EMERGING INFECTIOUS DISEASES, VOLUME 1, NUMBER 4

Editors

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

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

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

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

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

Editorial and Computer Support

Emerging Infectious Diseases receives editorial and computer
support from the Office of Planning and Health Communication,
National Center for Infectious Diseases.

Editorial Support
Maria T. Brito
Anne D. Mather, M.A.
Carol D. Snarey, M.A.

Production
Rita M. Furman, M.A.

Electronic Distribution
Carol Y. Crawford
Cheryl G. Shapiro, M.S.


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
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
Centers for Disease Control
  and Prevention (CDC)
Atlanta, Georgia, USA

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

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

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

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

Philip 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

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

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

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

Emerging Infectious Diseases

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

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

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

Use of trade names is for identification only and does not imply
endorsement by the Public Health Service or by the U.S.
Department of Health and Human Services.


Electronic Access to Emerging Infectious Diseases

If you have Internet access, you can retrieve the journal
electronically through file transfer protocol (FTP), electronic
mail, or World-Wide Web (WWW).

The journal is available in three file formats: ASCII, Adobe
Acrobat (.pdf), and PostScript (.ps). The ASCII version of the
journal does not contain figures. Both the .pdf and .ps files,
however, contain graphics and figures and are true
representations of the hard copy of the journal. The Adobe
Acrobat format requires an Adobe Reader. This reader is available
in DOS, Windows, UNIX, and Macintosh versions. Installation
instructions come with the Adobe software.

Access Methods 

FTP: Download the journal through anonymous FTP at ftp.cdc.gov.
The files can be found in the pub/Publications/EID directory in
each of the file types listed above.

WWW: Launch WWW browser for the Internet and connect to the
following address: http://www.cdc.gov. Your WWW software will
allow you to view, print, and retrieve journal articles.

ListServer (e-mail lists): You may have the table of contents sent to your
e-mail box by subscribing to the EID-TOC mailing list. When you
subscribe to this list, you automatically receive the table of
contents and will be able to receive individual journal articles
by FTP or e-mail.

If you choose to receive the entire journal, you may subscribe to
one of three other lists. EID-ASCII sends the journal in ASCII
format. EID-PDF sends the journal in Adobe Acrobat format. You
can get the free Adobe Acrobat Reader by subscribing to the list.
EID-PS sends the journal in PostScript format. However, because
of the large file sizes and the complexity of sending the journal
to different e-mail systems, it is strongly recommended that if
you have FTP capabilities, you choose to access EID through FTP
rather than by e-mail lists.

To subscribe to a list, send an e-mail to lists@list.cdc.gov with
the following in the body of your message: subscribe listname
(e.g., subscribe EID-ASCII). Once you have requested a
subscription, you will receive further instructions by e-mail.

For more information about receiving Emerging Infectious Diseases
electronically, send an e-mail to eidhelp@cidod1.em.cdc.gov.

Instructions to Authors

Editorial Policy and Call for Articles

The goals of Emerging Infectious Diseases (EID) are to promote the
recognition of new and reemerging infectious diseases and to improve the
understanding of factors involved in disease emergence, prevention, and
elimination. EID has an international scope and is intended for
professionals in infectious diseases and related sciences. We welcome
contributions from infectious disease specialists in academia, industry,
clinical practice, and public health as well as from specialists in
economics, demography, sociology, and other disciplines whose study
elucidates the factors influencing the emergence of infectious diseases.

EID will be published in English and will feature three types of articles:
Perspectives, Synopses, and Dispatches. The purpose and requirements of
each type of article are described in detail below.

Instructions to Authors

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

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

Perspectives: Contributions to the Perspectives section are welcome from
scientists and professionals in all disciplines and should address factors
known to contribute to 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.
The section should begin with an introduction outlining the relationship of
the issues discussed in the paper to the emergence of infectious diseases.
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. The section should begin with an introduction outlining
the relationship of the issues discussed in the paper to the emergence of
infectious diseases. 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 of
no more than 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.

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.

Send documents in hardcopy (Courier 10-point font), on diskette, or by
e-mail. Acceptable electronic formats for text are ASCII, WordPerfect,
AmiPro, DisplayWrite, MS Word, MultiMate, Office Writer, WordStar, or
Xywrite. Send graphics documents in Corel Draw, Harvard Graphics,
Freelance, .TIF (TIFF), .GIF (CompuServe), .WMF (Windows Metafile), .EPS
(Encapsulated Postscript), or .CGM (Computer Graphics Metafile). The
preferred font for graphics files is Helvetica. If possible, convert
Macintosh files into one of the suggested formats. Submit photographs in
camera-ready hardcopy.

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


CONTENTS, EMERGING INFECTIOUS DISEASES, VOL 1., NO. 4

Synopses

The Ascension of Wildlife Rabies: A Cause for Public Health Concern or
Intervention? Charles E. Rupprecht, Jean S. Smith, Makonnen Fekadu,
and James E. Childs

Diagnosis of Tuberculosis in Children: Increased Need for Better
Methods, Ejaz A. Kahn and Jeffrey R. Starke

Data Management Issues for Emerging Diseases and New Tools for
Managing Surveillance and Laboratory Data, Stanley M. Martin and Nancy
H. Bean

Dispatches

Helicobacter hepaticus, a Recently Recognized Bacterial Pathogen,
Associated with Chronic Hepatitis and Hepatocellular Neoplasia in
Laboratory Mice, Jerry M. Rice

Hemolytic Uremic Syndrome Due to Shiga-like Toxin Producing
Escherichia coli 048:H21 in South Australia, Paul N. Goldwater and
Karl A. Bettelheim

Does Treatment of Bloody Diarrhea due to Shigella dysenteriae Type 1
with Ampicillin Precipitate Hemolytic Uremic Syndrome? Abdulaziz A. A.
Bin Saeed, Hassan E. El Bushra, and Nasser A. Al-Hamdan

An Outbreak of Hemolytic Uremic Syndrome Associated with Antibiotic
Treatment of Hospital Inpatients for Dysenstery, Sami Al-Qarawi,
Robert E. Fontaine, and Mohammed-Saeed Al-Qahtani

Epidemic Cholera in the New World: Translating Field Epidemiology into
New Prevention Strategies, Robert V. Tauxe, Eric D. Mintz, and Robert
E. Quick

Are North American Bunyamwera Serogroup Viruses Etiologic Agents of
Human Congenital Defects of the Central Nervous System?, Charles H.
Calisher and John L. Sever

Lymphocytic Choriomeningitis Virus: An Unrecognized Teratogenic
Pathogen, Leslie L. Barton, C.J. Peters and T.G. Ksiazek

Commentary

Hemolytic Uremic Syndrome, Mary Beers and Scott Cameron

News and Notes

Guidelines on the Risk for Transmission of Infectious Agents During
Xenotransplants, Louisa E. Chapman

Emerging Infectious Diseases Featured at ICAAC/IDSA Meeting

Building a Geographic Information System (GIS) Public Health
Infrastructure for Research and Control of Tropical Diseases, Allen W.
Hightower and Robert E. Klein

APHA Session Features Emerging Infections, Martin S. Favero
 
Southeast Asia Intercountry Consultative Meeting on Prevention and
Control of New, Emerging, and Reemerging Infectious Diseases, Samlee
Plianbangchang

SYNOPSES

(Page 107)

The Ascension of Wildlife Rabies: A Cause for Public Health
Concern or Intervention?

Charles E. Rupprecht, V.M.D., Ph.D., Jean S. Smith, M.S. Makonnen
Fekadu, D.V.M., Ph.D., and James E. Childs, Sc.D.
Centers for Disease Control and Prevention, Atlanta, Georgia, USA

ABSTRACT

The epidemiology of rabies in the United States has changed
substantially during the last half century, as the source of the
disease has changed from domesticated animals to wildlife,
principally raccoons, skunks, foxes, and bats. Moreover, the
changes observed among affected wildlife populations have not
occurred without human influence. Rather, human attraction to the
recreational and economic resources provided by wildlife has
contributed to the reemergence of rabies as a major zoonosis.
Although human deaths caused by rabies have declined recently to
an average of one or two per year, the estimated costs associated
with the decrease in deaths amount to hundreds of millions of
dollars annually. In future efforts to control rabies harbored by
free-ranging animal reservoirs, public health professionals will
have to apply imaginative, safe, and cost-effective solutions to
this age-old malady in addition to using traditional measures.


Introduction

Rabies virus is the type species (serotype 1) of the Lyssavirus
genus, a group of morphologically similar, antigenically and
genetically related, negative-stranded RNA viruses, with a near
global distribution (1). The lyssaviruses (Table 1) are well
adapted to particular mammalian species (2) and rarely initiate
panzootics. The public health threat of rabies as a preeminent
zoonosis relates to the acute, incurable encephalitis that
results from transmission of the virus by the bite of an infected
animal. An estimated 40,000 to 100,000 human deaths are caused by
rabies each year worldwide; in addition, millions of persons,
primarily in developing countries of the subtropical and tropical
regions (3), undergo costly postexposure treatment (PET).
Although the number of human rabies cases has been significantly
reduced in the United States, the total number of animal rabies
cases approached historical limits in 1993. To appreciate the
public health significance that lyssaviruses continue to play as
persistent and emerging infectious agents, one must understand
certain human activities, such as recent animal translocations
(i.e., the natural or purposeful change by humans of  the normal
home range or geographic distribution of an animal) and animal
ecology.


Table 1. Recognized members of the genus Lyssavirus, family
Rhabdoviridae

Lyssavirus    Rabies 

Reservoir     Found worldwide, except for a few island nations,   
              Australia, and Antarctica. Endemic and sometimes   
              epidemic in a wide variety of mammalian species,
              including wild and domestic canids, mustelids,     
              viverrids, and insectivorous and hematophagous     
              bats; >25,000 human cases/year, almost all in areas
              of uncontrolled domestic dog rabies. 
              
History      Descriptions of clinical disease in Greek and Roman 
             documents. In the late 1800s, Pasteur attenuated
             the virus by serial passage and desiccation to
             vaccinate humans and animals. Pathognomonic 
             inclusions in nerve cells described by Negri in 
             1903. An immunofluorescence test for rabies viral 
             antigen developed in the 1950s.

Lyssavirus   Lagosbat

Reservoir   Unknown, but probably fruit bats. 10 cases          
            identified to date, including 3 in domestic         
            animals, in Nigeria, South Africa, Zimbabwe,        
            Central African Republic, Senegal, and Ethiopia. No 
            known human deaths.
              
History     Isolated in 1956 from brain of Nigerian fruit bats
            (Eidolon helvum) at Lagos Island, Nigeria, but not
            characterized until 1970; 3 cases in domestic
            animals initially diagnosed as rabies, but weak
            immunofluorescence led to suspicion of 
            "rabies-related" virus, later confirmed by typing
            with monoclonal antibodies or nucleotide sequence 
            analysis. Marginal cross-protection with rabies
            vaccines.

Lyssavirus  Mokola

Reservoir   Mokola, Unknown, but probably an insectivore or 
            rodent species. Cases identified in Nigeria, 
            South Africa, Cameroon, Zimbabwe, Central African 
            Republic, and Ethiopia; 17 cases known, including
            9 domestic animals and 2 human cases. 
              
History     First isolated from Crocidura sp. shrews trapped
            in Mokola Forest near Ibadan, Nigeria, in 1968.
            Characterized in 1970. Like Lagos bat virus,
            evidence of infection with Mokola was recognized
            only by poor reaction with anti-rabies reagents.
            7 domestic animal cases in Zimbabwe in 1981 and
            1982 prompted serologic survey and identification
            of antibodies to Mokola in rodents, especially
            bushveld gerbils (Tatera leucogaster). No
            cross-protection with rabies vaccines.

Lyssavirus  Duvenhage

Reservoir     Unknown, but probably insectivorous bats. Cases
              identified in South Africa, Zimbabwe, and Senegal; 
              4 cases known, including 1 human death. No cases 
              in domestic animals. 
              
History       First identified in 1970 in rabies-like
              encephalitis in man bitten by an insectivorous bat
              near Pretoria, South Africa. Virus named after the
              victim. Although Negri bodies detected in
              histologic examination of brain tissue, negative
              immunofluorescence tests led to suspicion of
              rabies-related virus, subsequently confirmed by
              antigenic and genetic typing. Marginal
              cross-protection with rabies vaccines.

Lyssavirus    European bat Lyssavirus 1 (EBLV1) 

Reservoir     European insectivorous bats (probably Eptesicus 
              serotinus); >400 cases in bats. 1 confirmed human 
              case in 1985 and a suspect case in 1977. No known 
              domestic animal cases. 

History       Although cases in European bats were reported as
              early as 1954, identification of the virus was 
              not attempted until 1985, when the first of 
              100 infected bats was reported in Denmark and 
              Germany. Almost all cases are in the common 
              European house bat, E serotinus. Marginal 
              cross-protection with rabies vaccines.

Lyssavirus    European bat Lyssavirus 2 (EBLV2) 

Reservoir     European insectivorous bats (probably Myotis 
              dasycneme); 5 cases identified, including 1 human
              death. No known domestic animal cases. First 
              identified in isolate from Swiss bat biologist 
              who died of rabies in Finland. Marginal cross-
              protection with rabies vaccines.

Historical Perspectives
The history of rabies in the New World reflects the interaction
of chance, evolutionary constraint, ecologic opportunism, and
human surveillance activities. Rabies may have existed in the
United States before European colonization and the introduction
of domestic animals incubating the disease. Various pathogens
could have migrated during the exchanges of fauna and human
populations over the Bering Strait some 50,000 years ago;
folklore of a rabies-like malady among native people throughout
the Pacific Northwest supports this notion (4). Records at the
time of the Spanish conquest in Middle America associate vampire
bats with human illness (5). If chiropteran rabies viruses were
present and well established in the New World at the time of
continental interchange, terrestrial virus counterparts also
could have been present. Nonetheless, the first indication of
terrestrial rabies did not surface until 1703 in what is now
California (5). Dog and fox rabies outbreaks, reported commonly
in the mid-Atlantic colonies throughout the late 1700s (4), were
probably exacerbated by the introduction of dogs and red foxes
(Vulpes vulpes), imported for British-style fox hunting,
throughout New England in the 1800s; fox rabies epizootics ensued
and spread to the eastern United States by the 1940s to 1950s
(5,6). Skunk rabies reports were also frequent throughout the
western states by the 19th century, and they were replete with
cowboy tales of "phobey cats" (5).

Although individual reports document a high incidence of dog
rabies at the beginning of the last century, no national
surveillance system existed. Human deaths from rabies in the
United States were not commonly reported; the highest official
record was of 143 cases, from a survey of death certificates in
1890. During 1938, when rabies in humans and other animals became
a nationally reportable disease, the total number of rabies cases
reported was 9,412 per year (mostly in domesticated species),
with 47 human deaths. These numbers are certainly underestimates,
since surveillance was limited, and sensitive diagnostic tests
for human and animal rabies were not developed until the
mid-1950s.

An epizootiologic transition began in the United States in the
1920s, when rabies prevention efforts were no longer focused
exclusively on human vaccination but began to include programs
for the control of rabies in dogs. Domestic animal cases
gradually declined, largely as a result of local dog rabies
control programs that included vaccination, stray animal removal,
and leash and muzzle ordinances. However, as such cases
decreased, surveillance systems designed to track the source of
infection for residual domestic animal foci detected increased
cases in wild species. By 1960, rabies was diagnosed more
frequently among wildlife than among domesticated animals. In
1971, rabies was reported for the first time from all 48
contiguous states and Alaska. Skunks (primarily the striped
skunk, Mephitis mephitis) formed the major animal reservoir from
1961 to 1989, until they were unexpectedly supplanted by the
raccoon (Procyon lotor) during the rabies outbreak in the
mid-Atlantic and northeastern states (7). This epizootic is
believed to have started during the late 1970s by the
translocation of infected animals from a southeastern focus of
the disease.

The epidemiology of human rabies has also changed considerably
over the last 50 years (8,9). From 1946 to 1965, 70% to 80% of
human rabies cases occurred after a known exposure (most often a
dog bite), and 50% of the cases before 1975 occurred after
treatment with suboptimal vaccines. Over the last decade, 80% of
rabies-related human deaths were among persons who had no
definitive history of an animal bite (Table 2), and none resulted
from postexposure prophylaxis failures. Almost all the recent 
human cases occurred after an animal exposure that was
unrecognized by the patient as carrying a risk for rabies
infection. The apparent source of human rabies has also changed:
14 of the 18 cases acquired in the United States since 1980
involved rabies variants associated with insectivorous bats (10).

The latest report, in March 1995, typifies recent trends. A bat,
subsequently found to be rabid, was found in the bedroom of a
4-year-old girl in Washington State. The child denied any contact
with the bat, and no postexposure treatment was initiated. A
bat-associated rabies virus variant was later identified in
biopsy specimens from the child and from the bat's carcass (11).
Despite the current prominence of raccoons as the largest
wildlife reservoir in the United States (12), no documented human
rabies cases have been associated with this ubiquitous carnivore.

Table 2. Human rabies cases in the United States by exposure
category, 1946-1995*

               Exposure source 

Years     Domestic  Wildlife  Other  Unknown (%) Case total   
1946-1955    86        8        0     26 (22)       120
1956-1965    21        7        0     10 (26)        38
1966-1975     6        7        1      2 (13)        16
1976-1985     6        1        2     11 (55)        20
1986-1995*    2        2        0     14 (78)        18

* Through Oct. 1995.

The Cost of Prevention
Rabies prevention and control strategies in the United States
have succeeded in lowering the number of human rabies deaths to
an average of one to two per year. However, the reason for this
low mortality level is a prevention program estimated to cost
$230 million to $1 billion per year (13-15). This cost is shared
by the private sector (primarily the vaccination of companion
animals) and by the public (through animal control programs,
maintenance of rabies laboratories, and subsidizing of rabies
PET).

Accurate estimates of these expenditures are not available. The
number of PETs given annually in the United States is unknown,
although the total must be substantially greater than the
minimum of 20,000 estimated in 1980 to 1981 (16) when vaccine
distribution was more tightly regulated. As rabies becomes
epizootic or enzootic in a region, the number of PETs
increases (17). Although the cost varies, a course of rabies
immunoglobulin and five doses of vaccine given over a 4-week
period typically exceeds $1,000. Potential exposure to a single
rabid kitten in New Hampshire recently led to the treatment of
more than 650 persons at an estimated cost of $1.5 million (18).
Surveillance-related costs also rise as rabies becomes entrenched
in wildlife. During 1993, the New York State rabies diagnostic
laboratory received approximately 12,000 suspected animal
submissions. This compares with approximately 3,000 submissions
in 1989, before raccoon rabies became epizootic. In New Jersey,
rabies prevention expenditures in two counties increased from
$768,488 in 1988, before the raccoon epizootic, to $1,952,014 in
1990, the first full year of the epizootic (15); vaccination of
pet animals accounted for 82% of this total. Vaccinated domestic
animals are normally administered a booster vaccine dose after a
known or suspected rabid animal exposure (19). This further
increases costs, as wildlife rabies epizootics escalate. The cost
per human life saved from rabies ranges from approximately
$10,000 to $100 million, depending on the nature of the exposure
and the probability of rabies in a region (20).

What's more, most economic analyses do not take into account the
psychological trauma caused by human exposure to rabies, the
subsequent euthanasia of pets, or the loss of wildlife resources
during rabies outbreaks. Rabies in wildlife has now reached
historically high levels in the United States (12), and the costs
of preventing human rabies are mounting.

Human Influences and the Role of Translocation
The colonization of the New World had a profound effect upon
native fauna and consequent rabies epizootiology. Large-bodied
carnivores, such as bears, cougars, wolves, and wolverines, were
perceived as dangerous and killed outright. A few Carnivora have
persisted and flourished. For example, the coyote (Canis
latrans), a highly adaptable canid and the subject of many
unsuccessful control programs, has been gradually expanding its
range northward and eastward. Despite their widespread
distribution and abundance (even in suburban neighborhoods),
rabid coyotes have been reported rarely and sporadically, except
for a brief period from 1915 to 1917, when an extensive outbreak
occurred in portions of Utah, Nevada, California, and Oregon.

While dog rabies has been largely controlled, a region of
southern Texas that borders Mexico has persisted as a focus of
both dog and coyote rabies. The number of cases of coyote rabies
has gradually risen in this area since the late 1980s, accounting
for 46 of the 50 cases of coyote rabies reported in the United
States during 1991, 70 of 75 cases in 1992, and 71 of 74 cases in
1993 (12). The outbreak of coyote rabies has spread to the
vicinity of San Antonio. One of the dangers of this outbreak is
the continued spillover into the domestic dog population (21); at
least 25 rabid dogs were reported from the area in 1991, 41 in
1992, and 54 in 1993 (12). Human rabies closely parallels the
disease in domestic animals; at least two human deaths (in 1991
and 1994), probably due to coyote-dog interactions, have been
associated with this canid outbreak in Texas (10,22).

The translocation of infected coyotes from the south Texas focus
is believed to be responsible for the transmission of this rabies
variant to dogs in at least two other states: a single hunting
dog in Alabama during 1993 (12) and at least seven cases of
apparent dog-to-dog rabies transmission in Florida in 1994 (21).
Expanded surveillance similar to that done in 1977 with the
raccoon rabies focus in the mid-Atlantic region (7) is warranted
for this canid virus. In this effort, state health departments
should monitor unusual occurrences (such as the increased
submission of canid specimens to the diagnostic facility),
tracking of their time and location, and establishment of
suitable public health interventions. These would include
restricting further animal movements and enforcing mandatory
companion animal rabies vaccination. Assessing control efforts is
an important component of any intervention. In addition to the
problems posed by the emergence of the coyote as a reservoir for
rabies, the potential translocation of other species should be
recognized.

Since the transmission of rabies by a bat was first reported in
1953, rabid insectivorous bats have caused an average of 700 to
800 cases annually, and have been found throughout the United
States, excluding Alaska and Hawaii (12). The discovery of these
cases, coincident with the marked reduction of canine rabies
cases, has afforded a certain epidemiologic luxury to enhance
surveillance among wildlife. Similar to the Carnivora, the
chiropteran families most important in rabies perpetuation (e.g.,
Vespertilionidae, Molossidae) have several species that are
highly adaptable, abundant, and widespread. Rabies virus variants
maintained by insectivorous bats appear to be exchanged largely
independently from those in terrestrial mammalian reservoirs
(23), despite documented spillovers. A similar epidemiologic
situation exists among European bats, but with Lyssavirus
genotypes (24) that can be readily differentiated from New World
rabies isolates. The role of bats in Africa (25,26) in Lyssavirus
maintenance is less clear (Table 1). Infections with non-rabies
lyssaviruses have resulted in rabies vaccine failures (27). Such
infections raise the specter of potentially serious public health
consequences if introduced and subsequently established in
susceptible bat populations. How probable is this scenario?

The distances between Africa, Eurasia, Pacific Oceania, and the
New World mitigate against the dispersal, migration, and
introduction of healthy bats without human intervention (28).
However, several recent events illustrate the opportunity for the
transoceanic transfer of rabies-infected bats. In March 1986,
researchers from Canada inadvertently shipped a big brown bat
(Eptesicus fuscus) that was incubating rabies virus to colleagues
in Tubingen, Germany. When the bat became ill and was euthanized,
a diagnosis of rabies was made (29). A similar event occurred
when Boston researchers collected a dozen wild big brown bats
from Massachusetts during July 1994 and exported them to
researchers in Denmark. By December 1994, six of the imported
bats had died and were confirmed as positive for rabies virus by
the Danish Veterinary Services, State Institute for Virus
Research (L. Miller, pers. comm.).

Commercial enterprises also serve as vehicles for the accidental
translocation of animals infected with rabies virus. The first
confirmed nonindigenous case of rabies in Hawaii resulted from
the accidental introduction of a big brown bat (30). In March
1991, a bat was captured within a transport container unloaded
from a ship in Honolulu harbor. The container held automobiles
from Michigan loaded into the container ship in California. The
local department
of health laboratory diagnosed rabies; this was later confirmed,
and the virus was characterized antigenically at the Centers for
Disease Control and Prevention. The strain was a variant common
to E. fuscus in the midwestern and western United States. None of
the three instances cited above appear to have resulted in
secondary cases or establishment of the virus in foreign animal
populations.

No unintended importations of non-rabies lyssaviruses to the
United States have been documented. The likelihood of accidental
introduction, escape, survival, and perpetuation of infected
exotic bat species into the United States is remote. However,
other more recent deliberate translocation activities may
significantly enhance the probability of such introductions.

During 1994, a number of improperly issued federal permits
allowed as many as several thousand wild bats to be imported to
the United States for sale in the commercial pet trade. These
animals were primarily Egyptian tomb bats (Rousettus
aegyptiacus), although several other bat species were imported as
well. Sales of imported bats (and their offspring) to private
collectors or as pets in the United States are prohibited,
according to the Foreign Quarantine Regulations (42 CFR 71.54).
Animals that may be vectors of diseases of public health concern
are eligible for entrance only for restricted uses at accredited
zoos or research institutions, where contact with the general
public is limited. Imported bats that will be legally displayed
normally undergo an extended quarantine period.

Although no reports of lyssaviruses isolated from Egyptian fruit
bats exist, active surveillance for such viruses has not been
conducted. These bats are relatively common and widespread
throughout the area that extends from Turkey and Cyprus to
Pakistan, the Arabian peninsula, Egypt, and most of sub-Saharan
Africa (31). Because they may roost by the thousands in a wide
variety of habitats, there is ample opportunity for interaction
with other Chiroptera, such as the widely distributed
straw-colored (Eidolon helvum) or epauletted (Epomophorus
wahlbergi) fruit bats; both of these species have been implicated
in Lyssavirus epizootiology in Africa (25,26). The adaptability
of Egyptian fruit bats should be a cause for concern because of
the potential for survival and interaction among indigenous bat
fauna, particularly in the southern United States. Additionally,
beyond the obvious public health risks and foreign animal disease
introduction, imported bat species should not be released into
the wild because they may cause serious harm to local agriculture
and may displace native species.

Bats serve many critical ecologic functions worldwide and
generally avoid contact with humans. However, they may be
infected with many pathogens without demonstrating obvious
clinical signs of infection. When bats are placed in a private
household or pet shop, the hazard of disease transmission to
humans is greatly increased. Persons currently possessing
imported bats should be advised not to display them in settings
where human contact can occur.

