate participation by member states that
have ceased complying with what they regard as an outdated system (16). An
operations research effort by WHO and its member states could facilitate
the adoption of these suggested improvements by testing the sensitivity and
specificity of syndromic outbreak reporting.

Similarly, to enhance the effectiveness of the regulations, the acceptable
sources of information on disease outbreaks and health measures put in
place by member states under the regulations should be broadened to include
other reliable sources. WHO's use and acceptance of reliable outside
information for such determinations will provide more accurate information
and may also prompt more timely reporting by the affected countries faced
with preemption by WHO and others.

The complex issues arising in international relations are discussed
elsewhere (14,23-26), but the history of noncooperation with the
regulations does not preclude potential for improved compliance in the
future. If the regulations are substantially and meaningfully revised
(14,15) and key countries are sufficiently concerned about the dangers of
emerging diseases to press for compliance with the revised scheme,
compliance should improve (14).

Step Two: Expand Policy and Programmatic Collaboration

International organizations other than WHO deal more directly with the
underlying issues affecting the transmission of disease. The expansion of
international regulatory provisions in all areas has increased the
potential for overlapping policies and even regulation.

Interorganizational and Interagency Consultations
Revising the regulations presents an opportunity to establish
interorganizational and interagency consultations and address potential
contributions of agencies and organizations that can most directly affect
the factors involved in the emergence and control of infectious disease. A
first step would be an interorganizational summit led by WHO to examine
potential joint or coordinated programs. The breadth and focus of the
summit will depend on the specific programs most compatible with such an
approach. WHO has a long record of cooperation with other agencies and
organizations, and joint activities are part of its programs addressing
infectious disease control (4). Contacts need not be limited to the
WHO-affiliated international organizations but can include other groups:
WHO will need to evaluate whether participation by such agencies,
nongovernmental organizations, and other entities would be beneficial in a
particular context, and if so, at what points in the process.

Establishing new connections and building on existing relationships between
WHO and other institutions and organizations (e.g., the World Trade
Organization, the International Civil Aviation Organization, and the
International Maritime Organization) present many benefits: WHO can bring
its influence closer to the underlying processes and organizational
entities directly involved in disease emergence and control; it can more
effectively use its limited resources; and it can address initiatives in
other organizations that may be insufficiently sensitive to key public
health concerns. From an international relations perspective, such contacts
may ultimately develop into direct links between the administration of WHO
regulations and that of other international or multilateral organizations.

World Trade Organization
Health-related trade restrictions are regulated by multilateral
organizations and agreements (Table 1), such as the recently established
World Trade Organization (WTO) and its related multilateral agreements,
including the General Agreement on Tariffs and Trade (GATT) and the
Agreement on the Application of Sanitary and Phytosanitary Measures (SPS
agreement). Like the WHO regulations, parts of this complex trade
regulatory scheme address the health-based trade constraints that countries
may implement to protect their citizens and territories from infectious
disease threats. Peru's appeal to the GATT Council for assistance in
protecting its exports from unjustified rejection during the 1991 cholera
outbreak at the same time that PAHO was directly involved in dealing with
the epidemic exemplifies this potential overlap of regulation (20).

The WTO agreements specify which health-based trade barriers that would
otherwise violate a trade rule of WTO may be justified under the exceptions
in GATT article XX(b) for measures "necessary to protect human, animal, or
plant life or health" and interalia under the related SPS agreement that
governs health measures pertinent to most infectious disease threats.
Therefore, a WTO member's "sanitary and phytosanitary measures" (certain
health-based trade restrictions) that meet the SPS agreement's detailed
requirements are deemed in compliance with GATT's XX(b) exception. The SPS
agreement addresses all sanitary and phytosanitary measures that a member
may apply that potentially affect international trade (27, arts. 1, 2.4)
The WTO agreements, in contrast to the WHO regulations, are not
disease-specific. The sanitary and phytosanitary measures described in the
SPS agreement focus on risks to humans from diseases carried by animals,
plants, and their products; the entry or spread of pests; and additives,
contaminants, toxins, and disease-causing organisms in food and beverages
(27,Annex A, par. 1). The SPS agreement contains measures focusing on, for
example, cholera as transmitted in international trade in food and
beverages and insect-borne disease risks in international trade. Measures
directed against many health risks to animals and plants are also covered
(27, Annex A, par. 1). Although the SPS agreement does not refer
specifically to epidemics, its stated scope is broad enough to cover them,
assuming the agreement's various requirements are met (27, arts. 5.7 and
Annex B, pars. 2,6). However, aspects of the relevant applications of the
SPS agreement are as yet not entirely clear. The WTO agreement and related
Dispute Settlement Understanding have been in force only since 1995.
Measures directed at trade-related infectious disease risks not covered
under the SPS agreement may be addressed by the Separate Agreement on
Technical Barriers to Trade, which concerns product standards, or by the
GATT article XX[b] exception itself (28).

Like those of the regulations, the general purposes of the SPS agreement
include limiting health-based restrictions to those that are necessary and
as minimally burdensome to trade as possible. The SPS agreement provides
detailed rules and standards for determining what sanitary or phytosanitary
measures are permitted on the basis of scientific support, risk assessment,
and other factors (27, arts. 2.2, 3.3, 5 and Annex A par. 4), as well as
numerous other provisions. The SPS agreement provides, for members' use,
where applicable, certain established "international standards, guidelines
or recommendations" (rather than their own standards) to promote
international consistency on these measures. A member must justify,
scientifically and otherwise, implementation of higher standards if they
result in a greater restriction on trade than the stipulated international
standard (27, art. 3 and Annex A, pars. 2-3). For example, regarding food
safety issues, the SPS agreement designates the "standards, guidelines and
recommendations" established by the WHO Food and Agriculture Organization
Codex Alimentarius Commission. The Codex Alimentarius is an extensive code
that addresses a broad range of food production issues including food
additives, limits on pesticide residues, food labeling requirements,
product composition, recommendations on food processing techniques, and
suggested inspection procedures regarding food products and production (27,
Annex A, par. 3[a]). However, the regulations do not mention the Codex
(although it is a key part of WHO guidelines on food issues).

WTO mechanisms for dispute resolution differ substantially from those of
the WHO regulations: among other differences, ultimate adjudication
authority remains within WTO. Under GATT articles XXII and XXIII and the
Understanding on Rules and Procedures Governing the Settlement of Disputes
(Dispute Settlement Understanding), if the disputing parties' consultation
and other preliminary steps do not resolve a dispute, they ordinarily
resort to adjudication before a WTO three-person panel (29). The panel's
report (including its recommendation if a violation has been found) is
adopted by the WTO members (sitting as the Dispute Settlement Body), unless
there is a consensus to reject it. There is also a potential appellate
procedure, before the seven-person Appellate Body, regarding legal issues.
If the challenged trade restriction is found to be unjustified (and the
related rulings, usually to bring the violating measure into compliance,
are not implemented nor is negotiated compensation obtained), the injured
member can be authorized to obtain compensation by retaliation: a
proportional reduction in a trade concession or obligation owed to the
violator. The overall value of this mechanism is controversial. However,
since its establishment in 1995, some 50 requests to start consultations on
disputes of all kinds have been initiated; several cases are before panels;
two panels have completed proceedings; there has been one appellate ruling;
and ten disputes have been resolved by consultation without resort to a
panel (30).

Given the parallels between the WHO and WTO regulatory systems and the
interplay between epidemics and trade, WHO consultations with WTO would
enhance coordination (or other more or less formal arrangements) on trade
issues related to disease threats. Coordination seems particularly
appropriate in light of the current revision of the regulations. As the
range of diseases and syndromes covered by the regulations is substantially
broadened, areas of potential overlap or parallel may grow (15).

The SPS agreement provides for consultation and coordination between the
WTO system and those of other international organizations (27, art. 11.3).
The Committee on Sanitary and Phytosanitary Measures established under the
SPS agreement has a mandate to consult with other international
organizations in the field of sanitary or phytosanitary protection
(including the Codex Alimentarius Commission); to obtain scientific and
technical advice for administering the agreement; to avoid unnecessary
duplication of efforts; and to identify other international standards,
guidelines, or recommendations relevant to sanitary and phytosanitary
measures that have a major impact on trade. (27, art. 12). Consultation is
also recommended for dispute resolution. The SPS agreement encourages
dispute resolution panels deciding cases that involve the agreement on
scientific or technical issues to seek advice from experts, including the
relevant international organizations (27, art. 11.2).

The WTO also has a Committee on Trade and Environment, which addresses the
relationship of regulation and policy on trade and Environment issues,
including issues concerning the SPS agreement (31-32). In an example of a
potential consultation between WHO and WTO, a bulletin of the Committee on
Trade and Environment indicates that the Conference of the Parties of the
Biodiversity Convention (an environmental treaty) had requested that its
Secretariat "liaise with the WTO Secretariat and invite it to provide input
in identifying the synergies and relationship between the objectives" of
the convention and one of the WTO agreements (33).

Collaboration and consultation on health-related trade issues will depend
on accommodating the many differences between the two organizations, as
well as WHO's constitutional provisions concerning such relationships (7,
arts. 70-71) and the more limited articles in the current regulations on
such contacts (1, arts. 46[3], 85). While establishing working
relationships with WTO might not be a panacea for the many trade-related
concerns under the WHO regulations, it would provide opportunities for
reinforcement of the legal and institutional bases for the prevention of
inappropriate trade restraints.

International Civil Aviation Organization (ICAO)
Because of the importance of international travel (as well as air
transportation of cargo) to disease emergence, ICAO is another important
collaborative partner. Unlike WTO, however, ICAO has had a long-standing
relationship with WHO, dating from the 1940s and including participation in
creating the regulations (8). The conflicting pressures of globalization of
world transport (and commerce) and sovereignty have affected the ability of
ICAO and WHO to regulate effectively (34).

In preventing infectious diseases, WHO and ICAO have overlapping areas of
interest, such as the disinsectization of aircraft and airport health and
sanitary facilities. Under the Convention on International Civil Aviation
and related instruments, ICAO addresses a variety of civil aviation issues,
including many relating to public health and international transmission of
disease. The convention provides that each member state "agrees to take
effective measures to prevent the spread by means of air navigation of
cholera, epidemic typhus, smallpox, yellow fever, plague, and such other
communicable diseases as the contracting States shall ... designate" and to
"keep in close consultation with the agencies (such as WHO) concerned with
international regulations relating to sanitary measures applicable to
aircraft" (35). The specific compulsory "Standards" and related
"Recommended Practices" in Annex 9 to the convention include those
applicable to public health, infectious disease transmission, and related
requirements (36). These provisions are fundamentally tied to WHO
recommendations and the regulations in Standard 8.12, which requires ICAO
member states to "comply with pertinent provisions of the current edition
of the [regulations]." Specific ICAO standards and recommendations also
refer to WHO recommendations and regulations in key areas, including
aircraft disinsectization, provision of safe food and water at airports and
on aircraft at international airports, proper facilities for disposal of
refuse, waste, wastewater, and other dangerous matter, and yellow fever
certificates.

WHO cooperation with ICAO is exemplified by the participation of ICAO in
the 1995 informal WHO consultation regarding revision of the regulations;
ICAO was the only such international organization to participate in the
consultation (15). The consulting group's recommendations suggested that
certain sections of the revised regulations concerning sanitation standards
at airports and seaports should refer to the applicable requirements
(exceptions being health care services for sick persons on arrival,
equipment necessary for disinfection, and disinsectization, and control of
animal-borne disease), under other international agreements, such as Annex
9. Although this specific recommendation may have flaws (for example, it
effectively renders the regulations incomplete in themselves as a reference
or guide to essential rules), it demonstrates ongoing attempts to link the
two organizations.

Coordination of WHO and ICAO in dealing with the inappropriate imposition
of health measures is particularly relevant now. At a 1995 session of the
ICAO division with jurisdiction over such public health issues,
"[d]elegates recommended that ICAO work with the World Health Organization
... to draft joint guidelines that, if followed, would prevent adoption by
Contracting States of excessive health measures that might disrupt
international air transport services in cases of outbreaks or epidemics of
diseases" (37).

International Maritime Organization (IMO)
Provisions under the regulations concerning deratting procedures (as noted
in the WHO informal consultation on revision of the regulations) (15) and
sanitary conditions at seaports also play an important role in maritime
health and in containing international disease transmission threats. The
regulations also address cholera-contaminated bilge water in certain
circumstances on arrival of the ship (1, art. 62[1]). Health authorities
are broadly authorized to take measures "to control the discharge from any
ship of sewage and refuse which might contaminate the waters of a port,
river or canal" (1, art. 29). IMO has recognized the global public health
problems of bacterial and viral diseases transmitted in discharges of
ballast water and sediment (38). A poll of IMO member states indicated that
such transmission is a major international problem expected to worsen (38).
The IMO assembly has accordingly adopted (generally nonmandatory)
guidelines to prevent the introduction of bacterial and viral pathogens in
ballast water and sediment. The IMO resolution traced this concern in part
to the 1973 International Conference on Marine Pollution, in which the
parties called for WHO, in collaboration with IMO, "to carry out research
into the role of ballast water as a medium for the spreading of epidemic
disease bacteria" (38).

A sound legal and policy framework is needed to support efforts against
emergent infections. Truly intersectoral, interagency, and
interorganizational collaboration in addressing the broad factors of
emergence and expanded reporting of disease are major steps in this
process. The challenge is broad, but in view of the increased pace of
emergence and the globalization of disease, the importance of a
comprehensive legal and policy framework cannot be overstated.

Acknowledgments
The authors acknowledge the helpful comments on many issues in this article
by Roy Prosterman, Professor of Law, University of Washington, and Tim
Hanstad, Lecturer in Law, University of Washington.

Mr. Plotkin is an attorney and legal consultant. He is currently a
candidate for Master of Laws degree in the Law of Sustainable International
Development Program. Among other professional activities, he has practiced
law in Seattle, Washington, and with the antitrust enforcement section of
the Washington State Attorney General's Office.

Dr. Kimball is cochair of the working group on emerging infections,
associate professor of health services, and epidemiology adjunct in
medicine at the University of Washington. She is also director of the
community medicine program. Her research interests include surveillance of
emerging infections in the Asia/Pacific region and health and prevention
services at the local level. She is contract quarantine physician for the
Seattle Tacoma airport and has served as Director of Washington State
HIV/AIDS program and national program support for the Global Program on
AIDS with the Pan American Health Organization.

Address for correspondence: Ann Marie Kimball, University of Washington,
Box 357660, Seattle, WA 98195-7660;
fax:206-543-3964;e-mail:akimball@u.washington.edu.

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---------------------------------------------------------------------------
 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
--------------------------------------------------------------------------- 

Synopses

Surface Antigens of the Syphilis Spirochete and Their Potential as
Virulence Determinants

David R. Blanco, James N. Miller, and Michael A. Lovett
UCLA School of Medicine, Los Angeles, California
---------------------------------------------------------------------------
 

      A unique physical feature of Treponema pallidum, the
      venereally transmitted agent of human syphilis, is that its
      outer membrane contains 100-fold less membrane-spanning
      protein than the outer membranes of typical gram-negative
      bacteria, a property that has been related to the chronicity
      of syphilitic infection. These membrane-spanning T. pallidum
      rare outer membrane proteins, termed TROMPs, represent
      potential surface-exposed virulence determinants and targets
      of host immunity. Only recently has the outer membrane of T.
      pallidum been isolated and its constituent proteins
      identified. Five proteins of molecular mass 17-, 28-, 31-,
      45-, and 65-kDa were outer membrane associated. The 17- and
      45-kDa proteins, which are also present in greater amounts
      with the T. pallidum inner membrane protoplasmic cylinder
      complex, had been previously characterized lipoproteins and
      are, therefore, not membrane-spanning but rather
      membrane-anchored by their lipid moiety. In contrast, the 28-,
      31-, and 65-kDa proteins are exclusively associated with the
      outer membrane. Both the purified native and an Escherichia
      coli recombinant outer membrane form of the 31-kDa protein,
      designated Tromp1, exhibit porin activity, thereby confirming
      the membrane-spanning outer membrane topology of Tromp1. The
      28-kDa protein, designated Tromp2, has sequence
      characteristics in common with membrane-spanning outer
      membrane proteins and has also been recombinantly expressed in
      E. coli, where it targets exclusively to the E. coli outer
      membrane. The 65-kDa protein, designated Tromp3, is present in
      the least amount relative to Tromps1 and 2. Tromps 1, 2, and 3
      were antigenic when tested with serum from infection and
      immune syphilitic rabbits and humans. These newly identified
      TROMPs provide a molecular foundation for the future study of
      syphilis pathogenesis and immunity.

Despite the fact that Treponema pallidum subsp. pallidum has remained
sensitive to penicillin for more than four decades, syphilis remains an
important cause of sexually transmitted disease, accounting for more than
14,000 new cases in 1995 in the United States alone (1). Although syphilis
is not an emerging infectious disease, its reemergence, from 1986 to 1990,
culminated in more than 100,000 reported cases in 1990 and an 80% increase
in the reported rate per 100,000 population. The reemergence of syphilis
has also been associated in certain areas with the use of crack cocaine,
and syphilis continues to be an important risk factor for acquiring and
transmitting human immunodeficiency virus.

Syphilis is characterized by months of clinical disease followed by years
of latency with the potential for relapse to debilitating or lethal late
disease if left untreated. The chronicity of infection may relate to a
striking property of the T. pallidum outer membrane, namely that it
contains 100-fold less membrane-spanning protein than the outer membranes
of typical gram-negative bacteria (2,3). This article reviews recent
progress in identifying and characterizing the rare outer membrane proteins
(TROMPs) of T. pallidum.

The interaction of host and pathogen leading to the establishment of
infection generally proceeds through the interactions of their respective
surface molecules. An understanding of the molecular basis for syphilis
pathogenesis and the identification of corresponding T. pallidum surface
molecules have long been hampered by the inherent difficulties associated
with the study of this organism. In contrast to that of many other
pathogenic spirochetes from the genera Treponema, Borrelia, and Leptospira,
the in vitro multiplication of T. pallidum is unsuccessful in artificial
media and is limited and impractical in tissue culture (4). Further, only
limited numbers of T. pallidum are attainable from testicular cultivation
in rabbits. Organisms acquired from rabbit infection are also bound with
host material including albumin (5), fibronectin (6), and
glycosaminoglycans (7); therefore, the separation and purification of T.
pallidum molecules are complicated. Host material associated with T.
pallidum has also been the basis for past theories of molecular mimicry
during syphilitic infection (6), of T. pallidum adherence to host cells and
tissues (8), and of the long recognized antigenic inertness of the surface
of this organism.

Past studies showed that the surface of T. pallidum behaves as if it lacks
proteins and antigens. These findings included the inability to detect
antibody bound to the surface of structurally intact treponemas by
immunofluorescence (9) or by immunoelectron microscopy (10,11), the limited
radiolabeling of surface proteins (12,13), and the observation that only
with extended in vitro incubation could proteins of intrinsically
radiolabeled T. pallidum bind specific antibody (14). One hypothesis
generated by these observations was that the surface of T. pallidum was
antigenically inert because of a limited number of available
surface-exposed antigens.

The inability to apply conventional gram-negative cell fractionation
procedures to T. pallidum to isolate its outer membrane and identify its
constituent proteins further hampered the identification of surface-exposed
proteins. As a result, alternative methods, which included detergent
solubilization, were used to achieve this goal. The detergent Triton X114
(TX114) was used with particular interest because of its inherent property
of a low temperature cloud point that results in phase separation yielding
both aqueous and detergent phases. It was found that 0.1% to 2% TX114 would
selectively solubilize the T. pallidum outer membrane and result in the
recovery of several prominent detergent-phase proteins (15,16), which were
speculated, at the time, to be part of the outer membrane and potentially
surface exposed. These proteins were subsequently found to be lipoproteins
(17) and were strongly antigenic when tested with serum from syphilitic
rabbits and humans (17). While surface exposed lipoproteins have been
demonstrated for other pathogenic spirochetes, including Borrelia (18) and
Leptospira (19), a surface exposed location for a strongly antigenic T.
pallidum lipoprotein was inconsistent with the surface antigenic inertness
of this organism.

          [Figures not available in ASCII version]
          Figure 1. Freeze-fracture electron microscopy of
          Treponema pallidum subsp. pallidum (A), and
          Escherichia coli (B). Concave ([concave symbol]) and
          convex ([convex symbol]) outer membrane (OMF) and
          inner membrane (IMF) fracture faces.

A major advance in the understanding of the T. pallidum outer membrane and
cell surface followed the results of freeze-fracture electron microscopy in
1989. The observation that the fracture faces of the T. pallidum outer
membrane contained only 170 particles/mm2 (2,3), which is approximately
100-fold less than what is contained in typical gram-negative bacterial
outer membranes (Figure 1), together with the finding of freeze-etch
analysis (3), has provided the most plausible explanation for the antigenic
inertness of the T. pallidum surface. The rare particles revealing T.
pallidum rare outer membrane proteins were designated TROMPs (2,20), and
because of their membrane-spanning topology, they were distinct from
lipoproteins, which do not span membranes to form particles in
freeze-fracture analyses (21). Moreover, a membrane-spanning protein by
definition should possess several surface exposed regions, suggesting that
TROMPs had surface exposed antigenic sites. This theory was confirmed by
freeze-fracture analysis when it was observed that incubation of T.
pallidum in serum from syphilitic rabbits immune to reinfection resulted in
the aggregation of TROMPs (20) (Figure 2). It was found that 8 to 16 hours
of incubation was required for this aggregation to occur, suggesting that
antibody-mediated aggregation of TROMPs is the rate limiting step for
complement activation and killing of T. pallidum (20).

       [Figures not available in ASCII version]
       Figure 2. Freeze-fracture electron microscopy of T. pallidum
       subsp. pallidum demonstrating TROMP aggregation. Concave (
       [concave symbol]) and convex ([convex symbol]) outer membrane
       fracture faces (OM). T. pallidum incubated in
       heat-inactivated normal rabbit serum for 16 hours (A), in
       immune rabbit serum for 2 hours (B), and in immune rabbit
       serum for 16 hours (C) and (D). Arrows show individual (A&B)
       and aggregated (C&D) TROMPs. Bar in each micrograph
       represents 0.1mm. Photograph reprinted with the permission of
       the Journal of Immunology, copyright 1990, The American
       Association of Immunologists.

The findings of a potentially protective immunogen in low molar amounts on
the outer membrane surface of T. pallidum have provided a further
attractive explanation for the prolonged period required for the induction
of acquired protective immunity during syphilitic infection. This is also
in accord with the repeated immunizations required for protective immunity
with T. pallidum attenuated by gamma irradiation (22), the only method of
successful vaccination ever reported. Thus, the identification and study of
TROMPs has become essential in understanding syphilis pathogenesis and
immunity in molecular terms.

Isolation of the T. Pallidum Outer Membrane

In 1994, we developed a procedure to isolate the outer membrane of T.
pallidum without detergents (23). This method used three key features: 1) a
Ficoll step gradient to purify T. pallidum from host contaminating
material; 2) octadecyl rhodamine B chloride salt, a lipid-conjugated
chromophore that intercalates into membranes and provides a visual marker
to monitor outer membrane release and recovery; and 3) a hypotonic 0.05 M
sodium citrate buffer, pH 3.0, which was found to selectively release the
T. pallidum outer membrane. Since it has been suggested that endoflagellar
filaments physically interact with the outer membrane in the process of
motility, the probable mechanism for outer membrane release by this
procedure is the depolymerization of endoflagella at low pH.

Purification of T. pallidum outer membrane vesicles in sucrose density
gradients showed that the membrane banded at the very low density of 1.03
g/ml (7% sucrose). This, however, was expected for properties of a membrane
that lacks lipopolysaccharide (24) and contains a small amount of protein.
Freeze-fracture electron microscopy demonstrated that the purified membrane
vesicles contained extremely rare intramembranous particles consistent with
the low particle density observed for the native outer membrane of T.
pallidum. The selective isolation of the T. pallidum outer membrane was
shown by the inability to detect T. pallidum penicillin binding proteins, a
marker of the cytoplasmic membrane. Further support for the selectivity of
the outer membrane isolation was the complete absence of a previously
characterized 19-kDa periplasmic protein termed 4D (15) and the abundant
47-kDa lipoprotein (17) and the presence of only trace amounts of
endoflagellar protein. The absence of the 47-kDa lipoprotein was
particularly important since it is widely accepted that this very abundant
protein is exclusively anchored to the inner membrane (25). The finding
that the 47-kDa lipoprotein was not detected in the purified outer membrane
material indicates that inner membrane anchored lipoproteins were not
released by this procedure.

Two dimensional immunoblot analysis was used to identify the protein
constituents present in purified T. pallidum outer membrane. Two strongly
antigenic species were found by syphilitic immune rabbit serum, one very
basic protein (pI > 7.0) at 17-kDa and the other having a pI of
approximately 4.5 at 45-kDa. The observation that the 17-kDa protein was
very basic, showed higher oligomeric forms, and was selectively partitioned
into the hydrophobic phase after Triton X114 detergent extraction was
consistent with properties reported for the 17-kDa lipoprotein of T.
pallidum (26). With specific monoclonal antibodies, the 45-kDa protein was
identified as the previously characterized lipoprotein termed TmpA (27). It
was also found that most of these two lipoproteins, well over 90%, are
associated with the inner membrane protoplasmic cylinder complex. It would
appear, however, that the association of the 17- and 45-kDa lipoproteins
with the outer membrane is specific, given the absence of the normally
abundant 47-kDa lipoprotein. The function of these two outer membrane
lipoproteins remains to be determined.

In addition to the strongly antigenic 17- and 45-kDa lipoproteins,
gold-stained two-dimensional blots of outer membrane showed four additional
T. pallidum proteins, including one each at 28- and 65-kDa and two at
31-kDa, with close but distinct pI migration patterns. All of these
proteins, including the 17- and 45-kDa lipoproteins, were present in
approximately equal amounts in the outer membrane. However, in contrast to
the 17- and 45-kDa lipoproteins, these four additional proteins required
very low dilutions (1:25) of immune syphilitic rabbit serum for detection
by immunoblot analysis. Moreover, when compared with stained
two-dimensional blots of total T. pallidum proteins, these four additional
outer membrane proteins were either not detectable, as was the case for the
65-kDa protein and the more basic of the two 31-kDa proteins, or
represented extremely minor species, as was the case for the 28-kDa protein
and the more acidic 31-kDa protein. In view of the paucity of TROMPs
determined from freeze-fracture analysis, these four proteins, which were
clearly enriched in our outer membrane preparation, became the leading
candidates for membrane-spanning outer membrane proteins of T. pallidum.

In 1995, a second T. pallidum outer membrane isolation method that used
plasmolysis of organisms in 20% sucrose was reported by Radolf et al. (28).
Outer membrane isolated by this method was also shown by freeze-fracture
analysis to have an extremely low density of membrane-spanning protein.
These studies showed that the lipid composition of the T. pallidum outer
membrane was similar to that of the protoplasmic cylinder inner membrane
complex, with the exception of cardiolipin, which was prominently detected
only in protoplasmic cylinders. Of further interest was the finding that
outer membrane phospholipids and glycolipids did not react with antibody
from infection-derived immune serum, suggesting that only outer membrane
proteins are target antigens. However, in contrast to the limited number of
protein species that we observed, silver stained gels of their outer
membrane material showed more than 20 proteins, including the 47-kDa
lipoprotein. One interpretation of these results is that many of the
proteins identified in their outer membrane preparation are possibly
contaminants of the periplasm or inner membrane. Thus, we believe that the
small number of protein species identified by our isolation method
represent a more accurate account of the outer membrane protein composition
of T. pallidum.

While lipoproteins are integral membrane proteins, their membrane
association extends only to integration of the lipid moiety in the bilayer.
In contrast to membrane-spanning proteins, the T. pallidum lipoproteins do
not span membrane lipid bilayers and, therefore, do not form particles upon
freeze-fracture electron microscopy (21). By comparison, one type of
membrane-spanning protein common to virtually all gram-negative outer
membranes are porins, which form water-filled channels through membranes
allowing for transport of small molecular weight solutes (29). Porin
proteins, like almost all outer membrane proteins, span membranes in a
betapleated sheet topology rather than in alphahelical hydrophobic regions,
which are common to inner membrane-spanning proteins (30). Porin activity
can be detected by several methods (29), including radioisotope efflux or
substrate uptake in proteoliposomes, liposome swelling, and the black lipid
bilayer assay, which measures the conductivity of ions through porin
channels. In the black lipid bilayer assay, a set voltage is applied across
an artificial lipid bilayer which, if a porin protein has been inserted,
results in an increase in electrical conductance. The degree of conductance
increase for many insertional events is used to calculate the average pore
channel size. When the black lipid bilayer assay was used with Triton X100
detergent solubilized proteins from our purified T. pallidum outer
membrane, porin activity was demonstrated (23). Two distinct average
conductance increases were observed at 0.4 and 0.76 nanosiemens (nS),
corresponding to pore channel sizes of 0.35 and 0.68 nm, respectively,
which are similar in size to porin channels found for other gram-negative
bacteria (29). At the time, the two distinct conductance measurements
suggested two different porin species, or alternatively, that the larger
activity could be the result of dimeric aggregates of the smaller activity
or the smaller activity could be a substate of the larger channel, possibly
caused by the application of a voltage (31). Regardless of the exact number
of T. pallidum porin species, these findings confirmed that at least some
of the particles observed by freeze-fracture electron microscopy of the T.
pallidum outer membrane were membrane-spanning porin proteins. This finding
was of particular importance since several gram-negative bacterial porins
play a role in pathogenesis by acting as adhesins (32,33) and as surface
targets of bactericidal antibody (34-37). Thus, the search for an outer
membrane T. pallidum porin protein was initiated to identify a first TROMP
species.

