Am. J. Trop. Med. Hyg., 65(4), 2001, pp. 329–334
Copyright 䉷 2001 by The American Society of Tropical Medicine and Hygiene
SPOTTED FEVER IN BRAZIL: A SEROEPIDEMIOLOGICAL STUDY AND
DESCRIPTION OF CLINICAL CASES IN AN ENDEMIC AREA IN THE
STATE OF SÃO PAULO
ELBA R. S. DE LEMOS, FÁTIMA B. F. ALVARENGA, MARIA L. CINTRA, MARCELO C. RAMOS,
CHRISTOPHER D. PADDOCK, TARA L. FEREBEE, SHERIF R. ZAKI, FÁTIMA C. C. FERREIRA,
ROBERTO C. RAVAGNANI, RAIMUNDO D. MACHADO, MARIA A. A. M. GUIMARÃES, AND J. R. COURA
Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Rio de Janeiro, Brazil; State University of Rio de Janeiro,
Rio de Janeiro, Brazil; University of Campinas, UNICAMP, Campinas, São Paulo, Brazil; Centers for Disease Control and
Prevention, Atlanta, Georgia; Division of Health of the Municipality of Pedreira, São Paulo, Brazil; Institute of Microbiology,
Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
Abstract. During 1985–1995, illnesses clinically and epidemiologically compatible with Brazilian spotted fever
were identified in 17 patients in the county of Pedreira, in the state of São Paulo, Brazil. Spotted-fever group rickettsial
infection was confirmed by serology and/or immunostaining of tissues in 10 of these patients. Immunostaining confirmed infection in a 37-year-old pregnant patient, although rickettsial antigens were not demonstrable in the tissues
of the fetus. A serosurvey was conducted in four localities in the county to determine the prevalence of subclinical
or asymptomatic infections with spotted fever group rickettsiae. Five hundred and twenty-five blood samples were
tested by an indirect immunofluorescence assay for antibodies reactive with Rickettsia rickettsii. Twenty-two (4.2%)
of these samples demonstrated titers ⱖ 1:64. The results indicate that Brazilian spotted fever is endemic within this
region of Brazil.
INTRODUCTION
MATERIALS AND METHODS
Brazilian spotted fever (BSF) is the most prevalent rickettsial disease in Brazil.1,2 As with Rocky Mountain spotted
fever (RMSF) in North America, the disease is caused by
Rickettsia rickettsii. In Central and South America, the Cayenne tick (Amblyomma cajennense) is the most important
vector for R. rickettsii, and humans are frequently bitten by
larvae and nymphs of this tick. The clinical presentation of
BSF closely resembles RMSF1–5 and typically presents as
sudden onset of fever, malaise, myalgia, headache, chills,
and conjunctival injection, followed several days later by a
maculopapular rash. Before the development of antibiotics
effective against rickettsiae (e.g., tetracyclines and chloramphenicol), case-fatality ratios of patients infected with R.
rickettsii were as high as 25–80%.2,6 Although BSF generally
occurs sporadically, geographic and family clusters of disease have been described.2,7
Following the initial description of BSF in 1920,8 relatively few cases were reported in Brazil in the subsequent
60 years. In the last two decades, however, more than 90
confirmed cases have been reported in the southeastern region of the country.9–11 In addition, many clinically compatible but unconfirmed cases have been reported, reflecting the
unavailability of laboratory diagnostic tests for this disease
in many regions of Brazil.
Between 1985–1995, 17 patients with disease clinically
and epidemiologically compatible with BSF, including eight
fatalities, were reported from the county of Pedreira, state
of São Paulo. To determine the background seroprevalence
of spotted fever group rickettsial infections in this region, a
serological survey was initiated among residents in the four
Pedreira localities where the majority of BSF cases occurred.
This paper summarizes the clinical, pathologic, and epidemiologic findings associated with cases over the 10-year
study period, and the results of the serosurvey.
Case finding for Brazilian spotted fever, 1985–1995.
