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CONTENTS, EMERGING INFECTIOUS DISEASES, VOL 1., NO. 2
Synopses
Streptococcal Toxic-Shock Syndrome: Spectrum of Disease,
Pathogenesis, and New Concepts in Treatment, Dennis L. Stevens
Spiral Bacteria in the Human Stomach: The Gastric Helicobacters,
Andre Dubois
Dispatches
HIV-1 Patients May Harbor Viruses of Different Phylogenetic
Subtypes: Implications for the Evolution of the HIV/AIDS
Pandemic, Danuta Pieniazek, Luiz M. Janini, Artur Ramos,
Amilcar Tanuri, Mauro Schechter, Jose M. Peralta, Anna C. P.
Vicente, Norman K. Pieniazek, Gerald Schochetman, and Mark A.
Rayfield
Simultaneous Infection of Ixodes ricinus Nymphs by Two Borrelia
burgdorferi Sensu Lato Species, Bruno Pichon, Edmond
Godfroid, Bernard Hoyois, Alex Bollen, Francois Rodhain, and
Claudine Prez-Eid
Epidemiologic and Evolutionary Relationships Between Romanian
and Brazilian HIV-Subtype F Strains, Claudiu I. Bandea, Artur
Ramos, Danuta Pieniazek, Rodica Pascu, Amilcar Tanuri, Gerald
Schochetman, and Mark A. Rayfield
Electronic Communication Facilitates Investigation of a Highly
Dispersed Foodborne Outbreak: Salmonella on the Superhighway,
Barbara E. Mahon, Dale D. Rohn, Sheila R. Pack, and Robert V.
Tauxe
Commentary
Reemergence of Ebola Virus in Africa, Anthony Sanchez, Thomas
G. Ksiazek, Pierre E. Rollin, Clarence J. Peters, Stuart T.
Nichol, Ali S. Khan, and Brian W. J. Mahy
Prospects for the Control of Bolivian Hemorrhagic Fever, Paul
E. Kilgore, Clarence J. Peters, James N. Mills, Pierre E. Rollin,
Lori Armstrong, Ali S. Khan, and Thomas G. Ksiazek
News and Notes
Conference on "Emerging Infectious Diseases: Meeting the
Challenge," James M. Hughes
Japanese Encephalitis Acquired in Australia,
USPHS and IDSA Collaborate on Guidelines to Prevent Opportunistic
Infections in HIV-Infected Persons, Jonathan E. Kaplan,
Henry Masur, and King K. Holmes
Recommendations for a Regional Strategy for the Prevention and
Control of Emerging Infectious Diseases in the Americas,
Daniel B. Epstein
Emerging Infectious Diseases Laboratory Fellowship Program
Tenth Annual ASTPHLD Conference on Human Retrovirus Testing,
James L. Pearson
__________
SYNOPSES
Streptococcal Toxic-Shock Syndrome:Spectrum of Disease,
Pathogenesis, and New Concepts in Treatment
Dennis L. Stevens, Ph.D., M.D.
Professor of Medicine, University of
Washington School of Medicine, Seattle,
Washington Chief, Infectious Disease Section,
Veterans Affairs Medical Center, Boise, Idaho,
USA
ABSTRACT
Since the 1980s there has been a marked increase in the
recognition and reporting of highly invasive group A
streptococcal infections with or without necrotizing
fasciitis associated with shock and organ failure. Such
dramatic cases have been defined as streptococcal
toxic-shock syndrome. Strains of group A streptococci
isolated from patients with invasive disease have been
predominantly M types 1 and 3 that produce pyrogenic
exotoxin A or B or both. In this paper, the clinical
and demographic features of streptococcal bacteremia,
myositis, and necrotizing fasciitis are presented and
compared to those of streptococcal toxic-shock
syndrome. Current concepts in the pathogenesis of
invasive streptococcal infection are also presented,
with emphasis on the interaction between group A
Streptococcus virulence factors and host defense
mechanisms. Finally, new concepts in the treatment of
streptococcal toxic-shock syndrome are discussed.
An emerging pathogen can be one that is totally new
(e.g., human immunodeficiency virus), one that was
known but has only recently been identified (e.g.,
Helicobacter pylori), or one that is old but has
learned new tricks. The last type is, as Dr. Stanley
Falkow contends, merely trying to "make a living" in a
changing environment. Regardless of environmental
pressures, many old pathogens have become major
clinical problems because of increased virulence or
antibiotic resistance (e.g., penicillin-resistant
pneumococcus, multidrug resistant Mycobacterium
tuberculosis, methicillin-resistant Staphylococcus
aureus, and vancomycin-resistant Enterococcus faecium).
Arguably, group A Streptococcus (GAS) is the
quintessence of an old organism that has become more
virulent. In this manuscript, the epidemiology,
clinical spectrum, and pathogenesis of GAS
infection are discussed in relation to the
streptococcal toxic-shock syndrome (TSS).
Current and Historical Perspectives on the Prevalence
and Severity of Streptococcal Infections
The British tabloids have recently coined the term
"flesh-eating bacteria" to describe invasive
necrotizing infections caused by GAS and have suggested
that epidemics of streptococcal infection
are imminent. Such aggrandizement is unfounded, yet it
has served to heighten public awareness of this
sporadic, but serious, infectious disease. Strictly
speaking, an epidemic is defined as an increase in the
prevalence of disease over a baseline endemic rate. In
this context, we are, in fact, experiencing an epidemic
of severe invasive GAS infections; however, few
concrete prospective population-based data support this
notion. Estimates suggest that the incidence of these
infections is 10 to 20 cases/ 100,000 population. Thus,
the stimulus for such public interest has not been the
incidence of the syndrome, but more likely, the
dramatic nature of these infections.
Whether these types of group A streptococcal infections
will decline, stay the same, or increase is not known.
History is replete with descriptions of epidemics of
GAS infections and their nonsuppurative sequelae. In
the 1600s, epidemics of scarlet fever spread from Italy
and Spain to Northern Europe (1), and in 1736, an
outbreak occurred in the American colonies, killing
4,000 people (2). Major epidemics of rheumatic fever
occurred in World War II in the U.S. military (3). Soon
afterward post-streptococcal glomerulonephritis struck
several regions of the United States (4,5).
Many of these epidemics waxed and waned before the
advent of antibiotics, suggesting that either changes
in socioeconomic conditions or variations in the
expression of virulence factors by the pathogen were
responsible. This concept is best exemplified by the
extraordinary mortality rate of scarlet fever
documented in the latter part of the 1880s in New York,
Chicago, and Norway; 25% to 30% of children with
scarlet fever died during that period (5,6). By 1900,
the mortality rate had dropped to under 2% in all three
locations. Since socioeconomic conditions likely did
not change markedly during that time and antibiotics
were not yet available, the decrease in mortality rates
must have been caused by reduced expression of a
streptococcal virulence factor or by the slow
acquisition of herd immunity to that factor.
The epidemiology of GAS infection is complex. More than
80 different M types of S. pyogenes exist, and five
separate and distinct scarlatina toxins, streptococcal
pyrogenic exotoxins (SPEs) (5) have also been
described; some of these can be transmitted to
different M types by bacteriophage. Minor drifts
in the antigenic or virulence properties of GAS could
account for the 5- to 6-year cycles of scarlet fever
documented by Kohler (9). In the same way as antigenic
shifts in influenza virus cause pandemics, major
alterations in GAS virulence properties could cause
major changes in clinical disease. The recent increases
in severe GAS infections, following a 50- to 60-year
span of relatively benign clinical disease, support
this notion.
Acute Life-Threatening Group A Streptococcal Infections
Streptococcal TSS
Recently, severe invasive GAS infections associated
with shock and organ failure have been reported with
increasing frequency, predominantly from North America
and Europe (8-18). These infections have been termed
streptococcal toxic-shock syndrome (TSS; Table 1) (19).
Persons of all ages are affected; most do not have
predisposing underlying diseases (11,20-25). This is in
sharp contrast to previous reports of GAS bacteremia,
in which patients were either under 10 or over 60 years
of age, and most had underlying conditions such as
cancer, renal failure, leukemia, or severe burns or
were receiving corticosteroids or other
immunosuppressing drugs (20-22). The complications of
current GAS infections are severe; bacteremia
associated with aggressive soft tissue infection,
shock, adult respiratory distress syndrome and renal
failure are common; 30% to 70% of patients die in spite
of aggressive modern treatments (Table 2) (1,8,24-26).
Table 1. Case definition of streptococcal toxic-shock syndrome
(streptococcal TSS) and necrotizing fasciitis*
____________________________________________________________
I. Streptococcal TSS
   A. Isolation of group A Streptococcus
      1. From a sterile site
      2. From a nonsterile body site
   B.Clinical signs of severity
      1. Hypotension
      2.  Clinical and laboratory abnormalities (requires two or
          more of the following):
         a)Renal impairment
         b)Coagulopathy
         c)Liver abnormalities
         d)Acute respiratory distress syndrome
         e)Extensive tissue necrosis, i.e., necrotizing fasciitis
         f)Erythematous rash
  Definite Case = A1 + B(1+2)
  Probable Case = A2 + B(1+2)
II. Necrotizing fasciitis
    A.Definite case
      1. Necrosis of soft tissues with involvement of the fascia
    PLUS
      2.Serious systemic disease, including one or more of the
        following:
        a)Death
        b)Shock (systolic blood pressure <90 mm of Hg).
        c)Disseminated intravascular coagulopathy
        d)Failure of organ systems
          a.respiratory failure
          b.liver failure
          c.renal failure
     3.Isolation of group A Streptococcus from a normally sterile
       body site
    B.Suspected case
    1.1+2 and serologic confirmation of group A streptococcal
      infection by a 4-fold rise against:
       a)streptolysin O
       b)DNase B
    2.1+2 and histologic confirmation:
      Gram-positive cocci in a necrotic soft tissue infection
_____________________________________________________________
*Streptococcal toxic-shock syndrome (streptococcal TSS) is
defined as any group A streptococcal infection associated with
the early onset of shock and organ failure.  Definitions
describing criteria for shock, organ failure, definite cases,
and probable cases are included below.
Source: reference 61.
__________________________________________________________
Table 2. Complications of group A streptococcal soft-tissue
infection
Complication                                  Percentage
                                              of Patients
Shock                                             95
Acute respiratory distress syndrome               55
Renal impairment                                  80
Irreversible                                      10
Reversible                                        70
Bacteremia                                        60
Death                                             30
________________________________________________________
Source: reference 1.
Acquisition of Group A Streptococcus
The portal of entry of streptococci cannot be proven in
at least half the cases (8) and can only be presumed in
many others.Patients with symptomatic pharyngitis
rarely develop streptococcal TSS, though such cases
have been reported,especially in the last year.
Procedures such as suctionlipectomy, hysterectomy,
vaginal delivery, bunionectomy and bone pinning have
provided a portal of entry in many cases (author's
unpublished observations). Most commonly, infection
begins at a site of minor local trauma, which
frequently does not result in a break in the skin (8).
Numerous cases have developed within 24 to 72 hours of
minor nonpenetrating trauma, resulting in hematoma,
deep bruise to the calf, or even muscle strain. Virus
infections, such as varicella and influenza, have
provided a portal in other cases. In some cases the use
of nonsteroidal antiinflammatory agents may have either
masked the early symptoms or predisposed the patient to
more severe streptococcal infection and shock (1). For
the most part, these infections have occurred
sporadically and have not been associated with clusters
of cases or minor epidemics, though outbreaks of severe
GASinfections have occurred in closed environments such
as nursing homes (27,28).
Clinical Symptoms
Pain the most common initial symptom of streptococcal
TSS is abrupt in onset and severe, and usually precedes
tenderness or physical findings. The pain usually
involves an extremity but may also mimic
peritonitis,pelvic inflammatory disease, pneumonia,
acute myocardial infarction, or pericarditis. Twenty
percent of patients have an influenza-like syndrome
characterized by fever, chills, myalgia, nausea,
vomiting, and diarrhea (8). Fever is the
most common early sign, although hypothermia may be
present in patients with shock. Confusion is present in
55% of patients, and in some, coma or combativeness is
manifest (8). Eighty percent of patients have clinical
signs of soft tissue infection, such as localized
swelling and erythema, which in 70% of patients
progressed to necrotizing fasciitis or myositis and
required surgical debridement, fasciotomy or amputation
(8). An ominous sign is the progression of soft tissue
swelling to the formation of vesicles, then bullae,
which appear violaceous or bluish. In such patients,
emergent surgical exploration should be performed
to establish the diagnosis and distinguish GAS
infection from other necrotizing soft tissue
infections. Among the 20% of patients without soft
tissue findings, clinical symptoms include
endophthalmitis, myositis, perihepatitis, peritonitis,
myocarditis, and overwhelming sepsis. A diffuse,
scarlatina-like erythema occurs in only 10% of
patients. Nearly 50% of patients may have normal blood
pressure (systolic pressure >110 mm Hg) on admission
but develop hypotension within the subsequent 4 hours
(8).
Laboratory Evaluation of Patients
On admission, renal involvement is indicated by the
presence of hemoglobinuria and by serum creatinine
values that are, on average, >2.5 times normal. Renal
impairment precedes hypotension in 40% to 50% of
patients (8). Hypoalbuminemia is associated with
hypocalcemia on admission and throughout the hospital
course. The serum creatinine kinase level is useful in
detecting deeper soft-tissue infections; when the level
is elevated or rising, there is a good correlation with
necrotizing fasciitis or myositis.  Though the
initial laboratory studies demonstrate only mild
leukocytosis, the mean percentage of immature
neutrophils (including band forms, metamyelocytes, and
myelocytes) is striking, reaching 40% to 50%. Blood
cultures are positive in 60% of cases (8).
Clinical Course
Shock is apparent at the time of admission or within 4
to 8 hours in virtually all patients (Table 2). In only
10% of patients does systolic blood pressure become
normal 4 to 8 hours after administration of
antibiotics, albumin, and electrolyte solutions
containing salts or dopamine; in all other patients,
shock persists. Similarly, renal dysfunction progresses
or persists in all patients for 48 to 72 hours in spite
of treatment, and many patients may require dialysis
(8). In patients who survive, serum creatinine values
return to normal within 4 to 6 weeks. Renal dysfunction
precedes shock in many patients and is apparent early
in the course of shock in all others. Acute respiratory
distress syndrome occurs in 55% of patients and
generally develops after the onset of hypotension (8).
Supplemental oxygen, intubation, and mechanical
ventilation are necessary in 90% of the patients
in whom this syndrome develops. Mortality rates vary
from 30% to 70% (1,8,24-26). Morbidity is also high; 13
of 20 patients in one series underwent major surgical
procedures, which included fasciotomy, surgical
debridement, exploratory laparotomy, intraocular
aspiration, amputation, or hysterectomy (8).
Clinical Isolates
M types 1, 3, 12, and 28 have been the most common
isolates from patients with shock and multiorgan
failure (8,29). Recently, 80% of strains in Sweden from
all types of GAS infection have been M type 1 (S. Holm,
pers. comm.). Pyrogenic exotoxin A and/or B was found
in most cases of severe infection. In the United
States, pyrogenic exotoxin A is most frequently
associated with these infections (8,23,29-33), while in
Sweden and the United Kingdom, exotoxin B has been most
common (12,25). Recently, streptococcal superantigen
(SSA), a novel pyrogenic exotoxin, was isolated from
an M 3 strain, albeit in small concentrations (34). In
addition, mitogenic factor (MF) has been demonstrated
in many different M types of GAS (35,36).
Necrotizing Fasciitis
Necrotizing fasciitis, a deep-seated infection of the
subcutaneous tissue that progressively destroys fascia
and fat but may spare the skin and muscle, can be
caused by GAS, Clostridium perfringens, or C. septicum.
