FEMS Immunology and Medical Microbiology 45 (2005) 435–441

www.fems-microbiology.org

Identification of the infectious source of an unusual outbreak of

histoplasmosis, in a hotel in Acapulco, state of Guerrero, Mexico
Maria Lucia Taylor a,*, Guillermo M. Ruı́z-Palacios b, Marı́a del Rocı́o Reyes-Montes a,
Gabriela Rodrı́guez-Arellanes a, Laura E. Carreto-Binaghi a,
Esperanza Duarte-Escalante a, Aurora Hernández-Ramı́rez a, Armando Pérez c,
Roberto O. Suárez-Alvarez a, Yuri A. Roldán-Aragón b, Rafael Romero-Martı́nez a,
Jorge H. Sahaza-Cardona a, José Sifuentes-Osornio b,
Luis E. Soto-Ramı́rez b, Gabriela R. Peña-Sandoval a
a
Laboratorio de Inmunologı́a de Hongos, Departamento de Microbiologı́a y Parasitologı́a, Facultad de Medicina,
Universidad Nacional Autónoma México (UNAM), Ciudad Universitaria s/n, México DF 04510, Mexico
b
Departamento de Infectologı́a, Instituto Nacional de Ciencias Médicas y Nutrición ‘‘Salvador Zubirán’’, México DF 14000, Mexico
c
Departamento de Biologı́a Celular y Tisular, Facultad de Medicina UNAM, México DF 04510, Mexico

Received 29 April 2005; accepted 27 May 2005

First published online 19 July 2005

Abstract

Three isolates of Histoplasma capsulatum were identified from mice lung, liver, and spleen inoculated with soil samples of the X
hotelÕs ornamental potted plants that had been fertilized with organic material known as compost. The presence of H. capsulatum in
the original compost was detected using the dot-enzyme-linked immunosorbent assay. Nested-PCR, using a specific protein Hcp100
coding gene sequence, confirmed the fungal identification associated with an unusual histoplasmosis outbreak in Acapulco.
Although, diversity between the H. capsulatum isolate from the hotel and some clinical isolates from Guerrero (positive controls)
was observed using random amplification of polymorphic DNA based-PCR, sequence analyses of H-anti and ole fragment genes
revealed a high homology (92–99%) between them.

2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Outbreak; Histoplasma capsulatum; Nested-PCR; RAPD-PCR; Gene sequencing

1. Introduction Microconidia together with small hyphal fragments are

In its natural environment, the dimorphic fungus
Histoplasma capsulatum var. capsulatum (Darling, 1906)
grows favorably in bat and/or bird guano producing a
multicellular filamentous form, characteristic of its
mycelial-phase, which presents macro- and microconidia.
*
Corresponding author. Tel.: +52 55 5623 2462; fax: +52 55 5573
5564.
E-mail addresses: emello@servidor.unam.mx, luciataylor@yahoo.
com.mx (M.L. Taylor).
the main fungal structures that infect susceptible hosts
through their inhalation in contaminated places, starting
the process of histoplasmosis infection [1].
In Mexico, this disease represents an occupational
and environmental health problem, and people in the
countryside are particularly affected, especially miners,
peasants, and guano collectors, due to their work in gi-
ven enclosed spaces where guano accumulates [2–6].
Histoplasmosis has been registered in every state of
the country, and when an outbreak suddenly appears,

0928-8244/$22.00
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsim.2005.05.017
436 M.L. Taylor et al. / FEMS Immunology and Medical Microbiology 45 (2005) 435–441

