Emerging Infectious Diseases
[Volume 5 No.2 / March - April 1999]
The MarchApril 1999 issue of the journal will contain the articles listed
below. Highlighted articles are currently available in electronic format.
International EditorsUpdate
   * Emerging Infectious DiseasesUnited Kingdom, D. Walford and N. Noah
Perspectives
   * The Next Influenza Pandemic: Lessons from Hong Kong, 1997, R. Snacken,
     A.P. Kendal, L.R. Haaheim, and J.M. Wood
   * Disparity in the Natural Cycles of Borrelia burgdorferi and the Agent
     of Human Granulocytic Ehrlichiosis, M.L. Levin, F. des Vignes, and D.
     Fish
Synopses
   * Malaria Reemergence in the Peruvian Amazon Region, J. Arambur Guarda,
     C. Ramal Asayag, and R. Witzig
   * Enteropathogenic E. coli, Salmonella, and Shigella: Masters of Host
     Cell Cytoskeletal Exploitation, D.L. Goosney, D.G. Knoechel, and B.B.
     Finlay
   * Bacterial Toxins: Friends or Foes? C.K. Schmitt, K.C. Meysick, and
     A.D. O'Brien
   * Clonal Differences among Erythromycin-Resistant Streptococcus pyogenes
     in Spain, E. Perez-Trallero, J.M. Marimn, M. Montes, B. Orden, and M.
     de Pablos
   * Air Evacuation under High-Level Biosafety Containment: The Aeromedical
     Isolation Team, G.W. Christopher and E.M. Eitzen, Jr.
   * emm Typing and Validation of Provisional M Types for Group A
     Streptococci, R. Facklam, B. Beall, A. Efstratiou, V. Fischetti, D.
     Johnson, E. Kaplan, P. Kriz, M. Lovgren, D. Martin, B. Schwartz, A.
     Totolian, D. Bessen, S. Hollingshead, F. Rubin, J. Scott, and G.
     Tyrrell
Research
   * Rapid Molecular Genetic Subtyping of Serotype M1 Group A Streptococcus
     Strains, N. Hoe, K. Nakashima, D. Grigsby, X. Pan, S. J. Dou, S.
     Naidich, M. Garcia, E. Kahn, D. Bergmire-Sweat, and J.M. Musser
Dispatches
   * Gnathostomosis, an Emerging Foodborne Zoonotic Disease in Acapulco,
     Mexico, N. Rojas-Molina, S. Pedraza-Sanchez, B. Torres-Bibiano, H.
     Meza-Martinez, and A. Escobar-Gutierrez
   * Acute Hemorrhagic Conjunctivitis Due to Enterovirus 70 in India, R.S.
     Maitreyi, L. Dar, A. Muthukumar, M. Vajpayee, I. Xess, R.B. Vajpayee,
     P. Seth, and S. Broor
   * Mycobacterium sp. as a Possible Cause of Hypersensitivity Pneumonitis
     in Machine Workers, B.G. Shelton, W.D. Flanders, and G.K. Morris
   * Evaluating Diagnosis and Treatment of Oral and Esophageal Candidiasis
     in Ugandan AIDS Patients, M. Ravera, A. Reggiori, A.M.Agliata, R.P.
     Rocco
   * Neospora caninum Infection and Repeated Abortions in Humans, E.
     Petersen, M. Lebech, L. Jensen, P. Lind, M. Rask, P. Bagger, C.
     Bjrkman, and A. Uggla
   * Lack of Association between First Myocardial Infarction and Past Use
     of Erythromycin, Tetracycline, or Doxycycline, L.A. Jackson, N.L.
     Smith, S.R. Heckbert, J.T. Grayston, D.S. Siscovick, and B.M. Psaty
   * An Epidemic of Bloody Diarrhea: Escherichia coli O157 Emerging in
     Cameroon? P. Cunin, E. Tedjouka, Y. Germani, C. Ncharre, R.Bercion, J.
     Morvan, and P.M.V. Martin
   * Genospecies Diversity of Lyme Disease Spirochetes in Rodent
     Reservoirs, D. Richter, S. Endepols, A. Ohlenbusch, H. Eiffert, A.
     Spielman, and F.R. Matuschka
Commentary
   * Preparing for Pandemic Influenza: The Need for Enhanced Surveillance,
     K.F. Gensheimer, K. Fukuda, L. Brammer, N. Cox, P.A. Patriarca, R.A.
     Strikas
Letters
   * An Outbreak of Gastroenteritis in Japan due to Escherichia coli O166,
     Y. Nishikawa, J. Ogasawara, A. Helander, and K. Haruki
   * Vibrio cholerae Outbreak in Italy, L.Cavalieri d'Oro, E. Merlo, E.
     Ariano, M.G. Silvestri, A. Ceraminiello, E. Negri, and C. La Vecchia
   * Shiga ToxinProducing Escherichia coli O157:H7 in Japan, J. Terajima,
     H. Izumiya, A. Wada, K. Tamura, and H. Watanabe
   * Streptococcus pyogenes Erythromycin Resistance in Italy, M. Bassetti,
     E. Mantero, G. Gatti, A. Di Biagio, and D. Bassetti
   * Estimated Incidence of Clostridium difficile Infection, F. Frost, J.S.
     Hurley, H.V. Petersen, and R.N. Casciano
   * Diphtheria in Eastern Nepal, H. Srinivasa, S.C. Parija, and M.P.
     Upadhyaya
   * Commercial Use of Burkholderia cepacia, J.J. LiPuma and E.
     Mahenthiralingam
   * Human Rabies in Israel, D. David, C.E. Rupprecht, J. Smith, I. Samina,
     S. Perl, and Y. Stram
   * Emerging Infections and Disease Emergence, M.E. Wilson
   * Malaria Control in South America, P.C. Matteson
   * Malaria Control in South AmericaResponse to Dr. Matteson, D.R.
     Roberts and L.L. Laughlin
   * On the Etiology of Tropical Epidemic Neuropathies, J. de la Fuente and
     M.P. Rodrguez
   * Risk for Ebola Virus Infection in Cte d'Ivoire, O. Kunii, P.
     Formenty, J. Diarra-Nama, and N. Nahounou
News and Notes
   * Automation in Threat Reduction and Infectious Disease Research: Needs
     and New Directions
   * Emerging Pathogens Initiative: An Automated Surveillance System
   * Erratum
____________________________________________________________________________
International EditorsUpdate
____________________________________________________________________________
Emerging Infectious DiseasesUnited Kingdom
Diana Walford and Norman Noah
Public Health Laboratory Service, London, United Kingdom
 		[fig]  	    Dr. Walford is          This review describes
                            director of the Public  some of the most
 Health Laboratory Service, which is responsible,   important new or
 through its Communicable Disease Surveillance      reemerging infectious
 Centre (CDSC), for the surveillance of infections  diseases in the United
 and communicable diseases in England and Wales.    Kingdom in the past
 Dr. Walford recently chaired a national committee  decade. Most of the
 that produced The Path of Least Resistance, a      statistics relate to
 report containing recommendations to minimize      England and Wales,
 the development of antimicrobial resistance        where the surveillance
 through the more prudent use of antimicrobial      system is operated by
 drugs in clinical practice. Professor Noah is a    the Public Health
 consultant epidemiologist at CDSC.                 Laboratory Service
                                                    (PHLS), through its
                                                    Communicable Disease
Surveillance Centre (CDSC). The statistics come from three main sources and
several more specialized ones. The most important is the laboratory-based
surveillance system, started more than 50 years ago, to which the national
network of 48 public health laboratories and the specialized reference
microbiology laboratories of PHLS and more than 150 National Health Service
laboratories contribute regularly. Most data collection is performed
electronically, through a computerized system that allows laboratory data
to be captured at the source, augmented with clinical or risk factor data,
and transmitted both centrally to CDSC and locally to the responsible
public health professionals. CDSC also runs, on behalf of the Office of
National Statistics, the long-established statutory notification system,
which is based on clinical reporting of a schedule of infectious and
communicable diseases including food poisoning. The third system is a
sentinel system based on general practice run by the Royal College of
General Practitioners Research Unit. These three systems complement each
other, and the data are collated, analyzed, and disseminated by CDSC and
are then extensively used to inform both government policies and local
activities for the prevention and control of communicable disease.
In addition to the "big three," the United Kingdom has several other more
specific surveillance systems, in particular, confidential clinical
reporting systems for AIDS and sexually transmitted diseases and
serosurveillance (including unlinked anonymous) systems for HIV infection.
In addition, active surveillance systems, developed jointly by PHLS and the
relevant professional associations, capture clinical case reports of rare
diseases, such as pediatric AIDS and Reye syndrome. These active systems
are particularly suited to the surveillance of rare and emerging diseases,
such as pediatric Creutzfeldt-Jakob (CJD) disease.
A growing number of disease-specific surveillance systems, operated on a
Europe-wide basis, are increasingly attracting other international
participants. CDSC coordinates Enter-Net, a Europe-wide scheme for
surveillance of salmonella and Escherichia coli O157. This network, which
comprises epidemiologists and reference microbiologists from each member
state of the European Union plus Switzerland and Norway, has led to the
successful detection and investigation of several international foodborne
outbreaks. A similar network coordinated by CDSC exists for
travel-associated Legionnaires' disease. A mainly European-based
surveillance network for meningococcal disease is also working well.
One of the side-effects of an efficient surveillance system is that because
new diseases or variants of diseases and outbreaks are detected earlier
than in countries with less comprehensive systems, such diseases are often
thought to have originated, or to predominate, in that country. This
happened, for example, with Salmonella Enteritidis contamination of hens'
eggs, which was, as it later became apparent, not confined to the United
Kingdom but was widespread throughout Europe. However, one emerging disease
that did have its origins in the United Kingdom is new variant (nv) CJD.
NvCJD
A previously undescribed encephalopathy in cows, bovine spongiform
encephalopathy, with characteristic clinical and histologic features, was
first identified in November 1986, although the first case may have
occurred as early as April 1985. Since then, the number of cases reported
in the United Kingdom has exceeded 166,000. However, the epidemic reached a
peak in 199293. The number of cases recorded has been diminishing since,
and the cattle epidemic, linked to the consumption of contaminated
ruminant-derived meat and bone meal, is predicted to be over by 2001 (1).
(The method of rendering protein changed shortly before the disease first
appeared in cows.) Surveillance of CJD in humans began in 1990, when the
National CJD Surveillance Unit was established in Edinburgh, Scotland.
The first human cases of nvCJD appeared in 1995, with onset dates from
February 1994 (2) (Table). NvCJD was distinguished from classic CJD on
clinical and histologic grounds and by the patients' young age. Although
evidence is mounting that nvCJD is the human form of bovine spongiform
encephalopathy (3,4), no direct association has yet been established
between the development of nvCJD and the consumption of beef.
HIV/AIDS             Table. Deaths of definite and probable U.K. cases due
                     to spongiform encephalopathy referred to the National
An estimated 25,000  Creutzfeldt-Jakob Disease Surveillance Unit(sup a),
persons in the       1995-1998(sup b)
United Kingdom are
infected with HIV,   ------------------------------------------------------
an overall                         Deaths of definite and probable cases
prevalence of
approximately 45 per              -----------------------------------------
100,000 population         Refer-  Spora- Iatro- Fami-  GSS nv-CJD
(5). At least 15,000 Year   rals    dic   genic  lial (sup c)(sup c)Total
are receiving care.
Disease prevalence   ------------------------------------------------------
varies considerably  1995    87      35     4      2     3     3    47
by geographic area,
with most cases in   1996    134     41     4      2     4    10    61
London and the       1997    161     58     6      4     1    10    79
southeast of         1998    150     39     3      2     0    12    56
England. National
prevalence is lower  ------------------------------------------------------
than in all but four (sup a)Data source: National CJD Surveillance Unit (set up
countries of Western in May 1990 by the Department of Health and the
Europe. As in most   Scottish Office Home and Health Department).
countries of         (sup b)To Dec. 31, 1998 (provisional); total number of
Northern Europe, sex  definite and probable cases of nvCJD=35.
between men remains  (sup c)GSS, Gerstmann-Straussler-Scheinker syndrome; nvCJD,
the predominant mode new variant Creutzfeldt-Jakob disease.
of HIV transmission.
In the UnitedKingdom, heterosexually acquired cases are the second largest risk
group. Most such infections are in persons from, or who have lived in, sub-Saharan
Africa. Some heterosexually acquired cases originate in other higher
prevalence areas of the world, as well as from within the United Kingdom,
but numbers are currently fairly small. Apart from cities in Scotland,
where outbreaks occurred in the mid-1980s, continuing HIV transmission
among injecting drug users appears low. Needle exchange programs, which
have contributed to limiting spread, have been in place in most urban areas
of the United Kingdom since the late 1980s. In this country, HIV infection
in pregnant women is still not adequately recognized, and thus steps to
greatly reduce the chance of transmission of infection from mother to baby
have not yet been taken. Recent guidance from the Department of Health
concerning the need for HIV screening in pregnancy is intended to address
this problem.
In spite of considerable efforts to reduce the spread of HIV in the United
Kingdom, the number of newly diagnosed cases of HIV infection has been
fairly constant in the 1990s. The rates among male homosexual attendees
younger than 25 years of age at sexually transmitted disease clinics have
shown no signs of decreasing, suggesting continued appreciable HIV
incidence in men who have sex with men. Furthermore, the rates of acute
sexually transmitted infections (markers of unsafe sex) in homosexual men
have increased again after a decline in the mid-1980s.
The incidence of AIDS (but not the incidence of HIV infections) and the
number of deaths have fallen dramatically in the last 2 years as a result
of the introduction of highly active antiretroviral therapies. This
decrease has been most marked in men who have acquired HIV through sex with
men. A consequence of the falling death rate, with sustained rates of new
diagnoses, is increasing prevalence of HIV infection.
Tuberculosis
After many years, tuberculosis is reemerging in this country as in many
other countries of the world (6). In England and Wales the annual number of
cases had been declining for over 150 years, reaching its lowest point of
5,085 reported cases (10.1 per 100,000 population) in 1987. Since then, the
number of cases has remained at a plateau of 5,000 to 6,000 cases per year.
However, increases were recorded in 3 recent consecutive years, 1995-1997.
Moreover, within these bare statistics, there are some disturbing trends.
Rates in certain ethnic groups have increased significantly, while those in
Caucasians have continued to fall. Cases of tuberculosis in persons of
black African origin accounted for 1% of all cases in 1988, and account for
more than 10% now. Multidrug-resistant tuberculosis remains at a low level,
although nosocomial transmission has been reported. An enhanced
surveillance system and new technology for rapid resistance testing have
been put in place.
E. coli O157
						 Extremely rare a decade ago, E. coli
                                     O157 infection is emerging as an
 		[fig]       		 important public health problem in
                                     the United Kingdom because of the
 Figure 1. Escherichia coli O157     severity of the illness it causes,
 central Scotland outbreak epidemic  even though the annual number of
 curve by date of onset of diarrhea. reported infections remains
                                     comparatively low (approximately
1,000). Undoubtedly the most important outbreak of E. coli O157 in the
United Kingdom originated in a butcher's shop in Lanarkshire, Scotland, in
November and December 1996 (7). Approximately 85 outlets in central
Scotland stocked the butcher's products, which made the task of outbreak
management and control extremely difficult. The date of onset of the first
known case was November 15 and of the last confirmed case December 15. The
number of cases escalated rapidly from November 15 and at the peak of the
outbreak, November 25, reached more than 40 cases (Figure 1) in 1 day; 496
persons were affected, and 18 persons died. One of the features of the
outbreak was the occurrence of several separate incidents, the largest of
which was a birthday party for 100 persons in a church hall. All isolates
of E. coli O157 belonged to phage type 25 and contained the verocytotoxin
gene VT2. Pulsed-field gel electrophoresis showed that all 262 strains
tested were indistinguishable. A group was convened by the Scottish Office
to consider the circumstances that led to the outbreak and the implications
for food safety. Among the many recommendations was the accelerated
introduction of the Hazard Analysis/Critical Control Point (HACCP) system
(8) for high-risk premises and the selective licensing of food premises.
Salmonella Food Poisoning and Other Gastrointestinal Infections
Although hardly an emerging disease,
salmonella food poisoning has reemerged both
in incidence and in importance in recent      		[fig}
years (Figure 2). Much of this increase is
attributable to S. Enteritidis, which has     Figure 2. Salmonella
accounted for approximately 70% of all        infections, 19811998.
salmonella infections in recent years, with
S. Enteritidis phage type 4 accounting for 45% of the total. These strains
are closely associated with eggs and poultry. In addition,
multidrug-resistant S. Typhimurium DT 104 has emerged as an important cause
of food-poisoning outbreaks. In 1997, the highest number of salmonella
infections was recorded (32,596), but in 1998 only 23,216 cases
(provisional) were reported, a fall of about 30%. It is too soon to say if
this decrease will be sustained and if it can be attributed to control
measures. Campylobacter infections are the major cause of
laboratory-confirmed bacterial gastrointestinal infections, while
small-round structured viruses now account for more than 40% of reported
pathogens associated with general outbreaks of infectious intestinal
disease in England and Wales (9).
Cryptosporidiosis
Cryptosporidium has emerged as a cause of waterborne outbreaks since the
1980s. The problem was considered serious enough for the government to
convene an expert group, which reported in 1990. An outbreak in the north
Thames area of England in 1997 had 345 confirmed cases and probably many
others, and 746,000 persons were advised to boil water before consumption
(10). This outbreak resulted from contamination of a filtered
borehole-derived public water supply. A period of heavy rainfall following
the driest spell for at least 200 years may have contributed.
Cryptosporidial oocysts need to be filtered out of water as they are not
affected by normal concentrations of chlorine, and the oocyst load was
probably too great for the filters at various times during this unusual
weather spell. This was the first reported cryptosporidiosis outbreak in
the United Kingdom caused by filtered borehole water and the third
involving groundwater supplies. It also resulted in the reconvening of the
expert group by the government. Mandatory continuous sampling for
Cryptosporidium in supplies at risk is under consideration (11).
						 In an important advance, PHLS
                                     researchers have recently
 		[fig]        		 demonstrated the existence of at
                                     least two genotypes of C. parvum.
 Figure 3. Meningococcal disease,    Evidence so far (12) suggests that
 19941998 (5-week moving average).  whereas human infections are
                                     associated with both genotypes,
livestock animal infections are associated with one genotype only. If
confirmed, these preliminary findings should make an important contribution
to elucidating the epidemiology of this still-emerging infection.
Meningococcal Disease
Meningococcal infection has produced a sustained and somewhat baffling rise
in incidence in the 1990s (Figure 3), with an even greater-than-expected
increase in the first few weeks of 1999. Although serogroup B disease
continues to predominate, the epidemiology is changing; the proportion of
infections caused by serogroup C is increasing, and these infections show
an increasing tendency to cause clusters and outbreaks in teenagers and
young adults. Conjugated vaccines against serogroup C are under evaluation
in the United Kingdom by PHLS and others.
Chlamydia trachomatis
In recent years, the public health importance of C. trachomatis infection
has been increasingly recognized. Reported rates of infection exceed 100
per 1,000,000 population in those ages 15 to 59 years, making it the most
common bacterial cause of sexually transmitted disease. It is estimated
that only 10% of cases are identified. Chlamydia is an important cause of
pelvic inflammatory disease and infertility in untreated women. Targeted
screening in groups at high risk is under consideration.
Syphilis
On average, 3,000 new cases and 1,000 late cases of syphilis are reported
each year in England and Wales. Approximately half of the
laboratory-confirmed cases appear to be indigenous infections; the
remainder are acquired abroad (13). A recent increase in infections
contracted in Russia has been noted. However, a large, prolonged outbreak
of indigenous syphilis occurred in Bristol in England between January 1997
and May 1998. Forty-five persons were infectedthe expected number was one
or two cases only. This was a heterosexually transmitted outbreak, and,
unusually, women outnumbered men by 26 to 19. An urgent coordinated public
health response identified many cases, but a number of sexual contacts were
untraceable, and the potential for ongoing transmission remains (14).
Diphtheria
Very few confirmed cases of infection with toxigenic Corynebacterium
diphtheriae are reported in the United Kingdom associated with travel to
disease-endemic areas. However, in recent years, cases of nontoxigenic C.
diphtheriae var gravis have emerged. In the wider European context,
diphtheria has reemerged as a major public health problem in the countries
of the former Soviet Union.
Antimicrobial Resistance
Levels of antimicrobial resistance, although lower than in many other
countries in Europe and elsewhere, are an increasing cause for concern. For
example, the numbers and geographic distribution of reported bloodstream
infections with methicillin-resistant Staphylococcus aureus have escalated.
Almost 32% of staphylococcal septicemias in 1997 were caused by
methicillin-resistant S. aureus, compared with only 2% in 1992.
Antimicrobial resistance has been the subject of two recent national
reports (15,16), and a comprehensive strategy is being developed to address
the problem (17).
Hepatitis C
"For the community as a whole, hepatitis C is a viral time-bomb which is
slowly destroying the health of large numbers of the world population"
(18). In England and Wales, only 57% of reported patients have known risk
factors. Of these, approximately 80% identified injecting drug use as the
main route of transmission, and 2.9% reported sexual exposure. Blood or
blood product recipients accounted for 7.2% of the cohort of cases
diagnosed between 1992 and 1996; this proportion is rapidly diminishing
(19) as a result of the screening of blood donors and the use of treated
blood products.
Influenza
Surveillance of influenza in England and Wales has reached a high level of
sophistication, with many sources of data now available, covering clinical
reports, reports of deaths from several different respiratory conditions,
and virus isolation and subtyping reports (20). In 1998, in the face of
concern that influenza might be reemerging because of the occurrence in
Hong Kong of 18 cases of infection (with 6 deaths) from a new influenza
subtype of avian origin, A(H5N1), PHLS activated the first phase of its
pandemic influenza plan. The chicken-related strain proved poorly
transmissible to humans, but the episode provided a useful practice run for
the contingency plan.
Measles
Given the existence of an effective vaccine against measles, mumps, and
rubella and a comprehensive immunization program, there might seem little
prospect of measles emerging once again as a serious public health problem
in the United Kingdom. However, vaccine coverage has fallen recently,
coinciding with adverse publicity over an alleged link between the vaccine
and both Crohn disease and autism (21). This illustrates the ever-present
potential for infections, even those close to elimination, to regain the
upper hand if preventive measures are disrupted.
References
  1. Maxwell RJ. An unplayable hand? BSE, CJD and the British Government.
     London: King's Fund Publishing; 1997.
  2. Will RG, Ironside JW, Zeidler M, Cousens SN, Estibeiro K, Alperovitch
     A, et al. A new variant of Creutzfeldt-Jakob disease in the UK. Lancet
     1996;347:921-5.
  3. Hill AF, Desbruslais M, Joiner S, Sidle KCL, Gowland I, Collinge J.
     The same prion strain causes vCJD and BSE. Nature 1997;389:448-50.
  4. Bruce ME, Will RG, Ironside JW, McConnell I, Drummon D, Suttie A, et
     al. Transmissions to mice indicate that "new variant" CJD is caused by
     the BSE agent. Nature 1997;389:489-501.
  5. Communicable Disease Surveillance Centre. The global HIV epidemic.
     Commun Dis Rep CDR Wkly 1998;8:227.
  6. Communicable Disease Surveillance Centre. Tuberculosis remains "the
     captain of all these men of death." Commun Dis Rep CDR Rev
     1997;7:R105-l6.
  7. The Pennington Group. Report on the circumstances leading to the 1996
     outbreak of infection with E. coli O157 in Central Scotland, the
     implications for food safety and the lessons to be learned. Edinburgh:
     The Stationery Office; 1997.
  8. FAO/WHO. Introducing the Hazard Analysis Critical Control Point
     system. Geneva: The Organization; 1997. Report: WHO/FSF/FOS/97.2.
  9. Evans HS, Madden P, Douglas C, Adak GK, O'Brien SJ, Djuretic T, et al.
     General outbreaks of infectious gastrointestinal disease in Scotland
     and Wales: 1995 and 1996. Communicable Disease and Public Health
     1998;1:165-71.
 10. Willcocks L, Crampin A, Milne L, Seng C, Susman M, Gair R, et al. A
     large outbreak of cryptosporidiosis associated with a public water
     supply from a deep chalk borehole. Communicable Disease and Public
     Health 1998;1:239-43.
 11. Casemore D. Cryptosporidium and the safety of our water supplies.
     Communicable Disease and Public Health 1998;1:218-9.
 12. Patel S, Pedraza-Diaz S, McLauchlin J, Casemore D. Molecular
     characterisation of Cryptosporidium parvum from two large suspected
     waterborne outbreaks. Communicable Disease and Public Health
     1998;1:231-3.
 13. Ratcliffe L, Nicoll A, Carrington D, Wong H, Egglestone SI, Lightfoot
     NF, et al. Reference Laboratory Surveillance of syphilis in England
     and Wales, 1994 to 1996. Communicable Disease and Public Health
     1998;1:14-21.
 14. Communicable Disease Surveillance Centre. Syphilis in Bristol 1997-8:
     an update. Commun Dis Rep CDR Wkly 1998;8:413.
 15. House of Lords Select Committee on Science and Technology. Resistance
     to antibiotics and other antimicrobials. Session l997-98,7th report.
     London: The Stationery Office; 1998.
 16. Standing Medical Advisory Committee Sub-Group on Antimicrobial
     Resistance. The path of least resistance. Department of Health, 1998.
 17. Government response to the House of Lords Select Committee on Science
     and Technology Report: Resistance to antibiotics and other
     antimicrobial agents. London: The Stationery Office; 1998.
 18. English R, Foster G. Living with hepatitis C. London: Robinson; 1997.
 19. Ramsay ME, Balogun MA, Collins M, Balraj V. Laboratory surveillance of
     hepatitis C virus infection in England and Wales: 1992 to 1996.
     Communicable Disease and Public Health 1998:1:89-94.
 20. Dedman DJ, Zambon M, Van Buynder P, Fleming DM, Watson JM, Joseph CA.
     Influenza surveillance in England and Wales: October 1997 to June
     1998. Communicable Disease and Public Health 1998;1:244-51.
 21. Communicable Disease Surveillance Centre. MMR vaccine coverage falls
     after adverse publicity. Commun Dis Rep CDR Wkly 1998;8:41.
_____________________________________________________________________________
Perspectives
_____________________________________________________________________________
Perspectives
The Next Influenza Pandemic: Lessons from Hong Kong, 1997
Ren Snacken,* Alan P. Kendal, Lars R. Haaheim, and John M. Wood
*Scientific Institute of Public Health Louis Pasteur, Brussels, Belgium;
The Rollins School of Public Health, Emory University, Atlanta, Georgia,
USA; University of Bergen, Bergen, Norway; National Institute for
Biological Standards and Control, Potters Bar, United Kingdom
---------------------------------------------------------------------------
      The 1997 Hong Kong outbreak of an avian influenzalike virus,
      with 18 proven human cases, many severe or fatal, highlighted
      the challenges of novel influenza viruses. Lessons from this
      episode can improve international and national planning for
      influenza pandemics in seven areas: expanded international
      commitment to first responses to pandemic threats; surveillance
      for influenza in key densely populated areas with large
      live-animal markets; new, economical diagnostic tests not based
      on eggs; contingency procedures for diagnostic work with highly
      pathogenic viruses where biocontainment laboratories do not
      exist; ability of health facilities in developing nations to
      communicate electronically, nationally and internationally;
      licenses for new vaccine production methods; and improved
      equity in supply of pharmaceutical products, as well as
      availability of basic health services, during a global
      influenza crisis. The Hong Kong epidemic also underscores the
      need for national committees and country-specific pandemic
      plans.
Influenza pandemics are typically characterized by the rapid spread of a
novel type of influenza virus to all areas of the world, resulting in an
unusually high number of illnesses and deaths for approximately 2 to 3
years. Such pandemics occurred in 1918, 1957, and 1968 (Table); in the most
severe pandemic (1918-20), at least 20 million people died, most
working-age adults (10-12). Most deaths occurred in developing nationsmore
than 10 million people died in India alone (M. Rammana, pers. comm.).
Pregnant women were also severely affected, particularly those from lower
socioeconomic groups (13,14). The age distribution of those who died
differed from that in later pandemics or epidemics, when deaths were higher
in the elderly and lower in other age groups, except possibly in very young
children.
Novel Influenza Viruses without Pandemics
In addition to true pandemics, false alarmsemergences of a novel strain
with few cases and little human transmissibility (Table)have occurred.
Several involved "swine influenza viruses" (4-6) antigenically related to
viruses circulating in some pig populations and linked to viruses of the
1918 pandemic (see below). These unusual infections may be more common than
reported, as laboratory diagnosis for influenza is rarely undertaken in the
absence of unusual illness or association with an outbreak.
Origin of Pandemic Viruses
Before influenza virus could be propagated in a laboratory, retrospective
measurement of antibodies to the influenza virus' major surface antigen
(hemagglutinin) in persons of different ages was used to identify viruses
causing pandemics. Additional use of antibody tests to the second surface
antigen (neuraminidase) confirmed earlier ideas that H1N1 subtype viruses
resembling classic swine influenza caused the 1918 pandemic (15).
 Table. Influenza landmarks in humans this century
 --------------------------------------------------------------------------
       Colloquial
          Name
 Year   (Subtype)              Source                     Impact
 --------------------------------------------------------------------------
 Pandemics
 1918 Spanish flu   Possible emergence from      Pandemic with >20 million
 (1)  (H1N1 viruses swine or an avian host of a  deaths globally
      like swine    mutated H1N1 virus
      flu)
 1957 Asian flu     Possible mixed infection of  Pandemic, H1N1virus
 (2)  (H2N2)        an animal with human H1N1and disappeared
                    avian H2N2 virus strains in
                    Asia
 1968 Hong Kong flu High probability of mixed    Pandemic, H2N2 virus
 (2)  (H3N2)        infection of an animal with  disappeared
                    human H2N2 and avian H3Nx
                    virus strains in Asia
 1977 Russian flu   Source unknown but virus is  Benign pandemic,
 (3)  (H1N1)        almost identical to human    primarily involving
                    epidemic strains from 1950.  persons born after the
                    Reappearance detected at     1950s. H1N1 virus has
                    almost the same time in      cocirculated with H3N2
                    China and Siberia            virus in humans since
                                                 1977
 Incidents with limited spread
 1976 Swine flu     United States/New Jersey.    Localized outbreak in
 (4)  (H1N1)        Virus enzootic in U.S. swine military training camp,
                    herds since at least 1930    with one death
 1986 (H1N1)        The Netherlands. Swine virus One adult with severe
 (5)                derived from avian source    pneumonia
 1988 Swine flu     United States/Wisconsin.     Pregnant woman died after
 (6)  (H1N1)        Swine virus                  exposure to sick pig
 1993 (H3N2)        The Netherlands. Swine       Two children with mild
 (7)                reassortant between old      disease. Fathers
                    human H3N2 (1973/75-like)    suspected to have
                    and avian H1N1               transmitted the virus to
                                                 the children after having
                                                 been infected by pigs.
 1995 (H7N7)        United Kingdom Duck virus    One adult with
 (8)                                             conjunctivitis
 1997 Chicken flu   Hong Kong Poultry virus      18 confirmed human case,
 (9)  (H5N1)                                     6 deaths
 --------------------------------------------------------------------------
Molecular biologic analysis of viral nucleic acid supports the hypothesis
that animals (particularly birds and pigs) may have been the source for
(and possibly are a continuing reservoir of) the hemagglutinin and other
genes found in viruses from the above pandemics (16). Some animal viruses
containing these genes (e.g., H1, H2, H3) might infect humans directly and
become adapted to the human host; alternately, through reassortment of the
genes in different animal or human influenza viruses, the genetic
information might reappear in an infectious human virus (17). The Hong Kong
experience, however, showed that an animal virus with another HA subtype
(H5) could directly infect humans and cause illness. The H5 virus, however,
did not evolve into a form that is readily transmitted from person to
person, and its potential for this kind of transmission remains unknown.
Reports in 1957, 1968, and 1977 indicated China and nearby areas as places
where outbreaks of novel viruses often first occur (18). Close contact
occurs in such regions between humans and animals (e.g., ducks, pigs)
raised for food. Surveillance data show that because of the different
seasonality of influenza in northern and southern China, human influenza
infections normally occur every month of the year (19). Thus, many
opportunities exist in China for viruses to cross-infect different animal
species and humans, which may explain why it and nearby areas are the
origin of many influenza pandemics.
Avian Influenza Virus in Humans in Hong Kong
In May 1997, a 3-year-old boy in Hong Kong contracted an influenzalike
illness, was treated with salicylates, and died 12 days later with
complications consistent with Reye syndrome. Laboratory diagnosis included
the isolation in cell culture of a virus that was identified locally as
influenza type A but could not be further characterized with reagents
distributed for diagnosis of human influenza viruses. By August, further
investigation with serologic and molecular techniques in the Netherlands
(9, 20, 21) and in the United States (22) had confirmed that the isolate
was A/Hong Kong/156/97 (H5N1), which was very closely related to isolate
A/Chicken/Hong Kong/258/97 (H5N1). The latter virus was considered
representative of those responsible for severe outbreaks of disease on
three rural chicken farms in Hong Kong during March 1997, during which
several thousand chickens had died. Molecular analysis of the viral
hemagglutinins showed a proteolytic cleavage site of the type found in
highly pathogenic avian influenza viruses.
Because no further cases of human infection with H5 viruses were seen in
Hong Kong during the summer, the case in May was considered an isolated
incident, with little or no person-to-person spread. However, surveillance
for influenza was increased, and local capability was established to test
for H5 subtype among human patients.
As summarized on their Internet disease surveillance site, the Hong Kong
Special Administrative Region Department of Health
(http://www.info.gov.hk/dh/diseases/flu_1997.htm) detected new cases of
human illness caused by H5 virus during November 1997. By late December,
the total number of confirmed new cases had climbed to 17, of which 5 were
fatal (one in a 13-year-old child and four in adults, 25, 34, 54, and 60
years of age). Including the fatal index case in May, the case-fatality
rates were 18% in children and 57% in adults older than 17 years.
Investigation of the circumstances surrounding each case was undertaken by
the local authorities with assistance from the World Health Organization
Collaborating Centers in the United States and Japan. Except for one
doubtful unconfirmed case, all illnesses or laboratory evidence of
infection was in patients who had been near live chickens (e.g., in market
places) in the days before onset of illness, which suggests direct
transmission of virus from chicken to human rather than person-to-person
spread. On December 28, 1997, veterinary authorities began to slaughter all
(1.6 million) chickens present in wholesale facilities or vendors within
Hong Kong, and importation of chickens from neighboring areas was stopped.
Subsequently, no more human cases caused by avian influenza virus were
detected. Because these cases occurred at the beginning of the usual
influenza season in Hong Kong, public health officials were concerned that
human strains might cocirculate with the avian influenza to generate human
and avian reassortant viruses with capacity for efficient person-to-person
spread.
Response to Emerging Influenza Pandemics. Lessons from Hong Kong
Pandemic planning has been proceeding in various countries and at WHO for
several years (23). Now, 1 year after the Hong Kong episode ended, a period
during which several countries have had severe local outbreaks or epidemics
of interpandemic variant A/Sydney/5/97 (H3N2)-like viruses, lessons from
Hong Kong could be incorporated in existing or new pandemic response plans.
Improve International Response
When the Hong Kong episode occurred, WHO had been developing formal
guidelines for addressing pandemic situations. The draft guidelines were
revised after the Hong Kong episode, taking into consideration two
strategic steps especially important in the outbreak: risk assessment,
which encompasses two components, data collection (investigating the
circumstances of the initial infection and subsequent infections, and
searching for further evidence of spread) and data evaluation (interpreting
and communicating the significance of the threat based on the available
data); and risk management, which is a process of continuously considering
and updating alternative courses of action as new action is obtained,
defining potential risks and benefits of each approach, and selecting the
next step, or series of steps, recommended for appropriate authorities.
Having already established a Pandemic Task Force by 1997, WHO was able to
initiate technical investigation and evaluation of the Hong Kong situation.
Only a very few organizations, from the United States and Japan, rapidly
committed staff to join local authorities in collecting information needed
for risk assessment. The widespread local and international consequences of
the situation in Hong Kong, including impact on commerce and travel,
compounded the already large pressures on the investigating team to gather
evidence about the risk for an epidemic or pandemic. Further pressure was
exerted on the investigating team, WHO Task Force, and collaborating
organizations because much work was urgently needed on a contingency basis
to expand capabilities of international surveillance laboratories to detect
H5 influenza viruses elsewhere and to support preliminary steps necessary
for developing a vaccine against the Hong Kong virus.
Because influenza pandemic threats affect more than one country,
facilitating multicountry studies could save critical time in the risk
assessment process. Hence, increasing international involvement in both
phases of risk assessment is desirableboth to expand resources for
investigations and to ensure that all regions of the world, including
developing nations, are represented during decision making. Advance
commitments could be made to rapidly expand the network of academic,
governmental, or other laboratories or disease-investigating organizations
that can conduct field investigations and analyze potentially large numbers
of isolates and other specimens. We suggest several ways for improving
international response. First, the WHO Task Force could develop formal
Terms of Reference for its own role and that of its investigating teams.
Second, National Health Authorities of WHO member nations might then make
these commitments: to invite WHO team(s) to carry out investigations of
pandemic threats without delay, agree with the Terms of Reference for the
task force and its investigating teams, and designate national
organizations to assist investigating teams. Such advance agreements should
facilitate the rapid deployment of investigating teams and the acceptance
of their work by WHO member nations, regardless of what countries appear to
be relevant sites for investigation of a pandemic threat or in what ways
the pandemic threat is first identified or affects local interests.
However, special questions will be raised regarding leadership,
communications, and internal cooperation as more countries become involved,
and these issues also should be addressed, if possible, in advance.
In setting Terms of Reference, data collection may be formally separated
from risk evaluation and risk management. Such separation would allow
technical experts to concentrate on organizing and conducting field and
laboratory investigations without being distracted by having to evaluate
the significance of findings or recommending responses to a pandemic threat
(24). Furthermore, the willingness of some countries to receive WHO
investigating teams may be enhanced if the Terms of Reference specify that
data collected for the WHO Task Force will be evaluated by an independent
advisory group composed of infectious disease and public health experts
representing all WHO regions, including developing nations. Such a tiered
approach would be consistent with ways many other public health policy
decisions are made about epidemics.
Enhance Human and Veterinary Surveillance
Human influenza epidemics may be evaluated through death data (25-27), but
weekly illness reports from sentinel primary-care practices, coupled with
laboratory diagnosis, provides more timely detection of early isolates as
well as epidemics (28,29). First detection of influenza outside the normal
influenza season, however, may come from unsystematic
samplingepidemiologic investigations of reports of unusual outbreaks
(e.g., most recently among tourists during summer in different parts of the
United States [30], the events in Hong Kong in 1997).
The current WHO global influenza program, with the help of four
collaborating centers (Atlanta, London, Melbourne, and Tokyo) and 110
national influenza centers, aims to centralize world data, study the
epidemiology of the disease, and rapidly obtain new circulating strains to
make timely recommendations about the composition of the next vaccine (31).
However, many countries have only limited capabilities or resources to
systematically search for and investigate unusual occurrences of influenza.
The events in 1997 in Hong Kong show the need to expand routine
surveillance efforts. Had the H5 virus isolated in May 1997 from a sporadic
case not been identified in August, the reagents would not have been
available locally to rapidly diagnose the additional human cases of H5
influenza in humans in November and December. Without such diagnoses, and
the investigations which they stimulated, authorities might not have
addressed the issue of chicken influenza as they did. Transmission of the
H5 virus to humans could have continued into the normal influenza season in
Hong Kong, possibly developing into a human-transmissible form.
Therefore, priority should be given to the establishment of regular
surveillance and investigation of outbreaks of influenza in the most
densely populated cities in key locations, particularly in tropical or
other regions where urban markets provide opportunities for human-live
animal contact (e.g., swine and poultry[and possibly caged birds kept as
pets]). Communication and cooperative studies with veterinarians could
monitor influenza outbreaks in locations where large numbers of animals are
raised, exhibited, or held pending transport or sale, i.e., situations
increasing the potential for virus spread. International collaboration with
the WHO Collaborating Centers studying human influenza and the WHO
Collaborating Center on influenza ecology in lower animals and birds
(Memphis, USA) should be enhanced.
Develop Improved, Low-Cost, Laboratory Surveillance Techniques
For many years, influenza viruses have been isolated by injecting clinical
samples into embryonated chicken eggs. Viruses have been detected by
agglutination of erythrocytes and inhibited by using antisera provided
through WHO, thus keeping costs relatively low and methods relatively
simple. Laboratories in industrialized countries (including Hong Kong) have
the facilities to use tissue culture for virus isolation. However, when the
H5 viruses isolated in Hong Kong were injected into chicken eggs, they
caused high numbers of deaths, thus making eggs less suitable as the sole
host system for surveillance purposes. Thus, developing simple low-cost
techniques (with reagents appropriate for the task of detecting circulation
of animal influenza viruses) that can be used in places with limited
resources needs to be a priority.
Choices must be made whether such tests should be based on isolation of
infectious virus (which can immediately provide virus samples for biologic
characterization and development of reagents or vaccines) or on antigenic
or molecular methods (which may minimize laboratory capabilities needed).
In making the choice of tests, it should be remembered that the reported
isolation of an atypical virus by one or a very few laboratories may result
from contamination of diagnostic specimens by viruses used for research,
reagent production, or quality control; molecular techniques may be needed
to confirm unrecognized cases of contamination with live viruses (32,33).
It is unclear if diagnostic methods based on molecular methods will incur
fewer risks from specimen contamination.
Increase Laboratory Safety Capabilities
The episode of H5, a potentially highly pathogenic virus for humans as well
as for chickens and other avian species, also raised the issues of how to
contain new viruses and protect laboratory workers and the environment.
Although the 1918 pandemic strain was extremely pathogenic and was related
to classic swine influenza virus, influenza diagnostic laboratories around
the world do not use biologic containment procedures (biosafety level 3 or
greater) to handle specimens. The Hong Kong experience shows that there can
be no absolute certainty about the human pathogenicity or animal
transmissibility of any influenza specimen.
Training of laboratory staff in national centers and local laboratories
undertaking influenza surveillance, therefore, is needed to ensure that the
best practices are routinely used to reduce infection or transmission risk.
Contingency plans can be prepared to increase stringency of biological
safety procedures, should an unusually pathogenic new influenza subtype
again appear. Procedures would need to be appropriate for the technical
facilities that actually exist in laboratories in different locations.
Authorization to import and maintain supplies of an antiviral agent (e.g.,
rimantadine) could be organized in advance to protect laboratory workers
and others at high risk. Procedures for authorized shipment of potentially
hazardous strains to a reference center also can be planned in advance.
Experience in 1997 also showed that the same needs may extend to the
expanded network of laboratories likely to collaborate in investigations of
new influenza viruses, including laboratories using live field strains of
the virus for research, vaccine development, or reference material
preparation.
Enhance Electronic Communications about Influenza
In 1997, the Hong Kong authorities set a new standard in communications
about influenza by providing daily updates on a readily accessible Internet
site. Information was also accessible on the FluNet WHO Internet site
(http://www.who.ch/flunet/). Further examples of electronic influenza
information systems are the partial European system, which collects and
disseminates data from seven countries (34); weekly information from the
Centers for Disease Control and Prevention about influenza in the United
States (http://www.cdc.gov/ncidod/diseases/flu/weekly.htm); and electronic
(e)-mail by the Public Health Service in the United Kingdom, which
disseminates up-to-date information on influenza occurrence there. However,
these regional or national systems do not obviate the need for a single,
universally accessible, global system that would enable national or local
public health officials and laboratory workers to monitor influenza without
receiving multiple e-mail messages or having to connect to different
Internet sites that use varied formats, representations of data, and
possibly languages. Such a system could have reduced uncertainty in late
1997 about whether the lack of reports of H5 viruses outside Hong Kong was
due to lack of adequate searching for them or lack of their spread. This
concern also is hard to address until it becomes possible to receive
information electronically from, or provide technical guidance to, most
local or national health centers in developing nations undertaking disease
investigation and diagnosis.
Accordingly, development of a multifunctional electronic global influenza
information exchange system is suggested. (Such a system could also be used
to communicate about other important infectious diseases, so long as this
does not complicate widespread accessibility for influenza information
exchange.) This system would extend current capabilities beyond those of
the existing WHO Flu-Net by ensuring the existence of resources (e.g.,
connection by wired or wireless communication systems) and system
management procedures (e.g., authorization passwords and encryption) to
allow simple daily access by all national influenza centers; extending
access to local scientists and health officials in key cities within
participating countries who, because of their surveillance or diagnostic
capabilities, may have early information about possibly new influenza virus
cases or outbreaks; enabling users to send and receive information rapidly
within their own countries, as well as to or from WHO or the collaborating
centers; and providing access also to key national and international
scientists knowledgeable about occurrences of possible influenza outbreaks
in animals. For scientists at a local level to benefit from international
electronic information, translation into several major languages may be
needed, on line if possible or at international or national Internet sites.
Among other benefits, information from an electronic information exchange
system could enable local and national or international scientists to make
cooperative decisions about diagnostic sampling and needed epidemiologic
information, without the effort and expense of outside experts.
Furthermore, operators of public electronic information sites, such as WHO,
or a national authority, as was the case in Hong Kong, would be better able
to fulfill their task if such a system were in place for them to collect
and check information.
Enhance Vaccine Production Capabilities
Pathogenicity of the H5 virus for chickens and chicken eggs complicated the
preparation of seed virus for potential production of vaccine, even for
supplies for testing in humans; thus, a high-yielding production seed could
not be easily adopted. Alternative strategies (e.g., attenuation of the
virus by genetic manipulation, expression of the gene coding for the H5
virus into baculovirus-infected insect cells, or use of a nonpathogenic
virus antigenically close to the currently isolated strain) were envisaged.
However, even now, it is not clear that a practical way to mass-produce
vaccine to the H5 Hong Kong virus exists or could be established in a short
time, should a similar event occur. Thus, the rules for pandemic planning
need revision, recognizing that reliance on existing licensed techniques
for vaccine production could entail unacceptably long delays, should a
highly pathogenic strain of avian influenza emerge and lead to a strain
transmissible in humans (35).
Efforts begun in 1997 to find ways to mass-produce vaccine when the wild
virus is highly lethal for eggs should be continued. These include
producing vaccine with existing facilities (attenuating the effect of
vaccine virus on eggs) and developing alternative techniques (e.g., cell
culture grown virus, genetically engineered vaccines). Placing applications
to license new methods on the fast track for review by regulatory
authorities would be consistent with a basic tenet of pandemic
preparedness: the greater the interpandemic production and use of influenza
vaccine, the easier it will be to meet needs should a pandemic occur.
Modifying vaccine-control procedures to decrease delays in releasing
batches of vaccines in diverse countries with similar requirements in an
emergency is also important. (This issue is already being discussed in
Europe [J. Wood, pers. comm.].)
Improve Access to Vaccine or Antiviral Agents and Establish Support Systems
During the Hong Kong episode, a rapid local shortage of existing
antiinfluenza drugs was observed, and rimantadine was imported. Had
vaccines begun to be produced, no process existed for reaching agreements
about access by different countries. Waiting until a pandemic strikes to
determine access to prophylactic materials inevitably contributes to
inequities in supply for countries lacking facilities to produce antiviral
agents or vaccines or lacking resources to competitively purchase supplies
at a time of scarcity. The issue of equity cannot be resolved by individual
governments or manufacturers. Both vaccine and drug industry and
international organizations need to discuss how to encourage fair
distribution of scarce vaccines or other pharmaceutical drugs before a
pandemic crisis arises.
Regardless of vaccine supply issues, vaccines and antiviral agents are
unlikely to meet demand, even for industrialized countries able to purchase
them. Assuming that people in all countries will be similarly susceptible
to the next influenza pandemic virus and even though the elderly usually
constitute a smaller percentage of the population in developing than in
industrialized countries, during any future pandemic, the absolute number
of those dying in the developing world will likely equal or exceed the
number of those dying in industrialized countries, as in 1918. Other needs
for responding medically must also be considered, including methods to
ensure provision of basic nursing support and care when large numbers of
people become ill over a few-week period in community after community.
During the 1918 pandemic in the United States, for example, the Public
Health Service called on the Red Cross to assume responsibility for
mobilizing health workers and paying for them during the epidemic and
supplying hospitals when local authorities could not (36). Efforts were
mounted in many communities, even in remote areas with few facilities for
health care. In India, efforts by individual communities without government
directive were credited with saving many lives in 1918-19 (M. Rammana,
pers. comm.).
Conclusions
One year after concerns were raised in Hong Kong about another influenza
pandemic, are we really much further along in establishing the most
effective early warning systems and developing the ability to deal with a
true pandemic? WHO now has guidelines for responding to a pandemic (24).
New helpful relationships, procedures, and scientific knowledge were
undoubtedly established in 1997, particularly concerning international
efforts for virus surveillance and vaccine production. However, both
serious pandemic threats in recent years (1976, United States; 1997, Hong
Kong) raised unpredictable new issues related to vaccine supply, which
should not stand in the way of planning about the many predictable needs,
which extend well beyond producing and using vaccines. For example, had the
H5 viruses spread among the human population in Hong Kong (or any other
country), national authorities would have rapidly needed to obtain numerous
pharmaceutical products, to store and equitably distribute them, to manage
demand for basic health-care services, and to maintain social and economic
functions during a potential major health crisis (24). Because of the large
variety of tasks, the formation of National Pandemic Planning Committees
(NPPCs) has been suggested to develop the options for intervention
strategies appropriate to each country (37).
Establishment of NPPCs will likely raise procedural matters, such as
membership and chain of command. Unless these matters are resolved,
valuable time will be lost. As seen in Hong Kong, a pandemic threat arises
suddenly and rapidly becomes a public health concern. Yet very few
countries have formally established NPPCs and influenza pandemic plans (a
process requiring several years). Without increased urgency about this
matter, the next pandemic will find most of the world unprepared.
---------------------------------------------------------------------------
Acknowledgments
      The authors express their appreciation particularly to Dr. Daniel
Lavanchy, WHO, Geneva, for his numerous helpful comments.
      This document was an activity of the European Scientific Working
Group on Influenza.
      Dr. Snacken directs the influenza surveillance program at the
Scientific Institute of Public Health-Louis Pasteur. He also chairs the
European Scientific Working Group on Influenza.(ESWI).
      Address for correspondence: R. Snacken, Department of Virology,
Scientific Institute of Public Health Louis Pasteur, 14 J. Wytsman Street,
B-1050 Brussels, Belgium; fax 32-2-642-5654; e-mail: r.snacken@iph.fgov.be.
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     1998;351:460-1.
 36. La mobilisation de la Croix-Rouge Amricaine pendant l'pidmie de
     grippe 1918-1919. La Tribune de Genve, 1920;1-27 (archival source:
     International Federation of the Red Cross, box 19746).
 37. Ghendon Y. Introduction to pandemic influenza through history. Eur J
     Epidemiol 1994;10:451-3.
_______________________________________________________________________________
Perspectives
Disparity in the Natural Cycles of Borrelia burgdorferi and the Agent of
Human Granulocytic Ehrlichiosis
Michael L. Levin, Franka des Vignes, and Durland Fish
Yale School of Medicine, New Haven, Connecticut, USA
---------------------------------------------------------------------------
      We studied the prevalence of Borrelia burgdorferi and the agent
      of human granulocytic ehrlichiosis (HGE) among questing nymphal
      and adult Ixodes scapularis ticks of the same generation and
      the infectivity of wild white-footed mice for ticks feeding on
      them. The prevalence of B. burgdorferi infection in
      host-seeking ticks increased less than twofold from nymphal
      (31% to 33%) to adult (52% to 56%) stage, and 52% of
      white-footed mice were infected. Prevalence of the agent of HGE
      increased 4.5- to 10.6-fold from nymphal (1.5% to 1.8%) to
      adult stage (7.6% to 19.0%), while only 18% of mice were
      infectious to ticks. B. burgdorferi infection was more common
      in mouse-fed ticks than in ticks collected from vegetation,
      whereas the agent of HGE was half as common in mouse-fed ticks
      as in ticks collected from vegetation. The different prevalence
      in nature of these pathogens in ticks suggests that their
      maintenance cycles are also different.
The agent of human granulocytic ehrlichiosis (HGE) is nearly identical to
Ehrlichia phagocytophila, which causes tick-borne fever in sheep and goats
in Europe (1,2) and is transmitted by the tick Ixodes ricinus (3)the major
vector of Lyme disease (4). In the eastern and midwestern United States,
the agent of HGE is transmitted by I. scapularis (5-7), also a vector of
Borrelia burgdorferithe agent of Lyme disease. Both agents are perpetuated
in natural cycles between the tick vector and vertebrate hosts (8-10).