Necrotizing fasciitis caused by mixed organisms such as
aerobic gram-negative bacteria, anaerobes, and
microaerophilic streptococci may develop in diabetic
patients or patients with open wounds contaminated with
bowel contents. Though Meleney called infections caused
by hemolytic streptococci "streptococcal gangrene"
(37), the process has been renamed necrotizing
fasciitis. His patients' infections began at the site
of trivial or inapparent trauma. Within 24 hours of the
initial lesion which frequently was only mild erythema
swelling, heat, erythema, and tenderness rapidly
developed. During the next 24 to 48 hours, the erythema
changed from red to purple and then to blue, and
blisters and bullae, which contained clear yellow
fluid, appeared. On days 4 and 5, the purple areas
became gangrenous. From day 7 to day 10, the line of
demarcation became sharply defined, and the dead skin
began to separate at the margins or breaks in the
center, revealing an extensive necrosis of the
subcutaneous tissue. In more severe cases, the process
advance d rapidly until several large areas of skin
became gangrenous, and the intoxication rendered the
patient dull,unresponsive, mentally cloudy, or even
delirious. Meleney was the first to advocate aggressive
"bear scratch" fasciotomy and debridement. With this
treatment, together with irrigation with Dakains
solution, the mortality rate dropped to 20% (37).
These older reports of necrotizing fasciitis (6) differ
from reports of current necrotizing fasciitis cases
associated with streptococcal TSS (8). First, recent
cases have mainly occurred in young healthy persons who
had no underlying disease but sustained minor trauma to
an extremity. Earlier series describe older patients
with multiple medical problems (6). Meleney's cases
(reported from China) were probably among young healthy
persons who sustained minor trauma, though the major
difference between them and present cases is the low
mortality rate (20% vs 20% to 60% in streptococcal TSS)
(6,37) before antibiotics were available (37). Analysis
of Meleney's reports also suggests that most of his
patients did not have shock or organ failure, nor did
they require amputation. In contrast, present cases of
necrotizing fasciitis caused by GAS are invariably
associated with severe manifestations of systemic
illness and high morbidity despite the absence of
underlying disease and the use of antibiotics,
dialysis, ventilators, intravenous fluids, and improved
surgical techniques. In summary, the high mortality
rate among current cases of streptococcal necrotizing
fasciitis could be due to the emergence of more
virulent streptococci (8).
Streptococcal Myositis
Streptococcal myositis is an extremely uncommon GAS
infection. Adams et al. (38) documented only 21
reported cases from 1900 to 1985, and Svane (39) found
only four cases in more 20,000 autopsies. Severe pain
may be the only early symptom, and swelling and
erythema may be the only early physical findings,
though muscle compartment syndromes may develop rapidly
(8-10,38-41). Distinguishing streptococcal myositis
from spontaneous gas gangrene caused by C. perfringens
or C. septicum (42) may be difficult, though crepitus
or demonstration of gas in the tissue favors
clostridial infection (40). Patients with streptococcal
TSS may have both necrotizing fasciitis and
myositis (8,38). In published series, the case-fatality
rate for necrotizing fasciitis is 20% to 50%, whereas
GAS myositis has a fatality rate of 80% to 100% (6).
Aggressive surgical debridement is extremely important
for establishing a diagnosis and removing devitalized
tissue.
Bacteremia
Streptococcal bacteremia has occurred most commonly in
the very young and in the elderly (5). Among children,
predisposing factors (other than scarlet fever) include
burns,varicella, malignant neoplasm,immunosuppression,
and age less than 2 years (5). In patients with scarlet
fever, the pharynx is the most common source of GAS.
Frequently such patients have complications, such as
extension of infection into the sinuses, peritonsillar
tissue, or mastoids (septic scarlet fever
or scarlet fever anginose); yet documented bacteremia
occurs in only 0.3% of febrile patients (43). Among the
children with varicella studied by Bullowa and Wischik
(43), GAS bacteremia occurred in only approximately
0.5% of patients. In elderly patients the source of GAS
infection is invariably the skin and is associated with
cellulitis or erysipelas (5). GAS sepsis in the elderly
(mean age, 50 to 60 years) has also been associated
with diabetes, peripheral vascular disease, malignancy,
and corticosteroid use. Not surprising, mortality
rates of 35% to 80% have been described in this patient
population. In the past, GAS bacteremia was rare among
persons 14 to 40 years of age; puerperal sepsis
accounted for most bacteremia in this age group.
Recently, intravenous drug abuse has emerged as a
leading cause of GAS bacteremia in this age group (5).
Martin and Hoiby have comprehensively demonstrated that
the prevalence of GAS bacteremia in Norway in the late
1980s increased in all age groups, but the greatest
increase (600% to 800%) was in adolescents and young
adults (10). Thus, the demographics of invasive
streptococcal infection have changed dramatically in
the past 4 to 6 years.
Current Hypotheses Regarding Mechanisms of Shock and
Tissue Destruction Caused by Virulent Group A
Streptococci
Pyrogenic exotoxins cause fever in humans and animals
and also help induce shock by lowering the threshold to
exogenous endotoxin (5). Streptococcal pyrogenic
exotoxins A and B induce human mononuclear cells to
synthesize not only tumor necrosis factor-
(TNF) (44) but also interleukin- (IL-1) (45)
and interleukin-6 (IL-6) (45), suggesting that TNF
could mediate the fever, shock, and tissue injury
observed in patients with streptococcal TSS (8).
Pyrogenic exotoxin C has been associated with mild
cases of scarlet fever in the United States
(author's observations) and in England (46). The roles
of two newly described pyrogenic exotoxins, SSA and MF
(see section on "Clinical Isolates"), in streptococcal
TSS have not been elucidated.
M protein contributes to invasiveness through its
ability to impede phagocytosis of streptococci by human
polymorphonuclear leukocytes (47). Conversely,
type-specific antibody against the M protein enhances
phagocytosis (47). After infection with a  particular M
type, specific antibody confers resistance to challenge
to viable GAS of that M type (47). While M types 1 and
3 strains have accounted for most strains isolated
from cases of streptococcal TSS, many other M types,
including some nontyp-able strains, have also been
isolated from such cases. M types 1 and 3 are also
commonly isolated from asymptomatic carriers, patients
with pharyngitis, and patients with mild scarlet fever
(7,29).
Could streptococcal TSS be related to the ability of
pyrogenic exotoxin or M proteins type 1 or 3 to act as
"super antigens"(48)? Data suggest that this exotoxin
and a number of staphylococcal toxins (toxic shock
syndrome toxin-1 [TSST-1] and staphylococcal
enterotoxins A, B, and C) can stimulate T-cell
responses through their ability to bind to both
the Class II major histocompatibility ability complex
of antigen-presenting cells and the Vb region of the
T-cell receptor (48). The net effect would be to induce
T-cell stimulation with production of cyto- kines
capable of mediating shock and tissue injury. Recently,
Hackett and Stevens demonstrated that pyrogenic
exotoxin A induced both TNF and TNF from mixed
cultures of monocytes and lymphocytes (49), supporting
the role of lymphokines (TNF) in shock associated with
strains producing that exotoxin. Kotb et al. (50) have
shown that a digest of M protein type 6 can also
stimulate T-cell responses by this mechanism; however,
the role of specific superantigens in this or
any other infectious disease has not been proven. Proof
would require demonstration of massive expansion of
T-cell subsets bearing a V repertoire specific for the
putative superantigen. However, quantitation of such
T-cell subsets in patients with acute streptococcal TSS
demonstrated deletion rather than expansion, suggesting
that perhaps the life span of the expanded
subset was shortened by a process of apoptosis (51). In
addition, the subsets deleted were not specific for
streptococcal pyrogenic exotoxins A, B, C, or mitogenic
factor, suggesting that an as yet undefined
superantigen may play a role (51).
Cytokine production by less exotic mechanisms likely
contributes as well to the genesis of shock and organ
failure. Peptidoglycan, lipoteichoic acid (52), and
killed organisms (53,54) are capable of inducing TNFa
production by mononuclear cells in vitro (6,54,55).
Exotoxins such as streptolysin O (SLO) are also potent
inducers of TNF and IL-1. Pyrogenic exotoxin B, a
proteinase precursor, has the ability to cleave
pre-IL-1 to release preformed IL-1 (56). Finally, SLO
and exotoxin A together have additive effects in the
induction of IL-1 by human mononuclear cells (49).
Whatever the mechanisms, induction of cytokines in vivo
is likely the cause of shock, and these two exotoxins,
cell wall components, and the like, are potent inducers
of TNF and IL-1.
The mere presence of virulence factors, such as M
protein or pyrogenic exotoxins, may be less important
in streptococcal TSS than the dynamics of their
production in vivo. Recently, Cleary et al. proposed a
regulon in GAS that controls the expression of a group
of virulence genes coding for known virulence factors
such as M protein and C5 peptidase (57). When
DNA fingerprinting was used, differences were shown
between M1 strains isolated from patients with invasive
disease and strains from patients with noninvasive GAS
infections (58). Finally, genetic information coding
for exotoxins A or C may be introduced to strains of
GAS by certain bacteriophage; after lysogenic
conversion,synthesis of exotoxin A would occur during
growth of the streptococcus (31,59,60). Multilocus
enzyme electrophoresis demonstrates two patterns that
correspond to the M1 and M3 type organisms that produce
pyrogenic exotoxin A, a finding that supports
epidemiologic studies implicating these strains in
invasive GAS infections (33).
The interaction between these microbial virulence
factors and an immune or nonimmune host determines the
epidemiology, clinical syndrome, and outcome. Since
horizontal transmission of GAS in general is well
documented, the only explanation for the absence
of a high attack rate of invasive infection is
significant herd immunity against one or more of the
virulence factors responsible for streptococcal TSS.
This hypothetical model explains why epidemics have not
materialized and why a particular strain of GAS can
cause different clinical manifestations in the same
community (8,61) (Figure 1 cannot be viewed in ASCII
format.)
Treatment
Antibiotic Therapy-Cures and Failures with Penicillin
S. pyogenes continues to be exquisitely susceptible to
-lactam antibiotics, and numerous studies have
demonstrated the clinical efficacy of penicillin
preparations for streptococcal pharyngitis. Similarly,
penicillins and cephalosporins have proven efficacy in
treating erysipelas, impetigo, and cellulitis,
all of which are most frequently caused by S. pyogenes.
In addition, Wannamaker et al. (6) demonstrated that
penicillin therapy prevents the development of
rheumatic fever following streptococcal pharyngitis if
therapy is begun within 8 to 10 days of the onset of
sore throat. Nonetheless, some clinical failures
of penicillin treatment of streptococcal infection do
occur. Penicillin treatment of S. pyogenes has failed
to eradicate bacteria from the pharynx of 5% to 20% of
patients with documented streptococcal pharyngitis
(62-64). In addition, more aggressive GAS infections
(such as, necrotizing fasciitis, empyema, burn wound
sepsis, subcutaneous gangrene, and myositis)
respond less well to penicillin and continue to be
associated with high mortality rates and extensive
morbidity (6,8,9,12,15,38,65). For example, in a recent
report, 25 cases of streptococcal myositis had an
overall mortality rate of 85% in spite of penicillin
therapy (38). Finally, several studies in experimental
infection suggest that penicillin fails when large
numbers of organisms are present (66,67).
The Efficacy of Penicillin, Compared to Clindamycin, In
Fulminant Experimental S. pyogenes Infection
In a mouse model of myositis caused by S. pyogenes,
penicillin was ineffective when treatment was delayed
up to 2 hours after initiation of infection (67).
Survival of erythromycin-treated mice was greater than
that of both penicillin-treated mice and untreated
controls, but only if treatment was begun within 2
hours. Mice receiving clindamycin, however, had
survival rates of 100%, 100%, 80%, and 70%, even if
treatment was delayed 0, 2, 6, and 16.5 hours,
respectively (67,68).
Eagle suggested that penicillin failed in this type of
infection because of the "physiologic state of the
organism"(66). This phenomenon has recently been
attributed to both in vitro and in vivo inoculum
effects (69,70).
Inoculum Size and the "Physiologic State of the
Organism": Differential Expression of
Penicillin-Binding Proteins
Penicillin and other -lactam antibiotics are most
efficacious against rapidly growing bacteria. We
hypothesized that large inocula reach the stationary
phase of growth sooner than smaller inocula both in
vitro and invivo. That high concentrations of S.
pyogenes accumulate in deep-seated infection is
supported by data from Eagle et al. (66). We compared
the penicillin-binding protein patterns from membrane
proteins of group A streptococci isolated from
different stages of growth, i.e., mid-log phase and
stationary phase. Binding of radiolabeled penicillin by
all penicillin-binding proteins was decreased in
stationary cells; however, PBPs 1 and 4 were
undetectable at 36 hours (69). Thus, the loss of
certain penicillin-binding proteins during
stationary-phase growth in vitro may be responsible for
the inoculum effect observed in vivo and may account
for the failure of penicillin in treatment of both
experimental and human cases of severe streptococcal
infection.
The Greater Efficacy of Clindamycin in Experimental S.
pyogenes Infections: Mechanisms of Action
The greater efficacy of clindamycin is likely
multifactorial: First, its efficacy is not affected by
inoculum size or stage of growth (69,71); secondly,
clindamycin is a potent suppressor of bacterial toxin
synthesis (72,73); third, it facilitates
phagocytosis of S. pyogenes by inhibiting M-protein
synthesis (73); fourth, it suppresses synthesis of
penicillin-binding proteins, which, in addition to
being targets for penicillin, are also enzymes involved
in cell wall synthesis and degradation (71); fifth,
clindamycin has a longer postantibiotic effect than
-lactams such as penicillin; and lastly, clindamycin
causes suppression of LPS-induced monocyte synthesis of
TNF (74). Thus, clindamycin's efficacy may also be
related to its ability to modulate the immune response.
Other Treatment Measures
Though antibiotic selection is critically important,
other measures, such as prompt and aggressive
exploration and debridement of suspected deep-seated S.
pyogenes infection, are mandatory. Frequently, the
patient has fever and excruciating pain. Later,
systemic toxicity develops, and definite evidence of
necrotizing fasciitis and myositis appears. Surgical
debridement may be too late at this point. Prompt
surgical exploration through a small incision with
visualization of muscle and fascia, and timely Gram
stain of surgically obtained material may provide
an early and definitive etiologic diagnosis. Surgical
colleagues should be involved early in such cases,
since later in the course surgical intervention may be
impossible because of toxicity or because infection has
extended to vital areas impossible to debride (i.e.,
the head and neck, thorax, or abdomen).
Anecdotal reports suggest that hyperbaric oxygen has
been used in a handful of patients, though no
controlled studies are under way, nor is it clear that
this treatment is useful.
Because of intractable hypotension and diffuse
capillary leak, massive amounts of intravenous fluids
(10 to 20 liters/day) are often necessary. Pressors
such as dopamine are used frequently, though no
controlled trials have been performed in streptococcal
TSS. In patients with intractable hypotension,
vasoconstrictors such as epinephrine have been used,
but symmetrical gangrene of digits seems to result
frequently (author's unpublished observations), often
with loss of limb. In these cases it is
difficult to determine if symmetrical gangrene is due
to pressors, infection, or both.
Neutralization of circulating toxins would be
desirable; however, appropriate antibodies are not
commercially available in the United States or Europe.
Two reports describe the successful use of intravenous
gamma globulin in treating streptococcal TSS in
two patients (75,76).