Intervention
Widespread, sustained population reduction of mammalian
reservoirs to eliminate rabies is not justified (32) for
ecologic, economic, and ethical reasons. Given the multispecies
complexity and considerable geographic areas affected by wildlife
rabies, and the opportunities for translocation, what alternative
preventive strategies exist? Recent progress in implementing
terrestrial wildlife rabies control programs elsewhere in the
world has public health relevance for the United States. Oral
rabies vaccination of the red fox with vaccine-laden baits is an
integral aspect of rabies control throughout southeastern Canada
and Europe, where more than 75 million doses of vaccine have been
distributed over 5 million km2 during the past two decades (33).
Consequently, rabies incidence among wild and domestic animals
has fallen, as have PETS for human rabies.

The raccoon rabies epizootic in the eastern United States
provided renewed impetus for reconsidering oral vaccination
technology, first conceived at the Center for Disease Control in
the 1960s (34). The shift of the vaccination and baiting methods
from a fox model to the raccoon involved extensive field and
laboratory research during the 1980s. The existing attenuated
rabies vaccines for foxes were shown to be less effective for
raccoons and other carnivores (35,36). Additionally, studies of
new candidate vaccines raised safety issues regarding
vaccine-induced disease in wildlife (36).

In 1983, a vaccinia-rabies glycoprotein (V-RG) recombinant virus
vaccine was developed (37) that has proven to be an effective
oral immunogen in raccoons and various other important reservoir
species (38); vaccine advantages include improved thermostability
and an inability to cause rabies. (Only the gene for the surface
glycoprotein of a vaccine strain of rabies virus was included in
the recombinant virus.) When vaccine-laden baits are offered
under natural conditions, contact with them by nontarget wildlife
species cannot be totally excluded. However, studies of V-RG
virus have shown no vaccine-associated morbidity, mortality, or
gross pathologic lesions in more than 40 warm-blooded vertebrate
species examined. Moreover, with rare exceptions, there has been
no contact-transfer of vaccine between vaccinated and control
animals housed together (38); viral recovery has been limited to
a few anatomical sites over a 48-h interval (39).

While laboratory evaluations of target and nontarget species
proceeded during 1987 to 1989, small-scale trials of V-RG were
conducted in Belgium and France, with promising results (40). The
first North American V-RG vaccine field trial began on August 20,
1990, on Parramore Island off the eastern shore of Virginia
(41,42). This limited field trial demonstrated vaccine safety.
Efficacy was also suggested: more than 80% of field-vaccinated
raccoons survived a severe laboratory rabies challenge (7 months
after V-RG release) to which more than 90% of control raccoons
succumbed (43).

A 1991 Pennsylvania study site closely approximated the ecologic
communities of the eastern United States targeted for use of V-RG
vaccine, while still maintaining relative biosecurity through its
geographic barriers. The study at this site evaluated the rate of
vaccine-laden bait contact and potential vaccine-related adverse
effects among nonraccoon species, including rodents, carnivores,
insectivores, and opossum. Raccoons and other furbearers
demonstrated no adverse effects associated with vaccine contact.
Examination of more than 750 nontarget individuals, representing
35 species, failed to demonstrate gross lesions suggestive of
V-RG contact.

After these safety trials, the first efficacy field experiments
began in New Jersey during 1992 (44). Between spring 1992 and
autumn 1994, more than 100,000 vaccine-laden fishmeal polymer
baits were distributed by hand and helicopter over an area of
56,000 hectares. This trial attempted to create a population of
immunized raccoons across the northern Cape May Peninsula to
prevent the spread of epizootic raccoon rabies from affected
portions of the state. Surveillance demonstrated a significant
decrease in the rate of spread and overall rabies incidence in
the target and other monitored areas (44), suggesting the
potential effectiveness of this strategy.

In the United States, oral vaccination of raccoons is now under
way in Massachusetts (45), New York (46), and Florida, and an
experimental extension of the program to coyotes is under way in
south Texas. However, the future of such vaccination for wildlife
in the United States may be seriously questioned. For oral
vaccination to become an adjunct to traditional methods, the
following major questions need to be answered: 1) What is the
relationship between animal population density and the minimum
density of vaccine/baits needed? 2) What level of herd immunity
is
necessary to eliminate rabies under various environmental
circumstances? 3) What bait distribution techniques are optimal?
4) How can these methods be generalized from foxes and raccoons
to other species, such as skunks, mongooses and dogs? 5) What
long-term funding sources are available? 6) What are the various
costs of rabies control and prevention methods? Given the
problems inherent in wildlife control, the greater issue of
extending these methods to the control of dog rabies in the
developing world will be a challenge well into the next century.

Dr. Rupprecht is chief of the Rabies Section, Ms. Smith is a
research microbiologist, Dr. Fekadu is a research veterinary
medical officer, and Dr. Childs is chief of the Epidemiology
Section, Viral and Rickettsial Zoonoses Branch, Division of Viral
and Rickettsial Diseases, National Center for Infectious
Diseases, Centers for Disease Control and Prevention, Atlanta,
Georgia.

Acknowledgment
The authors gratefully acknowledge the technical expertise of the
staff of the Rabies and Epidemiology Sections, Viral and
Rickettsial Zoonoses Branch, Division of Viral and Rickettsial
Diseases, National Center for Infectious Diseases, Centers for
Disease Control and Prevention, without whose assistance this
work would not have been possible.

Address for correspondence: Charles E. Rupprecht, Centers for
Disease
Control and Prevention, 1600 Clifton Rd., MS G33, Atlanta, GA
30333,
USA; fax 404-639-1058; e-mail cyr5@ciddvd1.em.cdc.gov.

References
1. World Health Organization. World survey of rabies 28 for the
year 1992. Geneva: World Health Organization, 1994.
2. Wandeler A, Nadin-Davis SA, Tinline RR, Rupprecht CE. Rabies
epizootiology: an ecological and evolutionary perspective. In:
Rupprecht CE, Dietzschold B, Koprowski H, editors. Lyssaviruses.
New York: Springer-Verlag, 1994:297-324.
3. Meslin FX, Fishbein DB, Matter HC. Rationale and prospects for
rabies elimination in developing countries. In: Rupprecht CE,
Dietzschold B, Koprowski H, editors. Lyssaviruses. New York:
Springer-Verlag, 1994:1-26.
4. Winkler WG. Foxrabies. In: Baer GM, editor. The natural
history of rabies. 1st ed. New York: Academic Press, 1975:3-22.
5. Baer GM. Rabies -- an historical perspective. Infectious
Agents
and Disease 1994;3:168-80.
6. Carey AB, Giles RH, McLean RG. The landscape epidemiology of
rabies in Virginia. Am J Trop Med Hyg 1978;27:573-80.
7. Rupprecht CE, Smith JS. Raccoon rabies -- the re-emergence of
an
epizootic in a densely populated area. Semin Virol 1994;5:155-64.
8. Held JR, Tierkel ES, Steele JH. Rabies in man and animals in
the United States, 1946-65. Public Health Rep 1967;82:1009-18.
9. Anderson LJ, Nicholson KG, Tauxe RV, Winkler WG. Human rabies
in the United States, 1960 to 1979: epidemiology, diagnosis, and
prevention. Ann Intern Med 1984;100:728-35.
10. Centers for Disease Control and Prevention. Human rabies --
Alabama, Tennessee, and Texas, 1994. MMWR 1995;44:269-72.
11. Centers for Disease Control and Prevention. Human rabies
Washington state, 1995. MMWR 1995; 44:625-7.
12. Krebs JW, Strine TW, Smith JS, Rupprecht CE, Childs JE.
Rabies surveillance in the United States during 1993. J Am Vet
Med Assoc 1994;205:1695-709.
13. Stehr-Green JK, Schantz PM. The impact of zoonotic diseases
transmitted by pets on human health and the economy. Vet Clin
North Am Small Anim Pract 1987;17:1-15.
14. Fishbein DB, Arcangeli S. Rabies prevention in primary care:
a four-step approach. Postgrad Med 1987;82:83-90.
15. Uhaa IJ, Dato VM, Sorhage FE, et al. Benefits and costs of
using an orally absorbed vaccine to control <M%-1>rabies in
raccoons. J Am Vet Med Assoc<D%0> 1992;201:1873-82.
16. Helmick CG. The epidemiology of human rabies postexposure
prophylaxis, 1980-1981. JAMA 1983;250:1990-6.
17. Centers for Disease Control and Prevention. Raccoon rabies
epizootic: United States, 1993. MMWR 1994;43:269-73.
18. Centers for Disease Control and Prevention. Mass treatment of
humans exposed to rabies -- New Hampshire, 1994. MMWR
1995;44:483-6.
19. Centers for Disease Control and Prevention. Compendium of
animal rabies control, 1995. MMWR 1995;44:(RR-2):1-9.
20. Fishbein DB, Robinson LE. Rabies. N Engl J Med
1993;329:1632-8.
21. Centers for Disease Control and Prevention. Translocation of
coyote rabies -- Florida, 1994. MMWR 1995;44:580-1, 7.
22. Centers for Disease Control and Prevention. Human rabies --
Texas, Arkansas, and Georgia, 1991. MMWR 1991;40:765-9.
23. Smith JS, Orciari LA, Yager PA. Molecular epidemiology of
rabies in the United States. Semin Virol (in press).
24. Bourhy H, Kissi B, Lafon M, Sacramento D, Tordo N. Antigenic
and molecular characterization of bat rabies virus in Europe. J
Clin Microbiol 1992;30:2419-26.
25. Swanepoel R, Barnard BJH, Meredith CD, et al. Rabies in
southern Africa. Onderstepoort J Vet Res 1993;60:325-46.
26. King AA, Meredith CD, Thomson GR. The biology of southern
Africa lyssavirus variants. In: Rupprecht CE, Dietzschold B,
Koprowski H, editors. Lyssaviruses. New York: Springer-Verlag,
1994:267-96.
27. Foggin CM. Mokola virus infection in cats and a dog in
Zimbabwe. Vet Rec 1983;113:115.
28. Wiles GJ, Hill JE. Accidental aircraft transport of a bat to
Guam. J Mamm (full title) 1986;67:600-1.
29. World Health Organization Collaborating Center for Rabies
Surveillance and Research. Bat rabies cases in the Federal
Republic of Germany. World Health Organization Rabies Bulletin
Europe 1986;10:8-9.
30. Sasaki DM, Middleton CR, Sawa TR, Christensen CC, Kobayashi
GY. Rabid bat diagnosed in Hawaii. Hawaii Med J 1992;51:181-5.
31. Nowak RM. Walkers mammals of the world. 5th ed. Baltimore:
Johns Hopkins University Press, 1991:198.
32. Debbie JG. Rabies control of terrestrial wildlife by
population reduction. In: Baer GM, editor. The natural history of
rabies. 2nd ed. Boca Raton, FL: CRC Press, 1991:477-84.
33. World Health Organization. Oral immunization of foxes in
Europe in 1994. Wkly Epidemiol Rec 1995;70:89-91.
34. Baer GM. Oral rabies vaccination: an overview. Rev Infect Dis
1988;10 (Suppl 4):S644-8.
35. Rupprecht CE, Dietzschold B, Cox JH, Schneider L. Oral
vaccination of raccoons (Procyon lotor) with an attenuated
(SAD-B19) rabies virus vaccine. J Wildl Dis 1989;25:548-54.
36. Rupprecht CE, Charlton KM, Artois M, et al. Ineffectiveness
and comparative pathogenicity of attenuated rabies virus vaccines
for the striped skunk (Mephitis mephitis). J Wildl Dis
1990;26:99-102.
37. Wiktor TJ, Macfarlan RI, Reagan KJ, et al. Protection from
rabies by a vaccinia virus recombinant containing the rabies
virus glycoprotein gene. Proc Natl Acad Sci USA 1984;81:7194-8.
38. Rupprecht CE, Hanlon CA, Hamir AN, Koprowski H. Oral wildlife
rabies vaccination: development of a recombinant virus vaccine.
Transactions of the North American Wildlife Natural Resources
Conference 1992;57:439-52.
39. Rupprecht CE, Hamir AN, Johnston DH, Koprowski H. Efficacy of
a vaccinia-rabies glycoprotein recombinant virus vaccine in
raccoons (Procyon lotor). Rev Infect Dis 1988;10 (4
Suppl):S803-9.
40. Aubert MFA, Masson E, Artois M, Barrat J. Oral wildlife
rabies vaccination field trials in Europe, with recent emphasis
on France. In: Rupprecht CE, Dietzschold B, Koprowski H, editors.
Lyssaviruses. New York: Springer-Verlag, 1995:219-44.
41. Hanlon CA, Hayes DE, Hamir AN, et al. Proposed field
evaluation of a rabies recombinant vaccine for raccoons Procyon
lotor: site selection target species characteristics and placebo
baiting trials. J Wildl Dis 1989;4:555-67.
42. Hanlon CA, Buchanan JR, Nelson E, et al. A vaccinia-vectored
rabies vaccine field trial:ante- and post-mortem biomarkers. Rev
Sci Tech 1993;99-107.
43. Rupprecht CE, Hanlon CA, Niezgoda M, Buchanan JR, Diehl D,
Koprowski H. Recombinant rabies vaccines: efficacy assessment in
free-ranging animals. Onderstepoort J Vet Res 1993;60:463-8.
44. Roscoe DE, Holste W, Niezgoda M, Rupprecht CE. Efficacy of
the V-RG oral rabies vaccine in blocking epizootic raccoon
rabies. Presented at the 5th Annual International Meeting of
Rabies in the Americas, Niagara Falls, Ontario, Canada, 1994,
Abstract, p.33.
45. Robbins AH, Niezgoda M, Levine S, et al. Oral rabies
vaccination of raccoons (Procyon lotor) on the Cape Cod Isthmus,
Massachusetts. Presented at the 5th Annual International Meeting
of Rabies in the Americas, Niagara Falls, Ontario, Canada, 1994;
Abstract, p. 29.
46. Hanlon CA, Trimarchi C, Harris-Valente K, Debbie JG. Raccoon
rabies in New York State: epizootiology, economics, and control.
Presented at the 5th Annual International Meeting of Rabies in
the Americas, Niagara Falls, Ontario, Canada, 1994; Abstract,
p.16.




(Pages 115-23)

Diagnosis of Tuberculosis in Children: Increased Need for Better
Methods

Ejaz A. Khan, M.D., and Jeffrey R. Starke, M.D.
Baylor College of Medicine, Houston, Texas, USA

ABSTRACT

In the last decade tuberculosis (TB) has reemerged as a major
worldwide public health hazard with increasing incidence among
adults and children. Although cases among children represent a
small percentage of all TB cases, infected children are a
reservoir from which many adult cases will arise. TB diagnosis in
children usually follows discovery of a case in an adult, and
relies on tuberculin skin testing, chest radiograph, and clinical
signs and symptoms. However, clinical symptoms are nonspecific,
skin testing and chest radiographs can be difficult to interpret,
and routine laboratory tests are not helpful. Although more rapid
and sensitive laboratory testing, which takes into account recent
advances in molecular biology, immunology, and chromatography, is
being developed, the results for children have been
disappointing. Better techniques would especially benefit
children and infants in whom early diagnosis is imperative for
preventing progressive TB.


Despite the availability of effective preventive measures and
chemotherapy, the prevalence of tuberculosis (TB) is increasing
in the developing world and in much of the industrialized world
as well (1-4). According to World Health Organization (WHO)
estimates, in 1990 there were 8 million new cases of TB and 3
million deaths due to the disease worldwide; 1.3 million new
cases and 450,000 deaths were among children under 15 years of
age (5). WHO projects that 90 million new cases and 30 million
deaths -- including 4.5 million deaths among children -- will occur in
the 1990s (6,7). In developing countries, the risk for TB
infection and disease is relatively uniform in the population;
annual rates of infection often exceed 2% (5,6). In
industrialized countries, risk is more uneven and depends on the
individual's past or present activities and exposure to persons
at high risk for the disease (Table 1). From 1987 to 1991, the
number of TB cases among children under 5 years of age in the
United States increased by 49% from 674 cases to 1006 (8).
Although cases among children represent a small percentage of all
TB cases, infected children are a reservoir from which many adult
cases will arise. The risk for infection by Mycobacterium
tuberculosis among children depends primarily on the level of
risk of developing infectious TB for the adults in their
immediate environment, especially their household. Because most
current diagnostic tests for TB infection and disease have low
specificity and therefore low positive predictive values,
epidemiologic investigation continues to be important in
establishing the diagnosis of TB in children. In industrialized
countries, clinicians and public health professionals in TB
services must always ask: Has the child been exposed to an adult
with infectious pulmonary TB? 

Table 1. Persons at high risk for Mycobacterium  tuberculosis
infection in industrialized countries
 
Persons likely to be exposed to or become infected with M.
tuberculosis

     Close contacts of a person with infectious tuberculosis (TB)

     Foreign-born persons from high-incidence areas (e.g., Asia, 
     Africa, Latin America)

     The elderly

     Residents of long-term care facilities (e.g., correctional 
     facilities and nursing homes)

     Persons who inject drugs

     Other groups identified locally as having increased
     prevalence of TB (e.g., migrant farm workers or homeless     
     persons)

     Persons who may have occupational exposure to TB

Persons at high risk of developing TB disease once infected

     Persons recently infected with M. tuberculosis (within 
     the past 2 years)

     HIV-infected persons

     Persons with immunosuppressing conditions or medication use

     Persons with a history of inadequately treated TB

     Infants


Natural History of TB in Children

The natural history of TB in children follows a continuum;
however, it is useful to consider three basic stages: exposure,
infection, and disease (1). Exposure implies that the child has
had recent and substantial contact with an adult or adolescent
who has suspected or confirmed contagious pulmonary TB (a source
case). Exposed children are usually identified during followup
investigations for persons with suspected pulmonary TB by public
health workers (9); the child's tuberculin skin test (TST) is
nonreactive, the results of the chest radiograph are normal, and
the child is free of physical signs or symptoms of TB. Some -
exposed children are infected with M. tuberculosis. The clinician
cannot know immediately which exposed children are infected
because the development of delayed-type hypersensitivity to
tuberculin may take up to 3 months. Unfortunately, in children
under 5 years of age, severe TB -- especially meningeal and
disseminated disease -- can occur in fewer than 3 months, before the
TST becomes reactive (10). Young children in the exposure stage
should receive chemotherapy, usually isoniazid, until infection
can been excluded.

TB infection is first signaled by a reactive Mantoux TST. In this
stage, there are no signs or symptoms, and the results of the
chest radiograph are either normal or show only fibrotic lesions
or calcifications in the lung parenchyma or regional lymph nodes.
In developing countries, TB infection is rarely discovered and
almost never treated. In most industrialized countries, children
with a positive TST receive isoniazid for 6 to 12 months.

TB disease occurs when signs and symptoms or radiographic
manifestations caused by M. tuberculosis appear. Radiographic
abnormalities and clinical manifestations in infected children
probably are influenced by the host inflammatory reaction more
than by the number of organisms. Studies show that in 40% to 50%
of infants with untreated TB infection disease develops within 1
to 2 years (11). The risk decreases to 15% among older
children. In 25% to 35% of children TB is extrapulmonary and more
difficult to confirm bacteriologically.

In adults, the distinction between TB infection and disease is
usually clear because most disease is caused by reactivation of
dormant organisms years after infection. Disease in adults is
usually accompanied by symptoms, and patients frequently are
infectious. In children, who most often have primary disease, the
interval between infection and disease can be several months to
several years, and radiographic abnormalities often are not
accompanied by symptoms; moreover, these children are rarely
infectious. The major reason for separating infection from
disease in children is that the perception affects the approach
to treatment: infection is generally treated with a single
anti-TB drug, whereas active disease is treated with two or more
drugs. The rationale for the difference in treatment is that the
likelihood of emergence of resistance to a drug increases as the
bacillary population increases (3). This distinction is somewhat
artificial in children since infection and primary disease are
parts of a continuum. Because anti-TB medications are well
tolerated by children and are relatively inexpensive in
industrialized countries, the usual paradigm of infection and
disease encourages overtreatment rather than undertreatment.
Asymptomatic lymphadenopathy and mild lung parenchymal changes
are labeled and treated as disease.

When evaluating new diagnostic tests the basic differences
between the pathophysiology of TB in adults and children should
be considered. Among children with recent TB infection, active
multiplication of mycobacteria occurs with or without the
presence of radiographic abnormalities or clinical symptoms. For
example, gastric aspirate cultures yield M. tuberculosis from a
small proportion of recently infected children with normal chest
radiographs. One can anticipate that most diagnostic tests
designed to detect M. tuberculosis in adults with TB disease will
be positive in some proportion of children who have what is
usually called TB infection. It will take careful consideration
and investigation to determine if and how the results of these
new tests should influence the definitions and treatment of TB
infection and disease in children.

Established Diagnostic Methods

Tuberculin Skin Test (TST)

The Mantoux TST, which uses five tuberculin units of purified
protein derivative, is the standard method for detecting
infection by M. tuberculosis. The reaction is measured as
millimeters of induration after 48 to 72 hours. Since TST is the
only way to determine asymptomatic infection by M. tuberculosis,
the false-negative rate cannot be calculated. A negative TST does
not rule out TB disease in a child. Approximately 10% of
otherwise normal children with culture-proven TB do not react to
tuberculin initially (12,13). Most of these children have
reactive skin tests during treatment, which suggests that TB
disease contributed to the immunosuppression. In most cases, the
anergy occurs to all antigens, but in some cases, reactions to
tuberculin are negative but reactions to other antigens remain
positive (13). The rate of false-negative TST is higher in those
who are tested soon after becoming infected with severe TB; in
children with debilitating or immunosuppressive illnesses,
malnutrition, or viral and certain bacterial infections; in
infants; and if poor technique is used (1,14). The rate of
false-negative TST in children with TB who are infected with
human immunodeficiency virus (HIV) is unknown, but it is
certainly higher than 10%.

False-positive reactions to TST are often attributed to
asymptomatic infection by environmental nontuberculous
mycobacteria (NTM). Vaccination with M. bovis can cause transient
reactivity to a subsequent TST, but the association is weaker
than commonly recognized. Most 80% to 90% in several
studies -- children who received BCG as infants have a nonreactive
TST at 5 years of age (15-17). Among older children or
adolescents who receive BCG, most develop a reactive skin test
initially; however, by 10 to 15 years postvaccination, 80% to 90%
have lost tuberculin reactivity (18,19). Skin test reactivity can
be boosted, probably by antigenic stimulation, by serial testing
in many children and adults who received BCG (20). Various
factors determine the TST reaction size after receipt of BCG (1).
Many recipients of BCG have a reactive TST because they are
infected with M. tuberculosis and are at risk for disease,
especially if they have had recent contact with an infectious TB
patient (18). In general, TST reaction should be interpreted in
the same manner for persons who have received BCG (20) and for
unvaccinated persons.

The relatively low sensitivity and specificity of TST make the
test very useful for persons at high risk for TB infection or
disease but undesirable for use in persons at low risk (21,22).
The predictive values of TST can be improved by varying the size
of induration considered positive according to epidemiologic risk
factors for infection (Table 2). However, most of even 15-mm
reactions in children at low risk are false-positive results, and
testing of persons at low risk should be discouraged. Although
the scheme in Table 2 is scientifically and mathematically valid,
it assumes that the clinician and family are willing and able to
develop an accurate history for TB risk factors for children and
adults in their environments.

Table 2. Cut-off size of reactive area for a positive Mantoux 
tuberculin reaction                          

5 mm                   10 mm                     15 mm

Persons who had         Foreign-born persons       No risk
  contact with            from high-prevalence     factors
  infectious persons      countries

Persons with an         Residents of prisons, 
  abnormal chest          nursing homes,
  radiograph              institutions

HIV-infected and        Persons who inject
  other immuno-           drugs
  suppressed persons
                        Persons with other 
                          medical risk factors

                        Health-care workers

                        Locally identified 
                          populations at high risk

                        Children in contact with 
                          adults at high risk

                        Infants

Clinical Signs and Symptoms

Two scenarios lead the clinician to suspect that a child has TB
disease. The first occurs when TB is considered during the
differential diagnosis for an ill child. This is a common
scenario in the developing world but is less common in developed
countries. Infants are more likely to be symptomatic than older
children with pulmonary TB. The most common symptoms are cough,
fever, wheezing, and failure to gain weight (13). Clinical signs
are surprisingly meager, but rales and wheezes over the affected
lung field are most common. Signs and symptoms of extrapulmonary
TB are referable to the involved organ. The sensitivity and
specificity of signs and symptoms are extremely low and can lead
to both overdiagnosis and underdiagnosis when radiographs and
other tests are not available.

The second scenario occurs when evaluating a child who has had
significant contact with an adult with suspected or confirmed TB.
Usually the TST is applied first and is reactive. A subsequent
chest radiograph or physical examination leads to discovery of
early disease. The child is usually relatively asymptomatic. In
the United States, about 50% of childhood TB cases are discovered
in this manner (13). 

Radiologic Studies

Evidence of pulmonary TB in chest radiographs varies (23,24), but
usually radiographs show enlargement of hilar, mediastinal, or
subcarinal lymph nodes and lung parenchymal changes (Figure 1 cannot 
be viewed in ASCII format). Most of the radiographic abnormalities 
are caused by a combination of lung disease and the mechanical 
changes induced by partial or complete airway obstruction resulting 
from enlarging intrathoracic nodes. The most common findings are 
segmental hyperinflation, then atelectasis, alveolar consolidation,
interstitial densities, pleural effusion, and, rarely, a focal
mass. Cavitation is rare in young children but is more common in
adolescents, who may develop reactivation disease similar to that
seen in adults.

The development of radiographic techniques, such as computed
tomography (CT) scanning, illustrates some of the issues that
arise when newer and more sensitive diagnostic tests become
available (24). A CT scan may show enlarged or prominent
mediastinal or hilar lymph nodes in some children with recent TB
infection and a normal chest radiograph (25). In the absence of a
CT scan, the child's disease stage would be called TB infection,
and single drug therapy would be used. Many studies, involving
thousands of children have shown this treatment to be successful.
However, when the CT scan shows mild adenopathy, the clinician
may consider this finding indicative of TB disease and treat with
several drugs, although this probably is not necessary in the
absence of drug resistance. These findings reinforce the idea
that pediatric TB is a continuum, and the distinction between 
infectionand disease is somewhat artificial. There is no current
role for the CT scan in the evaluation of the asymptomatic
TB-infected child with a normal chest radiograph. This scan can
be helpful in selected cases to demonstrate endobronchial
disease, pericardial invasion, early cavitation, and
bronchiectasis resulting from pulmonary TB when the chest
radiograph is abnormal but the pathologic process is not clear.