The study was conducted in the county of Pedreira, located
in the southeast region of São Paulo, Brazil. Clinical and
epidemiological data were obtained from clinical records of
all patients admitted to Pedreira County Hospital, Hospital
of the Campinas University, and Amparo County Hospital
between 1985 and 1995. Data were summarized on all patients evaluated during this period who presented with an
acute febrile illness associated with rash and a history of
recent tick exposure.
Serologic information. Acute-phase serum samples were
available for evaluation for 12 patients. Three patients were
tested for febrile agglutins by using the Weil-Felix (WF) test.
The cut-off for a positive result was considered a titer ⱖ 1:
80. Serum specimens from nine patients were evaluated for
IgM and IgG antibodies reactive with R. rickettsii by using
an indirect immunofluorescence assay (IFA) as previously
described.12 Endpoint titers were recorded as the reciprocal
of the highest dilution exhibiting specific rickettsial fluorescence. Patients with titers ⱖ 1:64 were considered seropositive.
Histopathological study. For patients with clinically and
epidemiologically compatible disease identified by case finding, a review of pathology files from the three hospitals was
conducted to identify formalin-fixed, paraffin-embedded biopsy and autopsy specimens obtained between 1985 and
1995 from suspect BSF patients. Tissues from six patients
with presumed BSF were evaluated by using routine hematoxylin and eosin (HE) stain and by specific immunofluorescence and immunohistochemical stains.
Indirect immunofluorescence (IF) staining for spotted fever group rickettsiae was performed by using a previously
described method,13,14 with some modifications. Briefly, after
a blocking step using 10% bovine serum albumin for 20 min
at room temperature, sections were incubated with human
329
330
LEMOS AND OTHERS
anti-R. rickettsii antiserum obtained from a seropositive patient (IFA titer of ⱖ 1:1,024) diluted 1:2 in PBS, pH 7.4 for
90 min at room temperature. After being washed, slides were
incubated at room temperature in fluorescein-labeled rabbit
antihuman IgG (Sanofi Diagnostic Pasteur, Paris, France) diluted 1:5, for 60 min. For each test, a series of three controls
were run in parallel with the patient tissue: a) omission of
the human anti-R. rickettsii serum; b) omission of the fluorescein-labeled rabbit anti-human IgG conjugate; and c) substitution of normal human sera (inactivated at 56⬚C for 30
min) for the primary antibody. As an additional control, paraffin-embedded tissues from a patient with fatal, culture-confirmed meningioccocemia were evaluated with each run.
Patient tissues were additionally evaluated at the Centers
for Disease Control and Prevention (CDC) by using an indirect immunoalkaline phosphatase (IHC) stain for spotted
fever group rickettsiae, as described previously.15 Briefly, tissue sections were digested with proteinase-K and then incubated with a polyclonal rabbit anti-R. rickettsii antibody.
This step was followed by incubation in biotinylated swine
anti-mouse and anti-rabbit antibody, followed by an incubation in alkaline phosphatase-conjugated streptavidin. Rickettsial antigens were detected using a naphthol/fast red substrate.
Serosurvey. During 1994, whole blood samples were collected from apparently healthy volunteers at four study sites
selected for the survey because of their proximity to the
locations of the majority of identified BSF cases (Figure 1).
These included The Workers’ Colony of Nadir Figueiredo
Industry (NF), Fortaleza Farm (FF), Jaguari Farm (JF), and
the village of São Nilo (SN), located along Jaguari River, 3
km from Pedreira City. Serum samples were collected from
a total of 525 persons in NF, FF, and JF, and were represented
by 93% (379), 95% (20), and 82% (18) of the total inhabitants from each study site, respectively. In the village of São
Nilo serum specimens were collected from 108 (23%) of 470
total inhabitants who agreed to participate in this study. Serum samples were separated by centrifugation and stored at
⫺20⬚C until tested. Serum was evaluated by using IFA, as
previously described.
FIGURE 1. Map of Pedreira County, São Paulo, Brazil and localities studied.