Neither agent is maintained transovarially (11,12); thus, horizontal
transmission involving a susceptible vertebrate host is necessary. The
white-footed mouse (Peromyscus leucopus) plays an important role as a
reservoir for B. burgdorferi (13). Animal species that serve as the main
source(s) of the agent of HGE for ticks have not been determined.
Rodents serving as natural hosts for the tick species that transmits both
agents can be coincidentally exposed to the two agents. Indeed,
white-footed mice from Connecticut have been shown to carry antibodies to
both agents simultaneously (14). Granulocytic ehrlichia have been found in
wild rodents (10,15). Furthermore, the white-footed mouse and several
strains of laboratory mice (Mus musculus) are susceptible to the agent of
HGE in laboratory experiments (5,10,12). Antibodies to the agent of HGE
have been detected in various rodent species from California, Colorado,
Connecticut, Florida, New Jersey, New York, Maryland, Minnesota, and
Wisconsin (14,16-18). These findings allowed the authors to suggest that
small rodents, particularly the white-footed mouse, play the same role in
perpetuating the agent of HGE in North America that they do in perpetuating
B. burgdorferi (10,14,16,18,19). We present results of a 2-year field study
that provide evidence to the contrary.
The Study
We studied the prevalence of B. burgdorferi and the agent of HGE among
questing nymphal and adult I. scapularis of the same generation, in natural
foci with concurrent circulation of both pathogens in Connecticut. In 1996,
we collected ticks at two study sitesBridgeport and
Woodbridgeapproximately 30 km apart. In 1997, we collected ticks at the
Bridgeport site only; ticks feeding on white-footed mice and from
vegetation were collected and flagged in June (nymphs) and October
(adults). Mice were trapped in Sherman live-traps twice per month from June
through August and were held for several days in the laboratory in
individual wire-mesh cages over water to allow all naturally attached ticks
to feed to repletion. While in the laboratory, mice were provided with food
and drinking water ad libitum. Engorged ticks were collected daily. A serum
sample was collected from each mouse before release at the capture site.
Serum samples from 121 mice were tested for specific antibodies to the
agent of HGE by indirect immunofluorescence assay (IFA) (Aquila
Biopharmaceuticals, Worcester, MA). Antigen derived from human promyelocyte
cell culture (HL-60) infected with the agent of HGE obtained from
Westchester County, New York. Sera were screened at a dilution of 1:40 in
1X phosphate-buffered saline (pH 7.4). Several studies examined the
specificity of IFA for the agent of HGE (including an assay involving the
antigen produced by Aquila Biopharmaceuticals) and found no considerable
serologic cross-reactivity between the agent of HGE and rickettsial
organisms outside the E. phagocytophila group at dilutions 1:16 and higher
(8,20,21). Thus, screening samples at 1:40 dilution ensures that sera
testing HGE-positive were from mice actually exposed to that agent.
Host-seeking nymphal and adult I. scapularis collected from vegetation were
tested individually by polymerase chain reaction (PCR) for infection with
B. burgdorferi and the agent of HGE. Engorged ticks were allowed to molt to
the next stage and were identified by species. I. scapularis nymphs derived
from mouse-fed larvae were tested in poolsone pool (up to 10 ticks) per
mouseto assess the infectivity of individual mice for feeding larvae.
Adult I. scapularis ticks derived from mouse-fed nymphs were tested
individually, and the prevalence of each infection among these ticks was
compared with the prevalence of each infection among questing adult ticks
collected from vegetation at the same site.
For PCR testing, individual adult or nymphal ticks, or pools of nymphs,
were placed in sterile 1.5 cc plastic vials, deep-frozen in liquid
nitrogen, ground with a sterile plastic pestle, and resuspended in 50 ml of
Tris-EDTA buffer. DNA was extracted from samples by using the IsoQuick
Nucleic Acid Extraction Kit (ORCA Research Inc., Bothell, WA) to maximize
sensitivity (22). Briefly, guanidine thiocyanate, a proprietary extraction
matrix, and sodium dodecyl sulfate solution were added to a suspension, and
the mixture was incubated at 65C for 10 min. After separation of phases by
centrifugation, DNA was precipitated with sodium acetate and isopropanol
and washed with 70% ethanol. The final DNA pellet was resuspended in 50 ml
of RNase-free water, and a 2.5-ml aliquot was used for each PCR. Primers
EHR521 (5'-TGT AGG CGG TTC GGT AAG TTA AAG-3') and EHR747 (5'-GCA CTC ATC
GTT TAC AGC GTG-3') were used to amplify a 247-bp fragment of 16S rDNA from
the agent of HGE (6). Primers FLA297 (5'-CGG CAC ATA TTC AGA TGC AGA
CAG-3') and FLA652 (5'-CCT GTT GAA CAC CCT CTT GAA CC-3') developed in the
laboratory of Dr. Erol Fikrig (Yale School of Medicine) were used to
amplify a 378-bp fragment of the flagellin gene of B. burgdorferi. The
amplification products were visualized in 2% agarose gels.
Prevalence of Infections
The prevalence of B. burgdorferi infection in both questing nymphal and
adult I. scapularis was similar between the two study sites in 1996 and
remained stable for 2 consecutive years at the Bridgeport site (Table 1).
Within a generation, the proportion of Borrelia-infected ticks increased
less than twofold from nymphal (32% to 33%) to adult stage (52% to 56%) at
both sites and in both years.
Prevalence of ehrlichial infection in I. scapularis nymphs was also similar
at the two study sites and between years at the Bridgeport site (Table 1),
while in questing adult ticks, ehrlichial prevalence varied. In 1996, the
prevalence of the agent of HGE in adult ticks at the Bridgeport site
(12.5%) was higher than at the Woodbridge site (8.3%), although not
significantly (p = 0.11). The proportion of adult ticks infected with the
agent of HGE in Bridgeport was significantly higher in 1997 than in the
previous year (p = 1.44x10-7). Within the same generation, the proportion
of Ehrlichia-infected ticks increased fivefold in Woodbridge in 1996 and
10.4- to 10.6-fold in Bridgeport in 1996 and 1997, respectively.
An average of    Table 1. Prevalence of infection with Borrelia
0.7 engorged I.  burgdorferi and the agent of human granulocytic
scapularis       ehrlichiosis among questing nymphal and adult Ixodes
nymphs (0 to 7)  scapularis of the same generation, Connecticut
and 16.7 (0 to
113) engorged I. ----------------------------------------------------------
scapularis                                      % Borrelia-   % Ehrlichia-
larvae were      Study site             Ticks    infected      infected
collected per    (year)         Stage   tested    ( SE)        ( SE)
mouse. The       ----------------------------------------------------------
highest mean
density (       Woodbridge    Nymphs      442  32.9  2.3     1.6  0.6
standard error)  (1996)        Adults      251  52.6  3.2     8.3  1.9
of nymphal       Bridgeport    Nymphs      164  31.7  2.2     1.2  0.5
infestation in   (1996)        Adults       48  56.3  3.2    12.5  2.1
P. leucopus      Bridgeport    Nymphs      110  32.7  4.5     1.8  1.3
(1.30.2) was    (1997)        Adults      100  55.0  3.8    19.0  3.0
recorded in late ---------------------------------------------------------
June, with mean
larval density peaking in late August (34.61.7). Overall, 113 of
121 mice were infested with either nymphal or larval I. scapularis.
Of 40 mice with nymphs, 32 (80.0%)produced adult ticks infected with
B. burgdorferi, and 7 (17.5%) yielded ticks infected with the ehrlichial
agent. Of 108 mice infested with I. scapularis larvae, 56 (51.4%)
produced B. burgdorferi-infected nymphal ticks, and 20 (18.4%)
produced nymphs infected with the agent of HGE. Of the 108 mice,
13 (11.9%) infected feeding ticks with both pathogens.
Prevalence of infectivity in mice did not differ by month.
Of 121 mice trapped from June to August 1997 in Bridgeport and tested by
IFA, 46 (38.0%) had antibodies against the agent of HGE. The relatively
high sera dilution (1:40) used for screening allows the possibility that
some samples with low antibody titers were missed and that an even higher
proportion of the mouse population had actually been exposed to the agent
of HGE.
The proportion of HGE-seropositive mice decreased from 47% (n = 49) in late
June and July to 32% (n = 72) in August, perhaps because of a loss of
antibody by mice over time, the recruitment of naive young mice into the
population, or both. Of the 20 mice that produced nymphs infected with the
agent of HGE, 17 were also seropositive. Evidently, these mice remained
infectious for ticks, despite the presence of specific antibodies. The
other three infectious mice were HGE-seronegative, which most likely
indicated recent infection. Thus, only 18.4% of P. leucopus were capable of
infecting ticks with the agent of HGE, although at least twice as many (as
determined by IFA) had been exposed to this agent. Apparently, mice exposed
to ticks infected with the agent of HGE may develop an immune response to
the pathogen but not become infectious for xenodiagnostic ticks. These
findings suggest that a high prevalence of the specific antibody against
the agent of HGE in rodent populations does not necessary reflect the scope
of rodents' involvement in transmitting the ehrlichial agent.
 Table 2. Prevalence of infection with Borrelia             Prevalence of
 burgdorferi and the agent of human granulocytic            B. burgdorferi
 ehrlichiosis among adult Ixodes scapularis ticks           infection among
 collected as engorged nymphs from wild-caught Peromyscus   adult ticks
 leucopus and from vegetation at the                        derived from
 same site (Bridgeport, Connecticut)                        mouse-fed
                                                            nymphs was
 ---------------------------------------------------------- higher than
                                                  % Con-    among questing
                                                currently   ticks collected
                     % Borrelia-  % Ehrlichia-              from vegetation
 Origin      Ticks    infected      infected    infected    (p = 0.0051)
 of ticks    tested     ( SE)       ( SE)      ( SE)     (Table 2).
 ---------------------------------------------------------- Conversely,
                                                            prevalence of
 P. leucopus   76     64.5  3.6    9.2  2.2   6.6  2.5   infection with
                                                            the agent of
 Vegetation    100    55.0  3.8   19.0  3.0  14.0  3.5   HGE in mouse
 ---------------------------------------------------------- -fed ticks
                                                            was not quite
half that of adult ticks collected from vegetation (p = 0.0022). Concurrent
infection in mouse-fed ticks was also half that in adult ticks collected
from vegetation (Table 2). The same trend was observed when prevalence of
ehrlichial infection was compared among Borrelia-infected adult ticks. In
the Borrelia-infected cohort collected from vegetation, 14 (25.5%) of 55
ticks were simultaneously infected with the agent of HGE, but in the cohort
collected from mice, only 5 (10.2%) of 49 were simultaneously infected with
the agent of HGE.
Mice that collect and feed many nymphs have a high probability of finding
an infected tick and becoming infected. Mice that feed large numbers of
ticks after infection would increase the rate of pathogen transmission. We
tested the hypothesis that mice exposed to or currently infected with the
agent of HGE are continuously infested with large numbers of ticks and thus
are capable of increasing the prevalence of infection in a natural tick
population. When we compared tick densities between mice infectious for
ticks, mice seropositive for the agent of HGE, and mice without specific
antibodies or infected ticks, nymphal and larval infestation densities did
not differ significantly (Table 3).
Conclusions
Because nymphal I. scapularis  Table 3. Exposure to the agent of human
naturally feed on vertebrate   granulocytic ehrlichiosis in white-footed
animals (small rodents,        mice, as detected by xenodiagnosis and
medium-sized and large         indirect immunoflorescence assay (IFA),
mammals, birds, and reptiles)  and their infestation with Ixodes
(23) that may vary in their    scapularis nymphs and larvae (Bridgeport,
ability to acquire and         Connecticut)
transmit a pathogen, the       --------------------------------------------
prevalence of infection in                            Mean no.   Mean no.
different portions of an adult Status         Mice    of nymphs  of larvae
tick population would differ   of mice        tested   ( SE)      SE)
depending on which host        --------------------------------------------
species the tick had fed on.
Thus, the prevalence of        Infectious(sup a)20   0.70.1    19.51.6
infection for the tick         IFA-positive     46   0.90.1    14.90.9
population as a whole is an    IFA-negative,    72   0.70.1    17.81.9
contributions of individual    noninfectious
host species. We compared      --------------------------------------------
average prevalence to the      (sup a)Mice infectious for ticks at the time
average resulting from          of study.
prevalence of infection in ticks derived from a particular host species to
assess the relative contribution of that host species to amplification of a
pathogen. For example, the prevalence of B. burgdorferi infection in
mouse-fed ticks is considerably higher than the average prevalence of the
infection in a general tick population, which suggests that the
white-footed mouse is an important amplifying reservoir of B. burgdorferi.
The prevalence of the agent of HGE in mouse-fed ticks is lower than the
average, suggesting that the white-footed mouse is not as effective an
amplifying reservoir for this agent as are other host species. An important
contribution from non-Peromyscus host species that infects a large
proportion of feeding nymphs with the agent of HGE appears necessary to
account for the average prevalence of infection in host-seeking adult
ticks.
The prevalence of B. burgdorferi infection in ticks increased less than
twofold from nymphal to adult stage, while more than 50% of the
white-footed mouse population was infected with B. burgdorferi and
transmitted the pathogen to feeding ticks. At the same time, prevalence of
infection with the agent of HGE in ticks regularly showed a 4.5- to
10.6-fold increase from nymphal stage to adult stage, although only 18% of
mice were infectious for feeding ticks. This steep increase in prevalence
of ehrlichial infection in ticks also suggests the involvement of other
susceptible host species that maintain the natural transmission cycle of
the agent of HGE at the observed level.
Dissimilarities between two pathogensin the increase of infection in ticks
from nymphal to adult stage and in prevalence of infection in the
host-seeking population versus a subpopulation of mouse-fed tickssuggest
that natural cycles of the agents of Lyme disease and HGE differ. They
involve the same vector-species, but the principal amplifying hosts for the
two pathogens are not the same.
Although the white-footed mouse is susceptible to infection with both
agents, this species alone cannot account for the observed prevalence of
the agent of HGE in adult ticks. Our data suggest that most nymphal I.
scapularis acquire the agent of HGE from non-Peromyscus hosts.
---------------------------------------------------------------------------
Acknowledgments
      We thank Yukiko Otsuka for assistance in both the field and
laboratory and Richard Coughlin for providing IFA slides for our assays.
      This research was sponsored by grants from G. Harold and Leila Y.
Mathers Charitable Foundation, the National Institutes of Health, National
Institute of Allergy and Infectious Diseases (AI28956), and U.S. Department
of Agriculture cooperative agreement 58-1265-5023.
      Dr. Levin is a postdoctoral associate, Department of Epidemiology and
Public Health, Yale School of Medicine. His areas of expertise are ecology
and epidemiology of tick-borne zoonoses. His current research focuses on
the dynamics of circulation of the agents of Lyme disease and human
granulocytic ehrlichiosis in nature, reservoir competence of vertebrate
animals, and multiple pathogen interactions.
      Address for correspondence: Michael L. Levin, Department of
Epidemiology and Public Health, Yale School of Medicine, 60 College Street,
#600, P.O. Box 208034, New Haven, CT, 06520-8034, USA; fax: 203-785-4782;
e-mail: Michael.Levin@Yale.edu.
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  2. Sumner JW, Nicholson WL, Massung RF. PCR amplification and comparison
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  5. Des Vignes F, Fish D. Transmission of the agent of human granulocytic
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  6. Pancholi P, Kolbert CP, Mitchell PD, Reed KD Jr, Dumler JS, Bakken JS,
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  8. Magnarelli LA, Stafford KC, Mather TN, Yeh MT, Horn KD, Dumler JS.
     Hemocytic rickettsia-like organisms in ticks: serologic reactivity
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     granulocytic ehrlichiosis by PCR. J Clin Microbiol 1995;33:2710-4.
  9. Reed KD, Mitchell PD, Persing DH, Kolbert CP, Cameron V. Transmission
     of human granulocytic ehrlichiosis [letter; comment]. JAMA
     1995;273:23.
 10. Telford SR, Dawson JE, Katavolos P, Warner CK, Kolbert CP, Persing DH.
     Perpetuation of the agent of human granulocytic ehrlichiosis in a deer
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 11. Patrican LA. Absence of Lyme disease spirochetes in larval progeny of
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     Med Entomol 1997;34:52-5.
 12. Hodzic E, Fish D, Maretzki CM, de Silva AM, Feng S, Barthold SW.
     Acquisition and transmission of the agent of human granulocytic
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 13. Barbour AG, Fish D. The biological and social phenomenon of Lyme
     disease [review]. Science 1993;260:1610-6.
 14. Magnarelli LA, Anderson JF, Stafford KC, Dumler JS. Antibodies to
     multiple tick-borne pathogens of babesiosis, ehrlichiosis, and Lyme
     disease in white-footed mice. J Wildl Dis 1997;33:466-73.
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     for human granulocytic ehrlichiosis agent in urban white-footed mice
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 18. Nicholson WL, Muir S, Sumner JW, Childs JE. Serologic evidence of
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     Serologic cross-reactions among Ehrlichia equi, Ehrlichia
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     1995;33:1098-103.
 21. Nicholson WL, Comer JA, Sumner JW, Gingrichbaker C, Coughlin RT,
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 22. Schwartz I, Varde S, Nadelman RB, Wormser GP, Fish D. Inhibition of
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______________________________________________________________________________
Synopses
______________________________________________________________________________
Synopses
Malaria Reemergence in the Peruvian Amazon Region
Javier Arambur Guarda,* Csar Ramal Asayag,* and Richard Witzig
*Loreto Department of Public Health, Iquitos, Peru; and Centers for
Disease Control and Prevention, Atlanta, Georgia, USA
---------------------------------------------------------------------------
      Epidemic malaria has rapidly emerged in Loreto Department, in
      the Peruvian Amazon region. Peru reports the second highest
      number of malaria cases in South America (after Brazil), most
      from Loreto. From 1992 to 1997, malaria increased 50-fold in
      Loreto but only fourfold in Peru. Plasmodium falciparum
      infection, which has increased at a faster rate than P. vivax
      infection in the last 3 years, became the dominant Plasmodium
      infection in the highest transmission areas in the 1997 rainy
      season. The vector Anopheles darlingi has also increased during
      this epidemic in Loreto. Moreover, chloroquine and
      pyrimethamine-sulfadoxine drug-resistant P. falciparum strains
      have emerged, which require development of efficacious focal
      drug treatment schemes.
The Peruvian Department of Loreto is
the last part of the greater Amazon
region to report the reemergence of
epidemic malaria after the eradication
efforts of the 1960s. Loreto, which           [fig]
comprises nearly one-fourth of the land Figure 1: The department of
mass of Peru, has the ecologic          Loreto and the city of Iquitos in
characteristics of the Amazon lowlands  Peru.
(Figure 1). In central Loreto, the
Maraon and Ucayali Rivers combine to
form the nascent Amazon River. Iquitos (population 345,000), the only large
urban center in Loreto, is accessible only by air or river. The rural
population of 474,000 is clustered in towns and villages throughout the
Amazon tributary system. Loreto's economy relies on basic agriculture,
fishing, lumber, commercial activities, and petroleum.
Epidemiology
Since 1941, the highest number of malaria cases in Peru was 95,000 in 1944,
and the lowest was 1,500 cases in 1965 (after the malaria eradication
campaign) (1). Plasmodium falciparum infections (chloroquine-sensitive)
were confined to the northwestern coastal areas of Peru bordering Ecuador,
with occasional reports of cases from the north and eastern Loreto border
areas with Ecuador, Colombia, and Brazil.
 						 In 1988, no cases of P. falciparum
                                     were reported in Loreto. In 1991, 140
 						 cases were reported; the number
        [fig]				 increased annually until 1997 when
 Figure 2: Malaria incidence in      54,290 slide-confirmed P. falciparum
 Loreto, 19921997.                  cases and 85 deaths were reported
                                     (Figure 2). In 1997, a total of
                                     121,268 malaria cases were reported in
Loreto; the number indicates an equally dramatic rise in P. vivax malaria.
P. malariae is infrequently reported (44 cases in 1997). An additional
36,864 persons were treated for probable malaria (slide-negative or no
available laboratory diagnosis) in 1997. Therefore, the number of malaria
cases in 1997 reported by slide or clinical definition was 158,115 (2).
More Plasmodium infections occurred in males (60.5%) than in females
(39.5%). Malaria transmission has been seasonal in the Loreto epidemic,
with peaks in the rainy season from November to June (Figure 3).
 		[fig]                               [fig]
 Figure 3. Malaria (all species)      Figure 4: Annual parasite index in
 incidence in Loreto by month,        Loreto, by district (per 1,000
 19941997.                           population).
                                                                                                                                                    most
Malaria around the city of Iquitos accounts for most cases in Loreto.
Indigenous P. vivax malaria was initially reported in the
Iquitos area (Rumococha and Zungarococha) in April 1991, and P. falciparum
was first detected (Padrecocha) in November 1994. A small number of P.
falciparum cases have been diagnosed in patients who had not traveled
outside Iquitos in the 2 months before illness. Entomologic investigations
have confirmed that malaria transmission inside Iquitos is possible. Two
high transmission zones surround Iquitos: communities on the Nanay River
(which empties into the Amazon after bordering northern and eastern
Iquitos) and communities along the first asphalt extension road from
Iquitos (the unfinished Iquitos-Nauta road). Two additional high
transmission zones include communities on the Yavar River and the Pastaza
River. The annual parasite index (number of malaria cases per 1,000 persons
per year), with malaria transmission by districts in Loreto, is shown in
Figure 4. The global annual parasite index for Loreto for 1997 was 148 per
1,000 persons.
Age-specific attack rates of P. falciparum in most of Loreto are consistent
with hypoendemic malaria (defined as parasite or spleen rates less than 10%
[Figure 5A]), although in 1997 in the highest transmission areas,
mesoendemic malaria measurements were transiently documented in prevalence
studies (11% to 50% parasite rates) (3). The high parasitemia rates in
adults in Loreto emphasize the environmental risks for malaria infection to
adults and the lack of immunity in this epidemic. Age-specific death rates
have documented a substantial risk for death among those under 5 years of
age and among those 60 years of age and older, although the number of
deaths in children under 5 years declined in 1997 (Figure 5B). The death
rate from P. falciparum malaria has been decreasing (from 1.8 to 1.3 per
1000) in the last 4 years (1994 to 1997).
Because of the current epidemic, Peru
reports the second highest number of
malaria cases in South America (after
Brazil)most from Loreto (in 1997, Loreto
reported 67.2% of the malaria cases in    	[fig]
Peru) (4). From 1992 to 1997, malaria     Figure 5. A. Age-adjusted
increased 50-fold in Loreto and fourfold  incidence rates for Plasmodium
in Peru. In 1992, 1.6% of malaria cases   falciparum malaria in Loreto,
in Peru were due to P. falciparum; in     1996-1997. B. Age-adjusted
1997 (in Loreto), 44.8% of malaria cases  death rates for Plasmodium
were due to P. falciparum (5,6).          falciparum malaria in Loreto,
                                          1996-1997.
Vector Dynamics
Paralleling the malaria epidemic has been an increase in the highly
competent and anthropophilic malaria vector, Anopheles darlingi, the
principal P. falciparum vector in the Brazilian Amazon (7,8). An. darlingi
was not found in the Iquitos area in 1991 (9) when indigenous P. vivax was
detected but has now been found in all areas of Loreto and even within
Iquitos (10-12). The abundance of An. darlingi, already established when P.
falciparum was first reported from the peri-Iquitos area in 1994, is
clearly linked to the marked rise in P. falciparum transmission.
An. darlingi larvae habitats around Iquitos include small pools on cleared
land, fish hatcheries, areas of poor sanitation, swamps, and the edges of
small rivers. An. darlingi in the Iquitos area bite indoors or outdoors
from dusk to midnight, with a small dawn peak. An. darlingi represent more
than 90% of the anophelines in the peri-Iquitos area in the rainy season;
they decrease but remain the major anopheline species in the dry season.
An. benarrochi is the dominant malaria vector in western Loreto, while An.
triannulatus is the dominant vector in eastern Loreto. Other Anopheles
species in Loreto known to be malaria vectors are An. oswaldoi, An.
nuneztovari, and An. rangeli.
Climatic Associations
Loreto has a climate typical of the Amazon region, with a rainy season from
November to May and a second precipitation peak in September in the dry
season. The El Nio phenomenon in 1997 extended the dry season in Loreto,
but caused torrential rains along coastal Peru.
The level of the Amazon River in Iquitos varies 10 meters annually (from
108 to 118 meters above sea level); flooding is usual on the smaller
tributaries but infrequent on the main Amazon River. The mean annual
temperature is 28C, the warmest months are September and October, and the
humidity is persistently higher than 87% (2).
 				    Climatic indexes of river level, rainfall,
                            temperature, and humidity at Iquitos were
 				    analyzed to determine association with malaria
 	     			    prevalence. The height of the Amazon River at
 	[fig]                 Iquitos depends on rainfall on the eastern
 Figure 6: Plasmodium       side of the Andes Mountains as well as
 falciparum malaria         precipitation in Loreto. The height of the
 incidence in Loreto. A.    Amazon is related to malaria prevalence; the
 Average Amazon River level highest river level precedes the malaria
 at Iquitos by month. B.    prevalence peak by 2 months, and the lowest
 Precipitation at Iquitos   level precedes the malaria prevalence low by 2
 by month. C. Average       months (Figure 6A). Precipitation peaked twice
 temperature (C) at        in 1997: 3 months before and the same month
 Iquitos by month.          that malaria cases reached their highest
                            number (Figure 6B). The mean temperature was
                            27C to 29C and was inversely associated with
malaria cases (Figure 6C). Relative humidity was 87% to 93% and was not
associated with malaria prevalence.
Drug Resistance and Drug Treatment
Clinical resistance of P. falciparum parasites to chloroquine and to the
combination antifolate drug pyrimethamine-sulfadoxine is a growing public
health problem in Peru. In an epidemic characterized by limited population
immunity, parasitologic resistance (persistence or reemergence of
parasites) inevitably leads to clinical resistance (reemergence of malarial
symptoms).
Multiple regional in vivo drug susceptibility testing for chloroquine (10%
to 70% resistance) and pyrimethamine-sulfadoxine (10% to 63% resistance)
has documented significant focal differences in resistance patterns
(13-15). Three P. falciparum strains have converged on Loreto and Iquitos:
(Brazilian-pyrimethamine-sulfadoxine and chloroquine, complete resistance;
Loretanapyrimethamine-sulfadoxine, variable resistance; chloroquine
resistant; and Pastazan/coastal-chloroquine susceptible). These strains
were likely introduced and disseminated in different ways (e.g., routine
travel, illegal narcotrafficking), but the increased abundance of An.
darlingi in Loreto has allowed them to thrive. The highest percentage of
strains resistant to multiple drugs (both chloroquine and pyrimethamine-
sulfadoxine resistant) have been found in the border areas of Colonia
Angamos on the Yavar River, communities on the Blanco River, and in Santa
Clara near Iquitos.
From July through November 1996, we performed in Iquitos two prospective
studies of P. falciparum drug resistance in patients from periurban and
rural areas in Loreto. Twenty-eight day in vivo drug susceptibility testing
of P. falciparum to pyrimethamine-sulfadoxine was performed in 62 patients.
The WHO in vitro microtest method was used to evaluate P. falciparum
susceptibility to mefloquine, chloroquine, pyrimethamine-sulfadoxine, and
quinine in pretreatment specimens from the same patients (16). Results
showed significant pyrimethamine-sulfadoxine resistance in Loreto (31% in
vivo and 67% in vitro) and documented in vitro resistance of chloroquine
(78% resistance). P. falciparum parasites were susceptible to mefloquine
and quinine (17).
A recent study in Loreto showed that in vivo resistance to
pyrimethamine-sulfadoxine correlated with specific point mutations in P.
falciparum dihydrofolate reductase at amino acid positions 51, 108, and
164, and dihydropteroate synthase mutations at positions 437, 540, and 581
(18). Further studies mapping the molecular resistance patterns of P.
falciparum in Loreto are under way.
Malaria diagnosis and treatment are
provided free of charge by the
National Malaria Program. Focal
treatment schemes have been designed    		[fig]
depending on the local resistance       Figure 7: Initial Plasmodium
patterns as documented by in vivo       falciparum malaria treatment
methods. Depending on local efficacy,   schemes in Loreto by district.
first-line treatment of chloroquine,    Treatment efficiency* (cases
second-line of                          cured/cohort number x 100) and
pyrimethamine-sulfadoxine, or           efficacy** (cases cured/[cases
third-line of quinine with clindamycin  cured + resistant cases] x 100)
or tetracycline is administered         shown for each treatment scheme
(Figure 7). In 1998, treatment with     region. Treatment schemes:
mefloquine was initiated in selected    Primary-chloroquine; secondary-
communities with tertiary treatment     pyrimethamine-sulfadoxine;
schemes. All treatment lines include    tertiary-quinine.
one dose of primaquine to eradicate P.
falciparum gametocytes.
Control Schemes
Control strategies used by the Loreto Public Health Department and the
National Malaria Program have included source, chemical, and biologic
reduction strategies. Source reduction has consisted of community
participation in identifying and filling in larval sites. Chemical control
includes the use of insecticides in spatial fogging and domiciliary
spraying and on bed nets. Pyrethroids (e.g., cyfluthrin) are used for
fogging and domiciliary residual spraying. Since 1988, DDT has not been
used in Loreto. An important challenge is that many Amazonian houses have
open eaves and windows that allow mosquitoes to enter and exit without
touching surfaces (except for a person's skin). An. darlingi behavioral
studies have not yet been performed in Loreto to determine if the
mosquitoes rest inside or outside after feeding. Larvaciding is performed
with temephos (tetramethyl-thiodiphenylene phosphorothioate) placed at the
surface of the larval breeding sites. Biologic control has been
investigated with Bacillus sphaericus and thuringiensis in larval breeding
pools. These bacteria have a short residual effect of 7 to 15 days in
Loreto and require optimal conditions of shade for maximum efficacy.
Bed nets (mosquiteros) are ubiquitous in Loreto because of the many biting
mosquito species. Local bed nets are made principally of cotton and sell
for US$10 to $15. Insecticides were used in 1997 in the form of spraying
(with cyfluthrin or deltamethrim) and impregnation (with either permethrim
or deltamethrim) (19). Preliminary results show a decrease in malaria
prevalence where insecticide-treated bed nets are in use.
Conclusions
Malaria began to reemerge in the upper Pastaza River and eastern border
areas of Loreto in the early 1990s. P. falciparum exceeded P. vivax
infections in the early part of the Loreto epidemic (1992-1993). The
abundance of An. darlingi starting in 1993 set the stage for explosive
growth of both P. falciparum and P. vivax transmission, especially around
Iquitos, which had previously not been affected. The increase in the number
of malaria cases and the erosion of P. vivax dominance over P. falciparum
occurred in spite of governmental interventions (treatment schemes,
fumigation, larviciding, and domiciliary spraying). While P. vivax
infections decreased slightly from 1996 to 1997, P. falciparum cases
continued to increase by 60%. However, the rate of increase in total
malaria cases slowedfrom a factor of 2.23 from 1995 to 1996 to 1.19 in
1996 to 1997. The predominance of malaria infection in men is likely the
result of occupational risks (e.g., working in recently cleared areas,
logging, and night fishing and hunting).
The death rate of epidemic P. falciparum in Loreto appears to be low. The
oft-quoted 1% death rate for P. falciparum infection (3) has been reduced
seven times, perhaps because of determined efforts at the regional and
national levels to provide rapid and appropriate treatment in malarious
areas. Future efforts are planned to continue and expand coverage to
further reduce deaths due to P. falciparum.
Although positive correlations were found between malaria transmission
periods and Amazon River level and rainfall, the negative correlation with
temperature may not be as significant as in other areas where higher
temperatures are positively correlated with malaria cases (20). The mean
temperature in the rainy season in Loreto is still within the optimal range
for anopheline development, and so the observed temperature correlation may
have little biologic influence on malarial prevalence.
Analysis of environmental factors indicates that the optimal timing of
malaria control activities in 1998 was 2 months before the rainy season.
Integrated malaria control activities began in the dry season with the
strategy to intervene before high transmission season. In 1998,
slide-documented malaria cases decreased 30.7% to 84,059, with P. vivax
cases decreasing 15.3% to 56,710 and P. falciparum cases decreasing 49.6%
to 27,336. The longer dry season in 1997, apparently caused by El Nio, may
have assisted the Loreto Public Health Department and National Malaria
Program efforts to control malaria in 1998.
Danger exists for further expansion of malaria in Loreto, especially
through expansion of An. darlingi (into areas where it is not yet the
dominant vector) and evolution of P. falciparum drug resistance.
Consequently, more malaria control research is needed, particularly
environmental control program studies, Anopheles behavioral studies, drug
resistance testing, and community bed net trials.
---------------------------------------------------------------------------
Acknowledgments
      We thank Ernesto Coln Bernal, Jorge Quintana Zurita, and Mercy
Panduro Gaviria for technical assistance for the in vitro drug resistance
studies, Henrry Echeanda Rossell for computer design, ernesto Berrocal
Vilchez for entomologic coordination,  Cristiam Carey Angeles and Carlos
Calampa del Aguila for logistical support, and the workers at the Loreto
Public Health Department for malaria control data. In memoriam Javier
Benzaquen Garcia, a young Loreto malariologist (died in October 1998).
Support for this work was provided by a National Foundation for Infectious
Diseases-Merck Postdoctoral Fellowship in Emerging Infectious Diseases (Dr.
Witzig).
Dr. Javier Arambur Guarda is chairman of Surveillance of Infectious
Diseases, General Office of Epidemiology, Ministry of Health of Per. His
research focuses on antimalarial resistance, integrated malaria control,
and malaria control policies.
      Address for correspondence: Javier Arambur Guarda, Oficina General
de Epidemiologa, Ministerio de Salud, Per, Avenida Camilo Carrillo 402,
Jess Mara, Lima11, Per; e-mail: jaramburu@oge.sld.pe.
References
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  2. Malaria Control Program. Malaria statistics 1992-1997. Iquitos,
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     London: Edward Arnold; 1993. p. 129,136.
  4. Status of malaria programs in the Americas: XLIV Report. Washington:
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  8. Branquinho MS, Lagos CBT, Rocha RM, Natal D, Barata JMS, Cochrane AH,
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 10. Fernandez R, Carbajal F, Quintana J, Chauca H, Watts DM. Presencia del
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_______________________________________________________________________________
Synopses
Enteropathogenic E. coli, Salmonella, and Shigella: Masters of Host Cell
Cytoskeletal Exploitation
Danika L. Goosney, Derek G. Knoechel, and B. Brett Finlay
University of British Columbia, Vancouver, British Columbia, Canada
------------------------------------------------------------------------
      Bacterial pathogens have evolved numerous strategies to exploit
      their host's cellular processes so that they can survive and
      persist. Often, a bacterium must adhere very tightly to the
      cells and mediate its effects extracellularly, or it must find
      a way to invade the host's cells and survive intracellularly.
      In either case, the pathogen hijacks the host's cytoskeleton.
      The cytoskeleton provides a flexible framework for the cell and
      is involved in mediating numerous cellular functions, from cell
      shape and structure to programmed cell death. Altering the host
      cytoskeleton is crucial for mediating pathogen adherence,
      invasion, and intracellular locomotion. We highlight recent
      advances in the pathogenesis of enteropathogenic Escherichia
      coli, Salmonella Typhimurium, and Shigella flexneri. Each
      illustrates how bacterial pathogens can exert dramatic effects
      on the host cytoskeleton.
Enteropathogenic Escherichia coli (EPEC): A Model for Studying Bacterial
Attachment and Effacement
Pathogenic E. coli strains remain a leading cause of severe and persistent
infant diarrhea in developing countries. Although EPEC is recognized as a
major diarrheal pathogen, until recently our understanding of how it causes
disease lagged behind that of other pathogenic E. coli, such as
enterotoxigenic E. coli or enteroinvasive E. coli.
EPEC is one of a class of pathogens identified as causing attaching and
effacing (A/E) lesions on intestinal cells (1). A/E pathogens typically
reside on a pedestal on the surface of the host epithelial cell and
ultimately cause severe disruption of the microvilli brush border (Figure
1A). Other pathogens displaying similar histopathologic features include
Hafnia alvei, Citrobacter rodentium (formerly C. freundii biotype 4280),
and enterohemorrhagic E. coli, the causative agent of hemolytic uremic
syndrome.
Bacterial Factors Involved in EPEC-Induced A/E Lesion Formation
The interactions between EPEC and host cells have been divided into three
stages. Initial adherence to cultured epithelial cells is mediated by the
formation of type IV fimbriae known as bundle forming pili (BFP) (2). While
not essential for forming the characteristic A/E lesions, initial adherence
helps bring the bacteria in close contact with the host cell. BFP mediate
bacterial-bacterial interactions in a human intestinal organ culture model
(3).
All the genes necessary for the formation of A/E lesions by EPEC are
contained within a 35-kbp pathogenicity island termed the locus of
enterocyte effacement (LEE) (Figure 1B) (4,5). These include the esps (E.
coli-secreted protein), escs (E. coli secretion), sep (secretion of E. coli
proteins), eae (E. coli attaching and effacing that encodes intimin), and
tir (translocated intimin receptor) genes (6).
					[fig]
 Figure 1. A. Transmission electron micrograph of an A/E lesion formed by
 rabbit enteropathogenic Escherichia coli (EPEC) infecting rabbit
 intestinal epithelial cells (micrograph provided by Dr. Ursula Heczko,
 Biotechnology Laboratory, University of British Columbia). B. Effects of
 EPEC infection on host intestinal epithelial cells. EPEC initially adheres
 to the host cell by its bundle-forming pili, which also mediate bacterial
 aggregation. Following initial attachment, EPEC secretes several virulence
 factors by a type III-secretion system. Signal transduction events occur
 within the host, including activation of phospholipase C (PLC) and protein
 kinase C (PKC), inositol triphosphate (IP[sub 3])fluxes, and Ca(sup 2+) release
 from internal stores. The bacterium intimately adheres to the cell by
 secreting its own receptor, Tir, into the host and binding to it with its
 outer membrane ligand, intimin. Intimin can also bind 1-integrins. Several
 cytoskeletal proteins are recruited to the site of EPEC attachment,
 including actin, alpha-actinin, talin, and ezrin. Cytoskeletal
 rearrangements occur following Tir-intimin binding, resulting in the
 formation of a pedestal-like structure upon which the pathogen resides.
The second stage of EPEC pathogenesis involves the secretion of bacterial
proteins, some into the host cell, including EspA, EspB, and EspD (7,8). The
expression of these proteins is maximal at the host body temperature (9) and
at conditions similar to those found in the which implies that they may
be involved in virulence. The translocation of these proteins is essential
for activating a number of signal transduction pathways (7), although their
precise role in pathogenesis is not well defined. EspA makes filamentous
appendages outside the bacterium and may be part of the translocation
machinery involved in delivering other virulence proteins (11). EspB is
translocated into the host cytosol and membrane, where it may effect
changes in the host cell's signaling pathways (12). All of these effector
proteins are secreted by a type-III secretion system encoded by the esc and
sep genes (6). Type-III secretion systems also play an important role in
other gram-negative pathogenic bacteria such as Yersinia, enabling
virulence factors to be translocated directly from the bacterial cytoplasm
to the host-cell membrane or cytoplasm (13).
The third stage of EPEC infection is characterized as intimate attachment
with the host cell. Intimin, a 94-kDa outer membrane protein encoded by the
eae gene (14), binds to a 90-kDa tyrosine phosphorylated protein in the
host membrane (15). This receptor, originally thought to be a host protein,
has recently been found to be of bacterial origin and has been designated
as the translocated intimin receptor (Tir) (16). As the name suggests, Tir
is translocated from the bacterial cell into the host membrane, where it
becomes phosphorylated on one or more tyrosine residues and functions as a
receptor for its binding partner, intimin. The resultant tight association
is accompanied by the formation of actin pedestals up to 10 m in length
(15). Purified intimin also binds 1 integrins, which suggests that intimin
may be binding more than one receptor on the epithelial cell (17). Although
integrins are not present on the apical surface of enterocytes, they are
located on the apical surface of microfold cells found in Peyer's patches
along the intestinal lumen (18).
Host-Cell Factors Involved in A/E Formation
The host cell undergoes a number of changes during infection by EPEC
(Figure 1B). The most striking change in the cellular structure of the host
cell is the formation of characteristic actin pedestals. Within 3 hours of
infection by EPEC, host-cell actin, [alpha.gif (53 bytes)]-actinin, talin,
erzin, and villin accumulate directly under the bacteria (19,20). The
latter four cytoskeletal components are involved in cross-linking of actin
microfilaments. Localized actin accumulation is so distinct that it forms
the basis of an in vitro diagnostic test for EPEC, which uses
fluorescein-tagged phalloidin to detect actin accumulation within infected
cells (21). The actin pedestals are not static; instead they lengthen and
shorten, resulting in apparent movement of EPEC along the host-cell surface
(20). The pedestals resemble microvilli in the distribution of actin and
villin (20). Microtubule and intermediate filament structures are not
affected by EPEC virulence factors (19).
Intracellular calcium levels also seem to play a role in EPEC pathogenesis.
EPEC-infected HEp-2 cells show significant elevation of intracellular
calcium levels (22), and buffering of these levels can prevent or delay the
formation of A/E lesions (23). Increases in intracellular calcium levels
can result in the depolymerization of actin by villin (a calcium-dependent
microvillus protein) and a breakdown of the host cytoskeleton not unlike
that seen in EPEC-infected cells (24). Inositol triphosphate (IP[sub 3]) is
involved in the release of Ca(sup 2+)from intracellular stores, and increased
levels of IP(sub 3) (25) and inositol phosphate fluxes (26) have been observed in
EPEC-infected cells. EPEC interactions with PLC-gamma1 HeLa epithelial cells
activate a number of proteins, including phospholipase C-gamma1 (PLC-gamma1)
(27). Phosphorylation of PLC-gamma1 leads to the IP(sup 3) and Ca(sup 2+)
fluxes  mentioned above, underscoring the importance of this signaling event.
Cytosolic protein kinase C also gets activated upon EPEC infection and
translocates to the plasma membrane (28).
Despite the dramatic changes induced by EPEC in the cytoskeleton, there
appears to be little involvement of the Rho family of small GTP-binding
proteins normally involved in cytoskeletal rearrangements (29). Inhibition
of Rho, Rac, and Cdc42 by compactin and Clostridium difficile ToxB, as well
as dominant negative alleles, had no effect on pedestal formation by EPEC,
which suggests that this pathogen uses a nontraditional mechanism to
rearrange actin.
Salmonella Typhimurium: A Model for Studying Bacterial Invasion
S. Typhimurium is a gram-negative bacterium that causes a variety of
diseases, from gastroenteritis in humans to typhoid fever in mice. S.
Typhimurium infections are contracted by oral ingestion and penetration
into the intestinal epithelium before induction of systemic (invasive)
disease. Invasion into the host intestinal cells results in dramatic
morphologic changes to the cell that are due to exploitation of the host
cytoskeleton.
Once in close contact with the epithelium, Salmonella induces degeneration
of enterocyte microvilli (30). Loss in microvillar structure is followed by
profound membrane ruffling localized to the area of bacterial-host cell
contact (Figure 2A) (29-31). Membrane ruffling is accompanied by profuse
macropinocytosis, which leads to the internalization of bacteria into the
host cells (32). The entire process occurs within minutes and when
completed, Salmonella resides within membrane-bound vesicles, and the
cytoskeleton returns to its normal distribution (33).
Bacterial Factors Involved in Salmonella Invasion
Salmonella entry into nonphagocytic epithelial cells requires several
chromosomal genes (inv/spa) clustered in a pathogenicity island termed SPI1
(Salmonella pathogenicity island 1) (34). Like EPEC, SPI1 encodes a type
III-secretion system and several potential virulence factors secreted by
this machinery. The type III-secretion system is activated upon host-cell
contact and allows export of virulence determinants directly into the host
cell, where they effect bacterial uptake (35,36). Recently, SptP, a
bacterial protein encoded within SPI1, has been shown to be translocated
into the host epithelial cell, where it modulates the host actin
cytoskeleton through its tyrosine phosphatase activity (37) (Figure 2B).
Disruption of a critical Cys residue in the catalytic domain of SptP
results in loss of phosphatase activity (38). It is hypothesized that SptP
may function in disrupting host actin stress fibers, thereby facilitating
membrane ruffling and subsequent bacterial uptake into host cells.
					[fig]
 Figure 2. A. Transmission electron micrograph of Salmonella-induced
 membrane ruffling in polarized Caco-2 epithelial cells. B. Salmonella
 invasion into host epithelial cells. Salmonella secrete virulence
 proteins, including SopE and SptP, into host cells by the type
 III-secretion system. SopE functions as a guanidine exchange factor for
 small GTP-binding proteins, probably mediating the exchange of GDP for GTP
 on a Rho subfamily member, CDC42. SptP is a tyrosine phosphatase required
 for invasion, probably by disrupting the cytoskeleton. Invasion also
 stimulates phospholipase C (PLC) activity, leading to inositol
 triphosphate (IP[sub 3]) and Ca(sup 2+) fluxes, which in turn may be
 involved in cytoskeletal rearrangements leading to membrane ruffling and
 Salmonella internalization.
Other bacterial factors are not encoded next to the secretion apparatus
but instead on the genome of a cryptic bacteriophage found in the Salmonella
chromosome. Recently, a virulence factor encoded within this genome, SopE,
has been shown to be required for efficient bacterial entry into host
cells (39). SopE requires the type III-secretion system to be translocated
into the host cell, where it can directly stimulate actin cytoskeletal
rearrangements. It acts as a guanidine exchange factor for members of the
Rho subfamily of small GTPases. sopE mutants exhibit less extensive actin
cytoskeletal rearrangements upon entry into epithelial cells than do wild-
type Salmonella (40). This discovery clearly illustrates how pathogens
which contain no primary sequence homology with host proteins) can craftily
subvert the host's own signaling machinery within the cell by mimicking
host proteins.
Host Factors Involved in Salmonella Invasion
The massive restructuring of the host cytoskeletal components during
Salmonella entry requires many host factors. A Rho subfamily member, Cdc42,
is needed for mediating bacterial uptake through membrane ruffling (41). It
is believed that the guanidine exchange activity of SopE is responsible for
the stimulation of Cdc42 in the host. The pathogen also activates host PLC
upon bacterial contact, leading to the production of two second messengers,
which further initiate signaling events (42). As a consequence, the host
cell's Ca(sup 2+)levels are altered to trigger cytoskeletal rearrangements
resulting in Salmonella invasion. Although EPEC and Salmonella use some of
the same signaling components (PLC, Ca[sup 2+]fluxes), the cytoskeletal
changes induced in the host cell by each pathogen are quite different.
This could be the result of different upstream or downstream effectors
in the signaling pathway. Several cytoskeletal components involved in
invasion have been identified. These include alpha-actinin, tropomyosin,
ezrin, and talin (19). The specific roles of these proteins in Salmonella
invasion are not defined.
Shigella flexneri: A Model for Intracellular Motility
S. flexneri, a gram-negative bacillus that causes bacillary dysentery in
humans, directs its own uptake into the colonic mucosa through membrane
ruffling and macropinocytosis in a manner similar to Salmonella uptake
(43,44). After engulfment, the pathogen is surrounded by a membrane-bound
vacuole within the host. Unlike Salmonella, however, Shigella rapidly lyses
the surrounding vacuole and is released into the cytosol, where it grows
and divides (45). Once the microbe has escaped from the vacuole, it quickly
becomes coated with filamentous actin and ultimately forms an actin tail at
one pole of the bacterium (Figure 3A) (46,47). This actin polymerization
propels the bacterium through the cytoplasm at speeds reaching 0.4 m/sec
(48). When the pathogen reaches the plasma membrane of the cell, it forms a
long protrusion into the neighboring cell, which subsequently internalizes
the microbe (49). The bacterium again breaks out of the vacuole, thereby
starting a new cycle of infection in a new host cell (50). This process
allows Shigella to move from cell to cell without ever contacting the
extracellular milieu.
					[fig]
 Figure 3. A. Immunofluorescence micrograph showing Shigella (red)
 propelling itself through the cytoplasm by polymerizing actin (green)
 (Philippe Sansonetti, Institut Pasteur, reprinted with permission from
 Trends in Microbiology, 1996). B. Shigella-mediated cytoskeletal
 rearrangements. The outer membrane protein, IcsA, is sufficient to drive
 actin-based motility of Shigella. IcsA directly binds two proteins,
 vinculin and neural-Wiskott-Aldrich Syndrome protein (N-WASP). Vinculin
 undergoes proteolysis within the host cell upon Shigella infection,
 producing a 90-kDa fragment that can bind to IcsA and to the
 vasodilator-stimulated phosphoprotein (VASP). VASP in turn can recruit
 profilin to the bacterial surface, which can provide actin for tail
 construction. N-WASP binding of IcsA can also recruit profilin to the
 bacterial surface and may be another means of obtaining monomeric actin
 for tail formation and subsequent bacterial motility.
Bacterial Factors Involved in Shigella Motility
Analysis of mutants deficient in intracellular motility and cell-to-cell
spread has identified a bacterial gene, icsA, necessary for Shigella
locomotion (46,51,52). IcsA (also called VirG) is a 120-kDa outer membrane
protein that hydrolyzes ATP and is localized to one pole of the bacterium,
at the junction between the microbe and the actin tail (Figure 3B) (53).
IcsA expression on the surface of Shigella is sufficient to direct actin-
based motility (54,55). In fact, E. coli expressing IcsA can synthesize
actin tails in cytoplasmic extracts (54,55).
During infection, IcsA is also detected as a 95-kDa amino-terminal fragment
of the 120-kDa full-length protein (53). This proteolytic cleavage of IcsA
is due to a bacterial protease, SopA (IcsP) (56,57). Cleavage is required
for polarized distribution of IcsA on the bacterial surface and for proper
actin-based motility of Shigella in infected cells (56-58).
Host Factors Involved in Shigella Motility
IcsA expression on the Shigella surface promotes rapid accumulation of
actin around the bacterium. Following bacterial division and IcsA
polarization, actin tails begin to form on one end of the bacterium.
Several host cytoskeletal proteins are involved in tail formation,
including alpha-actinin (48), filamin (59), fimbrin (59), vasodilator-
stimulated phosphoprotein (VASP) (60), vinculin (49,61), and
neural-Wiskott-Aldrich syndrome protein (N-WASP) (63). Of these proteins,
only vinculin and N-WASP are able to directly bind IcsA (61,62).
Shigella infection results in the cleavage of intact vinculin (120 kDa) to
produce a 90-kDa fragment (63). This proteolysis unmasks an actin-based
motility 1 site on vinculin, which contains a polyproline region capable of
binding VASP. VASP recruitment to the bacterial surface in turn allows the
recruitment of other cytoskeletal proteins, such as actin and profilin, and
forms the basis of an actin-based motor for Shigella movement.
Recently, N-WASP was shown to be required for Shigella motility (62); like
vinculin, it can bind IcsA directly. It is possible that N-WASP, in
addition to VASP, can recruit profilin and actin to the surface of
Shigella, thereby mediating actin polymerization. Furthermore, N-WASP
contains an actin depolymerization factor/cofilin homologous region, which
could be used for severing actin filaments at the pointed ends and
increasing the monomeric actin concentration. The precise mechanisms
involved in Shigella-driven actin polymerization, however, are unclear.
Conclusions
Bacterial pathogens have evolved several mechanisms to hijack host-cell
signaling machinery and disrupt the cytoskeleton. EPEC mediates its effects
on the host cell from the cellular surface. It secretes its own receptor,
Tir, into the host and then binds intimately to it by its outer membrane
protein, intimin. Tir-intimin binding results in a dramatic reorganization
of the cytoskeleton to form the pedestal upon which EPEC resides.
Salmonella, on the other hand, actively invades intestinal epithelial cells
by inducing membrane ruffling and macropinocytosis. Invasion is dependent
on the secretion of virulence proteins, including SptP and SopE, into the
host cell, and mediates its effects on the host from within a
membrane-bound vesicle. Shigella is also an invasive pathogen but lyses the
phagocytic vacuole and initiates intracellular actin-based locomotion to
spread from cell to cell in the cytoplasm. This motility is dependent on
the bacterial outer membrane protein IcsA, which recruits several
actin-associated proteins to the bacterial surface. Despite the outward
differences between each mode of pathogenesis, EPEC, Salmonella, and
Shigella have effectively managed to subvert the host cytoskeleton for
their own purposes and cause substantial diarrheal disease.