In summary, if a wild "flesh-eating strain" has
recently emerged, a major epidemic with a high attack
rate would normally be expected. Clearly, epidemics of
streptococcal infections, including impetigo,
pharyngitis, scarlet fever, and rheumatic  fever have
occurred in the past. However, in the last decade,
subsequent to early reports of streptococcal TSS, we
have observed that the incidence has remained
relatively low. I hypothesize that large outbreaks have
not occurred because 1) most of the population probably
has immunity to one or more streptococcal virulence
factors (6,25); 2) predisposing conditions (e.g.,
varicella, and use of NSAIDs) are required in a
given patient; and 3) only a small percentage of the
population may have an inherent predisposition to
severe streptococcal infection because of
constitutional factors such as HLA Class II
antigen type (77,78), B-cell (79), or specific Vb
regions on lymphocytes. This last hypothesis is further
supported by the observation that secondary cases of
streptococcal TSS, though reported (80), have been
rare.
Dr. Stevens is chief, Infectious Diseases Section,
Veterans Affairs Medical Center, Boise, Idaho, and
professor of medicine, University of Washington School
of Medicine, Seattle.  He is a member of CDC's Working
Group on Streptococcal Infections and a consultant to
the National Institutes of Health and the World Health
Organization on Streptococcal Infections. On July 1994,
he testified before Congress on Severe Streptococcal
Infections and is currently President of the American
Lancefield Society.
Address for correspondence: Infectious Disease Section
Veterans Affairs Medical Center, 500 West Fort
Street (Bldg 6), Boise, ID 83702, USA, fax: 208-389-7965
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SYNOPSES
Spiral Bacteria in the Human Stomach: The Gastric Helicobacters
Andre Dubois, M.D., Ph.D.
Digestive Diseases Division,
Department of Medicine,
Uniformed Services University of the Health Sciences
Bethesda, Maryland, USA
ABSTRACT
During the past decade, Helicobacter pylori has become recognized
as one of the most common human pathogens, colonizing the gastric
mucosa of almost all persons exposed to poor hygienic conditions
from childhood. It also is often found, albeit with a lower
frequency, in groups of high socioeconomic status. H. pylori
causes chronic active gastritis and is a major factor in the
pathogenesis of duodenal ulcers and, to a lesser extent, gastric
ulcers. In addition, the presence of this bacterium is now
recognized as a risk factor for gastric adenocarcinoma and
lymphoma. Nevertheless, most infections appear without clinical
consequences. In this second decade of intensive research, it is
important to understand why H. pylori is sometimes a dangerous
pathogen, and to determine how it can be eradicated in those at
highest risk for severe disease.
At the end of the 19th century, several types of spirochetes and
spirilla were observed for the first time in the stomach of
animals (1,2). Beginning at the turn of the 20th century, similar
spiral bacteria were found in gastrectomy specimens from patients
with gastric cancer and peptic ulcer disease (3,4). In addition,
gastroenterologists and surgeons noted but could not explain the
almost universal presence of antral gastritis in patients with
duodenal ulcers and the frequent presence of atrophic gastritis
in patients with gastric ulcer and cancer. Nevertheless, the
possibility that peptic ulcer disease or gastric cancer might be
caused by an infectious agent was generally discounted. The
observation made in 1975 that gram-negative bacteria were present
in 80% of patients with gastric ulcer (5) was largely ignored by
the scientific community which, at the time, was busily
developing potent antiulcer agents (6). Skepticism remained the
overwhelming reaction to the 1983 reports describing the frequent
association between antral gastritis and the presence of
Campylobacter-like bacteria (7), as well as of their culture and
isolation from patients with gastritis (8). A similar reaction
followed the subsequent demonstration that these
Campylobacter-like bacteria were present in almost all patients
with gastric and duodenal ulcers, and were generally associated
with antral gastritis (9). In the past decade, however, a number
of studies have confirmed and extended these early observations.
A consensus regarding the major role of this bacterium, now named
Helicobacter pylori, in causing gastroduodenal ulceration was
formally presented in 1994 (10).
Furthermore, in June 1994, the International Agency for Research
on Cancer Working Group stated , "H. pylori plays a causal role
in the chain of events leading to cancer," referring to
adenocarcinoma and lymphoma of the stomach as well as to the more
benign mucosal-associated lymphoid tissues (MALT) (11-13).
An important consequence of the considerable interest generated
by these clinical observations is that extensive bacteriologic
and molecular studies have been performed on this bacterium and
similar organisms. 16S rRNA gene sequence analysis has revealed
important differences between H. pylori and the closely related
Campylobacter, Flexispira, and Wolinella genuses. These
differences have necessitated the creation of the genus
Helicobacter, which, to date, includes eight gastric, three
intestinal, and two hepatic species (14). Each of these
Helicobacter species colonizes different, or a spectrum of,
mammalian species.
This review summarizes our current knowledge of the two
Helicobacter species that have been observed in the human stomach
and reported on extensively in the literature: H. pylori, the
type strain, and H. heilmannii, also known as Gastrospirillum
hominis (15,16).
Characteristics of Gastric Helicobacters Observed in Humans
H. pylori, a gram-negative bacterium with a curved, spiral, or
gull-wing shape, is 2.5 to 3.5 m long and 0.5 to 1.0 m in
diameter and has a periodicity of 1 to 2 mm. It has smooth
surfaces, and one to six polar-sheathed flagellae emerge from one
of its rounded ends. Since it is morphologically similar to C.
jejuni, it was initially named "pyloric Campylobacter" and
subsequently C. pyloridis and C. pylori before finally being
named H. pylori. This organism colonizes only the
non-acid-secreting mucosa of the stomach and is not found where
parietal cells are numerous. Thus, it may be observed in the
gastric antrum and the cardia, but also in the corpus, when
atrophic gastritis is present, and attached to the gastric
epithelial cells found in the duodenum, when gastric metaplasia
is present.
G. hominis (H. heilmannii) is tightly spiraled, and is 3.5 to 7.5
mm in length and 0.9mm in diameter; it has a periodicity of 0.8
to 1 mm and up to 12 flagellae at each pole. 16S rRNA indicates
that this organism belongs to the genus Helicobacter, and is more
closely related to a Helicobacter sp. isolated from the stomach
of cats (H. felis) than to H. pylori (17). The name H. heilmannii
was proposed in honor of the late German pathologist Heilmann.
However, the subsequent examination of the rRNA of different
clinical isolates indicates that there is enough heterogeneity
among isolates tentatively identified as H. heilmannii that it is
premature to propose an official name (17) . This bacterium
colonizes only the parietal cell area of the gastric mucosa and
may be found within parietal cells (18,19).
Diagnosis
H. pylori infection may be diagnosed by harvesting gastric biopsy
specimens during endoscopy, by culturing and isolating the
bacterium under microaerobic conditions (90% N2, 5% O2, and 5%
CO2), and by characterizing the enzymes (urease, catalase, and
oxidase) it produces. Visualization of the bacterium by light
microscopy on slides stained with hematoxylin and eosin, Gram,
Giemsa, Genta, or Warthin-Starry stain is also of great benefit
since it allows the concurrent diagnosis of the extent of the
antral chronic-active gastritis that H. pylori causes. However,
because H. pylori colonization is focal, negative biopsy results
do not exclude the possibility of infection in areas not sampled.
Infection also may be diagnosed by determining plasma and
salivary immunoglobulin (Ig) G or IgA levels with enzyme-linked
immunosorbent assays (20,21). This latter technique is
noninvasive, specific, and sensitive and is believed to reflect
the mucosal and systemic immunity induced by H. pylori infection.
Two other tests, which rely on the production of urease, also can
be used to identify H. pylori. One is the CLO (for
Campylobacter-like organisms) test, which is performed by placing
a mucosal biopsy specimen in medium containing urea and a pH-
sensitive dye that changes color in the presence of OH- ions. The
second test is the noninvasive 14C or 13C breath test following
the oral administration of 14C- or 13C-urea. Neither of these
tests is specific for H. pylori since G. hominis, which generates
urease, also gives a positive reaction. Until specific methods
based on the polymerase chain reaction (PCR) amplification of 16S
rRNA (17) become widely available, the diagnosis of G. hominis
infection must rely on histologic morphologic characteristics;
histologic identification must be confirmed by transmission
electron microscopy since other spiral organisms, e.g.,
Flexispira rappini, also may be present in the stomach of humans
(22).
Epidemiology
The seroepidemiology of H. pylori has been extensively studied in
the United States and in other countries (23). The high frequency
of seropositivity (up to 100% in some age groups in Albania) and
acquisition of the infection during infancy are characteristic of
disadvantaged socioeconomic groups living in crowded or poor
hygienic conditions and appears to be independent of gender and
ethnic origin. In adults of higher socioeconomic groups, the rate
of seroconversion is estimated at 0.5% per year, although the
frequency of seropositivity increases with age and may be as high
as 40%. A longitudinal study has indicated that the high
frequency of seropositivity in older adults might be due to a
higher rate of H. pylori infection in Western countries in the
years between the two world wars than during recent years (cohort
effect) (24). Alternatively, the increase in frequency of
infection in older adults might be due to years of low but
cumulative risk for infection. Although the route of transmission
for this infection is not known, the contamination of drinking
water may play a role in certain developing countries (25). In
the United States and in other regions, direct contact and/or
consumption of food or water contaminated by saliva (26), gastric
contents,  or feces (27) may be major factors. The recent
observation that H. pylori can be isolated from cats (28)
suggests that transmission from pets to humans (or humans to
pets) is also possible.
The epidemiology and route of transmission of G. hominis are
largely unknown. The frequency of this infection appears to range
from less than 1% of the population in industrialized countries
(29) to 3% to 8% in developing countries (30). Although the
detection of spirilla in the stomach of cats and dogs suggests
possible transmission from pets, marked morphologic differences
exist between these spirilla and the organism found in the
stomach of humans.
Pathogenicity
H. pylori is considered a pathogen because its presence is always
associated with chronic active gastritis, and eradication of the
bacterium is always followed by resolution of gastritis. In
addition, nearly all patients with duodenal ulcer disease have H.
pylori gastritis, and ulcer relapse is exceptional after H.
pylori eradication. Thus, the presence of H. pylori seems
necessary for the production of duodenal ulcers, with the
exception of ulcers attributed to the use of nonsteroidal
antiinflammatory agents or to the Zollinger-Ellison syndrome
(10). The association with gastric ulcers is not as strong,
although H. pylori infection is present in 80% of patients with
gastric ulcers who do not consume nonsteroidal anti-inflammatory
agents (10). However, most H. pylori-infected persons do not
report any clinical symptoms. This may be because these persons
are colonized by less virulent strains or because other host or
bacterial cofactors are required for overt disease.
In addition, three prospective cohort studies have demonstrated
that H. pylori-infected persons have an increased risk of
developing intestinal-type, but not undifferentiated, gastric
adenocarcinoma (10). In fact, the association of H. pylori with
either gastric ulcer or gastric cancer may be underestimated in
these studies: the atrophic gastritis that follows long-term
infection makes the gastric niche less hospitable for the
bacterium, which may either eliminate H. pylori or make it
difficult to detect. Nevertheless, atrophic gastritis per se is
believed to be a precancerous lesion that leads to carcinogenesis
without the presence of H. pylori.
The pathogenicity of G. hominis is unclear. The organism has been
associated with upper gastrointestinal complaints, and its
carriage is generally accompanied by gastritis, although the
inflammation and gastric atrophy are less than noted with H.
pylori (31,32). In addition, G. hominis was observed in gastric
cancer patients (3) as well as in patients with only minimal
gastritis (29). In this relatively small number of cases, the
frequent concurrent infection with H. Pylori makes interpreting
the respective pathogenic role of either bacterium difficult. It
is probable that G. hominis will turn out to be at least somewhat
pathogenic, as it makes urease and products of urease action that
have been implicated in inflammation.
Colonization and Virulence Factors
H. pylori multiplies with great efficiency in the hostile
environment within the stomach but survives poorly in the gastric
lumen; it is mainly found where the pH ranges between 4 and 7,
i.e., under the mucous layer and in close proximity, or even
attached, to gastric superficial epithelial cells. The virulence
and the ecologic niche of G. hominis are unknown, although its
presence within parietal cells of patients with gastrointestinal
complaints (18,19) suggests that it is even more resistant to
acid than H. pylori.
The production of urease was the first putative colonization or
virulence factor studied. The production of this enzyme is shared
by the two organisms, and it may explain their extraordinary
ability to survive in an environment previously considered
sterile because of the presence of proteolytic enzymes, as well
as the low pH of gastric contents. Because the ecologic niches of
these bacteria are rich in urea, urease generates OH- ions that
neutralize gastric acid. Although the neutralization of gastric
acid benefits the two bacteria, the production of hydroxide ions
also is toxic to gastric epithelial cells in vivo, as indicated
by in vitro experiments (33).
Two other important virulence factors shared by H. pylori and G.
hominis are their spiral shape and the motility of their
flagellae, which render them resistant to peristaltic flushing of
the gastric contents and enable them to persist in the mucous
layer. Because G. hominis appears to infect fewer persons than H.
pylori, a more important role might be attributable to
characteristics that are unique to H. pylori; these include the
production of other enzymes (catalase, oxidase, protease, and
phospholipase), as well as the synthesis of specific adhesin
proteins that enable them to adhere to mucous and epithelial
cells, both in vivo and in vitro (34-36).
The putative virulence factor of H. pylori that has commanded the
most attention during the past few years has been its vacuolating
cytotoxin (vacA gene product). Intragastric administration of the
toxin to mice causes some (but not all) of the tissue damage seen
in H. pylori-infected persons (37). In addition, cytotoxin
production is highly correlated with the production of a high
molecular weight (120 to 128 kilodaltons) major protein antigen
that is called cytotoxin-associated protein (cagA) and is not the
toxin itself (38).
Diversity of H. pylori
H. pylori isolates may differ with respect to each of the
virulence factors described above; this diversity is likely to
contribute to variation in colonization or disease. For example,
urease-negative strains have been isolated, and the vacuolating
cytoxin is produced by only a subset of H. pylori strains (vacA+
or tox+ strains)(39-41). This observation is probably clinically
relevant because most or all strains from duodenal ulcer
patients, and many strains from gastric cancer patients, produce
cytotoxin, whereas only a fraction of strains from patients with
gastritis alone produce the cytotoxin (42,43). This phenotypic
diversity is mirrored in great diversity on the DNA level. Thus,
only cytotoxin-producing strains contain the gene for this
cytotoxin-associated protein (cagA)(38,42), although genetic
tests have shown that cagA protein is not needed for toxin
production (44). Strains that do not produce the 128-kDa cagA
protein generally lack the entire cagA gene and additional
neighboring genes. Although the function of the cagA region is
unknown, its presence or absence is easily scored by
hybridization or PCR and thus serves as an easy marker for
probable cytotoxin production and possible virulence of H. pylori
strains.  Additional virulence factors are likely to be present.
For example, another recently discovered region constitutes at
least 21 kilobases of the H. pylori genome in hybridization
experiments, and its presence is highly correlated with the
presence of cagA: 39 of 40 strains lacking cagA also lacked this
region, and 50 of 52 strains containing cagA contained this
region. This newly discovered region is being called cagII, and
the effort to sequence it is nearly complete (D. E. Berg, pers.
comm.). Preliminary searches have identified several open reading
frames with strong homologies to virulence functions from other
microbes (45).
In addition to these extensively studied genes, genetic diversity
of various H. pylori strains can be demonstrated by the use of
two sensitive, efficient, and reliable PCR-based methods (46,47).
This approach is particularly useful because it allows tracing of
strains in epidemiologic studies.
Infection and Immune Response
One of the most puzzling aspects of gastric infection with H.
pylori is its persistence despite intense local and systemic
immune responses. These immune responses are extremely complex
and vary among infected humans. The systemic response is
characterized by a marked increase in plasma IgG, which remains
present for months after the infection has been cured. The local
response includes the production of IgA, which binds to the
surface antigens of H. pylori in vitro and coats the bacterium in
vivo. In addition, infection is consistently associated with an
intense inflammatory response and the infiltration of cells into
the gastric mucosa. Although polymorphonuclear cells are often
present, most cells in such infiltrates are mononuclear cells.