the lethality percentage gets higher. However, the pres-
ence of fungus propagules in urban areas may be also
important to explain clinical cases that do not refer
any exposure to high risk infection sites [2].
In 2001, the Department of Health of the USA noti-
fied the Centers for Disease Control and Prevention
(CDC) of an outbreak of histoplasmosis affecting Amer-
ican tourists in Acapulco. Morgan et al. [7] reported a
cohort study of this outbreak of acute pulmonary histo-
plasmosis among American travelers, mostly college stu-
dents, who were vacationing during their spring break in
Acapulco, Mexico, from March to May 2001. Frequent
use of the hotelÕs stairwells, where construction was
ongoing, was associated with increased risk of illness.
However, the source of infection of the outbreak was
not accurately confirmed.
The present work reports the identification of the
source of infection in an outbreak that occurred in Sep-
tember 2001, in a hotel in Acapulco, Guerrero (GR),
Mexico, which most likely was the same source of infec-
tion of the outbreaks that occurred in March and May
in the same hotel in 2001.
lected (Table 1) in October. Each sample (1 g) was
prepared in 10 ml phosphate buffered saline (PBS), pH
7.2, supplemented with 50 lg ml
1 streptomycin and
100 IU ml
1 penicillin (Lakeside, Mexico City, MX),
and allowed to precipitate. Finally, 0.5 ml of each suspen-
sion was intraperitoneally inoculated into inbred male
BALB/c mice (4-weeks old), using five mice per sample.
2.2. Bio-collector mice and mice processing
Sixty BALB/c mice were used as targets of infection
and distributed, in special boxes, in 12 different sites of
the hotel to expose them to natural infection overnight.
One box of these different sites was randomly chosen as
control (see Table 2).
All mice were kept under observation to find histo-
plasmosis signs, and retro-orbital bleeding was per-
formed on days 10 and 17 to separate sera to detected
anti-H. capsulatum antibodies using dot-enzyme-linked
immunosorbent assay (dot-ELISA), as described by Pa-
pas et al. [8]; after the last bleeding, mice were killed for
fungal isolation and histopathology assessment.

2.3. Fungal isolation and Histoplasma capsulatum

2. Materials and methods identification

2.1. Samples
Nineteen environmental samples from different places
of the X hotel in Acapulco, GR, and three compost
(organic fertilizer) samples from a commercial plant
nursery, located near the Acapulco airport, which sup-
plied compost material to the hotel during 2001, were col-
The guts, lungs, livers, and spleens were processed for
fungal isolation, as previously described by Taylor et al.
[9]. Briefly, homogenates of each organ sample were ob-
tained under sterile conditions, and mixed in 5 ml PBS,
pH 7.2, supplemented with antibiotics. Tissue homoge-
nates were centrifuged at 300g for 15 min, and 0.1 ml
supernatant of each homogenate sample was inoculated

