Am. J. Trop. Med. Hyg., 87(4), 2012, pp. 675–680
Copyright © 2012 by The American Society of Tropical Medicine and Hygiene
Profile of Trypanosoma cruzi Reactivity in a Population at High Risk for Endemic
Pemphigus Foliaceus (Fogo Selvagem)
Joaquim X. Sousa Jr., Luis A. Diaz, Donald P. Eaton, Gu ¨nter Hans-Filho, Elder Lanzani de Freitas, Livia Delgado,
Ligia Maria F. Ichimura, Fla ´via Cristaldi, Renata Orlandi, Norival Kesper Jr., Eufrosina S. Umezawa, Evandro A. Rivitti,
Valeria Aoki,* and The Cooperative Group on Fogo Selvagem Research
Department of Dermatology, University of Sa ˜o Paulo Medical School, Sa ˜o Paulo, Brazil; Department of Dermatology, University of
North Carolina at Chapel Hill, North Carolina; Wildlife Conservation Society, Campo Grande, MS, Brazil; Federal University of
Mato Grosso do Sul, Brazil; Instituto de Medicina Tropical, University of Sa ˜o Paulo Medical School, Sa ˜o Paulo, Brazil
desmoglein 1 (Dsg1), a desmosomal glycoprotein. In certain settlements of Brazil, a high prevalence of FS (3%) is
reported, suggesting environmental factors as triggers of the autoimmune response. Healthy individuals from endemic
areas recognize nonpathogenic epitopes of Dsg1, and exposure to hematophagous insects is a risk factor for FS. Fogo
selvagem and Chagas disease share some geographic sites, and anti-Dsg1 has been detected in Chagas patients. Indeter-
minate Chagas disease was identified in a Brazilian Amerindian population of high risk for FS. In counterpart, none of
the FS patients living in the same geographic region showed reactivity against Trypanosoma cruzi. The profile of anti-
Dsg1 antibodies showed positive results in 15 of 40 FS sera and in 33 of 150 sera from healthy individuals from endemic
FS sites, and no cross-reactivity between Chagas disease and FS was observed.
Fogo Selvagem (FS) is an autoimmune bullous disease with pathogenic IgG autoantibodies recognizing
Pemphigus foliaceus (PF) is an autoimmune bullous derma-
tosis driven by immunoglobulin G (IgG) autoantibodies that
recognize glycoproteins involved in epidermal adhesion. The
clinical expression of the autoimmune process is blister for-
mation, consequent to epidermal detachment (acantholysis).
These autoantibodies bind to the extracellular domains of
desmoglein 1 (Dsg1), a cadherin located in the desmosomal
core of the keratinocyte surface.1–4
There are two main forms of PF: the classic one, with uni-
versal distribution, and the endemic form, also known as Fogo
Selvagem (FS), prevalent in certain regions of Brazil and other
Latin American countries.1,5Main differences between the
classic and the endemic presentation include peculiar epidemi-
ological features, which are unique to FS, such as the presence
of familial cases, involvement of children and young adults, and
specific endemic settlements.6
The peak of FS in Brazil occurred in the first half of the
20th century. Aranha-Campos reported 604 cases through
1880 to 1940, where 26.5% were blood related7: a decline of
the disease, concurrent with the development of the settle-
ments has been observed. New foci in the Midwestern Brazilian
States (Goia ´s, Mato Grosso, Mato Grosso do Sul) reported
yearly incidences varying from 0.09 cases/10,000 inhabitants
to 0.83 cases/10,000 inhabitants.8Frequency of 30.7 FS cases/
year through 1990 to 1999 in the State of Mato Grosso do Sul
has been detected.9Endemic sites of PF were also found
in other countries such as Colombia, Venezuela, Paraguay,
Fogo Selvagem has a complex pathogenesis, which includes
genetic, immunological, and environmental factors. A Brazilian
Amerindian Terena reservation, located at Limao Verde,
Aquidauana, State of Mato Grosso do Sul (MS), with a high
prevalence of FS (3%), has been closely followed up, once its
main features includes a geographic, limited distribution of
FS cases, that exhibit familial and temporal clustering.5,15–17
The immune response in FS is characterized by pathogenic
IgG4 auto-antibodies that are driven to the extracellular
1 and 2 domains of Dsg1 (EC1-2).18Interestingly, 55% of
healthy individuals living in endemic FS areas generate anti-
Dsg1 antibodies that recognize the extracellular 5 domain of
Dsg1 (EC-5), a nonpathogenic epitope of the molecule. In
those genetic predisposed individuals, intra-molecular spread-
ing may occur, leading to an EC1-2-oriented IgG4 response,