Both B and T cells are present, and recent studies have indicated
that the natural killer activity of peripheral blood lymphocytes
can be increased by H. pylori, possibly by its stimulating the
production of interferon and other cytokines (48). Thus, the
long-term carriage of the infection may be related to the ability
of the bacterium to influence the T-cell response. Fragmentary
evidence also suggests that this infection can be abortive and
cure spontaneously without the use of antibiotics (A. Dubois and
D. E. Berg, unpublished).
On the other hand, the mucosal response may promote colonization,
as indicated by the observation that patients with acquired
immunodeficiency syndrome (AIDS) tend to have a lower rate of
infection than aged-matched subjects who are negative for human
immunodeficiency virus (49,50). The latter study (50) also
demonstrated that AIDS patients had a different pattern of
gastritis, characterized by greater mononuclear cell responses,
fewer lymphoid follicles, and a greater prevalence of intestinal
metaplasia. The immune response may also prevent the invasiveness
of H. pylori, as suggested by the anecdotal but puzzling
observation of invasive H. pylori infection in a patient with
AIDS (51).
Treatment
Although H. pylori is sensitive to many antimicrobial drugs  in
vitro, it is difficult to eradicate from the stomach. This may be
ascribed to antibiotic breakdown by gastric acid, clearance by
gastric emptying, and the difficult-to-penetrate mucous layer in
which the bacterium resides. Resistance of H. pylori to specific
antibiotics, especially metronidazole, is also frequent.
Therefore, it is generally accepted that a combination of at
least two, and possibly three, antimicrobial agents should be
given for a minimum of 1 week. The regimen found to be most
effective is the administration of amoxicillin (or tetracycline)
plus metronidazole and bismuth subsalicylate 2 to 4 times a day
for 2 to 3 weeks (52). The use of one antibiotic associated with
an antisecretory agent, such as a histamine H2 receptor
antagonist, has given disappointing results. In contrast, the
combination of a proton pump inhibitor (H+-K+ ATPase antagonist)
with amoxicillin or acid-stable macrolides (clarythromycin or
roxithromycin) appears more promising; a number of studies are
being conducted to determine the optimal dose, duration,
concomitant therapy, and cost-effectiveness of these compounds
(53,54). Recently, it was shown that at least a 7-day course of
any of these regimens is required to obtain a high (90%) cure
rate, but that continuing treatment for more than 10 days does
not significantly improve its efficacy. Finally, topical therapy
for 1 h was recently tried with excellent results, albeit in only
one center at this time (55). This treatment involves a 2-day
administration of a mucolytic agent to dissolve the mucous layer
and of a proton pump inhibitor. On the third day, a balloon is
introduced into the second portion of the duodenum under
fluoroscopic control, and a solution of pronase, amoxicillin,
metronidazole, and bismuth subsalicylate is injected into the
stomach, where it is left for 1 h. The presence of the duodenal
balloon appears to prevent emptying of the antibiotics and the
mucolytic agent, thus ensuring maximum efficacy of the therapy.
Future Research
The past 12 years have seen extensive progress in research on H.
pylori as a cause of chronic active gastritis, duodenal ulcer
disease, and gastric cancer. This has been largely due to an
unusual collaboration among gastroenterologists, pathologists,
molecular geneticists, bacteriologists, and immunologists.
However, our understanding of how H. pylori colonizes and causes
diseases is far from complete, and it will benefit from studies
performed in animal models that can be experimentally infected
with H. pylori (56-59). In addition, no easily administered
treatment leading to eradication of this bacterium in all
patients is yet available, although a better knowledge of its
physiology may lead to the development of such a "silver bullet."
Studies in animals that are not naturally infected with H. pylori
suggest possibilities for vaccines (56,57), and ongoing trials in
nonhuman primates are exploring the possibility of immunizing
hosts that can be naturally infected with this organism. Although
the elimination of peptic ulcer disease and of certain forms of
gastric cancer will require extensive and coordinated efforts
from public health authorities, this goal now appears to be
within the reach of the scientific and medical community.
Acknowledgments
The author thanks Drs. P. Baker and D.E. Berg for their helpful
comments and suggestions during the preparation of this review.
Dr. Dubois is professor of medicine and surgery (research),
assistant director, Digestive Diseases Division, and chief,
Laboratory of Gastrointestinal and Liver Studies, F. Edward
Hbert School of Medicine of the Uniformed Services University of
the Health Sciences, Bethesda, Maryland. He studies the
physiology and pathophysiology of gastric secretion and gastric
emptying as well as the role of gastric infection with H. pylori
in gastroduodenal diseases.
Address for correspondence: Andre Dubois, Department of
Medicine, Uniformed Services University, 4301 Jones Bridge
Road, Bethesda, MD 20814-4799, USA; fax: 301-295-3676 or
-3557; e-mail dubois@usuhsb.usuhs.mil.
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gastric disease. J Exp Med 1994;179:1653-8.
38. Tummuru MK, Cover TL, Blaser MJ. Cloning and expression of
a high-molecular-mass major antigen of Helicobacter pylori:
evidence of linkage to cytotoxin production. Infect Immun
1993;61:1799-809.
39. Figura N, Gugliemetti P, Rossolini, et al. Cytotoxin
production by Campylobacter pylori strains isolated from patients
with peptic ulcers and from patients with chronic gastritis only.
J Clin Microbiol 1989;27:225-6.
40.Cover TL, Dooley CP, Blaser MJ. Characterization of and
human serologic response to proteins in Helicobacter pylori broth
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42. Covacci A, Censini S, Bugnoli M, et al. Molecular
characterization of the 128-kda immunodominant antigen of
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43. Cover TL, Glupczynski Y, Lage AP, et al. Serologic detection
of infection with cagA+ Helicobacter pylori strains. J Clin
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44. Tummuru MK, Cover T, Blaser M. Mutation of the
cytotoxin-associated cagA gene does not affect the vacuolating
cytotoxin activity of Helicobacter pylori. Infect Immun
1993;62:2609-13.
45. Akopyants NS, Kersulyte D, Berg DE. cagII, a new multigenic
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B-90.
46. Akopyanz N, Bukanov NO, Westblom TU, Kresovich S, Berg DE.
DNA diversity among clinical isolates of Helicobacter pylori
detected by PCR-based rapid fingerprinting. Nucleic Acid Res
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47. Akopyanz N, Bukanov NO, Westblom TU, Berg DE. PCR-based RFLP
analysis of DNA sequence diversity in the gastric pathogen
Helicobacter pylori. Nucleic Acids Res 1992;20:6221-5.
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with Helicobacter pylori augments natural killer cell activity
and induces production of interferon. Infect Immun
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DA. Helicobacter pylori-associated gastritis is rare in AIDS:
antibiotic effect or a consequence of immunodeficiency? Am J
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gastritis in Zambian patients with the acquired immunodeficiency
syndrome. Gastroenterology 1995;108:A921.
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Campylobacter pylori gastritis in the acquired immunodeficiency
syndrome. Gastroenterology 1988;95:209-12.
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efficacy of antibiotic therapy in eradicating Helicobacter
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of Helicobacter pylori with clarythromycin and omeprazole. Gut
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the treatment of Helicobacter pylori infection. Am J
Gastroenterol
1995;90:60-3.
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of BALB/c mice against Helicobacter felis infection with
Helicobacter pylori urease. Gastroenterology 1994;107:1002-11.
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Ghiara P. Development of a mouse model of Helicobacter pylori
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gastric Campylobacter pylori infection in the neonatal
gnotobiotic piglet. Infect Immun 1987;55:2789-96.
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infection with Helicobacter pylori in monkeys. A model for human
infection with spiral bacteria. Gastroenterology
1994;106:1405-17.
DISPATCHES
HIV-1 Patients May Harbor Viruses of Different Phylogenetic
Subtypes: Implications for the Evolution of the HIV/AIDS Pandemic
The virus variants isolated from HIV-infected persons worldwide
share remarkable diversity, especially in the envelope
glycoprotein, gp120. Phylogenetic studies have clustered HIV-1
isolates into eight subtypes (A-H). Nevertheless, even within a
single infected person, HIV is present as a "quasi-species," or a
swarm of closely related variants. This genetic diversity, which
in the case of HIV-1 accumulates at a rate of approximately one
nucleotide substitution per genome per replication cycle, gives
the virus an enormous flexibility to respond to a wide array of
in vivo selection pressures. As a consequence, drug-resistant and
immunologic escape mutants are rapidly generated in infected
persons through all stages of infection. On a global scale, the
HIV pandemic is recognized as consisting of many separate
epidemics, each with characteristic geography, affected
populations, and predominant viral strain type. With an estimated
15 million infected persons, the geographic distribution of viral
subtypes is becoming more dispersed, and these demarcations are
further confounded by growing evidence of mixed infections.
The epidemic emergence of mixed heterotypic infections with HIV-1
and HIV-2 variants has been recognized for some time in the
geographic areas where both types of viruses are present. We
reported these infections in Cte d'Ivoire and Brazil (1,2);
they have also been reported from India (3). In contrast,
homotypic mixed infections of distinct HIV-1 variants have only
recently been suggested by the presence of broadly reactive sera
and evidence of HIV recombinants from geographic regions in which
multiple HIV-1 subtypes are circulating. Dual HIV-1 infection in
two patients from Thailand has been demonstrated by viral DNA
sequence analysis (4).
As the HIV-1 pandemic has grown, the simultaneous presence of
multiple subtypes in a region has become common. As a
consequence, an increased frequency of HIV-1 mixed infections
could be expected. Thus, there is a need to estimate the
prevalence and geographic distribution of this type of infection.
Sequence analysis of HIV proviral DNA has been the method of
choice to characterize HIV genetic diversity. However, because
even relatively limited sequence determinations of small
polymerase chain reaction (PCR)fragments are time-consuming and
very labor-intensive, this method is not particularly practical
for large-scale molecular epidemiologic studies. To address this
problem, we have developed a genetic method based on restriction
site polymorphism to screen for homotypic HIV-1 mixed infections
within infected populations. The concept of this assay is based
on the observed correlation between the restriction maps of HIV-1
isolates with their phylogenetic classification, which is based
on the sequence data. Thus, certain restriction enzymes may be
used to predict the phylogroup of HIV-1 infected samples. The
differences in electrophoretic mobility of endonuclease
digestion products result from restriction site polymorphisms in
the selected region of the HIV-1 genome and allow for quick
recognition of the distinct phylogenetic subtypes. A 297 bp pol
fragment spanning the entire viral protease gene is used for our
analysis. The viral gene is amplified by nested PCR using DNA
templates from uncultured peripheral blood mononuclear cells
(PBMC) or virus culture. Preliminary classification of HIV-1
strains to well defined subtypes A, B, C, D, and F is done by
sequential endonuclease restriction analysis. AluI restriction
polymorphism in a PCR-amplified protease gene segregates viral
strains into two groups: subtypes B and D belong to one group,
and subtypes A, C, and F to another (Figure 1A cannot be viewed
in ASCII). Further differentiation of HIV-1 subtypes within those
two groups is accomplished by analysis of HinfI, BclI, MaeI,
SpeI, an ScaI restriction enzyme digestion patterns of the
protease gene (Pieniazek et al., manuscript in preparation).
The electrophoretic migration patterns visualized by ethidium
bromide staining or by radiolabeled probes are then determined
on a 10% acrylamide gel. In single infections, a single
restriction pattern is detected, whereas in multiple infections
involving HIV-1 strains of distinct subtypes, complex digestion
patterns are observed in infected persons. As an example, in
Figure 1A, we present three distinct AluI restriction patterns
of the protease gene that are characteristic for single
infections by viruses of subtypes A, C, and F (pattern #1)
and by subtypes B and D (patterns #2 and #3). In Figure 1B,
we show a typical combination of two distinct AluI restriction
patterns (#1 and #2) found in a patient infected with two
viral strains of subtypes F and B. Basing our analysis on
the conserved protease gene region, we should detect most
HIV-1 strains; however, some highly divergent isolates could
escape PCR amplification as a result of primer mismatches.
Moreover, since a single nucleotide substitution could either
generate or destroy a restriction site, sequence analysis
remains the ultimate tool in identifying variants of multiple
infections. Nevertheless, this assay can be conveniently
applied to screen a large number of samples.
By using this method, we have screened HIV-1 proviral DNA from
208 specimens collected from countries in South America, Africa,
and Asia where HIV-1 strains of distinct subtypes are found. We
observed the simultaneous presence of two distinct digestion
patterns in PCR amplified protease gene (Figure 1B cannot be viewed
in ASCII) in 31 samples; our observation suggests
superinfection with HIV-1 strains of distinct origin.
To eliminate the potential for laboratory cross-contamination,
we analyzed the restriction patterns of the protease gene
from multiple aliquots of the patient's PBMC.
In addition, the analysis was repeated on DNA
from a second collected blood sample from each of the patients.
The analyses for the first five of 31 patients were completed,
and data are summarized here (details are in Janini et al.,
manuscript in preparation). Sequence and phylogenetic analysis of
the viral protease gene (Figure 2 cannot be viewed in ASCII.) in PBMC from those five
patients confirmed dual infections caused by HIV-1 strains of
subtypes B and F in one person (Br5), subtypes F and D in another
patient (Br22), and subtypes C and D in a married couple (Br19
and 20). Moreover, in the child (Br30) of this couple, two
distinct AluI digestion patterns were also found; the major
HIV-1 strain clustered among subtype C viruses of the parents.
The minor strain of this child is likely to represent subtype D,
but there was not sufficient material for cloning and further
sequencing of this strain.
Detection of naturally occurring heterotypic and homotypic
multiple infections may have important implications for
immunotherapies because infection with one HIV subtype may not
fully protect against subsequent superinfections with distinct
HIV strains. However, we do not know if the acquisition of
viruses in the dually infected adult patients was sequential or
simultaneous. Nevertheless, the consequences of mixed infections
may profoundly affect the ability of the virus to change and may
modify the direction of the pandemic through altered patterns of
viral pathogenesis, increased genetic variation through
recombination, and the generation of pseudotype virions,
including phenotypically mixed virus particles. It is to be
anticipated that such events would ultimately broaden the
cellular tropism for HIV and mandate the designed polyvalent
immunotherapies. Finally, our data together with recently
published genetic analysis for HIV-1 and HIV-2 (5) suggest that
multiple homotypic infections with divergent HIV strains may be
more common than previously thought. The screening assay
described here will be useful in estimating incidences of
such HIV-1 infections. We believe that this information is
crucial for both evaluating the pandemic and developing
intervention strategies.
Danuta Pieniazek,* Luiz M. Janini,*#  Artur Ramos,*#  Amilcar
Tanuri,# Mauro Schechter,& Jose M. Peralta,# Anna C.P. Vicente,
Norman J. Pieniazek,* Gerald Schochetman,* and Mark A. Rayfield*
*National Center for Infectious Diseases, Centers for Disease
Control and Prevention, Atlanta, Georgia, USA; #Universidade
Federal do Rio de Janeiro, and &Hospital Clementino Fraga Filho,
and Instituto Oswaldo Cruz, Rio de Janeiro, Brazil
References
1. Rayfield M, De Cock K, Heyward W, et al. Mixed human
immunodeficiency virus (HIV) infection in an individual:
demonstration of both type 1 and type 2 proviral sequences by
using polymerase chain reaction. J Infect Dis 1988:158:1170-6.
2. Pieniazek D, Peralta JM, Ferreira JA, et al. Identification of
mixed HIV-1/HIV-2 infections in Brazil by polymerase chain
reaction. AIDS 1991;5:1293-9.
3. Grez M, Dietrich U, Balfe P, et al. Genetic analysis of Human
Immunodeficiency Virus type 1 and 2 (HIV-1 and HIV-2) mixed
infections in India reveals a recent spread of HIV-1 and HIV-2
from a single ancestor for each of these viruses. J Virol
1994;68:2161-8.