Mycobacterial Detection and Isolation

Despite recent advances, early mycobacteriologic diagnosis of TB
still relies primarily on examination of acid-fast-stained smears
from clinical specimens. It is the easiest, least expensive, and
most rapid procedure for obtaining preliminary information.
However, children under 12 years of age with pulmonary TB rarely
produce sputum and are usually unable to expectorate voluntarily.
When sputum samples cannot be obtained, gastric aspirate samples
are used for detection and isolation of M. tuberculosis. Even
though an acid-fast bacilli (AFB) stain of sputum is positive in
up to 75% of adults with pulmonary TB, fewer than 20% of children
with TB have a positive AFB smear of sputum or gastric aspirate
(26,27). The newer fluorochrome stains, such as auramine and
rhodamine, are superior to classic carbolfuchsin stains (28). The
rates of positive AFB stain from body fluids and tissues in
children with extrapulmonary TB also are low, and false-positive
results caused by NTM disease are common, especially in cervical
lymph nodes.

For most children with pulmonary TB, culture confirmation is not
needed. Diagnosis is made on the basis of a positive TST,
clinical and radiographic findings suggestive of TB, and history
of contact with an adult source case. The drug-susceptibility
test results from the source case isolate can be used to design
the optimal treatment for the child. However, cultures should be
obtained from the child if the source patient is unknown or has a
drug-resistant organism and if the child is immunocompromised or
has extrapulmonary TB.

The best specimen for culture from children with suspected
pulmonary TB is the early morning gastric aspirate obtained in
the hospital by using a nasogastric tube before the child arises
and peristalsis empties the stomach of the respiratory secretions
swallowed overnight (29,30). Three consecutive morning gastric
aspirates yield M. tuberculosis in only 30% to 50% of cases,
although the yield from infants is as high as 70% (14). The
culture yield from other body fluids or tissues from children
with extrapulmonary TB is usually less than 50% (13). Gastric
aspiration is inconvenient, expensive, and uncomfortable. The
culture yield from random, outpatient gastric aspirates has not
been determined recently. Therefore, this procedure cannot be
recommended but should be studied.

Bronchoscopy

The role of bronchoscopy in evaluating children for TB is
controversial. The culture yield is lower from bronchoscopy
specimens than from properly obtained gastric aspirates (29,31).
Most children do not need flexible fiberoptic bronchoscopy, but
the procedure may be useful in diagnosing endobronchial TB and
excluding other causes of pulmonary abnormality, particularly in
immunocompromised children, such as those with HIV infection in
whom other opportunistic infections may coexist with or mimic TB.
In a recent study of 36 children with pulmonary TB, bronchoscopy
showed endobronchial involvement in 42%; most (63%) of these
children had no clinical or radiographic evidence of
endobronchial TB (31). This technique may be used to determine if
a child might benefit from corticosteroid therapy, but guidelines
for making this decision have not been established.

Clinical Scoring System

TB is an enormous problem in developing countries, where about
95% of cases occur (6). Cost, technical difficulties, and lack of
resources make TB diagnosis in children very difficult in these
countries. Various clinical scoring systems have been proposed on
the basis of available information and tests (32,33) (Table 3).
Although helpful, many of these systems have low sensitivity and
specificity. However, even in industrialized countries, the triad
of a positive tuberculin skin test, an abnormal radiograph, and a
history of exposure to an adult with TB remains the most
effective method for diagnosing TB in children.

Table 3. A set of criteria for the diagnosis of pulmonary 
tuberculosis (TB) in children when culture is not available       

A. Positive acid-fast stain of sputum or gastric aspirate

   or 

B. Two or more of the following:

    History of contact with a tuberculous adult

    Cough lasting longer than 2 weeks

    A reactive tuberculin skin test

        10 mm in children without prior BCG vaccination

        15 mm in children with prior BCG vaccination

    Radiographic findings compatible with TB

    Response to anti-TB therapy (increased body weight by 10% 
     after 2 months, decrease in symptoms)


Source: ref. 33.

New Diagnostic Techniques

Polymerase Chain Reaction (PCR)

Diagnostic PCR is a technique of DNA amplification that uses
specific DNA sequences as markers for microorganisms (34). In
theory, this technique can detect a single organism in a specimen
such as sputum, gastric aspirate, pleural fluid, cerebrospinal
fluid, or blood. Recent publications show that various PCR
techniques, most using the mycobacterial insertion element IS6110
as the DNA marker for M. tuberculosis-complex organisms, have a
sensitivity and specificity greater than 90% for detecting
pulmonary TB in adults (35,36). However, these tests are not
performed correctly in all clinical laboratories (36) and may
offer little advantage over high-quality microscopic examination
of sputum (34). The cost involved and the need for sophisticated
equipment and scrupulous technique to avoid cross-contamination
of specimens preclude the use of PCR techniques in many
developing countries. PCR may have a special role in the
diagnosis of extrapulmonary TB and pulmonary TB in children since
sputum smears are usually unrevealing in these cases.

Use of PCR for detecting M. tuberculosis in children has not been
evaluated extensively. Pierre et al. (37) used an IS6110-based
PCR to detect M. tuberculosis in gastric aspirate samples from 22
children with pulmonary TB. They found that 15 (25%) of 59
samples were positive; however, testing multiple samples or
testing samples at least twice improved the sensitivity. When
three samples from the same patient were tested two times each,
two or more positive results were obtained from 9 of 15 children
with TB, but from 0 of 17 controls. However, 2 of 65 single
samples from controls were positive by PCR. Using an IS6110-based
PCR assay, Starke et al. (38) tested gastric aspirates from 35
hospitalized children with pulmonary TB and 30 controls to detect
M. tuberculosis. When compared with the clinical diagnosis, PCR
had a sensitivity of 40% and specificity of 80%. Six controls had
false-positive PCR results; one had a recent TB infection, two
had NTM disease, and three had conditions unrelated to
mycobacterial infection. Delacourt et al. (39) studied 199
specimens from 68 children with suspected TB. An IS6110-based PCR
identified M. tuberculosis in clinical samples from 83% of
children with disease compared to the low yield from positive AFB
smears (21%) and positive cultures (42%) (39). PCR identified 70%
of children with clinical pulmonary TB but no other microbiologic
proof of the infection. However, 39% of children with infection
but no radiographic or clinical disease also had positive PCR
results. These results again demonstrate the arbitrariness of the
distinction between TB infection and disease in children.

It appears that PCR may have a useful but limited place in
evaluating children for TB. A negative PCR result never
eliminates TB as a diagnostic possibility, and a positive result
does not confirm it. PCR's major use will be in evaluating
children with significant pulmonary disease, when the diagnosis
is not easily established by clinical or epidemiologic grounds.
PCR may be particularly helpful in evaluating immunocompromised
children with pulmonary disease, although published reports of
PCR performance in such children are lacking. PCR may also aid in
establishing the diagnosis of extrapulmonary TB, though only rare
case reports have been published. However, performing PCR on
gastric aspirates is not a useful test to distinguish between TB
infection and disease and should not be used for children with
normal chest radiographs.

Serology and Antigen Detection

Despite dozens of studies published over the past several
decades, serology has found little place in the routine diagnosis
of TB in adults or children. Several recent studies have used the
enzyme-linked immunosorbent assay (ELISA) to detect antibodies to
various purified or complex antigens of M. tuberculosis in
children. Rosen (40) used mycobacterial sonicates in an ELISA on
samples from 31 children with clinical TB and found a sensitivity
of 26% and a specificity of 40%. This ELISA was influenced by
recent BCG vaccination in children under 5 years of age. Barrera
et al. (41) used an ELISA that detects antibodies to purified
protein derivative and found a sensitivity of 51% for
culture-positive pulmonary TB cases in children, but the
sensitivity was only 28% for the clinical cases. Hussey et al.
(42) used an autoclaved suspension of M. tuberculosis to detect
antibodies in serum from 132 children with clinical pulmonary TB;
the test was 62% sensitive and 98% specific. Higher sensitivity
was obtained among patients with positive culture results (69%, n
= 35), miliary TB (100%, n = 6), tuberculous meningitis (80%, n 
= 15), and pleural effusion (78%, n = 16). No correlation was
observed with the tuberculin skin-test result, BCG vaccination,
or nutritional status whereas duration of therapy, increasing age
and chronicity of infection were positively correlated. Delacourt
et al. (43) used an ELISA to detect IgG and IgM antibodies
directed against mycobacterial antigen A60 in children with TB.
At a chosen specificity of 98%, IgG was detected in 68% of
children with clinical disease when results were highly
controlled for age and prior BCG vaccination. IgM detection had
only a 19% sensitivity. However, using the same anti-A60 ELISA at
a defined specificity of 95%, Turneer et al. (44) found the IgG
sensitivity to be 26% for past TB, 6% for asymptomatic primary
TB, 14% for symptomatic TB, and 9% for NTM adenitis. No available
serodiagnostic test for TB has adequate sensitivity, specificity,
or reproducibility under various clinical conditions to be useful
for diagnosing TB in children.

Mycobacterial antigen detection has been evaluated in clinical
samples from adults, but rarely from children (45,46). Two recent
assays detecting M. tuberculosis-specific antigens yielded high
sensitivity and specificity in various clinical specimens from
adults with TB (47,48). Measurement of tuberculostearic acid, a
mycobacterial mycolic acid, has been used to detect M.
tuberculosis in clinical specimens (49). Brooks et al. (50)
demonstrated a sensitivity of 95% and specificity of 91% when
chromatographic profile of carboxylic acids and detection of
tuberculostearic acid were combined and compared with culture
results and clinical findings in adults with pulmonary TB;
however, these techniques require technically advanced equipment
and expertise, which are not available where TB in children is
most common. Their sensitivity and specificity in children are
unknown.

Implications for HIV Infection and Drug Resistance

The resurgence of TB over the last decade has coincided with the
HIV pandemic. HIV-infected infants and children are in close
contact with their caregivers, who may be infected with HIV and
M. tuberculosis and are at high risk of developing infectious TB
as they become immunocompromised. None of the available
diagnostic tests for TB infection or disease in children has been
evaluated systematically in children with HIV infection and
pulmonary disease or suspected TB. In adults with HIV infection
and TB, the sensitivity of diagnostic tests that rely on the host
immune response, such as the TST or serology, is much lower than
in nonimmunocompromised TB patients. It is likely that the tests'
sensitivity also will be lower in HIV-infected children with TB.
Tests that directly detect M. tuberculosis, such as PCR or
antigen detection assays, may be particularly important for
HIV-infected children. The culture yield of M. tuberculosis from
children with HIV infection and TB is unknown but appears to be
similar to that from non-HIV-infected children. The most
important diagnostic clue for detecting TB in HIV-infected
children is a history of contact with an adult who has infectious
TB. Since TB may not have yet been diagnosed in this adult, a
rapid and aggressive evaluation for TB in adults who care for the
child is a critical part of the evaluation of the child.

The current prevalence of drug resistance among M. tuberculosis
isolates in the United States is 8% to 14% (51, 52). Drug
resistance is most common in patients who received treatment, are
not responding to therapy, do not adhere to treatment, live in
developing countries, are immunocompromised, are prisoners, are
homeless, or are children exposed to adults at increased risk for
drug resistance. Drug-resistant TB has increased significantly
among children (52). Because of low culture yields from children
with TB, the clinician must often rely on the antimicrobial
susceptibility results for the M. tuberculosis isolate obtained
from the adult source case who presumably infected the child.
This again emphasizes the crucial need to identify and evaluate
the source case for every child with TB. The rapid identification
of drug-resistant organisms is necessary for control of
drug-resistant TB. Various new methods, such as high-performance
liquid chromatography or PCR and DNA sequence analysis, may help
to identify and test for antimicrobial susceptibility within a
few days of diagnosis, but these techniques remain experimental.

Summary

Most recently developed sensitive and specific diagnostic tests
have not found a place in the routine evaluation of children with
suspected TB. Clinical criteria, particularly skin-test results,
radiographic changes, and documented exposure to an infectious
adult remain standard diagnostic methods. In industrialized
countries, the local public health entity is a crucial partner to
the clinician in establishing the diagnosis in the child and
determining if drug resistance is present. As new diagnostic
tests are developed, they must be evaluated against clinical
criteria. The basic differences in pathophysiology of TB in
adults and children must be considered before new tests are
applied in pediatrics. It will be crucial to study the new
techniques in children and not simply extrapolate from results
for adults with TB.

Dr. Khan is a postgraduate fellow in pediatric infectious
diseases at Baylor College of Medicine, Houston, Texas. Dr.
Starke is an associate professor at Baylor College of Medicine
and current chairman of CDC's Advisory Committee for the
Elimination of Tuberculosis.

Address for correspondence: Jeffrey R. Starke, Texas Children's 
Hospital, MC 3-2371, 1102 Bates Street, Houston, tX 77030, USA;
fax 713-770-4347; e-mail jstark@msmailpo2.is5.tch.tmc.edu.


References

1. Starke JR, Correa AG. Management of mycobacterial infection
and disease in children. Pediatr Infect Dis J 1995;14:455-70.
2. Styblo K, Rouillon A. Tuberculosis in developing countries:
burden, intervention and cost. Bull Int Union Against Tuber Lung
Dis 1990;65:6-24.
3. Starke JR, Jacobs R, Jereb J. Resurgence of tuberculosis in
children. J Pediatr 1992;120:839-55.
4. Cantwell M, Snider D Jr, Cauthen G, Onorato I. Epidemiology of
tuberculosis in the United States, 1985 through 1992. JAMA
1994;272:535-9.
5. Kochi A. The global tuberculosis situation and the new control
strategy of the World Health Organization. Tuber Lung Dis
1991;72:1-6.
6. Raviglione MC, Snider DE, Kochi A. Global epidemiology of
tuberculosis. Morbidity and mortality of a worldwide epidemic.
JAMA 1995;273:220-6.
7. Dolin P, Raviglione M, Kochi A. Global tuberculosis incidence
and mortality during 1990-2000. Bull World Health Organ
1994;72:213-20.
8. Barnes PF, Borrows, SA. Tuberculosis in the 1990s. Ann Intern
Med 1993;119:400-10.
9. Hsu KHK. Contact investigation: a practical approach to
tuberculosis eradication. Am J Public Health 1963;53:1761-9.
10. Nolan RJ Jr. Childhood tuberculosis in North Carolina: a
study of the opportunities for intervention in the transmission
of tuberculosis to children. Am J Public Health 1986;76:26-30.
11. Brailey ME. Tuberculosis in white and negro children. II. The
epidemiologic aspects of the Harriet Lane study. Cambridge, MA:
Harvard University Press, 1958.
12. Steiner P, Rao M, Victoria MS, et al. Persistently negative
tuberculin reactions: their presence among children culture
positive for M. tuberculosis. Am J Dis Child 1980;134:747-50.
13. Starke JR, Taylor-Watts KT. Tuberculosis in the pediatric
population of Houston, Texas. Pediatrics 1989;84:28-35.
14. Vallejo J, Ong LT, Starke JR. Clinical features, diagnosis
and treatment of tuberculosis in infants. Pediatrics 1994;94:1-7.
15. Lifschitz M. The value of the tuberculin skin test as a
screening test for tuberculosis among BCG-vaccinated children.
Pediatrics 1965;36:624-7.
16. Landi S, Ashley MJ, Grzybowski S. Tuberculin sensitivity
following the intradermal and puncture methods of BCG
vaccination. Can Med Assoc J 1967;97:222-5.
17. Joncas JH, Robitaille R, Gauthier T. Interpretation of the
PPD skin test in BCG-vaccinated children. Can Med Assoc J
1975;113:127-8.
18. Johnson H, Lee B, Kelly E, McDonnell T. Tuberculin
sensitivity and the BCG scar in tuberculosis contacts. Tuber Lung
Dis 1995;35:113-7.
19. Menzies R, Vissandjee B. Effect of bacille Calmette-Guerin
vaccination on tuberculin reactivity. Am Rev Respir Dis
1992;141:621-5.
20. Sepulveda RL, Burr C, Ferrer X, Sorensen RU. Booster effect
of tuberculin testing in healthy 6-year-old school children
vaccinated with bacille Calmette-Guerin at birth in Santiago,
Chile. Pediatr Infect Dis J 1988;7:578-82.
21. American Thoracic Society. Diagnostic standards and
classification of tuberculosis. Am Rev Respir Dis
1990;142:725-35.
22. American Academy of Pediatrics Committee on Infectious
Diseases. Screening for tuberculosis in infants and children.
Pediatrics 1994;93:131-4.
23. Schaaf HS, Beyers N, Gie RP, et al. Respiratory tuberculosis
in childhood: the diagnostic value of clinical features and
special investigations. Pediatr Infect Dis J 1995;14:189-94.
24. Parisi MT, Jensen MC, Wood BP. Pictorial review of the usual
and unusual roentgen manifestations of childhood tuberculosis.
Clin Imag 1994;18:149-54.
25. Delacourt C, Mani TM, Bonnerot V, et al. Computed tomography
with normal chest radiograph in tuberculous infection. Arch Dis
Child 1993;69:430-2.
26. Strumpf IJ, Tsang AY, Syre JW. Reevaluation of sputum
staining for the diagnosis of pulmonary tuberculosis. Am Rev
Respir Dis 1979;119:599-602.
27. Lipsky BA, Bates J, Tenover FC, Plorde JJ. Factors affecting
the clinical value of microscopy for acid-fast bacilli. Rev
Infect Dis 1984;6:214-22.
28. Kent PT, Kubica GP. Public health mycobacteriology   a guide
for the level III laboratory. Atlanta, GA; Centers for Disease
Control, 1985.
29. Abadco DL, Steiner P. Gastric lavage is better than
bronchioalveolar lavage for isolation of Mycobacterium
tuberculosis in childhood tuberculosis. Pediatr Infect Dis J
1992;11:735-8.
30. Carr DT, Karlson AG, Stillwell AA. A comparison of cultures
of induced sputum and gastric washings in the diagnosis of
tuberculosis. Mayo Clinic Proc 1967;42:23-5.
31. Chan S, Abadco DL, Steiner P. Role of flexible fiberoptic
bronchoscopy in the diagnosis of childhood endobronchial
tuberculosis. Pediatr Infect Dis J 1994;13:506-9.
32. Glidey Y, Hable D. Tuberculosis in childhood: an analysis of
412 cases. Ethiop Med J 1983;21:161-7.
33. Migliori AB, Borghesi A, Rossanigo P et al. Proposal for an
improved score method for the diagnosis of pulmonary tuberculosis
in childhood in developing countries. Tuber Lung Dis
1992;73:145-9.
34. Schluger NW, Rom WN. Current approaches to the diagnosis of
active pulmonary tuberculosis. Am J Respir Crit Care Med
1994;149:264-7.
35. Eisenach KD, Sifford MD, Cane MD, Bates JH, Crawford JT.
Detection of Mycobacterium tuberculosis in sputum samples using a
polymerase chain reaction. Am Rev Respir Dis 1991;144:1160-3.
36. Noordhock A, Kolk A, Bjune G, et al. Sensitivity and
specificity of polymerase chain reaction for detection of
Mycobacterium tuberculosis: a blind comparison study among seven
laboratories. J Clin Microbiol 1994;32:277-84.
37. Pierre C, Oliver C, Lecossier D, Bousssougant Y, Yemi P,
Hance AJ. Diagnosis of primary tuberculosis in children by
amplification and detection of mycobacterial DNA. Am Rev Respir
Dis 1993;147:420-4.
38. Starke JR, Ong LT, Eisenach KD, et al. Detection of M.
tuberculosis in gastric aspirate samples from children using
polymerase chain reaction. Am Rev Resp Dis 1993;147(Suppl):A801.
39. Delacourt C, Poveda J-D, Churean C, et al. Use of polymerase
chain reaction for improved diagnosis of tuberculosis in
children. J Pediatr 1995;126:703-9.
40. Rosen EU. The diagnostic value of an enzyme-linked
immunosorbent assay using adsorbed mycobacterial sonicates in
children. Tubercle 1990;71:127-30.
41. Barrera L, Miceli I, Ritacco V, et al. Detection of
circulating antibodies to purified protein derivative by
enzyme-linked immunosorbent assay: its potential for the rapid
diagnosis of tuberculosis. Pediatr Infect Dis J 1989;8:763-7.
42. Hussey G, Kibel M, Dempster W. The serodiagnosis of
tuberculosis in children: an evaluation of an ELISA test using
IgG antibodies to M. tuberculosis, strain H37RV. Ann Trop
Paediatr 1991;11:113-8.
43. Delacourt C, Gobin J, Gaillard J-L, de Blic J, Veran M,
Scheinmann P. Value of ELISA using antigen 60 for the diagnosis
of tuberculosis in children. Chest 1993;104:393-8.
44. Turneer M, Nerom EV, Nyabenda J, Waelbroeck A, Duvivier A,
Toppet M. Determination of humoral immunoglobulins M and G
directed against mycobacterial antigen 60 failed to diagnose
primary tuberculosis and mycobacterial adenitis in children. Am J
Respir Crit Care Med 1994;150:1508-12.
45. Sada E, Ruiz-Palacios AM, Lopez-Vidal Y, et al. Detection of
mycobacterial antigens in cerebrospinal fluid of patients with
tuberculous meningitis by enzyme-linked immunosorbent assay.
Lancet 1983;2:651-2.
46. Radhakrishnan VV, Sehgal S, Mathai A. Correlation between
culture of Mycobacterium tuberculosis and detection of
mycobacterial antigens in cerebrospinal fluid of patients with
tuberculous meningitis. J Med Microbiol 1990;33:223-6.
47. Wadee AA, Boling L, Reddy SG. Antigen capture assay for
detection of a 43-kilodalton Mycobacterium tuberculosis antigen.
J Clin Microbiol 1990;28:2786-91.
48. Sada E, Aguilar D, Torres M, et al. Detection of
lipoarabinomannan as a diagnostic test for tuberculosis. J Clin
Microbiol 1992;30:2415-18.
49. Brooks JB, Daneshvar MI, Fast DM, et al. Selective procedures
for detecting femtomole quantities of tuberculostearic acid in
serum and cerebrospinal fluid by frequency-pulsed
electron-capture gas-liquid chromatograph. J Clin Microbiol
1987;25:1201-6.
50. Brooks JB, Daneshvar MI, Harberger RL, et al. Rapid diagnosis
of tuberculous meningitis by frequency-pulsed electron-captive
gas-liquid chromatography detection of carboxylic acids in
cerebrospinal fluid. J Clin Microbiol 1990;28:989-97.
51. Centers for Disease Control and Prevention. National action
plan to combat multidrug-resistant tuberculosis. MMWR
1992;41:5-50.
52. Bloch AB, Cauthen GM, Onorato IM, et al. Nationwide survey of
drug-resistant tuberculosis in the United States. JAMA
1994;271:665-71.



(Pages 124-8)

Data Management Issues for Emerging Diseases and New Tools for
Managing Surveillance and Laboratory Data

Stanley M. Martin, M.S., Nancy H. Bean, Ph.D.
Centers for Disease Control and Prevention, Atlanta, Georgia, USA

Data Management Issues for Emerging Diseases

Since 1976, when Legionnaires' disease affected attendees at the
American Legion Convention in Philadelphia (1), the scope of
public health has expanded. During the 1976 outbreak
investigation, public attention was drawn to news accounts of the
increasing numbers of cases and deaths as well as to speculations
about diseases causes and prevention. After the outbreak, public
health officials contended with volumes of information, including
clinical data, epidemiologic survey results, and records of
specimens collected from patients and the environment. This
information was managed on mainframe computers.

In 1980, a cluster of cases of unrecognized illness, primarily
affecting young women, created a data management situation
similar to that surrounding the Legionnaires' disease outbreak. A
major epidemiologic investigation, which included examining a
multitude of laboratory specimens and analyzing volumes of data,
was undertaken by a large team of federal, state, and local
public health officials, as well as numerous academic
institutions and private industries. The problems with
establishing databases and implementing a data management system
for toxic shock syndrome (2) were essentially the same as the
data management problems of Legionnaires' disease, except that
computer technology had crept forward slightly in public health
offices.

During the spring of 1993, a cluster of cases of another unknown
illness, eventually attributed to hantavirus (3), occurred in the
southwestern United States. The reaction to this unknown disease
by public health officials reflected a startling fact: even
though the epidemiologic and laboratory methods for curtailing
the outbreak were in place, a consistent data management strategy
had not been established. Ad hoc databases built by outbreak
investigators for a multitude of purposes began to bog down the
investigation. Cases were recorded in multiple databases that did
not recognize duplicate reports of cases. Updates of data about
cases were done in some, but not all, databases. Laboratory data
about specimens from patients were not linked to other clinical
and epidemiologic data about a patient. No single database was
available with well-edited, complete data about all the cases.
Parallel, fragmented data management efforts evolved in at least
15 locations, with no coordinated mechanism to integrate them
into one system.

Introducing a single system for data management in the midst of
the hantavirus outbreak involved more than the data management
issues encountered in the earlier outbreaks. Previously, computer
technology was viewed as a solution that, although somewhat
cumbersome, enabled officials to move from data management by
hand to electronic management. However, during the hantavirus
outbreak, computer technology became part of the problem; it
initially prevented good data management and may have hindered
some of the laboratory and epidemiologic efforts to control the
outbreak. Data were essentially being locked into various
databases and could not be adequately analyzed or merged with
data in other databases. In some instances, this peculiar
circumstance caused investigators to perform analyses by hand
using printouts from electronic databases or entering data again
into other systems.

In recent years, legal considerations, such as the Privacy Act
enacted in 1974 and the Freedom of Information Act enacted in
1966 (4,5), have also complicated data management. These acts, in
their efforts to protect individual privacy and ensure
availability of data, have in some cases, constrained public
health responses to emergency situations and subsequent
surveillance efforts by enforcing strict database design and
handling requirements.

Data Management Requirements

In epidemiologic investigations, disease problems are generally
characterized by person, place, and time, whether the problem
concerns the emergence of a new disease, a change in the
resistance pattern of a known pathogen, an emergency response to
an outbreak, or a routine disease surveillance program. The
principles of data gathering, management, and analysis are
essentially the same for all these purposes. Computer systems
developed to manage data associated with these problems should be
regarded as tools for the epidemiologic characterization of
pathogens, syndromes, cases, and risk factors. Therefore,
laboratory data management and reporting systems must be able to
handle data about all of these.

The most stringent requirements for data management are imposed
by data from laboratory testing of specimens from patients, human
and nonhuman sources, and the environment. A system having a
relational data model adequate to properly handle the laboratory
data requirements will almost certainly be adequate to handle the
clinical, exposure, and demographic data requirements.

Two primary data management functions can satisfy the laboratory
data demands with multiple requirements in each function. The
first function, internal laboratory data management, consists of
entering test results and tracking specimens. The second,
surveillance, includes gathering data and moving data beyond the
electronic files of the laboratory to appropriate sites for
analysis. A data management system should be able to perform
these functions not only during an outbreak but throughout the
period of surveillance as well.

