Emerging Infectious Diseases
[Volume 5 No.3 / May - June 1999]

International EditorsUpdate

   * Bacterial Resistance to Antimicrobial Agents: Selected Problems in
     France, 1996 to 1998, H. Aubry-Damon and P. Courvalin

Perspectives

   * The Cost Effectiveness of Vaccinating against Lyme Disease, M.I.
     Meltzer, D.T. Dennis, and K.A. Orloski
   * Use of Antimicrobial Growth Promoters in Food Animals and Enterococcus
     faecium Resistance to Therapeutic Antimicrobial Drugs in Europe, H.C.
     Wegener, F.M. Aarestrup, L.B. Jensen, A.M. Hammerum, and F. Bager
   * Bacterial Vaccines and Serotype Replacement: Lessons from Haemophilus
     influenzae and Prospects for Streptococcus pneumoniae, M. Lipsitch
   * Iron Loading and Disease Surveillance, E.D. Weinberg

Synopses

   * Human Herpesvirus 6: An Emerging Pathogen, G. Campadelli-Fiume, P.
     Mirandola, and L. Menotti
   * Emergence of a Unique Group of Necrotizing Mycobacterial Diseases,
     K.M. Dobos, F.D. Quinn, D.A. Ashford, C.R. Horsburgh, and C.H. King
   * Respiratory Diseases among U.S. Military Personnel: Countering
     Emerging Threats, G.C. Gray, J.D. Callahan, A.W. Hawksworth, C.A.
     Fisher, and J.C. Gaydos
   * Q Fever in Bulgaria and Slovakia, V. Serbezov, J. Kazr, V.
     Novkirishki, N. Gatcheva, E. Kovcov, and V. Voynova
   * Adhesins as Targets for Vaccine Development, T.M. Wizemann, J.E.
     Adamou, and S. Langermann

Research

   * Tuberculosis in the Caribbean: Using Spacer Oligonucleotide Typing to
     Understand Strain Origin and Transmission, C. Sola, A. Devallois, L.
     Horgen, J. Masetti, I. Filliol, E. Legrand, and N. Rastogi
   * Human Rabies Postexposure Prophylaxis during a Raccoon Rabies
     Epizootic in New York, 1993 and 1994, J.D. Wyatt, W.H. Barker, N.M.
     Bennett, and C.A. Hanlon

Dispatches

   * Factory Outbreak of Escherichia coli O157:H7 Infection in Japan, Y.
     Watanabe, K. Ozasa, J.H. Mermin, P.M. Griffin, K. Masuda, S. Imashuku,
     and T. Sawada
   * First Case of Yellow Fever in French Guiana since 1902, J.M. Heraud,
     D. Hommel, A. Hulin, V. Deubel, J.D. Poveda, J.L. Sarthou, and A.
     Talarmin
   * Risk for Rabies Transmission from Encounters with Bats, Colorado,
     1977-1996, W.J. Pape, T.D. Fitzsimmons, and R.E. Hoffman
   * Australian Bat Lyssavirus Infection in a Captive Juvenile Black
     Flying-Fox, H. Field, B. McCall, and J. Barrett
   * Bordetella holmesii-Like Organisms Isolated from Massachusetts
     Patients with Pertussis-Like Symptoms, W.K. Yih, E.A. Silva, J. Ida,
     N. Harrington, S.M. Lett, and H. George
   * New Cryptosporidium Genotypes in HIV-Infected Persons, N.J. Pieniazek,
     F.J. Bornay-Llinares, S.B. Slemenda, A.J. da Silva, I.N.S. Moura, M.J.
     Arrowood, O. Ditrich, and D.G. Addiss
   * Fatal Case due to Methicillin-Resistant Staphylococcus aureus Small
     Colony Variants in an AIDS Patient, H. Seifert, C. von Eiff, and G.
     Ftkenheuer
   * Application of Data Mining to Intensive Care Unit Microbiologic Data,
     S.A. Moser, W.T. Jones, and S.E. Brossette
   * Sentinel Surveillance for Enterovirus 71, Taiwan, 1998, T.-N. Wu,
     S.-F. Tsai, S.-F. Li, T.-F. Lee, T.-M. Huang, M.-L. Wang, K.-H. Hsu,
     and C.-Y. Shen
   * Chlorine Inactivation of Escherichia coli O157:H7, E.W. Rice, R.M.
     Clark, and C.H. Johnson
   * Fulminant Meningococcal Supraglottitis: An Emerging Infectious
     Syndrome?, E. Schwam and J. Cox
   * Genetic Evidence of Dobrava Virus in Apodemus agrarius in Hungary,
     J.J. Scharninghausen, H. Meyer, M. Pfeffer, D.S. Davis, and R.L.
     Honeycutt
   * Bacterial Resistance to Ciprofloxacin in Greece: Results from the
     National Electronic Surveillance System, A.C. Vatopoulos, V.
     Kalapothaki, Greek Network for the Surveillance of Antimicrobial
     Resistance, and N.J. Legakis
   * Emergence of Related Nontoxigenic Corynebacterium diphtheriae Biotype
     mitis Strains in Western Europe, G. Funke, M. Altwegg, L. Frommelt,
     and A. von Graevenitz

Letters

   * First Case of Human Ehrlichiosis in Mexico, R.A. Gongra-Biachi, J.
     Zavala-Velquez, C.J. Castro-Sansores, and P.Gonzles-Martnez
   * HIV-1 Subtype F in Single and Dual Infections in Puerto Rico: A
     Potential Sentinel Site for Monitoring Novel Genetic HIV Variants in
     North America, I. Flores, D. Pieniazek, N. Morn, A. Soler, N.
     Rodrguez, M. Alegra, M. Vera, L.M. Janini, C.I. Bandea, A. Ramos, M.
     Rayfield, and Y. Yamamura
   * Paratyphoid Fever in India: An Emerging Problem, S. Sood, A. Kapil, N.
     Dash, B.K. Das, V. Goel, and P. Seth
   * Hepatitis C Virus RNA Viremia in Central Africa, N. Cancr, G.
     Grsenguet, F.-X. Mbopi-Kou, A. Kozemaka, A.S. Mohamed, M. Matta,
     J.-J. Fournel, and L. Blec
   * Immunization of Peacekeeping Forces, R. Steffen
   * Sexually Transmitted Diseases in Ukraine, D.I. Ivanov
   * Yellow Fever Vaccine, S.C. Arya
   * Yellow Fever VaccineReply to S. Arya, T.P. Monath, J.A. Giesberg, and
     E.G. Fierros

News and Notes

   * 54th International Northwestern Conference on Diseases in Nature
     Communicable to Man

_________________________________________________________________________
International EditorsUpdate
_________________________________________________________________________

Update

International Editors

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Bacterial Resistance to Antimicrobial Agents: Selected Problems in France,
1996 to 1998
Helene Aubry-Damon* and Patrice Courvalin
*Institut de Veille Sanitaire, Saint-Maurice, France; and Centre National
de Rfrence des Antibiotiques, Institut Pasteur, Paris, France

           [picture]       Dr. Aubry-Damon is a     Surveillance of
                           microbiologist           antibiotic resistance
 specializing in antimicrobial resistance at the    in human pathogens has
 National Institute for Public Health               long been performed in
 Surveillance, Saint-Maurice, France. The           France. Existing
 institute has recently carried out an exploratory  surveillance relies on
 study of a national program for surveillance of    national reference
 antimicrobial resistance in France. Dr.            centers dedicated to
 Aubry-Damon also collaborates closely with the     various bacterial
 French National Reference Center for Antibiotics.  genera and on networks
 (No photo available)                               of volunteer medical
                                                    microbiologists, mainly
 Dr. Courvalin is professor at the Institut         in general hospitals
 Pasteur, where he directs the French National      but also in private
 Reference Center for Antibiotics and heads the     laboratories. Regional
 Antibacterial Agents Unit. Dr. Courvalin           data (often initiated
 specializes in the genetics and biochemistry of    at the request of and
 antibiotic resistance. His research has led to     funded by the
 revision of the description of the natural         pharmaceutical
 dissemination of antibiotic resistance genes. His  industry) have been
 laboratory demonstrated that pathogenic bacteria   available since the
 can promiscuously exchange genetic material        early 1950s. Because of
 conferring antibiotic resistance, documented that  the major health
 conjugation could account for dissemination of     problems caused by
 resistance determinants between phylogenetically   antibiotic resistance
 remote bacterial genera, elucidated the            in the last few years,
 transposition mechanism of conjugative             attempts have been made
 transposons from gram-positive cocci, and          to organize a national
 recently, obtained direct gene transfer from       surveillance program
 bacteria to mammalian cells.                       similar to those being
                                                    established in other
                                                    European countries.
Although this update reviews recent data from France, the
representativeness of the data has not been assessed. In addition, these
are raw data, and their clinical importance remains to be seen; for
example, the contribution of bacterial isolates to infection or
colonization is, in most instances, unknown.

Evolution of Antibiotic Resistance in Hospitals

Dissemination of -Lactamases in Gram-Negative Bacilli

In France, -lactamases and fluoroquinolones are the most frequently
prescribed antibiotics in Enterobacteriaceae infections. A multicenter
study (14 hospitals) across the country analyzed the antibiotic
susceptibility of 2,507 and 2,312 consecutive, nonrepetitive enterobacteria
responsible for infection in 1996 and 1997, respectively (1,2). Strains
were isolated from inpatients (86%) in intensive care (ICU) (12%), surgical
(17%), medical (37%), and geriatric (9%) units. The majority of isolates
were from urine (71%), pus (9%), and bronchopulmonary specimens (8%).
Escherichia coli (64%) was isolated most frequently, mainly in outpatients,
whereas Klebsiella spp. and Enterobacteriaceae with inducible -lactamases
predominated in ICUs. Resistance of E. coli to amoxicillin and cefotaxime
was 47% and 0.5%, respectively. In 1997, the frequency of isolates
producing an extended-spectrum -lactamase varied by species: in
Enterobacter aerogenes, 56%; in Klebsiella pneumoniae, 15%; and in E. coli,
0.5%. The incidence differed within and between hospitals. Such strains
arise in response to the selective pressure exerted by use of
extended-spectrum cephalosporins (3); infections with such strains have
also been associated with hospitalization in ICUs. Production of
-lactamases resistant to -lactam-enzyme inhibitor combinations in E. coli
was approximately 3.5% (2). Susceptibility to fluoroquinolones was high
(66%-97% ciprofloxacin-susceptible) except in E. aerogenes and Serratia
marcescens (35%-52% ciprofloxacin-susceptible) (1).

The organisms most frequently isolated in ICUs in 1995 belonged to the
family Enterobacteriaceae (59%) and Pseudomonas aeruginosa (25%) (4). In
1997, 1,362 nonrepetitive P. aeruginosa (5% of all clinical isolates) were
collected in 13 teaching hospitals (5). The lowest rates of susceptibility
to ceftazidime (<75%), amikacin (70%), and ciprofloxacin (65%) were
observed with serotype 12 (the fourth main serotype). Among
penicillinase-producing strains, the percentages of resistance to amikacin
and ciprofloxacin were 80% and 93%, respectively; these figures were
substantially higher in -lactam-resistant P. aeruginosa than in
susceptible strains.

Spread of Methicillin-Resistant and Gentamicin-Susceptible Staphylococcus
aureus

Methicillin-resistant S. aureus (MRSA) is one of the most frequent
nosocomial pathogens in France as in the rest of the world. Surveys
conducted in hospitals in Paris and surroundings found that MRSA decreased
from 42% in 1992 to 37% in 1997 and that the incidence of MRSA
colonization-infection (approximately 0.65 per 100 admissions) also
decreased after national recommendations against dissemination of
multidrug-resistant bacteria were implemented (6,7). However, a survey of
26 geographically representative hospitals found that the incidence of
gentamicin-susceptible MRSA progressively increased because of the presence
of a predominant clone (H. Lelivre, G. Lina, M.E. Jones, et al., unpub.
obs.). The epidemiologic situation in France is complex. The endemic
aminoglycoside-resistant MRSA strain persisted while new clones became
endemic in hospitals, perhaps after changes in the use of aminoglycosides
(decrease of gentamicin and increase of amikacin consumption) (8). The
first vancomycin-intermediate S. aureus was isolated in a French hospital
in 1995; no other cases of MRSA with reduced susceptibility to vancomycin
have been reported (9).

Most Frequent Macrolide-Resistance Mechanisms among Staphylococci

Over 3 weeks in 1995, 607 staphylococci were collected in 32 hospitals
(10). Of these, 45.5% of the S. aureus and 54% of the coagulase-negative
staphylococci were resistant to methicillin, and 71.5% of MRSA were
resistant to macrolides. Of these MRSA strains, 75% were constitutively
resistant, whereas 76% of MSSA were inducibly resistant. A similar
distribution (61% vs. 27.5%) was observed among coagulase-negative
staphylococci. Resistance to at least one of the macrolide, lincosamide,
and streptogramin antibiotics (88%) was due to the presence of the ermA and
ermC genes, which confer resistance by modifying the ribosomal target. The
ermA gene was more common in MRSA (57.6%) than in MSSA (5.6%), where ermC
was predominant (20.1%). ermC was also common among methicillin-susceptible
coagulase-negative staphylococci (14%). Only a few strains had the ermB
gene, which is found in animal strains. Macrolide resistance by efflux due
to acquisition of the msrA gene was more prevalent in coagulase-negative
staphylococci (14.6%) than in S. aureus (2.1%). The incidence of
lincomycin-resistant but macrolide- and streptogramin-susceptible
staphylococci was low: 0.2% in S. aureus and 4.6% in Staphylococcus
epidermidis (11). The prevalence of pristinamycin-resistant (and also most
probably quinupristin/dalfopristin-resistant) strains remained low because
of the low incidence of resistance to streptogramins type A (pristinamycin
has limited use in France).

Dissemination of Resistance in the Community

Effect of Antibiotics on Oropharyngeal Flora

     Antibiotic Resistance in Streptococcus pneumoniae

The National Reference Center for Pneumococci determined the susceptibility
to antibiotics of 2,837 S. pneumoniae isolated in 1997. The incidence of S.
pneumoniae with reduced susceptibility to penicillin G increased from 3.8%
in 1987 to 48% in 1997 (12). Whereas 53% of all strains were resistant to
macrolides, 80% of penicillin-resistant strains were macrolide-resistant;
15% of all strains (versus 51% of penicillin-resistant strains) were
resistant to tetracycline, and 10% (versus 66%), respectively, were
resistant to trimethoprim-sulfamethoxazole (F. Goldstein et al., unpub.
data). According to a 1997 survey of 18 regional laboratories in France
(11,757 strains collected), 27% had intermediate levels of resistance to
penicillin G, and 13.5% were fully resistant. The rates varied considerably
by region (highest in southwest and central France), age (highest [37.4%
penicillin G-intermediate and 21.5% resistant] in children <16 years old),
and specimen source (highest in middle ear and sinus specimens) (13).

     Resistance to Macrolides in -Hemolytic Streptococci

In 1995, a national survey in 98 hospitals of invasive infections due to
Streptococcus pyogenes found that 5.2% to 9.8% of the strains isolated from
blood were erythromycin-resistant (A. Bouvet, pers. comm.) (14).

   Vaccination against and Resistance in Haemophilus influenzae Type b
     

To monitor the trends in H. influenzae meningitidis and the prevalence of
resistance, the National Reference Center conducted a survey of
approximately 80 hospitals (15). Since vaccination for Hib invasive
infections began in 1993, the percentage of capsulated isolates has
decreased 5% per year. Moreover, resistance to antimicrobial drugs
decreased among Hib and increased among noncapsulated strains isolated from
upper and lower respiratory tract infections. The percentage of
-lactamase-producing H. influenzae increased progressively from 22% in
1992 to 35% in 1997, with a similar evolution for kanamycin resistance.
Tetracycline and chloramphenicol resistance remained stable in 1997less
than 10% and 2%, respectively (15,16).

     Antibiotic Resistance in Neisseria meningitidis

Meningococcal resistance to antibiotics is emerging in France. The
incidence of N. meningitidis with reduced susceptibility to penicillin G
(MICs from 0.125 mg/L to 1 mg/L) increased from less than 1% in 1991 to 18%
in 1996 (17,18). The strains belonged to various serogroups; most belonged
to serogroup B, none produced a -lactamase, and all were susceptible to
cefotaxime and ceftriaxone. Resistance to rifampin, used for prophylaxis of
secondary cases in France, remained low (0.02% in 1996).

Effect of Antibiotics on Digestive Flora

     Antimicrobial Resistance in Helicobacter pylori

Susceptibility testing of H. pylori from 535 patients with a positive CLO
test was performed in 1997 (19). Depending on the method, the percentages
of clarithromycin resistance (disk-agar diffusion or MIC determination by
agar dilution) and metronidazole resistance (breakpoint method at 8 mg/L or
MIC determination) varied from 14.3% (95% confidence interval [CI]
11.5-17.6) to 14.0% (95% CI 11.2-17.3) and from 30.5% (95% CI 25.6-34.5) to
23.6% (95% CI 20.1-27.5), for the two antibiotics, respectively. No
resistance to amoxicillin was observed.

     Fluoroquinolone Resistance in Campylobacter and Salmonella Hadar

The evolution of antimicrobial resistance in Campylobacter jejuni and C.
coli is worrisome. Between 1986 and 1997, 2,713 strains of C. jejuni (68%
of total Campylobacter isolates) were isolated from stool (94%) and blood
(4%) and studied (20). Between 1993 and 1997, fluoroquinolone resistance
increased from 7.4% to 32% in C. jejuni and from 11.8% to 52% in C. coli.
The high resistance rate to quinolones makes them ineffective in therapy of
Campylobacter infections. These resistance rates are similar to those in
other countries (e.g., Spain, the United Kingdom) (21,22). However, the
prevalence of macrolide-resistant strains remains low (3.6%). The high
incidence of multidrug-resistant Salmonella Typhimurium DT104 (12
atypical), with 82% resistance to ampicillin, streptomycin, sulphonamide,
tetracycline, and chloramphenicol, is the most serious epidemiologic
problem of the last decade in France (23). The incidence of Salmonella
Hadar is increasing, and the percentages of amoxicillin- and
fluoroquinolone-resistant strains in 1997 were 72% and 75%, respectively.
Fluoroquinolone resistance had not been observed before 1987 in France,
Spain, and the United Kingdom. This was before concomitant introduction of
ciprofloxacin into clinical use and enrofloxacin into veterinary use (in
particular in the poultry industry) in the late 1980s. More than 50% of C.
jejuni and S. Hadar, the most frequent serotype associated with poultry,
are now fluoroquinolone-resistant in these countries. The situation is
different in Sweden, where fluoroquinolones are not readily available.
Therefore, guidelines for the prudent use of antibiotics (in prophylaxis or
therapy) should be developed that respect the indigenous flora of humans
and animals.

     New Types of Resistance in Enterococci

The increase in the incidence of glycopeptide-resistant enterococci (GRE)
isolated from hospitalized patients throughout the United States has not
been observed in France. A multicenter study in 1993 showed a very low
incidence of GRE: 0.2% among 251 enterococcal clinical isolates and 7.5%
among Enterococcus faecium (24). Study of 24 ICUs in 1994 determined that
the prevalence of GRE colonization in patients' fecal flora was
approximately 2%, 30% of which had been present at admission. No nosocomial
infection due to GRE was observed (25). GRE have been identified in human
food of animal origin (40% of GRE were isolated from uncooked meat) in a
French study conducted in military cafeterias in 1997 (26). Thus, food may
represent a major source of human colonization with GRE in France. GRE
strains isolated in France were also resistant to ampicillin, tetracycline,
and macrolides. However, the percentage of high resistance levels to
gentamicin among GRE was comparable to that among glycopeptide-susceptible
enterococci.

     Antibiotic Resistance in Bacteroides fragilis

Studies of antibiotic resistance in anaerobic pathogens indicate stability
of resistance to carbapenems (imipenem) and nitroimidazole antibiotics
(27,28). In 1998, fewer than 2% of all B. fragilis from 39 hospitals were
resistant to metronidazole (MICs >8 mg/L), and the number of
imipenem-resistant strains remained low. However, this gene reservoir
requires surveillance of resistance in B. fragilis infections because of
the use of these antibiotics in therapy.

Other Bacteria

     Antibiotic Resistance in Neisseria gonorrhoeae

The number of N. gonorrhoeae strains identified by the National Reference
Centre for Sexually Transmitted Diseases fell sharply from 1986 to 1990 (by
81%) and more slowly from 1990 until 1999 (by 55%) (29). The number of
anorectal gonococcal infections reached a plateau from 1995 to 1997 but
increased again in 1998, mostly in the Paris/Ile-de-France region (V.
Goulet, P. Sednaoui, et al., unpub. data). An increasing percentage of N.
gonorrhoeae displayed diminished sensitivity to penicillin G and to
tetracycline. In 1997, 15% and 30% of N. gonorrhoeae were resistant to
penicillin G (MIC >/= 2 mg/L) and tetracycline (MIC 2 >/= mg/L and < 16 mg/L),
respectively, by chromosomal mutation. In contrast, the percentage of
strains with plasmid-mediated resistance to penicillins and tetracycline
has remained stable at approximately 15% since 1994. No ceftriaxone,
spectinomycin, or ciprofloxacin resistance was found until 1997, when the
first ciprofloxacin-resistant strains (MIC=1 mg/L) were isolated.

Conclusions

Antibiotic resistance trends in France are for the most part similar to
trends in other European countries but with some peculiarities. For
instance, fluoroquinolone resistance in Salmonella spp. and Campylobacter
spp. is a problem throughout Europe. However, methicillin resistance in
Staphylococcus is more common in France than in the Scandinavian countries,
although it has started to decrease because of reinforcement of hygiene
measures since 1992. Also, heavy use of 16-membered macrolides has selected
for resistance in gram-positive cocci by ribosomal modification rather than
by efflux. In pneumococci, decreased susceptibility to penicillins is as
common in France as in Spain, but the incidence of resistance to macrolides
is the highest in Europe. The public health problems caused in France by
bacterial resistance to antibiotics are clearly distinct from those in
North America. The incidence of enterobacteria producing extended-spectrum
-lactamases and glycopeptide-resistant enterococci remains rather low in
France, as in most other European countries. In the United States, the high
incidence of nosocomial GRE infections is probably caused by the heavy
nosocomial use of vancomycin, particularly in hematology wards and for the
prevention of colitis due to Clostridium difficile. In contrast, no
intestinal carriage of such strains is found in the general population. The
situation in Europe mirrors that in the United States. In Europe, the
prevalence of nosocomial GRE infections remains low, but colonization of
the population is substantial, possibly because of the use of a
vancomycinlike antibiotic (avoparcin) as an animal food additive. This
example stresses the need for a multidisciplinary approach to surveillance
of bacterial resistance to antibiotics.

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      Address for correspondence: P. Courvalin, Unit des Agents
Antibactriens, Institut Pasteur, 28, rue du Dr. Roux, 75724 Paris Cedex
15, France; fax: 33-1-45-68-83-19; e-mail: pcourval@pasteur.fr.

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 23. Breuil J, Armand-Lefevre L, Casin I, Dublanchet A, Collatz E and The
     College de Bacteriologie-Virologie-Hygiene des Hpitaux Gnraux
     Franais. Surveillance de la sensibilit aux antibiotiques des
     salmonelles et shigelles isoles dans 77 hpitaux franais. Bulletin
     Epidmiologique Hebdomadaire 1998;51:219-21.
 24. Schmit JL, Leclercq R, Scheimberg A, Landauer D. Approche
     pidemiologique et clinique des entrocoques: rsultat d'une enqute.
     Medecine et Maladies Infectieuses 1994;24S:141-8.
 25. Boisivon A, Thibault M, Leclercq R, and The College de
     Bacteriologie-Virologie-Hygiene des Hpitaux Gnraux Franais.
     Colonization by vancomycin-resistant enterococci of the intestinal
     tract of patients in intensive care units from French general
     hospitals. Clin Microb Infect 1997;3:175-9.
 26. Perrier-Gros-Claude JD, Courrier PL, Breard JM, Vignot JL, Masseront
     T, Garin D, et al. Entrocoques rsistants aux glycopeptides dans les
     viandes. Bulletin Epidmiologique Hebdomadaire1998;12:50-1.
 27. Breuil J, Podglajen I, Collatz E. Susceptibility testing of anaerobic
     pathogens: rational and results. Proceedings of the 38th Interscience
     Conference on Antimicrobial Agents and Chemotherapy; 1998 Sep 24-27;
     San Diego, California. Washington: American Society for Microbiology;
     1998. p. 636.
 28. Reysset G, Trinh S, Carlier JP, Sebald M. Bases gntiques de la
     rsistance aux 5-nitroimidazoles des Bacteroides spp. Medecine et
     Maladies Infectieuses 1996;26 Suppl:1-7.
 29. National network on gonococcal infections. Les gonococcies en France
     en 1997, le rseau RENAGO. Bulletin Epidmiologique Annuel 1998
     report. France: National Institute for Public Health Surveillance. In
     press 1999.

_________________________________________________________________________
Perspectives
_________________________________________________________________________

Perspectives

The Cost Effectiveness of Vaccinating against Lyme Disease

Martin I. Meltzer, David T. Dennis, and Kathleen A. Orloski
Centers for Disease Control and Prevention, Atlanta, Georgia, USA

---------------------------------------------------------------------------
      To determine the cost effectiveness of vaccinating against Lyme
      disease, we used a decision tree to examine the impact on
      society of six key components. The main measure of outcome was
      the cost per case averted. Assuming a 0.80 probability of
      diagnosing and treating early Lyme disease, a 0.005 probability
      of contracting Lyme disease, and a vaccination cost of $50 per
      year, the mean cost of vaccination per case averted was $4,466.
      When we increased the probability of contracting Lyme disease
      to 0.03 and the cost of vaccination to $100 per year, the mean
      net savings per case averted was $3,377. Since few communities
      have average annual incidences of Lyme disease >0.005, economic
      benefits will be greatest when vaccination is used on the basis
      of individual risk, specifically, in persons whose probability
      of contracting Lyme disease is >/=0.01.

Lyme disease, caused by infection with Borrelia burgdorferi, is the most
common tick-borne disease in the United States and Europe (1-3). In the
United States, the disease has spread slowly, and the number of cases in
disease-endemic areas has increased (4-6). Most Lyme disease patients
become infected with B. burgdorferi near their homes, while engaged in
property maintenance, recreation, and relaxation (7). Occupational and
recreational activities away from home may also pose a risk (8). Lyme
disease prevention based primarily on avoidance of tick bites, use of
repellants, early detection and removal of attached ticks, and tick control
has not substantially reduced disease incidence (4-6). Therefore,
preventive vaccines have been of considerable interest. Results of
randomized and blinded phase-III field trials with recombinant B.
burgdorferi outer surface protein A (rOspA) vaccines indicate that they are
safe and efficacious (9,10). On December 21, 1998, the U.S. Food and Drug
Administration licensed one of the vaccines (LYMErix, SmithKline Beecham
Biologicals, Reixensart, Belgium) for use in the United States (11).

We present the results of an analytic model that evaluates the cost
effectiveness of using a vaccine to protect against Lyme disease in the
United States.

The Model

Using a computer-based spreadsheet (Excel 5.0 for Windows, Microsoft), we
constructed a decision tree (12) to evaluate the cost per case averted
(cost effectiveness) to society of vaccinating against Lyme disease (Figure
1). Many data needed to determine the cost effectiveness of vaccinating
against Lyme disease are unvalidated, unavailable, or available only from
very small databases. Thus, rather than calculate a single estimate of cost
per case averted, we examined the effect of combinations of six inputs:
cost of vaccination; annual probability of contracting Lyme disease; costs
of successfully treating either early symptoms of Lyme disease or one of
three sequelae (cardiovascular, neurologic, arthritic); probability of
diagnosing and treating early symptoms; probability of sequelae due to
early infection; probability of sequelae due to late, disseminated
infection.

					    Mathematically, we examined the effect
	   [Fig]                    of altering the values of the inputs by
                                  using specialized computer software
Fig 1. Decision tree to model     (@Risk, Palisade Corp., Newfield, NY)
the cost effectiveness of         (13) that employs Monte Carlo methods
vaccinating a person against      (14-16). To use these methods, the
Lyme disease.                     researcher defines probability
                                  distributions for selected inputs by
                                  using available data and enters them
into the computer program. For each iteration of the program, an algorithm
selects input values from the probability distributions, calculates the net
result (here, the cost per case averted), and stores that result. After all
iterations (typically 1,000 to 5,000) are completed, the program produces a
probability-based distribution of the net result, which can then be used to
report statistics such as mean, median, and 5th and 95th percentiles.

Cost Effectiveness Formula

The formula used to calculate the cost per case of Lyme disease averted was
as follows:
Cost per case averted  = ($ of vacc+$of LD with vacc-$ of LD without vacc)/
(Prob LD without vacc-Prob LD with vacc) where $ = cost; vacc. = vaccination; 
LD = Lyme disease; and prob. = probability. The numerator is the cost of 
vaccination less any savings resulting from the reduced probability of 
contracting the disease (decreased incidence) due to vaccination. If the 
vaccine is not 100% effective in preventing Lyme disease (i.e., if the term Prob. 
LD with vacc. >0), treatment costs may still be incurred after vaccination. 
The cost of a case of Lyme disease is the weighted average cost of all health 
outcomes (Figure 1), where the weights are the probabilities of those outcomes 
12). The denominator reflects the change in the probability of Lyme disease due
to vaccination.

Vaccine Timeline

Although experiments have shown that a Lyme disease vaccine using rOspA is
safe and immunogenic in both animals and humans (17-23), no data have been
published concerning the decrease in antibody levels over more than 20
months (9). Phase-III vaccine field trials used a 0-, 1-, and 12-month
immunization schedule, and antibody levels dropped almost 10-fold between
the month after the second dose and just before the third dose at month 12
(9). The third dose at month 12 boosted antibodies to levels higher than
measured at month 2, but these declined by half by month 20 (9). We
assumed, therefore, that an annual booster dose would be required and that
the cost-effectiveness model would be repeated annually. When calculating
annual benefits, however, we included the discounted savings of preventing
Lyme disease that may generate multiyear sequelae.

Lyme Disease Symptoms and Sequelae

The most common symptoms of infection with B. burgdorferi can be
categorized as early localized disease (stage I); early disseminated
disease (stage II); and later stage sequelae of disseminated
infection-(stage III) (24). Stages I and II correspond to the branches
labeled "Recognize early LD? Yes" in Figure 1, and stage III corresponds to
the branches labeled "Recognize early LD? No." Most early symptoms of Lyme
disease respond promptly and completely to short courses of oral
antibiotics (25-27). Later-stage sequelae, however, may require costly,
more prolonged treatment, sometimes repeated courses of treatment using
intravenous cephalosporins, and may not be completely eliminated (28).

If a person, vaccinated or unvaccinated, contracts Lyme disease, the model
allows for one of four possible categories of outcomes (Figure 1) (29-31):
cardiovascular sequelae (e.g., high-grade atrioventricular blocks);
neurologic sequelae (e.g., isolated cranial nerve palsy, meningitis);
arthritic or rheumatologic/musculoskeletal sequelae (e.g., episodic
oligoarticular arthritis, arthralgia); and case resolved (after a course of
an oral antibiotic such as doxycycline) with no further complications.

The disseminated stages of Lyme disease may be manifested weeks to months
after infection (24). However, few data concerning the duration of such
sequelae are available. One study, for example, involving 38 patients
showed that their long-term clinical sequelae lasted a mean of 6.2 years
from onset of disease (32). The use of health-care resources, however, by
those patients during that time was not reported. We assumed that
cardiovascular sequelae would be treated and resolved in an average of 1
year and that late neurologic and arthritic sequelae would both take an
average of 11 years to diagnose and satisfactorily treat to full resolution
(initial year of diagnosis and treatment plus 10 years of additional
treatment). These assignments of average time are arbitrary and longer than
any published average, which maximizes estimated economic benefits of using
a vaccine.

Probabilities

We selected three probabilities (0.005, 0.01, and 0.03) of contracting Lyme
disease (Table 1) on the basis of data concerning disease incidence in Lyme
disease-endemic areas ((33-36); the probability of 0.03 is among the
highest reported. (Before the risk for Lyme disease was widely recognized,
a one-time annual incidence of 10% was reported in a community of 190
people living next to an open nature preserve [37].) Vaccine efficacy in
preventing Lyme disease was 50% (95% confidence intervals [CI]: 14% to 71%)
after the first two doses and 78% (95% CI: 59% to 88%) after three doses
(9,11).

Table 1. Probabilities and their statistical distributions
---------------------------------------------------------------------------
Item                           Values               Type of distribution(sup a)
---------------------------------------------------------------------------
Probability of contracting
LD(sup b)                      0.005, 0.01, 0.03    Fixed intervals (sup c)

Effectiveness of vaccine       0.85                 Fixed
Probability of early                                Fixed intervals (sup c,d)
detection of LD                0.6 - 0.9
Probability of sequelae (sup e)
if detect LD early
             Cardiac           0 - 0.01             Uniform (sup f)

             Neurologic        0 - 0.02             Uniform (sup f)

             Arthritic         0.02-0.05-0.07       Triangular (sup g)

             Case resolved     Residual (sup h)     N/A
Probability of sequelae if do not detect LD early
            Cardiac            0.02-0.03-0.06       Triangular
            Neurologic         0.02-0.15-0.17       Triangular
            Arthritic          0.5-0.6-0.62         Triangular
            Case resolved      Residual (sup h)     N/A
---------------------------------------------------------------------------
(sup a) Statistical distribution used in Monte Carlo simulations (14-16).
(sup b) LD = Lyme disease.
(sup c) Iterations are run by using different combinations of the probabilities
of infection and cost of
  treatment (Table 2).
(sup d) The interval between the minimum and the maximum is divided into 0.1
increments.
(sup e) See text for description of sequelae.
(sup f) Uniform distribution implies that there is an equal chance that any
number between, and including, the   minimum and maximum will be used for a 
given iteration.
(sup g) Triangular distribution is defined by points of minimum, most likely, and
maximum.
(sup h)The probability of an LD case being successfully resolved (i.e., no
further sequelae) is 1 - (sum of the probabilities of cardiac + neurologic 
+ arthritic symptoms).

We assumed Lyme disease vaccine to be 85% effective, which is near the
upper end of the 95% confidence limits and thus maximizes estimated
economic benefits. We selected 0.6 to 0.9 as the range of probability of
early diagnosis and treatment on the basis of a study on the economic cost
of Lyme disease, which included data from an expert panel (38). For the
Monte Carlo simulations (14-16), we constructed the distributions
describing the probabilities of having one of the three sequelae (due to
either early or late disseminated disease) using data from the previously
mentioned expert panel (Table 1) (38). The distributions describing cardiac
and neurologic complications associated with early Lyme disease are
uniform, defined by using minimum and maximum values (39) and reflecting
the uncertainty regarding a most likely value (38). All other distributions
are triangular (39), with minimum, most likely, and maximum values (Table
1).

Vaccination Costs

Although a Lyme disease vaccine has been licensed (11), data are not
available on the actual cost of vaccination, which includes costs of the
vaccine, its administration, time spent in receiving the vaccine, travel,
and treatment of adverse side-effects of vaccination. To allow for
variation caused by variables such as location of provider, type of
provider, and type of third-party payer, we estimated cost effectiveness by
using three costs: $50 per person per year, $100 per person per year, and
$200 per person per year.

Few data are available on the costs of treating a case of Lyme disease;
only one study (29) has documented the charges in 1989 dollars associated
with some sequelae. To adjust charges reported in that study to 1996
prices, we multiplied the charges by a factor of 1.528 (medical care
component of the consumer price index) (40). These 1996 prices, however,
reflected health-care charges paid by health insurance companies and not
necessarily actual economic costs (41,42). Thus, to reflect economic costs,
the adjusted prices were multiplied by cost-to-charge factor (the weighted
average of the urban and rural hospital cost-to-charge ratios used by the
U.S. Federal Health Care Finance Administration [43]) of 0.53. Data
describing indirect costs, particularly lost productivity, associated with
sequelae were unavailable. We therefore assumed that Lyme diseaserelated
cardiac sequelae would cause 14 days of lost productivity, and neurologic
and arthritic sequelae would each cause 21 days of lost productivity per
year. Each day of lost productivity was valued at $100 (the average income
of a workday [1990 dollars inflated to 1996 values] weighted by the age and
sex composition of the U.S. workforce) (44). Because we assumed that
late-stage neurologic and arthritic complications may take up to 11 years
to completely resolve, the 1-year cost estimates for treating these
sequelae were replicated over 11 years and then discounted at 3% to the
base year (Table 2).

Table 2: Costs of treating one case of Lyme                        We also
disease and the sequelae due to early and late                     altered
disseminated disease                                               the
                                                                   estimate
-----------------------------------------------------------------  of Magid

                                    Cost/     Length    Total      et al.
                                    year     of treat- costs(sup a)(29) of
Item                                 ($)        ment      ($)      charges
                                                                   for
-----------------------------------------------------------------  resolving
Case resolved: no sequelae                                         a case
         Antibiotics                    14                         of Lyme
                                                                   disease
        Office visits (2)               50                         without
        Laboratory tests                35                         complications
        5 hrs lost work time            62                         by
                                                                   doubling
              Total                   161     2-3 wks        161   the
Sequelae (sup b)due to early and                                   number
  late disseminated disease                                        of
                                                                   office
       Cardiac-direct(sup c)      5,445                            visits

       Cardiac-indirect (sup d)   1,400                            to two
                                                                   ($25
       Cardiac-total               6,845       </= 1 yr    6,845   each
       Neurologic-direct (sup c)   4,865                           visit)
                                                                   and
       Neurologic-indirect (sup d) 2,100                           allowing
                                                                   for 5
       Neurologic-total            6,965       11 yrs     61,243   hours of
       Arthritic-direct (sup c)    1,804                           lost
                                                                   productivity
       Arthritic-indirect (sup d)  2,100                           ($62)

       Arthritic-total             3,904       11 yrs     34,354   for a
                                                                   total of
-----------------------------------------------------------------  $161
(sup a)All costs that occur over more than 1 year are discounted   (Table
  at a rate of 3% per year.                                        2). In
(sup b)See text for description of the sequelae.                   comparison,
                                                                   a recent
(sup c)Direct costs are for all medical costs and are derived from study
  the 1-year charges reported by Magid et al. (29), inflated to    concerning
  1996 dollars (factor of 1.528) (40), and then adjusted by a      Lyme
  cost-to-charge ratio of 0.53 (43) (see text for details).        disease
(sup d)Indirect costs are the valuation of lost productivity due   on the
  to Lyme disease-related illness, with each day lost valued at    eastern
  $100. For cardiac-related sequelae, it was assumed that 14       shore of
  workdays were lost, and for neurologic and arthritic-related     Maryland
  sequelae, it was assumed that 21 workdays were lost each year.   found
                                                                   the
median charge for the diagnosis and treatment of Lyme disease was $199
(45); this figure represents charges, and actual economic costs are likely
lower than this amount (41,42).

Sensitivity Analyses

To allow for uncertainty caused by lack of data, we conducted multivariate
sensitivity analyses in which we simultaneously altered the assumed
effectiveness of the vaccine and the cost of treating sequelae. We altered
vaccine effectiveness to either 0.75 or 0.95 (compared with 0.85 in the
base case [Table 1]), and we multiplied the total costs for treating
sequelae (Table 2) by 0.5 or 1.5. For example, for neurologic sequelae, the
latter multiplier is equivalent to increasing the days of lost productivity
(indirect costs) from 21 days per year to 31.5 days per year. We set the
probability of identifying and successfully treating early Lyme disease at
0.80 and the cost of vaccination at $100 per year. The estimates generated
by these sensitivity analyses were compared with those generated using the
base costs, with an assumed vaccine effectiveness of 0.85, and with the
same assumptions for probability of identifying and treating early Lyme
disease and cost of vaccination as used in the sensitivity analyses.

Findings

Assuming a 0.005 probability of contracting Lyme disease, a 0.80
probability of diagnosing and treating early Lyme disease, and a $50 per
year cost of vaccination, the mean cost per case averted was $4,466 (5th
percentile = $5,408; 95th percentile = $3,587) (Figure 2). The 5th and 95th
percentiles were calculated as part of the Monte Carlo simulations (14-16).
To enhance clarity, the 5th and 95th percentiles were not plotted on Figure
2. Increasing the cost of vaccination to $100 per year increased the mean
cost per case averted to $16,231 (5th = $17,267; 95th = $15,298) (Figure
2). At a cost of vaccination of $200 per year, the mean cost per case
averted was $39,761 (5th = $40,858; 95th = $38,830) (Figure 2).

With a 0.01 probability of                  
contracting Lyme disease and a 0.80                  [Fig]
probability of correct diagnosis and
treatment of early disease, the mean        Fig. 2. Average cost
savings per case averted was $1,416         effectiveness of vaccinating a
when the cost of vaccination was $50        person against Lyme disease
per year. Vaccination resulted in a         with changes in the cost of
net cost of $4,467 when the cost of         vaccination, probabilities of
vaccination was $100 per year and a         identifying and treating early
net cost of $16,231 when the cost of        Lyme disease, and
vaccination was set at $200 per year        probabilities of contracting
(Figure 2).                                 Lyme disease. A negative value
                                            indicates that vaccinating a
When we set the probability of              person will result in a net
contracting Lyme disease at 0.03 and        cost to society, while a
used the same probability of                positive value indicates a net
diagnosis as before (0.80), the mean        savings to society. The
savings per case averted was $5,337         results shown are the means
when the cost of vaccination was $50        from Monte Carlo simulations
per year and $3,377 when the cost of        (see Table 1 and text for
vaccination was $100 per year. The          further details). Vaccine
net cost per case averted was $545          assumed 85% effective (Table
when the cost of vaccination was $200       1).
per year (Figure 2).

When the costs of treating sequelae were reduced by half of base costs, at
a 0.01 probability of contracting Lyme disease, the average cost of
averting one case was $9,684 when vaccine effectiveness was assumed to be
0.75 and $6,877 when vaccine effectiveness was assumed to be 0.95 (Table
3). These are 117% and 54% higher, respectively, than the costs calculated
using the base costs (Table 3).

 Table 3. Sensitivity analyses: Cost or savings per case averted (5th, 95th
 percentiles) by altering  assumed vaccine effectiveness and the cost of 
 treating Lyme disease sequelae (sup a)

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

                 Base treatment     Base	       Base treatment 
                  costs (sup b)    treatment      costs (sup b) 
                 x 0.5            costs (sup c)    x 1.5

             --------------------------------------------------------------
                   Vaccine
 Probability   effectiveness (sup d)           Vaccine effectiveness (sup d)
 of Lyme     -------------------            -------------------------------
 disease       0.75      0.95       0.85         0.75            0.95
 --------------------------------------------------------------------------
 0.005        23,018    17,404     16,231       15,720          10,105
             (23,527;  (17,947;   (17,283;
             22,556)   16,927)    15,261)   (17,249;14,286) (11,641;8,703)

 0.01         9,684     6,877      4,467         2,386       Net savings (sup e)

             (10,178;  (7,372;    (5,531;                   (1,220; save (sup e))
              9197)     6,412)     3,487)    (3,846; 958)
                         Net        Net
 0.03          795     savings    savings    Net savings    Net savings
			      (sup e)   (sup e)     (sup e)        (sup e)
             (1,303;    (385;      (save;    (save; save   (save; save
               330)     save      save        (sup e))       (sup e))
				(sup e))  (sup e))
 --------------------------------------------------------------------------
 (sup a)These results were generated by setting the probability of detecting and
 successfully treating  early Lyme disease at 0.80 and the cost of vaccination 
 at $100 per year.
 (sup b) Base treatment costs are given in Table 2. The data presented in this
 table were generated by multiplying the costs in Table 2 by either 0.5 (i.e., 
 reducing costs by half) or by 1.5 (i.e., increasing costs by half).
 (sup c) For comparison, the results using the base costs (Table 2) are presented
 here, assuming a vaccine effectiveness of 0.85. Figure 2 presents the complete 
 set of results using the base costs.
 (sup d)The initial assumed level of vaccine effectiveness was 0.85 (Figure 2).
 (sup e)Net savings are generated when a person is vaccinated against Lyme
 disease and the costs saved by not having to treat a case of Lyme disease 
 are higher than the costs of vaccination plus the costs of having to treat a 
 case of Lyme disease that occurs after vaccination. The net savings range from
 $140 (probability of Lyme disease = 0.03, vaccine effectiveness = 0.95,
 cost of treating Lyme disease sequelae = 0.5 x base costs) to $7,438 
 (probability of Lyme disease = 0.03, vaccine effectiveness = 0.95, cost of 
 treating Lyme disease sequelae =1.5 x base costs). Note also that in
 some instances where mean net savings are calculated, the 5th percentiles
 are net costs.

When the costs of treating sequelae were increased to 1.5 times base costs,
the equivalent cost per case averted was $2,386 at a vaccine effectiveness
of 0.75, while a vaccine effectiveness of 0.95 was estimated to generate
cost savings (Table 3). The former estimate represents a 47% decrease in
cost per case averted compared with the base case (Table 3).

These results show that, as the weighted average cost of treating a case of
Lyme disease decreases (increases), the cost per case averted through
vaccination increases (decreases). An inspection of the formula to
calculate the cost per case of Lyme disease averted, presented in the Model
section, shows that, as the term $ of LD w/out vacc (in the numerator)
decreases, the cost per case averted must increase.

Conclusions

Because of either lack of data or wide variability in some key variables
(e.g., cost of vaccination, risk for Lyme disease), a single answer
regarding the cost effectiveness of vaccinating a person against Lyme
disease cannot be calculated. The methods we used allow physicians,
health-care decision makers, and public health authorities to use Figure 2
and Table 3 to determine the cost effectiveness of vaccination for their
specific situations. This simple model can be rerun to provide estimates
per case averted for situations not covered in the results presented (e.g.,
lower or higher probabilities of Lyme disease). The estimates do not
include any valuation of a person's willingness to pay for the vaccination.

Relative Importance of Input Variables

The probability of contracting Lyme disease is the most important factor in
determining the economic benefit of vaccinating against Lyme disease
(Figure 2). The results from Figure 2 and from the sensitivity analyses
concerning the costs of treating sequelae and vaccine effectiveness (Table
3) indicate that the next most important variables are the cost of treating
sequelae and the probability of early detection and treatment of Lyme
disease.

Research Priorities

Given the importance of treatment costs in assessing the cost effectiveness
of Lyme disease vaccine, accurate data regarding the cost of treating
sequelae should receive high priority when setting a research agenda for
Lyme disease. Data concerning the duration of the various forms of
long-term sequelae and the indirect costs borne by patients are also
important. For both items, research should not focus on obtaining a mean
value but rather on collecting sufficient data to describe the probability
distribution of these input variables, which could either replace the
assumed distributions (Table 1) or be added to the model to further refine
the results.

Implications for Public Health Policy

Very few communities have an annual incidence of Lyme disease of 0.005 or
higher. From 1992 to 1996, approximately 47% (1,483) of U.S. counties
reported at least one case of Lyme disease. However, 148 counties (almost
all in the northeastern and northcentral United States [CDC, unpub. data;
1]) reported 90.3% of cases. Connecticut and Rhode Island had the highest
cumulative annual incidences of reported Lyme disease, equivalent to
probabilities of contracting Lyme disease of 0.000949 and 0.000539,
respectively (1996 data) (46). Two studies (47,48) have shown that cases
have been underreported in areas where the disease is highly endemic.
However, the range of probabilities in our model allows for both
underreporting and overdiagnosis.

The benefits are likely highest if both community-level incidence of Lyme
disease and individual risk for exposure to tick bites and infection (38)
can be considered in using the vaccine. The Advisory Committee on
Immunization Practices, Public Health Service, U.S. Department of Health
and Human Services, recently agreed with this conclusion and, voted in
February 1999, to recommend the use of Lyme disease vaccine on the basis of
a combination of both community-level and individual risk. These
recommendations will be published soon (49).

Ours is not the only study to suggest that the vaccine not be used
universally. A forthcoming Institute of Medicine report (50) uses cost per
quality-adjusted life year (QALY) saved to examine vaccine priorities. The
authors estimate that it would cost more than $100,000 per QALY saved if
the vaccine were given ". . . to resident infants born in, and immigrants
of any age to, geographically defined high risk areas." This result led the
authors to rank Lyme disease vaccine as "less favorable," their lowest
ranking in terms of priorities for vaccine development.

Our model also considers the relative value of two interventions:
vaccination and the detection and treatment of early Lyme disease.
Communities with average individual probabilities of contracting Lyme
disease of less than 0.01 may benefit from interventions that improve the
probability of early diagnosis and treatment of Lyme disease.

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

      Dr. Meltzer is senior health economist, Office of the Director, NCID,
CDC. His research interests focus on assessing the economics of public
health interventions such as oral raccoon rabies vaccine, Lyme disease
vaccine, and hepatitis A vaccine, as well as estimating the economic burden
of bioterrorism, dengue, pandemic influenza, and other infectious diseases.
His research uses various methods, including Monte Carlo modeling,
willingness-to-pay surveys (contingent valuation), and the use of
nonmonetary units of valuation, such as disability adjusted life years
(DALYs).

Address for correspondence: Martin I. Meltzer, National Center for
Infectious Diseases, Centers for Disease Control and Prevention, 1600
Clifton Road, Mailstop C12, Atlanta, GA 30333, USA; fax: 404-639-3039;
e-mail: qzm4@cdc.gov.

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Perspectives

Use of Antimicrobial Growth Promoters in Food Animals and Enterococcus
faecium Resistance to Therapeutic Antimicrobial Drugs in Europe

Henrik C. Wegener, Frank M. Aarestrup, Lars Bog Jensen, Anette M.
Hammerum, and Flemming Bager
Danish Veterinary Laboratory, Copenhagen, Denmark

---------------------------------------------------------------------------
      Supplementing animal feed with antimicrobial agents to enhance
      growth has been common practice for more than 30 years and is
      estimated to constitute more than half the total antimicrobial
      use worldwide. The potential public health consequences of this
      use have been debated; however, until recently, clear evidence
      of a health risk was not available. Accumulating evidence now
      indicates that the use of the glycopeptide avoparcin as a
      growth promoter has created in food animals a major reservoir
      of Enterococcus faecium, which contains the high level
      glycopeptide resistance determinant vanA, located on the Tn1546
      transposon. Furthermore, glycopeptide-resistant strains, as
      well as resistance determinants, can be transmitted from
      animals to humans. Two antimicrobial classes expected to
      provide the future therapeutic options for treatment of
      infections with vancomycin-resistant enterococci have analogues
      among the growth promoters, and a huge animal reservoir of
      resistant E. faecium has already been created, posing a new
      public health problem.

Vancomycin-Resistant Enterococcus faecium (VRE)

In addition to being a member of the normal gut flora of nearly all
warm-blooded animals (including humans), E. faecium has the ability to
cause a wide range of infections, primarily serious infections in hospital
patients (particularly in intensive care units). Increasing incidence of E.
faecium infections has been associated with use of third-generation
cephalosporins in hospitals (1). Enterococci are resistant to many
antibiotics. In an increasing number of cases, vancomycin is the only
treatment drug that remains effective. Because E. faecium was untreatable
with practically all other antibiotics, the emergence of the first high
level vancomycin-resistant E. faecium was of particular concern (2). By
1997, more than 15% of nosocomial enterococcal infections in U.S. hospitals
were due to VRE (3).

The vancomycin-resistant strain of E. faecium (VRE) contains the VanA gene
cluster located at a mobile genetic element, a transposon designated
Tn1546. Although other mechanisms and determinants of glycopeptide
resistance have been found in E. faecium, in this article, VRE will refer
to strains containing the vanA gene and Tn1546. First isolated in France in
1986, VRE were subsequently found in the United States in 1989, where they
rapidly became a frequent cause of hospital infections (3). Like many other
nosocomial pathogens, VRE were believed to originate and be maintained in
hospitals and to have little, if any, association with the community.
Nosocomial outbreaks and clonal spread in the United States supported this
assumption (4). In Europe, however, even though serious incidents have
occurred in some countries, VRE-associated hospital infections have not
increased at the same rate and to the same proportion as in the United
States (5). The first indication that the epidemiology of VRE may differ
from that of other gram-positive organisms capable of causing hospital
infections (e.g., methicillin-resistant Staphylococcus aureus) came from
the United Kingdom, where Bates et al. (6) reported isolating VRE from pig
herds as well as from the farm environment. These scientists suggested that
a community source of VRE may exist. Soon afterwards, Klare et al. (7) in
Germany showed that VRE could be cultured frequently from pigs, poultry,
and humans in the community and suggested that VRE may be associated with
the use of glycopeptides as growth promoters in food animals.

Antimicrobial Growth Promoters

Antimicrobial growth promoters (AGPs) are antibiotics added to the feed of
food animals to enhance their growth rate and production performance. The
mechanism by which AGPs work is not clear. AGPs reduce normal intestinal
flora (which compete with the host for nutrients) and harmful gut bacteria
(which may reduce performance by causing subclinical disease). The effect
on growth may be due to a combination of both fewer normal intestinal flora
and fewer harmful bacteria. The class of antimicrobial drugs used and the
animal species involved may determine the relative importance of each
mechanism (8). The quantity used in feed varies with each antimicrobial
agent. In the European Union (EU), avoparcin 20 mg/kg and 40 mg/kg was
approved for different age groups of pigs and chickens; the concentration
is often referred to as "subtherapeutic" (not to be confused with sub-MIC
levels). The resulting concentration in the gastrointestinal tract of the
animal is sufficient to inhibit the susceptible bacteria and markedly
affect the composition of the bacterial gut flora (8).

In Denmark, as well as in other countries, only a few glycopeptides have
been used; for humans vancomycin and (to a lesser extent) teicoplanin have
been used, and for animals avoparcin has been used exclusively as a feed
additive for growth promotion. Avoparcin has not been used in animals in
Sweden since 1986 because of a national prohibition of AGPs; in the United
States, avoparcin was never approved because of its carcinogenic effects
(9).

Few countries have accurate data on the use of antibiotics in animals and
humans. In Denmark, 24 kg of active vancomycin was used for human therapy
in 1994. In comparison, 24,000 kg of active avoparcin was used as a feed
additive for animals (10). In Austria, an average of 582 kg of vancomycin
was imported for medical purposes and 62,642 kg of avoparcin for animal
husbandry per year from 1992 to 1996 (11). Thus, although there are more
food animals than humans, the selective pressure favoring VRE in Europe can
be estimated to be much higher in food animals than in humans. Data on the
yearly use of vancomycin in the United States and in major European
countries were recently published by Kirst et al. (12). Denmark, a small
country, used more glycopeptide growth promotant (avoparcin) than all of
Europe and the United States used for treating ill humans (vancomycin).
Difference in denominators implies huge difference in use (10).

Use of Avoparcin as a Growth Promoter and the Occurrence of VRE in Food
Animals

The high selective pressure by the use of glycopeptides as growth promoters
could explain the presence of VRE in food animals. We have conducted a
number of studies to investigate the association between the use of
avoparcin as a growth promoter and the occurrence of VRE.

In one study, eight poultry flocks raised conventionally and six raised
without growth promoters were compared (13). No VRE was found in birds
raised without growth promoters, whereas five out of eight conventional
flocks contained VRE. Isolation rates in positive flocks were as follows:
of five fecal samples tested, one to four (20%80%) were positive.
Twenty-two pig herds and 24 poultry flocks, half of which had used
avoparcin and half of which had not, were compared by occurrence of VRE in
fecal samples collected from animals of the herds and flocks. A strong and
statistically highly significant association between the presence of VRE
and the use of avoparcin was observed (14). Of 12 pig herds using feed with
avoparcin, 8 had VRE, while of 10 herds not using avoparcin, 2 had VRE (p =
0.043, risk ratio [RR] 3.3; 95% confidence interval [CI]: 1.1, 10.0). In
broiler farms where avoparcin was used, VRE was isolated from 11 of 12
fecal samples. In farms where avoparcin was not used, VRE was isolated in 2
of 12 samples (p <0.0006; RR 5.5; 95% CI: 2.2, 13.9).

The association observed at the flock and herd level has also been observed
at the country level. In countries where avoparcin had been used as a
growth promoter, VRE could frequently be cultured from food animals,
whereas in countries where avoparcin had not been used, VRE were not
detected (Table 1). These findings are consistent with the hypothesis that
use of avoparcin has created a reservoir for VRE in food animals.

Table 1. Avoparcin as a growth promoter      Transmission of VRE from
in countries where the occurrence of         Animals to Humans
vancomycin-resistant enterococci (VRE)
in animal husbandry has been investigated    Can an animal reservoir in
                                             itself be regarded a public
-------------------------------------------- health risk? What are the
                            VRE in           chances that VRE or the
                Avoparcin   animal           resistance genes will be
Country            used    husbandry  Ref.   transmitted from animals to
-------------------------------------------- humans? A public health risk
                                             must be assumed to exist when
Belgium              +         +       15    transfer from animals to
Denmark              +         +     13, 14  humans can be shown directly
Finland              +         +       16    or indirectly.
France               +         +       17    VRE are frequently present in
Germany              +         +       7     food produced in Denmark as
                                             well as in food imported into
Great Britain        +         +       6     Denmark from other European
The Netherlands      +         +       18    countries (23,24). Thus,
Norway               +         +       19    exposure to humans from
                                             insufficiently heated food or
Sweden                               20    cross-contaminated
United States                      21, 22  ready-to-eat food takes place.
                                             Unlike studies in the United
States (21,25), European studies reported that humans frequently are fecal
carriers of VRE (26-29). This suggests that VRE can be ingested from food
in Europe. Furthermore, VRE was not detected in strict vegetarians in The
Netherlands, supporting the view that the source of VRE is contaminated
meat (Table 2) (28).

Molecular typing shows a very high diversity of VRE types in animals as
well as humans (30). Nevertheless, similar or related types have been shown
to occur in animals and humans on a number of occasions, supporting the
assumption that transfer of VRE between humans and animals does take place
(18,19). We have recently compared 84 isolates of E. faecium from swine,
chickens, and humans in Denmark by SmaI generated macrorestriction profiles
and EcoRI ribotyping. Similarity analysis by unweighted pair group method
with arithmetic averagesderived dendrograms did not indicate a higher
degree of similarity among E. faecium isolates (VRE as well as non-VRE)
from humans than from animals. This finding indirectly supports the
hypothesis that E. faecium from different food animals and humans are not
discrete populations but belong to a common pool of E. faecium shared by
animals and humans (data not shown).

The VanA gene cluster      Table 2. Prevalence of vancomycin-resistant
encoding for vancomycin    Enterococcus faecium in fecal samples of
resistance in animal and   residents in a vegetarian and nonvegetarian
human VRE is located on a  nursing home, The Netherlands (28)
transposon designated
Tn1546 (32,33). Tn1546 can -------------------------------------------------
easily spread from one                                No. E.
enterococcal species to                  No. persons  faecium    No. VRE
another as well as from                  investigated positive positive(sup a,b)
enterococci to S. aureus   -------------------------------------------------
(33,34). Recent
investigations have        Vegetarians         42         23         0
documented that in vivo    Nonvegetarians      62         32         6
transfer of Tn1546 can     -------------------------------------------------
take place in the
mammalian intestinal tract(sup a)P<0.05.
(A. Sundsfjord, pers.     (sup b)All VRE-positive samples were E. faecium.
comm.). Furthermore,
animal VRE can colonize the human intestinal tract for at least 3 weeks
after experimental ingestion of 10(sup 7) CFUs of a single strain (35). This
indicates that vancomycin resistance can spread in the gastrointestinal
tract from transiently colonizing animal VRE to E. faecium strains of the
resident human gut flora.

The VanA gene cluster consists of several genes. We investigated the genes
and the regions between them by sequencing of selected areas, polymerase
chain reaction amplification of other areas, and hybridization with
specific probes (36,37). Thirteen different types were observed. Most
differences arose from the presence of insertions or deletions in noncoding
intergenic regions. One nucleotide difference was observed in the coding
sequences; this point mutation occurred in the vanX gene at position 8234,
where a G in the reference VRE strain was substituted for a T in some
isolates.

In human VRE isolates, this mutation was evenly distributed, whereas in
poultry isolates from different countries only the G variant occurred; in
isolates from swine from different countries the T variant occurred in
nearly all isolates (Table 3). Although we have no explanation for the
uneven distribution of subtypes between different animals, the finding of
both types in humans does support the hypothesis that animals are a primary
source of vancomycin resistance genes in humans, whereas humans apparently
do not serve as reservoir for animals, in which case both types would be
expected to occur in both animal species. In the same investigation, we
found that all human isolates from a Muslim country belonged to the poultry
subtype (37). The absence of pork variant types in a Muslim country
suggests that food of animal origin is a major reservoir for VRE in humans.

Table 3. Variations in Tn1546-    The European/American Paradox
like elements of vancomycin-
resistant Enterococcus faecium    Even though the greater frequency of VRE
isolates of animal and human      infections in U.S. than in European
origin (37)                       hospitals would seem to contradict it
                                  (12), the hypothesis that animals could
--------------------------------- serve as reservoirs of human VRE

Source  No.      T       G        infections is supported by several lines
        isolates variant variant  of indirect evidence.
---------------------------------
                                  Heavy use of vancomycin (and probably
Humans    45     16        29     also third-generation cephalosporins) is
Pigs      33     32          1    a prerequisite for frequent VRE
Poultry  193      0       193     infections in hospitals. Heavy use of
                                  vancomycin and third generation
Total    271     48       223     cephalosporins is more frequent in U.S.
--------------------------------- than in European hospitals (12,18). Thus,
                                  the problem in Europe, irrespective of a
high carrier rate of VRE in the community, has not grown to the same
proportions as in the United States. VRE infections in the United States
are probably due to the heavy use of antibiotics in hospitals and the
eventual spread of VRE within and among hospitals by carrier personnel
(38).

Another prerequisite for high incidence of VRE hospital infections is a
source of VRE. In the United States, primary sources of VRE to hospitals
include travelers returning from abroad, tourists, and imported food. Once
inside the hospital, VRE can cause nosocomial outbreaks because of its high
potential to colonize and persist in the environment, which facilitates its
persistence and spread (4). An alternative hypothesis could be that some
clones of VRE have a higher potential to cause infections and that such
clones are more prevalent in United States. This hypothesis, however, is
contradicted by the high number of different types of VRE causing
infections in Europe and the United States, which suggests that pathogenic
potential is not limited to a few clones of VRE (38).

Avoparcin was approved for growth promotion in Europe in 1974. The first
VRE was detected in a human patient in France in 1986. Does this suggest
that VRE was not present in animals before 1986? Historically, resistance
to growth promoters in animal bacteria was not monitored, and with few
exceptions the studies conducted have looked at bacteria other than
enterococci because enterococci were not considered foodborne pathogens.
Thus, if VRE were not detected in food animals, it may be because they were
not looked for.

The Effect of Prohibiting Use of Avoparcin as a Growth Promoter

Because of public health concerns about resistance to glycopeptide
antibiotic drugs, avoparcin was banned in Denmark in 1995. In 1996, Germany
took a similar step, and finally in 1997, avoparcin was banned in all EU
member states. After the ban in Denmark, a marked reduction in the
occurrence of VRE in Danish poultry flocks has been observed at slaughter
(from 82% in 1995 to 12% of flocks in 1998; x(sup 2) = 68.3 on 5 df.; 
p <0.0001), whereas in swine only a minor reduction has been observed 
(Figure) (39). In Germany, a decrease in the incidence of VRE in poultry 
meat and in fecal samples from humans in the community was observed after 
discontinuation of avoparcin use in animal husbandry (40). In poultry meat 
the proportion of VRE-positive samples were reduced from 100% in 1994 to 25% 
in 1997, and in fecal samples from humans in the community, the carrier rate 
decreased from 12% in 1994 to 3% in 1997.

Similar                      
Problems Related 				    	     [fig]			                                                                Figure.
to Other                      Fig. Trend in the proportion of Enterococcus
Antimicrobial                 faecium isolates resistant to vancomycin
Growth                        (VRE) during successive half-year periods
Promoters                     from second half of 1995 to first half of 1998
                              (39).
Most of the                                                               
different growth promoters approved in the EU are active against gram-positive                                                             
bacteria. With increasing resistance in gram-positive pathogenic bacteria,                                                                 
antimicrobial drugs used as growth promoters have attracted renewed                                                                   
attention as potentially useful for human therapy. More than 10 years after 
the first VRE was discovered, the first drug for humans with good clinical 
effect against VRE infections is ready to be marketed. This drug is a 
combination of two streptogramins, quinupristin and dalfopristin (Synercid).

For decades, virginiamycin, which belongs to the group of streptogramins,
has been used as a growth promoter in the European Union as well as in the
United States, primarily for poultry production. Investigations in the
United States, The Netherlands, and Denmark have frequently found
Synercid-resistant E. faecium in poultry (41-43). As for VRE, we have no
data on the prevalence of streptogramin-resistant E. faecium in animal
husbandry before virginiamycin was used as a growth promoter. No monitoring
has been carried out. Moreover, the gene (satA) conferring resistance to
virginiamycin and Synercid have been found in animals and humans (44), and
in vivo transfer of these genes from resistant to sensitive strains of E.
faecium in the mammalian gastrointestinal tract has been shown (45). Thus,
the events associated with avoparcin and vancomycin may be recurring for
Synercid and virginiamycin. Furthermore, the drug anticipated to be next in
line after Synercid, a compound called Ziracin, belonging to the class of
everninomicins, is practically identical to another growth promoter called
avilamycin, which has primarily been used in poultry.

We have detected avilamycin resistance in 69% of E. faecium isolates from
poultry in Denmark (43). Moreover, preliminary investigations show that
resistance to avilamycin gives cross-resistance to Ziracin and that a
transferable genetic element may be involved (46). Thus, again use of an
antimicrobial drug as a growth promoter may have created a major animal
reservoir of resistant E. faecium, threatening to shorten the life span of
a new promising drug when it is put to use in humans.

Future Perspectives

At the core of the VRE issue appears to be the way antimicrobial drugs are
being developed. New classes of antimicrobial drugs are not available.
Instead, old drugs are being modified that may have been used in
agriculture as growth promoters for decades because they were not
considered useful for humans. Now that physicians are searching for more
options in antibiotic treatment, the older drugs may no longer be viable.
The use of antimicrobial drugs and development of resistance in animals and
humans are interrelated. Therefore, systems to monitor antimicrobial
resistance in pathogenic and commensal bacteria should be established. Such
systems should cover relevant bacteria from the entire farm-to-fork chain
and monitor resistance towards antimicrobial drugs used in both animals and
humans, including growth promoters (47-49).

Finally, antimicrobial agents should not be used for growth promotion if
they are used in human therapeutics or are known to select for
cross-resistance to antimicrobial drugs used in human medicine (47).
Antimicrobial agents are too valuable to be used as a tool in animal
production because any antimicrobial drug may be useful for human therapy
in the future even if not used therapeutically today. Adherence to the
World Health Organization recommendations (47) will ensure a systematic
approach toward replacing antimicrobial growth promoters with safer
nonantimicrobial drug alternatives. The EU countries entered this process
in December 1998 when four growth promoters (tylosin, spiramycin,
bacitracin, and virginiamycin) were banned because of their structural
relatedness to therapeutic antimicrobial drugs used for humans (50).

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

      Dr. Wegener is head of The Danish Zoonosis Centre. His research
interests are veterinary public health, microbiology, epidemiology,
molecular biology; control of zoonoses, particularly the so-called "modern"
bacterial zoonoses; and potential public health consequences of the use of
antimicrobial drugs in animal husbandry.

      Address for correspondence: Henrik C. Wegener, Danish Zoonosis
Centre, Danish Veterinary Laboratory, Blowsvej 27, DK-1790 Copenhagen V,
Denmark; fax: +45-35-300-120; e-mail: hcw@svs.dk.

References

  1. Edmond MB, Ober JF, Weinbaum DL, Pfaller MA, Hwang T, Sanford MD, et
     al. Vancomycin-resistant Enterococcus faecium bacteremia: risk factors
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     vancomycin and teicoplanin resistance in Enterococcus faecium.
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     Tn1546 from vancomycin-resistant enterococci from humans, pigs and
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 41. Welton LA, Thal LA, Perri MB, Donabedien S, McMahon J, Chow JW, et al.
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 42. Van den Bogaard AE, Mertens P, London NH, Stobberingh EE. High
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---------------------------------------------------------------------------------

Perspectives

Bacterial Vaccines and Serotype Replacement: Lessons from Haemophilus
influenzae and Prospects for Streptococcus pneumoniae

Marc Lipsitch
Emory University, Atlanta, Georgia, USA

---------------------------------------------------------------------------
      Conjugate vaccines have reduced the incidence of invasive
      disease caused by Haemophilus influenzae, type b (Hib), in
      industrialized countries and may be highly effective against
      Streptococcus pneumoniae. However, the serotype specificity of
      these vaccines has led to concern that their use may increase
      carriage of and disease from serotypes not included in the
      vaccine. Replacement has not occurred with the use of Hib
      vaccines but has occurred in trials of pneumococcal vaccines.
      Mathematical models can be used to elucidate these contrasting
      outcomes, predict the conditions under which serotype
      replacement is likely, interpret the results of conjugate
      vaccine trials, design trials that will better detect serotype
      replacement (if it occurs), and suggest factors to consider in
      choosing the serotype composition of vaccines.

Conjugate vaccines are a major advance in the control of diseases caused by
two members of the normal bacterial flora of the human nasopharynx,
Streptococcus pneumoniae (pneumococcus) and Haemophilus influenzae type b
(Hib). In the absence of widespread vaccination, pneumococci have been
responsible for an estimated 7 million cases of otitis media, 500,000 cases
of pneumonia, 50,000 cases of bacteremia, and 3,000 cases of meningitis
each year in the United States (1). Before the widespread use of conjugate
vaccines, Hib caused invasive disease in an estimated 1 in 200 children <5
years of age in the United States (2). Conjugate vaccines have reduced the
incidence of invasive Hib disease by 90% or more in industrialized
countries (2,3). After promising phase-II clinical trials (4-7), the first
results from a phase-III trial of a pneumococcal conjugate vaccine have
shown very high efficacy against invasive disease (8).

In addition to protecting against disease, conjugate vaccines protect
against asymptomatic carriage of the target organisms (4-7,9). H.
influenzae and S. pneumoniae are frequently found in the normal
nasopharyngeal flora of healthy persons, with invasive disease being
relatively rare compared with asymptomatic carriage (10,11). Asymptomatic
carriers are also responsible for most transmission of these organisms
(10,11); in contrast to many other vaccine-preventable infections, disease
caused by these organisms seems to contribute little to the process of
transmission (12-14). By reducing the rate of carriage of targeted
bacteria, conjugate vaccines also reduce their transmission and should
thereby offer protection to unvaccinated contacts of vaccinated persons. It
has been shown (in the case of Hib) and suggested (in the case of
pneumococcus) that the use of conjugate vaccines results in herd immunity
(15). Herd immunity may explain why the reduction in invasive Hib disease
in some populations has exceeded the fraction of the population that
received the vaccine (3) and why Hib invasive disease declined even in age
groups that had not yet received the vaccine (16).