RESULTS
Case findings of BSF patients, 1985–1995. Seventeen
patients identified from a review of hospital records demonstrated clinical and epidemiological findings compatible
with BSF. A laboratory diagnosis of spotted fever group
rickettsial infection was available for 10 patients, including
3 by Weil-Felix assay, 5 by IFA, and/or 5 by IF or IHC
staining of tissues (Tables 1 and 2). Except for a single patient (patient 13), who was a visitor to the area, all patients
resided in the county of Pedreira. When a population denominator obtained by averaging the population of Pedreira
in the census between 1980 and 199116 was used, the median
annual rate of occurrence of confirmed cases of BSF was
3.68/100,000/year, from 1985 through 1995. When all the
cases (probable and confirmed) were considered, the median
annual rate of occurrence of BSF was 6.07/100,000/year
with the highest rate in males who showed an annual rate
of 11.41/100,000/year. Only three patients were female (annual rate of occurrence of 2.45/100,000/year). All the pa-
tients were white and 16 (94%) of the patients became ill
between June and October. Patients ranged in age from 3 to
59 years (median of 28 years).
All the patients were febrile. Headache, rash, and myalgias were reported for 16 (94.1%), 15 (88%), and 10 (77%)
of patients, respectively (Table 1). Three surviving patients
(cases 1, 11, and 16) had illnesses complicated by pneumonia, digital and scrotal gangrene, and acute renal failure.
One or more findings of encephalopathy (e.g., headache, seizures, confusion, and/or coma) were observed in 16 (94%)
patients. Six patients had leukocytosis (leukocyte count ⬎
10⫻109/L, median 14.3⫻109/L). Three of seven patients tested demonstrated thrombocytopenia (platelets ⬍ 150⫻109/L,
median 41.3⫻109/L). One patient (patient 16) showed
marked elevations in hepatic enzyme levels, including an
aspartate aminotransferase level of ⬎ 800 U. Of nine patients
treated with chloramphenicol, eight became afebrile within
the first five days of treatment and one patient (case 13),
who received therapy on the tenth day of illness, died. None
331
SPOTTED FEVER IN BRAZIL
TABLE 1
Epidemiology and clinical featuresa of Brazilian spotted fever from County of Pedreira, São Paulo, 1985–1995
UPN/Age/Sex
date of onset
skin rash
myalgia
splenomegaly
edema
jaudice
coma/shock
IF
WF
IHC
outcome
01/35/Mb,c
02/27/M
03/57/M
04/11/Fb,d
05/12/M
06/27/M
07/57/M
08/56/Mb
09/59/Me
10/03/Fd
11/29/M
12/12/Me
13/37/Fe,f
14/28/M
15/20Md
16/44/M
17/04/Me,g
09/85
09/85
09/85
06/86
08/86
08/86
08/86
06/87
07/87
10/88
07/93
08/93
09/93
09/93
09/94
04/95
10/95
⫹
⫹
⫹
NA
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫹
⫹
⫹
⫹
NA
NA
NA
NA
⫹
⫹
⫺
⫹
⫹
⫹
⫺
⫹
⫹
⫺
⫹
NA
NA
NA
NA
NA
NA
⫺
⫹
⫺
⫹
NA
⫺
⫺
⫺
⫹
⫺
⫹
⫺
⫹
⫹
⫹
⫹
⫹
⫺
⫺
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫹
⫹
⫺
⫹
NA
⫹
⫹
NA
⫺
⫹
⫺
⫹
⫹
⫺
⫺
⫹
⫹
⫺
⫹
⫺
⫹
⫹
⫹
⫹
NA
⫺
⫺
⫺
⫹
⫹
⫹
⫹
⫹
⫹
⫺
ND
ND
ND
ND
ND
ND
ND
⫹
⫹
ND
⫹
⫺
⫺
⫺
⫺
⫹
⫹
⫹
⫹
ND
ND
ND
ND
ND
ND
ND
⫹
ND
ND
ND
ND
ND
ND
ND
NA
NA
NA
NA
⫺
⫹
NA
⫹
NA
NA
⫹
NA
⫹
NA
NA
⫹
NA
R
R
D
D
D
D
R
R
R
R
R
D
D
D
D
R
R
a Tick exposure and fever were observed in all the patients. Only patient 2 did not have nervous manifestations (headache, convulsion, stupor, mental confusion); b conjunctivitis; c necrotic
gangrene of the distal phalanx of the toes with auto-amputation; d arthalgia; e vomiting; f immunohistochemical stain was positive in the pregnant patient tissues at autopsy examination, but
negative in fetal tissues; g sore throat; UPN ⫽ unique patient number; M ⫽ male; F ⫽ female; immunofluorescent assay; WF ⫽ Weil Felix test; IHC ⫽ immunohistochemical staining; NA
⫽ not available; ND ⫽ not done; D ⫽ death; R ⫽ recovery; Patients 1, 2 and 3 ⫽ members of a family who lived in the village of São Nilo. Patients 4, 5, 6 and 7 ⫽ members of a family
who lived in Jaguari Farm.