------------------------------------------------------------------------
Acknowledgments
      We thank Ursula Heczko for providing the T.E.M. of rabbit EPEC
pedestals.
      This work was supported by Natural Sciences and Engineering Research
Council of Canada postgraduate scholarships to D.L.G. and D.K. and
operating grants from the Medical Research Council of Canada and a Howard
Hughes International Scholar award to B.B.F.
      Danika Goosney is a Ph.D. candidate in Dr. B. Brett Finlay's
laboratory in the Department of Microbiology and Immunology and the
Biotechnology Laboratory at the University of British Columbia. She is
currently investigating EPEC-induced cytoskeletal rearrangements in
cultured epithelial cells.
      Address for correspondence: B. Brett Finlay, Biotechnology Laboratory
and the Departments of Microbiology and Immunology and Biochemistry and
Molecular Biology, University of British Columbia, Vancouver, British
Columbia, Canada V6T 1Z3; fax: 604-822-9830; e-mail: bfinlay@unixg.ubc.ca.
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______________________________________________________________________________
Synopses
Bacterial Toxins: Friends or Foes?
Clare K. Schmitt, Karen C. Meysick, and Alison D. O'Brien
Uniformed Services University of the Health Sciences, Bethesda, Maryland,
USA
---------------------------------------------------------------------------
      Many emerging and reemerging bacterial pathogens synthesize
      toxins that serve as primary virulence factors. We highlight
      seven bacterial toxins produced by well-established or newly
      emergent pathogenic microbes. These toxins, which affect
      eukaryotic cells by a variety of means, include Staphylococcus
      aureus alpha-toxin, Shiga toxin, cytotoxic necrotizing
	factor type 1, Escherichia coli heat-stable toxin, botulinum
	and tetanus neurotoxins, and S. aureus toxic-shock syndrome
	toxin. For each, we discuss the information available	on
	its synthesis and structure, mode of action, and contribution
	to virulence. We also review the role certain toxins have
	played in unraveling signal pathways in eukaryotic cells
	and summarize the beneficial uses of toxins and toxoids.
      Our intent is to illustrate the importance of the analysis of
      bacterial toxins to both basic and applied sciences.
Since diphtheria toxin was isolated by Roux and Yersin in 1888 (1),
microbial toxins have been recognized as the primary virulence factor(s)
for a variety of pathogenic bacteria. Bacterial toxins have been defined as
"soluble substances that alter the normal metabolism of host cells with
deleterious effects on the host" (2). Indeed, the major symptoms associated
with disease caused by Corynebacterium diphtheriae (diphtheria), Bordetella
pertussis (whooping cough), Vibrio cholerae (cholera), Bacillus anthracis
(anthrax), Clostridium botulinum (botulism), Clostridium tetani (tetanus),
and enterohemorrhagic Escherichia coli (bloody diarrhea and hemolytic
uremic syndrome) are all related to the activities of the toxins produced
by these organisms. With the recognition of the central role of toxin in
these and other diseases has come the application of inactive toxins
(toxoids) as vaccines. Such toxoid vaccines have had an important positive
impact on public health.
In this review, we provide a summary overview (Table) of a variety of
bacterial toxins categorized according to mode of action: damaging cell
membranes, inhibiting protein synthesis, activating second messenger
pathways, inhibiting the release of neurotransmitters, or activating the
host immune response. We also describe in detail seven toxins:
Staphylococcus aureus alpha-toxin, Shiga toxin (Stx),
cytotoxic necrotizing factor type 1 (CNF1), E. coli heat-stable toxin (ST),
botulinum and tetanus neurotoxins, and toxic-shock syndrome toxin (TSST)
produced by S. aureus. We emphasize these toxins because they are produced
by emerging (Stx of enterohemorrhagic E. coli) or reemerging (alpha-toxin
of multidrug-resistant S. aureus) pathogens or illustrate different
structures or modes of action (ST, CNF1, neurotoxins, and TSST).
 Table. Characteristics of bacterial toxins(sup a)
 ---------------------------------------------------------------------------------------------
                                                                                     Toxin
                                                                                  implicated
 Organism/toxin               Mode of action       Target             Disease  in disease(sup b)
 ---------------------------------------------------------------------------------------------
 Damage membranes
    Aeromonas                 Pore-former          Glycophorin        Diarrhea        (yes)
    hydrophila/aerolysin
    Clostridium               Pore-former          Cholesterol        Gas               ?
    perfringens/                                                      gangrene(supc)
    perfringolysin O
    Escherichia coli/         Pore-former          Plasma membrane    UTIs            (yes)
    Listeria monocytogenes/   Pore-former          Cholesterol        Foodborne       (yes)
    listeriolysin O                                                   systemic
                                                                      illness,
                                                                      meningitis
    Staphyloccocus aureus/    Pore-former          Plasma membrane    Abcesses(sup c)  (yes)
      alpha-toxin
    Streptococcus             Pore-former          Cholesterol        Pneumonia(sup c) (yes)
     pneumoniae/
     pneumolysin
    Streptococcus pyogenes/   Pore-former          Cholesterol        Strep            ?
    streptolysin O                                                    throat Sf(sup c)
  Inhibit protein synthesis
    Corynebacterium           ADP-                 Elongation factor  Diphtheria      yes
     diphtheriae/              ribosyltransferase   2
     diphtheria toxin
    E. coli/Shigella          N-glycosidase        28S rRNA           HC and HUS      yes
     dysenteriae/
     Shiga toxins
    Pseudomonas               ADP-                 Elongation factor  Pneumonia(sup c)(yes)
    aeruginosa/               ribosyltransferase   2
    exotoxin A
 Activate second messenger pathways
    E.coli
    CNF                       Deamidase            Rho G-proteins     UTIs            ?
    LT                        ADP-                 G-proteins         Diarrhea       yes
                              ribosyltransferase
    ST(sup d)                 Stimulates guanylate guanylate cyclase  Diarrhea       yes
                               cyclase              receptor
    CLDT(sup d)			G2 block             Unknown            Diarrhea      (yes)
    EAST                      ST-like?             Unknown            Diarrhea        ?
    Bacillus anthracis/       Adenylate cyclase    ATP                Anthrax        yes
    edema factor
    Bordetella pertussis/
      dermonecrotic toxin     Deamidase            Rho G-proteins     Rhinitis      (yes)
      pertussis toxin         ADP-                 G-protein(s)       Pertussis      yes
                              ribosyltransferase
    Clostridium botulinum/    ADP-                 Monomeric G-actin  Botulism        ?
    C2 toxin                  ribosyltransferase
    C. botulinum/C3 toxin     ADP-                 Rho G-protein      Botulism        ?
                              ribosyltransferase
    Clostridium difficile/
    toxin A                   Glucosyltransferase  Rho G-protein(s)   Diarrhea/PC   (yes)
    toxin B                   Glucosyltransferase  Rho G-protein(s)   Diarrhea/PC     ?
    Vibrio cholerae/cholera   ADP-                 G-protein(s)       Cholera        yes
    toxin                     ribosyltransferase
 Activate immune response
    S. aureus/
       enterotoxins           Superantigen         TCR and MHC II     Food           yes
                                                                      poisoning(sup c)
       exfoliative toxins     Superantigen (and    TCR and MHC II     SSS(sup c)     yes
                              serine protease?)
       toxic-shock toxin      Superantigen         TCR and MHC II     TSS(sup c)     yes
    S. pyogenes/pyrogenic     Superantigens        TCR and MHC II     SF/TSS(sup c)  yes
    exotoxins
 Protease
    B. anthracis/lethal factorMetalloprotease      MAPKK1/MAPKK2      Anthrax        yes
    C. botulinum/neurotoxins  Zinc-metalloprotease VAMP/synaptobrevin Botulism       yes
      A-G                                            SNAP-25 syntaxin
    Clostridium tetani/       Zinc-metalloprotease VAMP/synaptobrevin Tetanus         yes
    tetanus toxin
 ---------------------------------------------------------------------------------------------
 (sup a)Abbreviations: CNF, cytotoxic necrotizing factor; LT, heat-labile toxin; ST, heat-stable
 toxin; CLDT, cytolethal distending toxin; EAST, enteroaggregative E. coli heat-stable toxin;
 TCR, T-cell receptor; MHC II, major histocompatibility complex class II; MAPKK,
 mitogen-activated protein kinase kinase; VAMP, vesicle-associated membrane protein; SNAP-25,
 synaptosomal-associated protein; UTI, urinary tract infection; HC, hemorrhagic colitis; HUS,
 hemolytic uremic syndrome; PC, antibiotic-associated pseudomembranous colitis; SSS, scalded
 skin syndrome; SF, scarlet fever; TSS, toxic-shock syndrome.
 (sup b)Yes, strong causal relationship between toxin and disease; (yes), role in pathogenesis
 has been shown in animal model or appropriate cell culture; ?, unknown.
 (sup c)Other diseases are also associated with the organism.
 (sup d)Toxin is also produced by other genera of bacteria
When It Rains, It Pores
Many bacterial exotoxins have the capacity to damage the extracellular
matrix or the plasma membrane of eukaryotic cells. The damage not only may
result in the direct lysis of cells but also can facilitate bacterial
spread through tissues. Toxins that mediate this cellular damage do so by
either enzymatic hydrolysis or pore formation. Bacterial hyaluronidases,
collagenases, and phospholipases have the capacity to degrade cellular
membranes or matrices. Specific examples of these types of toxins include
the alpha-toxin of Clostridium perfringens, which has
phospholipase C activity; Streptococcus pyogenes streptokinase, which can
hydrolyze plasminogen to plasmin and dissolve clots; and the clostridial
collagenases (3-5). Pore-forming toxins, as the name suggests, disrupt the
selective influx and efflux of ions across the plasma membrane by inserting
a transmembrane pore. This group of toxins includes the RTX (repeats in
toxin) toxins from gram-negative bacteria, streptolysin O produced by S.
pyogenes, and the S. aureus alpha-toxin (described below).
S. aureus alpha-toxin can be considered the prototype of
oligomerizing pore-forming cytotoxins. The alpha-toxin
gene resides as a single copy on the chromosome of most pathogenic S.
aureus strains, and its expression is environmentally regulated at the
transcriptional level by the staphylococcal accessory gene regulator (agr)
locus (6,7). The alpha-toxin is synthesized as a 319 amino
acid precursor molecule that contains an N-terminal signal sequence of 26
amino acids. The secreted mature toxin, or protomer, is a hydrophilic
molecule that lacks cysteine residues and has a molecular mass of
approximately 33 kDa (6-8). Recently, the crystallographic structure of the
fully assembled alpha-toxin pore was solved (9). On the
plasma membrane, seven toxin protomers assemble to form a 232 kDa
mushroom-shaped heptamer comprising three distinct domains (Figure 1A)
(9,10). The cap and rim domains of the alpha-toxin
heptamer are situated at the surface of the plasma membrane, while the stem
domain serves as the transmembrane channel.
 		[fig]		    	    Figure 1. Diagrammatic representation of
           				    the mode of action of several bacterial
 					    toxins. A. Damage to cellular membranes
                                  by Staphylococcus aureus
                                  alpha-toxin. After
 binding and oligomerization, the stem of the mushroom-shaped
 alpha-toxin heptamer inserts into the target cell and
 disrupts membrane permeability as depicted by the influx and efflux of
 ions represented by red and green circles. B. Inhibition of protein
 synthesis by Shiga toxins (Stx). Holotoxin, which consists of an
 enzymatically active (A) subunit and five binding (B) subunits, enters
 cells through the globotriasylceramide (Gb3) receptor. The N-glycosidase
 activity of the A subunit then cleaves an adenosine residue from 28S
 ribosomal RNA, which halts protein synthesis. C. Examples of bacterial
 toxins that activate secondary messenger pathways. Binding of the
 heat-stable enterotoxins (ST) to a guanylate cyclase receptor results in
 an increase in cyclic GMP (cGMP) that adversely effects electrolyte flux.
 By ADP-ribosylation or glucosylation respectively, the C3 exoenzyme (C3)
 of Clostridium botulinum and the Clostridium difficile toxins A and B (CdA
 & CdB) inactivate the small Rho GTP-binding proteins. Cytotoxic
 necrotizing factor (CNF) of E. coli and the dermonecrotic toxin (DNT) of
 Bordetella species activate Rho by deamidation.
Alpha-toxin is cytolytic to a variety of cell types, including human
monocytes, lymphocytes, erythrocytes, platelets, and endothelial cells
(6,8). For alpha-toxin to damage cellular membranes, three
sequential events are required. Toxin protomers must first bind to target
membranes by either unidentified high-affinity receptors or through
nonspecific absorption to substances such as phosphotidylcholine or
cholesterol on the lipid bilayer (6-8). Second, membrane-bound protomers
must oligomerize into a nonlytic prepore heptamer complex. Third, the
heptamer must undergo a series of conformational changes that create the
stem domain of the toxin, which is then inserted into the membrane (9,10).
The alpha-toxin pore allows the influx and efflux of small
molecules and ions that eventually lead to the swelling and death of
nucleated cells and the osmotic lysis of erythrocytes. Pore formation has
also been shown to trigger secondary events that could promote development
of pathologic sequelae. These events include endonuclease activation,
increased platelet exocytosis, release of cytokines and inflammatory
mediators, and production of eicosanoids (6,8). Several animal models have
demonstrated that alpha-toxin is required for S. aureus
virulence in these systems (6,8); however, the precise role of
alpha-toxin in staphylococcal diseases in humans remains
unclear.
Stop, in the Name of Toxin
A second class of toxins intoxicates target cells by inhibiting protein
synthesis. Substrates for toxins in this group are elongation factors and
ribosomal RNA. Diphtheria toxin and Pseudomonas exotoxin A act by
ADP-ribosylating elongation factor 2 (EF2) (11,12). The modified EF2 is no
longer able to function in protein synthesis. Stxs, also called verotoxins,
are produced by Shigella dysenteriae serotype 1 and the emerging pathogens
designated Stx-producing E. coli (STEC). Stxs inactivate ribosomal RNA (by
a mechanism described below) so that the affected ribosome can no longer
interact with elongation factors (13,14). The inhibition of protein
synthesis by this group of toxins ultimately results in death of the target
cell.
Stxs are potent cytotoxins that can be divided into two antigenically
distinct groups that share 50% to 60% homology: Stx/Stx1 and Stx2 (15-17).
Stx and Stx1 are elaborated by S. dysenteriae serotype 1 and E. coli,
respectively, and differ at only one amino acid. Stx2-type toxins have been
found only in E. coli isolates and are quite diverse. While Stx2 is
considered the prototype of this group, variants have been found that
differ antigenically, in receptor specificity and in activation by
intestinal mucus. Some of these attributes are the result of only one or
two nucleotide differences in the toxin genes.
The stx of S. dysenteriae is invariably chromosomally located. The genes
that encode Stx1 and Stx2 are carried chromosomally or by lysogenic
bacteriophages. The genes that code for the A and B subunits of Stxs, stxA
and stxB, are organized within an operon. The operator region of Stx/Stx1
(but not Stx2) contains a consensus fur box that is responsible for the
iron-regulation of Stx and Stx1 production. Neither iron nor any other
environmental factors examined affect the expression of Stx2. However,
intestinal mucus enhances the activity of some Stx2 variants (18). The
Stxs, which carry typical N-terminal leader sequences, are not actively
secreted from the bacterial cell and are thought to be released into the
milieu during cell lysis.
Stxs display an AB-toxin structure; an enzymatically active A subunit is
noncovalently associated with a binding, or B, component. The crystal
structures of the Stx1 B pentamer (19) and the Stx holotoxin have been
solved (20) (Figure 2). Other toxins that share this AB structure are the
E. coli heat-labile toxin (21), cholera toxin, and pertussis toxin (22)
(Figure 2). The molecular masses of mature Stx A and B monomeric subunits
are approximately 35 kDa and 7.5 kDa, respectively, although holotoxin
contains five B subunit molecules. The B subunit pentamer directs the
binding of the holotoxin to sensitive eukaryotic cells via specific
glycolipid receptors. Once internalized, the A polypeptide is cleaved into
an enzymatically active A(sub 1) portion and an A(sub 2) portion; these fragments
remain associated through a disulfide bond. The A(sub 2) portion serves to link
the A(sub 1) fragment and the B pentamer.
The enzymatic A subunit acts as a
specific N-glycosidase to cleave a
single adenine residue from 28S				[fig]
ribosomal RNA (13,14). This             Figure 2. Ribbon crystal structures
depurination ultimately results in the  of Shigella dysenteriae Shiga toxin
inhibition of protein synthesis within  (20), Escherichia coli heat-labile
intoxicated cells (Figure 1B).          toxin I (LT-I) (21), and pertussis
Prokaryotic ribosomes are as sensitive  toxin (22). The Shiga toxin figure
to the N-glycosidase activity of Stx as was contributed by Marie Frasier.
eukaryotic ribosomes (23).              The LT-I and pertussis figures were
                                        contributed by Ethan Merritt. The
STEC are considered emerging pathogens  figures were drawn in MOLSCRIPT
(24) because they were first described  (75).
less than 20 years ago, during a 1983
outbreak of hemorrhagic colitis
associated with undercooked hamburger (25,26). STEC O157:H7 causes
approximately 20,000 cases of hemorrhagic colitis each year in the United
States (27). Approximately 1,000 cases of the life-threatening sequelae
hemolytic uremic syndrome and approximately 100 deaths are also attributed
to E. coli O157:H7 annually in the United States (27).
Don't Shoot the Messenger
Bacterial toxins can also target and alter the function of a variety of
cellular proteins without directly killing the intoxicated cell. Toxin
activation or modification of secondary messengers can cause dramatic
alterations to signal transduction pathways critical in maintaining a
variety of cellular functions. To demonstrate the diversity among the
toxins that belong to this category, we will describe CNF type 1 and the
heat-stable enterotoxins.
Cytotoxic Necrotizing Factor (CNF)
CNF types 1 and 2 (CNF1/2) from E. coli belong to a group of bacterial
toxins that modify Rho, a subfamily of small GTP-binding proteins that are
regulators of the actin cytoskeleton (28,29). Most members of this toxin
family, which includes the large clostridial cytotoxins and the C3
exoenzyme of C. botulinum, inactivate Rho (29). CNF1, CNF2, and the
dermonecrotic toxins from Bordetella species form a unique subset in this
family, since these toxins have the capacity to activate Rho (Figure 1C)
(29-32). CNF1 and CNF2 share 99% amino acid similarity; however, we will
discuss only CNF1 in detail because of its association with extraintestinal
E. coli infections in humans, most notably urinary tract infections.
The gene for CNF1 is chromosomally encoded and resides on a pathogenicity
island in uropathogenic E. coli (33,34). The toxin is synthesized as a
hydrophilic polypeptide of approximately 115 kDa that remains primarily
cytoplasmic because of the lack of a signal sequence (33). Recent structure
and function analysis of CNF1 indicates that the toxin has distinct binding
and enyzmatic domains (35). The N-terminal half of CNF1, which includes two
potential transmembrane domains, contains the cellular binding domain. This
region of the molecule shows amino acid similarity to the Pasteurella
multicoda toxin, a potent mitogen thought to be the etiologic agent of
progressive atrophic rhinitis in pigs (33,35). The C-terminal portion of
CNF1 represents the toxin's enzymatic domain and shows homology with
dermonecrotic toxins in a 100-amino acid stretch that may represent the
active site of the toxin (33,35).
Eukaryotic cells intoxicated with CNF1 exhibit membrane ruffling; the
formation of focal adhesions and actin stress fibers; and DNA replication
in the absence of cell division, a phenomenon that results in enlarged
multinucleated cells (Figure 3). The drastic changes apparent in
CNF1-treated cells are a result of the toxin's capacity to modify Rho
(29,30,32). This modification has recently been identified as a deamidation
of the glutamine residue at position 63 of Rho to a glutamic acid. This
amino acid change produces a dominant active Rho protein unable to
hydrolyze bound GTP (30,32). In vivo, CNF1 causes necrosis in rabbit skin
following intradermal injection and persistent inflammation in a mouse
footpad assay (36). Epidemiologic data support the role of CNF1 as a
virulence factor in human extraintestinal infections, although direct proof
of the toxin's role in disease remains to be determined (29,37).
					    Heat-Stable Toxin (ST)
 				     	    Two families of diarrheagenic STs have
					    been described: STa (or STI) and STb (or
        [fig]                     STII). Distinct STas are produced by a
 Figure 3. The effect of          variety of enteric pathogenic organisms:
 cytotoxic necrotizing factor     enterotoxinogenic E. coli (ETEC) (the
 type 1 (CNF1) on eukaryotic      focus of this section), V. cholerae,
 cells. A. HEp-2 cells,           Vibrio mimicus, Yersinia enterocolitica,
 magnification 10X. B. HEp-2      Citrobacter freundii, and Klebsiella.
 cells intoxicated with CNF1,
 magnification 10X.               Strains of ETEC associated with human
                                  disease may produce either STa,
heat-labile toxin I, or both. STas from ETEC isolates are related but
distinct toxins (38). STh is produced by strains of human origin, while STp
is found predominantly in porcine strains. The STa genes (estA) of ETEC are
encoded within a transposable element and have been found on a variety of
replicons (39,40). STa is translated as a precursor molecule of 72 amino
acids and undergoes two cleavage events before the secretion of the mature
form into the culture supernatant. Mature STs are small peptides that range
from 17 to 53 amino acids. STh and STp contain 19 and 18 residues,
respectively. STas share a conserved C-terminal region of 13 amino acids
essential for toxicity and the heat-stable nature of the toxin. Six
cysteine residues are present within this domain, and the three disulphide
bonds formed between the cysteine residues are necessary for toxicity of
the molecule. Binding of STa to its cellular receptor results in the
stimulation of membrane-bound guanylate cyclase, which in turn leads to an
increase in intracellular cyclic GMP (Figure 1C) (41). This increase in
cyclic GMP affects electrolyte flux in the bowel; sodium absorption is
inhibited and chloride secretion is stimulated. These ion flux changes
result in the secretory diarrhea characteristic of ETEC infection. ETEC
cause traveler's diarrhea and are a major source of childhood diarrhea in
many parts of the world.
The Nerve of Some Toxins
The C. botulinum neurotoxins (BoNTs, serotypes A-G) and the C. tetani
tetanus neurotoxin (TeNT) constitute another category of bacterial toxins
on the basis of similarities in structure, enzymatic activity, and the
targeting to cells of the nervous system. BoNTs are most commonly
associated with infant and foodborne botulism and exist in nature as large
complexes comprised of the neurotoxin and one or more associated proteins
believed to provide protection and stability to the toxin molecule while in
the gut (42,43). TeNT, which is synthesized from vegetative C. tetani in
wounds, does not appear to form complexes with any other protein components
(42,43).
The BoNTs and TeNT are either plasmid encoded (TeNT, BoNTs/A, G, and
possibly B) or bacteriophage encoded (BoNTs/C, D, E, F), and the
neurotoxins are synthesized as inactive polypeptides of 150 kDa (44). BoNTs
and TeNT are released from lysed bacterial cells and then activated by the
proteolytic cleavage of an exposed loop in the neurotoxin polypeptide (45).
Each active neurotoxin molecule consists of a heavy (100 kDa) and light
chain (50 kDa) linked by a single interchain disulphide bond (42,45). The
heavy chains of both the BoNTs and TeNT contain two domains: a region
necessary for toxin translocation located in the N-terminal half of the
molecule, and a cell-binding domain located within the C-terminus of the
heavy chain (45,46). The light chains of both the BoNTs and TeNT contain
zinc-binding motifs required for the zinc-dependent protease activities of
the molecules (45,46).
The cellular targets of the BoNTs and TeNT are a group of proteins required
for docking and fusion of synaptic vesicles to presynaptic plasma membranes
and therefore essential for the release of neurotransmitters. The BoNTs
bind to receptors on the presynaptic membrane of motor neurons associated
with the peripheral nervous system. Proteolysis of target proteins in these
neurons inhibits the release of acetylcholine, thereby preventing muscle
contraction (47,48). BoNTs/B, D, F, and G cleave the vesicle-associated
membrane protein and synaptobrevin, BoNT/A and E target the
synaptosomal-associated protein SNAP-25, and BoNT/C hydrolyzes syntaxin and
SNAP-25 (42,45,46). TeNT affects the central nervous system and does so by
entering two types of neurons. TeNT initially binds to receptors on the
presynaptic membrane of motor neurons but then migrates by retrograde
vesicular transport to the spinal cord, where the neurotoxin can enter
inhibitory interneurons (45,47). Cleavage of the vesicle-associated
membrane protein and synaptobrevin in these neurons disrupts the release of
glycine and gamma-amino-butyric acid, which, in turn, induces muscle
contraction (47,48). The contrasting clinical manifestations of BoNT or
TeNT intoxication (flaccid and spastic paralysis, respectively) are the
direct result of the specific neurons affected and the type of
neurotransmitters blocked (45-47).
Bacterial Superantigens: Too Much of a Good Thing
Several bacterial toxins can act directly on the T cells and
antigen-presenting cells of the immune system. Impairment of the
immunologic functions of these cells by toxin can lead to human disease.
One large family of toxins in this category are the pyrogenic toxin
superantigens (PTSAgs), whose hallmark biological activities include potent
stimulation of the immune cell system, pyrogenicity, and enhancement of
endotoxin shock (49-51). These stable, secreted toxins of 22 kDa to 30 kDa
include staphylococcal enterotoxins serotypes A-E, G, and H; group A
streptococcal pyrogenic exotoxins serotypes A-C and F; group A
streptococcal superantigen; and staphylococcal TSST-1, which we discuss
below.
All PTSAgs share common biological activities, but TSST-1 is the most
divergent member of the toxin family, with less than 30% amino acid
homology to other family members (52-54). TSST-1 is chromosomally encoded,
and the tst gene is located in a variable genetic element in S. aureus
(49,52,55). The toxin is synthesized as a precursor molecule of 234
residues with the first 40 amino acids acting as a signal sequence that is
cleaved to generate the mature 22 kDa toxin (49). Expression of TSST-1
depends on oxygen, temperature, pH and glucose levels, and is regulated by
the S. aureus agr locus (49,51). On the basis of crystallographic analysis,
TSST-1 appears structurally similar to several other PTSAgs in that the
toxin consists of two distinct domains; however, unlike other family
members, TSST-1 does not require a zinc cofactor (51-54). Domain A of
TSST-1 (amino acid residues 1-17 and 90-194) exists as a -grasp motif, and
domain B consists of a five-stranded -barrel motif that forms an
oligosaccharide/oligonucleotide binding fold.
In general, the potent immunostimulatory properties of PTSAgs are a direct
result of toxin binding to distinct regions outside the peptide binding
cleft of the major histocompatibility class II molecules (expressed on the
surface of antigen-presenting cells) and to specific V elements on the
T-cell receptor. In particular, the domain B of TSST-1 binds primarily to
the alpha-chain of human leukocyte antigen-DR1 molecules, while domain A
specifically binds to human T-cell receptor V2 elements (51-53,56).
Binding of TSST-1 to V2 T-cell receptor elements results in a massive
proliferation of up to 20% of peripheral T cells, an event that drastically
skews the T-cell V repertoire (53,56). T cells that undergo this expansion
can subsequently exist in a state of anergy or undergo apoptosis (56).
Concomitant to T-cell proliferation is a massive release of both lymphocyte
(interleukin [IL]-2, tumor necrosis factor , gamma interferon)-derived and
monocyte (IL-1, IL-6, tumor necrosis factor alpha-derived cytokines (51,56).
These cytokines serve as mediators of the hypotension, high fever, and
diffuse erythematous rash that are characteristic of toxic-shock syndrome.
Long established as a key substance in causing staphylococcal toxic-shock
syndrome, TSST-1 has more recently been linked with Kawasaki syndrome, a
leading cause of acquired heart disease in children in the United States
(50,54).
Dr. Jekyll or Mr. Hyde?
Some of these powerful disease-causing toxins have been exploited to
further basic knowledge of cell biology or for medical purposes. For
example, cholera toxin and the related labile-toxin of E. coli, as well as
B. pertussis toxin, have been used as biologic tools to understand the
mechanism of adenylate cyclase activation and the role of cyclic AMP as a
second messenger in the eukaryotic cell (57-59). Derivatives of some of
these toxins, cholera toxin and labile toxin, have also been incorporated
into human vaccines because of the adjuvant properties of these molecules
(60,61).
Similarly, the activities of several potent cytotoxins have been harnessed
as potential therapies for certain cancers. Such toxins can either be used
directly in treatment or as components of immunotoxins (62-64). For
example, Stx binds to the cell surface glycolipid CD77, which is expressed
by B cells in certain B-cell lymphomas (65,66). This finding led to studies
that showed that Stx can purge murine (and potentially human) bone marrow
of malignant CD77+ B cells before an autologous bone marrow transplant
(67). Other toxins that inhibit protein synthesis, such as diphtheria
toxin, Pseudomonas exotoxin A, or the plant toxin ricin, are frequently
engineered as the cell-killing component of immunotoxins. These "magic
bullets," hybrids of the enzymatically active portion of a toxin molecule
and monoclonal antibodies (or a receptor), are in clinical trials for the
treatment of persons with B-cell lymphomas, leukemia, and bone marrow
transplants.
Several clinical applications have also been found for the powerful
botulinum neurotoxin type A (BoNT/A) (46,68). The disorders that respond to
BoNT/A involve muscle hyperactivity. A minuscule amount of purified toxin
injected into specific sites results in paralysis of the target muscle and
ablation of the muscle spasm. Therapy must be continual since the effect of
the toxin usually lasts for no more than several months. The first maladies
treated with BoNT/A were eye movement abnormalities (69). However, the
therapeutic value of BoNT/A has been shown for many other disorders
including cervical and laryngeal dystonia, writer's cramp, hemifacial
spasm, tremors, and tics (46,68). BoNT/A is also used cosmetically to
reduce deep wrinkles caused by the contraction of facial muscles (70).
Another toxic bacterial product with medical applications is streptokinase,
a potent plasminogen activator produced by several pathogenic streptococcal
strains. The proteolytic activity of streptokinase is used to clear blocked
arteries in patients who have heart attacks (71,72).
Vaccinate, Don't Procrastinate
Vaccines directed at the toxic component of bacterial pathogens have proven
quite effective in preventing certain diseases. Most licensed toxoid
vaccines are relatively crude, but effective, preparations. These vaccines
consist of partially purified toxin preparations obtained from culture
supernatants of bacteria such as C. diphtheriae, C. tetani, or B.
anthracis. Formaldehyde treatment is used to detoxify the diphtheria and
tetanus toxins for vaccine formulation. The anthrax vaccine contains the
protective antigen and small amounts of the lethal factor and edema factor
toxins. The current botulinum vaccine is an investigational drug composed
of crude preparations of five botulinum toxoids and is distributed by the
Centers for Disease Control and Prevention to researchers that work with
the toxin or organism. Acellular pertussis vaccines that contain pertussis
toxoid, alone or as one of several components, are as effective as killed
whole-cell vaccines but less reactogenic (73); such vaccines have recently
been approved for use in infants as well as older children.
New vaccines aimed at toxins are in various stages of development: research
and development, preclinical, phase I, phase II, or phase III (74). The
next generation of toxoid vaccines falls into three general categories:
purified toxoids that have been inactivated by chemical or genetic means;
live, attenuated strains of the causative agent that produce a genetically
derived toxoid; or live, attenuated unrelated bacterial vector strains,
such as V. cholerae or Salmonella, that produce the target toxoid. Examples
of each of these approaches and progress in development of specific toxoid
vaccines are described annually in the Jordan Report (74).
Antitoxins raised against diphtheria, tetanus, and botulinum toxoids have
also been used for many years to treat seriously ill patients. Antiserum
specific for the Stx toxins produced by E. coli O157:H7 and other STEC is
under development for the treatment and prevention of hemolytic uremic
syndrome, a life-threatening sequela of these infections.
Summary
Microbial toxins capable of interrupting or hyperstimulating many essential
functions and pathways of eukaryotic cells have evolved along with the
carrier bacterium. Presumably these toxins confer some benefit to the
bacterium, either during a stage of the host-parasite interaction or in
some environmental niche encountered by the bacterium. Certain bacterial
toxins act on the target cell surface to irreparably damage the cell
membrane or alter normal cellular signal transduction. Other toxins exhibit
enzymatic activity once the molecule has gained access to the cytoplasm of
the sensitive cell by endocytosis. Yet other bacterial toxins act by either
turning off or locking on a normal host cell function.
Although detrimental to the susceptible host during an infection, the
activities of several bacterial toxins have been exploited as probes of
eukaryotic cellular pathways and for medicinal applications. Thus, research
on a microbial toxin produced by an established, emerging, or reemerging
pathogen is likely to yield novel information about the role of that toxin
in disease as well as the properties of host cells that are subverted by
the toxin.
---------------------------------------------------------------------------
Acknowledgments
We thank Drs. Ethan Merritt and Marie Frasier for generating and
contributing the figures on toxin crystal structures.
Work in Dr. O'Brien's laboratory is supported by grants from the National
Institutes of Health (AI20148-16, AI33525-5, AI38281-3), the Department of
Agriculture (97-35201-4578), and the Uniformed Services University of the
Health Sciences (R073EQ).
     Dr. Schmitt is a research assistant professor in the Department of
Microbiology and Immunology, Uniformed Services University of the Health
Sciences, Bethesda, Maryland, USA. Research interests include virulence
factors and pathogenesis of disease caused by Shiga toxin-producing E. coli
and regulation of Salmonella virulence.
Address for correspondence: Alison D. O'Brien, Uniformed Services
University of the Health Sciences, Department of Microbiology and
Immunology, 4301 Jones Bridge Road, Bethesda, MD 20814, USA; fax:
301-295-3773; e-mail: aobrien@mxb.usuhs.mil.
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_____________________________________________________________________________
Synopses
Clonal Differences among Erythromycin-Resistant Streptococcus pyogenes in
Spain
Emilio Perez-Trallero,* Jos Mara Marimn,* Milagrosa Montes,* Beatriz
Orden, and Manuela de Pablos
*Universidad del Pas Vasco, San Sebastin, Spain; Centro Especialidades
"Argelles," Madrid, Spain; Hospital Txagorritxu, Vitoria, Spain
---------------------------------------------------------------------------
      The aim of this study was to determine whether the high levels
      of erythromycin resistance in Streptococcus pyogenes found in
      Spain are due to the introduction and spread of one or more
      clones. Phenotypic and genotypic techniques were used to
      characterize all erythromycin-resistant S. pyogenes (ErR)
      isolated in Gipuzkoa, Spain, in the last 10 years and 128 ErR
      isolated in Vitoria and Madrid during 1996. Of 437 ErR, 97% had
      the M phenotype; all 283 of the strains studied had the mefA
      determinant of resistance. After biotyping, T serotyping, emm
      typing, and genotyping, four major clones were detected. Clones
      B (biotype I, type T4, emm4, pulsed-field gel electrophoresis
      [PFGE] II) and D (biotype V, type T8.25, emm75, PFGE IV)
      comprised 78.8% of all ErR. The resistance of S. pyogenes to
      erythromycin was mainly due to an efflux mechanism of
      resistance (M phenotype); few clones were responsible for it.
The Lancefield group A streptococci (Streptococcus pyogenes), major
causative agents of human disease (1), can produce both mild (e.g.,
pharyngitis) and severe (e.g., life-threatening "toxic shock-like
syndrome," necrotizing fasciitis) infections. During the last few years,
erythromycin-resistant S. pyogenes (ErR) has been reported in different
parts of the world (2-4). Two distinct mechanisms of erythromycin
resistance are described among group A streptococci. One consists of
target-site modification by erm methylase (5,6) strains that express the
MLSb phenotype of resistance; the other (recently described) consists of an
active drug efflux that pumps 14- and 15-membered macrolides out of the
cell (7). This novel mechanism of macrolide resistance is encoded by the
gene mefA (8), and strains show the M phenotype. In the last few years,
increased resistance to erythromycin in S. pyogenes has been detected in
Spain (9-11). Therefore, we performed an epidemiologic investigation to
determine the biotypes, serotypes (T-agglutination patterns), emm types,
and pulsed-field gel electrophoresis (PFGE) patterns of chromosomal DNA and
their relationship to macrolide resistance.
The Study
Sources of Bacterial Isolates
From 1988 to 1997, 2,561 nonduplicated isolated strains of S. pyogenes were
collected from throat swabs and extratonsilar samples at the Nuestra Seora
de Arnzazu Hospital and at primary-care centers in two districts of
Gipuzkoa (approximately 300,000 residents). In 1996, two other samples were
collected and included in this study; 33 ErR strains from Vitoria (Hospital
Txagorritxu) and 95 from Madrid (Centro de Especialidades Argelles).
Gipuzkoa Province (San Sebastian is its capital) is located in the
northeastern area of the Basque country of Spain, bordered by the Cantabric
Sea and France to the north; Madrid is located in the center of Spain (415
km from San Sebastian); and Vitoria (in Alava Province) is located in the
north of Spain (110 km from San Sebastian).
Identification, Susceptibility, Typing, and Clone Definition
Group A streptococci were identified by colony morphology, beta-hemolysis
on blood agar, and commercial latex-agglutination techniques (Streptex,
Wellcome, Dartford, UK, or Phadebact, Boule Diagnostics AB, Huddinge,
Sweden). The confirmation of S. pyogenes and biotyping were done with a
commercially available identification system: rapid ID 32 STREP
(BioMerieux, La Balme-les-Grottes, France). Biotyping was performed
according to Bouvet et al. (12).
All S. pyogenes were tested for susceptibility to erythromycin and other
antibiotics by broth microdilution. ErR strains were restudied by agar
dilution and by agar diffusion (erythromycin induction of resistance) to
determine macrolide and lincosamine resistance phenotypes (11).
T-protein types were determined by slide agglutination of trypsin-digested
suspensions of bacteria with rabbit type-specific antiserum (SEVAC,
Prague)(13).
The emm gene type of T-serotyped strains was determined by polymerase chain
reaction (PCR)-enzyme-linked immunosorbent assay (ELISA), as described by
Saunders et al. (14). Capture probes for the emm gene not described by
Saunders et al. were selected from the DNA sequences encoding the
N-terminal hypervariable region of strains of types emm2, emm9, emm48, and
emm75 (GenBank accession nos. X56608, U12002, U11961, and U11993).
PFGE was done (15) with the following modifications. Cells were resuspended
to an optical density (OD)(sub 560nm) = 1.0, and 4 ml of the adjusted suspension
was centrifuged. Pelleted cells were resuspended in 200 l of Pett IV
buffer (10 mM Tris, pH 7.2, 20 mM NaCl, 50 mM EDTA) and mixed with 100 l
of 2% low-melting agarose; incubation of plugs with lysozyme solution (1
mg/ml) was reduced to 3 hours. Slices of plugs were digested at 50C for 16
hours with 30 UI of the enzyme SfiI since it has proven satisfactory in
differentiating DNA fragments of S. pyogenes (15,16). Digested inserts were
electrophoresed by using a CHEF-DR III apparatus (BioRad), along with DNA
size standards (BioRad) under the following conditions: 22 hours with an
initial switch time of 20 seconds, rising on linear ramp to 75 seconds at
6V/cm, with an included angle of 120C. Gels were stained with ethidium
bromide and visualized under UV light with Imagestore 5000 ver.7.12
(Ultra-Violet Products Ltd, Cambridge, England). Similarities among PFGE
patterns were established using the Dice coefficient and Lane-Manager 2.1
(TDI, Madrid, Spain) commercial computer software.
A clone was defined as a group of strains expressing both the same
characteristic phenotype and genotype (PFGE pattern similarity >/= 90%).
Erythromycin Resistance
We studied 2,561 strains of S. pyogenes isolated from 1988 to 1997 in
Gipuzkoa; 309 (12.1%) were resistant to erythromycin. A report of ErR in
Gipuzkoa from 1984 to 1996 (11) showed that until the end of 1990,
erythromycin resistance in S. pyogenes was low (1.2%, 13 of 1,060); after
1990, resistance increased, reaching 34.8% (87 of 250) of all S. pyogenes
isolated in 1995. In 1997, resistance decreased to 13.7% (57 of 417). In
Madrid and Vitoria, resistance to erythromycin in 1996 was 22.4% (126 of
563) and 31.6% (43 of 136), respectively.
Among the ErR strains isolated in Gipuzkoa, two phenotypes of resistance
were found: 8 (2.6%) strains showed the classic MLSb phenotype, while the
other 301 (97.4%) strains expressed the M phenotype, as shown by
susceptibility testing and confirmed by the presence of the mefA gene (11).
Of the 128 ErR isolated in Madrid and Vitoria, 5 (3.9%) strains (all from
Madrid) displayed the MLSb phenotype, while the remaining 123 (96.1%)
showed the M phenotype. The presence of the mefA gene was searched for in
283 ErR with the M phenotype; it was detected in all.
Biotyping, T Serotyping, emm Typing, and PFGE of M-Phenotype ErR
Among the 424 ErR with the M phenotype, only four biotypes (of 10 possible)
were identifiedbiotypes I, II, III, and V. In Gipuzkoa, biotype III was
the only biotype found until 1990; between 1991 and 1997, biotypes I and V
comprised 275 (93.8%) of the 293 resistant strains. Seven T-agglutination
patterns were found in Gipuzkoa, each one correlating with an emm type
except for TB3264 (T1 emm1, T2 emm2, T4 emm4, T8.25 emm75, T12 emm12, and
T28 emm28). TB3264 biotype III was emm2, but TB3264 biotype I was not
typeable with any of the 14 emm types assayed. Until 1990, type T12 emm12
was the only type found. Between 1991 and 1997, type T4 emm4 and T8.25
emm75 comprised 92.2% (270 of 293) of all isolates with the M phenotype of
resistance. In Vitoria and Madrid, T4 emm4 and T8.25 emm75 types were also
the most frequently found.
Fifteen different PFGE patterns were found among the 424 M phenotype ErR;
92% of these strains belonged to four patterns (clones A-D) (Table, Figure
1). Among ErR with the MLS(sub b) phenotype, eight PFGE patterns were found. Each
biotype/T-serotype/emm-type combination corresponded with one PFGE pattern
except on three occasions. Three different PFGE patterns that could be
established among ErR belonged to biotype III/T12/emm12, two PFGE patterns
belonged to I/T4/emm4, and another two patterns belonged to V/T8.25/emm75.
The types of other clones, their annual distribution, and a dendrogram
showing their similarities are given in the Table and Figure 2.
 Table. Annual distribution of clones of M-phenotype erythromycin resistant
 Streptococcus pyogenes
  -----------------------------------------------------------------------------
							Clones(sup a)
                        Total M-                             E-O
 Year                   phenotype  A  B   C    D  (biotype/T type/emm type)
 -----------------------------------------------------------------------------
 Gipuzkoa 1988-1990         8      7
 Gipuzkoa 1991-1994        80         76            1 E (I/T1/emm1)
                                                    1 F (I/T28/emm28)
 Gipuzkoa 1995             83         31  18  29    1 G (I/TB3264/emm nt[sup b])
                                                    1 H (III/TB3264/emm2)
 Gipuzkoa 1996             74         19  23  25    2 H
                                                    1 E
                                                    1 I (I/T4/emm4)
 Gipuzkoa 1997             56      1  4   3   40    2 H
                                                    2 J (V/T8 25/emm75)
                                                    1 K (II/T2/emm2)
                                                    1 L (III/Tnt/emm3)
 Madrid 1996               90         12  2   68    6 M (III/T12/emm12)
                                                    2 N (III/T12/emm12)
 Vitoria 1996              33      1  30       1    1 O (III/Tnt/emm3)
 Total                    424(supc)9 172  46  163 23
 -----------------------------------------------------------------------------
 (sup a)Clone A: biotype III, type T12 emm12, pulsed-field gel electrophoresis
 (PFGE) pattern I; clone B: biotype I, type T4 emm4, PFGE pattern II; clone
 C: biotype I, type T4 emm4, PFGE pattern III; clone D: biotype V, type
 T8.25 emm75, PFGE pattern IV.
 (sup b)nt: nontypeable.
 (sup c)Eleven strains were not typed.
				[fig]
 Figure 1. Pulsed-field gel electrophoresis of SfiI restriction
 fragments of Streptococcus pyogenes DNAs of erythromycin-resistant S.
 pyogenes. Lanes 1-2, MLS (sub b) phenotype  strains; lane 3,
 clone F (biotype I, type T28, emm28); lanes 4-5, clone A
 (biotype III, type T12, emm12); lanes 6-7, clone B (biotype I,
 type T4, emm4); lanes 8-9, clone C (biotype I, type T4, emm4);
 lane 10, clone D (biotype V, type T8.25, emm75); lanes
 11-12, clone E (biotype I, type T1, emm1); lane 13, DNA size standards
 (lambda ladder, 48.5 to 1,018 kb); lanes 14 and 16, clone H (biotype
 III, type TB3264, emm2); and lane 15, clone G (biotype I, type
 TB3264 emm not typeable).
 				[fig]
 Figure 2. A dendrogram showing the genetic relationship of 15 clones of
 M-phenotype erythromycin-resistant Streptococcus pyogenes and two
 erythromycin-sensitive strains (ErS)established from pulsed-field gel
 electrophoresis patterns obtained after SfiI digestion by using the
 Dice coefficient and UPGMA and Lane Manager 2.1 software. Clone A
 (III/T12/emm12[sup a]); Clone B (I/T4/emm4); Clone C (I/T4/emm4);
 Clone D (V/T8,25/emm75); Clone E (I/T1/emm1); Clone F (I/T28/emm28);
 Clone G (I/ TB3264/emm nt[sup b]); Clone H (III/TB3264/emm2); Clone I
 (I/T4/emm4); Clone J (V/T8,25/emm75); Clone K (II/T2/emm2); Clone L (III/T
 nt/ emm nt); Clone M (III/T12/emm12); Clone N (III/T12/emm12); Clone O
 (III/Tnt/emm nt); ErS T12 (III/T12/emm12); ErS T4 (I/T4/emm4).
 (sup a)Biotype/T-serotype/emm type.
 (sup b)nt = nontypeable.
Genetic Relationship of ErR and Erythromycin-Sensitive S. pyogenes
The genetic relationship of ErR and erythromycin-sensitive S. pyogenes
with the same biotype and T serotype was analyzed by PFGE. In a sample of
360 erythromycin-sensitive S. pyogenes, biotypes I and III were
the most frequent (66.7%); T1 and T28 were the most frequent T
serotypes (30.6%). Only 8 (2.2%)erythromycin-sensitive strains of
biotype I, serotype T4 were found. None of the 19 T8.25
erythromycin-sensitive strains found were biotype V (18 biotype II, emm
75 and 1 biotype I, emm nontypeable). Infrequent biotype and
T-serotype combinations among ErR, such as I/T1 and I/T28, were
frequently found among erythromycin-sensitive strains.
Similarities between PFGE patterns of most of the
erythromycin-sensitive and -resistant strains with the same
biotype and T-serotype combination was less than 75%. Exceptions to
this were several III/T12, I/T1, and I/T28 erythromycin-sensitive strains
that had a close similarity (>90%) with ErR of the same biotype and
T-serotype combination.
Four major clones of ErR were detected: clone A (T12, emm12,
biotype III, PFGE I), which was present in Gipuzkoa until 1990;
clone B (T4, emm4, biotype I, PFGE II), which was introduced in 1991;
and clones C (T4, emm4, biotype I, PFGE III) and D (T8,25, emm75,
biotype V, PFGE IV), which were introduced in 1995 and persisted
during 1996 and 1997. In Madrid and Vitoria, 89.4% (110) of the 123
M-phenotype strains isolated in 1996 belonged to clones B and D.
Conclusions
In 1990, a new phenotype of erythromycin resistance (first
designated NR and later M) was found in group A streptococci in
Finland (7,17). ErR with the M phenotype had a
low level of erythromycin resistance (8 mg/l to 16 mg/l) and showed
cross-resistance with the 14- and 15-membered macrolides; however, they
showed the same susceptibility to the 16-membered macrolides and to
clindamycin as the erythromycin-susceptible strains (11). This M phenotype
was prevalent among S. pyogenes in Europe and was the predominant phenotype
of resistance among ErR isolated in Finland (18,19), Sweden (20), Austria
(21), and Spain (9-11). In Italy, a high prevalence of
erythromycin-resistant S. pyogenes was reported, but the prevalence of the
M-phenotype strains among ErR varied by geographic area (22-24). Among the
437 ErR in our study in Spain, 424 (97%) showed the M phenotype; the mefA
gene was detected and studied in 283 of these strains.
The epidemiologic surveillance of S. pyogenes can be done by using
phenotypic or genotypic methods or both (as we did). Biotyping does not
need specialized personnel or equipment, and it is useful, in combination
with serotyping methods, for a first approximation of the epidemiologic
characterization of S. pyogenes. In this study, the most prevalent T
serotypes among ErR were infrequent among erythromycin-sensitive strains
and vice versa. M serotyping is a classic typing method with at least 74
types recognized, but because it is a very specialized method and reagents
are not available commercially, it is restricted to a few reference
centers. A rapid PCR-ELISA to determine the emm gene type was an accessible
alternative to serology for M-antigen typing.
Although the discriminatory power of biotyping and serotyping is considered
poor because different genotypes may express the same phenotypic
characteristics (15,25,26), these tests were of great value in our
epidemiologic surveillance. The biotype and T-serotype combination was able
to discriminate between ErR and erythromycin-sensitive strains and
delimited most clones among ErR.
Genomic typing methods have rarely been used in characterizing the
epidemiology of noninvasive S. pyogenes. Among these methods, restriction
endonuclease analysis of genomic DNA (REA), random amplified polymorphic
DNA (RAPD), ribotyping, PFGE of chromosomal DNA, and DNA sequence analysis
have been used with varying degrees of success (15,16,19,26-28). PFGE
patterns confirmed the results of biotyping and serotyping and further
distinguished among isolates within the same biotype and T-serotype
combination. However, PFGE is a complex methodresults take at least 4 days
to obtainand expensive equipment and specialized personnel are needed.
Although the polyclonal nature of the ErR strains was established in this
study and previously (18), most ErR belonged to only a few clones. In
Finland, 91% of the ErR isolated in 1994 were serotype T4 M4 and 88%
constituted one clone by RAPD and REA (19). In our study, many clones were
detected during the 10-year period. Two of the four main clones comprised
78.8% of ErR. The clonal distribution of ErR in Spain could be due to the
introduction and spread of ErR from other locations or to mutations in S.
pyogenes previously present in our environment. ErR with the M phenotype
already existed in Gipuzkoa before 1990, and serotype T12 was the only one
found. From 1980 to 1988, serotype T12 was predominant among ErR with the M
phenotype in Sweden (20). In Gipuzkoa, the first serotype T4 ErR strain was
isolated in 1991; until the end of 1994, 93.8% of ErR isolated belonged to
the same clone (clone B: biotype I, type T4, emm4, PFGE II). Only three
biotype I serotype T4 erythromycin-sensitive strains were detected before
1991. Clone B probably did not emerge in Gipuzkoa from a mutation of one of
these uncommon sensitive strains. Apart from the strains described in
Finland, Sweden, and Spain, strains with M phenotype were isolated in Great
Britain before 1990 (4,29). Among these British and Finnish strains, type
T4 M4 was frequently isolated (4,18,29). In a 1986 outbreak of 10
associated cases in Somerset, Great Britain, isolates were group A, type M4
and resistant to erythromycin (MIC 8 mg/l) but sensitive to clindamycin
(29). We do not know whether the strains of clone B isolated in Spain are
the same as the ErR type T4 M4 found in Great Britain and Finland before
1991, but it is probable. In Italy, serotypes T4 and T8.25 were found among
M-phenotype strains (22), which suggests that a few clones have spread
across Europe and caused a regional epidemic. No erythromycin-sensitive
strains were detected in Gipuzkoa with the same biotype/T-type/emm type
combination as clone D.
We believe that clones B and D, the most frequent among ErR in Spain, were
of epidemic origin and that clone B probably came from northern or western
Europe.
---------------------------------------------------------------------------
Dr. Perez-Trallero is a clinical microbiologist and infectious disease
consultant. He is head of the Microbiology Department at Complejo
Hospitalario Donostia and assistant professor of Preventive Medicine and
Public Health at the Facultad de Medicina at the Basque Country University.
His research focuses on antimicrobial resistance, epidemiology of
transmissible diseases, and methods for their prevention and control.
Address for correspondence: Emilio Perez-Trallero, Servicio de
Microbiologa, Apartado 477, 20080 San Sebastin, Spain; fax:
34-943-007112; e-mail: labmikro@teleline.es.