Although the reduction in carriage achieved by conjugate vaccines is
beneficial from the perspective of herd immunity, it has raised concerns
about the possibility of serotype replacement. Both H. influenzae and
pneumococci are characterized by extensive antigenic diversity in their
polysaccharide capsules. In H. influenzae, six capsular types are known, in
addition to a large group of nontypeable (unencapsulated) variants. Before
vaccination, serotype b was responsible for most invasive diseases, with
minor contributions from the other encapsulated types. Because of the
importance of Hib in invasive disease, vaccination efforts have
concentrated on the b serotype (16). Pneumococci are even more diverse,
with 90 recognized serotypes; many of these serotypes are capable of
causing invasive disease. To accommodate this greater diversity,
pneumococcal conjugate vaccines have incorporated multiple serotypes.
Because the protection offered by conjugate vaccines is specific to the
capsular type(s) included in the vaccine, it has been suggested that
reducing carriage of these vaccine types may leave open an ecologic niche
that will be filled by serotypes not included in the vaccine (5,17-20).

Hib conjugate vaccines served as a model for the development and testing of
pneumococcal vaccines. However, pneumococci are epidemiologically different
from Hib, and results of clinical trials with pneumococcal conjugates
suggest that the two bacteria differ in their response to vaccination,
especially with respect to serotype replacement. This article describes how
mathematical models can be used to elucidate these contrasting outcomes,
specify the conditions under which serotype replacement is likely,
interpret the results of conjugate vaccine trials, design trials that will
be better able to detect serotype replacement (if it occurs), and suggest
factors to consider in choosing the serotype composition of vaccines.

Serotype Replacement: Hib and Pneumococcal Conjugate Vaccines

Serotype replacement has not been detected since the introduction of Hib
conjugate vaccines. Studies of H. influenzae carriage in 700 children in
Finland (21) and 364 families in the United Kingdom (15,22) found no
evidence of increased carriage of non-b H. influenzae as a result of
vaccination. Although increases in invasive disease from other
nasopharyngeal bacteria have been reported since Hib vaccination began
(23,24), no evidence of a causal link to Hib vaccination has been observed
(22,23). Furthermore, a recent study in the United States showed that the
net impact of Hib vaccination has been a 68% reduction in invasive disease
from all H. influenzae between 1986 and 1995 (25); therefore, any increase
in disease from non-b serotypes is small compared with the reduction in
disease from type b.

In contrast, pneumococcal conjugate vaccine studies show considerable
evidence of serotype replacement, as measured by nasopharyngeal carriage of
nonvaccine type organisms. Increases in the carriage of nonvaccine
serotypes have occurred in three major ongoing clinical trials of
pneumococcal conjugate vaccines. In Gambia, carriage of nonvaccine
serotypes was 79% in children receiving three doses of a pneumococcal
conjugate vaccine (compared with 42.5% in controls) (5). In trials of a
9-valent vaccine in South Africa, carriage of nonvaccine serotypes
increased from 21% in controls to 39% in vaccine recipients (6). Serotype
replacement was observed in the second of two large studies in Israel
(4,7); the reason for the difference in outcome between the two studies
remains unclear. In the first phase-III trial for which data were
presented, no increase was observed in invasive disease from nonvaccine
types (8). While this result is encouraging, it may not be indicative of
what will occur as conjugate vaccines enter widespread use in a variety of
communities.

A Mathematical Model of Vaccination against Colonizing Bacteria

Mathematical models can be useful in defining the extent of serotype
replacement in various contexts, optimizing the design of clinical trials
to discern whether such replacement occurs, and interpreting the results of
these trials. With these goals in mind, I constructed and analyzed a
mathematical model of the transmission dynamics of colonizing bacteria with
multiple serotypes, such as pneumococci, and the effect of vaccination on
these dynamics. The model is similar in structure to the compartmental
models used to design and predict the effects of vaccination programs
against other infectious diseases (26).

The main distinguishing feature of this model is that it simultaneously
considers the transmission of two (or more) strains of the same organism.
The model is designed to analyze the effects of competitive interactions
between these strains, in which carriage of one serotype reduces the
probability that a host will be colonized with another serotype. If such
competitive interactions occur, serotype replacement is possible, because
vaccine-induced reductions in some serotypes will increase the
opportunities for others to spread in the population. Epidemiologic studies
have provided indirect evidence of such competitive interactions (27-29),
while laboratory studies have suggested mechanisms by which different
species of streptococci (30,31) or different strains of H. influenzae (32)
might compete in the nasopharynx. At present, however, little is known
about the precise nature of these interactions, and perhaps the most
compelling evidence that competition occurs comes from the replacement
observed in pneumococcal conjugate vaccine studies.

The assumptions and structure of the model are as follows. In the absence
of vaccination, the model (Figure 1) assumes that humans are born into the
susceptible (X) compartment at a particular rate and are removed from that
compartment (and all other compartments) at a specific per capita death (or
maturation) rate. Two pneumococcal serotypes (designated 1 and 2) are
present, and susceptible hosts may be colonized by either type; colonization
moves the host into the Y(sub 1) or Y(sub 2) compartment, respectively.
The incidence of colonization with each type is proportional to the total
number of persons carrying that type. Colonization has average duration
1/gamma. While carrying one serotype, a host may be colonized by the other
type, which moves the host into the dually colonized compartment (Y(sub 12)).
This secondary colonization also occurs at a rate proportional to the
prevalence of the colonizing type, but a rate that is c(sub j) (j = 1 or 2) times
the rate at which a susceptible person would be colonized by the same type.
Thus, c(sub j) is an inverse measure of the competitive inhibition of type j by
the resident type in a host.

                                When vaccination begins, a fraction f of
         [Fig]			  all persons are assumed to be vaccinated at
                                birth. In the model, these persons are born
Fig. 1. The structure of the    into the vaccinated (V) compartment. It is
mathematical model described    assumed that vaccination completely
in the text and in greater      protects a person against carriage of type
detail (30).                    1 (this is done to simplify the analysis of
                                the model; if only partial protection were
                                offered, the effects would be similar to
those observed at a lower level of vaccine coverage f). To consider the
effects of including more than one bacterial serotype in the vaccine, the
model can accommodate vaccines that are effective only against type 1
(monovalent vaccines), as well as those that give either partial or full
protection against type 2 (bivalent vaccines). The parameter k represents
the degree of protection offered by the vaccine against serotype 2.

By varying the parameters of the model, it is possible to compare the
effects of different levels of vaccine coverage (fractions of the
population vaccinated), different assumptions about the competitive
interactions among pneumococcal serotypes, and different types of vaccines
(monovalent vs. bivalent) (33). In summary, the major predictions of the
model are as follows.

  1. If there is competition between different pneumococcal serotypes to
     colonize hosts, vaccination against serotype 1 alone will increase the
     prevalence of serotype 2. The extent of replacement, measured as the
     increase in the prevalence of serotype 2, will be greatest when
     vaccine coverage is high and when serotype 2 is strongly inhibited
     from colonizing persons who carry serotype 1. Serotype replacement may
     take either of two forms: an increase in prevalence of a type already
     present in the population or the appearance and spread of types
     previously absent from the population because they were unable to
     compete with the vaccine type(s).
  2. Bivalent (or polyvalent) vaccines can also cause replacement if the
     protection offered against different serotypes is uneven. In
     particular, if a vaccine has relatively low efficacy against serotype
     2 but very high efficacy against serotype 1, use of a bivalent vaccine
     may increase the prevalence of type 2.
  3. If only two serotypes interact in a population, the amount of
     replacement that can occur is limited. Specifically, the increase in
     the prevalence of serotype 2 will always be less than or equal to the
     decrease in the prevalence of serotype 1. Thus, for example, if the
     prevalences of serotypes 1 and 2 before vaccination are 15% and 20%,
     respectively, then the prevalence of serotype 2 after vaccination will
     be no more than 35%.
  4. If more than two types are competing to colonize hosts, this
     limitation need not hold. In the presence of more than two types,
     vaccination can increase the prevalence of a single, nonvaccine type
     more than it reduces the prevalence of the vaccine type.
  5. Although replacement is of concern, it may also be beneficial. If
     serotypes compete to colonize hosts, increases in the prevalence of
     the nonvaccine types will help reduce the prevalence of the serotypes
     included in the vaccine. Thus, replacement will augment the effects of
     herd immunity in reducing the exposure of all members of the
     population to vaccine serotypes. This results in a tradeoff between
     the breadth of coverage of a vaccine (number of serotypes covered) and
     the effectiveness of the vaccine in reducing carriage of each serotype
     at the population level.

The model's predictions have several implications for the interpretation of
existing data from the use of conjugate vaccines, the design of vaccine
trials, and the choice of vaccine composition.

Why Has Replacement Carriage Occurred with Pneumococcal Conjugate Vaccines
but Not with Hib Vaccines?

As noted above, the absence of serotype replacement observed with the use
of Hib in industrialized countries contrasts with the findings of
considerable serotype replacement in two studies of pneumococcal vaccines.
What might account for this difference?

The mathematical model suggests an explanation. The model predicts that, in
a pairwise interaction between two serotypes, the increase in prevalence of
a nonvaccine type will be no more than the reduction in prevalence of a
vaccine serotype. This principle is illustrated in Figure 2, which presents
data from a study of Hib conjugate vaccine in the United Kingdom (15). In
the figure, the white bars show the prevalence of each of three H.
influenzae serotypesb, e, and fin vaccinated persons, and the black bars
show the prevalence of each of these serotypes in controls. If one assumes
that Hib interacts independently with each of the two nonvaccine serotypes
(e and f), one can use the two-serotype model to calculate the maximum
prevalence of these nonvaccine types in vaccinees that would be expected if
these serotypes compete very strongly with serotype b. The striped bars
show the maximum prevalence of types e and f expected in the study, where
only a small fraction of the community was vaccinated; the shaded bars
indicate the equivalent figure if the whole community had been vaccinated.
As is clear from the figure, the increase in nonvaccine-type carriage in
vaccinees would be minuscule and statistically undetectable in a study of
this kind (indeed, the study from which these data were drawn was not
designed to detect replacement; data on the prevalence of types e and f
were used to control for general changes in the prevalence of H. influenzae
that could have been attributable to factors other than vaccination [15]).
The reason is that the prevalence of Hib was so low before vaccination that
even its complete removal by widespread vaccination would have little
effect on competing bacteria. The prevalence of Hib carriage in other
industrialized countries is similar to that measured in the UK study.
Therefore, the model suggests that the lack of replacement, even after
widespread use of the Hib conjugate vaccine in industrialized countries,
may be a simple result of the low prevalence of Hib carriage.

If this interpretation is correct, then  
serotype replacement would be more       		   [Fig]
likely to occur in areas where the
prevalence of Hib is higher or for       Fig. 2. Carriage of three
vaccination against other organisms      serotypes of Haemophilus
whose prevalence is higher. This         influenzae in children vaccinated
difference could account for the         against serotype b (white bars)
contrasting outcomes of vaccination      and in controls (black bars) (14).
against Hib and pneumococci. Differences Error bars indicate 95% confidence
in the biology of colonization or in the interval (binomial approximation).
interactions between bacterial types may Shaded bars show the maximum
also have a role in these contrasting    carriage of serotypes e and f in
outcomes. Distinguishing the relative    vaccine recipients that could
importance of these two explanations     result from replacement in a
will require further research into the   population where only a small
biologic interactions of bacterial       proportion of susceptibles are
populations in the nasopharynx, as well  vaccinated (as in the study).
as studies of the effects of conjugate   Striped bars show the equivalent
Hib vaccination in areas where Hib's     figures in a hypothetical study in
prevalence is higher.                    which virtually all susceptibles
                                         were vaccinated.
Detection of Replacement: The Design of
Clinical Trials

If used by a large fraction of the human population in a community, a
conjugate vaccine may alter the composition of the bacterial population,
not only in vaccinated, but also in unvaccinated persons in that community.
Vaccination may reduce the prevalence of serotypes included in the vaccine,
thereby protecting unvaccinated persons against exposure to these serotypes
(herd immunity). Similarly, if serotype replacement occurs and vaccinated
persons become more likely to carry nonvaccine serotypes, the exposure of
unvaccinated persons to these serotypes will increase. As a result of these
indirect effects, strain replacement will be magnified in communities where
large numbers of persons are vaccinated.

This process is also evident from Figure 2. There, the striped bars show
the model's prediction of the maximum increase in non-vaccine type carriage
in vaccine recipients in a community in which the vaccine is used only on a
very small proportion of the population, while the shaded bars show the
same increase in a community where everyone is vaccinated. As is clear from
the figure, replacement will be most easily observed in communities where
the level of vaccine coverage is high.

Therefore, one would expect that the extent of serotype replacement when
vaccines enter widespread use in a community may be much greater than that
observed in clinical trials where a relatively small fraction of the
community is immunized. This is one important reason why the failure to
observe an increase in invasive disease from nonvaccine-type pneumococci in
the Northern California trial (8), while promising, may not be indicative
of the potential for replacement once the vaccine is used on a large scale.
If one is interested in designing a clinical trial that simulates the
selective pressures exerted by communitywide use of a conjugate vaccine,
and therefore maximizes the chances of observing serotype replacement
during the trial, then community-randomized clinical trials will be
superior to individually randomized ones. Studies of pneumococcal vaccines
in which communities are the units of randomization are under way in Native
American communities in the southwestern United States (K. O'Brien, pers.
comm.).

Vaccine Composition: Replacement Revisited

For an organism like pneumococcus, in which a number of serotypes can cause
disease, the choice of serotypes for inclusion in a conjugate vaccine is
critical. One strategy would be to include as many serotypes as possible to
achieve the broadest possible protection. In addition to some clinical
limitations on the number of serotypes that can be included in a single
vaccine, there are other reasons why such a strategy would not be ideal. As
noted above in the last prediction from the model, serotype replacement can
augment the effectiveness of a vaccination program in a community. This
occurs because increases in the prevalence of nonvaccine serotypes
competitively inhibit carriage of vaccine serotypes. Ideally, then, one
would like to design a vaccine that maximizes these beneficial effects
while minimizing the risk of added disease from increased carriage of
nonvaccine serotypes.

The question is how to accomplish such a balance. So far, the model
describes only carriage of various serotypes; it does not directly address
the problem of disease. The effect of vaccination on disease will depend
both on changes in patterns of carriage of different serotypes and on the
propensity of the individual serotypes to cause disease. Serotypes of H.
influenzae and S. pneumoniae vary considerably in their pathogenicity, as
manifested by experimental evidence (34) and by differences between the
frequency of particular serotypes in carriage isolates and their frequency
in disease isolates (17,35). If these serotype associations were stable,
the ideal vaccine could simply include the most pathogenic serotypes but
exclude those that tend to be avirulent, thereby taking advantage of any
increases in the prevalence of the avirulent serotypes to augment the
effect of the vaccine (36).

This approach has several limitations. First, the model predicts that
widespread use of a vaccine may result in the appearance of bacterial types
which, before vaccination, had been absent from the population because of
competition from vaccine types. The virulence of these novel types would be
difficult to predict, since competitive inferiority to existing types need
not be correlated with low virulence (12,13). Second, both species
discussed here are highly transformable. Although capsular type seems to be
very closely associated with virulence in H. influenzae (34,37),
transformation studies in pneumococci have shown complicated interactions
between capsular type and other genes in determining virulence (38), so the
existing associations between virulence and capsular type in pneumococci
(39,40) may change in response to conjugate vaccine-induced selective
pressure. If such vaccines are used on a widespread scale, surveillance of
shifts in the serotype associations of invasive disease should be
maintained.

Serotype replacement has been discussed primarily as it applies to
serotypes not included in the vaccine. However, if the vaccine is only
weakly effective in immunizing against carriage of some of the serotypes
included in it, even these serotypes may increase in prevalence after
vaccination is introduced. This can occur if the efficacy of the vaccine
against these serotypes is outweighed by its effect in removing competing
serotypes. Results of trials published thus far indicate that the
protection offered by the vaccine against included serotypes taken together
is considerably lower than 100%. Therefore, the results of future trials
should be monitored to determine whether prevalence of any of the
individual vaccine serotypes is increasing in vaccinated hosts.

Interpreting Replacement: Is It Real?

Studies of pneumococcal carriage are typically performed by sampling the
nasopharyngeal flora of vaccinated and unvaccinated persons, plating the
samples on agar, and serotyping one or a few colonies. This technique
typically identifies the most abundant pneumococcal serotypes carried by a
person, and possibly a minority type if it is present in large numbers.
However, many people carry more than one pneumococcal type (27,41), and
when the pneumococci are studied in detail, the minority type may be much
less plentiful than the majority typeat a frequency of 10% or less (41).
Therefore, current methods are likely to have very low sensitivity for the
detection of minority types.

This creates a problem in measuring serotype replacement during
pneumococcal vaccine trials. Vaccinated persons, who are protected against
carriage of vaccine types, may become more susceptible to carriage of
pneumococcal types not included in the vaccine. This is serotype
replacement, a phenomenon that vaccine trials are intended, in part, to
detect. In addition, nonvaccine type pneumococci, even if they are not more
plentiful, may be more readily detected in vaccinated persons. Some
unvaccinated persons carry both vaccine-type and nonvaccine-type
pneumococci, and in some of them, the vaccine-type will be in the majority.
Because minority populations of pneumococci are difficult to detect, the
nonvaccine-type pneumococci carried by these persons is masked by the
vaccine types, resulting in an underestimate of the prevalence of
nonvaccine-type pneumococci in the unvaccinated population. Vaccinated
persons, by contrast, are less likely to carry vaccine-type pneumococci, so
their nonvaccine-type pneumococci are more likely to be detected. This is
known as unmasking. Figure 3 illustrates the distinction between serotype
replacement and unmasking. Unmasking is an artifact of sampling, and one
would like to be able to determine whether a finding of higher
nonvaccine-type carriage rates in vaccinated persons reflects true serotype
replacement, unmasking, or a combination of these phenomena.

I have recently developed a statistical procedure to answer this question
(M. Lipsitch, submitted for publication). The procedure attempts to detect
serotype replacement by attempting to reject a null model that incorporates
the effect of unmasking alone. In short, if the increase in nonvaccine type
carriage in vaccinees, compared to controls, is greater than can be
accounted for by this null model, then one concludes that additional
factors, presumably serotype replacement, must be responsible for the
observed increase. This technique has been applied to two datasets, one
from South Africa (6) and one from Gambia (5). In both cases, the observed
increase was greater than that expected from unmasking alone. In the South
African case, the difference was statistically significant (p = 0.02), but
it was not in the Gambian dataset (p = 0.085). However, the Gambian dataset
was extremely small and some information was unavailable for this dataset
that might have improved the power of the test. The test is simple to
perform using the BUGS software (42,43) available free on the World Wide
Web (http://www.mrc-bsu.cam.ac.uk/bugs/Wel come.html) and a program
available from the author; thus, it may be readily applied to future
datasets.

                                   Limitations of Mathematical Models
	    [Fig]    
                                   The mathematical models described here,
Fig. 3. Two hypotheses explain     like all such models, involve a number
the observation of higher rates    of simplifications. In some cases, these
of carriage of nonvaccine          simplifications are introduced to make
serotypes in vaccine recipients    the model more tractable and focus
than in controls. Large circles    attention on fundamental processes of
represent plated samples from      transmission and competition between
controls (top) andvaccine          serotypes. In other cases, the
recipients (bottom). The left      simplifications are necessary because
side shows true serotype           much remains unknown about the
replacement; here a control        biologyand especially the immunologyof
carries vaccine types (white       carriage of these organisms. The
colonies), while a vaccine         assumptions of the model are discussed
recipient does not, and (possibly  at greater length (33). One of these
as a result of decreased           assumptions will be considered here in
competition) now carries only      greater detail to highlight some areas
nonvaccine types (black            where additional knowledge of the
colonies). The right side shows    biology of pneumococcal-host
the unmasking phenomenon, which    interactions is most needed.
is an artifact of sampling. Here,
both vaccinees and controls carry  The model assumes that bacteria of
nonvaccine types, but because      different serotypes compete via direct
only one colony is sampled in      interactions in the nasopharynx. These
each, the vaccinee does not        interactions may take the form of
appear to carry nonvaccine types.  competition for resources, such as
                                   attachment sites or nutrients, or they
                                   may take the form of interference
competition, in which a resident type produces substances toxic to other
bacteria that may attempt to colonize the same host. Apart from the few
studies cited above, little is known about either the intensity or the
mechanisms of such inhibition. There are some epidemiologic data that
indirectly indicate the existence of such competitive interactions. A study
of military personnel in 1946 (27) used a very sensitive technique, mouse
inoculation, to detect nasopharyngeal carriage of one or more pneumococcal
serotypes. The numbers of persons carrying one, two, three, or four
serotypes are given, and although the published data do not provide all of
the information necessary for formal statistical inference, the pattern
suggests that interference between serotypes may have occurred.

The model does not take into account acquired immunity to carriage of these
bacteria, or the possibility that carriage of one serotype may inhibit
future carriage of another serotype, even after the first is no longer
carried. It is unclear to what degree it is realistic to ignore acquired
immunity to carriage. While carriage has been shown to induce a serum
antibody response in at least one report (44), it is less clear whether
such responses affect carriage at the nasopharyngeal mucosa. The success of
conjugate vaccines in reducing carriage indicates that some antibody
responses can affect carriage. However, it remains to be demonstrated
whether such responses are induced by natural exposure through the
respiratory route, whether natural exposure induces responses to other,
more conserved antigens or only to the capsular antigen, and whether
natural exposure induces long-lived immunologic memory. Preliminary results
of mathematical models that incorporate naturally acquired immunity to
carriage suggest that the expected effects of vaccination on the serotype
composition of the population may be different from those expected under
the models described here. Therefore, further research into the
microbiology and immunology of the host-bacterial relationship in the
nasopharynx will be critical to understanding and predicting the
population-wide effects of conjugate vaccines.

Additional Considerations

The choice of serotypes for inclusion in conjugate vaccines has been
different in different locations but has generally been designed to cover
serotypes that are most often implicated in invasive disease. Often, these
types coincide with serotypes showing the greatest levels of antibiotic
resistance (45,46). As a result, conjugate vaccination has led to a
reduction in the percentage of antibiotic-resistant pneumococci carried by
vaccinees (4,6).

In principle, replacement could occur with bacteria that differ from the
vaccine targets not only in serotype but in species. Indeed, one of the
studies of bacterial antagonism in the nasopharynx concentrated on
interactions between species rather than between serotypes of the same
species (30). Furthermore, even if replacement is limited to members of the
same species, the serotypes that increase may tend to cause a disease
different from that caused by vaccine-type organisms (e.g., otitis rather
than pneumonia or bacteremia). Therefore, as conjugate vaccines are used,
changes in diseases attributable to organisms that colonize the nasopharynx
should be monitored.

Finally, capsular polysaccharide is not the only possible target for
vaccination. Several pneumococcal vaccines based on protein antigens are in
various stages of testing (47). Because these protein antigens show
considerably less variation among pneumococcal isolates, vaccines based on
them should be less vulnerable to serotype replacement and may be useful as
complements or alternatives to polysaccharide conjugate vaccines.

Conclusions

The occurrence of serotype replacement in three trials of pneumococcal
conjugate vaccines confirms the validity of concerns expressed in
anticipation of these trials. As the results of more clinical trials become
available, it will become clearer how general this phenomenon is.
Mathematical models are useful in suggesting ways to improve the design of
these trials and the interpretation of their results.

The extent and importance of serotype replacement will depend on many
locally variable factors, the prevalence of vaccine-type organisms before
vaccination, and the level of vaccine coverage. This prediction underscores
the need for continuing studies of vaccination in different communities and
for at least some studies in which a substantial fraction of a community
receives the vaccine. Furthermore, the epidemiologic findings of these
studies should be the impetus for further research into the role of
serotype and other factors in determining the variation in pneumococcal
virulence, the nature of immune responses to organisms like the
pneumococcus at the nasopharyngeal mucosal surface, and other questions in
the biology of bacterial carriage.

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

Acknowledgments

      The author thanks Dr. R. Moxon for introducing him to the problem of
serotype replacement and Drs. K. O'Brien, B.R. Levin, O. Levine, R. Dagan,
D. Stephens, M.E. Halloran, and G. Carlone for helpful discussions.

      Marc Lipsitch's work is supported by grant # GM19182 from NIH.

      Dr. Lipsitch completed this research while he was a postdoctoral
fellow with Dr. Bruce Levin at Emory University and a visiting scientist
with Dr. George Carlone at CDC. His research includes experimental,
epidemiologic, and mathematical modeling studies of vaccination and
antimicrobial resistance. In September 1999, he will begin as an assistant
professor of epidemiology at the Harvard School of Public Health.

      Address for correspondence: Marc Lipsitch, Department of
Epidemiology, Harvard School of Public Health, 677 Huntington Avenue,
Boston, MA, 02115, USA; e-mail: lipsitch@epinet.harvard.edu.

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

Perspectives

Iron Loading and Disease Surveillance

Eugene D. Weinberg
Indiana University, Bloomington, Indiana, USA

---------------------------------------------------------------------------
      Iron is an oxidant as well as a nutrient for invading microbial
      and neoplastic cells. Excessive iron in specific tissues and
      cells (iron loading) promotes development of infection,
      neoplasia, cardiomyopathy, arthropathy, and various endocrine
      and possibly neurodegenerative disorders. To contain and
      detoxify the metal, hosts have evolved an iron withholding
      defense system, but the system can be compromised by numerous
      factors. An array of behavioral, medical, and immunologic
      methods are in place or in development to strengthen iron
      withholding. Routine screening for iron loading could provide
      valuable information in epidemiologic, diagnostic,
      prophylactic, and therapeutic studies of emerging infectious
      diseases.

Excessive iron in specific tissues (iron loading) promotes infection,
neoplasia, cardiomyopathy, arthropathy, and a profusion of endocrine and
possibly neurodegenerative disorders (1-5). An array of behavioral,
medical, and immunologic methods are being developed to decrease iron
loading or its detrimental effects. Routine screening for iron loading in
populations exposed to certain diseases can provide valuable epidemiologic,
diagnostic, prophylactic, and therapeutic information.

Hazards of Iron Loading

Iron can contribute to disease development in several ways. Excessive
amounts of the metal in specific tissues and cells can hinder the ability
of proteins, such as transferrin and ferritin, to prevent accretion of free
iron. Moreover, in infectious diseases, inflammatory diseases, and
illnesses that involve ischemia and reperfusion, iron causes reactions that
produce superoxide radicals (6). Nonprotein bound ferric ions are reduced
by superoxide, and the ferrous product is reoxidized by peroxide to
regenerate ferric ions and yield hydroxyl radicals, which attack all
classes of biologic macromolecules. Hydroxyl radicals can depolymerize
polysaccharides, cause DNA strand breaks, inactivate enzymes, and initiate
lipid peroxidation (6).

Iron can also increase disease risk by functioning as a readily available
essential nutrient for invading microbial and neoplastic cells. To survive
and replicate in hosts, microbial pathogens must acquire host iron. Highly
virulent strains possess exceptionally powerful mechanisms for obtaining
host iron from healthy hosts (7). In persons whose tissues and cells
contain excessive iron, pathogens can much more readily procure iron from
molecules of transferrin that are elevated in iron saturation. In such
cases, even microbial strains that are not ordinarily dangerous can cause
illness. Markedly invasive neoplastic cell strains can glean host iron more
easily than less malignant strains or normal host cells (3). Moreover,
iron-loaded tissues are especially susceptible to growth of malignant cells
(Table 1).

How Microbes Acquire       Table 1. Iron loading in specific tissues and
Iron: A Determinant of     increased risk for disease
Host Range and of Tissue
Localization              -------------------------------------------------
                          Tissue type          Disease
The number of infectious  -------------------------------------------------
disease agents whose
virulence is enhanced by  Alveolar macrophages Pulmonary neoplasia
iron continues to                                and infection
increase (Table 2). To
obtain host iron,         Anterior pituitary   Gonadal and growth
                                                 dysfunction
successful pathogens use  Aorta; carotid and
one or more of four         coronary arteries  Atherosclerosis
strategies: binding of
ferrated siderophilins    Colorectal mucosa    Adenoma, carcinoma
with extraction of iron
at the cell surface;      Heart                Arrhythmia,
                                                 cardiomyopathy
erythrocyte lysis,
digestion of hemoglobin,  Infant intestine     Botulism, salmonellosis,
and heme assimilation;                           sudden death
use of siderophores that  Joints               Arthropathy
withdraw iron from
transferrin; and          Liver                Viral hepatitis, cirrhosis,
                                                 carcinoma
procurement of host
intracellular iron.       Macrophages          Intracellular infections

Microbial strains that    Pancreas             Acinar and beta cell
                                                 necrosis, carcinoma
use siderophilin binding  Plasma and lymph     Extracellular infections
often have a very narrow
host range (7).           Skeletal system      Osteoporosis
Bacterial receptors       Skin                 Leprosy, melanoma
recognize siderophilins
generally from a single   Soft tissue          Sarcoma
or closely related host   Substantia nigra     Parkinson's disease
species. Strains of       -------------------------------------------------
Haemophilus somnus, for
example, form receptors for bovine but not for human transferrin; these
bacteria are virulent for cattle but not for humans (9). The human
pathogen, Neisseria meningitidis, can bind ferrated transferrins from
humans and such hominids as chimpanzees, gorillas, and orangutans, but not
from monkeys or nonprimate mammals (10,11). Actinobacillus pleuropneumoniae
synthesizes a swine-specific transferrin receptor and causes pneumonia only
in hogs (12).

Each of the above three pathogens, as well as other organisms that use
siderophilin binding, can often obtain iron from heme. Helicobacter pylori,
for instance, first obtains iron from human ferrated lactoferrin in the
gastric lumen. Then, as it migrates into intercellular junctions of
epithelial cells in the gastric wall, its sole source of iron is heme. This
pathogen binds neither bovine ferrated lactoferrin nor human, bovine, or
equine ferrated transferrin (13).

However, not every pathogen that uses siderophilin binding has a narrow
host range. For example, Staphylococcus aureus can be virulent for a
variety of mammalian species. Strains of this organism can bind human, rat,
and rabbit transferrins and, much less efficiently, bovine, porcine, and
avian transferrins (14). Moreover, isolates of S. aureus also may produce
siderophores (15,16). These small molecules can withdraw iron from
transferrins synthesized by a variety of host species. The siderophore,
staphyloferrin A, removes iron from both human and porcine transferrin;
thus, the metal can be available to invading cells in humans and in hogs.
Erythrocyte lysis, digestion of hemoglobin, and heme assimilation are
available to strains of S. aureus. Bacterial hemolysins generally are
active against erythrocytes from several, although not from all, potential
host species.

Table 2. Microbial genera with strains whose growth in body fluids, cells,
tissues,and intact vertebrate hosts is stimulated by excess iron (8)
---------------------------------------------------------------------------
                              Gram-positive
                              and
                              acid-fast
Fungi             Protozoa    bacteria           Gram-negative bacteria
---------------------------------------------------------------------------
Candida           Entamoeba   Bacillus         Acinetobacter   Klebsiella
Cryptococcus      Leishmania  Clostridium      Aeromonas       Legionella
Histoplasma       Naegleria   Corynebacterium  Alcaligenes     Moraxella
Paracoccidioides  Plasmodium  Erysipelothrix   Campylobacter   Neisseria
Pneumocystis      Toxoplasma  Listeria         Capnocytophaga  Pasteurella
Pythium           Trypanosoma Mycobacterium    Chlamydia       Proteus
Rhizopus                      Staphylococcus   Ehrlichia       Pseudomonas
Trichosporon                  Streptococcus    Enterobacter    Salmonella
                                               Escherichia     Shigella
---------------------------------------------------------------------------

Virulent streptococci are examples of bacteria that neither bind
siderophilins nor produce siderophores yet proficiently invade and
replicate in many tissues in diverse host species. The cellulytic
activities of these pathogens enable them to access such intracellular
sources of host iron as hemoglobin, myoglobin, catalase, and ferritin (17).

The remarkable versatility for host species shown by Listeria monocytogenes
illustrates the adeptness of this organism in procuring iron. Although
mainly a saprophyte that lives in the plant-soil environment, L.
monocytogenes can be acquired by humans and other mammals through ingestion
of undercooked tissue of other mammals, birds, fish, and Crustacea, as well
as from raw vegetables. Unable to bind siderophilins or form siderophores,
L. monocytogenes obtains iron by using either exogenous siderophores of
other microorganisms or natural catechols, such as dopamine and
norepinephrine, in host tissues. The pathogen expresses a cell surface
ferric reductase that recognizes the siderophoric chelated iron site; the
metal is then reduced and assimilated (18). Furthermore, in contrast to
saprophytic strains, systemic pathogenic strains of L. monocytogenes are
hemolytic.

To grow within host cells, pathogens apparently are not required to
synthesize siderophilin binding sites or form siderophores. For instance,
unlike the wild type, siderophore-minus mutants of Salmonella Typhimurium
cannot grow in extracellular compartments of the host. However, both the
wild and mutant strains replicate within host cells (19). Possible sources
of intracellular iron are heme, iron released from transferrin at pH 5.5-6,
and ferritin.

For at least two pathogens, Francisella tularensis and Legionella
pneumophila, the host intracellular niche is obligatory. Like the mutant
strain of S. Typhimurium, these organisms are unable to access iron in
extracellular fluids and tissues. Culturing these bacteria in laboratory
media requires markedly elevated concentrations of iron (20,21).

In host intracellular niches, growth of microbial pathogens is stimulated
by elevation and depressed by decrease of iron. Indeed, at least one
bacterial pathogen, Ehrlichia chaffeensis, induces elevation of iron in its
host cells; intracellular inclusions of the organism cause the host cell to
upregulate expression of the transferrin receptor mRNA (22).

Iron Withholding Defense System

Hosts use several mechanisms (Table 3) to withhold iron from invading
microbial and neoplastic cells: stationing of potent iron binding proteins
at sites of impending microbial invasion; lowering iron levels in body
fluids, diseased tissues, and invaded cells during invasion; and
synthesizing immunoglobulins to the iron acquisition antigens of microbes.

Table 3. The iron withholding defense system (1,8)
---------------------------------------------------------------------------
Constitutive components
     Siderophilins

          Transferrin in plasma, lymph, cerebrospinal fluid

          Lactoferrin in secretions of lachrymal and mammary
          glands and of respiratory, gastrointestinal, and
          genital tracts

     Ferritin within host cells

Processes induced at time of invasion
     Suppression of assimilation of 80% of dietary iron(sup a)

     Suppression of iron efflux from macrophages that have digested
     effete erythrocytes to result in 70% reduction in plasma iron(sup a)

     Increased synthesis of ferritin to sequester withheld iron(sup a)

     Release of neutrophils from bone marrow into circulation and then
     into site of infection(sup a)

     Release of apolactoferrin from neutrophil granules followed by
     binding of iron in septic sites

     Macrophage scavenging of ferrated lactoferrin in areas of sepsis
     and of tumor cell clusters

     Hepatic release of haptoglobin and hemopexin (to bind
     extravasated hemoglobin and hemin, respectively)

     Synthesis of nitric oxide (from L-arginine) by macrophages to
     disrupt iron metabolism of invadersb

     Suppression of growth of microbial cells within macrophages via
     downshift of expression of transferrin receptors and enhanced
     synthesis of Nrampl (23) by the host cells(sup b)

     Induction in B lymphocytes of synthesis of immunoglobulins to
     iron-repressible cell surface proteins that bine either heme,
     ferrated siderophilins, or ferrated siderophores

---------------------------------------------------------------------------
(sup a)Activated by interleukin-1 or -6 or by tumor necrosis factor-alpha
(sup b)Activated by interferon-gamma.

High concentrations of iron not only benefit invading cells, they may also
mediate antimicrobial activities of defense cells. In in vitro studies, 150
M iron augmented macrophage killing of Brucella abortus (24) and, without
altering phagocytosis, 250 M iron enhanced anti-Candida activity of
microglia (25). In the latter system, the metal suppressed synthesis of
nitric oxide but not of tumor necrosis factor A. By generating
oxidant-sensitive mediators, iron may focus influx of neutrophils to sites
of infection (26). Iron loading of staphylococci increased their killing by
peroxide, macrophages, and neutrophil-derived cytoplasts but not by
neutrophils (27). Certain conditions can impair iron withholding (Table 4);
numerous studies have presented evidence that risk for infection or
neoplasia is increased significantly in persons with these conditions.

Table 4. Conditions that can compromise iron                     Detection
withholding (1,3)                                                of Iron
--------------------------------------------------------------   Loading
Excessive intake of iron through intestinal absorption
     Behavioral and nutritional factors                          Screening
                                                                 of large
          Accidental ingestion of iron tablets                   populations
                                                                 for iron
          Adulteration of processed foods with inorganic         loading
            iron or blood                                        can be
                                                                 accomplished
          Excessive consumption of red meats                     with
            (heme iron)                                          inexpensive,
                                                                 noninvasive
          Excessive intake of alcohol (HCl secretion             methods. A
            enhanced)                                            useful
                                                                 indicator
          Folic acid deficiency                                  of iron
                                                                 loading is
          Ingestion of ascorbic acid with inorganic iron         marked
                                                                 elevation
          Use of iron cookware                                   of serum
                                                                 ferritin
     Genetic and physiological factors                           (sFt).
                                                                 However,
          African siderosis                                      sole
                                                                 reliance
          Asplenia (mechanism unknown)                           on this
                                                                 measurement
          Pancreatic deficiency of bicarbonate ions              can be
                                                                 misleading
          Porphyria cutanea tarda                                because
                                                                 sFt
          Regulatory defect in mucosal cells in                  increases
            hemochromatosis                                      moderately
                                                                 during
          Thalassemia, sicklemia, other                          inflammatory
            hemoglobinopathies                                   episodes.
                                                                 Accordingly,
Parenteral iron                                                  concurrent
     Intramuscular and intravenous iron saccharate injections    determination
       in excess                                                 of the
                                                                 percentage
     Multiple transfusions of whole blood or erythrocytes        of iron
       in excess                                                 saturation
                                                                 of serum
Inhaled iron                                                     transferrin
     Exposure to amosite, crocidolite, or tremolite asbestos     (%TS)
                                                                 provides
     Exposure to urban air particulates                          useful
                                                                 information
     Mining iron ore, welding, grinding steel                    (29). In
                                                                 iron
     Painting with iron oxide powder                             loaded
                                                                 persons,
     Tobacco smoking (1-2 g iron inhaled per cigarette pack)    hyperferritinemia
                                                                 generally
Release of body iron from compartments into plasma               is
     Efflux of erythrocyte iron in hemolytic diseases            accompanied
                                                                 by an
     Efflux of hepatocyte iron in hepatitis                      elevation
                                                                 in %TS. In
Deficit in iron withholding                                      contrast,
     Transferrin                                                 in
                                                                 patients
     Decreased synthesis                                         with an
                                                                 inflammatory
          Congenital defect                                      process,
                                                                 hyperferritinemia
          Lack of dietary amino acids in kwashiorkor or          generally
            in jejunoileal bypass                                is
                                                                 accompanied
          Decreased activity in acidosis                         by a
                                                                 reduction
     Lactoferrin                                                 in %TS.

          Neutropenia                                            Iron
                                                                 loading is
          Substitution of bovine milk or milk formula for        associated
            human milk in nursling nutrition                     also with
                                                                 moderate
     Haptoglobin                                                 depression
                                                                 of a third
          Decreased synthesis in persons with haplotype          variable,
            2-2 (28)                                             serum
                                                                 transferrin
--------------------------------------------------------------   receptor
                                                                 (sTfR).
The ratio of sTfR/sFt, apparently independent of inflammation, is
significantly reduced in persons with high levels of iron (5).

Strengthening the Iron Withholding Defense

A considerable array of behavioral, medical, and immunologic methods are in
place or in development for strengthening iron withholding (Table 5) (3).
Additional precautions are indicated for persons who are known to be (or
have a tendency to become) iron loaded. For example, persons with elevated
iron due to either hemochromatosis or alcoholism are cautioned to avoid
eating raw oysters, which may contain Vibrio vulnificus (30). Another
pathogen that likewise causes severe systemic infection in hosts with
elevated iron is Capnocytophaga canimorsis. Accordingly, persons who have
hemochromatosis, alcoholism, or asplenia are advised to receive prompt
antibiotic therapy if they are exposed to a dog bite (31).

De-ironing by phlebotomy is effective in lowering risk for cardiovascular
diseases (32,33) and various neoplasms (34), as well as in therapy for
hepatitis C (35). Interfering with iron metabolism by administering gallium
can be useful in suppressing growth of lymphoma and bladder cancer cells
(36). The antineoplastic action of monoclonal antibodies against ferrated
transferrin receptors has been examined (37). Combinations of the iron
chelator, deferoxamine, with gallium or with antibodies against ferrated
transferrin receptors increase effectiveness against tumor cells.

The natural iron scavenger, lactoferrin, has been shown to remove free iron
from synovial fluid aspirated from joints of rheumatoid arthritic patients
(38). Recombinant human lactoferrin, which is indistinguishable from native
breast milk lactoferrin with respect to its iron binding properties, is now
available (39) and could become a very useful addition to our array of
de-ironing pharmaceutical products.

A recently discovered integral membrane phosphoglycoprotein, Nrampl, is
expressed exclusively in macrophages and is localized to phagolysosomes.
The protein suppresses replication of intramacrophage microbial invaders
apparently by altering iron availability (23). A second protein, Nramp2, is
involved in enhancement of intestinal iron absorption (40). Future research
might develop useful medical procedures for modulation of the actions of
these proteins.

Potential vaccines that incorporate iron acquisition antigens of pathogens
in the families Neisseriaceae and Pasteurellaceae are being developed by
several research groups. For example, in Moraxella catarrhalis, the
recombinant transferrin binding protein B (TbpB) has been shown to elicit
bactericidal antibodies (41) In N. meningitidis, antisera to TbpA and TbpB
were bactericidal for both homologous and heterologous strains (42,43).
Because the antigenic proteins function at the cell surfaces of the
pathogens, the receptors are potentially ideal vaccine candidates. For
synthesis of the receptors, the organisms must be cultured in
iron-restricted media.

Table 5. Methods of strengthening the iron withholding defense system
---------------------------------------------------------------------------
Reduction of excessive intake of ingested iron
     Decreased consumption of red meats (heme iron)

     Avoidance of processed foods that have been adulterated with
     inorganic iron or with blood

     Decreased consumption of alcohol and ascorbic acid

     Elimination of iron supplements unless an iron deficiency has
     been correctly diagnosed

Reduction of excessive intake of parenteral iron
     Inject iron saccharates only if unequivocally justified

     Transfuse blood or erythrocytes only if unequivocally justified

     Substitute erythropoietin (+ minimal amount of iron) for whole
     blood transfusions when possible

Reduction of excessive inhalation of iron
     Eliminate use of tobacco

     Use iron-free chrysotile in place of iron-loaded amosite,
     crocidolite, tremolite varieties of asbestos

     Use mask to avoid inhalation of urban air particulates

     Use mask and protective clothing when mining or cutting
     ferriferous substances

Reduction of iron burden by regular depletion of whole blood or
erythrocytes
     Avoidance of premature hysterectomy

     Routine ingestion of aspirin

     Regular donations of whole blood or erythrocytes

     Vigorous exercise

Increased use of iron chelators
     Use human milk (high in lactoferrin, low in iron) rather than
     milk formula (lacking in lactoferrin, high in iron) in nursling
     nutrition

     Use tea (iron-binding tannins) and bran (iron-binding phytic acid)

     Continue research and development (R&D) of potential iron
     chelator drugs (e.g., recombinant human lactoferrin;
     hydroxpyridones; pyridoxal isonicotinoyl hydrazones)

Initiation of prompt therapy of chronic infections and neoplastic diseases
to forestall saturation of iron withholding defense system
     Continued R&D of cytokines such as interferon g that induce
     cellular iron withholding

     Continued R&D of passive and active methods of immunization
     against surface receptor proteins used by microbial and
     neoplastic cells to obtain iron

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

Perspectives and Conclusions

There is growing awareness that transmissible agents are involved in
diseases not earlier suspected of being infectious (44-46). A recent review
contains a list of 34 degenerative, inflammatory, and neoplastic diseases
associated in various ways with specific infectious agents (44). Other
chronic inflammatory diseases, such as sarcoidosis, inflammatory bowel
disease, rheumatoid arthritis, systemic lupus erythematosus, Wegener
granulomatosis, diabetes mellitus, primary biliary cirrhosis, tropical
sprue, and Kawasaki disease may also have infectious etiologies (45).
Excessive iron is correlated with synovial damage in rheumatoid arthritis
(47) and with impaired glucose metabolism in diabetes (48). The association
of Chlamydia pneumoniae (49) and excessive iron (5) with cardiovascular
disease is well established. Growth of this pathogen is strongly suppressed
by iron restriction (50).

Proving the role of infection in chronic inflammatory diseases and cancer
presents challenges (46). The means by which pathogens suppress, subvert,
or evade host defenses to establish chronic or latent infection have
received little attention. However, the association and causal role of
infectious agents in chronic inflammatory diseases and cancer have major
implications for public health, treatment, and prevention (44-46).

Iron loading is a risk factor in these illnesses, as well as in classic
infectious diseases. Because the prevalence of iron loading in various
populations can be remarkably high, routine screening of iron values in
host populations could provide valuable information in epidemiologic,
diagnostic, prophylactic, and therapeutic studies of emerging infectious
diseases.

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

Acknowledgment

      Dedicated to Jerome L. Sullivan, pioneer and leader in our awareness
of the role of iron in cardiovascular disease.

      Support for this review was provided by the Office of Research and
the University Graduate School, Indiana University, Bloomington, IN, USA.

      Dr. Weinberg is professor emeritus of microbiology in both the
College of Arts and Sciences and the School of Medicine at Indiana
University, Bloomington, IN. His studies on iron were initiated in 1952.
Since retiring from teaching in 1992, he has devoted full time to research.

      Address for correspondence: E.D. Weinberg, Jordan Hall 142, Indiana
University, Bloomington, IN 47405, USA; fax: 812-855-6705; e-mail:
eweinber@indiana.edu.

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_____________________________________________________________________________
Synopses
_____________________________________________________________________________

Synopses

Human Herpesvirus 6: An Emerging Pathogen

Gabriella Campadelli-Fiume, Prisco Mirandola, and Laura Menotti
University of Bologna, Bologna, Italy

---------------------------------------------------------------------------
      Infections with human herpesvirus 6 (HHV-6), a -herpesvirus of
      which two variant groups (A and B) are recognized, is very
      common, approaching 100% in seroprevalence. Primary infection
      with HHV-6B causes roseola infantum or exanthem subitum, a
      common childhood disease that resolves spontaneously. After
      primary infection, the virus replicates in the salivary glands
      and is shed in saliva, the recognized route of transmission for
      variant B strains; it remains latent in lymphocytes and
      monocytes and persists at low levels in cells and tissues. Not
      usually associated with disease in the immunocompetent, HHV-6
      infection is a major cause of opportunistic viral infections in
      the immunosuppressed, typically AIDS patients and transplant
      recipients, in whom HHV-6 infection/reactivation may culminate
      in rejection of transplanted organs and death. Other
      opportunistic viruses, human cytomegalovirus and HHV-7, also
      infect or reactivate in persons at risk. Another disease whose
      pathogenesis may be correlated with HHV-6 is multiple
      sclerosis. Data in favor of and against the correlation are
      discussed.

The Discovery of Human Herpesvirus 6 (HHV-6)

Initially designated HBLV, for human B-lymphotropic virus, HHV-6 was
isolated fortuitously in 1986 from interleukin 2-stimulated peripheral
blood mononuclear cells (PBMCs) of patients with AIDS or
lymphoproliferative disorders (1). The PBMC cultures exhibited an unusual
cytopathic effect characterized by enlarged balloonlike cells. The
causative agent was identified as a herpesvirus by electron microscopy and
lack of cross-hybridization to a number of human herpesviruses (2). The GS
strain is the prototype of the first isolates. Two additional isolates of
lymphotropic human herpesviruses, U1102 and Gambian, genetically similar to
HBLV, were obtained 1 year later from PBMCs of African AIDS patients. All
of the isolates could grow in T cells (CEM, H9, Jurkat), in monocytes
(HL60, U937), in glial cells (HED), as well as in B-cell lines (Raji,
RAMOS, L4, WHPT) (3,4). A new variant, Z29, subsequently shown to differ in
restriction endonuclease pattern from GS-like strains, was isolated from
PBMCs of patients with AIDS (5). The cells supporting virus growth were
characterized as CD4+ T lymphocytes (6). The designation HHV-6 was proposed
1 year after discovery of the first isolate to comply with the rules
established by the International Committee on Taxonomy of Viruses (7).

More than 100 additional HHV-6 strains have been isolated from PBMCs of
children with subitum or febrile syndromes (8), from cell-free saliva of
healthy or HIV-infected patients (9,10), from PBMCs of patients with
chronic fatigue syndrome (CFS) (11), and from PBMCs of healthy adultsthese
PBMCs were cultivated for human herpesvirus 7 (HHV-7) isolation (12).

The Virus

HHV-6 and HHV-7 belong to the Roseolovirus genus of the -herpesvirus
subfamily; HHV-6 species are divided into two variants: HHV-6A and HHV-6B.
The virion particle is 160 nm to 200 nm and has the morphologic features
typical of herpes virion particles (a central core containing the viral
DNA, a 90-nm to 110-nm capsid, and a tegument layer surrounded by a
membrane structure) (13). We summarize briefly key features.

                                  HHV-6A genomes are 159 kbp to 170 kbp
	  [fig]                     long. As sequenced, the genome of U1102
                                  strain is 159 kbp long (14); the HHV-6B
Fig. 1. Schematic                 genome has been sequenced only partially.
representation of HHV-6 and       Seven gene blocks in the central region
HHV-7 genomes. The genomes are    (I-VII) designated as herpesvirus core
colinear. Homologies are 46.6%    genes are common to all Herpesviridae.
to 84.9%. Red blocks represent    Another block, spanning open reading
the herpesvirus core genes,       frames (ORFs) U2 to U14, contains genes
numbered from I to VII. Yellow    specific to -herpesviruses. A further
blocks represent -herpesvirus    region, encompassing ORFs U15-U25,
subfamily-specific genes (from    contains genes specific to Roseolovirus
U2 to U14). Green blocks          genus. Three genes (U22, U83, U94) are
indicate genes present only in    specific to HHV-6 and absent from HHV-7
the Roseolovirus genus, i.e.,     (Figure 1). The closest homology and
in HHV-6 and HHV-7. Only three    similarity in genome organization is to
ORFs (U22, U83 and U94) are       HHV-7 and next to human cytomegalovirus
present in HHV-6 and absent       (HCMV). Amino acid similarity to HHV-7 is
from HHV-7 (modified from         46.6% to 84.9% and to HCMV 41.0% to 75.8%
[15]).                            (14,16). The HHV-6 genome is composed of
                                  a unique sequence (85% to 90% of the
                                  genome) bracketed by direct repeats (10%
to 15% of the genome) that contain the cleavage and packaging sequences
pac-1 and pac-2 and a single origin of replication (OriLyt) located at 70
kbp of the genome. The number of predicted ORFs, 102 or 85, varies
depending on the values used to define an ORF and was attributed mainly on
the basis of the similarity with HCMV (14) or HHV-7 (17) counterparts. Few
gene products have been characterized so far. They include the
immediate-early gene IE-A, which together with IE-B constitutes the IE
locus, a highly spliced region with an arrangement similar to that of HCMV
(18); the U3 gene, a homolog of the HCMV UL24 gene, with transactivating
activity (19); the origin binding protein (20), the U53 protease (21); and
p100, also designated p101, highly immunogenic, and most probably a
constituent of the tegument (22,23). In addition, HHV-6 (but not HHV-7)
carries a homolog of the adenoassociated type 2 parvovirus rep gene (24),
which is transcribed in latently infected cells (25). Recently, the U12
protein was recognized as a -chemokine receptor (26). A major focus has
been in the glycoprotein field. Five glycoproteins were identified: gB
(U39, gp116) (27-30), gH (U48, gp100) (31), and gL (U82, gp80), which form
at least a heterodimer, gM (U72), and gp82-105 (U100) (29,30,32,33). gB and
gH/gL were shown to be virion constituents, and antibody to gH can
neutralize virion infectivity and syncytia formation, suggesting a role of
gH in virus entry and in virus-induced cell fusion (31). The HHV-6 genome
sequence predicts a locus of glycoproteins U20-U24 and U85 that are
specific to the Roseolovirus genus (14), but the proteins have not yet been
identified. U20 and U85 have a predicted immunoglobulin structure.

Variant A and Variant B HHV-6 Strains

Frenkel and co-workers (34), Ablashi et al. (35), and Aubin et al. (36)
were the first to discover that HHV-6 isolates display genetic and
phenotypic variations. All the strains derived so far segregate into two
groups, variant A and variant B, whose genome organization appears to be
overlapping. Viruses belonging to the two variants differ with respect to
several properties. Differences in restriction endonuclease cleavage sites
are scattered throughout the entire genomes. Extent of homologies at
nucleotide level varies from 99% to 95% for the most conserved genes
located in the center of the genome to approximately 75% for the most
divergent portions, located in the immediate-early region. Major
differences in biologic properties concern the in vitro cell tropism,
regulation of transcription and splicing patterns, reactivity to some MAbs
directed to variant-specific epitopes (29,34,35). Typically, variant A
viruses replicate in HSB-2 cells, whereas the variant B viruses grow in the
less differentiated Molt3 T-cell lines. Variant B viruses grow to higher
yields than variant A viruses in primary human fetal astrocytes and require
IL-2- and phytohemagglutinin-activated PBMCs. Differences between the two
variants affect the regulation of transcription of some ORFs of the
immediate early region-B and-A (U16, U17, U91) and the splicing pattern of
ORFs U18-U20 (37). The differences relative to infection in humans
(epidemiology, correlation with pathologic features, tissue tropism) are
detailed below and in the table.

All strains fall into one or the other variant group. There is no evidence
of recombination or a genetic gradient, which suggests that in vivo the two
groups of viruses occupy different ecologic niches. Any isolate
characterized for more than one marker has been unambiguously assigned to
one or the other variant group. The designation of the two groups as
variants has been highly debated and controversial (38). A key question is
whether the two variants fulfill the criteria defined by the International
Committee on Taxonomy of Viruses for classification as different species
(13). In our opinion, the information summarized above indicates that the
two variant groups may be different species; therefore, the issue of
nomenclature should be reconsidered.

Natural History of HHV-6 Infection

Infection with HHV-6 is very common, approaching 100% in seroprevalence.
Exceptions, if confirmed, are represented by countries (e.g., Morocco)
where seroprevalence is much lower (20%) (39). Antibody titers are high in
newborn children, drop at 3 to 9 months after birth, rise again briefly
thereafter, and remain elevated until the age of 60 or older. This pattern
indicates that newborns carry maternal antibodies and primary infection
occurs in the first 3 years of life, most frequently the first year.
Transplacental infections are very infrequent but may contribute to HHV-6
seropositivity in newborns (40).

Three stages can be     Table. Epidemiology and distribution of human
recognized in the       herpesvirus (HHV-6) variants
natural history of      ---------------------------------------------------
HHV-6 infection                                        Variant A Variant B
(Figure 2). The         ---------------------------------------------------
first is represented
by acute primary        Associated pathologic
infection in              conditions
infants. The second       Exanthem subitum               _(sup a) ++++
                            febrile syndromes(sup b)
occurs in healthy
children and adults;      Multiple sclerosis             ++        ++
the virus replicates      Lymphomas and neoplasies       ++        ++
in the salivary           Reactivation in transplants    ++        ++
glands and is             Reactivation in AIDS           ++        ++
secreted in saliva
(for HHV-6B) without    Tissue distribution

inducing any obvious      Peripheral blood                +        +++
pathology, remains          mononuclear cells
latent at least in        Salivary glands                 _       ++++
lymphocytes and           Skin                           ++        ++
monocytes, and            Brain                          ++        ++
persists in various       Lymph nodes                     _        ++++
tissues, possibly         Other tissues                   _       ++++
with a low-level          Serum from healthy persons      _         _
replication. The
third stage occurs        Serum from exanthem             _        ++++
infrequently,              subitum patients
typically in              Serum from other patients(supc)+++        +
immunocompromised         Saliva                          _       ++++
persons, and is           Cerebrospinal fluid            +++        +
linked to               ---------------------------------------------------
reactivation of         (sup a)Different degrees of HHV-6 positivity.
virus from latency      (sup b)Exception, Zambian children, 44% variant A.
or reinfection.         (sup c)Patients with AIDS, chronic fatigue syndrome, 
Other pathologic          and lymphomas.
conditions, mainly 
multiple sclerosis, tumors, and CFS have been linked to HHV-6.

Primary Infection

The unequivocal demonstration that primary infection with HHV-6B causes
roseola infantum was provided by Yamanishi et al. (8), who investigated the
correlation between seroconversion to HHV-6B and childhood infectious
diseases and found that seroconversion occurs concomitantly with roseola
infantum, also designated exanthem subitum or sixth disease, a common,
mild, acute febrile disease of infants. Fever lasts for a few days and is
sometimes followed by a maculopapular rash that resolves spontaneously.
Primary infection may be asymptomatic or may cause clinical manifestations
other than classic exanthem subitum. In four studies, children admitted to
emergency clinics with febrile illnesses were HHV-6-positive in
approximately 10% to 15% of cases and in one study in approximately 45% of
cases, as determined by viral isolation, seroconversion, or detection of
viral DNA sequences in PBMCs. Other than rash, symptoms included otitis,
gastrointestinal or respiratory distress, and seizures (41-44).
Complications of primary HHV-6 infections are uncommon and rarely fatal;
they were described mainly as case reports and include invasion of the
central nervous system (CNS) with seizures, hyperpyrexia, vomiting,
diarrhea, cough, emophagocytic syndrome, fulminant hepatitis, disseminated
infection, and hepatosplenomegaly. These complications suggest that the
virus may spread to a number of organs, which may represent potential sites
of virus persistence or latency and (subsequently) reactivation. For
example, seizures and other CNS complications are clear indications of
invasion of this organ and correlate well with neurotropism of HHV-6. HHV-6
primary infection accounts for 10% to 45% of cases in children admitted to
emergency clinics with febrile illness and 1% of cases in hospitalized
children (42).

HHV-6B is not the only causative agent of exanthem subitum. Occasionally,
HHV-7 may also cause fever with or without rash. Primary infection with
HHV-7 occurs at a somewhat later age than with HHV-6B. Initially, it was
proposed that pathologic manifestations seen during primary HHV-7 infection
were the consequence of HHV-6 reactivation by HHV-7. Evidence that HHV-7 by
itself causes an exanthematic disease, although less frequently than
HHV-6B, rests on the finding that children with exanthem subitum
seroconvert to HHV-7 but remain HHV-6B-negative (45,46).

                                    Virus replicated in the salivary glands
	    [fig]                     and secreted in saliva is the
                                    epidemiologically proven source of
Fig. 2. Stages of the natural       transmission. Other routes of
history of HHV-6 infection: I.      transmission have been suggested but
Primary infection occurs in         remain to be proven. HHV-6B DNA was
infants, may result in exanthem     recovered from cervical tissues and
subitum (rash on the child's        secretions (47-49), but children born
chest), and spreads to organs.      to mothers with positive cervical swabs
Question marks denote sites         did not acquire the infection.
where HHV-6 spread is likely but    Intrauterine transmission was suggested
not proven. II. In healthy          by polymerase chain reaction (PCR)
infants and adults, HHV-6 is        positivity of uncultured cord blood
present in a latent or              mononuclear cells (CBMCs) in 1.6% of
persistent form in lymph nodes      the cases and by a case of abortion of
and is produced asymptomatically    an HHV-6-positive fetus (40).
in salivary glands and shed in      Transmission through breastfeeding is
saliva, the most probable route     also doubtful since HHV-6 DNA, unlike
of transmission. III. HHV-6         HHV-7, is not found in breast milk
infection/reactivation occurs in    (50). Integration of the HHV-6 genome
persons undergoing therapeutic      in lymphoblasts from a leukemic patient
immunosuppression after organ       and his offspring raised the
transplant or in AIDS patients.     possibility of genetic transmission. As
                                    vertical transmission was not observed
                                    in other cases of genome integration,
the presence of HHV-6 DNA in offspring was alternatively interpreted as a
tendency of HHV-6 to integrate at specific chromosomal loci (51,52).

With the exception of a strong association of HHV-6A with febrile syndromes
in Zambian children (43), which could reflect an endemic variant A hot
spot, HHV-6A has rarely been isolated or detected in children with primary
HHV-6 infection (53,54). The age at which primary HHV-6A infection occurs
and the diseases clearly linked to it have not been determined.

HHV-6 in Healthy Persons

The second stage of HHV-6 infection occurs in healthy children and adults,
in whom the virus actively replicates in the salivary glands, is latent in
at least lymphocytes and monocytes, and persists in various tissues.
Replication in salivary glandsobserved for HHV-6B but not HHV-6A
(9,10,47)accounts for the route of transmission and for the high frequency
of detection and isolation of virus in saliva. Lymphocytes, and probably
monocytes, represent two known sites of latency, as the virus can be
reactivated from PBMCs and adherent monocytes upon cultivation (55), and
viral DNA sequences are detected in PBMCs of as much as 90% of the
population. Additional sites of latency likely exist since the virus or
viral sequences can be readily detected in a number of tissues. A form of
latent infection is represented by integration of the HHV-6 genome in the
host chromosomes (51,52). Persistence of HHV-6 in cells and tissues is
discussed in the section In Vivo Tropism.

A missing link in our understanding of the natural history of HHV-6
infection is the source of the virus that spreads to organs. Monocytes have
a short half-life; they may be vehicles of virus spread to organs, but they
themselves need to be infected. A possible source may be virus produced in
the salivary glands. In one case, early bone marrow progenitor cells were
found to be latently infected in vivo (56), which raises the possibility
that they may represent a site of latency, and by corollary, upon viral
reactivation from latency, an alternative source by which virus spreads to
tissues.

In immunocompetent adults, infection or reactivation of HHV-6 at sites
other than the salivary glands is rare. Occasionally, infection results in
lymphoadenopathy, fulminant hepatitis, mononucleosislike syndrome, or
generalized infection.

HHV-6 in the Immunosuppressed

The third stage of HHV-6 infection, which occurs in the immunosuppressed,
is responsible for the most serious clinical manifestations associated with
HHV-6 infection or reactivation. Persons at risk are recipients of bone
marrow, kidney, and liver transplants, in whom immunosuppression is induced
for therapeutic reasons. In these patients, HHV-6 infection or reactivation
may result in bone marrow suppression, pneumonitis, encephalitis,
encephalopathy, hepatitis, fever, and skin rash or may complicate
engraftment of the transplanted organ and culminate in rejection and death.
As the number of persons undergoing organ transplantation and,
consequently, subjected to therapeutic immunosuppression increases, the
number of persons at risk is increasing. Assessment of the contribution of
HHV-6 to posttransplant complications is made more difficult by the
presence of other opportunistic viruses and by the scarcity of thorough
studies on all the viruses present in these organs. Thus, most of the
reports on the presence of HHV-6 did not deal with the fact that HCMV
reactivation is frequent in transplant recipients (particularly kidney) and
may occur together with HHV-6 reactivation. When investigated in detail, a
synergistic effect of HHV-6 and HCMV was apparent in renal transplant
recipients, and the simultaneous detection of both HHV-6 and HCMV DNAs in
urine or serum or of immunoglobulin (Ig) M antibodies was the strongest
predictor of viral disease and of severity of disease (57,58). HHV-7 can
also reactivate in transplant recipients (59), again alone and in
association with HHV-6 or HCMV. Each of these viruses is a pathogen in its
own right, and in combination with the other, may produce disease far more
serious in outcome and clinical manifestations than it would alone. In many
studies, no effort was made to identify the HHV-6 variant. When the
variants were characterized, a rather heterogeneous pattern emerged. In
PBMCs, brain and lungs variant B strains were predominant (60-62), whereas
in spinal fluid and serum, variant A strains were prevalent (63,64). In
approximately 30% of bone marrow transplant recipients in whom pneumonitis
developed, both variants were simultaneously detected (62), an otherwise
rare occurrence.

AIDS patients are the second group of immunocompromised persons at risk for
HHV-6 and HCMV-related opportunistic viral infections. The overall
incidence of these infections has decreased substantially after the
introduction of highly active antiretroviral therapy. HHV-6
infection/reactivation in AIDS patients results in an increase in HHV-6
load both in lymph nodes and generalized, in viremia, disseminated
infection in many organs, active CNS infection, pneumonitis, and retinitis
and may contribute as a cause of death (65-67). These findings lead to the
proposal that HHV-6 acts as a cofactor in the progression of AIDS and in
the switch of HIV from the latent to the replicative state (68). Although a
significant increase in HHV-6 viral load was not observed in PBMCs of
HIV-seropositive persons (68,69), HHV-6 and HIV could interact in lymph
nodes. The possibility that HHV-6 acts as a cofactor in AIDS progression
boosted intense research on mutual interactions between HHV-6 and HIV in
cell cultures and cell-free systems. In addition to coinfection, observed
in vivo and in vitro, HHV-6 promotes HIV replication through upregulation
of cytokines (e.g., TNF-alpha and IL-1) and through
transactivation of the long terminal repeat by IE-A and IE-B (68). The
possibility that in vivo HHV-6 infection may lead to HIV reactivation was
examined recently in HIV-positive children. The children in whom AIDS
progressed rapidly acquired primary HHV-6 infection later than those in
whom HIV progressed slowly; however, in the rapid progress to AIDS the HIV
viral load did not increase at the outcome of HHV-6 infection. Late in
AIDS, HHV-6 detection in PBMCs is reduced, most likely because of T-cell
depletion (69). As a rule, the variant A strains are more frequently
associated with AIDS patients.

In Vivo Tissue Distribution

In addition to the salivary glands, HHV-6 has been frequently isolated from
cultured PBMCs from AIDS patients or children with exanthem subitum or
febrile illnesses. This led to the initial definition of HHV-6 as a
lymphotropic virus. In lymphocytes, the virus establishes a latent
infection, readily monitored by PCR amplification of viral DNA sequences in
uncultured PBMCs (47). Furthermore, productive infection has been monitored
in some cases by immunohistochemistry (e.g., in CD4+ T lymphocytes) (70).
In contrast with the earlier view of HHV-6 as a T-lymphotropic virus,
recent investigations detected HHV-6 in many tissues. Despite the wealth of
research on the presence of HHV-6 in humans, our knowledge is fragmentary.
By immunohistochemical staining, active HHV-6 infection was detected in
various cells (e.g., CD68+ cells of the monocyte/macrophage lineage in
Kaposi sarcoma [71], epithelial cells and lung macrophages, dendritic cells
and macrophages of lymph nodes and infiltrating lymphocytes of organs of
unselected patients who died of AIDS, tubular epithelial cells of kidney)
and in submandibular glands (67,72). Consistent with this wider host range,
HHV-6 DNA sequences were detected in a number of organs (e.g., skin,
spleen, lung, heart, kidney, adrenal gland, esophagus, duodenum, colon,
liver, and early bone marrow progenitor cells) from patients who died of
heart attack or accidents (47,56,65). Since in numerous studies detection
was performed by PCR, latent, persistent, or productive infections were not
differentiated, nor was the nature of the infected cells defined. Variant B
strains are more frequently found in both PBMCs and solid tissues. Variant
A viruses appear predominant in skin and can replicate in primary
fibroblast cultures, suggesting a preferential tropism for skin (47). HHV-6
is a brain commensal (see section entitled Neurotropism and Multiple
Sclerosis).

In Vitro Tropism

In vitro, HHV-6 infects and replicates at highest titers in PBMCs and
CBMCs. In these heterogeneous cultures, susceptible cells are the CD4+ T
lymphocytes but also the CD4-CD3+CD8+ and the CD4-CD3- natural killer cells
(68). Inasmuch as CD4 expression is not a requirement for susceptibility to
HHV-6 infection and soluble forms of CD4 and antibodies to CD4 fail to
inhibit virus infectivity (73), CD4 is not a necessary component of the
cellular receptor for HHV-6. In addition to primary T lymphocytes,
T-lymphocytic lines (e.g., HSB-2, SupT1, Molt3, JJhan, MT-4, ET62) support
HHV-6 growth. Viruses of the two variants display different host range, as
variant A strains generally do not replicate in Molt3 cells, whereas
variant B strains do not replicate in HSB-2 cells. Permissive cells of
lineages other than T lymphocytes are the liver cell line HepG2 (74) and a
number of human and nonhuman cell lines in which the virus generally grows
at very low yields (e.g., cervical cells lines, human primary astrocytes [B
variant does not replicate very well] neuroblastoma, human bowel-derived
cell monocytes, megakaryocytes endothelial cells, NBL-7 mink lung
epithelial cells, and PBMCs of several Macaca species) (75-77). Altogether,
in cell cultures as well as in vivo, HHV-6 appears to have a host range
wider than initially recognized, extending beyond T lymphocytes. While this
is meaningful with respect to studies on the natural history of the
infection, the practical use of these cells in the laboratory is hampered
by the very low virus yields. Even in the most permissive systems (PBMCs,
CBMCs, and T-cell lines), the virus yields are very low. In our experience,
CBMC cultures, the most productive cell type, do not yield more that 10(sup 4)
infectious units per ml, whereas the titer of a herpes simplex virus type 1
stock is generally as high as 10(sup 9) - 10(sup 1)0 plaque-forming units per ml.

Neurotropism and Multiple Sclerosis

HHV-6 is probably the most neurotropic virus known. Neuroinvasion has been
documented in infants with primary infection, in focal encephalitis, in
children and adults with AIDS, in recipients of bone marrow transplants, as
well as in immunologically competent children and adults. Challoner et al.
(78) reported viral DNA sequences in approximately two thirds of brain
specimens and viral antigen expression in a number of cell types (e.g.,
astrocytes, macrophages, epithelial cells, endothelial cells of blood
vessels) at very similar frequencies in specimens from healthy persons and
multiple sclerosis patients. Astrocytes were confirmed as a susceptible
cell population, although in a subsequent study only samples from AIDS
patients were positive (79).

Both variant viruses were detected in the brain of patients who died of
causes related or unrelated to HHV-6, which demonstrates that both variant
viruses can invade and be harbored in the brain (61,78-82). Although
studies on the differential distribution of the two variant groups provided
conflicting results (78,83), for HHV-6B, CNS invasion has been documented
at primary infection. Instead, for HHV-6A, the time of CNS invasion has not
been documented.

A possible correlation between active HHV-6 infection and multiple
sclerosis has been the focus of much attention in the past few years.
Multiple sclerosis is a severe CNS disease of young adults, characterized
by the progressive demyelination of nerves that leads to progressive
paralysis and eventually death. The disease appears to be an autoimmune
reaction to myelin, the coating of nerve fibers. Viruses have long been
suggested as etiologic agents of myelopathies, and DNA sequences from a
number of viruses, particularly herpesviruses, have been detected, although
not consistently. In addition, since multiple sclerosis is accompanied by a
characteristic increase in IgG titer in serum and spinal fluid, antibodies
to various viruses (including HHV-6) have been frequently searched for.
Even immunologic studies have been inconclusive, most probably because the
increase in antibody response reflects an immune dysfunction or different
genetic background together with damage of the blood-brain barrier, rather
than an epidemiologic correlation with any given virus.