4. Artenstein AW, VanCott TC, Mascola JR, et al. Dual infection
with HIV type 1 of distinct envelope subtypes in humans. J Infect
Dis 1995;171:805-10.
5. Gao F, Yue L, Robertson DL, et al. Genetic diversity of human
immunodeficiency virus type 2: evidence for distinct sequence
subtypes with differences in virus biology. J Virol
1994;68:7433-47.
6.Larsen N, Olsen GJ, Maidak BN, el al. The ribosomal database
project. Nucleic Acids Res 1993;21:3021-3.
Simultaneous Infection of Ixodes ricinus Nymphs by Two
Borrelia burgdorferi Sensu Lato Species: Possible
Implications for Clinical Manifestations
Data from European studies indicate that in
humans, particular Borrelia burgdorferi
genospecies may be associated with specific
clinical manifestations of Lyme disease.
Infections by B. burgdorferi sensu stricto
tend to lead to arthritic symptoms, whereas
infections by B. garinii appear to cause
neurologic complications. Late cutaneous
manifestations (acrodermatitis) appear to be
associated with B. afzelii (1). Mixed clinical
manifestations have also been described (2).
Recently it has been demonstrated, by using
polymerase chain reaction (PCR), that DNA from
more than one of the three Borrelia species
associated with Lyme disease in Europe was
present in the biological fluids of Lyme
disease patients (3). These data raise
questions concerning the relative growth of
the Borrelia species after a bite by a dually
infected tick, the clinical significance of
human infection caused by more than one
species of Borrelia, and the origin of these
multiple infections. This last point evokes
the following question: do they result from
successive bites by two infected ticks or from
a single bite by a tick infected by more than
one species?
To investigate whether ticks are infected by
different species of the B. burgdorferi
complex at the same time, we carried out a
survey of the vector Ixodes ricinus during the
spring of 1994, in Rambouillet Forest near
Paris. A total of 249 unfed nymphs, collected
from vegetation, were analyzed by PCR. The
ticks were then crushed in phosphate-buffered
saline, solubilized in 0.5% Tween 20, and
boiled for 10 min. The resulting lysate was
used as a template for the amplification
reactions by either the universal ospA-based
primers SL or the three pairs of
genospecific-based primers (3). These last
primers distinguish the three Lyme
disease-associated B. burgdorferi sensu lato
species, i.e., B. burgdorferi sensu stricto,
B. garinii, and B. afzelii. In some cases,
amplified DNA products were digested with
specific restriction enzymes to confirm the
typing of the Borrelia strain.
Thirty of the 249 nymphs were positive for B.
burgdorferi when SL universal primers were
used. Further testing of 5 of 30 nymphs by
PCR, using genospecific primer sets and
restriction analysis, did not confirm the
preliminary results with the universal
primers. This may have been due either to the
genotypic variability of B. burgdorferi sensu
lato or to the existence of other distinct
subgroups or genomic species included in B.
burgdorferi sensu lato, as other data appear
to indicate (4). Of the 25 other nymphs, 22
were analyzed by both restriction analyses and
the specific primers, and three by restriction
analysis alone. (The available tick material
was not sufficient to perform PCR with
genospecific primers.) Nineteen nymphs were
infected by a single species of Borrelia (four
by B. garinii, 15 by B. afzelii), and six were
infected by more than one (two by both B.
burgdorferi sensu stricto and B. garinii,
three by B. garinii and B. afzelii, one by B.
burgdorferi sensu stricto and B. afzelii).
From these results, it appears that when
nymphs are infected with one species, B.
afzelii is the most prevalent. This species
may actually be prevalent in this study area
or may have a greater tropism for dermal
tissue and/or for the peripheral circulatory
system of the vertebrate than the other two
species.  In infected nymphs, the simultaneous
presence of more than one genospecies in unfed
nymphs of I. ricinus was not exceptional
(24%), and all combinations of two species
were observed. The association of three
genospecies has not yet been detected.
Simultaneous infections in unfed nymphs could
have different explanations. The first is a
larval meal on a host infected by more than
one species. Recently, Apodemus speciosus
(field mice) infected by two different species
have been found (5). A second possibility is
successive infectious interrupted larval
meals. A third possibility is an infectious
larval meal by a previously transovarially
infected larva. The fourth possibility is a
mixed infection acquired   transovarially.
Bruno Pichon,* Edmond Godfroid,# Bernard
Hoyois,# Alex Bollen,# Francois Rodhain,* and
Claudine Prez-Eid*
* Unit d'Ecologie des Systmes Vectoriels,
Institut Pasteur 75724 Paris, France
# Laboratoire de Genetique Appliqu, Universit
Libre de Bruxelles, Rue de l'Industrie 24,
B-1400 Nivelles, Belgium
This work was supported by grants from the
Recherche & Partage Association, the Gould
Foundation, and the Conseil du Departement du
Val d'Oise. E. Godfroid, B. Hoyois, and A.
Bollen, were supported by a grant from the
Walloon Region of Belgium (Convention UIB,
Region Wallonne No. 2267).
References
1. Assous MV, Postic D, Paul G, Nevot P,
Baranton G. Western blot analysis of sera from
Lyme borreliosis patients according to the
genomic species of the Borrelia strain used as
antigens. Eur J Clin Infect Dis 1993;12:261-8.
2. Wienecke R, Neubert U, Volkenandt M.
Cross-immunity among types of Borrelia
burgdorferi. Lancet 1993;341:830-1.
3. Demaerschalck I, Ben Messaoud A, De Kesel
M, Hoyois B, Lobet Y, Hoet P, et al.
Simultaneous presence of different Borrelia
burgdorferi genospecies in biological fluids
of Lyme disease patients. J Clin Microbiol
1995;33:602-8.
4. Nohlmans LMKE, De Boer R, Van Den Boggard
AEJM, Van Boven CPA. Genotypic and phenotypic
analysis of Borrelia burgdorferi isolates from
the Netherlands. J Clin Microbiol
1995;33:119-25.
5. Nakao M, Miyamoto K. Mixed infection of
different Borrelia species among Apodemus
speciosus mice in Hokkaido, Japan. J Clin
Microbiol 1995;33:490-2.
Epidemiologic and Evolutionary Relationships between
Romanian and Brazilian HIV-1 Subtype F Strains
The initial classification of HIV-1 viruses as Western or
African strains has been replaced by phylogenetic
subtyping that uses nucleotide sequence data. Eight
distinct phylogenetic HIV-1 lineages or subtypes, A to H,
have been defined (1). Considering that the rate of HIV-1
genome evolution is estimated at 0.5% to 1% per year and
that the average genetic distance between the HIV-1
subtypes is approximately 20%, it is likely that these
subtypes originated before the HIV-1 pandemic (1). The
global mosaic of HIV-1 subtypes is consistent with the
hypothesis that most regional epidemics started with the
introduction of one or a few variants that diversified
locally rather than through radiant waves of already
diversified HIV-1 subtypes that spread from the place of
origin. In this report, we address the evolutionary and
epidemiologic relationships between the HIV-1 subtype F
viruses recently identified in two geographically
distinct regions, Romania and Brazil.
The HIV-1 epidemic among Romanian children living in
orphanages was recognized during 1989-1990 (2).
Epidemiologic studies have shown that most children
became infected by horizontal transmission of HIV-1
through blood transfusions or through the use of
unsterilized medical equipment. We have shown by
nucleotide sequence analysis that all HIV-1 isolates from
children in southeastern Romania are highly related
genetically (3). The average interperson nucleotide
distance within the C2-V3 region of the env gene was 0.9%
to 3.6%. Phylogenetic analysis of these sequences showed
that the Romanian HIV-1 strains clustered together with
a single Brazilian HIV-1 strain in a previously
unrecognized evolutionary clade later designated as the
F subtype. We now have the opportunity to augment this
comparison with additional F subtype sequences from the
two countries and to further address the relationship
between Romanian and Brazilian viruses.
In the phylogenetic analysis of the envelope C2-V3
nucleotide sequences (Figure 1 cannot be viewed in ASCII),
we included eight HIV-1 F subtype viruses isolated from
children in southeastern Romania (L19570-L119579) (3)
as well as two representative sequences of the HIV-1
viruses found in children living in northcentral Romania
(R18586 and R18598) (Bandea et al., manuscript in
preparation). From Brazil, we included, in addition
to the sequence of the first identified F subtype
strain (BRA7944)(4), three recently reported F sequences
(BZ126A, BZ162A, and BZ163A) (5) and four F strains
(BR46, BR57, BR58, and BR59) isolated in our laboratory
from patients in Rio de Janeiro. We also included two
HIV-1 strains from Cameroon, CA4 and CA20, that have
been tentatively classified as F subtype viruses (1,6)
and reference nucleotide sequences representing the other
HIV-1 subtypes.
The results of this phylogenetic analysis show that the
Brazilian and Romanian sequences cluster in two highly
related but separate groups. The genetic distance between
Brazilian subtype F sequences was 5% to 13.8% , which is
within the limits of the established intrasubtype
distance values (1). Among Romanian C2-V3 nucleotide
sequences this distance was 0.9% to 6.5%, and between the
two groups it was 7.5% to 12.9%. These values support the
inclusion of the Romanian and Brazilian groups within the
same F HIV-1 subtype. The reliability of these
phylogenetic results was verified by bootstrap analysis
(100 data sets) and by pruning, which consists of
sequential removing of different strains and rerunning
the analysis.
The two sequences from Cameroon associated only weakly
with Romanian and Brazilian groups, and this association
was not stable. The genetic distance between the Romanian
and Brazilian sequences and sequences from Cameroon was
16.5% to 24.1%, which is typical of intersubtype rather
than intrasubtype genetic distances. Our analysis does
not support a strong linkage between the Cameroonian
strains and the other F subtype viruses; however, in
evolutionary terms, these viruses may be closer to each
other than to the other subtypes, or they may have
undergone convergent evolution within the envelope region
that we analyzed. Analysis of additional sequences from
other regions of the HIV-1 genome may clarify the
relationship between the Cameroonian strains and the
other subtypes.
The amino acid GPGR motif at the tip of the V3 protein
loop has been considered a signature sequence for the
Brazilian F subtype viruses (5). This relatively
conserved motif was noticeable because it is
characteristic among B subtype viruses, whereas the GPGQ
sequence predominates among all the other HIV-1 strains,
including all the initial Romanian F viruses. Two of the
newly identified Brazilian strains (BR58 and BR59),
however, contain the GPGQ motif, and the Romanian strain
R18598 contains the GPGH motif (Figure 2 cannot be viewed
in ASCII). Phylogenetically, these sequences group with their
respective geographic clusters (Figure 1), which
indicates that independent mutations may have occurred at
this locus.
As indicated earlier, the genetic distance between
Romanian HIV-1 nucleotide sequences is very small, which
suggests a direct epidemiologic link among these strains
and a short period of evolution. The exclusive presence
of highly related viruses in two geographically distinct
provinces of Romania strongly suggests that the initial
pediatric HIV-1 epidemic in this country started from a
single infectious source. This suggestion is also
supported by the fact that F subtype viruses are uncommon
and, therefore, the chances for independent multiple
introduction of highly related F subtype strains in
Romanian children are very low. Although no nucleotide
sequence data are available about HIV-1 strains
circulating in the adult population in Romania, the
epidemiologic and serologic studies indicate that the
number of infections is small, and probably most
HIV-1-infected adults were infected though sexual contact
with foreign visitors or with Romanians that traveled
outside the country (2). It is expected, therefore, that
most of the HIV-1 strains infecting the adults represent
internationally prevalent subtypes. It is remotely
possible, however, that adults were infected with F
subtype viruses and could have served as the original or
intermediary carriers for HIV-1 transmission among the
groups of children living in distinct geographic regions
of Romania.
In Brazil, the relatively long genetic distance between
F subtype viruses could indicate that a single ancestor
was introduced during the early phases of the HIV-1
epidemic and diverged locally, or that multiple different
F strains were introduced to this country. The low
prevalence of F subtype viruses worldwide makes the
latter alternative less likely. However, no information
is available about the HIV-1 strains present during the
early phases of the epidemic in Brazil. Our ongoing
studies and the published data (4,5) indicate that the
HIV-1 F subtype infections represent roughly 10% of the
estimated number of cases. This relatively large number
of F subtype infections and the estimated rate of HIV-1
divergence suggest an early introduction of the F subtype
viruses in Brazil.
Because of the geographic position and the sparsity of
socioeconomic relations between Romania and Brazil, the
potential for an epidemiologic link between HIV-1 F
subtype viruses is small. A more compelling argument
against a direct epidemiologic link between F subtype
viruses from these two countries can be made on the basis
of the topology of the phylogenetic branches that set
apart the two groups of viruses (Figure 1). If a direct
epidemiologic link existed between the two groups, the
clustering would be integral with one group branching
from within the other. The tree topology shows the two
groups of viruses on separate branches with a relatively
distant common ancestor. Although limited in scope, our
findings support an evolutionary relationship between the
Romanian and Brazilian F subtype viruses and indicate
that the two regional epidemics arose independently.
Claudiu I. Bandea,* Artur Ramos,* Danuta Pieniazek,*
Rodica Pascu,& Amilcar Tanuri,# Gerald Schochetman,* and
Mark A. Rayfield*
*National Center for Infectious Diseases, Centers for
Disease Control and Prevention, Atlanta, Georgia, USA;
#Universidade Federal do Rio de Janeiro, Rio de Janeiro,
Brazil; &University of Medicine and Pharmacy, Tg-Mures,
Romania
References
1. Myers G, Korber B, Wain-Hobson S, Smith R, Pavlakis G,
eds. Human retroviruses and AIDS 1994: a compilation and
analysis of nucleic acid and amino acid sequences. Los
Alamos, NM: Los Alamos National Laboratory, 1993.
2. Hersh BS, Popovici F, Apetrei RC, et al. Acquired
immunodeficiency syndrome in Romania. Lancet
1991;338:654-9.
3. Dumitrescu D, Kalish ML, Klicks SC, Bandea CI, Levy
JA. Characterization of human immunodeficiency virus type
1 isolates from children in Romania: identification of a
new envelope subtype. J Infect Dis 1994;169:281-8.
4. Potts KE, Kalish ML, Lott T, et al. Genetic
heterogeneity of the V3 region of the HIV-1 envelope
glycoprotein in Brazil. AIDS 1993; 7:1191-7.
5. Louwagie J, Delwart EL, Mullins JI, McCutchan FE, Eddy
G, Burke DS. Genetic analysis of HIV-1 isolates from
Brazil reveals presence of two distinct genetic subtypes.
AIDS Res Hum Retroviruses 1994;10:561-7.
6. Nkengasong JN, Janssens W, Heyndrickx L, et al.
Genotypic subtypes of HIV-1 in Cameroon. AIDS
1994;8:1405-12.
7. Felsenstein J. PHYLIP-phylogeny interference package
(version 3.2). Cladistics 1989;5:164-6.
Electronic Communication Facilitates Investigation of a
Highly Dispersed Foodborne Outbreak: Salmonella on the
Superhighway
Widely dispersed foodborne disease outbreaks are an
emerging public health problem (1-3). Increasing
population mobility and the wide distribution of
centrally produced foods mean that when an outbreak of
foodborne disease occurs, the affected persons may be
distributed across the country or even the world. Widely
dispersed outbreaks challenge limited public health
resources; they can be difficult to detect, labor-intensive,
and time-consuming to investigate. We report the rapid,
efficient investigation of a widely dispersed interstate
outbreak through electronic communication between the
possible patients and public health workers.
On August 4, 1994, a resident of a western state
contacted the Centers for Disease Control and Prevention
(CDC) regarding a possible outbreak of foodborne illness.
