Dispatches

An Outbreak of Spotted Fever Rickettsiosis in U.S. Army Troops Deployed to
Botswana

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

Novel infectious diseases are recognized when nonimmune persons move into
the ecologic niche of a pathogen and become inadvertent hosts. One
population at frequent risk to such emerging pathogens is the military.
Historical examples of this phenomenon include outbreaks of scrub typhus
during World War II and Korean hemorrhagic fever during the Korean War (1).
In this report, we describe a modern example as we document an unusually
high attack rate of a rickettsial disease among U.S. troops camping for a
brief period in Africa.

In January 1992, U.S. soldiers participated in a 10-day training exercise
with the Botswana Defense Force near Shoshong, Botswana. Activities
included individual weapons training and a series of mock battles. The
terrain was generally flat with scattered bushes and small trees. The
climate was semiarid, and temperatures reached 120F during the day. During
the field exercise, one soldier sought medical assistance to remove small
insects crawling rapidly on his body. No specimens were reliably
identified in the field or preserved for subsequent examination. No unusual
illnesses were reported to medical personnel during the training exercise.
After the field-training exercises, the soldiers spent 2 days in Gaborone,
the capital of Botswana. Within 2 days of their return to their home
station, approximately 30% of the deployed soldiers sought medical
attention with symptoms of fever, headache, and regional lymphadenitis.
Several soldiers reported insect bites. An epidemiologic team from Walter
Reed Army Institute of Research was sent to Botswana to assist with the
outbreak investigation.

From airplane manifests, 169 soldiers were identified as having been
deployed to Botswana. One week after the soldiers returned to their home
station, a questionnaire requesting information on symptoms, personal
protective measures taken against arthropod vectors, reservoir exposures,
and other potential risk factors for infection was administered to all
available soldiers (n = 140); 132 soldiers underwent directed physical
examinations, in which skin lesions, lymphadenopathy, and right upper
quadrant tenderness were noted. Clothing and equipment used in the field
that had not been cleaned since the exercise were visually examined in
detail by a team of entomologists. Blood samples from 126 soldiers were
obtained at 1 week and 10 weeks postdeployment. Serum specimens were
divided into aliquots and frozen at -20C for laboratory analyses.

Sera were tested for antibodies to rickettsiae by using an indirect
fluorescence antibody technique (IFA) (2). Sera drawn at 10 weeks
postdeployment were initially screened for whole immunoglobulin reactivity
against Rickettsia conorii Moroccan strain, a member of the spotted fever
group of rickettsiae that cause boutonneuse fever. Subsequently, sera
showing titers >=1:64 were paired with their respective 1-week samples and
titered to the endpoint, defined as the highest dilution showing
discernible fluorescing organisms. The rickettsial species used as antigens
for antibody titration included cell culturepropagated R. conorii
(Moroccan, ATCC VR141), Rickettsia akari Hartford, and Rickettsia typhi
(Wilmington, ATCC VR144). The latter two microorganisms are the etiologic
agents of rickettsial pox and murine typhus, respectively. Slides were read
by one technician who was blinded to the clinical history corresponding to
the sample.

A case-patient was defined as a soldier who had either a fourfold or
greater rise in immunoglobulin G (IgG) titer to R. conorii within 9 weeks
to a titer >=1:128 by IFA, or clinically, by the presence of a tache noire
(eschar) and at least two of the following symptoms: regional
lymphadenitis, fever, chills, severe headache, and muscle or joint pains.
By this case definition, the overall attack rate was 23% (39 of 169
soldiers). Of 36 case-patients who provided paired serum samples, 24 (67%)
seroconverted during the postdeployment period. All 24 case-patients who
seroconverted were symptomatic, but six did not have an eschar. Of the 15
case-patients without documented fourfold rises in titer, all had at least
one eschar and regional lymphadenitis observed by a physician. Three of the
soldiers who did not seroconvert, but were identified as case-patients by
the clinical definition, had high standing titers, >=1:256.