Studies of HHV-6 infection or reactivation in multiple sclerosis patients
have provided controversial results. In initial reports, active infection
was suggested by an increase in IgG titer in both serum and spinal fluid
but was not confirmed by increase in PCR positivity of PBMCs (84). By
representational difference PCR, Challoner et al. (78) found that multiple
sclerosis specimens contained HHV-6B DNA sequences. HHV-6B antigen
expression was detected at higher frequency in multiple sclerosis plaques
than in histologically normal specimens. Nuclei of oligodendrocytes were
positive in multiple sclerosis samples (80%) but not in control samples
(0%) and were interpreted as a hallmark of the association between active
or reactivable HHV-6 infection and the disease. Attempts to reproduce the
immunohistochemical results were not successful, and viral expression as
documented by reverse transcriptase (RT)-PCR was also negative (85). In
favor of a correlation are subsequent findings that the frequency and titer
of anti-HHV-6 IgM antibodies are higher in samples from multiple sclerosis
patients than from controls (73% vs. 18%) and that the serum DNAemia was
specifically positive in multiple sclerosis patients (30% vs. 0%) (86).
HHV-6 DNA sequences had been detected in spinal fluid, but not in serum
from multiple sclerosis patients (87). Critical interpretation of these
data can be summarized as follows. The serologic analyses are difficult to
interpret as this disease is characterized by an immunologic dysregulation;
therefore, the increase in antibody titer may be a sign of the disease
rather than a cause. The PCR data were not confirmed. Thus, no statistical
difference was reported in DNA positivity of plaques (32% active vs. 17%
inactive plaques) (88), no DNA was detected in serum and cerebrospinal
fluid samples (89-91), and no viral RNA was found by RT-PCR in multiple
sclerosis brain specimens (85). The differences in PCR results may reflect
differences in PCR conditions (e.g., primers, number of cycles,
characteristics of the amplified sequences, nature, and conservation of the
specimens analyzed) but do not account for the observed discrepancies.
Therefore, correlation between active HHV-6 infection and multiple
sclerosis is still a controversial issue rather than a firmly established
conclusion.

Kaposi Sarcoma

Kaposi sarcoma is a multifocal angioproliferative disease localized
predominantly in the skin or mucous membranes and in other visceral organs
and lymph nodes. In addition to the classic, iatrogenic, and endemic forms,
the disease occurs frequently and aggressively in AIDS patients. Human
herpesvirus 8 (HHV-8) sequences were detected for the first time in Kaposi
sarcoma specimens (92,93) by representational difference analysis PCR;
HHV-8 is being investigated as the possible etiologic agent. Epidemiologic
studies had long suggested a viral etiology, and many viruses, including
HHV-6 and HHV-7, were detected in Kaposi sarcoma tissues. While neither
HHV-6 nor HHV-7 appears to contribute to its etiology, Kaposi sarcoma
represents a unique and interesting environment for these viruses, and they
may have a role in the progression of the tumor. By immunohistochemistry,
HHV-6B has been localized to CD68+ cells of the monocytic macrophage
lineage. These cells are either singly infected with HHV-6 or HHV-7 or
doubly infected with HHV-6 and HHV-7 (Figure 3) (71). Although some tissues
harbor both viruses, albeit in different cells (e.g., lungs and salivary
glands), cells doubly infected with HHV-6 and HHV-7 have not been detected
in any tissue other than Kaposi sarcoma lesions (94). In addition, in the
case of HHV-7, CD68+ cells are a cell type infected, singly or doubly, in
no other tissue but in this tumor (71). Data suggest that the particular
microenvironment of Kaposi sarcoma lesions, which is rich in chemokines and
cytokines, attracts circulating lymphocytes and monocytes that harbor HHV-6
and HHV-7 in a latent or persistent form, induces viral reactivation, and
promotes viral growth. In this peculiar environment two unusual situations
occur. Viral yields are high for both HHV-6 and HHV-7. This accounts for
the likelihood of double HHV-6 and HHV-7 infection, which most likely
appears to take place in the tumor itself. HHV-7 tropism is also not
restricted to T lymphocytes and extends to CD68+ cells, a lineage not
susceptible to HHV-7 infection in other tissues. A predicted chemokine
(U83) encoded by HHV-6 may contribute to dysregulating cellular chemokine
expression or signaling. In addition, the virus expresses its own chemokine
receptors encoded by the U12, and possibly U51 genes. Once HHV-6 is
reactivated and actively replicating, HHV-6 may play a role in tumor
progression through these molecules and mechanisms. Different studies
detected different variant strains in Kaposi sarcoma tumors (95,96). The
reason for this discrepancy is unknown.

Lymphoproliferative and Neoplastic               
Disorders                                        
                                                 
Initial isolation of HHV-6 from patients         
with lymphoproliferative disorders boosted       
studies on possible association of HHV-6       		    [fig]  
with proliferative diseases, particularly
of lymphoid origin, aimed at showing             Fig. 3. Expression of human
either a transforming potential of the           herpesvirus 6b (HHV-6B)
virus in cell cultures or epidemiologic          and HHV-7 antigens in serial
and molecular relationships between HHV-6        sections of Kaposi sarcoma
and various types of neoplasia.                  specimens. Panels A-C: In
                                                 Kaposi sarcoma environment,
In favor of a possible oncogenic potential       cells can be doubly infected
is the transforming ability of three viral       by HHV-6b and HHV-7: (A)
DNA fragments on mouse fibroblast cell           Staining with monoclonal 
lines or human epidermal keratinocytes.          antibody 5E1 to HHV-7
One encodes DR7 (97) and the other two           specific antigen pp85;
encompass the regions spanning U2-U20 and        (B) Staining with monoclonal
U31-U37. The derived cellular clones were        antibody to HHV-6B-specific
malignant and tumorigenic in athymic mice        antigen p101; (C) Overlaid
(98,99). DR7 and the two other loci also         serial sections show
contain genes with transactivating               colocolizations of HHV-6B
activity on the HIV LTR.                         and HHV-7 (71).
                                                 
Clinical and molecular investigations    
dealing with HHV-6 and various types of         
tumors have been reviewed (13). The             
overall importance of these findings             
remains controversial, mainly because the        
criteria for establishing an association        
between the virus and its oncogenic                                       
potential have not been met. Thus, the                                    
viral DNA sequences found in a tumor are                                  
expected to be the same as those with in                                  
vitro transforming potential, and in                                      
vitro-transformed cells should be                                         
tumorigenic in animals. In a given type of                                
tumor the viral sequences should                                          
constantly be the same. In                                                
vitro-transformed cells and tumors should                                 
express the same viral gene products. The                                 
oncogenic potential of the virus should be                                
demonstrated in a suitable animal model                                   
(which is lacking for HHV-6). Chromosomal                                 
integration of HHV-6 DNA in cells from                                    
lymphomas (51,52) may open a new scenario.                                
                                                                          
CFS                                                                       
                                                                          
CFS is an illness characterized by memory                                 
and attention impairment, muscle and                                      
multijoint pain, and unrefreshing sleep                                   
and weakness lasting longer than 6 months.                                
The etiology of the disease is unknown,                                   
and many viruses have been investigated as                                
possible causing agents. The overall                                      
scenario is in a way similar to that of                                   
multiple sclerosis. Serologic analysis on                                 
the presence of antibody to HHV-6 provided                                
inconclusive data. An increase in IgG and                                 
IgM titer in the sera of a large number of                                
CFS patients (119 of 154) was found                                       
relative to that in the control population                                
(77% vs. 12%) (100). However, this was not                                
specific, as an increase in antibodies to                                 
other viruses was also detected,                                          
reflecting probably an immunologic                                        
dysfunction. Molecular analysis showed a                                  
higher prevalence of HHV-6A but not HHV-6B                                
or HHV-7 in CFS patients (64,101,102), and
HHV-6A could also be isolated from these
patients (103). Whether this reflects an association or the consequence of
an immune dysregulation remains to be determined.

Conclusions

The epidemiologic and clinical investigations summarized here establish a
clear correlation between HHV-6B primary infection and exanthem subitum and
between HHV-6 infection/reactivation and a number of pathologic conditions
in immunocompromised patients and transplant recipients. A firm correlation
with other diseases remains doubtful. In the case of multiple sclerosis a
clearly established correlation may identify patients who might benefit
from specific anti-HHV-6 chemotherapy.

Yet another area deserving attention is the state of the virus in healthy
people, a key prerequisite to understanding virus behavior in pathologic
conditions. We have underlined that, in addition to establishing the true
latent infection recognized in earlier studies, HHV-6 persists in the host
through a combination of low-level persistent infection of various cells
and tissues, a situation similar to that reported recently for HHV-7 (94).
Sites of latency may represent a reservoir of the virus, which upon
reactivation may feed the sites of persistency.

The pathogenic mechanisms of HHV-6 at the molecular, cellular, and tissue
level remain largely obscure. Almost 10 years elapsed between the first
isolation of HHV-6 and publication of the sequence of the entire genome.
Now, single gene products can be studied in the context of the viral genome
and in heterologous expression systems. Although a system for mutagenesis
of the viral genome has yet to be developed, the stage is set to ask
questions on the molecular mechanisms underlying pathogenicity of the
virus. The forthcoming area of research will probably focus on links
between the function of single gene products and mechanisms of pathogenesis
and virus spread. A key feature of the HHV-6 life-style in the human host
is its ability to infect and survivein a latent or persistent formin the
cells of the immune system, and the pathogenic potential of HHV-6 is linked
to its ability to evade immune system control. Analysis of the genomic
sequence shows candidates for immune evasion strategies. Yamanishi and
colleagues reported that a 7-transmembrane protein encoded by U12 acts as a
-chemokine receptor (26). As -chemokines are key mediators of the immune
response, the -chemokine receptor may subtract these mediators in a
particular microenvironment. The immune evasion strategy must be more
complex, as analysis of the viral DNA sequence shows additional candidates,
e.g., a predicted chemokine encoded by ORF U83 and a second 7-transmembrane
proteina structural feature typical of chemokine receptorsencoded by ORF
U51. This latter protein has a very unusual cell-type-dependent trafficking
property (as it is transported to the plasma membrane in infected as well
as transfected T lymphocytes) but fails to be transported to the plasma
membrane in transfected human monolayer cells (104), raising the
possibility that its function is regulated in a cell-dependent fashion
through modulation of cell surface expression. Also U51 appears to
dysregulate cellular chemokine expression (105). Studies of single gene
products will probably lead to the identification of immunodominant
proteins and the development of standardized recombinant diagnostic
reagents.

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

      The work done in our laboratory was supported by grants from AIDS
Project from Istituto Superiore di Sanit, BIOMED2 BMH4 CT95 1016 grant
from UE, Target Project in Biotechnology, MURST 40%, University of Bologna
60% and pluriannual plan.

      Dr. Campadelli-Fiume is professor of microbiology, University of
Bologna. Her research interests focus on human herpesviruses 6 and 7;
identification, mapping, and expression studies of the major viral
glycoproteins and immunodominant tegument proteins; and definition of the
natural history of the infections. Her research on herpes simplex virus 1
deals primarily with mechanisms of virus entry into the cells and
identification of cellular receptors for entry and the mechanisms of HSV
transport and exit through the exocytic pathway.

      Address for correspondence: G. Campadelli-Fiume, Dipartimento di
Patologia Sperimentale, Sezione di Microbiologia e Virologia, Via San
Giacomo, 12, 40126 Bologna, Italy; fax: 39-051-354-747; e-mail:
campadel@kaiser.alma.unibo.it.

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Synopses

Emergence of a Unique Group of Necrotizing Mycobacterial Diseases

Karen M. Dobos,* Frederick D. Quinn, David A. Ashford, C. Robert
Horsburgh,* and C. Harold King*
*Emory University School of Medicine, Atlanta, Georgia, USA; Centers for
Disease Control and Prevention, Atlanta, Georgia, USA

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      Although most diseases due to pathogenic mycobacteria are
      caused by Mycobacterium tuberculosis, several other
      mycobacterial diseasescaused by M. ulcerans (Buruli ulcer), M.
      marinum, and M. haemophilumhave begun to emerge. We review the
      emergence of diseases caused by these three pathogens in the
      United States and around the world in the last decade. We
      examine the pathophysiologic similarities of the diseases (all
      three cause necrotizing skin lesions) and common reservoirs of
      infection (stagnant or slow-flowing water). Examination of the
      histologic and pathogenic characteristics of these mycobacteria
      suggests differences in the modes of transmission and
      pathogenesis, though no singular mechanism for either
      characteristic has been definitively described for any of these
      mycobacteria.

Diseases Caused by Emergence of Atypical Mycobacteria

Mycobacterial diseases cause substantial illness and death throughout the
world, despite years of public health control efforts. Although most
illnesses and deaths are due to tuberculosis (1), particularly in
developing countries and in association with the AIDS pandemic (2),
diseases caused by nontuberculous mycobacteria (NTM) have had a strong
impact on human populations in both developing and industrialized countries
(3). Many NTM diseases, such as those caused by Mycobacterium avium
complex, are considered opportunistic infections in patients with AIDS (4).
However, the rates of non-AIDS-associated NTM infections are also
increasing (5). Specifically, disease caused by M. ulcerans, M. marinum,
and M. haemophilum has increased in both healthy and immunocompromised
patients in the last decade. Moreover, these diseases have been reported
from previously unaffected geographic areas, which indicates an increase in
the geographic distribution of these organisms.

Of these three emerging NTM diseases, Buruli ulcer (BU), caused by M.
ulcerans, poses the greatest immediate public health threat. Indeed, BU is
rapidly becoming the third most prevalent mycobacterial disease, with an
impact soon to surpass that of leprosy (6). Although it was first
documented in Australia in 1947 (7), the disease was named after the Buruli
District of Uganda (8) after an investigation of superficial, ulcerative
lesions in Ugandan children. At the time, the disease was sporadically
reported throughout Central and West Africa and Australia. In the past
decade, incidence of this disease has dramatically increased, with cases
now reported in most of sub-Saharan Africa, Mexico, Surinam, Peru, Bolivia,
French Guiana, India, sporadically throughout southern Asia, and in Papua
New Guinea (6,9). In addition, several cases have been reported in Belgium,
Japan, Northern Ireland, and in the United States, resulting from
international travel (6,10,11).

A retrospective investigation over a 10-year period in the Daloa region of
Cte d'Ivoire was conducted to document increases in the incidence of BU.
Cases increased dramatically over a 10-year period, with some villages
demonstrating disease rates of 16% of the population at the end of the
study (12). Current rates are estimated at more than 22% in some of these
villages (6). Increases have also been reported in Australia, with
outbreaks of BU during the 1990s in areas where the disease had not been
previously seen (13). These data most likely underestimate BU occurrence as
there are no reliable tools for the surveillance and diagnosis of this
disease other than clinical signs and symptoms.

Disease caused by M. marinum was observed in clusters of cases between 1930
and 1970, and M. marinum was well accepted as a human pathogen before the
1980s. In contrast, M. haemophilum was a rarely identified pathogen before
1974. A retrospective study conducted in 1974 reported that, on the basis
of epidemiologic evidence, M. haemophilum was the causative agent of a
syndrome that included mycobacterial adenitis and skin lesions that
developed in 29 immunocompetent patients (14). In 1978, the bacterium was
identified as the cause of cutaneous ulcerating lesions in a woman with
underlying Hodgkin disease (15). Subsequently, M. haemophilum has been
described as causing cutaneous lesions in persons receiving
immunosuppressive therapy after a renal transplant (16).

The occurrence of M. marinum and M. haemophilum in human disease is likely
underreported, as diagnosis of the diseases caused by M. marinum and M.
haemophilum is frequently missed. Nonetheless, confirmed cases of these
diseases have been increasing, both internationally (5) and within the
United States. A national survey involving 46 state and local laboratory
centers, representing 33 states and the District of Columbia, was conducted
from 1981 to 1983 to determine the prevalence of NTM diseases. Fifty-three
cases of NTM disease caused by M. marinum and one case caused by M.
haemophilum were reported over the 3-year period (17), for a national
average number of cases of 40 and 0.76 respectively, per year.

In   			Table 1. Laboratory-confirmed cases of Mycobacterium
1993,			marinum in the five regions of the United States (40
a    			states reporting), by year, 19931996(sup a)
laboratory-based	-----------------------------------------------------------
surveillance                               No. cases (%)
system                   	-----------------------------------------------
(18) 			Region      1993         1994        1995       1996
began			-----------------------------------------------------------
to   			Northeast      21 (14)      40 (22)    34 (23)    28 (18)
asses			Southeast      58 (38)      66 (37)    41 (28)    64 (41)
the  			North central  43 (28)      38 (21)    27 (27)    38 (24)
recent		South central  17 (11)      17 (10)    17 (17)    14 (9)
prevalence		Mountain        8  (5)      11  (6)    13 (13)     7 (5)
of   			Pacific         5  (3)       7  (4)    15 (15)     6 (4)
NTM  			Total         152          179        147        157
disease		-----------------------------------------------------------
in   		      (sup a)These data are reported as part of the passive 
the  			laboratory-based surveillance system using the Public Health  
United		Laboratory Information System software developed by the  
States.		Centers for Disease Control and Prevention and the Association 
For  			of State and Territorial Public Health Laboratory Directors.
the
Mycobacterium 
module, the 
population under 
surveillance included patients in the United States who had a specimen submitted 
to the state laboratories for evaluation. It is not known what percentage of all 
mycobacterial isolates this represented. Only one isolate per person was recorded. 
The geographic distribution and number of cases of M. marinum disease (40
states reporting) and M. haemophilum disease (9 states reporting),
submitted from 1993 to 1996, are presented in Tables 1 and 2, respectively.
These data demonstrate that the number of cases of these two diseases in
the United States has increased from the past decade, with the estimated
national average number of cases of 198 and 35 per year, respectively. In
addition, cases of M. marinum in several states over the 4-year period of
this survey have increased (Table 3) (cases of M. marinum had not been
reported in Missouri previously [17]). Elsewhere, increases in M. marinum
disease have been reported throughout the world in temperate climates (5).

In the United States, most cases of M. haemophilum disease are still found
in the South; however, M. haemophilum disease has been described in the New
York City metropolitan area (19). In addition, the number of cases of M.
haemophilum disease in the United States is expanding (Table 2). Thought to
occur only in immunocompromised persons (with organ transplant patients and
persons with AIDS representing most of the patients) (20), M. haemophilum
disease was rarely reported even in these populations before 1990. By 1994,
40 cases of M. haemophilum disease associated with immunocompromised
persons had been reported worldwide (20). However, more recently, a report
by Saubolle and colleagues (19) described 10 cases of M. haemophilum
disease in Arizona (1984 to 1994), which included cases in two otherwise
healthy children and three in adults undergoing corticosteroid therapy for
rheumatoid arthritis or Crohn disease. Additional cases of M. haemophilum
disease in otherwise healthy children (21) and adults have recently been
observed (M.A. Saubolle, pers. comm.). Elsewhere, M. haemophilum disease
has been reported in Australia, Canada, France, Israel, and the United
Kingdom (19).

Clinical and   Table 2. Laboratory-confirmed cases of Mycobacterium
Histologic	    and  haemophilum in the five regions of the United States
Features	    Histologicates reporting), by year, 19931996(sup a)
    		    ----------------------------------------------------------
Buruli Ulcer                                       No. cases (%)
               			--------------------------------------------
M 		    Region          1993        1994       1995        1996
ulcerans	    Northeast      0           0         0           0
causes	    Southeast     11 (79)      0         0           2 (50)
a    		    North central  1 (7)       0         0           0
skin		    South central  0           1 (25)    0           0
disease	    Mountain       2 (14)      3 (75)    3 (100)     2 (50)			
commonly	    Pacific        0           0         0           0
known		    Total         14           4         3           4	
as   		   -----------------------------------------------------------	
BU.		   (sup a)These data are reported as part of the passive 
The  		   laboratory based suveillance system using the Public Health
incubation	   Laboratory Information System software developed by the Centers
period	   Centers for Disease Control and Prevention and the Association 
can  		   of State and Territorial Public Health Laboratory Directors.
be   
highly	
variable
but is generally less than 3 months (22). The ulcers are indolent and
necrotizing (9). Systemic signs and symptoms, such as fevers or weight
loss, and bacterial superinfection are rare (12,22). Erythema and
induration are present at the onset of infection but subside rapidly with
the beginning of ulceration. Healing usually takes 4 to 6 months and
involves extensive scar formation. This scarring frequently results in
deformity, particularly in children, in whom the result can be joint
contracture, subluxation, disuse atrophy, or distal lymphedema.
Circumferential cicatrization may lead to stunted limb growth. In one
series, 26% of patients were left with functional disability of a limb
(12). However, death from BU is rare, and no disseminated disease has been
reported in either healthy or immunosuppressed persons.

Histologically, M. ulcerans produces a circumscribed area of necrosis and
(unlike most other mycobacterial pathogens) infected tissues that primarily
contain extracellular bacilli, with microcolonies containing large numbers
of extracellular acid-fast bacilli (AFB) in the center of the lesions and
in association with adipose cells (Figure 1A, B). The effect of AFB at the
site of infection can be extensive during the preulcerative phase, with few
to no intracellular AFB present (Figure 1B). The lesions are symmetrical
with associated coagulation necrosis of the deep dermis and panniculus. The
lesions very rarely penetrate beyond the fascia to associate with the
underlying muscle. Necrosis occurs extensively beyond the central regions
with destruction of capillaries, larger vessels, and adipose cells (Figure
1A) (23). The AFB localize to the adipose tissue (Figure 1B) with necrosis
of the adipose tissues occurring at sites distant to the location of the
bacilli (Figure 1A) and extensive AFB in all preulcerative nodules and
early lesions. The necrosis and damage of the dermis lead to ulceration of
the overlying skin. As the ulceration spreads through the panniculus,
hypersensitivity granulomas, most likely stimulated by mycobacterial
antigens, develop in the dermis and other tissues surrounding the lesions.

Table 3. Laboratory-confirmed cases of Mycobacterium                   
marinum reported by individual states within the                       
United States, by year, 19931996(sup a)                               
---------------------------------------------------------------------- 
                                   No. cases (%)(sup b)                
               --------------------------------------------------------
                                                                       
State              1993         1994          1995           1996      
---------------------------------------------------------------------- 
Florida        15 (9.9)    13 (7.3)       13 (8.8)      24 (15.3)      
Maryland       15 (9.9)    24 (13.4)      21 (14.3)     22 (14.0)      
                                                                       
Minnesota      6 (4.0)       4 (2.2)        6 (4.1)       8 (5.1)      
Missouri       2 (1.3)       7 (3.9)        5 (3.4)       9 (5.7)      
Utah           3 (1.9)       2 (1.1)        5 (3.4)       4 (2.6)      
Virginia       7 (4.6)      13 (7.3)       12 (8.2)      11 (7.0)      
Wisconsin	   9  (5.9)       8 (4.5)        9 (6.1)       9 (5.7)    	
---------------------------------------------------------------------- 
(sup a)These data are reported as part of the passive laboratory-       
based surveillance system using the Public Health Laboratory            
Information System software developed by the Centers for        	      
Disease Control and Prevention and the Association of State and
Territorial Public Health Laboratory Directors.
(sup b)Percent (%) denotes contribution to cases reported nationally                             several                            months (24).
for the year.								      	
	                                                                                                                lesions are minimally painful and usually heal in 1 to 2 years without
M. marinum Disease

M. marinum casuses small ulcers or nodules usually on the extremeties
(24). The incubation is approximately 2 weeks to several months (24)
Thease lessions are minimally painful and usually heal in 1 to 2 years
without treatment. Main symptoms include slight tenderness and discharge 
from the necrotic sites. In fewer than 10% of cases, localized lymphangitic 
spread is noted, with sporotrichoid lesions and lymphadenitis. These 
lesions may result in scarring but are less extensive than those caused 
by M. ulcerans; deformity is unusual. Disease in patients with AIDS has 
been reported (25), and dissemination may occur in the immunosuppressed 
(26,27).

The bacilli are located throughout the necrotic lesions (Figure 2A), with
many bacilli observed as singular rods within cells and vacuoles (Figure
2B). These lesions swell progressively as the infection ensues until
nodules are formed. Tissue necrosis usually occurs at small sites within
these nodules and is observed only in close proximity to the AFB (Figure
2A). Unlike lesions of M. ulcerans, the histopathologic features of early
M. marinum lesions are similar to those of the lesions observed in
pulmonary tuberculosis (24). M. marinum lesions generally show nonspecific
inflammation followed by granuloma formation (24). Very few AFB are
observed in the lesions themselves; they are present as single or a few
bacilli without microcolonies (Figure 2A, B); however, the organism can be
cultured from the skin lesion.

									  M. haemophilum
									  Disease					
	  [Fig] 				 [Fig]	                      					
                                                       M. haemophilum
Fig. 1. Early-(A) and       Fig. 2 A and B. Active     generally causes
late- stage (B) disease     disease histopathologic    joint and cutaneous
histopatho- logic sections  sections of soft tissue    infections in
of the dermis stained for   stained for acid-fast      immunocompromised
acid-fast bacilli (AFB)     bacilli from a patient with patients and
from a patient with a       a Mycobacterium marinum    lymphadenitis and
Mycobacterium ulcerans      infection. In A, the arrow cutaneous lesions in
infection. In A, arrows     indicates localized        healthy children
indicate necrosis of        necrosis, and in B, the    (19,28). In
adipose tissue distant from arrow indicates            addition, a recent
the location of AFB, and in predominance of            report has described
B, the arrow indicates      intracellular bacilli.     cutaneous lesions
predominance of             (Slide courtesy of Arthur  arising from
extracellular bacilli and   B. Abt and Leslie Parent,  infection with M.
microcolonies. Patients'    Penn State Geisinger Health haemophilum in two
samples were obtained from  System and Pennsylvania    healthy men with no
the study conducted in Cte State University College ofother risk factors
d'Ivoire (12).              Medicine, Hershey Medical  for disease (19).
(Photos courtesy of         Center, Hershey,           Lesions often begin
National Center for         Pennsylvania.)             as raised,
Infectious Diseases, CDC,                              violaceous nodules,
Atlanta, Georgia.)                                     most commonly on the
                                                       extremities (19,28).
                                                       In one report, onset
of disease occurred approximately 16 months after the onset of AIDS (28).
Nodules frequently become erythematous and ulcerated, and recurrence of
ulcers may occur in patients in whom complete lesions were not excised
(19).

Unlike M. marinum lesions, M. haemophilum lesions are not sporotrichoid and
do not appear to localize to areas above the lymphoid tissues; they are
more frequently found above joints, especially appearing around
submandibular and cervical joints in infections in children. Mature lesions
are often extremely painful, in contrast to mature lesions caused by M.
marinum and M. ulcerans. The organism may also cause septic arthritis or
respiratory disease and can be associated with systemic symptoms such as
fevers and night sweats. Disseminated disease occurs in severely
immunodeficient persons (20). In one report, 9 of 13 immunocompromised
patients died, although death may have been due to conditions other than M.
haemophilum infection (28).

M. haemophilum-infected skin shows minute necrotic foci in the deep dermis
surrounded by granulocytes, lymphocytes, monocytes, fusiform cells, and a
few giant cells of the Langerhans type (29). Large numbers of bacilli are
generally observed both extracellularly and intracellularly as singular
cells within these necrotic foci (Figure 3A, B). Histopathologic
examinations also reveal poorly formed granulomas within the ulcerated skin
lesions (Figure 3B). Similarly, AFB are observed inside and outside the
cells and as microcolonies within these granulomas and in the surrounding
tissue (Figure 3A, B).

Pathogenesis

One hallmark of most diseases caused by mycobacteria is the ability of the
bacilli to grow within host cells. M. haemophilum and M. marinum grow
prolifically within fibroblast, epithelial cells (Figures 2, 3) (30,31) and
macrophages (29,32). In contrast, M. ulcerans primarily forms extracellular
microcolonies within necrotic tissues, is rarely found within host cells,
and disrupts macrophages and adipose cell monolayers in vitro in lieu of
growing within these cells (33; our unpub. obs.). Rastogi et al. (34)
showed that although M. ulcerans would infect and persist in murine
macrophages after 4 days, no intracellular growth occurred. Others have
demonstrated that culture filtrates from M. ulcerans suppressed
phagocytosis of the bacilli and speculated that this in vitro suppression
of phagocytosis is the reason that the bacilli are only rarely observed
within host cells in human disease (33).

The necrosis in the skin lesions of all three mycobacterial infections
suggests a secreted or somatic cytotoxin or other necrotic bacterial
component.

Buruli Ulcer

The pre-ulcerative and early ulcer stages of BU are characterized by a
central zone of microcolonies of AFB surrounded by a larger zone of
necrotic tissue with no evidence of host-derived inflammatory exudates that
might contribute to cytotoxicity (35). Further, culture filtrates from M.
ulcerans produce a cytotoxic effect on cultured fibroblasts (35). This
material simulated clinical and histopathologic changes similar to those in
BU when injected into guinea pigs (36). An initial analysis of the culture
filtrates identified a high molecular weight
phospholipoprotein-polysaccharide complex that retained the ability to
produce a cytotoxic effect on cell monolayers (37). Others have ascribed
the cytotoxic effect of M. ulcerans to a low molecular weight lipid (742
daltons) in the filtrates (38). Fractionation of the culture filtrates and
observation of the effects of each fraction on cultured L929 fibroblast
cells initially identified a lipid as the cytotoxic component. Further
purification and analysis of this lipid did not induce cell death, but
rather arrested the cellular growth (38).

Some suggest that the factors secreted by M. ulcerans may also possess
immunosuppressive properties indirectly contributing to the destruction of
human tissue (33). Others believe that the necrosis of tissue is due
primarily to infarction, with no contribution from cytotoxic bacterial
factors (23). Thus, the overall cytotoxic effects demonstrated by M.
ulcerans in human disease may result from multiple factors. No study has
addressed whether cytotoxic or immunosuppressive factors are released from
M. ulcerans during the early, active, or late stages of infection; the
mechanisms by which such factors might act on host tissues are also not
known.

M. marinum Disease

In M. marinum infection, bacilli are capable of invading and replicating
within cultured macrophages and epithelial cells (31). Intracellular growth
of M. marinum is limited at temperatures above 33C, as the bacilli do not
grow intracellularly at 37C. In one study, temperature-dependent growth
also correlated with cytotoxicity of macrophage monolayers, but no evidence
of secreted toxins was noted from supernatants of these infected tissue
cultures. The investigators suggested that intracellular growth and a
faster growth rate at 33C probably caused cytotoxicity (31).

M. marinum produces skin lesions in animal
models without prior induced immunosuppression.
Intravenous injection of M. marinum into normal
mice caused skin lesions similar to those
observed in humans, but no dissemination of the
organism occurred (40). Dissemination of M.
marinum was induced only when mice or leopard
frogs (Rana pipiens) were subjected to
conditions that lower the immune response, such 
as lower body temperatures or treatment with
hydrocortisone (40,41). A strain adapted to an
optimal growth temperature of 37C in broth		   [Fig]
culture produced immediate disseminated disease
when injected into the foot pads or tail veins
of normal mice (40), demonstrating that once
temperature restriction was removed as a
barrier to growth, the bacilli quickly
disseminated regardless of immune status of the
mice.                                           

M. haemophilum Disease 					 	   
                                                Fig. 3 A and B. Active
A putative cytotoxin from M. haemophilum has    disease histopathologic
not been reported, even though the presence of  sections of the epidermis
such a toxin is suggested by histopathologic    stained for acid-fast
examinations of infected tissues and in vitro   bacilli from a patient
tissue culture studies (30). In particular, an  infected with Mycobacterium
epithelial cell tissue culture model            haemophilum. In A and B,
demonstrated that M. haemophilum induced        the arrows indicate
substantial cytotoxicity in epithelial cells at localized necrosis and
33C but not at 37C, even though the bacilli   presence of intracellular
grew extracellularly in coculture with          and extracellular bacilli
epithelial cells at 37C (30). Filtered         and microcolonies and the
                                                presence of loose
supernatant from 33C infected tissue cultures  granulomas.
produced identical cytotoxic reaction when      (Slide courtesy of Michael
layered onto fresh monolayers. However, unlike  A. Saubolle, Good Samaritan
from M. ulcerans, broth medium from bacterial   Regional Medical Center,
culture was not cytotoxic. These studies        Phoenix, Arizona, and
suggest that a cytotoxin may be produced only   Department of Medicine,
upon infection of epithelial cells growing at   University of Arizona
33C. Intracellular growth occurred only during School of Medicine, Tucson,
infections at 33C, even though electron        Arizona.)
microscopy showed that the bacilli were capable
of invading these cells at 37C (30). This
temperature-specific cytotoxicity and intracellular growth mimic the
clinical signs of infection in humans. The bacilli grow in cooler,
superficial regions of the body where primary tissue destruction occurs;
subsequently, the bacilli may spread to deeper, warmer tissues of the host,
where little tissue disruption is observed and granulomas form around the
infected areas (19).

Systemic symptoms have been reported but were not likely caused by a
cytotoxin released by the bacteria (19). Animal studies suggest that
immunosuppression leads to disseminated M. haemophilum disease. The
development of skin lesions and dissemination by M. haemophilum do not
occur when the bacilli are injected intravenously into healthy mice.
However, upon inoculation with M. haemophilum, mice treated with steroids
to induce immunosuppression develop skin lesions and disseminated disease
similar to human disease (39).

Epidemiology

Buruli Ulcer

The reservoir of M. ulcerans is unknown. The organism has only been
recovered from lesions of humans or, in one case, a koala (42), and there
has been only one report of person-to-person spread (9). Thus,
environmental exposure, either by direct inoculation or an insect vector,
is the likely route of infection. Epidemiologic studies suggest that
proximity to water sources, such as freshwater lakes or rivers, predisposes
to disease, but specific contact with water that might lead to transmission
of the bacteria has not been identified. In fact, one study showed that BU
was more common during the dry season (43). Cultures of water near
BU-endemic areas have not yielded M. ulcerans (9,12,43,44), though testing
of water samples by polymerase chain reaction found M. ulcerans DNA
(44,45). Soil also has been considered a possible reservoir, but the
organism has not been isolated from soil samples.

BU is primarily a disease of children, with the highest rates found in
children ages 2 to 14 years (12). Boys and girls are equally affected. In
some areas, women also have an elevated risk of BU (12). No data are
available on disease rates by race, but the disease has been reported in
all racial groups. Although reported in persons with HIV (46,47), no
predilection for BU has been noted in HIV-infected persons or other
immunodeficient patients, despite the substantial rates of HIV in many
BU-endemic areas (9,48).

M. marinum Disease

Water is the source of infection by M. marinum in humans (24). Recovered
from unchlorinated swimming pools and salt and fresh water aquariums
associated with cases of disease, the organism is also a common pathogen of
fish; however, water is likely the reservoir, and fish are a susceptible
host. The organism may be transmitted through minimal trauma or abrasions
to the skin. Person-to-person spread has not been well documented, and no
geographic localization of the disease has been noted (41).

The mean age of persons with M. marinum disease is 35 to 42 years (17,24),
although cases have been reported in all age groups. Caucasians, men, and
urban residents are at highest risk. Like BU, the disease is not associated
with immunosuppression and is rare in AIDS patients, even though cases are
common in industrialized countries where AIDS is prevalent (25-27).

M. haemophilum Disease

Unlike M. ulcerans and M. marinum, most M. haemophilum infections occur in
immunocompromised patients (20). There is scant evidence of
person-to-person spread of M. haemophilum, similar to that observed for M.
ulcerans and M. marinum infections. M. haemophilum disease also appears to
be acquired from the environment, but the reservoir is unknown; the
organism has been isolated only from humans with disease (20,49). A
case-control study that included cases apparently acquired through
nosocomial transmission did not find common epidemiologic elements (28).
Another study considered the role of aerosolized pentamidine in the spread
of M. haemophilum. In this study, six patients at a cancer center
contracted M. haemophilum disease after prophylactic therapy with
aerosolized pentamidine, with two of these patients having M. haemophilum
pneumonia. The role of this therapeutic treatment in exposure could not be
clarified either, as numerous patients receiving this therapy did not
contract M. haemophilum, nor was the organism recovered from the
reconstituted pentamidine or water used before and after nebulization (19).
Because cases have occurred in persons residing near the ocean or large
lakes, water has been suggested as a possible reservoir (20,50). This
conjecture, however, does not explain the increase in the number of cases
observed in the southwestern United States, particularly Arizona (19).

The median age of persons with M. haemophilum disease is 31 years, and 80%
were immunosuppressed (20). Two thirds of cases were in men, and most were
in Caucasians.

Factors Relevant to the Mode of Transmission

Mycobacteria are widespread in the environment, particularly in aquatic
reservoirs. In one survey, more than 67% of water specimens collected from
natural, treated, and animal-contact sources contained mycobacteria,
including M. marinum (50). Mycobacteria also are commonly found in soil.
Wolinsky and Rynearson (51) identified at least one Mycobacterium species
from 86% of the samples they collected from several locations. M.
haemophilum was not recovered from any samples in this survey, probably
because the culture methods used were not suitable for the growth of this
Mycobacterium.

M. ulcerans

M. ulcerans grows slowly at all temperatures between 25C and 37C,
although greater proliferation is observed during growth at temperatures
between 30C and 33C. No colony variants have been reported, and the
bacterium apparently has a shorter doubling time in a medium enriched with
fatty acids, a phenomenon consistent with bacilli in lipid-rich areas
surrounding sites of infection (6,9).

Singular lesions involving areas of the body most susceptible to trauma
(i.e., upper and lower extremities) are frequently observed, and direct
inoculation is the most plausible mode of transmission for M. ulcerans
infection. Numerous case histories document skin trauma and abrasions
preceding the onset of BU (9,12). Others propose that an insect vector may
transmit M. ulcerans (54), although supporting evidence for this hypothesis
has not been reported.

M. ulcerans infections may be linked to environmental disturbances
(9,12,23,43). The first reported cases of M. ulcerans infection occurred 2
or 3 years after severe regional flooding and continued intermittently
until 1950 (7). In 1978, there again was severe flooding in this area, and
again approximately 2 years later, infections occurred, first in koalas and
later in humans. Cases of BU were also observed on the east side of the
Victoria Nile in Uganda between 1962 and 64 (43); this outbreak was also
likely associated with severe flooding in the region caused by heavy rains.
As in the outbreak observed in Australia, 2 or 3 years elapsed between the
flooding in Uganda and the first cases in the region. In Togo, infection in
children was related to seasonal flooding of rivers in proximity to the
local village (55). Cases were reported in one area in Liberia after swamp
rice was introduced to replace upland rice. This introduction was
associated with the construction of dams on a major river and the
artificial extension of wetlands (56). In addition, recent cases described
in Cte d'Ivoire occurred primarily in association with farming activities
near the main river (12). Therefore, a common feature of M. ulcerans
disease is that infected persons often reside near swampy areas, river
valleys, or lakes and coastal areas.

After a flood or some other environmental disturbance, mycobacteria may be
washed from their normal habitat into draining rivers or lakes. Given
favorable circumstances, such as relative stream stagnation, temperatures
of 27C to 33C, low salinity, low pH, and the presence of adequate
nutrients, survival and growth of the organism may be enhanced. With the
temperatures that are reached in the tropics, moderate temperatures in the
surface water and in moist silt beside lakes may be sufficient to sustain
the growth of M. ulcerans during the daytime for most of the year (57).
Moreover, M. ulcerans could be dispersed from a water environment in a
fashion analogous to that documented for aquatic M. avium, where droplet
aerosolization of the organism may result in infection (50,58), also
suspected in recent BU cases in Bairnsdale, Australia. Most of the patients
infected with M. ulcerans resided in a small region near one of the lakes
but showed no history of direct contact with the water (57). This type of
airborne dispersal would also explain the acquisition of BU in tree-living
koalas that may be exposed to contaminated aerosols generated in the
adjacent lake system (59). Additionally, airborne dispersal might explain
the prevalence of disease on a geographic continuum in all countries
bordering the Gulf of Guinea in West Africa (6). Finally, spray
aerosolization of M. ulcerans in recycled sewage water used to irrigate a
golf course has been proposed as the route of infection for another series
of cases in Australia (13).

M. marinum

Unlike that of M. ulcerans and M. haemophilum, the etiology of M. marinum
disease is well known. Of the three, M. marinum has the clearest
association with water as the source of the infection (60). In 1926, M.
marinum was first isolated and identified as the cause of saltwater
aquarium fish deaths (61). Tuberculoid skin lesions in users of a swimming
pool in 1939 and 1954 and granulomatous mycobacterial disease in freshwater
fish in 1942 also were ultimately attributed to M. marinum (62). Since
then, a variety of skin infections due to this organism have been observed
around the world, and names such as "mariner's TB," "aquarium granuloma,"
and "swimming pool granuloma" have been coined to describe the disease as
well as the source of the infection. However, when swimming pools are
properly chlorinated, this association has all but disappeared (60).
Nevertheless, virtually any water source and water-related activity is a
potential risk, including tending aquariums (62), fishing (63), skin diving
(62), and a number of other water-related activities (64).

M. haemophilum

M. haemophilum is fastidious, grows slowly, requires supplemental iron, and
has a lower incubation temperature for growth than most other mycobacteria.
This organism is usually grown on chocolate agar, on egg-based media, or on
Middlebrook media containing 15 g/mL to 25 g/mL ferric ammonium citrate,
0.4% hemoglobin, 60 mol/L hemin, or on X-factor (52). The temperature
growth range is 25C to 35C, but the optimal incubation temperature is
32C (52). Because of these requirements, M. haemophilum cannot be isolated
by the standard techniques used in clinical laboratories, and its
fastidiousness may account for the lack of isolation from environmental
sources and patient specimens.

The ecology of M. haemophilum is poorly understood, and the reservoir and
modes of transmission still need to be elucidated. However, one study
describing M. haemophilum isolates from different patients in the same
hospital that had identical fingerprint patterns (53) supports the
possibility that the patients were exposed to a common source, such as
water. Additional evidence that M. haemophilum grows over a wide pH range
(49) and can use chelated iron (52), characteristics common to other
aquatic bacteria, suggests an environmental niche. This organism survives
in cold water (our unpub. obs.) and is resistant to chlorine (50). Several
researchers have suggested using frogs as models for the study of M.
haemophilum systemic disease, as they (or other amphibians) have cooler
body temperatures and thus could be environmental sources for this organism
(15).

Possible modes of transmission for infection with M. haemophilum include
inhalation, ingestion, and skin inoculation. Since most infections occur on
the skin, direct inoculation may be most likely. However, patients were no
more likely than healthy persons from the area to recall injuries or skin
conditions before the onset of symptoms (19), and patients generally did
not report cutaneous injuries before illness (19). The isolation of M.
haemophilum from sputum and lung tissue suggests the possibility of
respiratory transmission; however, respiratory therapies, exposure to
irritants, or previous respiratory infections have rarely been associated
with infection (19). Interestingly, an early animal model demonstrated that
M. haemophilum can spread to the skin through dissemination in the blood
after intravenous inoculation, suggesting bloodborne transmission (39).
This study was performed on prednisolone-treated mice challenged
intravenously with M. haemophilum; skin lesions (predominantly on the
cooler regions of the body) developed in 12 of 30 mice.

This collective evidence illustrates a defined reservoir and mode of
transmission for M. marinum infections, and, although similar evidence is
described for M. ulcerans and M. haemophilum, the precise mode of
transmission of these infections remains undefined.

Conclusions

We have discussed the emergence of three skin diseases and presented the
pathophysiologic, epidemiologic, and environmental characteristics that may
contribute to their emergence. Disease caused by M. ulcerans and M. marinum
is primarily in immunocompetent persons, while the emergence of M.
haemophilum disease is primarily in immunosuppressed persons. All three
mycobacteria are thought to be acquired by inoculation during contact with
contaminated water, suggesting that changes in the environment may be
contributing to their emergence.

Histologic studies demonstrate that M. ulcerans has an apparent association
with adipose tissue, is not observed within host cells in vivo, and does
not grow intracellularly within cultured macrophages. In contrast, M.
marinum and M. haemophilum grow intracellularly within cultured macrophages
and epithelial cells and have apparent associations with epithelial tissues
in vivo, suggesting that different cellular tropisms occur between these
species after the dermis is initially infected. None of these organisms
readily cause disseminated disease in immunocompetent humans, and this
restriction may be related to the optimal growth temperature of these
mycobacteria (temperatures similar to that of the human dermis), although
all three can grow at warmer temperatures, albeit to a lesser extent in
vitro. M. marinum colony variants that grow best at 37C in vitro overcome
this apparent temperature restriction in vivo and disseminate in animals,
further demonstrating that while temperature-restricted growth likely
limits the spread of disease, this restriction can be surmounted by
acquired factors. M. haemophilum can become a disseminated disease in
immunosuppressed persons, suggesting that the organism can also overcome
temperature restrictions in a favorable environment.

The common source of infection for these three mycobacteria is likely water
(it is the definitive source for M. marinum). As with the source of
infection, the mode of transmission has been clearly defined as inoculation
after skin abrasion for M. marinum, while no clear association with skin
abrasion has been demonstrated for infection with M. ulcerans or M.
haemophilum in humans. Experimental inoculation of all three organisms into
the skin of animals produces pathologic effects similar to those in humans.
However, epidemiologic studies on M. ulcerans and M. haemophilum and
disease and laboratory experiments with these two organisms in animals
suggest different modes of transmission than M. marinum, including possible
aerosol transmission with subsequent hematogenous spread to the dermis.

Finally, comparisons of the pathogenic and histopathologic characteristics
of these three diseases suggest differences in the levels of virulence,
mechanisms of pathogenesis, and modes of transmission. As we learn more
about the ecology, epidemiology, and pathogenesis of these unique
mycobacteria through improvements in culture techniques, diagnostic tests,
and continued laboratory research, we may be able to identify contaminated
environments contributing to the source of these infections and elucidate
the mode of transmission. Such information is essential if we are to
develop strategies to prevent the further emergence of these diseases.

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

Acknowledgments

      The authors thank Barbara J. Marston and Jordan Tappero for their
advice and consultation regarding BU, Leslie Parent for histology slides of
M. marinum infection and her consultation, Michael A. Saubolle for
histology slides of M. haemophilum infection and his consultation, Bryan R.
Davis for his consultation regarding disseminated M. haemophilum
infections, Laura Povinelli for critical review of the PHLIS data, and
Jennifer Ekmark and Ellen Spotts for critical review of this submission.

      Dr. Dobos is a postdoctoral fellow working on the molecular
pathogenesis and serodiagnosis of Mycobacterium ulcerans infection at The
Emory University School of Medicine and CDC. She received her graduate
training at Colorado State University, where she was the first to describe
the glycosylation of a Mycobacterium tuberculosis protein.

      Address for correspondence: C. Harold King, Division of Infectious
Diseases, Department of Medicine, Emory University School of Medicine, 69
Butler Street SE, Atlanta, GA 30303; fax: 404-880-9305; e-mail:
cking01@emory.edu.

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Synopses

Respiratory Diseases among U.S. Military Personnel: Countering Emerging
Threats

Gregory C. Gray,* Johnny D. Callahan,* Anthony W. Hawksworth,* Carol A.
Fisher, and Joel C. Gaydos
*Naval Health Research Center, San Diego, California, USA; Naval Medical
Center, San Diego, California, USA; and Walter Reed Army Institute of
Research, Washington, DC, USA

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      Emerging respiratory disease agents, increased antibiotic
      resistance, and the loss of effective vaccines threaten to
      increase the incidence of respiratory disease in military
      personnel. We examine six respiratory pathogens (adenoviruses,
      influenza viruses, Streptococcus pneumoniae, Streptococcus
      pyogenes, Mycoplasma pneumoniae, and Bordetella pertussis) and
      review the impact of the diseases they cause, past efforts to
      control these diseases in U.S. military personnel, as well as
      current treatment and surveillance strategies, limitations in
      diagnostic testing, and vaccine needs.

Respiratory infections, the most common cause of acute infectious disease
in U.S. adults (1), are also the leading cause of outpatient illness and a
major cause (25% to 30%) of infectious disease hospitalization in U.S.
military personnel (2,3). Because of crowded living conditions, stressful
working environment, and exposure to respiratory pathogens in
disease-endemic areas, military trainees and newly mobilized troops are at
particularly high risk for respiratory disease epidemics (2, 4-6). For
example, before vaccines were used, more than 80% of military trainees had
respiratory infections, and as many as 20% were hospitalized during the 2
months of recruit training (7). Although respiratory disease control is
improved, epidemics continue to occur, and respiratory disease in military
trainees continues to exceed that in U.S. civilian adults (Figure 1). The
recent loss of adenovirus vaccine (types 4 and 7) production, changes in
the susceptibility of pathogens to antimicrobial drugs, and emerging
respiratory pathogens threaten to increase the military population's
vulnerability to respiratory diseases.

We review the changing epidemiology and   
control of six major respiratory disease
pathogens of special concern to the       
military.                                 
								[fig]
Adenoviruses                              Figure 1. Hospitalization
                                          rates for acute respiratory
Respiratory disease agents discovered in  disease per 10,000 persons,
adenoidal tissue in U.S. soldiers in the  1991 to 1994: U.S. army
1950s were associated with rhinitis,      recruits vs. young adults in
pharyngitis, conjunctivitis,              U.S. nonfederal hospitals.
pneumonitis, and atypical pneumonia and   U.S. army recruit estimates
were subsequently designated as           are converted from percentage
adenoviruses (14). In 1958, adenoviruses  febrile acute respiratory
caused hospitalization of an estimated    disease rates per 100
10% of military recruits (15).            trainee-week figures (8). On
Adenoviral disease was highest during     average, recruits were 19
winter, accounting for 90% of all         years old. U.S. national
recruits hospitalized with pneumonia      nonfederal estimates were
(16,17) and 72% of all respiratory        taken from first-listed
disease (17). Military recruits had a     diagnoses with the
greater chance of acquiring adenoviral    International Classification
infections than similar civilian          of Diseases codes 460 to 466
populations, with most infections         (9) among persons of ages 15
occurring during the first 3 weeks of     to 44 years (10-13).
military training (16,18,19). Of the 47
adenoviral serotypes, types 4 and 7 accounted for most military respiratory
disease epidemics. A 1965 study of a typical epidemic at Fort Dix, New
Jersey, established the need for vaccines (20).

In 1971, the Department of Defense (DoD) began routine use of live,
enteric-coated types 4 and 7 vaccines, which have remained very effective
(6). Vaccine development for other serotypes that cause only infrequent
epidemics was begun, but no vaccine became licensed. Recently, the sole
manufacturer of the adenovirus type 4 and type 7 vaccines ceased
production, so neither vaccine is available. The unavailability of
adenovirus vaccines threatens a sharp increase in numbers of acute
respiratory disease epidemics in the military, especially among recruits
(6). Recently, two recruit centers where the vaccines were not available
had large acute respiratory disease epidemics (21,22).

The ecologic and pathologic features  
of adenoviruses in military
populations are poorly understood     
(23,24). Most available surveillance  		[fig]
data are more than 20 years old
(7,20). To better understand the      Figure 2. Department of Defense
distribution of adenovirus            medical treatment facilities and
serotypes, risk for infection, and    recruit training camps
agent dynamics following vaccine      participating in surveillance
loss, triservice adenovirus           for emerging respiratory disease
surveillance has been established at  pathogens: invasive
five military training centers        Streptococcus pneumoniae (typing
(Figure 2) (25). Early data indicate  and antibiotic sensitivity
that types 4 and 7 vaccines remain    studies); Streptococcus pyogenes
effective, but nonvaccine serotypes   (typing and antibiotic
are prevalent and should be           sensitivity studies); and
considered in new vaccine             adenovirus (typing studies).
development strategies. More than
55% of 3,212 throat cultures from symptomatic trainees from October 1996 to
May 1998 yielded adenoviruses. Most prevalent were types 4 (46%), 7 (32%),
3 (13%), and 21 (5%). Among trainees with acute respiratory infection
symptoms, nonvaccinated personnel were at greater risk of having a culture
positive for adenovirus types 4 and 7 (odds ratio = 41.2; 95% confidence
interval = 18.7 to 113.2) than vaccinated personnel. Capability to isolate
and identify adenoviruses has improved, but simple rapid molecular
diagnostic techniques have not yet been developed.

Influenza

Since an annual influenza vaccine policy was adopted for active-duty
personnel in the 1950s, massive influenza epidemics have largely ended.
However, the potential for illness and death due to new viral strains
remains. During the last 3 months of 1918, an influenza A pandemic affected
106,897 (18.8%) of 569,470 navy personnel, with an estimated case-fatality
rate of 4.5%. The case-fatality rate was particularly high among military
trainees, especially those who had pneumonia. For example, during a 30-day
period beginning in September 1918, 9,623 (21.5%) of 44,605 navy trainees
(Illinois) had influenza, and 924 died; the case-fatality rate was highest
(48%) among those with pneumonia (26). At autopsy, streptococcal organisms
were often associated with pneumonia, which suggests that pathogens in the
training camps may have exacerbated the influenza illnesses and deaths
during this pandemic.

Even with annual use of influenza vaccine, laboratory-based surveillance is
critical. During February 1996, a U.S. navy ship with a 600-person crew had
an estimated 42% influenza A attack rate, although more than 95% of the
crew had received the annual influenza vaccine (K. Earhart, pers. comm.).
The annual vaccine for that winter (A/Johannesburg/33/94-like [H3N2] and
A/Texas/36/91-like [H1N1]) did not protect against the A/Wuhan/359/95
[H3N2] strain that infected the crew.

The recent outbreak of H5N1           
review of capability to detect new    
influenza strains (27); only the air 		[fig] 
force was conducting a
laboratory-based surveillance         Figure 3. Military sites in the
program (28). Since the Hong Kong     United States participating in
outbreak, a cooperative global        Department of Defense influenza
influenza surveillance network has    surveillance. The focus of
been formed. Worldwide, more than 20  surveillance at etiology-based
medical treatment facilities and      sites is to determine the viral
laboratories from all services are    causes of influenzalike
collecting influenza isolates for     illnesses; the focus of
typing (Figure 3)(29). Additionally,  population-based sites is to
in the United States, military        closely monitor for
training sites at high risk for       influenzalike illness epidemics.
influenza are monitored so that
epidemics might be quickly detected. This early warning system allows
public health officials to modify vaccine antigens, use antiviral drugs,
and take other measures to reduce illness.

Streptococcus pneumoniae

Before penicillin was introduced, complications of S. pneumoniae infections
were frequent and often fatal. Large epidemics of pneumonia occurred in
crowded military populations, particularly after influenza outbreaks,
especially during winter. In 1918, a 1-month epidemic of S. pneumoniae in a
military camp in Illinois resulted in 2,349 hospital admissions with a 50%
death rate (30). From the 1960s to the 1980s, military epidemics of
pneumococcal disease were very rare. However, in recent years, military
pneumococcal epidemics have occurred in southern California (31), in North
Carolina (32), and among a ship's crew in the Mediterranean Sea (4).

S. pneumoniae infections have various clinical features, including
pneumonia, meningitis, empyema, bacteremia, conjunctivitis, sinusitis,
arthritis, and otitis media. Since the introduction of penicillin,
epidemics of respiratory disease caused by S. pneumoniae are much less
frequent but remain a threat. To counter outbreaks, the military has used
mass prophylaxis with benzathine penicillin G (1.2 million units)
intramuscularly (31). However, the efficacy of this intervention and its
impact on antibiotic resistance have not been fully evaluated (33). In
1991, the Armed Forces Epidemiological Board recommended pneumococcal
vaccine (23-valent polysaccharide) for populations at high risk for S.
pneumoniae infection. However, because of the cost and uncertainty about
efficacy in healthy young adults, the vaccine is given only to trainees at
one marine corps installation.

During the last 20 years, penicillin-resistant S. pneumoniae (intermediate
and highly resistant strains), as well as multidrug-resistant strains, have
been reported with increasing frequency throughout the world. Recently,
investigators from Korea reported that 70% of 131 clinical civilian
pneumococcal isolates were penicillin-resistant (34). On the basis of
limited surveillance data, the public health threat of penicillin-resistant
S. pneumoniae to U.S. military personnel and their dependents is increasing
(35). Walter Reed Army Medical Center, Washington, D.C., reported an
increase in the percentage of penicillin-resistant S. pneumoniae isolates
from 0% in 1990 to 36.2% in 1994 (36).

In winter 1989-90, an outbreak among marine trainees at Camp Pendleton,
California, resulted in 128 reported cases of pneumonia, one of the largest
epidemics since the development of antibiotics. The epidemic triggered mass
prophylaxis with benzathine penicillin G and administration of pneumococcal
vaccine (31). Soon after, other smaller epidemics of pneumococcal pneumonia
occurred among U.S. army rangers (32) and among the crews of two navy ships
in Italian waters (4). As we write (March 1999), another pneumococcal
pneumonia outbreak among army trainees is under epidemiologic
investigation. These recent pneumococcal epidemics may be evidence of a
changing epidemiologic threat. Pneumococcal pneumonia, which was checked
when antibiotics became available in the 1950s, seems to have reemerged.

Increasing antibiotic resistance and epidemics prompted surveillance for
invasive S. pneumoniae disease (Figure 2). Early data from patients
hospitalized in military hospitals in the United States are consistent with
data from civilian U.S. populations. On average, 35% of isolates have full
or partial resistance to penicillin (35,37). Thus far, nearly all invasive
isolates are of types included in the 23-valent vaccine. A
cost-effectiveness analysis projected that using this vaccine among new
navy and marine corps personnel would result in a lifetime savings of
approximately $9 million (38).

Streptococcus pyogenes

U.S. military populations have frequently had large S. pyogenescaused
epidemics of pharyngitis and acute rheumatic fever, accompanied by other
concomitant diseases, such as pneumonia, sepsis, polymyositis, necrotizing
fasciitis, scarlet fever, and glomerulonephritis (5,39). Historically,
because of cramped living conditions, military recruits have been at high
risk for streptococcal disease (2,5,39,40). Illness was especially high in
World War II, with the navy reporting approximately one million
streptococcal infections and more than 21,000 cases of acute rheumatic
fever (5,41).

In 1948, Massell et al. (42) reported that the treatment of acute
pharyngitis infection with oral penicillin prevented acute rheumatic fever.
Further studies confirmed the effectiveness of a single intramuscular
injection of benzathine penicillin G in preventing a broad range of acute
and chronic sequelae of streptococcal infections (5,40,43). These early
successes led to mass antimicrobial prophylaxis with benzathine penicillin
G in training populations at high risk to interrupt and prevent outbreaks
of acute disease and their sequelae (44). This control strategy was
generally very effective. However, a 1989 epidemic of S. pyogenes
pharyngitis among marine corps trainees demonstrated that benzathine
penicillin G prophylaxis for nonpenicillin-allergic trainees alone might
not protect against epidemics in closely contained populations, especially
those with longer training periods, as unprotected penicillin-allergic
recruits may serve as S. pyogenes reservoirs. This finding led to the
navy's adoption of oral erythromycin as prophylactic therapy for
penicillin-allergic recruits (39,45). Another study has shown that 500 mg
of azithromycin taken orally each week is, by serologic evidence, an
effective prophylactic intervention against S. pyogenes (33).

Since the development of antibiotic prophylaxis, civilian and military
epidemics of S. pyogenes disease have declined and then reemerged
(39,46,47). Epidemics of acute rheumatic fever have occurred throughout the
United States (46-48). In addition, an estimated 10,000 cases of severe S.
pyogenes disease, such as necrotizing fasciitis and streptococcal toxic
shock, occur nationwide each year (49-52). Increases in invasive
streptococcal disease among some U.S. populations have been attributed to
changes in the prevalence of virulent strains of S. pyogenes(53).

Although antibiotic prophylaxis remains effective, S. pyogenes persists as
a leading cause of bacterial respiratory illness among military personnel
(5,39,47,48,54). Risk factors associated with S. pyogenes infection include
recent entry to the military, crowding, lack of prophylaxis, close contact
with an S. pyogenes carrier, and close contact with a trainee who has not
received antibiotic prophylaxis (39).

Prophylactic use of oral erythromycin or azithromycin may promote macrolide
resistance among endemic streptococci. The Naval Medical Center, San Diego,
California, found 5(10%) of 50 consecutive clinical isolates collected
during March and April 1997 resistant to erythromycin. While frequently
reported in Europe and Japan, macrolide resistance has been uncommon in
U.S. military populations (T. Ferguson, R. Haberberger, pers. comm.) and
infrequent in isolates from civilians in the United States (55).

Triservice surveillance has been established to define antibiotic
resistance patterns and determine which serotypes of S. pyogenes are
causing clinical disease (Figure 2). Data from eight sentinel military
medical treatment facilities will be used to monitor resistance and develop
alternate prophylactic strategies, rapid diagnostic tests, and vaccines.

Mycoplasma pneumoniae

During World War II, acute pneumonia in military personnel was frequently
milder than lobar pneumonia. Chest radiographs showed substantial pulmonary
involvement, yet patients did not have high fever, pleuritic chest pain, or
rigors characteristic of pneumonia caused by S. pneumoniae. In 1943, these
infections were recognized as primary atypical pneumonia, which accounted
for an estimated 68% of atypical pneumonias among marine trainees (56) and
infected as many as 44% of recruits over a 3-month training period (57). In
1944, samples from a patient with atypical pneumonia showed M. pneumoniae
(57,58), and soon thereafter, M. pneumoniae was identified as an important
cause of acute respiratory disease in U.S. military personnel (59).

A common cause of pharyngitis and bronchopneumonia, M. pneumoniae may also
cause fulminant pneumonia, cardiac disease, arthritis, dermatologic
conditions, and central nervous system disease (60). Crowded military
populations are at particularly high risk for infection. In the 1970s, up
to 57% of U.S. recruits had evidence of acute infection (61), and from the
1960s through the 1990s, as many as 56% of pneumonia cases among recruits
were due to M. pneumoniae (62-64). Because culture and diagnostic tests for
M. pneumoniae are not commonly available at military facilities, M.
pneumoniae is often not recognized, and ineffective antibiotics are
prescribed (62).

Few options are available for combating M. pneumoniae epidemics. More than
25 years ago, several studies suggested that preexisting antibody titers
might prevent infection (65,66), and vaccine candidates were tested with
mixed success (64,67,68). In 1965, preventing disease with a 10-day course
of oxytetracycline (69) (4 times a day) among close contacts was successful
but impractical. More recently, weekly oral azithromycin (500 mg) had a 64%
protective efficacy (by serologic tests) against M. pneumoniae in U.S.
marines (33).

Reliable diagnostic tests and enhanced surveillance efforts are needed to
assess the epidemiology and impact of M. pneumoniae on military
populations. With the exception of serologic tests, few rapid diagnostic
tests are commercially available.

Bordetella pertussis

Before vaccines were available, B. pertussis caused considerable illness in
children. With the effectiveness of whole-cell childhood vaccines, disease
incidence increased among older children and adults, whose childhood
vaccine immunity had waned (70-74). B. pertussis infection in adults, while
generally mild (75), can be incapacitating. No pertussis vaccines are
available for adults.

B. pertussis also affects military populations; a 1989 study of marine
trainees who reported 7 or more days of cough showed that 18% had acute B.
pertussis infection (73). The potential for military epidemics of B.
pertussis is demonstrated by outbreaks among other confined populations,
such as those receiving general or institutionalized medical care, which
have attack rates as high as 91% (76,77). Infection in adults is often
difficult to verify since culture and polymerase chain reaction diagnostic
tests may be negative (73). While often used epidemiologically, serologic
methods are not standardized, nor are they routinely performed by clinical
laboratories (78). Hence, many epidemics are monitored by clinical case
definitions.

Some clinicians have observed a prophylactic benefit in administering oral
erythromycin to close contacts of patients (78). However, erythromycin
prophylaxis is not without side effects, and its value has been questioned
(76,79). New acellular pertussis vaccines, now approved only for use among
infants and children, are being studied for use in adults (80).

Research and Disease Control

Trainees entering military service receive influenza vaccine and adenovirus
types 4 and 7 vaccines when available. Mass antibiotic chemoprophylaxis is
also often used to prevent acute respiratory disease and control epidemics,
particularly those caused by S. pyogenes infections. After initial
training, military personnel receive annual influenza vaccine and periodic
tuberculosis screening.

Table. Current capacity to control respiratory pathogens at most military
medical treatment facilities, United States
---------------------------------------------------------------------------
                             Rapid
Pathogen        Culture      diagnostic    Prophylaxis     Vaccine(sup a)
                             tests
---------------------------------------------------------------------------

                             Antigen       Benzathine
Streptococcus                detection     penicillin G (5),
pyogenes        Available    available and erythromycin      Needed
                             used          (45), or
                                           azithromycin (33)
                                                             Available
Streptococcus   Available                                    but  seldom
pneumoniae      but not      Needed        Azithromycin (33) used among
                sensitive                                    military
                                                             personnel

Mycoplasma      Not          Serologic
pneumoniae      available    tests are     Azithromycin (33) Needed
                             available
                                                             Vaccine
                Not                                          available and
Influenza   available(sup b) Needed        Amantadine
                                                             routinely
                                                             used
                                                             Types 4 and 7
                Not                                          vaccines
Adenovirus  available(sup b) Needed        Not available
                                                             effective but
                                                             not available
                                           Erythromycin
Bordetella      Available                  prophylaxis is of
pertussis       but not      Needed        questionable      Needed
                sensitive
                                           value (79)
---------------------------------------------------------------------------
(sup a)While a number of civilian populations, such as the institutionalized,
may have similar needs these vaccine needs are particularly urgent for
crowded military trainees.
(sup b)Some medical treatment facilities have access to culture support.

Almost all respiratory illnesses, including pneumonia, are treated
empirically (4,62), often with penicillin or a macrolide (62). Without
accurate laboratory diagnoses and an early warning system to detect changes
in acute respiratory disease rates and antibiotic resistance, more
respiratory disease epidemics are likely to occur in military populations.
A Global Emerging Infections Surveillance and Response System has been
established to address this problem. Surveillance data will be used to
direct acute respiratory disease research, training, and education. Under
the system, DoD has recently established modest surveillance programs for
influenza, adenovirus, S. pyogenes, and S. pneumoniae at a number of U.S.
military recruit training camps and special facilities, in collaboration
with other federal, state, and civilian organizations. Recruit sites were
chosen for their long history of respiratory disease epidemics and the
possibility of monitoring the impact of mass antibiotic prophylaxis.
Tertiary referral medical centers were chosen to participate because they
were more likely to detect unusual and antibiotic-resistant strains of
respiratory pathogens. Limited samples of clinical influenza A, adenovirus,
S. pyogenes, and S. pneumoniae isolates are being studied. However, new
diagnostic tools and vaccines are still needed (Table).

Conclusions

Military personnel, because of crowding and unique stressors, are subject
to respiratory disease epidemics. Their risk often exceeds that of their
civilian peers. Adenovirus, influenza virus, S. pyogenes, S. pneumoniae,
and B. pertussis are particularly problematic. Pathogen control measures,
many of which were developed more than 20 years ago, are threatened by loss
of vaccine production, changes in pathogen virulence, changes in pathogen
antibiotic sensitivity, changes in population immunity, and lack of
laboratory infrastructure to identify respiratory disease pathogens and
evaluate new diagnostic and control measures.

Strong, laboratory-based surveillance programs are needed to quickly
identify new problems. The surveillance programs must be supported by fast,
accurate diagnostic laboratory tests. Surveillance data must then be used
to direct the development and evaluation of new interventions, particularly
vaccines.

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

Acknowledgments

      We acknowledge the contributions of Drs. Richard Haberberger and Gale
Chapman, Tom Ferguson, Tim Driscoll, David Trump, Christie Beadle, and
Theodore Woodward.

      Collaborators in Department of Defense surveillance for respiratory
disease pathogens include Patrick Kelley, Lisa Keep, Ramy Mahmoud, Annette
Hamilton, Maria Hook, Beverly Watts, Mills McNeill, Laura Trent, Linda
Canas, William Corr III, Sandra Williams, Kelly McKee Jr., Debra Prantl,
Rose Marie Hendrix, Jane Lindner, Johnnie Conolly, Michael Escalara, Gerald
Sandifer, Robert Greenup, Barbara Workman, Denise Clayton, David Niebuhr,
Gretchen Demmin, Maritza Johnson, Jeffrey Gunzenhauser, Mary Meyers, Mark
Kotepeter, Crystal Chatman-Brown, Alice Washington, Megan Ryan, Thomas
Hatley, Becky Christian, Julie Wohlrabe, Sharon Urban, Stephanie Thorn,
Dennis Butterworth, James Bean, Beverly Southerland, John Newsome, Edward
Gastaldo, Juan Rivas, Walter Cole, Roger Batchelor, Marianne Jesse, Jim
Blanks, Roger Gibson, Ron Hale, Royce Brockett, Pulak Goswami, Marieta
Malasig, Marie Hudspeth, Julie Hochwalt, Mary Sorenson, Jason Unruh, Paul
Sato, Colleen McDonough, Heather Taylor, Rosana Magpantay, Tuan Pham, Chris
Barrozo, Pam Plobette, Debbie Kamens, Jeff LeClair, Cassandra Morn, Farukh
Khambaty, Janet Yother, and Susan Hollingshead.

      This represents report no. 98-22, supported by the Office of Naval
Research, Washington, DC, under DoD/HA reimbursable - 6609.

      Captain Gray is a medical epidemiologist and chief, Emerging Illness
Division, Naval Health Research Center, San Diego. His work involves
surveillance and epidemiologic studies of respiratory disease. He also
studies illness among veterans of the Persian Gulf War.

      Address for correspondence: Gregory C. Gray, Naval Health Research
Center, Emerging Illness Division, P.O. Box 85122, San Diego, CA
92186-5122, USA; fax: 619-553-7601; e-mail: Gray@nhrc.navy.mil.

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

Synopses

Q Fever in Bulgaria and Slovakia

V. Serbezov,* J. Kazr, V. Novkirishki,* N. Gatcheva,* E. Kovcov, and
V. Voynova*
*National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria;
Institute of Preventive and Clinical Medicine, Bratislava, Slovak
Republic; and Institute of Virology, Slovak Academy of Sciences,
Bratislava, Slovak Republic

---------------------------------------------------------------------------
      As a result of dramatic political and economic changes in the
      beginning of the 1990s, Q-fever epidemiology in Bulgaria has
      changed. The number of goats almost tripled; contact between
      goat owners (and their families) and goats, as well as goats
      and other animals, increased; consumption of raw goat milk and
      its products increased; and goats replaced cattle and sheep as
      the main source of human Coxiella burnetii infections. Hundreds
      of overt, serologically confirmed human cases of acute Q fever
      have occurred. Chronic forms of Q fever manifesting as
      endocarditis were also observed. In contrast, in Slovakia, Q
      fever does not pose a serious public health problem, and the
      chronic form of infection has not been found either in
      follow-ups of a Q-fever epidemic connected with goats imported
      from Bulgaria and other previous Q-fever outbreaks or in a
      serologic survey. Serologic diagnosis as well as control and
      prevention of Q fever are discussed.

Q fever, a widespread zoonosis recognized as a clinical entity in 1937 (1),
is caused by the obligate intracellular parasite, Coxiella burnetii. The
disease is endemic worldwide, occurring in different geographic regions and
climatic zones (2). New Zealand is probably the only large country without
Q fever (3). The principal vectors of C. burnetii are ticks, which transmit
the agent to wild animals (causing wildlife coxiellosis) or to domestic
animals (creating the livestock reservoir of C. burnetii) (4). The most
important reservoirs in nature are small wild rodents, but infection was
also demonstrated in insectivores, lagomorphs, carnivores, ungulates,
ruminants, marsupials, monkeys, bats, birds, and even reptiles and fish
(5,6). Infected domestic animals (cattle, sheep, and goats but also pet
animals, especially cats), frequently with persistent and subclinical
coxiellosis, represent the main source of C. burnetii infection for humans,
who become infected by direct contact with these animals, by environmental
contamination (from animal excrements), and (indirectly) through processing
or consuming animal products. Human infection is acquired most often by the
inhalation of contaminated aerosols but may also occur through the
digestive tract, through skin trauma, or by sexual contact. Mother-to-fetus
transmission may also occur (7).

