Endemic regions of paracoccidioidomycosis in Brazil: A clinical and epidemiologic study of 584 cases in the Southeast Region

Abstract

This paper describes the clinical-seroepidemiologic characteristics of patients with paracoccidioidomycosis (PCM) who visited the University Hospital at the State University of Campinas (Campinas, Sao Paulo, Brazil). The study group consisted of 584 individuals (492 males and 92 females) with ages ranging from 5 to 87 years. The highest incidence of the disease occurred between the ages of 41 and 50 years for men and between 11 and 40 years for women. Rural activities were the principal occupation of 46% of the patients. The diagnosis was confirmed by histopathologic examination and demonstration of fungus in scrapings, secretions, or in the sputum. Serologic test results for PCM were positive in 80% of the 584 patients studied. The significant number of patients, including 33 children less than 14 years old, indicates the presence of the fungus in the area and that this region is an important endemic area for PCM.

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390
Am. J. Trop. Med. Hyg., 61(3), 1999, pp. 390–394
Copyright
q
1999 by The American Society of Tropical Medicine and Hygiene
ENDEMIC REGIONS OF PARACOCCIDIOIDOMYCOSIS IN BRAZIL: A CLINICAL AND
EPIDEMIOLOGIC STUDY OF 584 CASES IN THE SOUTHEAST REGION
MARIA HELOISA S. L. BLOTTA, RONEI LUCIANO MAMONI, SARA JESUS OLIVEIRA, SIMONE A. NOUE
´R,
PRISCILA M. O. PAPAIORDANOU, ALEXANDRE GOVEIA,
AND
ZOILO PIRES DE CAMARGO
Department of Clinical Pathology, and Department of Infectious Disease, Faculty of Medical Sciences,
State University of Campinas, Campinas, SP, Brazil; Department of Microbiology, Immunology and Parasitology,
Federal University of Sa˜o Paulo, Sa˜o Paulo, SP, Brazil
Abstract. This paper describes the clinical-seroepidemiologic characteristics of patients with paracoccidioidomy-
cosis (PCM) who visited the University Hospital at the State University of Campinas (Campinas, Sa˜o Paulo, Brazil).
The study group consisted of 584 individuals (492 males and 92 females) with ages ranging from 5 to 87 years. The
highest incidence of the disease occurred between the ages of 41 and 50 years for men and between 11 and 40 years
for women. Rural activities were the principal occupation of 46% of the patients. The diagnosis was confirmed by
histopathologic examination and demonstration of fungus in scrapings, secretions, or in the sputum. Serologic test
results for PCM were positive in 80% of the 584 patients studied. The significant number of patients, including 33
children less than 14 years old, indicates the presence of the fungus in the area and that this region is an important
endemic area for PCM.
Paracoccidioidomycosis (PCM) is a systemic mycosis
caused by the thermally dimorphic fungus Paracoccidioides
brasiliensis. Almost all South and Central American coun-
tries have large regions where PCM is endemic, particularly
in Brazil, Colombia, Venezuela, and Argentina. The disease
has a limited geographic distribution from 23
8
N (southern
Mexico) to 34.5
8
S (Argentina and Uruguay).
1
The endemic
areas have mild temperatures (17–24
8
C), moderate rainfall
(900–1,810 mm/year), abundant forests, numerous small riv-
ers, and indigenous trees, and a short winter and rainy sum-
mer.
2
This disease typically manifests between the third and
sixth decades of life. Two main clinical forms of the disease
are recognized by the International Committee for PCM,
3
namely, an acute or subacute form and a unifocal or multi-
focal chronic form. The acute form affects young patients
of both sexes and involves mainly the reticuloendothelial
system, whereas the chronic form, is most prevalent in adult
males and has a predominant pulmonary and/or mucocuta-
neous involvement.
4, 5
The higher frequency of the disease
in men was initially attributed to their more intense exposure
to the fungus’ habitat (soil) through agricultural work. This
disease is less frequent in females once they reach puberty
because of the protective role of estrogen. This female hor-
mone inhibits the transition of conidia-mycelial propagules
to the yeast form, a critical step in the pathogenesis of the
disease.
6, 7
Since the reporting of PCM is not compulsory, it is dif-
ficult to establish the real prevalence of the disease. How-
ever, current data indicate a high incidence in Brazil, partic-
ularly in the State of Sa˜o Paulo and the surrounding southern
region, where there are various specialized health centers or
hospitals.
Patients suspected of having a systemic mycosis are usu-
ally sent to general hospitals where they undergo a precise
diagnosis and are advised about how to treat the condition.
The University Hospital at the State University of Campinas
(UH-UNICAMP), which is located in Campinas, a city of
approximately 1 million inhabitants in Sa˜o Paulo State,
serves 5 million people throughout the region.
The gold standard for the diagnosis of PCM is the direct
visualization of characteristic multiple-budding cells in bio-
logical fluids and tissue sections, or isolation of the fungus
from human specimens. Serologic tests generally provide re-
sults earlier than culture and histopathology and can be of
great use in diagnosing of the disease. The most common
test for this purpose is the immunodiffusion test (ID), which
uses a standardized exoantigen (Ag7) prepared from a 7-day
culture of P. brasiliensis B339 strain.
8
The ID test with Ag7
has a sensitivity of 97.1% and is specific when used with a
reference serum. However, the lack of a serologic response
does not exclude PCM, particularly in immunocompromised
patients.
This paper describes the clinical-seroepidemiologic char-
acteristics of patients with PCM who visited UH-UNICAMP
and indicates that the Campinas region is an important en-
demic area for PCM.
MATERIALS AND METHODS
The study was approved by the University of Campinas
Medical Science School Research Ethical Committee. In-
formed consent was not necessary because the study was
retrospective and no personal identifiers were used.
Sera. Sera from patients suspected of having PCM were
sent to the Clinical Pathology Laboratory at UH-UNICAMP
for serologic analysis over a period of 8 years (1988–1996).
Antigen preparation. A lyophilized exoantigen was pre-
pared from a yeast-form culture of P. brasiliensis B-339 as
described previously.
8
A peptone-rich medium supplemented
with thiamine and asparagine was used. After a 7-day in-
cubation, the cells were killed with merthiolate (0.2 g/L),
left at 4
8
C overnight, and then filtered through filter paper.
The crude filtrate was concentrated under vacuum at 45
8
C,
dialyzed for 48 hr against distilled water, and lyophilized.
This preparation is rich in gp43, the 43-kD glycoprotein that
is the immunodominant and specific molecule for use in the
immunodiffusion tests.
9
Immunodiffusion. This assay was performed as described
elsewhere.
8
Briefly, a 3-ml portion of a 1% solution of aga-
rose in phosphate-buffered saline was poured onto a glass
slide (75
3
25 mm). The pattern for the microimmunodif-

391
PARACOCCIDIOIDOMYCOSIS IN SOUTHEAST BRAZIL
F
IGURE
1. Distribution of patients with paracoccidioidomycosis
according to sex and age (years).
T
ABLE
1
Brazilian states from which the patients with paracoccidioidomy-
cosis came
State No. of patients %
Sa˜o Paulo
Minas Gerais
Rondoˆnia
Mato Grosso
Bahia
524
50
3
2
1
89.7
8.5
0.5
0.3
0.2
Mato Grosso do Sul
Parana´
Pernambuco
Rio de Janeiro
Total
1
1
1
1
584
0.2
0.2
0.2
0.2
100
T
ABLE
2
Residency of patients with paracoccidioidomycosis in Sa˜o Paulo
State, Brazil
Micro-regions No. of patients %
Campinas
Sa˜o Joa˜o da Boa Vista
Limeira
Jundiaı´
Piracicaba
251
62
60
43
32
47.9
11.9
11.5
8.1
6.1
Sorocaba
Braganc¸a Paulista
Rio Claro
Sa˜o Carlos
Itapeva
31
21
13
4
2
5.9
4
2.4
0.8
0.4
Presidente Prudente
Lins
Arac¸atuba
Ribeira˜o Preto
Total
2
1
1
1
524
0.4
0.2
0.2
0.2
100
fusion test consisted of a central well surrounded by six
wells, each 3-mm in diameter. The central well was filled
with 10
m
l of antigen, while the others received 10
m
lof
serum. The slides were incubated overnight in a moist cham-
ber at room temperature (20–25
8
C), washed for 1 hr in 5%
sodium citrate and then for 24–48 hr in saline. The slides
were subsequently dried, stained for 3–5 min with 0.15%
Comassie brilliant blue (Sigma, St. Louis, MO) in ethanol-
acetic acid-water (4:2:4 [v/v]), and destained in this solvent
mixture without dye whenever necessary. The slides were
air-dried and the precipitin lines were recorded.
Clinical and epidemiologic data. The age, sex, area of
residency, occupation, clinical forms of the disease, risk fac-
tors (alcoholism and tobacco smoking), chest radiographic
findings, histopathologic findings, and the results of direct
examination of clinical specimens were obtained from the
medical records of each patient.
RESULTS
Epidemiological data. The study group consisted of 584
individuals (492 males and 92 females) with ages ranging
from 5 to 87 years. The highest incidence of the disease
occurred between the ages of 41 and 50 years for men and
11 and 40 years for women (Figure 1). The male:female ratio
was 5.4:1. A predominance of males is characteristic of
PCM, although the number of women in this particular
group was high (16%) compared with other reports.
10
Most
of the patients (90%) lived in Sa˜o Paulo State (Table 1),
mainly in counties around Campinas (Table 2 and Figure 2).
Rural activities were the principal occupation of 46% of
the patients, although only 120 subjects were actually in-
volved with these activities when the symptoms appeared.
The second main professional activity was bricklaying and
masonry (Table 3).
Laboratory data. Serologic test results for PCM were
positive in 80% of the 584 patients studied, with titers rang-
ing from 1:2 to 1: 4,096. The diagnosis was confirmed by
histopathologic examination of tegumentary, ganglionary
and pulmonary biopsies, and by demonstration of fungus in
tegumentary lesions scrapings, lymph node secretions, or in
the sputum (direct examination) (Table 4). For 20% of the
patients, a positive serology was the only laboratory evi-
dence of the disease and, together with clinical and epide-
miologic findings compatible with PCM, was decisive for a
correct diagnosis. Radiographic results also contributed to
the diagnosis in 14 patients. The patients with negative se-
rology (20%) consisted of immunosuppressed subjects or in-
dividuals under treatment (7%) as well as patients with ac-
tive disease (13%).
Clinical data. The main complaints that initially led the
patients to seek medical help were oral ulceration, pain in
the mouth and throat, coughing with expectoration, and dys-
pnea.
The patients were classified as having one of the two
forms of the disease (acute or chronic). Mucosal involve-
ment was predominant followed by pulmonary, ganglionary,
and tegumentary complications in both the isolated and dis-
seminated chronic forms (Table 5). Most (
;
80%) of the pa-
tients lost weight (up to 10–15 kg) during the course of the
disease. Six women were pregnant during treatment for PCM
and in one case the fungus was found in the placenta, but
the child was not infected.
11
In young individuals (
,
30
years old, 21%), the reticuloendothelial system was severely
affected and the disease showed an acute course. The most
relevant associated pathologies were tuberculosis, Addison’s
disease, cancer, and autoimmune diseases.
DISCUSSION
This study involved 584 patients with PCM who visited
UH-UNICAMP. The male:female ratio of 5.4:1 was lower

392
BLOTTA AND OTHERS
F
IGURE
2. Residency of patients with paracoccidioidomycosis in Sa˜o Paulo State (Brazil). The distribution of patients/area is expressed as
a percentage of the total number of patients studied.
T
ABLE
4
Methods used to diagnose paracoccidioidomycosis in 584 patients
Diagnosis established by
Serology
Positive Negative
Biopsy*
Direct examination*
Biopsy plus direct examination*
Radiograph*
Only serology
Total
241
78
23
12
114
468
71
33
10
2
0
116
* Plus serology.
T
ABLE
3
Principal occupations among 584 patients with paracoccidioidomy-
cosis
Occupation
Male
No. %
Female
No. %
Total
No. %
Agricultural work
Construction*
Industry
Others
Total
241
113
39
99
492
49
23
8
20
100
28
2
0
62
92
30
2
0
68
100
269
115
39
161
584
46
20
7
27
100
* Bricklaying, masonry.
(thus, the number of females was higher) than in other stud-
ies in similar populations.
12,13
Mota
14
reported a male:female
ratio of 6.5:1 in Parana´ State (southern Brazil) and attributed
this to the number of women working on coffee plantations
in that region. In our group there was a higher number of
women and children with PCM. Almost 40% of the women
presented with the acute form, a more severe and many times
disseminated version of the disease; this would explain their
desire to attend a specialized hospital. The study groups in-
cluded 33 children less than 14 years old, the youngest being
a 5-year-old boy. Although the disease is uncommon in chil-
dren, when the mycosis is taken into consideration in the
differential diagnosis in endemic areas, cases of PCM in
childhood are more frequently recognized. The young pa-
tients in our group showed frequent involvement of the
lymph nodes and skin, in contrast to a low involvement of
the oropharynx and lungs. In patients
.
25 years old, mu-
cosal and pulmonary involvement was preponderant, fol-
lowed by tegumentary, ganglionary, and suprarenal compli-
cations, both in the isolated and disseminated forms. The
significant number of young people involved (98 patients
less than 25 years old, 16%) is an indication of the presence
of the fungus in the area studied. Children and young adults
are considered epidemiologic markers since they have a re-
stricted migratory profile.
The region of Campinas has acidic soil, long periods of
rainfall (8 months), a tropical climate, agricultural areas, and
planted pastures. The predominance of sugar cane, cotton,
and coffee plantations in the region favors contact between
the rural worker and soil particles and plants, in which the
fungus is found.
Several investigators have indicated that PCM is more
prevalent among rural workers engaged in intensive agri-
culture.
15–18
Most of the patients in our group stated that they
have lived and worked in rural areas during some period of
their lives. After moving to urban centers, they started new
jobs, many of them working as bricklayers. It is possible
that some infections may have occurred in the urban areas
during activities that involved contact with soil and wood.
Apparently innocuous activities, such as gardening, may also

393
PARACOCCIDIOIDOMYCOSIS IN SOUTHEAST BRAZIL
T
ABLE
5
Clinical forms of paracoccidioidomycosis among 584 patients
Clinical forms
Male
No. %
Female
No. %
Total
No. %
Acute unifocal
Acute multifocal
Chronic unifocal
Chronic multifocal
Total
49
29
143
271
492
10
6
29
55
100
14
25
21
32
92
15
27
23
34
100
63
54
164
303
584
11
9
28
52
100
give rise to the disease. In large cities, multiple construction
and demolition projects occur simultaneously, and individ-
uals exposed during such work may become infected.
Alcoholism and tobacco smoking were frequently asso-
ciated with the disease among our patients. The role of al-
cohol as a risk factor favoring an immunologic imbalance
and the reappearance of quiescent PCM has been reported
for the progressive form of the disease (Andrade JAF, 1987.
Avaliac¸a˜o da Frequ¨eˆncia de Micoses Sisteˆmicas e Oportun-
istas em Pacientes com Doenc¸as Pulmonares: Estudo Clı´nico
e Sorolo´gico no Hospital Ota´vio Mangabeira. Masters De-
gree Thesis. State University of Bahia, Salvador, Bahia, Bra-
zil).
19
Many diseases must be considered in any differential di-
agnosis of PCM. Neoplasms (carcinoma and lymphoma), in-
fections (tuberculosis and other fungi), noninfectious inflam-
mation (sarcoidosis and idiopathic pulmonary fibrosis) and
autoimmune diseases must be ruled out before a diagnosis
of PCM is accepted. Serologic tests are of great importance
as an aid in excluding these and other pathologies.
During the study period, there were situations in which
the serology was negative but the fungus was detected in
biopsy specimens or by direct examination. Some of these
cases involved immunosuppressed patients (human immu-
nodeficiency virus positive, cancer, and autoimmune disease)
in whom the mycosis remained serologically undiagnosed.
Patients at the end of their treatment may also have unde-
tectable levels of specific antibodies. However, for some pa-
tients with confirmed active disease, the negative serology
seems to be related to the inadequacy of the serologic test
used. The diagnosis of such cases may be possible using
antigens prepared from different strains of P. brasiliensis
and new immunologic approaches.
In contrast to public hospitals in Sa˜o Paulo, the individ-
uals who visited UH-UNICAMP were mainly from neigh-
boring counties. The towns involved are associated with the
hospital since the latter provides many of them with tertiary
medical care.
Since we studied a hospital population, the number of pa-
tients with PCM does not necessarily reflect the real inci-
dence of the disease in the area. In particular, the mild forms
of the disease are probably diagnosed and treated in health
centers and small hospitals near the homes of patients. Some
cases are likely to be missed because the disease frequently
is not given medical attention. Thus, since specialized cen-
ters tend to treat only the severe forms of PCM, an analysis
of such data may result in an erroneous epidemiologic pro-
file. Nevertheless, considering the increased number of pa-
tients admitted to our hospital and the areas from which the
patients come, we conclude that the region of Campinas is
an endemic zone for PCM. We have since initiated a detailed
epidemiologic study of this region, with particular attention
being paid to children with PCM since they serve as useful
markers for the distribution of the disease.
In conclusion, this study calls attention to PCM as a major
public health problem in the Campinas region of Brazil. Al-
though there are no control measures that may be applied to
prevent the disease, efforts should include increasing aware-
ness among clinicians and the public, especially people liv-
ing in rural areas where PCM is endemic.
Authors’ addresses: Maria Heloisa S. L. Blotta, Ronei Luciano Ma-
moni, Sara Jesus Oliveira, and Alexandre Goveia, Department of
Clinical Pathology, Faculty of Medical Sciences, State University of
Campinas, Campinas, SP, Brazil. Simone A. Noue´r and Priscila M.
O. Papaiordanou, Department of Infectious Disease, Faculty of Med-
ical Sciences, State University of Campinas, Campinas, SP, Brazil.
Zoilo Pires de Camargo, Department of Microbiology, Immunology
and Parasitology, Federal University of Sa˜o Paulo, Sa˜o Paulo, SP,
Brazil.
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