noalkaline phosphate staining for spotted fever group rickettsiae was positive in skin biopsies for two patients (cases
13 and 16), and negative for two patients (cases 8 and 11).
Autopsy tissues were available for three patients (cases 5,
6, and 13). Specific IF staining was detected in tissues of
one patient (case 6). Indirect immunofluorescence staining
of tissues from case 13 showed diffuse immunofluorescence
in the walls of blood vessels and was interpreted as inconclusive. Indirect immunoalkaline phosphate staining was
positive in autopsy tissues in two patients, case 6 (Figure
2A) and case 13 (Figure 2B), and negative in one patient
(case 5). Indirect immunofluorescence and IHC stains for
spotted fever rickettsiae were discordant for three patients
(cases 8, 11, 13; Tables 1 and 2).
Skin biopsy and autopsy specimens from a 37-year-old
pregnant woman (case 13) revealed intense immunostaining
for spotted fever group rickettsial antigen in multiple tissues,
including kidney, liver, pancreas, and skeletal muscle. Multiple tissues from the fetus (estimated gestational age of 8–
10 weeks) including kidney, liver, and lung showed no evidence of rickettsial infections when evaluated by using IHC
stain.
Serosurvey. Serum specimens were obtained from 525
inhabitants, representing 58% of all inhabitants from the four
of them was treated with tetracyclines. A total of eight
deaths occurred.
Weil-Felix agglutinin titers of ⱖ 1:200 (range 200–1,600)
were observed in three patients (patients 1, 2, and 10); each
of these patients received chloramphenicol and recovered.
Serum samples from five of nine patients tested by IFA had
antibody reactive with R. rickettsii at titers ⱖ 1:64 (range
64–1,024). Two patients who died one day after admission
to the hospital showed no significant anti-rickettsial antibodies by IFA (cases 12 and 13). Four deaths were associated
with clustering of seven cases of disease in two families
(patients 1–3 and 4–7), although laboratory confirmation of
a spotted fever group rickettsiosis was obtained for only one
patient in these deaths.
Histopathology and immunostaining of patients’ tissues. Skin biopsies were available from four patients (cases
8, 11, 13, and 16) (Table 2). Each biopsy revealed histopathology consistent with rickettsial infection, including mononuclear perivascular infiltrates, endothelial damage, or vasculitis. When IF techniques were used, three of the biopsies
showed small, coccobacillary bacteria compatible with rickettsiae in the endothelium and smooth muscle of dermal
blood vessels (patients 8, 11, 16). One biopsy (case 13)
showed no specific immunofluorescence. Indirect immu-
TABLE 2
Immunohistochemical staining of cases of Brazilian spotted fever from County of Pedreira, São Paulo, 1985–1995
Immunofluorescence staining
Tissue examined
Immunoalkaline phosphatase staining
UPN
Sex/age
5
6
8
11
13
M/12
M/27
M/56
M/27
F/37
lung
nervous tissues
skin*
skin*
skin* liver, and nervous tissues
negative
positive
positive
positive
not specific, inconclusive
16
M/44
fetal tissues
skin*
inconclusive
positive
UPN ⫽ unique patient number; * biopsy material.