T. Pallidum Rare Outer Membrane Protein 1 (Tromp1): an Outer Membrane Porin

Since most porin proteins have molecular masses of 28- to 48-kDa (29), our
focus was directed toward isolating the 28- and 31-kDa proteins identified
in purified T. pallidum outer membranes. Internal amino acid sequences from
the native 31-kDa protein were used to clone the encoding structural gene,
designated tromp1 (38). Antiserum generated to a recombinant Tromp1 fusion
protein has shown that both 31-kDa proteins identified by twodimensional
blot analysis of outer membrane are Tromp1. Analysis of the deduced amino
acid sequence showed an N-terminal hydrophobic region consistent with a
signal peptide. Two potential leader peptidase I cleavage sites are noted,
including threonine-histidine-alanine at residues 30 through 32 and
alanine-alanine-alanine at residues 38 through 40. Identification of the
cleavage site by N-terminal amino acid sequence of the native protein has
not been possible because of the limited amount of native protein
recoverable. However, other data discussed later in this review suggest
that alanine-alanine-alanine is the correct cleavage site. The deduced
Tromp1 amino acid sequence also supports the concept that Tromp1 topology
is in accord with the structural paradigms of other gram-negative outer
membrane proteins. Beta-moment analysis, which shows amphipathic sequence
regions, has predicted that Tromp1 has 14 membrane-spanning amphipathic
betasheet segments typical of gram-negative outer membrane proteins
(29,30).

The lethality for Escherichia coli transformants harboring the intact
tromp1 structural gene, which was found by immunoblot analysis to be
expressing Tromp1, is similar to that observed for many recombinant
gram-negative porin proteins expressed in E. coli (39). The tromp1 gene has
recently been found to be part of a putative transport operon that includes
an adenosine triphosphate binding protein and a hydrophobic membrane
protein (Hardham et al., Gordon Research Conference, 1996). To determine if
Tromp1 was a porin, native Tromp1 was purified from Triton X100 solubilized
T. pallidum by nondenaturing isoelectric focusing. The demonstration of
porin activity, by the black lipid bilayer assay, has confirmed that Tromp1
is a membrane-spanning outer membrane protein of T. pallidum, the first
such protein to be identified for this organism. Native Tromp1 showed two
distinct conductance distributions about means of 0.15 and 0.7 nS. The
larger of these channels is similar in mean conductance to the 0.76 nS
activity observed by using purified outer membrane. The smaller 0.15 nS
conductance channel had not been detected previously, and while two
channels of different sizes have been reported for other porins (40), this
may simply be the result of partial damage during isolation.

Because most gram-negative bacterial porins exist in the outer membrane in
either a sodium dodecyl sulfate (SDS) unstable or stable trimer
conformation (29), it was theorized that Tromp1 would also be organized in
an oligomeric form in the outer membrane of T. pallidum. Antiserum against
a Tromp1 fusion protein has in fact identified two higher molecular mass
forms of native Tromp1 on immunoblots of T. pallidum with sizes of
approximately 55- and 80-kDa. A 98-kDa size is further found to be the only
detectable form of native Tromp1 when T. pallidum is solubilized at room
temperature in a low concentration (0.02%) of SDS. These findings would
appear to be consistent with the idea that native Tromp1 exists in the T.
pallidum outer membrane in an SDS-unstable trimer conformation, a property
that would be common to several other gram-negative outer membrane porins
(29).

Expression of a Porin Active Recombinant Tromp1 (rTromp1)

Porin proteins of gram-negative pathogens not only function as portals for
nutrient acquisition across the outer membrane but also play a role in
pathogenesis by acting as adhesins (32,33) and targets for bactericidal
antibody (34-37). The membrane-spanning conformation of membrane porins is
critical for their biologic function. Many surface-exposed epitopes on
porins shown to be targets for bactericidal antibodies are conformational
(34,36). Thus, correct outer membrane protein conformation should be a key
consideration in the study of porins as they relate to bacterial virulence
and host immunity. The previous demonstration of immune serum antibody
mediated aggregation of TROMPs, as viewed by freeze-fracture electron
microscopy (20), suggested that Tromp1 could be a key surface exposed
target for antibody that mediates killing (20,41) or opsonization (42,43)
of T. pallidum. Because of these possibilities, studies have been initiated
to express and isolate a porin form of recombinant Tromp1 (rTromp1) with
native conformation and in amounts necessary to conduct functional studies.

The controlled nonlethal expression in E. coli of exported rTromp1 was
accomplished by inserting the tromp1 gene, including the region encoding
its native signal peptide, into a relatively low copy number expression
plasmid that has an inducible T7 promoter (44). Uninduced basal levels of
T7 RNA polymerase resulted in the stable expression, export, and outer
membrane localization of rTromp1. Expression detected in whole cell lysates
showed several forms of rTromp1, ranging in molecular mass from 31- to
35-kDa. These different sizes of rTromp1 may represent different
conformations of the protein as observed for other recombinant outer
membrane proteins expressed in E. coli. Only the 35-kDa form of rTromp1 is
detected in outer membranes isolated from E. coli. One explanation for the
higher molecular mass outer membrane form is its possible association with
lipopolysaccharides, known to form a complex with gram-negative outer
membrane porin proteins (45). Such an interaction would certainly be
noteworthy because the T. pallidum outer membrane does not contain
lipopolysaccharides (24).

The demonstration of porin activity by using rTromp1 isolated from E. coli
outer membranes has further confirmed the membrane-spanning conformation of
Tromp1. Single channel conductance measurements of rTromp1 in the black
lipid bilayer assay showed two distinct distributions of activity about 0.4
and 0.8 nS. The 0.8 nS conductance is clearly similar to the 0.7 nS
conductance determined for native Tromp1 (38) and the 0.76 nS conductance
determined for purified outer membrane, indicating that the poreforming
conformation of a portion of rTromp1 is similar if not identical to that of
the native protein. Although it is not known why rTromp1 also showed a 0.4
nS conductance measurement, one possibility is an altered conformation of
the protein after the isolation process.

A further question regarding the outer membrane localization of porin
active rTromp1 has been whether surface exposed antigenic sites are
present. Whole mount immunoelectron microscopy has demonstrated the surface
binding of immune rabbit serum antibody on E. coli expressing rTromp1 (44).
Antibody raised against a soluble fusion protein form of rTromp1, which was
found by immunoblot analysis to be 100-fold more sensitive in detecting
Tromp1 than the immune rabbit serum antibody, did not show surface binding
of antibody on E. coli expressing rTromp1. This soluble fusion protein form
of rTromp1 does not have porin activity when tested in the black lipid
bilayer assay. Thus, a reasonable explanation for the immunoelectron
microscopy observations is that the bound immune rabbit serum antibodies
recognize conformational surface epitopes on rTromp1, a possibility which
may also extend to native Tromp1 on the surface of T. pallidum.

The importance of porin protein conformation to biologic function has been
further demonstrated in studies using rTromp1. The primary amino acid
sequence of Tromp1 suggests two possible signal peptide processing sites
within the first 40 residues at threonine-histidine-alanine and at
alanine-alanine-alanine. Signal peptide fusion constructs at these two
possible cleavage sites using the OmpT signal peptide of E. coli result in
both forms of rTromp1 being exported and targeted to the E. coli outer
membrane. Only the OmpT signal fused at the alanine-alanine-alanine
position resulted in an exported product that has a molecular mass closest
to that of native Tromp1. Moreover, while the outer membrane form of
rTromp1 processed at threonine-histidine-alanine showed porin activity, the
average channel conductance was 3.2 nS, which is considerably larger than
the 0.70.8 nS channels observed for native and recombinant Tromp1 exported
with its native signal peptide. The greater lethality and limited recovery
of the OmpTTromp1 fusion construct processed at alanine-alanine-alanine has
precluded at this time the ability to test this outer membrane form for
porin activity. These findings suggest that alanine-alanine-alanine is the
likely processing site in the Tromp1 sequence for leader peptidase I. More
importantly, these findings have indicated that subtle changes in the
length of the primary amino acid sequence of Tromp1 can have significant
effects upon porin activity and are likely the result of an altered
conformation of the protein. For this reason and as described previously,
proper Tromp1 conformation is an important consideration for future studies
addressing its role in syphilis pathogenesis.

T. Pallidum Rare Outer Membrane Protein 2 (Tromp2)

In the search for additional TROMP species, we have recently focused on the
28-kDa protein identified in purified T. pallidum outer membrane
preparations (23). As in the protein for Tromp1, internal amino acid
sequences from the native 28-kDa protein were used to clone the encoding
structural gene, now designated tromp2 (46). Analysis of the deduced amino
acid sequence showed a 24-residue N-terminal hydrophobic region consistent
with a signal peptide and terminating in a typical leader peptidase I
cleavage site of leucine-alanine-alanine. As for Tromp1, the deduced amino
acid sequence for Tromp2 is also in accord with the structural paradigms of
other gram-negative outer membrane proteins. Beta-moment analysis has
predicted that Tromp2 has 9 membrane-spanning amphipathic betasheet
segments.

Recombinant expression and export of Tromp2 (rTromp2) in E. coli has
recently been accomplished by using the entire tromp2 structural gene in a
relatively low copy number plasmid that has an inducible T7 promoter (46).
In contrast to Tromp1, Tromp2 expression was not found to be lethal to E.
coli, even under maximum inducing conditions. Immunoblot analysis using
antiserum generated to rTromp2 has shown that virtually all of the
recombinant protein produced is targeted to the E. coli outer membrane.
When tested in the black lipid bilayer assay, rTromp2 isolated from E. coli
outer membranes showed only occasional channel formation. The total number
of porin insertional events was extremely low when compared with the amount
of rTromp2 tested. This is in contrast to both native and recombinant
Tromp1 where numerous insertional porin events were readily observed. Thus,
whether Tromp2 truly has porin function is not clear at this time.

As for rTromp1, a question regarding the outer membrane localization of
rTromp2 was the existence of surface exposed antigenic sites. The use of
whole mount immunoelectron microscopy demonstrated the surface binding of
immune rabbit serum antibody on E. coli expressing rTromp2 (46). By
comparison, antibody raised against a denatured form of rTromp2, which by
immunoblot analysis was found to be more sensitive in detecting rTromp2
than immune rabbit serum, did not show surface binding on E. coli
expressing rTromp2. This finding is again similar to that described above
for rTromp1 and again suggests that the bound immune rabbit serum
antibodies recognize conformational-surface epitopes on rTromp2, a
possibility which may extend to native Tromp2 on T. pallidum.

T. Pallidum Rare Outer Membrane Protein 3 (Tromp3)

Of the three outer membrane protein species identified (28-, 31-, and
65-kDa), the 65-kDa protein, tentatively termed Tromp3, is present in the
least amount on the basis of 2-dimensional SDS-PAGE gold-stained
immunoblots of purified outer membrane (23). In addition, the presence of
the 65-kDa protein from one outer membrane preparation to the next has been
inconsistent, suggesting that this protein may be differentially expressed.
The extremely small amount of Tromp3 has at this time precluded amino acid
sequencing and, therefore, the cloning of the gene that encodes this
protein.

The Potential Role of TROMPs in Acquired Immunity Against Syphilis

Freeze-fracture electron microscopy has demonstrated the ability of serum
from immune syphilitic rabbits to aggregate TROMPs (20). These studies have
been extended by using serum from infected rabbits with varying degrees of
challenge immunity and have shown that TROMP aggregation correlates
directly with the development of challenge immunity (Lewinski et al.,
unpub. obs., 1994). These findings are in accord with past studies that
have demonstrated a significant to complete level of protection of animals
after passive immunization with serum from immune donors (47). In a recent
study, we have used the small amount of attainable T. pallidum outer
membrane to immunize mice. We have found that serum from the immunized mice
possesses complement-dependent treponemicidal activity (100% killing of T.
pallidum) that is 30 times greater than that of serum from immune
syphilitic rabbits (Blanco et al., unpub. obs.). Immunoblot analysis using
this serum has shown that only the outer membrane associated proteins are
detected. We have also found that absorption of this serum to remove
antibodies to the T. pallidum lipoproteins does not diminish the titer of
the 100% killing activity. Such levels of serum treponemicidal activity
have heretofore never been generated by immunization of mice or rabbits
with either native or recombinant T. pallidum antigens. These observations
are consistent with the idea that TROMPs represent the key surface exposed
targets for treponemicidal antibody and perhaps a protective host immune
response.

Our recent ability to express in sufficient amounts recombinant Tromp1 and
Tromp2 provides an opportunity to address directly the ability of these T.
pallidum outer membrane proteins to elicit protective immunity in
experimental animals. However, the correct outer membrane conformation of
the recombinant TROMPs may prove a key factor in whether protective
immunity results, as is the case for several bacterial porins. We hope that
TROMP immunization will generate a humoral and/or cellular response capable
of efficiently killing T. pallidum and provide a significant level of
acquired resistance.

Acknowledgments

We thank Dr. Eldon M. Walker for the electron microscopy presented in this
review and Dr. Cheryl I. Champion for her extensive contributions to the
studies involving Tromp1 and Tromp2.

This paper is dedicated to Dr. Mary C. Pangborn, the discoverer of
cardiolipin at the Division of Laboratories and Research, New York State
Department of Health, for her 90th birthday, and to Dr. Thomas B. Turner,
the pioneer of treponemal research conducted at the Johns Hopkins School of
Medicine, for his 100th birthday.

Dr. Blanco is a member of the Department of Microbiology and Immunology,
UCLA School of Medicine, Los Angeles. He has been involved over the past 16
years in syphilis research focusing on structure-function relationships and
the immunobiology of surface and subsurface proteins.

Address for correspondence: David R. Blanco, Dept. Microbiology and
Immunology, CHS 43-239, UCLA School of Medicine, 10833 LeConte Ave., Los
Angeles, CA 90095; fax:310-206-3865;e-mail:
dblanco@microimmun.medsch.ucla.edu.

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---------------------------------------------------------------------------
 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
--------------------------------------------------------------------------- 

Synopses

Mycoplasmas: Sophisticated, Reemerging, and Burdened by Their Notoriety

Joel B. Baseman* and Joseph G. Tully
*The University of Texas Health Science Center at San Antonio, San Antonio,
Texas, USA; National Institute of Allergy and Infectious Diseases,
Frederick Cancer Research and Development Center, Frederick, Maryland, USA
---------------------------------------------------------------------------

      "Sit down before fact as a little child, be prepared to give
      up every preconceived notion, follow humbly wherever and to
      whatever abysses nature leads, or you shall learn nothing."
                                                  Thomas Henry Huxley

      Mycoplasmas are most unusual self-replicating bacteria,
      possessing very small genomes, lacking cell wall components,
      requiring cholesterol for membrane function and growth, using
      UGA codon for tryptophan, passing through
      "bacterial-retaining" filters, and displaying genetic economy
      that requires a strict dependence on the host for nutrients
      and refuge. In addition, many of the mycoplasmas pathogenic
      for humans and animals possess extraordinary specialized tip
      organelles that mediate their intimate interaction with
      eucaryotic cells. This host-adapted survival is achieved
      through surface parasitism of target cells, acquisition of
      essential biosynthetic precursors, and in some cases,
      subsequent entry and survival intracellularly. Misconceptions
      concerning the role of mycoplasmas in disease pathogenesis can
      be directly attributed to their biological subtleties and to
      fundamental deficits in understanding their virulence
      capabilities. In this review, we highlight the biology and
      pathogenesis of these procaryotes and provide new evidence
      that may lead to increased appreciation of their role as human
      pathogens.

No other group of procaryotes has been so embroiled in controversy and in
establishing a clear pathogenic niche as the mycoplasmas. Their virulence
determinants are undeniably complex, and their unique biological properties
likely challenge the host differently from typical bacterial pathogens
(1,2). Also, numerous Mycoplasma species appear to comprise the commensal
microbial flora of healthy persons (3), and the association of these
mycoplasmas with disease complicates the diagnosis and necessitates
extensive and highly specific serologic, nucleic acid, and epidemiologic
data. Nonetheless, mycoplasmas by themselves can cause acute and chronic
diseases at multiple sites with wide-ranging complications and have been
implicated as cofactors in disease. Recently, mycoplasmas have been linked
as a cofactor to AIDS pathogenesis and to malignant transformation,
chromosomal aberrations, the Gulf War Syndrome, and other unexplained and
complex illnesses, including chronic fatigue syndrome, Crohn's disease, and
various arthritides (4-8). Even with mounting evidence of their pervasive
and pathogenic potential, mycoplasmas still evoke the image of a group of
obscure or impotent microorganisms. Yet they are evolutionarily advanced
procaryotes (9-11), and their elite status as "next generation" bacterial
pathogens necessitates new paradigms in fully understanding their disease
potential.

Mycoplasmas, which lack cell walls but possess distinctive
sterol-containing plasma membranes, are taxonomically separated from other
bacteria and belong to the class Mollicutes (mollis, soft; cutis, skin).
Mollicutes, a term that includes the cell wall-less procaryotes assigned to
numerous genera under the class Mollicutes and is frequently used
interchangeably with mycoplasmas, are unusual for other biological reasons
as well. They are evolutionary descendants of the low G+C containing
gram-positive bacteria and, through chromosome reduction, represent the
smallest self-replicating life forms. Their streamlined genome size, which
illustrates extreme biological gene economy, imposes complex nutritional
requirements, such as dependence on external supplies of biosynthetic
precursors, including amino acids, nucleotides, fatty acids, and sterols.
This limited coding capacity dictates for mycoplasmas a parasitic way of
life that few pathogenic micro-organisms can claim. Therefore, the view
that pathogenic mycoplasmas can grow "independently" requires an
appreciation of their fastidious nature and their intimate dependence upon
the host. Because of these properties, pathogenic mycoplasmas are among the
most difficult micro-organisms to grow from clinical specimens and remain
frequent contaminants of primary and continuous eucaryotic cell lines and
tissue cultures (12). In some instances, mycoplasma contamination is
obvious since infected eucaryotic cells exhibit aberrant growth,
metabolism, and morphology. However, mycoplasmas often establish covert and
chronic infections of target cells that lead to either invalid and
misleading data or introduction of mycoplasmas or their products into
reagents dedicated to therapeutic or research purposes. The recent emphasis
on isolating viral agents, such as human immunodeficiency virus (HIV)-1,
from human primary lymphocytic cells has also demonstrated the frequent
cocultivation of mycoplasmas of human origin. Often, the unwanted sources
of exogenous mycoplasmas are serum products and filter-"sterilized"(450 nm)
solutions; cross-contamination by already infected cell cultures, viral
stocks, or immunologic preparations; breaks in technique, including
aerosols from the respiratory tract or by mouth pipetting; ignorance of the
mycoplasma problem; or scientific indifference.

Detailed up-to-date reviews describing the biological and pathogenic
properties of mycoplasmas have been published (1,2,13,14). Our intention
here is to provide a concise historical perspective of the role of
mycoplasmas in human disease; highlight the discoveries of new Mycoplasma
species and their association with human illness and host conditions that
present problems in detection and treatment; describe selected biological
properties of mycoplasmas consistent with their intimate host relationship
and possible mechanisms of pathogenicity; and address recent controversies
associated with mycoplasmas as emerging infectious agents. Renewed
attention to these issues may provide the impetus to demystify mycoplasmas
and improve their standing as genuine, card-carrying pathogens.

Historical Perspectives

The earliest reports of mycoplasmas as infectious agents in humans appeared
in the 1930s and 1940s. At that time, primary atypical pneumonia was
associated with an infectious agent that because of its minute size and
innate biological properties unknown at that time, passed through
bacteria-retaining filters, resisted penicillin and sulfonamide therapies,
and adapted to growth in embryonated eggs and tissue culture cells.
Correlations between the etiologic agent of "walking pneumonia" with
viruses, L-forms, and pleuropneumonia-like agents (referred to as PPLOs in
publications and textbooks of that era) were frequent and often misleading.
Finally, definitive studies in the early 1960s established Mycoplasma
pneumoniae as the singular cause of cold agglutinin-associated primary
atypical pneumonia (2). Today M. pneumoniae remains an important cause of
pneumonia and other airway disorders, such as tracheobronchitis and
pharyngitis (13,14), and is associated with extrapulmonary manifestations,
such as hematopoietic, exanthematic, joint, central nervous system, liver,
pancreas, and cardiovascular syndromes (15).

The confusion associated with M. pneumoniae-mediated infections has
recurred many times with other mycoplasmas, whose detection in clinical
specimens through culture, antibody, or DNA-based testing is frequently
dismissed as "only mycoplasmas" even when they appear to be the primary
pathogens. Two mycoplasmas commonly found in the urogenital tracts of
healthy persons are Mycoplasma hominis and Ureaplasma urealyticum. However,
over the years, the pathogenic roles of these mycoplasmas have been proven
in adult urogenital tract diseases, neonatal respiratory infections, and a
range of other diseases usually in immunocompromised patients (2).

Several recent examples illustrate the increasing impact of Mycoplasma
species on emerging diseases. Mycoplasma fermentans strains were first
isolated from the lower genital tract of both adult men and women in the
early 1950s, but their role in classic lower genital tract disease has not
been established (16). Reports in the 1970s of M. fermentans in the joints
of rheumatoid arthritis patients and in the bone marrow of children with
leukemia raised expectations for its pathogenic potential (17,18); these
findings have not been adequately confirmed. Sufficient evidence, however,
has accumulated recently to establish an important and emerging role for M.
fermentans in human respiratory and joint diseases. For example, M.
fermentans has been detected by specific gene amplification techniques such
as polymerase chain reaction (PCR) in the synovial fluid of patients with
inflammatory arthritis, but not in the joints of patients with juvenile or
reactive arthritis (19). In two other studies using PCR, M. fermentans was
identified in the upper respiratory tract of 20% to 44% of both healthy and
HIV-infected patients (20,21) and was associated with acute respiratory
distress syndrome in nonimmunocompromised persons (22).

Mycoplasma genitalium was detected in the urogenital tract of two patients
with nongonococcal urethritis in 1981 (23), but for more than a decade,
very little was known about its host distribution and pathogenicity. Early
experimental studies established that the organism caused lower genital
tract infections in both male and female chimpanzees, with extensive
urethral colonization in males and apparent tissue invasion, eventually
leading to overt bacteremia (24). However, the fastidious growth
requirements of M. genitalium from human hosts severely limited further
study until the advent of molecular detection techniques. Specific
sequences in the 140 kDa adhesin protein gene of M. genitalium were
selected as targets in a PCR-based detection assay (25,26). Subsequent
application of these techniques in cases of acute nongonococcal urethritis,
not including those of patients colonized or infected with Chlamydia
trachomatis, has provided mounting evidence for the involvement of M.
genitalium as an etiologic agent of this disease (27-29). Also, M.
genitalium has been suspected in chronic nongonococcal urethritis and
pelvic inflammatory disease (30).

The discovery in 1988 of M. genitalium strains in human nasopharyngeal
throat specimens, where they were frequently mixed with strains of M.
pneumoniae, not only changed dramatically the concept of host distribution
of M. genitalium but also prompted critical questions about the role of
this mycoplasma in human respiratory disease (31). However, the immunologic
cross-reactivity between M. genitalium and M. pneumoniae and the inability
of most conventional diagnostic serologic tests to conclusively identify M.
genitalium have complicated its delineation in acute human respiratory
disease. PCR assays specific for the organism have detected M. genitalium
in throat specimens of patients infected with HIV-1 (32). However, these
probes have not been applied to control groups and patients in outbreaks of
acute respiratory disease and/or pneumonia to determine whether M.
genitalium alone is an etiologic agent in respiratory infections.

M. genitalium has been implicated as an etiologic agent in certain human
joint diseases. This clinical correlation began with the observation of a
mixed infection of M. pneumoniae and M. genitalium in synovial fluid
specimens of a nonimmunocompromised patient after an acute respiratory
infection (33). A predominant role was not established for either
Mycoplasma species in the initial respiratory disease or in the joint
manifestations, although evidence to implicate postinfectious autoimmunity
to both organisms was described. These findings prompted a PCR assay on
synovial fluids from patients with various arthritic syndromes, which
presented case reports on two of 13 patients with M. genitalium detected in
joint fluids (34).

Another area of emerging mycoplasmal infections concerns immunodeficiency.
Although patients with congenital or acquired disorders of antibody
production are susceptible to a wide variety of microbial infections, the
unique susceptibility of such patients to mycoplasmal infections is a
growing concern, especially considering the number of occurrences, the
types of mycoplasmas involved, and the difficulties posed in the
therapeutic management of such infections. In addition, the increased use
of prolonged or permanent immunosuppressive chemotherapy required for
patients undergoing tissue or organ transplantation or treatment of various
malignant diseases has also increased the risk for mycoplasmal infections
from mycoplasmas that are part of the normal human mollicute flora to those
acquired through animal contact.

The association between immunodeficiency and mycoplasmal infections was
first reported in the mid 1970s in patients with primary
hypogammaglobulinemia and infection with U. urealyticum, M. pneumoniae,
Mycoplasma salivarium, and M. hominis that localized in joint tissue,
frequently with destructive arthritis. Similar joint infections in
hypogammaglobulinemic patients with these mycoplasmal species continue to
be reported (35). Since most of these mollicutes, with the possible
exception of M. pneumoniae, occur as part of the normal human flora, the
origin of such joint infections is considered endogenous. Patients with
hypogammaglobulinemia and other antibody deficiencies are also especially
susceptible to mycoplasmal infections of the upper respiratory and urinary
tracts caused most frequently by M. pneumoniae or U. urealyticum,
respectively (36).

Mycoplasmal infections following organ transplantation and
immunosuppressive chemotherapy were observed in the early 1980s, with both
M. hominis and U. urealyticum reported most often (37-39). Although these
infections most likely originated from the patient's normal microbial
flora, a recent report of donor transmission of M. hominis to two lung
allograft recipients (40) suggests that donor tissue may be a more
important factor in transplant infections than currently recognized.

While patients with antibody defects or those receiving immunosuppressive
drugs appear to be the most susceptible to infections with mycoplasmas
present in healthy tissues, emerging evidence indicates that contact with
other mycoplasmas in the environment is an important hazard. For example,
the direct isolation of a feline mycoplasma (M. felis) from the joint of a
hypogammaglobulinemic patient with septic arthritis was recently reported
(41), with suspected transmission occurring through a cat bite 6 months
before the onset of arthritis. Other examples include fatal septicemia
caused by M. arginini, a common animal mycoplasma, from blood and multiple
tissue sites in a slaughter house employee who had advanced non-Hodgkin's
lymphoma and hypogammaglobulinemia (42), and a septicemic infection with a
canine mycoplasma (M. edwardii) in a patient with advanced AIDS (M.K.York,
pers. comm.).

One of the most critical aspects of mycoplasmal infections in
immunodeficient patients is the frequent inability to control such
infections with appropriate broad spectrum antibiotics. Although the
tetracyclines and erythromycins are effective chemotherapeutic agents for
many mycoplasmal infections, M. fermentans and M. hominis strains are
usually resistant to erythromycin, and tetracycline-resistant strains of M.
hominis andU. urealyticum have been reported from the lower urogenital
tract of patients. However, these antibiotics and most other broad spectrum
agents have limited mycoplasmacidal activity in vivo, and their efficacy
eventually depends on an intact host immune system to eliminate the
mycoplasmas. Most hypogammaglobulinemic patients lack the ability to mount
a strong antibody response. Guidelines for managing such mycoplasmal
infections in patients with immune defects should include immediate in
vitro testing of the isolated mollicute against a wide range of
antibiotics; expeditious administration of the antibiotic by the most
appropriate route (intravenously, if warranted); prolonged therapy
terminated only if there is no rapid clinical or microbiological response;
and possibly administration of intravenous immunoglobulin (35,36). Clinical
management of mycoplasmal infections in transplant patients is more
difficult since immunoglobulins may enhance graft or organ rejection. In
the absence of suitable mycoplasmacidal chemotherapeutic agents, vigorous
and sustained chemotherapy with the most active antibiotic is the current
method of choice.

Mechanisms of Pathogenicity

Many mycoplasmal pathogens exhibit filamentous or flask-shaped appearances
and display prominent and specialized polar tip organelles that mediate
attachment to host target cells (43,44). These tip structures are complex,
composed of a network of interactive proteins, designated adhesins, and
adherence-accessory proteins (Figure 1, [14,43]). These proteins cooperate
structurally and functionally to mobilize and concentrate adhesins at the
tip and permit mycoplasmal colonization of mucous membranes and eucaryotic
cell surfaces, probably through host sialoglycoconjugates and sulfated
glycolipids (Figure 2, [14,43,45]). It appears that mycoplasmal
cytadherence-related proteins represent a superfamily of genes and proteins
that have been conserved through horizontal gene transfer from an ancestral
gene family. This protein network resembles a specialized cytoskeleton-like
apparatus, which may represent the precursor to mammalian cytoskeletal and
extracellular matrix-like complexes (14). Other Mycoplasma species lack
distinct tip structures yet are capable of cytadherence, and they may use
related genes or proteins or alternative mechanisms of surface parasitism.

 [Figure 1] [Figures not available in ASCII version]
 Figure 1. Transmission electron photomicrographs of the specialized tip
 organelle of cytadherence-positive M. pneumoniae demonstrating a)
 truncated structure with nap, b) clustering of cytadherence-related
 proteins (P1, B, C, P30) at the tip based on immunolabeling with ferritin
 and colloidal gold and crosslinking studies, and c) Triton
 X-100-resistant, cytoskeleton-like, structure with distinct bleb and
 parallel filaments (14,43,45,46).