On July 22, the day after returning from a conference in
Baltimore that included attendees from all 50 states, he
became ill with diarrhea, and Salmonella was isolated
from his stool. He contacted four other conference
attendees who had taken the same flight. One of these
also had culture-confirmed Salmonella infection; a
second, who was taking antibiotics for other reasons, had
a diarrheal illness with a negative stool culture, and
two had nonspecific diarrheal illnesses. Because of the
possibility of a multistate outbreak involving the
airline or the conference and affecting many people, we
initiated a survey of conference attendees to determine
the rate and correlates of diarrheal illness.
Traditionally, surveys of dispersed populations have been
conducted either by telephone, requiring many
person-hours of interviewing, or by mail, leading to many
days' delay while questionnaires are distributed and
returned. However, in this case, the organization that
sponsored the conference had an internal electronic mail
(e-mail) system. Each section of the organization,
although not each person, had a computer that could
receive e-mail messages. On August 5, 1994, the
organization's central office e-mailed a questionnaire,
developed in consultation with CDC, to all computers in
the organization with instructions to organization staff
to print and distribute the questionnaire to conference
attendees. The questionnaire contained items regarding
diarrheal illness, meal and flight exposures, and
demographic information. Because the organization's
e-mail system was only internal, responses could not be
made to CDC by e-mail. Therefore, attendees were
instructed to send their completed questionnaires to CDC
by fax. By August 12 (7 days later), sufficient responses
had been received to evaluate the flight and the
conference as sources of an outbreak of salmonellosis
(Figure 1 cannot be viewed in ASCII).
Of 390 persons registered at the conference, 86 (22%)
returned questionnaires by August 12. A questionnaire was
returned by the index patient who made the initial call
to CDC but not by the four other persons he contacted who
were passengers on the same flight. Six (7%) of the 86
respondents reported having diarrhea (three or more loose
stools in a 24-hour period) during the period beginning
12 hours after the conference started and ending 5 days
after the conference ended (July 20 to 26). Among
questionnaire respondents, only the index patient was
diagnosed with salmonellosis. Three respondents had taken
the initially suspect flight. Illness was not associated
with taking the same flight as the index patient (p = 0.20,
Fisher's Exact Test, 2-tailed).
To further investigate the reports of diarrhea, we
interviewed the six persons who reported diarrheal
illness by questionnaire, as well as the four persons
initially contacted by the index patient who had not
completed questionnaires. This group included the two
persons with known Salmonella infection, of whom one had
completed a questionnaire and one had not. Seven of the
other eight persons had mild, nonspecific symptoms of
less than 2 days' duration; the onset dates of their
illnesses spanned a 5-day period, and none sought medical
attention. Because few conference attendees or flight
passengers became ill with symptoms suggestive of
salmonellosis during a likely period, we thought that an
airplane- or conference-associated outbreak was
improbable. By September 9, questionnaires were returned
by 156 (40%) of the conference attendees. No additional
cases of diarrhea were reported, confirming our initial
conclusion that the Salmonella infections were not
associated with the flight or the conference.
The Salmonella isolates were identified at CDC as
Salmonella serotype Norwich, of the Salmonella serogroup
C1. S. Norwich is rare; in 1993 and 1994, respectively,
63 and 102 isolates of this serotype were reported to the
Public Health Laboratory Information System (PHLIS), a
nationwide electronic laboratory-based surveillance
system that collects and summarizes data on isolates from
state public health laboratories (4). Because infection
with S. Norwich is so uncommon, it still seemed likely
that the two infections could have a common source, such
as a restaurant.
Subsequent investigation focused on meals that the two
persons with salmonellosis shared outside the conference
and ultimately revealed the source, a restaurant in
Baltimore. In late July 1994, the Maryland Department of
Health and Mental Hygiene received reports that
Salmonella, serogroup C1, had been isolated from five
other persons who visited Baltimore around the time of
the conference. Two persons from one family had driven to
Baltimore on July 17, eaten only at one restaurant, then
returned to their home state of Pennsylvania. Three
persons in a second family, from a different part of
Pennsylvania, ate at the same Baltimore restaurant on
July 21 during a vacation trip.  The Salmonella isolates
from members of both families were initially
misidentified as other serogroup C1 serotypes. They were
retested because of this outbreak and were confirmed as
S. Norwich. The two conference attendees with S. Norwich
infection also ate at the implicated restaurant on July
21. No single menu item had been eaten by all ill
persons. In response to a complaint by the first family,
the restaurant had been inspected by the local health
department; multiple violations of food safety
regulations were found. S. Norwich was isolated from a
stool specimen from an employee who reported a diarrheal
illness beginning on July 22 and who ate the restaurant's
food. In the month following the inspection of Restaurant
A and subsequent corrective action, no further cases of
S. Norwich were reported to PHLIS from Maryland or
Pennsylvania.
E-mail can expedite questionnaire distribution,
especially when the population of interest is on one
network. The computer system used to send the e-mail
message in this outbreak was not linked to individual
conference attendees; therefore, we could not evaluate
the rates at which individual attendees obtained and
responded to the message. If we had been able to reach
attendees directly, our response rate may have been
higher, and we would have been able to send additional
messages to nonresponders. In the future, when outbreaks
occur among persons accessible by e-mail, it may be
possible to evaluate strategies to improve response rate
and to compare the effectiveness of the delivery of
questionnaires by e-mail and by more traditional means.
This outbreak illustrates the usefulness of rapid
electronic communication in a public health setting.
Isolation of a rare Salmonella serotype and national
electronic reporting to PHLIS assisted in the detection
and investigation of a widely dispersed multistate
outbreak of salmonellosis. Without the national
Salmonella serotyping system, the outbreak would not have
been recognized. Questionnaires were distributed rapidly
by e-mail; the utility of this method is likely to
increase as more people become accessible by e-mail. Fax
provided a means for respondents to return questionnaires
quickly. Continued on-line analysis of surveillance data
with PHLIS confirmed that the outbreak was controlled.
Rapid communication between public health workers in
Maryland and Pennsylvania and at CDC was also essential.
The usefulness of electronic communication is not limited
to outbreak investigation. New technologies will
undoubtedly continue to be useful in addressing emerging
public health problems.
Barbara E. Mahon,* Dale D. Rohn,# Sheila R. Pack,& Robert
V. Tauxe*
*National Center for Infectious Diseases, Centers for
Disease Control and Prevention, Atlanta, Georgia, USA;
#Epidemiology and Disease Control Program, Maryland
Department of Health and Mental Hygiene, Baltimore,
Maryland, USA; &Division of Acute Communicable Diseases
(now in Division of HIV Seroepidemiology), Baltimore City
Health Department, Baltimore, Maryland, USA
References
1. Tauxe RV. Salmonella: a postmodern pathogen. J Food
Protection 1991;54:563-8.
2. Hedberg CW, MacDonald KL, Osterholm MT. Changing
epidemiology of food-borne disease: a Minnesota
perspective. Clin Infect Dis 1994;18:671-82.
3.Hedberg CW, Levine WC, White KE, Carlson RH, Winsor DK,
Cameron DN, et al. An international foodborne outbreak of
shigellosis associated with a commercial airline. JAMA
1992;268:3208-12.
4. Bean NH, Martin SM, Bradford H. PHLIS: an electronic
system for reporting public health data from remote
sites. Am J Public Health 1992;82:1273-6.
COMMENTARY
Reemergence of Ebola Virus in Africa
Members of the family Filoviridae, which currently consists of
Ebola and Marburg viruses, cause severe and often fatal hemorrhagic
fevers in humans and nonhuman primates. The recent isolation and
identification of a new Ebola virus from a single nonfatal human
case in Cte d'Ivoire (1) and the more recent outbreak of Ebola
hemorrhagic fever in and around Kikwit, Zaire (2,3), have raised
concerns about the public health threat of these human pathogens.
Filoviruses are classified as biosafety level 4 agents because of
the extreme pathogenicity of certain strains and the lack of a
protective vaccine or effective antiviral drug. Moreover,
filoviruses are among the most mysterious groups of viruses known
because their natural history and reservoirs remain undefined and
their pathogenesis is poorly understood.
Ebola virus infections were first recognized in 1976, when
simultaneous but separate outbreaks of human disease caused by two
distinct virus subtypes erupted in northern Zaire and southern
Sudan (4) and resulted in hundreds of deaths. The Zaire subtype of
Ebola virus had a higher case-fatality, nearly 90%, while the Sudan
subtype had a case-fatality rate of approximately 50%. Before 1995,
the last identified outbreak of Ebola disease in Africa occurred in
1979, when the Sudan subtype of Ebola virus infected 34 persons
(5). In late 1989, in Reston, Virginia, a novel Ebola virus
infected a colony of cynomolgus macaques that had been imported
from the Philippines (6). The new virus, named Reston virus, was
shown by researchers at the Centers for disease Control and
Prevention (CDC) to be antigenically and genetically distinct from
the African Ebola viruses, yet despite its high pathogenicity for
nonhuman primates, it did not appear to cause disease in humans.
Several persons who handled the infected animals developed antibody
to Ebola virus but showed no signs of disease; one of these persons
was infected while performing an autopsy on an animal that had died
of a Reston virus infection. In 1992, a repeat of the 1989 Reston
episode occurred in Siena, Italy when macaques were received from
the same Philippine exporter; no evidence of a human infection was
found (7). The new Ebola virus recently isolated from a patient in
the Cte d'Ivoire has been shown to be genetically distinct from
previous Ebola isolates (A. Sanchez, unpublished data) and is the
first evidence of Ebola virus in West Africa.
Investigations of these outbreaks, as well as of those caused by
Marburg viruses, have yet to produce any substantial evidence for
the natural reservoir(s) of filoviruses. Filoviruses do not persist
in experimentally infected nonhuman primates; therefore, nonhuman
primates are likely not the natural reservoir. Like humans, these
species probably are infected when direct or indirect contact is
made with the natural host.
The recent news of a large Ebola outbreak in Kikwit, Zaire, alarmed
a worldwide audience already sensitized by an array of books,
magazine articles, television programs, and movies dealing with the
danger of Ebola virus disease. The public concern is underscored by
the potential for the spread of these viruses to far regions of the
world as a result of international commerce and jet travel. The
Kikwit outbreak was similar to the original 1976 episode in Zaire,
which was centered around the small village of Yambuku some 1000 km
to the north (8). As in the 1976 outbreak, secondary transmission
of the virus in Kikwit occurred through close personal contact with
infectious blood and other body fluids and was facilitated by the
lack of modern medical facilities and medical supplies that could
protect those giving care to the initially affected patients. The
chief difference between the Yambuku episode and this year's
outbreak is that Kikwit is a large and densely populated center
close to larger cities, such as Kinshasa and Brazzaville, and the
potential for communitywide transmission and spread to neighboring
areas is greater. Retrospective case surveillance suggests that the
index case may have been a charcoal maker that worked in the forest
outside Kikwit. Human to human transmission occurred without being
recognized until the end of April 1995. Ebola hemorrhagic fever was
suspected when nosocomial infections in the surgical teams and the
nursing staff followed repeated laparotomies on an infected
laboratory technician in Kikwit General Hospital. Specimens were
send to CDC through the Tropical Institute of Antwerpen (Belgium).
Teams of experts from CDC, the World Health Organization, Belgium,
France, South Africa, and Sweden traveled to the region to assist
in implementing safe patient care, management, and containment of
the Ebola virus outbreak. As of July 1, 1995, 233 deaths had been
reported among the 293 cases.
Rapid diagnosis and characterization of Ebola virus was performed
at CDC in Atlanta on blood specimens from 14 patients received on
May 9. Nine hours after the specimens had been delivered to CDC,
Ebola virus antigen and/or antibody to this virus was confirmed in
specimens from 13 of the patients. Four hours later, reverse
transcriptase-polymerase chain reaction (RT-PCR) assays targeting
conserved regions of filovirus polymerase or Ebola virus
glycoprotein genes each detected Ebola virus RNA in 12 of the
patients. Subsequent analysis of the genetic profile of the virus
was especially important to understanding the epidemiology of the
Kikwit outbreak. Within 48 hours of receiving the specimens,
sequence analysis on the PCR DNA (528 bp) amplified from the
glycoprotein gene derived from four different patients showed that
the Ebola virus was a Zaire subtype that differed from the original
1976 strain in four bases (<1%). No differences were seen when the
polymerase gene PCR products (~350 bp) from those four patients
were sequenced, which indicated that they had been infected with
the same virus. Three days later, sequence data from expanded
analysis of the entire glycoprotein gene were compared with those
of the original 1976 Yambuku isolate (9) and showed that the
overall difference between these Ebola viruses was less than 1.6%.
Such little change in viruses that caused outbreaks of disease at
extreme ends of Zaire separated by a span of nearly 19 years, may
indicate that the genomes of Ebola viruses (and filoviruses in
general) are unusually stable and have evolved to occupy special
niches in the wild.
The capability to rapidly diagnose and characterize filovirus
infections is critical to the ability of public health
professionals to identify and limit the spread of future outbreaks
of filovirus disease. A continued commitment to research and modern
disease-surveillance programs is necessary to minimize or preclude
filovirus outbreaks similar to that in Kikwit. The possibility of
outbreaks is increasingly likely given the continued human
incursions into the African forests and the vulnerability of large
impoverished populations to rapid transmission of disease as a
result of inadequate public health services. With the current
outbreak under control, CDC and collaborators have begun their
efforts to identify the natural reservoir by sending teams of
scientists to collect specimens from the area where the putative
index patient worked. Attempts to identify the reservoir after
outbreaks in 1976 and 1979 were handicapped by the lack of
satisfactory diagnostic tools that are critical to detecting small
quantities of the virus. However, now that sensitive enzyme
immunoassays and PCR assays have been developed for filoviruses,
the chances are much better that, if appropriate materials can be
collected in the field, the virus can be detected.
In conclusion, we want to alert physicians and public health
agencies who encounter persons that have clinical signs and
symptoms of hemorrhagic fever disease to the reemergence of Ebola
virus. Recommendations for the management of viral hemorrhagic
fevers attributable to filoviruses in the United States were
recently published in CDC's Morbidity and Mortality Weekly Report
(1995;44:475-79).
Anthony Sanchez, Thomas G. Ksiazek, Pierre E. Rollin,
Clarence J. Peters, Stuart T. Nichol, Ali S. Khan, and
Brian W. J. Mahy
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, Georgia, USA
References
1. 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
2.  Centers for Disease Control and Prevention. Outbreak of
Ebola viral hemorrhagic fever Zaire, 1995. MMWR 1995;44:381-2.
3. Centers for Disease Control and Prevention. Update: outbreak
of ebola viral hemorrhagic fever Zaire, 1995. MMWR 1995;44:399.
4. Bowen ETW, Platt GS, Lloyd G, Baskerville A, Harris WJ,
Vella EC. Viral haemorrhagic fever in southern Sudan and northern
Zaire: preliminary studies on the aetiologic agent. Lancet
1977;1:571-3.
5. Baron RC, McCormick JB, Zubeir OA. Ebola virus disease in
southern Sudan: hospital dissemination and intrafamilial spread.
Bull WHO 1983;62:997-1003.
6. Jahrling RB, Geisbert TW, Dalgard DW, et al. Preliminary
report: isolation of Ebola virus from monkeys imported to USA.
Lancet 1990;335:502-5.
7. World Health Organization. Viral haemorrhagic fever in
imported monkeys. Wkly Epidemiol Rec 1992;67:142-3.
8. World Health Organization. Ebola haemorrhagic fever in
Zaire, 1976. Bull WHO 1978;56:271-93.
9. Sanchez A, Kiley MP, Holloway BP, Auperin DD. Sequence
analysis of the Ebola virus genome: organization, genetic elements,
and comparison with the genome of Marburg virus. Virus Res
1993;29:215-40.