References
  1. Bisno A. Streptococcus pyogenes. In: Mandell GL, Bennett JE, Dolin R,
     editors. Principles and practice of infectious diseases. 4th ed. New
     York: Churchill Livingstone; 1995. p. 1786-99.
  2. Holmstrom L, Nyam B, Rosengren M, Wallander S, Ripa T. Outbreaks of
     infection with erythromycin-resistant group A streptococci in child
     day care centers. Scand J Infect Dis 1990;22:179-85.
  3. Stingmore N, Francis GRJ, Toohey M, McGenchie DB. The emergence of
     erythromycin resistance in Streptococcus pyogenes in Fremantle,
     Western Australia. Med J Aust 1989;150:626-31.
  4. Philips G, Parratt D, Orange GV, Harper I, McEwan H, Young N.
     Erythromycin-resistant Streptococcus pyogenes. J Antimicrob Chemother
     1990;25:723-4.
  5. Leclerq R, Courvalin P. Bacterial resistance to macrolide,
     lincosamide, and streptogramin antibiotics by target modification.
     Antimicrob Agents Chemother 1991;35:1267-72.
  6. Sepl H, Skurnik M, Soini H, Roberts MC, Huovinen P. A novel
     erythromycin resistance methylase gene (ermTR) in Streptococcus
     pyogenes. Antimicrob Agents Chemother 1998;42:257-62.
  7. Sutcliffe J, Tait-Kamradt A, Wondrack L. Streptococcus pneumoniae and
     Streptococcus pyogenes resistant to macrolides but sensitive to
     clindamycin: a common resistance pattern mediated by an efflux system.
     Antimicrob Agents Chemother 1996;40:1817-24.
  8. Clancy J, Petitpas J, Dib-Hajj F, Yuan W, Cronan M, Kamath AV, et al.
     Molecular cloning and functional analysis of a novel
     macrolide-resistance determinant, mefA, from Streptococcus pyogenes.
     Mol Microbiol 1996;22:867-79.
  9. Garcia-Bermejo I, Cacho J, Orden B, Alos JI, Gomez-Garces JL.
     Emergence of erythromycin-resistant, clindamycin-susceptible
     Streptococcus pyogenes isolates in Madrid, Spain. Antimicrob Agents
     Chemother 1998;42:989-90.
 10. Orden B, Perez-Trallero E, Montes M, Martnez R. Erythromycin
     resistant Streptococcus pyogenes in Madrid. Pediatr Infect Dis J
     1998;17:470-3.
 11. Perez-Trallero E, Urbieta M, Montes M, Ayestaran I, Marimon JM.
     Emergence of Streptococcus pyogenes resistant to erythromycin in
     Gipuzkoa, Spain. Eur J Clin Microbiol Infect Dis 1998;16:25-31.
 12. Bouvet A, Gelsin P, Kriz-Kuzemenska P, Blanc V, Devine C, Grimont F.
     Restricted association between biotypes and serotypes within Group A
     Streptococci. J Clin Microbiol 1994;32:1312-7.
 13. Johnson DR, Kaplan EL, Sramek J, Bicova R, Havlicek J, Havlickova H,
     et al. Determination of T-protein agglutination patterns. In:
     Laboratory diagnosis of group A streptococcal infections. Geneva:
     World Health Organization; 1996. p. 37-41.
 14. Saunders NA, Hallas G, Gaworzewska ET, Metherell L, Efstratiou A,
     Hookey JV, et al. PCR-enzyme-linked immunosorbent assay and sequencing
     as an alternative to serology for M-antigen typing of Streptococcus
     pyogenes. J Clin Microbiol 1997;35:2689-91.
 15. Single LA, Martin DR. Clonal differences within M-types of the group A
     Streptococcus revealed by pulsed field gel electrophoresis. FEMS
     Microbiol Lett 1992;91:85-90.
 16. Upton M, Carter PE, Morgan M, Edwards GF, Pennington TH. Clonal
     structure of invasive Streptococcus pyogenes in Northern Scotland.
     Epidemiol Infect 1995;115:231-41.
 17. Seppala H, Nissinen A, Yu Q, Huovinen P. Three different phenotypes of
     erythromycin-resistant Streptococcus pyogenes in Finland. J Antimicrob
     Chemother 1993;32:885-91.
 18. Seppala H, Nissinen A, Jarvinen H, Houvinen S, Henriksson T, Herva E,
     et al. Resistance to erythromycin in Group A Streptococci. N Engl J
     Med 1992;326:292-7.
 19. Kataja J, Huovinen P, Muotiala A, Vuopio-Varkila J, Efstratiou A,
     Hallas G, et al. Clonal spread of group A streptococcus with the new
     type of erythromycin resistance. Finnish Study Group for Antimicrobial
     Resistance. J Infect Dis 1998;177:786-9.
 20. Jasir A, Schalen C. Survey of macrolide resistance phenotypes in
     Swedish clinical isolates of Streptococcus pyogenes. J Antimicrob
     Chemother 1998;41:135-7.
 21. Kriebernegg I, Feierl G, Grisold A, Marth E. In vitro susceptibility
     of group A beta-haemolytic streptococci (GABHS) to penicillin,
     erythromycin, clarithromycin and azithromycin in Styria, Austria.
     Zentralbl Bakteriol 1998;287:33-9.
 22. Cornaglia G, Ligozzi M, Mazzariol A, Valentini M, Orefici G, the
     Italian Surveillance Group for Antimicrobial Resistance, et al. Rapid
     increase of resistance to erythromycin and clindamycin in
     Streptococcus pyogenes in Italy, 1993-1995. Emerg Infect Dis
     1996;2:339-42.
 23. Cocuzza C, Blandino G, Mattina R, Nicoletti F, Nicoletti G. Antibiotic
     susceptibility of group A streptococci in 2 Italian cities: Milano and
     Catania. Microb Drug Resist 1997;3:379-84.
 24. Borzani M, De Luca M, Varotto F. A survey of susceptibility to
     erythromycin amongst Streptococcus pyogenes isolates in Italy. J
     Antimicrob Chemother 1997;40:457-8.
 25. Haase AM, Melder A, Mathews JD, Kemp DJ, Adams M. Clonal diversity of
     Streptococcus pyogenes within some M-types revealed by multilocus
     enzyme electrophoresis. Epidemiol Infect 1994;113:455-62.
 26. Upton M, Carter PE, Orange G, Pennington TH. Genetic heterogeneity of
     M type 3 group A streptococci causing severe infections in Tayside,
     Scotland. J Clin Microbiol 1996;34:196-8.
 27. Seppala H, Vuopio-Varkila J, Osterblad M, Jahkola M, Rummukaien M,
     Holm SE, et al. Evaluation of methods for epidemiologic typing of
     group A streptococci. J Infect Dis 1994;169:519-25.
 28. Bert F, Branger C, Lambert-Zechovsky N. Pulsed-field gel
     electrophoresis is more discriminating than multilocus enzyme
     electrophoresis and random amplified polymorphic DNA analysis for
     typing pyogenic streptococci. Current Microbiology 1997;34:226-9.
 29. Scott RJD, Naidoo J, Lightfoot NF, George RC. A community outbreak of
     group A beta-haemolytic streptococci with transferable resistance to
     erythromycin. Epidemiol Infect 1989;102:85-91.
___________________________________________________________________________
Synopses
Air Evacuation under High-Level Biosafety Containment: The Aeromedical
Isolation Team (ft 1)
George W. Christopher and Edward M. Eitzen, Jr.
U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick,
Maryland, USA
---------------------------------------------------------------------------
      Military contingency operations in tropical environments and
      potential use of biological weapons by adversaries may place
      troops at risk for potentially lethal contagious infections
      (e.g., viral hemorrhagic fevers, plague, and zoonotic poxvirus
      infections). Diagnosis and treatment of such infections would
      be expedited by evacuating a limited number of patients to a
      facility with containment laboratories. To safely evacuate such
      patients by military aircraft and minimize the risk for
      transmission to air crews, caregivers, and civilians, the U.S.
      Army Medical Research Institute of Infectious Diseases
      maintains an aeromedical isolation team. This rapid response
      team, which has worldwide airlift capability designed to
      evacuate and manage patients under high-level containment, also
      offers a portable containment laboratory, limited environmental
      decontamination, and specialized consultative expertise. This
      article also examines technical aspects of the team's
      equipment, training, capabilities, and deployments.
Air evacuation of patients with potentially lethal, contagious infections
poses unique challenges and risks to air crews and medical personnel.
Evacuation of such patients is relevant to military contingency operations
because troops may be placed at risk for hemorrhagic fevers and other
infections during deployment to tropical environments or by adversaries'
use of biological warfare agents.
Evacuation of patients to the U.S. Army Medical Research Institute of
Infectious Diseases (USAMRIID) would afford the immediate availability of
biosafety level 4 laboratories (designed for the study of pathogens
requiring maximum biological containment for laboratory safety) and
facilitate rapid diagnosis of diseases due to pathogens posing
extraordinary laboratory safety hazards. Furthermore, USAMRIID has the only
fixed patient-care suite in the world designed for medical care under
maximum biological containment. To safely evacuate a limited number of
patients to the containment-care suite and provide medical care while
minimizing the risk for transmission to air crews, caregivers, and
civilians, USAMRIID maintains an aeromedical isolation team (1-3).
The Aeromedical Isolation Team
The purpose of the isolation team is to safely transport patients with
potentially lethal communicable diseases for which no effective vaccines,
chemoprophylaxis, or specific therapies exist. These would include patients
with an unknown disease pending identification of the pathogen, patients
with viral hemorrhagic fevers (notably those due to filoviruses and
arenaviruses), and those suspected of being affected by a biological attack
(Table 1) (3). Etiologic diagnosis and medical care would be provided at
USAMRIID.
 Table 1. Infections and conditions requiring       USAMRIID can
 containment care during transport                  simultaneously deploy
                                                    two teams, each
 ---------------------------------------------------consisting of one
 Arenavirus infection                               physician, one
    Argentine hemorrhagic fever (Junin virus)       registered nurse, and
                                                    four to six medics.
    Bolivian hemorrhagic fever (Machupo virus)      Each team can
    Brazilian hemorrhagic fever (Sabi virus)       transport and manage
    Lassa fever                                     one patient. In
    Venezuelan hemorrhagic fever (Guanarito virus)  addition, the team can
                                                    deploy a portable
 Bunyavirus infection                               containment laboratory
   Congo-Crimean hemorrhagic fever                  with rapid diagnostic
   	                                              assays, including
 Filovirus infection                                enzyme-linked
   Ebola                                            immunosorbent assays
   Marburg                                          and polymerase chain
                                                    reaction (PCR), as
 Orthopoxvirus infection                            well as standard
   Monkeypox                                        clinical laboratory
   Variola                                          support, for all
                                                    agents listed in Table
 Pneumonic plague until sputum cultures are         1 (in development:
 negative                                           Machupo, Sabi, and
                                                    Guanarito viruses).
 Any unknown virulent communicable disease          Deployable on rotary
   pending diagnosis                                and fixed-wing
                                                    military aircraft, the
 Suspected biologicalwarfarecaused infection      team conducts
 -------------------------------------------------  in-flight training
                                                    and can deploy withing
6 to 12 hours of notification. Although a military asset, the team has
been mobilized for situations involving civilians. Requests may be
forwarded through local and state health departments to the Centers
for Disease Control and Prevention, through the Federal Emergency Management
Agency, or through the Federal Bureau of Investigation and are then reviewed
by the Directorate of Military Support. Evacuation of non-U.S. citizens from
other countries to the United States would require coordination through the
Department of State, Bureau of Political-Military Affairs.
Biosafety Containment under Field Conditions
Maximum biological containment is designed to prevent transmission of
highly hazardous pathogens and is accomplished in two steps. First, the
health-care worker wears an impermeable suit consisting of a lightweight
polyvinyl chloride (PVC) coverall, a separate hood, and vinyl boots (Figure
1). A HEPA-filtered respirator powered by a rechargeable battery supplies
air under positive pressure for breathing and cooling. HEPA filters are
certified to remove 99.7% of particles 0.03 m to 3.0 m diameter; each
filter is tested with particulate aerosol challenge studies before
delivery. Air enters at a rate of 170 L/min through an intake port near the
top of the hood and exits through an exhaust valve at its base. Two-way
radios permit communication between team members and patients. The suit and
respirator ensemble has been tested by the manufacturer by particulate
aerosol challenge and meets the standards of the National Institute of
Occupational Safety and Health and the Occupational Safety and Health
Administration for working in environments with respiratory hazards.
Second, the patient is isolated within
a sealed container under negative air
pressure maintained by a
battery-powered HEPA-filtered           		[fig]
ventilation system providing five air  Figure 1: Aeromedical isolation
exchanges per hour (Figure 1). Two     team members in field-protective
isolators are used: the stretcher      suits equipped with battery-powered
isolator, a lightweight unit for       HEPA-filtered respirators
initial patient retrieval (Figure 1),  transporting the stretcher
and the Vickers aircraft transport     isolator, a light-weight unit
isolator (VATI), a larger unit for     designed for initial patient
definitive transport and in-flight     retrieval. The team trains on
care (Figure 2; Table 2).              several types of military aircraft,
                                       including the C-130 transport shown
                                       in the background.
 		[fig]
 Figure 2: The Vickers aircraft
 transport isolator (VATI), designed
 for prolonged patient
 transportation and in-flight care.
The design and construction of the isolators are similar to that of transparent
flexible PVC isolators for gnotobiotic animals in biomedical research; the
isolators were adapted both for in-patient and transport use (4-7).
Challenge studies have demonstrated the efficacy of containing aerosolized
T-2 bacteriophage during both hypobaric and isobaric conditions (8).
The HEPA filters are certified to remove 99.7% of all particles 0.3 m
to 3 m in diameter. Isolators have been used to treat in-patients with
suspected Ebola, Lassa, and Marburg hemorrhagic fevers (5,6). The utility
and safety of these isolators for in-patient care have been questioned (9),
and their use in hospitals is not recommended (10,11). However, transport
isolators, the only available technical means of reliably maintaining
airborne isolation in a military transport aircraft, have been successfully
used for the aeromedical evacuation of patients with suspected Ebola fever
(6) and suspected (7) and proven Lassa fever (9).
 Table 2. Dimensions of portable            Both isolators feature
 isolators                                  transparent PVC envelopes
                                            suspended from metal frames by
 ------------------------------------------ detachable plastic rings. Both
 Isolator   Length  Width  Height  Weight   envelopes include gloved
              (cm)   (cm)    (cm)   (kg)    sleeves, transfer and docking
 ------------------------------------------ ports for patient entry, and
                                            transfer and supply ports for
 Stretcher    221     69      86     45     introducing supplies.
 Vickers      221     91     152     112    Electrical current is supplied
  aircraft                                  by rechargeable batteries or
  transport                                 the aircraft electrical system.
 ------------------------------------------ Both isolators can be equipped
                                            with portable oxygen tanks,
intravenous fluids and tubing, medication, and portable defibrillators.
Aeromedical Evacuation Process
The patient must be evaluated and stabilized before transport to ensure
survival en route. Only patients likely to survive transport would be
evacuated. The physiologic effects of altitude, effect of confinement on
patient-care delivery, and psychologic effect of confinement within the
isolator must be considered. Mechanical ventilation cannot be provided in
the VATI, and suction capabilities are limited; therefore, acute
respiratory failure and presence of gas trapped within closed body cavities
that may pressurize at high altitudes (e.g., pneumothorax or intestinal gas
due to ileus or bowel obstruction) contraindicate evacuation. Evacuation of
patients with conditions requiring special in-flight management, e.g.,
hemodynamic instabilty and severe anemia (<2.5 million erythrocytes/cc or
<7.0 g hemoglobin/100 ml) (12), may also be contraindicated.
The patient is placed inside the stretcher isolator and carried to a
transfer point near the aircraft. There the stretcher isolator and team
members are decontaminated with a 5% hypochlorite solution. During the
decontamination procedure, the patient breathes portable oxygen from a
mask, and the ventilation intake port is sealed to prevent chlorine gas
from entering the isolator. The portals of the isolators are then connected
with an airtight sleeve, and the patient is transferred to the VATI (Figure
3). The sleeve is clamped at two points before being heat-sealed and cut,
maintaining air-tight seals throughout the transfer. The cut ends are
decontaminated and covered with PVC seals, which are then attached to the
isolators with pressure-sensitive tape. Both isolators are maintained under
negative air pressure until decontaminated at USAMRIID. Equipment is
removed, placed in bags, and returned to USAMRIID for decontamination of
respirators and radios and disposal or decontamination of coveralls.
The patient is transported on standard
military transport aircraft (C-130 or
C-141), which maintain an internal
cabin atmosphere equivalent to			[fig]
approximately 8,000 feet above sea     Figure 3: Patient's view of an
level while at altitude (26,000 feet   aeromedical isolation team member
to 35,000 feet). This level of air     providing care through a half-suit
pressure is considered adequate to     in the Vickers aircraft transport
protect commercial airline passengers  isolator (VATI).
(13) and results in an arterial blood
hemoglobin oxygen saturation of approximately 90% in healthy persons.
However, because the VATI maintains negative air pressure, the partial
pressure of oxygen inside the VATI is lower than that of ambient
atmosphere. This lower pressure would be hazardous for persons with
respiratory failure or chronic obstructive pulmonary disease. In addition,
rapid decompression could place the patient at further risk. Accordingly,
the VATI is deployed with portable oxygen tanks, tubing, and masks capable
of delivering 100% of needed oxygen.
Design features of the VATI that facilitate in-flight care include its
larger size, additional glove ports, two half-suits, 12 cones at the base
of the envelope for introducing wires and tubing, two sleeves for
intravenous therapy, and two large pockets for placing waste supplies.
Diagnosis and therapy, which can be delivered in the VATI, include
monitoring cardiac function, blood-pressure, and oxygen saturation of the
blood; providing oxygen supplementation, intravenous therapy, and
phlebotomy; and determining hemoglobin and hematocrit levels and serum
electrolytes (by using a portable hand-held laboratory analyzer) (Figure
3). Because the use of glove ports limits manual dexterity, team members
practice these skills on each other during on-ground and in-flight training
exercises. Endotracheal intubation, manual ventilatory assistance with a
bag and valve device, and cardiopulmonary resuscitation are practiced on
mannequins in the isolators. To minimize the risk of puncturing the
isolator, no glass bottles or instruments with rough or sharp edges are
used. Phlebotomy is minimized, and a needleless intravenous system is used.
After arriving at USAMRIID, the patient is transferred from the VATI into
the containment-care suite through a plastic sleeve connected to a port on
an outside wall.
Aeromedical Isolation Team Deployments
The first of several team deployments occurred during the October 1989
epizootic of Ebola hemorrhagic fever among cynomolgus monkeys (Maccaca
fascicularis) imported from the Philippines and held at a primate
quarantine facility in Reston, Virginia (14-16). Because Ebola virus had
been isolated only in association with epidemics of human disease in
Africa, which had death rates of 53% to 88%, potential transmission of
Ebola to animal handlers in the facility and secondary transmission to
other members of the community were of concern. Aeromedical isolation team
and additional personnel from USAMRIID were deployed. Animal handlers were
trained in the use of suits and respirators, containment methods,
decontamination, and waste disposal; 450 monkeys were humanely euthanized;
and team members obtained specimens of blood and tissue for histopathologic
and virologic studies and sealed and decontaminated the facility by
paraformaldehyde fumigation followed by conventional disinfectants (16).
Respiratory transmission was suggested by the epizootic spread among
monkeys housed in separate cages (with no opportunity for physical contact
[16]) and by subclinical human infections. Serologic evidence of recent
Ebola infection developed in four of the five animal handlers; only one had
percutaneous blood exposure. None became ill, which suggested that the
epizootic strain was not virulent for humans (17), and none of the 42
USAMRIID personnel participating became infected. The Ebola isolates from
infected primates represented a newly described strain, Ebola Reston,
genetically and taxonomically distinct from related human pathogens
identified in Africa.
In another episode, an aeromedical isolation team member was deployed to
Linkoping, Sweden, in January 1990 to assist in implementing biosafety
containment for a patient suspected of having a viral hemorrhagic fever
after returning from eastern Africa (1).
During November 1995, the team was deployed with a senior medical advisor
after construction workers at Wright-Patterson Air Force Base, near Dayton,
Ohio, uncovered a buried cache of biological munitions. Some of the
munitions, produced during the U.S. offensive biological warfare program
(19421969) (18), were intact, but most were perforated due to corrosion of
the munition casings. The munitions were brought inside a bunker by the
U.S. Army Technical Escort Unit and sampled inside the VATI. Samples of
liquid bomb fill and adjacent soil samples were transported in sealed
containers on ice packs in accordance with U.S. Department of
Transportation regulations by military aircraft to USAMRIID, the Naval
Medical Research Institute, Bethesda, Maryland, and the Armed Forces
Institute of Pathology, Washington, D.C. The bomb fill contained nonviable
gram-negative bacteria (identified as Brucella suis by strain-specific
PCR); soil samples tested positive for Brucella DNA and antigens and
cultures yielded normal commensal flora but no growth of Brucella sp.
Background soil and groundwater tested negative for Brucella DNA and
antigens. All munitions were drained, and the fill and casings were
sterilized by autoclave before disposal. Documents later retrieved
confirmed that the munitions were bomblets filled with B. suis and used at
the base from June to October 1954 to train personnel in viability testing
and handling of biological weapons. After training was completed, the
munitions were heated in a ground portable heater with an ambient
temperature of 104C for 4 hours each of 2 days, with the temperatures of
the innermost munitions reaching 70C to 74C, and then buried.
Although not deployed, the team was on alert during 1994 for a
laboratory-acquired Sabi virus infection (Brazilian hemorrhagic fever) at
Yale University (19) and during the 1995 Ebola epidemic in the former
Zaire.
(ft 1)An earlier version of this paper was presented at the 84th Panel Symposium
of the North Atlantic Treaty Organization Aerospace Medical Panel,
Aeromedical Support Issues in Contingency Operations, Session VII, Control
of Communicable Diseases, Rotterdam, The Netherlands, 1 October 1997.
---------------------------------------------------------------------------
Lt. Colonel Christopher is chief of the Containment Care Medicine
Department, Operational Medicine Division, USAMRIID. His interests include
medical care delivery under biological containment and the formulation of
medical practice guidelines for the care of biological warfare casualties.
Colonel Eitzen is chief of the Operational Medicine Division, USAMRIID. He
directs the division in multiple activities to enhance medical biological
defense, including medical education programs; collaboration of basic
scientific, medical, public health, and operational communities; and
consultative expertise for military and other government agencies.
Address for correspondence: George W. Christopher, USAMRIID, ATTN:
MCMR-UIZ-T, 1425 Porter Street, Fort Detrick, MD, 21702-5011, USA; fax:
301-619-2312; e-mail: george_christopher@detrick.army.mil.
References
  1. Hill EE, McKee KT. Isolation and biocontainment of patients with
     highly hazardous infectious diseases. Journal of the U.S. Army Medical
     Department 1991;PB 8-91-1/2:10-4.
  2. Wilson KE, Driscoll DM. Mobile high-containment isolation: a unique
     patient care modality. Am J Infect Control 1987;15:120-4.
  3. Franz DR, Parrott CD, Takafuji ET. The U.S. Biological Warfare and
     Biological Defense Programs. Textbook of military medicine, part I.
     In: Sidell FR, Takafuji E, Franz DR, editors. Washington: TMM
     Publications; 1997. p. 425-36.
  4. Trexler PC. An isolator system for the maintenance of aseptic
     environments. Lancet 1973;1:91-3.
  5. Trexler PC, Edmond RED, Evans B. Negative-pressure plastic isolator
     for patients with dangerous infections. BMJ 1977;559-61.
  6. Clausen L, Bothwell TH, Isaacson M, Koornhof HJ, Gear GHS, McMurdo J,
     et al. Isolation and handling of patients with dangerous infectious
     disease. S Afr Med J 1978;53:238-42.
  7. Clayton CJ. Containment aircraft transit isolator. Aviated Space
     Environ Med 1979;50:1067-72.
  8. Hutchinson JP, Gray J, Flewett TH, Emond RTD, Evans B, Trexler PC. The
     safety of the Trexler isolator as judged by some physical and
     biological criteria: a report of experimental work at two centres. The
     Journal of Hygiene 1978;81:311-9.
  9. Fisher-Hoch SP, Price ME, Craven RB, Price FM, Forthall DN, Sasso DR,
     et al. Safe intensive-care management of a severe case of Lassa fever
     with simple barrier nursing techniques. Lancet 1985;2:1227-9.
 10. Centers for Disease Control. Management of patients with suspected
     viral hemorrhagic fever. MMWR Morb Mortal Wkly Rep 1988;37:1-15.
 11. Centers for Disease Control and Prevention. Update: management of
     patients with suspected viral hemorrhagic feverUnited States. MMWR
     Morb Mortal Wkly Rep 1995;44:475-9.
 12. Johnson A Jr. Treatise on aeromedical evacuation: I. Administration
     and some medical considerations. Aviated Space Environ Med
     1977;48:546-9.
 13. National Academy of Sciences. The airliner cabin environment-air
     quality and safety. Washington: National Academy Press; 1986. p. 8.
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     primatesVirginia, 1989. MMWR Morb Mortal Wkly Rep 1989;38:831-9.
 15. Jahrling PB, Geisbert TW, Dalgard DW, Johnson ED, Ksiazek TG, Hall WC,
     et al. Preliminary report: isolation of Ebola virus from monkeys
     imported to USA. Lancet 1990;335:502-5.
 16. Dalgard DW, Hardy RJ, Pearson SL, Pucak GJ, Quander RV, Zack PM, et
     al. Combined simian hemorrhagic fever and Ebola virus infection in
     cynomolgus monkeys. Lab Anim Sci 1992;42:152-7.
 17. Centers for Disease Control. Update: filovirus infections among
     persons with occupational exposure to nonhuman primates. MMWR Morb
     Mortal Wkly Rep 1990;39:266-7.
 18. U.S. Dept of the Army. U.S. Army Activity in the U.S. Biological
     Warfare Programs. Washington: U.S. Dept of the Army; 24 February
     1977;2. Publication DTIC B193427 L.
 19. Barry M, Russi M, Armstrong L, Geller D, Tesh R, Dembry L, et al.
     Brief report: treatment of a laboratory acquired Sabi virus
     infection. N Engl J Med 1995;333:294-6.
_________________________________________________________________________
Synopses
emm Typing and Validation of Provisional M Types for Group A Streptococci (ft 1)
R. Facklam,* B. Beall,* A. Efstratiou, V. Fischetti, D. Johnson, E.
Kaplan, P. Kriz, M. Lovgren,# D. Martin,** B. Schwartz,* A. Totolian,
D. Bessen, S. Hollingshead, F. Rubin, J. Scott,## and G. Tyrrell
*Centers for Disease Control and Prevention, Atlanta, Georgia, USA;
Central Public Health Laboratory, London, United Kingdom; The Rockefeller
University, New York, New York, USA; University of Minnesota, Minneapolis,
Minnesota, USA; National Institute of Public Health, Prague, Czech
Republic, #National Center for Streptococcus, Edmonton, Alberta, Canada;
**Communicable Disease Group, ESR, Porirua, New Zealand; Institute of
Experimental Medicine, St. Petersburg, Russia; Yale University, New
Haven, Connecticut, USA; University of Alabama, Birmingham, Alabama, USA;
National Institutes of Health, Bethesda, Maryland, USA; and ##Emory
University, Atlanta, Georgia, USA
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      This report discusses the following issues related to typing of
      group A streptococci (GAS): The development and use of the 5'
      emm variable region sequencing (emm typing) in relation to the
      existing serologic typing system; the designation of emm types
      in relation to M types; a system for validation of new emm
      types; criteria for validation of provisional M types to new
      M-types; a list of reference type cultures for each of the
      M-type or emm-type strains of GAS; the results of the first
      culture exchange program for a quality control testing system
      among the national and World Health Organization collaborating
      centers for streptococci; and dissemination of new approaches
      to typing of GAS to the international streptococcal community.
The Lancefield M-typing system, a typical serologic system based on
antigen-antibody reactions, is dependent on the preparation of
type-specific antisera and extraction of a protein identified as M protein
on the surface of group A streptococci (GAS) (1). The antisera against the
M-protein antigens are produced with whole-cell streptococcal vaccines used
to immunize rabbits. Acceptable antisera contain specific-precipitin
antibodies and type-specific antibodies that must enhance the phagocytosis
of the strain used to immunize the rabbit (2,3). The precipitin antibodies
are made specific by absorption of the serum with streptococcal cells to
remove the carbohydrate group antibodies and any cross-reactive precipitin
antibody to heterologous M-type strains. Each rabbit antiserum is tested
for reaction with antigens of all known M types.
Approximately half of GAS strains produce an apoproteinase, an enzyme that
causes mammalian serum to increase in opacity. This reaction is called the
serum opacity factor reaction, and the responsible enzyme is referred to as
opacity factor (OF). The OF enzymes are OF type-specific because each M
type that produces OF can induce type-specific OF antibodies that can be
used in OF inhibition tests (3,4). Preparation of the OF antisera and
specific details of the OF tests are described elsewhere (3,4). Some
laboratories have used OF typing to predict M types in epidemiologic
investigations. Even though it is not uniformly agreed that OF typing
antisera results should be reported as M or provisional M-typing results,
anti-OF antisera in many situations predict the M type in a type-specific
manner. For reporting purposes, if cultures are identified with M- or
OF-typing antisera, they should be identified as M type or OF type.
Reference strains used to prepare the M antisera (several of which were
originally described by Griffith in 1935) were historically obtained from
the Lancefield collection, Rockefeller University, New York; however more
recently, reference strains have been available from other reference
laboratories. M-types 1 through 51 were designated in the laboratory of Dr.
Rebecca Lancefield between 1928 and 1945. M-types 52 through 81 were
submitted by various investigators to reference laboratories in Atlanta,
London, New York, and Prague between 1965 and 1976 for confirmation. Some
laboratories believe that certain strains in the Lancefield collection do
not adequately express M protein and are unsuitable for the production of
M-type antiserum. Preparing M-type antiserum or a typing system accurately
related to the Lancefield system requires documented reference strains from
one of the internationally recognized reference laboratories. (ft 2)
In the Lancefield typing system, strains representing types 7, 16, 20, and
21 (originally described as Griffith) are not GAS but belong to groups C
and G. M-type 10 is the same serotype as M12, M-type 24 is the same as M45,
and M-type 35 is the same as M49; thus, designations of serotypes 7, 10,
16, 20, 21, 35, and 45 are not included in the Lancefield M-typing system 1
to 81 for GAS.
The emm gene of S. pyogenes is the gene that encodes the M protein. The M
protein, responsible for the bacterium's capacity to resist phagocytosis,
is a major virulence factor in GAS. The 5' ends of emm genes are highly
heterogeneous and encode for the serotype specificity used for the M-typing
system developed by Dr. Lancefield in 1928 (1). Producing type-specific
M-typing antisera is difficult and specialized; no attempt has ever been
made to produce them commercially, and only a few international reference
laboratories prepare them. The resurgence of rheumatic fever cases in the
United States and the emergence of severe infections (streptococcal toxic
shock and necrotizing fasciitis) caused by GAS in the 1980s and 1990s
indicated the need to reassess typing strategies for GAS. Since production
of M-type precipitating antisera is very expensive and labor-intensive, the
potential usefulness of a nonserologic typing system for GAS sequencing the
5' end of the M protein (emm) gene toward a molecular-based typing system
was examined.
emm Typing System for GAS
Before an emm genotype-based typing scheme was developed for GAS, the
nucleotide sequence at the 5' ends of emm genes had been reported for many
strains representing M-types 1-81 and several provisional M types (PT)
(5,6). However, it was not always evident that true reference strains had
been used. The knowledge gained from these studies provided the impetus for
exploring the feasibility of an emm-based genotyping system. Subsequent
studies based on sequencing the 5' emm genes from GAS reference strains and
clinical culture specimens have now been published (7,8). In all studies
involving emm sequence typing, 160 to 660 bases were sequenced from the 5'
terminal end of the emm gene. The methods of emm sequencing and emm gene
amplicon profiling restriction pattern techniques have been described
(7,8). Two isolates are regarded as sharing the same emm sequence type if
they are >/= 95% identical over their 5' end 160 nucleotides (includes
approximately 50 bp of the moderately conserved leader peptide-coding
region), allowing for one frame shift or in-frame insertion/deletion of no
more than seven codons (8). The results of emm gene sequencing of Dr.
Lancefield's reference strains types 1 to 51 and reference strains of
M-types 52 to 81, submitted to the Centers for Disease Control and
Prevention (CDC) Streptococcus Reference Laboratory as potential new M
types from 1967 to 1976 by various international investigators, are
summarized below.
The 5' end emm sequences of the following CDC M-type reference strains
matched the following sequences in GenBank that were submitted by other
investigators: Types 1, 2, 3, 4, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 19,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 33, 36, 37, 39, 41, 43, 44, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
72, 73, 74, 75, 76, 77, 78, 80, 81. The accession numbers for these and
many other emm sequences deposited in GenBank are listed in references 5-8
and at http://www.cdc.gov/ncidod/biotech/infotech_hp.html.
The 5' emm sequences from the CDC reference strains of GAS for the
following M types were submitted to GenBank: M-types 13 (AF025950), 32
(L47325), 34 (L47324), 38/40 (L46817), 42 (L46799), 67 (AF025949), 68
(AF025948), 69 (AF035838), 70 (AF035838), 71 (L46652). The 5' emm sequences
that were different from those submitted to GenBank by previous
investigators (6) are M-types 13, 67, 68, and 79. For M-type 13, the emm
sequence in GenBank was not from a recognized Lancefield typing strain; emm
sequence AF025950 should be considered the emm sequence for Lancefield
M-type 13. For M-types 67 and 68, the emm sequences in GenBank did not
match those from CDC. The reasons for this are unknown; however, emm
sequence AF025949 (M-type 67) and emm sequence AF025948 (M-type 68) were
obtained from the reference strains submitted to CDC by the investigators
who originally described these M types. For M-type 79, the emm sequence
obtained by the CDC investigators matched the emm sequence in GenBank
labeled M-type 80. Personal communication with the investigators who
submitted the emm sequences for M-types 79 and 80 indicated a transcription
error during submission of the sequences. Sequence U12004 is the correct
sequence for M-type 79. The emm sequences from the CDC reference strains
representing these four M types were confirmed by an independent
laboratory.
CDC has additional data on emm typing of more than 1,500 GAS isolates from
population-based studies, as well as random cultures from the United States
and several other countries. Nearly 100% of the cultures could be genotyped
by the emm typing system. In addition to determining the emm types of
reference strains of M-types 1 to 81, several provisional type strains as
well as new sequence type strains were typed by the emm typing procedure.
Only one of more than 1,500 GAS isolates could not be emm typed by current
methods. The emm sequence type of clinical isolates representing 35
distinct serotypes match the emm sequence type of the corresponding
reference strainincluded in this set of analyses are clinical isolates
that underwent M or OF serotyping at one of the internationally recognized
reference laboratories or elsewhere (8;9; B. Beall and D. Bessen, unpub.
data).
The historical correlations between T type, OF reaction, and M type are
largely unchanged when emm sequence type is substituted for M type
(3,4,7,8). Because this observation is based upon the analysis of >3,000
clinical isolates, the T type and OF reactions together constitute an
invaluable second-tier method for further confirmation of the grouping of
closely related isolates, as well as for resolution of unrelated sets of
organisms (R. Beall and R. Facklam, unpub. data).
When comparing a phenotypic-based typing scheme to a genotypic-based
scheme, complete concordancy is not expected. Although for most types the M
serotype is paired with a unique emm sequence type, there are several
discrepancies. In five well-documented instances, 5' end emm sequences are
>95% identical for two distinct M serotypes: M-types 27L and 77, 38 and 40,
44 and 61, 50 and 62, and 65 and 69 (10). The molecular basis for a lack of
concordancy can differ for each unique M-type/emm-type pair. A few critical
nucleotide substitutions at the 5' emm region could result in new, dominant
antigenic epitopes that lead to the generation of a distinct serologic
type. Alternatively, emm genes can occasionally undergo horizontal exchange
and move onto a new genetic background, as appears to be the case for emm44
and emm61 (6); in this instance, additional dominant, polymorphic antigens
may exist that can also be detected by M-typing sera. For the five examples
cited above, introduction of a second typing scheme (T-agglutination and OF
reaction) has proven useful in distinguishing between pairs displaying
identical emm sequence types, but distinct M serotypes.
The discriminatory power of the genotypic emm sequence-typing scheme
approximates that of the phenotypic M-serotyping scheme. For most M
serotypes, there is a one-for-one relationship with a unique emm sequence
type. The selection of 95% sequence identity as the cutoff value for
defining the emm sequence type is based on empirical measures that best
match the level of resolution achieved by M serotyping. In several
examples, the emm sequence of one emm-type/M-type pair displays a
relatively high level of sequence identity (but <95%) to a second, unique
emm-type/M-type pair. For example, the emm3 and emm31 sequences share 91.3%
identity over their first 160 bases; emm2 and emm73 are 89% identical over
their first 160 bases of 5' end sequence, and this similarity is increased
to 92.3% identity over their first 326 5' bases. Conceivably, certain
genetic changes, such as a single bp insertion in the emm hypervariable
region followed by a single bp deletion much farther downstream, could
alter the reading frame of the gene and hence the antigenic structure and
serotype of the emm gene product; however, this kind of variant has rarely
been encountered in CDC surveys. Other genetic changes, such as synonymous
substitutions, have no effect on phenotype. In some instances a single
deletion or insertion of seven or fewer codons within the hypervariable 5'
end 160 bp had no effect on the predicted M serotype (B. Beall, unpub.
data). Therefore, an emm gene with less than 95% sequence identity to other
emm genes may confer a new M serotype specificity. Thus, two isolates are
regarded as sharing the same emm sequence type if they are >/= 95% identical
over their first 160 nucleotides, allowing for one frame shift or in-frame
insertion/deletion of no more than seven codons (8). The 95% identity
cutoff is not expected to match perfectly what can be achieved by serologic
methods.
Most of the GAS isolates that are deemed nontypable by serologic methods
can be genotyped through emm sequence determination. Furthermore, the M
serotype does not always match the emm sequence-type (9,11). However, among
the hundreds of strains analyzed by the streptococcal reference laboratory
at CDC no discrepancies were observed between M serotype and emm sequence
type. The full extent of putative M-serotype/emm sequence-type
discordancies is not known, and the explanations for such a lack of
congruency are numerous. A more complete understanding of the basis for
discrepancies will be forthcoming as the emm sequence-typing method becomes
more widely implemented.
The emm-typing system is a useful and reliable epidemiologic tool for
subdividing GAS. Because it is independent of emm gene expression and can
often discriminate between biologically distinct isolates that may be only
weakly antigenic or nontypeable, emm sequence typing has the potential to
classify isolates that have been difficult to type by serologic methods.
Designation of M, Provisional M, and emm Sequence Types
GAS strains fall into three categories: Validated M types, provisional M
types, and emm sequence types. If a laboratory has prepared antiserum to an
unknown strain and the serum has type-specific precipitating antibodies, as
well as bactericidal antibodies directed to that strain, verified by one of
the six original reference laboratories, an M-type designation can be
assigned to that strain. If two laboratories (at least one being one of the
six reference laboratories) produces type-specific precipitating and
bactericidal antiserum to the strain, the strain also qualifies as a new M
type.
When a laboratory has prepared antiserum as described above to an unknown
strain but the specificity of this antiserum has not yet been confirmed by
a second reference laboratory, that strain is designated a provisional
type. The requirements for conventional validation of new M types will be
described elsewhere. A third category are sequence types or emm types,
which are strains typed by sequencing the emm gene. If cultures are
identified by emm sequencing, they should be reported as emm type.
Validation Procedures and Nomenclature of New emm Sequence Type Strains
Published emm sequences from studies conducted in New Zealand and Australia
included several new emm sequences included in GenBank (12,13). In
addition, data from the CDC studies (B. Beall and R. Facklam,
http://www.cdc.gov/ncidod/biotech/infotech_hp.html) indicated that more
than 30 unknown emm sequences were identified among the 1,500 isolates of
GAS that had been emm sequenced. A working group of representatives of each
of the six international reference centers in Canada, New Zealand, Czech
Republic, United Kingdom, and United States, was charged with establishing
a definitive protocol both for submission of new emm sequences and for
subsequent validation of new emm types. As an interim measure, unique 5'
end emm sequences proposed as new emm types must be confirmed by a second
laboratory; at least one of the two laboratories should be the
streptococcal reference laboratory at CDC. If the uniqueness of the emm
sequence can be confirmed by the second laboratory, the original
investigator or one of the confirming laboratories will submit the findings
to the Working Group, which will determine whether the strain should be
assigned a new emm type number (e.g., emm94). In addition to sequence
uniqueness, additional factors may be considered by the working group when
making this decision; for example, previous requirements for assigning
regular M- and provisional M-type numbers to strains were restricted to
strains of particular clinical significance or to those occurring in a
population "with significant frequency." Another remaining unresolved issue
is whether or not all new emm reference strains should actively express M
protein. A lack of surface expression of this emm gene product will
preclude any possibility for correlation of emm type with classic serologic
type or subsequent evaluation of biological significance. The relationship
of emm sequence to biological function needs to be further explored.
CDC has validated six emm sequences submitted to GenBank by Australian
investigators (12-14) and one emm sequence from a strain submitted to CDC
by an investigator in the United States (15); these sequences should all be
considered for official status as new emm types. Four additional isolates
were examined at the CDC laboratory for which emm sequences had been
submitted to GenBank (12-14). Strains STBSB75, ST1293, ST87/156, and STNS27
were shown to have the same emm types as M-type 70, M-type 76, PT2110, and
PT5757, respectively. Therefore, these four emm sequences should not be
accepted as new emm types, and their sequences should be reidentified in
GenBank.
CDC has identified 10 new emm types from population-based studies of GAS
invasive disease (7,8, unpub. data). In addition, eight new emm sequences
from Brazil, six from Malaysia, three from Papua New Guinea, three from
India, two from Ethiopia, two from Gambia, and one each from New Zealand
and Chile have been confirmed by a second laboratory for emm sequence
uniqueness, for 36 potentially new emm types.
List of Reference Strains
The WHO Collaborating Laboratory for Reference and Research on Streptococci
in Prague has prepared a database of the reference type strains to be used
for research and antiserum production from information provided by the six
international Reference Centers in Canada, New Zealand, Czech Republic,
United Kingdom, and United States. Although all the reference strains on
the list at one time had demonstrated survival in the in vitro bactericidal
test (presumably reflecting functional M protein), the reference strains
should be retested for survival in the bactericidal test before use in
research. Dr. Lancefield's strains types 1 to 50 are listed at
http://www.rockefeller.edu/vaf/
Because listing of type strains may be slightly different for each culture
collection, when cultures are obtained, the strains should be properly
identified. The strains for reference types 1 to 50 should be traced to the
Lancefield collection, from which the M-typing system was derived. In the
past, if a reference strain lost the capacity to express M protein on the
bacterial cell surface, that strain was passaged in vivo and selected for
the increased presence of M protein; therefore, many derivatives of the
original reference cultures are in use by the reference laboratories, and
none are known to have undergone change in their emm gene nucleotide
sequence. The American Type Culture Collection (ATCC) has Lancefield's
strains 1 to 50, the UK National Collection of Type Cultures has types 1 to
81, as well as the provisional types, and the Czech Culture Collection in
Prague also has types 1 to 81 on deposit. Reference cultures 51 to 81 have
been deposited in the ATCC by the CDC investigators who will also deposit
the provisional type strains shortly. Plans are to continue the deposition
of cultures of new emm types as they are confirmed.
Additionally, the CDC Streptococcus Laboratory has established an emm-type
database at http://www.cdc.gov/ncidod/biotech/infotech_hp.html for use as a
sequence comparison tool and additional documentation of all of the
verified M and emm types. The database contains detailed information on the
CDC method of emm typing and provides additional background on each
reference strain and most known emm sequence types. Sequence comparisons
can be done by using BLAST (Basic Local Alignment Search Tool). The data
are prereviewed for errors and discrepancies, and all newly deposited
emm-types are first verified directly by the CDC reference laboratory. At
this time, until a complete validation protocol is established,
investigators who discover new emm sequence types should submit their
isolates to the CDC Streptococcus Laboratory to confirm uniqueness. As a
first step, investigators should search the CDC database for emm sequences;
Genbank can be used as a second step, since it is always possible that
neither database will necessarily have all known emm sequences of GAS at
any given time. Furthermore, the CDC database will include the accepted emm
type designation for those types, whereas a possible incorrect emm sequence
may have been submitted to GenBank by an investigator.
Validation of Provisional M Types to New M and emm Types
The Table lists the provisional M-type strains and the status of
validation. Collaborative investigations involving the six reference
laboratories included several other provisional M-type strains and has
confirmed the following: PT179 fulfills all the phenotypic criteria for an
M type; however, only anti-OF serum has been prepared. Therefore, the
status of this strain as a potential new M type remains on hold until
further supporting data can be provided. PT4854, Colindale Laboratory,
United Kingdom (UK), had a closely matching emm type as M-type 43; this
finding correlated to serologic tests at CDC and UK laboratories as M-type
43 that demonstrate that both strains reacted with M43 typing antiserum.
PT3800, Prague, Czech Republic (CZ), has the same emm type as M-type 65,
which correlated to serologic tests performed at the National Streptococcus
Laboratory in Edmonton, Canada, showing that both strains reacted with M65
typing antiserum. PTYE327 (CZ) has the same emm type as PT2841(UK), which
correlates to serologic tests performed in both the Colindale and Prague
laboratories. PT1437, Porirua Laboratory, New Zealand (NZ), has the same
emm type as PT4245 (UK), which correlates to serologic tests performed in
both the UK and NZ laboratories. ST2974.95 (CDC) has the same emm type as
PT5118 (NZ). ST2974.95 was shown to be PT5118 in serologic tests performed
in the Porirua Laboratory. In summary, PT4854, PT3800, PTYE327, PT1437, and
ST2974.95 should be identified as emm43 (M43), emm65 (M65), emm87 (M87),
emm89 (M89), and emm92, (M92) respectively. The sources of most provisional
types were first documented in 1985 (16).
 Table. Recommended action for M and emm                     The following
 designation for provisional M types(sup a)                  emm types were
                                                             isolated from
 ----------------------------------------------------------- patients with
 Provisional                      CDC ID        New emm      severe
 type                ID number    number       or M type     invasive
                                                             disease in the
 ----------------------------------------------------------- United States;
 PT180(UK)(sup b)    NCTC12062    SS-1395          82        emm82, emm83,
                                                             emm86, emm87,
 PT2110(UK)          NCTC12064    SS-1400          83        emm88, emm89,
 PT2233(UK)          R75/2233     SS-1449          84        and emm92.
 PT2612(UK)          R76/2612     SS-1447          85        These isolates
                                                             comprised
 PT2631(UK)          R76/2631     SS-1448          86        12.7% of all
 PT2841(UK)          NCTC12065    SS-1399          87        isolates
 PT3875(UK)          R67/3875     SS-1455          88        identified
                                                             during these
 PT4245(UK)          NCTC12067    SS-1397          89        studies. Types
 PT4931(UK)          NCTC12068    SS-1396          90        emm82, emm85,
                                                             emm87, and
 PT5757(UK)          NCTC12056    SS-1398          91        emm89 were
 PT5118(NZ)                       SS-1460          92        associated
 PTPotter41(UK)      R76/2631     SS-1493          93        with epidemic
                                                             investigations
 ----------------------------------------------------------- in the United
 (sup a)For definitions of M and emm designations, see       States. Many
 section in text, Designation of M, Provisional M, and emm   of these new
 Sequence Types.                                             emm types were
 (sup b)Abbreviations; UK= United Kingdom, NZ= New Zealand.  also
                                                             identified
                                                             from other
countries, including Argentina, Brazil, Bulgaria, Chile, Colombia, Denmark,
India, Korea, Malaysia, New Guinea, and Poland. Other reference
laboratories have reported these new M types (by using provisional type
identifiers) in a variety of infections, including rheumatic fever and
acute glomerulonephritis (17,18).
An External Quality Assurance Typing Program among International Reference
Laboratories
At the request of the World Health Organization (19), a quality assurance
program on GAS typing has been established by the Central Public Health
Laboratory, London, United Kingdom. Ten GAS isolates were examined by six
different laboratories, and the results of the first distribution showed a
very good correlation between laboratories. Emm typing by one laboratory
correlated very well with M typing by the other five laboratories. Only
minor differences were noted among the T- and OF-typing results; no errors
were found among the M-typing results. Responsibility for sending 10
cultures to the other five centers twice a year would be rotated among the
six reference centers and a report of the quality assurance program will be
presented at the next International Lancefield Society meeting in New
Zealand in the fall of 1999.
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(ft 1)Information in this report was first presented at an International
Workshop on Demonstration of emm Typing and Validation of Provisional M
types for Group A Streptococci held at the Centers for Disease Control and
Prevention, Atlanta, Georgia, August 26-28, 1997.
(ft 2)Centers for Disease Control and Prevention, Atlanta, Georgia, USA;
Central Public Health Laboratory, London, United Kingdom; The Rockefeller
University, New York, New York, USA; University of Minnesota, Minneapolis,
Minnesota, USA; National Institute of Public Health, Prague, Czech
Republic, National Center for Streptococcus, Edmonton, Alberta, Canada;
Communicable Disease Group, ESR, Porirua, New Zealand.
  Dr. Richard Facklam is chief of the Streptococcus Laboratory, Division of
Bacterial and Mycotic Diseases Division, National Center for Infectious
Diseases, CDC. His major fields of interest include improvement in
laboratory procedures for the diagnosis of acute respiratory tract
infections, taxonomy of streptococci and related gram-positive cocci,
identification of virulence factors associated with bacterial respiratory
pathogens, and development of new systems for epidemiologic study of the
transmission of bacterial respiratory pathogens.
      Address for correspondence: Richard R. Facklam, Centers for Disease
Control and Prevention, 1600 Clifton Road, Mail Stop C02, Atlanta, GA
30333, USA; fax: 404-639-1379; e-mail: rrf2@cdc.gov.
References
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  3. Johnson DR, Sramek J, Kaplan EL, Bicova R, Havlicek J, Havlickova H,
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  5. Kaufhold AA, Podbielski A, Blokpoel M, Schouls L. Rapid typing of
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  7. Beall BB, Facklam R, Thompson T. Sequencing emm-specific PCR products
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  8. Beall BB, Facklam R, Hoenes T, Schwartz B. Survey of emm gene
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____________________________________________________________________________
Research
____________________________________________________________________________
Research
Rapid Molecular Genetic Subtyping of Serotype M1 Group A Streptococcus
Strains
Nancy Hoe,* Kazumitsu Nakashima,* Diana Grigsby,* Xi Pan,* Shu Jun Dou,*
Steven Naidich, Marianne Garcia, Emily Kahn, David Bergmire-Sweat, and
James M. Musser*
*Baylor College of Medicine, Houston, Texas, USA; Naidich Space
Laboratory, Inc., New York, New York, USA; Texas Department of Health,
Austin, Texas, USA
---------------------------------------------------------------------------
      Serotype M1 group A Streptococcus, the most common cause of
      invasive disease in many case series, generally have resisted
      extensive molecular subtyping by standard techniques (e.g.,
      multilocus enzyme electrophoresis, pulsed-field gel
      electrophoresis). We used automated sequencing of the sic gene
      encoding streptococcal inhibitor of complement and of a region
      of the chromosome with direct repeat sequences to unambiguously
      differentiate 30 M1 isolates recovered from 28 patients in
      Texas with invasive disease episodes temporally clustered and
      thought to represent an outbreak. Sequencing of the emm gene
      was less useful for M1 strain differentiation, and restriction
      fragment length polymorphism analysis with IS1548 or IS1562 as
      Southern hybridization probes did not provide epidemiologically
      useful subtyping information. Sequence polymorphism in the
      direct repeat region of the chromosome and IS1548 profiling
      data support the hypothesis that M1 organisms have two main
      evolutionary lineages marked by the presence or absence of the
      speA2 allele encoding streptococcal pyrogenic exotoxin A2.