One week after returning from Botswana, the case-patients (n = 36) had a
geometric mean reciprocal (GMT) IgG titer to R. conorii of 43, which
increased to 185  9 weeks later (Student's t test paired, p < 0.0001). Over
the same period, the GMT of non-case-patients (n = 38) who had positive
titers on the initial whole immunoglobulin screening remained the same at
38. To determine whether early treatment impaired antibody production, the
case-patients were divided into two groups: those who received treatment
within 2 days of the start of their symptoms (n = 12) and those who
received treatment 3 or more days later (n = 21). The group that was
treated early had a lower mean GMT at follow-up, but the difference was not
statistically significant (early 136, 95% confidence interval [C.I.]: 77,
239: late 197, 95% C.I.: 114, 338).

A Western blot immunoassay was used (3). Rickettsial antigens (R. typhi
Wilmington, R. akari, Rickettsia africae F (4,5), R. conorii Malish (ATCC
VR613), and Israeli tick typhus rickettsiae (T487)) used in the
immunoblots, either boiled or solubilized at room temperature, were
electrophoresed on an 8% to 16% gradient PAGE gel. Renografin density
gradient purified antigen was applied at 20 g protein per lane. R. typhi
was yolk sacpropagated, whereas spotted fever group antigens were grown in
irradiated L cells. Patients' serum specimens were reacted with the
electrophoresed antigens at 1:250 dilution. Horseradish peroxidaselabeled
oat antibodies against human IgG (gamma-chain-specific) and IgM (
chain-specific) (Calbiochem, San Diego, CA) were used at 1:1000 dilution,
and bound antibody was detected with 4-Cl-1-naphthol-H2O2 reagent.
Prestained SDS-PAGE molecular weight standards (Bio-Rad, Melville, NY) were
used to estimate antigen sizes

Western blots were used to characterize the serologic reactivities of 38
case-patients and 37 noncase-patients. Serum specimens were considered
positive for reactivity with spotted fever rickettsial antigens if a
characteristic broad washboard of spotted fever group lipopolysaccharide
reactivity (SFG LPS) was observed in the 20- to 40-kDa region with anti-IgG
conjugate (Figure 1) [Figures not available in ASCII]. 
Most of these serum specimens also exhibited
pronounced IgM reactivity, which sometimes included R. typhi LPS. Serologic
specificity toward the individual spotted fever species was determined by
the relative IgG reactivity to species-specific antigens (SPAs) solubilized
at room temperature found at 125 to 145 kDa. Thirty-two of the 38
case-patient specimens tested by Western blot were positive for spotted
fever group IgG LPS.

On Western blot, the sera of 23 case-patients reacted most strongly with R.
africae SPA (not shown), one with R. conorii SPA, and eight showed no
specificity (Figure 1)[Figures not available in ASCII]. Nine of 37 soldiers 
who did not meet our case
definition were positive for spotted fever group IgG LPS. The specimens of
two reacted primarily with R. africae SPA, and specimens from the remaining
seven had nonspecific patterns. Five of these nine soldiers reported no
symptoms. Two soldiers reported a runny nose, another reported a rash on
the dorsal portion of the right upper arm, and another had a tender raised
papule. By Western blot criteria, these nine soldiers would be considered
infected and may represent asymptomatic cases. However, they did not meet
our case definition and were not counted as case-patients in our analyses.

Patients' sera showed expected broad cross-reactivity with other spotted
fever group rickettsiae by IFA. In sera collected at 10 weeks, 17 (51%) of
the case-patients had identical R. akari and R. conorii titers; a
difference of one dilution in titers was observed in 12 (36%) of the
case-patients. The remaining cases had R. akari titers that were fourfold
less than their R. conorii titer. However, no Western blots were positive
for R. akari. All but two 10-week serum samples from the case-patients were
nonreactive at a titer of <1:32 with R. typhi antigen by IFA, and none were
positive by Western blot. Serum from one case-patient had titers of 1:128
to R. akari, R. typhi, and R. conorii. Serum from the second case-patient
had titers of 1:512 to R. conorii and 1:64 to R. typhi.