Table 1
Dot-ELISA of processed samples
Number Type Place of collection Dot-ELISA
S-158 Guano (marine birds) Rock in the sea Negative
S-159 Soil (planter) Restaurant A (1st planter) Positive
S-160 Soil (planter) Restaurant A (4th planter) Positive
S-161 Soil (planter) Restaurant A (6th planter) Positive
S-162 Soil (planter) Restaurant A (exterior planter) Positive
S-163 Soil (planter) Auditorium (ornamental potted plants) Positive
S-164 Ventilator dust Restaurant A (5th ventilator) Negative
S-165 Soil (planter) Restaurant A (1st planter) Negative
S-166 Leaves (ornamental plants) Restaurant A (1st planter) Negative
S-167 Leaves (ornamental plants) Restaurant A (2nd planter) Negative
S-168 Leaves (ornamental plants) Restaurant A (3rd planter) Negative
S-169 Dust (air conditioner) Restaurant B Negative
S-170 Guano (pigeon) Swimming pool (planter) Negative
S-171 Mold (wall) Right lateral hotel wall Negative
S-172 Leaves (ornamental plants) Swimming pool (planter) Negative
S-175 Sand Restaurant B (path) Negative
S-176 Bat guano (scarce) Restaurant B (ceiling) Negative
S-177 Bat guano (scarce) Elevator shaft Negative
S-178 Dust (air conditioner) Restaurant B Negative
S-180 Compost (in the shade) Plant nursery Positive
S-181 Compost (in the sun) Plant nursery Negative
S-182 Compost/sand (mixture) Plant nursery Positive
 M.L. Taylor et al. / FEMS Immunology and Medical Microbiology 45 (2005) 435–441
Table 2
Dot-ELISA of bio-collector mice
Location Dot-ELISA
1 – Restaurant B (ceiling with accumulated bat Negative
guano)
2 – Restaurant B (ceiling near the kitchen) Negative
3 – Restaurant B (ceiling near the outside fissure) Negative
4 – Restaurant B (ceiling near the windows) Negative
5 – Restaurant B (central table, controls*) Negative
6 – Guest elevator shaft Negative
7 – Auditorium (ornamental potted plants) Positive
8 – Night club (air conditioner) Negative
9 – Restaurant A (1st planter) Positive
10 – Restaurant A (2nd planter) Negative
11 – Restaurant A (5th planter) Negative
12 – Restaurant A (6th planter) Positive
Serum samples were obtained on days 10 and 17 after exposing mice to
natural infection.
*
Restaurant B (central table) was chosen as a control because it had
very few reports of infected people, when a cohort study was made by
Mexican Epidemiologists (unpublished data).
in Petri-plates with Mycobiotic agar and Brain Heart
Infusion (BHI) agar (Bioxón, Mexico City, MX) supple-
mented with 0.05% cycloheximide and 0.005% chloram-
phenicol. Plates were incubated at 28 C and checked
daily for fungal growth during 2 to 3 weeks; suspected
colonies of H. capsulatum were cultured after serial dilu-
tions to separate and isolate each one.
Once the macro and microscopic morphology of
H. capsulatum had been observed, the mycelia-to-yeast
conversion was processed, at 37 C, in synthetic-liquid
medium [10] and in BHI broth (Bioxón) supplemented
with 0.1% L-cysteine and 1% glucose. Finally, the exoan-
tigen test of Kaufman and Standard [11] was performed
by the double immunodiffusion method [12] to confirm
antigenic identity of the fungus. A positive rabbit hyper-
immune serum and a standardized reference exoantigen
were used.
2.4. Histopathology
The organs from each inoculated mice were fixed with
Zamboni and De Martino solution [13] during 24 h
at least, and then processed to be paraffin embedded.
Tissue sections of 6 lm were stained for fungi by means
of the Grocott method.
2.5. Dot-enzyme-linked immunosorbent assay
A deproteinized polysaccharide-protein complex of
Histoplasma (DPPC-Histo), as described elsewhere
[14], was used as antigen for dot-ELISA. The purified
DPPC-Histo (50 ng 20 ll
1 of protein) was applied in
separate wells to a 0.45 lm nitrocellulose membrane
narrow strip (BioTrace, GelmanSciences, Inc. MI,
USA) using a 96-well microfiltration Bio-Dot apparatus
(Bio-Rad, Richmond, CA, USA). DPPC-Histo binding
437
to the membrane was favored by vacuum filtration, dur-
ing 15 min. The membrane was blocked with PBS, pH
7.2, plus 3% bovine serum albumin (PBS-A) for 1 h, to
prevent non-specific binding. The membrane was
washed with 0.05% Tween 20-PBS, and 1:50 or 1:100
PBS-A-diluted, of each mouse serum sample, was ap-
plied to different membrane narrow strips. Membranes
were incubated 20 h at 4 C. After membrane washing,
1:500 dilution of a biotinylated anti-mouse IgG anti-
body produced in goat (Sigma Chemical Co., St. Louis,
MO, USA) was added and incubation was allowed dur-
ing 1 h. After washing membranes again, they were incu-
bated for another hour with streptavidin-peroxidase
conjugate. Membranes were again washed and
immersed in a fresh mixture of 25 mg of 3 0 ,3 0 -diam-
inobenzidine-4HCl (Sigma) in 50 ml of 0.1 M Tris–
HCl buffer, pH 7.5, plus 50 ll of 30% H2O2. After
enzyme-substrate reaction, membranes were washed
and dried. Positive enzymatic reactions were visible as
brown spots. Sera from infected and non-infected mice
were processed as positive and negative controls. Crite-
ria to define positive reaction in sera from tested mice
were standardized taking into account that three out
of five mice revealed brown spots using the dot-ELISA.
2.6. DNA sampling and nested-PCR assay
The H. capsulatum isolate recovered from the X
hotel, where several histoplasmosis outbreaks were
detected, was characterized by nested-PCR, random
amplification of polymorphic DNA based-PCR
(RAPD-PCR), and sequencing assays. DNA samples
were extracted from one H. capsulatum isolate (EH-
554B) recovered from an infected mouse inoculated with
contaminated planter soil collected in the hotel and from
six H. capsulatum clinical isolates from different geo-
graphic origins in America (EH-46, EH-316, and EH-
557 from GR-Mexico; 01558 from Argentina; WCh
from Colombia; and H.1.02.W from Guatemala) used
as positive controls, as well as from Sporothrix schenckii
(DNA negative control of non-related strain). Isolates
were taken from the Fungal Immunology Laboratory
Culture Collection of the Department of Microbiology
and Parasitology, School of Medicine-UNAM. Proce-
dures for isolation of whole-cell DNA were performed
as described elsewhere by Reyes-Montes et al. [15]. Each
purified DNA was centrifuged at 15 000g for 5 min, and
the pellet was washed with 70% ethanol, dried and resus-
pended in 100 ll of water. The DNA was quantified
fluorometrically and checked against standard concen-
trations using agarose gel electrophoresis. Finally, it
was frozen at
20 C until required.
Nested-PCR, with minor modifications, was per-
formed as described by Bialek et al. [16]. DNA samples,
from the EH-554B isolate and from six positive H. capsu-
latum controls, as well as from one clinical isolate of
438 M.L. Taylor et al. / FEMS Immunology and Medical Microbiology 45 (2005) 435–441