and therefore precipitating FS onset.6,18There is also evidence
of other immunoglobulin classes in FS pathogenesis: circulat-
ing IgM autoantibodies directed against Dsg1 are found in
FS patients and in healthy individuals living in endemic
areas, indicating a role as serological markers for the dis-
ease19; moreover, an IgE-based immune response to Dsg1
was detected in the sera of 81% of FS patients.20These
findings lead to the hypothesis of continuous exposure to an
environmental antigen that may share epitopes to Dsg1, and
become a strong stimulus to nonpathogenic anti-Dsg1 IgM
and IgG production in areas at high risk for FS.20
The genetic influence on FS is characterized by a positive
association with the human leukocyte antigen alleles HLA-
DRB1-0404, -1402 or -1406, with a relative risk of 14. A
sequence of eight amino acids (LLEQRRAA) at the posi-
tions 67–74 in the third hypervariable domain of the DRB1
gene is shared by these alleles, conferring susceptibility to
In genetically predisposed individuals, there may be trig-
gers that initiate the immune response in FS through an
antigen mimicry process.16It is hypothesized that a break
of immune tolerance follows exposure to some environ-
mental factor(s) that include hematophagous insect bites, as
reported elsewhere.16,23,24The potential role of black fly trig-
gering the autoimmune response in FS is supported by two
main studies: exposure to simuliid bites as a risk factor for FS
(4.7 odds ratio),23and the predominance of a certain black
fly species (Simulium nigrimanum) in endemic areas, when
*Address correspondence to Valeria Aoki, Av. Dr. Ene ´as de Carvalho
Aguiar 255, Sala 3016, Departamento de Dermatologia-Hospital das
Clı ´nicas da Faculdade de Medicina da Universidade de Sa ˜o Paulo,
Sa ˜o Paulo, Brazil, CEP 05403-002. E-mail: email@example.com
compared with non-endemic areas of the Brazilian coast.24
Additional data on the environment triggers is suggested by
Aoki and others,16who reported a high frequency of black fly
(87%), kissing bugs (67%), and bed bugs (60%) bites in FS
patients, and showed that precarious living conditions rep-
resented a significant risk factor for FS. The sialotranscriptome
of S. nigrimanum, the most common black fly species in
endemic FS areas has been isolated, comprising over 70 dis-
tinct genes within over 30 protein families, offers an infinite
source for testing pemphigus patients.25
Some geographic areas of Brazilian vector-mediated tropical
diseases, such as cutaneous leishmaniasis and Chagas disease,
coincide with those endemic areas of FS. A previous work from
our group evaluated the prevalence of anti-desmoglein 1 anti-
body in patients with cutaneous leishmaniasis, onchocerciasis,
and Chagas disease, detecting a high prevalence of circulat-
ing autoantibodies directed against the nonpathogenic extra-
cellular domain 5 of Dsg1 in Chagas disease (58%),leishmaniasis
(43%), and onchocerciasis (81%).17
Chagas disease is one of the major causes of cardiac
chronic disease in Latin America, and is endemic in many
regions of Brazil. However, in the State of Mato Grosso do
Sul (MS), it is not considered an endemic disease26because
of the measures adopted for the vector’s control. Triatoma
infestans, the main vector for Chagas disease in Brazil, has a
low density in MS. Triatoma infestans was found among dif-
ferent regions in MS in the end of the 20th century (1980–
2000), but it has been seldom detected in the last decade. On
the other hand, different Triatominae species such as Triatoma
sordida, Panstrongylus geniculatus, and Rhodnius neglectus
with infection rates by Trypanosoma cruzi varying from 0.1%
to 3.2% have been reported in this geographic region.26
Information about the reactivity against T. cruzi of individ-
uals from endemic areas of FS in the State of Mato Grosso
do Sul is scarce. Therefore, this study aimed to characterize
the immune response to T. cruzi in a population at high risk
MATERIALS AND METHODS
Geographic location of the endemic site. Limao Verde
(LV) reservation is located 25 km Northeast of Aquidauana,
and 160 km West of Campo Grande, the capital of the State of
Mato Grosso do Sul (55°41¢07²W, 20°19¢00²S), as described
elsewhere.27It comprises a total of 1,712 hectares, with
two well-defined geographical regions, LV and Corrego Seco.