Median ages of the case-patients and noncase-patients were 23.5 and 22
years, respectively. Although a larger proportion of cases was found among
the older age groups, this trend was not significant (chi-squared trend, p =
0.28). Most of the soldiers (77%) were non-Hispanic white. There were too
few soldiers in other race-ethnic groups to make separate comparisons, but
when those from other ethnic backgrounds were combined into one group, the
group had an attack rate (22%) similar to that of the non-Hispanic white
soldiers (24%).

The illness observed in the soldiers was mild to moderately severe.
Although all cases were symptomatic, some came to medical attention only
through active case finding. Many case-patients had systemic complaints,
commonly described as flu-like. Each of the classic symptoms associated
with rickettsial diseases (i.e., fever, chills, headache, myalgia, and
arthralgia) was reported by at least 59% of the case-patients but by fewer
than 10% of the noncase-patients (Table 1). Gastrointestinal complaints of
stomach pain, anorexia, nausea, or vomiting were also reported more
frequently by the case-patients than by the noncase-patients. In contrast,
diarrhea and upper respiratory symptoms were reported at similar rates
among case-patients and noncase-patients. On physical examination,
lympadenitis was observed in 36 (92%) and right upper quadrant tenderness
in 17 (44%) of the case-patients. The skin lesions began as a tender
erythematous papule, which progressed over 24 to 36 hours to form one to
three clear vesicles. The fragile vesicles ruptured, exposing an
erythematous base, which darkened into a small eschar. The total duration
of the evolution was 72 to 96 hours; 34 (87%) of case-patients had at least
one eschar, and 10 of the case-patients had more than one. Of all eschars
reported during physical examinations, 50% were located on the trunk;
others were located on the head, neck, extremities, penis, and scrotum. No
soldiers were hospitalized, and all case-patients responded rapidly to
antibiotic treatment.

   Table 1. Symptoms of case-patients and noncase-patients during a
                        spotted fever outbreak.
----------------------------------------------------------------------
                 Casepatients; Noncase-patients; Risk 95% Confidence
 Symptoms            n=32(%)         n=107(%)     ratio    interval
----------------------------------------------------------------------
 Fever               19 (59)          8 (7)        6.06   3.44,10.68
 Chills              25 (78)          6 (6)       12.44   5.96,26.00
 Headache            23 (72)          8 (7)        8.90   4.61,17.21
 Muscle ache         26 (81)          10 (9)      12.40   5.56,27.66
 Joint pain          21 (66)          9 (8)        6.94   3.78,12.73
 Fatigue             27 (84)          6 (6)       17.35   7.26,41.43
 Lymphadenitis       27 (84)          3 (3)       19.62   8.27,46.57
 Abdominal pain      9 (28)           4 (4)        3.79    2.26,6.36
 Anorexia            11 (34)          3 (3)        4.68    2.90,7.53
 Nausea/vomiting     13 (41)          3 (3)        5.26    3.27,8.46
 Diarrhea            7 (22)          21 (20)       1.11    0.54,2.30
 Cough               4 (12)           9 (8)        1.38    0.58,3.33
 Nasal congestion    7 (22)          17 (16)       1.34    0.66,2.74
 Sore throat         6 (19)          16 (15)       1.23    0.57,2.63
----------------------------------------------------------------------

An incubation period of up to 6 days was estimated from the histories of
the first two case-patients (Figure 2) [Figures not available in ASCII]. 
The first case-patient reported
onset of symptoms on January 20, 6 days after he arrived in Botswana as a
member of an advance team. The second case-patient arrived in Botswana on
January 18 and had onset of symptoms 6 days later. Thus, assuming a
Botswana exposure, the time from infection to symptoms in these two
case-patients could not have exceeded 6 days, an incubation time consistent
with the 5- to 7-day period commonly reported for African spotted fever
rickettsioses. Finally, since most case-patients reported symptoms during
the 3 days before or the 4 days after leaving Botswana, we concluded that
the period of high risk exposures occurred during field training in
Shoshong and not in Gaborone.