Molecular genetic approaches that differentiate isolates of a pathogenic
microbial species have revolutionized contemporary epidemiologic
investigations of putative disease outbreaks. The human gram-positive
bacterium group A Streptococcus (GAS) has more than 80 M-protein serotypes,
but isolates expressing the M1 serotype are disproportionately represented
among invasive disease episodes in most case series (1). M1 organisms also
commonly cause pharyngitis. For reasons that are unknown, M1 isolates and
organisms expressing other M serologic types can undergo rapid temporal
variation in disease frequency and severity (1). Serotype M1 isolates have
been studied by several molecular typing approaches, including multilocus
enzyme electrophoresis; pulsed-field gel electrophoresis; rRNA gene
polymorphism typing (ribotyping); random amplified polymorphic DNA
analysis; and sequencing of the genes encoding streptokinase, C5a
peptidase, M protein, hyaluronidase, and pyrogenic exotoxin A, B, and C
(1-5). The common theme of these analyses is that most M1 isolates cultured
from patients with invasive disease episodes are closely allied in overall
chromosomal relationship as a consequence of sharing a recent common
ancestor (1,3,5). Lack of readily detectable chromosomal variation has
limited insights on the molecular origin of new virulent strains, velocity
of strain spread in human populations, and association of genetic subtypes
with certain clinical syndromes, including necrotizing fasciitis and acute
rheumatic fever.
Recently, Akesson et al. (6) identified a GAS extracellular protein made by
M1 strains that inhibits human complement. This streptococcal inhibitor of
complement (Sic) protein is incorporated into the membrane-attack complex
(C5b-C9) and inhibits target cell lysis by an undetermined mechanism.
Analysis of molecular diversity among 16 M1 GAS isolates from patients with
pharyngitis identified seven alleles of the sic gene (7). The high level of
sic polymorphism was unanticipated, given that other methods of molecular
analysis had failed to identify substantial variation among M1 isolates
(1-5). Subsequently, Stockbauer et al. (8) analyzed 165 M1 isolates from
diverse localities, identified 62 alleles, and documented a uniquely high
level of allelic variation in this gene. The molecular features of sic
variation indicated that structural change in Sic is mediated by natural
selection (8). Moreover, study of 70 M1 isolates from two temporally
distinct epidemics of streptococcal infections in the former East Germany
suggested that variation in sic contributed to fluctuations in GAS disease
frequency and severity (8).
The observation that the polymorphism in the sic gene greatly exceeded that
for all other genes examined in serotype M1 isolates suggested that sic
sequencing could be used as a rapid strategy to differentiate organisms
thought to be epidemiologically linked. A recent statistically significant
increase in cases of invasive GAS in Texas presented an opportunity to test
this hypothesis. We also tested whether molecular variation in a region of
the chromosome with multiple direct repeat (DR) nucleotide sequences and
restriction fragment length polymorphism (RFLP) analysis with insertion
elements IS1548 (9) and IS1562 (10) would differentiate M1 isolates.
Brief Overview of the GAS Epidemiology
Statistics gathered by the Texas Department of Health indicated that from
December 1, 1997, through March 5, 1998, 117 invasive episodes of GAS (and
26 deaths) had occurred statewide. Sixty of these cases and 14 deaths were
in central Texas (population 1.4 million). Concern was raised by community
physicians, lay individuals, and the media that an unusually virulent
strain was causing a disease outbreak. (A complete description of the
epidemiology of this outbreak will be presented elsewhere.) For molecular
analysis of the GAS causing recent cases, 100 isolates were sent to the
laboratory of J.M.M. at Baylor College of Medicine, Houston, TX. On
receipt, the bacteria were checked for purity by visual inspection and were
confirmed to contain beta-hemolytic organisms with a colony morphology
consistent with GAS. Chromosomal DNA was isolated as described (5).
Sequence Analysis of emm
To determine whether one or a few unusually virulent strains might account
for most of the invasive episodes, we sequenced the hypervariable part of
the emm gene encoding M-type specificity (5,11). After the sequence data
were edited electronically, they were used to search an emm database
maintained in the laboratory that contains at least one sequence of all
known M-protein serotypes and provisional serotypes (11). The database also
contains 33 emm1 allelic variants identified among serotype M1 organisms
from global sources (1,5,12) (Figure 1).
The most common M type identified
was M1 (n = 30 isolates) (Table).
Five emm1 alleles were identified in
the 30 M1 isolates, including four   		[fig]
(emm1.13, emm1.18, emm1.19, and      Figure 1. Alignment of inferred
emm1.24) not previously described    N-terminal amino acid sequences of 33
(Figure 1). Twenty-three Texas       alleles of emm1. The region shown
isolates had allele emm1.0, the most represents amino acids 27 through 110
common emm1 allele in M1 isolates    (GenBank accession number X07860).
globally (5). Three isolates had     Six of the emm1 alleles were
allele emm1.19, two organisms had    identified in this study, several
allele emm1.24, and one isolate each were described previously (1,5,12),
had allele emm1.13 and emm1.18       and others were from ongoing analysis
(Table). Compared with the emm1.0    of emm1 in M1 strains from global
allele encoding variant M1.0, each   sources. Amino acid residues
of these alleles is characterized by identical to those encoded by emm1.0
single nucleotide changes resulting  are represented by periods.
in single amino acid substitutions
in the resulting M1 protein (Figure 1). The additional 70 isolates were a
heterogeneous array of M types, including M3, M4, M5, M6, M12, M18, and
many others. A more detailed description of the bacteriologic features will
be presented elsewhere.
Analysis of speA Encoding Pyrogenic Exotoxin A
Because M1 isolates were a prominent cause of the invasive disease
episodes, we sought to determine the extent of genotypic heterogeneity
among the 30 M1 GAS isolates. First, polymerase chain reaction (PCR) was
used to test whether the organisms possessed the speA gene encoding
pyrogenic exotoxin A (scarlet fever toxin) (3,13). Most contemporary M1
isolates cultured from patients with invasive disease have this gene
(1,3-5), but some lack it because speA is bacteriophage encoded (13).
Possession of speA is therefore a variable trait among M1 organisms. All 30
M1 isolates had the speA gene, and sequence analysis of 11 random isolates
found that all had allele speA2 (14). Previous study of the speA gene in
several hundred contemporary M1 strains showed that all organisms had the
speA2 allele (1,14).
 Table. Characteristics of serotype M1 Group A Streptococcus
 isolates analyzed
 -----------------------------------------------------------------
                                   DR(sup c)
                                           DR se-
 MGAS                sic    emm1    PCR    quence  speA   IS1548
 no.     TDH no.    allele allele   (sup d) type    PCR    type
 (sup a) (sup b)                    (bp)            (sup e)
 -----------------------------------------------------------------
 6151    BE8-776     1.01   1.0      372     4.0     pos    1.0
 6168    BE-98-743   1.01   1.0      306     3.0     pos    1.0
 6184    BE8-873     1.01   1.0      306     NS(supf)pos    1.0
 6199    BE8-917     1.01   1.19     306     NS      pos    1.0
 6262    BE8-1085    1.01   1.19     306     NS      pos    1.0
 6264    BE8-1087    1.01   1.19     306     3.0     pos    1.0
 6181    BE-98-764   1.02   1.0      240     NS      pos    1.0
 6293    BE8-1339    1.02   1.0      306     NS      pos    1.0
 6294    BE8-1340    1.02   1.0      306     NS      pos    1.4
 6140    BE8-629     1.13   1.0      240     NS      pos    1.0
 6200    BE8-918     1.13   1.0      240     NS      pos    1.0
 6201    BE8-919     1.13   1.0      240     NS      pos    1.0
 6281    BE8-1149    1.13   1.24     306     NS      pos    1.3
 6137    BE8-563     1.32   1.0      306     3.0     pos    1.0
 6148    BE8-773     1.32   1.0      306     NS      pos    1.0
 6249    BE8-929     1.32   1.0      306     NS      pos    1.0
 6172    BE-98-751   1.34   1.0      306     NS      pos    1.0
 5997    BE8-191     1.36   1.0      240     NS      pos    1.0
 6135    BE8-548     1.36   1.0      240     2.2     pos    1.0
 6254    BE8-1021    1.36   1.24     306     NS      pos    1.0
 6189    BE8-88      1.66   1.13     306     NS      pos    1.0
 5999    BE8-208     1.99   1.0      306     3.0     pos    1.0
 6003    BE8-322     1.100  1.0      240     NS      pos    1.0
 6251    BE8-1000    1.100  1.0      240     2.1     pos    1.0
 6006    BE8-369     1.101  1.0      306     3.0     pos    1.0
 6138    BE8-566     1.118  1.0      240     2.2     pos    1.0
 6150    BE8-775     1.119  1.0      306     3.0     pos    1.0
 6154    BE8-792     1.120  1.18     240     2.1     pos    1.0
 6272    BE8-1111    1.179  1.0      306     NS      pos    1.0
 6299    BE8-1380    1.180  1.0      240     2.0     pos    1.0
 2221    NA          1.01   1.0      306     NS      pos    1.0
 5305    NA          1.01   1.0      306     3.0     pos    1.0
 5809    NA          1.01   1.0      305     3.01    pos    1.0
 2139    NA          1.02   1.0      306     3.0     pos    1.0
 2350    NA          1.09   1.0      306     3.0     pos    1.0
 1272    NA          1.35   1.0      306     NS      pos    1.5
 5297    NA          1.121  1.0      240     2.0     pos    1.0
 279     NA          1.08   1.3      570     7.0     neg    1.6
 1632    NA          1.08   1.3      570     7.0     neg    1.6
 1653    NA          1.19   1.3      570     7.0     neg    1.6
 326     NA          1.20   1.3      570     7.0     neg    1.6
 570     NA          1.21   1.3      570     7.0     neg    1.8
 1642    NA          1.24   1.3      504     6.1     neg    1.6
 6708    NA          1.225  1.6      504     6.0     neg    1.7
 (sup g)
 -----------------------------------------------------------------
 (sup a)MGAS, Musser group A Streptococcus strain number. All
 isolates had no known direct epidemiologic connection
 except MGAS 6199, 6264, and 6272 (associated household
 cases); MGAS 6140, 6200, and 6201 (blood and cerebrospinal
 fluid cultures of same patient); and MGAS 6293 and 6294
 (mother-neonate paired isolates).
 (sup b)TDH, Texas Department of Health strain number; NA, not
 applicable (control isolate).
 (sup c)DR, direct repeat.
 (sup d)PCR, polymerase chain reaction.
 (sup e)pos, PCR-positive for speA; neg, PCR-negative for speA.
 The speA gene in MGAS 1272, 6135, 6137, 6138, 6150, 6151,
 6154, 6168, 6251, 6264, 6272, and 6299 was sequenced and
 identified as allele speA2.
 (sup f)NS, not sequenced.
 (sup g)MGAS 6708 is also known as SF370. The genome of this
 organism is being sequenced at the University of Oklahoma.
Sequence Analysis of sic
Recent molecular genetic studies have documented that sic is a uniquely
hypervariable gene among M1 GAS strains (7,8). Our sic database consists of
252 distinct alleles identified by sequence analysis of ~1,200 M1 isolates
from worldwide sources and cultured from patients with a large array of GAS
diseases, including pharyngitis and invasive episodes (7;8; unpub. data).
sic allelic variation has not been identified during in vitro laboratory
passage, nor has variation been detected among strains that are
epidemiologically associated (8). These molecular features suggest that
automated sequencing of sic may be a convenient method for identifying M1
genetic subtypes and inferring epidemiologic relationships in potential
outbreaks. To test this idea, we sequenced the sic gene in the 30 M1
isolates and identified 15 sic alleles that differed from one another by at
least one nucleotide (Figure 2). Seven of the 15 alleles were not found
among the ~1,200 M1 isolates previously characterized for sic variation.
Eight new nucleotide substitutions were identified in eight codons, and one
codon had a new dinucleotide change; these changes would result in nine
amino acid substitutions in the expressed Sic proteins. As observed in
earlier analyses (7,8), the amino-terminal half of the Sic protein had many
insertions and deletions, all in frame (Figure 2).
RFLP Analysis with Insertion Sequences IS1548 and IS1562
IS1548, a recently described
insertion sequence, has been reported
to be polymorphic in copy number and
location in the chromosome of group A
and group B streptococci (9). IS1562
is an insertion sequence located in   		[fig]
the Mga regulon between the sic gene  Figure 2. Variation in the sic gene
and scpA gene encoding C5a peptidase  and Sic protein identified in M1
in some GAS (10). Relatively few GAS  group A Streptococcus isolates
strains have been analyzed by RFLP    characterized in the study. The
profiling with these elements, and    figure is a compilation of
their ability to differentiate among  variations found in the 15 distinct
isolates expressing the same M type   sic alleles in the sample. The
has not been assessed. Since          numbers at the top of the figure
insertion sequence profiling has      refer to the nucleotide sequence
helped elucidate transmission         position of a sic allele described
dynamics and evolutionary             in reference 6. Single-letter amino
relationships of Mycobacterium        acid abbreviations are used. SRR,
tuberculosis (15), Bordetella         amino-terminal short repeat region;
pertussis (16), Streptococcus         Roman numeral, short repeats I-V
pneumoniae (17), Escherichia coli     which recur in SRR; R2 and R3,
(18), and Salmonella Enteritidis      tandem repeats; MGAS strain, Musser
(19), we tested the hypothesis that   Group A Streptococcus strain number;
IS1548 or IS1562 subtyping would      X, presence of polymorphism.
provide additional epidemiologically
informative data regarding genetic
diversity among M1 isolates.
To determine whether the IS1548 element was present in M1 organisms
in our sample, PCR was performed on genomic DNA from 10 random isolates
by using the oligonucleotides (forward) 5'-TGCCGTTCATCAACTGATTTCAGTGG-3'
and (reverse 5'-CGACGATAACTGAGGTCTTTTTT AGGAAAT-3'(9). A PCR product
of the anticipated size of ~1 kb was obtained from all organisms, a
result indicating that the isolates had this element or a close relative.
The PCR-amplified fragment was subsequently used as a
probe for RFLP analysis by Southern blotting after EcoNI digestion and
electrophoretic separation of chromosomal DNA fragments. The data were
analyzed with a Bioimage Analyzer system interfaced with a Sun
Sparcstation. Four M1 isolates had the same 6-band IS1548 RFLP pattern,
which was distinct from the 3-band pattern obtained from three random
serotype M3 isolates (Figure 3A). Twenty-eight of the 30 M1 isolates
studied had the same IS1548 pattern (Figure 3B and data not shown). The
IS1548 RFLP patterns of the two other isolates were single-band variants of
the common M1 pattern, both characterized by the addition of one
hybridizing band (Figure 3B). One of the isolates (MGAS 6294) with a
variant IS1548 pattern was recovered from the blood of a neonate born to a
woman with GAS sepsis. The isolate (MGAS 6293) from the blood of the
infected mother had the common IS1548 pattern.
						To identify other IS1548 RFLP patterns
                                    in M1 GAS organisms, we analyzed 14
 	                           	non-Texas control isolates. These 14 M1
 	                              isolates were selected for analysis
  	[fig]                         because they have been well [fig]
 Figure 3. Representative           characterized by several molecular
 IS1548 RFLP fingerprint            techniques (5). The isolates also have
 patterns of M1 isolates.           many different sic alleles and include
 Panel A is a lane map showing      representatives of two major genetic
 results from analysis of           subclones of M1 organisms (5). IS1548
 three serotype M3 control          profiling of this group identified the
 isolates and four M1 isolates      common six-band pattern and also found
 with different sic alleles.        five organisms with a distinct subtype
 Lane 1, MGAS5892; lane 2,          with four bands (Figure 3C). All
 MGAS6004; lane 3, MGAS6005;        organisms with this profile were
 lane 4, MGAS5997; lane 5,          speA-negative. Interestingly, MGAS6708
 MGAS5999; lane 6, MGAS6003;        (SF370), the M1 strain whose genome is
 lane 7, MGAS6006. kb, 1-kb         being sequenced (20), had a unique
 DNA ladder. Panel B is a lane      five-band IS1548 fingerprint (Figure
 map showing results from           3C). The IS1548 profile for this strain
 analysis of eleven M1              was very similar to the four-copy
 isolates with eight different      pattern characteristic of most of the
 sic alleles. Lane 1,               speA negative organisms.
 MGAS6201; lane 2, MGAS6249;
 lane 3, MGAS6251; lane 4,          We next used PCR to determine whether
 MGAS6254; lane 5, MGAS6262;        IS1562 was present in the 30 M1
 lane 6, MGAS6264; lane 7,          organisms from Texas and in 11 of the
 MGAS6272; lane 8, MGAS6281;        14 non-Texas isolates by using
 lane 9, MGAS6293; lane 10,         oligonucleotide primers 3244 and 3267,
 MGAS6294; lane 11, MGAS6299.       as described by Berge et al. (10). A
 kb, 1-kb DNA ladder. Panel C       PCR product of the expected size of ~1
 is a lane map showing results      kb was obtained from all isolates. The
 from analysis of four              ~1-kb fragment was used to reprobe the
 speA-positive and seven            nylon membranes used for IS1548 RFLP
 speA-negative M1 isolates.         analysis. The results showed that all
 Lane 1, MGAS2350, lane 2,          M1 isolates tested had the identical or
 MGAS2221, lane 3, MGAS2139,        closely similar RFLP characterized by
 lane 4, MGAS1272, lane 5,          one copy of IS1562 (data not shown).
 MGAS6708, lane 6, MGAS1653,
 lane 7, MGAS1642, lane 8,
 MGAS1632, lane 9, MGAS570,
 lane 10, MGAS326, lane 11,
 MGAS279. kb, 1-kb DNA ladder.
PCR and Sequence Analysis of a Polymorphic Direct Repeat (DR) Chromosomal Region
Several years ago Groenen et al. (21) characterized an unusual region of
the M. tuberculosis chromosome that contains up to approximately 40 copies
of a 36-bp DR sequence interspersed with unique-sequence spacer regions 35 bp
to 41 bp in length. Subsequent analysis of this DR region in hundreds of M.
tuberculosis isolates by a method referred to as spacer oligotyping
(spoligotyping) has identified large numbers of distinct subtypes of this
pathogen (22), indicating that the DR region is highly polymorphic, even
among isolates closely related in overall chromosomal character (23). We
examined the M1 GAS genome database maintained by the University of Oklahoma
Advanced Center for Genome Technology and identified a region of the GAS
chromosome located on contig 208 (database as of February 22, 1999) that
consists of seven DR elements separated by six unique 30-bp spacer regions.
This area of the M1 chromosome is referred to as a DR region on the basis of
its shared structural features with the M. tuberculosis DR region.
To test the hypothesis that the DR region is polymorphic among M1 GAS
isolates, we analyzed the 14 control isolates by PCR with primers that
flank this region (DR003, 5'-GGGCTTTTCAAGACTGAAGTCTAGCTG-3' and DR004,
5'-TCCGACTGCTGGTATTAACCCTC TT-3'). Four sizes of PCR products were
identified (data not shown). Six of seven isolates previously identified as
RFLP type 1a (speA-positive, containing allele emm1.0) had an apparently
identical size PCR product of ~300 bp. A PCR product of ~240 bp was
identified in the remaining isolate. Two sizes of PCR products (~500 bp and
~570 bp) were also identified in the six organisms with RFLP type 1k
(speA-negative, allele emm1.3). Hence, the PCR results indicated that size
variation was present in the GAS DR region in M1 organisms and showed that
isolates of the RFLP types 1a and 1k categories did not share PCR fragment
sizes.
To examine nucleotide variation in this chromosomal region, we sequenced
the PCR products obtained from 12 of these control M1 isolates, including 5
with the ~240-bp or ~300-bp PCR product and 7 organisms with either the
~500-bp or ~570-bp PCR product. The one organism with the ~240-bp PCR
product, characterized by two identical DR elements and two nonidentical
spacer sequences, is arbitrarily designated DR type 2.0 (Figure 4). Three
of the four organisms with the ~300-bp PCR product had identical DR-region
sequences defined by the presence of three identical DR elements and three
nonidentical spacer sequences (Figure 4B). This molecular arrangement was
designated DR type 3.0 (Figure 4C). The DR element of the fourth isolate
differed from the other three by the absence of 1 base in the second spacer
region and is designated DR type 3.01 (Figure 4C). Consistent with the
difference in PCR fragment size, the sequences of the DR region in the
seven other organisms were distinct from the DR type 3.0 sequence. Five of
these seven isolates had an identical DR-region sequence that was
characterized by seven spacer regions (designated DR type 7.0). Two
organisms lacked one of the spacer regions present in the DR type 7.0
strains; these molecular variants were designated DR types 6.0 and 6.1
(Figure 4C).
We next analyzed the 30 M1 Texas
isolates by PCR of the DR region and
obtained three PCR fragment sizes:
products of ~240 bp (n = 11		  		[fig]
isolates), ~300 bp (n = 18 isolates), Figure 4. Polymorphism identified in
and ~370 bp (n = 1 isolate). We       the direct repeat (DR) region of
sequenced the PCR products from 12    serotype M1 group A Streptococcus.
organisms selected to represent an    The data were generated by automated
array of DR PCR fragment sizes and    DNA sequencing of polymerase chain
emm and sic alleles. Two additional   reaction products obtained with the
sequences (designated DR types 2.1    oligonucleotide primers DR003 and
and 2.2) were identified among the    DR004 described in the text. (A) The
five isolates with the DR region PCR  36-bp sequences of the two related
fragment size of ~240 bp. All six     DR and DR' elements. Multiple copies
isolates with the ~300-bp PCR product of the DR element present in
had the identical sequence (DR type   different M1 isolates all had the
3.0). The one isolate with the        identical sequence. (B) The 29-bp or
~370-bp PCR product had a unique      30-bp sequences of the 10 distinct
sequence (DR type 4.0) with four      spacer regions identified in the
spacer regions (Figure 4). The        analysis. (C) Arrangement of the DR
results showed that the DR region had elements and spacer sequences in
more molecular variation than emm.    nine distinct DR allelic variants.
However, the level of allelic         The DR types were given arbitrary
variation in sic exceeded that found  designations based in part on the
in either emm or the DR region.       number of DR elements present. Open
                                      or cross-hatched rectangles
Conclusions                           represent copies of the DR or DR'
                                      elements; arrows represent copies of
Our data underscore the importance of the spacer region sequences
molecular typing techniques in        connecting the DR elements. The
rapidly providing information about   numbers above the spacer region
the epidemiology of GAS infections    sequences refer to the spacers
(24). The emm sequence data indicated designated in part B of the figure.
that a heterogeneous array of GAS M
types was present in the sample of 100 GAS isolates; thus, we could rapidly
rule out the notion that the invasive cases had been caused by one or a few
distinct GAS strains. Moreover, molecular analysis of several other
polymorphic loci, including automated DNA sequencing of sic and a
chromosomal region with multiple DR sequences, showed that M1 organisms,
the most abundant serotype in the sample, had substantial levels of genetic
diversity. Of the molecular techniques used in this analysis, sequencing
the sic gene was the most effective for differentiating among M1 isolates
because it identified the most variants. RFLP-based typing with IS1548 and
IS1562 failed to provide extensive, or even adequate, resolving power among
the M1 organisms for epidemiologic purposes. Moreover, the variation in the
IS1548 RFLP profile we detected in two isolates (MGAS 6293 and MGAS 6294)
from a woman with puerperal sepsis and the blood of her newborn child
suggests that IS1548 can be mobile in host-pathogen interactions.
Instability in insertion sequence profiles has also been reported for
IS6110, an element commonly used for molecular subtyping of M. tuberculosis
(25).
Although sequence analysis of emm and the DR region provided some useful
molecular subtyping data for M1 strains, the level of polymorphism at these
loci was less than in sic. A rapid PCR-based subtyping system to index
polymorphism in the DR region could be formulated for M1 GAS that would be
similar to the method available for M. tuberculosis. However, this approach
would be less useful for M1 GAS than M. tuberculosis because in the latter
organism 43 distinct spacer regions have been described. Hence, the number
of polymorphic markers is considerably greater than in M1 GAS, in which
thus far only 13 spacer regions have been found (unpub. data).
Our work, recently reported results (7,8), and unpublished data obtained
from ongoing analysis of sic polymorphism in large samples obtained from
population-based studies demonstrate four emerging themes in the molecular
epidemiology and evolutionary biology of M1 organisms. First, several sic
variants are dispersed over broad geographic areas; some have achieved
intercontinental distribution. For example, M1 strains with the sic1.01
allele have been identified in 14 countries. This allele might be widely
disseminated because it is the ancestral condition in M1 organisms or
otherwise has had a long-standing association with the M1 serotype. Another
plausible hypothesis to explain its widespread dissemination is that
expression of Sic1.01 protein bestows greater fitness than do other Sic
variants. A third possibility is that the Sic1.01 variant marks an M1
subclone with an unusual propensity to survive and spread. In this regard,
we note that virtually all isolates with the sic1.01 allele are
speA-positive. GAS isolates with the speA gene are statistically
overrepresented among organisms recovered from children with pharyngitis
who have not been cured by oral antibiotic therapy (26). Bacterial survival
despite appropriate antibiotic therapy would likely enhance spread of the
organism to new hosts and, hence, assist widespread dispersal. We also note
that speA-positive M1 isolates are internalized efficiently by human
respiratory tract epithelial cells grown in culture (27,28), a process that
could provide access to a protective niche that enhances survival
capability.
A second important theme is that many sic alleles are confined to local
geographic areas (e.g., individual countries or communities). For example,
seven of the sic alleles identified in this study were unique to the Texas
M1 isolates. Several unique sic alleles also were found among organisms
cultured from patients in Mexico (7) and the former East Germany (8).
Because many sic alleles can be readily linked with one another by a single
molecular event such as a nucleotide substitution or one insertion or
deletion, some of the variants likely arise rapidly in local areas. Their
absence in other regions is explained by lack of sufficient elapsed time
required for widespread dispersal. Recent data obtained from study of M1
isolates recovered from population-based surveys in Finland (29), Ontario,
Canada (30), and Atlanta, Georgia (31) strongly support this explanation
(unpub. data).
The third theme is the remarkable polymorphism in the sic gene. Stockbauer
et al. (8) reported that virtually all changes in the sic gene result in
structural changes in the Sic protein and concluded that positive Darwinian
selection is mediating Sic variation. Our study confirmed these
observations. For example, all 10 new nucleotide changes identified would
result in amino acid substitutions in Sic, and all insertions and deletions
were in frame. Moreover, most of the amino acid changes were radical
replacements, that is, those producing charge changes or polar-nonpolar
substitutions. These types of amino acid replacements commonly result in
functional differences in the resulting proteins and are a hallmark of
positive selection (32).
Last, accumulating data suggest the existence of two genetically divergent
M1 subpopulations, which can be thought of as two evolutionarily distinct
lineages. Our study found that organisms with the speA gene and chromosomal
PFGE type 1a (5) have shorter DR-region sequences and an IS1548 profile
characterized by six hybridizing bands. In contrast, organisms that are
speA-negative usually have PFGE type 1k (5), longer DR sequences, and an
IS1548 fingerprint with four bands. In addition, we will show elsewhere
that the two M1 lineages each have distinct families of sic alleles.
Together, the data indicate that sufficient time has elapsed since a shared
common ancestor for members of the two lineages to have diverged at many
chromosomal loci. The data also indicate that transduction of the speA2
allele between members of the two lineages is apparently rare in natural
populations of GAS (5,14). As more comparative analyses are conducted,
additional genetic differences will probably be identified between isolates
of the two lineages.
In summary, automated sequence analysis of sic and a region of the
chromosome with DR sequences permitted rapid and unambiguous
differentiation among serotype M1 isolates during a period of a significant
increase in the number of invasive disease cases. Genetic analysis of these
polymorphic markers permitted us to rapidly rule out the idea that a single
unusually virulent strain of M1 GAS was responsible. The subtyping methods
described in this work will assist other outbreak investigations and
studies designed to understand the molecular basis of temporal variation in
disease frequency and severity of infections caused by M1 GAS isolates.
---------------------------------------------------------------------------
Acknowledgments
      We thank C. Stager, S. Rossman, K. Krause, and C. Baker for
generously providing strains.
      This work was supported by Public Health Service Grant AI-33119 to
J.M.M.
     Dr. Hoe is a research associate in the Institute for the Study of
Human Bacterial Pathogenesis, Baylor College of Medicine. Her main
interests are in the areas of molecular epidemiology and bacterial
pathogenesis.
      Address for correspondence: James M. Musser, Institute for the Study
of Human Bacterial Pathogenesis, Department of Pathology, Baylor College of
Medicine, One Baylor Plaza, Houston, Texas 77030, USA; fax: 713-798-4595;
e-mail: jmusser@bcm.tmc.edu.
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  2. Martin DR, Single LA. Molecular epidemiology of group A streptococcus
     M type 1 infections. J Infect Dis 1993;167:1112-7.
  3. Musser JM, Hauser JM, Kim MH, Schlievert PM, Nelson K, Selander RK.
     Streptococcus pyogenes causing toxic-shock-like syndrome and other
     invasive diseases: clonal diversity and pyrogenic exotoxin expression.
     Proc Natl Acad Sci U S A 1991;88:2668-72.
  4. Norgren M, Norrby A, Holm SE. Genetic diversity in T1M1 group A
     streptococci in relation to clinical outcome of infection. J Infect
     Dis 1992;166:1014-20.
  5. Musser JM, Kapur V, Szeto J, Pan X, Swanson DS, Martin DR. Genetic
     diversity and relationships among Streptococcus pyogenes strains
     expressing serotype M1 protein: recent intercontinental spread of a
     subclone causing episodes of invasive disease. Infect Immun
     1995;63:994-1003.
  6. Akesson P, Sjoholm AG, Bjorck L. Protein SIC, a novel extracellular
     protein of Streptococcus pyogenes interfering with complement
     function. J Biol Chem 1996;271:1081-8.
  7. Perea Mejia LM, Stockbauer KE, Pan X, Cravioto A, Musser JM.
     Characterization of group A Streptococcus strains recovered from
     Mexican children with pharyngitis by automated DNA sequencing of
     virulence-related genes: unexpectedly large variation in the gene
     (sic) encoding a complement inhibiting protein. J Clin Microbiol
     1997;35:3220-4.
  8. Stockbauer KE, Grigsby D, Pan X, Fu Y-X, Perea Mejia LM, Cravioto A,
     et al. Hypervariability generated by natural selection in an
     extracellular complement-inhibiting protein of serotype M1 strains of
     group A Streptococcus. Proc Natl Acad Sci U S A 1998;95:3128-33.
  9. Granlund M, Oberg L, Sellin M, Norgren M. Identification of a novel
     insertion element, IS1548, in group B streptococci, predominantly in
     strains causing endocarditis. J Infect Dis 1998;177:967-76.
 10. Berge A, Rasmussen M, Bjorck L. Identification of an insertion
     sequence located in a region encoding virulence factors of
     Streptococcus pyogenes. Infect Immun 1998;66:3449-53.
 11. Whatmore AM, Kapur V, Sullivan DJ, Musser JM, Kehoe MA. Non-congruent
     relationships between variation in emm gene sequences and the
     population genetic structure of group A streptococci. Mol Microbiol
     1994;14:619-31.
 12. Harbaugh MP, Podbielski A, Hugl S, Cleary PP. Nucleotide substitutions
     and small-scale insertion produce size and antigenic variation in
     group A streptococcal M1 protein. Mol Microbiol 1993;8:981-91.
 13. Johnson LP, Schlievert PM. Group A streptococcal phage T12 carries the
     structural gene for pyrogenic exotoxin type A. Mol Gen Genet
     1984;194:52-6.
 14. Musser JM, Kapur V, Kanjilal S, Shah U, Musher DM, Barg NL, et al.
     Geographic and temporal distribution and molecular characterization of
     two highly pathogenic clones of Streptococcus pyogenes expressing
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 15. Alland D, Kalkut GE, Moss AR, McAdam RA, Hahn JA, Bosworth W, et al.
     Transmission of tuberculosis in New York City. An analysis by DNA
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 16. van der Zee A, Mooi F, van Embden J, Musser J. Molecular evolution and
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 17. Robinson DA, Hollingshead SK, Musser JM, Parkinson AJ, Briles DE,
     Crain MJ. The IS1167 insertion sequence is a phylogenetically
     informative marker among isolates of serotype 6B Streptococcus
     pneumoniae. J Mol Evol 1998;47:222-9.
 18. Lawrence JG, Dykhuizen DE, DuBose RF, Hartl DL. Phylogenetic analysis
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 19. Stanley J, Jones CS, Threlfall EJ. Evolutionary lines among Salmonella
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     DNA polymorphism in the direct repeat cluster of Mycobacterium
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 23. Sreevatsan S, Pan X, Stockbauer KE, Connell ND, Kreiswirth BN, Whittam
     TS, et al. Restricted structural gene polymorphism in the
     Mycobacterium tuberculosis complex indicates evolutionarily recent
     global dissemination. Proc Natl Acad Sci U S A 1997;94:9869-74.
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     et al. Real-time molecular epidemiologic analysis of an outbreak of
     Streptococcus pyogenes invasive disease in US Air Force trainees. Arch
     Pathol Lab Med 1994;118:128-33.
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 26. Musser JM, Gray BM, Schlievert PM, Pichichero ME. Streptococcus
     pyogenes pharyngitis: characterization of strains by multilocus enzyme
     genotype, M and T protein serotype, and pyrogenic exotoxin gene
     probing. J Clin Microbiol 1992;30:600-3.
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     efficiently invade human respiratory epithelial cells. Proc Natl Acad
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 28. Cleary PP, McLandsborough L, Ikeda L, Cue D, Krawczak J, Lam H.
     High-frequency intracellular infection and erythrogenic toxin A
     expression undergo phase variation in M1 group A streptococci. Mol
     Microbiol 1998;28:157-67.
 29. Muotiala A, Seppala H, Huovinen P, Vuopio-Varkila J. Molecular
     comparison of group A streptococci of T1M1 serotype from invasive and
     noninvasive infections in Finland. J Infect Dis 1997;175:392-9.
 30. Davies DD, McGeer A, Schwartz B, Green K, Cann D, Simor AE, et al.
     Invasive group A streptococcal infections in Ontario, Canada. N Engl J
     Med 1996;335:547-53.
 31. Zurawski CA, Bardsley MS, Beall B, Elliott JA, Facklam R, Schwartz B,
     et al. Invasive group A streptococcal disease in metropolitan Atlanta:
     a population-based assessment. Clin Infect Dis 1998;27:150-7.
 32. Hughes MK, Hughes AL. Natural selection on Plasmodium surface
     proteins. Mol Biochem Parasitol 1995;71:99-113.
___________________________________________________________________________
Dispatches
___________________________________________________________________________
Dispatches
Gnathostomosis, an Emerging Foodborne Zoonotic Disease in Acapulco, Mexico
Norma Rojas-Molina,* Sigifredo Pedraza-Sanchez, Balfre Torres-Bibiano,*
Hector Meza-Martinez,* and Alejandro Escobar-Gutierrez
*Hospital Regional "Vicente Guerrero," Instituto Mexicano del Seguro
Social, Acapulco, Guerrero, Mxico; and Secretara de Salud, Mxico, DF,
Mxico
---------------------------------------------------------------------------
      Between 1993 and 1997, 98 gnathostomosis cases were clinically
      identified in Acapulco, Mexico. Intermittent cutaneous
      migratory swellings were the commonest manifestation. Larvae
      were identified in 26 cases, while in 72, final diagnosis was
      made on the basis of epidemiologic data, food habits, and
      positive enzyme-linked immunosorbent assay and Western blot
      results.
Gnathostomosis is a foodborne zoonotic disease caused by several species of
nematode Gnathostoma. The life cycle of this parasite is as follows: Adult
parasites of G. spinigerum are found in the stomach of mammals (e.g., dogs
and cats). feces containing ova reach the water (i.e., when domestic
parasitized animals live at the shore of a lagoon). Free-swimming
first-stage larvae are formed, which are ingested by the minute copepod
crustacean cyclops, and become second-stage larvae. Freshwater fish eating
cyclops are the second intermediate host. Larvae develop to the third state
(L3) in the fish muscles. Consumption of this fish by cats, dogs, or other
mammals results in development of adults in the gut, closing the cycle.
Humans acquire the infection by consuming raw or undercooked freshwater
fish. When a larva is ingested by a human host, no further development
occurs, but the larva migrates through subcutaneous tissue and internal
organs where it produces migratory swelling in the skin and other symptoms
depending on the site or organ affected. In most cases, symptoms are not
serious; however, if the parasite migrates to vital organs of the host, it
can cause severe illness or even death (1,2).
With the highest prevalence in Southeast Asia, gnathostomosis is now an
emerging public health problem in Peru, Ecuador and, since 1970, in Mexico
(3). In the 1970s and 1980s a few cases were reported in towns around
Presidente Miguel Aleman Dam and Papaloapan River basin in the Gulf of
Mexico (4). The parasites may have spread when new dams built on rivers of
the Pacific Ocean coast were unnoticeably seeded with infected fish. Until
now, G. spinigerum has been the only species of Gnathostoma identified in
Mexico (5).
Acapulco (estimated population 890,000), a resort on the southern Pacific
coast, is the major Mexican city where gnathostomosis cases have been
reported (5). Between December 1, 1993, and July 31, 1997, 98 patients with
symptoms compatible with gnathostomosis were identified at the outpatient
clinics of the Dermatology and Allergy Services, Hospital Regional "Vicente
Guerrero", IMSS. Because the hospital gives medical attention to
approximately 45% of the permanent residents of the city, the number of
gnathostomosis cases identified can be considered representative prevalence
in the local population.
A standardized questionnaire was administered to each patient, requesting
demographic data, medical history, clinical features, age, occupation,
years of residence at the city, and food habits. Baseline and diagnostic
investigations were made after obtaining a written consent. Gender and age
distribution were as follows: 50 were male and 48 female and all but two (5
and 10 years) were 20 to 45 years of age (mean age 36 years). Blood samples
were obtained for full blood and eosinophil counts. Serum total
immunoglobulin E (IgE) levels were measured by a commercial enzyme-linked
immunosorbent assay (ELISA) (Pharmacia Diagnostics, Uppsala, Sweden), and
the results were expressed as international units (IU)/mL. Biopsies were
taken from the progressing eruption and were examined for worms. Commonest
symptoms were intermittent episodes of localized migratory skin swelling,
with edema of variable size, slightly reddish, feverish at times,
accompanied by stabbing pain, burning sensation and pruritus. Initial edema
generally appeared on the abdomen. Recurring edema developed randomly,
mainly in the upper and lower extremities, gluteus, thorax, and face. The
duration of edema varied from 1 day to approximately 2 weeks. Only two
patients reported visceral manifestations, mainly epigastrial pain and
nausea, lasting approximately 2 days. Medium value for blood eosinophils
was 12%, and serum IgE was 302.6 Ul/mL (normal 10 to 180 Ul/mL). Although
identification of larvae by a biopsy is recommended for definitive
diagnosis, such identification was possible in only 16 cases, including
three in which the worms were easily excised after their outward migration
to the dermis as a consequence of treatment with albendazole as has been
recommended (6).
To assess the effectiveness of serologic tests for diagnosis, a
representative sample of patients were selected for antibody screening by
ELISA (7) and Western blot confirmation (8). Antigens used were two batches
of larval total extract. The first one was prepared with worms recovered
from patient's biopsies or from muscles of freshwater fishes (Dormilatum
latrifons) from laguna Tres Palos (a large freshwater lagoon 20 km
southeast Acapulco). The second batch was obtained from G. spinigerum
freeze-dried specimens donated by Dr. Wichit Rojekittkhum (Faculty of
Tropical Medicine, Mahidol University, Bangkok, Thailand). For ELISA,
protein concentration of the antigen was adjusted to 10 g/ml, and sera with
OD(sub 420) values >/= the OD(sub 420) average obtained in control sera plus
four standard deviations were considered positive. ELISA-positive results were
found in four of five parasitologically confirmed cases and 19 of 22
biopsy-negative patients. Immunoblots with sera from ELISA-positive
patients showed two strong bands (30 and 38 kDa) and two weak bands (35 and
43 kDa) not observed in sera from the control group. No differences between
the two batches of antigens were detected by ELISA or by immunoblotting.
However, by ELISA, higher OD(sub 420) values were seen when G. spinigerum
lyophilized antigen was used. Lyophilization may make the antigen better
for binding to the ELISA pates. No correlation was found with age, time of
onset, or parasitologic treatment in patients with negative serologic
results.
Although characteristic clinical manifestations could be very indicative of
gnathostomosis, up to now confirmation through identification of larvae has
been mandatory; nevertheless, larva identification is rare, and a
combination of such factors as permanent residence or recent visit to a
disease-endemic area, history of eating raw fish, exclusion of other
diseases, and results of serologic tests can be taken into account in
establishing a definitive diagnosis.
Inability to interrupt the parasite's life cycle and lack of effective
medical treatment (2) make preventive measures critical in controlling the
disease. Therefore, travelers to disease-endemic areas must be warned of
the possibility of acquiring gnathostomosis and be instructed to avoid
ingesting any form of raw fish. To protect the general population of
disease-endemic areas, public campaigns should be implemented and
encouraged against eating or selling raw or poorly cooked freshwater fish,
especially in the form of sushimi or "ceviche" (a spicy lime-marinated fish
salad of South American origin now very popular in Mexico).
---------------------------------------------------------------------------
      Dr. Rojas-Molina is an allergologist at Service of Allergy, Hospital
Regional "Vicente Guerrero," Instituto Mexicano del Seguro Social,
Acapulco, Guerrero, Mxico.
      Address for correspondence: Alejandro Escobar-Gutierrez, Departamento
de Investigaciones Inmunolgicas, Instituto Nacional de Diagnstico y
Referencia Epidemiolgicos, SSA, Carpio 470, Mxico, DF, 11340, Mxico; fax
525-341-3264; e-mail: indre@cenids.ssa.gob.mx.
References
  1. Rusnak JM, Lucey DR. Clinical gnathostomiasis: case report and review
     of the English-language literature. Clin Infec Dis 1993;16:33-50.
  2. Yoshimura K, 1998. Angiostrongylus (Parastrongylus) and less common
     nematodes. In: Cox EG, Kreier JP, Wakelin D, editors. Topley &
     Wilson's microbiology and microbial infections. 9th ed. Vol 5. London:
     Arnold; 1998. p. 635-59.
  3. Pelaez D, Perez-Reyes R. Gnatostomiasis humana en America. Rev
     Latinoam Microbiol 1970;12:83-91.
  4. Martnez-Cruz JM, Bravo-Zamudio R, Aranda-Patraca A, Martnez-Maran
     R. La gnatostomiasis en Mxico. Salud Pblica Mex 1989;31:541-9.
  5. Ogata K, Nawa Y, Akahane H, Daz Camacho SP, Lamothe-Argumedo R,
     Cruz-Reyes A. Short report: gnathostomiasis in Mexico. Amer J Trop Med
     Hyg 1998;58:316-8.
  6. Suntharasamai P, Riganti M, Chittamas S, Desakorn V. Albendazole
     stimulates outward migration of Gnathostoma spinigerum to the dermis
     in man. Southeast Asian J Trop Med Public Health 1992;23:716-22.
  7. Dharmkrong-at A, Migasena S, Suntharasamai P, Bunnag D, Priwan R,
     Sirisinha S. Enzyme-linked immunosorbent assay for detection of
     antibody to Gnathostoma antigen in patients with intermittent
     cutaneous migratory swelling. J Clin Microbiol 1986;23:847-51.
  8. Towbin H, Straehelin T, Gordon J. Electrophoresis transfer of proteins
     from polyacrylamide gels to nitrocellulose sheets: procedure and some
     applications. Proc Natl Acad Sci U S A 1979;76:4350-4.
________________________________________________________________________________
Dispatches
Acute Hemorrhagic Conjunctivitis Due to Enterovirus 70 in India
R.S. Maitreyi, L. Dar, A. Muthukumar, M. Vajpayee, I. Xess, R.B. Vajpayee,
P. Seth, and S. Broor
All India Institute of Medical Sciences, New Delhi, India
---------------------------------------------------------------------------
      An outbreak of acute hemorrhagic conjunctivitis occurred in
      Delhi, India, during August and September 1996. The etiologic
      agent was confirmed as enterovirus type 70 by a modified
      centrifugation-enhanced culture method followed by
      immunofluorescence and neutralization tests. After nearly a
      decade, this virus is reemerging as a cause of acute
      hemorrhagic conjunctivitis in India.
Acute conjunctivitis can be caused by viruses including enterovirus 70
(EV-70), coxsackievirus A24, and epidemic adenoviruses. These viruses may
also lead to acute hemorrhagic conjunctivitis (AHC), characterized by
photophobia, watering, and foreign body sensation, eyelid edema,
conjunctival hemorrhages, and superficial punctate keratitis. The disease
is self-limiting.
In 1996, an outbreak of AHC occurred in Delhi, north India, during the
rainy season (August and September). We conducted a study to identify the
etiologic agent by viral culture, immunofluorescence and neutralization
tests, and polymerase chain reaction (PCR).
The Study
We enrolled 13 patients with clinically diagnosed bilateral AHC who
attended the outpatient clinic of Rajendra Prasad Centre for Ophthalmic
Sciences during the outbreak. At the initial visit, all patients had a
complete ophthalmic examination, including slit-lamp biomicroscopy.
Conjunctival swabs were taken from the right eye of each patient. These
swabs were collected in 2 ml of Hanks balanced salt solution with
antibiotics and were transported on wet ice to the virology laboratory for
processing.
All samples were vortexed thoroughly and treated with antibiotics (1,000
IU/ml penicillin and 1,000 g/ml streptomycin). Specimens were clarified by
centrifugation at 700 x g for 10 minutes at 4C. Supernatant fluid was
separated and used for inoculation in cell culture. The samples were stored
at -70C until they were processed.
For virus culture, Hep-2cell monolayers were grown in 24-well plates,
containing 12-mm cover slips (Bellco Glass Inc., New Jersey, USA). Each
plate was seeded with 1 ml of Hep-2cell suspension, incubated at 37C in a
5% CO(sub 2) atmosphere, and used for a modification of the
centrifugation-enhanced viral culture technique (1).
Cover-slip monolayers were washed twice with phosphate-buffered saline
(PBS) before specimen inoculation. Two hundred microliters of each specimen
was inoculated in parallel in two 24-well plates containing Hep-2cell
monolayers grown onto 12-mm cover slips. Plates were centrifuged at 700 x g
for 60 minutes at room temperature. Inoculum was discarded and washed once
with PBS; infected cells were re-fed with 1.0 ml of Eagle's minimum
essential medium containing 2% fetal calf serum. The plates were reincubated
at 37C in a 5% CO(sub 2) atmosphere. Cover slips from one plate were
removed, washed twice with PBS, and fixed with chilled acetone at 48 hours
postinoculation for immunofluorescence. The parallel 24-well plate cultures
were observed for viral cytopathic effect, which included rounding and
refractility of cells and destruction of the monolayer at 2 to 4 days; the
resulting effect suggested enteroviral infection. Because a standardized
reverse transcriptase-PCR test for enteroviral RNA was available at the All
India Institute of Medical Sciences, New Delhi, India, we screened the
samples with this test.
Ten of the 13 clinical samples and the positive control showed the amplicon
band, while the negative control did not (Figure). The PCR results
suggested enteroviral infection, helped narrow the search for the etiologic
agent, and provided a rapid preliminary diagnosis.
Viral antigen was detected by indirect immunofluorescence staining (on the
fixed cover slips stored earlier) using specific antibodies to EV70,
coxsackievirus A24, and adenoviruses (Chemicon International Inc., CA,
USA). Infected cover-slip monolayers were stained with 25 l of monoclonal
antibodies, incubated in a moist chamber at 37C for 45 minutes, and washed
twice with PBS for 10 minutes each. Fluorescein-labeled anti-mouse
conjugate was added, and the cover slips were reincubated at 37C for 45
minutes, followed by a PBS washing step, as described above. The cover
slips were air-dried, mounted with glycerol buffer, and examined under
fluorescence microscopy.
		  [fig]    		 Figure: Reverse transcriptase-polymerase
                               chain reaction (RT-PCR) for enteroviral RNA
 from acute hemorrhagic conjunctivitis cases. Lane 1: pX174 DNA/Hae III
 marker; lane 2: negative control; lane 3: positive control (enteroviral
 RNA); lanes 4-6: clinical samples.
 For PCR, viral RNA was extracted by the guanidinium thiocyanate method
 (2) from 200 l of clinical specimen. The RNA pellet was resuspended in
 10 l of diethyl pyrocarbonate treated water. A 5-l volume was used for
 cDNA synthesis. The primers used for PCR amplification were selected from
 the highly conserved 5' noncoding region of enterovirus described by
 Rotbart et al. (3) and, using the same protocol, cDNA for PCR was
 synthesized by reverse transcription. Briefly, cDNA (10 Cl) was
 amplified in a PCR mix volume of 50 l, containing 100 ng of each primer,
 250 l of each dNTP, 1.5 mM MgCl(sub 2), 2.5 units Taq polymerase, and
 5 l of 10X PCR buffer. The tubes were subjected to 30 cycles at 94C for 1
 minute, 58C for 1 minute, and 72C for 2 minutes, respectively, followed
 by a final extension for 7 minutes at 72C. A 154-bp sized band of the
 PCR-amplified product was visualized under UV illumination on a 2%
 agarose gel after ethidium bromide staining.
The samples showing a viral cytopathic effect were given a second passage,
and if the effect was seen again, the 50% tissue infective dose of the
virus isolate was calculated. A virus neutralization test was performed
using 100 50% tissue infective doses of the isolate and virus-specific
antiserum.
Findings
All 13 patients enrolled in the study (9 male and 4 female, ages 14 to 37
years) had bilateral ocular involvement and described redness and watering
of the eyes, mild photophobia, and severe foreign body sensation. Ocular
examination showed severe conjunctival congestion, interspersed
subconjunctival hemorrhages, and superficial punctate epithelial keratitis.
No neurologic manifestations (as reported in an earlier epidemic) were
observed (4).
Cover-slip monolayers infected with a known EV-70 prototype (Kono)like
strain from a previous outbreak (5) and 10 of the 13 clinical specimens
showed specific cytoplasmic fluorescence with monoclonal antibodies to
EV-70. Infected monolayers tested with monoclonal antibodies to adenovirus
and coxsackievirus A24 yielded negative results. Neutralization test
results confirmed the identification, as all 10 clinical isolates were
neutralized by EV-70specific antisera. Also, the known EV-70 prototype
(Kono)like strain was neutralized by pooled convalescent-phase sera from
the patients in this outbreak. PCR was positive for EV-70 in 11 of the 13
cases, including the 10 culture-confirmed cases.
Conclusions
An outbreak of AHC was first reported from Ghana in 1969 and was referred
to as Apollo conjunctivitis (6). A new enterovirus (EV-70) was identified
as the etiologic agent of AHC (7); subsequently it spread to other parts of
Africa and Asia including India.
The first serologic evidence of EV-70 infection in India came from Bombay
(western India) in 1971-72 (4), and the first isolation was reported from a
single case during a small epidemic in southern India in 1975 (8). During
an epidemic in north India in 1981 (also during the rainy season), two
isolates of EV-70 prototype (Kono)like strain were reported from Delhi
(5), and antigen-positive cases were found by immunofluorescence in the
city of Chandigarh (9). The last reported outbreak of EV-70 from this
region (July to September 1986) was also confirmed only by demonstration of
antigen in cell scrapings by immunofluorescence (10). AHC due to EV-70
appears to have reemerged in north India after nearly a decade. During the
intervening period, a coxsackievirus A24 variant was circulating as a cause
of AHC in this region of India (11). However, a prime-type EV-70 isolate
was obtained from a case-patient during an outbreak in Pune, western India,
nearly 1,200 km from Delhi, in 1991 (12).
In this outbreak of AHC, we achieved an unusually high isolation rate of
EV-70 (10 of 13 cases) by using a modification of centrifugation-enhanced
culture. The results of neutralization tests indicate that the strains
circulating in this part of India continue to resemble the prototype strain
(also reported during the 1981 epidemic). PCR was useful because of its
rapidity and help in narrowing the search for an etiologic agent to
enteroviruses. With the availability of PCR based on EV-70 specific primers
(13), this highly sensitive centrifugation-enhanced technique is likely to
be used increasingly in the future.
---------------------------------------------------------------------------
      Dr. Maitreyi is a senior research fellow, Department of Microbiology,
All India Institute of Medical Sciences, New Delhi, India. Her areas of
expertise include tissue culture and molecular biology, and her research
focuses on respiratory viruses and enteroviruses.
      Address for correspondence: Shobha Broor, Department of Microbiology,
All India Institute of Medical Sciences, Ansari Nagar, New Delhi-110029,
India; fax: 91-11-686 2663; e-mail: broor@medinst.ernet.in.
References
  1. Pass RF, Britt WJ, Stagno S. Cytomegalovirus. Diagnostic procedures
     for viral, rickettsial and chlamydial infections, 7th ed. In: Lennette
     EH, Lennette DA, Lennette ET, editors. Washington: American Public
     Health Association; 1995.