Prospects for the Control of Bolivian Hemorrhagic Fever
Bolivian hemorrhagic fever (BHF) was first identified in 1959 as
a sporadic hemorrhagic illness in rural areas of Beni department,
Bolivia. Clusters of BHF patients were noted the same year, and
by 1962 BHF was recognized as a new epidemic infectious disease.
In 1963, Machupo virus (a member of the family Arenaviridae) was
first isolated from patients with acute hemorrhagic fever in San
Joaquin, Bolivia (1). Ecologic investigations established the
rodent Calomys callosus, which is indigenous to the
disease-endemic region of northern Bolivia, as the reservoir for
Machupo virus (2,3).
Machupo virus infection in C. callosus results in asymptomatic
infection with shedding of virus in saliva, urine, and feces; 50%
of experimentally infected C. callosus are chronically viremic
and shed virus in their bodily excretions or secretions (2).
Although the infectious dose of Machupo virus in humans is
unknown, exposed persons may become infected by inhaling virus
shed in aerosolized secretions or excretions of infected rodents,
by eating food contaminated with rodent excreta, or by direct
contact of excreta with abraded skin or oropharyngeal mucous
membranes (4). Reports of person-to-person transmission are
uncommon; however, hospital contact with a patient resulted in
person-to-person spread of Machupo virus to nursing and pathology
laboratory staff (5). In 1994, the fatal secondary infection of
six family members in Magdalena from a single naturally acquired
infection further suggested the potential for person-to-person
transmission (Ksiazek et al., manuscript in preparation).
The pathogenesis of BHF, which resembles that of other South
American hemorrhagic fevers due to Arenavirus infection (e.g.,
Argentine hemorrhagic fever), has been described in clinical and
pathologic investigations of naturally infected patients (6,7).
Experimental infection of rhesus monkeys with Machupo virus
demonstrated an incubation period of 7 to 14 days, which is
consistent with clinical observations in human infection (8).
Early clinical manifestations in humans are characterized by
nonspecific signs and symptoms including fever, headache,
fatigue, myalgia, and arthralgia. Later in the course of disease
(usually within 7 days of onset), patients may develop
hemorrhagic signs, including bleeding from the oral and nasal
mucosa and from the bronchopulmonary, gastrointestinal, and
genitourinary tracts.
During the BHF epidemics in the 1960s, rodent control was
recognized as the primary method for the prevention of Machupo
virus transmission (9). Since C. callosus was frequently found in
domestic and peridomestic environments, rodent control measures
(e.g., trapping, poisoning) resulted in an immediate reduction in
the number of C. callosus and control of BHF outbreaks; an
epidemic in 1964 ended after 2 weeks of continuous trapping for
C. callosus in homes of the affected community (10). Rodent
control programs became a new priority for health officials in
Bolivia, and active interventional programs were carried out for
many years by survivors of past BHF epidemics known to be immune
to Machupo virus (11).
From 1973 to 1992, no cases of BHF were reported, possibly
because of effective control of rodent reservoir populations
(12). Since the late 1960s, no epidemics of BHF have occurred
that involve rural communities, but recent sporadic cases have
been identified in the disease-endemic region (13). Although
patients with BHF have been treated at hospitals outside the
disease-endemic region, these patients had a history of exposure
to Machupo virus in the disease-endemic region or secondary
contact with BHF patients who became infected in the endemic
region. Additionally, no documented cases of BHF have been
exported to other countries.
Concurrently with the lack of identification of BHF patients
during the 1970s and 1980s, the emphasis on conducting rodent
control programs in the BHF-endemic areas also diminished.
Moreover, in recent years, Bolivian health officials have been
faced with numerous other public health problems, including
diarrheal disease, tuberculosis, Chagas' disease, sexually
transmitted diseases, and acquired immunodeficiency syndrome.
Thus, local health authorities are confronted with the challenge
of allocating limited health resources for the control of BHF as
the demand for work with other important diseases increases.
Agricultural activities dominate the economy of northern Bolivia
where many workers are employed in farming and animal husbandry
(14). Farm workers may reside for prolonged periods in rural
areas also inhabited by C. callosus, and farm houses constructed
with partially open walls may allow rodents access to living
areas. Thus, human exposure to infected rodents may occur in and
around farm workers' shelters or during work in the fields and
grasslands of the BHF-endemic region. Given the projected
economic growth in Bolivia, it is likely that agricultural
workers' risk for exposure to C. callosus will continue and even
increase as development modifies the natural habitat of the
rodent reservoir leading to increased contact with humans (e.g.,
focused rodent habitats with increased densities) (15).
Future efforts to control BHF may benefit from recent experience
in neighboring Argentina where ongoing work has led to the
control of Argentine hemorrhagic fever (AHF), caused by Junin
virus, an arenavirus genetically related to Machupo virus.
Extensive study of AHF by Maiztegui, Enria, and colleagues has
provided new insights into the epidemiology, pathogenesis,
treatment, and control of this disease (16,17) and has led to an
effective Candid #1 vaccine against Junin virus as well as phase
2 clinical trials that suggest ribavirin may be effective in
patients with AHF (18,19). The use of an effective vaccine
against AHF and evidence for its cross-protection against Machupo
virus suggest that vaccination may play a role in the prevention
of BHF for persons at highest risk, such as workers who trap
rodents for control programs (20). Intravenous ribavirin has
shown promise for the treatment of clinically diagnosed BHF cases
subsequently confirmed in the laboratory (Kilgore, manuscript in
preparation). Intravenous ribavirin also appeared effective in
the treatment of a laboratory-acquired infection with Sabi
virus, a related Arenavirus first isolated in Brazil (21).
Ribavirin could be administered to patients whose symptoms meet a
clinical case definition with subsequent laboratory confirmation
of Machupo virus infection. Local laboratory handling of
specimens or testing by effective rapid enzyme-linked
immunosorbent assays for antigen and IgM antibodies is ideally
performed under biosafety level 4 containment, but use of
biological safety cabinets and addition to samples of inexpensive
reagents such as Triton X-100, which reduce viral titers, allow
the development of capability for real time testing.
The family cluster of BHF patients and later sporadic cases in
September and October 1994 highlighted the diagnostic challenge
of BHF for clinicians. Even local physicians may rarely evaluate
BHF patients, and other diseases (e.g., malaria, dengue fever,
and yellow fever) that coexist in the BHF-endemic region may
resemble BHF in the early phases of illness. Moreover, no readily
available diagnostic tests exist locally to differentiate BHF
from other diseases (22). Bolivian health care providers and
public health officials recognized the need for education of
health care providers and subsequently established a training
program aimed at increasing clinicians' recognition of BHF
particularly in the disease-endemic region.
The cluster of patients in 1994 also focused public attention on
BHF because the illnesses had higher case-fatality rate than
other diseases in the region where BHF is endemic. The
underrecognition of these illnesses as dangerous and potentially
fatal in disease-endemic communities suggests the need for
increased public health education to reduce virus exposure and
transmission. Proven control measures must be reinforced even in
towns affected by large epidemics 30 years ago where younger
residents have no recollection of the heavy toll exacted by BHF.
Prevention of communitywide epidemics through rodent control
programs may be combined with the application of barrier
precautions (e.g., gloves, masks) in hospitals or clinics to
minimize secondary person-to-person transmission of Machupo
virus. After the familial cluster of BHF in 1994, results of
rodent trapping confirmed the absence of reinfestation in towns
and indicated that the density of rodent reservoirs was not
unusually high in areas of probable exposure for the index
patient. The absence of communitywide epidemics of BHF suggests
that focused rodent control in towns of the disease-endemic
region prevented large urban outbreaks. Prevention of sporadic
illness in farm workers through widespread elimination of
reservoirs may not be feasible, but other measures, such as the
administration of Candid #1 AHF vaccine to workers at high risk,
may offer a more realistic alternative. Finally, agricultural
workers in the disease-endemic region should be taught methods to
reduce exposure to rodent reservoirs, especially around rural
shelters as a means of reducing their risk of exposure to Machupo
virus in the environment.
Paul E. Kilgore, Clarence J. Peters, James N. Mills, Pierre E.
Rollin, Lori Armstrong, Ali S. Khan, and Thomas G. Ksiazek
National Center for Infectious Diseases, Centers for Disease
Control and Prevention, Atlanta, Georgia, USA
References
1. MacKenzie RB, Beye HK, Valverde L, Garron H. Epidemic
hemorrhagic fever in Bolivia: a preliminary report of the
epidemiologic and clinical findings in a new epidemic area in
South America. J Trop Med Hyg 1964;13:620-5.
2. Johnson KM, MacKenzie RB, Webb PA, Kuns ML. Chronic infection
of rodents by Machupo virus. Science 1965;150:1618-9.
3. Johnson KM, Kuns ML, MacKenzie RB, Webb PA, Yunker CE.
Isolation of Machupo virus from wild rodent, Calomys callosus. Am
J Trop Med Hyg 1966;15;103-6.
4. Johnson KM. Epidemiology of  Machupo virus infection: III.
Significance of virological observations in man and animals. Am J
Trop Med Hyg 1965;14:816-8.
5. Peters CJ, Kuehne RW, Mercado RR. Hemorrhagic fever in
Cochabamba, Bolivia, 1971. Am J Epidemiol 1974;99:425-33.
6. Stinebaugh BJ, Scholoeder FX, Johnson KM, MacKenzie RB,
Entwisle G, DeAlba E. Bolivian hemorrhagic fever: a report of
four cases. Am J Med 1966;40:217-30.
7. Child PL, MacKenzie RB, Valverde LR, Johnson KM. Bolivian
hemorrhagic fever: a pathologic description. Arch Pathol Lab Med
1967;83:434-45.
8. Kastello MD, Eddy GA, Kuehne RW. A rhesus monkey model for the
study of Bolivian hemorrhagic fever. J Infect Dis 1976;133:57-62.
9. Kuns ML. Epidemiology of Machupo virus infection: II.
Ecological and control studies of hemorrhagic fever. Am J Trop
Med Hyg 1965;14:813-6.
10. Mackenzie RB, Kuns ML, Webb PA. Possibilities for control of
hemorrhagic fevers in Latin America. Pan American Health
Organization; Scientific Publication No.147:260-265. First
International Conference on Vaccines against Viral and
Rickettsial Diseases of Man, 1966, Washington, D.C..
11. Mercado R. Rodent control programmes in areas affected by
Bolivian hemorrhagic fever. Bull WHO 1975;52:691-6.
12. Pan American Health Organization. Bolivian hemorrhagic fever.
Epidemiolog Bull; 1982;3:15-6.
13. Centers for Disease Control and Prevention. Bolivian
hemorrhagic fever El Beni Department, Bolivia. MMWR
1994;43:943-6.
14. United Nations. Statistical yearbook. 39th issue, department
for economic and social information and policy analysis,
statistical division. New York: United Nations, 1994;201-356.
15. United Nations. Economic and social indicators for latin
american countries, including industrialized/    agricultural
production. Statistical yearbook for Latin American countries and
the Caribbean, 1993. New York: United Nations, 1994:238-41.
16. Peters CJ, Johnson KM. Arenaviridae: lymphocytic
choriomeningitis virus, lassa virus, and other arenaviruses. In:
Mandell GLK, Bennett JE, Dolin R, eds. Principles and practice of
infectious diseases, 4th ed. New York: Churchill Livingston,
Inc., 1995.
17. Enria D, Garcia Franco S, Ambrosio A, Vallejos D, Levis S,
Maiztegui J. Current status of the treatment of Argentine
hemorrhagic fever. Med Microbiol Immunol 1986;175:173-6.
18. World Health Organization. Vaccination against Argentine
hemorrhagic fever. Wkly Epid Rec 1993;68: 233-4.
19. Enria DA, Maiztegui JU. Antiviral treatment of Argentine
hemorrhagic fever. Antiviral Res 1994;23:23-31.
20. Jahrling PB, Trotter RW, Barrero O, et al. Cross-
protection against Machupo virus with Candid 1 Junin virus
vaccine III. In: Kurstak E, ed. Proceedings of the second
international conference on the impact of viral diseases on the
development of Latin American countries and the Caribbean Region.
Mar del Plata, Argentina, 1988.
21. Barry M, Russi M, Armstrong L, et al. Brief report:
occupational exposure to a new arenavirus; Sabi virus clinical
course, treatment and biosafety management. N Engl J Med (in
press).
22. Webb PA, Maiztegui JI. Argentine and Bolivian hemorrhagic
fevers (South American hemorrhagic fevers). In: Gear JHS, ed.
Handbook of viral and rickettsial hemorrhagic fevers. Boca Raton,
FL: CRC Press, Inc., 1988.
NEWS AND NOTES
Conference on "Emerging Infectious Diseases: Meeting the Challenge"
Emerging infectious diseases, the leading cause of death worldwide,
continue to pose difficult challenges to clinicians, public health
professionals, and biomedical researchers in academic settings and
industry. Addressing these challenges requires a cohesive effort to
develop prevention strategies and to communicate them effectively
to the health care community, the public, and policy makers.
On June 5-6, 1995, the New York Academy of Medicine and the
New York State Department of Health convened to examine the
problem of emerging infections. The speakers addressed four
themes:  1) emerging infectious diseases: why and why now?
2) transmission of emerging infectious diseases: old modes,
new agents; 3) surveillance and sentinel systems for
infectious diseases; and 4) emerging infectious diseases:
what is to be done?
The first three themes were addressed through presentations by 20
experts. The fourth was divided into six segments focusing on
diagnosis, the role of the microbiology laboratory in surveillance,
other surveillance issues, approaches to epidemic investigations,
risk perception, and global issues.
Speakers consistently alluded to recent complacency about
infectious diseases in the United States and stressed the need for
the clinical, public health, and research communities to work with
the biomedical industry in confronting emerging infectious disease
challenges in this era of transition to managed health care. In his
opening address Joshua Lederberg from Rockefeller University
reminded participants that the struggle between humans and microbes
could be characterized as a battle of "wits versus genes." Margaret
Hamburg, Commissioner of the New York City Department of Health,
emphasized that plague in India and Ebola virus infection in Zaire
were reminders that the world is a global village, that considering
domestic and international diseases as separate entities is an
outmoded concept, and that many conditions that contribute to
disease emergence or reemergence in the developing world are also
present in the United States, adding to our domestic vulnerability
to emerging infections.
Other speakers focused on the evolution of virulence, the molecular
basis of pathogenesis, observations on factors contributing to the
plague epidemic in India in 1994 and the Ebola outbreak in Zaire in
1976; foodborne and waterborne diseases; airborne diseases;
zoonoses; sexually transmitted and blood-borne diseases and the
increasing problem of antimicrobial resistance in both hospital and
community settings. Concerns were expressed about the possibility
of a "post-antimicrobial era" in which available drugs are no
longer effective against common bacterial infections. Other
speakers focused on innovative approaches to surveillance at the
local, state, national, and international levels. James LeDuc from
the World Health Organization (WHO) provided an update on the
emerging infections resolution passed by the World Health Assembly
in May 1995 and other WHO activities related to detecting and
responding to emerging and reemerging diseases.
Among the themes recurring throughout the conference were the
challenges that microbes will continue to pose; the critical role
of the modern microbiology laboratory in detecting and responding
to emerging and reemerging infections; and the limitations of
existing capacity at the local, state, national, and international
level to respond to these challenges. Human resource, equipment,
diagnostic reagent, and facility needs were addressed, and resource
needs were emphasized. Training needs of medical students,
clinicians, epidemiologists, microbiologists, entomologists,
mammalogists, behavioral scientists, and other researchers were
also stressed. Additional emphasis was placed on the critical
importance of communicating alerts about clusters of illness, data
on disease trends, and guidelines for disease prevention; the need
for educating professionals, the public, and policy makers about
the critical importance of these issues; the need for strengthening
existing partnerships and developing new ones, particularly with
health maintenance organizations, the pharmaceutical industry, and
nongovernmental organizations (including medical missionary
organizations); and the need to carefully identify priorities.