The internal laboratory function, universally similar among most
public health laboratories, includes data entry tailored for
individual laboratories at the site; retrieval/query ability; and
ability to add or delete tests, manage aliquots, share data input
in different laboratories of the site, track the status of every
specimen regardless of which laboratory tested it, develop
reports for specimen submitters, and in some cases assign costs
for laboratory tests performed and prepare invoices for
submitters. 

Requirements for the surveillance function include, in addition
to certain critical laboratory data, the following facilities: to
record clinical, exposure/risk factor, and demographic data about
patients; to include data about multiple specimens and aliquots
related to the same person, regardless of the interval separating
the specimen dates; and to change questions or test results that
are recorded for each specimen.

Although internal and surveillance functions are clearly
separate, they are not independent. Data entered into databases
for the internal function should be available without additional
effort for the surveillance function. In fact, when the internal
function is not electronic or when the internal electronic system
is inadequate, the system performing electronic surveillance
should also perform to some extent the internal functions. Good
laboratory data management does not address the internal function
at the exclusion of the surveillance function. 

If a laboratory data management system is to be useful for
emergency situations, it must provide mechanisms for adapting
quickly to the emergency situation. For example, it must provide
a way to immediately create an electronic data collection
instrument and to incorporate this new instrument into the system
at all reporting sites electronically. For the surveillance
function, these electronic features must include communications
facilities to move data electronically from one location to
another, mechanisms for sending messages or files, functions for
simple analysis, and methods for preparing and printing reports.
While some systems perform some of these functions, most systems
do not provide all of them. 

With appropriate systems in hand, data management plans for both
urgent and routine events can be approached in a sequential
fashion. With consensus among all participating investigators,
epidemiologists must decide what data (both laboratory and
epidemiologic) are needed so that data field characteristics can
be defined. Consensus should be reached in the early phase of the
outbreak investigation; otherwise participants in the
investigation will of necessity begin developing ad hoc data
management systems. The more thoroughly and carefully this task
is performed, the more stable the data will ultimately become.

In a well-designed system, the initial definitions in an
emergency situation can include projections about which data
fields will be needed. However, for routine surveillance these
can be more thoroughly planned. Thus, the data system should
allow fields to be deleted if not needed and to be added if they
become important. These modifications should 1) be handled
without having to alter the system, 2) use simple menu-driven
functions requiring no computer programmer intervention,  3) 
accomplish the changes immediately, 4) be distributed to all
investigators without disrupting their other functions during the
investigation, and 5) be incorporated automatically.

Next, all known participants in the investigation must be
identified. These should include local, state, and federal
officials as well as academic or private participants who may
provide reports to the central data repository. These
participants must be identified to the system specifically by
person and by site for system security. Appropriate state and
federal offices should be informed concerning the computer system
and the rules for its use well before an emergency occurs;
therefore, sites will be on the system in advance of an urgent
problem. However, the system must allow for additional sites to
be added quickly. In an emergency, a temporary agreement must be
drawn for all participants to cooperate with the demands of the
situation, i.e., to use a particular software system and operate
under a standard set of rules for collecting and reporting data
for the emergency. This agreement may occasionally stipulate that
participants share data temporarily in a common database for the
sake of data integrity.

Entering clinical, epidemiologic/risk factor, and laboratory data
about the same cases into the same database, rather than merging
separate databases after the data are collected, provides such
great payoffs in time savings and data integrity that the effort
to obtain cooperation for a common database during an urgent
situation is worthwhile. Although merging multiple databases
during routine surveillance is feasible, emergency situations do
not lend themselves to this type of data management. Therefore,
the system to be used for these situations must accommodate a
common database and provide a means of connecting the reporting
sites to the database. When the reporting system is activated and
data begin arriving at a central location, the system should
facilitate analysis at every reporting site and provide a
mechanism to export data (e.g., ASCII or .dbf files) for external
analysis.

Emergency situations create unusual demands for epidemiologic and
laboratory resources; therefore, data management should not
disrupt or threaten to divert resources devoted to these other
purposes. As the system is implemented, before emergencies occur,
discussions of the resources required should be held with -
participants. Participants must devote some resources to data
management, but these should be minimized. This is consistent
with implementing a single system in the beginning of the
outbreak investigation and continuing with it into the routine
surveillance follow-up. Incorporating data into a second system
for surveillance could waste resources.

Although internal data management does not need to change to
accommodate an outbreak, laboratories must implement systems that
can directly feed data into the master reporting system database,
either through an import function contained in the master system
or by a direct interface between the internal laboratory system
and the surveillance reporting system.

Data management considerations during outbreak investigations and
surveillance in the United States include the political concerns
of the participants. Political and legal constraints of all
participants must be addressed before the need to deal with them
arises. On a global scale, this consideration is equally
important, especially in countries whose economies may be
adversely affected by news of a dangerous disease situation.
Individual country sovereignty must not be violated by data
reporting, and the cooperation of each participating country or
political entity (e.g., World Health Organization [WHO], Pan
American Health Organization [PAHO]) must be obtained in an
atmosphere of confidentiality. All attempts to obtain, share, or
combine data on a regional or global basis must include a
well-defined set of rules agreed upon by all participants. For
example, data for scientific purposes might be received at an
office of  WHO or PAHO but not sent beyond these organizations.

Most often, for the sake of surveillance on a regional or global
scale, data management considerations must focus first on
establishing in-country data management infrastructures. This
means that regional or global surveillance will first translate
into establishing a master system, or at least compatible systems
in individual participating countries. In most cases, data
management systems available to developing countries do not
provide the relational model needed by the laboratory. Therefore,
efforts should be initiated to introduce and establish systems
that can meet these needs in countries desiring to use them.

A plan for regional or global surveillance must include tools to
respond to outbreaks and provide for the computing equipment and
modems or other means of transmitting the data electronically.
Today's environment demands that most data management be done on
personal computers located at critical sites where data can be
input. However, data volume may ultimately require that the
system provide for archiving data onto another medium. This does
not preclude the use of personal computers for data management
but simply recognizes that current technology limits the volume
of data that can practically be managed and analyzed on personal
computers.

The initial data management plan for a country should include a
section on reporting procedures and the appropriate medium for
archiving data. To handle an immediate, urgent situation the
system should contain, at a minimum, a personal computer with
large hard-disk capacity (at least 1-2 gigabytes at the central
level and possibly 300-500 megabytes at each reporting site),
large memory (at least 4 megabytes of RAM at every reporting
site), adequate speed (at least 33 megahertz at every reporting
site), and fast modems if appropriate. For sites located in areas
with inadequate telephone lines, other provisions for electronic
transmissions should be planned (e.g., diskettes). Until security
can be assured on the Internet, we do not recommend using this
medium for electronic transmission of laboratory clinical data
for outbreak investigations and surveillance.

New Tools for the Management of Surveillance and Laboratory Data

The Public Health Laboratory Information System (PHLIS)

To address the need for a data management system for outbreak
investigations and surveillance, the National Center for
Infectious Diseases, CDC, in cooperation with the Association of
State and Territorial Public Health Laboratory Directors in the
United States, developed PHLIS. With this system, data entry
screens (modules) are created and distributed to all reporting
sites electronically, and data are input and reported within
hours, without involving computer programmers. PHLIS provides the
capacity for a hierarchical reporting scheme involving reports to
multiple, successively higher reporting levels; a database is
created at every reporting level so that all data reported to a
site or input at the site are included in the database at that
site.

The most recent version of PHLIS (Version 3.0), is a menu-driven
system based on a relational data model sufficient for the needs
outlined in the first part of this report. The system allows for
a patient record to be input only one time and links multiple
specimens for that patient record. This is true even if specimens
for the same patient are entered in different disease modules, or
if the patient's name is to be added into a module that contains
only epidemiologic data (no laboratory specimens). PHLIS provides
a core set of data to be collected on every patient. In addition,
each disease module can be customized by adding additional fields
to the core data if needed. The system can accommodate data for
epidemiologic, laboratory, survey, and case-control studies, and
for other public health needs.

Field staff can rapidly add their own data fields to existing
disease modules to customize the data entry for special needs at
each data reporting site. During an outbreak, a new module can be
rapidly developed and electronically transmitted to all
participating reporting sites.

The system, which includes data communication software, is
configured so that data flow in a pyramid reporting structure:
that is, data are reported from lower level reporting sites
through higher level reporting sites and ultimately to a single
central site. As data are passed to each successively higher
level, they are automatically assimilated into that site's
database. Thus, databases are built and updated at successively
higher reporting sites. Additional information about a case or
specimen may be added at any reporting site; if desired, these
additional data are also transmitted to the next higher reporting
site.

To meet the need for feedback, PHLIS has a menu-driven option to
transmit files or messages up and down the reporting chain, with
these files and messages being transmitted automatically when
connections are established for each data transmission. This
facility is flexible enough to allow any valid user in the
reporting chain to transmit files or messages to any other user
in the reporting chain. For example, in the United States, a
county health official who is included in the reporting system in
one state can send messages or files to a participating county
official in another state. The feedback system does not mimic
electronic mail because these files and messages are sent along
the reporting chain in the same communications configuration as
data reporting. Therefore, successful arrival of these messages
at their destination(s) depends upon each member of the reporting
chain between the sender and the receiver to establish a
connection for reporting purposes. However, the system provides
an alternative mechanism for sending files and messages directly
to any other reporter having the capacity to receive them without
going through the reporting chain.

PHLIS is used in all 50 state public health laboratories, as well
as the District of Columbia and Guam. Disease modules included
are animal rabies, Campylobacter, Escherichia coli O157:H7, Lyme
disease, mycobacteria, respiratory and enteric viruses, human
Salmonella, nonhuman Salmonella, Shigella, and drug-resistant
Streptococcus pneumoniae.

PHLIS can be implemented independently: organizations can develop
their own PHLIS pyramid reporting system. For example, PHLIS is
currently being implemented at the Caribbean Epidemiology Center
(CAREC) in Trinidad and in its member countries for the reporting
of HIV/STD infections with the expectation that the reporting
system will be expanded to accommodate other diseases. CAREC can
receive reports from the member countries as each country is
added to the reporting structure.

Laboratory Information Tracking System (LITS)

The second system, LITS, is a PC local area network-based system
for tracking laboratory specimens. The system allows specimen
information to be entered at a central specimen receiving site;
additional information about the specimen can be entered into the
system in any of the laboratories performing tests on that
specimen. Although modules are customized for each laboratory's
needs, laboratorians can add additional tests or delete obsolete
ones. Furthermore, users can examine all the data about a
specimen, including data from all laboratories that performed
tests on the specimen. Other features in the system include cost
billing, user defined reports, user defined query, and specimen
or patient tracking and security. For emerging diseases, LITS
provides a mechanism to standardize laboratory protocol across
organizations and a mechanism to share data about specimens
within an organization.

Acknowledgments

We thank Tim Kuhn for leading the programming team; Bruce Wilson,
Dana Crenshaw, Joe Bates, and Neil Jones for programming support;
Tim Day for user support; Kathleen Maloney, Joy Goulding, Lori
Hutwagner, and Cecile Ivey for evaluating program integrity;
Brian Plikaytis for his early involvement with LITS; and Cheryl
Shapiro for financial management.

References
1. Fraser DW, Tsai TR, Orenstein W, Parkin WE, Beecham HJ,
Sharrar RG. Legionnaires' disease: description of an epidemic of
pneumonia. N Engl J Med 1977;297:1189-97.
2. Shands KN, Schlech WF III, Hargrett NT, Dan BB, Schmid GP,
Bennett JV. Toxic shock syndrome: case-control studies at the
Centers for Disease Control. Ann Intern Med 1982;96:895-8.
3. CDC. Outbreak of acute illness southwestern United States,
1993. MMWR 1993;42:421-4.
4. Administrative Conference of the U.S. Privacy Act. In: Federal
Administrative Procedure Sourcebook, 2nd ed. Office of the
Chairman, 1992:863-979.
5. Administrative Conference of the U.S. Freedom of Information
Act. In: Federal Administrative Procedure Sourcebook, 2nd ed.
Office of the Chairman, 1992:633-61.

DISPATCHES

(Pages 115-23)

Helicobacter hepaticus, a Recently Recognized Bacterial Pathogen,
Associated with Chronic Hepatitis and Hepatocellular Neoplasia in
Laboratory Mice

Gastric carcinoma, one of the most prevalent human cancers
worldwide, is among the neoplasms for which epidemiologic
evidence of environmental causes is strongest. The exact nature
of these environmental causes was obscure until mounting evidence
recently linked chronic infection of the gastric antrum mucosa by
Helicobacter pylori (a microaerobic, gram-negative, spiral
bacterium) with elevated cancer risk (1). It is now recognized
that gastric B-cell lymphoma of mucosa-associated lymphoid tissue
is also closely linked to gastric H. pylori infection, and
eradication of the infection with antibiotics can result in
regression of the lymphoma (2,3). This startling finding has
stimulated intense interest in the genus Helicobacter and related
organisms; as a result, additional species of Helicobacter are
now frequently isolated and characterized from many non-human
hosts. Until 1994, however, only H. pylori was known to be
associated with tumor development, in humans or in any other
animal species.

In 1992, at the National Cancer Institute's Frederick Cancer
Research and Development Center (FCRDC) in Frederick, Maryland, a
high prevalence of liver disease was observed among certain
strains of mice; these mice were untreated controls in long-term
chemical carcinogenesis experiments. Affected strains, notably
A/JCr, had been bred at FCRDC under pathogen-free conditions and
were free of known serologically detectable murine viruses and
parasites; moreover, they had no histologically demonstrable
hepatic abnormalities, except for a very low incidence (1% to 2%)
of hepatocellular tumors in mice 15 months of age or older. Over
a very short period, the prevalence of a histologically
distinctive form of hepatitis increased to virtually 100% in male
mice at 1 year of age (Table 1). The earliest demonstrable
lesions were small, undistinctive foci of hepatic necrosis seen
in young mice aged 2 to 6 months. In older mice, aged 6 to 10
months, there was a highly distinctive pericholangitis,
consisting of abundant mononuclear cell infiltrates around bile
ducts within portal triads. The biliary epithelium within
affected ducts was focally swollen, and the luminal surfaces of
damaged epithelial cells were poorly defined in hematoxylin and
eosin-stained sections (4,5). In livers with extensive lesions,
bile ductular (oval cell) hyperplasia was also prominent.
Moreover, mice with hepatitis usually had hepatocellular tumors,
often multiple, that included both adenomas and carcinomas (4).

Hepatocellular tumors in mice are one of the most common
endpoints in bioassays for chemical carcinogens. They were not,
at that time, known to be associated with infectious agents.
Accordingly, initial efforts to identify the cause of the
hepatitis/hepatocellular tumor syndrome were directed toward
possible sources of chemical exposure. The possibility of
accidental exposure to experimental substances within the
research animal facilities was ruled out when liver disease was
identified in mice that had never left the breeding areas which
are located in separate buildings. Extensive chemical analyses of
food, bedding, water, and other possible sources of toxic
substances had negative results.

Detailed pathologic examination by light microscopy of tissue
sections from diseased livers was continued, and many special
stains were used. One such stain, Steiner's silver impregnation
procedure for spirochetes (6), revealed in hepatic tissue uniform
bodies that were consistent in size and shape with bacteria.
Homogenates of fresh liver tissue from diseased mice proved
effective in transmitting hepatitis to A/J mice purchased from
commercial sources outside FCRDC, when given by intraperitoneal
injection (5). In addition, from these homogenates, a motile,
spiral bacterium could be cultivated on blood agar plates
incubated at 37 degrees C under anaerobic or microaerobic
conditions.

This organism was subsequently characterized by ultrastructural
morphologic examination, biochemical characteristics, and 16S
rRNA gene sequence. Determined to be a new species related to H.
pylori, it was given the name H. hepaticus (7). The bacterium is
motile and gram negative, 0.2 to 0.3 m in diameter, 1.5 to 5.0
m long, and curved to spiral in shape, with one to several
spirals; it has bipolar sheathed flagella (one at each end) but
lacks the periplasmic fibers that envelope the bacterial cells in
other mouse Helicobacter species. H. hepaticus has strong urease
activity, is oxidase and catalase positive, produces H2S, reduces
nitrate to nitrite, and grows microaerobically at 37 degrees C
but not at 25 degrees C or 42 degrees C. It is resistant to
cephalothin and nalidixic acid but sensitive to metronidazole.
Photographs illustrating its morphologic structure by light (4,5)
and electron (4,5,7) microscopy have been published. The
species-defining characteristic of the organism, the nucleotide
sequence of its 16S rRNA gene, has been used to develop a
diagnostic assay based on polymerase chain reaction (8).

Systematic examination of rodents of all species and strains
produced at FCRDC, especially retired breeders, showed that the
characteristic hepatitis and associated bacteria were present in
mice of several strains (A/JCr, DBA/2NCr, C3H/HeNCr) and that
within these strains, the male mice were more severely affected
than the female. Mice with severe combined immunodeficiencies
were especially vulnerable. The precise location of organisms
demonstrable by Steiner stain within infected liver parenchyma
was shown by transmission electron microscopy to be invariably
extracellular and characteristically within bile canaliculi
(4,5). No liver disease was seen in some strains (e.g.,
C57BL/6NCr) or in F1 hybrids between sensitive and resistant
strains (e.g., B6C3F1). Rodent species other than mice (e.g.,
rats, Syrian hamsters, and guinea pigs) were not affected.

In infected mice with severe combined immunodeficiency, cecal
inflammation was histologically demonstrable (5), and organisms
were isolated from the mucosa of the large intestine (7), which
may mean that the usual ecologic niche occupied by H. hepaticus
is that of a commensal colonizer of the intestinal tract (8).
Since mice are coprophagic, it appears highly likely that natural
transmission of the organisms is the oral-fecal route. Why and
how H. hepaticus invades the liver in mice of certain strains
remain to be determined. Hepatitis is also characteristic of
certain other enteric pathogenic bacteria, such as Campylobacter
jejuni (9) that, unlike H. hepaticus, have not been associated
with liver tumor development. The tissue damage that accompanies
persistent infection by H. hepaticus, H. pylori, and certain
other Helicobacter species may be due, at least in part, to a
soluble, trypsin-sensitive cytotoxin of high molecular weight
produced by these organisms (10). There is no precedent for any
direct role of such a toxin in carcinogenesis. On the other hand,
chronic infections by viruses, bacteria, or certain parasites are
recognized risk factors for human cancers at various sites. The
hypothesis that chemically reactive, potentially genotoxic,
substances of low molecular weight (including nitric oxide and
active oxygen species) generated by inflammatory cells at the
site of chronic infection may initiate or enhance carcinogenesis
has been examined (11). The hypothesis is under active
investigation in the context of H. hepaticus-associated liver
disease.

H. hepaticus is susceptible to a number of antibiotics; treatment
of susceptible, naturally infected 8- to 10-week-old strain A/JCr
mice with single or combined antimicrobial agents has been
evaluated for efficacy in eradicating established infections
(12). Amoxicillin, metronidazole, and tetracycline administered
singly failed to eradicate bacteria from the gastrointestinal
tract, but either amoxicillin or tetracycline, in combination
with metronidazole and bismuth, was effective in eradicating H.
hepaticus from the liver, cecum, and colon when given by oral
gavage for a period of 2 weeks (12). The effect of antibiotic
therapy on the carcinogenic process, or in older animals, remains
to be established.

The importance of H. hepaticus to humans is not yet completely
known. The organism clearly has the potential to confound
bioassays for chemical carcinogens, but this potential has no
direct effect on humans. Even though most Helicobacter species
identified to date are characteristically associated with (and
named after) specific mammalian host species in which they
generally inhabit the gastrointestinal tract (with or without
causing gastritis or other chronic inflammatory disease), the
potential host range for some species is quite broad. H. pylori,
originally isolated from humans, has recently been isolated also
from the domestic cat; this raises the possibility that
Helicobacter pylori may be a zoonotic pathogen that can be
transmitted from companion animals to humans (13). Exploring the
possibility of zoonotic transmission of H. pylori, H. hepaticus,
or any other Helicobacter species would require isolation of the
organism in question by culture methods. Serologic methods have
not yet been refined to the level of species specificity. Humans
infected with H. pylori mount a serum antibody response to the
bacteria that is readily detected by enzyme-linked immunosorbent
assays and is considered evidence of ongoing disease (1); mice
infected with H. hepaticus similarly produce serum antibodies to
that species that have been demonstrated by Western blotting (5).
Antisera to H. pylori can be used to visualize H. hepaticus in
mouse liver tissue sections stained by the avidin-biotin complex
immunohistochemical procedure (5). The cross- reactivity between
these two species precludes use of available serologic methods to
establish whether H. hepaticus has infected humans.

Regardless of whether H. hepaticus is itself capable of infecting
humans, it serves to demonstrate that liver tissue can be
persistently infected by at least one member of the genus
Helicobacter, and that liver cancer can be a long-term
consequence of such infection. This discovery raises questions
about the existence of a comparable relationship between liver
cancer in humans and unrecognized bacterial infections. Reviews
are under way of tissue blocks from pathology archives in search
of organisms demonstrable by the Steiner stain in liver sections
from human populations at high risk for liver cancer.

Jerry M. Rice
Laboratory of Comparative Carcinogenesis, National Cancer
Institute, Frederick, Maryland, USA


Table 1. Increasing prevalence of hepatitis and hepatocellular 
neoplasia in control male A/JCr mice at the National Cancer 
Institute's Frederick Cancer Research and Development Center, 
1989-1992(a)  

                Number   Age,      Mice with       Mice with
Date killed    of mice   in weeks  hepatitis (%)   liver tumors (%)
___________________________________________________________________
Jan-Mar 1989     48       47+or3     0                 0

May-Jul 1989     47       70+or60    0                 1 (2)

Jan-Apr 1992      6       36+or4     2 (33)            0

Jul 1992         16       54         16 (100)           1 (6)

Aug-Oct 1992      6       64+or3     5 (83)            3 (50)

Dec 1992         12       77         12 (100)          11 (92)
_______________________________________________________________ 
(a)Adapted from ref. 4.

References

1. Parsonnet J, Friedman GD, Vandersteen DP, Chang Y, Vogelman
JH, Orentreich N, et al. Helicobacter pylori infection and the
risk of gastric carcinoma. N Engl J Med 1991;325:1127-31.
2. Wotherspoon AC, Doglioni C, Diss TC, Pan L, Moschini A, de
Boni M, et al. Regression of primary low-grade B-cell gastric
lymphoma of mucosa-associated lymphoid tissue type after
eradication of Helicobacter pylori. Lancet 1993;342:575-7.
3. Bayerdrffer E, Neubauer A, Rudolph B, Thiede C, Lehn N, Eidt
S, et al. Regression of primary gastric lymphoma of
mucosa-associated lymphoid tissue type after cure of Helicobacter
pylori infection. Lancet 1995;345:1591-4.
4. Ward JM, Fox JG, Anver MR, Haines DC, George CV, Collins MJ
Jr, et al. Chronic active hepatitis and associated liver tumors
in mice caused by a persistent bacterial infection with a novel
Helicobacter species. J Natl Cancer Inst 1994;86:1222-7.
5. Ward JM, Anver MR, Haines DC, Benveniste RE. Chronic active
hepatitis in mice caused by Helicobacter hepaticus. Am J Pathol
1994;145:959-68.
6. Garvey W, Fathi A, Bigelow F. Modified Steiner for the
demonstration of spirochetes. J Histotechnology 1985;8:15-7.
7. Fox JG, Dewhirst FE, Tully JG, Paster BJ, Yan L, Taylor NS, et
al. Helicobacter hepaticus sp. nov., a microaerophilic bacterium
isolated from livers and intestinal mucosal scrapings from mice.
J Clin Microbiol 1994;32:1238-45.
8. Battles JK, Williamson JC, Pike KM, Gorelick PL, Ward JM,
Gonda MA. Diagnostic assay for Helicobacter hepaticus based on
nucleotide sequence of its 16S rRNA gene. 1995;33:1344-7.
9. Kita E, Oku D, Hamuro A, Nishikawa F, Emoto M, Yagyu Y, et al.
Hepatotoxic activity of Campylobacter jejuni. J Med Microbiol
1990;33:171-82.
10. Taylor NS, Fox JG, Yan L. In-vitro hepatotoxic factor in
Helicobacter hepaticus, H. pylori and other Helicobacter species.
J Med Microbiol 1995;42:48-52.
11. Ohshima H, Bartsch H. Chronic infections and inflammatory
processes as cancer risk factors: possible role of nitric oxide
in carcinogenesis. Mutat Res 1994;305:253-64.
12. Foltz CJ, Fox JG, Yan L, Shames B. Evaluation of
antibiotic therapies for eradication of Helicobacter
hepaticus. Antimicrob Agents Chemother 1995;39:1292-4.
13. Handt LK, Fox JG, Dewhirst FE, Fraser GJ, Paster BJ, Yan LL,
et al. Helicobacter pylori isolated from the domestic cat: public
health implications. Infect Immun 1994;62:2367-74.


(Pages 132-3)

Hemolytic Uremic Syndrome Due to Shiga-like Producing Escherichia
coli O48:H21 in South Australia

Enterohemorrhagic Escherichia coli (EHEC) other than serotypes
O157:H7 are increasingly recognized in association with hemolytic
uremic syndrome (HUS) (1) and have been reported in Australia
(2). While detecting strains of O157:H7 has become easier over
the years, identifying the expanding number of other serotypes of
EHEC also associated with HUS, with other conditions, and with
healthy domestic animals is still very difficult.

Cases of HUS have been reported in Australia over a number of
years. The most common serotype found was O111:H-, and
Australia's recently reported first HUS outbreak (3) was caused
by EHEC O111:H-. We wish to report a case of severe HUS due to
serotype O48:H21, which, as far as we know, has not been
previously reported as a cause of HUS. This case occurred in
1993, before surveillance of HUS had been initiated; after this
case, between July and December 1994, 10 cases of HUS (from which
four isolates were obtained; two were EHEC O111) were reported to
the Australian Paediatric Surveillance Unit (E. Elliott, pers.
comm.).

The patient in the 1993 case was an 8-year-old girl, living in a
rural setting in the outskirts of Adelaide, South Australia. Her
home was adjacent to a farm on which cows, sheep, and ducks were
kept. A kelpie/healer cross puppy was in the house in November
1993. Also kept were a pet galah (Australian cockatoo) and pet
fish. She was well until 23 December 1993, when she had diarrhea
described as very smelly and watery "like the juice of tinned
crab." The diarrhea became bloody on 2 January 1994 and was
associated with severe abdominal pains which made the patient
draw up her legs. She was having bowel movements six times a day,
had become very weak, and was unable to stand. She was admitted
to Adelaide Children's Hospital on 3 January 1994, and her
condition progressed to anuric renal failure over the next few
days. Serum biochemistry on 7 January showed a urea level of 23.3
mmol/L and creatinine level of 539 mol/L. Her hemoglobin level
fell from 157 g/L on 3 January to 86 g/L on 10 January. Her
hematocrit fell from 48% to 24%, and her platelet count fell from
463 x 109/L to 47 x 109/L on these dates, respectively. The blood
film showed microangiopathic hemolytic anemia with fragmented red
cells. She required hemodialysis for 3 weeks and was discharged
from the hospital on 31 January 1994.