Result
Tissue examined
lung, nervous tissues
nervous tissues
skin*
skin*
skin* kidney, pancreas, large
vein, liver, and skeletal muscle
fetal tissues
skin*
Result
negative
positive
negative
negative
positive
positive
negative
positive
332
LEMOS AND OTHERS
FIGURE 2. A). Photomicrograph of vascular endothelium from the hippocampus of a patient with fatal Brazilian spotted fever (BSF) (Case
#6), showing intracytoplasmic staining of rickettsial antigens. B). Photomicrograph of the kidney of patient with fatal BSF (Case #13) demonstrating positive staining for spotted fever group rickettsiae in a renal interstitial vessel. Both samples were stained with immunoalkaline
phosphatase stain using naphthol phosphate/fast red substrate and hematoxylin counterstain, original magnification ⫻ 158.
SPOTTED FEVER IN BRAZIL
localities combined. The majority of participants were white
(94.5%); 257 (49%) were males and 268 (51%) were females. Five hundred and seven (97%) had lived in the study
area for at least three years. Twenty-two (4.2%) individuals
demonstrated serum antibody reactive with R. rickettsii at a
titer of ⱖ 1:64 (range 64–512). Of the 22 seropositive individuals, 19, 2, and 1 individuals were from Nadir Figueiredo, São Nilo, and Fortaleza Farm, respectively. The seroprevalence of the infection in males and females was 3.9%
and 4.5 %, respectively. Only one (4.5%) of 22 seropositive
individuals recalled a spotted fever-like illness preceding the
survey.
DISCUSSION
The first case of laboratory-confirmed BSF was identified
in the county of Pedreira in 1985, although anecdotal evidence suggests that this rickettsiosis has existed in the region
for at least 100 years.2,17 The Pedreira County Bills of Mortality from the late 1890s describe fatal disease caused by
‘‘tick typhus’’ and ‘‘big measles’’ (a colloquial name for
BSF).2 However, BSF appears to have been unreported in
this area of Brazil between the late 1930s and 1985. The
absence of formally reported cases of BSF in Pedreira during
this period is intriguing and may be due in part to masking
of cases by the widespread use of chloramphenicol and tetracycline for acute febrile illnesses. It is also possible that
cases of BSF have been missed because of waning clinical
suspicion and the paucity of confirmatory diagnostic resources. Finally, the apparent emergence (or reemergence)
of BSF in this region may reflect changes in the number of
human exposures to naturally infected tick populations,
mainly A. cajennense, resulting from decreases in the development of farmlands, increasing suburbanization, and increasing human incursions into tick-infested habitats.18
The clinical characteristics of BSF seen in the study area
did not differ appreciably from the clinical features of the
disease described in patients from other regions of Brazil.1,3,7,8,11 Complications, notably renal failure, coma, and
thrombocytopenia, were observed in patients who either delayed seeking treatment or who had initial diagnoses other
than BSF. The majority of patients needed prolonged hospitalization, parenteral antibiotic therapy, and supportive
care. The high mortality rate (47%) was associated with misdiagnosis and subsequent delays in treatment. None of the
eight fatal cases received effective anti-rickettsial therapy
and all died between the tenth and fourteenth day of illness.
These data reaffirm the importance of early recognition of
disease and prompt initiation of appropriate anti-rickettsial
therapy to prevent serious complications and mortality attributable to R. rickettsii.1,19,20
For all 17 patients, the clinical and epidemiologic findings
were compatible with BSF. The BSF diagnosis was supported by one or more laboratory tests in 10 of 14 patients for
whom testing was available. Only two of eight fatalities were
confirmed by laboratory tests. Serum and/or tissues were unavailable for testing for three of the seven unconfirmed, and
four of the seronegative patients expired relatively early in
the course of infection, presumably before the development
of specific antibody reactive with R. rickettsii.15 However,
because many other infectious diseases mimic spotted fever
333
infection, it is possible that one or more of the seven unconfirmed patients had diseases other than BSF.