No significant differences were found between case-patients and others in
reported exposure to vectors, such as mosquitoes (68% vs. 62%; chi-squared, p =
0.53) and chiggers (19% vs 10%; chi-squared, p = 0.14). However, case-patients
(68%) were more likely than noncase-patients (40%) to report tick bites (
chi-squared, p = 0.007). Only one soldier reported finding an engorged tick on
his body. Fewer than 50% of all deployed soldiers reported using a
DEET-containing insect repellent at any time during the exercise, and fewer
than 10% of the soldiers reported using permethrin (anacaricide and
repellent applied to clothing). No statistical differences were found
between case-patients and noncase patients in reported uses of these
personal protective measures. Self-reported exposures of case-patients (n =
31) vs non-case-patients (n = 101) to potential reservoirs of R. conorii
(dogs: 52% vs 56%; rodents: 10% vs 35%; antelopes: 3% vs 7%; cattle: 81% vs
87%; horses:16% vs 25%; sheep: 42% vs 38%; and goats: 90% vs 87%) were not
statistically different.

More than 85% (n = 117) of the soldiers slept on sleeping pads placed
directly on the ground. Few (n = 18) soldiers reported sleeping on cots,
litters, hammocks, or pallets. The attack rate was approximately twice as
high among men sleeping on the ground (25%) as among those who did not
(11%). Because of the small number who slept off the ground, this
difference, was not statistically significant (risk ratio = 2.23; 95% C.I.:
0.58, 8.56). The attack rate among soldiers who maneuvered in the field
during daily training exercises was lower than the rate among soldiers who
remained at base camp (24% vs. 33%, respectively), but the difference was
also not statistically significant (odds ratio = 1.40, 95% C.I.: 0.77,
2.56).

This report documents the occurrence of a large focal outbreak of a spotted
fever group rickettsiosis among American soldiers participating in a short
field-training exercise in Botswana. Little is known about the epidemiology
of rickettsial diseases in Botswana. However, during the civil unrest that
occurred in neighboring Rhodesia (now Zimbabwe) in the late 1970s, several
thousand cases of tick typhus were reported among military personnel. In
Zimbabwe, ticks removed from 100 soldiers were all larvae of Amblyomma
hebreum (6). In another study, 33% to 75% of A. hebraeum ticks, collected
from animals or vegetation in four regions of Zimbabwe, were heavily
infested with rickettsia-like organisms (7). Only the recently
characterized new rickettsia R. africae, but not R. conorii, the etiologic
agent of boutonneuse fever, was detected in these ticks (4,7). In contrast,
the dog ticks Rhipicephalus simus and Haemaphysalis leachi were less
heavily infected (0% to 21%) and were only infected with R. conorii. R.
africae has also been isolated from a hospitalized patient with fever,
severe headache, and regional lymphadenopathy but no rash other than an
erythematous tick bite site (8).

On the other hand, in his original 1934 clinical description of African
tick bite fever, Pijper (9) described a patient with a rash and eschar,
whose condition he carefully distinguished from boutonneuse fever. He
clearly associated this milder disease with the hardly visible swarming
larval veld ticks (little pests) and not dog ticks. He also noted that
even if the disease was not severe, it needed to be recognized by
physicians and not confused with other potentially more devastating
illnesses requiring more aggressive therapy.

Recent surveys of human antibodies in eight African countries have
suggested a seroprevalence of antibodies to spotted fever rickettsiae of 0%
to 52%, which parallels the distribution of Amblyomma ticks (10,11). The
etiologic agent appeared to be R. africae, based on Western blot analysis
(10). IFA analysis has been found inadequate in distinguishing human
infections with R. africae and R. conorii (11).