Apart from the patient's 5-year-old brother, who had loose bowel
movements for 1 day on 28 December 1993, no other family members
were affected. An adequate dietary history was not obtained;
however, no food had been eaten from commercial food outlets.

Stool samples were collected on 4 and 5 January 1994. The samples
were probed for Shiga-like toxin (SLT)-I and SLT-II genes by
polymerase chain reaction (PCR), and the results were positive.
Approximately 80% of lactose-fermenting colonies on MacConkey
agar were also SLT positive. No sorbitol-negative colonies were
observed on sorbitol-MacConkey agar. In addition to being
cultured for E. coli, the stools were also routinely cultured for
Shigella, Salmonella, Yersinia, Vibrio, and Clostridium. In
addition, stained concentrates were examined for Giardia lamblia
and Entamoeba histolytica with negative results. Four typical E.
coli strains were subjected to further tests. They were typical
E. coli, positive in the indole and ONPG tests, negative in the
Voges-Proskauer, citrate, TDA, malonate, urease, gelatine, and
H2S tests. The strains fermented glucose, lactose, mannitol,
xylose, rhamnose, arabinose, sorbitol, sucrose, and melibiose.
They did not ferment inositol, adonitol, salicin, raffinose, or
amylose. They decarboxylated arginine, lysine, and ornithine. All
the strains produced enterohaemolysin (4). The strains were O and
H serotyped (5, 6) and found to be serotype O48:H21. Supernatant
preparations were tested on Vero cells (7) and found to give
typical verocytotoxic reactions in titers of 103 to 104. The
supernatants were also tested by enzyme-linked immunosorbent
assay (ELISA) by using monoclonal antibodies 13C4 and 11E10
directed against SLT-I and SLT-II, respectively, and strong
reactions with both antibodies were noted, confirming the
presence of both SLTs.

Stool samples taken from the patient on 8 February 1994 were
negative for SLT-I and SLT-II genes by PCR and were not cultured
further. Stool samples from the patient's brother and local
animals were not forthcoming.

That all four E. coli isolates tested were of serotype O48:H21
and demonstrated identical toxigenicity by both PCR and ELISA and
the fact that SLT-positive organisms were not found in the stools
collected during the patient's convalescence strongly suggest
that this serotype was the causative organism. The toxicity,
virulence, and part of the molecular structure of the SLT-II gene
derived from the EHEC O48:H21 strain reported here (and whose
novel serotype was discovered by the authors) have recently been
described elsewhere (8).

Paul N. Goldwater,* Karl A. Bettelheim+
*Microbiology and Infectious Disease Services,
  Women's & Children's Hospital, 
Adelaide, South Australia, 5006; 
 Biomedical Reference Laboratory,
+Victorian Infectious Diseases Reference Laboratory, 
Fairfield Hospital, Victoria, North Australia,
Australia 3078

References

1. Bokete TN, O'Callahan CM, Clausen CR, Tang NM, et al.
Shiga-like toxin producing Escherichia coli in Seattle children:
a prospective study. Gastroenterology 1993; 105: 1724-31.
2. Goldwater PN, Bettelheim KA. The role of enterohaemorrhagic
Escherichia coli serotypes other than O157:H7 as causes of
disease.
Communicable Disease Intelligence 1995;19:2-4.
3. Centers for Disease Control and Prevention. Community outbreak
of hemolytic uremic syndrome attributable to Escherichia coli
0111:NM South Australia, 1995. MMWR 1995;44:550-1, 557-8.
4. Beutin L, Montenegro MA, Orskov I, Orskov F, Prada J,
Zimmerman S, et al. Close association of verocytotoxin
(Shiga-like toxin)production with enterohemolysin production in
strains of Escherichia coli. J Clin Microbiol 1989;27:2559-64.
5. Bettelheim KA, Thompson CJ. New method of serotyping
Escherichia coli: implementation and verification. J Clin
Microbiol 1987;25:781-6.
6. Chandler ME, Bettelheim KA. A rapid method of identifying
Escherichia coli "H" antigens. Zentralblatt fur Bakteriologie,
Mikrobiologie und Hygiene 1. Abteilung Originale. A
1974;129:74-9.
7. Konowalchuk J, Speirs JL, Stavric S. Vero response to a
cytotoxin of Escherichia coli. Infect Immun 1977;18:775-9.
8. Paton AW, Bourne AJ, Manning PA, Paton JC. Comparative
toxicity and virulence of Escherichia coli clones expressing
variant and chimeric Shiga-like toxin type II operons. Infect
Immun 1995;63:2450-8.



(Pages 134-7)

Does Treatment of Bloody Diarrhea due to Shigella dysenteriae
Type 1 with Ampicillin Precipitate Hemolytic Uremic Syndrome?

Diarrhea-associated hemolytic uremic syndrome (HUS), the most
common cause of acute renal failure in infancy and childhood, is
often associated with infection by organisms producing Shiga
toxin (ST) or Shiga-like toxin (SLT), mainly
verocytotoxin-producing Escherichia coli (VTEC O157:H7) and
Shigella dysenteriae type 1 (1,2). Although antibiotics are
believed to be essential in treating shigellosis, treatment of S.
dysenteriae type 1 patients with antibiotics to which the
organism is resistant has been considered a risk factor for HUS
(3,4).

Until 1993, HUS was rarely reported from Saudi Arabia. Four cases
of diarrhea-associated HUS due to S. dysenteriae type 1 were
identified in 1989 (J. Hibbs and A. Mishkas, unpublished report),
and one case of HUS attributed to plasma transfusion was
documented in 1988 (5).

In May 1993, four dysentery-associated HUS cases in two families
were reported from northwestern Saudi Arabia (Tabuk). S.
dysenteriae type 1 was isolated from the stool of each HUS
patient. The organism was also isolated from 6 of the other 10
members of the two families who had dysentery. All isolates were
resistant to trimethoprim-sulfamethoxazole, chloramphenicol,
tetracycline, and ampicillin but sensitive to nalidixic acid. The
two families had just returned from a 1-week visit to relatives
in two neighboring villages in Gizan. This densely populated
region in southwestern Saudi Arabia has about 1.2 million people
living in more than 4,000 villages; the population is relatively
poor and uneducated, and environmental sanitation is generally
inadequate.

We defined a case of HUS as any case of bloody diarrhea (BD) that
had all of the following: acute renal failure (serum urea
nitrogen, 18 mg/dL (or 6.3 mmol/L); or creatinine, 1.3 mg/dL (or
115 mmol/L)); thrombocytopenia (platelet count 130,000/mm3); and
hemolytic anemia (hemoglobin level less than 10 g/dL; or
hematocrit less than 30%; or appearance of fragmented red cells
on direct microscopy). We standardized the treatment of BD in the
Gizan region as follows: No antibiotics were given for treatment
of BD at the primary health care centers (PHCCs) before a stool
specimen was taken for culture and sensitivity testing. After
reviewing the preliminary results, we either recommended use of
nalidixic acid for treatment of BD or were guided by the results
of the stool culture. This protocol was followed for management
of BD in the entire region.

Parasitologic, bacteriologic, and biochemical tests and drug
treatment regimens were obtained for all patients admitted with
BD or HUS to the regional referral hospital or five district
hospitals in the outbreak area. BD cases were identified through
hospital admission records, visits to PHCCs in the affected
villages, interviews with family members of the identified
patients, and school visits. We visited the houses of all HUS and
BD patients and interviewed family members to ascertain which
antibiotic was used to treat the BD patients; mothers were shown
bottles and boxes of antibiotics and were asked to identify the
antibiotic used for treating the children with BD.

We identified 233 cases of BD occurring from February through
July 1993 among 79 families scattered over 19 contiguous
villages. Affected villages were predominantly in southern Gizan
region near the Yemeni border. One hundred ninety patients
(81.5%) consulted PHCCs; of those, 97 (51%) were referred to
hospitals, and 81 (43%) were admitted. Thirty-four other BD
patients were admitted directly to hospitals (a total of 115
admissions). In nine BD cases patients did not seek medical care
including seven (3%) who used traditional treatment (the Wicka
plant). In 23 (10%) patients, 13 male and 10 female, BD developed
into HUS. Four isolates of S. dysenteriae type 1 that showed the
same antibiotic susceptibility described earlier were obtained
from four patients with BD in different villages in the middle of
the outbreak. We used Cary-Blair transport medium for
transporting stool specimens collected before antibiotic
treatment from newly recognized patients with BD. However,
community-and hospital-based interviews showed that the sequence
of symptoms was almost identical in all of the 233 BD cases: the
condition started with colicky abdominal pain and tenesmus (69%),
followed by watery diarrhea (60%), which rapidly became only
mucus and blood (83%) or blood-streaked (17%). Seven patients
(3.0%) had rectal prolapse. S. dysenteriae type 1 was not
isolated from any of the 23 HUS patients; however, all stool
specimens were taken during antibiotic treatment.

Most BD cases (92.3%) were among Saudis; the remaining 7.7% were
among Yemeni patients. No HUS case occurred among patients over
11 years of age. The male/female ratio for both BD and HUS was
1.3:1. Three boys and three girls with confirmed HUS died
(case-fatality rate = 26.1%); none of the patients with
uncomplicated BD cases died.

Of the 23 HUS patients, 18 (78%) became ill with the disease 2 to
14 days after hospital admission for uncomplicated BD. This
compares with a hospital admission rate of 40 (27%) of 147 for
children of the same age with BD from the same villages (odds
ratio = 9.6, 95% confidence interval 3.1-35). Five children, aged
8 to 16 months, got HUS either before or on the day of hospital
admission; all had received oral ampicillin at home for 5 to 7
days before their illness progressed to HUS. In comparison, two
of nine children of the same age, with BD, who were not
hospitalized, received ampicillin at home (OR = infinite, p value
= 0.02, Fisher's exact test).

Eighteen HUS cases occurred after the patients were admitted to
Samtah and Abu-Arish, two out of five district hospitals. The
demographic, clinical, and laboratory profiles of BD and HUS
cases are shown in Table 1A-C. Six different antibiotics were
used in various combinations for treating BD patients in these
hospitals Table 1B. Treatment with ampicillin prescribed alone or
with other antibiotics except nalidixic acid (Table 2), was
associated with development of HUS. However, three BD patients
who received nalidixic acid developed HUS. Of 12 BD patients
(including eight children under 12 years of age) who received no
antibiotic therapy, none got HUS.

These results support the implication from Bangladesh and from a
parallel investigation in Saudi Arabia that inpatient antibiotic
treatment of children with dysentery due to S. dysenteriae type 1
may precipitate HUS. We have extended these observations to show
the same association for antibiotic treatment at home. Although
Abu-Arish and Samtah hospitals received similar numbers of BD
patients, more HUS cases were reported from Abu-Arish hospital,
which used ampicillin to treat patients with BD. The various
combinations of antibiotics used to treat BD patients could be
explained by the presence of doctors from parts of the world that
have different prescription practices. Three BD patients got HUS
despite the use of nalidixic acid. Resistance to nalidixic acid
among S. dysenteriae isolates was reported from Bangladesh;
resistance increased from 2.1% in 1986 to 57.9% in 1990 (6).

We recommend a laboratory-based surveillance system to identify
and promptly contain emerging outbreaks. Physicians need to be
informed continuously about emerging resistant strains of
bacteria and be cautious when using antibiotics to treat patients
with dysentery unless the causative organism and the resistance
pattern have been identified. Parents of children with BD need to
be educated to take their children to the nearest health facility
as soon as possible.

S. dysenteriae is a delicate bacterium that does not withstand
adverse conditions (e.g., heat and dryness); prompt plating,
preferably at bedside, is recommended (7). Failure to isolate S.
dysenteriae type 1 from dysenteric stool specimens during this
outbreak could be attributed to delayed plating of specimens,
lack of appropriate transport media, and treatment with
antibiotics before stool specimens were obtained. Even with
direct inoculation of stool specimens in pediatric wards, HUS
resulted in a low yield of S. dysenteriae type 1 (8,9).

Treatment of BD due to S. dysenteriae type 1 with ampicillin may
precipitate HUS. It would be valuable to retrospectively examine
this association in other countries where both dysentery due to
S. dysenteriae type 1 and HUS are reported. 

Abdulaziz A. A. Bin Saeed, Hassan E. El Bushra, Nasser A.
Al-Hamdan Field Epidemiology Training Program, Department of
Preventive Medicine, Ministry of Health, Riyadh, Kingdom of Saudi
Arabia 
References

1. Neild GH. Haemolytic uraemic syndrome in practice. Lancet
1994;343:398-401.
2. Robson WLM, Leung AKC, Kaplan BS. Hemolytic uremic syndrome.
Curr Prob Pediatr 1993;23:16-33.
3. Butler T, Islam MR, Azad MAK, Jones PK. Risk factors for
development of hemolytic uremic syndrome during shigellosis. J
Pediatr 1987;110:894-7.
4. Al-Qarawi S, Fontaine RE, Al-Qahtani MS. An outbreak of
hemolytic uremic syndrome associated with antibiotic treatment of
hospital inpatients for dysentery. Emerging Infectious Diseases
1995;1:138-40.
5. Kher K, Hussein M. Severe haemolytic syndrome: report of a
child treated with fresh frozen plasma infusions and dialysis.
Saudi Med J 1988;9:205-7.
6. Bennish ML, Salam MA, Hussein MA, Myaux J, Khan EH,
Chakraborty J, et al. Antimicrobial resistance of Shigella
isolates in Bangladesh, 1983-1990: increasing frequency of
strains multiply resistant to ampicillin, trimethoprim
sulfamethoxazole, and nalidixic acid. Clin Infect Dis
1992;14:1055-60.
7. Keusch GT, Formal SB, Bennish M. Shigellosis. In: Warren KS,
Mahmoud AAF, eds. Tropical and geographical medicine 2nd ed. New
York: McGraw-Hill, 1990:760.
8. Khin-Maung-U, Myo-Khin, Tin-Aye, Myo-Min-Aung, Soe-Soe-Aye,
Thane-Oke-Kyaw-Myint, et al. Clinical features, including
hemolytic uremic syndrome, in Shigella dysenteriae type 1
infection in children of Rangoon. J Diarrhoeal Dis Res
1987;3:175-7.
9. Srivastava RN, Moudgil A, Bagga A, Vasudev AS. Hemolytic
uremic syndrome in children in northern India. Pediatr Nephrol
1991;5:284-8.

Table 1.

A. Profiles of children admitted to hospitals with bloody diarrhea (BD) 
or hemolytic uremic syndrome (HUS)

                        Places of hospitalization of BD and HUS cases   
                    __________________________________________________
                    Samtah   Abu-Arish    KFH       Bysh     Sabia
______________________________________________________________________

No. of BD cases      43         42         13         9         8

No. of HUS cases      8         14          -         -         1(a)

Percentage of 
  HUS cases          18.8       33.3        0         0        12.5

Median age in 
  years: BD cases     4.0        5.0        7.0       4.0       4.0
        HUS cases     1.8        2.8        -         -         0.8

Mean (SD) of 
  duration (in 
  days) between 
  onset of symptoms 
  and admission to 
  hospital:           
    BD                3.8 (1.9)  5.5 (1.6)  4.5 (0.4) 3.7       3.9 (2.0)
    BD complicated 
      with HUS        5.2 (2.6)  4.5 (2.5)  -         -         -
    HUS diagnosed 
      on admission   13.0 (2.8)  7.5 (0.7)  -         -         6.0 (0.0)
________________________________________________________________________

B. Percentage of 110 BD patients treated with antibiotics(b,c)

Ampicillan           36.6        70.0       15.4      11.1       100.0
Metroniadazole       14.6        55.0       7.7       0          100.0
Gentamicin           22.0        22.5       0         55.6       42.9
Nalidixic Acid       70.7        57.5       61.5      66.7       0
Claforna             2.4         20.0       0         11.1       0
Amikacin             9.8         0          7.7       22.2       28.6       
___________________________________________________________________________

C. Laboratory values of children hospitalized with BD or HUS

Laboratory test 
made on the day  Non-HUS cases    HUS diagnosed 2-14       HUS diagnosed
of admission to                         days              on admission to
the hospital(d)                                              hospital
              ____________________________________________________________
               Mean (N)e    SD      Mean (N)   SD       Mean (N)    SD
__________________________________________________________________________
Serum 
creatinine     60 (16)      46      63 (3)     79       279 (3)     94
Blood urea 
nitrogen (BUN) 5.4 (23)     5.8     15.3 (3)   12.0     23.0 (3)    6.1
Serum sodium   129 (33)     9       137 (3)    4.6      127 (3)     14
Serum 
potassium      3.8 (47)     0.8     3.9 (7)    1.1      4.6 (5)     1.1
(Leukocytes 
[WBC] count)   14.2 (48)    6.0     34.0 (6)   24.7     41.2 (5)   18.8
Hemoglobin     11.3 (50)    2.3     12.0 (1)   1.5      6.8 (4)    2.5
Hematocrit     36.1 (13)    3.9     NA         NA       15 (2)     7.1
Thrombocytes 
(platelets)    322 (5)      250     154 (1)    -        NA         NA
(Body 
temperature 
on admission)  37.8C (75)  0.8     37.9C     0.8      37.9C (5) 0.9
                                    (18)
_______________________________________________________________________                              
a Community case of HUS.
b Percent of cases receiving the corresponding antibiotics. A patient may
receive more than one antibiotic. Totals do not add up to 100%. 
c This table does not include 5 cases diagnosed as HUS on admission and
treated as cases of BD. 
d Creatinine in mg/dL, blood urea nitrogen in mg/dL, sodium in mmol/L, potassium in mmol/L, WBC (white blood cell count) in thousands/mm, hemoglobin level in g/dL, hematocrit, platelets in thousands/mm3. Values shown are for children under 12 years of age 
e N = number of cases of BD diagnosed in the hospital. NA = not available. 


Table 2. Risk of developing hemolytic uremic syndrome (HUS) by antibiotic 
combination used for treatment of bloody diarrhea (BD), Gizan, Saudi 
Arabia, 1993
_________________________________________________________________________


                           Dysentery 
                           patients       BD cases  
                         --------------   admitted
Antibiotic  Antibotic                     to Samtah
comb-       combi-                        and Abu-                    95%
ination     nations        Total  Devel-  Arish      HUS      Risk   confidence
group                     (N=110) oping   Hospitals  rate(%)  ratio  interval
_____________________________________________________________________________                                                    
Nalidixic   N(3/23),N+      41      3        28       7.3      1    0.23-4.34
acid with   G+N+C+A(0/1),
without     M+N=C(0/2),
other       N+C(0/4)
antibiotics
but no 
ampicillan(b)

No          No               12     0         6       0.0     NC(e)  p value=
antibiotic  antibiotic (0/12)                                        0.629(d)

Antibiotic  A(0/1),C(1/0),    4     1         4       25.0    1.58   0.22-
other than  G(0/1), M+G(0/1)                                         11.58(e)
nalidixic acid
or ampicillin

Ampicillin                   28     10        19      35.7    6.90   0.98-
with or                                                              48.68
without other
antibiotics 
but no
nalidixic acid

Ampicillin  P (3/3)           6      3          5     50.0    6.11   1.31-
only                                                                 28.54
                             
Ampicillin  P+G(1/2),P+M     22      7         14     31.8   10.50   0.54-
with other  (2/3),P+M+A                                              205.39
antibiotics (0/2), P+M+C
but no      (1/0) P+M+G
nalidixic   (3/8)
acid

Ampicillin  P+M+G+N(1/1),    25      4         24     16.0     1.77  0.31-
and         P+M+N(0/10),                                             10.21
nalidixic   P+M+N+C(1/0).
acid with   P+N(1/8), 
or without  P+N+A(1/2)
other 
antibiotics
____________________________________________________________________________
HUS rate (%) calculated from total number of BD in the five district 
hospitals using corresponding antibiotic. (a) Mantel-Haenszel weighted 
relative risk adjusted to hospital (EpiInfo, version 6.02). Analysis 
restricted for data from Samtah and Abu-Arish hospitals. HUS was not 
reported from the other three district hospitals. (b) Reference group. 
A = amikacin, C = claforan, g = gentamicin, N = nalidixic acid, 
M = metronidazole, P = ampicillin. Numbers between parentheses in the 
second column (antibiotic combination) indicate the (number of patients 
with HUS who took the corresponding antibiotic combination number or 
patients with BD who took the same antibiotic combination but did not 
develop HUS in the five district hospitals). (c) NC = not calculated. 
(d)One-tailed Fisher's exact test. (e) Adjusted Mantel-Haenszel 
relative risk could not be calculated.



(Pages 138-40)

An Outbreak of Hemolytic Uremic Syndrome Associated with
Antibiotic Treatment of Hospital Inpatients for Dysentery 

Shiga toxin (ST) from Shigella dysenteriae type 1 is accepted as
a cause of hemolytic uremic syndrome (HUS); however, the reasons
why HUS develops in only some infected patients are not clear
(1). The possibility that antibiotic therapy is associated with
the development of HUS has been explored for S. dysenteriae type
1 and for Escherichia coli O157:H7 (2-4).  In May 1993, during an
outbreak of S. dysenteriae type 1 in Gizan, Saudi Arabia, an
association between antibiotic treatment and HUS was also
observed (5). The strain of S. dysenteriae type 1 was resistant
to ampicillin, tetracycline, chloramphenicol, and
trimethoprim-sulfamethoxazole (TMP-SMX) and sensitive to
nalidixic acid. We report here some of our preliminary
observations for a concurrent outbreak.

In response to the Gizan outbreak, a circular was sent to all
regions of Saudi Arabia requesting immediate reports of S.
dysenteriae type 1. One region reported three cases of S.
dysenteriae type 1 with the same antibiotic resistance pattern.
The patients were visitors from the Najran region, on the Yemen
border. The affected family reported that many other persons in
their community of Barshash, home of about 6,000 Yemeni
immigrants, had recently had dysentery. The regional hospital in
Najran confirmed that several local children had been treated for
HUS, and an investigation was initiated.

Beginning in February 1993, the numbers of dysentery patients at
the Barshash Primary Health Care Center began to increase from 6
patients per week to 110 in mid May. After control measures were
initiated, weekly incidence decreased to zero by late June.
According to policy, the Primary Health Care Center treated
dysentery patients with oral rehydration, whereas those admitted
to the regional hospital received antibiotics. At the regional
hospital, S. dysenteriae type 1 was isolated from four Barshash
residents and one resident of another community. 

Between March and May, the illness of 10 (of 42) children
admitted to the regional hospital for dysentery progressed to HUS
from 2 to 10 days (median 5) after admission. For all 10
children, physicians noted HUS onset as a sudden change in the
clinical condition characterized by pallor, puffy face,
peripheral edema, or oliguria. Within 2 days of clinical onset,
blood urea nitrogen (BUN) levels of all 10 children were 10
mmol/L. In nine children, the hematocrit fell to below 75% of its
value on admission. One child had microcytic hypochromic anemia
on admission and was transfused with one unit of packed red
cells; his hematocrit fell from 29.8% after the transfusion to
24.6% the day after HUS onset; his BUN level rose from 3.1 to
23.5 mmol/L; and his thrombocyte count fell from 529,000/mm3 to
75,000/mm3. Red cell fragments were reported for seven patients.
Thrombocytopenia, developed in seven patients; in two,
thrombocyte counts decreased from admission values. A
leukemoid reaction 30,000 leukocytes/mm3) developed in seven
patients.

HUS cases included six children from Barshash, one from a
contiguous district, one with relatives in Barshash, and two
visiting from southern Gizan region where the concurrent outbreak
was occurring (5). No child was admitted from the community with
HUS. Reasons given for admission were either dysentery or bloody
diarrhea with mild or moderate dehydration. Two dysentery
patients who developed HUS were admitted because no one was
available to care for them at home. Characteristics on hospital
admission of dysentery patients who did or did not develop HUS
were similar withthe exception of serum sodium level and BUN
(Table 1). Elevations in creatinine (63 to 133 mmol/L) and urea
(6.1 to 7.9 mmol/L) levels on admission were mild, and after
intravenous rehydration returned to normal. No enteric pathogens
were isolated in stool specimens from 35 patients.

All 42 dysentery patients received a variety of antibiotic
combinations composed of ampicillin, TMP-SMX, nalidixic acid,
gentamicin, erythromycin, and metronidazole. Higher rates of HUS
were observed among patients receiving antibiotics to which the
locally circulating S. dysenteriae type 1 was resistant
(ampicillin or TMP-SMX) or antibiotics that are ineffective
against Shigella (metronidazole, erythromycin, gentamicin) than
among patients treated with nalidixic acid (with or without
metronidazole) (Table 2). Stratification of this analysis by
elevated creatinine level (62 mmol/L) or BUN (6.0 mmol/L) on
admission or by weight for age above and below the fifth
percentile had no effect on the magnitude or statistical
significance of these associations. However, stratification by
serum sodium on admission above and below 130 mmol/L yielded
increased risk ratios of 15 (95% confidence interval, 1.6 to 147)
for any ineffective antibiotic and 15 (95% confidence interval,
2.3 to 99) for ampicillin without nalidixic acid (Mantel-Haentzel
analysis).

These strong associations of HUS with prior antibiotic therapy
suggest that the antibiotics may be influencing the progression
of S. dysenteriae type 1 to HUS (2,5). The Najran patients had
milder disease on admission, and the risk ratios were higher than
in the other two reports. The assumption that S. dysenteriae type
1 caused the dysentery is supported by the isolation of S.
dysenteriae type 1 from community members during a concomitant
dysentery outbreak and by the absence of other ST-producing
organisms. However, because culture results are lacking, it is
possible that some hospitalized dysentery patients were infected
with other organisms. 

This investigation was retrospective, and without randomization, 
the selection of patients for treatment by
severity of illness was managed with stratified analysis. All
possible indicators of dehydration or severity of dysentery on
admission were not available for stratification. However, these
were also not available to the physicians for selection of
patients. Moreover, nalidixic acid was considered effective
therapy, and physicians did not indicate in the medical records
that they were selecting nalidixic acid for less severely ill
patients. Because of the variety of antibiotics prescribed, the
effect of antibiotics given to most patients (metronidazole) in
combination with other antibiotics could not be evaluated. The
effectiveness of those given to a few patients (TMP-SMX) could
not be assessed.