 [Figure 2] [Figures not available in ASCII version]
 Figure 2. Transmission electron photomicrograph of a hamster trachea ring
 infected with M. pneumoniae (43). Note the orientation of the mycoplasmas
 through their specialized tiplike organelle, which permits close
 association with the respiratory epithelium. M, mycoplasma; m,
 microvillus; C, cilia.

The family of mycoplasmal genes and proteins involved in cytadherence has
been studied most extensively in M. pneumoniae (14,43,46-48).
Noncytadhering phenotypes that arise through spontaneous mutation at high
frequency have been categorized into mutant classes on the basis of
distinct protein profiles. These noncytadhering mycoplasmas cannot
synthesize specific cytadherence-related proteins or are unable to
stabilize them at the tip organelle, which leads to abnormal anatomical tip
structures and avirulence (43). Spontaneous reversion to the cytadhering
phenotype is accompanied by the reappearance of the implicated proteins,
restoration of structurally and functionally intact tips, and return of
full infectivity (43). Similar cytadherence-related genes and proteins have
been reported for M. genitalium on the basis of biochemical, immunologic,
and genetic analyses (25,49,50). Furthermore, striking similarities exist
in the order of operons that comprise the cytadherence-related genes and
the organization of these genes within each operon of M. pneumoniae and M.
genitalium (14,50,51). These similarities reinforce the unexpected
coisolations of M. genitalium, along with M. pneumoniae, from the
nasopharyngeal throat swabs of patients with acute respiratory diseases and
from synovial fluids of patients with arthritis as described in the
previous section (31,33). The isolation of M. pneumoniae from the human
urogenital tract (52) further suggests that these mycoplasmas have evolved
parasitic strategies that include overlapping tissue tropisms as determined
by the genetic and chemical relatedness of their cytadherence genes and
proteins (14,25,43,50,51). The recent use of transposon mutagenesis to
generate M. pneumoniae and M. genitalium transformants displaying
cytadherence-deficient phenotypes should further clarify the relationships
between the cytadherence-related genes and proteins and identify additional
sites previously unlinked to cytadherence (46,53).

An interesting feature of specific M. pneumoniae and M. genitalium adhesins
is their multiple gene copy nature (14,43,54,55,56). Although only one
full-length copy of the adhesin structural genes exists in adhesin-related
operons, precise regions of these adhesin genes are detected as single
genomic copies, while other regions occur as closely homologous, but not
identical, multiple copies. In other words, multiple truncated and
sequence-related copies of the adhesin genes are dispersed throughout the
genome, which could generate adhesin variation through homologous
recombination. Consistent with this possibility is the existence of
restriction fragment length polymorphisms in the adhesin genes of human
clinical isolates of M. pneumoniae and M. genitalium, reflected by sequence
divergence in the multiple-copy regions of the adhesin genes (56-59). It
appears that a repertoire of partial adhesin-related gene regions serves as
a reservoir to regulate the structural and functional properties of
mycoplasmal adhesins through recombination events, which may lead to
circumvention of the host immune response. Mechanisms of phase and
antigenic variation are likely to occur in which mycoplasmal adhesins
exhibit altered specificities and affinities, as determined by the
organization of constant and variable adhesin gene sequences. Therefore,
despite their small genomes, pathogenic mycoplasmas facilitate DNA
rearrangements through repetitive gene sequences, thus promoting genetic
diversity and maximizing the coding potential of their limited genomes. The
immunodominant epitopes of the mycoplasmal adhesins appear not to be
identical to the adherence-mediating domains (13). The latter are in part
encoded by single copy regions of the adhesin genes and are highly
conserved, which reinforces their essential role in mycoplasmal recognition
of host cell receptors and colonization (60,61). Host immunoresponsiveness
directed at the noncytadherence-mediating variable regions is unlikely to
generate effective cytadherence-blocking antibodies, which may in part
clarify the observed high reinfection rates of patients. Thus, the grouping
of clinical isolates of M. pneumoniae into two categories, on the basis of
sequence divergence in the multiple-copy regions of the adhesin gene
(56-59), along with the immune status of the population, may explain the
epidemiologic patterns of M. pneumoniae reported over the years.

Another characteristic of the cytadherence-related proteins is their
proline-rich composition, which markedly influences protein folding and
binding. Several reports have established the importance of these
proline-rich domains in mycoplasmal cytadherence and virulence
(47,48,62,63), and recent evidence further suggests that mycoplasmal
peptidylprolyl isomerases, i.e., cyclophilins, are critical in regulating
the conformation and function of the mycoplasmal cytadherence-related tip
organelle, colony morphology, and growth (14,64). In addition to this
proline-rich property, one of the most unusual features of the adhesins is
their extensive sequence homology to mammalian structural proteins
(1,14,33,43,47,48). This molecular mimicry is especially interesting since
it has been suggested for decades that mycoplasmas provoke an antiself
response that triggers immune disorders, although the basis for the
induction has been elusive (65). Patients with documented M. pneumoniae
respiratory infections demonstrate seroconversion to myosin, keratin, and
fibrinogen (33) and exhibit extrapulmonary manifestations, such as
exanthems and cardiac abnormalities. Furthermore, a classic example of
bacteria-mediated autoimmune disorders is the development of acute
rheumatic fever following streptococcal infection (66). Antistreptococcal
antibodies reactive against ahelical coiled-coil regions of the M protein
crossreact with heart myosin, tropomyosin, and mycoplasmal adhesins
(14,66). In the latter case, these mycoplasmal adhesins exhibit amino acid
sequence homologies with human CD4 and class II major histocompatibility
complex lymphocyte proteins, which could generate autoreactive antibodies
and trigger cell killing and immunosuppression (67,68). Also, mycoplasmas
may serve as B-cell and T-cell mitogens and induce autoimmune disease
through the activation of antiself T cells or polyclonal B cells. The
multiorgan protean manifestations of mycoplasmal infections in humans are
consistent with the pathogenesis of autoimmunity. Furthermore, the ability
of mycoplasmas to induce a broad range of immunoregulatory events, mediated
by cytokine production and direct effects on macrophages, B and T cells,
and glial cells, is evidence that mycoplasmas possess the attributes of
primary mediators of pathogenesis (1,2,12,69). For example, cytokine
production and lymphocyte activation may either minimize disease through
the activation of host defense mechanisms or exacerbate disease through
lesion development (69,70). Also, a superantigen derived from Mycoplasma
arthritidis, a mycoplasma pathogenic for rodents, induces arthritis and
chronic disease manifestations (69). It has been suggested that related
superantigen-like molecules may exist in mycoplasmas of human origin
triggering autoimmune and other inflammatory pathologies.

It appears that cytadherence is the initial step in the virulence process
of pathogenic mycoplasmas (Figure 2) and precedes a spectrum of subtle or
overt host cell responses. In specific instances, distinct cytopathology
correlates with the infecting Mycoplasma species, the number of adherent
mycoplasmas, the length of coincubation, the induction of proinflammatory
cytokines, and the age and immune status of the patient. For example, the
exacerbation of clinical syndromes may correlate with a history of
mycoplasmal infection as observed in patients with recurrent M. pneumoniae
exposures (2,13). Also, the elevated expression of proinflammatory
cytokines associated with mycoplasmal disease pathogenesis may coincide
with the intensity of the symptoms. In other cases, chronic disease or no
obvious signs or symptoms of disease accompany mycoplasmal infection.

Other biological properties of mycoplasmas have been implicated as
virulence determinants and include 1) generation of hydrogen peroxide and
superoxide radicals by adhering mycoplasmas, which induces oxidative
stress, including host cell membrane damage; 2) competition for and
depletion of nutrients or biosynthetic precursors by mycoplasmas, which
disrupts host cell maintenance and function; 3) existence of capsule-like
material and electron-dense surface layers or structures, which provides
increased integrity to the mycoplasma surface and confers immunoregulatory
activities; 4) high-frequency phase and antigenic variation, which results
in surface diversity and possible avoidance of protective host immune
defenses; 5) secretion or introduction of mycoplasmal enzymes, such as
phospholipases, ATPases, hemolysins, proteases, and nucleases into the host
cell milieu, which leads to localized tissue disruption and disorganization
and chromosomal aberrations; and 6) intracellular residence, which
sequesters mycoplasmas, establishes latent or chronic states, and
circumvents mycoplasmicidal immune mechanisms and selective drug therapies
(1,2,71,72). Whether pathogenic mycoplasmas enter and survive within
mammalian cells has been debated for many years. Consistent with this
possibility, mycoplasmas exhibit limited biosynthetic capabilities; are
highly fastidious and dependent upon the host microenvironment and complex
culture medium for growth; have been observed in intimate contact with
mammalian cell surfaces and within target cells; may be capable of
initiating fusion with host cells through their cholesterol-containing unit
membranes; and survive long-term recommended antimicrobial treatment in
humans and tissue cultures. Recent sightings of intact mycoplasmas
throughout the cytoplasm and the perinuclear regions of tissue cells from
infected patients and in cell cultures, along with evidence that
mycoplasmas are capable of long-term intracellular survival and replication
in vitro, offer an additional dimension to the pathogenic potential of
mycoplasmas (4,14,72,73).

The Latest Controversies: Food for Thought or the Twilight Zone

On the basis of the above information, the virulence strategies displayed
by mycoplasmas are likely the summation of a multitude of biological
activities (1). Since no obvious single or group of mycoplasmal properties
inextricably correlates with disease manifestations, the proof that
mycoplasmas are card-carrying pathogens necessitates thorough and highly
specific microbiological, epidemiologic, and diagnostic criteria; detailed
descriptions of biochemical, genetic, and immunologic characteristics that
distinguish virulent and avirulent mycoplasmas; and reproducibility of the
symptoms of disease in experimental animal models or in the natural spread
of infection among susceptible populations. The portfolio of available
evidence concerning mycoplasma-mediated disease pathogenesis is limited.
These scientific shortcomings precipitate misconceptions concerning
mycoplasmas as singular agents of infectious diseases, as putative
cofactors in the progression of other diseases, and as universal
contaminants of cell cultures. Clearly, multiple pathways of interaction
with target cells appears to be the modus operandi of the Mycoplasma
species. With this conceptual scientific framework as a background, five
recently proposed and controversial associations of mycoplasmas to human
diseases are worth noting.

AIDS

The role of mycoplasmas in accelerating the progression of AIDS could not
have begun under more baffling and circuitous conditions. A virus-like
agent that arose through transfection of NIH 3T3 cells with DNA from Kaposi
sarcoma tissues of AIDS patients was later shown to be M. fermentans. The
spotted history of M. fermentans in rheumatoid arthritis and leukemia and
its frequent contamination of cell cultures, along with its contemporary
link to AIDS, have been considerable impediments to overcome in its
elevation to pathogenic status. However, careful and convincing independent
studies by several laboratories have implicated M. fermentans as a cause of
systemic infections and organ failure in AIDS patients (4,74). The
isolation of M. fermentans from blood and urine samples of HIV-infected
persons, its detection by PCR and immunohistochemistry in multiple tissue
sites at various stages of AIDS, and its ability to stimulate CD4+
lymphocytes and other immunomodulatory activities implicate this Mycoplasma
species as a cofactor in AIDS. Consistent with this possibility, M.
fermentans has been shown to act synergistically with HIV to enhance
cytopathic effects on human CD4+ lymphocytes. Coincident with these
studies, a new Mycoplasma species, Mycoplasma penetrans, also has emerged
as a potential cofactor in AIDS progression (75,76). Its isolation almost
exclusively from the urine of HIV-infected patients, the extraordinarily
high prevalence of antibodies against this mycoplasma in HIV-infected
patients and not in HIV-seronegative persons, and its capacity to invade
target cells and activate the immune system of HIV-infected patients at
various stages of disease correlate with a synergistic role with HIV. Other
mycoplasmas, including M. genitalium and Mycoplasma pirum, have also been
isolated from AIDS patients and implicated as potential cofactors. However,
the proposed role of mycoplasmas as infectious agents and cofactors in
AIDS-related disorders still remains a hypothesis without definitive proof.
If cofactors of HIV are essential to the development of late stages of
HIV-mediated disease, mycoplasmas possess all the prerequisite properties
of the consummate helper. Their ability to establish covert or overt
chronic and persistent infections with concomitant activation of the immune
system, stimulation of cytokine production, and induction of oxidative
stress correlate with increased HIV replication and disease progression.
Are mycoplasmas irrelevant to AIDS, or are the clinical and microbiological
correlations sufficient to imply intimate relationships between HIV and
mycoplasmas, especially as the infected host undergoes immunologic
distress?

Malignant Transformation

As early as the mid-1960s, mycoplasma-infected cell lines were associated
with chromosomal aberrations, altered morphologies, and cell transformation
(77,78). These abnormal oncogenic cell traits continued even after the
apparent elimination of mycoplasmas, and evidence implied increased
tumorigenicity of these transformed cells in animals. This issue has been
revisited in studies demonstrating that longterm, persistent mycoplasmal
infection of mouse embryo cells initiated a multistage cellular process
that resulted in irreversible cell transformation, karyotypic alterations,
and tumorigenicity in nude mice (6). Do these oncogenic events associated
with mycoplasma-mammalian cell coincubation relate to the ontogeny of human
cancers?

Gulf War Syndrome

One of the most controversial current medical issues is whether the
multiple acute and chronic symptoms found in veterans of the Persian Gulf
War were caused by chemical exposure, infectious agents, or psychological
problems, or whether a Gulf War Syndrome exists at all. The clinical
illness comprises a collection of symptoms, including chronic fatigue,
joint pain, headaches, and skin rashes. One study suggests that pathogenic
mycoplasmas are responsible for a large number of cases among veterans, on
the basis of DNA hybridization and the responsiveness of veterans to
prolonged antibiotic treatment (5). Even though the experimental evidence
is sparse and incomplete and well-controlled and detailed studies by
independent laboratories are needed, if the Gulf War Syndrome has
infectious causes, mycoplasmas with their requisite biological credentials
are potential candidates.

Crohn's Disease

Several epidemiologic studies correlate respiratory infections with
exacerbation of Crohn's disease and other chronic inflammatory bowel
diseases (7,79). Acute onset gastrointestinal symptoms in patients with
these diseases are accompanied by seroconversion to specific viral or M.
pneumoniae antigens. As indicated earlier, mycoplasmas can elicit
pleiotropic immune responses and are difficult to eliminate in patients
despite appropriate antibiotic treatment. Steroid therapy to control
gastrointestinal symptoms in these patients, along with the multifaceted
biological properties associated with pathogenic mycoplasmas, may
precipitate the onset of acute exacerbations of chronic inflammatory bowel
disease.

Rheumatoid Arthritis and Other Human Arthritides

The occurrence of various Mycoplasma and Ureaplasma species in joint
tissues of patients with rheumatoid arthritis, sexually transmitted
reactive arthritis, and other human arthritides can no longer be ignored
(8). A clinical trial of longterm (6 to 12 months) antibiotic (doxycycline)
therapy before cartilage destruction might prove beneficial in managing
such frequent and often debilitating infections.

Extensive clinical and microbiological evidence indicates that mycoplasmas
alone can elicit a spectrum of illness for which no other agents are
incriminated. The eradication of these pathogenic mycoplasmas from various
tissue sites requires an intact and functional immune system, although
persons with fully competent immune systems may have difficulty eliminating
mycoplasmas, even with recommended prolonged drug therapy. Nonetheless,
mycoplasmas are still viewed as subordinates to other infectious agents and
are relegated to a category of commensals that unwittingly cause disease in
patients whose immune systems offer little resistance to microbial stress
and overload.

The fundamental importance of mycoplasmas in specific diseases of humans,
animals, insects, and plants is irrefutable, and their unique biological
properties are consistent with their intimate association with host target
cells. These remarkable bacteria must continue to receive the scientific
attention of mycoplasmologists, cell culturists, clinicians, immunologists,
and DNA sequencers who most recently are compiling extensive databases that
may eventually dissect every approachable mycoplasmal element that defines
their biological and genetic being. Nonetheless, mycoplasmas remain
mysterious and enigmatic, and the available data and proposed hypotheses
that correlate mycoplasmas with disease pathogenesis range from definitive,
provocative, and titillating to inconclusive, confusing, and heretical.
Controversy seems to be a recurrent companion of mycoplasmas, yet good
science and openmindedness should overcome the legacy that has burdened
them for decades.

Acknowledgments
This study was supported in part by NIH grants AI 27873, AI 32829 and AI
41010.

Dr. Baseman is professor and chair, Department of Microbiology, University
of Texas Health Science Center, San Antonio. His research focuses on
pathogen-host cell interactions with special interest in defining the
biology and virulence determinants of mycoplasmas pathogenic for humans.

Dr. Tully heads the Mycoplasma Section, Laboratory of Molecular
Microbiology, National Institute of Allergy and Infectious Diseases,
Frederick, Maryland. His interest covers the host distribution,
pathogenicity, and taxonomy of mollicutes.

Address for correspondence: Joel B. Baseman, Department of Microbiology,
The University of Texas Health Science Center at San Antonio, 7703 Floyd
Curl Drive, San Antonio, TX 78284-7758; fax: 210-567-6491; e-mail:
baseman@uthscsa.edu.

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---------------------------------------------------------------------------
 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
---------------------------------------------------------------------------
Synopses

Fluoroquinolone Resistance in Neisseria gonorrhoeae

Joan S. Knapp,* Kimberley K. Fox,* David L. Trees,* and William L.
Whittington
*Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Center for AIDS and STD, University of Washington, Seattle, Washington,
USA
---------------------------------------------------------------------------

      Fluoroquinolones and broad-spectrum cephalosporins are the
      most effective antimicrobial agents for the treatment of
      gonorrhea. However, clinically significant resistance to
      fluoroquinolones has emerged in Neisseria gonorrhoeae.
      Fluoroquinolone-resistant strains account for approximately
      10% of all gonococcal strains in Hong Kong and the Republic of
      the Philippines. As many as 50% of strains from some Far
      Eastern countries exhibit decreased susceptibility
      (intermediate resistance) to fluoroquinolones. Strains with
      intermediate resistance and clinically significant resistance
      are being isolated sporadically in North America, where
      resistant strains have been associated with an outbreak and
      with failure of infections to respond to treatment with doses
      of ciprofloxacin and ofloxacin recommended by the Centers for
      Disease Control and Prevention; strains exhibiting decreased
      susceptibility to these agents are endemic in at least one
      metropolitan area. Monitoring for fluoroquinolone resistance
      is now critical for ensuring adequate treatment of infections
      with resistant strains and for maximizing the time during
      which fluoroquinolones may be used to treat gonorrhea.

Gonorrhea is among the most prevalent sexually transmitted diseases
throughout much of the world. The emergence of resistance to antimicrobial
agents in Neisseria gonorrhoeae is a major obstacle in the control of
gonorrhea. In 1989 and 1993, in response to the increasing frequency of
isolation of penicillin-, tetracycline-, and spectinomycin-resistant
strains of N. gonorrhoeae in the United States, the Centers for Disease
Control and Prevention (CDC) recommended the use of broad-spectrum
cephalosporins or fluoroquinolones for the primary treatment of
uncomplicated gonorrhea (1,2). However, resistance to fluoroquinolones has
now emerged in N. gonorrhoeae (3-8). Because ciprofloxacin and ofloxacin
are more frequently used to treat gonorrhea than other fluoroquinolones,
this synopsis will focus on these agents.

Fluoroquinolone Therapeutic Regimens, Therapy Failure, and Susceptibility
Tests

In 1993, CDC recommended single-dose, oral therapy with ciprofloxacin (500
mg) or ofloxacin (400 mg) as two of the primary regimens for the treatment
of uncomplicated gonorrhea (2). Enoxacin (400 mg), lomefloxacin (400 mg),
and norfloxacin (800 mg) were recommended among alternative regimens (2).
In some countries, gonococcal infections have been treated with a single,
orally administered dose of 250 mg ciprofloxacin (8). The failure of
gonococcal infections to respond to treatment with 250 mg ciprofloxacin has
been reported in the United Kingdom since 1990 (8-11). The failure of
infections to respond to single-dose therapy with 500 mg ciprofloxacin or
400 mg ofloxacin has been reported in the United Kingdom, Australia,
Canada, Hong Kong, and the United States (3-7,12).

Different methods for determining in vitro antimicrobial susceptibilities
of N. gonorrhoeae have been developed in several countries. Differences
between these methodsthe medium on which the susceptibilities are
determined, the concentrations of antimicrobial agents tested, the
concentration of antimicrobial agents in disks used to determine zone
inhibition diameters, or the inoculum sizecomplicate the interpretation of
susceptibility test results. For example, in the United Kingdom, Australia,
and Hong Kong, susceptibilities are usually determined on Isosensitest
medium (Oxoid, Basingstoke, United Kingdom) (3,8,13-15). In the United
States (where criteria for interpreting susceptibilities of N. gonorrhoeae
are established by the National Committee for Clinical Laboratory Standards
[NCCLS]) and Canada, susceptibilities are determined on a Difco or Becton
Dickinson base medium (Difco Laboratories, Detroit, MI; Becton Dickinson,
Cockeysville, MD) (16-19). Antimicrobial susceptibilities determined on
Oxoid base medium may be one concentration lower than those determined on
supplemented Difco or Becton Dickinson base media (15). Neither method is
right or wrong; nor is one method better than the other; they are
different. The method used in different countries is influenced by the
commercial availability of the base medium. Thus, criteria for interpreting
susceptibilities in the United States and Canada may differ slightly from
those used to interpret susceptibilities in the United Kingdom, Australia,
and Hong Kong, reflecting the difference in susceptibilities obtained on
different media. Similarly, when susceptibilities are measured on media
different from those described above it may be necessary to establish
"local" criteria using reference strains with known susceptibilities.

Gonococcal strains associated with therapy failure to 250 mg ciprofloxacin
have had minimum inhibitory concentrations (MICs) of  >= 0.06 to 0.25 g/ml
of ciprofloxacin (8,11); posttreatment strains from infections that failed
to respond to treatment with 500 mg ciprofloxacin or 400 mg ofloxacin have
had MICs  >= 1.0 g/ml and  >= 2.0 g/ml, respectively (4-6,12,15).

Criteria for Interpreting Fluoroquinolone Resistance

Before the emergence of fluoroquinolone resistance in N. gonorrhoeae, NCCLS
established interpretive criteria to differentiate between susceptible
strains and those exhibiting decreased susceptibility to selected
fluoroquinolones including ciprofloxacin, ofloxacin, lomefloxacin, and
enoxacin (16,17). Criteria for the interpretation of clinically significant
resistance of gonococcal strains to 500 mg ciprofloxacin and 400 mg
ofloxacin have been proposed (6,18).

Criteria for interpreting susceptibilities of N. gonorrhoeae to
antimicrobial agents should be based on treatment outcome and strain
susceptibility data. However, because many infections caused by
fluoroquinolone-resistant strains were treated with broad-spectrum
cephalosporins and few observations linked fluoroquinolone therapy outcome
and antimicrobial susceptibilities (MICs), CDC proposed criteria for
interpreting fluoroquinolone resistance in N. gonorrhoeae that were based
on theoretical predictions of the MICs at which gonococcal infections may
fail to respond to CDC-recommended doses of selected fluoroquinolones (18).
On the basis of the therapeutic index (calculated by dividing the peak
level of the agent in serum by the MIC of the infecting strain), CDC
proposed criteria for interpreting susceptibilities to selected
fluoroquinolones (Table) (18,20). These criteria were consistent with the
MICs of isolates from the observed treatment failures to 500 mg
ciprofloxacin and 400 mg ofloxacin documented at the time of the study
(4,5,12). Recently, Kam et al. proposed interpretive criteria based on the
susceptibilities of many strains that did not respond to treatment with
ofloxacin in Hong Kong (Table) (6). CDC and Kam et al. proposed identical
MICs for interpreting resistance to ciprofloxacin and ofloxacin: MICs of
 >= 2.0 g/ml and  >= 1.0 g/ml of ofloxacin and ciprofloxacin,
respectively. These criteria have also been adopted in Australia (15).

 Table. Criteria for interpreting susceptibilities of Neisseria gonorrhoeae
 strains to the fluoroquinolones ciprofloxacin, ofloxacin, enoxacin,
 lomefloxacin, and norfloxacina,b

 --------------------------------------------------------------------------------
  Therapeutic    Agent    Zone Inhibition    Equivalent MICc (mg/ml)   Reference
     agent,     Tested,      Diametersc
      Dose       Disk     nearest whole mm
               Content
                 (g)      R     I     S       R        I        S
 --------------------------------------------------------------------------------
 Ciprofloxacin,          <= 24               >= 0.03
 250 mg        Cip, 1                                                  8
 Ciprofloxacin,          <= 29        >= 36  >= 1.0           <= 0.06
 500 mg        Cip, 5          30-35                0.13-0.5           18

               Cip, 5    <= 22               >= 1.0                    6

 Ofloxacin, 400          <= 24        >= 31  >= 2.0           <= 0.25
 mg            Ofx, 5          25-30                0.5-1.0            18

               Ofx, 5    <= 24               >= 1.0                    6

 Enoxacin, 400           <= 31        >= 32  >= 1.0           <= 0.25
 mg            Enx, 10                              0.5                18
 Lomefloxacin,           <= 26        >= 36  >= 2.0           <= 0.125
 400 mg        Lom, 10         27-35                0.25-1.0           18
 Norfloxacin,            <= 32        >= 36  >= 1.0           <= 0.25
 800 mg        Nor, 5          33-35                0.5                18
 --------------------------------------------------------------------------------
 aThese criteria have not been recommended by the National Committee for
 Clinical Laboratory Standards (NCCLS); they are provided to guide
 interpretation of susceptibilities until the NCCLS establishes criteria.
 bAbbreviations: MIC, minimal inhibitory concentration (g/ml); R, resistant; I,
 intermediate resistance; S, susceptible; Cip, ciprofloxacin; Ofx, ofoxacin;
 Enx, enoxacin; Lom, lomefloxacin; Nor, norfloxacin.
 cWith the exception of norfloxacin, criteria for the susceptible categories are
 those designated by the NCCLS (16,17); criteria for the interpretation of the
 susceptible category for norfloxacin were proposed by CDC (18).

In the United Kingdom, where 250 mg ciprofloxacin has been used to treat
gonorrhea, strains with MICs of 0.06 to 0.25 g/ml have been isolated from
infections that did not respond to treatment (8,11). Ciprofloxacin in a
250-mg dose produces a peak serum level of approximately 1.2 g/ml; thus,
strains with MICs of  >= 0.25 to 0.5 g/ml would produce therapeutic
indices of 4.8:1 and 2.4:1, respectively. These calculated MICs suggest
that strains with MICs >= 0.25 g/ml should be considered resistant to
treatment with 250 mg ciprofloxacin. Criteria for interpreting
susceptibilities of gonococcal strains to treatment with 250 mg
ciprofloxacin have been determined to ciprofloxacin (1 g disks) or
nalidixic acid (30 g disks) (10,21). Measurement of nalidixic acid
resistance may be useful for detecting strains causing infections that may
not respond to treatment with 250 mg ciprofloxacin. However, because
naladixic acid-resistance indicates decreased susceptibility to
ciprofloxacin and ofloxacin, this test does not differentiate between
strains with intermediate resistance and clinically significant resistance
to treatment with 500 mg ciprofloxacin or 400 mg ofloxacin (18).

Geographic Distribution, Frequency, and Diversity of
Fluoroquinolone-resistant N. gonorrhoeae

Strains exhibiting intermediate resistance (MICs 0.125 to 0.5 g/ml of
ciprofloxacin; MICs 0.5-1.0 g/ml of ofloxacin) have been reported from
many geographic areas including Australia, Canada, the Canary Islands, Hong
Kong, Japan, the Republic of the Philippines, mainland Spain, Thailand, the
United Kingdom, the United States, and the West Indies (4,7,9-15,21-32).
Fluoroquinolone resistance (MICs of  >= 1.0 g/ml or  >= 2.0 g/ml of
ciprofloxacin or ofloxacin, respectively) has been reported most frequently
from the Far East, Republic of the Philippines, Hong Kong, Japan) and less
frequently from Australia, Canada, Spain, Thailand, United Kingdom, and the
United States (3,4,6,7,9,12,15,22-32). Ofloxacin- and
ciprofloxacin-resistant strains had been isolated sporadically in Hong
Kong, the Republic of the Philippines, and Thailand for some years (22-24).
The frequency of fluoroquinolone-resistant strains has increased
dramatically since the early 1990s. For example, in Hong Kong,
fluoroquinolone-resistant strains were isolated intermittently during 1990
to 1992 (13) but have increased dramatically from an estimated 0.5% in late
1992 to 10.4% in late 1994 (14).

From 1994 to 1995, strains exhibiting decreased susceptibility to
ciprofloxacin and ofloxacin accounted for approximately 36%, 54%, and 22%
of strains in Hong Kong, the Republic of the Philippines, and Thailand,
respectively (13,14,29,31,32). During the same period,
fluoroquinolone-resistant strains accounted for approximately 10%, 12%, and
1% of all strains in Hong Kong, the Republic of the Philippines, and
Thailand, respectively (13,14,29,31,32). Strains with ciprofloxacin MICs of
 >= 8.0 g/ml were first isolated in 1994 (10,12,29,31).