C. burnetii may cause acute and chronic forms of Q fever in humans, though
in many cases infection is asymptomatic and confirmed by serologic
diagnosis only (8). The acute form of Q fever manifests usually as a
flulike illness or atypical pneumonia, but often it has a protean character
with a clinical picture resembling that of nearly any infectious disease
(9). Whereas acute Q fever is usually self-limited, chronic Q fever is a
serious and often fatal illness with death rates exceeding 65% (10).
Illness occurs months to years after the acute infection in 1% to 11% of
patients and usually manifests as endocarditis. Infections of arterial
aneurysm or prosthesis; bone infection; pseudotumor of the lung; hepatitis;
cutaneous, musculoskeletal, or renal involvement; and placentitis in
pregnancy with miscarriage are also possible (11).

The first human cases of Q fever in Europe appeared in the Balkans during
World War II when strange, febrile, influenzalike infections, named
Balkangrippe, were observed among German troops (12,13). Similar infections
occurred among allied troops during operations in the Mediterranean area
(14,15). In the 1940s, Q fever was recognized in Romania (16), Greece (17),
and Bulgaria (18). The Balkans thus became the territory in which C.
burnetii could circulate in nature, be transmitted to humans, and be spread
to other parts of Europe; for example, Q fever was probably introduced to
Slovakia through infected sheep from Romania (6).

Q Fever in Bulgaria

Though the first human Q-fever cases in Bulgaria were described as early as
1949 (18), thorough epidemiologic and epizootologic studies started later
in connection with the unification of land and livestock farms into state
premises and agricultural cooperative units. Concentration of domestic
animals (especially cows and sheep) and conditions favoring circulation and
maintenance of C. burnetii in nature (ticks, reservoir animals) turned the
country into a huge natural focus of Q fever. The occurrence of C. burnetii
infection in different parts of the country was 6% to 100% in sheep, 5% to
31% in cattle, and 7% to 34% in goats (19), as confirmed serologically by
complement fixation (CF). Infestation of ticks with C. burnetii reached 26%
in southwest and 22% in northeast Bulgaria (20).

The situation changed dramatically in the 1990s as the large state premises
and cooperative farms collapsed and the number of cows and sheep decreased
(e.g., sheep from 8 million in 1990 to 3 million in 1997). As individual
farmers started to raise goats for easily accessible food, the number of
goats increased from 430,000 in 1990 to more than 1 million in 1997.
Moreover, the proportion of sera containing antibodies to C. burnetii from
domestic animals from five regions of Bulgaria in 1996 to 1997 also
changed; more (90% of 140) samples from goats than from sheep (73% of 118)
tested positive. At the same time, the rate of infestation of Dermacentor
marginatus, Ixodes ricinus, and Rhipicephalus sanguineus with C. burnetii
as demonstrated by the hemocyte test (21) was also very high (25 of 29
ticks tested were infected).

These changes also influenced the occurrence and seasonality of human
Q-fever cases in Bulgaria. Until 1990, more than 20 Q-fever outbreaks
occurred in several regions of the country, some with hundreds of patients
(e.g., 725 cases near Knyezha from 1982 to 1985 [22] and as many as 630
cases in Pavlikeni in 1985 only). These Q-fever epidemics among farmers
explain the sharp peak of Q fever incidence in 1985 (Figure 1). A sudden
drop and continual decrease thereafter could reflect a return to a
nonepidemic situation; the gradual increase in the 1990s was probably
associated with an increase in the number of goats (Figure 1). Depicting
the seasonality of the disease, the number of goats shifted from winter and
spring months (December to May) with a peak in January in the 1980s to
spring and summer months (March to August) with a peak in May and June in
the 1990s (Figure 2).

Several factors probably contributed to this shift. 1) A change in
insemination practices. Until 1990, artificial insemination of ewes was
carried out in early autumn, but in the 1990s, this practice was abandoned.
2) Sheep deliver in January and February, whereas goats deliver in March
and April. 3) Herds of cattle and flocks of sheep, though concentrated in
larger numbers, were separated from most of the population and served as
the source of C. burnetii infection usually among farmers and others
exposed to animals and their products. 4) An increased number of goats have
been kept in very close contact with goat owners and their family members.
5) Goats have been pastured daily (from March until October), so they cross
the streets of villages and small towns twice a day. Thus, at present in
Bulgaria, goats (rather than sheep or cattle) seem to be an important
source of C. burnetii infections for humans. Not only have great numbers of
goat owners with their families been exposed to C. burnetii (when tending
the goats or consuming their unpasteurized milk products), but also
occasional bystanders could contract C. burnetii infection from exposure to
the contaminated aerosols created from excrements of the goats, as they
repeatedly passed through villages and small towns to their pastures or
from exposure to infectious particles generated during parturition or
abortion. For example a Q-fever outbreak in Val de Bagnes, Switzerland,
which affected 415 persons, occurred 3 weeks after 12 flocks of sheep
descended from the Alpine pastures to the valley (8).

     [Fig]					[Fig]

Fig. 1. Incidence of        Fig. 2. Seasonal
human Q fever in            distribution of acute
Bulgaria, 19831996.        human Q-fever cases in
                            Bulgaria in 1983 to 1989
                            (dashed line) and 1990 to
                            1996 (full line).

The largest Q-fever outbreak was registered in Panagyurische (central part
of southern Bulgaria) in the 1990s, after an influenza epidemic (end of
1992, beginning of 1993). From January to June 1993, a second epidemic wave
with more than 2,000 cases of an acute flulike respiratory illness and
bronchopneumonia occurred. Atypical pneumonia was diagnosed in 589 cases by
X-ray examination, and 254 patients were admitted to the regional hospital.
Q fever was confirmed serologically only at the end of the epidemic. Of
more than 500 persons who recovered from Q-feverlike disease, 60% were
seropositive; several laboratories confirmed significant titers (from 80 to
640 in CF and microimmunoflourescence [MIF] tests) of antibodies to phase
II C. burnetii. Though most of the patients and those with positive
serologic tests were adults 20 to 59 years of age, high positivity was also
noticed in children <6 years of age (19%) and in persons 7 to 19 years of
age (23%). Most of the patients were not employed in agriculture or the
processing of animal products. Serologic examination of domestic animals by
CF test gave similar results for goats (26% of 969) and sheep (28% of 421).
However, sheep did not seem to serve as a source of C. burnetii
infectionthey went to pastures far away from the town from February to
March. The highest number of human Q-fever cases was observed from April to
June when goats delivered their kids and sporadic goat abortions occurred.

A Q-fever outbreak occurred in Panagyurische again in April to June 1995 as
evidenced serologically in 78% of 89 patients admitted to the local
hospital with bronchopneumonia. Acute Q fever was diagnosed in 28 (31%) on
the basis of seroconversion or fourfold rise of antibody titers. The
results indicate that Q fever appears to be endemic in the Panagyurische
area (with seasonal spring occurrence) and goats are probably the main
source of human infection. Nevertheless, such a seasonal Q-fever occurrence
has not been restricted to this area of Bulgaria (Table) but occurred in
all the regions (Ichtiman and Elin Pelin in the West, Stara Zagora in the
South, Blagoyevgrad in the Southwest, Vratza in the Northwest, and Varna in
the Northeast) of the country in which it had been followed. Of 252
patients with bronchopneumonia and acute flulike symptoms, 66% (46%-100%)
had antibodies to phase II C. burnetii detected by CF or microagglutination
(MA) tests. Acute Q fever was confirmed in 73 (29%) patients tested (13% to
60% in different regions). In all these regions, sheep, cattle, and goats
have been raised not only in villages surrounding the above-mentioned
towns, but also in their suburbs.

Table. Phase II-Coxiella burnetii antibodies in patients with
bronchopneumonia and acute respiratory symptoms, Bulgaria
---------------------------------------------------------------------------
                                            No. positive/     % positive
Town (region)            Testing period      No. tested(sup a)(% acute)
---------------------------------------------------------------------------
Ichtiman (West           April-June 1995        19/21          90.5 (38)
Elin-Pelin (West)        April 1995              9/9          100.0 (44)
Blagoyevgrad (Southwest) March-June 1996        26/44          59.1 (34)
Blagoyevgrad (Southwest) March-June 1997        24/30          80.0 (60)
Stara Zagora (South)     March-June 1997        24/52          46.2 (28)
Vraca (Northwest)        March-June 1997         37/6          60.7 (13)
Varna (Northeast)        Feb-March 1997         28/35          80.0 (14)
 Total                                         167/252         66.3 (29)
---------------------------------------------------------------------------
(sup a)Sera from Ichtiman and Elin-Pelin were examined by complement fixation
test, remaining sera by microagglutination. All sera were from patients
with bronchopneumonia, except those collected in Varna, which were from
patients with acute respiratory flulike symptoms. Sera with titers >/= 10 in
either test were considered serologically positive. Acute Q-fever diagnosis
was based on seroconversion or fourfold rise of antibody titers.

Similar results were observed when testing human sera from the serum bank.
Of 224 randomly chosen sera collected in nine localities (Gabrovo from the
North, Razgrad and Dobrich from the Northeast, Sofia from the West,
Blagoyevgrad from the Southwest, and Stara Zagora, Pazardzhik, Haskovo, and
Sliven from the South of Bulgaria), 87 (38%) reacted positively with phase
II C. burnetii in MA or MIF tests. Serologic positivity varied from 6% in
Sliven to 60% in Blagoyevgrad, except for Razgrad, where none were positive
(however, only four sera were tested).

Chronic Q-fever cases manifesting as endocarditis were confirmed
serologically by high titers from 640 to 1 ml of phase I- and phase II- C.
burnetii immunoglobulin (Ig)G antibodies in MIF, by demonstration of
specific immunofluorescence in the cuts of aortal valves, and by C.
burnetii isolation from the replaced prosthesis in three patients before
1990 (23). Two additional cases of Q-fever endocarditis were diagnosed
serologically by MA and MIF tests from 1996 to 1997.

Antibodies to phase II C. burnetii by MA were found in 16 of 18 aborting
women with titers of 10 to 320, which indicates the possibility of acute
Q-fever infection during pregnancy. In two cases, in paired sera collected
in 23-day intervals, a shift from the titer of 160 to seronegativity (titer
<10) was observed. Even though abortion tissues were not cultured or tested
for C. burnetii, these findings deserve further study, since the possible
adverse effects of C. burnetii infection during pregnancy has also been
suggested by other authors (24).

Q Fever in Slovakia

In Slovakia, Q fever has been known since 1954 when outbreaks occurred
among agricultural workers who contracted the infection from sheep imported
from Romania and among workers of a textile plant who were exposed to
contaminated imported cotton (6). From that time until the 1980s, the waves
of epizootics and small epidemics appeared in factories processing cotton,
wool, and hides from Mongolia and China, in a sheep farm with imported
breeding rams from England, and in various agricultural premises often
connected with excursions of workers to cattle or sheep farms in which C.
burnetii infections could have occurred. Veterinary and serologically
uncontrolled movement of cattle within the country also contributed to the
establishment of domestic coxiellosis (25). Some areas of the southern part
of central Slovakia became a natural focus of Q fever, with the D.
marginatus tick as the main vector of C. burnetii (6).

Since the 1980s, only sporadic cases of Q fever have been reported from
different parts of the country, though almost 3% of approximately 7,000
ticks collected in all districts of Slovakia were found (by the hemocyte
test) to harbor C. burnetii, and attempts to recover C. burnetii from
pooled positive ticks resulted in the isolation of 10 virulent C. burnetii
strains from five ticks, mostly I. ricinus species (26). On the other hand,
C. burnetii strains isolated from cow milk were of lower virulence for
guinea pigs and mice (Kovcov et al., submitted for publication).
Circulation of such low virulent strains among livestock and large-scale
vaccination of cattle by inactivated phase I-C. burnetii corpuscular
vaccine (one subcutaneous dose consisting of 500 g of highly purified C.
burnetii cells) carried out in the 1970s and 1980s, together with improved
veterinary control of domestic animal transport within the country, could
explain a decrease in the occurrence of human Q fever in Slovakia. This
explanation is supported by results of a serologic survey (carried out from
1989 to 1996) for Q-fever antibodies in groups of farmers or in patients
with suspected C. burnetii infection. Of 21,197 human sera tested, 655 (3%)
reacted with phase II- C. burnetii antigen in the CF test (until 1992) or
(later on) in enzyme-linked immunosorbent assay (ELISA). Acute Q fever (as
individual or clustered cases) was diagnosed in 23 sera, not including 113
from the Q-fever epidemic discussed below, on the basis of seroconversion
or IgM antibody detection. During the same period, phase-II C. burnetii
antibodies were detected in 11% of cattle and in 3% each of sheep and
goats.

Improved veterinary control of domestic animal transport within the
country, however, cannot exclude the possibility of introducing C. burnetii
infection through imported domestic animals or raw materials not tested
properly. The use of CF, which is much less sensitive to Q-fever antibodies
than other serologic tests (e.g., ELISA [27]), to screen the goats imported
to Slovakia from Bulgaria is unsatisfactory, as confirmed in 1993 by the
largest reported Q-fever epidemic in Slovakia (28). The epidemic started
suddenly during the spring as an outbreak of respiratory infection in
inhabitants of a village in West Slovakia. A total of 113 persons were
affected from the beginning of March until May 18, as confirmed
serologically (seroconversion, detection of IgM antibodies, and high
phase-II antibody titers, respectively) by CF, MA, MIF, and ELISA. Of 42
patients admitted to the hospital, 33 had atypical pneumonia (diagnosed by
X-ray examination), and 27 had hepatic involvement (diagnosed on the basis
of the increased values of liver transaminases). As many as 103 were male
patients who used to visit the local pub, in which they contracted
infection by the aerosol created from the heavily contaminated garments of
boys tending aborting goats. C. burnetii infection in incriminated goats
was confirmed serologically (46 of 216 goat sera tested were positive by
ELISA) and by seroconversion in mice inoculated with spleen, lung, and
liver suspension from an aborted kid.

In contrast to the situation in Bulgaria, a 4-year follow-up of patients
from this Q-fever epidemic did not result in clinical or serologic
confirmation of any chronic form of the disease (Kovcov  et al.,
submitted for publication). In addition, evidence of chronic Q fever was
obtained neither in the serologic survey carried out in Slovakia from 1989
to 1996, nor in testing of more than 200 patients with chronic
cardiovascular disease (some of them exposed to C. burnetii infection
through their work). Similarly, observation of patients from other Q-fever
epidemics (including those with 98 cases in a cotton-processing plant in
nearby Southern Moravia in 1980 [29]) was also negative. Whether this can
be explained by C. burnetii strains of different virulence circulating in
Bulgaria and Slovakia, respectively, remains to be seen, though in the
latest Q-fever epidemic in West Slovakia, Bulgarian C. burnetii strains
were presumably involved. However, whereas C. burnetii strains of tick and
domestic animals origin isolated in Slovakia may differ, no data are known
on the virulence of Bulgarian strains. A total number of human C. burnetii
cases can also be important. Whereas in Bulgaria more than 1,000 patients
were affected, in Slovakia tens of human cases occurred, so the probability
of developing the chronic form of Q fever in Slovakia was lower. The small
number of patients in Slovakia could also be explained by earlier diagnosis
and proper antibiotic treatment at the early stage of infection. The
patients' history, e.g., previous rheumatic disease, should be also taken
into consideration.

Lessons Learned from Q-Fever Outbreaks in Bulgaria and Slovakia

Epidemiologic and serologic investigations in Bulgaria and Slovakia
indicate that an increase in human Q fever in Bulgaria in the 1990s and
Slovakia in 1993 was associated with goats. The data on the propensity of
goats to transmit C. burnetii to humans from Greece, Cyprus, France, the
United States, and even a trans-Pacific cargo ship transporting dairy
goats, were summarized by Lang (4). More recently, a cluster of human C.
burnetii infections associated with exposure to vaccinated goats and their
unpasteurized products was reported from France (30). Goats may pose a
threat to human health as a source of C. burnetii infection in every
country in which they are raised extensively and are in close contact with
humans. However, Q fever can also be contracted from other sources of
infection and has been, even in Bulgaria and Slovakia.

Reporting of Q fever in a given territory depends on the attention of
public health authorities and the availability of diagnostic methods. Apart
from C. burnetii isolation (mainly in cell cultures by a shell-vial method
[31] or direct detection, preferably by polymerase chain reaction [32]),
these diagnostic methods are based mostly on serologic tests. Sensitivity
of serologic tests for screening Q-fever antibodies increased from CF to MA
and from MIF to ELISA (27). The cut-off values for individual tests may
differ between laboratories and antigens used; for CF and MA tests, 1:8 and
1:16 serum dilutions were acceptable (in Slovakia) and for either test 1:10
serum dilution was acceptable (in Bulgaria). For a more sensitive MIF and
ELISA allowing also detection of immunoglobulin classes, diagnostic titers
were set at the phase-II IgG >/= 200 and phase-II IgM >/= 50 in MIF (33) and at
>/= 128 for the IgM and IgG phase-I responses, but >/= 512 for the IgM and >/=
1,024 for the IgG response to phase II C. burnetii in ELISA (34),
respectively. Serologic diagnosis of acute Q fever relies on seroconversion
from negativity to positivity or at least fourfold rise of phase-II
antibodies in paired (acute- and convalescent-phase) serum samples and
demonstration of IgM antibody response or high titers (e.g., >/= 128 in CF
and 200 in MIF) of phase-II antibodies in a single serum sample. For
diagnosis of chronic Q fever, high titers (i.e., >/= 200 in CF and >/= 800 of
in MIF) of phase I antibodies, occurring rarely and in low titers in acute
Q-fever cases, are required (9).

Q-fever control and prevention measures have been reviewed (35). Apart from
the thorough control of imported domestic animals, raw materials, and
movement of domestic animals within a country, prevention measures should
include adequate disinfection and disposal of animal products of conception
and strict hygienic measures in cattle, sheep, and goat farms; plants
processing products of these animals; boiling or pasteurization of milk at
62.8C for 30 minutes or at 71.7C for 15 seconds; and vaccination. At
present, three types of Q-fever vaccine are available for human use: a
Formalin-inactivated whole-cell phase-I C. burnetii vaccine used in
Australia (36), a chloroform-methanol residue subunit of phase-I C.
burnetii recommended by American authors (37), and Q-fever chemovaccine (a
soluble subunit vaccine obtained by treatment with trichloroacetic acid of
phase-I cells) developed and used in Romania (38) and the former
Czechoslovakia (39). For vaccination of domestic animals, corpuscular phase
I (in Slovakia) or phase II (e.g., in France) were used. The fact that
phase-II vaccine did not protect goats from shedding C. burnetii in milk
(30) confirmed that an effective Q-fever vaccine should consist of or be
prepared from phase-I C. burnetii (40). Efficient recombinant vaccines,
however, should also be pursued.

Mass vaccination of cattle in Slovakia in the 1970s, followed by selective
vaccination of cattle in serologically positive herds and elimination of
positive reactors in the 1980s could lessen not only distribution of C.
burnetii among domestic animals, but also its transmission to humans.
However, absence of vaccination of domestic animals in Bulgaria could
contribute to the maintenance of C. burnetii and therefore to increased
possibility of human infection, though basic natural conditions for
circulation of this agent in either country have been similar. Moreover,
gradual changes in agriculture in Slovakia during the 1990s resulted in
reduced numbers of cattle and sheep but not in the dramatic increase in
goat numbers seen in Bulgaria after the collapse of state farms and
cooperative units. One can conclude that in Bulgaria there is a permanent
threat of more Q-fever outbreaks unless preventive measures, including
improvement of veterinary services and vaccination of domestic animals,
particularly goats, are established. In Slovakia, because of surveillance,
veterinary control, and vaccination of domestic animals, the situation is
much better; however, attention should still be paid to avoid introduction
of C. burnetii by imported animals and raw materials and the possibility of
coxiellosis outbreaks among domestic animals and consequently Q fever in
humans.

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

      Dr. Serbezov is consultant at the National Center of Infectious and
Parasitic Diseases in Sofia.

      Address for correspondence: Jn Kazr, Institute of Preventive and
Clinical Medicine, Limbov 14, 833 01 Bratislava, Slovak Republic; fax:
421-737-3906; e-mail: kazar@upkm.sanet.sk.

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Synopses

Adhesins as Targets for Vaccine Development

Theresa M. Wizemann, John E. Adamou, and Solomon Langermann
MedImmune, Inc., Gaithersburg, Maryland, USA

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      Blocking the primary stages of infection, namely bacterial
      attachment to host cell receptors and colonization of the
      mucosal surface, may be the most effective strategy to prevent
      bacterial infections. Bacterial attachment usually involves an
      interaction between a bacterial surface protein called an
      adhesin and the host cell receptor. Recent preclinical vaccine
      studies with the FimH adhesin (derived from uropathogenic
      Escherichia coli) have confirmed that antibodies elicited
      against an adhesin can impede colonization, block infection,
      and prevent disease. The studies indicate that prophylactic
      vaccination with adhesins can block bacterial infections. With
      recent advances in the identification, characterization, and
      isolation of other adhesins, similar approaches are being
      explored to prevent infections, from otitis media and dental
      caries to pneumonia and sepsis.

The ultimate aim of any vaccine is to produce long-term protective immune
responses against a pathogen. These responses include systemic humoral
antibodies that neutralize invasive organisms and cytotoxic T cells, which
are required to clear certain infections, particularly chronic viral
infections such as HIV (1). Helper T-cell and cytokine responses also
influence humoral and cellular immune responses (2). For most bacteria and
viruses, the first encounter with their host involves attachment to a
eukaryotic cell surface, which results in colonization of the host prior to
disease. In such cases, induced antibody responses at the mucosal surface
could prevent attachment and abrogate colonization. The ideal target for
such antibodiessurface proteins known as adhesinsmediate microbial
attachment to host tissue (3). We review recent advances in the
identification, isolation, and purification of adhesins (and putative
adhesins) that could serve as vaccines to elicit such responses. Studies of
at least one adhesin, the FimH protein from uropathogenic Escherichia coli,
show that antiadhesin antibodies can block microbial attachment and
subsequent disease. Furthermore, while specific induction of immune
responses along the mucosal surface by mucosal immunization may have its
advantages (4-7), the FimH studies demonstrate that immunoglobulin (Ig) G
antibodies alone, which transudate into secretions after parenteral
vaccination with the FimH adhesin, are sufficient to block colonization and
infection.

The Role of Adhesins in Microbial Pathogenesis

To initiate infection, bacterial pathogens must first be able to colonize
an appropriate target tissue of the host (8,9). This tropism (ability to
gain access to a niche within the body), in association with the ability of
the bacterium to breach mucosal barriers and invade the host, distinguishes
pathogenic from commensal organisms. Colonization begins with the
attachment of the bacterium to receptors expressed by cells forming the
lining of the mucosa (Figure 1A, 1B). Certain species of bacteria are
restricted in terms of the hosts and tissues they infect and the diseases
they cause. In many cases, tropism for specific tissues has been
corroborated in the laboratory by in vitro binding assays with isolated
epithelial cells collected from sites of infection or from infection-prone
hosts.

Attachment is mediated by adhesin proteins; bacterial lectins are the most
common type of adhesin among both gram-negative and gram-positive bacteria
(3,10-12). Adhesins, such as the FimH adhesin produced by most
Enterobacteriaceae (including uropathogenic E. coli), are highly conserved
proteins (13). This lack of major variation is most likely due to the
requirement that all pathogenic strains recognize invariant host receptors.
Although minor changes in the adhesin protein have been observed (2%
divergence) and correlate with decreased or increased affinity for binding
to sugars (14), antibodies against a single FimH protein cross-react with
>90% of E. coli strains expressing the FimH adhesin and block binding to
bladder cells in vitro (15,16). Furthermore, antibodies against FimH from a
single isolate protect against in vivo colonization by >90% of
uropathogenic strains in a murine model for cystitis (unpub. obs.). This
high degree of antigenic conservation is another reason why adhesins may
serve as ideal vaccines.

Figure 1. Four mechanisms of bacterial adherence where anti-adhesin vaccines 
could potentially block colonization and infection. A shows pili or fibrillae
protruding from the bacterial surface. These proteinaceous appendages bind 
to host cell surface molecules, usually carbohydrates, by adhesin proteins 
located at the distal tip of the pilus/fibrillar organelle. Antibodies 
targeting the adhesin protein block the bacterial/host interaction. B 
demonstrates a similar process of bacterial/epithelial cell interactions 
mediated by afimbrial adhesin proteins. In this case, antibodies directed 
against the bacterial surface proteins should also block attachment and 
colonization by impeding the ability of the bacteria to associate with 
mucosal tissues. C illustrates that some bacteria establish intimate 
associations with eukaryotic cells byintimin proteins, resulting in 
cytoskeletal rearrangements, host cell signaling, possible internalization 
of the bacteria, and in many cases systemic disease. Blocking the intimate 
association/adherence may also be another strategy to prevent bacterial 
infections. D shows a novel mechanism whereby bacteria secrete their own 
receptor protein, which is internalized by the target host cell, 
phosphorylated, and embedded in the eukaryotic cell as a new receptor 
for tight binding by the bacterium. Theoretically, blocking the secreted 
receptor (Hp90) before it is internalized by the host cell could provide 
another mechanism to block bacterial adherence and infection.

Aside from mediating colonization of the host, bacterial attachment often
results in the up-regulation or expression of many other virulence genes
encoding various proteins that allow for invasion of the host (Figure 1C)
(17-23). The proteins can mediate tighter associations with epithelial
cells, trigger epithelial cellactin filament rearrangements, and induce
changes in host-cell signaling and function. In some cases, the bacteria
may also secrete a protein that inserts into mammalian cells and serves as
a receptor for its own intimate adherence with the host (Figure 1D) (24).
Given that cross-talk between pathogenic bacteria and host cells after
microbial attachment may trigger expression of virulence factors leading to
local inflammation or invasive disease, vaccines that block bacterial
attachment may have multiple advantages.

Many studies have demonstrated the utility of vaccines against bacterial
surface components in blocking attachment in vitro as well as in vivo
(25-28). However, as understanding of the mechanisms of attachment has
evolved and characterization of specific adhesin molecules has been
refined, new opportunities have emerged for the development of
adhesin-based vaccines.

Bacterial Adhesins: Gram-Negative Organisms

One of the best understood mechanisms of bacterial adherence is attachment
mediated by cell surface structures called pili or fimbriae. Pili are long,
flexible structures that extend outward from the bacterial surface of many
species of bacteria and allow for contact between the bacteria and the host
cell. Originally pili were thought to be homopolymeric structures composed
of approximately 1,000 copies of a single structural subunit (fimbrin)
packed in a helical array. However, for many pili, such as the highly
characterized Type 1 and Pap pili expressed on E. coli and other
Enterobacteriaceae, they are heteropolymeric structures with minor tip
fibrillae proteins located at the distal end of the organelle (11). The
specific interaction with receptor architectures on host cell surfaces is
mediated by one of these tip proteins, called adhesins. For example, FimH
adhesin mediates attachment to alpha-D-mannosides (29,30)
by type 1 pili, while PapG mediates binding to
alpha-D-gal(1-4)-D-Gal-containing receptors on host cells
by Pap pili (31,32) (Table 1). The specificity of interaction may be
involved in conferring tropism to the bladder and kidney tissues,
respectively (33,38). Pilus-associated adhesins have been identified in a
number of other bacteria as well (Table 1).

Not all adhesins are associated with pili. Bordetella pertussis expresses
at least two putative adhesins on its surface: filamentous hemagglutinin
(FHA) and pertactin (34). FHA is thought to mediate attachment to sulfated
sugars on cell-surface glycoconjugates, although it may also have other
properties. Pertactin is thought to mediate binding by the Arg-Gly-Asp
triplet binding sequence characteristic of integrin-binding proteins,
although the role of this binding activity in the pathogenesis of
Bordetella infections is unclear. Both FHA and pertactin are components in
the recently approved acellular pertussis vaccine.

 Table 1. Adhesins of gram-negative bacteria
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 Adhesin       Strain          Ligand                         Reference
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 Pili family(sup a)                                           Hultgren
                                                              (33)

    PapG       Escherichia     Gala(1-4)Gal in globoseries of
               coli            glycolipids
    SfaS       E. coli         a-sialyl-2 3-b-galactose
    FimH       E. coli         Mannose-oligosaccharides

    HifE       Haemophilus     Sialylyganglioside-GM1
               influenzae

    PrsG       E. coli         Gala(1-4)Gal in globoseries of
                               glycolipids

    MrkD       Klebsiella      Type V collagen
               pneumoniae

 FHA           Bordetella      Sulfated sugars on             Brennan
               pertussis       cell-surface glycoconjugates   (34)

 Pertactin     B. pertussis    Integrins                      Brennan
                                                              (34)

 HMW1/HMW2     H. influenzae   Human epithelial cells         St. Geme
                                                              (35)

 Hia           H. influenzae   Human conjunctival cells       Barenkamp
                                                              (36)
 Le(sup b)-    Helicobacter    Fucosylated Le(sup b) histo-blood
   binding                                                    Ilver (37)
 adhesin       pylori          group antigens
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 (sup a)Representative examples from the large family of pilus-associated
 adhesins.
 FHA, filamentous hemagglutinin; HMW, high molecular weight; Hia, H.
 influenzae adhesin

In Haemophilus influenzae, two families of nonpilus adhesins have been
identified: high-molecular weight adhesion proteins (HMW1 and HMW2) and
immunogenic high molecular-weight surface-exposed proteins, the prototypic
member of which has been designated Hia for H. influenzae adhesin (35).
Both families are expressed by nontypable H. influenzae, which colonize the
respiratory tract and cause such diseases as otitis media, pneumonia, and
bronchitis. The HMW proteins, which share homology with the B. pertussis
FHA protein, mediate specific attachment of H. influenzae to different
types of human epithelial cells in vitro and have been implicated in
directing respiratory tract tropism for these organisms. The Hia protein,
in contrast, mediates tight association with human conjunctival cells and
is present only in H. influenzae strains deficient in HMW1/HMW2 expression
(36). Given the dichotomy among nontypable strains expressing either HMW or
Hia-like adhesins and the serologic conservation at least among the HMW1
and HMW2 proteins, a vaccine based on a combination of such proteins may be
protective against disease caused by most nontypable H. influenzae.

Another nonpilus adhesin has been identified and purified from Helicobacter
pylori. The adhesin, called Le(sup b)-binding adhesin, mediates bacterial
adherence to fucosylated Lewis b (Le(sup b)) histoblood group antigens, which
are expressed along the mucosal surface of the gastric epithelium (37). The
Leb-binding adhesin may be involved in conferring tropism for stomach
epithelium and allowing pathogenic bacteria to establish an ecologic niche
within the gastrointestinal tract. In association with other virulence
determinants expressed by H. pylori in the stomach, the colonization
process ultimately results in ulcer formation.

A unique mechanism has been identified by which certain strains of
enteropathogenic E. coli that cause severe diarrhea target cells for
attachment: Enteropathogenic E. coli express and insert their own receptor
(Hp90) into mammalian cell surfaces, thereby allowing the bacteria to
attach and establish intimate contact with the epithelial cells (24)
(Figure 1D). Although a bacterially encoded receptor for a cognate adhesin
protein (intimin), Hp90 is expressed and secreted by enteropathogenic E.
coli before colonization; therefore, it may also serve as a target for
vaccine development.

Bacterial Adhesins: Gram-Positive Organisms

Some of the most well-characterized colonization factors in gram-positive
bacteriathe polypeptides of the antigen I/II familybind to salivary
glycoproteins in a lectinlike interaction (11) and promote adhesion to the
tooth surface (Table 2). These proteins include the original AgI/II from
Streptococcus mutans, also known as SpaP, P1, or PAc, and the Streptococcus
sobrinus SpaA and PAg proteins (39). Streptococcus gordonii expresses two
antigen I/II polypeptides, SspA and SspB, products of tandem chromosomal
genes (39).

Surface proteins of the antigen I/II family contain alanine-rich repeats,
which adopt an a-helical coiled-coil structure, proline rich repeats, and a
carboxy-terminal region that includes the gram-positive cell wall anchor
motif LPXTG (12,49). Binding activity to salivary glycoprotein has been
attributed to both the highly conserved alanine rich repeats (50,51) and
the proline-rich repeating sequences (52). In addition to salivary
glycoprotein binding activity, SspA has been implicated in coaggregation of
S. gordonii with Actinomyces (39). Such bacterial coaggregation may be
involved in dental plaque formation.

Two other S. gordonii proteins, CshA and CshB, have also been implicated in
coaggregation with Actinomyces. These adhesins may play a role in adherence
of S. gordonii to immobilized fibronectin in vitro (52) and in colonization
in vivo (40).

Staphylococcus aureus also expresses fibronectin-binding adhesins. Two
genes encoding for fibronectin-binding proteins have been identified in S.
aureus fnbA and fnbB (41). Fibronectin binding activity is critical in
pathogenesis because it allows the bacteria to adhere to extracellular
matrix components including fibronectin and collagen (53). This can result
in cutaneous infections and in life-threatening bacteremia and endocarditis
(54).

 Table 2: Adhesins of gram-positive bacteria
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 Adhesin           Strain            Ligand             Reference
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 Antigen I/II                                           Family Demuth (39)
    Ag I/II, SpaP,

    P1, PAc        Streptococcus     Salivary
                   mutans            glycoprotein
                                     Salivary
    SspA, SspB     Streptococcus     glycoprotein
                   gordonii
                                     Actinomyces

    SpaA, PAg      Streptococcus     Salivary
                   sobrinus          glycoprotein

 CshA , CshB       S. gordonii       Actinomyces,       McNab (40)
                                     fibronectin

 FnbA, FnbB        Staphylococcus    Fibronectin        Jonsson (41)
                   aureus

 SfbI, Protein F   Streptococcus     Fibronectin        Talay (42) Hanski
                   pyogenes                             (43)

 LraI gamily                                            Burnett-Curley et
                                                        al. (44)
                                                        Jenkinson and
                                                        Lamont (12)
                                     Salivary
    FimA           Streptococcus     glycoprotein
                   parasanguis
                                     fibrin

    PsaA           Streptococcus     Unknown
                   pneumoniae
    ScaA           S. gordonii       Actinomyces

    SsaB           Streptococcus     Salivary
                   sanguis           glycoprotein

    EfaA           Enterococcus      Unknown
                   faecalis
 CbpA/SpsA/PbcA/   S. pneumoniae     Cytokine activated Rosenow (45)
   PspC                                                 Hammerschmidt et
                                       epithelial and   al. (46)
                                       endothelial      Briles et al. (47)
                                     cells, IgA         Smith et al. (48)
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Binding to fibronectin is also essential for the attachment of S. pyogenes
to respiratory endothelial cells. S. pyogenes, a group A streptococcus, has
been implicated in various diseases from skin and throat infections to
sepsis and shock (55). This binding activity is mediated by several
fibronectin binding proteins. Six different genes encoding proteins with
fibronectin binding activity have been identified (12). The most well
characterized are two closely related proteins, SfbI (42) and Protein F
(43). Both proteins are directly involved in the fibronectin-mediated
adherence to epithelial cells.

Another group of extensively studied streptococcal adhesins is the LraI
family of proteins. Included in this group are FimA from S. parasanguis,
PsaA from S. pneumoniae, ScaA from S. gordonii, ScbA from S. crista, SsaB
from S. sanguis, and EfaA from Enterococcus faecalis (44,56). These
membrane-bound lipoproteins are part of a larger family of ATP-binding
cassette metal permeases, involved in the acquisition of manganese (57).

The adhesins FimA and SsaB have high affinity for salivary glycoprotein on
tooth surfaces and are involved in colonization of the oral cavity (58).
The FimA adhesin for S. parasanguis has been localized to the tip of
peritrichous surface fimbria on these bacteria (58). FimA is also a major
virulence factor of S. parasanguis and binds fibrin (44). The ability to
bind fibrin has been implicated in the pathogenesis of infective
endocarditis.

The S. pneumoniae homologue of FimA, PsaA, also has a role in pathogenesis
(59,60). While it commonly colonizes the nasopharynx of healthy persons, S.
pneumoniae is a common pathogen in children and older adults and a leading
cause of otitis media, bacterial pneumonia, sepsis, and meningitis. Like
its homologues, PsaA may function as an adhesin, according to initial
evidence (61). Insertion inactivation of psaA resulted in pneumococcal
mutants that exhibited reduced adherence to alveolar epithelial cells in
vitro (60). However, the PsaA protein may be a permease involved in the
regulation of adherence rather than functioning as an adhesin per se
(57,61).

Another pneumococcal protein thought to be an adhesin is CbpA (45) (also
known as SpsA [46] and PspC [47]), one of a family of choline binding
proteins (CBPs) (i.e., surface proteins noncovalently associated with the
phosphocholine on the lipoteichoic acid). CbpA was initially isolated from
a mixture of pneumococcal proteins that were able to bind to a choline
affinity column. The CBP mix was purified from a strain with an inactivated
pspA gene (45). The exogenous CBP mix inhibited adherence of pneumococci to
type II pneumocytes and endothelial cells in vitro, suggesting that one or
more of these proteins may act as adhesins. CbpA was the most abundant
component in this mix and was shown to be on the surface of intact
pneumococci in an indirect fluorescent-labeling assay. The CbpA protein
also reacted strongly with a pool of human convalescent-phase serum. CbpA
is thought to mediate adherence of S. pneumoniae to sialic acid and
lacto-N-neotetraose ligands present on cytokine-activated epithelial and
endothelial cells in vitro (45). cbpA-defective mutants did not colonize
the nasopharynx of infant rats, further supporting its function as an
adhesin and potential usefulness as an adhesin-based vaccine (45). In
addition, others have shown that this choline binding protein (which they
called SpsA) has IgA-binding properties, though the relevance of this
function to pneumococcal pathogenesis is unclear (46). Yet another group
has demonstrated complement protein C3-binding activity for this protein,
which they termed PbcA (48). Whatever its role in pathogenesis, we have
demonstrated that the gene for CbpA (SpsA/PbcA/PspC) is highly conserved
among the most common pneumococcal isolates, further enhancing its use as a
vaccine candidate (62).

Adhesins as Vaccines: FimH as a Paradigm for Adhesin-Based Vaccines To
Block Colonization

One of the key aspects of proving the potential efficacy of an
adhesin-based vaccine in vivo is the development of an animal model of
disease that relies on bacterial colonization of the mucosal epithelium
mediated by the specific adhesin of interest. Although seemingly
straightforward, testing for protection in small animal models of disease
is difficult for various reasons: large doses of in vitro grown bacteria
are required to establish mucosal colonization in animals, which does not
necessarily mimic the course of infection in humans; specific glycoprotein
receptors for some adhesins are lacking in animal mucosal tissues that
correspond to the site of colonization in humans; and some bacterial
adhesins that are usually expressed as part of a larger structure on the
bacterial cell surface (e.g., tip adhesins associated with whole pili) are
difficult to purify. Despite these difficulties, adhesin-based vaccines
have demonstrated efficacy in protecting against infection, thus proving
the usefulness of such molecules as subunit vaccines. Research using the
FimH adhesin from E. coli provides one such example.

Type 1 pili have long been implicated in bacterial urinary tract infections
in humans (63,64). In a murine cystitis model, colonization of the bladder
by E. coli was shown to depend on growth conditions that favored expression
of type 1 pili and in particular required FimH (15,65). Thus, the murine
model was a valid small-animal model to prove whether adhesin-based
vaccines might block colonization.

Although purifying large amounts of pilus-associated adhesin is difficult
(because most adhesins are proteolytically degraded when expressed as
independent moieties), Hultgren et al. demonstrated that the FimH adhesin
could be stabilized in an active conformation by the periplasmic chaperone
FimC, making it possible to purify full-length FimH protein. Vaccination
with the FimCH complex elicited long-lasting immune responses to FimH. Sera
from mice vaccinated with the FimH vaccine inhibited uropathogenic strains
of E. coli from binding to human bladder cells in vitro. Vaccination with
the FimH adhesin-vaccine reduced in vivo colonization of the bladder mucosa
by >99% in the murine cystitis model (15). Furthermore, the FimH vaccine
protected against colonization and disease by uropathogenic strains of E.
coli capable of expressing multiple adhesins. IgG specific for FimH was
detected in the urine of protected mice, consistent with our original
hypothesis that antibodies directed against an adhesin protein might
protect along the mucosal surface. Subsequent studies in a primate model of
cystitis have corroborated these findings (Langermann et al., unpub. data).
Furthermore, in primate studies we demonstrated a direct correlation
between the presence of inhibitory antibodies in secretions and protection
against colonization and infection.

While IgG antibodies elicited against adhesins are protective, induction of
immune responses along the mucosa can be augmented by a variety of antigen
delivery systems that specifically target mucosa-associated lymphoid tissue
and activate the mucosal immune system (4-7,66). These delivery systems
include whole-inactivated or live-attenuated bacterial and viral vectors,
biodegradable microspheres, liposomes, transgenic plants, and antigens
conjugated to or coadministered with the cholera toxin B subunit or
attenuated forms of heat labile toxin from E. coli. Many of these systems
hold promise for future vaccine strategies, but only a few have been tested
in humans for safety and adjuvanticity. As these mucosal adjuvants progress
further toward approval for use in humans, testing should be done with
adhesin antigens to determine if induction of local immune responses
enhances the protective efficacy of adhesin-based vaccines as compared with
conventional parenteral vaccination. Such studies are under way with the
FimH vaccine.

Given the preclinical data with the FimH vaccine, similar efforts should be
directed at developing adhesin-based vaccines for a wide range of
pathogens. In this regard, additional efforts should also be focused on
developing mucosal models of infection. The availability of such models
should allow for appropriate screening of adhesin-based vaccines to prevent
infections by streptococci, staphylococci, and other pathogens for which
vaccine coverage is absent or inadequate.

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Acknowledgments

      We thank Luis Branco for his illustration (Figure 1) and Scott Koenig
and Jennifer Bostic for reviewing the manuscript.

      Dr. Wizemann served as Streptococcus pneumoniae Vaccine Project Team
Leader at MedImmune from 1996-98.

      Address for correspondence: Solomon Langermann, MedImmune, Inc.,
Department of Immunology and Molecular Genetics, 35 West Watkins Mill Road,
Gaithersburg, MD 20878, USA; fax: 301-527-4200; e-mail:
langermanns@medimmune.com.

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_________________________________________________________________________
Research
_________________________________________________________________________

Research

Tuberculosis in the Caribbean: Using Spacer Oligonucleotide Typing to
Understand Strain Origin and Transmission

Christophe Sola, Anne Devallois, Lionel Horgen, Jrme Masetti, Ingrid
Filliol, Eric Legrand, and Nalin Rastogi
Institut Pasteur de Guadeloupe, Pointe  Pitre, Guadeloupe

---------------------------------------------------------------------------
      We used direct repeat (DR)-based spacer oligonucleotide typing
      (spoligotyping) (in association with double-repetitive
      elementpolymerase chain reaction, IS6110-restriction fragment
      length polymorphism [RFLP], and sometimes DR-RFLP and
      polymorphic GC-rich sequence-RFLP) to detect epidemiologic
      links and transmission patterns of Mycobacterium tuberculosis
      on Martinique, Guadeloupe, and French Guiana. In more than a
      third of the 218 strains we typed from this region, clusters
      and isolates shared genetic identity, which suggests
      epidemiologic links. However, because of limited epidemiologic
      information, only 14.2% of the strains could be directly
      linked. When spoligotyping patterns shared by two or more
      isolates were pooled with 392 spoligotypes from other parts of
      the world, new matches were detected, which suggests imported
      transmission. Persisting foci of endemic disease and increased
      active transmission due to high population flux and
      HIV-coinfection may be linked to the recent reemergence of
      tuberculosis in the Caribbean. We also found that several
      distinct families of spoligotypes are overrepresented in this
      region.

Sequencing of the Mycobacterium tuberculosis H37Rv genome (1) has
facilitated the study of the biodiversity of M. tuberculosis around the
world (2). We investigated M. tuberculosis epidemiology and biodiversity in
the Caribbean region, where sequencing data are not available. Caribbean
islands possess independent and shared characteristics that justify
investigation of both the molecular epidemiology (3) and the evolutionary
history of M. tuberculosis (4). We used spacer oligonucleotide typing
(spoligotyping), double-repetitiveelement (DRE) polymerase chain reaction
(PCR) and IS6110-restriction fragment length polymorphism (RFLP) (5-7) to
understand the molecular epidemiology and reconstruct the phylogeny of M.
tuberculosis. The first step was to demonstrate that the chosen molecular
methods differentiated isolates with respect to their molecular clonality.
The second step was to demonstrate that under proper epidemiologic
circumstances, clonality implies active transmission. To eliminate sample
bias, we used all data for Guadeloupe during a 3-year period (1994-96) and
for Martinique and French Guiana during a 2-year period (1995-96).

Table 1. Demographic characteristics of Guadeloupe, Martinique, and        
French Guiana                                                              
-------------------------------------------------------------------------  
                                                                           
Data                    Guadeloupe   Martinique    French       Total      
                                                   Guiana
------------------------------------------------------------------------- 
Surface area (km2)          1,705       1,100       91,000      93,805    
Population (1990)         387,034     359,579      114,808     861,421     
Population (1996)         422,290     388,340      151,780     962,410     
Density (inhabitants          248         353            2          10
  /km(sup 2))                                                                                            
Birth rate/1,000                                                           
inhabitants                  16.8          14.4         29.2        17.8      
AIDS(sup a)                 700           383          561       1,644    
HIV-positive (%)(sup b)       1.4           0.8          1.8         1.3       
-------------------------------------------------------------------------  
(sup a)Cumulated cases on June 30, 1996.                                         
(sup b)On the basis of 7,087 serologic tests performed in departmental 
clinics in 1994.

M. tuberculosis may have developed through the domestical of cattle, and if
so, M. bovis was its most probable precursor (8). On the basis of
restricted allelic diversity, the speciation of M. tuberculosis is
estimated to have occurred 15,000 to 20,000 years ago (4). It is thought
that the spread of tuberculosis (TB) began in Europe around the middle ages
and reached the new world in the 16th century, Africa in the 19th century,
and only recently, remote regions such as Papua-New Guinea (mid-20th
century) and the Amazon (last quarter of the 20th century) (8). The study
of the genetic biodiversity of M. tuberculosis might help reconstruct this
evolutionary scenario. Previous work from our laboratory showed that a
significant proportion of patients had conserved (or ancient) strains of
tubercle bacilli (6), and analysis based on multiple genetic markers showed
genetic relatedness between some clusters of bacilli within Guadeloupe (9).

Demographics and Molecular Typing Methods

Guadeloupe,        Table 2. Epidemiologic and clinical data for 410
Martinique, and    tuberculosis (TB) cases reported to the health
French Guiana      authorities from 1994 to 1996 in the French
have major         West Indies
demographic        ---------------------------------------------------
differences
(Table 1).                            Guade-  Marti-  French   Total
                                      loupe   nique  Guiana
Guadeloupe and     ---------------------------------------------------
Martinique are
densely populated  TB cases             136     89     185      410
islands in the     Pleuropulmon         115     71     156      342
center of Lesser     -nary cases
Antilles; they     Total TB              11      8.2    40.6     14.20
have similar         incidencea
population sizes   Sex ratio              1.7    1.9     1.5      1.65
(417,000 and       PDR to INH (%)         4.4    2.2     3.5      3.4
388,000) and       PDR to RIF (%)         1.4     -       -        -
areas (1,705 
(km(sup 2)         Foreign-born TB       24.5   20.3    68       43.2
and 1,100 km(sup 2)  patients (%)
and homogeneous    TB-HIV coin-          28     19      25       24.6
populations (93%   fection (%)b
French
nationals).        ---------------------------------------------------
French Guiana is   (sup a)New cases/100,000 inhabitants, 1994 to 1996.
sparsely           (sup b)Total number of known HIV-positive cases among
populated          TB patients was 101. However, foreign-born patients
(150,000), its     represented 50% of all the TB-HIV coinfected patients
inhabitants        in Martinique, 60% in Guadeloupe, and 80% in French
scattered          Guiana. The % shown is a minimal estimation as HIV
throughout a       serology results were available only for 49% of
large territory    patients in Martinique, 75% in Guadeloupe, and 80%
(91,000 km2).      in French Guiana. 
Guadeloupe and     PDR, primary drug resistance; INH, isoniazid; RIF
Martinique are     rifampin.
characterized by   
an insular         
population,        
whereas French     
Guiana, which
borders Surinam and Brazil, is populated by persons of diverse geographic
and ethnic origin (many immigrants from South and Central America). The
distribution of TB cases among these three French overseas territories
reflects their individual demographics. In Guadeloupe and Martinique, 27%
and 25% of all TB cases, respectively, were imported, while in French
Guiana, 68% of all cases were imported (10). The basic epidemiologic data
(incidence, age, sex, nationality, HIV coinfection) for all TB cases
reported to health authorities describe a 3-year period (1994-96) (Table
2).

Of 136 TB cases reported in Guadeloupe, 107 had smear- or culture-positive
isolates, with culture available in 100 cases; we typed 95 (95%) of 100
isolates. Of 52 cases reported in Martinique, 41 had smear- or
culture-positive isolates, and a culture was available in 34 cases. We
typed 31 (91%) isolates. Of 124 cases reported in French Guiana, 96 had
culture- or smear-positive isolates, with a culture available in 76 cases;
we typed 73 (96%) of 76 isolates, along with three that grew in 1995 but
came from pathologic specimens received in 1994.

The isolates were first studied by spoligotyping (11), which is based on
polymorphism of the DR locus of M. tuberculosis (12); spoligotyping results
were analyzed by using the Recognizer and Taxotron software (Institut
Pasteur, Paris). The 1-Jaccard Index was calculated, and strains were
compared by the unweighted pair-group method using arithmetic averages
(UPGMA) (13) or by the neighbor-joining method (14). Secondary typing was
performed by DRE-PCR (15) or IS6110-RFLP (Table 3) (5). To determine
clonality between isolates, we used the following algorithm: strains were
considered clonal only when a combination of spoligotyping plus IS6110-RFLP
or DRE-PCR indicated they were identical. Data from DR-RFLP (12) and
SmaI-RFLP that used a polymorphic GC-rich probe (PGRS) (16) and available
epidemiologic information are also included in Table 3.

Table 3. Molecular fingerprinting and epidemiologic information from
spoligotyping-defined clusters shown in Figure 7.(sup a)
----------------------------------------------------------------------------
                    No. of strains typed   Summary of
                             by             results
Spoligo-  No. of                          obtained by      Clinical and
type      strains  IS6110   DR     DRE     molecular   epidemiologic data,
no.     harboring   RFLP    RFLP    PCR      typing             origin,
meth. 1  this type meth.2  meth.3 meth.4    methods(sup b)  observations
----------------------------------------------------------------------------
     1        2        2      2       2  Different by  Found in Surinam and
                                         meth. 2,      Guadeloupe
                                         Beijing IS6110
                                         pattern
     2        9        9      2       8  No subcluster 2 patients from
                                         by PGRS-RFLP: hospital A and 2
                                         all strains   from B + 2 patients
                                         identical by  with same surnames
                                         meth. 2 and 4
                                         IS-type J and
                                         by PGRS-RFLP
     3        3        3      1       3  2/3 strains   Found in Surinam and
                                         identical by  Guadeloupe, 2
                                         meth. 2, 3,   patients from
                                         and 4, IS-typehospital A
                                         P
     5        2        1      1       2  2/2 strains   Found in Martinique
                                         identical by  and Guadeloupe, no
                                         meth. 2 and 4,evident
                                         IS type not   epidemiologic link
                                         yet defined
   12         2        2      2       1  One spacer    Very common pattern,
                                         difference    represent both
                                         with type 14, active transmission
                                         identical by  and reactivation
                                         meth. 2 and 3,
                                         IS-type A
   13         2        2      2     ND   One spacer    Suspicion of
                                         difference    cross-contamination
                                         with type 14, (sampling in the
                                         identical by  same hospital in 3
                                         meth. 2 and 3,days)
                                         IS-type A
   14       15       15      13       8  Identical by  Very common pattern,
                                         meth. 2 and 3,represent both
                                         identical by  active transmission
                                         method 4 (one and reactivation
                                         band), IS-type
                                         A
   15         2        2      2       2  2/2 strains   Imported cluster
                                         identical by  (Surinam or
                                         meth. 2, 3,   Dominican Republic)
                                         and 4, IS-type
                                         C
   17         6        6      1       5  Subclustered  17B found in 2
                                         by PGRS-RFLP: Guadeloupean
                                         17B, 2 strainspatients
                                         identical by  hospitalized in same
                                         meth. 2, 3 andhospital B
                                         4, IS-type N
   29         5        5      4       5  5/5 strains   3 of 5 Guadeloupean
                                         identical by  patients
                                         meth. 2, 3,   hospitalized in same
                                         and 4, IS-typehospital B
                                         B
   30        2         2      2       2  2/2 strains   2 patients from the
                                         identical by  same part of
                                         meth. 2, 3,   Guadeloupe
                                         and 4
   31         2        2      1       1  2/2 strains   2 Found in French
                                         different by  Guiana and in
                                         meth.         Guadeloupe, no
                                                       epidemiologic link
   33         2      ND      ND       2  2/2 strains   Found in French
                                         different by  Guiana, no
                                         meth. 4       epidemiologic link
   34         2        2      1       2  2/2 strains   Patients from French
                                         identical by  Guiana, suspected to
                                         meth. 4,      be epidemiologically
                                         method 2:     linked
                                         inconclusive,
                                         under
                                         investigation
   42         3        3      1       3  3/3 strains   Found in Guadeloupe
                                         different by  (one patient) and
                                         meth. 2 and 4 French Guiana (2
                                                       patients), no link
   44         2        2     ND       2  2/2 strains   2 patients from St.
                                         identical by  Maarten (couple)
                                         meth. 2 and 4
   45         6       1      ND       4  4/6 strains   5 patients from
                                         identical by  Martinique, one from
                                         meth. 4. (2:  Guadeloupe, under
                                         pending)      investigation
   46         3       1      ND       3  2/3 strains   Found in a
                                         identical by  Martinique and a
                                         meth. 4       Guadeloupe patient,
                                                       under investigation
   50(sup c) 29       16       8     20  Subclustered  Imported clusters
                                         by meth. 2, 4 (Hati), other links
                                         and PGRS-RFLP,under investigation
                                         IS-type E (3
                                         pat.) and F (2
                                         pat)
   51         4        3      2       3  2/4 strains   Imported cluster
                                         identical by  (Hati) for 2
                                         meth. 2 and 4 patients, other
                                         (2: pending)  links under
                                                       investigation
   53c      29       10       8     20   Subclustered  Patients from
                                         by meth. 2, 4 cluster T come from
                                         and PGRS,     the same ward of
                                         IS-type K (2  hospital A, 1996
                                         pat.) and T (3
                                         pat.)
   61         2        2      1       1  2/2 strains   Found in French
                                         identical by  Guiana in
                                         PGRS-RFLP, IS Guadeloupe, under
                                         results: underinvestigation
                                         investigation
   63        2         2      1       2  2/2 strains   2 patients from
                                         identical by  hospital A in
                                         meth. 2 and 4,Guadeloupe, under
                                         IS-type R     investigation
   64         1        1     ND       1  ***           2 isolates from one
                                                       single patient
   65         2      ND      ND     ND   (Results      Found in French
                                         pending)      Guiana, under
                                                       investigation
   66         2        2     ND       2  2/2 strains   Found in French
                                         identical by  Guiana, same surname
                                         meth. 2 and 4
   67         2        1     ND       2  2/2 strains   Found in French
                                         identical by  Guiana, under
                                         meth. 2 and   investigation
                                         PGRS-RFLP
   68         2        1     ND     ND   (Results      Found in one
                                         pending)      Martinique patient
                                                       and in Barbados,
                                                       under investigation
----------------------------------------------------------------------------
(sup a)Of 218 isolates typed, 145 isolates were grouped in 27 distinct
spoligo-defined clusters, which were further analyzed by one or more typing
methodsIS6110-RFLP (meth. 2), DR-RFLP (meth. 3), and DRE-PCR (meth.4), and
sometimes PGRS-RFLP when DRE-PCR or IS6110-RFLP results were inconclusive
or unavailable.
(sup b)Isolates with matching spoligotypes and matching IS6110 patterns (meth. 1
and 2) or with matching spoligotypes and matching DRE-PCR patterns (meth. 1
and 4) were considered to make up a cluster of epidemiologically associated
strains.
(sup c)Noninformative spoligotype patterns that lack any discriminating power.
*** Two clinical isolates from a single patient.
RFLP, restriction fragment length polymorphism; DR, direct repeat; DRE,
double-repetitive element; PCR, polymerase chain reaction; PGRS,
polymorphic GC-rich probe; ND, not done.

Fig. 1. Distribution of
tuberculosis cases reported to
health authorities in Guadeloupe,
Martinique, and French Guiana,		[fig]
1994-96, by nationality. DomR =
Dominican Republic; Dom =
Commonwealth of Dominica.

TB Epidemiology

Epidemiologic data for the three territories have been described in
detail (Table 2) (10). The mean incidence of cases per 100,000
inhabitants for the period studied was 11 in Guadeloupe, 8.2 in Martinique,
and 40.6 in French Guiana. The sex ratio varied from 1.5 in French Guiana
to 1.9 in Martinique. In each territory, the highest proportion of 
TB cases was in persons more than 65 years old; most were reactivated 
infections. The highest number of cases was in adults 25 to 44 
years old; most were new cases due to active disease
transmission and the high rate of TB-HIV coinfections. Pleuropulmonary
disease was most common (80% to 85% of cases), and drug resistance was
rare; primary resistance to isoniazid was 2.2% to 4.4%; single drug
resistance to rifampin was not observed in Martinique and French Guiana and
was limited to 1.4% of cases in Guadeloupe. Four cases of
multidrug-resistant TB (MDR-TB, defined as resistance to at least isoniazid
and rifampin) were diagnosed; these cases of secondary drug-resistance were
caused by noncompliance to standard antituberculosis chemotherapy. In
addition, the rate of TB-HIV coinfection in these three territories was
high (19% of total TB cases in Martinique, 25% in French Guiana, and 28% in
Guadeloupe). Coinfected patients were most likely men 25 to 44 years of age
and of foreign origin (1 of 2 TB-HIV coinfected patients was a foreign
national in Martinique, compared with 6 of 10 in Guadeloupe and 8 of 10 in
French Guiana). These figures agree with the epidemiologic data concerning
the distribution of patients on the basis of their nationality (Figure 1).

						[fig]
In the        Fig. 2. Nomenclature of the spoligotype database (from 1 to
last 20       69) based on published spoligotypes (n = 393) and on
years, TB     spoligotypes generated during this investigation (n = 218).
incidence     The corresponding hybridization patterns for oligonucleotides
has           1 to 43 (black square, hybridizing; empty square,
decreased     nonhybridizing) are shown. Type 1 is unique for the Beijing
considerably  type pattern, whereas type 69 is unique for the Manila type
in these      pattern. Bold characters illustrate patterns that have so far
territories.  been noticed only in Caribbean and neighboring Central
For           American isolates (specific types). Unbolded characters
example,      illustrate patterns common to those reported elsewhere
the           (ubiquitous types), whereas italicized characters with an
incidence     asterisk illustrates patterns not yet found in the Caribbean.
of new        BCG and H37Rv spoligotypes are shown as controls.
cases in
Guadeloupe,   
36 per
100,000
inhabitants in 1975, declined to 10 per 100,000 inhabitants in the late
1980s. If the same trend continued, TB would vanish by 2000; however, this
decline has slowed and incidence has remained at 10 to 12 per 100,000 since
1989. This reversal of decline is linked to poor economic conditions,
increase in unemployment, increase in drug and alcohol abuse, and
persistent immigration from countries with a high incidence of TB and
ongoing HIV epidemics.

The Spoligotype Database

To define predominant genotypes and trace the origin of strains and their
potential movement, we compared spoligotypes of Caribbean isolates with
those of other geographic regions. We built a database of 610 spoligotypes
(218 from our own investigation and 392 from other countries) with Excel
software. The database contains 167 patterns from Goyal et al. (17); 118
from Kamerbeek et al. (11); 106 from Goguet et al. (18); and a single
pattern, named the Manila family, from Douglas et al. (19). In this
database, 69 spoligotypes shared by more than two patients in any region of
the world were numbered types 1 to 69 (Figure 2): types 1-54 denote 395
spoligotypes that were initially ordered from empty to full squares read
from left to right, and types 55 to 69 represent 15 new shared types in
chronologic order and correspond to 215 spoligotypes investigated later.
The nomenclature is temporary until international guidelines are
established. Fourteen shared types might be specific for the Caribbean and
bordering Central American regions (patterns 5, 12, 13, 14, 15, 17, 29, 30,
54, 63, 64, 66, 67, 68 [bolded in Figure 2]); 23 shared types were found
both in the Caribbean region and in other parts of the world (in regular
font in Figure 2). The remaining 32 patterns were not present in the
Caribbean (highlighted by an asterisk in Figure 2).

                             Fig. 3. A dendrogram represents
                             spoligotyping results of 95 Mycobacterium
                             tuberculosis isolates from Guadeloupe (shared
                             patterns are shown by bold characters). From
                             top to bottom; type 53, ubiquitous; type 44,
                             described by Goguet et al. (two cases) (18);
                             type 54, which does not appear, is found only
                             in isolate 94142, a pattern also found in
                             French Guiana; type 37, which also does not
                             appear, described by Kamerbeek et al. (three
                             cases) (11); type 50, ubiquitous; type 63,
                             specific; type 61, which does not appear is
                             found only in isolate 96131, a pattern also
                             found in Barbados and French Guiana; type 51,
  	[fig]                  described by Goguet et al. (one case) (18);
                             isolate 96095, a M. bovis BCG clinical
                             isolate; types 12 to 14, specific types; type
                             5, which does not appear, is found only in
                             isolate 95068, a pattern also found in
                             Martinique; type 31, which does not appear, is
                             found only in isolate 94112, a pattern also
                             found in French Guiana and already reported by
                             Goguet et al. (two cases) (18); type 46, which
                             does not appear, is found only in isolate
                             95087, a pattern also found in Martinique and
                             was already reported (18); type 17, specific;
                             type 30, specific; types 45 and 15, which do
                             not appear, are found only for isolates 96129
                             and 94127, respectively, and share patterns
                             with isolates in Martinique and Surinam; type
                             29, specific; type 2, previously reported by
                             Goguet et al. (one case) (18); type 3, also
                             found in Surinam, has been described by
                             Kamerbeek et al. (one case) (11); type 1,
                             which does not appear, is found only in 95076,
                             a pattern also found in Surinam, and described
                             (20) as the Beijing type.

Fig. 4. A dendrogram illustrating spoligotyping results 
of 76 Mycobacterium tuberculosis isolates from French 
Guiana (shared patterns are shown in bold). Top to bottom; 
patterns 50 and 53, ubiquitous; types 54 and 36, which do not 
appear, are found only for isolates IPC99 and IPC57, 
respectively; type 34, a ubiquitous type that was limited 		[fig]
to French Guiana in this study; types 66 and 67, specific; 
type 42, ubiquitous; type 17, specific; type 33,
ubiquitous; type 51, ubiquitous; type 31, which does
not appear, is found only for isolate IPC23 and is shared
with 94112 in Guadeloupe; type 2, shared by four isolates in
Guadeloupe.

                     
Population Structure of M. tuberculosis in Guadeloupe

Of 95 isolates from Guadeloupe (Figure 3), 34 were a unique spoligotype, and
61 shared 13 patterns (six patterns were shared by only two isolates, and
seven patterns were shared by the remaining 49 [3 to 13 isolates per pattern]).
Patterns 2 (4 isolates), 14 (13 isolates), 29 (5 isolates), 50 (10 isolates), 
and 53 (12 isolates) were the major shared patterns from Guadeloupe and 
made up 72% of clustered isolates. The interpretation of the population 
structure from Guadeloupe shows the presence of important nodes on the 
dendrogram. As illustrated in Figure 3 (from bottom to top), three 
distinct patterns show strain 95076 (designated type 1 in our database and 
identical to the Beijing type commonly found in Asia [20]), the recently 
reported pattern 3 (11), and pattern 2 (18), respectively. Above the 
patterns 1-3 (pattern 1 is not shown) lies the previously undescribed 
pattern 29, which might be specific to our region. Two very different 
groups can be seen on the next node (the "lower" and "upper" groups). 
The "lower" group, which comprises 20 isolates and two shared 
patterns (17 and 30), shows a stepwise polymorphism. This
group contains many ubiquitous patterns (reported from at least three
geographic regions) shared by a variety of isolates from around the world
and concerns isolates 95016, 94105, and 94041 of shared types 20, 42, and
47, respectively. The "upper" group, which comprises the remaining 63
isolates and eight shared spoligotypes (types 12, 13, 14, 51, 63, 50, 44,
53), can be further divided into two subgroups: "upper ubiquitous," which
lies above M. bovis BCG strain 96095 and the "upper specific," which lies
below the BCG strain (Figure 3). The "upper specific" subgroup is
homogeneous (shared types 12, 13, 14, and strain 95068 of shared type 5)
and probably has been present in Guadeloupe for a long time (except in a
single isolate with pattern 5 from the neighboring island of Martinique).
The "upper ubiquitous" subgroup seems considerably closer to M. bovis BCG
than do other isolates of M. tuberculosis from Guadeloupe.

Population Structure of M. tuberculosis in French Guiana

The first population structure of M.    			[fig]
tuberculosis isolates from French       
Guiana is shown in Figure 4. Of 76
isolates, 30 had a unique spoligotype.  Fig. 5. A dendrogram illustrating
The remaining 46 shared a total of 11   31 spoligotyping results of
patterns: eight patterns (51, 33, 17,   Mycobacterium tuberculosis isolates
42, 67, 66, 65, 34) were shared by      from Martinique. From top to
only two isolates and three major       bottom, types 50 and 53,
patterns (pattern 2, 4 isolates;        ubiquitous; types 52 and 49 are
pattern 53, 9 isolates; pattern 50, 17  represented by a single isolate,
isolates) were shared by the remaining  respectively, M35 and M30, and are
30 isolates. The three major patterns   ubiquitous; isolates M10 and M7
represented 65% of clustered isolates   belong to specific types 17 and 68,
in French Guiana. Most clusters were    respectively found in Guadeloupe
common to those found in Guadeloupe,    and Barbados; M25, belongs to
as well as other regions of the world   specific type 5 observed in
(types 2, 17, 33, 34, 42, 50, 51, 53,   Guadeloupe; type 45, ubiquitous;
65), except two patterns that have      type 46, ubiquitous; isolate M23
been so far only reported from French   shares type 2, with isolates in
Guiana (types 66, 67). No isolate was   Guadeloupe and French Guiana.
of the Beijing type, which is
unexpected, considering the number of
persons of Chinese origin in French Guiana. A high degree of heterogeneity
was observed in French Guiana, which is not surprising given the large
surface area, high number of immigrants and persons of various ethnic
origins, and high TB incidence rate.

Population Structure of M. tuberculosis in Martinique

Of 31 isolates studied in Martinique (Figure 5), 19 had an unshared
spoligotype, and 12 shared 4 patterns (pattern 46, 2 isolates; pattern 45,
5 isolates; pattern 53, 2 isolates; pattern 50, 3 isolates). All the
clusters in Martinique were also found in Guadeloupe (45, 46, 50, and 53).
Pattern 45 is overrepresented in this population, which could suggest
active transmission of this clone of tubercle bacilli in Martinique. Other
patterns common in Guadeloupe are only poorly represented in Martinique:
type 2 (isolate M23), type 5 (isolate M25), and type 17 (isolate M10). Type
5 is specific only to Martinique and Guadeloupe. On the contrary,
Martinique shares some patterns with the rest of the world that are not
found in Guadeloupe or French Guiana (type 49, isolate M30; type 52,
isolate M35). Despite the small sample size of isolates from Martinique,
many isolates (clustered or unclustered) share published patterns from the
rest of the world (18 of 31 isolates studied).

Population Structure of M. tuberculosis in Other Caribbean Regions

      				   We also investigated 16 additional M.
         [fig]                   tuberculosis isolates (12 from Surinam and
                                 4 from Barbados). Although not
Fig. 6. A dendrogram             representative of the population studied,
illustrating spoligotyping       spoligotyping of these isolates allowed
results of 16 Mycobacterium      detection of some interesting links
tuberculosis clinical            (Figure 6). Fourteen unique types and one
isolates from Barbados and       shared type (pattern 53, two isolates)
Surinam. From top: type 53,      were found. Among the unclustered
ubiquitous; isolates Barb.3      isolates, strain 94030 from Surinam
and 94030 belong to specific     matched isolate 94127 from Guadeloupe
types 68 and 15,                 (type 15), strain 94018 from Surinam
respectively; isolates 94018,    matched type 19 from Holland (11), and
94020, 94034, and Barb.1         strain 94034 from Surinam matched isolate
belong to ubiquitous types       95047 (type 3) from Guadeloupe.
19, 1, 3, and 61,                Furthermore, two strains from Barbados
respectively.                    shared patterns with isolates from other
                                 geographic areas: Barb 3 to M7 (type 68)
from Martinique and strain Barb 1 to the pattern 61 from France (18).

Predominant Genotypes and Strain Origin and Transmission

The dendrogram representing the global structure of the population studied
(Figure 7) shows the potential historical or epidemiologic interregional
flux of M. tuberculosis between Caribbean and neighboring Central American
regions. Of the 218 isolates, 145 (66.5%) had 29 shared types. Twenty-five
isolates were not initially grouped but were clustered only when a
dendrogram of all 218 isolates was drawn. These interregional links defined
eight new types in the database (patterns 1, 5, 15, 31, 54, 61, 64, and 68)
for 15 isolates; the remaining 10 isolates enriched patterns already
present (Figure 7). These links, made by finding clonal strains in distant
geographic territories, do not necessarily define epidemiologic
relationships between these strains (21) but are unlikely to be due to
independent genetic evolution. The dendrogram is an indirect picture of the
common history of TB spread in this part of the world.

For example, spoligotype 2 was recently proposed to have originated in
Latin America (22). Our recent investigations favor this hypothesis as we
have traced this pattern in all three territories investigated (Martinique
[one isolate]), Guadeloupe [four isolates], and French Guiana [four
isolates]). Isolates from all nine patients were further investigated by
IS6110-RFLP, DRE-PCR, and SmaI-PGRS typing (23), and the results confirmed
the clonality of this specific cluster. Searching for this genotype in
other Latin American countries may help elucidate its origin and
distribution.

To define the clonality of the isolates and to    
be in accordance with the current practice for           [fig]
molecular typing of M. tuberculosis using         
spoligotyping as a first-line method (7, 11, 17,
18), we systematically performed a study of a     Fig. 7. A cumulative
spoligotyping-defined cluster by a second method  dendrogram for the 218
(IS6110-RFLP or DRE-PCR). When corroborated by    Caribbean isolates of
epidemiologic information, the clusters so        Mycobacterium
defined were considered evidence of ongoing       tuberculosis. New types
active transmission of TB (Table 3). However, in  not visible on individual
numerous cases, the epidemiologic data were not   dendrograms can now be
conclusive, and molecular clonality was not       observed (types 1, 5, 15,
considered direct proof of a link. Thus, despite  31, 54, 61, 64, and 68).
suspected links in 81 (37%) of 218 of cases on
the basis of molecular typing alone, a direct
epidemiologic link was demonstrated in 31 (14.2%) of 218 of cases. However,
future prospective studies including active case-finding and source tracing
may help increase the number of epidemiologically linked cases in our
region.