Antibiotic therapy may be associated with HUS in various ways.
The antibiotics given may have been ineffective, so the
infections were allowed to run their natural course to HUS.
However, one would have expected at least a few cases to have
developed at home among the hundreds of Barshash residents with
dysentery. Another possibility is that ineffective antibiotics
suppressed competing microbial flora, allowing less restrained
proliferation of  S. dysenteriae type 1. This does not account
for the 12% HUS attack rate in patients treated with nalidixic
acid. An in-vitro study of enhanced Shiga-like toxin I (SLT-1)
production by E. coli O157:H7 suggests another explanation.
Subinhibitory concentrations of antibiotics resulted in up to a
400% increase in SLT-I recovery relative to controls (6). This
effect was maximal at maximum subinhibitory - concentrations of
antibiotics, and a quinoline antibiotic, ciprofloxacin, yielded
the greatest increase of toxin. These findings are most
consistent with the epidemiologic findings in this outbreak,
including the occurrence of HUS in patients treated with
nalidixic acid and the absence of HUS in untreated patients in
the community. With these considerations, antibiotic treatment of
dysentery from S. dysenteriae type 1 should be approached with
caution. 

Sami Al-Qarawi,* Robert E. Fontaine,  and Mohammed-Saeed
Al-Qahtani*
*Saudi Arabian Field Epidemiology Training Program, Ministry of
Health, Saudi Arabia;  Epidemiology Program Office, Centers for
Disease Control and Prevention, Atlanta, Georgia, USA. 


Table 1. Characteristics on hospital admission of dysentery
patients whose illness did or did not develop into hemolytic -
uremic syndrome (HUS) during hospitalization, Najran, Saudi
Arabia, March through May 1993

                    Dysentery patients   
                  _________________________________
                                        Did not
Characteristic    Developed HUS(10)  develop HUS(32)  p value(a)
________________________________________________________________

Age(yr mean)          4.4                4.8            0.71

Male sex              7(70%)             23(72%)        1.0

Percentile weight
for ageb (median)     15.4               16.3           0.57

 Below fifth          6(60%)             12(38%)        0.29
 percentile

Days with dysentery
(median)              3                  3             0.70

 Range                2-8                1-14

Stools on first
hospital day          12.5               9.0           0.23
(median)

 Range                2-26                2-43

Temperature (mean)    38.2C              38.0C       0.64

 Range                36.8C-39.4C      36.5C- 39.4C
                                                 
Hematocrit            38.9%3             8.8%          0.94

Below 35.5%           4(40%)             7(23%)        0.43

Thrombocytes/mm       555,000            501,000       0.62

Below 150,000         0                  0

Leucocytes/mm (mean)  11,440             12,300        0.62

Above 12,000          7(70%)             22 (69%)      1.0

Above 18,000          1(10%)             4(13%)        1.0

Serum sodium, 
mmol/L(mean)          130                134           0.02
Below 130 mmol/L      4(40%)             7 (22%)       0.41

Serum potassium,
mmol/L (mean)         3.4                3.4           0.86

Below 3.5 mmol/L      5(50%)             16(50%)       1.0

Serum creatinine,
mmol/L (mean)         72                 62            0.62

Above 62 mmol/L       5(50%)             13(41%)       1.0

BUN, mmol/L(mean)     4.1                3.2           0.06

Above 6.0 mmol/L      2(20%)             2(6%)         0.24
____________________________________________________________________

(a)Differences in means by Student's t-test, in medians by the
Kruskal-Wallis test, and in proportions by Fisher's exact test 
(two-tailed). (b) National Center for Health Statistics.


Table 2. Risk of developing hemolytic uremic syndrome (HUS) by 
antibiotic combinations used for in-hospital treatment of 
dysentery during a community outbreak of antibiotic-resistant 
Shigella dysenteriae type 1, Najran, Saudi Arabia, March through May
1993

                          Dysentery patients
                          __________________                             95%
                         Developed               HUS        Risk       confidence
Antibiotic combination   HUS(10)     Total(42)   rate/100   ratio(a)   intervalb
_________________________________________________________________________________
Ineffective(c) antibiotics
without nalidixic acid(d-j)  6         12          5          4.3        1.3-15

Ampicillin combinations
without nalidixic acid(d-f)  5          7         71          6.2        1.9-20

Ineffective antibiotics
without nalidixic acid
or ampicilling (j)           1          5         20          1.7        0.22-14

Ampicillin and nalidixic
acid with (2) or without
(3) metronidazole            1          3         33          2.9        0.40-20

Trimethoprim-
sulfamethoxazole, nalidixic
acid and metronidazole       0          1          0           0

Nalidixic acid with (24)
or without (2) metronidazole 3         26         12          1.0         Reference
(reference)
____________________________________________________________________________________
(a)Relative to the reference antibiotic combination (nalidixic acid with or 
without metronidazole). (b) Taylor series approximation standard. 
(c) Antibiotics to which the outbreak strain of S. dysenteriae type 
1 was resistant (ampicillin, trimethoprim-sulfamethoxazole) or which are 
ineffective against shigella (metronidazole, gentamicin, or erythromycin). 
(d) Ampicillin and metronidazole (4 patients). (e) Ampicillin, 
metronidazole, and gentamicin (2 patients). (f) Ampicillin only (1 patient). 
(g) Trimethoprim-sulfamethoxazole and metronidazole (1 patient). (h)
Trimethoprim-sulfamethoxazole, erythromycin, and metronidazole (1 patient). 
(i) Metronidazole only (2 patients). (j) Erythromycin and metronidazole 
(1 patient).


References

1. Moake JL. Haemolytic uremic syndrome: basic science. Lancet
1994;343:393-401.
2. Butler T, Islam MR, Azad MAK, Jones PK. Risk factors for
development of hemolytic uremic syndrome during shigellosis. J
Pediatr 1987;110:894-7.
3. Ostroff SM, Kobayahi JM, Lewis JH. Infections with Escherichia
coli O157:H7 in Washington State: the first year of statewide
disease surveillance. JAMA 1989;262:355-9.
4. Pavia AT, Nichols CR, Green DP, et al. Hemolytic-uremic
syndrome during an outbreak of Escherichia coli O157:H7
infections in institutions for mentally retarded persons:
clinical and epidemiologic observations. J Pediatr
1990;116:544-51.
5. Bin Saeed AAA, El Bushra HE, Al-Hamdan NA. Does treatment of
bloody diarrhea due to Shigella dysenteriae type 1 with
ampicillin precipitate hemolytic uremic syndrome? Emerging
Infectious Diseases 1995;4:134-8.
6. Walterspiel JN, Ashkenazi S, Morrow AL, Cleary TG. Effect of
subinhibitory concentrations of antibiotics on extracellular
shiga like toxin I. Infection 1992;20:25-9.




(Pages 141-6)

Epidemic Cholera in the New World: Translating Field Epidemiology
into New Prevention Strategies

Cholera, a devastating diarrheal disease, has swept through the
world in recurrent pandemics since 1817. The seventh and ongoing
pandemic began in 1961 when the El Tor biotype of Vibrio cholerae
O1 emerged in Indonesia. This pandemic spread through Asia and
Africa and finally reached Latin America early in 1991 (1). After
explosive epidemics in coastal Peru, it spread rapidly and
continues throughout Latin America (Figure 1 cannot be viewed in
ASCII). Because of underreporting, the more than 1,000,000 
cholera cases and 10,000 deaths reported from Latin America 
through 1994 (Table 1) (2) represent only a small fraction of 
the actual number of infections. Molecular characterization of 
V. cholerae O1 strains from Peru has shown that they do not 
match strains from anywhere else in the world; therefore, the 
source of the Peruvian epidemic strains remains unknown (3). 
Moreover, other strains have since
appeared in Latin America. At least one of these, a strain
resistant to multiple antimicrobial drugs, was first identified
in Mexico and elsewhere in the world in mid-1991 and has since
spread widely throughout Central America (4). The introduction of
strains into new areas illustrates the rapid global transfer of
pathogens. V. cholerae O139 Bengal, which emerged as a new cause
of epidemic cholera in Asia in 1992, could also appear in Latin
America (5).

Such introductions are not easy to prevent, because they may
follow the arrival of travelers who are not aware of their
infection or of ships carrying contaminated ballast water. The
key to controlling epidemic cholera lies in limiting its spread
by using measures that prevent sustained transmission. One
measure might be using an inexpensive and effective vaccine to
provide lasting protection; however, no such vaccine yet exists,
although progress in vaccine development is being made (6-8).
Another measure is interrupting transmission so that the
causative organism never reaches the human host. This approach to
prevention successfully controlled many epidemic diseases in the
industrialized world, including cholera, typhoid fever, plague,
and malaria, before vaccines or antibiotics were developed. Over
the last century, a large engineering infrastructure, built in
industrialized nations, has provided safe water and sewage
treatment for nearly all people in these nations and has made
sustained transmission of cholera in those countries extremely
unlikely. Despite sporadic cases along the U.S. Gulf Coast and
repeated introduction of the epidemic organisms by travelers,
epidemic cholera has not occurred in the United States since the
nineteenth century (9,10).

To prevent cholera by interrupting transmission of the organism
to the host, it is important to understand precisely how the
bacteria are transmitted. John Snow demonstrated waterborne
transmission of cholera during a large epidemic in London in 1856
(11). He and many others since have suspected that other routes
of transmission are also important. Epidemiologic investigations
during the seventh pandemic have documented a variety of specific
food and water pathways by which the bacteria reach the host,
some of which were new and unsuspected (12). The El Tor biotype
of V. cholerae O1, for example, multiplies rapidly in moist foods
of neutral acidity (13). This bacterium also persists in the
estuarine environment in niches that are poorly understood but
may involve the plankton on which shellfish feed. This means that
raw seafood can be contaminated naturally before it is harvested.
Understanding these pathways of transmission in detail has been
central to devising successful control measures to block them.
For example, advice to drink only boiled or bottled water would
be of little use in outbreaks where the source was actually
contaminated food, such as shellfish or leftover rice. On at
least one occasion, such advice actually worsened the situation
because the bottled water was itself contaminated (14).

When epidemic cholera appeared in Latin America, after an absence
of more than 100 years, we conducted a series of eight rapid
field investigations in collaboration with national public health
authorities and the Pan-American Health Organization to define
the pathways of disease transmission and the priorities for
prevention. Conducted in various settings between February 1991
and August 1993, these investigations guided the initial
emergency prevention efforts and the development of sustained
prevention measures (Table 2) (15-21). 

The same case-control method was used for each investigation. We
first interviewed a few patients in great detail about what they
had ingested in the 3 days before they became ill, probing for
known or potential vehicles of cholera. We then constructed a
standardized interview questionnaire that asked about possible
exposures. Using this questionnaire, we interviewed patients
recovering from cholera as well as healthy persons of the same
age and sex who lived in the same neighborhood. By comparing the
frequency of positive and negative responses among the ill and
well persons, we could identify the exposures most strongly
associated with disease. For example, if 25 of 35 patients, but
only 5 of 35 matched controls, reported eating sliced mango from
street vendors in the 3 days before the illness began, the
probability of observing this difference in proportions by chance
alone is 0.000045, and the ratio of the odds of exposure is 15, a
measure of the strong association between disease and consuming
sliced mangos. When disease is statistically associated with more
than one exposure, multivariate analysis can identify truly
independent risk factors. In the above example, "eating food from
a street vendor" would not be independent from "eating sliced
mango" if one usually got sliced mango from a street vendor. This
case-control method can be rapidly carried out in the field at
low cost. 

Investigations showed that cholera was being transmitted by
several distinct mechanisms. The predominant route of
transmission in a given setting depends largely on the degree of
sanitation already achieved. Therefore, a multifaceted approach
to prevention is needed. Emergency measures, such as advice to
boil drinking water or to heat all foods from street vendors, are
difficult to sustain because of their inconvenience and high
cost. Moreover, the cost of building large-scale water treatment
and sanitation systems is extraordinary, estimated at $200
billion for all of Latin America (22). The challenge of cholera
prevention lies in devising low-cost alternatives that are both
effective and sustainable.

Waterborne transmission was identified in seven of the eight
investigations. In three of these, the implicated water came from
municipal systems or from tanker trucks that reportedly obtained
water from municipal systems. Water delivered through poorly
maintained municipal water systems can be contaminated by sewage
because of leaky pipes, frequent pressure drops, and the lack of
residual chlorine disinfectant in the water. In developing
countries, water is rarely available 24 hours a day so it is
usually stored in the home, where further contamination can
easily occur. For example, when we measured the increase in
contamination of the water as it was distributed and stored in
Trujillo, Peru, fecal coliform counts, an index of sewage
contamination, were 1/100 ml in water collected at the source
well, 2/100 ml at public taps, and 20/100 ml in water stored in
the home (13). In four investigations, the implicated water was
collected from rivers or ponds, where direct sewage contamination
was likely. Specific protective practices were also noted in
these investigations, including treating water in the home by
boiling it or by adding chlorine bleach, using a small-mouthed
vessel to store water, pouring water out of the storage vessel
rather than scooping it out with a cup, and having hand soap in
the home. In one investigation on the Amazon, we found that the
local common practice of adding citrus juice to water to improve
its taste was protective because the acid in the fruit killed
Vibrio bacteria (14). This observation gave local authorities a
new, inexpensive, and immediately available emergency control
measure.

The first stage of prevention, then, is providing safe drinking
water. As a result of the above findings, we have developed and
are testing simple and inexpensive methods of domestic water
disinfection and storage that would also prevent other diseases
transmitted by the same route. A pilot trial in a periurban area
of Bolivia showed that disinfecting household water with a
calcium hypochlorite solution and storing it safely in a special
narrow-mouthed container was acceptable to a community of Aymara
Indians (20). Compliance, as measured by chlorine residuals in
stored water, was high among families using the intervention. The
concentration of Escherichia coli bacteria in the stored water, a
measure of fecal contamination, was significantly lower in
households that used the intervention than in neighboring
households that used traditional water handling methods (23). A
field trial in rural Bolivia showed that villagers could generate
their own disinfectant solution by using a simple electrolytic
apparatus (24); with the disinfectant and the special water
container the villagers provided clean water in their homes.
Households using this intervention had 40% fewer diarrheal
episodes than randomly selected neighboring families who used
traditional water-handling methods. The combination of
point-of-use disinfection and safer water storage containers
could have broad effects, including local empowerment for
production of potable water, safer infant foods, and new
microindustries for the production of disinfectant solutions and
water vessels (25). Cost-benefit analysis indicates that this
strategy is cost saving if it prevents more than 20% of diarrheal
illnesses (26). Inexpensive disinfectant generators are now being
manufactured for this purpose in Ecuador. In Colombia, the number
of cholera cases has dropped dramatically since late 1992 when
chlorine disinfectant tablets were distributed for treating
household water.

The second major route of cholera transmission is food
contaminated in the market or home. This includes food and
beverages sold by street vendors, leftover rice, and unwashed
fruits and vegetables. This was an important route in four of the
eight investigated areas, including Guatemala City, where there
was no evidence of waterborne transmission (21). Foods and
beverages sold by street vendors are a fixture of urban life
throughout the developing world; they are often prepared in
unhygienic ways and then held at ambient temperatures for hours,
which permits rapid bacterial multiplication. Other problems
associated with food and beverages from street vendors included
using unsafe ice to chill beverages and selling homemade frozen
drinks. Leftover rice is an excellent growth medium for V.
cholerae O1 and eating leftover rice without reheating it was
associated with illness in three investigations. In one
investigation, illness was associated with eating unwashed
produce that was probably splashed with river water while being
transported to market in small boats.

Thus, the second stage of cholera prevention is to improve food
handling, particularly for foods and beverages sold by street
vendors. Many countries in Latin America have begun educating
street vendors in fundamental food safety and linking this
education to licensing (27). By itself, however, education may
not improve food safety if clean water to prepare foods and
beverages is not available and handwashing and dishwashing with
soap and water are not routine. We are field testing a combined
strategy of point-of-use disinfection, handwashing with soap, and
use of a special water/beverage container to improve the
microbial quality of beverages sold by street vendors. Because
street vendors are responsive to customer demand, teaching
consumers to look for street vendors that are visibly practicing
better hygiene may reinforce more hygienic conditions. In
addition to these efforts to improve food sold on the street,
health authorities should advise the public to reheat leftover
rice and wash fruits and vegetables before eating. In Santiago,
Chile, suspicion that cholera was caused by vegetables irrigated
with fresh sewage led to a ban on this practice; the ban not only
prevented cholera transmission by this route, but also decreased
the incidence of typhoid fever and hepatitis A dramatically (28).

Transmission through seafood (identified in two of the eight
investigations) is a third major route of cholera transmission,
distinct from other foodborne mechanisms, because it requires
different prevention strategies. One investigation implicated
both uncooked seafood and cooked crab, and another implicated
cooked seafood eaten without reheating. Contaminated seafood also
caused three outbreaks of travel-associated cholera in the United
States. In the most dramatic, at least 75 persons contracted
cholera after taking a flight from Latin America to California
(29); illness was associated with eating cold seafood salad that
was loaded onto the plane in Lima, Peru. Two other outbreaks
followed the informal transport of cooked crabs from Ecuador to
the United States in travelers' suitcases (30,31). Marine
creatures may harbor V. cholerae O1 before they are harvested or
may be contaminated by seawater used in seaside processing
plants. In areas where raw and undercooked seafood are popular,
seafood-associated cholera may occur even if the general level of
sanitation and hygiene is high (32). Vibrios survive light
cooking and can subsequently grow if the seafood is held for many
hours before eating (33).

Preventing seafood-associated cholera in the long term will
depend on maintaining sewage-free harvest beds and improving
sanitation in processing plants. In coastal areas where the
organism persists in the environment, even in the absence of
sewage contamination, education to discourage the consumption of
raw or undercooked shellfish is also needed. Thorough cooking
provides the greatest security, but it is sometimes resisted by
local populations for cultural reasons. "Ceviche," a popular
Latin American dish prepared from seafood that is marinated in
citrus juice for variable lengths of time, is a case in point.
Prolonged marination in acidic liquid is likely to inactivate
vibrios if the acid can penetrate throughout the flesh and deep
organs of the fish or shellfish (34). Further evaluation of this
approach is needed, but for the present, encouraging the use of
ceviche recipes that provide sufficient marination time may be a
practical intervention.

In Latin America, as in other parts of the world, epidemiologic
field investigations of cholera have defined the local routes of
transmission, identified unsuspected and correctable control
points, and quantified the effects of emergency measures. The
results of investigations also have generated specific control
strategies targeted to blocking the predominant routes. While
this multistage portrait of transmission is complex, it is being
translated into action and change. The longstanding deficits in
basic urban infrastructure and the need for new efforts to
correct them have never been more apparent (35,36). Workable
prevention strategies include better domestic water storage
containers, point-of-use water disinfection, attention to the
education and hygiene of street vendors, and simple modifications
of traditional recipes. Many other diseases are transmitted by
these same waterborne and foodborne routes, so these control
measures may prevent other infections in addition to cholera. If 
it becomes a catalyst for long overdue improvements in the safety
of water and food, epidemic cholera can have a far-reaching
impact on the public health of Latin America.

Robert V. Tauxe, M.D., M.P.H., Eric D. Mintz, M.D., M.P.H.,
Robert E. Quick, M.D., M.P.H.
National Center for Infectious Diseases, Centers for Disease
Control and Prevention, Atlanta, Georgia, USA

Table 1. Cholera cases by country, as reported to the Pan
American Health Organization, 1991 through 1994
                                          
                                 Number of reported cases         
                                 ______________________________
      
                Date of
Country        first report     1991      1992      1993    1994  
________________________________________________________________
Argentina      Feb. 5, 1992       0        553     2,080     889
Belize         Jan. 9, 1992       0        159       135       6
Bolivia        Aug. 26 1991     206     22,260    10,134   2,710
Brazil         Apr. 8, 1991   2,101     30,054    56,286  49,455
Chile          Apr. 12,1991      41         73        32       1
Colombia       Mar. 10,1991  11,979     15,129       230     996
Costa Rica     Jan. 3, 1992       0         12        14      38
Ecuador        Mar. 1, 1991  46,320     31,870     6,833   1,785
El Salvador    Aug. 19,1992     947      8,106     6,573  11,739
French Guiana  Dec. 14,1992       1         16         2     NRa
Guatemala      July 24,1991   3,652     15,686    30,605   4,227
Guyana         Nov. 5, 1992       0          5        66       0
Honduras       Oct. 13,1991      17        388     2,290   4,965
Mexico         June 13,1991   2,690      8,162    10,712   4,059
Nicaragua      Nov. 12,1991       1      3,067     6,631   7,821
Panama         Sept.10,1991   1,178      2,416        42       0
Paraguay       Jan. 25,1993       0          0         3       0
Peru           Jan. 23,1991 322,562    210,836    71,448  23,887
Suriname       Mar. 6, 1992       0          2         0       0
United States  Apr. 9, 1991      26        103        22      34
Venezuela      Nov. 29,1991      13      2,842       409       0

Total                       391,734    352,300   204,547 112,612
_________________________________________________________________
(a) NR = no reports received.
Source: ref. 2.



Table 2. Mechanisms of transmission of epidemic cholera in Latin
America, as determined in eight epidemiologic - investigations, 1991-1993(a_

           Peru                     Ecuador   El        Bolivia  Brazil     Guatemala
          Trujillo   Piura  Iquitos Guayaquil Salvador  Saipina  Fortaleza  Guatemala City
          Urban      Urban  Urban   Urban     Rural     Rural    Rural      Urban
           3/91       3/9    7/91    7/91      3/91      2/92     6/93+     7/93         
           (15)       (16)   (17)    (18)      (19)      (20)      (b)      (21)
           
Transmission
mechanism
____________________________________________________________________________________
    
Waterborne
Municipal   +         +                 +                      
water                            
   
Surface water                +                   +        +          +
                        
Putting            
hands in
water vessel               

Foodborne

Street vendors'      +      
foods                        
                                                                                +
Street vendors'      +                                                          +
beverages                    
                                
Street vendors'      +                                                          +
ice/ices   
                                    
Leftover rice        +            + 
                                            

Fruits/vegetables                            +

Seafood                                      

Uncooked seafood                              +
    

Cooked seafood                                +            +
                                                
________________________________________________________________________________
(a)Reference numbers are in parentheses.
(b) CDC Unpublished data.

References

1. Centers for Disease Control. Cholera Peru, 1991. MMWR
1991;40:108-9.
2. Pan-American Health Organization. Cholera in the Americas.
Epidemiol Bull 1995;16:11-3.
3. Wachsmuth IK, Evins GM, Fields PI, Olsvik, Popovic T, Bopp
CA, et al. The molecular epidemiology of cholera in Latin
America. J Infect Dis 1993;167:621-6.
4. Evins GM, Cameron DN, Wells JG, Greene KD, Popovic T,
Giono-Cerezo S, et al. The emerging diversity of the
electrophoretic types of Vibrio cholerae in the Western
Hemisphere. J Infect Dis 1995;172:173-9.
5. Ramamurthy T, Garg S, Sharma R, Bhattacharya SK, Nair GB,
Shimada T, et al. Emergence of a novel strain of Vibrio cholerae
with epidemic potential in southern and eastern India. Lancet
1993;341:703-4.
6. Mekalanos JJ, Sadoff JC. Cholera vaccines: fighting an ancient
scourge. Science 1994;265:1387-9.
7. Sanchez JL, Vasquez B, Begue RE, Meza R, Castellares G,
Cabezas C, et al. Protective efficacy of oral
whole-cell/recombinant- B- subunit cholera vaccine in Peruvian
military recruits. Lancet 1994;344:1273-6.
8. Levine MM, Tacket CO. Recombinant live oral vaccines. In:
Wachsmuth IK, Blake PA, and Olsvik  , editors. Vibrio cholerae
and Cholera. Washington, DC: American Society for Microbiology,
1994:395-413.
9. Rosenberg CE. The cholera years: The United States in 1832,
1849, and 1866. Chicago: University of Chicago Press, 1987. 
10. Blake PA. Epidemiology of cholera in the Americas.
Gastroenterol Clin North Am 1993;22:639-60.
11. Snow J. On the mode of communication of cholera. The
Commonwealth Fund. London: Oxford University Press, 1936.
12. Mintz ED, Popovic T, Blake PA. Transmission of Vibrio
cholerae O1. In: Wachsmuth IK, Blake PA, and Olsvik O, editors.
Vibrio cholerae and cholera. Washington, DC: American Society for
Microbiology, 1994:345-56.
13. Kolvin JL, Roberts D. Studies on the growth of Vibrio
cholerae biotype El Tor and biotype classical in foods. J Hyg
(Cambridge)
1982;89:243-52.
14. Blake PA, Rosenberg ML, Florencia J, Costa JB, Quintino L do
P, Gangarosa EJ. Cholera in Portugal, 1974. II. Modes of
transmission. Am J Epidemiol 1977;105:344-8.
15. Swerdlow DL, Mintz ED, Rodriguez M, Tejada E, Ocampo C,
Espejo L, et al. Waterborne transmission of epidemic cholera in
Trujillo, Peru: lessons for a continent at risk. Lancet
1992;340:28-32.
16. Ries AA, Vugia DJ, Beingolea L, Palacios AM, Vasquez E, Wells
JG, et al. Cholera in Piura, Peru: a modern urban epidemic. J
Infect Dis 1992;166:1429-33.
17. Mujica OJ, Quick RE, Palacios AM, Beingolea L, Vargas R,
Moreno D, et al. Epidemic cholera in the Amazon: The role of
produce in disease risk and prevention. J Infect Dis
1994;169;1381-4.
18. Weber JT, Mintz ED, Caizares R, Semiglia A, Gomez I,
Semprtegui R, et al. Epidemic cholera in Ecuador: multidrug
resistance and transmission by water and seafood. Epidemiol
Infect 1994;112:1-11.
19. Quick RE, Thompson BL, Zuniga A, Dominguez G, de Brizuela EL,
de Palma O, et al. Epidemic cholera in rural El Salvador: risk
factors in a region covered by a cholera prevention campaign.
Epidemiol Infect 1995;114:249-55. 
20. Gonzales O, Aguilar A, Antunez D, Levine W. An outbreak of
cholera in rural Bolivia: rapid identification of a major vehicle
of transmission. 32nd Interscience Conference on Antimicrobial
Agents and Chemotherapy, Anaheim, 1992. Washington, DC: American
Society for Microbiology 1992; Abstract 937.
21. Koo D, Aragon A, Moscoso V, Gudiel M, Bietti L, Carrillo N,
et al. Epidemic cholera in Guatemala, 1993: transmission of a
newly introduced epidemic strain by street vendors. Epidemiol
Infect 1995; (in press).
22. de Macedo CG. Presentation of the PAHO regional plan.
Proceedings of the Conference: Confronting cholera, the
development of a hemispheric response to the epidemic; 1991 Jul
8-9; Miami: University of Miami, 1991;39-44.
23. Quick R, Venczel L, Gonzales O, Damiani E, Highsmith A,
Espada A, et al. Impact of narrow-necked water vessels and home
chlorination on fecal coliform and E. coli counts in drinking
water. 33rd Interscience Conference on Antimicrobial Agents and
Chemotherapy, New Orleans, 1993. Washington, DC: American Society
for Microbiology, 1993.
24. Quick R, Venczel L, Mintz E, Bopp C, Soleto L, Bean N, et al.
Diarrhea prevention in Bolivia through safe water storage vessels
and locally-produced mixed oxidant disinfectant. 35th
Interscience Conference on Antimicrobial Agents and Chemotherapy,
San Francisco, 1995. Washington, DC: American Society for
Microbiology, 1995; Abstract 1347.
25. Mintz ED, Reiff FM, Tauxe RV. Safe water treatment and
storage in the home: a practical new strategy to prevent
waterborne disease. JAMA 1995;273:948-53.
26. Miller M, Quick R, Mintz E, Tauxe R, Teutsch S. Solid stools
and solvent citizens: an effective solution for preventing
diarrhea in developing countries. 34th Interscience Conference on
Antimicrobial Agents and Chemotherapy, Orlando, 1994. Washington,
DC: American Society for Microbiology, 1994. Abstract 1244.
27. Arambulo P, Almeida CR, Cuellar J, Bellotto AJ. Street food
vending in Latin America. Bull PAHO 1994;28:244-54.
28. Alcayaga S, Alcagaya J, Gassibe P. Changes in the morbidity
profile of certain enteric infections after the cholera epidemic.
Rev Chil Infect 1993;1:5-10.
29. Centers for Disease Control and Prevention. Cholera
associated with an international airline flight, 1992. MMWR
1992;41:134-5.
30. Finelli L, Swerdlow D, Mertz K, Ragazzoni H, Spitalny K.
Outbreak of cholera associated with crab brought from an area
with epidemic disease. J Infect Dis 1992;166:1433-5.
31. Centers for Disease Control. Cholera New York, 1991. MMWR
1991;40:516-8.
32. Lowry PW, Pavia AT, McFarland LM, Peltier BH, Barrett TJ,
Bradford HB, et al. Cholera in Louisiana: widening spectrum of
seafood vehicles. Arch Intern Med 1989;149:2079-84.
33. Blake PA, Allegra DT, Snyder JD, Barrett TJ, McFarland L,
Caraway CT, et al. Cholera a possible endemic focus in the United
States. N Engl J Med 1980;302:305-9.
34. Mata L. Efecto del jugo y de la pulpa de frutas acidas sobre
el Vibrio cholerae. In: El Clera: Historia, prevencin y
control. San Jos, Editorial Universidad Estatal a Distancia -
Editorial de la Universidad de Costa Rica, 1992, 275.
35. Sepulveda J, Gomez-Dantes H, Bronfman M. Cholera in the
Americas: an overview. Infection 1992;20:243-8.
36. Witt VM, Reiff FM. Environmental health conditions and
cholera vulnerability in Latin America and the Caribbean. J
Public Health Policy 1991;12:450-63.