In other geographic areas, strains exhibiting intermediate resistance and
resistance have been isolated only sporadically, although with increasing
frequency. In Sydney, Australia, fluoroquinolone-resistant strains were
isolated infrequently in 1991 to 1994, but with dramatically increasing
frequency in early 1995 (15). The pattern of isolation of
fluoroquinolone-resistant strains in Australia, i.e., infrequent and
sporadic isolations for a number of years followed by increasing frequency
of isolation over a short period, may be anticipated in other countries
unless the fluoroquinolone-resistant strains are controlled in the Far
East, where they are now prevalent. In the CDC-sponsored Gonococcal Isolate
Surveillance Project in the United States, the frequency of strains with
intermediate resistance has increased significantly from 0.3% (17/5,238) in
1991 to 1.3% (65/4,996) in 1994 (p <= 0.001); however, resistant strains
accounted for only 0.04% (2/4,996) of strains in 1994 (33,34). In the
United States, the increase in strains with intermediate resistance is
associated largely, but not exclusively, with the persistence of such
strains in Cleveland, Ohio. First detected in 1992, these strains accounted
for 16% to 17.5% of isolates in Cleveland in 1994 (26,35). In addition, a
sustained outbreak caused by ciprofloxacin-resistant strains has been
reported from Seattle, Washington, in 1995; these strains had MICs of 8.0
g/ml of ciprofloxacin and ofloxacin (5).

Many different strains, as defined by penicillin/tetracycline resistance
phenotype and auxotype/serovar (A/S) class, exhibit intermediate resistance
and resistance to fluoroquinolones (4,5,15,25,26,31,32,36). Fluoroquinolone
resistance has been identified frequently in strains that produce
-lactamase and strains exhibiting chromosomally mediated resistance to
penicillin and tetracycline and less frequently in strains that are
susceptible to penicillin and tetracycline (4,31,32). Fluoroquinolone
resistance has not been documented in strains possessing the
25.2-megadalton TetM-containing plasmid (TRNG) alone or in TRNG strains
possessing a -lactamase plasmid (31). It should not, however, be assumed
that these strains may not develop fluoroquinolone resistance, but rather
that resistant strains have not been detected at this time. Many strains,
as defined by A/S class, are associated with fluoroquinolone resistance;
fluoroquinolone resistance is not associated with the epidemic spread of
one or two strains (15,31,32). In Australia, strains exhibiting
intermediate resistance and resistance to fluoroquinolones have belonged to
27 different A/S classes; strains with MICs 8.0-16.0 g/ml of ciprofloxacin
have belonged to 6 A/S classes (15). In the Republic of the Philippines,
strains belonging to 27 A/S classes exhibited decreased susceptibility to
ciprofloxacin; strains belonging to 10 A/S classes had MICs  >= 1.0 g/ml
of ciprofloxacin (31). In Thailand, strains belonging to 13 A/S classes
exhibited decreased susceptibility to ciprofloxacin (32).

The Gonococcal Isolate Surveillance Project monitors the susceptibilities
of N. gonorrhoeae isolates to fluoroquinolones in the United States and
provides prototype reference strains for quality assurance of
susceptibility testing of these agents. Information about antimicrobial
resistance in N. gonorrhoeae in the United States is available on the
Internet at http://www.cdc.gov/ncidod/dastlr/gcdir/gono.html.

Mechanisms of Resistance to Fluoroquinolones

Fluoroquinolones inhibit the replication of DNA; they are believed to bind
to the GyrA region of DNA gyrase, which is attached to DNA, and inhibit the
enzyme from supercoiling the DNA (37). Resistance to fluoroquinolones in N.
gonorrhoeae is associated with mutations that result in amino acid changes
in the A subunit (GyrA) and the B subunit (GyrB) of the DNA gyrase, and in
the parC-encoded subunit of topoisomerase IV (37-40). Although mutations in
gyrB confer low-level resistance to naladixic acid, high-level quinolone
resistance is associated with mutations in the quinolone
resistance-determining region of gyrA (41). Topoisomerase IV, encoded by
parC and parE in Escherichia coli and believed to be located in the
cytoplasmic membrane, is involved in DNA replication but is not as
sensitive to fluoroquinolone inhibition as is DNA gyrase (37). No parE
analog has been detected in N. gonorrhoeae (37). Mutations in gyrA and parC
are most relevant when considering clinically significant levels of
fluoroquinolone resistance in N. gonorrhoeae (37,39,40). Similar results
have been obtained in studies of gyrA mutations in both laboratory-adapted
strains and clinical isolates (37,39,40): ciprofloxacin-susceptible strains
(MICs, <0.03 g/ml) had no mutations in gyrA and strains with MICs, >= 0.5
g/ml of ciprofloxacin may have changes in nucleotides 272 and 283 of gyrA.
In addition, strains with MICs  >= 2.0 had mutations in parC. Mutations in
parC were observed only in strains with at least one mutation in gyrA
(37,39) and appeared to be associated with an MIC higher than would be
expected with the gyrA mutation alone (39). Mutations in gyrA and parC may
be characterized by polymerase chain reaction and DNA sequencing (37,39).
The transfer of gyrA and parC mutations between gonococcal strains has been
demonstrated in vitro (37). The presence of transformation sequences just
downstream from the gyrA sequences suggests that transformation may play a
role in the spread of gyrA mutations between gonococcal strains in vivo
(37). The opportunity for transformation of genes between gonococcal
strains, which depends on concurrent infections with multiple strains, has
been documented for women and homosexual men (42,43).

In addition to mutations in gyrA and parC, reduced permeability of the
cytoplasmic membrane may contribute to low-level resistance to
fluoroquinolones in N. gonorrhoeae, e.g., increasing the MIC of one
recipient strain from  <= 0.002 to 0.06 g/ml of ciprofloxacin (3,44). This
resistance may also be transferred between gonococcal strains by
transformation (3).

Guidance

Continued reports of the isolation of fluoroquinolone-resistant strains of
N. gonorrhoeae indicate the need for heightened awareness of the potential
for increasing prevalence of strains with clinically significant
fluoroquinolone resistance. In areas where a fluoroquinolone is used to
treat gonorrhea, the following steps are recommended to monitor and control
the spread of fluoroquinolone-resistant strains of N. gonorrhoeae.

  1. Susceptibility testing should be performed to detect fluoroquinolone
     resistant strains. In geographic areas where interpretive criteria
     have not been proposed, criteria should be developed for results
     obtained with local test procedures that use strains with known
     susceptibilities to ciprofloxacin or ofloxacin.
  2. Ideally, routine surveillance for emerging fluoroquinolone resistance
     should be performed in centers where fluoroquinolones are used widely
     to treat gonorrhea; e.g., a sample of approximately 20 to 50
     consecutive isolates should be tested periodically. If
     ciprofloxacin-resistant isolates are detected, the use of alternative
     therapies for gonorrhea should be considered; if isolates are
     ciprofloxacin-susceptible, ciprofloxacin or ofloxacin may be used to
     treat gonorrhea.
  3. Susceptibilities of isolates from individual patients whose infections
     did not respond to treatment with a fluoroquinolone should be
     determined.
  4. A cluster of infections unresponsive to fluoroquinolone therapy may
     indicate an outbreak caused by a resistant strain.
  5. If treating gonorrhea with a fluoroquinolone, e.g., ciprofloxacin or
     ofloxacin, never use less than the recommended dose. In the United
     States, 500 mg of ciprofloxacin or 400 mg ofloxacin should be used.
  6. Because gonococcal infections caused by fluoroquinolone-resistant
     strains have been acquired frequently in the Far East, clinicians may
     wish to treat infections possibly acquired in the Far East with 125 mg
     ceftriaxone, intramuscularly, or 400 mg cefixime, orally, the current
     CDC-recommended doses for treating gonococcal infections.

The importance of the emergence and spread of fluoroquinolone resistance in
N. gonorrhoeae cannot be overstated. Of antimicrobial agents available for
treating gonorrhea, broad-spectrum cephalosporins are the only agents to
which N. gonorrhoeae is not resistant, and exclusive use of these agents,
particularly the orally administered cephalosporins such as cefixime, may
result ultimately in the emergence of resistance to these agents. Thus, it
is critical to take measures to ensure that fluoroquinolones remain
effective for the treatment of uncomplicated gonorrhea for as long as
possible.

Dr. Knapp is chief, Gonorrhea, Chlamydia, and Chancroid Branch, Division of
AIDS, STD, and TB Laboratory Research, National Center for Infectious
Diseases, CDC. Her research interests are antimicrobial resistance in N.
gonorrhoeae, the molecular epidemiology of gonococcal strain populations,
and the taxonomy of Neisseria spp.

Address for correspondence: Joan S. Knapp, Ph.D., Centers for Disease
Control and Prevention, 1600 Clifton Road NE, MS G39, Atlanta, GA 30333,
USA; fax: 404-639-3976; e-mail: jsk2@cdc.gov.

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---------------------------------------------------------------------------
 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
--------------------------------------------------------------------------- 

Synopses

Infectious Diseases and Immunity: Special Reference to Major
Histocompatibility Complex

Neeloo Singh,* S. Agrawal, and A.K. Rastogi*
*Department of Biochemistry, Central Drug Research Institute, Lucknow,
India; Department of Medical Genetics, Sanjay Gandhi Post Graduate
Institute of Medical Sciences, Lucknow, India
---------------------------------------------------------------------------

      Human leukocyte antigens (HLAs) are an inherent system of
      alloantigens, which are the products of genes of the major
      histocompatibility complex (MHC). These genes span a region of
      approximately 4 centimorgans on the short arm of human
      chromosome 6 at band p 21.3 and encode the HLA class I and
      class II antigens, which play a central role in cell-to-cell
      interaction in the immune system. These antigens interact with
      the antigen-specific cell surface receptors of T lymphocytes
      (TCR) thus causing activation of the lymphocytes and the
      resulting immune response. Class I antigens restrict cytotoxic
      T-cell (CD8+) function thus killing viral infected targets,
      while class II antigens are involved in presentation of
      exogenous antigens to T-helper cells (CD4+) by antigen
      presenting cells (APC). The APC processes the antigens, and
      the immunogenic peptide is then presented at the cell surface
      along with the MHC molecule for recognition by the TCR. Since
      the MHC molecules play a central role in regulating the immune
      response, they may have an important role in controlling
      resistance and susceptibility to diseases. In this review we
      have highlighted studies conducted to look for an association
      between HLA and infectious diseases; such studies have had a
      variable degree of success because the pathogenesis of
      different diseases varies widely, and most diseases have a
      polygenic etiology.

Major Histocompatibility Complex (MHC) and Immune Response

Because of its remarkable power to deal with infection, the immune system
is central to the prevention and control of infectious disease. Immune
responsiveness is affected, even controlled, by gene products of the major
histocompatibility system (1). Many diseases are associated with human
leukocyte antigens (HLAs) (2,3). Moreover, in some infectious diseases
(4-6), the host immune reactivity, which is responsible for the pathologic
manifestation of disease, has been correlated with HLA specificities.

The discovery of the human MHC dates from the mid 1950s when
leukoagglutinating antibodies were found in the sera of patients who
received multiple transfusions and in the sera of 20% to 30% of multiparous
women. In humans, the entire histocompatibility complex is termed the HLA
complex. Genes coding for HLAs occupy a segment of approximately 4
centimorgans on the short arm of chromosome 6. The HLA-A, -B, and -C
genetic loci determine class I antigens; HLA-DR, -DP, -DQ genetic loci
determine class II antigens. Class I antigens are found on virtually every
human cell; class II antigens are found chiefly on the surfaces of
immunocompetent cells, including macrophages/monocytes, resting T
lymphocytes, activated T lymphocytes, and particularly B lymphocytes.

The MHC molecule provides a context for the recognition of antigens by T
lymphocytes. The polymorphic binding site of MHC class I and class II
molecules is composed of a -pleated sheet flanked by two alpha helixes.
They form a groove that accommodates one single microbial peptide ligand.

MHC class I molecules bind to peptides produced by the intracellular
degradation of viral proteins and display them on the cell surface for
recognition by CD8+ T lymphocytes. A class of white blood cells, the CD8 T
lymphocytes, bear receptors specific for the HLA class I antigens and route
pathogens such as viruses. Surface expression of class I MHC molecules
depends on the availability of peptides that bind MHC molecules in the
endoplasmic reticulum. A peptide transporter, associated with antigen
processing (TAP), plays an important role in maintaining adequate levels of
peptide (7). The transporter is a heterodimer encoded by two genes, TAP1
and TAP2, located in the MHC class II region. TAP genes belong to the
adenosine triphosphate (ATP) binding cassette super family of transport
proteins, which have two ATP-binding cassette domains and two transmembrane
domains. TAP genes are polymorphic (8), and allelic MHC differences may be
associated with disease by altering the peptides that bind class I MHC
molecules. Since human TAP genes are located between HLA-DP and HLA-DQ, TAP
alleles could result in an apparent disease association with class II HLA
alleles. Class I, i.e., HLA-A, -B, and -C molecules, play an important role
in viral infections in the lysis of target cells by cytotoxic killer T
lymphocytes.

MHC class II molecules are highly polymorphic membrane glycoproteins that
bind peptide fragments of proteins and display them for recognition by CD4+
T lymphocytes. The white blood cells known as CD4 T lymphocytes are of
central importance in defeating the bacteria and other parasites that live
within cells. The CD4 T lymphocytes are called helper T cells because they
secrete substances that amplify and control virtually all aspects of
immunity. These T cells have receptor molecules that can recognize one
particular peptide-HLA class II antigen combination. The binding capability
of any given peptide to MHC class II molecules depends on the primary
sequence of the peptide and allelic variation of the amino acid residues in
the binding site of the MHC receptor. Anchor residues defining
allele-specific peptide motifs have been identified in the class II binding
peptides. The proposed anchor residues combining with MHC pockets through
their side chains seem to be a primary requirement for peptide-MHC
interaction. The invariant chain (Ii) plays a critical role in the
assembly, intracellular transport, and function of MHC class II molecules
(9). In intracellular parasites (e.g., Leishmania infections of
macrophages), it is the class II MHC molecules that specifically bind to
receptors on these microbes.

HLA Association with Infectious Diseases

Infectious diseases are associated with impaired immunity. Some persons
mount very effective immune responses when given vaccines, while others
respond to vaccines poorly or not at all. The level of response is
determined by several factors: intensity of infection, factors related to
the intensity of the host immune response, T-cell state, T-cell function,
and perhaps most important, the genetic factor that interacts with the
other factors to determine the outcome of the disease. Infectious disease
research is now focusing on genetic markers such as allelic forms of HLA
molecules.

HLA Association with Mycobacterial Infections
Genetic factors may control host responses to Mycobacterium tuberculosis
(10-12). Several investigators have conducted population studies to
determine an association between pulmonary tuberculosis (TB) and HLA
specificities. HLA-DR2 is associated with the development of multibacillary
forms of both TB and leprosy (13,14); molecular subtyping of DR2 showed
that the majority of the allele in patients and controls was DRB1*1501 and
DRB1*1502. The frequency of these molecular subtypes of DR2 in patients was
not skewed, suggesting that the entire DR2 molecule or its closely linked
gene(s) may govern patient susceptibility to pulmonary TB and,
particularly, to drug-resistant TB. When the three-dimensional structure of
the HLA-DR molecule is elucidated (15), sequencing of class II alleles in
patients with pulmonary TB and drug-resistant TB could identify an amino
acid residue(s) critical for the binding of a M. tuberculosis-derived
pathogenic peptide(s) responsible for the detrimental or protective immune
response.

HLA alleles also modulate the immune response that determines the form of
leprosy (a heterogeneous disease caused by Mycobacterium leprae) that
develops in each patient (16,17). At one pole of the spectrum of leprosy
are the multibacillary lepromatous leprosy (LL) patients, who are anergic
to the antigens of M. leprae, and at the other extreme are the
paucibacillary tuberculoid leprosy (TT) patients, who exhibit a good
cell-mediated immune response. Humoral immunity is present throughout the
spectrum but does not seem to provide protection. Between the two poles are
patients with intermediate features as seen in the borderline lepromatous,
borderline leprosy (BB), and borderline tuberculoid forms (18). An
increased frequency of HLA-DR2 and -DQ1 in LL patients (19) and of HLA-DR3
in TT patients has been reported (20). These antigens can be further
subdivided into alleles defined by their amino acid sequence. A single
amino acid substitution may give rise to alleles with different immunologic
properties. The allele DRB1*1501 showed a stronger association with LL
patients than with TT patients (p < 0.00001). In addition, DQB1*0601 was
found significantly more often in LL patients than in controls (p <
0.00001); DQA1*0103 was more frequent in the LL group than in the
tuberculoid leprosy group; and DQA1*0102 was selectively increased in
patients with borderline lepromatous leprosy (Table 1). However, DRB1*0701,
DQB1*0201, and DQA1*0201 were decreased in LL patients compared with TT
patients and controls, and DQB1*0503 was selectively decreased in TT
patients, suggesting that these alleles might modulate the immune response
that determines the form of leprosy that develops in each patient (21).

              Table 1. Frequency of HLA class II alleles with
              significant differences between leprosy patients
              and healthy controls
              ------------------------------------------------
                          Healthy   Leprosy
                         controls  patients
                         --------------------
                 HLA       N=47      N=93
               alleles    N    %   N    %    RR(a)     p
              ------------------------------------------------
              DRB1*15    10 21.3   70 75.3  11.3   <0.00001
              DRB1*1501  6  12.8   49 52.7  7.6    <0.00001
              DRB1*1502  5  10.6   26 27.9  3.2    <0.05
              DRB5*0101  6  12.8   49 52.7  7.6    <0.00001
              DRB5*0102  5  10.6   26 27.9  3.2    <0.05
              DQA1*0102  9  19.1   38 40.9  2.9    <0.05
              DQA1*0103  13 27.6   48 51.6  2.8    <0.05
              DQB1*0601  8  17.0   56 60.2  7.4    <0.00001
              DRB1*0404  5  10.6   0  0.0   0.04   <0.01
              DRB1*0701  13 29.8   11 11.8  0.3    <0.05
              DRB1*1401  4  8.5    0  0.0   0.005  <0.05
              DQB1*0503  16 36.2   14 16.1  0.3    <0.05
              ------------------------------------------------
              (a)RR = relative risk

HLA Association with Parasitic Infections
Because there are significant differences between malaria-exposed and
-unexposed populations in the frequencies of HLA genes at the A and B loci,
the HLA complex may protect populations in endemic-disease areas who are
exposed to malaria parasites. The adaptative mechanisms may be expressed by
HLA-associated genes that control immune responsiveness to malaria
antigens. The association between the HLA class I antigen HLA-B53 and
protection from severe malaria has been well established (5). This link
might be mediated by HLA class I restricted cytotoxic T lymphocytes (CTL)
during the liver stage of the parasite's life cycle (22). The protective
association between HLA-B53 and severe malaria was investigated by
sequencing peptides eluted from this molecule before testing candidate
epitopes from preerythrocytic-stage antigens of Plasmodium falciparum in
biochemical and cellular assays. Among malaria-immune Africans, HLA-B53
restricted CTL recognized a conserved nonamerpeptide from liver
stage-specific antigen, but no HLA-B53 restricted epitopes were identified
in antigens from other stages (5). These findings indicate a possible
molecular basis for this HLA disease association and support the candidacy
of liver stage-specific antigen as a malaria vaccine component.

The association between HLA-DR/-DQ phenotypes and immune response to
circumsporozoite protein of the human malaria parasite were investigated in
Thai adults (23). Evidence suggests that human T- and B-cell responses to a
major P. falciparum antigen (Pf RESA) in persons primed by repeated
infections are genetically regulated (24). To associate T-cell and antibody
responses with the donors' MHC class II genotypes, genomic HLA class II
typing of DQ antigens of leukocytes from 145 donors living in
endemic-disease regions of Africa were performed by restriction fragment
length polymorphism (24). These data imply that the impact of MHC class II
gene products on specific immune responses to Pf 155/ RESA epitopes is weak
and hard to demonstrate in outbred human populations naturally primed by
infection. The relationship between class II HLA and immune recognition of
three candidate antigens for a vaccine against P. falciparum was
investigated in persons extremely heterozygous for HLA class II alleles
living in an endemic-disease area of West Africa (25). One class II DQA-DQB
combination (serologic specificity DQw2) was particularly common among
these persons. This haplotype was significantly associated with higher than
average levels of antibody to a peptide epitope (EENV)6 of Pf RESA. There
was little evidence of association between HLA class II genotype and
cellular proliferation responses to the antigen tested.

The frequency of HLAs was studied in 62 patients with scabies and 27
patients with cutaneous leishmaniasis to evaluate the role of HLA antigens
as genetic markers in the pathogenesis of these parasitic skin diseases. A
significant statistical association was found between HLA-A11 antigen and
scabies and between HLA-A11, -B5, and -B7 antigens and diffuse cutaneous
leishmaniasis (26). In another study, 24 families with one or more cases of
localized cutaneous leishmaniasis from an endemic-disease region with the
highest incidence of localized cutaneous leishmaniasis in Venezuela were
typed for HLA-A, -B, -C, -DR, and -DQ antigens and complement factors. The
parental HLA haplotypes segregated at random among healthy and affected
siblings, but in backcross families significantly higher frequencies of
HLA-A28, -Bw22 or -DQw8 were present in infected compared with healthy
siblings (27). In addition, HLA-B15 showed a higher frequency among healthy
siblings. Haplotypes Bw22, DR11, DQw7 were also significantly more frequent
in infected than in healthy siblings. No HLA linkage with a putative
localized cutaneous leishmaniasis susceptibility gene(s) could be
demonstrated in this study (27). A case/control comparison of 26 unrelated
localized cutaneous leishmaniasis patients and healthy persons of the same
ethnic origin confirmed the association of HLA-Bw22 and DQw3 with this
disease. The relative risk reached 12.5 for Bw22 and 4.55 for DQw3.
HLA-DQw3 apparently makes the major contribution as a genetic risk factor
for localized cutaneous leishmaniasis at the population level. In another
study, a statistically significant association was found between HLA-B5 and
-DR3 and schistosomiasis (28).

A study of the association of HLA class I antigen frequencies in 52
patients with kala-azar and 222 unrelated healthy controls in Iran found
HLA-A26 to be statistically significant (p = 0.004) (29). This indicates a
high risk of contracting the disease for HLA-A26 positive persons and a
remarkable influence of this antigen on the prevalence rate of kala-azar.

The significance of susceptibility/protection correlations between HLA and
parasitic diseases has been established by serologic typing methods. To
improve the accuracy of MHC-disease associations, we have used a DNA-based
HLA typing method, namely polymerase chain reaction with sequence specific
oligonucleotide probes, for the molecular typing of kala-azar patients in
India (30). To study the possible association at the molecular level of HLA
class I (A and B) as well as class II (DR) antigens in kala-azar patients,
we typed patients with kala-azar by polymerase chain reaction with sequence
specific oligonucleotide probes and compared the antigen frequencies with
healthy family-based controls. On the basis of the distribution of alleles
in each sample, percentage phenotype and genotype frequencies were
calculated for both control and kala-azar patients. Statistical analysis
using the Transmission Disequilibrium Test was carried out to assess the
association of different HLA allelic specificities with kala-azar patients.
No significant association between any of the HLA class I or class II
antigens was found. We will conduct a linkage analysis study based on the
data from typing the above-mentioned case/controls. The findings might lead
to a new dimension in the study of HLA association with parasitic
infections: genetic markers, such as HLA, that are sufficiently polymorphic
(as measured by their heterozygosities) can be used in linkage and
association analysis to detect Mendelian segregation underlying disease
phenotypes (31).

Comprehensive analysis of HLA associations with infectious diseases has
allowed precise definition of susceptibility and protective alleles in
large populations of different ethnic origins. Of great interest in the
fine dissection of molecular mechanisms leading to parasitic diseases,
these studies also provide the genetic basis for identification of the
subset of persons at risk for subsequent infection. Infectious diseases may
have exerted significant pressure on the development and maintenance of HLA
polymorphism (32). Widespread and frequently fatal parasitic diseases such
as malaria have selectively maintained certain gene frequencies in
endemic-disease areas (33).

Although HLA associations with parasitic diseases have provided clues to
pathogenesis, the molecular basis of these associations has not yet been
defined. The determinant selection hypothesis, which states that
associations result from the ability of a particular HLA type to present a
critical antigenic peptide, has been difficult to investigate because, for
most disease associations, the relevant antigen is unknown. Recently, the
identification of characteristic sequence features in peptides eluted from
HLA class I molecules (34,35) suggested that the relevant antigen might be
identifiable by assessing cellular immune responses to peptides containing
such motifs among antigens that are candidates for mediating HLA disease
associations. With the development of modern techniques of the HLA assembly
assay (36), relevant peptides can be synthesized; it can then be determined
whether they have any function as CTL epitopes during immune responses.
Such studies will elucidate the HLA associations with parasitic infections
and the molecular basis of these associations and facilitate the
development of vaccines for these infectious diseases.

HLA Association with Viral Infections
The associations of viral diseases with HLA alleles have not been studied
extensively. However, mechanisms by which HLA molecules determine the
immune response to viral peptides have been well studied as part of efforts
to develop safe and efficient virus vaccines. Successful development of
vaccines against viral infections depends on the ability of inactivated and
live virus vaccines to induce a humoral immune response and produce
antiviral neutralization antibodies. Additionally, virus vaccines that
induce a cellular immune response leading to the destruction of
virus-infected cells by CD8+ CTLs may be needed to provide protection
against some viral infections. Antiviral CD8+ CTLs are induced by viral
peptides presented within the peptide binding grooves of HLA class I
molecules on the surface of infected cells. Studies in the last decade have
provided an insight into the presentation of viral peptides by HLA class I
molecules to CD8+ T cells.

Herpesvirus saimiri, an oncogenic, lymphotropic, gamma-herpesvirus,
transforms human and simian T cells in vitro and causes lymphomas and
leukemias in various species of New World primates. An open reading frame
of the H. saimiri genome encodes a heavily glycosylated protein that is
secreted and binds to heterodimeric MHC class II HLA-DR molecules (37).
These results indicate that the open reading frame can function as an
immunomodulator that may contribute to the immunopathology of H. saimiri
infection.

Cytotoxic T cells that recognize dengue virus peptides have been reported
(38). Analysis of HLA class I haplotype-restricted peptides showed that
HLA-A2 and -A68 motifs were abundant compared with nonpeptides with
HLA-A24, -B8, and -B53 motifs. Studies by Zeng et al. (39) suggest that the
T-cell response to dengue virus is restricted by the HLA-DR15 allele.
Becker (40) developed an approach to priming antiviral CD8+ CTLs that may
provide cellular immune protection from flavivirus infection without
inducing the humoral immune response associated with dengue fever shock
syndrome. He proposed using synthetic flavivirus peptides with an amino
acid motif to fit with the HLA class I peptide binding group of HLA
haplotypes prevalent in a given population in an endemic-disease area as an
immunogen. These synthetic viral peptides may be introduced into the human
skin by using a lotion containing the peptides (Peplotion) and substances
capable of enhancing the penetration of these peptides into the skin to
reach Langerhans cells. The peptide-treated Langerhans cells, professional
antigen presenting cells, may bind the synthetic viral peptides by their
HLA class I peptide binding grooves. Antigens carrying Langerhans cells can
migrate and induce the cellular immune response in the lymph nodes.

Transmission of human immunodeficiency virus 1 (HIV-1) from an infected
woman to her offspring during gestation and delivery is influenced by the
infant's MHC class II DRB1 alleles. Forty-six HIV-infected infants and 63
seroreverting infants, born with passively acquired anti-HIV antibodies but
not becoming detectably infected, were typed by an automated
nucleotide-sequence-based technique (41). One or more DR-13 alleles,
including DRB1*1301, 1302, and 1303 were found in 15.2% of those becoming
HIV-infected and 31.7% of seroreverting infants (p = 0.048); this
association was influenced by ethnicity. Upon examining for other allelic
associations, only the DR2 allele DRB1*1501 was associated with
seroreversion in Caucasian infants. Among these infants, the DRB1*03011
allele was positively associated with HIV infection.

Molecular mimicry, where structural properties borne by a pathogen
"imitate" or "simulate" molecules of the host, also appears to be an
important mechanism in the association of HLA molecules with viral disease.
Molecular mimicry takes different forms in the molecular biology of HIV-1
(42). Molecular mimicry between HIV envelope proteins and HLA class II
molecules may lead to autoimmunity against CD4+ T cell expressing class II
molecules (43). Bisset (44) states that both the HIV-1 gp 120 envelope and
Mycoplasma genitalium adhesion proteins share an area of significant
similarity with the CD4-binding site of the class II MHC proteins.
Interaction with this triad could contribute to T-cell dysfunction, T-cell
depletion, Th1-cell-Th2-cell shift, B-cell proliferation,
hyperglobulinemia, and antigen-presenting cell dysfunction.

HLA-DR has been evaluated as a marker for immune response related to human
cytomegalovirus infection (45); this virus plays a role in chronic
inflammatory reaction in inflammatory abdominal aortic aneurysm. In the
fibrously thickened adventitia of this aneurysm, human
cytomegalovirus-infected cells and HLA-DR positive cells were more
frequently encountered than in that of atherosclerotic aneurysms and
control cases (p < 0.01).