Distribution of Spoligotypes Bearing Geographic Specificities

To reconstruct the evolutionary tree of M. tuberculosis on the basis of 610
different spoligotypes (mostly from the United Kingdom, Holland, France,
the Caribbean, and the neighboring Central American region), we performed a
similarity search (Figure 2). Although the tree may not reflect the full
diversity of spoligotypes, it suggests the existence of distinct families
of spoligotypes with geographic specificities (Figure 8). Some strain
families may be related to specific populations, geographic regions, and
the history of TB spread. For example, the Beijing genotype, which is most
divergent in the tree shown in Figure 8 (type 1), would have undergone the
most extensive genetic evolution since the origin of M. tuberculosis. This
information is consistent with the mechanism of evolution of the DR locus,
which appears to proceed through losses of direct repeats (24,25).

Although in an unrooted tree, the positions of patterns 50 and 53 correlate
well with the isolates most widely represented internationally (116 of 610
isolates studied; 54 of 218 isolates from the Caribbean and neighboring
Central American region and 62 of 392 published spoligotypes from different
parts of the world) and may constitute a candidate root for the
interpretation of distances between isolates.

Genetic Evolution of Tubercle Bacilli

                                  The genetic evolution of tubercle
          [fig]                   bacilli is closely associated with the
                                  past and present of its host.
Fig. 8. A preliminary             Consequently, human migration,
phylogenetic tree obtained by     population mixing, and other
the neighbor-joining algorithm    sociodemographic factors have long
on the basis of the 1-Jaccard     played an important role in the spread
index (Sj=a/a+c, where a is the   and subsequent evolution of the M.
number of simultaneously          tuberculosis genome. The insular model
positive characters and c is the  of the Caribbean, in which human
number of discrepancies), which   migration (hence the introduction of the
is not exhaustive for all         disease) was estimated to occur only
existing phylogenetic links. A    approximately 400 years ago, is
total of 70 shared spoligotypes   particularly interesting for discovering
were analyzed (69 types shown in  conserved strains of tubercle bacilli
Figure 2 and Mycobacterium bovis  and for detecting rare M. tuberculosis
BCG).                             genotypes. In this context, the DR locus
                                  is a unique chromosomal region
                                  characteristic of the M. tuberculosis
complex, which shows a high degree of polymorphism that involves homologous
recombination and IS-mediated transposition (24). The high degree of
internal homology within the DR region of M. tuberculosis is likely to
favor such genetic rearrangements. Similarly, transposition of IS6110 is
instrumental in generating new subclones of M. tuberculosis (26).

Following the principle of Dollo parsimony, which assumes that losses of
genes are much more common and likely than independent evolutionary origins
(27), the evolutionary process of M. tuberculosis can be speculated to have
involved the loss of DR repeats. However, the tree in Figure 8 does not
perfectly represent all phylogenetic links between isolates as the
mechanisms of loss of DR elements (by homologous recombination or
replication slippage) could involve simultaneous loss of >1 of the 43
building blocks. The present phylogenetic analysis will be extended to
other multiple genetic markers to include variables recently proposed by
Sreevatsan et al. (4). However, construction of such trees on the basis of
the simultaneous feeding and computer analysis of multiple mycobacterial
markers remains cumbersome and constitutes a priority research topic for
the studies of M. tuberculosis genome evolution.

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

Acknowledgments

      The authors thank F. Pfaff, L. Schlegel, P. Levett, and P. Prabhakar
for sending the cultures for identification to the Institut Pasteur,
Guadeloupe, and F. Prudent, M. Berchel, and K.S. Goh for their expert
technical help.

      This project was financed by research grants provided by Dlgation
Gnrale au Rseau International des Instituts Pasteur et Instituts
Associs, Institut Pasteur, Paris, and Fondation Franaise Raoul Follereau,
Paris, France.

      Dr. Sola is Charg de Recherche at the Tuberculosis and Mycobacteria
Unit at the Pasteur Institute of Guadeloupe, which is headed by Dr.
Rastogi. The authors work closely with public health agencies in
Guadeloupe, Martinique, and French Guiana to support the tuberculosis
control program. They provide expertise and training on the genetic
characterization and laboratory diagnosis of mycobacteria. Their current
research interests include molecular diagnostics and epidemiology, drug
resistance, and mechanisms of mycobacterial pathogenicity.

      Address for correspondence: Nalin Rastogi, Unit de la Tuberculose &
des Mycobactries, Institut Pasteur, Morne Jolivire, B.P. 484, F-97165
Pointe  Pitre-Cedex, Guadeloupe; fax: 590-893-880; e-mail:
rastogi@ipagua.gp.

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     JDA. Insertion element IS987 from Mycobacterium bovis BCG is located
     in a hot-spot integration region for insertion elements in
     Mycobacterium tuberculosis complex strains. Infect Immun
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Research

Human Rabies Postexposure Prophylaxis during a Raccoon Rabies Epizootic in
New York, 1993 and 1994

Jeffrey D. Wyatt,* William H. Barker,* Nancy M. Bennett, and Cathleen A.
Hanlon
*University of Rochester School of Medicine & Dentistry, Rochester, New
York, USA; Monroe County Department of Health, Rochester, New York, USA;
Centers for Disease Control & Prevention, Atlanta, Georgia, USA

---------------------------------------------------------------------------
      We describe the epidemiology of human rabies postexposure
      prophylaxis (PEP) in four upstate New York counties during the
      1st and 2nd year of a raccoon rabies epizootic. We obtained
      data from records of 1,173 persons whose rabies PEP was
      reported to local health departments in 1993 and 1994. Mean
      annual PEP incidence rates were highest in rural counties, in
      summer, and in patients 10 to 14 and 35 to 44 years of age. PEP
      given after bites was primarily associated with unvaccinated
      dogs and cats, but most (70%) was not attributable to bites.
      Although pet vaccination and stray animal control, which target
      direct exposure, remain the cornerstones of human rabies
      prevention, the risk for rabies by the nonbite route (e.g.,
      raccoon saliva on pet dogs' and cats' fur) should also be
      considered.

Raccoon rabies, present in the southeastern United States since the 1950s,
became responsible for an epizootic in the U.S. mid-Atlantic region during
the 1970s after raccoons were translocated there for hunting (1). The
introduction of the variant of rabies virus associated with raccoons into a
rabies-naive raccoon population caused the most intensive animal rabies
outbreak on record, in part because of the abundance of raccoons in
suburban environments throughout the mid-Atlantic and northeastern
metropolitan areas. Raccoon rabies affects approximately one million square
kilometers of the eastern United States with a human population of
approximately 90 million.

Since the mid-Atlantic raccoon rabies epizootic entered New York State in
1990, the number of rabid animals increased from 54 in pre-epizootic 1989
to 2,746 (89% raccoons) in 1993the largest number of rabid animals ever
reported from any state (2). Despite traditional public health measures for
rabies control (e.g., pet vaccination, stray animal control, public
education), human rabies postexposure prophylaxis (PEP) rates inevitably
increased with the arrival of the epizootic front (3). Preliminary data
from New York documented a 4,000% increase in the absolute number of
persons receiving PEP, from 81 (1989) to 3,336 (1993) (4). The
epidemiologic trends of human PEP in New York State remain largely
undescribed.

One of the Healthy People 2000 objectives formulated by the U.S. Public
Health Service is to reduce by 50% the need for human rabies PEP by the
year 2000 (5). A reduction in the number of PEP cases, which are not
reportable, appears unattainable without first defining the numerator, as
well as the epidemiologic characteristics of precipitating events leading
to suspected rabies exposure and inappropriate treatments.

We describe demographic and animal exposure data associated with human
rabies PEP in an area with epizootic raccoon rabies. The epidemiologic
description is intended to assist medical practitioners and public health
officials in reducing the incidence of human and domestic animal exposure
to rabid animals and thus in minimizing the need for PEP in communities
affected by the raccoon rabies epizootic.

The Study

Setting

Four contiguous upstate New York counties (Monroe, Wayne, Cayuga, and
Onondaga) were first affected by the raccoon rabies epizootic between
December 1992 and June 1993 (Figure 1). Monroe and Onondaga Counties,
encompassing the cities of Rochester and Syracuse, are predominantly
urban-suburban, with human population densities of 414 per square kilometer
and 230 per square kilometer, respectively. Wayne and Cayuga Counties are
predominantly rural-suburban, with relatively lower population densities of
57 and 45 people per square kilometer, respectively. The four-county region
in western upstate New York comprises 7,090 square kilometers and has an
estimated human population of 1,354,377.

Data Characteristics and Sources                                          
                                                                          
We considered all human rabies PEP                                        
cases reported in 1993 and 1994 for 
study area. The PEP capture rate was                                      
believed high because local health                                        
units were responsible for providing
funds for any treatment expenses not                                      
covered by health insurance, and a                                        
completed, rabies report form was                                         
required before reimbursement of the                   [fig]                   
local health unit from state funds.                                       
                                                                          
The New York State Sanitary Code
requires physicians to report potential      Figure 1. New York State raccoon                        rabies human exposure to rabies and PEP      epizootic progression (1990-94).
administration to county health              The raccoon rabies epizootic first                     
departments. We abstracted data from         affected Monroe, Wayne, Cayuga, and                   
these standardized reports and patient	   Onondaga Counties between December
records. Data were grouped by patient        1992 and June 1993.                    
demographics, animal characteristics,                                     
and exposure details. Exposure source                                     
was defined as the suspected- or                                          
confirmed-rabid animal that directly or                                   
indirectly resulted in one or more                                        
potential human exposures to rabies.
Direct contact exposure consisted of                                      
direct contact (e.g., bite, scratch) or                                   
contamination of mucous membranes with                                    
potentially infectious material from a                                    
rabid animal. Indirect contact                                            
consisted of contamination from a
fomite (e.g., through racoon saliva on                                    
a pet's fur with a pet owner's open                                       
wounds or mucous membranes).                                              

Analyses                                                                  
                                                                          
Population figures from the 1990 New                                      
York State census were used to                                            
calculate the incidence of PEP by                                         
county, age, and gender (6).
Descriptive analyses of data elements
were made through queries of Microsoft Access relational database. Each PEP
contributed to the denominator of the analyses. Since multiple PEP cases
occurred from exposure to a single animal, data for individual animals were
also summarized. Chi-square tests were performed with Epi-Info Version 5
software.

Findings

PEP Incidence

The annual PEP incidence for the study area increased from <1 case per
100,000 residents in pre-epizootic 1992 to 35 cases in 1993 and 52 cases in
1994. Of 1,173 cases of human rabies PEP in the study areas, 474 were
reported in 1993 and 699 cases were reported in 1994. The mean annual
incidence of PEP was 32 cases per 100,000 for the urban counties (Monroe
and Onondaga; 315 residents per square kilometer) and 123 cases per 100,000
for the two rural counties (Wayne and Cayuga; 51 residents per square
kilometer).

Season

The number of PEP cases peaked in summer to early autumn (Figure 2). During
1993, the highest number of PEP cases occurred approximately 4 to 6 months
(August through November) after the invasion of raccoon rabies during March
through June 1993; in 1994, the highest number occurred in summer (June
through August).

Gender and Age

Gender and age data were available for 100% and 95% of all patients,
respectively. Of 1,173 PEP cases, 642 (55%) were administered to male and
531 (45%) to female patients. The mean annual incidence of PEP in male and
female patients was 47 and 38 per 100,000, respectively. The PEP rates were
highest in persons 10 to 14 years of age (165 per 100,000) and 35 to 44
years of age (113 per 100,000) (Figure 3). The median age was 29 and 31
years for male and female patients, respectively. No significant
relationship was observed between gender and age groups for the study area.

       [fig]                          [fig]

Figure 2. Human rabies          Figure 3. Human rabies
postexposure                    postexposure
prophylaxis in four New         prophylaxis in four New
York State                      York State
counties (Cayuga, Monroe,       counties (Cayuga, Monroe,
Onondaga, and Wayne),           Onondaga, and Wayne),
1993-1994,                      1993-94:
by month.                       incidence by gender and
                                age.

Exposure Source Species

Exposure to wild animals accounted for 783 (67%) of all PEP cases (Table
1). Among wildlife, raccoons were by far the leading source of exposure,
accounting for 589 (75%) of 779 PEP cases due to wildlife exposure. The
other sources of wildlife exposure were bats (54 cases), skunks (35 cases),
foxes (28 cases), white-tailed deer (13 cases), woodchucks (12 cases),
small rodents (9 cases), sika deer (4 cases), and other wild species (39
cases). Of 390 domestic animal exposures resulting in PEP, 205 were
attributed to cats, 165 to dogs, 12 to cattle, 5 to pet rabbits, and 3 to
horses. Among PEP cases resulting from exposure to cats and dogs, 66% and
67%, respectively, were initiated after contact with stray animals
unavailable for the recommended 10-day confinement and observation to rule
out rabies or euthanasia and testing. Dog exposures were disproportionately
higher in urban (137 [18%] of 753) than in rural counties (28 [7%] of 420)
(p <0.001) (Table 2). In urban areas, dog exposure was primarily due to
stray or unowned dogs (95 [69%] of 137). In rural areas, stray or unowned
dogs accounted for 11 (39%) of 28 dog exposures (p <0.01). Only one dog (in
a rural county) tested positive for rabies in the study area in 1993 and
1994. Of 68 pet cats resulting in human exposure, 61 (90%) were not
vaccinated against rabies compared with 14 (24%) of 58 pet dogs (p <0.001).

Table 1. Human rabies postexposure prophylaxis (PEP), New York State,
1993-94(sup a)
----------------------------------------------------------------------------------
                                    Nonbite (N=818)
                      -------------------------------------------
                                  Direct(sup b)
                     -----------------------------                 
              Bite                                     Indirect(sup c)   Overall 
Animal       (N=355) Scratch  Saliva   NT    Blood         Saliva       total PEP 
source        N  (%)   N ( %)  N  (%)  N (%)  N  (%)       N   (%)     N      (%)
-----------------------------------------------------------------------------------
Raccoon      37 (10)  18 (33)  44(29)  4 (67) 13 (93) 472(sup d)(79) 589(sup e)(50)
Bat (all     29  (8)   3  (6)  12 (8)  1 (17)  0  (0)       9   (2)    54       (5)
 species)            
Other wild 
 species     24  (7)   5 (11)  21(14)  0  (0)  1  (7)      89   (15)  140      (12)
All wild                      
 species     90 (25)  26 (47)  77(51)  5 (84) 14(100)    570   (96)   783      (67)

Cat         114 (32)  29 (53)  41(28)  0  (0)  0  (0)     21    (4)   205      (17)
Dog         151 (43)   0 (0)   13 (9)  0  (0)  0  (0)      1   (<1)   165      (14)
                        
Other
 domestic        
 species      0 (0)    0 (0)   19(13)  1 (17)  0  (0)      0    (0)    20       (2)                   
All
domestic     
species     265(75)   29(53)   73(49)  1 (17)  0  (0)     22    (4)   390      (33)
                        
Total        355 (30) 55(55)  150(13)  6(0.5) 14  (1)    592   (51) 1,173     (100)
                                
-----------------------------------------------------------------------------------
(sup a)Data are from Cayuga, Monroe, Onondaga, and Wayne Counties.
(sup b)Direct contamination of an open wound or mucous membrane with potentially
infectious material such as saliva, nervous tissue (NT), or blood (mixed
with other body fluids), from a rabies-suspect or known-rabid animal.
(sup c)No direct contact with a rabid or suspect-rabid animal. Indirect exposure
through possible conveyance of saliva on an animal (i.e., pet dog or cat)
or inanimate object resulting in contamination of an open wound or mucous
membrane.
(sup d)p < 0.001. More people received PEP after indirect exposure to saliva
from raccoons than from any other species (472 PEP cases due to indirect
contact with 261 raccoons).
(sup e)Total PEP cases with raccoon as an exposure source (includes one case
with no reported route of exposure).

Table 2. Human rabies postexposure prophylaxis                    Type of
(PEP) in New York State, 1993-94: urban and rural                 Exposure
settings(sup a)
                                                                  Of 1,173
----------------------------------------------------------------- PEP
                                                      All four    cases,
                           Urban         Rural        counties    355
                       ---------------------------------------    (30%)
Animal source              N  (%)         N  (%)      N   (%)     resulted
                                                                  from
--------------------------------------------------------------    animal
Dog(sup b)                137 (18)       28  (7)     165 (14)     bites
Cat                       130 (17)       75 (18)     205 (17)     and 817
                                                                  (70%)
Other domestic                                                    from
  species(sup c)            5 (<1)       15  (4)      20  (2)     nonbite
All domestic                                                      encounters
  species                 272 (35)      118 (28)     390 (33)     (Table
Raccoon                    41 (45)      248 (59)     589 (50)     1). A
                                                                  route of
Bat (all species)          41  (5)      13   (1)      54  (5)     exposure
Striped skunk              29  (4)       6  (<1)      35  (3)     not
Fox 19 (3)                  9  (2)      28   (2)                  reported
Other wild                                                        in one
  species(sup d)           51  (4)      26   (2)      77  (7)     case
                                                                  involved
All wild species          481 (65)     302  (72)     783  (67)    a
Total                     753          420         1,173          raccoon.
Rate per                                                          Suspected
  100,000 pop.             32          123            43          contact
                                                                  with
--------------------------------------------------------------    animal
(sup a)haracteristics of human rabies PEP cases reported to the   saliva
health departments of the two relatively urbanized counties,      (148
Onondaga and Monroe, and the two relatively rural counties        cases
Cayuga and Wayne, during 1993 and 1994.                           from
(sup b)p < 0.00. Human PEP rates due to dog exposures were        direct
significantly higher in urban counties.                           contact
(sup c)Other domestic species include 2 and 3 PEP cases due and   594
to cow                                                            from           
and horse exposure in the urban counties and 10 and 5 cases due   indirect
to cow and domestic rabbit exposure in the rural counties,        contact)
respectively.                                                     was
(sup d)Other wild species includes 17, 6, 4, 2, 2, and 1 PEP      responsible
cases due  to an unknown animal type, wild rodent (other than     for 742
woodchuck), 4 Sika deer (exotic, captive species), opossum,      (91%) of
coyote, and mink in the urban counties and 17 and 3 PEP cases     due to
due to an unknown    animal type and wild rodent (other than      nonbite
woodchuck) in the rural  counties, respectively.                  817 PEP                                                                  
											cases
exposure; contact with nervous tissue (6 PEP cases) or blood (14 PEP cases)
accounted for 2% of cases due to nonbite exposure. Fifty-five (7%) of 817
nonbite exposures were attributable to scratches from 23 cats (responsible
for 29 PEP cases), 16 raccoons (18 cases), 3 bats (3 cases), 2 wild rodents
(2 cases), and 3 other wild animals (3 cases). Of 355 bite exposures, 265
(75%) involved domestic animals (151 due to 150 dogs, 114 due to 108 cats);
90 (25%) involved bites from wild animals34 raccoons (responsible for 37
PEP cases), 27 bats (29 cases), 9 rodents (9 cases), and 13 other wild
species (15 cases).

Mode of Contact

Of 1,173 cases, 594 (51%) occurred because of possible indirect contact
with a suspect rabid animal; 583 (98%) of 594 occurred after suspected
exposure to saliva from a rabid (or suspect rabid) animal on the fur of a
nonsuspect dog, cat, or other animal. In 9 (2%) of these cases, PEP was
administered after suspected exposure by possibly contaminated fomites
including door knobs, traps, arrows, a flashlight, and a wire. Possible
indirect exposure to dogs with potentially infectious material on their fur
resulted in 507 (85%) PEP cases, while suspected indirect exposure by cats
resulted in 70 (12%) cases. Other suspected exposure sources were a horse,
rabbit, pet duck, chicken, wild bird, captive exotic sika deer, and a
person.

Group Exposure

Exposure of one person to a suspect rabid animal precipitated 625 (53%) PEP
cases; the remaining 548 (47%) occurred after more than one person was
exposed to the same suspected animal (Table 3). Exposure of a single person
was more likely associated with a bite (p <0.001), while group exposure
(involving two or more persons) was more likely associated with nonbites (p
<0.001). Wild animal species accounted for most group exposureswith three
exceptions. The largest group exposures (involving 12, 13, and 14 people)
were associated with the handling of rabid domestic animals (before
diagnosis) by veterinary clinic employees.

Rabies Status

The laboratory diagnosis of rabies in the exposing animal was associated
with 540 (46%) of all PEP cases (445 due to wildlife and 95 due to domestic
animals). Eighty-nine percent of PEP cases attributed to rabid wildlife
involved raccoons. In 88 cases, PEP was initiated after contact with
animals eventually proven nonrabid. In 544 cases PEP was administered after
contact with animals not tested for rabies. Confirmation of rabies in
suspect domestic animals occurred in association with 91 (23%) of 390 PEP
cases resulting from exposure to domestic species, including 40 due to 5
pet cats, 23 to 5 stray cats, 13 to 1 pet dog, 5 to a domestic rabbit, 7 to
a cow, and 3 to a horse. Conversely, in 88 (8%) of all cases PEP was given
after encounters with 81 animals subsequently proven nonrabid (35 due to 33
cats, 32 to 32 dogs, 9 to 8 raccoons, 3 to 3 bats, 5 to 2 skunks, 1 to 1
woodchuck, 1 to 1 squirrel, and 2 to 1 muskrat).

Table 3. Human rabies postexposure prophylaxis (PEP) in New York State,
1993-94: Epidemiologic characteristics(sup a)
---------------------------------------------------------------------------
                                           Group size
                      ------------------------------------------------------
                         1        2       3        4       5         6+
                      ------------------------------------------------------
Characteristic          N    %   N   %   N    %   N   %   N    %   N    %
---------------------------------------------------------------------------
Number                 625  53  180  15  84   7  112  10  60   5  112  10
                                                               
No. of sources         625  79   90  11  28   4   28   4  12   2   13   2
                                                               
Route of exposure
    Bite(sup b)        328  52   17   9   4   5    3   3   0   0    3   3
                                                               
    Nonbite            296  47  163  91  80  95  109  97  60 100  109  97
    Unknown              1   0.2  0   0   0   0    0   0   0   0    0   0
                                                               
Source of exposure
    Dog or cat         273  44    16  9   3    4   4   4  10  17   64  57
    Other domestic
     species             2   0.3   4  2   3    4   4   4   5   8    6   5
    Raccoon            235  38   124 69  57   68  96  86  35  58   42  38
    Bat                 39   6    12  7   3    4   0   0   0   0    0   0
                                                               
   Other wild
    species             76  12    24 13  18    21  8   7 10   17    0   0
Mean age (yr)           33.4      32.5   24.8     22.5   21.8      26.0
---------------------------------------------------------------------------
(sup a)PEP data are from Cayuga, Monroe, Onondaga, and Wayne Counties.
(sup b)Probability of bite exposure for PEP involving single person vs. 
group of >1 PEP cases, p <0.001.

Of 540 cases of PEP associated with animals proven to be rabid, 505 (94%)
were due to suspected saliva exposure; 22 (4%) and 13 (2%) involved bites
or scratches, respectively. Conversely, 71 (81%) of 88 PEP cases associated
with nonrabid animals (i.e., laboratory-confirmed as negative or confined
and observed to be healthy) occurred after bite exposures. Of the 544 PEP
cases associated with animals of unknown rabies status, 48% were due to
bites, 45% to suspected saliva contacts, and 7% to scratches.

Wild animals accounted for 98% of the 690 animals submitted and testing
positive for rabies in the study area for 1993-94; 613 were raccoons. If
animals testing positive for rabies are used as a surrogate for the true
incidence, an approximately 20-fold increase in PEP cases per rabid
domestic animal compared with each rabid wild animal, regardless of rural
or urban region, is seen (data not shown).

Provoked Exposures

A provoked exposure was characterized by intentional, human-initiated
interaction with a suspect rabid animal. Cases resulting from provoked
exposure accounted for 392 (33%) of 1,173 of all PEP cases; 248 (63%)
involved domestic animals. Most cases resulting from provoked exposure of
domestic animals involved cats (162 [65%] of 248) and less frequently, dogs
(62 [25%] of 248). Wild animals accounted for 144 (37%) PEP cases from
provoked exposure.

Time of PEP Initiation

The interval between exposure to suspect rabid animals and initiation of
PEP was 0 to 43 days (median 2 days). Bite exposures were associated with
no delay in treatment; nonbite exposures were associated with a 3- to 4-day
interval (p <0.001).

PEP Regimen

In 1993 and 1994, postexposure biologic products licensed for use in the
United States were rabies vaccine adsorbed, human diploid cell vaccine
(Imovax), and human rabies immune globulin (HRIG; Hyperab or Imogam). As
recommended by the Advisory Committee on Immunization Practices (ACIP), PEP
for the rabies-naive person consists of HRIG (20 IU/kg) on day 0 and five
doses of rabies vaccine administered on days 0, 3, 7, 14, and 28 (7).
Scheduling information was unavailable for our cases.

Administration of PEP biologic products was recorded as complete in 1,016
(87%) of 1,173 PEP cases. Information regarding completion of the treatment
series was not available in 15 cases (1%). Appropriate PEP for preimmunized
persons consists of two vaccine doses on days 0 and 3 (7) and was
administered to 26 persons, accounting for 2% of all cases. Among
preimmunized persons, 17 (65%) of 26 PEP cases occurred after occupational
exposures by 11 veterinary staff personnel (including two group exposures
to proven rabid cats), four wildlife rehabilitators, one health department
employee, and one police officer.

In 54 (5%) instances, PEP was discontinued because of lack of clinical
signs in 29 dogs (29 PEP cases) and 23 cats (25 PEP cases) confined for the
recommended 10-day observation period. Moreover, 34 (3%) PEP cases were
discontinued because of rabies-negative laboratory results in 10 cats (10
PEP cases), 7 raccoons (8 cases), 2 skunks (5 cases), 4 dogs (4 cases), 3
bats (3 cases), 1 muskrat (2 cases), 1 woodchuck (1 case), and 1 squirrel
(1 case).

After PEP was initiated, 29 (2%) of 1,173 refused to complete the series;
two cited adverse reactions. In nine cases PEP deviated from ACIP
recommendations: apparently inadvertent scheduling and administration of
six total vaccine doses in four patients and intentional omission of HRIG
in the treatment regimen of five patients.

Adverse Effects

The categories available for characterizing adverse effects on the state
rabies report form were none, slight, moderate, severe, or unknown. In 596
(51%) of 1,173 PEP cases, no information was recorded. Of 577 responses,
495 (86%) reported no adverse effects resulting from PEP. Adverse effects
were characterized as slight by 67 (12%) persons. Moderate adverse
reactions including vomiting, nausea, fever, aches, and weakness were
reported by 13 (2%) persons. Serious systemic adverse reactions, recorded
as anaphylactic shock and serum sickness, occurred in two (0.2%) persons.
Both of these patients had received HRIG; PEP was discontinued after one
and two vaccine doses in each case.

Conclusions

The most important finding of this study was that in most cases PEP was
administered because of suspected nonbite, indirect exposure to animal
saliva, a route conventionally thought of nearly negligible risk in rabies
transmission (7,8). Because of effective PEP, public health personnel and
health-care workers are primarily challenged with the assessment of
exposure to rabies, rather than with treatment of human cases of the
disease. Assessment of nonbite saliva exposures are particularly
time-consuming and should consist of a thorough, but nonleading,
history-taking that elicits the probability or confirmation of mucous
membrane or nonintact skin contact and a realistic assessment of the
potential presence of infectious saliva on surfaces or pets. Given the
invariably fatal outcome of clinical rabies, the tendency may be to
administer PEP, even without clear indication of exposure. This tendency
may be unwisenot only for economic reasons, but also because, despite
their relative innocuity and high potency, modern rabies biologic products,
are not risk-free, nor is their supply unlimited.

The first descriptive study of PEP cases associated with the mid-Atlantic
raccoon rabies epizootic during 1982-83 (133 patients) also documented that
most PEP cases were due to nonbite exposures; however, these principally
involved direct exposure to the suspect rabid animal (1).

A 1980-81 nationwide survey of 5,634 PEP cases found an increased risk for
occupational and recreational exposure to animals in a rural setting (9).
The absolute mean annual PEP rate described in our report of 43 per 100,000
was nearly 10-fold higher than the rate of 4.7 per 100,000 reported in that
study. A rate of 66 per 100,000 was reported from two counties (93 people
per square kilometer) in New Jersey at the raccoon rabies epizootic front
in 1990 (10,11). The incidence of human rabies PEP in New Jersey and this
study exceeded by 10- to 20-fold the rates in areas reporting rabies in
skunks (12,13), raccoons (14), and mixed wildlife (9,15). The disparity may
be partially explained by regional epizootic versus enzootic status of
wildlife rabies and subsequent variations in the comparative intensity of
disease in wildlife populations, as well as recent increases in both human
and animal population densities and their close association in suburban
settings (2). The previous PEP studies involved communities in which rabies
had been enzootic in terrestrial wildlife for decades (9,12-16). However,
the mid-Atlantic raccoon rabies epizootic comprises the emergence of a
terrestrial rabies variant into areas that had, for the most part, been
free of terrestrial rabies. The exceptions were sporadic cases of spillover
from geographically widespread, but low-level, bat rabies into terrestrial
animals and occasional incursions of red fox rabies from Canada into New
York, Vermont, and other northern states (2,16).

Previous studies of PEP trends in the United States identified bites from
dogs and cats as the most common animal encounter, accounting for 65% to
84% of PEP cases (7,9,12-15). By contrast, only 23% of PEP cases in this
study were associated with dog or cat bites. In view of current
epidemiologic trends in canine rabies-free areas of the United States, if a
biting dog appears clinically normal and can be confined and observed for
signs of rabies, the decision to administer PEP may be based on suggestive
clinical signs and a prompt diagnostic evaluation that confirms rabies
rather than on presumptively initiating PEP. Given that cats are now the
leading rabid domestic animal in the United States (17), and more
specifically that 12 of the 13 domestic species confirmed rabid from the
four-county study area during 1993 and 1994 were cats, rabies vaccinations
for cats should become more prevalent. Among exposures to owned domestic
pets that resulted in human PEP, 9% of cats (versus 76% of dogs) were
vaccinated against rabies. Moreover, most of the encounters with dogs that
precipitated PEP in urban counties involved bites from stray dogs,
indicating the need for enhanced programs for urban dog control.

The economic impact of a new terrestrial rabies variant is substantial
(2,18). In 1994, the New York State Department of Health increased its
reimbursement to local health units for mandated rabies control activities
from $75,000 to $1,080,000 to assist in the expense associated with human
rabies PEP, animal rabies testing (11,896 specimens in 1993), and pet
immunization clinics (114 in 1993) (4). Local health units in New York
State provide funds for treatment expenses not covered by health insurance.
With the cost of rabies biologic products alone exceeding $1,500 per
treatment series, an exponential increase in the incidence of PEP, as
documented in this study, taxes the public health infrastructure. Moreover,
unlike red fox rabies, which periodically reinvades northeastern New York
from adjoining areas of Canada and Vermont but then dies out, raccoon
rabies is expected to persist in affected areas of New York State, as it
has in the southeastern United States for the past 5 decades and in the
mid-Atlantic and northeastern states more recently (17).

Control of canine rabies in the United States and other industrialized
countries was achieved by eliminating the susceptible reservoir population
(through stray dog control and mandatory vaccination) (16). Applications of
this concept to wildlife is problematic because of the difficulty in
capturing wild animals for vaccination or for applying lethal measures.
Population reduction alone is not sufficient to control or eliminate
terrestrial wildlife rabies variants over large geographic areas (16). An
emerging alternative is oral rabies vaccination of free-ranging reservoir
populations, although current methods are still in their infancy and the
cost-benefit of such interventions warrants further investigation (10).

During the enzootic raccoon rabies in the southeastern states since the
early 1950s or the current mid-Atlantic/northeastern United States
epizootic, this variant has not been known to cause human rabies deaths.
Yet its potential lethality for humans is supported by ample spillover into
other wild animal species (predominantly skunks, but also red foxes,
bobcats, and woodchucks) and into domestic animals (predominantly cats, but
also dogs, cows, horses, goats, and rabbits). Substantial amounts of
infectious rabies virus have been identified in the salivary glands of
rabid raccoons (19). No biologic or epidemiologic data suggest unique
attenuation or change in virulence of this particular rabies variant that
would account for a lack of identified human deaths. Instead, epidemiologic
data regarding PEP after suspected exposure to raccoon rabies indicate that
PEP frequently is administered even when no exposure has been identified.
Also, a bite, scratch, or other exposure, such as gross contamination of an
open wound or mucous membrane with moist, infectious material from a small
carnivore such as the raccoon, would unlikely be unrecognized or ignored.
The apparent liberal administration of effective PEP after known bites,
scratches, and other suspected exposures from rabid raccoons may have
resulted in complete prevention of human deaths due to this variant of
rabies virus associated with raccoons.

Although the Healthy People 2000 goal to reduce PEP is worthwhile, better
understanding of the circumstances leading to human exposure and
formulating ways to reduce exposure is required to meet this objective.
Until then, it will be particularly difficult to reduce PEP during an
ever-expanding raccoon rabies epizootic.

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

Acknowledgments

       We thank the following public health nurses and rabies coordinators
for data collection and their generosity and enthusiasm in sharing data:
Terri Hogan, Lynn Crane, Linda Thompson, and Hope Albino.

      Dr. Wyatt is chair of the University of Rochester School of Medicine
and Dentistry's Division of Laboratory Animal Medicine and chief
veterinarian at the Seneca Park Zoo in Rochester, New York. His research
interests include the epidemiology of zoonoses in the environment and the
workplace.

      Address for correspondence: Jeff Wyatt, University of Rochester, Box
674, Rochester, New York 14642, USA; fax: 716-273-1085; e-mail:
jeff_wyatt@urmc.rochester.edu.

References

  1. Jenkins SR, Winkler WG. Descriptive epidemiology from an epizootic of
     raccoon rabies in the mid-Atlantic states 1982-1983. Am J Epidemiol
     1987;126:429-37.
  2. Rupprecht CE, Smith JE, Fekadu M, Childs JE. The ascension of wildlife
     rabies: a cause for public concern or intervention? Emerg Infect Dis
     1995;1:107-14.
  3. Centers for Disease Control and Prevention. Raccoon rabies
     epizooticUnited States, 1993. MMWR Morb Mortal Wkly Rep
     1994;43:269-73.
  4. Hanlon CA, Trimarchi C, Harris-Valente K, Debbie JG. Raccoon rabies in
     New York State: epizootiology, economics and control. In: Proceedings
     of the 5th Annual International Meeting. Rabies in the Americas,
     coping with invading rabies epizootics. Niagara Falls, Canada; 1994
     Nov 16-19; p. 16.
  5. Public Health Service. Healthy People 2000: national health promotion
     and disease prevention objectives. Washington: U.S. Department of
     Health & Human Services; 1991. DHHS publication no. (PHS) 91-50213.
  6. U.S. Department of Commerce, Economics and Statistics Administration,
     Bureau of the Census, 1990. Census of population, general population
     characteristics. New York: The Department; 1990. CP-1-34.
  7. Centers for Disease Control and Prevention. Rabies preventionUnited
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     Committee. MMWR Morb Mortal Wkly Rep 1991;40:R-3:1-19.
  8. Ashfar A. A review of non-bite transmission of rabies virus infection.
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  9. Helmick CG. The epidemiology of human rabies postexposure prophylaxis,
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     vaccine to control rabies in raccoons. J Am Vet Med Assoc
     1992;201:1873-82.
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     1969;84:1069-77.
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     Epidemiology of antirabies treatment in Georgia, 1967-71. Public
     Health Rep 1975;90:435-9.
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     States, 1972. Public Health Rep 1979;94:166-71.
 16. Baer GM. The natural history of rabies. 2nd ed. Boston: CRC Press;
     1991.
 17. Krebs JW, Strine TW, Smith JS, Noah DL, Rupprecht CE, Childs JE.
     Rabies surveillance in the United States during 1995. J Am Vet Med
     Assoc 1996;209:2031-44.
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     human exposure to rabies in New Hampshire: exposures, treatment, and
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_________________________________________________________________________
Dispatches
_________________________________________________________________________

Dispatches

Factory Outbreak of Escherichia coli O157:H7 Infection in Japan

Yoshiyuki Watanabe,* Kotaro Ozasa,* Jonathan H. Mermin, Patricia M.
Griffin, Kazushige Masuda, Shinsaku Imashuku, and Tadashi Sawada*
*Kyoto Prefectural University of Medicine, Kyoto, Japan; Centers for
Disease Control and Prevention, Atlanta, Georgia, USA; Public Health
Bureau, Kyoto City Government, Kyoto, Japan; and Kyoto City Institute of
Health and Environmental Sciences, Kyoto, Japan

---------------------------------------------------------------------------
      To determine the cause of a July 1996 outbreak of Escherichia
      coli O157:H7 among factory workers in Kyoto, Japan, we
      conducted cohort and case-control studies. Eating radish sprout
      salad during lunch at the factory cafeteria had been linked to
      illness. The sprouts were traced to four growers in Japan; one
      had been associated with an outbreak of E. coli O157:H7 among
      6,000 schoolchildren in Sakai earlier in July.

During May through August 1996, approximately 10,000 cases of Escherichia
coli O157:H7 infection associated with at least 14 separate clusters were
reported in Japan (1,2). Most cases occurred in school-age children. One
cluster was a large outbreak in Sakai City, Osaka Prefecture, involving
more than 6,000 primary school children. The outbreak started on July 13,
1996, and an investigation suggested that radish sprouts were the most
likely cause (2,3).

An outbreak also occurred in a factory in Kyoto, approximately 50 km from
Sakai City. On July 17, 1996, a 24-year-old male factory worker went to a
local clinic with diarrhea. The next day, a second worker came to the
clinic with diarrhea. Bloody diarrhea and hemolytic uremic syndrome (HUS)
subsequently developed in both patients, and stool cultures from each
yielded E. coli O157:H7. On July 21, a third worker died of HUS-associated
encephalopathy; his stool culture later yielded E. coli O157:H7. All three
workers had recently eaten meals at the factory cafeteria. To identify a
possible food vehicle, we conducted an epidemiologic investigation.

The Study

On July 19, factory officials requested that ill workers report to factory
health-care workers any symptoms from the beginning of July. Stool samples
from workers with diarrhea were cultured for E. coli O157:H7 and other
bacterial pathogens (e.g., Salmonella and Shigella). Surveillance continued
until the end of July. A culture-confirmed case was defined as a stool
culture yielding E. coli O157:H7 from a factory worker who had onset of
diarrhea during July 15 to 22, 1996. A clinical case was defined as
diarrhea with one or more loose stools per day with onset during July 15 to
22, 1996.

During their shifts, workers could eat any of the meals served at the
factory cafeteria, which was operated by an outside company. Data on the
date, time, and type of meal purchased at the cafeteria were routinely
recorded by computer, and the cost of meals was deducted from employees'
salaries. Workers could not pay by cash. We analyzed these data for visits
to the cafeteria during July 8 to 14 (2 to 8 days before the date of
symptom onset for the first case of culture-confirmed infection). All
factory workers were included in analyses implicating a specific date of
eating at the cafeteria. Only factory workers who purchased food at the
cafeteria on a particular day were included in analyses of a particular
meal for that day.

Two set lunches with prespecified food items were served for the same price
in the cafeteria each day. Because the lunches could not be distinguished
by computer records, a self-administered questionnaire was completed during
September 24 to 27, 1996, by the 47 workers who had reported diarrhea and
300 randomly selected workers who had eaten at the factory cafeteria on the
suspected exposure days (July 11 or 12) and had not reported diarrhea in
July. A computer record of the meals purchased by each of the workers was
included with each questionnaire to assist with recall.

Published methods were used to calculate odds ratios (ORs), 95% confidence
intervals (CIs), and p values (4). P values were calculated by a chi-square
test: p values of <0.05 were considered significant, and those of 0.05 to
0.09 were considered borderline significant. Multivariate conditional
logistic regression analysis was conducted with Statistical Analysis System
(SAS) software (SAS Institute, Cary, North Carolina, USA, 1990).

After reports of the first three cases, fecal samples from ill factory
workers were cultured in sorbitol indole pyruvic acid bile salts agar (SIB)
medium at 35 C to 37 C for 18 to 24 hours at the Kyoto City Institute of
Health and Environmental Sciences. To differentiate E. coli O157:H7 from
other bacteria, colonies were examined on triple sugar iron agar, sulfide
indole motility medium, lysin indole motility semisolid agar,
Voges-Proskauer semisolid medium, and Simon's citrate agar. Cultures that
conformed to the biochemical pattern of E. coli O157:H7 were then
serotyped. The presence of Shiga toxin 1 or 2 was confirmed by reversed
passive latex agglutination and polymerase chain reaction (PCR). Stored
food samples were homogenized, and a portion was cultured in modified E.
coli broth before culturing in SIB medium. To differentiate strains of E.
coli O157:H7, pulsed-field gel electrophoresis (PFGE) and random amplified
polymorphic DNA-PCR (RAPD) assays were performed as previously described
(5,6).

On July 18, the regional public health center examined the factory
cafeteria kitchen facilities for deficiencies. All 25 food handlers were
asked questions regarding abdominal symptoms and provided stool samples for
bacteriologic testing. All food served was traced to the distributor and
grower as far back as possible.

Findings

Of the 3,155 employees of the factory, 74 reported gastrointestinal
symptoms in July; stool samples were obtained from these workers. Illness
in 47 persons met the case definition: 42 cases were clinically defined,
and 5 were culture-confirmed. The peak date of symptom onset was July 17
(Figure). Six workers had only abdominal pain, fever, or general fatigue,
and 21 had onset of diarrhea outside the defined period. HUS developed in
three workers with culture-confirmed E. coli O157:H7 infection; two fully
recovered; one died. One clinical case-patient and four culture-confirmed
case-patients had bloody diarrhea. The proportion of cases with bloody
diarrhea was 11% among all patients. The median age of case-patients was 30
years (18 to 61). Of the 47 case-patients, 45 (96%) (including all
culture-confirmed cases) had eaten at the factory cafeteria during July 8
to 14. Of the 47 case-patients, 39 (83%) were male, and eight (17%) were
female. No information on sex and age of the other factory workers was
available.

Because the five   			  [fig]
workers with
culture-confirmed  Figure. Escherichia coli O157:H7 infection by date of
E. coli O157:H7    symptom onset, July 15-21, 1996.
infection had no
common eating exposure except the factory cafeteria, we first analyzed the
association between illness and date of eating at the cafeteria. Eating in
the cafeteria any day during July 8 to 13 was associated with illness by
univariate analysis. On multivariate logistic regression analysis, this
association was significant or borderline significant only for July 11, 12,
and 13. The ORs (95% CI, p value) of eating on July 11, 12, and 13 were
2.58 (0.91 to 7.36, 0.08), 2.84 (1.02 to 7.94, 0.05), and 3.19 (1.03 to
9.86, 0.04), respectively. Because 81% of the patients ate in the cafeteria
on July 11 and 12 compared with 23% on July 13, July 11 and 12 were
considered the most likely days of exposure. On multivariate analysis of
the six meal times on July 11 and 12, only eating lunch in the cafeteria on
July 11 was associated with illness. The rate of diarrhea for 1,134 workers
who ate lunch on July 11 was 3.0%, compared with 0.6% for 2,021 workers who
did not (OR = 3.04, 95% CI = 1.08, p = 0.04).

Of the 47 case-patients, 44 (94%) responded to the questionnaire. In 31
patients who answered the question regarding symptoms, diarrhea lasted for
a median of 3 days (1 to 10 days), and four (13%) reported bloody diarrhea.
Of 300 potential controls randomly selected from factory workers who ate in
the cafeteria on July 11 or 12, 291 (97%) responded to the questionnaire.
Among the respondents, 16 (5%) reported gastrointestinal symptoms in July
(of these, four [25%] had diarrhea during July 15-22, but none reported
bloody diarrhea), and three did not respond to the question regarding
symptoms; 272 respondents who reported no illness were adopted as controls.
The median age was similar for case-patients (30 years [18 to 61 years])
and controls (32 years [20 to 65 years]). Eighty percent of case-patients
and 83% of controls were male.

Among the participants, 29 patients and 164 controls responded that they
clearly remembered if they had eaten the radish sprout salad. Seventeen
(59%) of 29 patients and 64 (39%) of 164 controls reported eating radish
sprout salad (OR = 2.21, 95% CI = 0.99 to 4.94, p = 0.08) (Table). No other
food item served on July 11 or 12 was eaten by >50% of patients and had a
higher odds ratio than radish sprout salad. Among the five patients with
culture-confirmed infection, computer records indicated that four patients,
including the one who died, ate radish sprouts. Radish sprout salad
(consisting of radish sprouts, mayonnaise, cauliflower, and fish paste) was
served with both lunches the cafeteria served on July 11, the only time
sprouts were served during July 8 to 14.

 Table. Factory cafeteria foods associated with illness, July 11, 1996,
 Kyoto, Japan
 --------------------------------------------------------------------------
                 Case-patients       Controls
                  exposed/total    exposed/total    Odds ratio(sup a)
 Food                  (%)              (%)           (95% CI)     p value
 --------------------------------------------------------------------------
 Radish sprout
 salad             17/29(58.6)      64/164(39.0)   2.21(0.99-4.94)  0.08
 Boiled beef
 with soy sauce    8/28(28.6)       24/152(15.8)   2.13(0.84-5.40)  0.18
 Scrambled eggs    10/28(35.7)      31/150(20.7)   2.11(0.89-5.04)  0.18
 --------------------------------------------------------------------------
 (sup a)Odds ratio>2.00; CI: confidence interval.

All five patient isolates of E. coli O157:H7 produced Shiga toxins 1 and 2.
E. coli O157:H7 was not detected in any of the frozen food samples
(including radish sprout salad) leftover from cafeteria meals during July
11 to 15. Both PFGE and RAPD patterns of the E. coli O157:H7 isolates from
this outbreak and the outbreak in Sakai City during the same time were
indistinguishable (1-3,7).

Examination of the factory cafeteria kitchen facilities on July 18 by the
regional public health center found no deficiencies. One female food
handler had diarrhea with onset July 17, but E. coli O157:H7 was not
cultured from her stool specimen or from specimens of any of the other food
handlers.

The radish sprouts served at the cafeteria on July 11 were supplied by a
single distributor that received the sprouts from four growers, one of whom
also supplied the radish sprouts suspected as the source of E. coli O157:H7
infections in the Sakai City school outbreak. Radish sprouts used at the
primary schools in Sakai City and at the factory cafeteria had been shipped
by the grower on July 9 (3); however, the sprouts used at the factory
cafeteria had been purchased along with radish sprouts from different
growers.

Conclusions

Our data indicate that the outbreak of E. coli O157:H7 infection among
Kyoto factory workers was most likely caused by contaminated radish
sprouts: the factory outbreak began during the week following the Sakai
City outbreak; the factory used radish sprouts from the same grower; they
were shipped on the same day as those served to school children in the
Sakai City outbreak; and isolates from both outbreaks had indistinguishable
PFGE and RAPD patterns (1-3,5,7). The PFGE patterns of earlier outbreaks in
Okayama Prefecture (Oku-cho), Gifu Prefecture, Hiroshima Prefecture, Aichi
Prefecture, and Okayama Prefecture (Niimi City) were indistinguishable from
each other and different from the PFGE patterns of isolates from the
outbreaks in Sakai City and the Kyoto factory (1-3,5). E. coli O157:H7 was
not isolated from radish sprouts; however, the process of freezing sprouts
or pooling them with other food items may have decreased the number of
organisms to an undetectable level.

Although radish sprouts had never been linked to E. coli O157:H7 infection,
they are plausible vehicles. Most outbreaks of E. coli O157:H7 infections
have been linked to ground beef (8), but other items, including
unrefrigerated sandwiches (9), apple cider (10), mayonnaise (11),
cantaloupe (12), lettuce (13), and alfalfa sprouts (14,15) have been
implicated. In addition, some sprout types, including alfalfa sprouts (16)
and mung bean sprouts (17), have been linked to Salmonella outbreaks. In
1997, E. coli O157:H7 was isolated from radish sprouts collected from two
different outbreaks of E. coli O157:H7 infections in Japan (1).

Three cases of HUS (6%) among 47 cases of clinically or laboratory-defined
cases of E. coli O157:H7 infection in the factory outbreak is comparable to
rates described in other outbreaks (18,19). The proportion of workers
reporting bloody diarrhea was low, possibly because infection with E. coli
O157:H7 follows a more benign course in adults than in children (20) or
because the amount of bacterial contamination was low.

Several reasons might explain the small proportion of workers who ate lunch
on July 11 and reported illness. First, some ill workers might not have
informed the factory health-care personnel about gastrointestinal symptoms
for fear of decreasing their chance for future promotion. This seems
plausible because 16 (5%) of 291 potential controls in the case-control
study mentioned unreported gastrointestinal symptoms. If this percentage of
underreporting occurred for the 3,155 workers in the factory, an additional
173 infections may have been missed. Second, the pathogens might have been
diluted because only a part of the radish sprout shipment was contaminated.
The latter hypothesis is supported by the fact that four growers, including
the one implicated in the Sakai City outbreak, supplied the radish sprouts
eaten at the factory cafeteria on July 11. Third, the contamination of
radish sprouts may have been reduced by washing. Although the association
between eating radish sprout salad and illness among workers who ate lunch
on July 11 was of borderline significance, it was the only item associated
with illness that was consumed by more than 50% of case-patients. Moreover,
the next lowest p value was 0.18, far from that of radish sprout salad.

The radish sprout salad contained other food items; therefore, the
individual risk for each food item could not be ascertained. However,
radish sprouts and mayonnaise were the only uncooked ingredients. Although
mayonnaise is a possible vehicle, no reports implicated it in other
outbreaks in Japan in 1996 (1).

Recall bias could have occurred in the case-control study because workers
were asked about meals they had eaten 8 weeks earlier. Providing with the
questionnaire a printout of food items purchased on July 11 and 12 may have
assisted recall. In addition, as a result of the outbreak, the cafeteria
stopped serving food on July 19 and had not resumed service at the time of
the case-control study. This may have assisted respondents in remembering
what food items they had eaten during the last week of dining in the
cafeteria.

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

Acknowledgments

   The authors thank the workers and staff of the Kyoto factory
where the outbreak occurred for their cooperation in the survey; Drs. Yurie
Kanamoto, Hiroyuki Imai, Shouji Tateishi, Mitsuaki Nishibuchi, Jiro
Imanishi, Jun Fukuda, Yusaku Matsui, Yoichi Mori, and Kozo Yokota, and
collaborators in the Kyoto City Institute of Health and Environmental
Sciences; Mr. Akio Kuroda and Ms. Hisae Takenobu; and Drs. Robert Hyams and
Robert Tauxe for helpful discussions and for facilitating the
investigation.

   Dr. Watanabe is professor, Department of Social Medicine and Cultural
Sciences, Research Institute for Neurological Diseases and Geriatrics,
Kyoto Prefectural University of Medicine. His research focuses on the
epidemiology of gastrointestinal and neurologic diseases and geriatrics.

  Address for correspondence: Yoshiyuki Watanabe, Department of Social
Medicine and Cultural Sciences, Research Institute for Neurological
Diseases and Geriatrics, Kyoto Prefectural University of Medicine,
Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan; fax:
81-75-251-5770; e-mail: watanabe@basic.kpu-m.ac.jp.

References

  1. National Institute of Health and Infectious Diseases Control Division,
     Ministry of Health and Welfare of Japan. Verocytotoxin-producing
     Escherichia coli (entero-hemorrhagic E. coli) infections, Japan,
     1996-June 1997. Infectious Agents Surveillance Report 1997;18:153-4.
  2. National Institute of Health and Infectious Diseases Control Division,
     Ministry of Health and Welfare of Japan. Enterohemorrhagic Escherichia
     coli (verocytotoxin-producing E. coli) infection, 1996-April 1998.
     Infectious Agents Surveillance Report 1998;19:122-3.
  3. Study report on the cause of the outbreak of diarrhea due to E. coli
     O157:H7 among primary school students in Sakai City. Tokyo: Ministry
     of Health and Welfare in Japan, Environmental Health Bureau, Food
     Sanitation Division; 1996. (In Japanese).
  4. Rothman KJ. Modern epidemiology. Boston: Little, Brown and Company;
     1986. p. 153-76.
  5. Watanabe H, Wada A, Inagaki Y, Itoh K, Tamura K. Outbreaks of
     enterohaemorrhagic Escherichia coli O157:H7 infection by two different
     genotype strains in Japan, 1996. Lancet 1996;348:831-2.
  6. Izumiya H, Terajima J, Wada A, Inagaki Y, Itoh K, Tamura K, et al.
     Molecular typing of Escherichia coli O157:H7 isolates in Japan by
     using pulsed-field gel electrophoresis. J Clin Microbiol
     1997;35:1675-80.
  7. Report on the cause of the outbreak of E. coli O157:H7 in Kyoto City
     in 1996. Kyoto, Japan: Public Health Bureau, Kyoto City Government;
     1997. (In Japanese).
  8. Boyce TG, Swerdlow DL, Griffin PM. E. coli O157:H7 and the
     hemolytic-uremic syndrome. N Engl J Med 1995;333:364-8.
  9. Carter AO, Borczyk AA, Carlson JA, Harvey B, Hockin JC, Karmali MA, et
     al. A severe outbreak of Escherichia coli O157:H7-associated
     hemorrhagic colitis in a nursing home. N Engl J Med 1987;317:1496-500.
 10. Besser RE, Lett SM, Weber JT, Doyle MP, Barrett TJ, Wells JG, et al.
     An outbreak of diarrhea and hemolytic uremic syndrome from Escherichia
     coli O157:H7 in fresh-pressed apple cider. JAMA 1993;269:2217-20.
 11. Keene WE, Mcanulty JM, Williams LP, Hoesly FC, Hedberg K, Fleming DW,
     et al. A two-restaurant outbreak of Escherichia coli O157:H7 enteritis
     associated with consumption of mayonnaise [abstract]. In: Proceedings
     of the 33rd Interscience Conference on Antimicrobial Agents and
     Chemotherapy; 1993 Oct 17-20; New Orleans, Louisiana. Washington:
     American Society for Microbiology; 1993. p. 354.
 12. Yet another outbreak of hemorrhagic colitisCorvallis. Portland (OR):
     Center for Disease Prevention and Epidemiology, Oregon Health
     Division, Department of Human Resources. CD Summary 1993;42:1-2.
 13. Mermin JH, Hilborn ED, Voetsch A, Swartz M, Lambert-Fair MA, Farrar J,
     et al. A multistate outbreak of Escherichia coli O157:H7 infections
     associated with eating mesclum mix lettuce [abstract]. In: Proceedings
     of the 3rd International Symposium and Workshop on Shiga Toxin
     (verocytotoxin)-producing Escherichia coli infections; 1997 Jun 22-26;
     Baltimore, Maryland. The Lois Joy Galler Foundation; 1997. p. 9.
 14. Outbreaks of Escherichia coli O157:H7 infection associated with eating
     alfalfa sproutsMichigan and Virginia, Jun-Jul 1997. MMWR Morb Mortal
     Wkly Rep 1997;46:741-4.
 15. Mohle-Boetani JC, Werner SB, Farrar JA, Abbott S, Bryant R, Vugia DJ.
     Outbreak of toxigenic E. coli O157:H7: non-motile (NM) associated with
     a clover-alfalfa mix [abstract]. In: Program and Abstracts of the
     Infectious Diseases Society of America 36th Annual Meeting; 1998 Nov
     12-15; Denver, Colorado. 536 Fr, 178.
 16. Mahon BE, Ponka A, Hall WN, Komatsu K, Dietrich SE, Siitonen A, et al.
     An international outbreak of Salmonella infections caused by alfalfa
     sprouts grown from contaminated seeds. J Infect Dis 1997;175:876-82.
 17. O'Mahoney M, Cowden J, Smyth B, Lynch D, Hall M, Rowe B, et al. An
     outbreak of Salmonella saint-paul infections associated with bean
     sprouts. Epidemiol Infect 1990;104:229-35.
 18. Bell BP, Goldoft M, Griffin PM, Davis MA, Gordon DC, Tarr PI, et al. A
     multistate outbreak of Escherichia coli O157:H7associated bloody
     diarrhea and hemolytic uremic syndrome from hamburgers: the Washington
     experience. JAMA 1994;272:1349-53.
 19. Akashi S, Joh K, Tsuji A, Ito K, Hoshi H, Hayakawa T, et al. A severe
     outbreak of haemorrhagic colitis and haemolytic uraemic syndrome
     associated with Escherichia coli O157:H7 in Japan. Eur J Pediatr
     1994;153:650-5.
 20. Rodrigue DC, Mast EE, Greene KD, Davis JP, Hutchinson MA, Wells JG, et
     al. A university outbreak of Escherichia coli O157:H7 infection
     associated with roast beef and an unusually benign clinical course. J
     Infect Dis 1995;172:1122-5.
--------------------------------------------------------------------------

Dispatches

First Case of Yellow Fever in French Guiana since 1902

J.M. Heraud,* D. Hommel, A. Hulin, V. Deubel, J.D. Poveda, J.L.
Sarthou,* and A. Talarmin*
*Institut Pasteur de la Guyane, Cayenne, French Guiana;General Hospital,
Cayenne, French Guiana; and Institut Pasteur, Paris, France

---------------------------------------------------------------------------
      The first case of yellow fever in French Guiana since 1902 was
      reported in March 1998. The yellow fever virus genome was
      detected in postmortem liver biopsies by seminested polymerase
      chain reaction. Sequence analysis showed that this strain was
      most closely related to strains from Brazil and Ecuador.

Yellow fever (YF) is a serious public health problem in many tropical
countries in Africa and South America. In South America, most infections
are sporadic, affecting unvaccinated persons who enter the forest; monkeys
are the primary reservoirs, and Haemagoggus sp. are the vectors. French
Guiana, located between Brazil and Surinam in the Amazonian forest, has had
many epidemics of YF. However, no case has been reported in French Guiana
since 1902, although a serologic survey in 1951 found circulating virus; of
430 persons younger than 50 years of age (who had not been affected by the
1902 epidemic), 9% of those living in the coastal area and 29% of those
living inland had significant titers of neutralizing YF antibodies (1). A
study conducted at the same time in Surinam showed even higher rates of YF
seropositivity in persons who had not been exposed to previous epidemics
and confirmed that YF viruses were circulating in the region (2). In French
Guiana, YF immunization became compulsory in 1967.

Case Report

In March 1998, an Amerindian woman living in a forest area on the Maroni
River was admitted to the health center in Maripasoula, French Guiana, with
fever, headache, abdominal pain, vomiting, and diarrhea. She was treated
for malaria because of a Plasmodium falciparumpositive blood smear. Two
days later, the patient's fever increased (40.2C), she became jaundiced,
and she was evacuated to the intensive care unit (ICU) at Cayenne Hospital
with multiple visceral failure: shock syndrome, renal failure (blood urea
level 32 mmol/l, creatinine level 656 mol/l), and liver failure (total
bilirubin level 314 mol/l, alanine aminotransferase 2048 IU/l, aspartate
amino transferase 6256 IU/l, prothrombin level 23%). No hemorrhages were
noted. Despite symptomatic treatment, the condition of the patient
deteriorated rapidly, and she died a few hours after admission to ICU.

Blood cultures were negative for bacterial pathogens. Because of anuria,
urine cultures were not possible, and albuminuria could not be tested.
Examination of peripheral blood smears showed no parasites on admission to
ICU and titers of antibodies to leptospira were low.

Microscopy examination of postmortem liver biopsies showed histopathologic
changes characteristic of YF: midzonal necrosis with a small rim of a few
viable periportal and pericentral hepatocytes and centrilobular cells with
microsteatosis and eosinophilic degeneration with round, eosinophilic
cytoplasmic structures (Councilman bodies).

A serum sample collected before death and a serum sample obtained from the
patient in 1994 during a seroepidemiologic study on HTLV-I infection and
stored at -80C at the Institut Pasteur de la Guyane, French Guiana, were
tested for immunoglobulin (Ig) G and IgM specific for three flaviviruses
(YF, dengue, and Saint Louis encephalitis), two alphaviruses Tonate and
Mayaro), and a new world arenavirus (Tacaribe), by enzyme-linked
immunosorbent assay (3). A plaque reduction neutralization test was also
used to detect YF-neutralizing antibodies (4). The serum collected in 1998
contained IgM (but not IgG) that specifically recognized YF antigens. IgM
specific for other flavivirus antigens (dengue, Saint Louis encephalitis)
were not found. The neutralizing antibody titer of the 1998 serum as
assessed by plaque reduction neutralization test was 20. In the serum
collected in 1994, no YF virusspecific antibodies were detected by any
technique. IgG to Mayaro virus was detected in both sera; no other
antibodies to alphaviruses and arenaviruses were detected.

Serum and homogenized liver samples from the patient were diluted 10-fold
in Leibowitz medium containing 3% fetal calf serum, and dilutions were
injected into subconfluent AP 61 cell cultures (5). After 7 days, cells
were harvested and tested for YF virus by an indirect immunofluorescence
assay using a monoclonal antibody from the Centers for Disease Control and
Prevention, Fort Collins, CO, USA. YF virus was not isolated by cell
culture from either blood or liver samples.

Reverse transcriptionpolymerase chain reaction (RT-PCR) tests were
performed on RNA extracted from serum and liver (6). RNA of the YF virus
was detected by RT-PCR after seminested PCR only in the liver sample. The
542-bp PCR product was purified and directly sequenced with an automatic
sequencing system (ACTgene, EuroSequence Gene Services, Evry, France). The
first 309 nucleotides of the 3' noncoding sequence were aligned with those
of sequenced YF strains from Genbank (7,8). Sequences were aligned with the
Clustal W program. Bootstrap confidence limits were calculated from 100
replicates with the program SEQBOOT. Phylogenetic analyses were performed
by maximum parsimony by using the DNAPARS program with uniform character
weights and a heuristic search option. All branch lengths were drawn to
scale by the program Treetool. The sequence of the YF strain isolated in
1998 was deposited in Genbank (accession number: AF121952).

The sequence of the amplified gene differed at 11 positions (3.5%
nucleotide divergence) from that of a Brazilian strain isolated in 1935 and
at 21 positions (6.8% nucleotide divergence) from that of a strain isolated
in Ecuador in 1979. The sequence diverged more from strains isolated in
Peru (1995) and Trinidad (1979) (8.1% and 10% divergence, respectively). As
expected, African strains differed more, with those of West Africa (from
Nigeria and Senegal) being less distant than those from East and Central
Africa (Uganda and Central African Republic). The nucleotide sequence
downstream from the NS5 stop codon in the 3' noncoding region of the YF
virus was deleted in the French Guianese strain, as in several South
American YF viruses (9). Phylogenetic analysis of the sequences confirmed
that the virus was most closely related to those isolated in Brazil and
Ecuador (Figure).

Conclusions

			[Fig]                                  The
                                                         histopathologic
Figure. Phylogenetic tree generated from 309             changes of the
nucleotides of the 3' noncoding region of the strain     liver were
of yellow fever (YF) isolated in French Guiana in 1998   characteristic of
(in bold) and of 14 other YF strains by using the        YF but also of
DNAPARS program. Numbers indicate bootstrap values for   other hemorrhagic
groups to the right. One l (30 ng) of primer VD8        fever viruses. The
(5'-GGGTCTCCTCTAACCTCTAG-3') was mixed with RNA          IgMs were only
resuspended in 10 l of distilled water; the mixture     slightly above the
was heated at 95C for 2 minutes and placed on ice.      cut-off values
cDNA was synthesized in a mixture containing reverse     used in the
transcriptase (RT) incubation buffer (provided by the    laboratory.
manufacturer), 0.2 mM of each of the four dNTPs, 20      Although YF was
units RNasine, and five units of AMV RT (Promega,        highly probable,
Charbonnires, France) by incubation for 1 hour at       the diagnosis
42C. cDNA was amplified by PCR. Four l of the cDNA     required
sample was added to 46 l of a mixture containing Taq    confirmation by
polymerase buffer (provided by the manufacturer), 2 mM   detection of the
MgCl2, 0.5 mM of each of the 4 dNTPs, 300 ng of primer   virus or its
VD8 and of degenerate primer EMF1                        genome. Indeed, in
(5'-TGGATGACSACKGARGAYATG-3') (S = C, G; K = G, T; R =   1990 a suspected
A, G; Y = C, T), and 0.5 unit of Taq polymerase          case of YF was
(Promega, Charbonnires, France). After 5 minutes of     reported to the
denaturation at 95C, the mixture was subjected to 30    World Health
polymerase chain reaction (PCR) cycles: 95C for 30      Organization
seconds, 53C for 90 seconds, and 72C for 60 seconds,   because of
followed by a final 10-minute polymerization step at     characteristic
72C. Four l of a 1 in 100 dilution of the PCR          histopathologic
products was used for seminested PCR using primers VD8   changes, but the
and NS5YF (5'-ATGCAGGACAAGACAATGGT-3'). After the        case was never
denaturation step, DNA was amplified by 25 cycles of     confirmed
PCR: 94C for 30 seconds, 55C for 90 seconds, and       (presence of IgG
72C for 120 seconds, followed by a final extension      but no IgM
step at 72C. Negative controls (serum from healthy      specific for YF,
persons) were included in the series. The positive       no detection of
control (supernatant of infected mosquito cells) was     the virus in liver
tested separately to avoid any contamination. The        or serum); later
phylogenetic analysis was conducted by the Pasteur       it was shown that
Institute in Paris.                                      the patient had
                                                         been vaccinated
                                                         against YF 1 year
before (10). However, because YF requires health authorities to take
specific measures, confirmation of the diagnosis is important, especially
when YF is not prevalent. The case we described was confirmed by RT-PCR
from liver only, because the serum sample was taken on day 6 when viremia
is usually resolved and because the sensitivity of cell culture for virus
in liver samples is very low (probably because of biliary salts toxicity).
This case underscores the need for postmortem liver biopsies for detecting
the viral genome to confirm diagnosis.

This patient did not leave her village the months before infection; she was
probably infected while working in forest clearings. The detection of a YF
virus in French Guiana nearly a century after the last report is notable;
however, the absence of reported cases during the previous years is
surprising because no natural borders exist between this country and
northern Brazil, where YF is not uncommon (11). A severe YF outbreak would
have easily been detected, but sporadic cases can be misdiagnosed as other
fevers or as hepatitis (when jaundice is present) and may be not tested for
YF even though serologic and YF virus detection tests are performed for
each suspected case.

No other case was diagnosed in the patient's family or neighborhood, but
sporadic cases are common in South America (12), probably because of poorly
anthropophilic vectors. Furthermore, in this area, approximately 90% of the
population have been vaccinated at least once (R. Pignoux, unpub. data).
Outbreaks are common among nonhuman primates, but no epidemic has occurred
in the area where the patient lived. However, YF incidence increased in
northern Brazil in 1998 (13).

This case calls attention to vaccination problems in French Guiana,
especially along the rivers. Our patient had been vaccinated in 1985, but
the absence of neutralizing antibodies in 1994 indicates that the
vaccination was not effective. Although this patient may have had a poor
antibody response, more likely inadequate storage of the vaccine before use
was responsible. In 1985, YF vaccines were less thermostable than now, and
the cold chain was difficult to maintain. In response to this case, an
immunization campaign was initiated in this area in May.

Vaccination of the population must continue since YF can reappear. The
immunization program implemented in French Guiana (compulsory vaccination
of children older than 1 year of age, booster YF vaccination every 10
years, and required vaccination certificate before entering school) should
avert the threat of outbreaks in urban areas, which have a vaccine coverage
rate of approximately 80%. However, the risk for sporadic cases in
unvaccinated persons will persist, and so active serologic and virologic
surveillance of YF remains necessary.

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

Acknowledgment

      The authors thank Michel Favre for his help in the phylogenetic
analysis of the yellow fever virus sequences.

      This study was supported by a grant from the Programme Hospitalier de
Recherche Clinique.

      Jean-Michel Heraud is a Ph.D. student working in the virology
laboratory of the Pasteur Institute of French Guiana. His areas of
expertise are virology and hematology with a focus on arbovirus
epidemiology and physiopathology.

      Address for correspondence: A. Talarmin, Institut Pasteur de la
Guyane, 23 Avenue Pasteur, BP 6010, 97306 Cayenne Cedex, Guyane Franaise;
fax: 0594-309-416; e-mail: atalarmin@pasteur-cayenne.fr.

References

  1. Floch H, Durieux C, Koerber R. Enqute pidmiologique sur la fivre
     jaune en Guyane franaise. Annales Institut Pasteur 1953;84:495-508.
  2. Wolff JW, Collier WA, De Roever-Bonnet H, Hoekstra J. Yellow fever
     immunity in rural population groups of Surinam. Tropical and
     Geographical Medicine 1958;325-31.
  3. Lhuillier M, Sarthou JL. Intrt des IgM anti-amariles dans le
     diagnostic et la surveillance pidmiologique de la fivre jaune.
     Annales de Virologie (Institut Pasteur) 1983;134E:349-59.
  4. Lindsey HS, Calisher CH, Matthews JH. Serum dilution neutralization
     test for California group virus identification and serology. J Clin
     Microbiol 1976;4:503-10.
  5. Reynes JM, Laurent A, Deubel V, Telliam E, Moreau JP. The first
     epidemic of dengue hemorrhagic fever in French Guiana. Am J Trop Med
     Hyg 1994;51:545-53.
  6. Deubel V, Huerre M, Cathomas G, Drouet M-T, Wuscher N, Le Guenno B, et
     al. Molecular detection and characterization of yellow fever in blood
     and liver specimens of a non-vaccinated fatal human case. J Med Virol
     1997;53:212-7.
  7. Hahn CS, Dalrymphe JH, Strauss JH, Rice CM. Comparison of the virulent
     Asibi strain of yellow fever virus with the 17D vaccine strain derived
     from it. Proc Natl Acad Sci U S A 1987;84:2019-23.
  8. Wang E, Ryman KD, Jenings AD, Wood DJ, Taffs F, Minor PD, et al.
     Comparison of the genomes of the wild-type French viscerotropic strain
     of yellow fever virus with its vaccine derivative French neurotropic
     vaccine. J Gen Virol 1995;76:2749-55.
  9. Wang E, Weaver SC, Shope RE, Tesh RB, Watts DM, Barett ADT. Genetic
     variation in yellow fever virus: duplication in the 3' noncoding
     region of strains from Africa. Virology 1996;225:274-81.
 10. World Health Organization. Yellow fever in 1989 and 1990. Wkly
     Epidemiol Rec 1992;67:245-51.
 11. World Health Organization. Yellow fever in 1994 and 1995. Wkly
     Epidemiol Rec 1996;71:313-8.
 12. Tolou H. La fivre jaune: aspects modernes d'une maladie ancienne. Md
     Trop 1996;56:327-32.
 13. World Health Organization. Yellow fever in Brazil. Wkly Epidemiol Rec
     1998;7:351.
--------------------------------------------------------------------------

Dispatches

Risk for Rabies Transmission from Encounters with Bats, Colorado, 19771996

W. John Pape,* Thomas D. Fitzsimmons,* and Richard E. Hoffman*
*Colorado Department of Public Health and Environment, Denver, Colorado,
USA; and Centers for Disease Control and Prevention, Atlanta, Georgia, USA

---------------------------------------------------------------------------
      To assess the risk for rabies transmission to humans by bats,
      we analyzed the prevalence of rabies in bats that encountered
      humans from 1977 to 1996 and characterized the bat-human
      encounters. Rabies was diagnosed in 685 (15%) of 4,470 bats
      tested. The prevalence of rabies in bats that bit humans was
      2.1 times higher than in bats that did not bite humans. At
      least a third of the encounters were preventable.