(Pages 147-51)

Are North American Bunyamwera Serogroup Viruses Etiologic Agents
of Human Congenital Defects of the Central Nervous System?

In 1941 Gregg provided the first evidence that rubella virus
(family Togaviridae, genus Rubivirus) causes human congenital
defects (1). Although rubella virus infection usually causes a
mild disease comprising fever and rash, rubella epidemics have
been associated with congenital defects in children of women who
became infected during their first trimester of pregnancy (2).
The risk of in utero rubella infection was reduced by the
introduction of safe and effective vaccines for women of
child-bearing age. Congenital abnormalities in fetal or neonatal
ruminants also are related to exposure of pregnant dams to
various viruses, including bovine viral diarrhea virus (family
Togaviridae, genus Pestivirus), the arthropod-borne bluetongue
viruses (family Reoviridae, genus Orbivirus), Wesselsbron virus
(family Flaviviridae, genus Flavivirus), Rift Valley fever virus
(family Bunyaviridae, genus Phlebovirus), Nairobi sheep disease
virus (family Bunyaviridae, genus Nairovirus), and Akabane and
Aino (family Bunyaviridae, genus Bunyavirus, Simbu serogroup)
viruses (3-10). Infections of livestock with these viruses may
produce low-titer viremia with no apparent clinical disease, or
high-titer viremia and severe clinical illness in the dam.  In
utero infections may result in malformations of the developing
fetus, fetal death with resorption, mummification, or
miscarriage. Stillborn ruminants may show various musculoskeletal
and central nervous system defects, including a syndrome of
arthrogryposis with hydranencephaly (AGH).

Bunyamwera serogroup viruses (family Bunyaviridae, genus
Bunyavirus) have been isolated from humans, and some, including
Cache Valley (CV) and Tensaw (TEN) viruses, have been isolated
from symptomatic and asymptomatic large mammals (11). Antibodies
to CV virus and other viruses of the Bunyamwera serogroup are
prevalent in livestock and large wild mammals and in humans in
the Western Hemisphere from Alaska to Argentina (11). Viruses of
this serogroup are isolated primarily from mosquitoes of the
genera Aedes and Anopheles. These viruses have focal geographic
distributions, although some are found over great expanses. CV
virus, a common North American bunyavirus, has been isolated
principally from mosquitoes of the genera Culiseta, Aedes, and
Anopheles. The geographic distribution of this virus includes all
North America, except the extreme southeastern states and
southern Mexico (11). In the southeastern United States, TEN
virus, also isolated from mosquitoes of the genera Anopheles and
Aedes, is the only known representative of the Bunyamwera
serogroup, probably because of the range of the principal vectors
and vertebrate hosts; mutual exclusion of these two viruses
likely occurs because of cross-protectivity between them (12).

Serologic and temporal associations of infection with CV virus
and congenital malformations, including primarily AGH, were
observed in sheep near San Angelo, Texas, between December 1986
and February 1987, suggesting that this virus causes AGH (13).
Subsequent outbreaks of similar congenital defects occurred in
sheep in Illinois in 1988 (J. Pearson, pers. comm.) and in North
Dakota, Pennsylvania, Maryland, Michigan, and Nebraska in 1986
and 1987 (14). Antibody to CV virus (but not to other viruses)
was found to correlate significantly with the occurrence of AGH
and other congenital anomalies during the Texas outbreak (15,
16), and IgM antibody to CV virus was detected in colostrum-free
neonates with AGH (C.H. Calisher, unpublished data). (Neither
maternal IgM nor maternal IgG crosses the placenta in sheep;
therefore, antibody in fetuses or in neonates before they
received colostrum indicates fetal exposure to an infectious
agent [17].)

In 1976, antibody to CV virus was detected in serum from cattle
that had delivered calves with AGH in Saskatchewan, Canada, in
1975; however, the prevalence of antibodies to CV virus in the
bovine population of that area was not investigated (R.E. Shope,
pers. comm.). In Texas, in 1981, CV virus was isolated from a
sick sheep and from a healthy cow in a herd with reproductive
problems (18). This virus also was isolated in Texas in 1988 from
a sentinel sheep in pasture where an outbreak of congenital
defects had occurred in 1986 to 1987. These historical data
suggest that CV-virus-related congenital malformations may be
more widespread than has been recognized.

Experimental infections have provided further evidence that CV
virus causes embryonic death and multiple congenital
malformations of sheep (19) and cattle (J. Edwards, pers. comm.).
No association of TEN virus and disease in livestock, wild
animals, or humans has been reported.  To determine whether
infection with selected Bunyamwera serogroup viruses, CV and TEN,
is associated with certain human congenital defects, a serosurvey
was done with serum samples from mothers of children with
microcephaly or macrocephaly, and the results were compared with
those from age- and location-matched controls.  The results,
reported here, provide the first evidence that these Bunyamwera
serogroup viruses may be etiologic agents of certain congenital
defects of the human central nervous system.

Two groups of 500 each human serum samples were selected at
random from an archival collection stored at the National
Institutes of Health (NIH), Bethesda, Maryland, and tested for
neutralizing antibody. These samples were part of a collection of
serum specimens obtained between 1959 and 1964, at delivery or
post partum, (from about 50,000 women enrolled under defined
protocols in a prospective study of congenital rubella syndrome.
Specimens had been collected from all pregnant women cared for at
the particular institution or from women randomly selected by
using the last digit of their hospital registration number.

For a first series of tests, samples were selected from 200
mothers of children with macrocephaly (head size at least two
standard deviations above the mean) and from 50 mothers of
children with microcephaly (head size at least two standard
deviations below the mean). The initial specimen from mothers
with the respective defects was selected for testing. An equal
number of mothers of babies without obvious central nervous
system defects were selected as controls, which were age- ( + or
= 2 years) and site-matched, were registered for the study in the
same month, and were of the same race as mothers of children with
either macrocephaly or microcephaly. Serum samples comprising
this first group had been collected from pregnant women in
Boston, Massachusetts (104), Providence, Rhode Island (20), New
York, New York (72), Philadelphia, Pennsylvania (36), Baltimore,
Maryland (38), Buffalo, New York (50), Minneapolis, Minnesota
(42), Charlottesville, Virginia (38), Memphis, Tennessee (52),
New Orleans, Louisiana (36), and Portland, Oregon (12); these
were tested for neutralizing antibody to eight bunyaviruses
(Table 1). 

An additional 500 samples were tested; 250 paired samples,
selected as above, from the same archival collection, two each
from women in Boston (120), Buffalo (40), New Orleans (8), New
York City (46), Baltimore (8), Charlottesville (12), and
Minneapolis (16), including controls (selected as above). These
were tested for neutralizing antibody to CV virus only, to follow
up on results of tests with the first set of samples. One sample
from each of these women had been collected in the first
trimester of pregnancy, and the second had been collected at
least 3 months later. Serum specimens were stored frozen at -20
degrees C until they were shipped on dry ice (-70 degrees C) to
the Centers for Disease Control laboratory at Fort Collins,
Colorado.

Twenty-nine of the first 500 serum specimens tested contained
neutralizing antibody to CV, 29 had antibody to TEN, 29 to
Jamestown Canyon, 26 to La Crosse, nine to Lokern, and six to
Buttonwillow viruses. None had antibody to Main Drain or Mermet
virus. No significant differences were observed in antibody
prevalences to La Crosse, Jamestown Canyon, Lokern, and
Buttonwillow viruses between mothers of microcephalic and
macrocephalic infants and age- and location-matched controls
(Table 1). Cases were not reviewed for other defects.

The prevalence of antibody to CV virus in mothers of
microcephalic infants was not significantly different from the
prevalence of such antibody in their matched control, but the
presence of antibody to CV virus in mothers was significantly
correlated with macrocephaly in their infants ( 2, d.f. = 1, n =
400, 4.797 p <0.05) (Table 1).

None of the samples with neutralizing antibody to CV or TEN virus
(titers ranged from 10 to 80) had IgM antibody. To determine
whether there were statistically significant differences between
prevalences of antibody to CV or TEN virus in mothers of
macrocephalic infants and in their age- and site-matched
controls, McNemar's chi-square was used. No significant
difference was found for the presence of antibody to CV virus (p
>0.05), but the presence of antibody to TEN virus (p <0.05 or
antibody to either CV or TEN virus (p <0.02) was related to the
occurrence of macrocephaly in the infants of these mothers (Table
2). 

Table 3 summarizes the presence of antibody to CV and to TEN
virus in women by hospital location and birth outcome. When
prevalence of antibody to CV or TEN virus in these women was
analyzed by location and birth outcome, no statistically
significant differences were determined (comparative data not
shown). 

When the second set of 500 specimens (250 paired early- and
late-pregnancy samples) was tested, specimens from eight women
had neutralizing antibody to CV virus. No diagnostically
meaningful change in titer was detected between six sample pairs,
but fourfold rises in titer were detected in two others, (10 to
80,  <10 to 40), indicating recent infections with CV virus or
with a closely related Bunyamwera virus group. Six of the eight
women in this group with antibody to CV virus delivered
macrocephalic infants, including the two (one in New Orleans and
one in New York City) whose specimens showed rises in titer to CV
virus.

These analyses provide the first evidence that Bunyamwera
serogroup viruses in North America are associated with congenital
defects in humans: the occurrence of macrocephaly in infants was
positively correlated with antibody to CV virus. Antibody to TEN
virus and to either CV or TEN virus correlated with microcephaly
and with macrocephaly. The presence of antibody to CV and to TEN
viruses corresponded with the known geographic distributions of
these viruses within the United States. Antibody to either of
these viruses in a woman living in an area where that virus is
not known to occur may reflect the close antigenic relationships
and considerable cross-reactivity of these Bunyamwera serogroup
viruses (22), differences between local virus strains and
prototype viruses, or travel to an area in which the virus does
occur (23). The two women with diagnostically significant rises
in antibody titer to CV virus (one from New Orleans, where TEN
virus has been isolated, and one from New York, where CV virus
has been isolated) are, as far as we know, the first two persons
with documented rises in antibody to a Bunyamwera serogroup virus
in North America. Whether either of them had an associated
illness could not be determined from the records. That among all
the women tested they were the only ones with rises in antibody
titer and that both gave birth to macrocephalic infants is, at
the least, a fascinating coincidence. IgM antibody in human
infections caused by other bunyaviruses may not persist for much
more than a few months after infection (21); IgM antibody has not
been detected in humans with antibody to Bunyamwera serogroup
viruses in North America. Therefore, the absence of IgM antibody
in specimens with neutralizing antibody likely indicates that
these infections were not acute, i.e., they occurred months
before the specimens were collected.

A fundamental problem in establishing a relationship between
infection of mammals with CV or TEN virus and attendant
congenital anomalies is the inherent inadequacy of controls. The
presence of antibody in humans or other animals with normal
offspring does not necessarily argue against the hypothesis that
CV virus causes congenital defects in humans because infection
could have occurred before pregnancy. Additionally, even if this
virus can be an etiologic agent of congenital anomalies,
preexisting antibody to this virus could provide immunity for the
mother and protect the fetus from viral infection. Thus, it
cannot be determined with certainty whether the presence of
antibody to a virus is coincidental to, or a cause of, the
observed congenital anomalies.

Review of NIH records for this relatively small set of samples
suggested that macrocephaly occurred somewhat more often when the
first trimester of pregnancy included the months April and May
for women living in New Orleans (4/16), Memphis (5/16), and
Charlottesville (3/12) and in late summer-early autumn for women
living in Boston (6/49), Minneapolis (3/11), Portland (2/6), and
New York City (9/33). In each instance, these periods coincide
roughly with the appearance of populations of Culiseta, Aedes, or
Anopheles mosquitoes, the vectors of CV and TEN viruses. However,
CV virus cannot be implicated in infections in New Orleans
because this virus is not known to occur there, although TEN
virus does.

The relatively small sample sizes in this study allow statistical
interpretation but do not provide sufficient support to warrant
statements as to the biological significance of the findings;
therefore, we consider these data merely preliminary. Determining
whether these data have merit awaits the results of additional
studies of mothers of children with congenital defects and their
offspring. More extensive studies are also needed to investigate
the influence of gestational phase and fetal development on
congenital defects; establish relationships between peak
abundance of arthropod vectors and first trimesters of
pregnancies; sequence the genomes of CV and TEN virus strains
from various geographic areas and establish relationships between
different gene sequences and virulence in livestock; and develop
diagnostic capacity by using monoclonal antibodies, hybridization
assays, polymerase chain reaction, and Western blotting
techniques. 

Given that many members of the family Bunyaviridae cause
congenital defects in naturally and experimentally infected
livestock, or may have such a potential (24,25), it would be
worthwhile to continue investigations with domestic animals and
to develop laboratory models to assess the teratogenic potential
for humans of CV, TEN, and other viruses of the family
Bunyaviridae. 

It is also important to determine the roles of CV and TEN viruses
in inducing human congenital defects and the relationships
between prevalence of antibody to CV and TEN viruses, prevalence
of congenital defects, and conception dates, all with respect to
local environmental conditions.

Charles H. Calisher, Ph.D.,* and John L. Sever, M.D.^
*Colorado State University, Fort Collins, Colorado, USA; 
^George Washington University Medical Center, Children's National
Medical Center, Washington, D.C., USA

Acknowledgments

The authors thank Dr. Maneth Gravell and Ms. Dorothy O'Neill,
National Institutes of Health, Bethesda, Maryland, for selecting,
sorting, and shipping serum samples; Mr. Raymond E. Bailey,
Centers for Disease Control and Prevention, Fort Collins,
Colorado, for statistical advice; Dr. Thomas P.C. Monath,
Division of Vector-Borne Infectious Diseases, CDC, Fort Collins,
for his encouragement and support; and Drs. Barry Beaty, Colorado
State University, Fort Collins, and John Edwards, Texas A&M
University, College Station, Texas, for their editorial
suggestions.


Table 1. Antibody to Cache Valley, Tensaw, La Crosse, Jamestown
Canyon, Lokern, or Buttonwillow viruses in mothers of
microcephalic or macrocephalic infants and matched controls

Antibody to virus     Infant's condition      Xc( )

Cache Valley           Microcephaly           3.840(0.05)
                       Macrocephaly           4.797(<0.05)
                       Either                 0.915(>0.20)

Tensaw                 Microcephaly           4.891(<0.05)
                       Macrocephaly           4.071(<0.05)
                       Either                 0.329(>0.20)

Cache Valley           Microcephaly           5.983(<0.02)
 or Tensaw             Macrocephaly           4.806(<0.05)

La Crosse              Microcephaly           0.211(>0.20)
                       Macrocephaly           1.481(>0.20

Jamestown Canyon       Microcephaly           0.709(>0.20)
                       Macrocephaly           0.037(>0.20)

Lokern                 Microcephaly           1.010(>0.20)
                       Macrocephaly           2.041(>0.10)

Buttonwillow           Microcephaly           2.041(>0.10)
                       Macrocephaly          <0.001(>0.20)
__________________________________________________________________________
Bunyaviruses used for all tests were prototypes: (Bunyamwera serogroup) 
CV (strain 6V-633), TEN (A9-171b), Lokern (FMS-4332), Main Drain 
(BFS-5015), (California serogroup) La Crosse (Original), Jamestown 
Canyon (61V-2235), (Simbu serogroup) Buttonwillow (A-7956), and 
Mermet (AV-782). Samples were tested for antibody by serum
dilution-plaque reduction neutralization (20). Briefly, diluted and heat-
inactivated o C/30 min) serum was added to an equal volume of virus
containing approximately 200 plaque-forming units (PFU), such that the final 
virus dilution was 100 PFU. Fresh human serum at a final concentration of 8% was added to
all virus suspensions. Serum-virus mixtures were incubated at 4oC for 18 h, and 0.1-ml
aliquots were dropped in the center of Vero cultures grown in 6-well plastic plates. After
a 45-min period of adsorption, cells were overlaid with medium containing 2% agar and
further incubated for 70 to 75 h at 37 degrees C in 5% CO2-95% air. A second overlay,
containing medium, 2% agar, and 1:25,000 neutral red was then added, and the plates again
were incubated until plaques were readable, usually 12 to 36 h later.
A serum specimen was considered positive when it reduced the number of plaques 90%
relative to control titrations, which ranged from 80 to 150 plaques. Samples that were
positive in a screening (1:10) test were titrated for end-point, and the serum titer was
taken as the highest twofold dilution of serum that reduced the number of
plaques 90%.

Although a human positive control for detecting IgM antibody by capture enzyme-linked
immunosorbent assays was not available, serum specimens were tested by a modification of a
published technique for detecting IgM antibody to California serogroup viruses (21). Mouse
or sheep serum samples containing IgM antibody to CV virus or mouse serum with IgM
antibody to TEN virus served as positive and
negative IgM antibody controls. Statistical analyses were done by chi-square 
or McNemar's chi-square.




Table 2. Presence of antibody to Cache Valley and Tensaw viruses in mothers of
macrocephalic infants and in matched controls

Mothers of                         Antibody-positive     Antibody-negative
macrocephalic infants    No.       controls              controls  
_____________________________________________________________________________            

Antibody to CV virus      16             3                     13
No antibody to CV virus  184             5                    179
McNemar's test(a)

Antibody to TEN virus     15             1                     14
No antibody to TEN virus 185             5                    180
McNemar's test(b)

Antibody to CV or TEN     19             3                     16
virus (or both)        
                                  
No antibody to CV        181             5                    176
or TEN virus            
McNemar's test(c)
____________________________________________________________________________ 
(a) (13-5)2/18 = 3.556 (d.f. = 1), p > 0.05. 
(b) (14-5)2/19 = 4.263 (d.f. = 1), p <0.05.
(c) (16-5)2/21= 5.762 (d.f. = 1), p <0.02.


Table 3. Antibody to Cache Valley and Tensaw viruses in women, by location and birth
outcome

             Antibody to CV virus                    Antibody to TEN virus      
             _____________________________________   ______________________________________
Location     In women with             In            In women with                In       
             macrocephalic infants:    controls:     macrocephalic infants:     Controls:
             no.(%)                    no.(%)        no.(%)                      no.(%)   
_____________________________________________________________________________
Boston         2/49(4.1)               1/49(2.0)      1/49(2.0)                 1/49(2.0)
Providence     0/7                     1/7(14.3)      0/7                       1/7(14.3)
New York City  2/33(6.1)               1/33(3.0)      1/33(3.0)                 1/33(3.0)
Philadelphia   0/15                    2/15(13.3)     1/15(6.7)                 2/15(13.3)
Minneapolis    1/11(9.1)               0/1            1/11(9.1)                 0/11 
Charlottes-    2/12(16.7)              0/12           2/12(16.7)                0/12 
 ville
Memphis        6/16(37.5)              3/16(18.8)     5/16(31.3)                1/16(6.3)
New Orleans    1/16 (6.3)              0/1            2/16(12.5)                0/16 
Portland       2/6 (33.3)              0/6            2/6(33.3)                 0/6 




References 

1. Gregg N McA. Congenital cataract following German measles in
the mother. Trans Ophthal Soc Austral 1941;3:35-46.
2. Best JM, O'Shea S. Rubella virus. In: Schmidt NJ, Emmons RW,
editors. Diagnostic procedures for viral, rickettsial and
chlamydial infections, 6th ed. Washington, DC: American Public
Health Association 1989:731-95.
3. Oberst RD. Viruses as teratogens. Vet Clin North Am; Food Anim
Prac 1993:9:23-31.
4. Erasmus BJ. The history of bluetongue. In: Barber TL, Jochim
MM, editors. Bluetongue and related orbiviruses. New York: AR
Liss, 1985:7-12.
5. Calisher CH, Monath TP. Togaviridae and Flaviviridae: the
alphaviruses and flaviviruses. In: Lennette EH, Halonen P, Murphy
FA, editors. Laboratory diagnosis of infectious diseases, vol. 2.
New York: Springer-Verlag, 1988:414-34.
6. Calisher CH, Shope RE. Bunyaviridae: the bunyaviruses. In:
Lennette EH, Halonen P, Murphy FA, editors. Laboratory diagnosis
of infectious diseases, vol. 2. New York: Springer-Verlag,
626-46.
7. Kurogi H, Inaba Y, Takahashi E, Sato K, Omori T, Miura Y, et
al. Epizootic congenital arthrogryposis- hydranencephaly
syndrome in cattle: isolation of Akabane virus from
affected fetuses. Arch Virol 1976;51:67-74. 
8. Kurogi H, Inaba Y, Takahashi E, Sato K, Satoda K, Goto Y, et
al. Congenital abnormalities in newborn calves after inoculation
of pregnant cows with Akabane virus. Infect Immun 1977:17:338-43.
9. Parsonson IM, Della-Porta AJ, Snowdon WA, Murray MD.
Congenital abnormalities in foetal lambs after inoculation of
pregnant ewes with Akabane virus. Austral Vet J 1975:51:585-6.
10. Coverdale OR, Cybinski DH, St. George TD. Congenital
abnormalities in calves associated with Akabane and Aino virus.
Austral Vet J 1978:54:151-2.
11. Calisher CH, Francy DB, Smith GC, Muth DJ, Lazuick JS,
Karabatsos N, et al. Distribution of Bunyamwera serogroup viruses
in North America, 1956-1984. Am J Trop Med Hyg 1986;35:429-43. 
12. Calisher CH. Evolutionary significance of the taxonomic data
regarding bunyaviruses of the family Bunyaviridae.  Intervirology
1988;29:268-76.
13. Crandell RA, Livingston CW. Laboratory investigation of a
naturally occurring outbreak of arthrogryposis-hydranencephaly in
Texas sheep. J Vet Diagn Invest 1988;1:62-5.
14. Rook JS, Yamini B, Steficek B. AGH syndrome: cooperation
answers questions. National Wool Grower 1988(April):24-5.
15. Edwards JF, Livingston CW, Chung SI, Collisson EC. Ovine
arthrogryposis and central nervous system malformations
associated with in utero Cache Valley virus infection:
spontaneous disease. Vet Pathol 1989;26:33-9.
16. Edwards JF. Cache Valley virus. Vet Clin North Am; Food Anim
Prac 1994;10:515-24.
17. Campbell SG, Siegel MJ, Knowlton BJ. Sheep immunoglobulins 
and transmission to the neonatal lamb. N Z Vet J 1977;25:361-5.
18. McConnell S, Livingston C Jr, Calisher CH, Crandell R.
Isolations of Cache Valley virus in Texas, 1981. Vet Microbiol
1987;13:11-8. 
19. Chung SI, Livingston CW Jr, Edwards JF, Gauer BB, Collisson
EW. Congenital malformations in sheep resulting from in utero
inoculation of Cache Valley virus. Am J Vet Res 1990;51:1645-8.
20. Lindsey HS, Calisher CH, Mathews JH. Serum dilution
neutralization test for California group virus identification and
serology. J Clin Microbiol 1976;4:503-10.
21. Calisher CH, Pretzman CI, Muth DJ, Parsons MA, Peterson ED.
Serodiagnosis of La Crosse virus infections in humans by
detection of immunoglobulin M class antibodies. J Clin Microbiol
1986;23:667-71.
22. Calisher CH, Lazuick JS, Lieb S, Monath TP, Castro KG. Human
infections with Tensaw virus in south Florida: evidence that
Tensaw virus subtypes stimulate the production of antibodies
reactive with closely related Bunyamwera serogroup viruses. Am J
Trop Med Hyg 1988;39:117-22.
23. Calisher CH, Sabattini MS, Wolff KL, Monath TP.
Cross-neutralization tests of South American isolates of Cache
Valley virus revealing the existence of multiple subtypes. J Trop
Med Hyg 1988;39:202-5.
24. Parsonson IM, Della-Porta AJ, Snowdon WA. Developmental
disorders of the fetus in some arthropod-borne virus infections.
Am J Trop Med Hyg 1981;30:660-73.
25. McPhee DA, Parsonson IM, Della-Porta AJ, Jarrett RG.
Teratogenicity of Australian Simbu serogroup and some other
Bunyaviridae viruses: the embryonated chicken egg as a model. 
Infect Immun 1984;43:413-20.