sporotrichosis (negative control), were processed. Two 2.8. DNA sequencing and data analysis
sets of primers were used, corresponding to gene encod-
ing for a 100-kDa protein (Hcp100) unique to H. capsu- DNA fragments of two H. capsulatum nuclear genes
latum [17]. The outer primer set Hc I (5 0 -GCGTTCCGA- were compared between the EH-554B H. capsulatum iso-
GCCTTCCACCTCAAC-3 0 ) and Hc II (5 0 -ATGT- late from the X hotel and all clinical isolates from GR
CCCATCGGGCGCCGTGTAGT-3 0 ) delimit a 391 (EH-46, EH-316, and EH-557) and were PCR-amplified
nucleotides sequence of the gene. The inner primers Hc as described by Kasuga et al. [19]. Selected primers
III (5 0 -GAGATCTAGTCGCGGCCAGGTTCA-3 0 ) (Operon Technologies) were: For H-anti gene (H antigen
and Hc IV (5 0 -AGGAGAGAACTGTATCGGTGGC- precursor), H-anti3 (5 0 -CGCAGTCACCTCCATAC-
TTG-3 0 ) delimit a specific 210-nucleotides sequence, the TATC-3 0 ) and H-anti4 (5 0 -GCGCCGACATTAACCC-
primers were supplied by Operon Technologies Inc. 3 0 ); for ole gene (delta-9 fatty acid desaturase), ole3
(Alameda, CA, USA). DNA amplification was per- (5 0 -TTTAAACGAAGCCCCCACGG-3 0 ) and ole4 (5 0 -
formed on a Perkin–Elmer Cetus DNA thermal cycler CACCACCTCCAACAGCAGCA-3 0 ). The PCR prod-
(Emeryville, CA, USA) and the first PCR was performed ucts were sent to the Institute of Cellular Physiology,
in a 50 ll reaction mixture containing 200 lM of dNTPs UNAM-Mexico, for sequencing in an ABI-automated
(Applied Biosystems Inc., Foster City, CA, USA), 1 mM DNA sequencer (Applied Biosystems). Sequences were
MgCl2, 100 pmol of each outer primer, 1.5 U Taq DNA generated for a single strand. They were edited, aligned,
polymerase (Applied Biosystems), and 10 ng DNA. and compared by means of the BLAST program, version
Cycling conditions were as follow: one cycle at 94 C
for 5 min; 35 cycles at 94 C for 30 s, 50 C for 30 s and
72 C for 1 min, one cycle at 72 C for 5 min. For the
second (nested) PCR, the mixture was 50 lM dNTPs,
0.25 mM MgCl2, 50 pmol of each primer, 1 U Taq
DNA polymerase, and 1 ll of the first reaction product.
Cycling conditions were as follows: one cycle at 94 C for
5 min; 30 cycles at 94 C for 30 s, 65 C for 30 s, 72 C for
1 min, and one cycle at 72 C for 5 min.
Amplification products were electrophoresed through
1.5% agarose in Tris-borate-EDTA 0.5· buffer. Electro-
phoresis was conducted at 90 V for 60 min. The 123 bp
DNA Ladder (Gibco Laboratories, Grand Island, NY,
USA) was used as molecular marker. The bands were
visualized with a UV transilluminator after ethidium
bromide (0.5 lg ml
1) staining. They were captured with
a documentation system (GeneCam; Syngene, Cam-
bridge, MA, USA) and printed with a thermal printer
(Sony 650, Tokyo Japan).