An estimated 250 million people throughout the world are chronically
infected with hepatitis B virus, the primary cause of chronic hepatitis,
cirrhosis, and hepatocellular carcinoma in endemic-disease areas (46,47).
Because HLA class I antigens contain viral peptides, they may be important
targets for immune mediated hepatocytolysis by CD8+ CTLs in hepatitis B
virus infection (48). Davenport et al. (49) have shown that HLA-DR13 is
associated with resistance to hepatitis B virus infection. Prognosis may be
quite different among patients infected with hepatitis C virus: a chronic
liver disease occurs in half the patients, while the other half exhibits no
signs of histologic progression of liver damage. The host immune responses
may play an important role in such different outcomes. To identify human
CTL epitopes in the NS3 region of hepatitis C virus, Kurokohchi et al. (50)
modified an approach using recombinant protein and the ability of short
peptides to bind to class I MHC molecules. They identified a cytotoxic
T-cell epitope presented by HLA-A2 in the hepatitis C virus NS3 region. A
study conducted by Peano et al. (51) establishes that HLA-DR5 antigen
appears as a protective factor against a severe outcome of hepatitis C
virus infection.

Epstein-Barr virus, a member of the herpesvirus family, has been associated
with virus replication (infectious mononucleosis, oral hairy leukoplakia)
as well as neoplastic conditions such as nasopharyngeal carcinoma, B-cell
lymphoma, and Hodgkin disease associated with viral latency. An influence
of CTL response on Epstein-Barr virus evolution was first suggested by the
finding that virus isolates from highly HLA-A11-positive Asian populations
were specifically mutated in two immunodominant A11 restricted CTL epitopes
(52). Additionally, B35.01-restricted CTL responses in white donors
reproducibly map to a single peptide epitope (53). However, most
Epstein-Barr virus isolates from a population where B35.01 was prevalent
(in the Gambia) either retained the CTL epitope sequence or carried a
mutation that conserved antigenicity; changes leading to reduced
antigenicity were found in only a minority of cases. Two epitopes for
Epstein-Barr virus specific CTLs restricted by the common allele HLA-B7
were identified by Hill et al. (54).

The level of serum HLA class I antigens markedly increases during the
course of viral infections such as those caused by cytomegalovirus,
hepatitis B virus, hepatitis C virus, HIV-1, and varicella-zoster virus
(55-57). During HIV-1 infection, the level of serum HLA class I antigens
correlates with disease stage and represents a good prognostic marker of
disease progression (55).

HLA Association with Bacterial Infections
Vaccines based on recombinant attenuated bacteria represent a potentially
safe and effective immunization strategy. A carrier system was developed by
Verjans et al. (58) to analyze in vitro whether foreign T-cell epitopes,
inserted in the outer membrane protein PhoE of Escherichia coli and
expressed by recombinant bacteria, are efficiently processed and presented
through HLA class I and II molecules by infected human macrophages.

A well-defined HLA-B27 restricted cytotoxic T-cell epitope and an HLA-DR53
restricted T-helper epitope of the fusion protein of measles virus were
genetically inserted in a surface-exposed region of PhoE, and the chimeric
proteins were expressed in E. coli and Salmonella typhimurium. Macrophages
infected with recombinant bacteria presented the T-helper epitope to a
specific CD4+ T-cell clone but failed to present the CTL epitope to the
specific CD8+ T-cell clone. Phagocytic processing of intact bacteria within
infected macrophages was essential for antigen presentation by HLA class
II. Nascent HLA class II molecules were also required for the presentation
of the T-helper epitope to the CD4+ T-cell clone by infected macrophages.

HLA associations may also link various diseases; for example the HLA-B27
association for ankylosing spondylitis, Reiter disease, reactive
arthropathy, and acute anterior uveitis indicate that these disorders may
share a pathogenic pathway. According to the molecular mimicry hypothesis,
antigens carried by a particular pathogen may resemble a certain HLA
allomorph. As the person carrying this allomorph is unresponsive to it, it
is susceptible to the disease caused by the pathogen. For example, some
investigators believe that one of the antigens of Klebsiella resembles
HLA-B27 and that pathogen is responsible for ankylosing spondylitis (59).
In most patients who have an acute attack of anterior uveitis, a common
ocular disease characterized by inflammation of the iris and ciliary body,
the only clues to the pathogenesis of this disease are its close
association with the genetic marker HLA-B27 and the likely triggering role
of a variety of gram-negative bacteria (60). HLA-B27 acute anterior uveitis
appears to be a distinct clinical entity frequently associated with the
seronegative arthropathies, such as ankylosing spondylitis and Reiter
syndrome.

Sasazuki (61) showed that low responsiveness to streptococcal cell wall
antigen was inherited as an HLA-linked dominant trait. The immune
suppression gene for streptococcal cell wall was in strong linkage
disequilibrium with particular alleles of the HLA-DQ locus. This shows that
the HLA-linked immune suppression genes exist in humans to control low
response to natural antigens.

Table 2 lists the associations that have been established between various
HLA factors and certain infectious diseases. Only the antigens showing
statistically significant associations are indicated. Because some persons
are unresponsive to certain critical epitopes of the pathogens presumably
responsible for certain infectious diseases, particular HLA alleles occur
more frequently in patients with certain infectious diseases than in
healthy persons; therefore, researchers associate these diseases with
certain HLA alleles. This article has summarized the findings from
population genetic analysis and from studies of the association of immune
response mechanisms of infectious diseases and HLA.

            Table 2. Association between human leukocyte
            antigen (HLA) and some infectious diseases
            ----------------------------------------------------
                                          HLA
            Disease                       Association
            ----------------------------------------------------

            Bacterial
            ---------
            Ankylosing spondylitis        B27
            Reiter disease                B27
            Acute anterior uveitis        B7

            Mycobacterial
            -------------
            Tuberculosis and leprosy      DR2
                (multibacillary forms)    (DRB1*1501, 1502)
                lepromatous leprosy       DR2 and DQ1
                paucibacillary
            tuberculoid                   DR3

            Viral
            -----
            Dengue fever virus            DR15
            Human immunodeficiency        DR13
                virus 1                   (DRB1*1301, 1302,
                                          1303)
                                          DR2
                                          (DRB1*1501)
                                          DRB1*03011
            Hepatitis B virus             DR13
            Hepatitis C virus             A2
                                          DR5
            Epstein-Barr virus            B35.01
                                          A11
                                          B7

            Parasitic
            ---------
            Malaria                       B53
            Scabies                       A11
            Diffuse cutaneous             A11, B5, B7
                leishmaniasis
            Localized cutaneous           A28, Bw22, DQw8
                leishmaniasis             Bw22, DR11, Qw7
                                          Bw22, Dqw3
            Schistosomiasis               B5, DR3
            Visceral leishmaniasis        A26
            ----------------------------------------------------

      Acknowledgments

      We are grateful to Dr. Derek Middleton for critically reading
      the manuscript and Dr. Martin Curran and Mr. J.C. Sharma for
      their help in the literature survey. This work was supported by
      a grant from the Department of Science and Technology, India
      (No. SR/OY/MB-10/92).

Dr. Singh is a scientist in the Department of Biochemistry, Central Drug
Research Institute, Lucknow. Her research interest is visceral
leishmaniasis, or kala-azar, as it is known in eastern India, where the
disease is now epidemic. She is developing PCR-based diagnostics for the
disease focusing on kinetoplast DNA, studying the molecular mechanisms of
drug resistance, and striving to answer the most important question: are
host genetic factors, like HLA, involved in susceptibility to kala-azar in
India.

Address for Correspondence: Neeloo Singh, Department of Biochemistry,
Central Drug Research Institute, Lucknow, India;fax: 91-522-223-405;
e-mail: root@cscdri.ren.nic.in.

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---------------------------------------------------------------------------
 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
--------------------------------------------------------------------------- 

Synopses

Cryptosporidiosis: An Emerging, Highly Infectious Threat

Richard L. Guerrant
University of Virginia School of Medicine
Charlottesville, Virginia, USA
---------------------------------------------------------------------------

      Cryptosporidium parvum, a leading cause of persistent diarrhea
      in developing countries, is a major threat to the U.S. water
      supply. Able to infect with as few as 30 microscopic oocysts,
      Cryptosporidium is found in untreated surface water, as well
      as in swimming and wade pools, day-care centers, and
      hospitals. The organism can cause illnesses lasting longer
      than 1 to 2 weeks in previously healthy persons or
      indefinitely in immunocompromised patients; furthermore, in
      young children in developing countries, cryptosporidiosis
      predisposes to substantially increased diarrheal illnesses.
      Recent increased awareness of the threat of cryptosporidiosis
      should improve detection in patients with diarrhea. New
      methods such as those using polymerase chain reaction may help
      with detection of Cryptosporidium in water supplies or in
      asymptomatic carriers. Although treatment is very limited, new
      approaches that may reduce secretion or enhance repair of the
      damaged intestinal mucosa are under study.

An emerging infection comes to our attention because it involves a newly
recognized organism, a known organism that newly started to cause disease,
or an organism whose transmission has increased. Although Cryptosporidium
is not new, evidence suggests that it is newly spread (in increasingly used
day-care centers and possibly in widely distributed water supplies, public
pools, and institutions such as hospitals and extended-care facilities for
the elderly); it is newly able to cause potentially life-threatening
disease in the growing number of immunocompromised patients; and in humans,
it is newly recognized, largely since 1982 with the AIDS epidemic.
Cryptosporidium is a most highly infectious enteric pathogen, and because
it is resistant to chlorine, small and difficult to filter, and ubiquitous
in many animals, it has become a major threat to the U.S. water supply.
This article will focus on the recognition and magnitude of
cryptosporidiosis, the causative organism and the ease with which it is
spread, outbreaks of cryptosporidiosis infection, and its pathogenesis,
diagnosis, and treatment.

Recognition and Magnitude of Cryptosporidiosis

First recognized by Clarke and Tyzzer (1) at the turn of the century and
well known to veterinarians, Cryptosporidium was reported as a human
pathogen in 1976 by Nime (2). From 1976 until 1982, seven cases of
cryptosporidiosis were reported in humans, five of which were in
immunosuppressed patients. Since 1982, cryptosporidiosis has been
increasingly recognized as a cause of severe, life-threatening diarrhea in
patients with AIDS as well as in previously healthy persons (3). Of the
first 58 cases of cryptosporidiosis described in humans by 1984, 40 (69%)
were in immunocompromised patients who contracted severe, often
irreversible, diarrhea (lasting longer than 4 months in 65%); of these 40
patients, 33 (83%) had AIDS (4-6); 55% of the 40 immunocompromised patients
died.

A review of 78 reports of more than 131,000 patients and more than 6,000
controls showed Cryptosporidium infection in 2.1% to 6.1% of
immunocompetent persons in industrialized and developing countries,
respectively, vs. 0.2% to 1.5% in controls (Table 1). A review of an
additional 22 reports of nearly 2,000 human immunodeficiency virus
(HIV)-infected persons showed Cryptosporidium infection in 14% to 24% of
HIV-infected persons with diarrhea vs. 0% to 5% of HIV-infected controls
without diarrhea (7). Seroepidemiologic studies suggest that 17% to 32% of
nonimmunocompromised persons in Virginia, Texas, and Wisconsin, as well as
nonimmunocompromised Peace Corps volunteers (before travel), have serologic
evidence of Cryptosporidium infection by young adulthood. In contrast, more
than half of the children in rural Anhui, China, had serologic evidence of
cryptosporidial infection by 5 years of age, and more than 90% of children
living in an impoverished area of Fortaleza, Brazil, had serologic evidence
of cryptosporidial infection in their first year of life (Figure) (8-11).

The Organism

Among protozoa, C. parvum is the major human pathogen that is also found in
numerous mammals. It is slightly smaller than the murine Cryptosporidium,
C. muris, and is also distinguished from the other Cryptosporidium species
commonly seen in birds, turkeys, snakes, and fish. Infection begins when a
person ingests chlorine-resistant, thick-walled oocysts (7). These hardy
oocysts appear to be infectious, with an estimated ID50 (from studies in
humans) of one isolate containing only 132 oocysts (12). Infections may
occur with ingestion of as few as 30 oocysts; some infections have occurred
with just one oocyst (13).

              Table 1. Rates of Cryptosporidium infection
              among immunocompetent and HIV-positive persons
              in industrialized and developing areas(abc)
              ------------------------------------------------

                                Patients          Controls
                              with diarrhea       without
                                                  diarrhea
              ------------------------------------------------
              Immuno-
              competent
                  Industr.   2.2%(0.26%-22%)   0.2%(0%-2.4%)
                  areas     [n=2232/107,329]    [n=3/1941]

              Developing    6.1%(1.4%-40.9%)  1.5% (0%-7.5%)
                  areas      [n=1486/24,269]    [n=61/4146]
              HIV-positive
                  Industr.     14%(6%-70%)       0%(0%-0%)
                  areas       [n=148/1074]       [n=0/35]

              Developing      24%(8.7%-48%)    5%(4.9%-5.3%)
                  areas        [n=120/503]       [n=5/101]
              ------------------------------------------------
              (a)From 100 reports of 133,175 patients with
              diarrhea and 6,223 controls. (b)Ranges given in
              parentheses. (c)Data from reference (7).

When the oocysts reach the upper small bowel, the proteolytic enzymes and
bile salts enhance the excystation of four infectious sporozoites, which
enter the brush border surface epithelium and develop into merozoites
capable of replicating either asexually or sexually beneath the cell
membrane (but extracytoplasmically) in the brush border epithelial cell
surface. Sexual stages combine to form new oocysts, some of which (perhaps
20% as thin-walled oocysts) may sporulate and continue infection in the
same person, while others (thick-walled oocysts) are excreted. Although few
organisms may enter through M cells, systemic infection essentially does
not occur; the occasional biliary tract or respiratory tract infections in
immunocompromised patients probably reached these sites through the lumenal
surface.

[Figure 1] [Figures not available in ASCII version]
Figure. Prevalence of IgG antibodies to Cryptosporidium parvum, by age, in
Brazil, China, and the United States.

Cryptosporidiosis Outbreaks

Numerous well-documented outbreaks of cryptosporidiosis have occurred. Most
of these often waterborne outbreaks have involved subtle problems in the
flocculation and/or filtration process (17-21). These outbreaks culminated
in the huge waterborne outbreak in Milwaukee, which was initially thought
to be viral gastroenteritis, reported to the State Health Department on
April 5, 1993, diagnosed on April 7, and followed by an advisory note that
evening to the public to boil all drinking water (Table 2). This became the
largest waterborne outbreak in U.S. history and affected an estimated
403,000 persons, thus constituting a 52% attack rate among those served by
the South Milwaukee water works plant. Several immunocompromised patients
died, and many previously healthy persons became ill. The mean duration of
illness was 12 days with a range of 1 to 55 days, and the average maximum
number of watery diarrheal stools was 19 per day at the peak of illness.
While watery diarrhea was the predominant symptom among 93% of confirmed
cases, other symptoms such as abdominal pain, low-grade fever, and vomiting
were not infrequent; 75% of infected nonimmunocompromised persons had an
average 10-lb weight loss.

Additional outbreaks involving public swimming pools and wade pools have
further documented the ability of Cryptosporidium to cause infection even
when ingested in relatively small amounts of fully chlorinated water
(22-26). While the leading causes of 129 drinking and recreational water
outbreaks in the United States from 1991 through 1994 were Giardia and
Cryptosporidium, cryptosporidiosis accounted for substantially more cases
(even if the Milwaukee outbreak were excluded) (23,24,26). In addition,
although Cryptosporidium oocysts cannot multiply in the environment, an
outbreak of foodborne cryptosporidiosis, affecting 54% of those ingesting
incriminated freshly pressed apple cider, has been reported (27). In this
outbreak, Cryptosporidium oocysts were found in the cider press, as well as
in a calf on the farm from which the apples were obtained. There was also a
15% secondary attack rate in households involved in this outbreak. The
apparent person-to-person spread in households and institutions such as
day-care centers and hospitals further documents the highly infectious
nature of Cryptosporidium. In an urban slum area in northeastern Brazil,
secondary household infections occurred in 58% of households with an
infected child (index case) despite the 95% prevalence of antibody in
children more than 2 years of age (28).

               Table 2. Symptoms of 205 patients with
               confirmed cases of cryptosporidiosis during
               the Milwaukee outbreak(a)
               ---------------------------------------------
                          Symptom              Percent(%)
               ---------------------------------------------
               Watery diarrhea                    93
                   mean=12d; med=9d (1-55d)
                   mean=19/d; med=12/d
               (1-90)
                   39% recurred after 2d
               free
               Abdominal cramps                   84
               Weight loss                        75
                   (med=10lb, 1-40lb)
               Fever                              57
                   (med=38.3, 37.2-40.5)
               Vomiting                           48
               ---------------------------------------------
               (a)Data from reference (21)

The spread of cryptosporidiosis in day-care centers is well documented,
with 14 outbreaks reported in the United States, as well as others in the
United Kingdom, France, Portugal, Australia, Chile, and South Africa (29).
Illnesses usually occurred in the summer and early fall, especially during
August and September in the United States and Portugal. Attack rates were
13% to 90%, with the highest rates found among nontoilet-trained toddlers
and staff caring for children in diapers. Overall prevalence rates were
usually in the 1.8% to 3.8% range; however, rates as high as 30% in
day-care homes were reported (30). During outbreaks, 3.7% to 22.9% of
infected children may not have diarrhea; infectious oocysts may be excreted
for up to 5 weeks after diarrheal illness ends (31). In addition, numerous
nosocomial outbreaks of cryptosporidiosis have occurred among healthcare
workers as well as patients in bone marrow transplant units, pediatric
hospitals, and patient wards with HIV-infected patients (32-37).
Furthermore, elderly hospitalized patients may also be at risk for
Cryptosporidium infection (38). In one Pennsylvania hospital, 45% of
nurses, medical students, and house staff caring for an HIV-positive
patient with cryptosporidiosis seroconverted (39).

Numerous potential animal and water sources have been found to be infected
with Cryptosporidium. In the Gonalves Dias slum in Fortaleza, Brazil, 10%
of animals (including dogs, pigs, donkeys, and goats), 6.3% during the dry
season to 14.3% during the wet season, had Cryptosporidium in their stool
specimens. In addition, 22% of drinking water sources studied were infected
with Cryptosporidium oocysts (40). Furthermore, LeChavalier et al. have
documented that Cryptosporidium oocysts were present in 27% of 66 drinking
water samples obtained from 14 states and one Canadian province (mean of
0.18 NTU) (41,42).

Pathogenesis and Impact

C. parvum does not infect tissue beyond the most superficial surface of the
intestinal epithelium; however, it can derange intestinal function.
Although a parasite enterotoxin has been extensively sought and some
reports have suggested that one may exist (43), this issue remains
controversial, and the source of substances in the stools of infected
animals and patients that induce secretion remains unclear (44). Extensive
studies in a piglet model of cryptosporidiosis by Argenzio and colleagues
demonstrate the loss of vacuolated villus tip epithelium (approximately
two-thirds of the villus surface area), accompanied by an approximate 50%
reduction in glucose-coupled sodium cotransport. What remains is a
predominance of transitional junctional epithelium, in which increased
glutamine metabolism drives a sodium-hydrogen exchange, to which is coupled
chloride transport. Thus, glutamine drives neutral sodium chloride
absorption in an apparent prostaglandin-inhibitable manner in
Cryptosporidium-infected piglet epithelium (45). Furthermore, Argenzio and
colleagues have demonstrated increased macrophages that produce increased
tumor necrosis factor (TNF) in the lamina propria of
Cryptosporidium-infected piglets (46). Although TNF did not directly affect
epithelial transport, when a fibroblast monolayer was added, an
indomethacin-inhibitable secretory effect was noted with TNF (46).
Consequently, the researchers propose a prostaglandin-dependent secretory
effect, which occurs 1) through a bumetanide-inhibitable chloride secretory
pathway, predominantly from crypt cells; and 2) through the inhibition of
neutral sodium chloride absorption through the amiloride-sensitive
sodium:hydrogen exchanger, predominantly in the junctional or transitional
epithelium during active cryptosporidial infection. Reduced xylose and B-12
absorption are among the effects described in humans and animals with
cryptosporidiosis (47-49). Disruption of intestinal barrier function with
strikingly increased lactulose to mannitol permeability and absorption has
been documented during active symptomatic cryptosporidial infection in
children and in HIV-infected adults (Lima et al., unpublished observations)
(50).

Cryptosporidium appears to be one of the leading causes of diarrhea,
especially persistent diarrhea, among children in northeastern Brazil
(51,52). In addition, the incidence of diarrhea has been nearly double for
many months in young children after symptomatic cryptosporidial infections,
suggesting that the disrupted barrier function in infected children leaves
residual damage resulting in increased susceptibility of injured epithelium
to additional diarrheal illnesses (Agnew et al., unpub. obs.).

Recognition and Diagnosis

The diagnosis of C. parvum in patients with diarrhea is usually made by
using acid-fast or immunofluorescence staining on unconcentrated fecal
smears. Appropriate concentration methods may enhance detection when small
numbers of oocysts are present, but some methods such as formalin-ethyl
acetate concentration may result in loss of many oocysts (52,53). While
several enzyme-linked immunosorbent assay methods are available for
etection of fecal cryptosporidial antigen with 83% to 95% sensitivity in
diarrheal specimens, these methods are less sensitive in formed specimens
and require more time. Microscopy using immunofluorescence antibody is
slightly more sensitive and may be faster (54,55).

Polymerase chain reaction (PCR) provides a new method that may help detect
Cryptosporidium in water supplies or asymptomatic carriers. A genomic DNA
library has been constructed in the plasmid pUC18 for propagation in
Escherichia coli. After sequencing a 2.3 kilobase C. parvum-specific
fragment, a 400-base sequence with a unique Sty I site has been amplified
by using primers of 26 nucleotides each (56). Laxer et al. then used a
20-base probe labeled with digoxigenin-11-dUTP to detect C. parvum DNA in
fixed, paraffin-embedded tissue (57). In addition, primers for a 556 BP
Cryptosporidium-specific region of the small subunit 18s ribosomal RNA gene
have been used to produce a PCR product with unique Mae 1 sites that
distinguish C. parvum from C. baileyi and C. muris (58). Available methods
for detection of viable oocysts in environmental samples are relatively
insensitive and under active investigation.

Treatment and Prevention

Despite numerous attempts at examining transfer factor, hyperimmune
colostral antibody, and more than 100 antiparasitic and antimicrobial
agents, few agents have shown modest benefit in controlled studies;
paromomycin is one of them. Although this agent does not eradicate the
parasite in immunocompromised patients, it slightly reduces parasite
numbers (from 314 x 106 to 109 x 106 oocysts shed per day) and decreases
stool frequency, with a tendency toward improved Karnofsky scores and
reduced stool weight (59). In view of its effectiveness in driving sodium
cotransport (60) and its success in studies of experimental animals, we are
examining a new approach to speeding repair of disrupted intestinal barrier
function by using glutamine and its derivatives.

The ability of the thick-walled oocysts to persist and spread in the
environment and their well-documented resistance to chlorine are
responsible for the spread of Cryptosporidium even in fully chlorinated
water supplies that meet existing turbidity standards in drinking water and
swimming pools. Although some scientists have noted that 9,600 parts per
million (mg/l) of chlorine for one minute of exposure are required to
decontaminate water (14), others have noted that even after exposure to
full-strength household bleach (5.25% sodium hypochlorite; 50,000 parts per
million) for 2 hours, the oocysts still remained infectious for
experimental animals (15). While Giardia are 14 to 30 times more
susceptible to chlorine dioxide or ozone, respectively, ozone is probably
the most effective chemical means of inactivating Cryptosporidium oocysts
(16). Consequently, eradication of the organism from drinking water
supplies depends on adequate flocculation and filtration, rather than
chlorination. Although previous turbidity requirements were based on the
removal of larger parasite cysts such as those of Giardia lamblia or
Entamoeba histolytica, the smaller C. parvum oocysts are more difficult to
remove. Several waterborne outbreaks, including the recent outbreak in
Milwaukee, have occurred with turbidity levels in the 0.45 to 1.7
nephelometry turbidity units (NTU) range. In a study of waterborne
cryptosporidiosis, predominantly among HIV-positive adults in Clark County,
Nevada, Goldstein et al. (1996) report that the outbreak was associated
with a substantial number of deaths and that the turbidity of the
implicated water never exceeded 0.17 NTU (much lower than the new standard
of 0.5 NTU required for 95% of measurements each month, with no spikes over
1.0 NTU) (5).

New approaches to the eradication of infectious oocysts from water supplies
are needed, possibly using reverse osmosis, membrane filtration, or
electronic or radiation methods, instead of the ineffective chemical or
difficult filtration techniques currently used. Ideally, these new methods
would provide low cost, effective treatment that could be applied in
developing areas as well. Meanwhile, an improved understanding of the
pathogenesis and impact of Cryptosporidium infections should aid the
development of improved treatment and control of this ubiquitous, highly
infectious threat to the water supply and to the people it
serves, especially malnourished children and immunocompromised patients
around the world.

Acknowledgments
Much of our work on cryptosporidiosis is supported by an International
Collaboration in Infectious Diseases Research Grant #2 U01 AI26512 from the
National Institute of Allergy and Infectious Diseases, National Institutes
of Health. Some of these materials were presented as a part of an American
Society for Microbiology symposium on emerging infections at the 95th
general meeting in Washington, D.C.

Dr. Guerrant is Thomas H. Hunter Professor of International Medicine and
director, Office of International Health, University of Virginia School of
Medicine. He holds several patents on innovative approaches to the
diagnosis and treatment of common gastrointestinal illnesses. In addition
to his many other contributions, Dr. Guerrant is instrumental in shaping
tropical medicine training in the United States.

Address for correspondence: Richard L. Guerrant, Division of Geographic and
International Medicine, University of Virginia School of Medicine, Health
Sciences Center #485, Bldg. MR-4, Rm 3146, Charlottesville, VA 22908; fax:
804-977-5323; e-mail: rlg9a@virginia.edu.

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     et al. Cryptosporidiosis in patients with hematologic malignancies.
     Reviews of Infectious Diseases 1991;13:842-6.
 36. Navarrete S, Stetler HC, Avila C, Aranda JAG, Santos-Preciado JI. An
     outbreak of Cryptosporidium diarrhea in a pediatric hospital. Pediatr
     Infect Dis J 1991;10:248-50.
 37. Ravn P, Lundgren JD, Kjaeldgaard P, Holten-Anderson W, Hjlyng N,
     Nielsen JO, et al. Nosocomial outbreak of cryptosporidiosis in AIDS
     patients. BMJ 1991;302:277-80.
 38. Neill MA, Rice SK, Ahmad NV, Flanigan TP. Cryptosporidiosisan
     unrecognized cause of diarrhea in elderly hospitalized patients. Clin
     Infect Dis 1996;22:168-70.
 39. Koch KL, Phillips DJ, Aber RC, Current WL. Cryptosporidiosis in
     hospital personnel. Ann Intern Med 1985;102:593-6.
 40. Newman RD, Wuhib T, Lima AA, Guerrant RL, Sears CL. Environmental
     sources of Cryptosporidium in an urban slum in northeastern Brazil. Am
     J Trop Med Hyg 1993;49:270-5.
 41. LeChevallier MW, Norton WD, Lee RG. Occurrence of Giardia and
     Cryptosporidium spp. in surface water supplies [published erratum
     appears in Appl Environ Microbiol 1992 Feb;58:780]. Appl Environ
     Microbiol 1991;57:2610-6.
 42. LeChevallier MW, Schulz W, Lee RG. Bacterial nutrients in drinking
     water. Appl Environ Microbiol 1991;57:857-62.
 43. Guarino A, Canani RB, Casola A, Pozio E, Russo R, Bruzzese E, et al.
     Human intestinal cryptosporidiosis: secretory diarrhea and enterotoxic
     activity in Caco-2 cells. J Infect Dis 1995;171:976-83.
 44. Sears CL, Guerrant RL. Cryptosporiodiosis: the complexity of
     intestinal pathophysiology. Gastroenterology 1994;106:252-4.
 45. Argenzio RA, Rhoads JM, Armstrong M, Gomez G. Glutamine stimulates
     prostaglandin-sensitive Na(+)-H+ exchange in experimental porcine
     cryptosporidiosis [see comments]. Gastroenterology 1994;106:1418-28.
 46. Argenzio RA, Lecce J, Powell DW. Prostanoids inhibit intestinal NaCl
     absorption in experimental porcine cryptosporidiosis. Gastroenterology
     1993;104:440-7.
 47. Modigliani R, Bories C, LeCharpentier Y, Salmeron N, Messing B, Galian
     A. Diarrhea and malabsorption in acquired immune deficiency syndrome:
     a study of four cases with special emphasis on opportunistic protozoan
     infestations. Gut 1985;26:179-87.
 48. Gardner AL, Roche JK, Weikel CS, Guerrant RL. Intestinal
     cryptosporidiosis: pathophysiologic alterations and specific cellular
     and humoral immune responses in rnu/+ and rnu/rnu (athymic) rats. Am J
     Trop Med Hyg 1991;44:49-62.
 49. Heine J, Moon HW, Woodmansee DB. Persistent Cryptosporidium infection
     in congenitally athymic (nude) mice. Infect Immun 1984;43:856-9.
 50. Goodgame RW, Kimball K, Ou CN, White AC, Jr, Genta RM, Lifschitz CH,
     et al. Intestinal function and injury in acquired immunodeficiency
     syndrome-related cryptosporidiosis. Gastroenterology 1995;108:1075-82.
 51. Fang G, Lima AAM, Martins CC, Nataro JP, Guerrant RL. Etiology and
     epidemiology of persistent diarrhea in northeastern Brazil: a
     hospital-based prospective case control study. J Pediatr Gastroenterol
     Nutr 1995;21:137-44.
 52. Wuhib T, Silva TM, Newman RD, Garcia LS, Pereira ML, Chaves CS, et al.
     Cryptosporidial and microsporidial infections in human
     immunodeficiency virus-infected patients in northeastern Brazil. J
     Infect Dis 1994;170:494-7.
 53. Weber R, Bryan RT, Bishop HS, Wahlquist SP, Sullivan JJ, Juranek DD.
     Threshold of detection of Cryptosporidium oocysts in human stool
     specimens: evidence for low sensitivity of current diagnostic methods.
     J Clin Microbiol 1991;29:1323-7.
 54. Newman RD, Jaeger KL, Wuhib T, Lima AA, Guerrant RL, Sears CL.
     Evaluation of an antigen capture enzyme-linked immunosorbent assay for
     detection of Cryptosporidium oocysts. J Clin Microbiol 1993;31:2080-4.
 55. Kehl KS, Cicirello H, Havens PL. Comparison of four different methods
     for detection of Cryptosporidium species. J Clin Microbiol
     1995;33:416-8.
 56. Laxer MA, Timblin BK, Patel RJ. DNA sequences for the specific
     detection of Cryptosporidium parvum by the polymerase chain reaction.
     Am J Trop Med Hyg 1991;45:688-94.
 57. Laxer MA, D'Nicuola ME, Patel R. Detection of Cryptosporidium parvum
     DNA in fixed, paraffin-embedded tissue by the polymerase chain
     reaction. Am J Trop Med Hyg 1992;47:450-5.
 58. Awad-El-Kariem FM, Warhurst DC, McDonald V. Detection and species
     identification of Cryptosporidium oocysts using a system based on PCR
     and endonuclease restriction. Parasitology 1994;109:19-22.
 59. White AC, Jr, Chappell CL, Hayat CS, Kimball KT, Flanigan TP, Goodgame
     RW. Paromomycin for cryptosporidiosis in AIDS: a prospective,
     double-blind trial. J Infect Dis 1994;170:419-24.
 60. Lima AAM, Soares AM, Freire JE, Jr, Guerrant RL. Cotransport of sodium
     with glutamine, alanine and glucose in the isolated rabbit ileal
     mucosa. Braz J Med Biol Res 1992;25:637-40.