Most cases occurred between the months of June and October, coincident with peak larval and nymphal feeding activities of A. cajennense.21 Familial clustering was observed
among seven patients in two separate families; four deaths
were noted in these clusters. Although three of these deaths
were unconfirmed by laboratory tests, positive immunostaining for rickettsiae in autopsy tissues from one of the family
members and positive serologic test in two surviving patients
suggested a rickettsial etiology for all four deaths. Clusters
of rickettsial spotted fever involving families have been reported in Brazil since the beginning of this century.1,2,7,11
Serologic survey of inhabitants from regions of presumed
BSF endemicity in the county of Pedreira revealed that 4.2%
of the population had prior exposure to R. rickettsii or to an
antigenically related spotted fever group rickettsiae, indicating that spotted fever group rickettsiaes are endemic in this
region of Brazil.
For three patients, results of IF staining and IHC staining
of skin biopsies for R. rickettsii did not agree. For one patient for whom the IF stain was positive and IHC stain was
negative, tests were performed on separate tissue blocks. For
another patient for whom IF staining was negative, yet IHC
was positive, the biopsy consisted of only a scant fragment
of tissue, making interpretation difficult. These discrepant
results may reflect the characteristically focal distribution of
rickettsial antigens observed between separate biopsies, or
even within separate sections from the same biopsy.14,15,22,23
Autopsy specimens from a pregnant woman who died
from laboratory-confirmed BSF demonstrated intense positive IHC staining for spotted fever group rickettsial antigen
in multiple tissues. However, rickettsiae were not observed
in similarly stained tissues of the fetus. Rickettsial infections
during pregnancy are rarely reported, and the direct effects
of maternal R. rickettsii infection on the fetus are unknown.
However, the findings in this study suggest that this pathogen
did not initiate infection in the fetus. Although the transplacental passage of rickettsiae has been suggested,24–26 limited
data from human and laboratory animal infections indicate
that maternal rickettsial infections are not transmitted to the
fetus.27–30
Information obtained in this study, coupled with reports
of seropositive domestic and wild animals31,32 and isolations
of spotted fever group rickettsia from ticks collected in this
region,33 emphasize the need for increased awareness of rickettsial infections in this region of Brazil. Patients presenting
with flu-like illnesses between June and October in this region and in other R. rickettsii-endemic regions in Brazil
should be carefully evaluated for rickettsial infection. Working from a presumptive diagnosis and implementing prompt,
specific anti-rickettsial therapy may reduce the high mortality observed with spotted fever cases in Brazil.
Acknowledgments: The authors wish to thank the Mayor’s Office
and the population of Pedreira for their cooperation in the fieldwork;
Santa Casa ‘‘Ana Cintra’’ and Dr. Sebastião AN Souza for providing
information and/or autopsy material of patients 4, 5, and 10; and Dr.
James Olson (CDC) for providing the antigen used in this study.
Financial support: This work was supported in part by Oswaldo Cruz
Foundation and National Council of Scientific and Technologic Development (grant no. 840244/95–9).
334
LEMOS AND OTHERS
Authors’ addresses: Elba R. S. de Lemos, José R. Coura, Departamento de Virologia, Instituto Oswaldo Cruz, FIOCRUZ, Avenida
Brasil 4365, 21045–900, Rio de Janeiro, RJ, Brazil. Fátima B. F.
Alvarenga, Fátima C. C. Ferreira, Departamento de Patologia, Universidade Estadual do Rio de Janeiro, Avenida Vinte e Oito de Setembro, s/n, Vila Isabel, Rio de Janeiro, RJ, Brazil. Maria L. Cintra,
Marcelo C. Ramos, Disciplina de Moléstias Infecciosas e Departamento de Anatomia Patológica, Faculdade de Ciências Médicas,
UNICAMP, Barão Geraldo, s/n, Campinas, São Paulo, Brazil. Christopher D. Paddock, Tara L. Ferebee, Sherif R. Zaki, Division of
Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, NE, Atlanta, GA 30333. Roberto C.