The collective observations parallel our clinical, epidemiologic, and
serologic findings on the Botswana outbreak and are most consistent with
disease caused by R. africae. R. africae is closely related to R. conorii
but has been proposed as a new species (5). Whether we consider this a
newly emerging rickettsiosis or, in deference to the pioneering work of
Pijper, a disease reemerging into our consciousness, African tick bite
fever caused by R. africae may be epidemiologically distinct from urban R.
conorii infections.

We have established the potential for high attack rates of this spotted
fever rickettsiosis, but much more needs to be understood about the
clinical severity of this disease and its potential for asymptomatic
infections. Physically fit, healthy, young soldiers may not represent a
particularly good model for assessing the severity of disease in
populations residing in areas where the disease is endemic.

Bonnie L. Smoak, M.D., Ph.D.*; J. Bruce McClain, M.D.; John F. Brundage,
M.D.*; Laurel Broadhurst, M.D.*; Daryl J. Kelly, Ph.D.; Gregory A. Dasch,
Ph.D.; and Richard N. Miller, M.D.*

*Divisions of Preventive Medicine and Communicable Diseases and
Immunology, Walter Reed Army Institute of Research, Washington, D.C., USA;
U.S. Army MEDDAC, Vicenza, Italy; Viral and Rickettsial Diseases Program,
U.S. Naval Medical Research Institute, Bethesda, Maryland, USA

Acknowledgment

This investigation was supported in part by the Naval Medical Research and
Development Command, Research Task No. 61102A.010BJX.1293.

References

  1. Institute of Medicine. Emerging infections: microbial threats to
     health in the United States. Washington, DC: National Academy Press,
     1992:110-2.
  2. Philip RN, Casper EA, Burgdorfer W, et al.. Serologic typing of
     rickettsiae of the spotted fever group by microimmunofluorescence. J
     Clin Microbiol 1978;3:51-61.
  3. Raoult D, Dasch GA. Line and Western blot immunoassays for diagnosis
     of Mediterranean spotted fever. J Clin Microbiol 1989;27:2073-9.
  4. Kelly PJ, Beati L, Matthewman LA, Mason PR, Dasch GA, Raoult D. A new
     pathogenic spotted fever group rickettsia from Africa. J Trop Med Hyg
     1994;97:129-37.
  5. Kelly PJ, Beati L, Mason PR, Matthewman LA, Roux V, Raoult D.
     Rickettsia africae sp. nov., the etiological agent of African tick
     bite fever. Int J Syst Bacteriol 1996;46:611-4.
  6. Yunker CE, Norval RAI. Observations on African tick typhus (tick-bite
     fever) in Zimbabwe. In: Fivaz B, Petney T, Horak I, editors. Tick
     vector biology: medical and veterinary aspects. Berlin: Springer
     Verlag 1989:143-7.
  7. Beati L, Kelly PJ, Mattewman LA, Mason PR, Raoult D. Prevalence of
     rickettsia-like organisms and spotted fever group rickettsiae in ticks
     (Acari: Ixodidae) form Zimbabwe. J Med Entomol 1995;32:787-92.
  8. Kelly P, Mattewman L, Beati L, Raoult D, Mason P, Dreary M, et al.
     African tickbite fever: a new spotted fever group rickettsiosis under
     an old name. Lancet 1992;340:982-3.
  9. Pijper A. Tickbite fever: a clinical lecture. SA Med J 1934;8:551-6.
 10. Dupont HT, Brouqui P, Faugere B, Raoult D. Prevalence of antibodies to
     Coxiella burnetii, Rickettsia conorii, and Rickettsia typhi in seven
     African countries. Clin Infect Dis 1995;21:1126-33.
 11. Kelly PJ, Mason PR, Matthewman LA, Raoult D. Seroepidemiology of
     spotted fever group rickettsial infections in Zimbabwe. J Trop Med Hyg
     1991;94:304-9.