Although no cases of human rabies have been reported since 1931 in
Colorado, rabies remains a health risk in this state because of the
frequency with which Coloradans have contact with bats. The first objective
of this study was to determine the prevalence of rabies in bats that were
submitted for laboratory testing in Colorado over a 20-year period,
including an analysis by bat species. The second objective was to
characterize the circumstances of confirmed bat-human encounters during
this same period and to evaluate how this information could be used to
prevent human rabies.

Data Sources

Laboratory Records

Rabies diagnosis was conducted by two laboratories in Colorado: the
Colorado Department of Public Health and Environment (CDPHE) Laboratory and
the Colorado State University (CSU) Veterinary Diagnostic Laboratory. Bats
were accepted for testing from public and private sources if they had had
contact with a person or a domestic pet, if the possibility of contact
could not be excluded, or if the bat exhibited abnormal behavior. County
agencies were also permitted to submit up to three bats per week (usually
found dead from no apparent cause or exhibiting aberrant behavior) for
local surveillance. None of the submissions were for studies of rabies
prevalence among bats with a normal appearance or in their natural habitat.

Records from both laboratories were maintained by the CDPHE Epidemiology
Division and made up the first dataset we analyzed. Information extracted
from these records included rabies test date, test result, and bat bite
information. For bats sent to CDPHE (but not CSU), laboratory technicians
identified the bats by species, and the data were included in the analysis.

Possible Rabies Exposure Memoranda

A second dataset consisted of memoranda describing any animal exposure
reported to CDPHE resulting in rabies postexposure prophylaxis (PEP).
Memoranda were not written for encounters in which the animal tested
negative for rabies, even if a person was bitten. Infrequently, CDPHE staff
wrote memoranda before learning that an animal had tested negative for
rabies or when PEP was recommended but not administered. Four persons in
the Epidemiology Division worked on zoonosis control during the 20-year
study period; two of them wrote 90% of the memoranda. A bat encounter was
defined as bat contact or possibility of bat contact with a person. A wound
was defined as a visible puncture, scratch, bleeding, or a sensation of
sharp pain during the encounter. No attempt was made to distinguish bite
wounds from claw marks or scratches.

The analysis of circumstances was restricted to memoranda in which the
presence of a bat was documented. Memoranda that described encounters with
bats were identified, and data on person, place, and time were extracted.
The circumstance of encounter was listed as one of 13 general categories
that best described the event. Any person who initiated the standard rabies
PEP series was designated as having received treatment.

Findings

Laboratory Records

From 1977 through 1996, 4,502 bats were submitted for testing. CDPHE
received 4,394 bats (98%), and CSU received 108 bats (2%). These bats
represented 15 (83%) of 18 species present in Colorado (1) (Table 1).
Thirty-two bats were excluded from further analysis because either the test
result or the bite status was unrecorded. Rabies was diagnosed in 685 (15%)
bats and accounted for 98% of all animal rabies cases in Colorado during
the study period. Of the 233 bats that bit people, 69 (30%) had rabies. Of
the 4,237 bats that did not bite people, 613 (14%) had rabies. The
prevalence of rabies among bats that bit humans was 2.1 times higher (95%
confidence interval 1.7 to 2.5) than in bats not involved in human bites.
None of the persons bitten by bats got rabies. Human rabies has not been
reported in Colorado since 1931 (CDPHE, unpub. data, 1998).

Table 1. Prevalence of rabies in bats submitted for                  Species
testing, Colorado, 19771996                                         data
                                                                     were
-------------------------------------------------------------------  available
                                             No.                     for
                                 No.       that did                  4,182
                              that bit     not bite     Total no.    (94%)
                               humans       humans       tested      bats.
Species                       (% rabid)    (% rabid)    (% rabid)    Three
-------------------------------------------------------------------  speciesbig
                                                                     brown
Big brown bat                    122         2,013        2,135      bats
  Eptesicus fuscus               (27)         (16)         (17)      (Eptesicus
Myotis genus group(sup a)         35          722          757       fuscus),                                                                					   (14)          (6)          (7)      hoary                         											   bats
Silver-haired bat                 28           628          656      (Lasiurus
                                                                     cinereus),
  Lasionycteris                  (14)          (5)          (5)      and
    noctivagans                                                      silver-haired
Hoary bat                          13          452          465      bats
                                                                     (Lasionycteris
  Lasiurus cinereus              (77)         (39)         (40)      noctivagans)accounted
Long-eared bat                      8           38          46       for
  Myotis evotis                  (88)         (21)         (33)      73% of
                                                                     total
Brazilian free-                     0           41          41       submissions,
tailed bat                                                           84% of
                                   (0)        (12)         (12)      the
  Tadarida                                                           rabies-positive
   brasiliensis                                                      specimens,
                                                                     and
Red bat                              1          25          26       70% of
  Lasiurus borealis             (100)          (8)        (12)       bats
                                                                     involved
Pallid bat                           0          21          21       in
  Antrozous pallidus               (0)         (5)          (5)      bite
Big free-tailed bat                 2           19          21       incidents.
                                                                     The
  Nyctinomops                    (50)         (11)        (14)       prevalence
   macrotis                                                          of
Townsend's big-                     1           13         14        rabies
   eared bat                      (0)          (0)          (0)      in
                                                                     silver-haired
  Plecotus townsendii                                                bats
Species data                      23          265          288       (5%)
                                                                     was
  unavailable                    (35)         (9)         (11)       lower
Total                            233         4,237        4,470      than
                                 (30)         (14)          (15)     in big
                                                                     brown
-------------------------------------------------------------------  bats
(sup a)Includes six species in the genus Myotis that could not be	   (17%) 
easily distinguished by inspection: M. lucifugus, M. volans, M.      or
thysanodes, M. californicus, M. ciliolabrum, and M. yumanesis.       hoary
                                                                     bats(40%).

Memoranda

During the 20-year study period, 271 memoranda described possible
encounters with bats; 240 (89%) memoranda documented the presence of a bat
and were included in the analysis. Of the 131 bats tested, 99 had rabies.

Of the 240 persons who encountered bats, 141 (59%) were male and 99 (41%)
were female. From the 195 (81%) records that recorded the person's age, the
range in age was 10 months to 81 years, and the median age was 25 years. Of
the 182 (76%) persons reporting that they were wounded, the most common
wound site was the hand (59%), followed by the arm (14%), head/neck (12%),
leg/foot (9%), torso (2%), or multiple sites (2%).

Not enough information was available to characterize the time of day of the
encounters. Two hundred (83%) encounters occurred between June and
September, corresponding to peak activity periods and seasonal migratory
patterns of bats in Colorado.

In the 217 records that noted location of bat encounters, 117 (54%)
occurred outdoors, chiefly on home properties and park and recreation areas
(none were reported in caves). Of the 100 (46%) bat encounters inside
buildings, 83 were in private homes (37 of these in bedrooms). Big brown
bats, colonial bats that commonly roost in houses and buildings, were
encountered more frequently outside than inside (60% vs. 39%) when rabid (n
= 46). All 11 rabid hoary bats, solitary tree dwellers, were encountered
outdoors. However, rabid silver-haired bats, another solitary tree-roosting
species, were encountered equally indoors (n = 3) and outdoors (n = 3).

The four most frequent circumstances in which people encountered bats,
accounting for 62% of the encounters, were a bat landing on an awake person
(19%), a person picking up a grounded bat outside (18%), a person awakening
to find a bat in the room (15%), and a person trying to remove a bat from
inside a structure (10%). The remaining nine circumstances occurred
repeatedly but less frequently (Table 2).

Of the 240 persons who had encounters with bats, 216 (90%) initiated PEP;
nine of these stopped treatment after the bat tested negative for rabies.
The bat tested negative in 17 of the 24 cases in which PEP was not
administered, but a memorandum was written before the test results were
available. The remaining seven persons did not receive prophylaxis because
they or their physician did not believe that the contact warranted
treatment. In three of these seven encounters (which occurred before 1983),
the bat was found to be rabid, but no definite wound was observed.

The time from bat   Table 2. Circumstances in which humans encountered
encounter to        bats, Colorado, 19771996
initiation of
treatment could be  -------------------------------------------------------
calculated for 199                             Bat captured
(92%) of the 216                                and tested    Bat    All
patients who                                           Not    not   encoun
received PEP and    Circumstances               Rabid  rabid tested -ters
was 1 hour to 28
days. Fifty percent -------------------------------------------------------
of patients         Bat landed on person         17       2    27      46
received their      Person picked up             24       5    15      44
first dose of         bat outdoors
vaccine within 24
hours of exposure;  Person awoke to              17       4    14      35
75% started           find bat in room
treatment within 72 Person tried to remove         5      2    17      24
hours. Of the 18      bat from indoors
patients who
initiated treatment Person inadvertently           3      5      8     16
7 or more days        touched hidden bat
after the           Person handled               12       0      1     13
encounter, nine did   captured bat
not do so until     Child found alone              4      3      2      9
advised by an         with bat
acquaintance or
physician of the    Person handled bat             6      1      1      8
possible rabies       as part of job
risk.               Person stepped on bat          3      0      3      6
                    Person bitten while            2      1      3      6
Silver-Haired Bats    taking bat from pet
Although the        Person bitten by pet that      1      4      1      6
silver-haired bat     had bat in mouth
rabies virus        Person attributed wound        0      2      0      2
variant was           to bat they saw
isolated from 15 of
the 21 persons who  Other circumstances            0      0      6      6
died of             Unspecified in report          5      3     11     19
bat-associated      Total                         99     32    109    240
rabies in the
United States from  -------------------------------------------------------
1980 through 1997, we observed that silver-haired bats in Colorado had
neither the greatest frequency nor the highest species-specific rate of
rabies. Our findings are consistent with tabulations from New York (1988 to
1992) and Arkansas, Virginia, and West Virginia (1990 to 1994), which
showed that silver-haired bats made up a small proportion of bats submitted
for rabies testing; only a small number of submitted silver-haired bats
were rabies positive (2,3). Nonetheless, Arkansas (1991), New York (1993),
and West Virginia (1994) each had human cases associated with the rabies
virus variant common to silver-haired bats (4-6). Because the frequency of
human encounters with this species is apparently low and the prevalence of
rabies in tested silver-haired bats is small, other factors must explain
the silver-haired bats' association with human rabies cases. One hypothesis
is that silver-haired bats are more aggressive than other bats (1).
Additionally, one study has demonstrated that the rabies virus variant of
silver-haired bats replicates in nonneuronal tissue more efficiently than a
coyote rabies virus variant (7). This attribute might explain how a small
dermal inoculum of silver-haired variant rabies virus from a seemingly
superficial bite could cause infection. As silver-haired and hoary bats are
tree dwellers that favor old growth forest habitat, it should be unexpected
to encounter them indoors. None of the 12 hoary bats (11 rabid) included in
this series were encountered inside. In contrast, three of the nine
encounters with silver-haired bats were indoors. All three bats were rabid.

Conclusions

Bats that interact with humans are far more likely to have rabies than bats
that avoid humans, and rabies prevalence is highest in bats that bite.
Conversely, rabid bats appear to interact more frequently and to be more
prone to bite than nonrabid bats. This behavior is consistent with clinical
manifestations of rabies in wildlife species in which the animal exhibits
abnormal behavior, loses its natural fear of humans, and acts aggressively
(8).

Encounters with bats resulted from a relatively small number of recurring
situations. At least a third of the encounters (picking up grounded bats,
handling captured bats, and trying to remove bats from structures or from
pets' mouths) were preventable; however, most encounters in which a person
inadvertently touched a hidden bat or a bat landed on a person were
probably unavoidable.

Delays in treatment suggest that some people may not be aware of the risk
for rabies transmission from contact with a bat. In the United States, in
nearly half of the cases of human rabies associated with bat variant rabies
virus, the person had no history of contact with a bat (9-11). Although
unrecognized exposures may have occurred, the persons involved probably did
not understand the risk after exposure to a bat and therefore did not seek
medical care. Even when specifically asked about animal exposures, some
patients and their families initially did not report bat contact (11,12).

This study has several potential limitations. First, we do not know whether
the prevalence of rabies in tested bats is representative of all bats that
encounter humans. The laboratory testing was a passive surveillance system,
dependent on the submission of bats by persons involved in encounters. We
calculated a lower limit estimate of the prevalence of rabies among bats
that bit humans by using data from the memoranda. If one assumed that the
bats that bit humans and later escaped and thus were not tested (90) were
rabies-free, the prevalence of rabies among bats that bit people would
decrease from 30% (69 of 233) to 21% (69 of 323), still significantly
higher than the prevalence in bats that did not bite people (p < .001).

The circumstances were categorized for a small proportion of all bat
encounters. Memoranda were written for only 131 of 4,502 bats submitted for
testing and for 109 encounters in which the bat escaped. The number of
unreported encounters cannot be estimated, and information from those
encounters could alter the frequencies of the type of encounter presented
in this study.

Finally, administration of rabies PEP is not reportable in Colorado and,
therefore, the study may not have included all persons who received PEP
after a bat encounter. Because the state health department was the primary
source of rabies biological supplies for medical providers in the state
from 1977 through 1985, nearly all exposures requiring PEP would have come
to the department's attention. Rabies biological supplies were more widely
available from other sources after 1985. Although the number of reported
PEP administrations remained stable, some exposures may not have been
reported.

Two findings of this study support recent revisions of the Advisory
Committee on Immunization Practices (ACIP) recommendations (13-15): bats
that encountered humans had a high prevalence of rabies, and the third most
frequently reported circumstance was a person awakening to find a bat in
the room. The ACIP stated in October 1997 that PEP may be appropriate even
in the absence of demonstrable bite, scratch, or mucous membrane exposures
in situations in which such exposure is likely to have occurred (e.g., a
sleeping person awakes to find a bat in the room or an adult finds a bat in
a room with an unattended child, a mentally deficient person, or an
intoxicated person) (14). Of 35 instances reported in this study in which a
bat was found in the room by a person upon awakening, 17 bats were rabid,
and 23 persons had evidence of a bite.

The Colorado Department of Public Health and Environment, the Colorado
Division of Wildlife, and the Colorado Bat Society recently collaborated to
publish an educational pamphlet that describes methods to prevent rabies
exposure from a bat and measures to take if a person encounters a bat (16).

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

Acknowledgments

      We thank rabies laboratory workers for invaluable service over the
past 20 years, especially Larry Briggs and Jane Carman. We also thank John
Emerson for his diligence in investigating and documenting bat encounters
from 1977 to 1986.

      John Pape is an epidemiologist with the Communicable Disease Program,
Colorado Department of Public Health and Environment. He has statewide
responsibility for zoonotic disease surveillance and control. In addition
to bat rabies, his research focuses on the epidemiology of zoonotic
diseases in the western United States, including plague, hantavirus, and
tick-borne relapsing fever.

      Address for correspondence: W. John Pape, 4300 Cherry Creek S,
Denver, CO 80246, USA; fax: 303-782-0904; e-mail: john.pape@state.co.us.

References

  1. Armstrong DM, Adams RA, Navo KW, Freeman J, Bissell SJ. Bats of
     Colorado: shadows in the night. 2nd ed. Denver (CO): Colorado Division
     of Wildlife; 1996. p. 11.
  2. Childs JE, Trimarchi CV, Krebs JW. The epidemiology of bat rabies in
     New York state, 1988-92. Epidemiol Infect 1994;113:501-11.
  3. Dreesen DW, Orciari LA, Rupprecht CE. The epidemiology of bat rabies
     in the southeastern United States 1990-1994 [abstract]. In:
     Proceedings from 7th Annual International Meeting of Advances Towards
     Rabies Control in the Americas; 1996 Dec 9-13; Atlanta, Georgia. p.
     44.
  4. Centers for Disease Control. Human rabiesTexas, Arkansas, and
     Georgia, 1991. MMWR Morb Mortal Wkly Rep 1991;40:765-9.
  5. Centers for Disease Control and Prevention. Human rabiesNew York,
     1993. MMWR Morb Mortal Wkly Rep 1993;42:799,805-6.
  6. Centers for Disease Control and Prevention. Human rabiesWest
     Virginia, 1994. MMWR Morb Mortal Wkly Rep 1995;44:86-7,93.
  7. Morimoto K, Patel M, Corisdeo S, Hooper DC, Fu ZF, Rupprecht CE, et
     al. Characterization of a unique variant of bat rabies responsible for
     newly emerging human cases in North America. Proc Natl Acad Sci U S A
     1996;93:5653-8.
  8. Kaplan C, Turner GS, Warrell DA. Rabies: the facts. 2nd ed. Oxford:
     Oxford University Press; 1986. p. 72-4.
  9. Centers for Disease Control and Prevention. Human rabiesMontana and
     Washington, 1997. MMWR Morb Mortal Wkly Rep 1997;46:770-4.
 10. Centers for Disease Control and Prevention. Human rabiesTexas and New
     Jersey, 1997. MMWR Morb Mortal Wkly Rep 1997;47:1-5.
 11. Centers for Disease Control. Human rabiesTexas, 1990. MMWR Morb
     Mortal Wkly Rep 1991;40:132-3.
 12. Centers for Disease Control and Prevention. Human rabiesConnecticut,
     1995. MMWR Morb Mortal Wkly Rep 1996;45:207-9.
 13. Advisory Committee on Immunization Practices. Revised ACIP rabies
     post-exposure prophylaxis (PEP) statement1997 Oct 22.
 14. Constantine DG. Bat rabies in the southwestern United States. Public
     Health Rep 1967;82:867-8.
 15. Rabies preventionUnited States, 1991: recommendations of the
     Immunization Practices Advisory Committee (ACIP). MMWR Morb Mortal
     Wkly Rep 1991;40:1-19.
 16. Colorado Division of Wildlife, Colorado Department of Public Health
     and Environment, Colorado Bat Society. Bats and rabies [pamphlet].
     Denver (CO): The Department; 1997.
--------------------------------------------------------------------------
Dispatches

Australian Bat Lyssavirus Infection in a Captive Juvenile Black Flying Fox

Hume Field,* Brad McCall, Janine Barrett
*Queensland Department of Primary Industries, Moorooka, Australia;
Brisbane Southside Public Health Unit, Upper Mount Gravatt, Australia; and
The University of Queensland, Brisbane, Australia

---------------------------------------------------------------------------
      The newly emerging Australian bat lyssavirus causes rabieslike
      disease in bats and humans. A captive juvenile black flying fox
      exhibited progressive neurologic signs, including sudden
      aggression, vocalization, dysphagia, and paresis over 9 days
      and then died. At necropsy, lyssavirus infection was diagnosed
      by fluorescent antibody test, immunoperoxidase staining,
      polymerase chain reaction, and virus isolation. Eight human
      contacts received postexposure vaccination.

Australia was considered free of rabies and the rabieslike viruses of the
genus Lyssavirus until the recognition in 1996 of Australian bat lyssavirus
(ABL) as the cause of a rabieslike disease in a black flying fox (Pteropus
alecto)(1) and a wildlife caretaker (2). While serotypic, antigenic, and
sequence analysis show that ABL is closely related to classic rabies virus
and European bat lyssavirus (1), phylogenetic analysis has clearly
demonstrated that ABL represents a new genotype, genotype 7 (3). Australia
is still considered free of terrestrial (genotype 1) classic rabies (4).
Rabies vaccine and antirabies immunoglobulin protect laboratory animals
against ABL infection (5), and their use pre- and post-ABL exposure is
recommended for humans (6,7).

On the morning of December 8, 1997, two juvenile black flying foxes were
found clinging to each other and vocalizing in a residential area near an
urban flying fox colony. An experienced, rabies-vaccinated wildlife
caretaker retrieved the two animals from an unusually low tree roost. On
the basis of body weight and forearm measurements, their age was estimated
at 2 to 3 weeks, an age of total maternal dependency. Their physical
condition was normal.

Both animals, Bat 1 (male) and Bat 2 (female), remained with the original
caretaker for 2 days before being placed with two different caretakers for
hand-rearing. Bat 1 was communally housed with another orphaned black
flying fox, and Bat 2 was housed with two others. For the next 5 weeks, all
the bats were clinically normal. However, in week 6, Bat 1 began to exhibit
signs of neurologic disease. The caretaker first observed the bat's sudden
and progressive aggression toward its companion and separated them.
Throughout day 1 of illness, the bat periodically "frothed at the mouth"
and had repeated lordotic spasms, during which it vocalized loudly.
Treatment with oral amoxycillin was initiated. On day 2, the bat was calmer
but still vocal, attempting to bite objects and eating little. On day 3, it
was no longer aggressive and was only able to eat pulped food and milk. On
day 4, it was seen by a veterinarian, who noted severe pharyngitis, and
administered injectable dexamethasone. The bat was much more alert that
evening and ate solid food well. The dexamethasone injection was repeated
on day 5; the bat remained alert and ate solid food overnight. On day 6, it
was dysphagic and was again offered pulped foods and liquids. On days 7 and
8, it was unable to roost normally, lay supine, was progressively
dysphagic, had diarrhea, and was losing weight. On day 9, it rapidly got
worse and died. The carcass was submitted to the Queensland Department of
Primary Industries Animal Research Institute for necropsy.

The emaciated carcass had poor pectoral muscle development and no
perirenal, pericardial, or mesenteric fat reserves. ABL infection was
diagnosed by fluorescein-labeled antirabies monoclonal globulin (CENTOCOR)
in a fluorescence antibody test (FAT) on impression smears of fresh brain.
With the absence of other lyssaviruses in Australia (1), a positive
reaction with this lyssavirus genus-specific antibody is considered
diagnostic for ABL. Sections of brain showed nonspecific, nonsuppurative
meningoencephalitis with perivascular cuffs of mononuclear cells and
widespread focal gliosis. Numerous neurons contained eosinophilic inclusion
bodies, which are highly suggestive of lyssavirus infection.

Immunoperoxidase staining of formalin-fixed, paraffin-embedded sections of
brain with a monoclonal antinucleoprotein antibody (Clone HAM, provided by
R. Zanoni, Berne University, Switzerland) detected lyssavirus antigen in
neurons of the frontal cortex, hippocampus, brain stem, and cerebellum,
including Purkinje cells. This antigen distribution is consistent with
previous reports of rabies (8). The diagnosis was independently confirmed
at the Commonwealth Scientific and Industrial Research Organisation's
Australian Animal Health Laboratory by FAT, immunoperoxidase staining,
virus isolation in murine neuroblastoma cells, and sequence analysis of
polymerase chain reaction (PCR) product (P. Daniels, pers. comm.). No blood
was available for serologic testing.

After ABL infection was diagnosed in Bat 1, the Brisbane Southside Public
Health Unit received information that up to eight persons had been bitten
or scratched by the bat in the weeks before and during its illness. Six
were bat handlers who had received postexposure treatment with five doses
of rabies human diploid cell vaccine (HDCV) during a 1996 campaign that
followed the diagnosis of the first human case of ABL (2,7). Two were
unvaccinated members of the principal bat caretaker's household. Despite
recommendations that unvaccinated members of bat caretakers' households not
handle bats, the two had come into regular contact with the bat and may
have been scratched during that time.

Lyssavirus prophylaxis was commenced in accordance with Australian
recommendations (6,7). All eight persons provided blood for rabies
serologic testing (indirect-enzyme linked immunosorbent assay [ELISA]). The
six vaccinated bat handlers had titers of 1.130 IU/ml to >8.80 IU/ml 12
months after their initial vaccinations (World Health
Organization_recommended protective level for rabies = 0.5 IU/ml [9]). Each
of these received two further intramuscular doses of 1.0 ml of HDCV,
according to the Australian recommendations for postexposure treatment of
vaccinated persons. The two unvaccinated persons had titers of <0.13 IU/ml
(nonimmune) and received the standard postexposure treatment for
unvaccinated persons: 20 IU per kg of human rabies immune globulin (HRIG)
by intramuscular injection and five intramuscular doses of 1.0 ml of HDCV.
All eight remain well 10 months after the incident.

Throughout this episode, Bat 2 and the other three bats that had been
directly or indirectly in contact with the infected bat remained healthy.
After the diagnosis of ABL in Bat 1, these four animals were quarantined
for observation at the Animal Research Institute for 11 weeks and then
euthanized. All were antibody-negative (<0.5 IU/ml) by rabies rapid
fluorescent focus inhibition test when quarantined and remained so during
the observation period. Brain impression smears from the four were negative
for lyssavirus antigen by FAT.

This case of naturally occurring ABL infection is of particular interest
for several reasons. First, the astute observations of the bat caretaker
provide possibly the first record of the clinical course of natural ABL
infection in a flying fox. Second, to our knowledge, this is the first
recorded case of ABL disease in a maternally dependent juvenile. Third, the
case history provides the first indication of the incubation period and
length of clinical disease in naturally infected flying foxes. Natural in
utero infection with lyssaviruses is not known to occur (10), and the four
flying foxes in contact with Bat 1 were unlikely to be the source of
infection as they subsequently tested negative for ABL antibody and
antigen. The infection appears to have occurred in the 2 to 3 weeks before
the rescue and, after an incubation period of 6 to 9 weeks, produced 9 days
of clinical disease. Infection in Bat 1 most probably resulted from a bite
from an ABL-infected bat. This bat may have been Bat 1's dam.

This episode demonstrates the necessity of examining for ABL any flying fox
that has bitten or scratched a person and of improving community and
professional awareness of the disease and associated risks. Costly
postexposure treatment with HRIG can be avoided if only vaccinated persons
handle Australian bats.

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

Acknowledgments

      We thank Helen Luckhoff, Jill Nelson, Jennifer Hawyes, and Merle
Thomas for reporting the case and making their records available; Barry
Rodwell for performing the fluorescence antibody test; Natasha Smith and
Craig Smith for technical assistance; Greg Smith, Ina Serafin, and Alan
Westacott for performing ELISA (human) serology; Ross Lunt, Peter Hooper,
and Alan Gould for fluorescence antibody test and immunoperoxidase
staining, rapid fluorescent focus inhibition test (flying fox) serology,
virus isolation, and sequence analysis of PCR product; and the general
practitioners and the staff of Queen Elizabeth II Hospital for collecting
serum samples and administering postexposure treatments.

      Hume Field is a veterinary research officer with the Animal Research
Institute, Queensland Department of Primary Industries. His particular
research interest is the epidemiology of wildlife diseases and the role of
wild species as reservoirs of infection for domestic animals and humans. He
is investigating the natural history of Australian bat lyssavirus and
equine morbillivirus (Hendra virus), two recently emerged zoonotic diseases
in Australia.

      Address for correspondence: Janine Barrett, School of Veterinary
Science and Animal Production, The University of Queensland, Brisbane,
Queensland, Australia 4072; fax: 61-7-3362-9420; e-mail:
j.barrett@mailbox.uq.edu.au.

References

  1. Fraser GC, Hooper PT, Lunt RA, Gould AR, Gleeson LJ, Hyatt AD, et al.
     Encephalitis caused by a lyssavirus in fruit bats in Australia. Emerg
     Infect Dis 1996;2:327-31.
  2. Allworth A, Murray K, Morgan J. A human case of encephalitis due to a
     lyssavirus recently identified in fruit bats. Commun Dis Intell
     1996;20:504.
  3. Gould AR, Hyatt AD, Lunt R, Kattenbelt JA, Hengstberger S, Blacksell
     SD. Characterisation of a novel lyssavirus isolated from Pteropid bats
     in Australia. Virus Res 1998;54:165-87.
  4. Office International des Epizooties, 10 Nov 1998, [computer program].
     Handistatus (download). Version 1.39. Microsoft Internet Explorer 4.0.
     http://www.oie.int/A_html.htm.
  5. Hooper PT, Lunt RA, Gould AR, Samaratunga H, Hyatt AD, Gleeson LJ, et
     al. A new lyssavirusthe first endemic rabies-related virus recognised
     in Australia. Bulletin Institut Pasteur 1997;95:209-18.
  6. Rabies and bat lyssavirus infection. In: Watson C, editor. The
     Australian Immunisation Handbook. 6th ed. Canberra: Australian
     Government Publishing Service; 1997. p. 162-8.
  7. Prevention of human lyssavirus infection. Commun Dis Intell
     1996;20:505-7.
  8. Feiden W, Kaiser E, Gerhard L, Dahme E, Gylstorff B, Wandeler A, et
     al. Immunohistochemical staining of rabies virus antigen with
     monoclonal and polyclonal antibodies in paraffin tissue sections.
     Zentralbl Veterinarmed [B] 1988;35:247-55.
  9. World Health Organization recommendations on rabies post-exposure
     treatment and the correct technique of intradermal immunization
     against rabies. Geneva: The Organization; 1997.
 10. Constantine DG. Absence of prenatal infection of bats with rabies
     virus. J Wildl Dis 1986;22:249-50.
--------------------------------------------------------------------------
Dispatches

Bordetella holmesii-Like Organisms Isolated from Massachusetts Patients
with Pertussis-Like Symptoms

W. Katherine Yih, Ellen A. Silva, James Ida, Nancy Harrington, Susan M.
Lett, and Harvey George
Massachusetts Department of Public Health, Boston, Massachusetts, USA

---------------------------------------------------------------------------
      We isolated Bordetella holmesii, generally associated with
      septicemia in patients with underlying conditions, from
      nasopharyngeal specimens of otherwise healthy young persons
      with a cough. The proportion of B. holmesii-positive specimens
      submitted to the Massachusetts State Laboratory Institute
      increased from 1995 to 1998.

Bordetella holmesii is a recently described gram-negative, asaccharolytic,
nonoxidizing, soluble, brown-pigment-producing rod previously known as CDC
nonoxidizer group 2 (NO-2) (1). This group consists of 15 closely related,
biochemically similar strains of fastidious nonmotile bacteria isolated
from human blood cultures. In establishing NO-2 as a species, Weyant et al.
(1) performed 16S rRNA sequencing of one NO-2 strain and the type strains
of B. pertussis, B. parapertussis, B. bronchiseptica, and B. avium. They
found a high degree of homology among them (>98% over 1,525 bases) and
confirmed a close relatedness between NO-2 and Bordetella species by DNA
relatedness studies (hydroxyapatite method). Biochemically, the lack of
oxidase activity and the production of a brown soluble pigment
differentiate B. holmesii from B. pertussis, B. bronchiseptica, and B.
avium; the lack of urease activity differentiates it from B. parapertussis
(1).

Unlike B. pertussis, which causes whooping cough, B. holmesii has been
associated most often with septicemia in patients with underlying
conditions (1-4). It also has been isolated from sputum from one patient
with respiratory symptoms (3). Van den Akker (5) suggested that the
difference in lipopolysaccharide expression (important in bacterial
pathogenesis) between the closely related B. pertussis and B. holmesii
might help explain their observed difference in propensity to infect
respiratory tract epithelium verses causing opportunistic bacteremia.

In Massachusetts, however, we have seen B. holmesii associated with a
different clinical picture. From January 1995 (when the article describing
B. holmesii [1] was published) through December 1998, the Massachusetts
State Laboratory Institute (SLI) isolated B. holmesii from 34 clinical
specimens: 33 nasopharyngeal specimens from patients suspected of having
pertussis and one blood culture specimen from a 45-year-old patient with
septicemia. Of the 33 patients with respiratory symptoms, 30 (91%) were 11
to 29 years old, 1 (3%) was an infant, and 2 (6%) were 10 years old; most
were otherwise healthy.

B. holmesii is confirmed by its biochemical patterns and cellular fatty
acid analysis or the DNA transformation test will definitively separate B.
holmesii from Acinetobacter, neither procedure is performed at SLI. Three
of the initial isolates were sent to the Centers for Disease Control and
Prevention, Atlanta, Georgia, for definitive identification and were
confirmed as B. holmesii by cellular fatty acid analysis. The remaining 31
isolates were biochemically and morphologically identical to those.

B. holmesiipositive nasopharyngeal specimens have increased both in
absolute number and as a percentage of the total nasopharyngeal specimens
processed at SLI (Table). The number rose from 3 (0.1% of total specimens
submitted for pertussis culture) in 1995, to 6 (0.2%) in 1996, to 9 (0.4%)
in 1997, and to 15 (0.6%) in 1998. A chi-square analysis for linear trend
of proportions from 1995 to 1998 found the trend significant (chi-square =
11.6, p <.001), possibly indicating a rise in prevalence. (When such an
analysis was applied to the proportion of nasopharyngeal specimens testing
positive for pertussis during the same period, no trend was apparent
([chi-square = 0.8, p = .36].) A growing awareness of the species by
laboratory personnel may be contributing to the observed increase.

Table. Bordetella species isolated from nasopharyngeal (NP) specimens   
at the Massachusetts State Laboratory Institute, 19941998              
                                                                        
----------------------------------------------------------------------  
                     1994      1995     1996      1997      1998        
                     -------------------------------------------------- 
                                                                        
                   No.  %    No.  %   No.   %   No.  %    No.  %        
---------------------------------------------------------------------- 
B. pertussis       75  4.2  140  5.8  325  8.9 132  5.6  165   6.6      
B. parapertussis    7  0.4   20  0.8   32  0.9  11  0.5   NA   NA       
B. holmesii         0  0  3(sup a)0.1   6  0.2   9  0.4   15   0.6     
Total NP specimens 1,792     2,399   3,653     2,375     2,508          
reported                                                                
----------------------------------------------------------------------  
(sup a)Does not include the one case of B. holmesii isolated from 	
 blood.

To ascertain what symptoms were associated with B. holmesii isolation, 
we investigated the 23 cases identified in 24 months from January 1997 
through December 1998. We called providers and patients for disease 
histories and demographic information. Pertussis case report forms, 
modified to include more possible symptoms and underlying conditions, 
were used to record the information collected in the interviews.

Nineteen (83%) of the 23 B. holmesii-positive cases were in adolescents (11
to 19 years), 2 (9%) in young adults (20 and 29 years), 1 (4%) in a
10-year-old child, and 1 (4%) in an infant. All had cough. In addition, 14
(61%) had paroxysms, 2 (9%) had whoop, and 6 (26%) had posttussive
vomiting. No other symptoms were identified by patients or providers.
Fourteen (61%) of the 23 had no underlying conditions, 8 (35%) had minor
conditions such as occasional asthma or allergies, and 1 (4%) had chronic
fatigue.

The fact that cultures were taken from 20 (87%) of the 23 case-patients
within 14 days of cough onset generally excluded convalescent-stage
pertussis as a cause of symptoms. However, B. pertussis had been confirmed
in a 14-year-old girl, who had occasional asthma, 3 months before B.
holmesii was confirmedshe received a reculture because of a persistent
cough that had not resolved since the original infection. She had had
paroxysms and vomiting associated with the pertussis infection but no
symptoms other than cough at the time B. holmesii was isolated. Cultures
were taken on the same day from two sisters, 15 and 9 years old, each with
cough of fewer than 14 days. B. holmesii was culture-confirmed in the
15-year-old; B. pertussis was culture-confirmed in the 9-year-old. This
raises the question of whether B. pertussis and B. holmesii might
cocirculate.

At least 11 (48%) of the 23 cases were found during active surveillance for
pertussis in school and university settings, which may explain the age
profile of the cases. Three case-patients, with cough onset dates of April
1, 1997; February 27, 1998; and March 9, 1998, were students at the same
university. These cases, though not epidemiologically linked to each other,
were in symptomatic contacts of pertussis patients and were cultured as
part of an azithromycin efficacy study. At least eight other cases were
also in contacts of confirmed patients with pertussis and were detected
through active surveillance. No cases were definitively linked. Peak months
of cough onset were November and December (8 of the 23 cases), as is true
for pertussis in Massachusetts, with a smaller peak in March and April (6
of the 23 cases). The observed peak in November-December may be due to the
role of active surveillance for pertussis in ascertaining cases of B.
holmesii colonization.

The clinical profile of the 21 cases in adolescent and adult patients
infected with B. holmesii was compared with that of 122 culture-confirmed
pertussis cases in patients between 11 and 29 years of age with cough
onsets in the same period, i.e., 1997 through 1998. (Relevant clinical
information was not available for an additional 42 culture-confirmed
pertussis case-patients in this age group.) We did not consider cough
duration, because of imprecise data, but rather focused on the presence or
absence of three classic pertussis symptoms: paroxysms of cough, whoop, and
posttussive vomiting. Cases were categorized as patients with 0, 1, or 2 to
3 of these symptoms. (No separate category for three symptoms was used due
to a cell size of 0 in the case of B. holmesii.) On applying the chi-square
test for independent proportions, we found B. holmesii infection milder
(i.e., accompanied by fewer of the above three pertussis symptoms) than B.
pertussis infection (chi-square = 10, p <.01).

To rule out the possibility that the difference was due to more frequent
cultures for severe than for milder pertussis cases, we compared the 21
adolescent and adult B. holmesii patients with the 577
SLIserology-positive pertussis patients 11 to 29 years of age with cough
onsets in 1997 or 1998 for whom sufficient clinical data were available.
The SLI pertussis serology test is a single-serum enzyme-linked
immunosorbent assay for immunoglobulin G to pertussis toxin, available
since 1987. The assay is for use in persons > 11 years of age and is
optimally sensitive at 2 to 8 weeks after cough onset. By the same methods
as for the previous comparison, we found that the B. holmesii cases were
milder at a higher level of significance (chi-square = 69, p <10(sup -8).

Without knowing the prevalence of B. holmesii carriage in asymptomatic
persons, we cannot say with certainty that B. holmesii is the causative
agent for the respiratory symptoms of the patients from whom it was
isolated. Approximately half the cases were discovered through active
surveillance for pertussis. This, together with the fact that the symptoms
associated with B. holmesii were relatively mild, suggests that the
organism may not have been causing disease. On the other hand, B. holmesii
may be the etiologic agent, given that it is closely related to B.
pertussis and the associated symptoms (like those of B. parapertussis) are
similar. B. pertussis is the only Bordetella species known to produce
pertussis toxin, although B. parapertussis and B. bronchiseptica have
silent copies of the toxin gene (6). We do not know whether B. holmesii has
the toxin gene. However, since B. parapertussis can cause disease (albeit
not as severe as B. pertussis [7]), the presence of pertussis toxin is not
necessary for the development of symptoms.

Continued investigation, including conducting diagnostic tests for agents
such as Chlamydia and Mycoplasma and culturing symptomatic and asymptomatic
contacts, is warranted to ascertain the degree to which B. holmesii is
pathogenic in the respiratory system and contagious. If it is contagious,
antibiotic susceptibility testing is also needed. B. holmesii is
susceptible to some 15 antibiotics of a variety of classes (2,3), but
whether erythromycin, the drug of choice for pertussis, is effective is not
known. SLI is establishing routine erythromycin susceptibility testing of
B. pertussis and will also test B. holmesii isolates.

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

Acknowledgment

      We thank Colin D. Marchant for early suggestions regarding data
analysis.

      Dr. Yih is epidemiology coordinator for vaccine-preventable diseases
at the Massachusetts Department of Public Health. Her research interests
include ecologic and evolutionary aspects of infectious disease.

      Address for correspondence: W. Katherine Yih, State Laboratory
Institute, 5th floor, 305 South Street, Boston, MA 02130, USA; fax:
617-983-6840; e-mail: Katherine.Yih@state.ma.us.

References

  1. Weyant RS, Hollis DG, Weaver RE, Amin MFM, Steigerwalt AG, O'Connor
     SP, et al. Bordetella holmesii sp. nov., a new gram-negative species
     associated with septicemia. J Clin Microbiol 1995;33:1-7.
  2. Lindquist SW, Weber DJ, Mangum ME, Hollis DG, Jordan J. Bordetella
     holmesii sepsis in an asplenic adolescent. Pediatr Infect Dis J
     1995;14:813-5.
  3. Tang Y-W, Hopkins MK, Kolbert CP, Hartley PA, Severance PJ, Persing
     DH. Bordetella holmesii-like organisms associated with septicemia,
     endocarditis, and respiratory failure. Clin Infect Dis 1998;26:389-92.
  4. Morris JT, Myers M. Bacteremia due to Bordetella holmesii. Clin Infect
     Dis 1998;27:912-3.
  5. van den Akker WMR. Lipopolysaccharide expression within the genus
     Bordetella: influence of temperature and phase variation. Microbiol
     1998;144:1527-35.
  6. Arico B, Rappuoli R. Bordetella parapertussis and Bordetella
     bronchiseptica contain transcriptionally silent pertussis toxin genes.
     J Bacteriol 1987;169:2847-53.
  7. Mastrantonio P, Stefanelli P, Giuliano M, Herrera Rojas Y, Ciofi degli
     Atti M, Anemona A, et al. Bordetella parapertussis infection in
     children: epidemiology, clinical symptoms, and molecular
     characteristics of isolates. J Clin Microbiol 1998;36:999-1002.
--------------------------------------------------------------------------
Dispatches

New Cryptosporidium Genotypes in HIV-Infected Persons

Norman J. Pieniazek,* Fernando J. Bornay-Llinares,* Susan B. Slemenda,*
Alexandre J. da Silva,* Iaci N. S. Moura,* Michael J. Arrowood,* Oleg
Ditrich, and David G. Addiss*
Centers for Disease Control and Prevention, Atlanta, Georgia, USA; and
Institute of Parasitology AS CR, Cesk Budjovice, Czech Republic

---------------------------------------------------------------------------
      Using DNA sequencing and phylogenetic analysis, we identified
      four distinct Cryptosporidium genotypes in HIV-infected
      patients: genotype 1 (human), genotype 2 (bovine)
      Cryptosporidium parvum, a genotype identical to C. felis, and
      one identical to a Cryptosporidium sp. isolate from a dog. This
      is the first identification of human infection with the latter
      two genotypes.

Protozoan apicomplexan parasites from the genus Cryptosporidium infect a
wide variety of hosts (1). The parasites are transmitted to humans through
contaminated drinking water (2), contact with infected animals, and contact
with infected persons (3). In the immunocompetent, cryptosporidiosis
manifests itself as self-limited diarrhea, sometimes accompanied by nausea,
abdominal cramps, fever, and vomiting. In the immunodeficient, however,
cryptosporidiosis may be severe, chronic, and life-threatening (4).

Cross-infection experiments, in which Cryptosporidium oocysts were obtained
from animals of one species and fed to animals of another species, have
investigated the host specificity of this parasite (5). The differences
observed in the host range of putative C. parvum isolates led to a proposal
to establish the Cryptosporidium isolate originating from guinea pigs,
morphologically indistinguishable from C. parvum, as a new speciesC.
wrairisolely on the basis of experimental infection (6). The possibility
of many Cryptosporidium species fostered the development of techniques
suitable for typing isolates. Commonly used techniques are isoenzyme
analysis (7), Western blotting (8,9), random amplified polymorphic DNA
analysis (10-12), polymerase chain reaction restriction fragment length
polymorphism (PCR-RFLP) analysis (13,14), and PCR followed by DNA
sequencing (11,15-19).

Early studies of the polymorphism of isolates classified as C. parvum found
significant geographic variation among isolates (20) in the region coding
for the small subunit ribosomal RNA (SSU-rRNA), commonly used for taxonomic
classification. Recently, it has been shown (21) that one of the sequences
used in this analysis (22) was erroneously identified as a C. parvum
sequence, while in fact it was C. muris. More recent work (e.g., Le Blancq
et al. [23] and GenBank entry AF040725) has shown that the SSU-rRNA region
of the C. parvum zoonotic (bovine) genotype does not show heterogeneity and
is practically identical to the sequence submitted to GenBank in 1993
(accession number L16996, [24,25]).

Recently, consistent results of typing bovine and human C. parvum isolates
led to unequivocal recognition of two genotypes of C. parvum. These two
genotypes were reproducibly differentiated by sequencing the SSU-rRNA
coding region (16), sequencing the Cryptosporidium thrombospondin-related
adhesion protein (TRAP-C2) gene (17), and PCR-RFLP analysis of the
Cryptosporidium oocyst wall protein (COWP) gene (15). Thus far, the
anthroponotic genotype (genotype 1) has been found only in infected humans,
while the zoonotic genotype (genotype 2) has been found both in infected
humans and in livestock, e.g., cows, lambs, goats, and horses. The
published partial SSU-rRNA sequence of the C. parvum genotype 1 (16) is
identical to the Cryptosporidium SSU-rRNA sequence that we observed in
PCR-amplified DNA from a patient with AIDS (GenBank accession number
L16997). That the data from different laboratories for these two genotypes
were reproducible calls into question the recent conclusion of Tzipori and
Griffiths (26) that "there are no clearly defined and fully characterized
reference `strains' of Cryptosporidium."

Other new Cryptosporidium genotypes have been identified in various animals
(pigs, mice, cats, and koalas) by sequencing the region coding for SSU-rRNA
(16,27,28). The cat genotype is thought to represent C. felis (28). When
morphologic and other data are available for these new genotypes, they may
be recognized as new Cryptosporidium species.

We present results of typing Cryptosporidium isolates by direct sequencing
the variable region of the SSU-rRNA amplified from fecal specimens with
Cryptosporidium genus-specific diagnostic PCR primers (25). Applying this
method to a set of specimens from a 3-year longitudinal study on the risk
for enteric parasitosis and chronic diarrhea in immunodeficient patients
with a low CD4+ count, we found the first human cases of infection by C.
felis and by a newly identified zoonotic Cryptosporidium species possibly
originating from a dog.

The Study

All analyzed specimens were collected from January 1991 through September
1994 in a study assessing the impact of enteric parasite-associated
diarrhea in persons infected with HIV (29). Study participants answered
comprehensive questionnaires concerning clinical and epidemiologic
information and provided stool specimens monthly. All stool specimens were
examined for C. parvum by Kinyoun carbol-fuchsin modified acid-fast stain
(30) and direct immunofluorescence (31). Stained slides were examined by
observing 200 oil-immersion fields. C. parvum was associated with 18 (5.1%)
of the 354 acute episodes and 36 (12.9%) of the chronic episodes of
diarrhea reported by the participants. All specimens from this study were
preserved in separate vials of 10% formalin and polyvinyl alcohol (Para-Pak
Stool System; Meridian Diagnostics, Inc., Cincinnati, OH) and thus were not
suitable for molecular analysis. Some specimens were originally aliquoted
and stored without preservation at -80C. Of this set, we selected 18
available specimens for 10 randomly chosen Cryptosporidium-positive
patients for this study.

An aliquot of approximately 300 l of each stool specimen was suspended in
1 ml of 0.01 M phosphate-buffered saline, pH 7.2, containing 0.01 M of EDTA
(PBS-EDTA), and the suspension was centrifuged at 14,000 x g, 4C for 5
minutes. The pellet from this centrifugation was washed two more times
under the same conditions. The pellet was resuspended in 300 l of PBS-EDTA
and used for DNA extraction, performed with the FastPrep disrupter and the
FastDNA kit (BIO 101, Inc., Vista, CA) (32). Extracted DNA was stored at
4C until PCR amplification.

Cryptosporidium genus-specific primers (CPBDIAGF and CPBDIAGR) were used to
amplify the Cryptosporidium SSU-rRNA variable region (24,25). The
conditions for PCR were 95C for 15 minutes; 45 cycles of denaturation at
94C for 30 seconds, annealing at 65C for 30 seconds, extension at 72C
for 1 minute, 30 seconds; followed by extension at 72C for 9 minutes; and
finished with a hold step at 4C.

PCR products were analyzed by electrophoresis on 2% SeaKem GTG agarose
(Cat. No. 50074, FMC Bioproducts, Rockland, ME), stained with ethidium
bromide, and visualized on UV transilluminator. The size of the diagnostic
fragment amplified with these primers for C. parvum genotype 1 (GenBank
accession number L16997) was 438 bp, for C. parvum genotype 2 (L16996) 435
bp, for C. baileyi (L19068) 428 bp, and for C. muris (L19069) 431 bp.

PCR products were purified by using the Wizard PCR Preps kit (Cat. No.
A7170, Promega, Madison, WI). Sequencing reactions were done with the
Perkin Elmer Big Dye kit (Cat. No. 4303149, PE Biosystems, Foster City, CA)
and analyzed on the Perkin Elmer ABI 377 automatic DNA sequencer. Sequences
were assembled by using the program SeqMan II (DNASTAR Inc., Madison, WI).
Sequences were aligned with the program MACAW (33) and analyzed by programs
from the PHYLIP (phylogeny inference program) package (34).

Findings

After the 18 fecal specimens were retrieved from storage and processed for
PCR with the CPBDIAGF/CPBDIAGR diagnostic primer set, visualization of PCR
products on 2% agarose gels found two different sizes of diagnostic bands.
The size of the Cryptosporidium diagnostic bands for patients 53, 75, 119,
124, 153, 278, and 554 did not differ significantly from the size of the
standard band (approximately 435 bp) obtained with cloned SSU-rRNA for the
C. parvum genotype 2 (Figure 1, lane 1). On the other hand, the
Cryptosporidium diagnostic bands in samples from patients 84, 184, and 485
were visibly larger, approximately 450 bp (Figure 1, lane 3 shows data for
patient 84).		

               [Fig]     
							    DNA sequence analysis showed
Figure 1. Agarose gel (2%) visualization      four types of sequences
of diagnostic polymerase chain reaction       (Table 1). The sequence of
products of four Cryptosporidium              the diagnostic band from
genotypes. Lane S, standard 100-bp ladder;    patient 53 was 435 bp long
lane 1, patient 53, zoonotic genotype 2;      and was identical to the
lane 2, patient 119, Cryptosporidium sp.      corresponding fragment of the
(zoonotic, canine genotype); lane 3,          C. parvum genotype 2 (GenBank
patient 84, C. felis (zoonotic, feline        accession number L16996)
genotype); lane 4, patient 75,                SSU-rRNA. The diagnostic
anthroponotic genotype 1.                     bands in patients 75, 124,
                                              153, 278, and 554 were 438 bp
                                              long and were identical to
the corresponding fragment of the C. parvum genotype 1 SSU-rRNA (GenBank
accession number L16997). The sequence from patient 119 was 429 bp long;
the diagnostic bands from patients 84, 184, and 485 were 455 bp long and
were identical to each other. Sequence similarity searches of GenBank
database and molecular phylogeny analysis of the two latter types of
sequences showed that, while they were not identical to any sequences in
GenBank, they clustered within other representative SSU-rRNA sequences from
the genus Cryptosporidium (results not shown). In addition, these sequences
were significantly different from recently reported partial SSU-rRNA
sequences (16,19,27,28). The 429-bp-long diagnostic fragment in patient 119
was identical to a recently identified canine C. parvum isolate SSU-rRNA
sequence (GenBank accession number AF112576). The longest sequence (455 bp)
from patients 84, 184, and 495 was identical in a 100-bp overlap to an
updated sequence (GenBank accession number AF097430) for C. felis (also
called feline C. parvum genotype) (28). Alignment of these sequences is
shown in Figure 2. In the hypervariable alignment region (from position 101
to 110), all genotypes display a variable number of thymidines. Genotype 1
has the longest stretch, 11 thymidines, while the Cryptosporidium sp.
(canine genotype) has only one. The sequence for C. felis has five
insertions at alignment positions 45, 86, 111, 187, and 218, as well as one
deletion of two thymidines at positions 197 and 198. Apart from the
difference in the hypervariable region, the Cryptosporidium sp. (canine
genotype) has a deletion of two bases at positions 49 and 50.

Results of genotyping the 18 specimens are in Table 2. For patients 53,
124, 153, 184, 278, and 485, only a single specimen was available. For
other patients (e.g., patient 554), four specimens collected during 12
months were available. The same Cryptosporidium genotype persisted
throughout a patient's infection.

Table 1. Cryptosporidium genotypes for 10 selected
patients in this study
-----------------------------------------------------------
Patient no.    Band size(sup a)(bp) C. parvum genotype
-----------------------------------------------------------
53                    435           Zoonotic (genotype 2)
75, 124, 153          438           Anthroponotic
  278, 554                            (genotype 1)
119                   429           Cryptosporidium sp.
                                      (canine genotype)
84, 184, 485          455           C. felis
-----------------------------------------------------------
(sup a)Size of the Cryptosporidium diagnostic band obtained 
with the CPBDIAGF/CPBDIAGR PCR primer pair

		
				[Fig]
     
Figure 2. Alignment of the Cryptosporidium small subunit ribosomal DNA
(SSU-rRNA) diagnostic fragments obtained with the CPBDIAGF/CPBDIAGR
polymerase chain reaction primer pair for the four genotypes. Only the first
300 columns of the alignment are shown, as the remaining columns were
identical for all genotypes. Gaps are shown with dashes (-), and bases
identical to the base in the first row (C. felis) are shown with dots (.).
Numbers to the right of the alignment show sequence positions for each
genotype. The sequences for the SSU-rRNA diagnostic fragment of all four
genotypes were submitted to GenBank and were assigned accession numbers
AF087574, AF087575, AF087576, and AF087577.

Table 2. Persistence of Cryptosporidium genotypes in
patients(sup a)
-------------------------------------------------------------
                                 Month of follow-up
             No. of     -------------------------------------
Patient     available
no.         specimens    1  2  3  4  5 6  7  8 9  10  11  12
-------------------------------------------------------------
  53            1        B

  75            2        H  -  -  H

  84            4        F  -  F  -  F F

119             2        C  -  C

124             1        H

153             1        H

184             1        F

278             1        H

485             1        F

554             4        H  -  - -   - -  -  H -   H   -  H

-------------------------------------------------------------
(sup a)B, C. parvum genotype 2 (zoonotic, bovine); C,
Cryptosporidium sp. (zoonotic, canine); F, C. felis
(zoonotic, feline); H, C. parvum genotype 1 (anthroponotic);
-, specimen not available.

Conclusions

Using the sequence of a diagnostic fragment of SSU-rRNA, as well as two
well-established genotypes of C. parvum (anthroponotic genotype 1 and
zoonotic genotype 2), we detected two new Cryptosporidium genotypes. The
first, in patients 84, 184, and 485, was identical to the feline
Cryptosporidium genotype (28), also described as C. felis. The second, in
two specimens from patient 119, represents the newly identified
Cryptosporidium sp. found in a sequence originating from a dog (GenBank
accession number AF112576).

New taxons may be established within the genus Cryptosporidium on the basis
of the isolate's host range together with molecular data, even though
morphologic criteria are apparently lacking. We propose to use C. felis
Iseki, 1979, instead of feline C. parvum genotype; we believe that the
anthroponotic genotype 1 of C. parvum and the Cryptosporidium sp. (canine
genotype) described here should be named as distinct species. Although the
taxonomy of protists is morphology-based, the taxonomy of bacteria is being
reevaluated on the basis of molecular data (35). There is no reason to use
different approaches to these two kingdoms, nor is it necessary to
postulate that the basis for speciation of Cryptosporidium remains
ambiguous or that molecular data lend little support to separation of
Cryptosporidium into distinct, valid species (26). Further, we see no
evidence for the unconventional conclusions of Tzipori and Griffiths that
"...genetic markers in C. parvum can change upon passage to a different
host, possibly through a selective mechanism favoring different
populations" (26) nor for those of Widmer (21) that "...genetic studies and
experimental infections suggest that a selective mechanism triggered by a
change in the intestinal environment might be involved in shaping the
genetic make-up of C. parvum populations."

The finding of new Cryptosporidium genotypes in immunodeficient patients
might suggest a unique susceptibility to infections by divergent
Cryptosporidium species circulating in companion animals or livestock.

However, recent identification of C. felis in a cow (36) may indicate a
complex pattern of flow of different Cryptosporidium species in the
environment. When more data on the distribution of these species become
available, public health and preventive measures will need to be
reevaluated, and isolates may need to be renamed to reflect their natural
history. Finally, it is clear that Kinyoun carbol-fuchsin modified
acid-fast stain (30) and direct immunofluorescence (31) could detect all
genotypes, but this may not be true for diagnostic reagents that may be
affected directly (PCR) or indirectly (Ab) by the genetic composition of
the new Cryptosporidium isolates reported here. Thus, discovery of these
new Cryptosporidium genotypes in human cryptosporidiosis should cause
existing diagnostic tools to be reevaluated.

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

Acknowledgment

      The authors thank Jane L. Carter for her expert help with running the
DNA sequencer.

      Dr. Pieniazek is a microbiologist with the National Center for
Infectious Diseases, CDC. His research interests include molecular
reference diagnosis of parasitic diseases and molecular systematics.

     Address for correspondence: Norman J. Pieniazek, Division of Parasitic
Diseases, Centers for Disease Control and Prevention, 4770 Buford Highway
NE, Mail Stop F13, Atlanta, GA, 30341-3724, USA; fax: 770-488-4108; e-mail:
nxp3@cdc.gov.

References

  1. Fayer R, Speer CA, Dubey JP. The general biology of Cryptosporidium.
     In: Fayer R, editor. Cryptosporidium and cryptosporidiosis. Boca Raton
     (FL): CRC Press; 1997. p. 1-41.
  2. MacKenzie WR, Schell WL, Blair KA, Addiss DG, Peterson DE, Hoxie NJ,
     et al. Massive outbreak of waterborne Cryptosporidium infection in
     Milwaukee, Wisconsin: recurrence of illness and risk of secondary
     transmission. Clin Infect Dis 1995;21:57-62.
  3. Navin TR. Cryptosporidiosis in humans: review of recent epidemiologic
     studies. Eur J Epidemiol 1985;1:77-83.
  4. Arrowood MJ. Diagnosis. In: Fayer R, editor. Cryptosporidium and
     cryptosporidiosis. Boca Raton (FL): CRC Press; 1997. p. 43-64.
  5. Lindsay DS. Laboratory models of cryptosporidiosis. In: Fayer R,
     editor. Cryptosporidium and cryptosporidiosis. Boca Raton (FL): CRC
     Press; 1997. p. 209-23.
  6. Vetterling JM, Jervis HR, Merrill TG, Sprinz H. Cryptosporidium wrairi
     sp. n. from the guinea pig Cavia porcellus, with an emendation of the
     genus. Journal of Protozoology 1971;18:243-7.
  7. Awad-el-Kariem FM, Robinson HA, Dyson DA, Evans D, Wright S, Fox MT,
     et al. Differentiation between human and animal strains of
     Cryptosporidium parvum using isoenzyme typing. Parasitology
     1995;110:129-32.
  8. McLauchlin J, Casemore DP, Moran S, Patel S. The epidemiology of
     cryptosporidiosis: application of experimental sub-typing and antibody
     detection systems to the investigation of water-borne outbreaks. Folia
     Parasitol 1998;45:83-92.
  9. Nichols GL, McLauchlin J, Samuel D. A technique for typing
     Cryptosporidium isolates. Journal of Protozoology 1991;38:237S-40.
 10. Carraway M, Widmer G, Tzipori S. Genetic markers differentiate C.
     parvum isolates. J Eukaryot Microbiol 1994;41:26S.
 11. Morgan UM, Constantine CC, O'Donoghue P, Meloni BP, O'Brien PA,
     Thompson RCA. Molecular characterization of Cryptosporidium isolates
     from humans and other animals using random amplified polymorphic DNA
     analysis. Am J Trop Med Hyg 1995;52:559-64.
 12. Shianna KV, Rytter R, Spanier JG. Randomly amplified polymorphic DNA
     PCR analysis of bovine Cryptosporidium parvum strains isolated from
     the watershed of the Red River of the North. Appl Environ Microbiol
     1998;64:2262-5.
 13. Bonnin A, Fourmaux MN, Dubremetz JF, Nelson RG, Gobet P, Harly G, et
     al. Genotyping human and bovine isolates of Cryptosporidium parvum by
     polymerase chain reaction-restriction fragment length polymorphism
     analysis of a repetitive DNA sequence. FEMS Microbiol Lett
     1996;137:207-11.
 14. Widmer G, Tzipori S, Fichtenbaum CJ, Griffiths JK. Genotypic and
     phenotypic characterization of Cryptosporidium parvum isolates from
     people with AIDS. J Infect Dis 1998;178:834-40.
 15. Spano F, Putignani L, McLauchlin J, Casemore DP, Crisanti A. PCR-RFLP
     analysis of the Cryptosporidium oocyst wall protein (COWP) gene
     discriminates between C. wrairi and C. parvum, and between C. parvum
     isolates of human and animal origin. FEMS Microbiol Lett
     1997;150:209-17.
 16. Morgan UM, Constantine CC, Forbes DA, Thompson RCA. Differentiation
     between human and animal isolates of Cryptosporidium parvum using rDNA
     sequencing and direct PCR analysis. J Parasitol 1997;83:825-30.
 17. Peng MM, Xiao L, Freeman AR, Arrowood MJ, Escalante AA, Weltman AC, et
     al. Genetic polymorphism among Cryptosporidium parvum isolates:
     evidence of two distinct human transmission cycles. Emerg Infect Dis
     1997;3:567-73.
 18. Chrisp CE, LeGendre M. Similarities and differences between DNA of
     Cryptosporidium parvum and C. wrairi detected by the polymerase chain
     reaction. Folia Parasitol 1994;41:97-100.
 19. Carraway M, Tzipori S, Widmer G. Identification of genetic
     heterogeneity in the Cryptosporidium parvum ribosomal repeat. Appl
     Environ Microbiol 1996;62:712-6.
 20. Kilani RT, Wenman WM. Geographical variation in 18S rRNA gene sequence
     of Cryptosporidium parvum. Int J Parasitol 1994;24:303-6.
 21. Widmer G. Genetic heterogeneity and PCR detection of Cryptosporidium
     parvum. Adv Parasitol 1998;40:223-39.
 22. Cai J, Collins MD, McDonald V, Thompson DE. PCR cloning and nucleotide
     sequence determination of the 18S rRNA genes and internal transcribed
     spacer 1 of the protozoan parasites Cryptosporidium parvum and
     Cryptosporidium muris. Biochim Biophys Acta 1992;1131:317-20.
 23. Le Blancq SM, Khramtsov NV, Zamani F, Upton SJ, Wu TW. Ribosomal RNA
     gene organization in Cryptosporidium parvum. Mol Biochem Parasitol
     1997;90:463-78.
 24. Johnson DW, Pieniazek NJ, Rose JB. DNA probe hybridization and PCR
     detection of Cryptosporidium parvum compared to immunofluorescence
     assay. Water Science and Technology 1993;27:77-84.
 25. Johnson DW, Pieniazek NJ, Griffin DW, Misener L, Rose JB. Development
     of a PCR protocol for sensitive detection of Cryptosporidium oocysts
     in water samples. Appl Environ Microbiol 1995;61:3849-55.
 26. Tzipori S, Griffiths JK. Natural history and biology of
     Cryptosporidium parvum. Adv Parasitol 1998;40:5-36.
 27. Morgan UM, Sargent KD, Deplazes P, Forbes DA, Spano F, Hertzberg H, et
     al. Molecular characterization of Cryptosporidium from various hosts.
     Parasitology 1998;117:31-7.
 28. Sargent KD, Morgan UM, Elliot A, Thompson RCA. Morphological and
     genetic characterization of Cryptosporidium oocysts from domestic
     cats. Vet Parasitol 1998;77:221-7.
 29. Navin TR, Weber R, Vugia DJ, Rimland D, Roberts JM, Addiss DG, et al.
     Declining CD4+ T-lymphocyte counts are associated with increased risk
     of enteric parasitosis and chronic diarrhea: results of a 3-year
     longitudinal study. J Acquir Immune Defic Syndr Hum Retrovirol
     1999;20:154-9.
 30. Melvin DM, Brooke MM. Laboratory procedures for the diagnosis of
     intestinal parasites. 3rd ed. Atlanta: Centers for Disease Control;
     1982. Publication no. (CDC) 85-8282.
 31. Garcia LS, Shum AC, Bruckner DA. Evaluation of a new monoclonal
     antibody combination reagent for the direct fluorescent detection of
     Giardia cysts and Cryptosporidium oocysts in human fecal specimens. J
     Clin Microbiol 1992;30:3255-7.
 32. da Silva AJ, Bornay-Llinares FJ, Moura INS, Slemenda SB, Tuttle JL,
     Pieniazek NJ. Fast and reliable extraction of protozoan parasite DNA
     from fecal specimens. Molecular Diagnosis. In press 1999.
 33. Schuler GD, Altschul SF, Lipman DJ. A workbench for multiple alignment
     construction and analysis. Proteins 1991;9:180-90.
 34. Felsenstein J. PHYLIPphylogeny inference package. Cladistics
     1989;5:164-6.
 35. Roth A, Fischer M, Hamid ME, Michalke S, Ludwig W, Mauch H.
     Differentiation of phylogenetically related slowly growing
     mycobacteria based on 16S-23S rRNA gene internal transcribed spacer
     sequences. J Clin Microbiol 1998;36:139-47.
 36. Bornay-Llinares FJ, da Silva AJ, Moura IN, Myjak P, Pietkiewicz H,
     Kruminis-Lozowska W, et al. Identification of Cryptosporidium felis in
     a cow by morphologic and molecular methods. Appl Environ Microbiol
--------------------------------------------------------------------------
Dispatches

Fatal Case Due to Methicillin-Resistant Staphylococcus aureus Small Colony
Variants in an AIDS Patient

Harald Seifert,* Christoph von Eiff, and Gerd Ftkenheuer
*University of Cologne, Cologne, Germany; and Westflische
Wilhelms-Universitt, Mnster, Germany

---------------------------------------------------------------------------
      We describe the first known case of a fatal infection with
      small colony variants of methicillin-resistant Staphylococcus
      aureus in a patient with AIDS. Recovered from three blood
      cultures as well as from a deep hip abscess, these variants may
      have resulted from long-term antimicrobial therapy with
      trimethoprim/sulfamethoxazole for prophylaxis of Pneumocystis
      carinii pneumonia.

Staphylococcus aureus causes acute and often fatal infections. Small colony
variants (SCVs), which are subpopulations of S. aureus, are implicated in
persistent and recurrent infections (in particular osteomyelitis, septic
arthritis, respiratory tract infections in patients with cystic fibrosis,
and deep-seated abscesses) (1-4). These phenotypic variants produce small,
slow-growing, nonpigmented, nonhemolytic colonies on routine culture media,
making correct identification difficult for clinical laboratories.
Biochemical characterization of these variants suggests that they are
deficient in electron transport activity (5).

We report a fatal case of a persistent deep-seated hip abscess due to
methicillin-resistant S. aureus SCVs that led to osteomyelitis and
bloodstream infection in a patient with AIDS.

Case Report

A 36-year-old man with AIDS came to the Cologne University Hospital,
Cologne, Germany, in June 1997 with fever and progressive pain (of 6 weeks
duration) in his right hip. HIV infection had been diagnosed in 1986. In
1994, his CD4 cell count was 250/L, and oral zidovudine therapy was
started. His medical history included Pneumocystis carinii pneumonia,
pulmonary tuberculosis, and recurrent oral thrush; his medication included
zidovudine, lamivudine, fluconazole, and trimethoprim/sulfamethoxazole. In
September 1996, he was in a traffic accident and had severe cerebral trauma
resulting in spastic hemiparesis with occasional seizures. After an
intramuscular injection 2 months before admission, pus was surgically
drained to treat recurrent abscesses of his right hip. Specimens for
culture were not obtained.

Physical examination found limited mobility of his right thigh and a
tender, nondraining scar at the site of surgical drainage. Neither warmth
nor swelling was observed over his right hip. Vital signs were temperature,
38.2C; respiration rate, 28; and heart rate, 108. He was awake and alert
and had spastic paresis in his right arm.

Laboratory studies performed on admission showed hemoglobin, 10.8 g/dL;
leukocyte count, 3,000/L with a normal differential; CD4 cell count,
20/L; platelet count, 131,000/L; C-reactive protein, 184 mg/L; and
alkaline phosphatase, 1490 U/L. Radiographs of the chest and a plain film
of the pelvis were normal. A triple-phase bone scan showed an area of minor
tracer accumulation in the acetabulum region of the right hip. Blood
cultures were drawn, but antimicrobial therapy was withheld until culture
results became available.

On hospital day 2, one of two blood cultures drawn on admission yielded
nonhemolytic staphylococci that were clumping factornegative. The organisms
were initially misidentified as coagulase-negative staphylococci and were
considered contaminants. Empiric antistaphylococcal therapy with
clindamycin (600 mg q8hr) was instituted. On hospital day 4, two sets of
blood cultures obtained on hospital day 2 yielded phenotypically identical
organisms, which on the basis of a positive tube coagulase test were
identified as oxacillin-resistant S. aureus. The colony morphology was
suggestive of an SCV of S. aureus. The patient was started on parenteral
vancomycin treatment (1 g q12hr). However, his condition deteriorated
rapidly, and he died of refractory septic shock 6 days after admission.

Autopsy showed a large (12 x 10 x 8 cm), deep-seated abscess of the right
hip and osteomyelitis of the ischial tuberosity. Both SCVs and typical
large colony forms of S. aureus were cultured from postmortem specimens of
the abscess and the bone.

Findings

S. aureus SCVs were recovered from one of two blood culture sets obtained
on admission and from two of four blood culture sets obtained on hospital
day 2. Growth was not detected until the blood culture bottles had been
incubated 24 hours. S. aureus with a normal phenotype was recovered from
nose and throat specimens but not from blood cultures, whereas both SCVs
and typical S. aureus phenotypes were isolated from the deep hip abscess
(Figure 1) before death, as well as from a postmortem specimen. All
isolates were clumping factornegative but showed a delayed positive
reaction in the tube-coagulase test at 24 hours. The results of the ID 32
staph test did not unambiguously identify SCVs as S. aureus because the
tests for urease and trehalose were negative. Both the nuc gene and the coa
gene were identified by polymerase chain reaction (PCR) amplification.
Methicillin resistance was confirmed for both small and large colony forms
by PCR amplification of the mecA gene.

When cultured without supplementation, all SCVs were nonpigmented and
nonhemolytic. Supplementation with hemin, thymidine, or menadione
identified two SCVs showing thymidine auxotrophy and a combined thymidine
and menadione auxotrophy, respectively. All SCVs were stable on repeated
subculturing.

Epidemiologic typing by PCR analysis of inter-IS256 spacer length
polymorphisms (Figure 2) and pulsed-field gel electrophoresis of genomic
DNA (data not shown) showed identical banding patterns for both SCVs and
large colony forms, which indicates that the phenotypically different S.
aureus isolates represented a single strain. Antimicrobial susceptibility
testing was performed by microbroth dilution, according to the National
Committee for Clinical Laboratory Standards guidelines. Susceptibility to
trimethoprim/sulfamethoxazole was tested with Etest (AB Biodisk, Solna,
Sweden). In contrast to current standards, the MICs for SCVs were
determined after 48 hours of incubation at 35C. Susceptibility testing
showed that all S. aureus isolates were resistant to penicillin (MIC, >8
g/mL), ampicillin (MIC, >32 g/mL), oxacillin (MIC, >8 g/mL),
erythromycin (MIC, >32 g/mL), clindamycin (MIC, >32 g/mL), ciprofloxacin
(MIC, >8 g/mL), gentamicin (MIC, >500 g/mL), and
trimethoprim/sulfamethoxazole (MIC, >32 g/mL) and susceptible to
vancomycin (MICs, 1-2 g/mL), teicoplanin (MICs, 0.5-1 g/mL), and
quinupristin/dalfopristin (MICs, 0.5-1 g/mL). No differences in MICs were
observed between S. aureus SCVs and S. aureus isolates with normal
phenotype.