---------------------------------------------------------------------------
 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
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Dispatches

Isolation and Phylogenetic Characterization of Ebola Viruses Causing
Different Outbreaks in Gabon

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

      Three outbreaks of Ebola hemorrhagic fever have recently
      occurred in Gabon. Virus has been isolated from clinical
      materials from all three outbreaks, and nucleotide sequence
      analysis of the glycoprotein gene of the isolates and virus
      present in clinical samples has been carried out. These data
      indicate that each of the three outbreaks should be considered
      an independent emergence of a different Ebola virus of the
      Zaire subtype. As in earlier Ebola virus outbreaks, no genetic
      variability was detected between virus samples taken during an
      individual outbreak.

Since the first recognized outbreaks of human Ebola virus hemorrhagic fever
in Africa in the 1970s, biological and genetic differences have been
described among the four distinct Ebola viruses isolated: Zaire, Sudan, and
Cte d'Ivoire, all isolated from humans, and Reston virus isolated from
macaque monkeys from the Philippines (1-5).

Serologic evidence has suggested the presence of Ebola virus in Gabon since
1982 (6). Since late 1994, three apparently independent outbreaks of Ebola
virus hemorrhagic fever have occurred among humans in northeastern Gabon,
in the forested areas of equatorial Ogoou-Ivindo province. The first,
which started in December 1994 in gold-panner encampments of far
northeastern Gabon, in the Minkouka area (between 124' and 144' North and
1249' and 1259' East, Figure 1) near the Nouna River, had several
laboratory-confirmed cases (7). The second, which began in early February
1996 in Mayibout village (107' North, 1306' East, Figure 1) on the Ivindo
River, resulted in 37 Ebola hemorrhagic fever cases (8). The only means of
transportation between these two areas is by boat; Makokou, the closest
town to them (033' North, 1250' East, Figure 1), has the provincial
hospital to which patients and contacts were transferred. The third
outbreak, which is ongoing, started in July 1996 in the village of Boou
(006' South, 1157' East, Figure 1), where most of the cases occurred;
however, scattered cases have been diagnosed in surrounding villages and
towns. Some patients have even been transported to Libreville, probably
during the incubation period of the disease. One patient was treated in
South Africa, where a fatal nosocomial infection was subsequently reported
in a health care worker (9,10); 43 deaths due to Ebola hemorrhagic fever
have already been reported during this prolonged outbreak (11).

             [Figure 1] [Figures not available in ASCII version]
             Figure 1. Geographic distribution of the three
             Ebola virus hemorrhagic fever epidemics and site
             of the infected chimpanzee in Gabon.

We report here the Ebola virus glycoprotein (GP) gene sequences obtained
from human samples collected during each of the three Gabonese epidemics.
The Gab280 sequence comes from blood collected 1 day before the death of a
patient, from the Nouna area, during the 1994 outbreak. The Gab282 and
Gab283 sequences were derived from blood collected during the spring 1996
outbreak, from two primary patients who were infected while butchering a
chimpanzee they found dead in the forest. The Gab281 sequence was derived
from blood collected from what appears to have been a secondary case during
the same outbreak; the patient was probably infected by contact with one of
the index patients while visiting a traditional doctor (nganga) who lived
near Mayibout village. Sequences Gab275 to Gab279 were derived from blood
specimens collected from two patients during the fall 1996 outbreak:
Gab275, Gab276, and Gab277 sequences are from one patient (a serum specimen
collected on October 27, a primary tissue culture passage from that
specimen, and a primary tissue culture passage from another blood sample
drawn on October 25, respectively). The Gab278 and Gab279 sequences were
derived from plasma (collected during the acute phase of illness) from
another patient and its primary tissue culture passage, respectively. The
isolation of Ebola virus in Vero E6 cell culture from human blood samples
collected during the three different outbreaks was easily accomplished in a
single passage.

RNA was extracted from blood or primary tissue culture samples (Table 1) by
using a commercial kit (RNaid Kit , BIO 101, Inc., Vista, CA) as directed
by the instruction manual. Viral sequences were amplified from RNA by using
the reverse transcriptase-polymerase chain reaction (RT-PCR) technique, and
DNA products were sequenced as described elsewhere (5). Briefly, amplified
products were subjected to agarose electrophoresis and were stained and
visualized with ethidium bromide; DNA bands were then excised and extracted
by using the QIAEX II kit (QIAGEN, Inc., Chatsworth, CA). In some cases,
nested PCR with internal primers was performed, using first-round products.
Amplified products were directly sequenced by using an automated
nonisotopic method (ABI PRISM dye-terminator cycle sequencing kit,
Perkin-Elmer, Foster City, CA). Centri Sep columns (Princeton Separation,
Inc.) were used to remove excess dye-labeled dideoxynucleotide terminators,
and reaction products were analyzed on an ABI 377 or 373 DNA sequencer
(Perkin-Elmer).

Initially, a 783-base-pair fragment of the GP gene, which encompasses the
most variable region of the gene, was sequenced. A consensus sequence was
established by aligning all the Ebola from Gabon, the Zaire 1976 and 1995
Ebola virus sequences, as well as the sequence of the virus obtained from a
nurse in South Africa who was infected by a patient from Gabon in November
1996 (10). Viruses from the same outbreak had identical sequences, but
unique virus sequences were found in each of the three different outbreaks
in Gabon. Although the viruses causing the Gabonese outbreaks clearly
belong to the Zaire subtype, they were distinct from viruses that had
caused disease in Zaire. No differences were observed between
tissue-culture-passaged and clinical-material-derived sequences or between
primary or secondary case sequences. RNA extracted from a single
representative of each outbreak (sequences Gab277, Gab280, and Gab283) was
then used to generate the entire GP gene sequence for the Gabon Ebola
viruses. The GP from the Gabon spring 1996 viruses differed from the
sequence of the Gabon fall 1994 viruses by four nucleotides. The GP
sequence from the Gabon fall 1996 viruses differed from the sequence of the
Gabon spring 1996 virus by four additional nucleotides (Figure 2).

  Table 1. Origin and identification of the sequence used in the analysis
  ------------------------------------------------------------------------
                                               Date of
  Outbreak           Patient   Specimen        original    Laboratory
                     #                                     #
                                               specimen
  ------------------------------------------------------------------------
  Gabon, Fall 94     1         Blood           Dec 28,94   Gab280
  Gabon,             1         Blood           Feb 20,96   Gab281
  Spring 96          2         Blood           Feb 8,96    Gab282
                     3         Blood           Feb 8,96    Gab283
  Gabon,             1         Serum           Oct 27,96   Gab275
    Fall 96                    Vero E6         Oct 27,96   Gab276
                                 Passage 1
                               Vero E6         Oct 25,96   Gab277
                                 Passage 1
                     2         Plasma          Oct 27,96   Gab278
                               Vero E6         Oct 27,96   Gab279
                                 Passage 1
  South Africa,      1         Vero E6         Nov 9,96    SA253
    Fall 96                      Passage 1
  ------------------------------------------------------------------------

Maximum parsimony analyses were performed by using the entire coding
regions of the GP genes of the Gabon viruses, together with those of
previously characterized Ebola viruses (5). In addition, the sequence of a
1980 Marburg virus isolate (Musoke strain) was included to provide an
outgroup in the analysis. The Phylogenetic Analysis Using Parsimony version
3.1.1 software, using the branch-and-bound search option (12), was run on a
Power Macintosh 8100/110 (Apple Computer, Inc.). A 4:1
transversion:transition weighting was used in analyses. A single most
parsimonious tree was obtained (Figure 2), and bootstrap analysis strongly
supports a common evolutionary origin for the viruses associated with
disease in Gabon and Zaire. Overall, these data indicate that the three
Gabon outbreaks should be considered independent events, likely originating
from different sources.

             [Figure 2] [Figures not available in ASCII version]
             Figure 2. Phylogenetic tree showing the
             relationship between the Ebola viruses that
             caused outbreaks of disease in Gabon and
             previously described filoviruses (5). The entire
             coding region for the glycoprotein gene of the
             viruses shown was used in maximum parsimony
             analysis, and a single most parsimonious tree was
             obtained. Numbers in parentheses indicate
             bootstrap confidence values for branch points and
             were generated from 500 replicates (heuristic
             search). Branch length values are also shown.

The presence of stable virus sequences and the lack of genetic variability
between strains isolated within an outbreak was previously seen during the
outbreak of Ebola hemorrhagic fever in Kikwit, Zaire, in 1995 (Rodriguez et
al., unpublished data), and despite the small number of isolates tested, is
again suggested in Gabon.
            [Figure 3]
            Figure 3. Immunostaining of Ebola virus antigens
            (red) within vascular endothelial cells in skin
            biopsy of the chimpanzee found dead in the forest
            near Boou. Note also the presence of
            extracellular viral antigens. (Rabbit anti-Ebola
            virus serum, napthol/fast red with hematoxylin
            counterstain, original magnification x250).

During a 20-month period, Gabon has had three different outbreaks of Ebola
virus hemorrhagic fever. The first and the second episodes apparently
started during the rainy season (December and February), while the third
began during the dry season (July). The deaths of nonhuman primates were
associated with all three outbreaks. Minkouka area inhabitants reported
finding dead chimpanzees and gorillas in the forest during the fall of
1994. All the primary human patients in the spring 1996 outbreak were
infected while butchering dead chimpanzees. For the third outbreak, the
investigation has indicated an index patient who was a hunter, living in a
forest camp in the Boou area. During the same period, an Ebola
virus-infected dead chimpanzee was found in the forest (011' South, 1136'
East) near Boou; Ebola virus infection in the chimpanzee was confirmed
using a newly developed immunohistochemical Ebola skin biopsy test (Figure
3). A long term surveillance for Ebola virus using this test is under way
in Africa (13,14).

In Cte d'Ivoire in 1994, an investigator was infected with Ebola virus
while performing necropsy on a dead chimpanzee (15). Primates are unlikely
to be the reservoir of Ebola virus since experimental or natural infection
is quickly fatal. A better knowledge of the ecology of great apes,
particularly their food preferences and habitats, may lead to the
identification of the virus reservoir. Gabon's equatorial forests, where
three independent outbreaks have occurred in less than 2 years, offer an
excellent opportunity for these investigations.

Marie-Claude Georges-Courbot,* Anthony Sanchez, Chang-Yong Lu,* Sylvain
Baize,* Eric Leroy,* Joseph Lansout-Soukate,* Carole Tvi-Bnissan,* Alain
J. Georges,* Sam G. Trappier, Sherif R. Zaki, Robert Swanepoel, Patricia
A. Leman, Pierre E. Rollin, Clarence J. Peters, Stuart T. Nichol, and
Thomas G. Ksiazek
* Centre International de Recherches Mdicales, BP 769 Franceville, Gabon;
Centers for Disease Control and Prevention, Atlanta, Georgia, USA;
National Institute for Virology, Sandringham, South Africa

References

  1. Cox NJ, McCormick JB, Johnson KM, Kiley MP. Evidence for two subtypes
     of Ebola virus based on oligonucleotide mapping of RNA. J Infect Dis
     1983; 147:272-5.
  2. Fisher-Hoch SP, Brammer TL, Trappier SG, Hutwagner LC, Farrar BB, Ruo
     SL, et al. Pathogenic potential of filoviruses: role of geographic
     origin of primate host and virus strain. J Infect Dis 1992;166:753-63.
  3. Jahrling PB, Geisbert TW, Jaax NK, Hanes MA, Ksiazek TG, Peters CJ.
     Experimental infection of cynomolgus macaques with Ebola-Reston
     filoviruses from the 1989-1990 US epizootic. Arch Virol
     1996;11(Suppl):115-34.
  4. Peters CJ, Sanchez A, Rollin PE, Ksiazek TG, Murphy F. Filoviridae:
     Marburg and Ebola viruses. In: Fields BN, Knipe DM, Howley PM,
     editors. Virology, 3rd ed., Philadelphia: Lippincott-Raven
     Publishers,1996:1161-76.
  5. Sanchez A, Trappier SG, Mahy BWJ, Peters CJ, Nichol ST. The virion
     glycoproteins of Ebola viruses are encoded in two reading frames and
     are expressed through transcriptional editing. Proc Natl Acad Sci
     USA 1996;93:3602-7.
  6. Ivanoff B, Duquesnoy P,Languillat G, Saluzzo J-F, Georges A, Gonzalez
     J-P, et al. Haemorrhagic fever in Gabon. I. Incidence of Lassa, Ebola
     and Marburg viruses in Haut-Ogoou.Trans R SocTrop Med Hyg
     1982;76:719-20.
  7. Georges AJ, Renaut AA, Bertherat E, Baize S, Leroy E, LeGuenno B, et
     al. Recent Ebola virus outbreaks in Gabon from 1994 to 1996:
     epidemiologic and control issues. Proceedings of the International
     Colloquium on Ebola Virus Research; 1996 Sept 4-7; Antwerp, Belgium.
  8. World Health Organization.Outbreak of Ebola haemorrhagic fever in
     Gabon officially declared over. Wkly Epidemiol Rec 1996;71:125-6.
  9. World Health Organization. Ebola haemorrhagic feverGabon. Wkly
     Epidemiol Rec 1996;71:320.
 10. World Health Organization. Ebola haemorrhagic fever-South Africa. Wkly
     Epidemiol Rec 1996;71:359.
 11. World Health Organization. Ebola haemorrhagic fever. Wkly Epidemiol
     Rec 1997;72:7.
 12. Swofford DL, 1991. PAUP: phylogenetic analysis using parsimony,
     version 3.1.1. Champaign (IL): Illinois Natural History Survey.
 13. Zaki SR, Greer PW, Goldsmith CS, Coffield LM, Rollin PE, Callain P, et
     al. Ebola virus hemorrhagic fever: pathologic, immunopathologic and
     ultrastructural studies. Proceedings of the International Colloquium
     on Ebola Virus Research; 1996 Sept 4-7; Antwerp, Belgium.
 14. Lloyd E, Zaki SR, Rollin PE. Long term surveillance for Ebola in
     Zaire. Proceedings of the International Colloquium on Ebola Virus
     Research; 1996 Sept 4-7; Antwerp, Belgium.
 15. LeGuenno 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.

---------------------------------------------------------------------------
 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
--------------------------------------------------------------------------- 

Dispatches

The Epidemiology of Creutzfeldt-Jakob Disease in Canada: A Review of
Mortality Data

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

      Creutzfeldt-Jakob disease (CJD), and particularly its
      transmissibility through blood and blood products, has become
      a focus of concern in Canada. The recent identification of new
      variant CJD led to a review of the Canadian mortality database
      to identify any clustering of CJD by age, sex, or geographic
      location.

The study by Holman and colleagues (1), which uses mortality data and
active surveillance methods, has provided further information on the
epidemiology of Creutzfeldt-Jacob disease (CJD). The study concluded that
the incidence of the disease in the United States has remained stable and
is similar to crude incidence worldwide at about one case per million
annually. In addition, the study found no evidence that new variant CJD is
present in the U.S. population. These findings are of interest because the
epidemiology of CJD in Canada is not well described, and concerns have been
raised there about the transmissibility of CJD through transfusion of blood
and blood products or through tissue and organ transplants. In addition,
many Canadians travel to and from the United Kingdom, where new variant CJD
was first identified and linked to the bovine spongiform encephalopathy
epidemic (2).

We report here our findings on the epidemiology of CJD in Canada, which are
derived from published mortality data (underlying cause of death by
standard 5-year age group and sex, for all Canadian residents). The
Statistics Canada mortality reports for the years 1979 to 1993 were
reviewed for CJD deaths by sex and age group for each province and
territory. Reports before 1979 were not used because CJD (ICD-9 code 46.1)
was not listed as a cause of death before this time.

Overall, 334 deaths attributed to CJD were recorded in Canada in the
15-year period from 1979 to 1993, ranging from 14 to 34 deaths per year,
with a 1.1:1 male-to-female ratio (Figure 1). Eighty-five percent of the
deaths were among persons at least 60 years of age and 50% among the 60- to
69-year-old age group, which corresponds to the peak age of onset of
sporadic-type CJD (3-5). Eleven deaths (3%) were reported among persons 30
to 44 years of age. Of these, one death was reported in the 30- to
34-year-old age group, four in the 35- to 39-year-old group, and the
remaining six in the 40- to 44-year old age group. No more than one
CJD-attributed death was reported per year in the 30- to 44-year-old age
groups, with the exception of two deaths reported in 1993. No CJD deaths
have been reported among persons under 30 years of age.

[Figure 1] [Figures not available in ASCII version]
Figure 1. Age and sex distribution for Creutzfeldt-Jakob disease in Canada,
1979-1993.

[Figure 2] [Figures not available in ASCII version]
Figure 2. Age-adjusted mortality rate for Creutzfeldt-Jakob disease in
Canada, 1979-1993.

Figure 2 shows the mortality rate for CJD in Canada, standardized to the
1979 population. The age-adjusted mortality rate increases from a low of
1.1 deaths per million population in 1979 to a high of 2.1 per million in
1992, dropping to 1.8 per million in 1993. The increase is most marked from
1986 onward. This may be related to increased case recognition following
publication of the discovery that CJD was linked to human growth hormone of
pituitary origin. A real increase in incidence may also be present, but
this graph must be interpreted with caution as the absolute numbers are
small and the validity of CJD on death certificates has not been determined
in Canada.

Familial-type CJD has been documented in Canada (6,7). As in other
countries, most cases of CJD in Canada are of the sporadic type (3-5). One
case of iatrogenic CJD was reported in Canada, in a dura mater recipient
(8). A review of the Canadian growth hormone recipient database, containing
information on the 800 patients who received human growth hormone shows no
cases of CJD (Dr. Heather Dean, pers. comm., 1996). Human growth hormone
was used in Canada from 1965 until April 1985 (9).

There is one report of a possible cluster of CJD cases in Canada; between
April 1989 and October 1990, six cases were reported in the province of
Ontario, from a population of 9.5 million (1986 census figure). Two of the
patients had come from areas of Czechoslovakia with a high incidence of
familial-type disease, but no other risk factors were associated with these
cases (7).

In conclusion, the epidemiology of CJD in Canada is not well defined, as
current data sources are limited to aggregated mortality data and the
annual total case numbers are small. However, several projects have been
initiated to provide further information on the transmission of the
disease, including an examination of death certificates to identify
space/time clustering, active surveillance for CJD and new variant CJD, and
a case control study of CJD and blood transfusion.

Elizabeth Stratton, Maura N. Ricketts, and Paul R. Gully
Laboratory Centre for Disease Control, Health Canada,Ottawa, Ontario,
Canada

References

  1. Holman C, Khan AS, Belay ED, Schonberger LB. Creutzfeldt-Jakob disease
     in the United States 1979-1994: using national mortality data to
     assess the possible occurrence of variant cases. Emerg Infect Dis
     1996;2:333-7.
  2. Will RG, Ironside JW, Zeidler M, Cousens SN, Estibeiro K, Alperovitch
     A, et al. A new variant of CJD in the UK. Lancet 1996; 347:921-5.
  3. Brown P, Gajdusek DC. The human spongiform encephalopathies: Kuru,
     Creutzfeldt-Jakob disease, and the Gerstmann-Straussler-Scheinker
     syndrome. Curr Top Microbiol Immunol 1991;172:1-20.
  4. Brown P, Calatha F, Castaigne P, Gajdusek DC. Creutzfeldt Jacob
     disease: clinical analysis of a consecutive series of 230
     neuropathologically verified cases. Ann Neurol 1986;20:597-602.
  5. Will, RG. Epidemiology of Creutzfeldt-Jakob disease. Br Med Bull
     1993;49:960-70.
  6. Goldfarb LG, Brown P, Mitrova E, Cervenakova L, Goldin L, Korczyn AD.
     Creutzfeldt-Jakob disease associated with the PRNP codon 200LYS
     mutation: an analysis of 45 families. Eur J Epidemiol 1991;7:477-86.
  7. Nosal R, Kapoor A, Shanin R. Cluster of cases of Creutzfeldt-Jakob
     disease - Ontario. Canada Diseases Weekly Report 1991;17:12.
  8. Brown P. Environmental causes of human spongiform encephalopathy. In:
     Baker H, Ridley RM, editors. Methods in Molecular MedicinePrion
     Diseases. Totowa (NJ): Humana 1995;139-54.
  9. Dean HJ and Friesen HG. Growth hormone therapy in Canada: end of one
     era and beginning of another. Can Med Assoc J 1986;135:297-301.

---------------------------------------------------------------------------
 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
---------------------------------------------------------------------------
 
Dispatches

Exudative Pharyngitis Possibly Due to Corynebacterium pseudodiphtheriticum,
a New Challenge in The Differential Diagnosis of Diphtheria

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

      Corynebacterium pseudodiphtheriticum has rarely been reported
      to cause disease in humans, despite its common presence in the
      flora of the upper respiratory tract. We report here a case of
      exudative pharyngitis with pseudomembrane possibly caused by
      C. pseudodiphtheriticum in a 4-year-old girl. The case
      initially triggered clinical and laboratory suspicion of
      diphtheria. Because C. pseudodiphtheriticum can be easily
      confused with Corynebacterium diphtheriae in Gram stain,
      clarification of its role in the pathogenesis of exudative
      pharyngitis in otherwise healthy persons is of public health
      importance. Simple and rapid screening tests to differentiate
      C. pseudodiphtheriticum from C. diphtheriae should be
      performed to prevent unnecessary concern in the community and
      unnecessary outbreak control measures.

Among the pathogenic nondiphtheria corynebacteria, Corynebacterium
pseudodiphtheriticum has rarely been reported to cause disease in humans,
despite its frequent presence in the flora of the upper respiratory tract
(1,2). C. pseudodiphtheriticum was first isolated in humans by Von
Hoffmann-Wellenhof from the throat of a patient in 1888 (2). The organism
exhibits little pleomorphism. It appears as short gram-positive rods that
often lie in parallel rows on smear preparation. We report here a case of
exudative pharyngitis in a 4-year-old girl with a pseudomembrane possibly
caused by C. pseudodiphtheriticum, which initially triggered suspicion of
diphtheria, and provide a summary of previously published case reports.

Case Report

On November 6, 1994, a 4-year-old girl was admitted to the Emergency Room
of St. Mary's Hospital in Rogers, Arkansas, with fever and generalized
lymphadenopathy. Two other children in her home day care, including the
patient's sibling, also had lymph node enlargement and fever. Her mother
reported that the patient had not been immunized against any of the common
childhood diseases but had an unremarkable medical history, with the
exception of hand-foot-and-mouth disease at 3 years of age; she had never
received antibiotics. Her developmental milestones were achieved on time.
The family included the patient, her mother, father, and a 3-year-old
sibling.

The patient's posterior pharynx was erythematous with swelling of the
posterior palate and with a thick grayish white membranous exudate adhering
to the posterior wall of the pharynx. The pseudomembrane had almost
completely detached and was gone, leaving behind ulcerated tissue. A
discrete adenopathy in the neck and palpable nodes in the axillary,
epitrochlear, and inguinal regions were observed.

The initial white blood cell count was 7,000 per ml, with 50%
polymorphonuclear cells and 17% lymphocytes. A rapid screening test for
group A streptococci and a Paul-Bunnell test for Epstein- Barr virus had
negative results. Routine throat and nasopharyngeal swabs were collected
and submitted to the hospital's clinical microbiology laboratory, where
they were injected into blood agar and blood chocolate agar (special media
for diphtheria culture were unavailable). From the throat swab, there was
light growth of Streptococcus pneumoniae and light growth of gram-positive
rods; a mixture of normal flora was also present. Gram-positive rods were
also isolated from the nasopharyngeal swab. Subsequently, when subcultured
on cystine-tellurite agar, these gram-positive rods grew as black colonies.
Subcultures were then sent to the state laboratory, which confirmed them as
C. pseudodiphtheriticum. After ambulatory treatment with cefprozil and
erythromycin, the patient recovered in 4 days.

  Investigation of Contacts
Because of its resemblance with C. diphtheriae, a C. pseudodiphtheriticum
isolate was not immediately identified, and the case was initially thought
to be diphtheria. Consequently, 36 children and adults who had recently
been in contact with the patient in day care, at the fitness center, or at
the church were cultured for diphtheria. C. diphtheriae was not found, but
C. pseudodiphtheriticum was found in the patient's 3-year-old sibling and
in three additional persons: a 1-year-old girl and her 3-year-old brother,
who lived in the same building as the patient and shared her day care, and
a 54-year-old woman who attended the same church.

  Microbiology and Molecular Characterization
The C. pseudodiphtheriticum isolate from the initial patient was forwarded
to the Centers for Disease Control and Prevention (CDC), where it was
assayed by phenotypic and genotypic methods. The identification was
confirmed on the basis of growth characteristics and biochemical
properties. The isolate was assayed by polymerase chain reaction (PCR),
which detects 248 base pair fragments of the A subunit of the diphtheria
toxin gene (3), and the result was negative. Additional PCR assays were
performed with 12 sets of primers that cover the entire diphtheria toxin
gene (tox) and diphtheria toxin regulatory element (dtxR); 560 base pairs
of the dtxR were detected. However, the PCR results of its five prime and
three prime ends were negative, indicating that the gene was nonfunctional.

Previous Case Reports 1

Since 1932, only 83 cases of disease possibly caused by C.
pseudodiphtheriticum in humans have been reported. Until 1981, the only
reported disease associated with this organism was endocarditis in the
presence of anatomic abnormalities of the heart. Since then, this organism
has been increasingly recognized as a pathogen of the lungs and bronchi,
particularly in patients with underlying immunosuppressive conditions and
preexisting pulmonary diseases and in patients undergoing endotracheal
intubation.

Among reported cases, 19 were endocarditis (4-17); 61 involved infections
of the lungs, trachea, or bronchi (18-32); one was a urinary tract
infection (33); one involved a skin infection (34); and one was a vertebral
discitis (35). All but three patients had underlying medical conditions,
including functional or anatomic abnormalities of the heart (N=27), lung
and tracheobronchial diseases (N=28), endotracheal intubations (N=3), and
immunosuppressive conditions including prolonged steroid use (N=6) and AIDS
(N=4).

Most patients showed good clinical response to various antimicrobial drugs:
penicillin, ampicillin, cefazolin, vancomycin, gentamicin, tobramycin,
norfloxacin, and others. Nineteen (23%) deaths were reported overall.
Information on sex was available for 80 cases; 54 (68%) were in males. Only
five (6%) cases occurred in children <18 years of age; no cases were
reported among children <5 years of age.

      1 A table listing all published case reports is available upon
      request from Dr. Hector Izurieta, Mail Stop A32, Centers for
      Disease Control and Prevention, 1600 Clifton Rd., Atlanta, GA
      30333.

Since the early 1990s, diphtheria has returned in epidemic proportions in
the former Soviet Union and is likely to be imported into other countries.
Two cases of exudative upper respiratory tract infections with a
pseudomembrane, in which the main organism isolated was C.
pseudodiphtheriticum, have been reported. The first case was in a
54-year-old man with necrotizing tracheitis in 1991 (32). Santos et al.
(36) reported isolation of C. pseudodiphtheriticum from a 32-year-old male
Uzbek national who had a severe sore throat and dysphagia of 2 days
duration. His tonsils were enlarged bilaterally, and a grayish-white
exudate extended from the tonsil to the posterior pharyngeal wall. The
uvula and soft palate were erythematous and edematous, and tender cervical
lymphadenopathy was present. The presumptive clinical diagnosis in this
patient was respiratory diphtheria.