  2. Chomczynski P, Sacchi N. Single step method of RNA isolation by acid
     guanidinium thiocyanate phenol chloroform extraction. Ann Clin Biochem
     1987;162:156-9.
  3. Rotbart HA. Enzymatic amplification of the enteroviruses. J Clin
     Microbiol 1990;28:438-42.
  4. Kono R, Miyamura K, Tajiri E, Shiga S, Sasagawa A, Irani PF, et al.
     Neurological complications associated with acute haemorrhagic
     conjunctivitis virus infection and its serological confirmation. J
     Infect Dis 1974;129:590-3.
  5. Manjunath N, Balaya S, Mahajan VM. Isolation of enterovirus 70 during
     conjunctivitis epidemic in Delhi in 1981. Indian J Med Res
     1982;76:653-5.
  6. Chatterjee S, Quarcoopome CO, Apenteng A. Unusual type of
     conjunctivitis in Ghana. Br J Ophthalmol 1970;54:628-30.
  7. Kono R, Sasagawa A, Ishii K, Sugiura S, Ochi M, Matsumiya H, et al.
     Pandemic of new type of conjunctivitis. Lancet 1972;i:1191-4.
  8. Christopher S, John J, Charles V, Ray S. Coxsackie virus A24 variant
     EH 24/70 and enterovirus type 70 in an epidemic of acute haemorrhagic
     conjunctivitis-a preliminary report. Indian J Med Res 1977;65:593-5.
  9. Pal SR, Szucs Gy, Melnick JL, Kaiwar R, Bharadwaj G, Singh R, et al.
     Immunofluorescence test for the epidemiological monitoring of acute
     haemorrhagic conjunctivitis cases. Bull World Health Organ
     1983;61:485-90.
 10. Kishore J, Manjunath N, Bareja U, Verma LK, Broor S, Seth P. Study of
     an outbreak of epidemic conjunctivitis in Delhi in 1986. Indian J
     Pathol Microbiol 1989;32:266-9.
 11. Broor S, Kishore J, Dogra V, Satapathy G, Seth P. An epidemic of acute
     haemorrhagic conjunctivitis caused by Coxsackie A24 variant. Indian J
     Med Res 1992;95:253-5.
 12. Bhide VS, Prasad SR, Gogate SS. Isolation of a variant of enterovirus
     70 from a patient during an epidemic of acute haemorrhagic
     conjunctivitis in Pune in 1991. Acta Virol 1994;38:245-6.
 13. Uchio E, Yamazaki K, Aoki K, Ohno S. Detection of enterovirus 70 by
     polymerase chain reaction in acute haemorrhagic conjunctivitis. Am J
     Ophthalmol 1996;122:273-5.
____________________________________________________________________________
Dispatches
Mycobacterium sp. as a Possible Cause of Hypersensitivity Pneumonitis in
Machine Workers
Brian G. Shelton,* W. Dana Flanders, and George K. Morris*
*PathCon Laboratories, Norcross, Georgia, USA; and Emory University,
Atlanta, Georgia, USA
---------------------------------------------------------------------------
      Hypersensitivity pneumonitis (HP) in workers exposed to metal
      removal fluids (MRFs) is increasing. This study supports the
      hypothesis that aerosolized mycobacteria colonizing the MRFs
      likely cause the disease. Three case studies of HP outbreaks
      among metal workers showed potentially high exposures to a rare
      and newly proposed Mycobacterium species. Retrospective review
      of samples submitted to our laboratory showed an association
      between presence of mycobacteria and HP.
Interest in occupational hypersensitivity pneumonitis (HP) or extrinsic
allergic alveolitis is increasing (1,2). HP, a rare group of diseases
characterized by recurrent dyspnea, cough, and systemic signs such as
myalgia and fever, is caused by repeated exposure and subsequent
sensitization to various antigens. Recognized antigens are often fungal or
bacterial. The disease process is thought to involve
lymphocyte-sensitization and cell-mediated immune response that ultimately
results in alveolitis. Types of occupational HP include farmer's lung, bird
fancier's lung, and mushroom worker's lung.
HP has recently been recognized among metal workers, and as evidenced at a
recent meeting of the Automotive Manufacturers Association in Detroit
(September 1997), controversy exists as to its possible cause(s).
Researchers from the National Institute for Occupational Safety and Health
recently reported that four of six outbreaks of HP in metal-working
facilities yielded unusual flora (e.g., Mycobacterium chelonae) in
contaminated MRFs (2,3). They discussed acid-fast bacteria, nontuberculous
mycobacteria, gram-positive bacteria, and fungi as possible causes (3) but
more recently concluded that the specific etiologic agent(s) for HP among
workers exposed to (MRF) aerosol remain(s) unknown (2). Advances in metal
removal fluid technology have led to the use of synthetic, semisynthetic,
and soluble fluids, as opposed to traditional oil coolants. These
water-based coolants are typically recycled and often become colonized by
microorganisms. We present case reports and observations that further
suggest mycobacteria may be a cause of occupational HP.
Case Report 1
We investigated a single case of HP in an employee who worked in an engine
production facility in 1997. Other than contaminated fluids, no chemicals
or substances known to cause HP were used at the facility. This 45-year-old
male nonsmoker, in otherwise good health, worked in a wet machining area of
the shop. His symptoms, which manifested themselves while he was at work,
included cough, dyspnea, and hoarseness. One episode, which included
malaise and fever of 102F, led to hospitalization. In-hospital evaluation
included a chest X-ray, which showed bilateral interstitial infiltrates, a
high resolution computed tomography scan consistent with alveolitis, normal
spirometry with a decreased diffusing capacity for carbon monoxide, and
nonspecific transbronchial biopsy findings. A diagnosis of HP was made, and
the patient was treated with oral glucocorticoids and relocated to an
office environment at work. His symptoms gradually improved over several
weeks, and the steroids were discontinued.
Mycobacteria in the M. chelonae complex were identified in high numbers in
numerous bulk coolant samples (from nondetectable to 6.6x10[sup 6] CFU/ml) and
counts from 56 to 2,256 CFU/m(sup 3) in positive air samples around the colonized
machines. Other organisms identified in the bulk samples included
Pseudomonas pseudoalcaligenes, P. alcaligenes, and yeast with counts
ranging from nondetectable to 1.7x10(sup 6) CFU/ml. The Mycobacterium isolates
were identical to M. immunogen (4), a proposed rare and new species of
rapidly growing mycobacteria with similarities to both M. chelonae and M.
abscessus. The case-patient (Case 1) and an unaffected co-worker (Control
1) yielded a significantly elevated antibody immunoglobulin G (IgG) titer
against this Mycobacterium and other organisms isolated from coolant
samples at the facility. Five other unaffected workers (Controls 2-6) who
worked in the same area as the case-patient were tested, as were 23 other
controls comprising the reference population not known to have exposure to
the facility. The standard deviation index used in the Figure was
calculated by subtracting the mean IgG/ml level of the reference population
from the patient's IgG/ml. This value was then divided by the standard
deviation of the reference population to give the standard deviation index.
For example, the levels were 5 or more in the case-patient, indicating that
his levels were at least 5 standard deviations higher than the
corresponding mean antibody levels in the reference population. The serum
antigen-specific antibody levels were measured by enzyme-linked
immunosorbent assay.
 					[fig]
 Figure. Immunoglobulin G (IgG) antibody titer vs. microbial extract from
 metal removal fluid.
Case Report 2
We were also involved in the investigation of HP at an engine manufacturing
plant that had numerous machines for cutting and grinding metal. The
facility employed approximately 700 machine workers. After HP was diagnosed
by a treating pulmonologist in two middle-aged male machinists, the
facility was inspected for known causes of HP. No causal exposures were
identified other than exposure to cutting fluids, which was common to the
two cases. Samples of machine fluid samples yielded Mycobacterium colony
counts of 10(sup 2) to 10(sup 7) per ml, with mycobacteria being the most
predominant organism in some fluids. Air samples near colonized machines
yielded high counts of mycobacteria, some of which exceeded the upper limits
of the sampler (>9,424 CFU/m[sup 3] of air). Other organisms in air samples
included Corynebacterium, Klebsiella-like organisms, Bacillus, Rhodococcus,
Hyalodendron, Cladosporium, and nonsporulating fungi at levels of 24 CFU/m(sup 3)
to 1,319 CFU/m(sup 3). No mycobacteria were detected immediately outside the
facility. The mycobacteria detected were rapid growers identical to the
newly proposed species M. immunogen. We evaluated a sister plant (in the
same state) that had no known cases of HP; no mycobacteria were detected
from samples of cutting fluids.
Case Report 3
We are currently investigating a cluster of five cases of
physician-diagnosed HP at a large metal working factory in the midwestern
part of the United States. Other than contaminated fluids, no chemicals or
other substances known to cause HP are used at the facility. Samples of
MRFs showed counts of mycobacteria ranging from nondetectable to >10(sup 6)
CFU/ml of sample. Again, the Mycobacterium species was identical to the
newly proposed species, M. immunogen. Other organisms identified in bulk
samples included Bacillus, Pseudomonas, Cladosporium, Trichoderma, and
nonsporulating fungi at concentrations of <10 CFU/ml to 6,000 CFU/ml.
Conclusions
All the cases we report occurred in men exposed to metal removal fluids
heavily colonized with mycobacteria. Moreover, we have documented high
airborne concentrations of mycobacteria in the workplace. At least five
other clusters of HP in workers exposed to mycobacteria have been reported
(2,3,5-7). In one outbreak of HP in metal workers (6,7), a sputum culture
sample from one of the six patients in the outbreak yielded M. chelonaea
possibly rare event in a person without pulmonary infection attributed to
that organism. By documenting further clusters of HP among workers exposed
to MRF contaminated with mycobacteria, our findings provide an additional
link suggesting a possible association between mycobacteria and HP.
It is possible that mycobacteria commonly contaminate MRFs even in the
absence of HP. To assess this possibility, we identified all samples of MRF
that had been submitted for culture to our laboratory since 1993 when we
routinely started testing these samples for mycobacteria. Of the seven
facilities from which we received samples as part of an investigation of
known HP, six had mycobacteria. Of the eight facilities without known HP,
only one had mycobacteria (odds ratio = 42, 95% confidence interval =
1.5-2194.47, p = 0.01 by Fisher's exact test). Thus, in facilities without
HP, mycobacteria were not commonly found in MRFs. Even though they
represent a review of existing records and not a prospectively designed
study, these data suggest that mycobacterial contamination of MRFs may
increase the risk for HP.
The hypothesis that mycobacterial contamination in cutting fluids is a
cause of HP has high biologic plausibility supported by several
observations: Cutting fluids and their contaminants can become easily
aerosolized thus providing a high opportunity for inhalation exposure;
mycobacteria have an acid-fast cell wall that is highly antigenic;
mycobacterium adjuvants, often used by immunologists to elicit a stronger
immunologic reaction, may lead to granuloma formation if used more than
once; elevated IgG antibody levels directed at mycobacteria were found in
patients with HPthe case-patient in case report 1 had elevated antibody
titers to several antigens including mycobacteria in the MRFs (although not
diagnostic of HP, this elevated titer documents exposure and immunologic
response to mycobacteria in this patient); outbreaks of HP in hot tubs have
been reported with mycobacteria (M. avium) as the suspect cause (8); and
animal models used to study HP have been produced by using antigenic
extracts from mycobacteria (9).
This hypothesismycobacteria as an antigen, or as an adjuvant that enhances
reactions to other antigens found in the environmentis biologically
plausible. Mycobacteria may be an important cause of HP in occupational
settings or other settings in which exposure to contaminated aerosolized
aqueous solutions is possible. These data and observations alone neither
prove nor disprove a causal link. However, given the available data from
the case studies presented here and elsewhere (2,3,5-7), the ability of
Mycobacterium to proliferate to high levels in certain water-based MRFs,
biologic plausibility, and the lack of use of other substances known to
cause HP at these facilities, exposure to mycobacteria should be considered
as a possible cause.
---------------------------------------------------------------------------
      Dr. Shelton is vice president of PathCon Laboratories, a private
laboratory specializing in the investigation of airborne environmental
microorganisms. His research interests are in the epidemiology of
environmental microorganisms in building water systems and indoor air. He
has conducted numerous outbreak investigations and worked on the prevention
and control of Legionnaires' disease. He was the first to use environmental
concentrations of Legionella to predict disease risk.
Address for correspondence: Brian G. Shelton, PathCon Laboratories, 270
Scientific Drive, Suite 3, Norcross, Georgia 30092, USA: fax: 770-446-0610;
e-mail: bshelton@pathcon.com.
References
  1. Centers for Disease Control and Prevention. Biopsy-confirmed
     hypersensitivity pneumonitis in automobile production workers exposed
     to metalworking fluidsMichigan, 1994-1995. MMWR Morb Mortal Wkly Rep
     1996;45:606-10.
  2. Centers for Disease Control and Prevention. Criteria for a recommended
     standard: occupational exposure to metalworking fluids. Cincinnati
     (OH): NIOSH; 1998. Publication No. 98-102.
  3. Kreiss K, Cox-Ganser J. Metalworking fluid-associated hypersensitivity
     pneumonitis: a workshop summary. Am J Ind Med 1997;32:423-32.
  4. Wilson RW, Steingrube VA, Bottger EC, SpringerB, Brown BA, Jost KC, et
     al. Recognition of a new taxon within the Mycobacterium
     abscessus-Mycobacterium chelonae complex and proposal of Mycobacterium
     immunogen sp. nov. [abstract #16]. Washington: American Society for
     Microbiology; 1998.
  5. Zacharisen MC, Kadambi AR, Schlueter DP, Kurup VP, Shack JB, Fox JL,
     et al. The spectrum of respiratory disease associated with exposure to
     metal working fluids. J Occup Environ Med 1998;40:640-7.
  6. Bernstein DI, Lummus ZL, Santilli G, Siskosky J, Bernstein IL. Machine
     operator's lung. A hypersensitivity pneumonitis disorder associated
     with exposure to metalworking fluid aerosols. Chest 1995;108:636-41.
  7. Muilenburg ML, Burge HA, Sweet T. Hypersensitivity pneumonitis and
     exposure to acid-fast bacilli in coolant aerosols [abstract #683]. J
     Allergy Clin Immunol 1993;91:311.
  8. Embil J, Warren P, Yakrus M, Stark R, Corne S, Forrest D, et al.
     Pulmonary illness associated with exposure to Mycobacterium-avium
     complex in hot tub water. Hypersensitivity pneumonitis or infection?
     Chest 1997;111:813-6.
  9. Richerson HB, Suelzer MT, Swanson PA, Butler JE, Kopp WC, Rose EF.
     Chronic hypersensitivity pneumonitis produced in the rabbit by the
     adjuvant effect of inhaled muramyl dipeptide (MDP). Am J Pathol
     1982;106:409-20.
____________________________________________________________________________
Dispatches
Evaluating Diagnosis and Treatment of Oral and Esophageal Candidiasis in
Ugandan AIDS Patients
Maurizio Ravera, Alberto Reggiori, Anna Maria Agliata, Roberto Pidoto Rocco
Regional Teaching Hospital, Hoima, Uganda
---------------------------------------------------------------------------
      A randomized cross-over clinical and endoscopic evaluation of
      85 Ugandan patients showed that esophageal candidiasis in AIDS
      patients with oral candidiasis could be managed without
      endoscopy and biopsies. Oral lesions, especially when
      accompanied by esophageal symptoms, were sufficient for
      diagnosis. Miconazole was more effective than nystatin in
      treating esophageal candidiasis and could be a valid
      alternative to more expensive azolic drugs in developing
      countries.
Candidiasis, a well-known opportunistic infection of AIDS patients, is the
leading cause of infectious esophagitis (1,2). Studies show similar
prevalence of Candida esophagitis in AIDS patients in the West (9.1% to
31%) (3-5) and in Africa (7.3% to 27%) (6-7). In most treated patients (80%
to 100%) Candida esophagitis recurs after 3 months (8,9). Nevertheless, in
patients with AIDS, candidiasis generally does not become systemic, and
thus, clinical cure is important (9). Defining the most effective
diagnostic and therapeutic approach to curing Candida esophagitis in AIDS
patients is especially important in developing countries, which often have
limited resources.
Diagnosis of esophageal candidiasis is usually based on the endoscopic
appearance of the typical mucosal lesions and on histopathologic studies
(10-12). Several western studies have shown that the diagnosis of this
disease in AIDS patients can be made on clinical findings alone because the
positive predictive value of esophageal symptoms as indexes of esophageal
infection is 71% to 100% (10,13,14). Such an evaluation has not yet been
made in African AIDS patients.
Several therapeutic regimens have been effective in treating oral and
esophageal candidiasis (8,15-23). For the past decade, oral nystatin
therapy has been considered effective in controlling Candida esophagitis
(11). In tropical countries, the efficacy of nystatin in treating this
disease is not well known, although a recent study in Zaire reported a cure
rate of less than 10% (24). We evaluated the diagnostic accuracy of
esophageal symptoms in predicting Candida esophagitis in Ugandan AIDS
patients with oral candidiasis and compared the effectiveness of miconazole
and nystatin in treating oral and endoscopically proven esophageal
candidiasis in these patients.
The Study
From September 1994 to December 1995, 320 consecutive AIDS patients were
observed at the Gastroenterology Department of Hoima Hospital in Uganda.
Among them, 85 (45 women, 40 men, mean age 27.1, standard deviation [SD]
5.3 years) fulfilled admission criteria: positive HIV test or clinical
diagnosis of AIDS and presence of oral patchy white plaques as markers of
oral candidiasis. The district medical officer and the hospital medical
superintendent granted approval for the study, and informed consent was
obtained from each patient. Patients were considered symptomatic if they
had any of the following symptoms: odinophagia, dysphagia, or retrosternal
burning pain.
All patients were hospitalized, and the upper digestive tract was examined
endoscopically. The diagnosis of esophageal candidiasis was made at the
examination. All patients had the same spectrum of lesions: patchy white
plaques, confluent pseudomembrane, and friable mucosa. Routine
histopathologic assessment was not performed, mainly because of cost.
Patients were randomly assigned to the nystatin or miconazole regimen; a
stratified randomization method was used to balance treatment groups by
esophageal symptoms, age, and sex (Table). Nystatin tablets were given at a
dose of 1,000,000 I.U. every 8 hours for 7 days (according to Uganda
Ministry of Health 1993 National Standard Treatment Guidelines), while
miconazole tablets were administered at a dose of 250 mg every 6 hours for
7 days (25). At a mean follow-up of 7.6 days (SD 0.9), the patients'
symptoms were reassessed, and the upper digestive tract was reexamined
endoscopically. The endoscopist was blind to the treatment used. Patients
given nystatin who still had candidiasis were placed on the miconazole
regimen and tested for candidiasis 1 week later.
 Table. Characteristics of patients participat-  Findings
 ing in the study
                                                 Most (90.8%) (42 female,
 ------------------------------------------------35 male, mean age 28.0 
                        No. Mean age     Sex     5.8 years) of the study
                            (yr) (S.D.)  M/F     participants had both
 ------------------------------------------------oral and esophageal
                                                 candidiasis. Forty
 Oral candidiasis       85  24.0 (6.7)   32 / 53 (47.1%) had esophageal
 Esophageal symptoms)   40               17 / 23 symptoms, and all had
  (sup a)                                        esophageal candidiasis at
   Nystatin group       20  23.9 (6.1)   7 / 13  endoscopy. Sensitivity,
   Miconazole group     20  23.7 (6.6)   8 / 12  specificity, and the
 Esophageal candidiasis 77  23.7 (6.4)   27 / 50 positive and negative
   Nystatin group       37  24.2 (6.5)   12 / 22 predictive values of
   Miconazole group     40  23.4 (6.1)   15 / 28 esophageal symptoms as
 ----------------------------------------------- markers of esophageal
 (sup a)Esophageal symptoms are any of the       infection were 83.3%
 following: odinophagia, dysphagia,              (confidence interval [CI]
 retrosternal burning pain.                      69.2 to 92.0), 100% (CI
                                                 88.3 to 100), 100% (CI
                                                 89.1 to 100), and 82.2%
                                                 (CI 67.4 to 91.5),
                                                 respectively.
Esophageal symptoms disappeared in 10 (27.0%) of the 37 patients in the
nystatin group and in 38 (95.0%) of 40 patients in the miconazole group
(Yates chi-square = 34.99, p <0.001). Oral candidiasis was cured in all
patients in both groups; esophageal candidiasis was cured in 8 (21.6%)
patients in the nystatin group and in 37 (92.5%) patients in the miconazole
group (Yates chi-square = 36.89, p <0.001). Of the 29 patients who did not
respond to nystatin, 27 (93.1%) were cured with miconazole (Figure). No
adverse effects were observed in either group.
More than 90% of AIDS patients with oral candidiasis in this study also had
esophageal candidiasis, thus confirming that such an association is also
very strong in Uganda (14,26). A little more than half (51.9%) of 77
patients with esophageal candidiasis also had esophageal symptoms. Our
findings and those of other studies support the observation that esophageal
candidiasis could be suspected if oral thrush is present, especially when
esophageal symptoms are associated (10,14,26,27). Thus, in tropical
countries, endoscopic assessment and biopsies might best be reserved for
patients who have esophageal symptoms after receiving prolonged antifungal
treatment to confirm diagnosis of candidiasis or to determine other
infectious causes of this symptoms (e.g., herpes simplex virus infection,
cytomegalovirus infection, cryptosporidiosis) (2,23).
Although our study was
not designed to detect 				[fig]
recurrence of             Figure. Esophageal candidiasis treatment flow.
candidiasis, esophageal
candidiasis is likely to recur in AIDS patients within 12 months from any
antifungal treatment; if it does, response to therapy is worse than
response to initial therapy (28). For this reason, as well as the
possibility of resistance to miconazole (sporadic cases have been reported
[29]), more expensive azolic drugs (e.g., fluconazole) should be reserved
for recurrences of the disease; the disease should be treated initially
with less expensive, but more effective drugs (e.g., miconazole).
In sum, AIDS patients with oral candidiasis in countries similar to Uganda
can be managed without endoscopic and bioptic assessments since oral
lesions are typical and a high prevalence of esophageal involvement is
expected (with or without symptoms) (>90% of cases in our study). In our
patients, nystatin had a very low cure rate in the treatment of esophageal
candidiasis in AIDS patients; however, it could still play a role in the
treatment of oral candidiasis, especially in nonimmunocompromised patients,
in whom concomitant esophageal involvement is less common. On the other
hand, miconazole, a medium-priced azolic drug, was very effective and could
be a valid alternative to more expensive azolic drugs in developing
countries.
---------------------------------------------------------------------------
Acknowledgments
      We thank Dr. G. Oundo, medical superintendent, and the staff of Hoima
Hospital.
      This study was funded by International Service Volunteers'
Association, Kampala, Uganda.
      Dr. Ravera is a specialist in gastroenterology and digestive
endoscopy. A researcher at the Italian National Health Institute, he serves
as site coordinator/monitor of the UNAIDS PETRA study at Nsambya Hospital,
Kampala, Uganda. His research interests include gastroenterology,
endoscopy, infectious diseases, and HIV/AIDS.
Address for correspondence: Maurizio Ravera, c/o AVSI P.O. Box 6785,
Kampala, Uganda; fax: 256-41-266967; e-mail: petra@swiftuganda.com.
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  2. Lopez Dupla M, Mora Sanz P, Pintado Garcia V, Valencia Ortega E, Uriol
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  7. Colebunders R, Latif AS. Natural history and clinical presentation of
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____________________________________________________________________________
Dispatches
Neospora caninum Infection and Repeated Abortions in Humans
Eskild Petersen,* Morten Lebech,* Lene Jensen, Peter Lind, Martin Rask,
Peter Bagger, Camilla Bjrkman, and Arvid Uggla
*Statens Serum Institut, Copenhagen, Denmark; State Veterinary Laboratory,
Copenhagen, Denmark; Rigshospitalet, Copenhagen, Denmark; and Swedish
University of Agricultural Sciences, Uppsala, Sweden
---------------------------------------------------------------------------
      To determine whether Neospora caninum, a parasite known to
      cause repeated abortions and stillbirths in cattle, also causes
      repeated abortions in humans, we retrospectively examined serum
      samples of 76 women with a history of abortions for evidence of
      N. caninum infection. No antibodies to the parasite were
      detected by enzyme-linked immunosorbent assay,
      immunofluorescence assay, or Western blot.
Neospora caninum, an intracellular protozoan parasite closely related to
Toxoplasma gondii (1,2), was first described in dogs in Norway in 1984 and
later in a wide range of other mammals including cattle, goats, horses, and
sheep. The life cycle of N. caninum is only partially known, but the dog
has recently been established as its definitive host (3). The pathogen's
only known natural route of transmission (which can occur during sequential
pregnancies in cattle) is transplacental (4).
N. caninum is now recognized as the most common cause of repeated abortions
and stillbirths in cattle, and infected herds have been reported in most
parts of the world, including Scandinavia (4-6). Infected, live-borne
offspring may have neurologic symptoms including progressive paralysis.
When experimentally transferred to pregnant nonhuman primates, N. caninum
has caused fetal infection. The fetal lesions closely resembled those in
congenital toxoplasmosis (7). N. caninum organisms are morphologically very
similar to T. gondii, the pathogen responsible for toxoplasmosis; however,
the two species have distinct antigenic characteristics and can be
distinguished by serologic and immunohistochemical methods (4).
No case of N. caninum infection has been described in humans. However,
because of the organism's close phylogenetic relationship to T. gondii and
its wide range of potential hosts, the possibility of human N. caninum
infection cannot be excluded. We investigated serologically the possible
presence of N. caninum infection in Danish women who had repeated abortions
of unknown cause.
The Study
The study included 76 women (mean age 30.8 years, range 19 to 41 years) who
had had repeated abortions or intrauterine death of the fetus. Blood
samples were obtained at the time of abortion or within 3 months of fetal
death. The study participants had been referred to the Department of
Gynecology and Obstetrics, Rigshospitalet, Copenhagen, Denmark, between 1
September 1991 and 31 October 1992 as part of a larger study of pregnant
women with repeated primary or secondary abortions or repeated intrauterine
fetal deaths. Serum specimens were tested for antibodies to N. caninum and
T. gondii as described below.
Findings
The absorbence values for the human serum samples were 0.10 to 1.24
absorbence units, whereas the mean value for the presumed N.
caninum-negative human control serum was 0.26 (0.13 to 0.56). The mean
absorbence values for the high-positive and low-positive control pig sera
were 1.73 (1.54 to 1.93) and 0.87 (0.85 to 1.07), respectively. As no true
N. caninum-negative or -positive human sera were available, serum specimens
with absorbencies 0.50 (n = 12) were selected for further investigation
(Table).
 Table. Results of enzyme-linked                  None of the 12 specimens
 immunosorbent assay (ELISA)(sup a) tests for     showed specific
 Neospora caninum and of the Sabin-               fluorescence in the
 Feldman dye test(sup b) for Toxoplasma           indirect fluorescence
                                                  antibody test (IFAT) at
 gondii                                           dilution 1:640 with N.
 ------------------------------------------------ caninum tachyzoites that
 Sample no.         ELISA mean OD      Dye test   had been cultivated in
                                                  vitro (6). (Sera had been
 ------------------------------------------------ diluted in twofold serial
 90                     0.720             0       dilutions from 1:20 in
                                                  phosphate-buffered
 107                    0.627             0       saline.) Of the 12, only
 262                    0.578            1:50     3 had T. gondii
 264                    1.238             0       antibodies. The
                                                  reactivities in the N.
 276                    1.043            1:50     caninum enzyme-linked
 279                    1.032             0       immunosorbent assay
 282                    0.647            1:10     (ELISA) were not
                                                  associated with the
 285                    0.656             0       presence of T. gondii
 287                    1.143           1:6250    antibodies (Table). Only
                                                  1 of the 12 human serum
 289                    0.818             0       specimens tested showed
 295                    0.583                     reactivity against the N.
 297                    0.541             0       caninum antigen by
                                                  Western blot analysis.
 ------------------------------------------------ This specimen, number
 (sup a)The ELISA was modified from Bjrkman      279, recognized an
 et al. (8,9). A T. gondii-negative               antigen with apparent
 human serum specimen was used as a               molecular weight of 60
 presumed N. caninum-negative control,            kDa (Figure, lane 11 and
 and serum specimens from                         12). This antigen was not
 experimentally N. caninum-infected               recognized by the N.
 pigs were used as positive controls. A           caninum-positive pig
 low-positive serum was collected from            sera, and serum 279 did
 a pig infected 11 days before sampling           not recognize any of the
 and a high-positive control serum was            low-molecular weight
 pooled from pigs infected for at least           antigens recognized by
 3 weeks (10). No reaction in human               the N. caninum-positive
 sera was definitely positive.                    pig sera. Three serum
 (sup b)The dye test was used to demonstrate      specimens reacted with
 antibodies to T. gondii as described             the T. gondii antigen;
 11) but using in vitro cultured T.               all were T.
 gondii.                                          (gondii-positive in the
                                                  dye test.
Because of the biologic similarities between N. caninum and the human
pathogen T. gondii, it has been speculated that N. caninum could be
transmissible to humans. Since repeated abortions and stillbirths are
common manifestations of neosporosis in cattle (4), women with a history of
repeated abortions seemed an obvious category to investigate for human N.
caninum infection. However, in this study of serum samples from women with
repeated abortions, no evidence of N. caninum infection was detected.
The assays we used were based on methods used for T. gondii analyses; we
used the same conjugates and serum dilutions found optimal in these
analyses. The N. caninum immunostimulating complex antigen has a high
specificity (14) and has been used for serologic investigations in
different animal species (8,9,15). It was therefore anticipated that it
would be applicable in a human system as well. However, because we could
not define a proper cut-off for the assay, we further investigated the
serum samples with the highest ELISA absorbence values by IFAT, regarded as
the reference test for N. caninum antibodies in different species (4), and
Western blot. None of the human sera investigated showed any reactivity in
IFAT. Only one of the specimens reacted with the N. caninum antigen in the
Western blot. However, because it only reacted with a band not recognized
by sera from the infected pigs, the reaction was considered unspecific, and
cross-reactivity between T. gondii and N. caninum was not found.
 					[fig]
 Figure. Western blot of Toxoplasma gondii or Neospora caninum antigen.
 Analysis was performed essentially as described by Sharma et al. (12) by
 using tachyzoites from in vitro culture of the N. caninum NC-1 isolate (13)
 and the T. gondii RH strain (10). Lanes 1-4 were probed with control sera
 and lanes 5-12 with human sera with high absorbencies in the N. caninum
 enzyme-linked immunosorbent assay.
 Lane 1: T. gondii-positive human serum and T. gondii antigen; Lane 2: N.
 caninum-positive pig serum and T. gondii antigen; Lane 3: T.
 gondii-positive human serum and N. caninum antigen; Lane 4: N.
 caninum-positive pig serum and N. caninum antigen; Lane 5 and 6: Serum 262:
 T. gondii and N. caninum antigens, respectively; Lane 7 and 8: Serum 264:
 T. gondii and N. caninum antigens, respectively; Lane 9 and 10: Serum 276:
 T. gondii and N. caninum antigens, respectively. Lane 11 and 12: Serum 279:
 T. gondii and N. caninum antigens, respectively.
That we found no evidence of N. caninum infection in women who had repeated
spontaneous abortions does not rule out the possibility that the infection
might occur in humans. The predominant effects of neosporosis in dogs are
primarily progressive neurologic signs including paralysis. It might,
therefore, be worthwhile to examine human patients with clinical symptoms
other than abortions, e.g., neurologic disorders of unknown etiology.
Furthermore, the possible presence of N. caninum in patients with weakened
immune systems should be considered. Researchers might continue the search
for N. caninum by using serologic tests, as we did, or, alternatively, by
using material collected at biopsy or autopsy for polymerase chain reaction
or immunohistochemical analysis.
---------------------------------------------------------------------------
Acknowledgments
      We thank Lisbeth Petersen, Lis Wassmann, and Ann Lene Andresen for
skillful technical assistance.
      Dr. Petersen is a specialist in infectious diseases and tropical
medicine at the Laboratory of Parasitology, Statens Serum Institut,
Denmark's national reference center for diagnosis and research of human
parasitic infections. His areas of expertise include immunology and
epidemiology, primarily applied to malaria and congenital toxoplasmosis.
      Address for correspondence: Eskild Petersen, Laboratory of
Parasitology, Statens Serum Institut, DK-2300 Copenhagen S. Denmark;
fax:45-3268-3033; e-mail: ep@ssi.dk.
References
  1. Dubey JP, Carpenter JL, Speer CA, Topper MJ, Uggla A. Newly recognized
     fatal protozoan disease of dogs. J Am Vet Med Assoc 1988;192:1269-85.
  2. Holmdahl OJM, Mattsson JG, Uggla A, Johansson K-E. The phylogeny of
     Neospora caninum and Toxoplasma gondii based on ribosomal RNA
     sequences. FEMS Microbiol Lett 1994;119:187-92.
  3. McAllister MM, Dubey JP, Lindsay DS, Jolley WR, Wills RA, McGuire AM.
     Dogs are definitive hosts of Neospora caninum. Int J Parasitol
     1998;28:1473-8.
  4. Dubey JP, Lindsay DS. A review of Neospora caninum and neosporosis.
     Vet Parasitol 1996;67:1-59.
  5. Holmdahl OJM, Bjrkman C, Uggla A. A case of Neospora associated
     bovine abortion in Sweden. Acta Vet Scand 1995;36:279-81.
  6. Agerholm JS, Willadsen CM, Nielsen TK, Giese SB, Holm E, Jensen L, et
     al. Diagnostic studies of abortion in Danish dairy herds. J Vet Med
     1997;A44:551-8.
  7. Barr BC, Conrad PA, Sverlow KW, Tarantal AF, Hendrickx AG.
     Experimental fetal and transplacental Neospora infection in the
     nonhuman primate. Lab Invest 1994;71:236-42.
  8. Bjrkman C, Lundn A, Holmdahl J, Barber J, Tress AJ, Uggla A.
     Neospora caninum in dogs: detection of antibodies by ELISA using an
     iscom antigen. Parasite Immunol 1994;16:643-8.
  9. Bjrkman C, Holmdahl OJM, Uggla A. An indirect enzyme-linked
     immunosorbent assay (ELISA) for demonstration of antibodies to
     Neospora caninum in serum and milk of cattle. Vet Parasitol
     1997;68:251-6.
 10. Jensen L, Jensen TK, Lind P, Henriksen SA, Uggla A, Bille-Hansen V.
     Experimental porcine neosporosis. Acta Pathologica et Microbiologica
     Scandinavica 1998;106:475-82.
 11. Sabin AB, Feldman HA. Dyes as microchemical indicators of a new
     immunity phenomenon affecting a protozoon parasite (Toxoplasma).
     Science 1948;108:660-3.
 12. Sharma SD, Mullenax J, Araujo FG, Erlich HA, Remington JS. Western
     blot analysis of the antigens of Toxoplasma gondii recognized by human
     IgM and IgG antibodies. J Immunol 1983;131:977-83.
 13. Dubey JP, Hattel AL, Lindsay DS, Topper MJ. Neonatal Neospora caninum
     infection in dogs: isolation of the causative agent and experimental
     transmission. J Am Vet Med Assoc 1988;193:1259-63.
 14. Bjrkman C, Lundn A. Application of iscom antigen preparations in
     ELISAs for diagnosis of Neospora and Toxoplasma infections. Int J
     Parasitol 1998;28:187-93.
 15. Huong LTT, Ljungstrm BL, Uggla A, Bjrkman C. Prevalence of
     antibodies to Neospora caninum and Toxoplasma gondii in cattle and
     water buffaloes in southern Vietnam. Vet Parasitol 1998;75:53-7.
____________________________________________________________________________
Dispatches
Lack of Association between First Myocardial Infarction and Past Use of
Erythromycin, Tetracycline, or Doxycycline
Lisa A. Jackson, Nicholas L. Smith, Susan R. Heckbert, J. Thomas Grayston,
David S. Siscovick, and Bruce M. Psaty
University of Washington, Seattle, Washington, USA
---------------------------------------------------------------------------
      To evaluate the association of prior treatment with antibiotics
      active against Chlamydia pneumoniae with the risk for incident
      myocardial infarction, we conducted a population-based
      case-control study. We found that use of erythromycin,
      tetracycline, or doxycycline during the previous 5 years was
      not associated with risk for first myocardial infarction. These
      results suggest little or no association between the use of
      these antibiotics and the risk for first myocardial infarction
      in the primary prevention setting.
Chlamydia pneumoniae has been associated with atherosclerotic
cardiovascular disease in seroepidemiologic studies, by detection of the
organism in atherosclerotic plaque, and in animal model studies (1-4). Two
small clinical trials to assess the effect of treatment with antibiotics
active against C. pneumoniae on cardiovascular disease outcomes have
indicated a possible effect of azithromycin (5) or roxithromycin (6) in the
secondary prevention of coronary heart disease. To evaluate whether past
use of antibiotics active against C. pneumoniae is associated with
decreased risk for first myocardial infarction (MI), we conducted a
retrospective, population-based case-control study of patients enrolled at
Group Health Cooperative of Puget Sound (GH), Seattle, WA, USA.
The Study
Case-patients were GH enrollees, ages 30 to 79 years, in whom an incident
fatal or nonfatal MI was diagnosed during July 1986 through December 1995.
Controls were a stratified random sample of GH enrollees frequency-matched
with the case-patients by age (within decade), sex, calendar year, presence
of treated hypertension, and menopausal status (post versus peri and
premenopausal). The case-patients and controls had been identified for two
previous cardiovascular studies, one of persons with pharmacologically
treated hypertension (7) and one of postmenopausal women (8,9), by using
methods previously reported. Therefore, men and women with treated
hypertension and women without hypertension were included as both
case-patients and controls, but men without hypertension were not included
in the study population. Medical records were reviewed for all study
participants to confirm the diagnosis of incident MI (case-patients) and
obtain information on other cardiovascular risk factors.
All study participants had an index date. For the hospitalized
case-patients, it was the date of admission for the first MI; for
out-of-hospital case-patients who died, it was the date of death; and for
the controls, it was a computer-generated random date within the calendar
year for which they had been sampled as controls. We excluded persons who
had been enrolled for fewer than 5 years or had fewer than four visits to a
GH provider before their index date.
The GH computerized pharmacy database, which contains records of all
prescriptions dispensed at GH pharmacies, was used to assess prescriptions
for antibiotics. A survey conducted in conjunction with the previous study
of postmenopausal women determined that 95% of study participants filled
all their prescriptions at a GH pharmacy (9). During the period of interest
to this study, newer macrolidelike antibiotics such as azithromycin and
clarithromycin were not routinely available at GH; thus, we selected
erythromycin, tetracycline, and doxycycline, antibiotics available for
routine use during this period, for evaluation because of their in vitro
activity against C. pneumoniae and their indication for treatment of C.
pneumoniae respiratory infections. To determine duration of use for each
agent, we defined 1 day of use as equivalent to 2 g erythromycin, 2 g
tetracycline, or 100 mg doxycycline. The total duration of therapy with
these drugs was calculated by using the sum of the quantities dispensed
during the 5 years before the index date.
The Findings
We identified 1,796 eligible case-patients with an incident fatal or
nonfatal MI and 4,882 eligible controls during the study period (Table 1).
At least one prescription for erythromycin, tetracycline, or doxycycline
was recorded for 775 (43%) of the case-patients and 2,061 (42%) of the
controls. In multivariate logistic regression models controlling for the
matching variables (age, sex, hypertension status, menopausal status, and
index year) or the matching variables and known cardiac risk factors
(smoking, diabetes, cardiovascular disease), risk for incident MI was not
associated with the cumulative duration of prescribed treatment with
erythromycin, tetracycline, doxycycline, or the three agents combined
(Table 2). In addition, risk for incident MI was not associated with
increasing cumulative duration of therapy across these categories for any
of the agents individually or for the three agents combined. Further, no
association was detected when assessment of the use of antibiotics was
restricted to 1 year before the index date (data not shown).
There are several possible
explanations for these        Table 1. Characteristics of case-patients
findings. Treatment with      with myocardial infarction and controls
antibiotics active against C.
pneumoniae may not affect the ---------------------------------------------
risk for heart disease,                       Case-patients    Controls
either because C. pneumoniae  Variable            n=1796        n=4882
does not play a causal role   ---------------------------------------------
in the atherosclerotic
process or because its effect Mean age (yr)        67.0          66.7
on that process cannot be
modified by antibiotics.      Male                  38%          41%
Although two published        Mean duration        16.8          17.4
clinical trials have            of enrollment
suggested a protective 		  in GH(sup a)(yr)
effect of antibiotics on the
secondary prevention of		Angina                26%          10%
coronary outcomes, these
findings are not conclusive.	Hypertension          75%          70%
The study of azithromycin
(5), which enrolled 80 men    Diabetes mellitus     27%          12%
with a history of MI who had
serologic evidence of C.      Current smoker        28%          15%
pneumoniae, was not a
randomized controlled trial   ---------------------------------------------
and so was subject to bias.  (sup a)Group Health Cooperative of Puget Sound,
The results reported from the Seattle, WA.
roxithromycin study (6),
which randomized patients
hospitalized for unstable
angina or non-Q wave infarctions to 1 month of treatment with roxithromycin
or placebo, were preliminary findings from the first 31 days of the 6-month
follow-up. Even if the protective effect indicated by the preliminary
analysis persists in the final analysis, further studies will be needed to
replicate and confirm those findings. Increasing evidence supports a causal
association of C. pneumoniae and atherosclerotic disease; however,
additional data are needed to validate this hypothesis.
Alternatively, treatment with antichlamydial antibiotics may be associated
with a protective effect, but this effect may vary depending on the
specific agent; the dose, duration, or timing of treatment; or the
patient's clinical status. Azithromycin and roxithromycin (both highly
active against C. pneumoniae in vitro) achieve much higher intracellular
levels, and in particular much higher levels in macrophages, than do
erythromycin, doxycycline, or tetracycline; they also have longer
half-lives than those agents (10,11). The beneficial effect of
roxithromycin in the published clinical trial may have been due to a
nonspecific antiinflammatory effect (12,13) rather than to a direct
antimicrobial effect. A true cardiovascular protective effect associated
with azithromycin or roxithromycin treatment may not, therefore, be seen
after treatment with erythromycin, tetracycline, or doxycycline because of
differences in pharmacodynamics or in the mechanisms of action of those
agents. Additionally, in our study population, the cumulative duration of
treatment with the antibiotics assessed was relatively limited; only 9% of
all participants had been prescribed more than a total of 28 days of
treatment with the three antibiotics combined during the 5-year study
period. Thus, the exposure to antibiotics in routine clinical care may be
insufficient to reduce risk. A protective effect of antibiotic treatment
may also be limited to the secondary, but not the primary, prevention
setting; to patients in the high-risk period after an acute event; or to
subsets of patients defined by factors that we could not evaluate, such as
seropositivity to C. pneumoniae. Further, if the organism plays a role
during the initiation or early progression of atherosclerotic lesions, but
not in later stages, treatment of older adults may not be effective.
 Table 2. Cumulative duration of prescribed treatment with erythromycin,
 tetracycline, doxycycline, and the three agents combined, and the risk for
 incident myocardial infarction
 --------------------------------------------------------------------------
             Cumulative
             duration    Case-patients   Controls  OR(sup b) OR(sup c)
 Drug       (day)(sup a)  (n=1796)(%)  (n=4,882)(%)(95% CI)  (95% CI)
 --------------------------------------------------------------------------
 Erythromycin     0        1,266 (70)     3,493  1.0          1.0
                                           (72)  reference    reference
                1-14         401 (22)     1,124  0.99         0.91
                                           (23)  (0.87-1.13)  (0.79-1.05)
                15-28          92 (5)   182 (4)  (1.08-1.82)  1.18
                                           1.41               (0.89-1.54)
                 29+           37 (2)    83 (2)  1.23         1.05
                                                 (0.83-1.83)  (0.69-1.59)
 Tetracycline     0        1,507 (84)     4,149  1.0          1.0
                                           (85)  reference    reference
                1-14         223 (12)  564 (12)  1.11         1.02
                                                 (0.94-1.31)  (0.86-1.22)
                15-28          39 (2)    88 (2)  1.24         1.04
                                                 (0.84-1.81)  (0.69-1.55)
                 29+           27 (2)    81 (2)  0.95         0.98
                                                 (0.61-1.47)  (0.62-1.54)
 Doxycycline      0        1,597 (89)     4,365  1.0          1.0
                                           (89)  reference    reference
                1-14           37 (2)    86 (2)  1.18         1.06
                                                 (0.79-1.74)  (0.71-1.59)
                15-28          85 (5)   244 (5)  0.94         0.90
                                                 (0.73-1.22)  (0.69-1.18)
                 29+           77 (4)   187 (4)  1.10         1.17
                                                 (0.84-1.45)  (0.88-1.56)
 Erythromycin     0        1,021 (57)     2,821  1.0          1.0
                                           (58)  reference    reference
 tetracycline   1-14         422 (24)     1,178  0.99         0.93
                                           (24)  (0.87-1.14)  (0.81-1.07)
 or                                              1.13         0.99
 doxycycline    15-28        185 (10)   454 (9)  (0.94-1.35)  (0.81-1.20)
                 29+          168 (9)   421 (9)  1.11         1.03
                                                 (0.91-1.34)  (0.84-1.26)
 --------------------------------------------------------------------------
 (sup a)For duration of each agent, 1 day is equivalent to 2 g erythromycin,
  2 g tetracycline, or 100 mg doxycycline.
 (sup b)Adjusted for sex, age, hypertension status, menopausal status, and
 index year.
 (sup c)Adjusted for sex, age, hypertension status, menopausal status,
 index year, smoking status, diabetes, and cardiovascular disease.
 OR = odds ratio; CI = confidence interval.
Lastly, while it is possible that our study may have failed to detect a
true beneficial effect of past antibiotic treatment, our sample size was
relatively large. Assuming a prevalence of exposure among the control group
of 42%, this sample size had 90% power (at a 95% confidence level) to
detect a 20% reduction in risk associated with antibiotic use.
In summary, even though the results of two small clinical trials have
suggested that newer macrolidelike antibiotics active against C. pneumoniae
may provide effective secondary prevention of coronary artery disease,
their effectiveness in the primary prevention setting has not been
evaluated prospectively. Our results suggest that treatment with
erythromycin, tetracycline, and doxycycline in doses commonly prescribed in
routine clinical practice is not associated with a reduction in the risk
for incident MI among our study population. Further clinical trials of the
newer agents for secondary prevention and further observational studies of
these agents for the primary prevention of heart disease are indicated.
---------------------------------------------------------------------------
Dr. Jackson is an assistant professor in the Department of Epidemiology,
University of Washington, and an assistant investigator in the Center for
Health Studies, Group Health Cooperative in Seattle. She is an infectious
diseases epidemiologist; her research focuses on Chlamydia pneumoniae,
vaccine evaluations, and the epidemiology of vaccine-preventable diseases.
      Address for correspondence: Lisa Jackson, 1730 Minor Avenue, Suite
1600, Seattle, WA 98101-1448; USA; fax: 206-287-4677; e-mail:
lajack@u.washington.edu.
References
  1. Saikku P, Leinonen M, Tenkanen L, Linnanmaki E, Ekman MR, Manninen V,
     et al. Chronic Chlamydia pneumoniae infection as a risk factor for
     coronary heart disease in the Helsinki Heart Study. Ann Intern Med
     1992;116:273-8.
  2. Campbell LA, O'Brien ER, Cappuccio AL, Kuo CC, Wang SP, Stewart D, et
     al. Detection of Chlamydia pneumoniae (TWAR) in human coronary
     atherectomy tissues. J Infect Dis 1995;172:585-8.
  3. Jackson LA, Campbell LA, Kuo CC, Rodriguez DI, Grayston JT. Isolation
     of Chlamydia pneumoniae from a carotid endarterectomy specimen. J
     Infect Dis 1997;176:292.
  4. Mhlestein JB, Anderson JL, Hammond EH, Zhao L, Trehane S, Schwobe EP,
     et al. Infection with Chlamydia pneumoniae accelerates the development
     of atherosclerosis and treatment with azithromycin prevents it in a
     rabbit model. Circulation 1998;97:633-6.
  5. Gupta S, Leatham EW, Carrington D, Mendall MA, Kaski JC, Camm AJ.
     Elevated Chlamydia pneumoniae antibodies, cardiovascular events, and
     azithromycin in male survivors of myocardial infarction. Circulation
     1997;96:404-7.
  6. Gurfinkel E, Bozovich G, Daroca A, Beck E, Mautner B. Randomised trial
     of roxithromycin in non-Q-wave coronary syndromes: ROXIS pilot study.
     Lancet 1997;350:404-7.
  7. Psaty BM, Heckbert SR, Koepsell TD, Siscovick DS, Raghunathan TE,
     Weiss NS, et al. The risk of myocardial infarction associated with
     antihypertensive drug therapies. JAMA 1995;274:620-5.
  8. Psaty BM, Heckbert SR, Atkins D, Lemaitre R, Koepsell TD, Wahl PW, et
     al. The risk of myocardial infarction associated with the combined use
     of estrogens and progestins in postmenopausal women. Arch Intern Med
     1994;154:1333-9.
  9. Heckbert SR, Weiss NS, Koepsell TD, Lemaitre RN, Smith NL, Siscovick
     DS, et al. Duration of estrogen replacement therapy in relation to the
     risk of incident myocardial infarction in postmenopausal women. Arch
     Intern Med 1997;157:1330-6.
 10. Dunn CJ, Barradell LB. Azithromycin. A review of its pharmacological
     properties and use as 3-day therapy in respiratory tract infections.
     Drugs 1996;51:483-505.
 11. Markham A, Faulds D. Roxithromycin. An update of its antimicrobial
     activity, pharmacokinetic properties and therapeutic use. Drugs
     1994;48:297-326.
 12. Anderson R, Theron AJ, Feldman C. Membrane-stabilizing,
     anti-inflammatory interactions of macrolides with human neutrophils.
     Inflammation 1996;20:693-705.
 13. Martin D, Bursill J, Qui MR, Breit SN, Campbell T. Alternative
     hypothesis for efficacy of macrolides in acute coronary syndromes.
     Lancet 1998;351:1858-9.
_____________________________________________________________________________
Dispatches
An Epidemic of Bloody Diarrhea: Escherichia coli O157 Emerging in Cameroon?
Patrick Cunin,* Etienne Tedjouka, Yves Germani, Chouabou Ncharre,
Raymond Bercion,* Jacques Morvan, and Paul M.V. Martin*
*Centre Pasteur du Cameroun, Yaound, Cameroon; Lomi Health District,
Lomi, Cameroon; Institut Pasteur, Bangui, Central African Republic; and
World Health Organization Representation, Yaound, Cameroon
---------------------------------------------------------------------------
      Between November 1997 and April 20, 1998, bloody diarrhea
      sickened 298 persons in Cameroon. Laboratory investigation of
      the epidemic (case-fatality rate, 16.4%) documented amoebiasis
      in one of three patients and three types of pathogens:
      multidrug-resistant Shigella dysenteriae type 1, S. boydii, and
      enterohemorrhagic Escherichia coli. We report the first
      isolation of E. coli O157:H7 in Cameroon and the second series
      of cases in the Central African region.
In December 1997, an epidemic of bloody
diarrhea was reported in Ngola, a
village of approximately 500[				 [fig]
inhabitants, approximately 400 km from   Figure 1. Progression of the
Yaound. Canoes and motorbikes are 	     bloody diarrhea epidemic,
necessary to reach Ngola, which is	     Cameroon.
linked to Lomi by a difficult road
across the Dja River. The remote zone to
the south of the river is inhabited by 2,610 persons, who belong to two
ethnic groups (Bantus and Baka Pygmies) and live in 22 villages (Figure 1).
The population of the outbreak area is 20,600. The sanitation system is
weak, latrines do not exist, and human feces are used as fertilizer. No
village has running water; drinking water, which is neither chlorinated nor
filtered, comes from wells or small streams.
Between December 1997 and March 1998, teams from Lomi Hospital and the
East Provincial Delegation of Health went to Ngola to treat patients and
make an inventory of the cases. After a March 19, 1998, wire service report
of a possible viral hemorrhagic fever epidemic in Ngola, a joint mission
of Centre Pasteur du Cameroun and the World Health Organization visited the
area March 25-28, 1998.