Ravagnani, Secretaria Municipal de Saúde de Pedreira, Rua Siqueira
Campos, s/n, Centro, Pedreira, São Paulo, Brazil. Raimundo D. Machado, Maria A. A. M. Guimarães, Universidade Federal do Rio de
Janeiro, Avenida Brigadeiro Trompowski, s/n, Rio de Janeiro, RJ,
Brazil.
REFERENCES
1. Dias E, Martins AV, 1939. Spotted fever in Brazil. A summary.
Am J Trop Med 19: 103—108.
2. Magalhães O, 1952. Contribuição ao conhecimento das doenças
do grupo tifo exantemático. Monografia do Instituto Oswaldo
Cruz 6: 968.
3. Monteiro JL, 1931. Estudos sobre o typho exanthemático de São
Paulo (1a/2a parte). Memórias do Instituto Butantan 6: 1–136.
4. Travassos J, Dias E, 1939. Febre maculosa. Identidade imunológica dos vı́rus de Minas Gerais, São Paulo e das Montanhas
Rochosas. Mem Inst Oswaldo Cruz 34: 149–179.
5. Walker DH, 1989. Rocky Mountain spotted fever: a disease in
need of microbiological concern. Clin Microb Rev 2: 227–
240.
6. Helmick CG, Bernard KW, DÁngelo LJ, 1984. Rocky Mountain
spotted fever, clinical, laboratory, and epidemiological features of 262 cases. J Infect Dis 150: 480–488.
7. Travassos J, 1948. Studies on rickettsial diseases in Brazil.
Whitlock C, ed. Proceedings of the 4th International Congress
of Tropical Medicine and Malaria. Washington, DC: US Government Printing Office, 414–421.
8. Cintra U, Piza JT, 1920. Sobre um caso de tifo exantemático
em São Paulo. Brasil Médico 34: 715–718.
9. Gonçalves AJR, Lopes PFA, Melo JCP, Pereira AA, Pinto
AMM, Lazera MS, Souza MLS, Teixeira CRV, Oliveira JC,
Duarte F, 1981. Rickettsioses—a propósito de quatro casos
diagnosticados no Rio de Janeiro de febre maculosa. Folha
Médica (BR) 82: 127–134.
10. Melles HHB, Colombo S, Silva MV, 1992. Febre maculosa:
isolamento de rickettsia em amostra de biópsia de pele. Rev
Inst Med trop São Paulo 34: 37–41.
11. Sexton DJ, Muniz M, Corey GR, Breitschwerdt EB, Hegarty
BC, Dumler S, Walker DH, Peçanha PM, Dietze R, 1993.
Brazilian spotted fever in Espı́rito Santo, Brazil: description
of a focus of infection in a new endemic region. Am J Trop
Med Hyg 49: 222–226.
12. Philip RN, Casper EA, Orsebee RA, Peackoc MG, Burgdorfer
W, 1976. Microimmunofluorescence test for the serological
study of Rocky Mountain spotted fever and typhus. J Clin
Microbiol 3: 51–61.
13. Huang S, Minassian H, More JD, 1970. Application of immunofluorescent staining on paraffin sections improved by trypsin digestion. Lab Invest 35: 383–390.
14. Walker DH, Cain BC, 1978. A method for specific diagnosis of
Rocky Mountain spotted fever on fixed, paraffin-embedded
tissue by immunofluorescence. J Infect Dis 137: 206–209.
15. Paddock CD, Greer PW, Ferebee TL, Singleton L Jr, McKechenie DB, Treadwell TA, Krebs JW, Clarke MJ, Holman
RC, Olson JG, Childs JE, Zaki SR, 1999. Hidden mortality
attributable to Rocky Mountain spotted fever: immunohistochemical detection of fatal, serologically unconfirmed disease.
J Infect Dis 179: 1469–1476.
16. IBGE, Brazilian Institute of Geography and Statistic. Census,
1991.