Conference participants resounded the message of the 1992 Institute
of Medicine Report, Emerging Infections: Microbial Threats to
Health in the United States, "Pathogenic microbes can be resilient,
dangerous foes. Although it is impossible to predict their
individual emergence in time and place, we can be confident that
new microbial diseases will emerge."  Particular future concerns
included a possible influenza pandemic, the emergence of vancomycin
resistance in Staphylococcus aureus, the occurrence of large dengue
hemorrhagic fever epidemics in the Western Hemisphere, and the
likelihood that additional chronic diseases will be found to have
infectious etiologies. Concerns were also expressed about the
possibility of a terrorist incident involving an infectious agent
and the potential difficulties in detecting and responding to such
an episode.
The New York Academy of Medicine plans to use the discussions
during the conference in formulating an agenda for further action.
James M. Hughes
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, Georgia, USA
Japanese Encephalitis Acquired in Australia
Japanese encephalitis (JE), a mosquito-borne flaviviral disease
of humans and animals, is a major public health problem in Asia,
where an estimated 50,000 cases occur each year. There has been
concern that the range of epidemic JE may be expanding.
On April 5, 1995, an outbreak of three cases of JE was recognized
in Australia. Two of the cases were fatal; all were among
residents of an island in Australia's Torres Strait, which lies
between mainland Queensland and Papua New Guinea. JE was
confirmed in two of the patients by polymerase chain reaction
(Jeffrey Hanna, Queensland Health, pers. comm.). No other cases
were reported. This is the first recognized episode of JE
acquired in Australia.
Control activities on the Australian island began on April 7. The
community was informed about the importance of personal mosquito
protection measures. In addition, larvicides were applied, and
areas were fogged to kill adult mosquitoes.
The patients were all male, aged 6 to 44 years. All were
hospitalized with symptoms that included fever (up to 40C),
stiff or painful neck, headache, and abdominal pain. Two patients
were unconscious at the time of admission.
Acute-phase sera showed elevated JE virus immunoglobulin M (IgM)
titers. Two of the patients also had detectable levels of Kunjin
and Murray Valley encephalitis virus IgM, but the JE IgM titers
were significantly higher in each case.
Flaviviruses have also been isolated from the sera of each of two
asymptomatic island residents. Preliminary tests suggest that
these are both JE virus. Blood taken from 10 horses and 12
domestic pigs living near humans on the island was also tested.
All 12 pigs and 9 of the horses had high JE titers by
hemagglutination inhibition assay. Neutralizing antibody to JE
virus was detectable in all the pigs and in four of the horses
tested to date.
Details of the index case are as follows: The patient, a
16-year-old male, was admitted to Thursday Island Hospital on
March 22, 1995. He was unconscious and was responsive only to
painful stimuli. His neck was stiff, and he showed a preference
for moving his right side. His illness had begun 3 days before.
The day before admission he complained of abdominal pain. This
patient had been mildly mentally retarded since birth and
occasionally had generalized seizures but was generally healthy.
He was transferred to Cairns Base Hospital, where a cerebral CT
scan showed a nonenhancing hypodense lesion in his posterior
right basal ganglia.
He had a leukocytosis of 17.3 x 109L, neutrophils, 15.2 x 109.
His cerebrospinal fluid contained 150 leukocytes/l with a
differential count of 50% polymorphs and 50% mononuclear cells.
He had a generalized seizure and 2 days after admission, required
mechanical ventilation. He never regained consciousness and died
on day 17 of hospitalization (April 8).
Adapted from Hanna J, Ritchie S, Loewenthal M, et al. Probable
Japanese encephalitis acquired in the Torres Strait. Communicable
Diseases Intelligence 1995;19:206-7.
USPHS and IDSA Collaborate on Guidelines to Prevent Opportunistic
Infections in HIV-Infected Persons
U.S. Public Health Service (USPHS)/Infectious Diseases Society of
America (IDSA) Guidelines for Preventing Opportunistic Infections
in HIV-Infected Persons will be published in an August 1995
supplement of Clinical Infectious Diseases. The guidelines, which
are intended for health care providers, are the result of
collaboration between the Centers for Disease Control and
Prevention (CDC), the National Institutes of Health, IDSA, numerous
federal and nonfederal organizations, community groups, and
HIV-infected persons. The guidelines are endorsed by the American
Academy of Pediatrics, the Infectious Diseases Society of
Obstetrics and Gynecology, and the Society of Healthcare
Epidemiologists of America. Jonathan E. Kaplan, M.D. (CDC), Henry
Masur, M.D. (NIH), and King Holmes, M.D., Ph.D. (University of
Washington), chaired the USPHS/IDSA Prevention of Opportunistic
Infections Working Group and are guest editors of the Clinical
Infectious Diseases supplement.
CDC initiated work on the guidelines in early 1994; meetings were
held in Atlanta in June and September to discuss and refine the
recommendations.
The USPHS/IDSA guidelines address 17 opportunistic infections from
three angles: 1) preventing exposure to opportunistic pathogens
(e.g., sexual, occupational, and environmental exposure as well as
exposure through pets, food, water, and international travel); 2)
preventing opportunistic disease by chemoprophylaxis and
vaccination; and 3) preventing disease recurrence. In this
document, new recommendations were made and earlier recommenda-
tions were updated. For example, new guidelines recommend that in
nonemergency situations, cyto-megalovirus (CMV)-seronegative
HIV-infected persons who require blood transfusions receive only
CMV-antibody-negative or leukocyte-reduced cellular blood products.
The guidelines also recommend that Toxoplasma-seropositive
HIV-infected persons who have a CD4+ lymphocyte count <100 cells/L
received chemoprophylaxis against toxoplasmosis (such
chemoprophylaxis is generally accomplished with anti-Pneumocystis
carinii medication). Earlier recommendations for chemoprophylaxis
against Pneumocystis carinii pneumonia and Mycobacterium avium
complex disease have also been updated.
In addition to disease-specific recommendations, the guidelines
include an overview article designed to prioritize the
recommendations for health care providers. This article provides an
approach to the initial and follow-up evaluations of the
HIV-infected patient and also contains sections on HIV-infected
pregnant women and HIV-exposed/infected children. The guidelines
are followed by 15 background articles, which provide the
information on which the recommendations were based and include
research priorities generated by the development of the prevention
recommendations.
The guidelines conclude with quality standards and implementation
steps on the most standard-of-care recommendations, such as
chemoprophylaxis against Pneumocystis carinii pneumonia. This final
section provides a mechanism by which health care facilities can
assess their degree of compliance with the recommendations, so that
they can detect and correct compliance-related problems.
An abbreviated version of the USPHS/IDSA Guidelines will be
published in CDC's Morbidity and Mortality Weekly Report in July.
Jonathan E. Kaplan
Centers for Disease Control and Prevention
Atlanta, Georgia, USA
Henry Masur
National Institutes of Health
Bethesda, Maryland, USA
King K. Holmes
University of Washington
Seattle, Washington, USA
Recommendations for a Regional Strategy for the Prevention and
Control of Emerging Infectious Diseases in the Americas
On June 14-15, 1995, a conference on "Combating Emerging Infectious
diseases: Challenges for the Americas" was held at the Pan American
Health Organization (PAHO) Headquarters in Washington, D.C. The
meeting was designed to shape a regional strategy for preventing
and controlling emerging infectious diseases that could pose
serious threats to the peoples of the Americas.
Participants, convened by PAHO, included top officials and
infectious disease experts from that organization as well as the
World Health Organization, the U.S. Centers for Disease Control and
Prevention, the Canadian Laboratory Center for Disease Control, the
U.S. Department of Defense, and several Latin American and
Caribbean countries.
This international group of experts noted that an increasing number
of new, emerging, and reemerging infectious diseases have been
identified in both developed and developing nations and that these
diseases threaten to increase in the near future. They include
human immunodeficiency virus/acquired immunodeficiency syndrome,
which emerged in the l980s and now affects some 16 million people
worldwide; and cholera, which returned to the Western Hemisphere
for the first time this century in 1991 and has caused more than 1
million cases and 9,000 deaths in the Americas. PAHO estimates that
it will take more than a decade and over $200 billion to control
the current pandemic of this disease.
The experts concluded that both early warnings of, and rapid
responses to, infectious disease threats are needed. The group made
several major recommendations to PAHO and its member states to
improve surveillance, research, and communications in developing
countries. They also issued more detailed recommendations in the
areas of antimicrobial resistance, outbreak control, and
information and communication. In addition, a plan of action is
forthcoming.
The group made the following recommendations for PAHO and its
member countries:
General Recommendations
Develop and frequently update prioritized disease-specific
guidelines for the prevention and control of diseases that are
emerging or reemerging, both at the public health and individual
levels. This should include biologic and behavioral change measures
and will require groups of experts for each disease as well as
communications experts. Diseases of interest include yellow fever,
dengue, antimicrobial-resistant organisms (malaria, tuberculosis,
and enteric diseases), measles, polio, cholera and other foodborne
and waterborne diseases, viral hemorrhagic fevers, plague, rabies
and other zoonoses, and trypanosomiasis and other vector-borne
diseases.
Identify points of contact in the field to receive and transmit
information in countries. These contacts should include
organizations and individuals outside the government.
Develop plans to distribute accurate and timely information to the
general public.
Develop plans to improve and make more efficient two-way
communication on reporting, control, and modification measures.
This may require contracting information management specialists to
identify and implement the most efficient means.
Make efficient use of the press, including radio, television and
newspapers, fliers, and other methods to educate the public and the
medical community, with an eye toward social mobilization of
communities to fight emerging diseases. This will require expertise
in communications and support to the countries in developing
information dissemination plans. Countries should define
populations at greatest risk and focus the information and control
measures in these populations.
Define different approaches for educating the public and the
medical community.
Focus efforts on intersectorial action, including education of
policy makers outside the health community.
Antimicrobial Resistance
The expert group recommended that both PAHO and its member
countries, where applicable, do the following:
Seek ways to reduce availability of over-the-counter antimicrobial
agents, including those used in veterinary medicine; this will
require efforts beyond the health care community and involve
education and dissemination of information to all sectors.
Intensify assistance to the countries in developing rational drug
policies.
Monitor sensitivity to antibiotics in each country to allow for
optimum antibiotic use for individual cases and to eliminate
antibiotics with little therapeutic value. Employ mechanisms such
as WHONET and PHLIS to centralize, analyze, and distribute
antimicrobial sensitivity data.
Develop and distribute specific recommendations to extend the
useful life of antimicrobial drugs.
Frequently revise the list of essential antimicrobials based on
sensitivity data.
Initiate educational campaigns on the cost-effectiveness of
rational drug use in hospitals.
Initiate collaboration with the pharmaceutical industry on rational
drug use, standardized labels and warnings, and ethical marketing
strategies.
Outbreak Control
The expert group endorsed the leadership role of PAHO in developing
and disseminating guidelines for outbreak evaluation and control
and recommended that PAHO
Make timely recommendations to coordinate response to outbreaks or
threats, including issues related to travel advice, quarantine, and
commerce.
Develop policies and standard operating plans for response to
outbreaks at the regional and country levels. Assist countries in
developing national outbreak response plans and assist in training
teams.
Identify and list individuals and groups with disease-specific
expertise, laboratories with disease-specific diagnostic
capabilities, and products, including diagnostic reagents, drugs,
and vaccines (both licensed and investigational products).
Frequently update these lists.
Establish a standard system for rapid procurement of vaccines,
reagents, insecticides and antimicrobial drugs for prompt response
to outbreaks.
Establish information management and dissemination procedures for
use during outbreaks, including accurate and frequent release of
information to the press and public.
Conduct formal evaluations of responses to each outbreak and use
the lessons learned to improve responses to subsequent outbreaks.
Information and Communication
The experts recommended communicating with high-level government
officials and emphasizing to  them the importance of a basic public
health infrastructure including improvements in water, sanitation,
and social and economic conditions in preventing diseases. The
group suggested disseminating more information about public health
implications of development (such as deforestation, dam
construction, urbanization, and other measures) and seeking
effective interaction with other sectors.
Other Recommendations
PAHO should
Create interagency task forces for emerging diseases at regional
and country levels.
Inform regional governments, other organizations, and the public
about the emerging disease initiative and strive for the highest
level of political support.
Solicit and allocate specific resources to deal with the emerging
diseases initiative, both at the regional and country levels. A
portion of these funds should be immediately available when
outbreaks are recognized.
For more information on these recommendations, the conference, or
its plan of action, contact PAHO.
Daniel B. Epstein
Office of Information & Public Affairs
Pan American Health Organization
Washington, D.C., USA
Emerging Infectious Diseases Laboratory Fellowship Program
A partnership has been established between the Association of
State and Territorial Public Health Laboratory Directors and the
Centers for Disease Control and Prevention (CDC) to develop and
initiate an emerging infectious diseases laboratory fellowship
program in January 1996. A goal of this fellowship program is to
strengthen local, state, and federal public health
infrastructures to support surveillance and implement prevention
and control programs. The fellowship program will help recruit
and train microbiologists for laboratories nationwide and provide
opportunities for doctoral level scientists to conduct
high-priority infectious disease research.
The emerging infectious diseases fellowship program will offer a
2-year laboratory research track for doctoral level scientists,
with emphasis on applied research or development in infectious
diseases and a 1-year advanced laboratory training track for
bachelor's and master's level scientists, with emphasis on the
practical application of emerging infectious diseases
technologies, methods, and practices. Fellow training and
research will take place at CDC and state and local public health
laboratories.
For applications or additional information, contact
Emerging Infectious Diseases Fellowship Program
Association of State and Territorial Public Health Laboratory
Directors
1211 Connecticut Avenue, Suite 608
Washington, D.C. 20036
Phone: 202-822-5227, Fax: 202-887-5098
Tenth Annual ASTPHLD Conference on Human Retrovirus Testing
The Tenth Annual Conference on Human Retrovirus Testing,
sponsored by the Association of State and Territorial Public
Health Laboratory Directors (ASTPHLD), was held March 6 to 9,
1995, in Reno, Nevada. The conference, which was attended by more
than 300 representatives of public and private sector
laboratories as well as test kit manufacturers, emphasized three
themes: new human immunodeficiency virus (HIV) variants,
international issues, and HIV testing of newborns. The topics
discussed included sequence data for type O isolates, the search
for new HIV variants, zidovudine (AZT) resistance, decreased
maternal-neonatal transmission due to AZT prophylaxis, results of
the national anonymous survey of HIV prevalence in the United
States, and the ethical concerns of perinatal screening.
An international perspective on HIV testing was brought to the
conference by presentations that focused on India and Latin
America. Results were given of a project, funded by a 12-month
study grant from the World AIDS Foundation, to provide training
on HIV testing to laboratories in India. Four Indian facilitators
were trained in the United States; they provided translation and
other assistance to eight ASTPHLD faculty, who gave workshops in
four training centers in India. This training, which focused on
enzyme immunoassay, linked trainees with staff from Indian
reference centers and established training materials and trainers
for future workshops to be conducted by Indian staff.
Laboratory aspects of HIV testing in Latin American and the
Caribbean were also discussed by a member of the Pan American
Health Organization (PAHO), who described the spectrum of HIV
incidence rates and testing algorithms. PAHO is asking countries
of the region to assess their algorithms in terms of sensitivity,
specificity, and cost. PAHO aims to support national laboratories
by providing guidelines and quality assurance. Proficiency
testing, which is encouraged, will be provided by the Centers for
Disease Control and Prevention.
ASTPHLD's 11th Annual Human Retrovirus Conference is set for
March 6-8, 1996, in Orlando, Florida. Requests for additional
information are available; FAX request to 202-887-5098.
James L. Pearson
Division of Consolidated Laboratory Services,
Commonwealth of Virginia, Richmond, Virginia, USA