Although the presence of a grayish pseudomembrane in the upper respiratory
tract associated with isolation of C. pseudodiphtheriticum in these two
cases and in the case we described is not proof that the isolated
corynebacterium caused the pharyngitis or tracheitis, these accumulating
case reports suggest that C. pseudodiphtheriticum may have played a
pathogenic role.

The isolate from our patient was nontoxigenic by PCR. The diphtheria toxin
is considered to be the main virulence factor of C. diphtheriae that causes
the formation of the pseudomembrane in faucial diphtheria (37). Several
possibilities could explain the presence of a pseudomembrane in infections
with nontoxigenic corynebacterium strains. First, toxigenic and
nontoxigenic colonies of the same species can coexist in a patient; this
phenomenon has been reported in diphtheria (38). Multiple colonies would
have to be tested to confirm this hypothesis in this case. Second,
nontoxigenic forms of C. diphtheriae can produce invasive disease (39,40).
It is possible that the symptoms in our patient were caused by a
nontoxigenic C. pseudodiphtheriticum and that the pseudomembrane was simply
an inflammatory exudate. This has been reported for Arcanobacterium
haemoliticum (formerly Corynebacterium haemoliticum), which has also been
associated with production of a grayish pharyngeal pseudomembrane (41,42).
In 1960, Barksdale et al. reported a pseudomembrane in two laboratory
workers contaminated with known nontoxigenic forms of C. diphtheriae (43).
Third, we cannot exclude the possibility that the symptoms were caused by
other organisms, including S. pneumoniae, which was found in the throat
culture of our patient (29). However, this is unlikely because the growth
of S. pneumoniae in our patient's throat culture was light, and the
organism was not found in the nasopharyngeal swab. Furthermore, S.
pneumoniae has not been reported to produce a pseudomembrane (44,45).

Because several other Corynebacterium spp. have been identified as
potentially pathogenic for humans, routine screening for C. diphtheriae and
other members of the Corynebacterium spp. in clinical samples of patients
with respiratory infections should be encouraged (46). Full identification
of gram-positive rods isolated from the respiratory tract, especially when
they appear on original plates as the predominant organisms or
copredominant with another species, should be carried out at the local,
state, or reference level. Simple screening tests, such as negative
cysteinase and positive pyrazinamidase, in addition to inability to ferment
glucose, maltose, and sucrose quickly differentiate C. pseudodiphtheriticum
from C. diphtheriae. These tests should be a part of improved training of
laboratory personnel. Clinical and laboratory experience gained through
this process will not only provide information about death rates for these
organisms, but would be invaluable in preventing unnecessary concern in the
community and the extraordinary measures needed to control dissemination of
diphtheria.

Acknowledgments

We acknowledge Drs. Cynthia Whitman and Rosalind Carter, New York City
Department of Health, and Dr. Robert S. Holzman, New York University
Medical Center, for sharing information regarding a suspected case of
diphtheria in New York; and Elizabeth Laborde, Alabama State Health
Department's Laboratory, and Mary Beth Young, St. Mary's Hospital
laboratory, Rogers, Arkansas, for sharing information regarding cultures
for C. pseudodiphtheriticum.

Hector S. Izurieta,* Peter M. Strebel,* Thomas Youngblood, Dannie G.
Hollis,* and Tanja Popovic*
*Centers for Disease Control and Prevention, Atlanta, Georgia, USA; Rogers
Pediatric Clinic, Rogers, Arkansas, USA

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     lysogeny in Corynebacterium diphtheriae. Ann NY Acad Sci
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---------------------------------------------------------------------------
 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
---------------------------------------------------------------------------

Dispatches

Mycoplasmal Conjunctivitis in Wild Songbirds: The Spread of a New
Contagious Disease in a Mobile Host Population

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

      A new mycoplasmal conjunctivitis was first reported in wild
      house finches (Carpodacus mexicanus) in early 1994. The
      causative agent was identified as Mycoplasma gallisepticum
      (MG), a nonzoonotic pathogen of poultry that had not been
      associated with disease in wild songbirds. Since the initial
      observations of affected house finches in the mid-Atlantic
      region, the disease has become widespread and has been
      reported throughout the eastern United States and Canada. By
      late 1995, mycoplasmal conjunctivitis had spread to an
      additional species, the American goldfinch (Carduelis
      tristis). This new disease exemplifies the rapid spread of a
      pathogen following introduction into a mobile wildlife
      population and provides lessons that may apply to emerging
      human diseases.

In February 1994, house finches with swollen or crusty eyelids and impaired
vision were observed at backyard bird feeders in suburban Washington, D.C.
(1). Severely affected birds lingered on bird feeders or on the ground
surrounding the feeders. These birds had chronic lymphoplasmacytic
conjunctivitis, sinusitis, and rhinitis. Microorganisms resembling
mycoplasmas were adhering to conjunctival epithelium, and Mycoplasma
gallisepticum (MG) was isolated from affected tissues (2,3). A Maryland
field survey of MG in house finches in late 1994 showed a strong
association between conjunctivitis and MG by culture and polymerase chain
reaction (PCR) (3). Subsequently the disease was reproduced by inoculation
of unaffected house finches with a finch-derived MG isolate (Fischer,
unpublished data).

[Figure] [Figures not available in ASCII version]
Figure. Case distribution of house finches with conjunctivitis, October
1994-June 1996.

Since the first reports from the mid-Atlantic states, mycoplasmal
conjunctivitis in house finches has spread rapidly to the north, south, and
west. The disease was first monitored by wildlife biologists with state and
federal wildlife resource agencies, and by October 1994, affected house
finches had been reported in nine states in the mid-Atlantic region
(Figure). Beginning in November 1994, backyard sightings of healthy and
diseased house finches have been recorded by private citizens participating
in a survey conducted by the Cornell Laboratory of Ornithology (4). The
percentage of participants reporting diseased house finches has steadily
increased since the survey began: House finches with conjunctivitis were
reported by 11% of 1,413 participants in November 1994, by 17.3% of 1,239
participants in March 1995, by 28.1% of 769 participants in November 1995,
and by 35.8% of 1,047 participants in March 1996. Most reports in November
1994 came from the mid-Atlantic region, and 30% to 40% of survey
participants in this area had reported diseased finches through March 1996.
The survey also has documented the dramatic spread of disease to house
finches in the Midwest and Southeast (Figure). In November 1994, diseased
finches were reported by only 0.4% of 229 participants in these regions,
but by March 1996, the percentage had climbed to 37% of 257 participants.
Mycoplasmal conjunctivitis now has been reported almost throughout the
eastern population of house finches. No diseased birds have been reported
within the western population of house finches, which occupies the historic
range of this species.

House finches are native to the western United States, where they were
captured during the 1930s for sale in eastern pet shops. House finches were
released in New York City in the early 1940s when regulations regarding the
commercial trade of domestic songbirds changed. The first nesting pair of
house finches was observed on Long Island in 1943, and by 1951 the area
held an estimated 280 birds (5). The eastern house finch population has
expanded dramatically from the original limited number of birds in New
York; it currently contains millions of finches over the entire eastern and
midwestern United States and southeastern Canada (6).

Mycoplasmal conjunctivitis apparently has spread to another wild finch
species in the eastern United States. During the winter and spring of 1995
to 1996, American goldfinches with inflamed eyelids were reported within
the range of diseased house finches in Georgia, Maryland, North Carolina,
South Carolina, and Tennessee. The goldfinches had lesions identical to
those of house finches with mycoplasmal conjunctivitis, and in March 1996,
MG was isolated from two diseased goldfinches from North Carolina (7).

Table. Test results for Mycoplasma gallisepticum in house finches and
American goldfinches, Southeastern Cooperative Wildlife Disease Study, May
1994-June 1996
----------------------------------------------------------------------------

                          Conjunctivitis            Mycoplasma gallisepticum
                                                               (a)
State
of     Species   Number BilateralUnilateralRhinitis Isolation PCR Serology(b)
Origin
----------------------------------------------------------------------------
AL     House     1      1        0         1        0/1      1/1   0/0
       finch
GA     House     6      3        3         2        3/6      3/3   6/6
       finch
       Am.       2      1        1         0        0/2      2/2   2/2
       goldfinch
KY     House     2      1        1         2        1/2      2/2   2/2
       finch
MD     House     9      4        5         6        0/7      6/6   6/6
       finch
       Am.       1      1        0         0        0/1      1/1   1/1
       goldfinch
OH     House     1      1        0         1        1/1      0/0   0/0
       finch
SC     Am.       1      1        0         0        0/1      1/1   0/0
       goldfinch
TN     House     12     9        3         6        2/7      2/2   4/6
       finch
       Am.       1      1        0         1        0/1      0/1   0/0
       goldfinch
TOTALS House     31     19       12        18       7/24     14/14 18/20
       finch
       Am.       5      3        1         1        0/5      4/5   3/3
       Goldfinch
----------------------------------------------------------------------------
(a) Data are presented as number positive/number tested.
(b) Positive = 2+ on rapid plate agglutination test.

House finches and goldfinches were submitted for diagnostic laboratory
examinations during this epornitic. At the Southeastern Cooperative
Wildlife Disease Study, gross and microscopic postmortem examinations,
serologic tests for antibodies against MG (3), mycoplasmal cultures, and
PCR testing for MG (3) were performed on 31 house finches and five
goldfinches from seven states (Table). Consistent pathologic findings in
both species included moderate to severe lymphoplasmacytic conjunctivitis
often with hyperplasia of associated lymphoid and epithelial tissues, and
rhinitis. Occasionally, keratitis and tracheitis were observed. Antibodies
against MG were detected by rapid plate agglutination tests in 91% of
tested birds, and positive PCR products were obtained from 95% of birds
tested. MG was isolated from only 24% of birds cultured; this percentage
probably reflects the fastidiousness of the strain affecting wild finches
and the condition of the specimens.

MG is a major pathogen of domestic poultry and causes infectious sinusitis
in turkeys, chronic respiratory disease in chickens, and subclinical
infections (8). MG also has been associated with conjunctivitis in chickens
(9) and farmed gamebirds (10). Clinical disease had not been associated
with MG in wild passerine birds, although MG (11) and antibodies against MG
(12) have been detected in these birds. Infections in poultry persist
despite antimicrobial treatment or the development of antibody response; MG
can be transmitted by direct contact, by airborne droplets or dust, and
vertically through eggs (8). Like other mycoplasmas, MG can alter its
antigenic surface components in vivo. This feature may contribute to its
ability to persistently infect by adapting to the host environment and
evading the host immune response (13). The possible role of this antigenic
variation in the adaptation to new host species is unknown.

The source of the MG affecting house finches and goldfinches and its
introduction into the eastern house finch population are under
investigation. Field isolates of MG collected during 1994 through early
1996 from house finches, goldfinches, and domestic poultry; three poultry
vaccine strains of MG; and three poultry reference strains of MG were
analyzed by random amplification of polymorphic DNA (7). Molecular
characterization showed that isolates from both finch species over a wide
geographic range had identical or nearly identical random amplification of
polymorphic DNA banding patterns, but they differed from reference strains,
vaccine strains, and isolates from commercial poultry (7). These results
suggested that the finch epornitic arose from a single source and was
caused by an MG strain that differed from strains commonly associated with
poultry disease or vaccination. The ultimate source of the outbreak strain
remains unknown; however, molecular studies have not shown any relationship
between wild finch and poultry strains of MG (7). Possible sources of MG
include unrecognized reservoirs of MG in the wild or small poultry
operations with poor biosecurity, such as backyard chicken flocks.

The remarkable spread of mycoplasmal conjunctivitis in wild finches
probably reflects both bird behavior and human activity. House finches are
well adapted to human land use practices; they nest and feed in open areas
around buildings and farms, as well as in suburban settings with ornamental
trees and shrubbery. They eat weed and grass seeds, as well as fruits and
berries of flowering trees and shrubs. During the winter, house finches
flock around backyard bird feeders (5). Although precise MG transmission
modes are unknown, the propensity of this highly gregarious species to
assemble at bird feeders may enhance contact with infected birds or with
surfaces contaminated with the causative agent. Unlike their western
counterparts, house finches within the eastern range of the species may
migrate several hundred miles (14) and thus disseminate an infectious agent
over a large geographic area.

Intentional and unintentional ecologic changes may have contributed
significantly to the current mycoplasmal conjunctivitis outbreak. The
affected species was introduced into a new range, and the birds have
flourished because they prefer the areas that humans increasingly provide.
Although the eastern house finch population is large and widely
distributed, the limited genetic pool from which it descended may have
contributed to its apparently high susceptibility to MG. The causative
agent is best known for its association with domestic poultry, and its
spread through house finches appears to have been enhanced by bird feeders,
which not only provide an opportunity for increased contact between
infected and uninfected birds, but also may prolong the lives of
infectious, diseased birds that otherwise would not have been able to feed.
The combination of these and other unknown factors has resulted in the
emergence of a severe infectious disease that has spread rapidly through
the susceptible host population and could become permanently established in
house finches and possibly other species.

Even though human activity has contributed to the emergence of this
disease, human efforts to control it have not been successful. Treatment
and immunization strategies to control infectious human and domestic animal
diseases are not available, and if they were available, they would likely
be impractical and costly. Treatment and subsequent release of individual
birds in rehabilitation facilities is of questionable value because the
effect may be minimal on a population basis. Furthermore, it is unknown if
birds released after treatment remain persistently infected with MG.
Moreover, the presence of multiple avian species in a rehabilitation clinic
may enhance the transmission of MG to other species (2).

The current epornitic of mycoplasmal conjunctivitis in house finches has
some interesting parallels to emerging human diseases, particularly
regarding the substantial roles that human activity, ecologic changes, and
the introduction of exotic species may play in the emergence of infectious
disease. Additionally, the impressive speed at which this pathogen moved
through the house finch population illustrates how rapidly a pathogen can
be disseminated throughout a large geographic area within a highly
gregarious and mobile host population. Finally, the lack of suitable
control methods for MG in house finch populations and the inability to
correct the ecologic conditions that contributed to its emergence provide
strong support for preventive rather than reactive measures in dealing with
the next potential wildlife or zoonotic disease.

Acknowledgments

We thank the National Biological Service, U.S. Fish and Wildlife Service,
Cornell Laboratory of Ornithology, as well as the state wildlife resource
agencies, colleges of veterinary medicine, veterinary diagnostic
laboratories, wildlife rehabilitation facilities, and private landowners
who helped with diagnostic investigations and surveillance. This work was
sponsored by the fish and wildlife agencies in Alabama, Arkansas, Florida,
Georgia, Kentucky, Louisiana, Maryland, Mississippi, Missouri, North
Carolina, Puerto Rico, South Carolina, Tennessee, Virginia, and West
Virginia. Funds were provided by the Federal Aid to Wildlife Restoration
Act (50 Stat. 917) and the Grant Agreement 14-45-0009-94-906, National
Biological Service, U.S. Department of the Interior. Support also was
received through Cooperative Agreement 95-9613-0032-CA, Veterinary
Services, Animal and Plant Health Inspection Service, U.S. Department of
Agriculture. Dr. Andre Dhondt was supported by the New York State Hatch
Fund.

John R. Fischer,* David E. Stallknecht,* M. Page Luttrell,* Andre A.
Dhondt, and Kathryn A. Converse
*Southeastern Cooperative Wildlife Disease Study, The University of
Georgia, Athens, Georgia, USA; Cornell Laboratory of Ornithology, Ithaca,
New York, USA; National Wildlife Health Center, National Biological
Service, Madison, Wisconsin, USA

References

  1. Fischer JR. Conjunctivitis in house finches. Proceedings of the 98th
     Annual Meeting of the USAHA; 1994 Oct 29-Nov 4; Grand Rapids (MI).
     Richmond (VA): U.S. Animal Health Association, 1994:530-1.
  2. Ley DH, Berkhoff JE, McLaren JM. Isolation of Mycoplasma gallisepticum
     from house finches with conjunctivitis. Avian Dis 1996;40:480-3.
  3. Luttrell MP, Fischer JR, Stallknecht DE, Kleven SH. Field
     investigations of Mycoplasma gallisepticum in house finches
     (Carpodacus mexicanus) from Maryland and Georgia. Avian Dis
     1996;40:335-41.
  4. Dhondt AA. Finch disease update. Birdscope 1996;10:4.
  5. Elliot JJ, Arbib RS Jr. Origin and status of the house finch in the
     eastern United States. Auk 1953;70:31-7.
  6. Hill GE. House finch (Carpodacus mexicanus). In: Poole A, Gill F,
     editors. Birds of North America, No. 46. Philadelphia: The Academy of
     Natural Sciences; Washington (DC): The American Ornithologists Union,
     1993:1-23.
  7. Ley DH, Berkhoff JE, Joyner K, Powers L, Levinsohn S. Molecular
     epidemiological investigations of Mycoplasma gallisepticum
     conjunctivitis in songbirds by random amplified polymorphic DNA (RAPD)
     analyses. Presented at the 11th International Congress of the
     International Organization for Mycoplasmology, Orlando, FL; 1996 July
     14-19; Programs and Abstracts, pp. 53-4.
  8. Yoder HS Jr. Mycoplasma gallisepticum infection. In: Calnek BW, Barnes
     HJ, Beard CW, Reid WM, Yoder HW Jr., editors. Diseases of poultry, 9th
     ed. Ames (IA): Iowa State Press, 1991:198-212.
  9. Nunoya T, Yagihashi T, Tajima M, Nagasawa Y. Occurrence of
     keratoconjunctivitis apparently caused by Mycoplasma gallisepticum in
     layer chickens. Vet Pathol 1995;32:11-8.
 10. Cookson, KC, Shivasprasad HL. Mycoplasma gallisepticum infection in
     chukar partridges, pheasants, and peafowl. Avian Dis 1994;38:914-21.
 11. Shimizu T, Numano K, Uchida K. Isolation and identification of
     mycoplasmas from various birds: an ecological study. Japanese Journal
     of Veterinary Science 1979;41:273-82.
 12. Stallknecht DE, Johnson DC, Emory WH, Kleven SH. Wildlife surveillance
     during a Mycoplasma gallisepticum epornitic in domestic turkeys. Avian
     Dis 1982;26:883-90.
 13. Levisohn S, Rosengarten R, Yogev D. In vivo variation of Mycoplasma
     gallisepticum antigen expression in experimentally infected chickens.
     Vet Microbiol 1995;45:219-31.
 14. Belthoff JR, Gauthreaux SA. Partial migration and differential winter
     distribution of house finches in the eastern United States. Condor
     1991;93:374-82.

---------------------------------------------------------------------------
 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
--------------------------------------------------------------------------- 

Dispatches

Knowledge-Based Patient Screening for Rare and Emerging
Infectious/Parasitic Diseases: A Case Study of Brucellosis and Murine
Typhus

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

      Many infectious and parasitic diseases, especially those newly
      emerging or reemerging, present a difficult diagnostic
      challenge because of their obscurity and low incidence.
      Important clues that could lead to an initial diagnosis are
      often overlooked, misinterpreted, not linked to a disease, or
      disregarded. We constructed a computer-based decision support
      system containing 223 infectious and parasitic diseases and
      used it to conduct a historical intervention study based on
      field investigation records of 200 cases of human brucellosis
      and 96 cases of murine typhus that occurred in Texas from 1980
      through 1989. Knowledge-based screening showed that the
      average number of days from the initial patient visit to the
      time of correct diagnosis was significantly reduced
      (brucellosisfrom 17.9 to 4.5 days, p = 0.0001, murine
      typhusfrom 11.5 to 8.6 days, p = 0.001). This study
      demonstrates the potential value of knowledge-based patient
      screening for rare infectious and parasitic diseases.

Knowledge-based medical decision support systems allow the user to relate
clinical and laboratory manifestations from which a list of possible
diagnoses can be generated. Studies have demonstrated that such a list is
extremely useful for alerting clinicians and epidemiologists to diseases
seen rarely, if ever, and to unusual clinical manifestations of diseases
(1,2).

This report describes the development and laboratory testing of a medical
decision support system designed to facilitate early recognition of rare
and unusual infectious and parasitic diseases. The assumption is made that
early diagnosis can improve the course of treatment as well as the overall
health of the patient.

              Table 1. Knowledge base of 223 diseases at the
              time of case study
              ------------------------------------------------
                      Information            Disease   Lines
                           axis             entities  of text
              ------------------------------------------------
              Etiology                     410
              Epidemiology(a)                         9724
              Immunodeficiency info                   352
              Diagnostic procedures        306
              Diagnostic procedure         637
              Occupational links(b)        37
              Geographic links
              (countries)(c)               191
              Vectors/Invertebrate hosts   703
              Symptoms/Clinical signs      1422
              Treatment/Prevention                    3162
              Special considerations(d)               308
              Bibliographic citations      838
              Descriptive notes(e)         322
              ------------------------------------------------
              (a)Includes specific etiologic information,
              reservoirs, portal of entry/escape, mode of
              transmission/dissemination, susceptible hosts,
              incubation period, seasonal distribution,
              age/sex/race/religion/occupation relationships,
              mortality and zoonotic classification.
              (b)Lists occupations with increased potential for
              exposure.
              (c)Lists countries where the disease is known to
              occur.
              (d)Describes additional disease attributes not
              fitting other categories.
              (e)Pop-up definitions of medical terms and
              concepts in the knowledge base.

Constructing a Knowledge Base

In this study, the knowledge base was restricted to the 223 diseases common
to humans and animals. Several disease information axes were identified for
inclusion in the medical knowledge base. For each disease, nomenclature
along with any recognized synonyms, etiology, epidemiologic descriptions,
diagnostic procedures, diagnostic procedure findings, and clinical signs
and symptoms were gathered and abstracted from recent editions of standard
medical texts. In addition, occupational risks, food consumption data,
travel history, and animal/insect exposure were included. Finally, basic
treatment, management, and prevention protocols were also recorded (3-10).
Bibliographic citations for all entries into the knowledge base were tied
to each information axis to authenticate the content and provide a quick
reference for additional reading. Glossaries of knowledge base entries
(e.g., signs, symptoms, occupation, diagnostic procedure results,
geography, animal/insect exposure) to be used in generating a differential
diagnosis were created to ensure the consistency of the lexicon. Audits
were performed to validate the knowledge base entries against the
glossaries. The resulting knowledge base contains the entries listed in
Table 1. A batch procedure indexing all findings to diseases for use by the
inference engine resulted in more than 12,000 finding/disease links. The
inference engine is the portion of the decision support system used to
derive useful clinical information (e.g., possible diagnoses). It is
composed of nine program modules containing more than 10,000 lines of C
language code linked into one executable program.

Differential Diagnosis Methods

The goal of the system is to remind the user of relationships between
clinical signs, symptoms, and findings and the diseases themselves.
Therefore, a simple, intuitive pattern-matching technique was employed. The
user sets a "closeness-of-fit" parameter in the system that determines how
tight the relationship between case signs/findings and diseases must be
before a disease can become a candidate for the differential diagnosis
list. A simple ratio is created with signs/findings entered that fit each
disease as the numerator and the total number of signs/findings entered as
the denominator. The ratio ranges from zero to 1.00 with 1.00 implying a
perfect fit. If this value is equal to or greater than the
"closeness-of-fit" parameter set by the user (also ranging from zero to
1.00), the disease is entered in the differential diagnosis list as a
candidate. The ratio for each disease shows how close the clinical
manifestations are to previously published descriptions of the disease. For
example, if seven clinical signs and findings are collected on a case and
six fit a particular disease by the pattern-matching algorithm, the case
fit would be 0.86. If the case fit parameter in the system is set to 0.86
or greater, that disease would be added to the differential diagnosis list.
If the case fit parameter is set to 1.00, only diseases that fit the
clinical manifestations perfectly would become differential diagnosis
candidates. Signs and findings are not weighted by the system.

Decision Support System Trial on Murine Typhus Cases

The purpose of this trial was to determine if the intervention of the
support system at the time of the first physician visit would significantly
alter the mean number of days to the correct diagnosis. Investigation
records of 96 cases of murine typhus occurring in Texas from 1979 to 1987
were obtained from the Texas Department of Health. All records were
complete and were used in the study. Each record contained the date of the
first contact with a physician (dp). The date on which the indirect
fluorescence antibody test for Rickettsia typhi was ordered was defined as
the date the correct diagnosis was suspected (ds). Therefore, the number of
days from the first contact with a physician (dp) to the correct diagnosis
without decision support system intervention (dc (subscript ni)) was
defined as follows: dc (subscript ni) = ds - dp.

The number of days till the correct diagnosis was suspected (dc (subscript
ni)) was calculated for all cases without support system intervention. A
case was classified as missed if dc (subscript ni) >13 days. This number
was derived from the worst-case scenario for a murine typhus test
turnaround from the Texas Department of Health. During 1979-1987, the
department had the only laboratory in Texas that used the indirect
fluorescence antibody test for confirming murine typhus. On the basis of
this criterion, 23 of the 96 original cases fell into the missed category.

The clinical history, symptoms, physical findings, and some preliminary
laboratory findings as noted in the investigation records for the 23 missed
patients were entered in the support system and processed at case fit
parameter values of 0.25, 0.50, 0.75, and 1.00. Specific historical data
such as occupation, travel, and animal/insect exposure, if available, were
also entered in the support system. The support system was said to have
suggested the correct diagnosis of murine typhus if the disease was
included in a differential diagnosis list of five or fewer diseases at a
case fit parameter of 0.50 or greater.

Testing for murine typhus was assumed to be ordered immediately if the
disease was suggested by the support system. This effectively set the
number of days to suspect the correct diagnosis with intervention (dc
(subscript wi)) to 1 day under these conditions.

The distributions of dc (subscript ni) and dc (subscript wi) were found to
be Gaussian (PROC UNIVARIATE, SAS). Therefore, parametric statistical
techniques were used for data analysis. A paired t-test was run on
(subscript ni) and (subscript wi) (mean time to suspecting murine typhus
with and without the system, respectively) to see if there was a
significant difference in the means (PROC MEANS, SAS) (14).

In 11 (48%) of the 23 cases defined as missed, murine typhus was clearly
suggested by the support system in a differential list of five or fewer
possibilities by using only clinical history, signs, symptoms, and
preliminary laboratory data if available. In six (26%) of these cases,
murine typhus was the only disease suggested by the support system. The
support system did not suggest the correct disease in 12 (52%) of the cases
classified as missed. Table 2 lists the most common diseases that also
appeared in the differential diagnosis lists for murine typhus cases.

         Table 2. Other diseases commonly listed in the differential
        diagnosis of murine typhus
        ------------------------------------------------------------
        1. Lyme borreliosis            7. Relapsing fever
        2. Tularemia                   8. Avian chlamydiosis
        3. Brucellosis                 9. Leptospirosis
        4. Zoonotic epidemic typhus    10. Salmonellosis
        5. Rocky Mountain              11. Babesiosis
             spotted fever             12. Malaria
        6. Rat bite fever              13. Q fever
        ------------------------------------------------------------

The mean number of days to suspect the correct diagnosis without support
system intervention ((subscript wi)) was 11.5 days. The mean number of
days to suspect the correct diagnosis with support system intervention
((subscript wi)) was 8.6 days, an improvement of 2.9 days (p = 0.001).

Decision Support System Trial on Brucellosis Cases

Investigation records of 342 cases of brucellosis occurring in Texas from
1980 to 1989 were also obtained from the Texas Department of Health. Of
these records, 13 were incomplete, and 129 involved cases of recrudescence
of the disease or had no specific date of onset of illness and were
excluded from the study. The remaining 200 records contained the date of
onset of illness. However, the day of the first physician visit was not
available and had to be extrapolated from the day of onset of illness. The
average number of days from onset of illness to the first physician visit
was assumed to be 4 days, on the basis of a study in Texas for patients
with murine typhus (12).

The day that the correct diagnosis was suspected was defined as the day on
which the serum agglutination test or a bacterial culture for brucellosis
was ordered. The number of days from the first contact with a physician to
the day the correct diagnosis was suspected without support system
intervention (dc (subscript ni)) was calculated as indicated above.

A case was classified as missed if dc (subscript ni) >11 days. This number
was derived from the worst-case scenario for a brucellosis test turnaround
from the Texas Department of Health. By this liberal criterion, 98 of the
200 original cases still fell into the missed category.

The clinical history, symptoms, physical findings, and some preliminary
laboratory findings as noted in the investigation records for the 98 missed
patients were entered in the support system and processed at case fit
parameter values of 0.25, 0.50, 0.75, and 1.00. Specific historical data
such as occupation, travel, and animal/insect exposure, if available, were
also entered in the support system. By definition, the system gave the
correct diagnosis of brucellosis if the disease was included in a
differential diagnosis list of five or fewer diseases at a case fit
parameter of 0.50 or greater.

It was assumed that the testing for brucellosis would be ordered
immediately if the disease was suggested by the support system. This
effectively sets the number of days to suspect the correct diagnosis with
intervention (dc (subscript wi)) to 1 day under these conditions. The
distributions of dc (subscript ni) and dc (subscript wi) were also Gaussian
and analyzed by paired t-tests on (subscript ni) and (subscript wi) (14).

In 86 (88%) of the 98 cases defined as missed, brucellosis was clearly
suggested by the support system in a differential list of five or fewer
possibilities by using only clinical history, signs, symptoms, and
preliminary laboratory data if available. In 69 (70%) of the cases,
brucellosis was the only disease suggested. The support system did not
suggest the correct disease in only 12 (12%) of the cases classified as
missed. Table 3 lists the most common diseases that also appeared in the
differential diagnosis of brucellosis.