In September 2011, 1,349 people among 295 families inhabited
LV reservation. The overall prevalence for FS of this endemic
site was 3% in previous reports.27Most of the houses have dirt
floors, adobe walls, thatched roofs, poorly fitted or nonexistent
doors, and no indoor plumbing or electricity. Poor toilet facili-
ties are a common feature. The majority of individuals sleep on
a raised platform covered by a variety of bedding material.16
Study design. We included 40 FS patients from the LV
Terena Reservation, MS, a FS focus where a clinical and
immunological surveillance has been started since 199317and
150 healthy individuals (selected at random from a total pop-
ulation of 1,349 inhabitants of LV) from September 2008 to
September 2011. Blood samples were obtained by venipunc-
ture and frozen at −70°C. All FS patients fulfilled clinical,
histological, and immunofluorescence criteria for PF.
All participants were informed about the study and signed
an informed consent, approved by the Ethics Committee
(CAPPesq) from our institution.
Serologic assays. The collected samples of the selected
individuals (FS patients and controls from LV) were ana-
lyzed for serological response against T. cruzi epimastigotes
(Biome ´rieux, Marcy l’Etoile, France), using enzyme-linked
immunosorbent assay (ELISA) and indirect immunofluores-
cence (IIF) with IgM and IgG (Biocientı ´fica SA, Buenos
Aires, Argentina) following standard procedures, according
to manufacturers’ instructions as described elsewhere.28,29
Immunoblotting with trypomastigote excreted-secreted
antigens (TESA blot), an assay used as a confirmatory test
for Chagas disease was performed in the five IIF positive
sera to rule out cross-reactivity with leishmaniasis, and is
briefly described as follows30:
The TESA from the Y strain of T. cruzi were obtained
as previously described.30Briefly, the supernatants of LLC-
MK2 cell cultures (in serum-free medium or with 2% fetal
calf serum) infected with T. cruzi were collected when
the concentration of trypomastigotes reached about 10–20+
106/mL. After being centrifuged at 1800+g for 15 min at
4°C, the supernatant containing TESA was then resubmitted
to a second centrifugation (7000+g for 5 min at 4°C) and
used directly without any further treatment or stored at −80°C
in small aliquots.
Proteins from TESA were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, transferred to nitro-
cellulose sheets, and blocked with phosphate buffered saline
(PBS) containing 5% fat-free milkfor 1 h at room temperature.
Membrane strips (5 mm) were incubated with human sera
(1:200) diluted in PBS with 1% milk for 2 h or overnight at
room temperature, washed, and the bound antibodies were
detected with horseradish peroxidase (HRP)-labeled anti-
human IgG (Sigma, St. Louis, MO), diluted (1:4000). The color
of detected bands was developed by addition of 0.1% hydrogen
peroxide and 4-chloro-1-naphthol. Samples were considered
positive when a large 150–160 kDa band and/or five bands
between 130 and 200 kDa were observed.
The serological profile of all sera (total IgG) against a recom-
binant form of human Dsg1, containing the entire extracellular
domain and a C-terminal His-tag, described elsewhere, was
tested by ELISA.6,18,31Briefly, the ectodomain of Dsg-1 with a
carboxy-terminal His-tag were produced in High Five insect
cells by infection with the recombinant baculovirus stock of
Dsg-1 (UNC Immunodermatology Laboratory, Department of
Dermatology, University of North Carolina at Chapel Hill, NC).
To generate the deglycosylated Dsg1, tunicamycin (Sigma) was
added to the culture medium (0.5 mg/mL) at the time of infec-
tion. The tunicamycin-treated (deglycosylated) and untreated
(glycosylated) recombinant Dsg1 was purified by nickel affinity
chromatography and used for ELISA assay. The ELISA plates
were coated with 200 ng/well of purified Dsg1 at 4°C overnight.
After washing with Tris-buffered saline containing 3.7 mM Ca++
and 0.05% Tween-20 (TBS/Ca++/T-20), the plate was blocked
with 1% bovine serum albumin (BSA) in TBS/Ca++/T-20 at
room temperature for 1h. The plate was then incubated with
duplicate 1:100 dilutions of serum samples for 1 h at room temp-
erature. Following wash, the plate was incubated with a
1:1000 dilution of HRP-labeled mouse anti-human IgG or with
1:2000 dilution of HRP-conjugated mouse anti-human IgG
(Zymed, San Francisco, CA). Results were expressed as index
SOUSA AND OTHERS
value units, and a cut-off value of 20 arbitrary units was used
to separate positive from negative sera; values > 20 were
Age and gender distribution. FS patients. There was a
slight predominance of male patients (22 male: 18 female) and
the median age was 32 years (ages ranging from 12 to 76 years).
Close familial clustering was present in 34 of 40 (85%) of the
patients, mostly parents/children or siblings. As for ethnic dis-
tribution, Terena Indians comprised the majority (32 of 40),
and eight individuals were mestizos of Terena origin.
Non-FS individuals. Inthis clustered group of 150 individuals
from LV, with a female predominance (89 female: 61 male),
ages varied from 4 to 92 years, median age of 22.5 years;
ethnic distribution revealed that 57% were Terena Indians
and 43% mestizos.
Chagas disease assays. ELISA. The ELISA assays using
T. cruzi epimastigotes for Chagas disease detected a negative
response in 39 but one FS patient (cutoff 0.303, median 0.304)
The ELISA assays using T. cruzi epimastigotes for Chagas
disease detected 5 of 150 non-FS patients from LV (cutoff
0.303, median 0.304) (Table 2).
IIF. None of the 40 FS sera showed reactivity against
T. cruzi epimastigotes by IIF. The IgG antibodies that recog-
nized T. cruzi epimastigotes were detected in 5 of 150 non-FS
sera, titers varying from IIF 1:320 to > 1:640. Three of five
individuals shared the same house, and were blood-related
to FS patients. One of five individuals showed both IgM and
IgG antibodies directed to T. cruzi.
TESA-blot. Five non-FS individuals confirmed indetermi-
nate Chagas disease by positive results using immunoblotting
with trypomastigote excreted-secreted antigens (Figure 1A
Recombinant desmoglein 1 ELISA assay (total IgG). FS
patients. Fifteen of 40 FS sera showed positive results by
rDsg1 (Table 1), index values varying from 23 to 422 (mean:
133). The FS sera with negative rDsg1 results by ELISA
corresponded to those in FS remission.
Non-FS individuals. Thirty-three healthy individuals out of
150 (22%) recognized rDsg1 by ELISA (Table 2), index
values ranging from 20 to 227 (mean: 86). It is noteworthy to
report that none of the non-FS individuals that recognized
T. cruzi antigens showed positive ELISAs for rDsg1.
Endemic PF or FS represents a unique model of an auto-
immune condition that may be triggered by environmental
factors in genetic-prone individuals. The antigenic target is
the extracellular portion of desmoglein 1, an adhesion mole-
cule of the cadherin superfamily, which is recognized by path-
ogenic IgG, especially of the IgG4 isotype.6,22
The recognition of Dsg 1 epitopes is not restricted to
patients with the disease, as previously reported.8In this
study, we detected 22% of reactivity against Dsg1 in non-FS
individuals, indicating a possible environmental stimulus in
the development of autoantibody formation. Some findings
concerning the nonpathogenic response towards Dsg1 are rel-
evant to reinforce the environmental hypothesis in FS, as
follows: the lower prevalence of the IgG anti-Dsg1 response
in normal subjects that live far away from the endemic sites
(13%),32the predominance of a certain black fly species,
Simulium nigrimanum, in FS regions,24an enhanced IgM or
IgE anti-Dsg1 immune response in FS,19,20and finally, the
epidemiological data emphasizing the relevance of housing
conditions and exposure to hematophagous insects, such as
bedbugs and kissing bugs of patients with the disease.16
Individuals with parasitic diseases that are vector-mediated,
such as onchocerciasis, cutaneous leishmaniasis, and Chagas
Chagas disease serology and autoantibodies anti-desmoglein 1 in
patients with Fogo Selvagem using ELISA*
Fogo Selvagem (rDsg1)
Total PositiveNegative Indetermined
*ELISA = enzyme-linked immunosorbent assay; rDsg1 = recombinant Desmoglein 1.
Chagas disease profile and autoantibodies anti-desmoglein 1 tested by
ELISA in non-Fogo Selvagem individuals from Limao Verde, MS
Non-FS individuals (rDsg1)
FS = Fogo Selvagem; rDsg1 = recombinant Desmoglein 1.
secreted antigens (TESA blot) polypeptides recognized by IgG
(lanes 1 to 5) from non-FS patients’ sera from Limao Verde (LV).
(B) Lanes 1¢and2¢, positive andnegative controls, respectively. Patients
in lanes 1, 2, 3, 4, and 5 were classified as having indeterminate forms
of Chagas disease, recognizing a 150- to 160-kDa band (chronic-phase
antigen). Molecular mass markers are on the left. The molecular mass
standards used were 205 kDa (rabbit muscle myosin); 116 kDa
(Escherichia coli galactosidase); 97 kDa (rabbit muscle phosphorylase
b); 66 kDa (bovine serum albumin).
(A) Immunoblotting with trypomastigote excreted-
T. CRUZI REACTIVITY IN ENDEMIC AREA OF FOGO SELVAGEM
disease, often possess circulating nonpathogenic anti-Dsg1
autoantibodies.17In the pre-clinical phase of FS, there is an
antigen-driven selection of anti-Dsg1 B cells,33but the real
source of this antigen remains to be determined.
Chagas disease is a major public health problem in many
Latin America countries, but there are differences about its
epidemiology in Amerindian populations living in these geo-
graphic sites.34In native South American populations located
in highlands (i.e., Bolivia, Argentina, Chile), records of Chagas
disease date since the pre-Columbian era.34Meanwhile, there
is no report of the disease among lowland Amerindians or of
Triatoma sp. living in their traditional houses.35
Among the native Brazilian population, there is no evi-
dence of Trypanosoma sp. in Xingu, Asurini Indians,
Karitiana and Surui Indians.35Coimbra36performed a sero-
logical survey with the Xavante Indians from Mato Grosso,
and found negative results in all 168 individuals tested for
It has been hypothesized that Chagas disease is endemic in
natives from the highlands because of features related to early
domiciliation of triatomines and maintenance of the domestic
cycle of T. cruzi.36,37On the other hand, Amerindians in the
lowland used to live in small settlements, with high village
mobility and absence of domestic animals, similar to the condi-
tions observed among native populations studied in Brazil.36
The Terena population of LV reservation shows closer fea-
tures with those populations of the highland Amerindians,
i.e., animal domestication, raising indoor chickens, and low
mobility. These conditions, when associated with the house
with thatched roofs or adobe walls (Figure 2) provide an
adequate environment to the domiciliation process of many
species of triatomines. Our entomologist (DPE) performed
an entomologic survey in LV, and detected four predominant
sordida, Rhodnius prolixus, and Panstrongylus geniculatus.
Rates of natural infection with T. cruzi have been not yet
recorded for those species (Eaton and others, unpublished
data). These data are corroborated by previous performed
studies on the profile of Reduviidae in a domestic environ-
ment in the State of Mato Grosso do Sul.26
Although there were reports of the presence of triatomine
inside the houses, and frequent exposure to kissing bugs in
previous studies performed in the LV reservation, no Chagas
disease has been so far detected in this endemic FS site, which
has been followed up since 1993.15,16To date, we did not find
cross-reactivity with desmoglein 1 among T. cruzi positive
sera in our samples, suggesting no relationship between
T. cruzi and FS. In Brazil, there is a single report on IgG
reactivity (38%) against trypomastigote forms of T. cruzi in
eight sera from PF patients by IIF. However, there was no
further information about those PF patients, and relevant
data such as geographic location and demographic character-
istics of these individuals were not available.38
The mechanisms involved in anti-Dsg1 autoantibodies for-
mation in patients with Chagas disease remain unknown. One
of the possibilities includes compounds of hematophagous
insect saliva inducing an immune response against Dsg1.17It is
interesting to note that antibodies of the IgG4 subclass
directed against Triatoma infestans salivary gland proteins are
produced by individuals living in triatomine-infested areas.39
Further studies are necessary to improve our understanding of
such mechanisms; Assumpc ¸a ˜o and others40recently described
sialotranscriptome of T. matogrossensis from high-risk areas
for FS, which may facilitate the identification of antigens with
potential role for triggering FS, as well as development of
biomarkers for low-level infestation of triatomines.