2.7. Two-primer RAPD-PCR assay

This assay was performed as described by Hu et al. [18]

using only H. capsulatum DNA samples in a 20 ll reac-
tion, using 10 ng H. capsulatum DNA, 2.5 mM MgCl2,
200 lM of each dNTPs (Applied Biosystems), 15 pmol
of each primer 1281 (5 0 -AACGCGCAAC-3 0 ) and 1283
(5 0 -GCGATCCCCA-3 0 ) supplied by Operon Technolo-
gies, and 1 U Taq DNA polymerase (Applied Biosys-
tems). PCR amplification was performed in a thermal
cycler, programmed as follows: one cycle of 7 min at
94 C followed by 45 cycles of 1 min at 92 C; 1 min at
35 C; and 1 min at 72 C. A final cycle of 5 min at
72 C ensured extension of all amplified products, and
samples were maintained at 4 C. The RAPD-PCR pat-
Fig. 1. Microscopic morphology of H. capsulatum isolated from a
terns were analyzed through digital images of ethidium BALB/c mouse inoculated with a sample from a planter (S-162) of
bromide-stained agarose gels (1.5%) captured with a restaurant A. (A) EH-554P from lung; (B) EH-554H from liver; (C)
documentation system (GeneCam). EH-554B from spleen. Bar = 10 lm.
 M.L. Taylor et al. / FEMS Immunology and Medical Microbiology 45 (2005) 435–441
2.0 [20] (National Center for Biotechnology Informa-
tion-NCBI- Databases).
3. Results and discussion
3.1. Histoplasma capsulatum identification
Three isolates were obtained from lung, liver, and
spleen of a mouse inoculated with the S-162 sample from
the exterior planter of restaurant A (Table 1). Isolates
developed brownish colonies with slow and limited
growth. Microscopic morphology revealed the presence
of thin hyphae, microconidia and the typical H. capsula-
tum tuberculate macroconidia (Fig. 1A–C); all isolates
converted to yeast-phase in 1–3 weeks, at 37 C, in
synthetic-liquid medium or in supplemented BHI broth.
Finally, exoantigen production confirmed the identifica-
tion of H. capsulatum isolates by the presence of specific
H and M precipitation lines, when exoantigens were
reacted with a rabbit hyperimmune serum in the double
immunodiffusion test. A register number was assigned to
each isolate, EH-554P, EH-554H, and EH-554B, respec-
tively, in order to incorporate them to the Histoplasma
capsulatum Culture Collection of Fungal Immunology
Laboratory, School of Medicine, UNAM.
3.2. Histopathology
Yeast cells were found in different organs of the mice
inoculated with the S-160 sample (Table 1). Intracellular
Fig. 2. Histopathological findings of mice inoculated with the S-160 sample. (A, B) Yeast-cells within KupfferÕs cells; (C, D) Yeast-cells within
epithelial cells from the bronchiole. Arrows indicate intracellular yeasts stained using the Grocott method. Bar = 10 lm.
439
yeasts compatible with H. capsulatum were observed in-
side KupfferÕs cells (Fig. 2A and B), as well as in bron-
chiolar epithelia (Fig. 2C and D). Inflammatory
responses associated to granulomas were mainly ob-
served in the liver (data not shown).
3.3. Dot-ELISA
The presence of anti-H. capsulatum antibodies in sera
of mice inoculated with different samples collected in the
X hotel (Table 1), as well as in sera of bio-collector mice
placed at strategic points that could be associated with
the source of infection (Table 2), were monitored using
the dot-ELISA. Results suggest that dot-ELISA was a
useful tool to detect infectious sources in the hotel plant-
ers and that the probable origin of infection was associ-
ated with the compost, from a plant nursery, which was
prepared to fertilize the hotel planters (Table 1), and ex-
plain the fungal isolation from organic material con-
tained in the S-162 planter sample (Fig. 1A–C).
Results of sera from bio-collector mice also support
the identification of the source of infection in planters
with soil and compost materials (Table 2). In addition,
it is important to emphasize that the analyzed compost
samples (S-180 to S-182) were taken from the plant
nursery that supplied the hotel during 2001.
3.4. Nested-PCR
The products revealed by the nested-PCR assay,
targeting the Hcp100 protein gene, showed that the
440 M.L. Taylor et al. / FEMS Immunology and Medical Microbiology 45 (2005) 435–441

Fig. 3. Identification of H. capsulatum by nested-PCR of fungal DNA

samples. The assay was performed with two sets of fungal-specific
primers of the 100-kDa protein gene of H. capsulatum (see details
under Section 2). PCR products were analyzed by electrophoresis
through 1.5% agarose gels containing ethidium bromide. First (A) and
nested (B) PCR reactions. M – molecular weight marker was estimated
from 123 bp DNA Ladder; WCh, 01558, H.1.02.W, EH-46, EH-316, Fig. 4. RAPD-PCR patterns of H. capsulatum isolates generated using
and EH-557 – H. capsulatum clinical isolates from different geographic two-primer assay. The reaction and PCR amplification are detailed
origins of the American Continent; EH-554B – H. capsulatum isolate under Section 2. Abbreviations are the same as in Fig. 3.
from the X hotel; Ss –S. schenckii (DNA negative control); C (
)
Negative control of the system. Abbreviations: CO – Colombia; AR –
Argentina; GT; Guatemala; MX/GR – Mexico/Guerrero. Table 3
DNA sequence analyses of two fragments of H. capsulatum nuclear
genes
H. capsulatum isolate from the hotel planters shares the
Isolates H-anti ole
same bands (0.391 and 0.210 Kb in the first and nested
amplifications, respectively) with six clinical isolates Identities Gaps Identities Gaps
(%) (%) (%) (%)
from different geographic origins (Fig. 3A and B).
DNA of S. schenckii, used as negative control, did not EH-554B and EH-46 92 2 96 2
EH-554B and EH-316 94 2 98 0
amplify. This type of PCR has been reported as sensitive EH-554B and EH-557 95 2 96 1
and highly specific for H. capsulatum. Although it is EH-46 and EH-316 96 0 98 0
more frequently used in samples where fungi are scarcely EH-46 and EH-557 96 0 97 (–)
found, its employ in Histoplasma DNA from cultures EH-316 and EH-557 99 0 98 0
was useful to confirm the molecular identification of The PCR products were sequenced taking into account the conditions
the H. capsulatum isolate sampled from the hotel described under Section 2. (
) Negative.
planters.
revealing no critical genome changes in the partial se-
3.5. RAPD-PCR and DNA sequences of two protein- quences of the two genes studied (Table 3).
coding genes The RAPD-PCR and DNA sequence analyses of two
nuclear gene loci distinguished between the H. capsula-
Fig. 4 shows distinct RAPD banding patterns among tum isolate from the hotel planters and the clinical
the studied H. capsulatum isolates. The EH-554B isolate strains from other geographic origins, including clinical
that was recovered from a mouse inoculated with soil isolates recovered from three patients from Acapulco,
sample from a planter of the X hotel differed undoubt- GR. This finding suggests that there could be more than
edly from the other RAPD patterns associated with iso- one molecular pattern of H. capsulatum in GR.
lates from the GR clinical cases, and also differed from
the DNA banding patterns of clinical isolates from
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