				[Fig]

Figure 1.
Staphylococcus aureus small colony variants(A) and S. aureus
with a normal phenotype(B) cultured on sheep blood agar after
24 hours of incubation at 35C. Staphylococci were identified
by conventional methods(6) and with the ID 32 Staph system
(bioMrieux, Marcy-L'Etoile, France) following the instructions 
of the manufacturer. The tube-coagulase test was read after
24 hours. S. aureus isolates were characterized as small
colony variants as described before(7-8). Auxotrophic              
requirements were evaluated with 10-g hemin disks, 1.5-g                                                       
menadione disks, and 1.5-g thymidine disks on Mueller-Hinton                                            
agar and on chemically defined medium (CDM) agar as well as                
on CDM agar supplemented with 1 g/mL hemin, 100 g/mL
thymidine, and 1 g/mL menadione, menadione,                                     

                    
				[Fig]

Figure 2. Fingerprint patterns obtained for Staphylococcus 
aureus small colony variants (lanes 3-5, bloodculture 
isolates; lanes 6 and 7, isolates from hip abscess; lane 8, 
postmortem specimen) and S. aureus isolates with a normal
phenotype (lanes 10 and 11, isolates from nose and throat; 
lanes 12 and 13,isolates from hip abscess and postmortem 
specimen) after polymerase chain reaction (PCR) 
analysis of inter-IS256 spacer length showing identical 
strains. Lane 1, 100-bp ladder; lanes 2, 9, and 16,
methicillin-resistant S. aureus (MRSA) reference strain;
lanes 14 and 15, epidemiologically unrelated MRSA strains. 
Strain relatedness of all isolates with different colony
morphologies and from different sources was analyzed by PCR 
analysis of inter-IS256 spacer length polymorphisms (9) and
pulsed-field gel electrophoresis after SmaI restriction (8). 
Minor modifications included the use of brain heart infusion
broth instead of trypticase soy broth to obtain sufficient 
growth of S. aureus small colony variants.

To our knowledge, this case represents the first of a serious S. aureus
infection in an AIDS patient in which all blood cultures yielded SCVs. The
SCVs' unusual morphologic appearance and slow growth delayed the correct
identification of these organisms as S. aureus. The empiric antimicrobial
regimen in our patient did not include a glycopeptide, because of the low
rate of methicillin resistance in community-acquired S. aureus infection in
Germany. Appropriate antistaphylococcal therapy was, therefore, not started
until hospital day 4. Delayed antimicrobial therapy on day 4 rather than on
day 2 may have contributed to the patient's death.

Proctor and colleagues recently reported five cases in which SCVs of S.
aureus were implicated in persistent and relapsing infections. They
identified only a single case reported in the previous 17 years and
ascribed this to insufficient ability of laboratories to identify these
organisms (8). In most cases, patients had received antibiotics.
Aminoglycoside treatment may have selected for S. aureus SCVs (10), and in
cases of osteomyelitis or deep-seated abscesses, persistence of these
variants in the intracellular milieu may have permitted evasion of host
defenses and allowed for the development of resistance to antimicrobial
therapy (7,11). Von Eiff and colleagues recently reported four cases of
chronic osteomyelitis due to SCVs of S. aureus in patients who had received
gentamicin beads as an adjunct to surgical therapy for osteomyelitis (2).
Kahl et al. described persistent infection with S. aureus SCVs in patients
with cystic fibrosis (4). All these patients had received long-term
trimethoprim/sulfamethoxazole prophylaxis. It may be tempting to speculate
that administration of trimethoprim/sulfamethoxazole for prophylaxis
against P. carinii pneumonia may have selected for SCVs within the
patient's large hip abscess. Further prospective studies are needed to
assess the role of S. aureus SCVs in HIV-infected patients on long-term
antimicrobial therapy.

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

      Dr. Seifert is assistant professor at the Institute of Medical
Microbiology and Hygiene, University of Cologne, Germany. His research
interests include the molecular epidemiology of nosocomial pathogens, in
particular Acinetobacter species, catheter-related infections, and
antimicrobial resistance.

      Address for correspondence: Harald Seifert, Institute of Medical
Microbiology and Hygiene, University of Cologne, Goldenfelsstrae 19-21,
50935 Cologne, Germany; fax: 49-221-478-3081; e-mail:
harald.seifert@uni-koeln.de.

References

  1. Proctor RA, Balwit JM, Vesga O. Variant subpopulations of
     Staphylococcus aureus as cause of persistent and recurrent infections.
     Infectious Agents and Disease 1994;3:302-12.
  2. von Eiff C, Bettin D, Proctor RA, Rolauffs B, Lindner N, Winkelmann W,
     et al. Recovery of small colony variants of Staphylococcus aureus
     following gentamicin bead placement for osteomyelitis. Clin Infect Dis
     1997;25:1250-1.
  3. Spearman P, Lakey D, Jotte S, Chernowitz A, Claycomb S, Stratton C.
     Sternoclavicular joint septic arthritis with small colony variant
     Staphylococcus aureus. Diagn Microbiol Infect Dis 1996;26:13-5.
  4. Kahl B, Herrmann M, Everding AS, Koch HG, Becker K, Harms E, et al.
     Persistent infection with small colony variant strains of
     Staphylococcus aureus in patients with cystic fibrosis. J Infect Dis
     1998;177:1023-9.
  5. von Eiff C, Heilmann C, Proctor RA, Woltz C, Peters G, Gtz F. A
     site-directed Staphylococcus aureus hemB mutant is a small colony
     variant which persists intracellularly. J Bacteriol 1997;179:4706-12.
  6. Kloos WE, Bannerman TL. Staphylococcus and micrococcus. In: Murray PR,
     Baron EJ, Pfaller MA, Tenover FC, Yolken RH, editors. Manual of
     clinical microbiology. 6th ed. Washington: American Society for
     Microbiology; 1995. p. 282-98.
  7. Balwit JM, van Langevelde P, Vann JM, Proctor RA. Gentamicin-resistant
     menadione and hemin auxotrophic Staphylococcus aureus persist within
     cultured endothelial cells. J Infect Dis 1994;170:1033-7.
  8. Proctor RA, van Langevelde P, Kristjansson M, Maslow JN, Arbeit RD.
     Persistent and relapsing infections associated with small colony
     variants of Staphylococcus aureus. Clin Infect Dis 1995;20:95-102.
  9. Deplano A, Vaneechoutte M, Verschraegen G, Struelens MJ. Typing of
     Staphylococcus aureus and Staphylococcus epidermidis by PCR analysis
     of inter-IS256 spacer length polymorphisms. J Clin Microbiol
     1997;35:2580-7.
 10. Pelletier LL Jr, Richardson M, Feist M. Virulent gentamicin-induced
     small colony variants of Staphylococcus aureus. J Lab Clin Med
     1979;94:324-34.
 11. Proctor RA, Kahl B, von Eiff C, Vaudaux PE, Lew DP, Peters G.
     Staphylococcal small colony variants have novel mechanisms for
     antibiotic resistance. Clin Infect Dis 1998;27 Suppl 1:S68-74.
--------------------------------------------------------------------------
Dispatches

Application of Data Mining to Intensive Care Unit Microbiologic Data(ft 1)

Stephen A. Moser, Warren T. Jones, and Stephen E. Brossette
The University of Alabama at Birmingham, Birmingham, Alabama, USA

---------------------------------------------------------------------------
      We describe refinements to and new experimental applications of
      the Data Mining Surveillance System (DMSS), which uses a large
      electronic health-care database for monitoring emerging
      infections and antimicrobial resistance. For example,
      information from DMSS can indicate potentially important shifts
      in infection and antimicrobial resistance patterns in the
      intensive care units of a single health-care facility.

We have defined a new exploratory data mining process for automatically
identifying new, unexpected, and potentially interesting patterns in
hospital infection control and public health surveillance data. This
process, and the system based on it, Data Mining Surveillance System
(DMSS), use association rules to represent outcomes and association rule
confidences to monitor changes in the incidence of those outcomes over
time. Through experiments with infection control data from the University
of Alabama at Birmingham Hospital, we have demonstrated that DMSS can
identify potentially interesting and previously unknown patterns. Future
work on prospective clinical studies to determine the usefulness of DMSS in
hospital infection control is needed, as is improved event presentation for
the user and strategies for handling larger datasets.

The statistical strategies developed for automatically detecting temporal
patterns in surveillance data require that analysts explicitly define
outcomes of interest before surveillance begins. The Data Mining
Surveillance System (DMSS), on the other hand, is not constrained to
monitoring changes in user-defined outcomes. In DMSS, complex outcomes are
represented by association rules, and outcome incidence is captured
monthly.

An early version of DMSS, along with association rules and early
experiments with a single organism, has been described (1). We briefly
describe a newer version of DMSS and experimental results obtained by using
it to analyze 1 year's data from intensive care units (ICUs) at the
University of Alabama at Birmingham Hospital.

DMMS uses the following definitions. An itemset is a subset of the set of
all items. The support of an itemset x, sup (x), is the number of records
that contain x. If sup (x) >/= FSST, where FSST is the frequent set support
threshold (FSST), then x is a frequent set. An association rule, A ==> B,
where A and B are frequent sets and the insection of A and B = , is a 
is a statement about how often the items of B are found with the items of A.
the incidence proportion of A ==> B, denoted ip(A ==> B), is equal to 
sup (union of A and B)/sup (A). The precondition support of association 
rule A ==> B is sup(A). The incidence proportion of an association rule 
A ==> B in data partition p(sub i)describes the incidence of the outcome, 
B, in the group, A, during time ti. A series of incidence proportions for 
A ==> B from partitions p(sub1), p(sub 2), ...., p(sub n)describes the 
incidence of the outcome B in group A from t(sub 1) through t(sub n). 
Therefore, by analyzing the series of incidence proportions of an 
association rule A==> B, it should be possible to detect important shifts
or trends in the incidence of B in A over time. In this way, surveillance
of B in A is possible.

Bacterial susceptibility and related demographic data of patients in the
University of Alabama at Birmingham Hospital ICUs (medical, surgical
[SICU], cardiac, neurologic [NICU]) during 1997 were extracted from the
PathNet laboratory information system. Each record describes a single
isolate and contains the following data elements: date of admission, date
of sample collection, date of results reported, source of isolate (e.g.,
sputum, blood), organism isolated, organism Gram stain and morphologic
features, patient's location in the hospital, and resistant (R),
intermediate (I), or susceptible (S) test results to relevant antibiotics,
according to the National Committee for Clinical Laboratory Standards MIC
breakpoints (2).

Duplicate records were removed so that for each patient, no more than one
isolate per organism per month was included. In each remaining record,
certain antimicrobial drug items were removed (only drugs to which the
organism is historically susceptible at least 50% of the time remained).
Additionally, items of the form S~Antimicrobial were removed so that only
I~Antimicrobial and R~Antimicrobial items remained. Finally, data were
divided into 1-month partitions (p(sub 1)....p(sub n)) before analysis. 
For each partition p(sub i), all frequent sets with support of at least 
3 (FSST >2) and association rules with precondition support greater than 
5 were generated. Both the frequent set discovery and association rule-
generating algorithms are beyond the scope of this review (3).

Each generated association rule must pass a set of rule templates that
describe families of interesting and uninteresting rules. Each template is
a construct of the form be(sub 1) ==> be(sub 2), where be(sub 1) and be(sub 2) 
are Boolean expressions over items and attributes. Association rule A ==> B 
satisfies rule template be(sub 1) ==> be(sub 2) if A satisfies be1 and B 
satisfies be(sub 2). Two types of association rule templates are used: 
include templates and exclude templates. An association rule A ==> B passes 
a set of rule templates if A ==> B satisfies at least one include template 
in the set and does not satisfy any exclude template in the set.

Rule templates are handcrafted by domain experts to eliminate inherently
uninteresting or nonsense rules. This is accomplished through iterative
experiments with representative data by initially using few templates and
then creating and modifying templates on the basis of pattern review.

History is a database that holds association rules and their incidence
proportions for different data partitions. In DMSS, the user specifies a
set of rule templates that contains any number of inclusive and restrictive
templates (Table 1). Only association rules that pass the rule templates
are included in the history. To establish a baseline for an association
rule, the incidence proportions of the rule for the three previous
partitions are obtained and stored in the history. Once stored in the
history, a rule is updated for each new partition regardless of whether or
not it is generated in the partition. Therefore, for every association
rule, the history contains an up-to-date time-series of incidence
proportions.

 Table 1. Templates used to filter association rules

 --------------------------------------------------------------------------
 Template
 type       Left (be(sub 1))  Right (be(sub 2))  Explanation

 --------------------------------------------------------------------------
 Exclude    (R~Antibiotic)    (Anything)         Want antibiotic
                                                 sensitivity info on the
                                                 right only.
 Exclude    (Anything)        (Source)           Source of infection is
                                                 not an outcome.
                                                 Therefore,
                                                   exclude all rules with
                                                 a source on the right.
 Exclude    (NS OR Org        (NS OR Org         NS, Org, and GrMp are
              GrMP)             OR GrMP          more informative if
                                                    kept together in
                                                 either a group or an
                                                 outcome.
 Exclude    (Loc)             (Org OR GrMp)      If the left contains
                                  AND            location, then exclude
                                                 rules that
                              (R~Antibiotic)        have Org and
                                                 R~Antibiotic or GrMp and
                                                 R~Antibiotic.
 Include    (Org OR Loc)      (R~Antibiotic OR   Include rules whose
                                GrMp OR Org)     groups are Org- or
                                 AND Not (Loc)   Loc-specific and
                                                    whose outcomes are
                                                 Antibiotic- or
                                                 GrMp-specific.
 --------------------------------------------------------------------------
 be(sub 1) and be(sub 2), Boolean expressions; R, resistant; NS, nosocomial; 
 OR, "or"; Org, organism; GrMp, Gram stain and morphology; Loc, Location.

Table 2. A sample event generated by the Data Mining Surveillance System
-----------------------------------------------------------------------------
          Association              P      P       P       P       P      P
            rule                (subc-5)(subc-4)(subc-3)(subc-2)(subc-1)(subc) 
                                (sup a)    
-----------------------------------------------------------------------------
(nosocomial   ==> {Acinetobacter 0/11    0/10    0/9     0/13    2/9    3/9
  SICU(sup b),     baumannii}
tracheal
  aspirate
-----------------------------------------------------------------------------
                                          w(subp)                w(subc)
                                          (sup c)
-----------------------------------------------------------------------------
(sup a)P(subc), current pair.
(sup b)SICU, surgical intensive care unit.
(sup c)w(subp), past window; w(subc), current window.

By analyzing information stored in the history, DMSS generates alerts that
describe an extreme change in the incidence of an outcome B in a group A
over time. For example, Table 2 describes the incidence of Acinetobacter
baumannii in a nosocomial tracheal aspirate and in SICU isolates over the
past six partitions. Clearly, a shift in incidence occurs between the first
4 months and the most recent 2 months of the series. If we call months 1,
2, 3, and 4 the past window, wp, and months 5 and 6 the current window, 
w(sub c), we can ask if there is an extreme change in the incidence between 
w(sub p) and w(sub c). We compute the cumulative incidence proportion for 
w(sub p) (0/43) and for w (sub c)(5/18) and compare the two by a statistical 
test of two proportions. To generate an alert for an association rule r, 
DMSS first constructs a current window (w(sub c)) and a past window (w(sub p))
on the series of incidence proportions of r (w(sub c)[r,0], w (sub p)[r,0] 
from the algorithm in the Figure). Second, it computes the cumulative 
incidence proportion for each window. Third, it compares the two cumulative 
incidence proportions by a test of two proportions. Finally, if the 
difference between the proportions is statistically extreme 
(p </= alpha = 0.01), it generates an alert. The value of alpha is 
user-defined and rather arbitrary. If an alert is not generated, the 
next set of current and past windows is formed (w(sub c)[r,1], w(sub p)
[r,1] from the algorithm in the Figure), and the cumulative incidence 
proportions are compared. Window pairs are generated for the same association 
rule until an alert is generated or no more window pairs remain to be formed. 
DMSS generates all alerts by executing the procedure described on every 
association rule in the history.

        [fig]                  Current and past window pairs are generated
  Fig. Algorithm used to       by the algorithm in the Figure. If n is the
  construct current and        number of incidence proportions in the
  past windows for             history for a given rule, (w(sub c)):w(sub p))
  association rule r.          pairs are generated for that rule in the
                               following order: (p(sub c):[p(sub c-1),
p(sub c-2)]}), ...,(p(sub c):[p(sub c-1),...,p(sub c-n)]]),([p(sub c),
p(sub c-1)],[p(sub c-2),p (sub c-3)]}),([p(sub c),p(sub c-1)]},[p(sub c-2),
p(sub c-3),p(sub c-4)]),([p(sub c),p(sub c-1)],[p(sub c-2),p(sub c-3),
p(sub c-4),...,p(sub c-n)]),([p(sub c),p(sub c-1),p(sub c-2)],[p(sub c-3),
p(sub c-4),p(sub c-5)]}),([p(sub c),p(sub c-1),p(sub c-2)]},[p(sub c-3),
p(sub c-4),p(sub c-5),p(sub c-6)]}),...,([p(sub c),p(sub c-1),p(sub c-2)]},
[p(sub c-3),p(sub c-4),p(sub c-5),p(sub c-6),...,p(sub c-n)]). For each pair,
w(sub p) must be at least as large as w(sub c).

The total number of events was reduced from 251, by including all rules, to
36, by using the templates in Table 1; thus, classes of inherently
uninteresting rules were eliminated. A retrospective look at the 155 events
eliminated by the rule templates showed that they were uninformative.
Therefore, the introduction of templates resulted in a more focused
presentation of DMSS output.

Of the 36 events, 18 were judged potentially interesting. Table 3 contains
several representative events, one per row. Each row contains the
association rule, the incidence proportions in w(sub c) (bold), and the 
incidence proportions in w(sub p)(nonbold). For example, event 1 in Table 3 
describes an increase in the number of Staphylococcus aureus resistant to 
oxacillin, clindamycin, and erythromycin isolated from tracheal aspirates 
in the fourth partition, and compared with those isolated in the 2nd and 3rd
partitions. Of the events identified by DMSS, only the NICU and SICU had
events that were location-specific (Table 3), while eight events were not.

The events identified by DMSS must be investigated by domain experts to
determine their actual importance. In this example, the data burden was
small since in a prospective analysis only a few events would be presented
to the user each month, thus allowing for the investigation of each event.

Table 3. Representative events identified and considered of potential 
interest
-----------------------------------------------------------------------
                                                Partition
                                   ------------------------------------
Left                    Right
Denominator          Numerator     1  2    3   4   5   6  7 Interpretation
----------------------------------------------------------------------------
Staphylococcus ==> R~Oxacillin       0/10 0/8 7/14          Increase in the
  aureus            (sup a,b)                               incidence of
 Source            R~Clindamycin                            oxacillin (ORSA),
 TRACHASP(sup c)   R~Erythromycin                           clindamycin and
                                                            erythromycin                                                                      
                                                            resistance in all
                                                            isolated from
                                                            tracheal
                                                            aspirates.
NSNoso(sup d)  ==> R~Ceftazidime              3/88 11/70    Increase in
                                                            incidence
                                                            of ceftazidime
                                                            resistance in all
                                                            nosocomial
                                                            isolates.
NP_GNR(sup e)  ==> R~Piperacillin             0/17 6/14     Increase in the
                                                            LocSICU incidence
                                                            of piperacillin
                                                            resistance in
                                                            non-pseudomonas
                                                            gram-negative
                                                            bacilli isolated
                                                            from NSNoso.
NP_GNR         ==> R~Piperacillin     1/12  0/14 4/11  4/8  Increase in the
                                                            LocSICU (sup f)
                                                            incidence
                                                            of piperacillin
                                                            resistance in
                                                            non-pseudomonas,
                                                            nosocomial gram-
                                                            negative bacilli
                                                            from the SICU.
NSNoso         ==> S. aureus 26  3/26 2/28  6/27 5/20  3/11 Increase in the
  LocNICUg                                                  incidence of
                                                            nosocomial  S.
                                                            aureus in
                                                            nosocomial
                                                            isolates from the
                                                            NICU.
------------------------------------------------------------------------------
(sup a)R, resistant.
(sup b)Oxacillin, resistance implies resistance to amoxycillin/clavulanic acid, 
cephalothin, and cefazolin.
(sup c)SourceTRACHASP, tracheal aspirates.
(sup d)NSNoso, nosocomial (3 days from admission).
(sup e)NP_GNR, non-pseudomonas gram-negative rod.
(sup f)LocSICU, location, surgical intensive care unit (SICU).
(sup g)LocNICU, location, neonatal intensive care unit (NICU).

We believe that this approach to surveillance will allow hospital infection
control programs to focus their limited resources on issues of probable
significance. We also believe that this approach is a step toward the
public health surveillance system described by Dean, Fagan, and
Panter-Conner (4).

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

This work was supported in part by cooperative agreement U47-CCU411451 with
the Centers for Disease Control and Prevention (SAM) and a predoctoral
research fellowship LM-00057 from the National Library of Medicine (SEB).

Dr. Moser is associate professor, Department of Pathology, University of
Alabama at Birmingham, and serves as director of Laboratory Information
Services, associate director of Clinical Microbiology for University
Hospital, and director of the Pathology Informatics Section. His research
interests are applied research in diagnostic microbiology and the
application of software as an aid to the intelligent analysis of medical
information, especially that generated in laboratory medicine.

Address for correspondence: Stephen A. Moser, University of Alabama at
Birmingham, Department of Pathology, P246, 619 19th St., South Birmingham,
AL 35233-7331, USA; fax: 205-975-4468; e-mail: moser@uab.edu.

(footnote 1)Presented in part at the International Conference on Emerging 
Infectious Diseases, March 8-11, 1998, Atlanta, Georgia.

References

  1. Brossette SE, Sprague AP, Hardin JM, Waites KB, Jones WT, Moser SA.
     Association rules and data mining in hospital infection control and
     public health surveillance. J Am Med Inform Assoc 1998;5:373-81.
  2. National Committee for Clinical Laboratory Standards. Methods for
     dilution antimicrobial susceptibility tests for bacteria that grow
     aerobically. 4th ed. Approved standard. NCCLS document M7-A4. Wayne
     (PA): The Committee; 1997.
  3. Brossette SE. Data mining and epidemiologic surveillance
     [dissertation]. Birmingham (AL): University of Alabama at Birmingham;
     1998.
  4. Dean AG, Fagan RF, Panter-Conner BJ. Computerizing public health
     surveillance systems. In: Teutsch SM, Churchill RE, editors.
     Principles and practice of public health surveillance. New York:
     Oxford University Press; 1994. p. 200-17.
--------------------------------------------------------------------------
Dispatches

Sentinel Surveillance for Enterovirus 71, Taiwan, 1998

Trong-Neng Wu,* Su-Fen Tsai,* Shu-Fang Li,* Tsuey-Fong Lee,* Tzu-Mei
Huang,* Mei-Li Wang,* Kwo-Hsiung Hsu, and Chen-Yang Shen
*Disease Surveillance and Quarantine Service, Ministry of Health, Taiwan,
Republic of China; Bureau of Communicable Disease Control, Ministry of
Health, Taiwan, Republic of China; and Institute of Biomedical Sciences,
Academia Sinica, Taipei, Taiwan, Republic of China

---------------------------------------------------------------------------
      Outbreaks of enterovirus 71 have been reported around the world
      since 1969. The most recent outbreak occurred in Taiwan during
      April-July 1998. This hand, foot, and mouth disease epidemic
      was detected by a sentinel surveillance system in April at the
      beginning of the outbreak, and the public was alerted.

Enterovirus type 71 (EV71), one of the etiologic agents of epidemic hand,
foot, and mouth disease (HFMD), has been associated with febrile rash
illness, aseptic meningitis, encephalitis, and a syndrome of acute flaccid
paralysis similar to that caused by poliovirus (1,2). EV71 was identified
in 1969 in the United States, when it was isolated from the feces of an
infant with encephalitis in California. By 1998, many EV71 outbreaks had
been reported around the world. In addition to the outbreak in California
in which one death was reported, four other outbreaks resulted in many
fatal cases involving clinical deterioration and death in young children
(Bulgaria, May-September 1975; Hungary, 1978; Malaysia, April-June 1997;
Taiwan, April-August 1998) (3-5). To the best of our knowledge, the
outbreak in Taiwan marked the first time that an EV71 outbreak was detected
by a surveillance system, which alerted the public about the epidemic of
HFMD. We describe how the EV71 outbreak was reported by a sentinel
surveillance system established in July 1989 by Disease Surveillance and
Quarantine Service (originally National Quarantine Services), Ministry of
Health, Taiwan.

In this sentinel surveillance system, public health officers contact local
and regional physicians weekly to actively collect disease information. On
the basis of information collected, disease incidence trends are predicted,
and, if necessary, the public is warned during the earliest stage of
disease outbreaks. Mumps, varicella, diarrhea, and upper-tract respiratory
infection are among the diseases subject to routine surveillance. For
convenience, we have established two channels of data collection: telephone
interviews and report cards mailed by physicians. Approximately 850
physicians (fewer than one tenth of the physicians) from every county in
Taiwan participate in the system: 258 in the northern region, 211 in the
central region, 296 in the southern region, and 85 in the eastern region
(Table); most of these are pediatricians, general practitioners, and family
physicians. Only a few are ear, nose, and throat specialists.

Table. Physician distribution, Sentinel      When an epidemic of fatal
Surveillance System, Taiwan                  myocarditis was reported in
                                             Sarawak, Malaysia, in 1997, we
-------------------------------------------- started to collect information
                      No. cards              regarding HFMD and vesicular
Region of             mailed by  Total  no.  pharyngitis (herpangina)
Taiwan     Interviews physicians physicians  through the sentinel
-------------------------------------------- surveillance system. Beginning
                                             in March 1998, some physicians
Northern       109       149        258      reported a notable increase of
Central         27       184        211      cases of HFMD, vesicular
Southern       175       121        296      pharyngitis (herpangina), and
                                             vesicular stomatitis exanthem.
Eastern         13        72         85      Furthermore, a dramatic
Total          324       526        850      upsurge of HFMD was seen in
                                             children in outpatient
-------------------------------------------- settings at the end of April
							   1998 (Figure 1).

Surveillance data provided the basis for immediate public health action.
When the number of weekly reported cases increased twofold at the end of
April, the public was informed about the epidemic of HFMD and herpangina
and the threat of enterovirus infection (May 12). Measures for preventing
the spread of infection (e.g., practicing good hygiene at all times,
confining infected children at home, avoiding contact with infected
children) were advised. However, the number of reported cases still
dramatically increased to approximately 3,000 in the following week.
Although some errors in reporting might have occurred, we are confident
that the reporting quality was adequate. The sentinel physicians, who have
voluntarily cooperated with us for 10 years, used a clinical case
definition of HFMD/herpangina we created when the surveillance started.

Monitoring                                       
incidence         
of the            	    [fig] 				  [fig]                              
fatal and         
most
severe            Figure 1. Total cases of       Figure 2. Number of
cases of          hand, foot, and mouth disease  hospitalizations and
HFMD              and herpangina reported from   severe cases of hand,
appeared          sentinel physicians in         foot, and mouth disease
critical;         Taiwan, March 19 to August     and herpangina in Taiwan,
therefore,        29, 1998.                      June-August, 1998.
another
report
system, designed for monitoring severe and fatal cases, was established
(May 29) to enroll all well-defined severe and fatal cases in 597 hospitals
and medical centers. This new system established a network of various
public health agencies, general and regional hospitals, and medical
centers. The difference between the two systems was that, while the
sentinel surveillance system was physician-based, the new system was
hospital-based. Severe and fatal cases were defined as HFMD with
complications, including aseptic meningitis, encephalitis, myocarditis,
acute flaccid paralysis, rapidly deteriorating clinical course, and death.
Both surveillance systems worked simultaneously from June 1998 onward. We
found that the trend peaked and declined earlier in the sentinel system
than in the hospital-based surveillance system (Figure 1, 2).

A case-control study was implemented, and enterovirus isolation data were
reviewed. Several enterovirus isolations, from patients with severe and
fatal cases, were in stool specimens, throat secretions, cerebrospinal
fluid, blood, and central nervous system tissue, including EV71, Coxsackie,
and ECHO. Additional studies comparing rates of EV71 isolation in different
years are in progress and will be reported separately. Most isolated
viruses were EV71 from all specimens in this epidemic. The epidemiologic,
clinical, and virologic evidence suggests an association between EV71
infection and this epidemic of HFMD. However, the causes of the severe
cases and deaths in Taiwan are yet to be defined (5).

A physician-based sentinel surveillance system can play an important role
in preventing emerging infectious diseases. Even though the data collected
may be rough, a sentinel surveillance system can provide necessary
information for monitoring communicable diseases, guiding further
investigation, and evaluating control measures, as well as early warning
for epidemics and rationale for public health intervention. Early detection
of communicable diseases and immediate public health intervention can
curtail the number of illnesses and deaths and reduce negative effects on
international travel and trade (6).

      Dr. Wu is vice superintendent of Municipal Hsiao-Kang Hospital,
Kaohsiung Medical College, Taiwan, Republic of China. He is also a
professor at both Kaohsiung Medical College, Kaosiung, Taiwan, and China
Medical College, Taichung, Taiwan.

      Address for correspondence: Su-Fen Tsai, Division of Disease
Surveillance, Disease Surveillance and Quarantine Service, Ministry of
Health, F6, No.6, Lin Shen S. Rd., Taipei, Taiwan, R.O.C.; fax:
886-2-23945312; e-mail: sftsai@net.dsqs.gov.tw.

References

  1. Melnick JL. Enterovirus type 71 infection: a varied clinical pattern
     sometimes mimicking paralytic poliomyelitis. Rev Infect Dis 1984;6
     Suppl:S387-90.
  2. Alexander JP Jr, Baden L, Pallansch MA, Anderson LJ. Enterovirus 71
     infection and neurologic diseaseUnited States, 1977-1991. J Infect
     Dis 1994;169:905-8.
  3. Shindarov LM, Chumakov MP, Voroshilova MK, Bojinov S, Vasilenko SM,
     Iordanov I, et al. Epidemiological, clinical, and pathomorphological
     characteristics of epidemic poliomyelitis-like disease caused by
     enterovirus 71. Journal of Hygiene, Epidemiology, Microbiology, and
     Immunology 1979;23:284-95.
  4. Nagy G, Takatsy S, Kukan E, Mihaly I, Domok I. Virological diagnosis
     of enterovirus type 71 infections: experiences gained during an
     epidemic of acute CNS diseases in Hungary in 1978. Arch Virol
     1982;71:217-27.
  5. Centers for Disease Control and Prevenetion. Deaths among children
     during an outbreak of hand, foot, and mouth diseaseTaiwan, Republic
     of China, April-July 1998. MMWR Morb Mortal Wkly Rep 1998;47:629-32.
  6. Heymann DL, Rodier GR. Global surveillance of communicable diseases.
     Emerg Infect Dis 1998;4:362-5.
--------------------------------------------------------------------------

Dispatches

Chlorine Inactivation of Escherichia coli O157:H7

Eugene W. Rice, Robert M. Clark, and Clifford H. Johnson
U.S. Environmental Protection Agency, Cincinnati, Ohio, USA

---------------------------------------------------------------------------
      We analyzed isolates of Escherichia coli O157:H7 (which has
      recently caused waterborne outbreaks) and wild-type E. coli to
      determine their sensitivity to chlorination. Both pathogenic
      and nonpathogenic strains were significantly reduced within 1
      minute of exposure to free chlorine. Results indicate that
      chlorine levels typically maintained in water systems are
      sufficient to inactivate these organisms.

Escherichia coli O157:H7 is becoming increasingly recognized as a
waterborne pathogen. Two recent outbreaks during summer 1998, one involving
a drinking water supply in Wyoming (1) and another involving recreational
water exposure at a water park in Georgia (2), have underscored the role of
water in transmission. Contaminated drinking water (3,4) and recreational
water have been associated with outbreaks of hemorrhagic colitis caused by
E. coli O157:H7 (5-7). Chlorination of water is one of the primary public
health measures used to ensure that both potable water and water used in
recreational settings are free of microbial pathogens. Our study was
undertaken to determine the chlorine resistance of E. coli O157:H7 and
compare this resistance with that of wild-type E. coli.

Seven strains of E. coli O157:H7, isolated from cattle from geographically
distinct areas (Florida, Idaho, Illinois, Missouri, Texas, Washington, and
Wisconsin), were obtained from the U.S. Department of Agriculture (D.
Miller, Ames, IA). The isolates exhibited the characteristic phenotypic
traits: sorbitol-negative, -glucuronidasenegative, lactose-positive,
indole-positive, and positive for glutamate decarboxylase (8). All
enterohemorrhagic isolates were active toxin producers, as determined by in
vitro enzyme immunoassay (Meridian Diagnostics, Inc., Cincinnati, OH).
These cattle isolates were chosen as representative strains that might
contaminate water supplies after surface run-off from pastures and fields.
Four wild-type E. coli isolates from cattle manure from a local dairy farm
(Ohio) were characterized by biochemical test kits (bioMerieux Vitek,
Hazelwood, MO). All bacterial cultures used in the disinfection experiments
were grown for 18 to 20 hours at 35C in brain heart infusion broth,
concentrated by centrifugation, and washed three times in phosphate buffer
(9) before testing.

The results of the disinfection experiments, including the rates of
inactivation, are shown in the Table. Initial levels for all isolates were
5.52 to 5.79 log(sub 10) CFU/ml. The mean chlorine levels at each exposure 
time were 1.1 mg/L free chlorine and 1.2 mg/L total chlorine. For both the
pathogenic and the wild-type strains, exposure to these levels of chlorine
for 1 minute reduced the viable populations by approximately four orders of
magnitude. The inactivation rates and corresponding correlation coefficient
(r[sup 2]) values are listed in the Table. Little difference was observed in the
rates of inactivation for the pathogenic and wild-type organisms.

Table. Chlorine inactivation of Escherichia coli O157:H7 and wild-type E.
coli(sup a)
---------------------------------------------------------------------------
                           Log(sub 10)CFU/ml
                   --------------------------------
                             After exposure time of
                           ------------------------
                    Initial                           Inactivation
Isolate             inoculum   30 sec 60 sec 120 sec rate(sec[sup -1])r(sup 2)
---------------------------------------------------------------------------
E. coli O157:H7
   N009-6-1           5.63      2.60  1.88   0.82        -2.96       0.82
   N6001-8-10         5.78      2.52  1.44   0.72        -3.06       0.68
   N6021-5-1          5.78      2.54  1.52   0.66        -3.06       0.54
   N60049-26-1        5.68      2.35  1.40   0.54        -3.00       0.86
   N6059-7-2          5.72      2.42  1.74   0.86        -3.02       0.72
   N6104-5-9          5.62      2.40  1.69   0.72        -2.96       0.89
   N6114-7-2          5.63      2.52  1.66   0.89        -2.96       0.82
   Mean               5.69      2.48  1.62   0.74        -2.93       0.82
E. coli (wild
type)
   A                  5.53      2.66  1.80   1.52        -2.51       0.61
   B                  5.79      2.60  1.48   0.81        -2.68       0.60
   C                  5.68      2.48  0.92   0.84        -2.61       0.61
   D                  5.52      2.34  0.95   0.39        -2.50       0.61
   Mean               5.63      2.52  1.28   0.89        -2.93        0.71
---------------------------------------------------------------------------
(sup a)In chlorine demand-free chlorinated (CDF) buffer, 5C, pH 7.0, 1.1 mg/L
free chlorine, 1.2 mg/L total chlorine. Duplicate chlorine inactivation
experiments were conducted in CDF buffer at pH 7.0. All experiments were
conducted at 5C in a recirculating, refrigerated water bath. The
chlorinated buffer was prepared by the addition of reagent-grade sodium
hypochlorite (Fisher Scientific, Fair Lawn, NJ). Reaction vessels were
continuously mixed (250 rpm) by using an overhead stirring apparatus
equipped with sterile stainless steel paddles. Chlorine concentrations
were determined by the N,N-dimethyl-p-phenylenediamine colorimetric method
(9). Samples were removed from the reaction vessels at the desired
exposure times, and the chlorine was immediately neutralized by the
addition of 0.5 ml of 10% (wt/vol) sodium thiosulphate. Vessels containing
CDF buffer without chlorine served as controls for determining unexposed
concentrations of the bacteria. Initial levels and the number of survivors
after chlorine exposure were determined by the membrane filtration
procedure using mT7 agar incubated for 22 to 24 hours at 35C. This medium
was chosen because of its ability to recover oxidant-stressed organisms
(9). Levels of bacteria were determined by duplicate filtrations of
appropriate dilutions for each exposure time.The log(sub 10)-transformed data
were used to determine the levels of inactivation for each isolate. The
means for the inactivation data for the E. coli O157:H7 isolates and for
the wild-type E. coli isolates at each exposure time were used to compare
the inactivation rates between the pathogenic and the wild-type organisms.
The following first order model was used to describe the inactivation
rate: y = y(sub 10)10(sup -at), where t = time in seconds, y = CFU/ml at 
any time t, y(sup 10)= CFU/ml at time zero, and a = the inactivation rate in 
sec(sup -1). The log transformation of this equation was used to calculate 
the inactivation rate. A regression analysis using least squares was 
conducted for experiments with each individual isolate and for the mean 
values for each of the two types of isolates (serotype O157 and wild-type) 
to determine the inactivation rates ("a" values).
---------------------------------------------------------------------------

These results indicate that the E. coli O157:H7 isolates used in this study
were sensitive to chlorination and were similar in resistance to that of
wild-type E. coli isolates. The biocidal activity of chlorine decreases
with decreasing temperature (not done in this study). The 5C temperature
we used represents a worst-case condition for both ground water or winter
surface-water temperature. A survey of disinfection practices in the United
States found that water utilities maintain a median chlorine residual of
1.1 mg/L and a median exposure time of 45 minutes before the point of first
use in the distribution system (10). At this level of chlorination, E. coli
O157:H7 is unlikely to survive conventional water treatment practices in
the United States. E. coli O157:H7 survives at a similar rate to that of
wild-type E. coli in nondisinfected drinking water (11). Survival patterns
and sensitivity to chlorination previously observed for the strains used in
this study suggest that wild-type E. coli could serve as an adequate
indicator organism for fecal contamination of water. Using wild-type E.
coli to indicate E. coli O157:H7 would be useful because most analytical
procedures for detecting E. coli in drinking water (e.g., assays for
lactose fermentation at 44C to 45C or production of the enzyme
-glucuronidase) cannot detect pathogenic E. coli O157:H7 strains (8).

Although chlorination appears to adequately control this pathogen, not all
municipal water supplies use chlorine disinfection. In addition, chlorine
residual can dissipate under adverse conditions, and exposure to sunlight
or organic chlorine-demand substances can greatly diminish chlorine levels.
Protection of organisms associated with particulate matter, such as fecal
material, can also readily decrease the biocidal activity of chlorine.
These considerations are particularly important in determining the efficacy
of chlorination in a recreational water setting. The results of this study
indicate that the isolates studied were sensitive to chlorination.
Evaluation of other isolates under differing environmental conditions would
be worthy of further consideration.

Acknowledgment

      We thank Dr. Robert V. Tauxe for his encouragement and guidance
regarding this project.

      Dr. Rice is a microbiologist in the Microbial Contaminants Control
Branch, Water Supply and Water Resources Division, National Risk Management
Research Laboratory, U.S. Environmental Protection Agency, Cincinnati,
Ohio. His research focuses on detection and inactivation of waterborne
pathogens and microbial indicator organisms.

      Address for correspondence: Eugene W. Rice, U.S. Environmental
Protection Agency, 26 West M.L. King Dr., Cincinnati, OH 45268, USA; fax:
513-569-7328; e-mail: rice.gene@epa.gov.

References

  1. Olsen J, Miller G, Breuer T, Kennedy M, Higgins C, McGee G, et al. A
     waterborne outbreak of E. coli O157:H7 infections: evidence for
     acquired immunity. In: Program and Abstracts of the 36th Annual
     Meeting of Infectious Diseases Society of America, Denver, Colorado;
     1998 Nov 12-15; [abstract 782]. Alexandria (VA): Infectious Disease
     Society of America; 1998. p. 62.
  2. Blake P. Escherichia coli O157:H7 outbreak among visitors to a water
     park. In: Program and Abstracts of the 36th Annual Meeting Infectious
     Diseases Society of America, Denver, Colorado; 1998 Nov 12-15;
     [abstract 537]. Alexandria (VA): Infectious Diseases Society of
     America; 1998. p. 178.
  3. Swerdlow DL, Woodruff BA, Brady RC, Griffin PM, Tippen S, Donnell HD,
     et al. A waterborne outbreak in Missouri of Escherichia coli O157:H7
     associated with bloody diarrhea and death. Ann Int Med
     1992;117:812-19.
  4. Dev VJ, Main M, Gould I. Waterborne outbreak of Escherichia coli O157.
     Lancet 1991;337:412.
  5. Ackman D, Marks S, Mack P, Caldwell M, Root T, Birkhead G.
     Swimming-associated hemorrhagic colitis due to Escherichia coli
     O157:H7 infection: evidence of prolonged contamination of a fresh
     water lake. Epidemiol Infect 1997;119:1-8.
  6. Brewster DH, Brown MI, Robertson D, Houghton GL, Bimson J, Sharp JCM.
     An outbreak of Escherichia coli O157 associated with a children's
     paddling pool. Epidemiol Infect 1994;112:441-7.
  7. Keene WE, McAnulty JM, Hoesly FC, Williams LP Jr, Hedberg K, Oxman GL,
     et al. A swimming-associated outbreak of hemorrhagic colitis caused by
     Escherichia coli O157:H7 and Shigella sonnei. N Engl J Med
     1994;331:579-84.
  8. Rice EW, Johnson CH, Reasoner DJ. Detection of Escherichia coli
     O157:H7 in water from coliform enrichment cultures. Lett Appl
     Microbiol 1996;23:179-82.
  9. American Public Health Association. Standard methods for the
     examination of water and wastewater. 19th ed. Washington: The
     Association; 1995.
 10. Water Quality Disinfection Committee. Survey of water utility
     disinfection practices. J Am Water Works Assoc 1992;84:121-8.
 11. Rice EW, Johnson CH, Wild DK, Reasoner DJ. Survival of Escherichia
     coli O157:H7 in drinking water associated with a waterborne disease
     outbreak of hemorrhagic colitis. Lett Appl Microbiol 1992;15:38-40.
--------------------------------------------------------------------------

Dispatches

Fulminant Meningococcal Supraglottitis: An Emerging Infectious Syndrome?

Eric Schwam* and Jeffrey Cox
*Sturdy Memorial Hospital, Attleboro, Massachusetts, USA; and Rhode Island
Hospital, Providence, Rhode Island, USA

---------------------------------------------------------------------------
      We report a case of fulminant supraglottitis with dramatic
      external cervical swelling due to associated cellulitis. Blood
      cultures were positive for Neisseria meningitidis. The patient
      recovered completely after emergency fiberoptic intubation and
      appropriate antibiotic therapy. We summarize five other cases
      of meningococcal supraglottitis, all reported since 1995, and
      discuss possible pathophysiologic mechanisms.

Neisseria meningitidis, a gram-negative diplococcus, can cause a broad
spectrum of clinical manifestations including acute meningitis,
meningococcemia, occult bacteremia, meningoencephalitis, pneumonia,
conjunctivitis, dermatitis-arthritis syndrome, and urethritis (1). We
describe a rare case of meningococcal supraglottitis (2-6) further
complicated by cervical cellulitis. Reports of five other cases suggest an
emerging clinical syndrome due to this pathogen.

Case Report

In January 1998, a 44-year-old woman became ill with rhinitis and sore
throat, which progressed to dysphagia, dyspnea, and neck swelling during
the night, requiring her to sleep sitting upright. The following morning,
she went to a community hospital emergency room.

The patient was alert but appeared toxic and had inspiratory stridor and a
muffled voice. She had routine dental cleaning 1 week before onset of
symptoms. She did not have a toothache and said she did not use tobacco or
alcohol. She had had no history of splenectomy or immune deficiency. Her
temperature was 38.1C, pulse 138, cuff blood pressure 120/74 mm Hg, and
respiratory rate 24. Room air pulse oximetry was 94%. The tongue was large,
but not edematous, and there was neither drooling nor sublingual swelling.
The soft palate and posterior pharynx, seen only with difficulty, were
diffusely swollen, protruding anteriorly, and covered with exudate. Massive
external swelling, tenderness, and erythema of the anterior neck extended
from the chin caudad to the midsternum, obliterating all cervical
landmarks. The patient did not have meningismus, crepitus, or jugular
venous distention. The lungs and heart were normal. No rash was present.

A portable lateral radiograph of the neck showed diffuse soft tissue
cervical and epiglottic swelling with a classic "thumb sign." White blood
count was 21.9 10(sup 9)/L with 0.70 polymorphonuclear leukocytes and 0.17
bands. Platelet count was 242 10(sup 9)/L. Hematocrit, electrolytes, glucose,
and urea nitrogen were normal. The patient was treated with oxygen and
nebulized epinephrine, ceftriaxone 1.0 gram intravenously (i.v.), and
clindamycin 600 mg i.v. She was taken to the operating room, where with
surgical standby, she was orally intubated with a 6.0 mm-endotracheal tube
over a fiberoptic laryngoscope.

The patient was transferred to a tertiary care hospital, where the
antibiotics were changed to ampicillin-sulbactam, clindamycin, and
gentamicin. Computed tomography (CT) scan of the neck and chest showed
extensive soft tissue swelling from the oropharynx to the supraglottic
region with obliteration of the airway surrounding the endotracheal tube.
The adjacent parapharyngeal and cervical soft tissues down to the upper
chest were also involved, but no discrete abscess was identified. Bilateral
pulmonary infiltrates, atelectasis, and small pleural effusions were
present, but the mediastinum was normal.

The next day, two blood cultures drawn before administration of antibiotics
grew N. meningitidis, serogroup Y (confirmed by the Massachusetts
Department of Public Health Laboratory). The organism was sensitive to
penicillin and ceftriaxone and resistant to tetracycline by the Kirby-Bauer
method. Sputum culture done by endotracheal tube (after the start of
antibiotics) was negative. Close family contacts and the community hospital
staff members involved with airway procedures were given prophylactic
antibiotics, according to guidelines (rifampin 600 mg PO bid x 4 doses,
ciprofloxacin 500 mg PO x 1 dose, or ceftriaxone 250 mg intramuscularly x 1
dose) (7).

Clindamycin and gentamicin were discontinued. Repeat CT scan on hospital
day 5 was unchanged, but by day 7, the swelling had resolved, and the
patient was extubated in the operating room. She recovered fully and was
discharged on hospital day 9; she received i.v. ampicillin-sulbactam at
home for an additional 7 days. She remained well, as determined by
telephone contact 13 months later. Complement testing (CH-50) 13 months
later was normal.

Conclusions

The diagnosis of supraglottitis is most consistent with the patient's
clinical picture of sore throat, dysphagia, fever, muffled voice, and
swollen supraglottic tissues, as seen on plain films, fiberoptic
laryngoscopy, and cervical CT scan (8). However, supraglottitis is
uncommonly accompanied by such dramatic external cervical swelling.
Ludwig's angina was initially considered, but this diagnosis was unlikely
because odontogenic infection and involvement of the sublingual or
submandibular spaces were lacking. Cervical abscesses can complicate
supraglottitis (9), but no abscess was detected on repeated CT scans.
Necrotizing fasciitis was a possibility, especially since the erythema and
swelling overlying the chest suggested mediastinal extension. In the one
reported case of supraglottitis with cervical necrotizing fasciitis (10),
fascial gas was demonstrated on CT scan. Surgical drainage was required for
cure; anaerobic organisms were the likely cause. Furthermore, N.
meningitidis, which rarely causes cellulitis, has not been reported to
cause necrotizing fasciitis. The pathologic process in bacterial
supraglottitis is cellulitis of the epiglottis and the surrounding upper
airway. In this patient, the cellulitis was so aggressive that it extended
posteriorly to the spine, antero-laterally to the cervical skin, cephalad
to the pharynx, and caudad to the chest.

Cultures of the epiglottis and pharynx were not performed, so the
meningococcal bacteremia may have reflected secondary or coincident
infection, not supraglottic infection. However, even when laryngeal
cultures are performed, organisms isolated from the blood and the throat
correlate poorly (11). Although the CT lung scan demonstrated pulmonary
infiltrates, bacteremic meningococcal pneumonia complicating supraglottitis
due to another organism is improbable.

A search of Medline (the National Library of Medicine's bibliographic
database of biomedical journals) from 1966 through mid-1999 located five
reports of meningococcal supraglottitis: two from Colorado (2,4), one from
Ohio (3), one from Singapore (5), and one from Helsinki, Finland (6).
Including our patient in the series of six, the ages of the patients (three
were women) were 44, 54, 60, 65, 81, and 95. Two patients had type 2
diabetes mellitus, but the others were otherwise healthy; all had fever,
sore throat, and evidence of upper airway compromise. No upper airway
cultures were reported, but blood cultures in all six cases were positive
for N. meningitidis: serogroup B (two), serogroup Y (three), and unreported
serogroup (one). No clinical evidence of meningitis or meningococcemia was
found in any of the cases. Only our patient had external cervical
cellulitis. All patients recovered with appropriate antibiotic treatment.
The possibility of complement deficiency was investigated and ruled out in
two of two patients. Two patients were treated with steroids i.v. Five of
the six patients required airway intervention (three, intubation; two,
urgent tracheostomy), a much higher proportion than that in a
population-based case series of adults with supraglottitis (11). Thus,
bacteremic meningococcal supraglottitis appears to be fulminant and
life-threatening.

Most meningococcal disease in the United States is sporadic. One third of
all cases occur in adults, who are usually immunocompromised (e.g., by
complement deficiency, corticosteroid use, or HIV infection). In one
population-based study, more than half the adults had neither rash nor
meningitis. Pneumonia, sinusitis, and tracheobronchitis were the main
sources of bacteremic meningococcal disease. Supraglottitis was not
observed (12). In particular, the proportion of meningococcal infections
due to serogroup Y has been increasing nationally in the last several
years. Serogroup Y is frequently associated with meningococcal pneumonia in
both civilian (13) and military populations (14).

Why N. meningitidis is not a more frequent cause of supraglottitis is not
known. The organism is a common colonizer of the upper airways of healthy
persons, and the pharynx is the suspected portal of entry of invasive and
disseminated disease (1). The organism may also cause simple pharyngitis
(15).

The pathogenic determinants of both meningococcal disease and
supraglottitis are complex and largely undefined, so we can only speculate
how meningococci might cause this syndrome. Meningococcemic syndromes seem
to require both epithelial and endothelial invasiveness so that the
organism can cross the nasopharyngeal mucosal barrier, enter the
bloodstream, and invade other blood vessel walls to produce the
characteristic vasculitic organ damage. In contrast, meningococcal isolates
from supraglottic syndromes seem to have relatively greater epithelial
invasiveness, a propensity for contiguous local inflammatory spread, and
decreased tropism for endothelial cells; these characteristics result in a
more locally aggressive but less disseminated disease. The presence of
various surface-expressed virulence factors (e.g., capsule, pili, cell
surface proteins, and lipo-oligosaccharides) that mediate the organism's
interaction with certain host cells may explain these differing
pathophysiologic properties. For example, certain Opa cell surface proteins
facilitate invasion of epithelial cells, while Opc proteins are more
efficient at promoting invasion of endothelial cells (16).

N. meningitidis can cause an inflammatory conjunctivitis that progresses to
septicemia in approximately 10% of cases (17). In an analogous manner, it
can (rarely) extend locally to produce a periorbital cellulitis. In one
such case, isolates from the blood and periorbital aspirate of the same
patient were identical except for their expression of Opa proteins and
their lipo-oligosaccharide phenotype (18).

Host factors (e.g., specific immune system deficiencies) could also be
responsible for different disease manifestations. The most well-known
example is the susceptibility of patients with terminal complement
component deficiencies to neisserial infections. However, these patients
have recurrent, but typical meningococcemia, so this deficiency would not
be expected to contribute to the supraglottitis syndrome (19).

While N. meningitidis has been known for nearly 2 centuries and blood
cultures have been routinely available for decades, meningococcal
supraglottitis had not been reported until 1995 (2). In contrast, incidence
of supraglottitis due to Haemophilus influenzae has remained constant in
adults 18 years of age or older (11). Furthermore, while one case was
reported each year from 1995 to 1997, three cases have been reported in
1998-1999, from three continents, suggesting the emergence of a new
meningococcal syndrome worldwide. Surveillance is needed to determine if
meningococcal supraglottitis will become more than just a rarity.

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

Acknowledgment

      We thank Dr. Gregory Jay for review of the manuscript.

      Dr. Schwam practices emergency medicine at Sturdy Memorial Hospital
in Attleboro, Massachusetts. He is clinical instructor in medicine at Brown
University Medical School and instructor in emergency medicine at the
University of Massachusetts Medical School.

      Dr. Cox practices emergency medicine at Rhode Island Hospital in
Providence, Rhode Island. He is assistant clinical professor of surgery at
Brown University Medical School.

      Address for correspondence: Eric Schwam, Emergency Care Center,
Sturdy Memorial Hospital, 211 Park Street, Attleboro, MA 02703-0963, USA;
fax: 508-236-7043; e-mail: schwam@massmed.org.

References

  1. Apicella MA. Neisseria meningitis. In: Mandell GL, Bennett JE, Dolin
     R, editors. Mandell, Douglas and Bennett's principles and practice of
     infectious diseases. 4th ed. New York: Churchill Livingston Inc.;
     1995. p. 1896.
  2. Crausman RS, Jennings CA, Pluss WT. Acute epiglottitis in the adult
     caused by Neisseria meningitidis. Scand J Infect Dis 1995;27:77-8.
  3. Nelson K, Watkins DA, Watanakunakorn C. Acute epiglottitis due to
     serogroup Y Neisseria meningitidis in an adult. Clin Infect Dis
     1996;23:1192-3.
  4. Donnelly TJ, Crausman RS: Acute supraglottitis: when a sore throat
     becomes severe. Geriatrics 1997;52:65-6, 69.
  5. Sivalingam P, Tully AM. Acute meningococcal epiglottitis and
     septicaemia in a 65-year-old man. Scand J Infect Dis 1998;30:198-200.
  6. Mattila PS, Carlson P. Pharyngolaryngitis caused by Neisseria
     meningitidis. Scand J Infect Dis 1998;30:200-1.
  7. Jafari HS, Perkins BA, Wenger JD. Control and prevention of
     meningococcal disease. MMWR Morb Mortal Wkly Rep 1997;46:RR-5.
  8. Frantz TD, Rasgon BM, Quesenberry CP Jr. Acute epiglottitis in adults:
     analysis of 129 cases. JAMA 1994;272:1358-60.
  9. Hebert PC, Ducic Y, Boisvert D, Lamothe A. Adult epiglottitis in a
     Canadian setting. Laryngoscope 1998;108:64-9.
 10. Nguyen R, Leclerc J. Cervical necrotizing fasciitis as a complication
     of acute epiglottitis. J Otolaryngol 1997;26:129-31.
 11. Mayo-Smith MF, Spinale JW, Donskey CJ, Yukawa M, Li RH, Schiffman FJ.
     Acute epiglottitis: an 18-year experience in Rhode Island. Chest
     1995;108:1640-7.
 12. Stephens DS, Hajjeh RA, Baughman WS, Harvey RC, Wenger JD, Farley MM.
     Sporadic meningococcal disease in adults: results of a 5-year
     population-based study. Ann Intern Med 1995;123:937-40.
 13. Racoosin J, Diaz PS, Samala U. Serogroup Y meningococcal
     diseaseIllinois, Connecticut, and selected areas, United States,
     1989-1996. MMWR Morb Mortal Wkly Rep 1996;45:1010-4.
 14. Koppes GM, Ellenbogen C, Gebhart RJ. Group Y meningococcal disease in
     United States Air Force recruits. Am J Med 1977;62:661-6.
 15. Pether JVS, Scott RJD, Hancock P. Do meningococci cause sore throats?
     Lancet 1994;344:1636.
 16. Nassif X, Magdalene SO. Interaction of pathogenic Neisseriae with
     nonphagocytic cells. Clin Microbiol Rev 1995;8:376-88.
 17. Moraga FA, Domingo P, Barquet N, Glasser I, Gallart A. Invasive
     meningococcal conjunctivitis. JAMA 1990;264:333-4.
 18. Patrick CC, Glen TF, Edwards M, Estabrook M, Blake MS, Baker CJ.
     Variation in phenotypic expression of the Opa outer membrane protein
     and lipooligosaccharide of Neisseria meningitidis serogroup C causing
     periorbital cellulitis and bacteremia. Clin Infect Dis 1993;16:523-27.
 19. Figueroa J, Andreoni J, Densen P. Complement deficiency states and
     meningococcal disease. Immunol Res 1993;12:295-311.
--------------------------------------------------------------------------

Dispatches

Genetic Evidence of Dobrava Virus in Apodemus agrarius in Hungary

Jerrold J. Scharninghausen,* Hermann Meyer, Martin Pfeffer, Donald S.
Davis,* and Rodney L. Honeycutt*
*Texas A&M University, College Station, Texas, USA; Federal Armed Forces
Medical Academy, Munich, Germany; and Ludwig Maximilians University,
Munich, Germany

---------------------------------------------------------------------------
      Using nested polymerase chain reaction, we sequenced Dobrava
      virus (DOB) from the rodent Apodemus agrarius in Hungary. The
      samples we isolated group with DOB samples previously isolated
      from A. flavicollis. This grouping may indicate host switching.

Hantaviruses, the causative agents of hemorrhagic fever with renal syndrome
and hantavirus pulmonary syndrome, are serologically related viruses of the
family Bunyaviridae and have a worldwide distribution. Unlike other
bunyaviruses, hantaviruses are not transmitted by arthropod vectors. The
virus is excreted in the saliva, urine, and feces of infected rodents.
Humans become infected by inhalation of aerosols of dried excreta,
inoculation through the conjunctiva, or entry through broken skin (1).

Each viral species within the genus Hantavirus is primarily associated with
a single rodent species, although accidental infections have been reported
in other mammals (2). Four primary reservoirs for hantaviruses are found in
Europe: Rattus norvegicus, Seoul virus; Clethrionomys glareolus, Puumala
virus (PUU); Apodemus flavicollis, Dobrava virus (DOB); and Microtus
arvalis, Tula virus (TUL) (3). An additional rodent species, Apodemus
agrarius, is the primary reservoir of Hantaan virus (HTN), the causative
agent of Korean hemorrhagic fever, which is found throughout Korea and
China but not in Europe. Recently, DOB was isolated from A. agrarius in
Estonia (4) and Russia (5).

Hantaviruses are present in Hungary; however, their particular virus
strains or species and their distribution are unknown. PUU has been
confirmed in western Hungary, but the species in eastern Hungary have not
been determined (6).

During field surveys of indigenous rodents in areas used by NATO forces
between 1995 and 1996, nine rodents were collected at Tazar Air Force Base,
outside Kaposvar, eastern Hungary. A total of 210 trap nights resulted in a
trap return of 4.28%. Two A. agrarius, four Microtus agrestis, and three
Mus musculus were captured. Animals were live-trapped and euthanized with
halazone. Tissue was collected from lungs and kidneys; urine, if present,
was also collected.

Total RNA was extracted from collected tissues by using the FastRNA
KitGreen (Bio 101), according to the supplier's recommendations. RNA was
converted to cDNA and amplified by the Titan One Tube RT-PCR System
(Boehringer, Mannheim, Germany), according to the manufacturer's
recommendations. Degenerate primers M-4 (ATGAARGCNGAWGA RNTNACMCCNGG) and
M-9 (TGRYCNAGYTG TATYCCCATWGATTG) were used to amplify a 583-bp section
corresponding to nt positions 605 to 1,188 in the S segment of HTN strain
X95077. No DOB strains were used in the laboratory.

A target sequence of 397 bp (nt from 692 to 1,089) was amplified by nested
polymerase chain reaction, and both strands of the amplicons were
sequenced. Amplified bands of the predicted size were demonstrable in all
tissue samples investigated from the two specimens of A. agrarius
(designated Tazar-2, Tazar-8). All other samples tested negative. Sequence
validity was confirmed by sequencing amplified lung and kidney products
from each infected animal. The respective sequences from Tazar-2 and
Tazar-8 differed in 3 of the 321 nucleotides examined (99% identity). Both
sequences were aligned with the corresponding S-segment section of several
DOBs and five other European and Asian hantaviruses.

The nucleotide sequence identities between Tazar virus (found in this
study) and related viral lineages (Figure) included Russian DOB from A.
agrarius, 88%; Estonian DOB from A. agrarius, 86%-87%; Bosnia DOB from A.
flavicollis, 88%; Greek DOB from A. flavicollis, 88%-85%; Sapporo rat
virus, 70%; HTN, 68%; Khabarousk, 57%; PUU, 56%; TUL, 53%; and Sin Nombre,
48%. These results indicate that, on the basis of sequence similarity, both
Tazar samples are hantaviruses most closely related to DOB. Although the
sequence data are limited, phylogenetic analyses linked Tazar-2 and Tazar-8
isolated from A. agrarius to a group of DOB previously isolated from A.
flavicollis. The A. agrarius DOB from Russia (5) did not support monophyly
(common ancestry) for DOB isolated from A. agrarius populations in Hungary
and Russia. Other representative hantaviruses, including PUU, TUL, HTN, and
Sapporo rat virus, were more basal in the phylogeny (Figure).

HTN infects A. agrarius populations in Asia but has not been isolated in
Europe. Direct enzyme-linked immunosorbent assay has demonstrated the
presence of Hantaan-like antigens in A. agrarius in the former republic of
Czechoslovakia (5.5%, [7]), the European regions of the former Soviet Union
(5.3%, [8]) (28.5%, [9]), and Serbia (2.2%, [10]). Because HTN sequences
have not been reported in Europe and HTN and DOB have similar immunologic
responses, the earlier findings of Hantaan-like antigens in Europe may
represent a more widespread occurrence of DOB in European populations of A.
agrarius. Recent verification of DOB in populations of A. agrarius in
Estonia (4), Russia (5), and now Hungary supports this conclusion.

                              

                              Figure. Cladogram derived from nucleotide
                              sequences of Tazar-2, Tazar-8, and other
                              hantaviruses. Numbers denote Genbank
                              accession numbers. The cladogram was derived
                              from the neighbor-joining estimated
                              phylogeny and bootstrap analysis using
                              p-distance estimates. The phylogenetic
	  [Fig]   			analysis was performed by using PAUP 3.1.1
                              (vers. 4.0.0d64). Numbers at each internode
                              or bifurcation represent bootstrap support
                              based on 1,000 replicates. A Sin Nombre
                              virus sequence (L37904) was used as the
                              outgroup to root the tree. Tazar 2 and Tazar
                              8 have been submitted to Genbank and
                              received accession numbers AF085336 and
                              AF085337, respectively.

The occurrence of DOB in both A. agrarius and A. flavicollis provides an
opportunity to evaluate the hypothesis concerning distribution of
hantaviruses in related rodent hosts. Phylogenetically different Sin
Nombrelike viruses have been found in different populations within species
of peromyscine rodents that vary ecologically and geographically throughout
their range (11). Although these authors found evidence of cospeciation
between the rodent host phylogeny and the host-borne hantavirus phylogeny,
evidence of host switching was observed with Peromyscus leucopusborne New
York virus grouped with P. maniculatusborne viruses rather than with other
P. leucopusborne viruses.

Our phylogenetic analysis (Figure) indicates a closer relationship between
the A. agrarius DOB from Hungary and A. flavicollis DOB, with
Russian/Estonian DOB representing a sister-group to this clade. This lack
of monophyly for the A. agrarius DOB may suggest host switching between A.
agrarius and A. flavicollis similar to that between P. leucopus and P.
maniculatus. Nevertheless, the relationships between the isolated DOB
lineages suggest a more basal position for the A. agrarius DOB than for A.
flavicollis DOB lineages.

A. flavicollis ranges throughout much of Western Europe eastward to the
Ural Mountains, and A. agrarius ranges from Eastern Europe eastward to the
Pacific Ocean, covering most of the Asian continent (12). Given the
extensive range of both species, an examination of other populations within
each species, as well as other species of Apodemus, might allow correlation
of the viral and rodent host phylogeny. The pattern of divergence for
hantaviruses in New World peromyscine rodent species may be mirrored in Old
World arvicoline rodents. Therefore, the existence of more than one
hantavirus in A. agrarius may reflect geographic variation within the
species or host switching in regions where two host species are potentially
sympatric.

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

      Dr. Scharinghausen, an active-duty Army captain, is completing his
Ph.D. at Texas A&M University in Wildlife and Fisheries Sciences. His area
of expertise is mammalogy, and his research interests include zoonoses and
molecular biology.

      Address for correspondence: Jerrold J. Scharninghausen, Department of
Wildlife & Fisheries Sciences, Texas A&M University, College Station, Texas
77843, USA; e-mail: 100304.2713@compuserve.com.

References

  1. Hjelle B, Jenison S, Goade D, Green W, Feddersen R, Scott A.
     Hantaviruses: clinical, microbiologic, and epidemiologic aspects. Crit
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     Atlanta: U.S. Department of Health and Human Services; 1998.
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     Hemorrhagic fever with renal syndrome in Czechoslovakia: detection of
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  9. Gavrilovskaya I, Apekina N, Myasnikov Y, Brenshtein A, Ryltseva E,
     Gorbachkova E, et al. Features of circulation of hemorrhagic fever
     with renal syndrome (HFRS) virus among small mammals in the European
     USSR. Arch Virol 1983;75:313-6.
 10. Gligic A, Obradovic M, Stojanovic R, Hlaca D, Antonijevic B,
     Arnautovic A, et al. Hemorrhagic fever with renal syndrome in
     Yugoslavia: detection of hantaviral antigen and antibodies in wild
     rodents and serological diagnosis of human disease. Scand J Infect Dis
     1988;20:261-6.
 11. Morzunov S, Rowe J, Ksiazek G, Peters CJ, St Jeor S, Nichol S. Genetic
     analysis of the diversity and origin of hantavirus in Peromyscus
     leucopus mice in North America. J Virol 1998;1:57-64.
 12. Nowak RM, editor. Walker's mammals of the world. 5th ed. Baltimore:
     Johns Hopkins University Press; 1991.
--------------------------------------------------------------------------

Dispatches

Bacterial Resistance to Ciprofloxacin in Greece: Results from the National
Electronic Surveillance System

A.C. Vatopoulos, V. Kalapothaki, Greek Network for the Surveillance of
Antimicrobial Resistance (ft1), and N.J. Legakis
Athens University, Athens (Goudi), Greece

---------------------------------------------------------------------------
      According to 1997 susceptibility data from the National
      Electronic System for the Surveillance of Antimicrobial
      Resistance, Greece has high rates of ciprofloxacin resistance.
      For most species, the frequency of ciprofloxacin- resistant
      isolates (from highest to lowest, by patient setting) was as
      follows: intensive care unit > surgical > medical > outpatient.
      Most ciprofloxacin-resistant strains were multidrug resistant.

Soon after the broad-spectrum, highly effective antibiotics
fluoroquinolones were introduced, their extensive use and misuse in
hospitals and communities, as well as in veterinary medicine, have led to
the emergence and spread of resistant strains (1,2). Highly divergent rates
of fluoroquinolone resistance in both community-acquired and nosocomial
pathogens have been reported worldwide (2). Many factors, including patient
characteristics, local epidemiologic factors, antibiotic policies,
over-the-counter use (which often leads to inadequate use), lower standard
of living in developing countries, lack of information on the prudent use
of antibiotics, and use of antibiotics in animal husbandry may contribute
to the emergence of quinolone-resistant organisms.

Surveillance is an integral part of controlling resistance, and local and
national surveys to identify, monitor, and study the epidemiology of the
emergence and spread of resistant isolates are needed (3). To identify
national trends and local differences in the epidemiology of quinolone
resistance in Greece, we report 1997 ciprofloxacin susceptibility data from
the National Electronic System for the Surveillance of Antimicrobial
Resistance.

The National Electronic System for the Surveillance of Antimicrobial
Resistance was introduced in Greece 3 years ago. Involving 17 hospitals
throughout Greece, the system analyzes the routine results of the
antibiotic sensitivity tests performed in hospital microbiology
laboratories by using WHONET software (4).

In our analysis we included 11,097 isolates (4,204 from medical wards,
2,897 from surgical wards, 1,724 from intensive care units [ICU], and 2,272
from outpatient departments) (Table 1). We focused on the bacteria most
frequently encountered in Greek hospitals (National Electronic System for
the Surveillance of Antimicrobial Resistance [www.mednet.gr/whonet]; N.J.
Legakis, Enare Sentry, unpub. data): Escherichia coli, Klebsiella
pneumoniae, Enterobacter species, Pseudomonas aeruginosa, Acinetobacter
baumanii, and Staphylococcus aureus. These species are also the most
important nosocomial pathogens in most parts of the world in terms of rate
of isolation, pathogenicity, and virulence (5,6).

Isolation     Table 1. Isolates included in the analysis(sup a)
and
identification-------------------------------------------------------------
were                                           Type of ward
performed by                    -------------------------------------------
standard                         Medi-  Surgi-           Outpa-
methods at    Species             cal     cal    ICU     tients      All
the                                             (sup b)
microbiology  -------------------------------------------------------------
laboratories  Escherichia         2,100   1,114    94     1,571     4,879
of each         coli
hospital      Pseudomonas           672    527    570       195     1,964
participating   aeruginosa
in the        Staphylococcus        452    467    318       248     1,485
network. The    aureus
susceptibility
testing       Enterobacter          396    332    198       142     1,068
methods were    spp.
Kirby-Bauer   Klebsiella            419    224    177        96       916
disk            pneumoniae
diffusion (7  Acinetobacter         165    233    367        20       785
hospitals);     spp.
Sensititre    All                 4,204  2,897  1,724     2,272    11,097
(Sensititre,
Salem, NH)    -------------------------------------------------------------
(1); Pasco    (sup a)One isolate per species per patient (the first isolated)
(Difco,        is shown. bICU, intensive care unit.
Detroit, MI)
(8); and VITEK (Bieux-Merieux Marcy l'Etoile, France) (1). The actual zone
diameters or MICs (not the interpretations of the tests) were entered into
WHONET. The chi-square test was used to evaluate differences in resistance
rates between types of wards, as well as between clinical specimens.
Pearson's correlation coefficients were calculated for possible
associations between resistance rates and hospital size.

The resistance rate to ciprofloxacin by type of ward, clinical specimen,
and bacterial species is shown in Table 2. There is a stepwise decrease in
the frequency of isolation of ciprofloxacin-resistant isolates
(ciprofloxacin resistance in isolates from ICU patients > isolates from
surgical patients > isolates from medical patients > isolates from
outpatients). These differences were significant (p <0.01), with the
exception of decreases in resistance rates for E. coli between surgical
wards and ICUs; for Enterobacter spp. between medical and surgical wards;
for Acinetobacter spp. between outpatients, medical, and surgical wards;
and for S. aureus between medical and surgical wards. Moreover, for P.
aeruginosa, the resistance rates were significantly higher in medical than
in surgical wards (p = 0.00097).

As for clinical specimens, each bacterial species followed a different
pattern (Table 2). In medical wards, enterobacterial strains isolated from
purulent infections were more often resistant to ciprofloxacin, but this
difference was statistically significant only for K. pneumoniae (p =
0.012). In surgical wards, blood and respiratory isolates were more often
resistant, but this difference was significant only for Enterobacter spp.
(p = 0.02). On the other hand, ciprofloxacin-resistant P. aeruginosa
strains were more frequently isolated (p = 0.0021) in medical wards from
urine and in surgical wards from urine and blood as opposed to all other
specimens (p = 0.0005). No significant differences were observed in the
rate of isolation of ciprofloxacin-resistant A. baumanii strains among the
various clinical specimens. S. aureus strains resistant to ciprofloxacin
were mostly methicillin-resistant (MRSA) (Table 2). Very low resistance
rates were observed in P. aeruginosa isolated from ear infections,
especially from outpatients.