The Investigation
Investigators defined a case as bloody diarrhea. Data on patients' age,
sex, village of origin, onset of disease, drugs received, and disease
diagnosis were obtained from the observation record book of the Lomi
medical officer. Standardized observation forms were filled out for 34
patients (from nine villages) seen during the mission. Serum specimens were
obtained from 21 patients (both current and former) in three different
villages, and stool specimens were obtained from 22 patients. Serum
specimens were immediately frozen in liquid nitrogen. Stool specimens were
cultured immediately in a field laboratory set up in each of the three
villages. Later, in the laboratories of Centre Pasteur in Yaound and the
Institut Pasteur of Bangui, the cultures were identified, their
susceptibility to antibiotics was determined (according to the diffusion
method on agar plates), and genetic studies were conducted. Hektoen agar
plates, purple bromocresol agar plates, MacConkey Sorbitol medium and
thiosulfate-citrate-bile- saccharose medium (Sanofi, Marnes la Coquette,
France) were used.
Isolates that agglutinated in O157 antiserum were confirmed biochemically
as Escherichia coli and screened for the lack of enzyme  glucuronidase,
using the substrate 4-methylumbelliferyl--D-glucuronide. Specific
anti-O157 and anti-H7 were obtained from Difco, USA. Toxin production was
assayed as follows. We used the Gb3 enzyme-linked immunosorbent assay
described by Ashkenazi and Cleary (1) for detection of Verotoxin.
Cytotoxicity assays and seroneutralization tests were performed according
to procedures (2) with Vero cells. The antisera to purified Verotoxin and
Shiga toxin had been used in previous studies (3,4) and were prepared in
New Zealand white rabbits to perform seroneutralization. The Verotoxin 1
preparations we used were purified from E. coli E40705 (O157:H7) provided
by the Public Health Laboratory Service, London, United Kingdom, by the
procedure described by O'Brien and LaVeck (5). Sera to Shiga toxin from
Shigella dysenteriae type 1 (provided by the Institut Pasteur, Paris,
France) were prepared according to a previously described procedure (4).
Virulence genes of E. coli were investigated by molecular biology
techniques; polymerase chain reaction was used to detect enterohemorrhagic
Shigalike toxins 1 and 2 genes and the attaching and effacing gene eae
(6-8).
Part of each stool specimen was conserved in a medium containing
Merthiolate-formalized iodine for further parasitologic analyses. Viral
hemorrhagic fever agents were sought by gene amplification and
immunoglobulin M detection.
The Outbreak
The first cases were reported at the end of November 1997 in two small
villages, Dounzok and Lamson, near Ngola. An additional two cases were
found retrospectively, at the end of November, one each in Djadom and
Yanebot, villages south of Ngola. The epidemic began to affect Ngola at
the beginning of December 1997 and then, traveling north along the roads,
spread to all villages in the area. It crossed the Dja River and reached
Messok on February 4 and Lomi, divisional headquarters of the district, on
April 3 (Figure 1). By April 20, 298 cases had been reported from 28
villages.
In the first villages affected, each with fewer than 100 inhabitants, the
epidemic lasted an average 3 months (57 days to 112 days), with an attack
rate of more than 50%. Villages south of the Dja had an attack rate of 9.7%
(253 of 2,610). In Ngola, the village with the most cases, more than a
fourth of the inhabitants were sick. The graph of the epidemic for the 271
patients with a known date of illness onset suggests person-to-person
transmission, with a slow increasing gradient consisting of successive
waves (Figure 2).
 					      Of 10 patients examined at Messok, 7
                                    belonged to the same family (of 12); 2
 		[fig] 			carried S. dysenteriae type 1, and 3
                                    carried enterohemorrhagic E. coli
 Figure 2. Cases of bloody          (EHEC). Disease onset spread out over 4
 diarrhea, by week, Cameroon, Nov.  weeks, with the index case in week 1,
 27, 1997March 23, 1998.           two cases in week 2, one case in week
                                    3, and three cases in week 4. The
median delay between illness onset was 6 days (range 3 to 8 days). A
relative who came to visit family members in the Messok Health Center was
the last case in this family. He became ill 7 days later; he had Shigella
and Entamoeba infections. The many Pygmies in the region (who live in
separate villages) were not affected by the epidemic. Both sexes in all age
groups became sick. The attack rate for the villages south of the Dja (with
known population sizes) was higher among female (11.4%) than among male
residents (8.1%) (p <0.02) and increased with age (Table 1). The
case-fatality rate was highest for women ages 60 years and older (39.7%).
Illness, Treatment, and Deaths
The most frequent      Table 1. Bloody diarrhea epidemic in 22 villages
signs of illness in    south of the Dja River, East Cameroon, 19971998
the 34 investigated
patients were          ----------------------------------------------------
abdominal pain (97%)                 Female              Male
and mucus in stools
(91%), followed by     Age    -------------------  -----------------
fever (53%), vomiting  group                Rate              Rate
(50%), and dehydration (yr) Cases No.(sup a)(%)Cases No.(sup a)(%)    p
(41%). The average     ----------------------------------------------------
number of stools per
day was 10; mucus and    0-4   16    221  (7.2)  14   218     (6.4) NS(sup b)
blood were observed in   5-9   16    191  (8.4)  11   191     (5.8)   NS
stools starting on the 10-19   25    321  (7.8)  18   313     (5.8)   NS
third day. The
quantity of blood in   20-29   22    211  (10.4) 25   207     (12.1)  NS
the stools varied;     30-39   15    127  (11.8) 14   154     (9.1)   NS
occasionally only
			     40-49   16      89 (18.0) 14   101     (13.9)  NS
blood was excreted.    50-59   11      63 (17.5)  4    70     (5.7)  <0.05
Stool specimens were   60 +    25      63 (39.7)  7    69     (10.1)<10(sup -4)
obtained from 12
female and 10 male     Total 146  1,286   (11.4)  107 1,324   (8.1)  <0.01
patients; their median p                <10(sup -8)           <0.05
age was 25 (2 years to
60 years), and the     ----------------------------------------------------
median length of       (sup a)Number of village inhabitants.
illness was 7 days (3  (sup b)NS, not significant.
days to 58 days).
African hemorrhagic
fever was not detected. Parasitologic examination of stool specimens showed
heavy infestation with numerous parasites. Hematophagous trophozoites of
Entamoeba histolytica histolytica were observed in 7 (31.8%) of 22 of the
cases. Bacteriologic studies isolated at least 1 enteropathogen in 20
(90.9%). EHEC were isolated in 12 (54.5%), S. dysenteriae type 1 in 9
(40.9%), and S. boydii in 2 (9.1%). Potentially dangerous associations were
common. Of the 22 patients with stool specimens, 7 (31.8%) had both E.
histolytica histolytica and one enteropathogenic bacterium; 3 (13.6%) had
both E. histolytica histolytica and S. dysenteriae type 1; and 2 (9.1%) had
both S. dysenteriae type 1 and EHEC. Simultaneous infection with all three
pathogensS. dysenteriae, EHEC, and E. histolyticawere found in patients
from all three villages, while dual infections with S. dysenteriae and EHEC
were observed in patients from two villages.
Drug susceptibility testing found that S. dysenteriae type 1 was resistant
to amoxicillin, amoxicillin in combination with clavulanic acid,
tetracycline, chloramphenicol, cefsulodin, and cotrimoxazole; it was
sensitive to nalidixic acid, piperacillin, cefalotin, ceftazidin,
gentamicin, and ofloxacin. E. coli O157:H7 was resistant to amoxicillin,
chloramphenicol, and cefsulodine, and sensitive to amoxicillin in
combination with clavulanic acid, tetracycline, cotrimoxazole, nalidixic
acid, and cefalotin. DNA amplification showed that E. coli O157:H7 strains
had genes that coded for Shigalike toxins 1 and 2 and the attaching and
effacing gene eaeA.
By April 1998, five different medications, depending on availability, had
been used, sometimes in combination. Of the 292 patients with known drug
regimens, 182 took cotrimoxazole (62.3%), 190 took metronidazole (65.1%),
34 took chloramphenicol (11.6%), and 19 took tetracycline (6.5%). Of these,
67 (22.9%) were given oral rehydration salts, while 71 (24.3%) received no
treatment or received traditional medicine. More female than male patients
were not treated, 51 (31.1%) of 164 versus 20 (15.6%) of 128 (p <0.01),
respectively. The groups that received the least or no drug treatment were
children younger than 5 years of age (12 [34.3%] of 35) and those ages 50
years and older (26 [46.4%] of 56). The proportion of patients treated with
drugs increased during the epidemic: 24 (54.5%) of 44 of the patients were
treated in December 1997, while 41 (68.3%) of 60, 85 (76.6%) of 111, and 44
(88%) of 50 were treated in January, February, and March, 1998,
respectively.
 Table 2. Bloody diarrhea epidemic in East Cameroon,        Of the 275
 case-fatality rate of by age and sex                       patients for
                                                            whom disease
 ------------------------------------------------------     outcome was
               Female              Male                     known, 45 died
                                                            (case-fatality
 Age    ------------------- -------------------             rate 16.4%).
 group                                                      The rate was
 (yr)   Cases Deaths  (%)   Cases Deaths   (%)    p         higher in
 ------------------------------------------------------     female (21.3%
   0-4    18     5  (27.8)    16     3   (18.8) NS(sup a)   [33 of 155])
                                                            than in male
   5-9    16     2  (12.5)    13     1     (7.7)  NS        patients, (10%
 10-19    25     1    (4.0)   20     1     (5.0)  NS        [12 of 120])
                                                            (p <0.02)
 20-29    24     1    (4.2)   27     1     (3.7)  NS        (Table 2).
 30-39    16     3  (18.8)    18     1     (5.6)  NS        Case-fatality
 40-49    17     3  (17.6)    12     1     (8.3)  NS        rates were
                                                            23.5% (8 of
 50-59    13     6  (46.2)      8    2   (25.0)   NS        34) among
 60 +     26    12  (46.2)      6    2   (33.3)   NS        those younger
                                                            than 5 years
 Total  155     33  (21.3)  120     12   (10.0) <0.02       old, 40% (22
 ------------------------------------------------------     of 55) among
 (sup a)NS, not significant.                                those 50 years
                                                            of age and
older, and 53.5% (38 of 71) among patients who received no treatment.
Conclusions
During the first half of 1997, an epidemic of bloody diarrhea with a
case-fatality rate of 19% was reported in villages near Mintom, a district
headquarters approximately 100 km west of Ngola. Then, the region of
Ngola in the East Province of Cameroon was hit by an extremely severe and
deadly epidemic of bloody diarrhea, which continued until July 1998. In
April 1998, 34 (11.4%) of 298 (the affected population) were examined, and
stool specimens were obtained from 22 patients with an active infection,
which represented 7.4% of the total epidemic population (298). Although the
sample was relatively small, three bacterial enteropathogens (S.
dysenteriae type 1, S. boydii, and EHEC) associated with numerous parasites
were isolated from the stool specimens. Thus, which of these pathogens were
responsible for the epidemic and its severity is not known with certainty.
The isolation of E. coli O157, in contrast to Shigella, is quite recent in
Africa. We believe that this is the first case of E. coli O157 described in
Cameroon and the second (after the 1996 epidemic in the Republic of Central
Africa) in the Central African region (9).
A few persons may have contracted the disease through food or drink.
However, the epidemic curve with a slow rise of successive waves does not
depict a common source of contamination; it suggests person-to-person
transmission. The spread of the disease followed roads, and women were the
most likely infected, probably because they live together in groups and
take care of the sick. Because of difficulties in communication and the
necessity to return to the laboratory to process the specimens, we could
not investigate in detail with a case-control study. The protracted course
of the illness, poor sanitation, and length of time patients were infected
with pathogens favored transmission. The bloody diarrhea was long-lasting
for some patients. In one patient with S. dysenteriae type I, the onset of
disease had begun 58 days before, while in another with EHEC, it had begun
39 days before.
In contrast to shigellosis, which is most often transmitted from person to
person (10) (often through the hands) (11) and affects mostly women (12),
E. coli O157 infection has been described in the northern hemisphere as
food poisoning (13). For example, in Japan an epidemic involving more than
5,000 cases was described in school children contaminated by food prepared
in the school canteen in 1996 (14). Sources of contamination most commonly
incriminated are meat in hamburgers (15) or sandwiches (16), milk (17),
drinking water (18), water absorbed during baths (19), nonpasteurized apple
juice (20), and salads (21). The main reservoir of the germ is cows and
other ruminants (22), but the association between E. coli O157 and cows is
not absolute (23). Its transmission from person to person has been observed
in a small number of cases, most often intrafamilially (24). In the
Republic of Central Africa during the first isolation of E. coli O157, food
was the suspected vehicle (9). The rain forest zone in South and East
Cameroon, like Ngola, has no cattle; breeding is limited to pigs, goats,
and chickens; and meat is not imported.
Deaths caused by the disease were quite high, with a case-fatality rate of
16.4%. In comparison, the case-fatality rate for cholera was globally
estimated at 4.7% in 1996 (25) and rose to 48% in the worst outbreak (the
beginning of the epidemic in Rwanda refugee camps in Goma in 1994) (26).
Shigellosis is one of the main causes of severe diarrhea in Africa,
accounting for 12% of all deaths in Kibue Sector in Burundi in 1992 (12)
and 19% of pediatric hospital deaths in KwaZulu-Natal in 1995 (27). The
portion of deaths due to shigellosis variesfrom 13% in hospital statistics
in children in KwaZulu-Natal to 3.8% in a Burundan refugee camp in Rwanda
in 1993 (28) and 0.25% during the first Shigella epidemic in Mozambique in
the same year (29). The severity of E. coli O157 is associated with its
complication, hemolytic uremic syndrome; an estimated 200,000 cases of this
syndrome per year occur in the United States, with 250 deaths and a
case-fatality rate of 0.1% (30). In the epidemic in the Republic of Central
Africa, the case-fatality rate was 3.7% (9).
The causes of death were unknown. The case-fatality rate diminished during
the course of the epidemic: 14 (31.8%) of 44 in patients in December 1997,
9 (15%) of 60 in January, 19 (18.3%) of 104 in February, and 2 (5%) of 40
in March 1998 (p <0.02), suggesting progressively better disease management
during the epidemic. The case-fatality rate was 3.4% in patients who
received either cotrimoxazole (6/174), metronidazole (6/176), or
chloramphenicol (1/29); 5.5% in those who were given tetracycline (1/18);
0% in patients who also received oral rehydration salts; and 53.5% (38/71)
in those who received no treatment. In Ngola, health facilities did not
have the resources to perform the classic hematologic and chemical
investigations. However, the case-fatality rate in patients who received
oral rehydration salts was zero, suggesting that fluid loss was a major
part of the disease. Other cofactors (e.g., anemia) could have played a
role in lethality. To determine the risk for death from the disease, we
constructed a logistic regression model that included age, sex, onset of
illness, and drug regimen received. Oral rehydration salts were not
included in the model (all rehydrated patients survived). The only factor
strongly associated with death was age >50 years (odds ratio [OR] = 5 ; 95%
confidence interval [CI]: 2.2-11.5); having received cotrimoxazole was a
protective factor (OR = 0.05, 95% CI: 0.02 - 0.13, p <10[sup -5]). Because
the treatment groups were not randomized, the decline in case-fatality rates
cannot be attributed to this drug. The use of other drugs, however, as well
as sex and month of onset of the illness, were not associated with death.
S. dysenteriae type 1 was resistant to cotrimoxazole, while E. coli O157
was susceptible.
The multiple resistance to antibiotics of S. dysenteriae type 1 strains
isolated from Ngola could also explain the high case-fatality rate.
Studies from Central and East Africa confirm that S. dysenteriae type 1 is
resistant to multiple drugs (31-34). There may, therefore, be a new
emerging disease due to the association of enterohemorrhagic E. coli
O157:H7 and S. dysenteriae type 1 in this region of Africa, an association
that could be as deadly as that of shigellosis and cholera. Finally, the
management of cases will be rendered difficult by the resistance of the
pathogens implicated to multiple drugs. After our microbiologic
investigations, the health authorities distributed nalidixic tablets and
ciprofloxacin. The case-fatality rate fell to 3.7% (8/213).
---------------------------------------------------------------------------
      Dr. Cunin, an epidemiologist and specialist in tropical diseases, is
affiliated with Coopration franaise and has worked in Africa (Morocco,
Cte d'Ivoire, Mauritania, Cameroon) since 1975. He now works at the
Pasteur Center in Cameroon. His research focuses on epidemic investigation,
infectious diseases (tuberculosis, arbovirus, schistosomiasis),
mother-to-child transmission of HIV, and maternal and child health.
      Address for correspondence: Paul Martin, Centre Pasteur du Cameroun,
BP 1274 Yaounde, Cameroun; fax: 237-231-564; e-mail: C.pasteur@camnet.cm.
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___________________________________________________________________________
Dispatches
Genospecies Diversity of Lyme Disease Spirochetes in Rodent Reservoirs
Dania Richter,* Stefan Endepols, Andreas Ohlenbusch, Helmut Eiffert,
Andrew Spielman, and Franz-Rainer Matuschka*
*Medizinische Fakultt der Humboldt-Universitt zu Berlin, Berlin, Germany;
Geschftsbereich Tiergesundheit, Bayer AG, Leverkusen, Germany;
Universittskliniken der Georg-August-Universitt Gttingen, Gttingen,
Germany; and Harvard School of Public Health, Boston, Massachusetts, USA
---------------------------------------------------------------------------
      To determine whether particular Borrelia burgdorferi s.l.
      genospecies associate solely with rodent reservoir hosts, we
      compared the genospecies prevalence in questing nymphal Ixodes
      ticks with that in xenodiagnostic ticks that had fed as larvae
      on rodents captured in the same site. No genospecies was more
      prevalent in rodent-fed ticks than in questing ticks. The three
      main spirochete genospecies, therefore, share common rodent
      hosts.
The several genospecies of the Lyme disease spirochete, Borrelia
burgdorferi sensu lato, that infect people in Eurasia produce a broad
spectrum of human disease. Particular genospecies have been associated with
characteristic symptoms; chronic skin disease, for example, results from
infection by Borrelia afzelii (1). Rodents (e.g., various Apodemus mice
[2], Norway rats, Rattus norvegicus [3,4], edible dormice, Glis glis [5])
serve unambiguously as reservoir hosts for Lyme disease spirochetes.
Although each major European genospecies has been associated with birds
(6), B. afzelii is thought to perpetuate in rodents and B. garinii is
thought to perpetuate in avian reservoir hosts (7,8). The European vector
of Lyme disease, Ixodes ricinus, maintains an unusual diversity of
pathogens in an extraordinarily broad array of hosts.
The vertebrates that are infested most frequently by arthropods generally
also serve as reservoir hosts for any pathogens transmitted by these
vectors. The transmission cycle would likely be broken if some vector
acquired a pathogen from one host but injected it into an ill-adapted host.
If B. afzelii thrived mainly in rodents (7), transmission might not be
sustained if a larval tick acquired these spirochetes from a mouse and
attached subsequently as a nymph to a bird. If B. garinii, on the other
hand, thrived mainly in birds, a corresponding diversion of its vector to a
rodent would similarly result in transmission failure. Efficient
perpetuation of rodent-borne B. afzelii as well as bird-borne B. garinii by
the same subadult I. ricinus vector ticks, therefore, would seem
paradoxical.
Because rodents may serve as reservoir hosts for both B. afzelii and B.
garinii, we determined whether the genospecies distribution of spirochetes
naturally infecting rodents corresponds to that in questing vector ticks.
We compared the genospecies diversity of spirochetes infecting questing
nymphal I. ricinus ticks with that of spirochetes infecting nymphs that had
fed as larvae on Norway rats or on yellow-necked mice, Apodemus
flavicollis, captured in the same site.
The Study
Norway rats and yellow-necked mice were captured in an urban park in
Magdeburg, Germany, from June through September 1994, by using apple- and
bread-baited Tomahawk traps (Tomahawk Live Trap Company, Tomahawk, WI) and
apple- and rodent chowbaited Longworth traps (Longworth Scientific
Instruments, Abingdon, UK) (4). Each captured rodent was caged over water
for 1 week to permit detachment of all ticks that had attached to these
rodents in the field. Subsequently, rodents were infested with noninfected
laboratory-bred I. ricinus larvae for xenodiagnosis. To confirm that larvae
used for xenodiagnosis were free of spirochetes, a sample of each batch was
routinely fed on a laboratory-bred mouse; no spirochetes were found when
midguts of 20 of the resulting nymphs were examined by dark-field
microscopy, and subsequent xenodiagnosis of the mouse showed no evidence of
spirochetes. The water was changed and inspected at least twice a day and
detached larvae were removed promptly. Engorged larvae were enclosed in
screened vials and kept at 202C in sealed desiccator jars over
supersaturated MgSO4 under a light-dark regimen (16:8) until they molted.
Questing I. ricinus ticks were collected from the vegetation in the study
site by using flannel flags. Collected ticks were confined in screened
vials and stored at 151C in sealed desiccator jars containing
supersaturated MgSO9(sup 4) until they were examined microscopically,
identified to stage, and prepared for polymerase chain reaction (PCR)
analysis.
DNA Extraction, Amplification, and Sequencing
Total DNA from the ear pinnae of field-caught rodents was prepared with a
QiAamp Tissue kit (Qiagen GmbH, Hilden, Germany). Engorged I. ricinus
larvae that served for xenodiagnosis were permitted to molt to nymphs. The
opisthosoma was then opened, andTthe contained mass of soft tissue was
dissected out into 400 l ice-cold Tris-EDTA buffer (pH 7.4, 10mM Tris, 1mM
EDTA). Suspensions of tick tissue were adjusted to 0.5% sodium dodecyl
sulfate, 0.2M NaCl, 10mM Tris, and 5mM EDTA at pH 8.0; proteinase K
(Boehringer Mannheim, Mannheim, Germany) was added (0.2mg/ml) and incubated
at 56C for 3 hours. DNA was extracted with phenol-chloroform.
Ethanol-precipitated DNA was resuspended in 50 l distilled water.
Borrelia genospecies were characterized by amplifying and sequencing a
400-nucleotide segment of the gene encoding the outer surface protein A
(OspA) (9-11). To increase the sensitivity for detection of spirochetal DNA
in ticks, we used nested PCR (10). Aliquots of DNA suspensions (20 l) were
diluted to 50 l by using 200 M each of deoxynucleoside triphosphate, 4mM
MgCl(sup 2), 10mM Tris at pH 8.3, 50mM KCl, 0.01% Tween-20, 0.01% gelatin, and
0.8 units Taq polymerase (Amersham, Braunschweig, Germany), as well as
10pmol of the outer primer pair or 20pmol of the inner primer pair. We used
the following primer sequences (5'-3') of the ospA gene (9): outer primers
GGTCTAATATTAGCCTT AATAGGCATG (positions 169-194) and TCAG
CAGCTAGAGTTCCTTCAAG (positions 665-643); inner primers CATGTAAGCAAAATGTTAG
CAGCC (positions 191-214) and CTGTGTATTCA AGTCTGGTTCC (positions 589-568).
The mixture was overlaid with mineral oil (Sigma, Deisenhofen, Germany),
placed in a thermocycler (Omnitech, Heidelberg, Germany), heated for 2 min
at 95C, and subjected to 40 cycles of 20 sec denaturation at 95C, 20 sec
each for the first annealing reaction at 59C, for the second at 61C and
with a 20-sec extension at 72C. After the first amplification with the
outer set of primers, 5 l of product was transferred to a fresh tube
containing 45 l of reaction mixture as described for the inner set of
primers. PCR products were detected by electrophoresis in a 1.5% agarose
gel stained with ethidium bromide. DNA was extracted, reaction vials were
prepared for amplification, and products were electrophoresed in separate
rooms. For comparison, each series of PCR amplification included two
laboratory-reared nymphs that had fed as larvae on B. afzelii-infected
jirds (Meriones unguiculatus) and two that had fed on noninfected jirds.
Each PCR amplification product was purified by using a QIAquick-Spin PCR
column (Qiagen). Amplified DNA fragments were directly sequenced in both
directions using the inner primers by the dideoxynucleotide
chain-termination method on an ABI 373 DNA-sequencer according to the
manufacturer's instructions (Applied Biosystems, Foster City, CA). Each
resulting sequence was compared with sequences of the same fragment
representing B. burgdorferi sensu stricto, B. afzelii, and five serotypes
of B. garinii (9-11); an exact fit was required. With this technique, each
of these genospecies can be detected with equal sensitivity; the technique
detects and identifies two different coinfecting spirochete genospecies,
even when one is five times as numerous as the other (10).
The Findings
We first described the frequency of spirochetal infection and the
distribution of spirochete genospecies infecting questing I. ricinus ticks
collected from vegetation. Although all three spirochete genospecies were
present in these ticks, B. garinii was somewhat more prevalent than B.
afzelii; B. burgdorferi s.s. was infrequent (Table 1). A similar
distribution of genospecies was found in questing adult ticks (data not
shown). Spirochetes of each of the major pathogenic European genospecies
infect questing vector ticks at our study site.
 Table 1. Borrelia genospecies diversity in questing nymphal
 Ixodes ricinus ticks and in nymphal ticks that had fed as
 larvae on Norway rats, Rattus norvegicus, or yellow-necked
 mice, Apodemus flavicollis                                      We then                                                               identified
 --------------------------------------------------------------- identified the
                                   Distribution of genospecies   spirochete
                Borrelia prevalence	 No.  			     genospecies
                  No.         %     ticks  % with Borrelia 	     that
 Tick source     tested  infected  tested afz(sup a)gar bur      naturally
 --------------------------------------------------------------- infected rodents
                                                                 transmitted to
 Questing          112      9       10      40      50   10      xenodiagnostic
 Fed on rat(sup b)  50     86       25      28      52   20      larvae.
 Fed on mouse(sup c)36     42       15      40      47   13      Spirochetes                                                            were
---------------------------------------------------------------  present 										     in virtually
 (sup a)afz,afzelii; gar, garinii; bur, burgdorferi sensu        all
  stricto.                                                       nymphal
 (sup b)Ticks fed on five infected rats.                         ticks
 (sup c)Ticks fed on four infected mice.                         that
                                                                 had
fed as larvae on rats captured at the study site and in approximately half
of those that had fed on mice (Table 1). Each of the three main genospecies
was present. The 1:2:1 ratio of B. afzelii to B. garinii to B. burgdorferi
s.s. in ticks that fed on rats did not differ from the 3:3:1 ratio in
nymphal ticks that fed on mice (Chi-square, p=0.7). The 2:3:1 overall ratio
of genospecies in xenodiagnostic ticks did not differ from the 4:5:1 ratio
in questing ticks (Chi-square, p=0.9). In contrast, only B. afzelii DNA was
amplified from the ear pinnae of these hosts (data not shown). The array of
spirochete genospecies acquired by ticks feeding on field-derived rodents
is similar to that in questing ticks but differs sharply from that present
in skin samples of these rodents.
To examine the transmissibility of the three genospecies of Lyme disease
spirochetes, we compared genospecies diversity in I. ricinus ticks that had
fed on individual rodents. At least two such infected ticks were available
for each of these rats and for all but one mouse; that mouse was excluded
from this analysis (Table 2). Although more than one spirochete genospecies
infected the cohort of ticks that fed on each rodent, no individual tick
was infected with more than one genospecies. The B. garinii genospecies
infected somewhat more ticks than did B. afzelii, and B. afzelii infected
somewhat more than did B. burgdorferi s.s. We found that no particular
spirochete genospecies is transmitted more frequently than any other.
Conclusions
                     Table 2. Natural infectivity for xenodiagnostic
Although B. garinii  Ixodes ricinus ticks of various genospecies of
spirochetes most	   Lyme disease spirochetes
frequently infect    ------------------------------------------------------
questing ticks in                            No. ticks infected by Borrelia
our German study     Infected        Rodent                  burgdorferi
site, the other two  rodent            no.   afzelii garinii sensu stricto
major genospecies    ------------------------------------------------------
predominate          Rattus             1       2      2          1
elsewhere in Europe. 	norvegicus      2       0      3          2
B. afzelii             (Norway rats)    3       1      4          0
predominates in				    4       3      2          0
questing ticks in      			    5       1      2          2
four European sites                    Total    7     13          5
(12-14) and B.	   Apodemus           1       3      3          1
burgdorferi s.s. in     flavicollis     2       3      2          0
four others (Table      (Yellow-        3       0      1          1
3) (7,15-17). In     	 necked mice)
three sites,			         Total    6       6         2
different            ------------------------------------------------------
combinations of two
genospecies
predominate
(7,15,18). No
regional pattern of
genospecies
diversity in questing ticks seems evident, nor does the sampling method
influence the genospecies ratio. If B. garinii were to perpetuate in an
avian reservoir (8,19) and B. afzelii in rodents (7), the relative
availability of these hosts to vector ticks would determine genospecies
distribution in a site. Alternatively, the founder's principle (i.e., in
the absence of some selective force, the present ratio randomly reflects
that of the past) may influence genospecies distribution. No available
longitudinal study, however, permits such a temporal interpretation. The
distribution of European B. burgdorferi s.l. genospecies appears to be
site-specific and may be random. For this reason, genospecies comparisons
designed to differentiate reservoirs of these spirochetes should be based
on diversity within a single site.
The method used to sample spirochetes from a mammal host may bias the
results in favor of one or another genospecies. When spirochetes are
isolated from European mice by culturing segments of their ear pinnae, the
B. afzelii genospecies seems to predominate (7). In direct identification
by PCR and sequence analysis, we confirmed the sole presence of this
genospecies in ear pinnae from Norway rats and yellow-necked mice captured
at our study site. A more diverse array of genospecies, however, infects
ticks questing at this site. Although this finding would suggest that B.
afzelii is the sole spirochete genospecies infecting these rodents,
xenodiagnostic observations indicate that samples based on ear biopsies do
not reflect the total diversity of spirochetes infecting such a rodent.
Interestingly, B. burgdorferi s.s. is readily detected in earpunch samples
taken from American mice (20). The apparent association of B. afzelii with
European rodent hosts may derive from a sampling artifact.
 Table 3. Borrelia genospecies diversity in questing            In contrast
 Ixodes ricinus ticks from various European sites               to the
                                                                reported
 -------------------------------------------------------------  association
                     Borrelia genospecies 			    of B.
              No.     							    afzelii with
           infected  Relative ratio	    %     Sampling          rodents, B.
 Site(sup a) ticks   afz   gar  bur   mixed   method    Ref.   garinii is
                    (sup b)                                     reported to
 -------------------------------------------------------------  depend on
 CH  1         7       1    2    2      8     Culture    7      avian
        2      6       0    1    5      0     Culture    7      reservoir
                                                                hosts
        v     50       1    6    9      0     Culture    17     (7,8,21).
 CR  v        56      19    3    1     20     Direct     13     Evidence for
                                                                such a
 D    1       52       5    11   1      2     Direct     1      genospecies-specific
       2      10       4    5    1      0     Direct   TA(sup c)avian
                                                                reservoir
 F    1       25      10    5    1     24     Direct     12     derives
 GB 1         16       0    10   1      0     Direct     8      originally
 IR  1        11       1    4    5      9     Direct     15     from
                                                                observations
       2      10       0    2    1      0     Direct     15     on an
       3      20       4    1    4     15     Direct     15     isolated
 NL 1         15       1    1    0     14     Direct     18     island site
                                                                where
       v      63       1    9    14     0     Culture    16     numerous
 SL 1         47       3    2    1      5     Culture    14     seabirds
                                                                nested and
       v      13      12    1    0      0     Culture    14     where no
 -------------------------------------------------------------  other
(sup a)CH, Switzerland; CR, Croatia; D, Germany; F, France; GB, spirochetes
Great Britain; IR, Ireland; NL, Netherlands; SL, Slovenia; v,   were
sum of various sites in a country.                              detected
(sup b)afz, Borrelia afzelii; gar, Borrelia garinii; bur,       (19). DNA
Borrelia burgdorferi sensu stricto.                             characteristic
(sup  c)This article.                                            of B.
                                                                garinii was
                                                                detected in
questing and seabird-feeding I. uriae ticks and in the footweb of a
razorbill. This genospecies also predominates in pheasants in a British
site in which no B. afzelii spirochetes are evident (8). Although B.
garinii infects bird-feeding I. persulcatus ticks in a Japanese site and B.
afzelii infects those feeding on rodents (21), another Japanese study
detected both genospecies in voles (22). Experimental studies, however,
suggest that birds are relatively incompetent as hosts of Lyme disease
spirochetes. Domestic chickens, for example, become only transiently
competent (23), and European blackbirds, Turdus merula, and Canary finches,
Serinus canarius, appear not to become infected (3,24). Spirochetes
(probably B. burgdorferi s.s.), however, have been detected in larval I.
dammini taken from particular North American birds (25). Evidence of
nonspecificity in avian hosts is provided by observations in Scandinavia,
where spirochetes of the three major genospecies infect larval ticks that
had fed on various passerine birds (6). Indeed, our finding that vector
ticks ingest B. garinii spirochetes from rodents at least as frequently as
B. afzelii argues against the concept of genospecies-reservoir specificity.
Surprisingly, few ticks questing in Europe appear to contain more than one
spirochete genospecies (Table 3). No more than one in four ticks is
multiply infected (12) (generally far fewer). None of the questing ticks
collected in our European study site appear to contain more than one kind
of spirochete, nor did any laboratory-reared ticks permitted to feed on
rodents captured in this site. These rodents, however, were multiply
infected: each rodent infected some of the ticks that fed on them with two
or more kinds of spirochetes. Although our diagnostic procedure may more
reliably detect DNA of the more abundant of two genospecies coinfecting a
tick, our findings may have a biologic basis. We suggest that a single
genospecies becomes established in a tick far more frequently than do
multiple genospecies.
Host specificity of a vector contributes powerfully to the intensity of
transmission of a tick-borne pathogen. Such a pathogen would fail to
perpetuate unless the host-range of its vector corresponds closely to that
of its reservoir. It would seem paradoxical if the same I. ricinus
population maintained spirochetes of one genospecies in birds, while
maintaining another in rodents. This "one-vector-one-reservoir" principle
is consistent with our discovery of a similar genospecies distribution in
rodents and in ticks questing at our study site. If the B. garinii
genospecies were to predominate there in birds (7), such spirochetes would
have been more prevalent in questing ticks than in ticks that had engorged
on sympatric rodents; however, this is not the case. Our site-specific
observation that the genospecies distribution in rodent-fed ticks reflects
that in questing ticks argues that all three pathogenic spirochete
genospecies share common reservoir hosts.
---------------------------------------------------------------------------
Acknowledgments
This study was supported by grants Ma 942/7-1 and Ma 942/10-1 from the
Deutsche Forschungsgemeinschaft. Dr. Richter was supported by a stipend
"Infektionsforschung" from the Bundesministerium fr Forschung und Technik.
Dr. Richter is a postdoctoral fellow conducting joint research in the
laboratories of Dr. Matuschka, Charit Medical School, Humboldt Universitt
zu Berlin, and Dr. Spielman at the Harvard School of Public Health. Her
research interests focus on the immunologic and molecular interface of the
host-vector-pathogen relationship in the epizootiology of Lyme disease.
Address for correspondence: Dania Richter, Institut fr Pathologie,
Charit, Medizinische Fakultt der Humboldt-Universitt zu Berlin,
Malteserstrae 74-100, 12249 Berlin, Germany; fax: 49-30-776-2085; e-mail:
dania.richter@ charite.de.
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____________________________________________________________________________
Commentary
____________________________________________________________________________
Commentary
Preparing for Pandemic Influenza: The Need for Enhanced Surveillance
K. F. Gensheimer,* Keiji Fukuda, Lynnette Brammer, Nancy Cox, Peter A.
Patriarca, and Raymond A. Strikas
*Maine Bureau of Health, Augusta, Maine, USA; Centers for Disease Control
and Prevention, Atlanta, Georgia, USA; and Food and Drug Administration,
Rockville, Maryland, USA
------------------------------------------------------------------------
Epidemic influenza, an age-old infectious disease, kills approximately
20,000 men and women in the United States every year. The emergence of
influenza viruses bearing novel surface antigens in 1918 (A/H1N1), 1957
(A/H2N2), and 1968 (A/H3N2) led to three worldwide pandemics of disease and
more than 600,000 deaths in the United States. During the influenza
pandemic of 1918-19, 500,000 deaths were reported, with persons younger
than 65 years of age accounting for 99% of all influenza-related deaths
(1).
Because it establishes the scientific foundation for a public health
response, surveillance is the single most important tool for identifying
new or reemerging infectious diseases with potential to cause serious
public health problems. Surveillance can be useful in rapidly identifying
and monitoring persons at highest risk, changes in disease rates, modes of
transmission, and groups at risk. Surveillance can help in planning and
evaluating disease prevention and control programs and can improve capacity
to control annual epidemics as well as the next influenza pandemic. The
outbreak of human infection with H5N1 (avian) influenza in Hong Kong in
1997 highlighted the potential of new and lethal pathogens to emerge
unexpectedly and questioned the capacity of local and state health agencies
to expand surveillance activities in response to a possible pandemic
influenza strain.
In 1993, a Working Group on Influenza Pandemic Preparedness and Emergency
Response (GrIPPE), which included influenza experts from the public and
private sectors, began to develop an updated, comprehensive blueprint for
an action plan for pandemic influenza for the United States (2). GrIPPE
identified surveillance as a key component of the pandemic plan. The group
has also recognized that to effectively address the threat of an influenza
pandemic, measures to reduce the impact of influenza must be in place and
operational at the state and local level now, during the prepandemic
period. Because more influenza-related illness and death occur in the
aggregate during regularly recurring influenza epidemics than during the
pandemics themselves, GrIPPE has attempted to link its plan to other
relevant public health initiatives such as those related to emerging
infections and adult immunization.
In 1994, the Council of State and Territorial Epidemiologists (CSTE) was
asked by GrIPPE to participate in the national pandemic influenza planning
process. As part of this effort, CSTE conducted a survey of state
epidemiologists in March 1995 to assess current influenza surveillance
systems; all 50 states and the District of Columbia responded. Questions
sought to determine the source and type of illness reports received, the
type of virologic surveillance activities performed, the state's perceived
preparedness for a pandemic, obstacles in detecting a new pandemic strain,
the need for a pandemic plan specific to the state's jurisdiction, whether
more influenza surveillance would be conducted if additional resources were
made available, and how much increased surveillance would cost.
All 51 respondents reported at least one source of influenza surveillance
information, and 39 (77%) identified sentinel physicians as the primary
source of disease reports. Forty-eight (94%) states had the capability,
through some private or public health laboratory, to identify influenza
viruses isolated in tissue culture. Of 47 laboratories that indicated to
what degree they could characterize influenza viruses, 37 (79%) could
subtype the viruses, while 10 (21%) could identify viruses only as
influenza A or B. Another 1995 survey by the Association of State and
Territorial Public Health Laboratory Directors of its membership was more
specific in defining influenza virologic resources available at each
state's public health laboratory, with 10 (20%) states indicating no state
public health laboratory capacity to isolate viruses and 13 (25%) state
public laboratories reporting no ability to subtype influenza isolates (A.
Di Salvo, pers. comm.).
In the CSTE survey, 34 (67%) states responded that their laboratory
surveillance system would be adequate to detect a new pandemic virus, with
29 (57%) states indicating that their disease surveillance system would be
adequate. Among the reasons given for the difficulty in detecting a new
pandemic strain were inadequate financial resources (20 [83%]), inadequate
personnel (19 [79%]), and low disease priority (10 [42%]).
Only 29 (59%) states reported that an influenza pandemic plan specific for
their jurisdiction was needed. Reasons for not developing a state pandemic
plan included lack of resources 4 (31%), insufficient interest 4 (31%), and
the perception that pandemic influenza did not pose an immediate threat 3
(23%). However, if targeted resources were made available, 44 (86%)
respondents indicated that they would increase laboratory surveillance for
influenza, and 39 (76%) indicated they would increase disease surveillance
activities. The estimates provided for increasing surveillance activities
were $2,000 to $100,000 (mean $37,602) for laboratory surveillance and
$2,000 to $100,000 (mean $40,914) for disease surveillance.
In the United States, public health services (including surveillance) are
provided most directly by municipal, county, and state health departments,
or a combination of all three. The 1995 CSTE survey found that many states
lacked surveillance activities dedicated to influenza; however, many states
would expand virologic and disease-based surveillance systems if nominal
resources were made available, despite the lack of urgency given to any
pandemic planning effort at the state level.
Several efforts have been undertaken at the national level to respond to
the states' needs and to promote enhanced preparedness for pandemic
influenza. A Pandemic Influenza Planning Guide for State and Local Health
Officials was developed as a result of these efforts. The guide provides a
checklist or set of guidelines for states to develop their own pandemic
plan. A draft guide was pilot-tested in six sites during February-March
1998.
The surveillance component of the planning guide calls for enhancements in
virologic and disease-based surveillance and improvements in surveillance
information systems. Specifically, the planning guide recommends that
during the prepandemic period 1) virologic surveillance capability be
improved by ensuring that at least one laboratory in each state or major
metropolitan area can isolate and subtype influenza viruses; 2)
disease-based surveillance capability be improved by expanding the existing
sentinel physician network, with the aim of establishing a population-based
system of approximately one sentinel physician per 250,000 population; 3)
contingency plans be developed for enhancing state and local virologic and
disease-based surveillance systems in the event of a novel virus alert or
pandemic alert; and 4) electronic and telecommunications capability with
neighboring jurisdictions and with the Centers for Disease Control and
Prevention (CDC) be enhanced.
Because the antigenic properties of influenza viruses change constantly,
surveillance to monitor the changes and their impact is necessary. One type
of surveillance information without the other is of limited value.
Although a disease-based surveillance system for influenza is in place in
the United States, the system is in jeopardy because of the misperception
that influenza is no longer an important public health problem and because
of continued erosion of resources supporting the public health
infrastructure at the state and local levels. Moreover, influenza
surveillance is compromised because influenza is not a nationally
notifiable disease.
One component of the existing surveillance system is weekly reports to
CDC's national notifiable disease system from each state epidemiologist
designating the level of influenza activity during the preceding week.
Levels of estimated activity are reported as widespread, regional,
sporadic, or nonexistent. The validity of these estimates has long been
questioned, since they may primarily reflect local interest or availability
of resources. Data on associated disease incidence are only rarely
collected.
Many states lack the financial resources for influenza virologic
surveillance, which is critical for monitoring antigenic drift and shift of
influenza viruses circulating among humans and is the basis for each year's
vaccine formulation. State public health laboratories that perform
virologic surveillance have a continuing need to culture and characterize
isolates on a timely basis. Submissions of specimens for virus isolation
are expected to decrease as rapid antigen test kits are improved and become
more widely available. Having fewer isolates for characterization is a
potential public health problem.
The demands of pandemic planning have prompted CDC and CSTE to begin
changing influenza surveillance. The premise of changes is that a solid
surveillance infrastructure must be in existence during the prepandemic
period. Disease- and laboratory-based surveillance is being reinforced and
other databases are explored as potential sources of additional qualitative
or quantitative data. Efforts are under way to upgrade the sentinel
physician network by enlisting and retraining more participants,
integrating influenza reporting with other state-based systems,
standardizing reporting procedures, and developing a semiautomated data
management system to provide rapid feedback. Twenty-eight states and the
District of Columbia pilot-tested steps to revise the existing sentinel
physician surveillance system during the 1997-98 influenza season. One
benefit of such efforts may be to increase the public's, medical
providers', and public health practitioners' understanding of influenza as
a potentially preventable disease.
A national plan is an important first step in highlighting the public
health problem posed by influenza and the need to identify ways in which
the federal, state, and local public health community can combine efforts
to address the problem.
Acknowledgments
The authors thank the many efforts of Lee Schmeltz and Willis Forrester.
References
  1. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ. Pandemic
     versus epidemic influenza mortality: a pattern of changing age
     distribution. J Infect Dis 1998;178:53-60.
  2. Patriarca PA, Cox NJ. Influenza pandemic preparedness plan for the
     United States. J Infect Dis 1997:176 (Supp 1):S4-7.
__________________________________________________________________________
Letters
__________________________________________________________________________
Letters
An Outbreak of Gastroenteritis in Japan due to Escherichia coli O166
To the Editor: Enteroaggregative Escherichia coli (EAggEC) heat-stable
enterotoxin 1 (EAST1) was originally found as an enterotoxin of EaggEC (1).
Recently, Yamamoto et al. (2) reported that the EAST1 gene, or its
variants, were present not only in EAggEC but in other diarrheagenic E.
coli, including some enteropathogenic E. coli (EPEC) and enterotoxigenic E.
coli (ETEC). Hedberg et al. (3) found that an outbreak of gastrointestinal
illness in 1991 had been caused by EAST1-producing E. coli that possessed
the EPEC gene locus for enterocyte effacement. We propose that E. coli
producing EAST1 but possessing no other identifiable pathogenic properties
may compose either a new group of diarrhea-associated E. coli or a new
subgroup of ETEC.
In an outbreak of gastroenteritis on July 23, 1996, in Osaka, Japan, 54 of
91 persons attending a meeting held in an office building on July 22, 1996,
became ill. The patients did not eat any common foods except the lunch
served at the office. Symptoms were diarrhea in 52 (96%); abdominal pain in
32 (59%); nausea in 8 (15%); fever in 8 (15%); and vomiting in 5 (10%). The
mean incubation period was 17 hours.
Stool specimens of 33 patients were examined, and E. coli O166 with an
unidentifiable H antigen were isolated from 29 specimens. Laboratory tests
for other bacterial pathogens and viruses were negative. The isolates
showed the same DNA banding pattern in pulsed-field gel electrophoresis
after treatment with the restriction enzymes Xba I or Not I.
The E. coli O166 organisms did not adhere to HEp-2 cells in a localized,
diffuse, or enteroaggregative manner and did not give mannose-resistant
hemagglutination of human or bovine red blood cells. Although the organisms
were further analyzed for expression of known ETEC colonization factors by
a dot-blot assay using specific monoclonal antibodies, they did not express
CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS7, CS17, PCFO159, PCFO166, or
CFA/III. In polymerase chain reaction (PCR) tests, the bacteria did not
have coding genes for verocytotoxin of enterohemorrhagic E. coli,
heat-labile, or heat-stable enterotoxin of ETEC, attachment and effacement
(eaeA) of EPEC, or invasion (invE) of enteroinvasive E. coli. Consequently,
they are not assigned to any of the recognized diarrheagenic groups of E.
coli: EPEC, ETEC, enterohemorrhagic E. coli, enteroinvasive E. coli,
EAggEC, and diffusely adhering E. coli. According to the PCR method of
Yamamoto et al. (2), however, the organisms possessed the EAST1 gene.
To our knowledge, this is the first report of an outbreak caused by
EAST1-producing E. coli that did not have other well-characterized
virulence genes. We believe that these strains should be assigned to a new
subgroup of ETEC. Such strains would not be detected in most current
surveys for diarrheagenic E. coli, as tests for EAST1 are rarely included.
The role of EAST1 in pathogenicity has been controversial. We propose that
diarrheal specimens be examined for EAST1-producing E. coli so that the
distribution of these organisms worldwide can be determined.
Yoshikazu Nishikawa,* Jun Ogasawara,* Anna Helander,  and Kosuke Haruki*
*Osaka City Institute of Public Health and Environmental Sciences, Osaka,
Japan; Gteborg University, Sweden
Acknowledgment
We thank Sylvia M. Scotland, who retired from the Laboratory of Enteric
Pathogens, Central Public Health Laboratory, London, for her critical
review of the manuscript.
References
  1. Savarino SJ, Fasano A, Robertson DC, Levine MM. Enteroaggregative
     Escherichia coli elaborate a heat-stable enterotoxin demonstrable in
     an in vitro rabbit intestinal model. J Clin Invest 1991;87:1450-5.
  2. Yamamoto T, Wakisaka N, Sato F, Kato A. Comparison of the nucleotide
     sequence of enteroaggregative Escherichia coli heat-stable enterotoxin
     1 genes among diarrhea-associated Escherichia coli. FEMS Microbiol
     Lett 1997;147:89-95.
  3. Hedberg CW, Savarino SJ, Besser JM, Paulus CJ, Thelen VM, Myers LJ, et
     al. An outbreak of foodborne illness caused by Escherichia coli
     O39:NM, an agent not fitting into the existing scheme for classifying
     diarrheagenic E. coli. J Infect Dis 1997;176:1625-8.
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Vibrio cholerae Outbreak in Italy
To the Editor: On 16 June, the microbiology unit of the Hospital of Lodi
communicated to the local public health unit that Vibrio cholerae had been
isolated and identified by standard biochemical tests in stool samples of
an outpatient whose clinical data were unknown. On the same day, we
contacted and interviewed the patient to investigate risk factors for
cholera. The patient reported abdominal pain starting on 6 June and then
severe diarrhea (10 to 12 stools per day) until 13 June; on that day the
patient went to his general practitioner, who gave him loperamide and
suggested a coproculture. The patient never traveled to cholera-endemic
areas; did not eat raw mussels, uncooked fish, or vegetables of uncertain
origin or from cholera-infected areas; and did not swim in rivers or lakes.
The patient reported that he ate a seafood salad in the canteen of his work
place on 5 June and that three out of the four persons who ate the same
kind of salad also had abdominal symptoms. Subsequently, the Istituto
Superiore di Sanit (ISS) in Rome confirmed isolation of toxinogenic V.
cholerae O1, biotype El Tor, serotype Ogawa, from stools from the index
patient.
The canteen where the index patient had eaten the seafood salad was 1 of 17
supplied by a single cooking center that used a precooked, frozen,
ready-to-eat product including shrimps, scallops, mussels, hen clams,
cuttlefishes, and squid. Each product was cooked and frozen in the country
of origin and mixed in Italy by an importer who packaged the seafood salad.
Tracking the products around the world was difficult, but we learned that
at least some had come from Far East countries where cholera is endemic.
Approximately 125 servings of the same food were distributed within our
local public health area (Azienda Sanitaria Locale) and more than 400 in
other areas.
We performed an epidemiologic case-control investigation beginning 18 June
involving 454 persons (94 who had eaten the seafood salad and 360 controls
who had eaten in the same canteen any food except seafood salad); 37 (39%)
of the persons who had eaten the seafood salad had had at least one episode
of diarrhea or other relevant gastrointestinal symptoms, as compared to one
(0.3%) of those who had not eaten it. We did not find symptomatic patients.
The corresponding odds ratio was 233 (95% confidence interval, 97 to 560).
No symptomatic person had to be hospitalized because of symptoms or
required intravenous treatment; three or more loose or watery stools during
a 24-hour period were reported in 24 cases. We performed coprocultures
(using TCBS medium) between 23 June and 3 July of all 94 persons who had
eaten the seafood salad. One positive coproculture for V. cholerae O1 Ogawa
(same strain) was identified on 25 June; the isolation was subsequently
confirmed by the ISS. This second patient with a positive culture worked in
a factory in a different town. She had severe diarrhea on 6, 7, and 8 June.
Her family doctor gave her rifaximin but did not ask for a coproculture,
but a specimen was obtained on 23 June. She did not report risk factors for
cholera infection, except having eaten seafood salad on June 5 in the
canteen of her work place. The delay between exposure to V. cholerae and
the coprocultures was longer than 1 week (median delay 26 days, range 19 to
31), and it is therefore not surprising that others who had eaten the
seafood salad did not have positive results. Both the culture-positive
index case-patient and the woman were recultured three more times; negative
results were obtained.
The identification of this cholera outbreak is a sentinel episode
confirming (1,2) that, if not adequately monitored, food preparation and
distribution can cause serious infectious diseases in industrialized
countries.
Luca Cavalieri d'Oro,* Elisabetta Merlo, Eugenio Ariano, Maria Grazia
Silvestri, Antonio Ceraminiello, Eva Negri,* and Carlo La Vecchia*
*Istituto di Ricerche Farmacologiche "Mario Negri," Milan, Italy; Azienda
Sanitaria Locale della Provincia di Lodi, Lodi, Italy; and Universit
degli Studi di Milano, Milan, Italy
References
  1. Centers for Disease Control and Prevention. Cholera associated with
     food transported from El SalvadorIndiana, 1994. MMWR Morb Mortal Wkly
     Rep 1995;44:385-6.
  2. Centers for Disease Control. Cholera associated with imported frozen
     coconut milkMaryland, 1991. MMWR Morb Mortal Wkly Rep 1991;40:844-5.
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Shiga ToxinProducing Escherichia coli O157:H7 in Japan
To the Editor: Shiga toxin-producing Escherichia coli (STEC) O157:H7
infection, which can cause hemolytic uremic syndrome and death, is a global
public health concern. Patients younger than 5 years of age are at high
risk for hemolytic uremic syndrome (1) and shed the organism longer than
adults (2). The public health importance of this symptomatic shedding in
transmission among preschool children is well established (3); however,
that of symptom-free shedding in adults is unknown. We report here that the
rate of symptom-free STEC O157:H7 shedding is higher in adults 30 to 49
years of age than in others.
STEC infections have been notifiable in Japan since August 1996. When STEC
is found in the feces of patients in schools, families, and hospitals,
local health centers and public health institutes must test (generally
using MacConkey sorbitol agar with cefixime-potassium tellurite medium) for
the pathogen in stool specimens of contacts of the patients. The pathogen
is also sought twice a month in the stool specimens of food-handlers. All
isolates from culture-positive patients are collected by Japan's National
Institute of Infectious Diseases.
In 1997, 1,412 STEC O157:H7 human isolates were examined for subtyping of
Shiga toxin genes stx1 and stx2 by polymerase chain reaction, for
genotyping by XbaI-digested pulsed-field gel electrophoresis (PFGE) (4,5),
and for their relationship with symptoms; 1,381 isolates (from
culture-positive persons with well-characterized clinical status) were
further analyzed. The rates by age group among STEC O157:H7-shedding
persons reporting one or more symptoms (vs. culture-positive persons
without symptoms) were as follows: 82% (475 of 576) younger than 10 years
old; 81% (145 of 178), 10 to 19 years; 63% (98 of 156), 20 to 29 years; 25%
(32 of 128), 30 to 39 years; 34% (34 of 100), 40 to 49 years; 54% (57 of
106), 50 to 59 years; 56% (38 of 68), 60 to 69 years; 68% (47 of 69), older
than 70 years. Culture-positive persons under 20 years of age, especially
children under 10 years of age, were more likely to have symptoms than
other age groups. Intermediate rates of symptom-free persons with positive
stool cultures occurred in young adults (20 to 29 years of age) and the
elderly (70 years of age), while the highest rates of stool-positive,
symptom-free persons were adults, especially those between 30 and 49 years
of age. In terms of pathogen virulence, we did not find significant
differences in the distribution of stx subtypes and PFGE genotypes between
strains shed by symptomatic and asymptomatic persons. These results suggest
that the rate of symptom-free STEC O157:H7 shedding may be associated with
age rather than organism-related factors. Possible age-related host factors
that could influence the presence of STEC O157:H7 in the stools of
symptom-free persons include qualitative and quantitative differences in
intestinal cross-reactive antibodies against STEC O157:H7, intestinal
bacterial flora, or the sensitivity to Stx toxins between children and
adults. Further investigations will be required to determine the relative
importance of these and other host factors. Our finding of a high rate of
asymptomatic shedding in adults may suggest the potential for secondary
transmission of the bacteria from symptom-free STEC O157:H7-shedding adults
to healthy children.
Acknowledgments
We thank investigators of prefectural and municipal public health
institutes for their collaboration.
This work was supported by a grant from the Ministry of Health and Welfare
of Japan.
Jun Terajima, Hidemasa Izumiya, Akihito Wada, Kazumichi Tamura, and Haruo
Watanabe
National Institute of Infectious Diseases, Tokyo, Japan
References
  1. Tarr PI. Escherichia coli O157:H7: clinical, diagnostic, and
     epidemiological aspects of human infection. Clin Infect Dis
     1995;20:1-8.
  2. Belongia EA, Osterholm MT, Soler JT, Ammend DA, Braun JE, MacDonald
     KL. Transmission of Escherichia coli O157:H7 infection in Minnesota
     child day-care facilities. JAMA 1993;269:883-8.
  3. Karch H, Russman H, Schmidt H, Schwarzkopf A, Heesmann J. Long-term
     shedding and clonal turnover of enterohemorrhagic Escherichia coli
     O157:H7 in diarrheal diseases. J Clin Microbiol 1995;33:1602-5.
  4. 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. Lancet 1996;348:831-2.
  5. Izumiya H, Terajima J, Wada A, Inagaki Y, Itoh K, Tamura K, et al.
     Molecular typing of enterohemorrhagic Escherichia coli O157:H7
     isolates in Japan by using pulsed-field gel electrophoresis. J Clin
     Microbiol 1997;35:1675-80.
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Streptococcus pyogenes Erythromycin Resistance in Italy
To the Editor: Streptococcus pyogenes resistance to erythromycin began to
emerge as a serious problem worldwide in the early 1990s. In some areas in
Italy, 30% to 40% of strains have beome resistant (1-3). Throughout Italy,
the use of macrolides, particularly the newest ones (azithromycin and
clarithromycin), has increased in the treatment of infections caused by
Group A streptococci. This therapeutic approach is contrary to current
guidelines, which recommend using betalactam antibiotics as first-choice
therapy and reserving macrolides only for patients allergic to betalactams.
In 1997 in Finland, a decrease was observed in the use of macrolide
antibiotics in ambulatory patients from 2.40 defined daily doses per 1,000
inhabitants in 1991 to 1.38 in 1992. Subsequently, the maintenance of doses
at 1.28 to 1.74 defined daily doses resulted in a substantial decrease in
the percentage of group A streptococcal resistance to erythromycin,
reported as 16.5% in 1992, 19% in 1993, 15.6% in 1994, 10% in 1995, and
8.6% in 1996 (4). These data prompted us to evaluate such phenomena in our
geographic area, the urban area of Genoa, Italy (approximately 120,000
inhabitants).
From January 1991 to June 1998, 311 (6.1%) of 5,117 strains of S. pyogenes
throat swabs from patients with pharyngotonsillitis were isolated. We
observed a higher number of group A streptococci isolates from throat swabs
starting in 1996 than we had in 1991 to 1995 (chi-square = 35.653, p
<0.0001). All isolates were tested for susceptibility to penicillin and
erythromycin by standard susceptibility tests (broth microdilution) as
recommended by the National Committee for Clinical Laboratory Standards.
All isolates were susceptible to penicillin. From 1991 to 1996, the
percentage of S. pyogenes resistant or with intermediate resistance to
erythromycin increased from 0% to 50% (1992, 6%; 1993, 13%; 1994, 14%;
1995, 24%; 1996, 50%). In 1997 and the first half of 1998, resistance to
erythromycin decreased to 39% and 34%, respectively. The number of
resistant strains before 1996 was significantly lower than from 1996 to
1998 (chi-square = 50.386, p <0.0001). Analysis of antibiotic consumption
in our district showed an increase in the use of macrolides (erythromycin
and the new compounds clarithromycin and azithromycin) from 0.445 defined
daily dose per 1,000 inhabitants in 1994 to 1.140 in 1996. In 1997 and in
the first half of 1998, consumption decreased to 0.9 and 0.8, respectively;
we observed a correlation between the number of resistant isolates and the
defined daily dose increase (correlation [R2] = 0.795, p = 0.0153).
S. pyogenes resistance to erythromycin rose from 6% to 50% in only 4 years
and then rapidly decreased from 50% to 34% in an 18-month period,
corresponding to a 57% decrease in defined daily dose (from 1.41 in 1996 to
0.8 in the first half of 1998). Our data suggest that S. pyogenes
resistance to erythromycin is associated with frequency of macrolide use.
Matteo Bassetti, Enrico Mantero, Giorgio Gatti, Antonio Di Biagio, and
Dante Bassetti
University of Genoa, G. Gaslini Children's Hospital, Genoa, Italy
References
  1. Cipriani P, Debbia EA, Gesu GP, Menozzi MG, Nani E, Nicolosi V, et al.
     Indagine policentrica AMCLI sull'incidenza di resistenze agli
     antibiotici in S. pyogenes. Microbiologia Medica 1995;10:171-4.
  2. Cornaglia G, Ligozzi M, Mazzariol A, Valentini M, Orefici G, Fontana
     R. Rapid increase of resistance to erythromycin and clindamycin in
     Streptococcus pyogenes in Italy, 1993-1995. Emerg Infect Dis
     1996;2:339-42.
  3. Cocuzza C, Blandino G, Mattina R, Nicoletti F, Nicoletti G. Antibiotic
     susceptibility of group A streptococci in 2 Italian cities: Milano and
     Catania. Microb Drug Resist 1997;3:379-84.
  4. Seppala H, Klaukka T, Vuopio-Varkila J, Muotiala A, Helenius H, Lager
     K, et al. The effect of changes in the consumption of macrolide
     antibiotics on erythromycin resistance in group A streptococci in
     Finland. Finnish Study Group for Antimicrobial Resistance. N Engl J
     Med 1997;337:441-6.
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Estimated Incidence of Clostridium difficile Infection
To the Editor: Since the publication of our article Increasing
hospitalization and death, possibly due to Clostridium difficile diarrheal
disease (1), we have received several requests to estimate the incidence of
C. difficile infection. Our original study included only hospitalized
patients treated at the Lovelace Medical Center from 1993 to 1996, and no
information on the incidence of C. difficile infection. In response to
these requests, we used inpatient and outpatient medical claims for the
Lovelace managed care population to calculate incidence rates. We searched
medical claims for the Lovelace Health Plan/Senior Plan (LHP) to identify
patients who had a C. difficile diagnosis between January 1, 1993, and
December 31, 1997. LHP members are residents of New Mexico, most residing
in or near Albuquerque.
LHP had approximately 713,000 person-years of enrollment from 1993 to 1997.
We identified 104 members with a C. difficile diagnosis on a claims record
during this period. This group includes most of the patients in our
original study. Most patients (62.5%) were identified exclusively from
inpatient records; another 15.4% had both an inpatient and an outpatient
record with a C. difficile diagnosis; and 22.1% had only an outpatient C.
difficile diagnosis. We calculated an age-adjusted rate of infection
(adjusted to the 1990 U.S. population), for each year and for the 5-year
period. The incidence of C. difficile infection for all members during 1993
to 1997 was 14.8 cases per 100,000 person-years of enrollment. The patients
rates for male and female were essentially the same (14.4 vs. 15.5,
respectively). The rates increased dramatically with age. For persons ages
0-4, the age-adjusted rate per 100,000 person-years of enrollment (number
of cases) was 5.3 (2); for 5-14, 2.7 (3), for 15-24, 2.2 (2); for 24-34,
6.4 (6); for 35-44, 9.2 (12); for 45-54, 15.7 (17); 55-64, 16.8 (10);
65-74, 38.5 (19); and 75+, 98.9 (33). The overall average rate of infection
was 15.4; there were 104 cases.
The rate of infection may have declined since 1993 in this population. The
1993 rate was 24.5 per 100,000 person-years of enrollment, declining to
11.1 in 1997 (1993, 24.4; 1994, 19.1; 1995, 9.9; 1996, 12.3; and 1997,
11.1).
Our method for estimating rates has some limitations. We did not examine
laboratory records to confirm the diagnosis. In addition, some
laboratory-confirmed infections may not have resulted in a claims record
with a C. difficile diagnosis. The Lovelace managed-care population is an
insured, generally healthy population that may not have the characteristics
of patients in other health care delivery settings or, because of its
geographic restriction, the characteristics of the general U.S. population.
Nevertheless, these estimates provide a basis for determining the magnitude
of the public health problem of C. difficile infection. Additional
surveillance studies are needed to better estimate the incidence of
infection and to determine whether the incidence has declined during recent
years.
Floyd Frost,* Judith S. Hurley,* Hans V. Petersen,* and Roman N. Casciano
*Southwest Center for Managed Case Research, Albuquerque, New Mexico, USA;
and Analytica Group, Ltd., New York, New York, USA
Reference
  1. Frost F, Craun GF, Calderon RL. Increasing hospitalization and death
     possibly due to Clostridium difficile diarrheal disease. Emerg Infect
     Dis 1998;4:619-25.
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Diphtheria in Eastern Nepal
To the Editor: Diphtheria, caused by Corynebacterium diphtheriae, was a
major childhood killer until the advent of the Expanded Program on
Immunization when diphtheria, pertussis, and tetanus (DPT) vaccination was
greatly increased; diphtheria gradually declined in many countries. We
report two cases of diphtheria identified at the B.P. Koirala Institute of
Health Sciences Hospital, Dharan, Nepal.
During April 1996, a 6-year-old patient had fever (for 5 days), difficulty
in swallowing and breathing, and change of voice (for 4 days). Throat
examination showed a grayish-white membrane over the right tonsil, uvula,
and adjacent soft palate. The membrane could not be removed, and the larynx
could not be examined. Swabs were taken from the membrane area and sent to
the laboratory, where smears were made and stained by Gram and Albert
stains. Gram-stained smears showed a large number of gram-positive bacilli
with the appearance of Chinese letters, and Albert stain showed bacilli
with numerous metachromatic granules. A diagnosis of faucial diphtheria,
with a strong possibility of laryngeal diphtheria, was made. The patient
was treated with parenteral penicillin and diphtheria antitoxin. His
condition improved after 6 days of therapy.
In December 1996, a 9-year-old patient sought treatment for chronic pain
and discharge in the left ear. On examination, he had mucopurulent
discharge, antral perforation, and mastoid tenderness. Throat examination
showed bilateral tonsilitis. A provisional diagnosis of acute mastoiditis
associated with acute septic tonsillitis was made. Throat swabs were
collected and sent to the laboratory; smear findings showed typical
organisms morphologically resembling C. diphtheriae. Culture done on 10%
defibrinated sheep blood agar and Loefflers serum slope grew colonies
consistent with C. diphtheriae. In addition to local antibiotic to the ear,
the patient was given parenteral penicillin, gentamicin, and metronidazole.
Because the patient had no features of systemic toxicity, no antidiphtheria
serum was administered. The patient became well and was discharged on day
4.
In the first case, a throat culture could not be done because the patient
had already received local antiseptic paint. However, the diagnosis was
clinically consistent with classic diphtheria with features of toxicity. In
the second case, diphtheria was suspected only after bacteriologic
examination. Unlike patient 1, patient 2 had no evident features of
systemic toxicity. Hence the isolate could be nontoxigenic. Localized
diphtheria due to nontoxigenic C. diphtheriae is known to occur (1).
The two patients did not give a complete history of immunization and may
not have been vaccinated (or may have been partially vaccinated) with DPT.
On the Indian subcontinent, DPT vaccination coverage is reported to be 80%.
However, it may not be so in all areas, and immunization may have decreased
to approximately 50% in certain areas of Southeast Asia (2). This may also
be true in certain areas of eastern Nepal. An immunization status survey
done in midwestern Nepal from 1989 to 1990 showed that DPT coverage was
unsatisfactory (3). Lack of sustained immunization may even result in
outbreaks. The recent epidemics of diphtheria in the Ukraine, Russian
Federation, and other countries of the former Soviet Union are examples of
resurgence due to ineffectively maintained immunization programs (4,5).
Diphtheria, still occasionally seen in many Southeast Asian countries
including India and Nepal, is thought to be declining in these areas.
However, accurate data have not been recently available, particularly from
Nepal, because reporting is infrequent, laboratory confirmation is not
available, and the extent of carriers is not clearly known (2).
These two cases show the persistence of diphtheria in a population in Nepal
immunized with DPT and underscore the need for careful surveillance,
laboratory documentation of clinical diphtheria, and increased immunization
of children in this area.
H. Srinivasa, S.C. Parija, and M.P. Upadhyaya
B.P. Koirala Institute of Health Sciences, Dharan, Nepal
References
  1. Dixon JMS, Noble WC, Smith GR. Diphtheria, other mycobacterial, and
     corynebacterial and coryneform infections. In: Topley and Wilson's
     principle of bacteriology, virology and immunology. Vol 3. 8th ed.
     London: Edwards Arnold; 1990. p. 55-79.
  2. Srinivasa H. Immunizing children in Southeast Asia: a critical
     appraisal of current EPI status and future prospects. In: Immunizing
     agents for tropics: success, failure and some practical issues (BPKIHS
     Monograph Series 1). B.P. Koirala Institute of Health Sciences
     (BPKIHS) Hospital, Dharan, Nepal; 1997. p. 1-8.
  3. Shrestha IB. Immunization status in mid-western region of Nepal. In:
     Health Research Abstract 1991-1994. Nepal Research Council 1995.
     Proceedings of Second National Seminar on Health Research in Nepal at
     Kathmandu, Nepal. 1994 Dec 20-22.
  4. The World Health Reports. Fighting disease, fostering development.
     Report of the Director General, World Health Organization, Geneva;
     1996.
  5. Begg N, Balraj B. Diphtheria: are we ready for it? Archives of
     Childhood 1995:568-92.
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Commercial Use of Burkholderia cepacia
To the Editor: In their review of the potential threat to human health by
the commercial use of Burkholderia cepacia, Holmes et al. (1) focus on the
biopesticidal uses of this bacterium in agriculture. By virtue of its
ability to antagonize a number of soilborne plant pathogens, B. cepacia is
an attractive natural alternative to currently used chemical pesticides,
such as captan, mancozeb, and metalaxyl. The replacement of these highly
toxic agents, which are among the mainstays of crop protection chemicals,
by safer products is a laudable goal. However, despite being nonpathogenic
to healthy humans (and thus classified as a Biosafety Level 1 species), B.
cepacia can cause life-threatening pulmonary infection in persons with
cystic fibrosis. Holmes et al. call for a moratorium on the use of B.
cepacia in agriculture until more is known about risks from such use.
Perhaps of greater concern than agricultural use is B. cepacia's use as a
bioremedial agent. Holmes et al. only briefly refer to the capacity of this
species to degrade chlorinated aromatic substrates such as those found in
certain pesticides and herbicides. By virtue of its extraordinary metabolic
versatility, B. cepacia can use such compounds as nutrient carbon energy
sources. In addition, some strains produce enzymes capable of degrading
nonnutritive substrates, such as trichloroethylene (TCE), a major ground
water contaminant used in the dry cleaning industry and in degreasing
solvents.
The degree to which B. cepacia is being used in bioremediation products is
unknown; however, the species has been used extensively to degrade ground
water TCE contamination in at least one large U.S. city. A number of
environment-friendly bioremediation products containing only naturally
occurring, nonpathogenic bacteria are being marketed for use in drain
opening and grease eradication systems. Because their formulations are
proprietary, it is not known if these products contain B. cepacia; however,
franchises that distribute such totally natural, noncorrosive, nontoxic
products specifically target fast-food restaurants, photo processing
facilities, and hospital radiology departments.
In the United States, the biopesticidal use of microorganisms such as B.
cepacia is regulated by the Environmental Protection Agency (EPA) under the
Federal Insecticide, Fungicide, and Rodenticide Act; however, the use of
naturally occurring, nonpathogenic bacteria as bioremedial agents is
essentially unregulated. Only new microorganisms (i.e., intergeneric or
formed by combining genetic material from organisms in different genera)
are regulated by EPA under the Toxic Substances Control Act (TSCA) (2).
Ironically, TSCA regulations provide a strong disincentive to the
development of safer microbiologic alternatives for use in bioremediation.
For example, although the genetic elements responsible for TCE degradation
by B. cepacia have been cloned, their recombination into another
nonpathogenic bacterial host (e.g., Escherichia coli) would constitute a
new microorganism, the licensure of which would be considered prohibitively
time-consuming and expensive by many companies.
In Canada, biopesticidal uses of microorganisms are regulated by the Pest
Management Regulatory Agency of Health Canada, under the Pest Control
Products Act (PCPA); bioremedial uses are regulated by Environment Canada
under the Canadian Environmental Protection Act (CEPA) (3). Both naturally
occurring and genetically engineered microorganisms are strictly controlled
under these acts. However, accurate species identification is the
cornerstone of all notification of products under the Canadian regulations.
This presents a further dilemma. At least five genomovars (discrete
species) constitute what has recently been designated the "B. cepacia
complex" (4). Insofar as the taxonomy of this group is poorly defined,
there are no conventional taxonomic designations to distinguish pathogenic
from nonpathogenic species. At present, it appears that all five B. cepacia
genomovars are capable of causing infections in vulnerable persons (4).
Because the epidemiology of B. cepacia complex infection in humans is
incompletely understood, the threat posed by the inclusion of this species
in biopesticides and bioremedial products is difficult to quantify.
However, we agree with Holmes et al. that such use should be approached
with considerable caution. In a broader context, the commercial use of B.
cepacia illustrates our incomplete understanding of nonpathogenic bacteria
and their potential to cause human disease. Regulations governing the use
of microorganisms in industry must constantly adapt to keep pace with the
emergence of infections due to nonpathogens and limit risk to human health.
John J. LiPuma* and Eshwar Mahenthiralingam
*MCP Hahnemann University, St Christopher's Hospital for Children,
Philadelphia, Pennsylvania, USA; and University of British Columbia,
Vancouver, British Columbia, Canada
References
  1. Holmes A, Govan J, Goldstein R. Agricultural use of Burkholderia
     (Pseudomonas) cepacia: a threat to human health? Emerg Infect Dis
     1998;4:221-7.
  2. Microbial products of biotechnology; final regulations under the Toxic
     Substances Control Act; final rule. Washington: U.S. Environmental
     Protection Agency, 1997. Federal Register. Vol 62. p. 17910-58.
  3. Guidelines for the notification and testing of new substances:
     organisms. Pursuant to the New Substances Notification Regulations of
     the Canadian Environmental Protection Act. Ottawa, Canada: Environment
     Canada, Health Canada, 1997.
  4. Vandamme P, Holmes B, Vancanneyt M, Coenye T, Hoste B, Coopman R, et
     al. Occurrence of multiple genomovars of Burkholderia cepacia in
     cystic fibrosis patients and proposal of Burkholderia multivorans sp.
     nov. Int J Syst Bacteriol 1997;47:1188-200.
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Human Rabies in Israel
To the Editor: Rabies, a major zoonotic disease in the Middle East, has two
main epidemiologic forms: urban and sylvatic. The last case of human rabies
in Israel was in the Golan Heights in 1971 (1). Twenty-five years later, in
1996, rabies was reported in a 20-year-old soldier, and then two cases were
documented in 1997.
The first case-patient, a soldier in the Golan Heights, was bitten on the
lip by an unidentified animal while sleeping. The wound was cleansed and
sutured; clinical signs started 39 days later with high fever and headache.
The patient was admitted to an emergency room with hallucinations,
difficulty in swallowing, and generalized weakness, and rabies was
considered in the differential diagnosis; 3 days later the patient became
comatose. Samples of saliva, serum, and cerebrospinal fluid; skin biopsy
tissue; and corneal impressions were sent to the Pasteur Institute, Paris,
France. Eight days after clinical signs developed, rabies was diagnosed by
the Kimron Institute by heminested reverse transcription-polymerase chain
reaction (hnRT-PCR) on the patient's saliva (2). The RT step was performed
with the specific primer 113 (5'-GTAGGATGATATATGGG-'3 at 1013-1030),
followed by PCR with the 509 (5'-GAGAAAGA ACTTCAAGA-'3 at 1156-1173) and
304 (5'-GAGT CACTCGAATATGTC-'3 at 1513-1533) primers. The hn-PCR was
performed with the 509 and 105 (5'-TTCTTATGAGTCACTCGAATA TGTCTTGTTTAG-'3 at
1393-1426) primers (3). The PCR results were confirmed by the Pasteur
Institute 3 days later, and the patient died 35 days after clinical
symptoms appeared.
The second case-patient was a 7-year-old girl admitted to the hospital
unconscious. Two months before admission, she had been scratched while
sleeping by an unidentified animal. On the second hospital day, generalized
convulsions and gasping occurred. During the following days, brain stem
function progressively deteriorated. Rabies was diagnosed by hnRT-PCR on
the saliva sample, and the diagnosis was confirmed by the Centers for
Disease Control and Prevention (CDC). The patient died despite supportive
care.
The third case-patient, a 58-year-old man with fever, headache, and sore
throat, was diagnosed as having pharyngitis and received an oral
antibiotic. The patient had been bitten 3 months earlier while sleeping. On
admission, the lumbar puncture, computerized tomography scan, and
electroencephalogram were normal. On the third hospital day, he had
respiratory arrest; during orotracheal intubation, acute laryngospasm with
copious amounts of salivation occurred. Rabies was suspected, and viral RNA
in the saliva was detected by hnRT-PCR. One day later the patient died.
We injected antemortem saliva and postmortem brain tissue from these
patients into suckling mice intracerebrally. Virus was isolated from saliva
samples of case-patients 1 and 3 but not from the sample of case-patient 2.
Rabies virus antigen in the brain tissue was confirmed by direct
immunofluorescence assay, and viral RNA was detected by RT-PCR.
For genetic analysis, we used brain samples from the three case-patients
and from animals that died of rabies near the location of the case-patients
to amplify and sequence a 328-bp (264 bp from the 3' of the N gene and 64
bp of the 3' NS-N region) fragment. On the basis of homologic results of
nucleotide sequences in the three case-patients and in virus isolates from
animals in the same regions, we concluded that a reservoir for rabies in
foxes is responsible for infection of all three humans.
The three human isolates were tested with a panel of 19 anti-N protein
monoclonal antibodies (CDC, Atlanta, GA, USA) and compared with those of
rabies isolates from the geographic vicinity of the human cases. Isolates
from case-patients 1 and 3 belonged to variant 1 (MAb C18 negative) and
were similar to virus isolates from 10 foxes, one jackal, and four cattle
in the same regions. Isolates from case-patient 2 belonged to antigenic
variant 2 (MAbs C2, C7, C12, C13, C18 negative) and were similar to
isolates from four foxes, one dog, and one cow in the vicinity of the
second case-patient.
Early antemortem diagnosis of virus in an infected human is very important.
Checking for virus in saliva eliminates the difficulty of tissue sampling
from humans with suspected cases of rabies, and the sensitivity of hnRT-PCR
makes it the technique of choice for detecting limited amounts of virus.
Previous work showed that a 200-bp region of the N gene had only one
nucleotide difference between them (4). Moreover, two samples from a region
in western Mexico, isolated 30 years apart, were identical in sequence (4).
Incorporation of the reference strains Pasteur and SAD B19 into our
phylogenetic tree indicated that the three human viruses we isolated belong
to lyssavirus genotype 1.
Dan David,* Charles E. Rupprecht, Jean Smith, Itzhak Samina,* Shmuel
Perl,* and Yehuda Stram*
*Kimron Veterinary Institute, Bet Dagan, Israel; and Centers for Disease
Control and Prevention, Atlanta, Georgia, USA
References
  1. Shimshony A. Veterinary public health in Israel. Revue Scientifique et
     Technique Office International Des Epizooties 1992;11:77-98.
  2. Heaton PR, Johnstone P, McElhinnely M, Coweley R, O'Sullivan E, Whitby
     JE. Heminested PCR assay for detection of six genotypes of rabies and
     rabies related viruses. J Clin Microb 1997;35:2762-6.
  3. Smith JS. Rabies virus. 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. 907-1003.
  4. Smith JS, Seidel HD, Warner CK. Epidemiology and historical
     relationships among 87 rabies virus isolates determined by limited
     sequence analysis. J Infec Dis 1992;166:296-307.
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Emerging Infections and Disease Emergence
To the Editor: Emerging infections have been defined as diseases whose
incidence in humans has increased within the last 2 decades or threatens to
increase in the near future (l). This definition, with minor variations,
has continued to be used, although occasional debate erupts over whether
one disease or another is truly emerging. Use of the term "emerging" has
facilitated communication about the changed pattern of infectious diseases
in recent years. While the study of infectious organisms and clinical
training about emerging infections are manifestly necessary, they are not a
sufficient foundation for understanding the process of disease emergence. I
would propose, specifically, that we distinguish between emerging
diseasesthe study of specific infections that are changingand the study
of disease emergence.
Studies of emerging infections typically rely on disease, organismic, or
syndromic approaches. Meetings on emerging infections typically cover newly
recognized or characterized organisms or diseases and update information
about the recognition, diagnosis, treatment, prevention, and control of
these infections. This ongoing education is essential for practicing
clinicians, who finished their formal training before AIDS, Lyme disease,
ehrlichiosis, Helicobacter pylori infection, cryptosporidiosis,
cyclosporiasis, and many other infections were described. These meetings
also help clinicians learn how to fit new information into their existing
knowledge base: What is the probability that a person with rash and fever
has ehrlichiosis and that a person with fever and pulmonary infiltrates has
hantavirus pulmonary syndrome?
By contrast, understanding the process of disease emergence involves
studying the origins and ecology of emerging infections. Many disciplines
relevant to disease emergence lie outside traditional infectious disease
training and research and include evolutionary biology, demography,
population dynamics, ecology, vector biology, climatology, epidemiology,
genetics, veterinary medicine, and behavioral sciences (2). Infectious
diseases of animals and plants have both a direct and indirect impact on
human health. The study of infectious diseases in other species may provide
important insights into understanding the process of disease emergence in
humans. The study is also relevant to understanding the species-to-species
spread of organisms.
Tools used to study and understand disease emergence include mathematical
modeling, geographic information systems, remote sensing, molecular methods
to study the genetic relatedness of organisms, and molecular phylogeny.
Paleobiology, paleoecology, and studies that allow the reconstruction of
past events may help inform future research and policy.
A major challenge is to reach people with relevant skills, knowledge, and
experience and develop a coherent framework to advance the understanding of
the process of disease emergence. No one institution, organization, or
country can itself prevent or manage emerging infectious diseases.
In the study of emerging infections we focus on the organism, the patient,
and the human population. The study of disease emergence must be at the
systems level and must look at ecosystems, evolutionary biology, and
populations of parasites and hosts, whatever their species. A primary goal
should be to identify conditions or combinations or sequences of events
that herald a changed pattern of infections so that preventive strategies
can be used.
Mary E. Wilson
Mount Auburn Hospital, Cambridge, Massachusetts, USA; Harvard Medical
School and Harvard School of Public Health, Boston, Massachusetts, USA
References
  1. Lederberg J, Shope RE, Oaks SC Jr., editors. Emerging infections:
     microbial threats to health in the United States. Institute of
     Medicine. Washington: National Academy Press, 1992.
  2. Wilson ME, Levins R, Spielman A, editors. Disease in evolution: Global
     changes and emergence of infectious diseases. Vol 4. New York: Ann N Y
     Acad Sciences; 1994.
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Malaria Control in South America
To the Editor: The article by Roberts et al. regarding DDT use and malaria
in South America (1) correctly observes that health policy makers have
shifted the emphasis of malaria control programs from vector control to
case detection and treatment and that malaria control has been woefully
underfunded in recent years. However, their conclusions that increased
malaria is due to decreased spraying of homes with DDT and that DDT is
still needed for malaria control do not withstand close scrutiny.
The authors did not mention several factors influencing malaria increase in
recent decades, including growing antimalarial-drug resistance, the
deterioration of public health systems responsible for malaria control, and
large-scale migration to areas at high risk for malaria (e.g., almost all
Brazilian malaria cases occur in the Amazon region) (2,3). Extradomiciliary
malaria transmission, poor housing conditions, and human behavior in
frontier areas such as the Amazon region limit the usefulness of
insecticides. Thus, the deduction of causality between less house spraying
with DDT and increased malaria incidence under those circumstances is
questionable.
Roberts et al. have not actually linked increased malaria with eliminating
DDT use but rather with eliminating house spraying altogether, without
implementing effective alternatives. Malaria's recent decline in Brazil is
due to a strategy that combines health education, aggressive case detection
and treatment, and environmental management to eliminate Anopheles breeding
sites (C. Cato Prates, unpub. data). A similar strategy has sharply
reduced malaria incidence and deaths in Colombia (W. Rojas, unpub. data).
In Mexico, use of two synthetic pyrethroid insecticides (deltamethrin and
lambda cyhalothrin) for bed-net treatment and house spraying is controlling
malaria at a much lower cost than the use of the alternative insecticides
tried earlier and mentioned by Roberts et al. (4). Far from being pursued
"without meaningful debate," the reduction and phaseout of DDT and other
persistent organic pollutants is the subject of a 3-year United
Nationsfacilitated global negotiation process begun in June 1998.
Roberts et al. assert that DDT applied indoors does not move easily from
the application site; however, a mass balance model indicates that 60% to
80% of the DDT ends up outdoors within 6 months (K. Feltmate, A model and
assessment of the fate and exposure of DDT following indoor application
[bachelor's thesis]. Ontario: Trent University; 1998). From there, DDT can
be transported long distances in air, waterborne sediments, and biota,
accumulating in humans and other nontarget species (5). Meanwhile,
residents of sprayed houses accumulate high, persistent body levels of DDT
through skin contact and food contaminated with DDT from air and dust (6).
Long considered a probable human carcinogen, DDT also is associated with
reduced lactation, premature births, absorbed fetuses, and lower birth
weights (7-9). In addition, recent animal research has raised the
possibility that exposure of human fetuses or infants to DDT may cause
permanent behavioral changes and impairment of body systems (10-12).
Synthetic pyrethroid insecticides used on bed nets or for house spraying
against malaria-infected mosquitoes seem safer for human health than DDT
because humans and other mammals possess the ability to hydrolyze the
pyrethroids rapidly and excrete them from the body (13-14). Nevertheless,
DDT and pyrethroids share known health risks, notably endocrine disruption,
and the possible transgenerational consequences of chronic human exposure
to pyrethroids have not yet been studied (10,15-16). Optimal protection of
human health requires the development of integrated malaria control
strategies that eliminate or reduce routine insecticide use by taking
maximum advantage of environmental management, biological controls, and
other nonchemical vector control measures (17).
Patricia C. Matteson
U.N. Food and Agriculture Organization Programme for Community Integrated
Pest Management in Asia, Hanoi, Vietnam
References
  1. Roberts DR, Laughlin LL, Hshieh P, Legters LJ. DDT, global strategies,
     and a malaria control crisis in South America. Emerg Infect Dis
     1997:3:295-302.
  2. Wirth DF, Cattani J. Winning the war against malaria. Technology
     Review 1997;Aug/Sep:52-61.
  3. World Resources Institute, United Nations Environment Programme,
     United Nations Development Programme, the World Bank. World Resources
     1998-99. New York: Oxford University Press; 1998.
  4. Matteson PC, editor. Disease vector management for public health and
     conservation. Washington: World Wildlife Fund-US; 1999.
  5. Wania F, Mackay D. Tracking the distribution of persistent organic
     pollutants. Environmental Science & Technology News 1996;30:390-6.
  6. Bouwman H, Cooppan RM, Becker PJ, Ngxongo S. Malaria control and
     levels of DDT in serum of two populations in Kwazulu. Journal of
     Toxicology and Environmental Health 1991;33:141-55.
  7. Rogan WJ, Gladen BC, McKinney JD, Carreras N, Hardy P, Thullen J, et
     al. Polychlorinated biphenyls (PCBs) and dichlorodiphenyl
     dichloroethan (DDE) in human milk: effects on growth, morbidity, and
     duration of lactation. Am J Public Health 1987;177:1294-7.
  8. Gladen BC, Rogan WJ. DDE and shortened duration of lactation in a
     northern Mexican town. Am J Public Health 1995;85:504-8.
  9. Toxicological profile for 4,4'-DDE, 4,4'-DDD (updated). Atlanta: U.S.
     Department of Health and Human Services, Public Health Service, Agency
     for Toxic Substances and Diseases Registry; 1994. Pub. no. TP-93/05.
 10. Eriksson P. Developmental neurotoxicity of environmental agents in the
     neonate. Neurotoxicology 1997;48:5719-26.
 11. Rehana T, Rao PR. Effect of DDT on the immune system in Swiss albino
     mice during adult and perinatal exposure: humoral responses. Bull
     Environ Contam Toxicol 1992;48:535-40.
 12. Banerjee BD, Saha S, Mohapatra TK, Ray A. Influence of dietary protein
     on DDT-induced immune responsiveness in rats. Indian J Exp Biol
     1995;33:739-44.
 13. Ray DE. Pesticides derived from plant and other organisms. In: Hayes
     WJ, Laws ER, editors. Handbook of pesticide toxicology. Vol 2. San
     Diego (CA): Academic Press; 1991. p. 585-636.
 14. Casida JE, Gammon DW, Glickman AH, Lawrence LJ. Mechanisms of
     selective action of pyrethroid insecticides. Annu Rev Pharmacol
     Toxicol 1983;23:413-38.
 15. Patro N, Misha SK, Chattopadhyay M, Patro IK. Neurological effects on
     deltamethrin on the postnatal development of cerebellum of rat.
     Journal of Biosciences 1997;22:117-30.
 16. Smolen MJ, Sang S, Liroff RA. Hazards and exposures associated with
     DDT and synthetic pyrethroids used for vector control. Washington:
     World Wildlife Fund-US;1999.
 17. Resolving the DDT dilemma: protecting biodiversity and human health.
     Toronto, Canada: World Wildlife Fund-Canada; 1998.
---------------------------------------------------------------------------
Malaria Control in South America Response to P.C. Matteson
To the Editor: Dr. Matteson, whose letter relies heavily on unpublished
information and nonrefereed publications, states that growing drug
resistance has contributed to increasing malaria. While drug resistance is
important, when DDT use declined below effective levels (1), the proportion
of Plasmodium falciparum infections (including infections with resistant
strains) compared with P. vivax infections (no resistance) did not
progressively increase (2). Moreover, malaria has increased in Central
America, where drug resistance is unknown (3-6). As for attributing
increasing malaria to deteriorating public health systems, the changes
imposed on developing countries (in organizational structures of malaria
control programs and prohibiting DDT [1,7]) correlate with increasing
malaria rates (1).
Dr. Matteson states that large-scale migration explains why almost all
Brazilian malaria cases occur in the Amazon Basin. However, DDT cleared
malaria from the more populated and temperate southern regions of the
country (8, unpublished report: U.S. Agency for International Development
review in 1973-74 of Brazil's malaria eradication program). When DDT was in
full use (pre-1980), large increases in malaria did not accompany
population movement (1). With the 1970s' colonization program of the Basin
came malaria problems, but not large population-based malaria increases.
DDT prevented that (1,9-11). However, since DDT has been eliminated,
persistent urban malaria is again becoming a problem (12-16).
Other factors (biting behavior, housing conditions, and human behavior),
which Dr. Matteson attributes to increasing malaria, have always thwarted
interdiction of malaria transmission in the Amazon Basin (17;18; an
unpublished report: U.S. Agency for International Development review in
1973-74 of the malaria eradication program in Brazil) and are no more
important today than they were before.
A UN-facilitated global negotiation process cited as a meaningful debate
for malaria control is an effort to provide a legally binding agreement for
global elimination of DDT and other persistent organic pollutants, not an
open forum for debate of DDT use for malaria control.
Dr. Matteson claims that DDT is associated with reduced lactation. In the
United States, where DDT has been banned for 26 years, mothers who stay
home breast-feed for an average of 25.1 weeksmothers who work parttime,
for 22.5 weeks (19). In Belize, mothers in urban areas, where DDT is not
used for malaria control, breast-feed less than 38.4 weeksmothers in rural
areas with lifetime exposures to DDT breast-feed more than 57.2 weeks (20).
The World Wildlife Fund's mass balance model of DDT sprayed in houses used
to refute our assessment that DDT does not readily move away from sprayed
houses also mentions that "There are few...data against which to validate
the results of this...model, although actual data...should not be difficult
to obtain." (21). Studies of DDT use in agriculture show that most DDT
settles where it is applied (22).
Studies have shown no meaningful population-based adverse health effects
from DDT use, despite more than 50 years' exposure, and evidence argues
forcefully that DDT does not cause breast cancer (23).
Donald R. Roberts and Larry L. Laughlin
The Uniformed Services University of the Health Sciences, Bethesda,
Maryland, USA
References
  1. Roberts DR, Laughlin LL, Hsheih P, Legters LJ. DDT, global strategies,
     and a malaria control crisis in South America. Emerg Infect Dis
     1997;3:295-302.
  2. Brasil. Registro de casos de malria1960 a 1997. Gerncia Tcnica de
     Malria/FNS-Brasilia, Brasilia, Brasil.
  3. Pan American Health Organization. Status of malaria programs in the
     Americas. XL report. Washington: The Organization; 1991. p. 145.
  4. Pan American Health Organization. Status of malaria programs in the
     Americas. XLII report. Washington: The Organization; 1994. p. 116.
  5. Pan American Health Organization. Status of malaria programs in the
     Americas. XLIII report. Washington: The Organization; 1995. p. 25.
  6. Pan American Health Organization. Status of malaria programs in the
     Americas. XLIV report. Washington: The Organization; 1996. p. 23.
  7. Roberts DR. Resurgent malaria: DDT and global control. U.S. Medicine
     1998;34:36-8.
  8. de Bustamante FM. Distribuio geogrfica e periodicidade estacional
     da malria no Brasil e sua relao com os fatores climticos. Situao
     atual do problema. Revista Brasileira de Malariologia e Doenas
     Tropicais 1957;9:181-90.
  9. Pinheiro FP, Bensabath G, Rosa APAT, Lainson R, Shaw JJ, Ward R, et
     al. Public health hazards among workers along the Trans-Amazon
     Highway. Journal of Occupational Medicine 1977;19:490-6.
 10. Smith NJH. Colonization lessons from a tropical forest. Science
     1982;13:755.
 11. Roberts DR. Health problems of colonists. Science 1982;217:484.
 12. Sandoval JJF, Diniz R, Saraiva MGG, da Silva EB, Alecrim WD, Alecrim
     MGC, et al. Histrico da malria na cidade de Manaus e proposta de
     controle integrado. Rev Soc Bras Med Trop 1998;31, Suplemento 1:141.
 13. Amaral JCOF, Machado RLD, Segura MNO, Oliveira GS, Povoa MM. Avaliao
     longitudinal da infeco causada por Plasmodium falciparum e/ou
     Plasmodium vivax na populao de duas localidades de Icoaraci,
     Distrito de Belem, Para. Rev Soc Bras Med Trop 1998;31 Suplemento
     1:16.
 14. da Silva EB, Costa MF, Melo YFC, Alecrim MGC. Inqurito
     soroepidemiologico numa rea urbana em fase de ocupao, na cidade de
     Novo Aryao-Amazonas-Brasil. Rev Soc Bras Med Trop 1998;31 Suplemento
     1:82.
 15. Ventura AM, Pinto AY, Uchoa R, Calvosa V, Santos MA, Filho MS, et al.
     Malria por Plasmodium vivax em crianas-I-aspectos epidemiologicos e
     clnicos. Rev Soc Bras Med Trop 1998;31 Suplemento 1:82.
 16. Suarez MC, Fe NF, Alecrim WD. Estudo do processo de transmisso da
     malria em uma rea de invaso recente na cidade de Manaus Amazonas.
     Estudo entomologico. Rev Soc Bras Med Trop 1998;31 Suplemento 1:15-6.
 17. Forattini OP. Entomologia medica: I volume parte Geral, Diptera,
     Anophelini. So Paulo (Brasil): Faculdade de Higiene e Sade Publica;
     1962. p. 414.
 18. Rachou RG. Some manifestations on behaviouristic resistance in Brazil.
     Semina Suscep. Insects to insecticides, Panama, Report.: WHO
     1958:208-95.
 19. Frank E. Breastfeeding and maternal employment: two rights don't make
     a wrong. Lancet 1998;352:1083-4.
 20. Central Statistical Office, Belize. 1991 Belize family health survey,
     final report. Reprinted by U.S. Dept of Health and Human Services;
     1992. p. 69.
 21. Resolving the DDT dilemma: protecting biodiversity and human health.
     Toronto, Canada: World Wildlife Fund-Canada; 1998.
 22. World Health Organization. DDT and its derivatives. Environmental
     health criteria 9. Geneva: The Organization; 1979. p. 194.
 23. Safe, SH. Xenoestrogens and breast cancer. N Engl J Med
     1997;337:1303-4.
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On the Etiology of Tropical Epidemic Neuropathies
To the Editor: In a recent report of an epidemic of optic neuropathy in Dar
es Salaam, Tanzania (1), Dolin et al. state that the disease is clinically
identical to one of the forms of epidemic neuropathy found in Cuba between
1991 and 1993 (2). Cases of peripheral neuropathy have been part of both
epidemics (1,2). Both epidemics occurred in nutritionally deficient
populations (1,3).
Dolin et al. state that the cause of the Tanzanian epidemic is unknown and
probably difficult to establish; however, we believe findings from the
Cuban epidemic could be used to study the etiology of this and other
tropical epidemic neuropathies.
In Cuba, several research groups isolated and characterized an enterovirus
in the cerebrospinal fluid (CSF) of epidemic neuropathy patients (4,5).
Enterovirus sequences were found in CSF of 40 (36%) of 111 epidemic
neuropathy patients versus 1 (8%) of 12 control surgical patients (p <
0.01, chi-square test with 2 x 2 contingency tables) (5). Recently, this
enterovirus has been shown to form quasispecies, which could account for
altered biologic properties (de la Fuente et al., submitted for pub.). We
thus propose that epidemic neuropathy has a nutroviral etiology:
Nutritional deficits and stress make the population more likely to become
ill after infection with enterovirus quasispecies with altered biologic
properties.
The relationship between the host's nutritional status and virus evolution
could be key in understanding the cause of epidemic neuropathy, the
Tanzanian epidemic of optic neuropathy, and other tropical epidemic
neuropathies. Etiologic factors must be identified before appropriate
intervention and treatment strategies can be implemented.
Jos de la Fuente and Mara P. Rodrguez
Centro de Ingeniera Gentica y Biotecnologa, Havana, Cuba
References
  1. Dolin PJ, Mohamed AA, Plant GT. Epidemic of bilateral optic neuropathy
     in Dar es Salaam, Tanzania. N Engl J Med 1998;338:1547-8.
  2. The Cuba Neuropathy Field Investigation Team. Epidemic optic
     neuropathy in Cubaclinical characterization and risk factors. N Engl
     J Med 1995;333:1176-82.
  3. Romn GC. On politics and health: an epidemic of neurologic disease in
     Cuba. Ann Int Med 1995;122:530-3.
  4. Ms P, Pelegrino JL, Guzmn MG, Comellas MM, Resik S, Alvarez M, et
     al. Viral isolation from cases of epidemic neuropathy in Cuba. Arch
     Pathol Lab Med 1997;121:825-33.
  5. Rodrguez MP, Alvarez R, Garca del Barco D, Falcn V, de la Rosa MC,
     de la Fuente J. Characterization of virus isolated from the
     cerebrospinal fluid of patients with epidemic neuropathy. Ann Trop Med
     Parasitol 1998;92:97-105.
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Risk for Ebola Virus Infection in Cte d'Ivoire
To the Editor: In Ta National Park, Cte d'Ivoire, where a new strain of
Ebola virus was isolated (1), the World Health Organization is conducting a
project to identify the reservoir of the virus and evaluate the risk for
its emergence in local populations. In March 1998, we conducted qualitative
and quantitative surveys of the villagers' awareness of and risk for Ebola
infection. In four villages close to Ta National Park (4 km to 10 km), we
carried out structured interviews with 150 villagers and in-depth
interviews with 17 villagers and three traditional healers.
Of the 150 villagers participating in the structured interviews, 18.0% had
heard of Ebola (90.7% had heard of yellow fever). Of those aware of Ebola,
96.3% thought it life-threatening; 65.4% of them thought it preventable.
When ill, 81.2% of the respondents generally relied on traditional healers
or herbal medicine. During in-depth interviews traditional healers
discussed their treatment practices. In one treatment, an incision is made
on the skin and medicinal herbs are applied to the incision. Such
traditional practices were implicated in the spread of Ebola virus in
Gabon, where a traditional healer and his assistant (who were infected with
Ebola virus) were suspected of spreading the virus to their patients
through an unsterilized blade (1). The same practices would seem to pose a
risk for virus transmission in Cte d`Ivoire.
Even though officially Ta National Park is protected from human activities
to preserve its natural ecology, 84.0% of the 150 respondents to our survey
often hunted or farmed in the park, 62.2% had encountered chimpanzees, and
53.3% had eaten chimpanzee meat. According to the in-depth interviews,
chimpanzee meat is available at bush meat markets and is thought safe for
eating, even though primates infected with Ebola virus have been linked
with human cases (2,3).
Our survey results show that, even though no large-scale Ebola outbreaks
have occurred in this area, villagers living near the park are at
particularly high risk for infection because they are not aware of Ebola
and do not know that their local customs and behavior may be putting them
at risk. To prevent future Ebola epidemics in Africa, information,
education, and communication (IEC) programs should be established (3).
Moreover, further sociocultural studies on perceptions and behavior should
be conducted in addition to exploring the nature of the virus and its cycle
in the wild (2,4,5).
Osamu Kunii,* Pierre Formenty, Jeanne Diarra-Nama, and Nol Nahounou
*International Medical Center of Japan, Tokyo, Japan;  WHO Ta Forest
Project, Abidjan, Cte d'Ivoire; Ministre de la Sant Publique et des
Affaires Sociales, Abidjan, Cte d'Ivoire
References
  1. Georges AJ, Leroy Em, Renault AA, et al. Ebola Hemorrhagic Fever
     Outbreaks in Bagon, 1994-1997. Epidemiologic and Health Control
     Issues. J Infect Dis 1999;179(Suppl1):S65-75.
  2. Le Guenno B, Formenty P, Wyers M, Gounon P, Walker F, Boesch C.
     Isolation and partial characterisation of a new strain of Ebola virus.
     Lancet 1995;345:1271-4.
  3. Morris K. Facing up to tomorrow's epidemics. Lancet 1997;349:1301.
  4. Georges-Courbot MC, Lu CY, Lansoud-Soukate J, Leroy E, Baize S.
     Isolation and partial molecular characterisation of a strain of Ebola
     virus during a recent epidemic of viral haemorrhagic fever in Gabon.
     Lancet 1997;349:181.
  5. Tukei PM. Threat of Marburg and Ebola viral haemorrhagic fevers in
     Africa. East Afr Med J 1996;73:27-31.
_________________________________________________________________________
News and Notes
_________________________________________________________________________
News and Notes
Automation in Threat Reduction and Infectious Disease Research: Needs and
New Directions April 29-30, 1999
A scientific colloquium entitled Automation in Threat Reduction and
Infectious Disease Research: Needs and New Directions will be held in
Washington, D.C., from April 29 to 30, 1999. Sponsors are the Association
for Laboratory Automation; Centers for Disease Control and Prevention;
Department of Energy; Department of Health and Human Services; Los Alamos
National Laboratory; National Academy of Engineering; and University of
California, Los Angeles.
The colloquium will focus on measuring, detecting, and monitoring in these
areas: 1) recognizing and addressing established and emerging infectious
diseases, 2) ensuring a safe food supply, 3) averting catastrophic
bioterrorism and biowarfare, and 4) advancing human genetics and molecular
medicine. It will also address automation, robotic, computer, information,
Internet, and microscale laboratory methods available to address needs in
these areas. The meeting's objective is to identify specific scientific
needs, assess research practices and their limitations, and then consider
strategic ways for integrating new high-throughput laboratory tools and
methods. In addition, the 2-day program has an educational component
designed for policy makers. The colloquium will emphasize a cross-cutting
approach, which recognizes that new tools and methods from one scientific
discipline can be applied to other scientific disciplines.
To obtain additional information or register for the colloquium, visit the
following web sites:
   * http://www.nae.edu/colloquium
   * http://labautomation.org/colloquium/home.html
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Emerging Pathogens Initiative: An Automated Surveillance System
The Veterans Health Administration, Department of Veterans Affairs (VA),
debuted its nationwide computer-based Emerging Pathogens Initiative on
October 1, 1998. The initiative is an automated surveillance system that
collects data from all 171 VA medical centers (from 146 reporting sites) on
14 specific pathogens or diseases: vancomycin-resistant enterococcus,
penicillin-resistant pneumococcus, Escherichia coli O157, Candida
bloodstream infections, Clostridium difficile, Cryptosporidium, dengue,
antibody-positive hepatitis C, Legionella, leishmaniasis, malaria,
tuberculosis, group A streptococcus, and Creutzfeldt-Jakob disease. Other
information (e.g., patient demographics, antimicrobial susceptibility where
appropriate, co-morbidities, and number of patients by facility) is also
collected and downloaded into a central database on a monthly basis. After
aggregate reports are compiled, the 22 VA patient-care networks will
receive network-specific data, along with national VA quartile rankings.
For further information, contact Gary A. Roselle, M.D., program director
for infectious diseases, VA Headquarters, at 513-475-6398.
 Erratum Vol. 5, No. 1
 In the article, "Genetic Diversity and Distribution of Peromyscus-Borne
 Hantaviruses in North America," by Martha Monroe et al., on page 85,
 reference 8 should read Mills JN, Ksiazek TG, Ellis BA, Rollin PE,
 Nichol ST, Yates TL, et al. Patterns of association with host and
 habitat: Antibody reactive with Sin Nombre virus in small mammals in the
 major biotic communities of the southwestern United States. Am J Trop
 Med Hyg 1997;56:273-84. We apologize to our readers for this error.
_________________________________________________________________________
Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA
URL: ftp://ftp.cdc.gov/pub/EID/vol5no2/ascii/vol5no2.txt
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