Conventional methods such as ELISA and IIF have been
established for the serologic diagnosis of Chagas disease. In
previous studies, ELISA sensitivity varied from 97.7% to
100%, and specificity, 93.3% to 100%; IIF showed sensitivity
from 72% to 100% and specificity from 96% to 100%. Accu-
racy of both assays displays the best results in indeterminate
and chronic stages, although there is frequent cross-reactivity,
especially with leishmaniasis.41,42Fortunately, TESA is a diag-
no cross-reaction for the 130- to 200-kDa antigen (acute-phase
antigens) or the 150- to 160-kDa antigens (chronic-phase anti-
gens).30In our samples, TESA blot (Figure 1) showed bands
that correspond tochronic-phaseantigens, confirmingthe inde-
terminate stage for all five positive non-FS individuals from
endemic areas of FS, previously tested by ELISA and IIF.
The description of reactivity against desmogleins 1 and 3
has been reported in patients from Tunisia, with visceral
leishmaniasis (22%) and hidatidosis (40%). In counterpart,
no significant difference was found in PF patients and con-
trols concerning the immune response against the above para-
sitic agents.43Similarly, Brazilian patients with mucocutaneous
leishmaniasis did show reactivity against Dsg1 in 43% of the
cases; however, all FS patients from LV, except one, developed
mucocutaneous leishmaniasis when tested by indirect immuno-
fluorescence and ELISA (Diaz and others, unpublished data).
Pathophysiology of Chagas disease is complex and has
been related to an autoimmune process. Immune response
in T. cruzi chronic infection has features of delayed-type hyper-
sensitivity with predominance of CD8+over CD4+T-cell sub-
sets. Although cell-mediated immunity plays a central role in
that process, humoral immunity may have participation through
IgG directed against self-antigens such as neurons, sciatic nerve
homogenates, and small nuclear ribonucleoproteins. Moreover,
complement membrane attack complexes were identified in
cardiac myocytes from Chagas disease patients.44
Similar to FS, molecular mimicry appears as an important
phenomenon in autoimmunity of Chagas disease. Trypanosoma
cruzi antigens such as B13 protein, microsomal fraction,
Typical house in Limao Verde - Terena Reservation,
SOUSA AND OTHERS
described as molecules that may induce cross-reactivity with
self-antigens found in human heart muscle (cardiac myosin,
human ribosomal protein) or nervous tissue. Yet, anti-neuron
autoantibodies found in Chagas disease may be linked to auto-
nomic nervous system dysfunction in those patients. Moreover,
T-cell clones sensitized to B13-protein in chronic Chagas
disease with cardiomyopathy have been identified, showing
multiple cross-reactive epitopes between T. cruzi B13 protein
and human cardiac myosin heavy chain.44
Our study revealed the occurrence of indeterminate Chagas
disease in an Amerindian Terena population at high risk for
FS in Brazil. Despite the absence of coexistence of the two
conditions, clinical, epidemiological, and immune surveillance
for FS and Chagas disease in this endemic area is mandatory,
once both conditions share the same environmental milieu.
glycolipids, and ribosomal proteinhave been
Received March 31, 2012. Accepted for publication June 22, 2012.
Financial support: This work was partially supported by a National
Institute of Health grant-NIAMSD 5 R01 AR032599-29 (LAD).
Disclaimer: The authors declare no conflicts of interest.
Authors’ addresses: Joaquim X. Sousa Jr., Elder Lanzani de Freitas,
Livia Delgado, Ligia Maria F. Ichimura, Fla ´via Cristaldi, Renata
Orlandi, Evandro A. Rivitti, and Valeria Aoki, Departamento de
Dermatologia-Hospital das Clı ´nicas da Faculdade de Medicina da
Universidade de Sa ˜o Paulo, Sa ˜o Paulo, Brazil, E-mails: jxsj@
yahoo.com, firstname.lastname@example.org/, email@example.com,
firstname.lastname@example.org, email@example.com, re_orlandi@hotmail
.com/, firstname.lastname@example.org, and email@example.com. Luis
A. Diaz, Department of Dermatology, University of North Carolina
at Chapel Hill, Chapel Hill, NC, E-mail: firstname.lastname@example.org. Donald
P. Eaton, Wildlife Conservation Society, Campo Grande, MS, Brazil,
E-mail: email@example.com. Gu ¨nter Hans-Filho, Department of
Dermatology, Federal University of Mato Grosso do Sul, Campo
Grande, Brazil, E-mail: firstname.lastname@example.org. Norival Kesper Jr.
and Eufrosina S. Umezawa, Instituto de Medicina Tropical, University
of Sa ˜o Paulo Medical School, Sa ˜o Paulo, Brazil, E-mails: nkesper@usp
.br and email@example.com.
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