(Pages 152-3)

Lymphocytic Choriomeningitis Virus: An Unrecognized Teratogenic
Pathogen

Lymphocytic choriomeningitis virus (LCMV), the first member of
the arenavirus family to be isolated, is the causative agent of a
zoonosis acquired from chronically viremic mice or hamsters (1).
The clinical spectrum of acquired human LCMV infection ranges
from inapparent and asymptomatic to, in rare instances, severely
symptomatic, systemic, and fatal central nervous system (CNS)
disease. Intrauterine LCMV infection has resulted in fetal or
neonatal death, as well as hydrocephalus and chorioretinitis in
infants (2-6). We have diagnosed congenital LCMV infection in
three infants (7) and have collated published and unpublished
data on three additional affected infants (8, G.R. Istre, pers.
comm.). This report briefly summarizes the salient features of
the infection in five of these six American infants and outlines
the similarities between these and features observed earlier in
Europe. We suggest that LCMV is a more frequent cause of CNS
disease in newborns than previously recognized.

Congenital LCMV infection was first recognized in Great Britain
in an infant who died at 12 days of age (3). Subsequently, fetal
infection with spontaneous abortion (2) and congenital infection
in liveborn infants with hydrocephalus and chorioretinitis were
documented in Germany (4), France (6), and Lithuania (5). We have
recently documented congenital LCMV infection in three infants
from Arizona (7) and have obtained information regarding three
additional neonates from Arizona, Nebraska (8), and Texas (G.R.
Istre, pers. comm.). Detailed clinical and laboratory data are
available for five of the six infants. All displayed
nonobstructive hydrocephalus with periventricular calcifications,
chorioretinitis, and psychomotor retardation. One of the five
infants had sensorineural deafness. None of the infants had
cardiac abnormalities. Two infants have had follow-up
ophthalmologic and audiologic examinations which have shown
neither the progression of chorioretinitis nor the development of
new auditory deficits. Toxoplasma gondii, cytomegalovirus, Herpes
simplex virus, rubella, enterovirus, and Treponema pallidum
infections were excluded by culture or serology in all infants.
The diagnosis of congenital LCMV infection was confirmed in all
infants by immunofluorescence antibody (IFA) and enzyme-linked
immunosorbent assays (ELISAs). In addition, serum, CSF, urine,
and throat wash specimens from two infants were injected into
Vero cell monolayers. Neither cytopathic effect nor LCMV antigens
were detected after incubation. Because virus isolation was only
attempted after the disease was first diagnosed when the children
were 10 months of age, failure to isolate LCMV was not
unanticipated.

Laboratory diagnosis of LCMV infection is generally made by
serologic techniques. IFA is a more sensitive diagnostic method
than either complement fixation or neutralizing antibody
techniques (9,10). The newer ELISAs are now being used to
evaluate congenitally infected infants. Testing the child's serum
and CSF and a simultaneously obtained serum specimen from the
mother yields the maximum information if done as soon after birth
as possible.

The mothers of four of the five infants in this report had a
history of febrile illness during pregnancy, in contrast to a
minority of mothers of affected infants previously reported.
Typical LCMV infection in adults is a biphasic disease with
fever, malaise, myalgias, anorexia, nausea, vomiting,
pharyngitis, cough, and adenopathy followed by defervescence and
a second phase of CNS disease. However, CNS symptoms may appear
without any prodrome or may never develop. Meningitis and
meningoencephalitis are the most frequent neurologic
manifestations of disease, although myelitis, Guillain-Barr
syndrome, and sensorineural deafness have been reported (11).
Between 1941 and 1958, 8% to 11% of viral CNS syndromes in
hospitalized patients in a Washington, D.C., medical center were
etiologically associated with LCMV (12). Arthritis, parotitis,
orchitis, myocarditis, and rash have also been noted (13).
Clinical interest in LCMV, however, has not been maintained, and
the disease is rarely considered despite improved serodiagnostic
methods.

Although a history of contact with rodents and their excreta is
of diagnostic utility, it is not universally present. A maternal
history of rodent exposure was elicited for three of our five
infants. Wild mice (Mus musculus) and hamsters infected in utero
with LCMV during maternal viremia develop both persistent viremia
and viruria. The virus is transmitted to humans by direct animal
contact; by contact with infected rodent saliva, nasal
secretions, urine, feces, semen, and milk; and by infectious
aerosols (1). Human-to-human transmission has not been
documented. The distribution of LCMV is highly variable within
mouse populations. Seasonal, annual, and cyclical variations in
rodent density and infection have been postulated but remain
inadequately studied (14). LCMV spreads to humans in rural
settings or when human habitats are substandard. Infected
laboratory and pet rodents have also been associated with disease
in humans (1). Serologic surveys and clinical studies have
documented both epidemic and endemic human infection in Europe
and the Americas. In Baltimore, 9.0% of house mice and 4.7% of
residents have had measurable LCMV antibody (15,16).

We hypothesize that congenital LCMV infection is generally
undiagnosed and may account for unexplained hydrocephalus with
microcephaly or macrocephaly, deafness, blindness, and mental
retardation (three of the five infants in this report were
referred for infectious disease consultation by pediatric
geneticists, and two were referred by pediatric neurologists). No
accurate data are available regarding the prevalence and
persistence of LCMV antibodies in unselected infants, children,
and adults in diverse geographic locales or in children with
unexplained visual and/or auditory deficits, microcephaly, and
retardation. Increased recreational activities in rural
environments, rehabilitation of and habitation in older
rodent-infected domiciles, and acquisition of unscreened rodents
for pets or laboratory use pose as yet undefined risks for LCMV
infection to the fetus, child, and adult. The need for further
research to define the frequency of LCMV infection in human and
animal populations is clear. LCMV infection can best be prevented
by educating the public and medical professionals on the hazards
of contact with infected rodents.

Leslie L. Barton, M.D.,* C.J. Peters, M.D.,+  T.G. Ksiazek,
D.V.M., Ph.D.+
*University of Arizona Health Sciences Center, Department of
Pediatrics and Steele Memorial Childrens Research Center, Tucson,
Arizona, USA; +National Center for Infectious Diseases, Centers
for Disease Control and Prevention, Atlanta, Georgia, USA

Acknowledgments
We thank Drs. Jane Wilson, Gregory Istre, Stephen Chartrand, and
Laurie Seaver for patients' information; Dr. Matafija Seinbergas
for his enthusiastic support; and Ms. Amy Sites for technical
support.

References
1. Jahrling PB, Peters CJ. Lymphocytic choriomeningitis: a
neglected pathogen of man. Arch Pathol Lab Med 1992;116:486-8.
2.  Ackermann R, Stammler A, Armbruster B. Isolierung von Virus
der lymphozytaren Choriomeningitis aus Abrasionsmaterial nach
Kontakt der Schwangeren mit einem Syrischen Goldhamster
(Mesocricetus auratus). Infection 1975;3:47-9.
3. Komrower GM, Williams BL, Stones PB. Lymphocytic choriomen-
ingitis in the newborn: probable transplacental infection. Lancet
1955;1:697-8.
4. Ackermann R, Korver G, Turss R, Wonne R, Hochgesand P.
Prenatal infection with the lymphocytic choriomeningitis virus.
Dtsch Med Wochenschr 1974;13:629-32.
5. Seinbergas MM. Hydrocephalus due to prenatal infection with
the lymphocytic choriomeningitis virus. Infection 1976;4:185-91.
6. Chastel C, Bosshard S, Le Goff F, Quillien MC, Gilly R, Aymard
M. Infection transplacentaire par le virus de la choriomeningite
lymphocytaire: resultats dune enguete serologique retrospective
en France. Nouv Press Med 1978;7:1089-92.
7. Barton LL, Budd SC, Morfitt WS, et al. Congenital lymphocytic
choriomeningitis virus infection in twins. Pediatr Infect Dis J
1993;12:942-6.
8. Larsen PD, Chartrand SA, Tomashek KY, Hauser LG, Ksiazek TG.
Hydrocephalus complicating lymphocytic choriomeningitis virus
infection. Pediatr Infect Dis J 1993;12:528-31.
9.Lewis VJ, Walter PD, Thacker WL, Winkler WG. Comparison of
three tests for the serological diagnosis of lymphocytic
choriomeningitis virus infection. J Clin Microbiol 1975;2:193-7.
10. Lehmann-Grube F, Kallay M, Ibscher B, Schwartz R. Serologic
diagnosis of human infections with lymphocytic choriomeningitis
virus: comparative evaluation of seven methods. J Med Virol
1979;4:125-36.
11. Lehmann-Grube F. Diseases of the nervous system caused by
lymphocytic choriomeningitis virus and other arenaviruses. In:
Handbook of clinical neurology. New York: Elsevier,
1989;12:355-81.
12. Meyer HM, Johnson RT, Crawford, IP, Dascomb HE, Rogers NG.
Central nervous system syndromes of "viral" etiology: a study of
713 cases. Am J Med 1960:334-47.
13. Lewis JM, Utz JP. Orchitis, parotitis and meningoencephalitis
due to lymphocytic-choriomeningitis virus. N Engl J Med
1961;265:776-80.
14.Childs JE, Peters CJ. Ecology and epidemiology of arenaviruses
and their hosts. In: The Arenaviridae. New York: Plenum,
1993:331-84.
15. Childs JE, Glass GE, Korch GW, Ksiazek TG, Leduc JW.
Lymphocytic choriomeningitis virus infection and house mouse (Mus
musculus) distribution in urban Baltimore. Am J Trop Med Hyg
1992;47:27-34.
16. Childs JE, Glass GE, Ksiazek TG, Rossi CA, Barrera Oro JG,
Leduc JW. Human-rodent contact and infection with lymphocytic
choriomeningitis and Seoul viruses in an intercity population. Am
J Trop Med Hyg 1991;44:117-21.


COMMENTARY

(Pages 154-55)

Hemolytic Uremic Syndrome

Along with a report of the first outbreak of hemolytic uremic
syndrome (HUS) caused by Shiga-like toxin (SLT) producing E. coli
in Australia (1), this issue of Emerging Infectious Diseases
presents three papers detailing the investigations of pediatric HUS
cases linked to Shiga toxin (ST) and SLT producing bacteria.
Goldwater and Bettelheim present a case of pediatric HUS associated
with SLT producing Escherichia coli (SLTEC) O48:H21 in South
Australia; this strain has not previously been recognized as an
SLTEC. Saeed et al. report on the increasingly common
identification of HUS in Saudi Arabia, its association with
multiple-antibiotic-resistant Shigella dysenteriae type 1, and the
inherent dangers of treating such patients with ampicillin and
nalidixic acid. Al-Qawari et al. report on the results of active
surveillance for dysentery and HUS in Saudi Arabia and discuss a
possibly elevated risk for HUS in patients with bloody diarrhea who
are hospitalized and treated with nalidixic acid during an outbreak
of S. dysenteriae type 1.

The three papers raise a number of important issues regarding HUS.
First, it is clear that a large number of SLT producing bacteria
have the potential to cause HUS, particularly among children.
Current research has focused on E. coli O157:H7, which since the
early 1980s has emerged as a major foodborne cause of bloody
diarrhea, hemorrhagic colitis, and HUS since the early 1980s (5-7).
However, the large outbreak of pediatric HUS in South Australia in
1995 caused by foodborne E. coli O111:HNM has demonstrated that
minor pathogens can emerge as major causes of HUS (1). Goldwater
and Bettelheim (5) and other researchers have identified a number
of E. coli serotypes isolated from patients with HUS (6,7).
A-Qawari et al. and Saeed et al. offer a timely reminder that S.
dysenteriae type 1, a pathogen with a human-only reservoir, is an
equally serious contender in HUS etiology and pathogenesis when
conditions facilitate the person-to-person transmission of
pathogens.

The second key issue raised by the latter two papers concerns
treatment of bloody diarrhea. Both discuss the potential for
antibiotic (ampicillin and nalidixic acid)-mediated HUS and
conclude that this issue should be carefully evaluated before
antibiotics are used to manage bloody diarrhea. Saeed et al. note
that the wide variety of antibiotics used to treat bloody diarrhea
in Saudi Arabia could be explained by the various prescription
practices of doctors recruited from different parts of the world.
Antibiotic resistance is a worrying component of the mechanisms of
emerging infectious diseases. Inappropriate antibiotic use is a key
factor in the development of resistance, and major efforts must be
directed towards educating physicians on effective prescribing
practices.
Central to all three papers is the need for surveillance of
organisms that cause bloody diarrhea, hemorrhagic colitis, and HUS,
as well as for knowledge of the local epidemiology of SLTECs, their
potential sources, and the optimal way to investigate and manage
outbreaks. Goldwater and Bettelheim discuss the characteristic
disappearance of E. coli from patients' stools after the
development of HUS and, therefore, the importance of early
detection in cases of bloody diarrhea. Laboratory testing of bloody
diarrheal specimens is clearly critical to understanding the
epidemiology of toxin-producing organisms that relate to the
development of HUS (8). However, testing can be difficult,
time-consuming and costly. Not all laboratories routinely test for
SLTECs or have the capacity to do so. In Australia, for example,
using polymerase chain reaction (PCR) technology to detect SLT
genes is expensive: a negative test result costs approximately
$A15, but if the results are positive, the cost rises to around
$A250 when the SLTEC is isolated and typed.
Human surveillance is essential to the early detection of outbreaks
and to the critical assessment of the impact on public health of
new approaches to food safety (2,8). We conservatively estimate
that the South Australian HUS outbreak has cost around $A20 million
in direct and indirect costs, with major impacts being felt by
industry. This must surely be considered when contemplating the
costs of surveillance. One approach may be to use PCR techniques on
all samples of bloody diarrhea in children under the age of 16
because if surveillance is to be effective, it must be specific
(9). Intermittent surveys, or the use of sentinel laboratories for
all cases of diarrhea (8) could be undertaken and mandatory
notification of HUS instituted.
Compulsory notification of Shigella infection is a requirement in
Australia, and including SLTECs on the list of notifiable diseases
is being considered. A national surveillance scheme for HUS was
established in 1994, although notification is not mandatory.
Without formal notification requirements, good reporting of HUS has
been associated with either clustering of cases or the fact that
few hospitals in a region have the capacity to manage these cases
(10).

Although more attention has been focused on E. coli O157:H7, S.
dysenteriae is likely the most common cause of HUS in children
worldwide and more attention needs to be given to this pathogen in
terms of surveillance and control. A strong and healthy public
health infrastructure is required to address the infectious disease
issues raised by HUS (11).

Mary Beers,* Scott Cameron 
*National Centre for Epidemiology and Population Health, and South
Australian Health Commission;  Communicable Disease Control Unit,
South Australian Health Commission, Adelaide, Australia

References:
1. Cameron AS, Beers MY, Walker CC, et al. Community outbreak of
hemolytic uremic syndrome attributable to Escherichia coli
O111:NM South Australia, 1995. MMWR 1995;44:550-8.
2.Waters JR, Sharp JC, Dev VJ. Infection caused by Escherichia coli
O157:H7 in Alberta, Canada, and in Scotland: a five-year review,
1987-1991. Clin Infect Dis 1994;19:834-43.
3. Bell BP, Goldoft M, Griffin P, Davis MA, Gordon DC, Tarr P, et
al. A multistate outbreak of Escherichia coli O157:H7-associated
bloody diarrhea and hemolytic uremic syndrome from hamburgers: the
Washington experience. JAMA 1994;272:1349-53.
4. Alexander ER, Boase J, Davis M, et al. Escherichia coli O157:H7
outbreak linked to commercially distributed dry-cured
salami Washington and California, 1994. MMWR 1995;44:157-60.
5. Goldwater PN, Bettelheim KA. The role of enterohaemorrhagic
Escherichia coli serotypes other than O157:H7 as causes of disease
in Australia. Communicable Diseases Intelligence 1995;19:2-4.
6. Caprioli A, Luzzi I, Rosmini F, Resti C, Edefonti A, Perfumo F,
et al. Communitywide outbreak of hemolytic-uremic syndrome
associated with non-O157 verocytotoxin-producing Escherichia coli.
J Infect Dis 1994;169:208-11.
7. Griffin PM, Tauxe RV. The epidemiology of infections caused by
Escherichia coli O157:H7, other enterohaemorrhagic E. coli, and the
associated hemolytic uremic syndrome. Epidemiol Rev 1991;13:60-98.
8. Alexander ER. Editorial response: surveillance of Escherichia
coli O157:H7 a necessity for the prevention of an emerging
infectious disease. Clin Infect Dis 1994;19:844-5.
9. Satcher D. Emerging infections: getting ahead of the curve.
Emerging Infectious Diseases 1995;1:1-6.
10. Siegler RL, Pavia AT, Christofferson RD, Milligan MK. A 20-year
population-based study of postdiarrheal hemolytic uremic syndrome
in Utah. Pediatrics 1994;94:35-40.
11. MacDonald KL, Osterholm MT. The emergence of Escherichia coli
O157:H7 infection in the United States: the changing epidemiology
of foodborne disease. JAMA 1993;269:2264-6.



NEWS AND NOTES

(Page 156)

Guidelines on the Risk for Transmission of Infectious Agents
During Xenotransplants

An increasingly critical shortage of human donors has limited the
availability and benefit of organ and tissue transplantation.
This chronic shortage, coupled with recent scientific and
biotechnological advances, has been a catalyst for new
therapeutic approaches directed at using animal tissues in
humans. The use of xenogeneic tissues and organs for
transplantation or perfusion has raised concerns about the
potential of both recognized zoonotic pathogens and unknown
xenogeneic agents to infect individual human recipients and then
spread through human populations.

Public health guidelines intended to minimize the risk for
transmission of known pathogens through human-to-human
transplantation exist. Similar guidelines addressing the issue of
infectious agents that may be associated with xenotransplantation
are being jointly developed by Public Health Service working
groups at the Centers for Disease Control and Prevention (CDC),
the Food and Drug Administration, and the National Institutes of
Health. A provisional draft of these guidelines will be published
in the Federal Register in late 1995. Public comment on the
proposed guidelines is invited. Critical review by members of the
transplant community is particularly sought. Publication of a
final version of these guidelines in CDC's Morbidity and
Mortality Weekly Report is planned for the spring of 1996.

Louisa E. Chapman
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, Georgia, USA



(Page 156)

Emerging Infectious Diseases Featured at ICAAC/IDSA Meeting

Emerging infectious diseases were highlighted recently at a joint
meeting of the Interscience Conference on Antimicrobial Agents
and Chemotherapy (ICAAC) and the Infectious Diseases Society of
America (IDSA) in San Francisco. In his opening address, IDSA
President Vincent Andriole stated that the topic of most pressing
concern to both organizations was new and reemerging pathogens.
Presentations were made on the following subjects: arenavirus
hemorrhagic fevers; changing virulence of streptococcal
infections; cryptosporidia, cyclospora, and microsporidia;
dengue/dengue hemorrhagic fever; emerging fungal pathogens;
epidemic diphtheria in the newly independent states; Escherichia
coli O157:H7; Helicobacter pylori; human ehrlichioses; new
lymphotropic herperviruses, and rabies.

Common themes emanated from the presentations. The speakers noted
that infectious diseases continue to occur throughout the world,
both sporadically and as outbreaks, because of  multiple factors.
They observed that the incidence and prevalence of infectious
diseases are increasing in certain populations, particularly
among immunocompromised persons. Additionally, new infectious
diseases and etiologic agents continue to be identified with
remarkable frequency and that microorganisms are being identified
as causes of chronic diseases, including cancer. Several
presenters expressed concern about the migrations of animal
reservoirs and arthropod vectors into new populations and
geographic areas. The speakers also called for additional support
for the public health infrastructure and for basic sciences that
provide the foundation for infectious disease prevention and
treatment.

(Page 156)

Building a Geographic Information Systems (GIS) Public Health
Infrastructure for Research and Control of Tropical Disease


A course on the use of Atlas GIS and associated peripherals, such
as digitizing tablets and global positioning systems (GPS) to
build a GIS public health infrastructure in Latin American
countries, was taught August 7 18, 1995, at the Centers for
Disease Control field station in Guatemala City, which includes
the Medical Entomology Research Training Unit housed at the
Universidad del Valle de Guatemala. The course was funded by the
Special Programme on Research and Training in Tropical Diseases
and presented by the Latin American Tropical Disease Research
Training Consortium. Course instructors included statisticians
and epidemiologists from the Division of Parasitic Diseases,
National Center for Infectious Diseases, Centers for Disease
Control and Prevention in Atlanta, Ga., USA, and in Guatemala
City, Guatemala, and staff from the National Aeronautic and Space
Administration, Center for Health Application of Aerospace
Related Technologies, Ames Research Center, Sunnyvale, Calif.,
USA.

Objectives of the training included mastery of the principles and
general concepts of all GIS systems; use of Atlas GIS/DOS to
associate map files with databases to produce thematic maps,
manipulate various layers (rivers, highways, village locations)
of the map files to produce customized maps, and create buffers
around geographic features and use them in simple analyses;
design georeferenced dBase data files that can be read by the
GIS; digitize paper maps to acquire new data for building a GIS;
use of GPS to obtain latitudes, longitudes, and elevations of
villages and other major landmarks and use this information in
GIS; and mastery of import/export databases and map files.

The course was designed to enable participants to set up and use
a GIS for research, planning, or operational purposes.
Participating were institutions from Mexico (two teams), Colombia
(two), Puerto Rico, Costa Rica, Venezuela, Guatemala (two),
Ecuador, and Brazil.  Each team came to the course with ideas,
maps, and data pertaining to an existing project that would be
continued at their home institution. Student project areas
included onchocerciasis, malaria, water sanitation,
leishmaniasis, and public health and natural resource
utilization/preservation. The students were taught digitizing
exercises and were asked to use Guinea worm surveillance data to
create their own GIS.

A full day was devoted to geographic analyses. Topics covered
included aggregating data from one geographic layer to another,
combining geographic features with common database values, and
combining selected features to form new map layers. A workshop on
remote sensing, GIS, and image classification explained that
satellite imagery and remotely sensed data are obtained by
measuring reflectance on seven spectral frequencies and that
ground cover can be partially deduced by the amount of
reflectance at each band. Field exercises to practice GPS' use in
the Lake Attilan area followed. Another workshop covered advanced
digitizing and gave each team a good start on the digitizing part
of their projects. Individual instructions were given on how to
import map files from other GIS programs into Atlas GIS. Lastly,
the Guatemalan Onchocerciasis GIS system was presented as a case
study.

In addition to the 2 weeks of training, each participating
institution received a copy of all lecture notes; the following
software, complete with documentation: Atlas GIS/DOS, Import-Export, and Arcview 2.1; and the critical hardware needed to
continue the project at home. An Internet-based discussion group
made up of class organizers and participants, as well as other
interested parties, is being planned.

Allen W. Hightower
Robert E. Kline
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, Georgia, USA, and Guatemala City, Guatemala


(Page 157)

APHA Session Features Emerging Infections

Emerging and reemerging infections will be the featured topic of
a two-part session at the annual meeting of the American Public
Health Association, October 29-November 2, in San Diego,
California.

The session, titled "Emerging Infections: Solving the Mysteries
in the Field and Laboratory," will focus on the worldwide impact
of new and reemerging infections from both an epidemiologic and
laboratory perspective.

Eight speakers from national and international health
organizations will discuss the following aspects of the public
health threat of these diseases: public health strategies for
controlling infectious diseases; social, geographic, ecologic,
and environmental factors that have allowed these diseases to
spread; the growing threat of antimicrobial resistance; the
increased need for accurate and meaningful disease surveillance;
and the challenge to apply the latest laboratory technology to
rapidly detect and characterize new infectious agents.


Martin S. Favero
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, Georgia USA



(Page 158)

Southeast Asia Intercountry Consultative Meeting on Prevention
and Control of New, Emerging, and Reemerging Infectious Diseases

An intercountry meeting to identify strategies and approaches for
tackling the problems of new, emerging, and reemerging infectious
diseases in the Southeast Asia region was held in New Delhi,
India, August 21 to 25, 1995.

Nine countries (Bangladesh, Bhutan, India, Indonesia, Maldives,
Myanmar, Nepal, Sri Lanka, and Thailand) participated. Experts
from these countries, England, and the United States, as well as
representatives from USAID, DANIDA, UNICEF, the World Bank, and
the World Health Organization (WHO) also attended.

Meeting participants expressed serious concern at the global and
regional spread of new, emerging, and reemerging infectious
diseases, especially in the Southeast Asia region. Reports from
various countries emphasized that these diseases not only have
worldwide health implications but also can disrupt commerce and
industry and set back important progress achieved in public
health during recent years. The spread of these diseases also has
major social and political implications.

Participants at the meeting underlined the importance of
surveillance, prompt epidemiologic investigation, and the
build-up of adequate laboratory capacities. The need for
maintaining ecologic and environmental integrity in various
developmental activities was also emphasized.  Member countries
were called upon to immediately review and strengthen capacities
in epidemic surveillance and response and to formulate
country-specific strategies and action plans to anticipate,
quickly recognize, and rapidly respond to the threat of emerging
infections.

To ensure program sustainability, participants stressed that
strategies to combat emerging diseases should be an integral part
of existing national infrastructures, particularly infectious
disease control programs, and should build on the capacities that
already exist. Four broad areas for tackling the challenge of
emerging diseases were identified: strengthening communicable
disease surveillance and response, strengthening the existing
infrastructure, capacity building for prevention and control, and
applied research.

A total of 12 actions were recommended. One, for example, was
that each country create a rapid response team to react to
epidemic situations. A second recommendation was that countries
develop linkages between their national reference laboratories
and WHO collaborating centers. This would be to strengthen
diagnostic capacities, facilitate quality assurance, and promote
national self-reliance in laboratory diagnosis.


Eight recommended actions for WHO to undertake were also
enumerated. These included assessing and monitoring microbial
susceptibility to antibiotics, and vector susceptibility to
insecticides.

For the complete list of conclusions and recommendations of this
meeting, contact WHO's Regional Office for South-East Asia.

Samlee Plianbangchang
WHO, Regional Office for Southeast Asia
World Health House
New Delhi-110 002, India