17. Travassos J, Vallejo-Freire A, 1944–1945. Criação artificial de
Amblyomma cajennense para o preparo da vacina contra a
febre maculosa. Memórias do Instituto Butantan 18: 145–235.
18. Smadel JE, 1959. Status of the rickettsiosis in the United States.
Ann Intern Med 51: 421–435.
19. Hattwick MAW, Retailliau H, O’Brien RJ, Slutzker M, Fontaine
RE, Hanson B, 1978. Fatal Rocky Mountain spotted fever.
JAMA 240: 1499–1503.
20. Kirkeland KB, Wilkinson WE, Sexton DJ, 1995. Therapeutic
delay and mortality in cases of Rocky Mountain spotted fever.
Clin Infect Dis 20: 1118–1121.
21. Lemos ERS, Machado RD, Coura JR, Guimarães MAA, Freire
NM, Amorim M, Gazeta GS, 1997. Epidemiological aspects
of the Brazilian spotted fever: seasonal activity of ticks collected in an endemic area in the State of São Paulo, Brazil.
Rev Soc Bras Med Trop 30: 181–185.
22. Procop GW, Burchette JL Jr, Howell DN, Sexton DJ, 1997. Immunoperoxidase and immunofluorescent staining of Rickettsia
rickettsii in skin biopsies. A comparative study. Arch Pathol
Lab Med 121: 894–899.
23. Silber JL, 1996. Rocky Mountain spotted fever. Clin Dermat
14: 245–258.
24. Giroud P, Capponi M, Dumas N, 1965. Les rickettsies ou des
éléments proches interviennent-ils dans les troubles de la gestation de la femme? Bull Soc Pathol Exot 58: 410–414.
25. Giroud P, Giroud A, Capponi M, Dumas N, 1965. Démonstrations concernant la culture élective de Rickettsia conori sur
les membranes de la ratte gestante au cours d’une maladie
inapparente. Bull Soc Path Exot 58: 157–159.
26. Giroud P, Giroud A, Capponi M, Dumas N, Martinet M, 1965.
Rickettsioses inapparentes chez la ratte gestante. Ann Inst
Pasteur 108: 711–722.
27. Surdan CT, 1968. La contribution Romaine a létude des infections latentes provoquées chez l’homme par les rickettsies et
les pararickettsies. Bull Soc Path Exot 5: 737–749.
28. Gallis HA, Agner RC, Painter CJ, 1984. Rocky Mountain spotted fever in pregnancy. North Carolina Med J 45: 187–188.
29. Shirai A, Chan TC, Gan E, Groves MG, 1984. Lack of transplacental infection with scrub typhus organisms in laboratory
mice. Am J Trop Med Hyg 33: 285–287.
30. Arango-Jaranillo S, Wisseman JR, Azad F, 1988. Newborn rats
in the murine typhus enzootic infection cycle: studies on
transplacental infection and passively acquired maternal antirickettsial antibodies. Am J Trop Med Hyg 39: 391–397.
31. Lemos ERS, Machado RD, Coura JR, Guimarães MAAM, Chagas N, 1996. Epidemiological aspects of the Brazilian spotted
fever: serological survey of dogs and horses in an endemic
area in the State of São Paulo, Brazil. Rev Inst Med trop São
Paulo 38: 472–480.
32. Lemos ERS, Machado RD, Coura JR, Guimarães MAAM, Serra-Freire NM, 1996. Infestation by ticks and detection of antibodies to spotted fever group rickettsiae in wild animals captured in the State of São Paulo, Brazil. A preliminary report.
Mem Inst Oswaldo Cruz 91: 701–702.
33. Lemos ERS, Melles HHB, Colombo S, Machado RD, Coura
JR, Guimarães MAA, Sanseverino SL, Moura A, 1996. Primary isolation of spotted fever group rickettsiae from Amblyomma cooperi collected from Hydrochaeris hydrochaeris in
Brazil. Mem Inst Oswaldo Cruz 91: 273–275.