Table 2. Ciprofloxacin resistance by specimen and type of ward(sup a)
-------------------------------------------------------------------------
                      Outpatients    Medical       Surgical       ICU
                     ------------- ------------  ------------- ----------
                      No.    %R   No.    %R     No.    %R    No.   %R
                           (sup b)
-------------------------------------------------------------------------
Escherichia coli

   Urine            1,191   5.0   1,572   5.5   597    8.5    39  10.2
   Blood               -            195   6.9    14   18.1     5   0.0
   Respiratory         -             56   2.1     -           23   9.0
   Pus                 -             33  12.1   203    8.4    11  27.8
   Other              380   4.5     244   7.5   300    6.5    16  20.0
   All              1,571   3.7   2,100   5.6 1,114    8.2    94  13.3
Salmonella spp.
   Stool              195   0.7
Klebsiella pneumoniae
   Urine               62   6.6     254  15.5    85   19.8    28  64.0
   Blood               -             45  11.3    10    9.8    18  72.3
   Respiratory         -             62   9.8    12   50.0    90  69.8
   Pus                 -             14  50.0    42   19.0     0   0.0
   Other               34    3.1     44  18.5    79   28.3    41  65.4
   All                 96    5.4    419  15.8   226   23.9   177  67.7
Serratia marcences
  All                               76    7.7                 20  45.2
                                   (sup c)(sup c)
Enterobacter spp.
  Urine                76   12.0   190   29.7    85   32.0    24  75.4
  Blood                -            37   21.8    13   54.2    24  66.6
  Respiratory          -            76    6.3    10   40.2    58  48.6
  Pus                  -              22 36.8   138   18.5    27  67.6
  Other                66   10.8    71   16.9    86   23.3    65  69.0
  All                 142   11.6   396   22.2   332   24.8   198  62.2
Pseudomonas
aeruginosa
  Urine                51   31.0   270   44.0   171   40.7    70  79.3
  Blood                 0    0.0    24   20.6    13   46.5    29  75.6
  Respiratory          11   18.2   258   34.4    29   44.6   379  62.9
  Pus                  18   11.3    35   31.6   147   22.6    16  69.5
  Ear                  72    1.7     7   47.3    30    3.7     0   0.0
  Other                43   18.8    78   26.9   137   25.9    76  66.9
  All                 195   16.7   672   37.5   527   28.2   570  66.4
Acinetobacter spp.
  Urine                -            72   62.6    32   65.9    34  94.4
  Blood                -            18   38.7    16   69.0    40  92.3
  Respiratory          -            38   49.7    11  100.0   190  91.0
  Pus                  -            13   61.8    87   60.1    19  94.8
  Other                -            24   62.5    87   69.1    84  78.9
  All                  20   45.1   165   56.8   233   66.6   367  88.4
Staphylococcus
aureus
  Urine                -            37   32.9    16   31.0   -
  Blood                -           101   51.0    15   67.0    40  62.7
  Respiratory          -           123   45.3    28   57.1   221  65.8
  Pus                 104   18.2    88   21.6   272   30.8    14  71.4  
  Ear                  52    3.8    -             -            -
  Other                92   10.3   103   25.6   136   31.4    43  67.4
  All                 248   12.8   452   30.5   467   33.0   318  63.6
  MRSAd                40   56.7   140   69.1   176   75.3   375  94.3
  MSSAe               184    1.7   256   12.4   219    6.5    92   4.6
-------------------------------------------------------------------------
(sup a)One isolate per patient (the first isolated) is shown. 
(sup b)R, resistant.
(sup c)Medical and surgical wards combined. 
(sup d)MRSA, methicillin-resistant S. aureus. 
(sup e)MSSA, methicillin-sensitive S. aureus.

Approximately 75% of K. pneumoniae, 87% of Enterobacter spp., 55% of P.
aeruginosa, 76% of A. baumanii, and 75% of MRSA strains were drug resistant
to at least three different classes (Table 3). However, 15% of the
ciprofloxacin-resistant E. coli were resistant only to this antibiotic, and
25% had additional resistance only to cotrimoxazole. Moreover, 48% of
ciprofloxacin-resistant but methicillin-sensitive S. aureus were resistant
only to chloramphenicol.

Table 3. Resistant phenotypes of ciprofloxacin-resistant isolates to other 
classes of antibiotics(sup a)
---------------------------------------------------------------------------
Klebsiella
pneumoniae              Enterobacter spp      Escherichia coli
-------------------     ------------------    -------------------
Phenotypeb No.   %      Phenotype No.   %     Phenotype No.  %
F           4   3.7     F           0     0    F          25 15.1
DBXF        9   8.4     IF          4   2.5    IDBXF      16  9.6
IDB F      16  15.0     IDB F       7   4.4    IXF        29 17.5
IDBXF      64  59.8     IDBXF     131  82.9    XF         42 25.3
all other  14  13.1     all other  16  10.1    all other  54 32.5
All       107 100.0     All       158 100.0    All       166 100.0
                    
Pseudomonas             Acinetobacter
aeruginosa              baumanii
-------------------     ------------------
Phenotype  No.   %      Phenotype No.   %
F          10   7.3     F           0   0.0
1DM F      14  10.2     SMD X       5  10.0
1DMNF      23  16.8     D XF       15  30.0
1 M F      40  29.2     MD XF      23  46.0
all other  50  36.5     all other   7  14.0
All       137 100.0     All        50 100.0

Staphylococcus aureus
MRSA                    MSSA
---------------------------------------------
Phenotype  No.   %      Phenotype No.   %
F           0   0.0     F           7  10.3
OG E F     23  11.3     E F         9  13.2
OG ECF     44  21.7     CF         33  48.5
OGXECF     84  41.4 
all other  52  25.6     all other  19  27.9
All       203 100.0     All        68 100.0
------------------------------------------------------------------------
(sup a)All wards, intensive care units isolates are not included. 
b1, piperacillin; B, tobramycin; C, chloramphenicol; D, ceftazidime; 
E, erythromycin; F, ciprofloxacin; G, gentamicin; I, cefoxitin; M,
amikacin; N, imipenem; O, oxacillin; S, amoxicillin/sulbactam; X, 
cotrimoxazole; MRSA, methicillin-resistant S. aureus; 
MSSA, methicillin-sensitive S. aureus.

When we plotted resistance rates to ciprofloxacin against the number of
beds in each hospital, we found no correlation (Figure). The rate of
isolation of ciprofloxacin-resistant isolates varied greatly by hospital
for all species examined: from 1% to 15% for E. coli, 1% to 23% for K.
pneumoniae, 1% to 33% for Enterobacter spp., 11% to 33% for P. aeruginosa,
29% to 73% for A. baumanii, and 11% to 48% for S. aureus. Ciprofloxacin
resistance was observed in hospitals throughout Greece.

In Europe and North America, a striking difference in the incidence of
bacterial resistance to quinolones has been observed between nosocomial and
community-acquired infections; resistance is only rarely encountered among
the latter (2,7). The incidence of resistance to fluoroquinolones in
bacteria isolated from hospital-acquired infections varies among bacterial
species, clinical settings, and countries and may be related to local
epidemic spread of a few clones (2). The highest incidence of resistance is
among P. aeruginosa, Acinetobacter spp., Serratia marcescens, and
particularly MRSA strains (8). Our results place Greece among the countries
with high resistance levels to quinolones. Although quinolones are among
the antibiotics restricted by the Greek Ministry of Health and Welfare, the
mean national level of quinolone resistance has increased in most bacterial
species during the last 5 years (9).

                                    The 3.7% quinolone resistance rate
            [fig]                   among E. coli isolated from
                                    outpatients is almost double that in
  Fig. Resistance rates to          other industrialized countries (2).
  ciprofloxavin in each hospital by This high rate may be due to the use
  number of beds and geographic     of quinolones, and especially
  area of the hospital. Only        norfloxacin, as a first-line
  hospitals with more than 20       antibiotic in Greece to treat
  isolates are included.            uncomplicated urinary tract infections
  (Isolates from all wards but      in the outpatient setting. Free access
  not intensive care units.)        to fluoroquinolones has also been
                                    incriminated in increased quinolone
                                    resistance in industrialized and
                                    developing countries (10,11). The low
                                    rate of quinolone resistance in
                                    salmonellas, compared with other
countries (12,13), may be due to infrequent use of quinolones in farm
animals in Greece. Among Enterobacteriaceae, quinolone resistance seems to
be higher in K. pneumoniae and Enterobacter spp. than in S. marcescens.

The high level of resistance in ICUs was expected since ICUs are well-known
focuses of antimicrobial resistance (14). Hospitalization in ICUs was an
independent risk factor for acquiring infection by multidrug-resistant
strains in Greece (15). Moreover, ICU patients are often colonized with
endemic, multidrug-resistant strains, which often spread to other wards
(16).

We found higher rates of isolation of quinolone-resistant strains of some
species in the surgical wards than in medical wards. Patients at high risk
for a resistant nosocomial infection (e.g., cancer patients,
immunosupressed patients) are usually in medical wards. High resistance in
the surgical wards could be the result of nursing practices or unnecessary
prophylactic administration of antibiotics, both of which should be further
evaluated.

Most quinolone-resistant strains in Greece are also resistant to other
clinically relevant antibiotics. The possible clinical and epidemiologic
importance of the newly described multidrug efflux pumps in multidrug
resistance, mainly in P. aeruginosa, is under investigation worldwide (17).
Moreover, the marginal susceptibility of S. aureus to quinolones and the
ease with which mutations affecting susceptibility can occur in this
species contribute to the observed high rates of quinolone resistance. MRSA
strains are no more likely to develop resistance to quinolones than other
staphylococci (8). In any case, the favorable accumulation of different
traits in quinolone-resistant strains or, alternatively, the favorable
potential for mutation to quinolone resistance in multidrug-resistant
strains has not been proved. Epidemiologic parameters, and more
specifically the sequential introduction of various antibiotic classes in
most of the world and in Greek hospitals, could explain multidrug
resistance. The extensive aminoglycoside and beta-lactamase use in the
1980s is responsible for the high prevalence of multidrug-resistant
plasmids and transposons found in the nosocomial strains of various
bacterial genera in Greek hospitals (18-20). The strains harboring these
plasmids can survive in the hospital environment and become the best
candidates for selection of resistant mutants under the pressure of
quinolones.

That quinolone-resistant strains are found in hospitals in all parts of
Greece and resistance is not associated with the size of the hospital or
its geographic area are consistent with the high prescription rate for
quinolones. However, the isolation rate of resistant strains varied
considerably by hospital, perhaps because of local epidemiologic factors
(e.g., prescribing or nursing habits) or possible (epidemic) spread of
strains among patients.

This study has limitations. First, it is based on routine data generated in
the microbiology laboratories of participating hospitals. Sometimes
different antibiotics are tested in each hospital, which limits the
possibility for interhospital comparisons. Moreover, different methods for
susceptibility testing are used in each hospital. Data such as antibiotic
consumption or days of hospitalization are not available since they are not
included as information in the WHONET software and they are difficult and
time-consuming to collect routinely.

Quinolone use is a well-proven independent risk factor for resistance
(21,22). Nevertheless, local differences indicate that other epidemiologic
parameters should be further evaluated.

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

      The National Electronic System for the Surveillance of Antimicrobial
Resistance has been supported in part by a grant from the Greek Ministry of
Health and Welfare.

      The following hospitals participate in the system: Polycliniki
General Hospital, Agia Olga General Hospital, Elpis General Hospital, First
IKA Hospital of Athens, Agios Savas Cancer Hospital, Sismanoglion General
Hospital, Hippocration General Hospital, Areteion University Hospital,
Venizelio General Hospital, University Hospital of Alexandroupolis,
University Hospital of Ioannina, General Hospital of Xanthi, Threassio
General Hospital, Tzannio General Hospital, Asclepeion Voulas General
Hospital, Theagenio Cancer Hospital, and Hippocration Hospital
Thessaloniki.

      Dr. Vatopoulos is a medical microbiologist and assistant professor in
the Department of Hygiene and Epidemiology, Medical School, Athens
University. His chief research interest is the molecular epidemiology of
antibiotic resistance in bacteria (mainly gram-negative). He is now
involved in the establishment and operation of an electronic network for
the surveillance of antibiotic resistance in Greece.

      Address for correspondence: A.C. Vatopoulos, Department of Hygiene &
Epidemiology, Medical School, Athens University, 115 27 Athens (Goudi),
Greece; fax: 30-1-7704225; e-mail: avatopou@cc.uoa.gr.

(footnote 1)G. Antoniadis, E. Arhondidou, S. Chatzipanagiotou, E. Chinou, 
A. Chrysaki, V. Daniilidis, G. Genimata, H. Gessouli, P. Golemati, E.
Kaili-Papadopoulou, A. Kansuzidou, D. Kailis, E. Kaitsa, M. Kanelopoulou,
Sp. Kitsou-Kyriakopoulou, Z. Komninou, E. Kouskouni, Chr. Koutsia-Karouzou,
S. Ktenidou-Kartali, V. Liakou, H. Malamou-Lada, H. Mercuri, C.
Nicolopoulou, A. Pagkali, E. Panagiotou, E. Papafragas, A. Perogamvros, C.
Poulopoulou, D. Sofianou, G. Theodoropoulou-Rodiou, S. Thermogianni, E.
Trikka-Graphakos, O. Vavatsi-Manou, M. Ventouri, E. Vogiatzakis, A.
Xanthaki, Chr. Zagora, E. Chatzidaki, G. Papoutsakis.

References

  1. Blondeau JM, Yaschuk Y, Canadian ciprofloxacin susceptibility study.
     Comparative study from 15 medical centers. Antimicrob Agents Chemother
     1996;40:1729-32.
  2. Acar JF, Goldstein FW. Trends in bacterial resistance to
     fluoroquinolones. Clin Infect Dis 1997;24:S67-73.
  3. Report of the American Society for Microbiology Task Force on
     Antibiotic Resistance. Washington: American Society for Microbiology;
     1995. p. 1-23.
  4. Stelling JM, O'Brien TF. Surveillance of antimicrobial resistance: the
     WHONET program. Clin Infect Dis 1997;24:S157-68.
  5. Emori TG, Gaynes RP. An overview of nosocomial infections, including
     the role of the microbiology laboratory. Clin Microbiol Rev
     1993;6:428-42.
  6. Wartz MN. Hospital-acquired infections: diseases with increasingly
     limited therapies. Proc Natl Acad Sci U S A 1994;91:2420-7.
  7. Goldstein FW, Acar JF. Epidemiology of quinolone resistance: Europe
     and North and South America. Drugs 1995;49:S36-42.
  8. Sanders CC, Sanders WE Jr, Thomson. Fluoroquinolone resistance in
     Staphylococci: new challenges. Eur J Clin Microbiol Infect Dis
     1995;Suppl 1:6-11.
  9. Legakis NJ, Tzouvelekis LS, Tsakris A, Legakis JN, Vatopoulos AC. On
     the incidence of antibiotic resistance among aerobic gram-negative
     rods isolated in Greek hospitals. J Hosp Infect 1993;24:233-7.
 10. Kresken M, Hafner D, Mittermayer H, Verbist L, Bergogne-Berezin E,
     Giamarellou H, et al. Prevalence of fluoroquinolone resistance in
     Europe. Study Group `Bacterial Resistance' of the Paul-Ehrlich-Society
     for Chemotherapy. Infection 1994;22:S90-8.
 11. Casellas JM, Blanco MG, Pinto ME. The sleeping giant: antimicrobial
     resistance. Infect Dis Clin North Am 1994;8:29-45.
 12. Tassios PT, Markogiannakis A, Vatopoulos AC, Katsanikou E, Velonakis
     EN, Kourea-Kremastinou J, et al. Molecular epidemiology of antibiotic
     resistance of Salmonella enteritidis during a seven year period in
     Greece. J Clin Microbiol 1997;35:1316-21.
 13. Tassios PT, Vatopoulos AC, Mainas E, Gennimata D, Papadakis J,
     Tsiftsoglou A, et al. Molecular analysis of ampicillin-resistant
     sporadic Salmonella typhi and Salmonella paratyphi B clinical
     isolates. Clinical Microbiology and Infection 1997;3:317-23.
 14. Archibald L, Phillips L, Monnet D, McGrowan JE, Tenover F, Gaynes R.
     Antimicrobial resistance in isolates from inpatients and outpatients
     in the United States: increasing importance of the intensive care
     unit. Clin Infect Dis 1997;24:211-5.
 15. Vatopoulos AC, Kalapothaki V, Legakis NJ, the Hellenic Antibiotic
     Resistance Study Group. Risk factors for nosocomial infections caused
     by gram-negative bacilli. J Hosp Infect 1996;34:11-22.
 16. Tassios PT, Gennimata V, Spaliara-Kalogeropoulou L, Kairis D, Koutsia
     C, Vatopoulos A, et al. Multiresistant Pseudomonas aeruginosa
     serogroup O:11 outbreak in an intensive care unit. Clinical
     Microbiology and Infection 1997;3:621-8.
 17. Nikaido H. Antibiotic resistance caused by gram-negative multidrug
     efflux pumps. Clin Infect Dis 1998;Suppl 1:S32-41.
 18. Vatopoulos A, Phillipon A, Tsouvelekis L, Komninou Z, Legakis NJ.
     Prevalence of a transferable SHV-5 type -lactamase in clinical
     isolates of Klebsiella pneumoniae and Escherichia coli in Greece. J
     Antimicrob Chemother 1990;26:635-48.
 19. Tsakris A, Johnson AP, George RC, Mehtar S, Vatopoulos AC.
     Distribution and transferability of plasmids encoding trimethoprim
     resistance in urinary pathogens from Greece. J Med Microbiol
     1991;34:153-7.
 20. Vatopoulos AC, Tsakris A, Tzouvelekis LS, Legakis NJ, Pitt TL, Miller
     GH, et al. Diversity of aminoglycoside resistance in Enterobacter
     cloacae in Greece. Eur J Clin Microbiol Infect Dis 1992;11:131-8.
 21. Richard P, Delangle MH, Merrien D, Barille S, Reynaud A, Minozzi C, et
     al. Fluoroquinolone use and fluoroquinolone resistance: Is there an
     association? Clin Infect Dis 1994;19:54-9.
 22. Carratala J, Fernandez-Sevilla A, Tubau F, Callis M, Gudiol F.
     Emergence of quinolone-resistant Escherichia coli bacteremia in
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     norfloxacin. Clin Infect Dis 1995;20:557-60.
--------------------------------------------------------------------------

Dispatches

Emergence of Related Nontoxigenic Corynebacterium diphtheriae Biotype mitis
Strains in Western Europe

Guido Funke,* Martin Altwegg,* Lars Frommelt, and Alexander von
Graevenitz*
*University of Zurich, Zurich, Switzerland; and Endo-Klinik, Hamburg,
Germany

---------------------------------------------------------------------------
      We report on 17 isolates of Corynebacterium diphtheriae biotype
      mitis with related ribotypes from Switzerland, Germany, and
      France. Isolates came from skin and subcutaneous infections of
      injecting drug users, homeless persons, prisoners, and elderly
      orthopedic patients with joint prostheses or primary joint
      infections. Such isolates had only been observed in
      Switzerland.

Nontoxigenic Corynebacterium diphtheriae strains were recovered from
approximately 1 per 1,000 throat swabs from immunized British military
personnel in Germany from 1993 to 1995 (1). Such nontoxigenic C.
diphtheriae biotype mitis isolates had been described in skin, throat, and
blood cultures of Swiss injecting drug users; 32 of the isolates belonged
to the same clone (2,3). Our study demonstrates that this clone and closely
related clones occurred between 1990 and 1997 in two other European
countries, and only sometimes in persons with poor hygiene.

Five C. diphtheriae isolates came from two laboratories in Zurich and Bern,
Switzerland; 11 from four laboratories in Hamburg, Germany; and 1 from
Paris, France (Table). They were identified in Zurich as C. diphtheriae
biotype mitis (4); that is, they were nonlipophilic, were nitrate
reductase-positive, and did not ferment glycogen. By polymerase chain
reaction techniques (5), the diphtheria toxin gene was not detected in any
isolate. For some isolates, the Elek test was also performed; it was
consistently negative. For ribotyping, DNA was isolated, digested with
either EcoRI or PvuII, electrophoresed, blotted, and probed for rDNA as
described elsewhere (2,3). Disk susceptibility testing to tetracycline and
MIC determinations were done according to National Committee for Clinical
Laboratory Standards methods (6). All other bacteria isolated were also
identified and serotyped in Zurich.

Table. Nontoxigenic Corynebacterium diphtheriae isolates

-----------------------------------------------------------------------------------------------------
                               Ribotyping
                               pattern(sup c)  
                               ----------- Patients    Clinical
Isolate  Date       Place of               sex, age   diagnosis/     Other bacteria     Underlying
no.    isolated    isolation   EcoRI PvuII   (yr)       source          isolated         conditions
		(sup a)   (sup b)  
-----------------------------------------------------------------------------------------------------
2012    1990       Paris         A     C     m, 6   Endocarditis/                    Skin lesions,
                                                    blood culture                     scabies

2410    2/1995     Hamburg       A     B    m, 30   Ulcus/lower    Staphylococcus     Prisoner
                                                    leg            aureus,
                                                                   Streptococcus
                                                                     pyogenes
2689    3/1995     Hamburg       A     A    f, 82   Puncture/hip   S. epidermidis,    Total hip joint
                                                    joint          Corynebacterium    endoprosthesis
                                                                   pseudodiphtherit-
                                                                   icum

1682    9/1995     Hamburg       A     B    m, 45   Wound swab     S. aureus,         Homelessness
                                                                   S. pyogenes

1935    3/1996     Hamburg       A     B    m, 58   Wound swab     S. aureus,         Alcoholism,
                                                                   S. pyogenes        homelessness

1836    3/1996     Hamburg       A     B    m, 45   Abscess/lower  S. aureus,         Injecting
                                                    leg            S. pyogenes        drug use

2661    3/1996     Hamburg       A     A    m, 35   Arthroscopy    Peptostreptococcus Osteosynthesis,
                                                    aspirate/knee
                                                    joint          prevotii,P. magnus arthrotomy,
                                                                                      knee joint
                                                                                      after trauma

2658    4/1996     Hamburg       A     A    f, 76   Fistula/hip    S. aureus          Total hip
                                                    joint                             joint
                                                                                      endoprosthesis
2674    5/1996     Hamburg       A     A    f, 62   Aspirate/hip   Streptococcus      Total hip joint
                                                    joint          group C
                                                                                      endoprosthesis
2670    7/1996     Hamburg       A     A    f, 81   Aspirate/knee                    Total knee
                                                    joint                             joint
                                                                                      endoprosthesis
2667    7/1996     Hamburg       A     A    m, 23   Swab/hip joint Coagulase-negative Total hip joint

                                                                   staphylococci      endoprosthesis
2413    11/1996    Hamburg       A     B    m, 36   Ulcus/lower    S. aureus,         Prisoner
                                                    leg            Streptococcus
                                                                   group C/G
2446    12/1996    Zurich        A     A    f, 38   Wound/upper    S. aureus, S.      Injecting drug
                                                    leg            pyogenes           use

2464    1/1997     Zurich        A     A    m, 39   Ulcera/lower   Escherichia coli,  Injecting drug
                                                    arm                               use
                                                                   S. aureus, S.
                                                                   pyogenes
2473    1/1997     Zurich        A     A    m, 38   Ulcera/leg     E. coli,           Injecting drug
                                                                   Streptococcus      use
                                                                   group C, G
2475    1/1997     Zurich        A     A    m, 31   Ulcus/pretibialPseudomonas        Injecting drug
                                                                   aeruginosa,  E.    use
                                                                   coli,
                                                                   Streptococcus
                                                                   group C, G

480     5/1997     Bern          A     A    m, 39   Ulcus/upper legS. aureus,         Injecting
                                                                   Clostridium        drug use
                                                                   perfringens,
                                                                   mixed
                                                                   anaerobic flora
-----------------------------------------------------------------------------------------------------
(sup a)Month/Year
(sup b)Paris, France; Hamburg, Germany; Zurich, Switzerland; Bern, Switzerland.
(sup c)DNA was isolated, digested with either EcoRI or PvuII, electrophoresed, blotted, and probed for
DNA. Three distinct ribopatterns emerged, each with eight bands: patterns A, B, and C.

Many (10 [59%] of 17) C. diphtheriae isolates were from skin or
subcutaneous infections (wounds, ulcers) in Swiss patients and were found
with Staphylococcus aureus, Streptococcus pyogenes, group C/G streptococci,
or (sometimes) with gram-negative rods. Most patients in this subgroup were
injecting drug users, homeless persons, prisoners, and (with one exception)
men with a mean age of 40 (30 to 58) years. Another subgroup (from Germany)
consisted of six patients with joint or bone infections (mentioned briefly
in an earlier publication on coryneform bacteria from such infections; a
seventh patient had C. diphtheriae biotype gravis [7]). The six had been
hospitalized at the Endo Clinic in Hamburg, which specializes in orthopedic
surgery. Four had had implantations of hip endoprostheses and had been
admitted for prosthetic infections (with coagulase-negative staphylococci
or S. aureus); one pure culture of C. diphtheriae biotype mitis was
obtained from a patient with a knee prosthesis, and one mixed culture with
Peptostreptococcus magnus and Peptostreptococcus prevotii was obtained from
a patient who had a purulent knee infection after a fracture. Their average
age was 60 (23 to 82) years and, to our knowledge, none was a drug user.
While the Hamburg isolates were recovered only once from every patient,
their isolation dates stretched from March 1, 1995, until July 26, 1996.
Three were isolated on the day of admission, and all of them were isolated
by different technologists. Finally, one child (from France) had
endocarditis with C. diphtheriae biotype mitis.

All isolates had identical antimicrobial susceptibility patterns. They were
susceptible to amoxicillin (MIC, 0.25 g/ml), amoxicillin-clavulanic acid
(0.06 g/ml), chloramphenicol (2 g/ml), ciprofloxacin (0.25 g/ml),
clarithromycin (0.03 g/ml), clindamycin (0.25 g/ml), imipenem (0.03
g/ml), penicillin (0.25 g/ml), and vancomycin (1 g/ml). In contrast, all
strains were resistant to tetracycline in the disk diffusion test
(inhibition zone diameter 10 mm to 11 mm); their MICs for tetracycline,
doxycycline, and minocycline were 64 g/ml, 16 g/ml, and 16 g/ml,
respectively. While this type of isolated tetracycline resistance was
typical for the isolates from Swiss injecting drug users (3), tetracycline
resistance in nontoxigenic C. diphtheriae has otherwise very rarely been
observed in Europe (8). It has, however, been reported in toxigenic C.
diphtheriae isolates from Indonesia and, rarely, from Canada (9). The
mechanism conferring this resistance in our strains has not been
investigated.

On analysis with restriction enzyme Pvu II, three different ribopatterns
with eight bands each were found among the strains (Figure). These
patterns, though distinct, shared five (A vs. C, B vs. C) and six (B vs. A)
bands, respectively, and, therefore, may be considered related, as they
would be if criteria for pulsed-field gel electrophoresis were applied
(10). This view is supported by the fact that all strains had identical
EcoRI patterns (not shown). All Swiss isolates were identical with the use
of both enzymes, whereas both the Swiss pattern and a second pattern were
found among the Hamburg isolates. The third pattern was found exclusively
in the single French strain. The staphylococcal and streptococcal strains
were not typed; they are no longer available to us.

Our isolates thus   
resemble those      Figure. Ribosomal RNA gene restriction patterns
reported from       obtained by using restriction enzyme Pvu II of
Switzerland between Corynebacterium diphtheriae isolates belonging to
1990 and early 1996 ribotypes B (lanes 1 to 3), A (lanes 4 to 6 and 8),
(2,3), which were   and C (lane 7).
also often
accompanied by S.
aureus or beta-hemolytic streptococci. Such nontoxigenic C. diphtheriae
mitis may cause endocarditis, arthritis, and osteomyelitis (11,12). Most of
the 52 isolates from France (11), examined with restriction enzymes
different from ours and not available to us, also belonged to one ribotype.
Their relatedness to our strains is unknown; however, they were largely
tetracycline-susceptible. The two throat isolates from St. Petersburg,
Russia, associated with a fatal diphtherialike disease (12) were not typed
or tested for antibiotic susceptibility.

The origin of the isolates we describe is unknown. They may have been
present (but unrecognized) in the population for a long time. The mode of
transmission is most likely common use of drug paraphernalia in the
injecting drug use cases and in the endocarditis case; transmission is very
difficult to explain in the orthopedic infection subgroup. Although the
Swiss and the Danish borders are not close, migration and contacts are not
uncommon among injecting drug users. The isolates may also have been
distributed through the drugs themselves, as recently reported for S.
pyogenes in Switzerland (13).

Our study may be representative for Switzerland and Germany, but since
submitting C. diphtheriae strains to a central reference laboratory in
these countries is not mandatory, we cannot estimate the frequency of these
strains. These strains may also have spread to other European countries;
this hypothesis can only be tested in a large multicenter study of European
diphtheria reference laboratories.

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

Acknowledgments

      We thank J. Lthy-Hottenstein and V. Pnter-Streit for excellent
technical assistance and P. Das for her help in securing the strains from
Hamburg. G. Funke is recipient of a European Society for Clinical
Microbiology and Infectious Diseases research fellowship.

      Dr. Funke is director of clinical microbiology at Grtner &
Colleagues Laboratories in Weingarten, Germany. His areas of expertise are
clinical and systematic bacteriology. Research interests include taxonomy
and disease associations of coryneform bacteria and actinomycetes, as well
as rapid methods for identification and susceptibility testing of medically
relevant bacteria.

      Address for correspondence: Alexander von Graevenitz, Department of
Medical Microbiology, University of Zurich, Gloriastra sse 32, CH-8028
Zurich, Switzerland; fax: 411-634-4906; e-mail: avg@immv.unizh.ch.

References

  1. Sloss JM, Hunjan RS. Incidence of non-toxigenic corynebacteria
     diphtheria in British military personnel in Germany. J Infect
     1996;33:139.
  2. Gubler J, Huber-Schneider C, Gruner E, Altwegg M. An outbreak of
     non-toxigenic Corynebacterium diphtheriae infection: single bacterial
     clone causing invasive infection among Swiss drug users. Clin Infect
     Dis 1998;27:1295-8.
  3. Gruner E, Zuber PLF, Martinetti-Lucchini G, von Graevenitz A, Altwegg
     M. A cluster of non-toxigenic Corynebacterium diphtheriae infections
     among Swiss intravenous drug abusers. Medical Microbiology Letters
     1992;1:160-7.
  4. Von Graevenitz A, Funke G. An identification scheme for rapidly and
     aerobically growing Gram-positive rods. Zentralbl Bakteriol
     1996;284:246-54.
  5. Martinetti-Lucchini G, Gruner E, Altwegg M. Rapid detection of
     diphtheria toxin by the polymerase chain reaction. Medical
     Microbiology Letters 1992;1:276-83.
  6. National Committee for Clinical Laboratory Standards. Performance
     standards for antimicrobial susceptibility testing; eighth
     informational supplement [NCCLS document M 100-S8]. Wayne (PA): The
     Committee; 1998.
  7. von Graevenitz A, Frommelt L, Pnter-Streit V, Funke G. Diversity of
     coryneforms found in infections following prosthetic joint insertion
     and open fractures. Infection 1998;26:36-8.
  8. Patey O, Bimet F, Emond JP, Estrangin E, Riegel P, Halioua B, et al.
     Antibiotic susceptibilities of 38 non-toxigenic strains of
     Corynebacterium diphtheriae. J Antimicrob Chemother 1995;36:1108-10.
  9. Rockhill RC, Sumarmo, Hadiputranto H, Siregar SP, Muslihun B.
     Tetracycline resistance of Corynebacterium diphtheriae isolated from
     diphtheria patients in Jakarta, Indonesia. Antimicrob Agents Chemother
     1982;21:842-3.
 10. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing
     DH, et al. Interpreting chromosomal DNA restriction patterns produced
     by pulsed-field gel electrophoresis: criteria for bacterial strain
     typing. J Clin Microbiol 1995;33:2233-9.
 11. Patey O, Bimet F, Riegel P, Halioua B, Emond JP, Estrangin E, et al.
     Clinical and molecular study of Corynebacterium diphtheriae systemic
     infections in France. J Clin Microbiol 1997;35:441-5.
 12. Rakhmanova AG, Lumio J, Groundstroem KWE, Taits BM, Zinserling VA,
     Kadyrova SN, et al. Fatal respiratory tract diphtheria apparently
     caused by nontoxigenic strains of Corynebacterium diphtheriae. Eur J
     Clin Microbiol Infect Dis 1997;16:816-20.
 13. Streptokokken-Infektionen bei Drogenschtigen in der Region Bern.
     Bulletin des Bundesamtes fr Gesundheit 1997;44:3.

______________________________________________________________________
Letters
______________________________________________________________________

Letters

First Case of Human Ehrlichiosis in Mexico

To the Editor: Ehrlichiosis is a zoonotic disease transmitted to humans
through the bite of infected ticks (1). The first recognized human
ehrlichial infection, Sennetsu fever, was described in Japan in 1954 (2).
The first case of human ehrlichiosis in the United States was recognized in
1986 and was reported in 1987 (3). The disease is caused by intracellular
gram-negative bacteria of the Ehrlichia genus. The bacteria can be found in
the monocytes and granulocytes of peripheral blood. Human monocytic
ehrlichiosis is caused by E. chafeensis, and human granulocytic
ehrlichiosis is caused by E. equi or E. phagocytophilia, which was first
recognized in 1994 (4). Most cases occur between April and September, and
the reservoirs are field animals such as rodents, deer, and dogs. The
clinical spectrum of the disease is similar to that of other febrile
illnesses; without adequate and timely treatment, approximately 5% of the
patients die (5).

In the United States, more than 400 cases of serologically confirmed E.
chaffensis infection have been documented since 1996 (6). No cases have
been reported in Mexico.

In February 1997, we evaluated a 41-year-old male patient from Merida. The
patient had been exposed to ticks during activity in a rural area 1 week
before the onset of illness. Clinical manifestations included frequent
hyperthermia, rash, myalgia, headache, anorexia, fatigue, and cough.
Physical examination showed bilateral cervical lymphadenopathy, and a chest
radiograph showed an interstitial bilateral infiltrate. Hematic cytometry
showed thrombocytopenia of 134 x 10(sup 3)/L and 3200 leukocytes (1440
neutrophils/L). Hepatic transaminases were elevated, with an aspartate
aminotransferase: 92 U/L (normal: 22 U/L), alanine aminotransferase: 48 U/L
(normal: 18 U/L), gamma-glutamyltranspeptidase: 278 U/L (normal: 28 U/L);
and globulins: 4.8 g/dL with a polyclonal pattern. No antibodies against
rickettsia, dengue virus, B-19 parvovirus, or HIV were detected. A serum
sample gave a positive reaction by indirect immunofluorescence assay
against E. chaffeensis at titers of 1:64 on week 2 and 1:128 on week 3. No
infected monocytes or granulocytes were observed in peripheral blood.
Remission of the clinical manifestations began on week 4 and was completed
on week 6.

This case indicates the existence of human ehrlichiosis in Yucatan, Mexico.
Reactivity to E. chaffeensis suggests human monocytic ehrlichiosis;
however, as antibody testing was not performed with E. phagocytophila or E.
equi, the possibility of human granulocytic ehrlichiosis cannot be
excluded. In any event, case reports indicate the need for deliberate
search for cases. Dengue is endemic in this area of Mexico, and
ehrlichiosis should be considered as a differential diagnosis.

Renn A. Gongra-Biachi,* Jorge Zavala-Velzquez,  Carlos Jos
Castro-Sansores,* and Pedro Gonzlez-Martnez*
*Centro de Investigaciones Regionales "Dr. Hideyo Noguchi," Mrida,
Yucatn, Mxico; and Facultad de Medicina, Universidad Autnoma de
Yucatn, Mrida, Yucatn, Mxico

References

  1. Dumler SJ, Bakken JS. Ehrlichial diseases of humans: emerging
     tick-borne infections. Clin Infect Dis 1995;20:1102-10.
  2. Schaffner W, Standaert SM. Ehrlichiosisin pursuit of an emerging
     infection. N Engl J Med 1996;334:262-3.
  3. Maeda K, Markowitz N, Hawley RC, Ristic M, Cox D, McDade JE. Human
     infection with Ehrlichia canis, a leucocytic rickettsia. N Engl J Med
     1987;316:853-6.
  4. Bakken JS, Dumler JS, Chen SM, Eckman MR, Van Etta LL, Walker DH.
     Human granulocytic ehrlichiosis in the upper midwest United States: a
     new species emerging? JAMA 1994;272:212-8.
  5. Walker D, Raoult D, Brouqui P, Marrie T. Rickettsial diseases. In:
     Fauci AS, Braunwald E, Isselbacher KJ, Wilson JD, Martin JB, Kasper
     DL, et al., editors. Harrison's principles of internal medicine. 14th
     ed. New York: The McGraw-Hill Companies; 1998. p. 1045-52.
  6. Walker DH, Dumler JS. Emergence of the ehrlichioses as human health
     problems. Emerg Infect Dis 1996;2:18-29.

HIV-1 Subtype F in Single and Dual Infections in Puerto Rico: A Potential
Sentinel Site for Monitoring Novel Genetic HIV Variants in North America

To the Editor: Although international efforts to systematically collect,
characterize, and classify HIV isolates from around the world have
increased considerably, data on HIV-1 genetic variations in Puerto Rico are
limited. This island (population 3.7 million) has one of the highest AIDS
incidence rates in the United States (53.3 cases per 100,000) (1). To
evaluate the potential for a multiple subtype distribution pattern in
Puerto Rico, we analyzed genetic variations between HIV-1 strains isolated
from peripheral blood mononuclear cells of 63 asymptomatic HIV-infected
female commercial sex workers from 12 communities. These participants were
part of 290 female commercial sex workers followed in a larger
cross-sectional study of risk behavior (2).

HIV-1 subtypes F (n = 4) and B (n = 44) strains were identified in persons
infected with a single viral subtype with a molecular screening assay based
on restriction fragment length polymorphism (RFLP) analysis and with DNA
sequencing of the viral protease gene-prot (3). The remaining 15 specimens
were classified by RFLP as potential dual infections. Further cloning and
sequencing of prot from three of these specimens confirmed one dual
infection involving subtypes F and B viruses and identified two infections
caused by genetically distinct quasispecies of subtype B variants.

In further detailed pairwise analysis of HIV-1 prot genes, a small
nucleotide divergence of 0.3% (0.0 to 1.1) within subtype F contrasted with
a typical value of 6.3% (5.1 to 7.8) for the intrasubtype distance within
subtype B prot sequences (4). The 99% similarity between prot subtype F
Puerto Rican sequences suggested an epidemiologic link or a recent
introduction of subtype F in Puerto Rico. Comparative sequence analysis of
the C2-V3 env is useful in establishing the time that elapsed from
infection on the basis of an annual nucleotide divergence of 0.5% to 1% in
this region (5). Such analysis has been used to study the epidemiologic
link between cases (4,6). Thus, we compared env sequences from two of five
persons infected with prot subtype F strains. This analysis provided
several observations. Env nucleotide divergence of 13.2% did not support a
direct epidemiologic link between these strains. Furthermore, the
relatively high intrasubtype diversity between env sequences suggested that
evolution from a common progenitor would have taken a minimum of
approximately 13 years. Phylogenetic analysis classified these two env
sequences as subtype B, indicating that at least some of Puerto Rican prot
subtype F viruses represent HIV-1 mosaics involving closely related prot F
and significantly divergent env B sequences. Overall, discrepancy in both
subtype assignment and nucleotide diversities within prot and env regions
may indicate that distinct F/B mosaics circulating in Puerto Rico were
likely the result of recombination between highly homogeneous subtype F of
relatively recent arrival and divergent resident subtype B viruses.

HIV-1 infections with subtype F strains including B/F mosaics have been
reported in Brazil (3,7). To evaluate a potential HIV-1 linkage between
Brazil and Puerto Rico, a comparative phylogenetic analysis was done on
subtype F viral prot sequences from these countries. This analysis
documented that HIV-1 subtype F strains in Puerto Rico are distinct from
both Brazilian and Romanian viruses. Furthermore, our results show that
genetic analysis of prot allows tracking of subtype F viruses of different
origin. Recently, by this approach, HIV-1 prot subtype F of Puerto Rican
origin and F prot/B env mosaic were identified in HIV-1-infected persons in
New York city (8). Observation of HIV-1 subtype F strains in Puerto Rico
together with the recent report describing the first cases of such
infections in New York indicates the potential for further emergence of
subtype F on the North American continent. The presence of a complex
distribution pattern of subtype F infections in Puerto Rico has serious
implications for the evaluation and development of HIV diagnostics and
vaccines.

Supported in part by grant G12RR-03050 (Y.Y.).

The nucleotide HIV-1 sequences obtained in this study were submitted to
GenBank; their accession numbers are AF096813-AF096833.

Idhaliz Flores,* Danuta Pieniazek, Nitza Morn,* Angel Soler,* Nayra
Rodrguez,* Margarita Alegra, Mildred Vera, Luiz M. Janini, Claudiu I.
Bandea, Artur Ramos, Mark Rayfield, and Yasuhiro Yamamura*
*Ponce School of Medicine, Ponce, Puerto Rico; Centers for Disease Control
and Prevention, Atlanta, GA; University of Puerto Rico, San Juan, Puerto
Rico

References

  1. Centers for Disease Control and Prevention. HIV/AIDS surveillance
     report no. 2; 1997;9.
  2. Drugs, HIV infection and risk behaviors among Puerto Rican sex
     workers, 1994-1996. Grant: NIAID/RCMI #G12RR03051 [Dr. Margarita
     Alegria]. Sociomedical Research Department, Graduate School of Public
     Health, University of Puerto Rico, Medical Sciences Campus, San Juan,
     Puerto Rico.
  3. Ramos A, Tanuri A, Schechter M, Rayfield MA, Hu DJ, Cabral MC, et al.
     HIV-1 dual infections are an integral part of the HIV epidemic in
     Brazil. Emerg Infect Dis 1999;5:65-74.
  4. Janini LM, Tanuri A, Schechter M, Peralta JM, Vicente AC, De la Torre
     N, et al. Horizontal and vertical transmission of human
     immunodeficiency virus type 1 dual infections caused by viruses of
     subtypes B and C. J Infect Dis 1998;177:227-31.
  5. Myers G, Korber B, Berzofski JA, Smith RF, and Database and Analysis
     Staff, editors. Human retroviruses and AIDS 1991: a compilation and
     analysis of nucleic acid and amino acid sequences. Los Alamos (NM):
     Los Alamos National Laboratory; 1991.
  6. Ou CY, Ciesielski C, Myers G, Bandea CI, Luo CC, Korber BT, et al.
     Molecular epidemiology of HIV transmission in a dental practice.
     Science 1992;256:1167-71.
  7. Sabino EC, Shpaer EG, Morgado MG, Korber BT, Diaz RS, Bongertz V, et
     al. Identification of human immunodeficiency virus type 1 envelope
     genes recombinant between subtypes B and F in two epidemiologically
     linked individuals from Brazil. J Virol 1994;68:6340-6.
  8. Weidle PJ, Ganea CE, Pieniazek D, Ramos CA, Ernst JA, McGowan JP, et
     al. Prevalence of HIV-1 group M, non-B-subtypes in Bronx, New York
     community: a sentinel site for monitoring of HIV genetic diversity in
     the United States. In: Proceedings of the 12th World AIDS Conference;
     1998 Jun; Geneva, Switzerland [abstract no. 13225].

Paratyphoid Fever in India: An Emerging Problem

To the Editor: Enteric fever is a major public health problem in India,
accounting for more than 300,000 cases per year, Salmonella typhi is the
most common etiologic agent (1), but Salmonella paratyphi A, the other
causative agent, causes more asymptomatic infections than S. typhi.
According to earlier reports from India, S. paratyphi A was implicated as a
causative agent in 3%-17% of enteric fever cases (2). However, a large
community-based study in an urban slum of Delhi during October 1995 to
October 1996 found that S. paratyphi A caused approximately 20%-25% of the
cases of enteric fever in this region (3). An outbreak of enteric fever due
to a single S. paratyphi A strain in an urban residential area was reported
in 1996 from New Delhi, where contaminated water was implicated as the
probable source (4,5). This outbreak prompted a retrospective analysis of
the laboratory records of the All India Institute of Medical Sciences, New
Delhi, over a 5-year period (1994-1998) to study the change, if any, in the
etiology of enteric fever in North India.

We evaluated all blood culture records from the institute's clinical
bacteriology laboratory for April to October (the months with the highest
number of enteric fever cases) each year. Records were from patients
residing in New Delhi and the surrounding areas of North India. The blood
was collected by a phlebotomist in the outpatient department or by a
resident doctor in hospital wards. Blood cultures were carried out by
standard laboratory technique (6). Five ml of blood was added to 50 ml of
brain heart infusion broth (Hi-Media Laboratory, India) under aseptic
conditions. Bacterial identification was accomplished by standard
microbiologic protocol (6). Susceptibility to antibiotics (amoxycillin,
chloramphenicol, cotrimoxazole, gentamicin, ciprofloxacin, and ceftriaxone)
was tested by the comparative disk diffusion method (Stokes method) (7).
Chi-square for trend was calculated, and the p value was determined.

The total number of blood cultures performed for enteric fever cases
(10,109 in 1994, 12,092 in 1995, 17,652 in 1996, 15,997 in 1997, and 17,012
in 1998) did not change significantly over this period. The isolation of S.
typhi changed little (Chi-square = 2.367; p = 0.123; statistically not
significant). However, the proportion of S. paratyphi A isolates rose from
6.5% in 1994 to 44.9% in 1998 (Chi-square = 22.20; p <0.001; statistically
significant). The proportion of S. paratyphi A isolations in enteric fever
cases from 1994 to 1998 was 6.5%, 21.2%, 50.5%, 30.7%, and 44.9%,
respectively. Even excluding the strains from the 1996 outbreak (4), we
found that the proportion of S. paratyphi A in enteric fever cases
increased compared with S. typhi (Chi-square = 30.528; p <0.001). With our
catchment area, case definition of enteric fever, and laboratory methods
remaining the same during this period, it appears that the etiology of
enteric fever in North India is changing significantly.

The age-wise distribution of S. typhi and S. paratyphi A showed that S.
typhi was a significant isolate from children < 5 years of age, while this
distribution was not observed for S. parathyphi A, which involved those > 5
years of age. Sex was not significantly associated (mean male to female sex
ratio was 32.4:18 for S. typhi and 15.8:10.6 for S. paratyphi A).

S. typhi has become increasingly sensitive to amoxycillin, chloramphenicol,
and gentamicin, increasing from 75.1% in 1994 to 96.6% in 1998 for
amoxycillin, from 71.9% in 1994 to 91.6% in 1998 for chloramphenicol, and
from 96.4% to 100% for gentamicin. S. paratyphi A strains have remained
uniformly sensitive (100%) to all antibiotics (amoxycillin,
chloramphenicol, and gentamicin, as well as ciprofloxacin and ceftriaxone)
used in the treatment of enteric fever. In light of reports of multidrug
resistance in S. typhi, especially to quinolones, continued surveillance
and monitoring of antimicrobial sensitivity of S. paratyphi A strains are
needed.

The increase in proportion of S. paratyphi A cases, which may be due to a
high degree of clinical suspicion (with mild fever cases investigated for
enteric fever), changing host susceptibility, or even change in the
virulence of the organism, should be further investigated.

Seema Sood, Arti Kapil, Nihar Dash, Bimal K. Das, Vikas Goel, and Pradeep
Seth
All India Institute of Medical Sciences, New Delhi, India

References

  1. Richens J. Typhoid and paratyphoid fevers. In: Oxford textbook of
     medicine. Weatherall DJ, Ledingham JGG, Warrell DA, editors. Vol 1.
     3rd ed. London:: Oxford Medical Publication; 1996. p. 560-8.
  2. Saxena SN, Sen R. Salmonella paratyphi A infection in India: incidence
     and phage types. Trans Royal Soc Trop Med Hyg 1966;603:409-11.
  3. Kumar R, Sazawal S, Sinha A, Sood S, Bhan MK. Typhoid fever:
     contemporary issues as related to the disease in India. Round Table
     Conference Series on Water Borne Diseases. 12th ed. Ranbaxy Science
     Foundation, New Delhi, 1997;2:31-6.
  4. Kapil A, Sood S, Reddaiah VP, Das BK, Seth P. Partyphoid fever due to
     Salmonella enterica serotype paratyphi A. Emerg Infect Dis 1997;3:407.
  5. Thong K, Nair S, Chaudhry R, Seth P, Kapil A, Kumar D, et al.
     Molecular analysis of Salmonella paratyphi A from an outbreak in New
     Delhi, India. Emerg Infect Dis 1998;4:507-8.
  6. Collee JG, Duguid JP, Fraser AG, Marmion BP. Mackie and Mc Cartney
     practical medical microbiology: laboratory strategy in the diagnosis
     of infective syndromes. 13th ed. London (UK): Churchill Livingstone;
     1989. 601-7.
  7. Stokes EJ, Ridgway GL. Clinical bacteriology: anti-bacterial drugs.
     5th ed. London: Edward Arnold; 1980. p. 205-19.

Hepatitis C Virus RNA Viremia in

Central Africa

To the Editor: Epidemiologic serosurveys have demonstrated high prevalence
(6%-15%) of hepatitis C virus (HCV) infection in adults in sub-Saharan
Africa (1-4). Although possible false-positive HCV serologic test results
have been reported in Africa, HCV prevalence rates suggest a high rate of
chronic infection among persons with anti-HCV antibodies (5,6). We have
focused on HCV RNA infectivity of blood from donors attending the National
Blood Center in Bangui, Central African Republic.

We prospectively tested all blood donors between February and April 1998
for serum anti-HCV antibodies by both an HCV third-generation enzyme-linked
immunosorbent assay (ELISA) (Abbott HCV EIA 3.0 test, Abbott, Chicago, IL,
USA), which was chosen as a reference test for immunoglobulin (Ig) G
antibodies to HCV, and by a simple membrane immunoassay system (Ortho HCV
Ab Quik Pack, Ortho Diagnostic Systems Inc., Tokyo, Japan) (7).
Anti-HCV-positive serum samples were further subjected to qualitative
detection of HCV RNA by reverse transcription-polymerase chain reaction
(AMPLICOR-HCV, Roche Diagnostic Systems, Inc., Branchburg, NJ, USA) (8). Of
163 serum samples (mean age  standard deviation, 308 years), 155 were
from male blood donors, 83 (51%) from first-time donors, and 125 (77%) from
donors in the recipient's family. Fifteen (9.2%; 95% confidence interval
[CI] 5%-15%) samples contained IgG to HCV by ELISA. Of the ELISA-positive
samples, 14 were positive by the Quik Pack assay (sensitivity, 93.0%); of
the 148 remaining ELISA-negative samples, 147 were negative by the Quik
Pack assay (specificity, 99.3%). The agreement between the results of the
two methods was 98.7%. Of the 163 samples, 10 (6.1%; CI 95%: 3%-11%) were
positive for HCV antibodies (by ELISA and rapid test) and for HCV RNA.

We confirmed a high prevalence of HCV-seropositivity among blood donors in
Bangui and the subsequent high rate of HCV RNA viremic blood donations. To
offset the major risk for transfusion-acquired HCV in Central Africa we
recommend screening donated blood for anti-HCV. When laboratory facilities
to perform ELISA are not available, the Quik Pack system, a simple reliable
method for detecting anti-HCV antibodies in human serum that requires
neither complex reagent preparation nor expensive instrumentation, could
prove useful.

Nicole Cancr,* Grard Grsenguet, Franois-Xavier Mbopi-Kou, Alain
Kozemaka, Ali Si Mohamed,* Mathieu Matta,* Jean-Jacques Fournel, and
Laurent Blec*
*Universit Pierre et Marie Curie, Hpital Broussais, Paris, France;
Centre National de Transfusion Sanguine, Bangui, Rpublique
Centrafricaine; London School of Hygiene and Tropical Medicine, London,
United-Kingdom; and Hpital de la Piti-Salptrire, Paris, France

References

  1. Ndumbe PM, Skalsky J. Hepatitis C virus infection in different
     populations in Cameroon. Scand J Infect Dis 1993;25:689-92.
  2. Xu LZ, Larzul D, Delaporte E, Brchot C, Kremsdorf D. Hepatitis C
     virus genotype 4 highly prevalent in Central Africa (Gabon). J Gen
     Virol 1994;75:2393-8.
  3. Fretz C, Jeannel D, Stuyver L, Herve V, Lunel F, Boudifa A, et al. HCV
     Infection in a rural population of Central African Republic (CAR):
     evidence for three additional subtypes of genotype 4. J Med Virol
     1995;47:435-7.
  4. Pawlotsky JM, Blec L, Grsenguet G, Desforges L, Bouvier M, Duval J,
     et al. High prevalence of hepatitis B, C and E markers in young
     sexually active adults from the Central African Republic. J Med Virol
     1995;46:269-73.
  5. Aceti A, Taliani D. Hepatitis C virus testing in African sera. Ann
     Intern Med 1992;116:427.
  6. Callahan JD, Constantine NT, Kataaha P, Zhang X, Hyams KC, Bansal J.
     Second generation hepatitis C virus assays: performance when testing
     African sera. J Med Virol 1993;41:35-8.
  7. Kodama T, Ichiyama S, Sato K, Nada T, Nakashima N. Evaluation of a
     membrane filter assay system, Ortho HCV Ab Quik Pack, for detection of
     anti-hepatitis C virus antibody. J Clin Microbiol 1998;36:1439-40.
  8. Young KKY, R. Resnick RM, Myers TW. Detection of hepatitis C virus RNA
     by a combined reverse transcription-polymerase chain reaction assay. J
     Clin Microbiol 1993;31:882-6.

Immunization of Peacekeeping Forces(sup 1)

To the Editor: The immunization status of military contingents arriving
from different nations for peacekeeping missions may vary widely. This
variation results from lack of information, coordination, and financial
support.

For larger missions, the United Nations (UN) Headquarters issues
recommendations about needed vaccines; recently, operations officers have
consulted World Health Organization experts before issuing recommendations,
and their advice, which takes into account epidemiologic data in the host
country, has improved. Medical officers who develop recommendations for
smaller missions must consider the pathogenic agent; environment; host
efficacy, safety, and price of preventive measures; and legal and ethical
aspects.

Data on the incidence of vaccine-preventable diseases within a military
population that had similar duties in the same location are rarely
available. When data from the respective region are not available, disease
incidence or prevalence in the host country may be substituted. These data,
however, may be misleading since the military often does not have the same
lifestyle as the native population. Plague, for instance, had an incidence
rate of 8 per 100,000 in Namibia, but not a single case was reported in the
South African Armed Forces (unpub. SAMS report: Disease Profile of South
West Africa, 1989). If epidemiologic documentation for a host country is
not available, data from neighboring countries may be useful.

Traveler's diarrhea is the most frequent health problem abroad (1,2).
Although the diarrhea is self-limited and lasts an average of 1 day with
appropriate treatment (4 days without), the unproductive time may be
detrimental to a military mission. Oral vaccines against the three most
frequent causes of traveler's diarrhea (enterotoxigenic Escherichia coli,
Campylobacter spp., and rotavirus [1,2]) are being developed; the latter
will be available soon (3). Hepatitis A, most frequent among the
vaccine-preventable diseases (4), is 10 to 100 times more frequent than
typhoid fever (4,5). Hepatitis B occurs mainly in expatriates, but
infections have also been observed in tourists who have had unprotected
casual sex (6). The incidence rate of rabies is unknown, but animal bites
that may result in rabies virus transmission and thus necessitate
postexposure prophylaxis are frequent (7). Only anecdotal cases of
diphtheria, tetanus, and tuberculosis have been reported (8).
Poliomyelitis, yellow fever, Japanese encephalitis, and plague occur only
in limited parts of the world (5). The situation may rapidly change as
epidemics occur (e.g., diphtheria in eastern Europe in the early and
mid-1990s) (9). If needed, the World Health Organization can provide
information on confirmed and unconfirmed epidemics on a weekly basis.

Travel and peacekeeping mission statistics share similarities. In Namibia,
the South African Armed Forces had most often observed hepatitis
(unspecified), with rare cases of tuberculosis, typhoid, and meningitis
(unpub. SAMS report: Disease Profile of South West Africa, 1989), as did
the UN mission to Namibia, where within 12 months and with 7,114 employees,
seven cases of hepatitis (mostly hepatitis A, some unspecified) occurred
(10). No other vaccine-preventable infections were diagnosed in this UN
mission.

Considering both risk (on the basis of incidence rates) and impact of
infection, the priority for immunization (from highest to lowest) is as
follows: hepatitis A, hepatitis B, rabies, poliomyelitis, yellow fever,
typhoid fever, influenza, diphtheria, tetanus, meningococcal disease,
Japanese encephalitis, cholera, and measles. To administer all vaccines
would be extremely costly and may also result in an increased rate of
adverse side-effects. Immunizations against the more frequent, more severe
infections should be given priority.

If a mission is limited to one season, environmental factors of that
respective season should be considered. This general rule is more important
for vector-borne than for vaccine-preventable infections, except for
influenza and meningococcal disease.

Persons who are already immune (because of previous immunization or
immunity after infection) need not be vaccinated. The latter cause is
particularly often true of hepatitis A; troops recruited in developing
countries have an anti-hepatitis A virus seroprevalence rate close to 100%
(11). Hepatitis B immunization, except for non- and low-responders,
probably grants lifelong protection (12); the same is likely for measles
vaccine.

Sometimes the host country may require proof of some specific vaccination
based on the International Health Regulations (13), currently under
fundamental revision to become a more effective tool in preventing the
spread of infections that may be a global hazard (14).

In addition to adequate epidemiologic information and coordination between
the military, international health organizations, and the host country,
successful intervention efforts require thorough knowledge of vaccine
characteristics with varying rates of efficacy and duration of protection.
Cost-benefit evaluations, which would be very desirable, are unlikely in
areas of political instability.

Robert Steffen
Institute for Social and Preventive Medicine of the University, Zurich,
Switzerland

(sup 1)Presented in part at the NATO Research & Technology Organization,
Aerospace Medical Panel Symposium on Aeromedical Support Issues in
Contingency Operations, Rotterdam, The Netherlands, 1 October 1997.

References

  1. DuPont HL, Ericsson C. Prevention and treatment of travelers'
     diarrhea. Drug Therapy 1993;328:1821-7.
  2. Farthing MJG, DuPont HL, Guandalini S, Keusch GT, Steffen R. Treatment
     and prevention of travellers' diarrhoea. Gastroenterology
     International 1992;5:162-75.
  3. Levine MM, Svennerholm A-M. Prioritization of vaccines to prevent
     enteric infections. In: DuPont HL, Steffen R, editors. Textbook of
     travel medicine. 1st ed. Hamilton: B.C. Becker Inc.; 1997. p. 370.
  4. Steffen R, Kane MA, Shapiro CN, Schoellhorn JK, Van Damme P.
     Epidemiology and prevention of hepatitis A in travelers. JAMA
     1994;272:885-9.
  5. World Health Organization. International travel and health. Geneva:
     The Organization; 1999.
  6. Steffen R. Risk of hepatitis B for travellers. Vaccine 1990;8:31-2.
  7. Hatz CF, Bidaux JM, Eichenberger K, Mikulics U, Junghanss T.
     Circumstances and management of 72 animal bites among long-term
     residents in the tropics. Vaccine 1994;13:811-5.
  8. Steffen R. Travel medicine prevention based on epidemiological data.
     Trans R Soc Trop Med Hyg 1991;85:156-62.
  9. Hardy IRB, Dittmann S, Sutter RW. Current situation and control
     strategies for resurgence of diphtheria in newly independent states of
     the former Soviet Union. Lancet 1996;347:1739-44.
 10. Steffen R, Desaules M, Nagel J, Vuillet F, Schubarth P, Jeanmaire C-H,
     et al. Epidemiological experience in the mission of the United Nations
     Transition Assistance Group (UNTAG) in Namibia. Bull World Health
     Organ 1992;70:129-33.
 11. Centers for Disease Control and Prevention. Hepatitis A immunization.
     MMWR Morb Mortal Wkly Rep 1996;45(RR-15):7.
 12. Hall AJ. Hepatitis B vaccination: protection for how long and against
     what. BMJ 1993;307:276-7.
 13. World Health Organization. International health regulations. 3rd
     annotated ed. Geneva: The Organization; 1983.
 14. World Health Organization. Revision of the international health
     regulations. Wkly Epidemiol Rec 1997;72:213-5.

Sexually Transmitted Diseases in Ukraine

To the Editor: With the political changes in eastern Europe in the last 10
years have come social and economic changes (1). Ukraine not only faces
almost insurmountable problems as it tries to form a new government, it
also faces many serious health issues including sexually transmitted
diseases (STDs).

Surveillance data from the Ukrainian STD Center from January 1, 1989,
through December 31, 1995, were analyzed on the basis of reports received
through 1997. In western Europe, the incidence of syphilis and gonorrhea
declined from 1980 to 1991 to less than 2% per 100,000 persons for syphilis
and less than 20% per 100,000 persons for gonorrhea. However, in Ukraine,
since 1989, the notification rate of syphilis has skyrocketedfrom 5 per
100,000 persons in 1990 to 170 in 1995. In some regions, this rate exceeds
220 cases per 100,000 persons. Moreover, cases among children younger than
14 years of age are also increasing. In 1995, the syphilis rate for persons
older than 30 years of age was 170 per 100,000; 600 per 100,000 girls
younger than 15 years of age; and 1,550 to 2,000 per 100,000 girls 15 to 16
years of age. The large number of girls with the disease is in part due to
teenage prostitution (1).

Most syphilis and gonorrhea cases are attributed to sexual transmission.
Explanations of this phenomenon include the rapid growth of the sex
industry, increasing numbers of homeless persons and refugees in Ukrainian
cities, poor diagnostic facilities, punitive legislation that reduces the
likelihood of going to treatment services, and limited or inadequate
treatment (2).

The Ukrainian government is reviewing its arrangements for the control of
STDs, including HIV/AIDS, to identify clear objectives and priorities.
Education and treatment would be effective in preventing the spread of STDs
in Ukraine, but these measures are inadequately funded (3). Evaluation and
risk reduction are also great weapons in preventing the spread of STDs (4).
However, the response of the local and world communities has been
inadequate in stemming a major STD epidemic in Ukraine.

United Nation's Children's Fund (UNICEF) is developing a long-term program
in Ukraine with a focus on STDs in adolescents and youth. This
comprehensive program will tackle not only STDs but other related issues,
such as HIV and teenagers' reproductive health (5).

Greater coordination of the agencies responsible for STD control in Ukraine
will be sought, together with an expansion of health promotion and
prevention projects for young persons and groups at high risk (6). An
effective strategy for the control of STDs in Ukraine will, therefore, need
to find ways to modify current programs and the way they interact to create
effective control interventions.

Dmitry I. Ivanov
University of Alabama at Birmingham, Birmingham, Alabama, USA

References

  1. Dittmann S, Gromyko A, Mikkelsen H, Schaumburg A, Adamian R,
     Khodakevich L, et al. Epidemic of sexually transmitted diseases in
     eastern Europe. Geneva: World Health Organization; 1996.
  2. Kobyshcha Y. HIV risk-related behavior of homo and bisexual men and
     STD patients in Ukraine. National AIDS Committee and Center
     1994;7:290-3.
  3. Normand J, Vlahov D, Moses LE. Preventing HIV transmission: the role
     of sterile needles and bleach. The effects of needle exchange
     programs. Washington: National Academy Press; 1995. p. 208-55.
  4. Spinhenko Y. Prevention of the spread of AIDS in the Ukrainian SSR.
     Lik Sprava 1988;9:1-3.
  5. Usenko A, Grazhdanov N, Stepanets V, Neshcheret E, Maksiutenko E.
     Effective knowledge propaganda in the chief strategy for preventing
     HIV infection among adolescents. Lik Sprava 1994;9:192-6.
  6. Tichonova L, Borisenko K, Ward H, Meheus A, Gromyko A, Renton A, et
     al. Epidemics of syphilis in the Russian Federation: trends, origins,
     and priorities for control. Lancet 1997;350:210-3.

Yellow Fever Vaccine

To the Editor: Monath et al. (1) outlined existing facilities for
distribution of yellow fever vaccines in the United States and pointed to
difficulties for prospective vaccinees in remote locations. Their
recommendation that primary health-care providers be allowed to dispense
yellow fever vaccination merits serious consideration. Acceptance of such a
strategy in the United States would inevitably be emulated elsewhere.
Nevertheless, before such a strategy is approved, vaccine potency should be
monitored at distribution points, and a sample of vaccine recipients should
be examined for vaccine-induced immune response.

In Nigeria, systematic investigation of yellow fever vaccine distribution
and transportation to remote locations has found loss in vaccine potency.
Vaccine in storage sites and immunization centers in Lagos was fully
potent, but potency in Osun and Oyo was 016 log(sub 10) to 0.22 log(sub 10) 
lower than the stipulated level (2). Furthermore, the titer of two vaccine 
lots that had been frozen after reconstitution from their lyophilized state 
dropped from the initial 3.15 log(sub 10) to 3.53 log(sub 10) to zero.

If the United States were to implement an extended strategy, similar
studies of vaccine lots should be conducted to determine whether every
vaccinee has received a full dose of yellow fever vaccine. In Illinois
during the early 1970s, weak links in maintenance of refrigeration
facilities and use of outdated vaccines in vials exposed to the sun for
long hours were reported for live poliovirus vaccines (3). In the Northern
Territory of Australia, examination of 144 vials of hepatitis B vaccine
formulations during transport to immunization centers showed that 47.5% had
been exposed to temperatures of -3C or lower (4).

Assays of the potency of yellow fever vaccine, as well as quantification of
vaccine-induced neutralizing antibody, is a multistep procedure that relies
on inoculation of mice or Vero or polysaccharide cells (5). The successful
"take" of yellow fever vaccine can be determined starting the second
postvaccination day by demonstrable viremia detected by
reverse-transcriptase polymerase chain reaction and by marked increases in
neopterin, beta2-microglobulin, and circulating CD8+ cells (6).
Alternatively, elevated levels of tumor necrosis factor and interleukin-1
receptor antagonists on day two after vaccination (7) could be used to
monitor the success of vaccinations by primary-care providers in remote
areas in the United States (1) and elsewhere.

During the 1990s, isolation of yellow fever virus was reported in persons
with a nonspecific febrile illness that did not meet the case definition of
yellow fever (8). Air travel by such persons to the United States, which
has areas infested by Aedes aegypti, could initiate yellow fever epidemics;
because these travelers would have a nonspecific febrile illness, they
would escape the existing surveillance network.

In conclusion, introducing yellow fever immunizations by primary
health-care providers would be ideal, only with a concurrent plan to
monitor vaccine potency at immunization centers and obtain in vitro
evidence of a successful vaccine take. Such a strategy would blunt yellow
feverassociated deaths, illnesses, and symptomless viral carriage in the
community.

Subhash C. Arya
Centre for Logistical Research and Innovation New Delhi, India

References

  1. Monath TP, Giesberg JA, Fierros EG. Does restricted distribution limit
     access and coverage of yellow fever vaccine in the United States?
     Emerg Infect Dis 1998:4:698-702.
  2. Adu FD, Adedeji AA, Esan JS, Odusanya OG. Live viral vaccine potency:
     an index for assessing the cold chain system. Public Health
     1996;110:325-30.
  3. Rasmussen CM, Thomas CW, Mulrooney RJ, Morrissey RA. Inadequate
     poliovirus immunity levels in immunised Illinois children. Am J Dis
     Child 1973;126:465-9.
  4. Miller NC, Harris MF. Are childhood immunization programmes in
     Australia at risk? Investigations of the cold chain in the Northern
     Territory. Bull WHO 1994;72:401-8.
  5. World Health Organization. Techniques for potency evaluation of yellow
     fever vaccine. Technical Report Series 1998;872:67-8.
  6. Reinhardt B, Jaspert R, Niedrig M, Kostner C, L'age-Stehr J.
     Development of viremia and humoral and cellular parameters of immune
     activation after vaccination with yellow fever virus strain 17D: a
     model of human flavivirus infection. J Med Virol 1998;56:159-67.
  7. Hacker UT, Jelinek T, Erhardt S, Eigier A, Hartmann G, Nothdurft HD,
     et al. In vivo syntheses of tumor necrosis factor-alpha in healthy
     humans after live yellow fever vaccination. J Infect Dis
     1998;177:774-8.
  8. Sanders EJ, Maffin AA, Tukei PM, Kuria G, Ademba G, Agata NN, et al.
     First recorded outbreak of yellow fever in Kenya, 1992-1993. I.
     Epidemiologic investigations. Am J Trop Med Hyg 1998;59:644-9.

Yellow Fever VaccineReply to S. Arya

To the Editor: Dr. Arya correctly points out that there have been problems
with degradation of live viral vaccines, including yellow fever vaccines,
that have not been properly handled and stored at the point of use.
However, in the United States and western Europe, yellow fever vaccines are
stabilized and require the same storage facilities at the point of use as
other vaccines routinely distributed by family physicians and
pediatricians. Varicella vaccine (and even measles vaccine) is less stable
than yellow fever vaccine but is distributed to all registered physicians
in the United States. Since vaccines and other perishable medicines are
typically shipped by overnight courier services using qualified methods
that ensure maintenance of low temperature, there is no barrier to use of a
similar system for yellow fever vaccine.

Empirical testing for antibody, viremia, or even surrogate markers of
T-cell activation may be useful; however, it is difficult and expensive,
involves unvalidated tests with unknown sensitivity and specificity, and is
unnecessary, except under very special circumstances. A more direct measure
of vaccine stability is direct potency measurement of samples stored at the
point of use, as was done in the cited study in Nigeria by Adu et al.
However, given the current controls on vaccine distribution in the United
States, we do not believe that there would be a need to validate vaccine
effectiveness at point of use in the event of a change of policy with
respect to vaccinating centers. The cold-chain infrastructure and the
training of medical personnel in vaccine storage and administration may not
provide the same assurances in other countries. While our suggested changes
to the system of yellow fever distribution may improve vaccine coverage and
have other desirable benefits in the United States, they would not be
appropriate for less stable systems for vaccine supply and use.

T.P. Monath, J.A. Giesberg, and E.G. Fierros
OraVax, Cambridge, Massachusetts, USA

________________________________________________________________________
News and Notes
________________________________________________________________________

News and Notes

54th International Northwestern Conference on Diseases in Nature
Communicable to Man

August 15-18, 1999

The 54th International Northwestern Conference on Diseases in Nature
Communicable to Man at Utah State University, Logan, UT, is scheduled for
August 15-18, 1999. Call for abstracts in the following general categories
are proposed, but may be modified: viral zoonoses, bacterial zoonoses,
water and foodborne infections, vector-borne diseases, and curiosa et
exotica. The deadline for abstracts is April 15, 1999.

The R.R. Parker Memorial Address will be presented by Jesse Goodman, Senior
Advisor for Medical Programs, U.S. Food and Drug Administration. To obtain
program announcements and registration forms, make hotel reservations, or
submit abstracts, contact one of the following persons: Dhitinut
Ratnapradipa, scientific program coordinator, e-mail:
dratnapr@health.utah.edu; or Robert Elbel, meeting coordinator, e-mail:
elbel@bioscience.utah.edu, tel:(801) 581-4816, fax: (801) 581-4668. Program
information is also available at the following web site:
http://www.mosquito.org./upcoming.html.
-----------------------------------------------------------------------

Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

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