               Table 3. Other diseases commonly listed in
               the differential diagnosis of brucellosis
               ---------------------------------------------
               1. Toxoplasmosis       6. Rat bite fever
               2. Q fever             7. Listeriosis
               3. Viral hepatitis A   8. Blastomycosis
               4. Salmonellosis       9. Trichinellosis
               5. Histoplasmosis      10. Lyme borreliosis
               ---------------------------------------------

The mean number of days to suspect the correct diagnosis without support
system intervention ((subscript wi)) was 17.9 days. The mean number of
days to suspect the correct diagnosis with support system intervention was
4.5 days, an improvement of 12.9 days (p = 0.0001). However, data were
derived by using an extrapolated day of first physician visit, and that may
have affected these comparisons.

The brucellosis investigation records screened showed that of nine cases,
three involved apparently healthy pregnancies, one a premature delivery,
one a miscarriage, one seizures, one weakness and chronic headaches, and
two deaths. In eight of the nine cases, the support system correctly
suggested brucellosis. In addition, 59 (30%) of the patients with
brucellosis were hospitalized, some for extended periods during the
diagnostic phase.

Twenty-one of 23 murine typhus patients were hospitalized, some for
extended periods during the diagnostic phase of the case. Furthermore, many
patients had to undergo expensive diagnostic testing, including 19
electrocardiograms, four computer-aided tomography scans, and a various
other procedures such as bone scans, liver scans, barium enemas, and renal
and pelvic sonograms. Earlier diagnoses of brucellosis and murine typhus
could have eliminated the need for many of the diagnostic procedures,
shortened hospital stays, and improved the course of treatment.

On the average, it took approximately 3 minutes of interaction with the
decision support system to construct a differential diagnosis. Therefore,
this type of screening could easily become part of the routine
history-taking and physical exam of a patient. Furthermore, this study
underlines the discriminatory power of the clinical history and physical
signs and symptoms in suspecting the presence of a certain disease in a
patient. Factors such as the patient's occupation, travel history,
animal/insect exposure, and unusual dietary habits can be especially
important in helping to diagnose many rare infectious and parasitic
illnesses.

The results of this evaluation indicate that for two rare diseases
(brucellosis and murine typhus), the decision support system appears to
perform well with high sensitivity) in suggesting the correct diagnosis in
a patient. However, the specificity of the system has not been evaluated. A
prospective double-blinded study in a general clinical or hospital setting
would make that determination.

Craig N. Carter,* Norman C. Ronald, James H. Steele, Ed Young, Jeffery
P. Taylor, Leon H. Russell, Jr.,* A. K. Eugster,* Joe E. West*

*Texas A&M University, College Station, Texas, USA; Texas Medical
Informatics, Inc., College Station, Texas, USA; University of Texas School
of Public Health, Houston, Texas, USA; Veteran Affairs Medical Center,
Houston, Texas, USA; and Texas Department of Health, Austin, Texas, USA

References

  1. Elkin PL, Barnett GO, Famigletti KT, Kim RJ. Closing the loop on
     diagnostic decision support systems. Proc Annu Symp Comput Appl Med
     Care, 1990;589-93.
  2. Johnston ME, Langton KB, Haynes RB, Mathieu A. Effects of
     computer-based clinical decision support systems on clinician
     performance and patient outcome. Yearbook of Medical Informatics
     1995:486-93.
  3. Acha PN, Szyfres B. Zoonoses and communicable diseases common to
     animals and man. 2nd ed. Washington (DC): Pan American Health
     Organization, 1987.
  4. Steele JH. CRC handbook series in zoonoses. 1st ed. Boca Raton (FL):
     CRC Press, 1981.
  5. Young EJ. Human brucellosis. Review of Infectious Diseases
     1983;5:821-41.
  6. Benenson AS. Control of communicable diseases in man. 15th ed.
     Washington (DC): American Public Health Association, 1990.
  7. Mandell GL, Douglas RG, Bennett JE. Principles and practice of
     infectious diseases. 3rd ed. New York: Churchill Livingstone, 1990.
  8. Greene CE. Infectious diseases of the dog and cat. 1st ed.
     Philadelphia: Saunders, 1990.
  9. Blood DC, Radostits OM. Veterinary medicine. 7th ed. London: Billiere
     Tindall, 1989.
 10. Smith BP. Large animal internal medicine. 1st ed. St. Louis: Mosby,
     1990.
 11. Hughes, C. The representation of uncertainty in medical expert
     systems. Med Inf 1989;14:269-79.
 12. Dumler JS, Taylor JP, Walker DH. Clinical and laboratory features of
     murine typhus in Texas, 1980-1987. JAMA 1991;266:1365-70.
 13. Buck C, Donner A. The design of controlled experiments in the
     evaluation of nontherapeutic interventions. Journal of Chronic
     Diseases 1982;35:531-8.
 14. Ott L. An introduction to statistical methods and data analysis 3rd
     ed. Boston: PWS-Kent Publishing, 1988.

---------------------------------------------------------------------------
 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
--------------------------------------------------------------------------- 

Letters

Emergence of Epidemic O'nyong-nyong Fever in Southwestern Uganda, After an
Absence of 35 Years

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

To the Editor: In July 1996, an uncommon disease suspected to be
O'nyong-nyong fever was recognized in the Rakai district of southwestern
Uganda. It was reported to have started in June 1996. The disease spread
into the neighboring Mbarara and Masaka districts of Uganda and in the
bordering Bukoba district of northern Tanzania.

The initial symptoms of O'nyong-nyong fever are high fever and generalized
maculopapular skin rash with crippling arthritis, primarily in the big
joints, in the absence of joint effusion. Other features are lymphadenitis,
eye pain and reddening with no discharge, chest pain, and general malaise.
The disease is self-limiting. All age groups and both sexes are equally
affected. In areas where the disease is epidemic, 60% to 80% of the people
are infected, and familial clustering is found in affected households. No
deaths have been reported, but two miscarriages have been associated with
infection.

The Ministry of Health (Uganda), in collaboration with the Uganda Virus
Research Institute, began epidemiologic and clinical investigations of the
epidemic in August 1996. Acute-phase serum samples were collected from
patients, and adult mosquitoes were collected from within and around
patients' homes. Virus isolates were made from acute-phase serum samples
from several patients by intracranial inoculation and passage in baby mice.
Attempted virus isolations from mosquito specimens are in progress. Serum
samples and aliquots of the virus isolates were sent to the Centers for
Disease Control and Prevention, Fort Collins, Colorado, USA, for
reisolation and identification. A portion of the capside and NS4 genes of
the virus isolates was sequenced and identified as O'nyong-nyong virus; the
virus was isolated and sequenced directly from another serum sample. Two
serum samples were positive for IgM antibody to O'nyong-nyong antigen.

O'nyong-nyong virus was responsible for a similar epidemic in 1959 to 1961,
which started in northern Uganda and spread south and eastward into Kenya,
Tanzania, and Zambia, and then northward from Tanzania into southwestern
Uganda, where it subsided. The disease has reemerged in this area after 35
years of absence.

E.B. Rwaguma,* J.J. Lutwama,* S.D.K. Sempala,* N. Kiwanuka, J. Kamugisha,
S. Okware, G. Bagambisa, R. Lanciotti, J. T. Roehrig, and D.J. Gubler

*Uganda Virus Research Institute, Entebbe, Uganda; Rakai Project Uganda
Virus Research Institute; Communicable Disease Control, Ministry of
Health, Uganda; Ministry of Health, Rakai, Uganda; Centers for Disease
Control and Prevention, Fort Collins, Colorado, USA

---------------------------------------------------------------------------
 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
--------------------------------------------------------------------------- 

Letters

Prostatitis and Benign Prostatic Hyperplasia: Emerging Infectious Diseases?

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

To the Editor: In their excellent article, Molecular Approaches to the
Identification of Unculturable Infectious Agents, Gao and Moore (1) point
out that molecular approaches should be unleashed on diseases such as
sarcoidosis, Kawasaki disease, and type I diabetes mellitus, which are
thought but not proven to be infectious. The authors, however, are
overlooking the more common and most likely infectious disease of unknown
etiology today--prostatitis.

According to the pathologist McNeal, the prostate gland is the most
commonly diseased internal organ of the human body (2). Prostatitis is the
most common prostate disease, resulting in more physician visits than
either benign prostatic hyperplasia or prostate cancer, according to the
National Institutes of Health (3). Despite its frequency, prostatitis as a
disease and as a histologic lesion is understudied (4).

By the Meares and Stamey culture localization procedure, in which the first
voided urine, a midstream urine, the expressed prostatic secretions, and a
final voided urine are compared, more than 90% of cases in patients with
chronic pelvic symptoms are labeled as "nonbacterial" prostatitis or
prostatodynia, both of which are thought to be incurable diseases (5).

The University of Washington has documented white blood cell counts as high
as 38,000 per mm3, in "nonbacterial" prostatitis patients (6). According to
urologist Thomas Stamey, up to 50% of all men experience symptoms of
prostatitis during their lifetimes (7). The prostatitis lesion was found in
40 (44%) of 91 men at random autopsy (8). In another study of 100
consecutive autopsies on men who died suddenly in automobile accidents and
from other causes, the prevalence of histologic signs of prostatitis
increased with age and was highest when benign prostatic hyperplasia was
also present. Prostatitis was present in 22% of men under 40 years of age
and in 60% of those over 40 years of age (9).

In fact, the line between benign prostatic hyperplasia and prostatitis is
blurred. Prostatitis as a histologic lesion has been found in 98% of
patients with benign prostatic hypertrophy (10). Microbial tests on benign
prostatic hyperplasia tissue have found significant rates of infectivity.
In another study, more than 70% of transurethral resection of the prostate
specimens showed clinical or laboratory signs of infection (11). Benign
prostatic hyperplasia and prostatitis cannot be distinguished by symptoms,
and some believe that they may be the same disease.

In these days of prostate specific antigen testing, more than 50% of men
who undergo biopsies for prostate cancer have a prostatitis lesion whether
they have cancer or not (Gottesman et al., unpublished data; McNeal,
personal communication, 1995). Prostatitis occurs at an early age, and
prostate cancer decades later, in the same part of the prostate gland, the
peripheral zone.

Why aren't DNA techniques being unleashed on what is apparently the most
common and most purulent unknown inflammatory disease in menan
inflammatory lesion that is associated with benign prostatic hyperplasia
and prostate cancer? Surely, DNA microbial testing has important
implications for all three major prostate diseasesprostatitis, benign
prostatic hyperplasia, and prostate cancer.

Brad Hennenfent
Director, The Prostatitis Foundation
Chicago, Illinois, USA

References

  1. Gao S-J, Moore PS. Molecular approaches to the identification of
     unculturable infectious agents DNA vaccines for emerging infectious
     diseases: what if? Emerg Infect Dis 1996;2:159-67.
  2. McNeal JE. The prostate gland: morphology and pathobiology. Monographs
     in Urology 1988;9:3.
  3. National Institutes of Health. The National Kidney and Urologic
     Diseases Advisory Board 1990 long-range plan. Bethesda (MD): U.S.
     Department of Health and Human Services, Public Health Service,
     National Institutes of Health, 1990.
  4. Hennenfent BR. The economics of urological care in the 21st century
     [letter]. Urology 1996;47:285-6.
  5. Weidner W, Schiefer HG, Krauss H, Jantos CH, Freidrich HJ,
     Altmannsberger M. "Chronic prostatitis" a thorough search for
     etilogically involved microorganisms in 1,461 patients. Infection
     1991;19:S119-25.
  6. Krieger JN, Egan KJ, Ross SO, Jacobs R, Berger RE. Chronic pelvic
     pains represent the most prominent urogenital symptoms of "chronic
     prostatitis." Urology 1996;48:715-22.
  7. Stamey TA. Pathogenesis and treatment of urinary tract infections.
     Baltimore: Williams and Wilkins, 1980.
  8. McNeal J. Regional morphology and pathology of the prostate. Am J Clin
     Pathol 1968;49:347-57.
  9. Bostrom K. Chronic inflammation of the male accessory sex glands and
     its affect on the morphology of the spermatozoa. Scand J Urol Nephrol
     1971;5:133.
 10. Kohnen PW, Drach GW. Patterns of inflammation in prostatic
     hyperplasia: a histologic and bacteriologic study. J Urol
     1979;121:755-60.
 11. Riedash G, Morhing K, Brkovic D. Concentration of ofloxacin in
     prostatic tissue during TURP. Drugs 1993;45 (Suppl. Preprint).

---------------------------------------------------------------------------
 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
---------------------------------------------------------------------------

Letters

Risk Factors for Severe Leptospirosis in the Parish of St. Andrew, Barbados

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

To the Editor: Leptospirosis, an important zoonosis in most warm-climate
areas, is endemic in most Caribbean countries (1). The disease was first
reported in Barbados 60 years ago (2), and since 1979 has been the subject
of continual study as the result of the establishment of the Leptospira
Laboratory by the governments of Barbados and the United Kingdom. The
annual incidence of severe leptospirosis in Barbados over the past 17 years
has been approximately 11.5 cases per 100,000 population with a death rate
of 13%. However, the incidence rate varies in the parishes of Barbados. For
the 12-year period from 1979 to 1991, the lowest incidence rates were in
St. Peter (9.5 cases per 100,000 population) and St. Michael (9.9 cases per
100,000 population), while the highest was in St. Andrew (40 cases per
100,000 population). This greater than fourfold difference in incidence
rates has been attributed to differences in rainfall (3). We performed a
retrospective case-control study to determine what other factors were
important.

We identified cases of leptospirosis from the records of the Leptospira
Laboratory and included them in the study if they occurred from January
1980 to December 1993, if the home address was in the Parish of St. Andrew,
and if laboratory evidence of leptospirosis was confirmed by one or more of
the following: an IgM ELISA titer 160 in a single sample, a titer in the
microscopic agglutination test (MAT) of 800 in a single sample, a fourfold
or greater rise in antibody titer between two samples tested by the same
method, or isolation of leptospires from blood or urine cultures (3).

Of the 36 cases of laboratory-confirmed leptospirosis and 41 controls
(selected for residence close to the case-patient), 22 patients and 38
controls were included in the study. For case-patients, the mean age at
onset of symptoms was 30.8 years (range 8 to 73 years); 28 (78%) of 36
cases occurred in males. The mean age of controls was 31.3 years (range 13
to 78 years); 15 (39.5%) of 38 controls were male. Controls were matched
for age, but because St. Andrew is a sparsely populated parish, and because
the survey was conducted during the day, it was difficult to recruit
sufficient male controls. The participants were administered a
questionnaire, and blood samples were taken and tested for leptospiral
antibodies. Serologic results were compared with the results obtained for
each of the patients during their acute illness and with the results of
previous followup studies conducted over several years.

Gardening was a significant risk factor (odds ratio [OR] 4.57, 95%
confidence level [CL] 1.09-20.36) and appeared to remain so whether gloves
were worn or not, as was the presence of dogs around the home (OR 7.82, 95%
CL 1.79-46.55). With few exceptions, the respondents kept dogs, and these
animals are an important risk factor for leptospirosis in Barbados (6). A
positive association was observed between illness and wearing boots in the
garden or yard (OR 8.5, 95% CL 1.93-42.55), but this may be because case
patients had changed their behavior since recovery, because they were
working in wetter areas than the controls, or because the male/female ratio
was lower among controls. We were unable to define the odds ratios for
walking barefoot some or all of the time because none of the controls
admitted to going barefoot. The most important risk factor we identified
was walking through ponds or stagnant water (OR 25.62, 95% CL
2.89-1151.84). Flooding is common during the rainy season in Barbados, and
people living in rural areas such as St. Andrew are often exposed in this
way. These risk factors bear a striking resemblance to those identified in
the outbreak in Nicaragua a few months after our study (7).

We conclude that almost all of the patients had multiple risk factors for
leptospiral infection. Few indicated a change in lifestyle since recovering
from leptospirosis. Serologic evidence of recent reexposure to
leptospirosis was detected in two (17%) of 12 case-patients.

The relatively high rainfall in St. Andrew may have contributed to their
risk for leptospirosis by enhancing the survival of leptospires in the soil
and water. The incidence of leptospirosis in St. Andrew shows a close
association with mean monthly rainfall, the highest incidence during the
period studied being October and November. However, when individual cases
were examined, a less strong correlation was observed between onset of
symptoms and rainfall in the preceding month and with rainfall in the
preceding 3-month period. No evidence was observed of clustering of cases
in months or years with rainfall above the mean. Similar findings have been
reported for the island as a whole (4,5). The incidence of leptospirosis
appeared to lag behind the rainfall, since rainfall tended to increase from
June to a peak in November, while leptospirosis incidence increased from
August to November. There was a marked decrease in rainfall in December
each year, with the dry season continuing until May. However, continuing
low incidence of leptospirosis was seen throughout the less wet months,
until during the months of May to July only one case occurred during the
study period.

On the basis of these findings, we conclude that the ground remains
sufficiently damp during the period from December through the early months
of the year for leptospires to survive. As the middle months of the year
are reached, the ground may become too dry for leptospires to survive. This
would also account for the apparent lag between the onset of the rainy
season and the rise in leptospirosis incidence, as the ground may take some
weeks of consistent rainfall to become saturated.

No clustering of cases in time was observed, which confirms that
leptospirosis in Barbados is endemic and that increases in incidence result
from multiple sporadic cases rather than microepidemics (5).Cases were
clustered geographically, but this may have been an artifact resulting from
variation in population density. Moreover, the place of residence is not
necessarily the place of exposure to leptospirosis.

We emphasize the importance of public education regarding the relative
risks, as a means of preventing exposure, and of continuing education of
physicians and primary healthcare workers to raise their awareness of the
seasonal distribution and early symptoms of leptospirosis.

Acknowledgments

We thank Mr. J. Charlery (Meteorological Department) for supplying the
rainfall data and Ms. C. Whittington and Ms. S. Branch (Leptospira
Laboratory) for their technical assistance.

C. P. Douglin,* C. Jordan, R. Rock, A. Hurley,* P.N. Levett*
*Leptospira Laboratory, St. Michael, Barbados; Maurice Byer Polyclinic,
St. Peter, Barbados; University of the West Indies, School of Clinical
Medicine and Research, Barbados

References

  1. Everard JD, Everard COR. Leptospirosis in the Caribbean. Reviews in
     Medical Microbiology 1993;4:114-22.
  2. Bayley HH. An investigation of the infectious jaundice of Barbados.
     Caribbean Medical Journal 1939;1:135-42.
  3. Everard COR, Edwards CN, Everard JD, Carrington DG. A twelve-year
     study of leptospirosis on Barbados. Eur J Epidemiol 1995;11:311-20.
  4. Everard COR, Bennett S, Edwards CN, Nicholson GD, Hassell TA,
     Carrington DG, et al. An investigation of some risk factors for severe
     leptospirosis on Barbados. Journal of Tropical Medicine and Hygiene
     1992;95:13-22.
  5. Bennett S, Everard COR. Absence of epidemicity of severe leptospirosis
     in Barbados. Epidemiol Infect 1991;106:151-6.
  6. Everard COR, Jones CJ, Innis VA, Carrington DG, Vaughan AW.
     Leptospirosis in dogs on Barbados. Israel Journal of Veterinary
     Medicine 1987;43:288-95.
  7. Spiegel RA, Ashford DA, Trevejo RT, Rigau-Perez JG, McClure EM, Amador
     JJ, et al. Leptospirosis outbreak associated with pulmonary
     hemorrhage--Nicaragua, 1996. Abstracts of the First Meeting of the
     International Leptospirosis Society; 1996 Sept; Nantes, France. Nance,
     France: International Leptospirosis Society, 1996.

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 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
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Letters

Electronic Communication and the Rapid Dissemination of Public Health
Information

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To the Editor: In the United States, communicable disease surveillance,
investigation, and control are the responsibility of the states. The
Centers for Disease Control and Prevention (CDC) provides epidemiologic and
laboratory support to the state and territorial epidemiologists (state
epidemiologists) and state public health laboratory directors (state
laboratory directors), who are located in each of the 50 states,
Washington, D.C., the Virgin Islands, the Federated States of Micronesia,
American Samoa, the Marianas Islands, and Puerto Rico. Historically,
communication between CDC and these state representatives has been
conducted by telephone, facsimile, or letter, and more recently by the
WONDER (1) electronic mail (e-mail) system. We examined the timeliness and
coverage of the WONDER system when used to contact state epidemiologists
and laboratory directors during two recent foodborne outbreaks.

The first outbreak was reported to CDC on February 10, 1995, by the
Communicable Disease Surveillance Centre (CDSC) in the United Kingdom. CDSC
had linked an outbreak of salmonellosis in the United Kingdom to a snack
food distributed to many countries including the United States (2). CDC
decided to notify all state epidemiologists about the outbreak immediately
so that they could take appropriate action to protect consumers and report
suspected cases. This e-mail message was ready to be accessed by all state
epidemiologists from 4:27 p.m. Eastern Standard Time (E.S.T.) on Friday,
February 10, 1995.

The second outbreak involved Salmonella serotype Stanley infections
associated with the consumption of alfalfa sprouts. In the United States,
the outbreak was recognized when a larger than expected number of isolates
of Salmonella Stanley for the first week of June 1995 was reported (3). CDC
notified state epidemiologists and laboratory directors about the outbreak
and requested that cases of Salmonella Stanley infection be reported and
Salmonella Stanley isolates be sent to CDC. This e-mail message was ready
to access from 9:41 a.m. E.S.T. on Friday, June 9, 1995.

These two e-mail messages were sent to two group codes maintained by the
Council for State and Territorial Epidemiologists and the Association of
State and Territorial Public Health Laboratory Directors on the CDC WONDER
e-mail system. The subject heading for these messages indicated that they
were urgent and from CDC. The messages were available for 22 days from the
day of posting, at which time unaccessed messages were automatically
returned to sender. Each message was sent with an automatic receipt
acknowledgment function.

Because many of the territories are not regularly connected to WONDER, only
the 50 states, the District of Columbia, and Puerto Rico were included in
the study. The time to receipt was calculated on the basis of working days
(Monday through Friday) only. E-mails accessed during a weekend were
attributed to the following Monday.

In February, 48 of 50 states were on the state epidemiologists WONDER
e-mail distribution list; 47 states, Puerto Rico, and the District of
Columbia accessed the e-mail message within 22 days; one state did not
access it within that period; 8 (16%) accessed the message the day it was
sent; 28 (57%) accessed it within 1 working daythree of these accessed the
message during the weekend; and 43 (88%) of 49 recipients accessed the
message within 1 week. While no additional cases were reported, e-mail
communication may have hastened product recall, thereby preventing further
cases.

In June, 49 states were on the state epidemiologists WONDER e-mail
distribution list; 48 states and Puerto Rico accessed the e-mail message
within 22 days; two did not access the message within that period; 25 (51%)
accessed the message the day it was sent; and 40 (82%) accessed the message
by the second working daytwo of these accessed the message on a weekend.

Thirty-eight states and Washington, D.C., were on the state laboratory
directors WONDER distribution list in June; 25 (64%) accessed the message
the day it was sent, and 32 (84%) of 38 accessed the message by the second
working dayone of these accessed the message on a weekend. All 38 states
and Washington D.C. accessed the e-mail message within the systems' 22-day
limit. The pattern for state laboratory directors was almost identical to
that for state epidemiologists.

Within 3 weeks of transmission of the June message (by June 30, 1995),
state health department laboratories had forwarded 55 Salmonella Stanley
isolates to CDC: 44(80%) of these were the outbreak strain. These reports
contributed to a traceback that implicated a single alfalfa seed
distributor.

The use of e-mail to communicate health related messages to epidemiologists
and laboratory directors was timely and highly successful in these
incidents. By the second working day, more than half of the intended
recipients had accessed the February message, and more than 80% had
accessed the June message. However, not all state epidemiologists and
laboratory directors access WONDER e-mail daily, and so other means of
communication would be necessary if contact were required within 1 working
day.

Because epidemiologists and laboratory directors have to dial into the
WONDER mainframe by modem to find out if they have new messages and to
receive them, retrieving WONDER e-mail messages can be less than timely;
there is no mechanism to alert users to incoming WONDER e-mail messages.
This delay is likely to be overcome as more epidemiologists and laboratory
directors become connected to the Internet by local area networks that
automatically check for incoming messages several times per hour. Some
epidemiologists and laboratory directors have been slower to access their
WONDER e-mail address because they also had an Internet address and thus
accessed the WONDER system less often.

Perhaps more than one person in each state office should be on the
distribution list to ensure message delivery when one representative is
absent. We confirmed only that the message had been accessed by someone
using the state epidemiologists' password; however, it is possible that
someone other than the state epidemiologists accessed the message on their
behalf adding to the delays.

Electronic communication by public health groups (e.g., Epi-net links
public health agencies in the United Kingdom, Salm-net links agencies
involved in foodborne disease surveillance and control in Europe) is
rapidly increasing (4). However, there is a need for a global network that
allows public health agencies of every country to rapidly communicate real
or potential emergent disease threats.

Craig B. Dalton, Patricia M. Griffin, and Laurence Slutsker
Centers for Disease Control and Prevention, Atlanta, Georgia, USA

References

  1. Fried A, Roser DH, Reid JA. CDC WONDER: A cooperative processing
     architecture for public health. J Am Med Inform Assoc 1994;1:303-12.
  2. An outbreak of Salmonella agona due to contaminated snacks. Commun Dis
     Rep CDR Wkly 1995 Feb 17;5:29,32.
  3. Martin SM, Bean NH. Data management issues for emerging diseases and
     new tools for better methods. Emerging Infectious Diseases
     1995;1:124-8.
  4. Vacalis DT, Bartlett CL, Shapiro CG. Electronic communication and the
     future of international public health surveillance. Emerging
     Infectious Diseases 1995;1:34-5.

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 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
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News and Notes

Emerging Infectious Diseases Conference

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"The Hot Zone 1997: Conference on Emerging Diseases" will be held in
Lexington, Kentucky, at the Hyatt Regency on June 27 and 28, 1997. The
conference is hosted by the University of Kentucky and the University of
Cincinnati. Topics include human immunodeficiency virus infection,
vector-borne diseases, viral diseases, tick-borne diseases, prion diseases,
and antibiotic resistance. Continuing medical education credit hours (10.5)
will be offered. For complete information contact Continuing Medical
Education, University of Kentucky, L007 Kentucky Clinic, Lexington, KY
40536-0284 or e-mail sbsize00@pop.uky.edu.

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 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
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News and Notes

"Emergent Illness and Public Scholarship" Fellowships

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

Emory University's Center for the Study of Public Scholarship announces
four semester-long fellowships for its program on Emergent Illness and
Public Scholarship to be held during the 1997-1998 academic year. Emergent
Illness and Public Scholarship is the first of a 3-year series of programs
funded by the Rockefeller Foundation and organized by the Center for Public
Scholarship. The center is directed by Dr. Ivan Karp and is housed in the
Institute for Liberal Arts. Emergent Illnesses and Public Scholarship is
being coorganized by Emory's Center for the Study of Health, Culture, and
Society, which is directed by Dr. Randall M. Packard.

Emergent illnesses, from Legionnaires' disease to AIDS, not only challenge
the scientific and medical communities to find new explanations and
treatments, but prompt patients and their caretakers and families to
challenge the authority and expertise of biomedical specialists. In various
settings globally, the formal scholarship of health scientists coexists
with, and is frequently challenged by, that of lay persons searching for
knowledge and explanations of their own illnesses.

Emergent Illness and Public Scholarship will address the politics of
knowledge as well as the mechanics by which information travels between
professionals and lay communities that simultaneously produce and consume
knowledge. Issues of education, technology, audiences, and the ownership of
knowledge will stand alongside more traditionally focused biomedical and
epidemiologic concerns as foci of the workshop.

Fellows in the program will spend part of the semester talking across
disciplinary and institutional boundaries about emerging illness; help
determine the agenda of the workshop series; plan one of its sessions; and
interact on a limited basis with Emory graduate students.

Especially welcome to the program are applicants working on health issues
outside academia, as the workshop's goal is to transcend the boundaries
that often mute discussions between producers of knowledge with differing
credentials.

For more information, contact: Michael McGovern, Center for the Study of
Public Scholarship, S-412 Callaway Building, Emory University, Atlanta, GA
30322.

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 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
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News and Notes

International Workshop on Molecular Epidemiology and Evolutionary Genetics

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

The 2nd International Workshop on Molecular Epidemiology and Evolutionary
Genetics of Pathogenic Microorganisms, will be held at ORSTOM, Montpellier,
France, May 26-28, 1997. The workshop is organized under the auspices of
ORSTOM (French agency for scientific research in developing countries),
CNRS (French agency for basic research) and the Centers for Disease Control
and Prevention, Atlanta, Georgia, USA. The goal of the workshop is the
integration of laboratory science and epidemiology, in using genetic
information to study evolution, emergence, reemergence, and dispersal of
microorganisms. The objectives of the workshop are to 1) integrate
molecular biologic and evolutionary genetics approaches in areas of
diagnosis, strain typing, species identification, pathogenesis, antigenic
variation, drug and vaccine resistance, and host and vector specificity; 2)
foster interaction between scientists working on parasites, yeasts and
fungi, bacteria, and viruses; and 3) provide health care providers, public
health professionals, and laboratory scientists an opportunity to discuss
the joint use of genetic tools and methods needed to meet the challenges of
diagnosis and management of emerging, reemerging, and endemic infectious
diseases.

The deadline for submitting abstracts to Dr. Michel Tibayrenc by fax
(33-467547800) or e-mail (tibayren@orstom.rio.net) is March 15, 1997. For
more information about the workshop, please contact Dr. Michel Tibayrenc or
Dr. Altaf Lal (aal1@.cdc.gov).
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Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA