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JCM05271-11 version 2 1
Development of specific SCAR markers for detecting Histoplasma capsulatum in clinical 2
and environmental samples 3
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María Guadalupe Frías De León1, Gabina Arenas López2, Maria Lucia Taylor1, Gustavo Acosta 5
Altamirano3, María del Rocío Reyes-Montes1* 6
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1Departamentos de Microbiología-Parasitología and 2Fisiología, Facultad de Medicina, 8
Universidad Nacional Autónoma de México (UNAM), Mexico City, 04510, Mexico, 3Hospital 9
Juárez de México, Mexico City, 07760, Mexico 10
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Running title: Specific-SCAR markers for H. capsulatum detection 12
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*Corresponding author. Mailing address: Laboratorio de Micología Molecular, Departamento de 14
Microbiología-Parasitología, Facultad de Medicina, UNAM, Mexico City, 04510, Mexico. 15
Phone: (55) 5623 2463. E-mail: [email protected] 16
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Copyright © 2011, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.05271-11 JCM Accepts, published online ahead of print on 21 December 2011
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ABSTRACT 18
Sequence-characterized amplified region (SCAR) markers, generated by randomly amplified 19
polymorphic DNA (RAPD)-PCR, were developed to detect Histoplasma capsulatum selectively 20
in clinical and environmental samples. A 1200-bp RAPD-PCR-specific band produced with the 21
1281-1283 primers was cloned, sequenced, and used to design two SCAR markers, 1281-1283220 22
and 1281-1283230. The specificity of these markers was confirmed by Southern hybridization. To 23
evaluate the relevance of the SCAR markers for the diagnosis of histoplasmosis, another 24
molecular marker (M antigen probe) was used for comparison. To validate 1281-1283220 and 25
1281-1283230 as new tools for the identification of H. capsulatum, the specificity and sensitivity 26
of these markers were assessed for the detection of the pathogen in 27 clinical (17 humans, as 27
well as nine experimentally and 10 naturally infected non-human mammals) and 20 28
environmental (10 contaminated soil and 10 guano) samples. Although the two SCAR markers 29
and the M antigen probe identified H. capsulatum isolates from different geographic origins in 30
America, the 1281-1283220 SCAR marker was the most specific and detected the pathogen in all 31
samples tested. In contrast, the 1281-1283230 SCAR marker and the M antigen probe also 32
amplified DNA from Aspergillus niger and Cryptococcus neoformans, respectively. Both SCAR 33
markers detected as little as 0.001 ng of H. capsulatum DNA, while the M antigen probe detected 34
0.5 ng of fungal DNA. The SCAR markers revealed the fungal presence better than the M antigen 35
probe in contaminated soil and guano samples. Based on our results, the 1281-1283220 marker can 36
be used to detect and identify H. capsulatum in samples from different sources. 37
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Keywords: Histoplasma capsulatum; RAPD-PCR; SCAR-markers; molecular diagnosis. 39
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Histoplasmosis is a widespread respiratory infection caused by the fungus Histoplasma 42
capsulatum (2). The disease is endemic in North America, mainly in the Ohio and Mississippi 43
river valleys, and frequent outbreaks occur in several countries of Latin-America. Histoplasmosis 44
has been registered in every state of Mexico (MX), and it has shown a variable prevalence in 45
endemic areas (14, 31, 47). High lethality has been recorded in several Mexican outbreaks (50). 46
The presence of fungal propagules in urban areas has been documented and may be important to 47
explain clinical cases not associated with any exposure to high risk infection sites (46). 48
Infection with the etiologic agent, the dimorphic fungus H. capsulatum, is initiated by 49
inhalation of aerosolized microconidia and mycelial fragments that convert into the virulent yeast 50
phase in the parasitized host. The yeast cells proliferate within the host phagocytes, mainly 51
dendritic cells and macrophages. Usually, the activation of cell-mediated immunity inhibits the 52
yeast’s intracellular multiplication (10). 53
A wide variety of tests are used in the laboratory for the diagnosis of histoplasmosis; however, 54
several of them have particular limitations (13, 41, 51-53). The diagnosis of this mycosis 55
regularly requires histologic examination and/or fungal culture from clinical specimens such as 56
blood, bone marrow, or bronchoalveolar lavage. Isolation of the pathogen requires three weeks 57
for fungal growth, which delays and complicates an accurate diagnosis. Furthermore, the 58
identification of the organism can only be performed in biosafety level three laboratories (41). In 59
addition, confirmatory tests are needed for organisms suspected to be H. capsulatum, because 60
some saprobic microorganisms mimic the morphological mold phase of this fungus. 61
Although several immunological and molecular methods for identifying H. capsulatum have 62
been reported (4, 5, 16, 19, 22, 26, 27, 30, 33, 38, 40, 42, 43, 49), some of them have distinct 63
limitations, such as low sensitivity and specificity (12). Among the molecular methods used for 64
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diagnosis, some fail to detect the fungal presence due to high genetic variability of H. 65
capsulatum. For these reasons, a specific fungal marker, such as a sequence characterized 66
amplified region (SCAR), could solve critical problems in histoplasmosis diagnosis (1, 8, 23, 24, 67
39). 68
In this study, a randomly amplified polymorphic DNA (RAPD)-PCR method was used to 69
screen polymorphic DNA bands in order to select a marker capable of distinguishing H. 70
capsulatum from related pathogenic microorganisms. A selected RAPD-PCR band was converted 71
to a SCAR marker with the aim of developing species-specific and sensitive PCR for H. 72
capsulatum in clinical and environmental samples to allow for a fast histoplasmosis diagnosis. 73
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MATERIALS AND METHODS 75
H. capsulatum. Forty isolates of H. capsulatum from different sources and geographic origins 76
were selected for this study. Twenty-four isolates were from MX: EH-53, EH-316, EH-323-EH-77
328, EH-355-EH-357, and EH-359 human clinical isolates; EH-372-EH-377, EH-383H, EH-78
384H, EH-391, EH-393, and EH-408H from infected bats; and EH-554B from compost. Three 79
isolates were from Guatemala (GT): the Cepa 3 clinical isolate and two isolates from bird guano 80
(L-100-91 and Cepa 2), provided by the Facultad de Ciencias Químicas, GT; four clinical isolates 81
(LA, Gli, DS, and RG) were from Colombia (CO), provided by the Corporación para 82
Investigaciones Biológicas, CO; six clinical isolates (951539, 01559, 01733-01735, and 01737) 83
were from Argentina (AR), provided by the Instituto Nacional de Enfermedades Infecciosas, 84
ANLIS "Dr. Carlos G. Malbrán", AR; the G-186B human reference strain was from Panama 85
(PA,) from the ATCC (American Type Culture Collection); and the G-217B and Downs human 86
reference strains were from the United States of America (USA), also from the ATCC. The 87
isolates and reference strains studied were deposited in the Histoplasma capsulatum Culture 88
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Collection of the Laboratorio de Inmunología de Hongos, Departamento de Microbiología-89
Parasitología, Facultad de Medicina, UNAM. This collection can be accessed at: 90
http://www.histoplas-mex.unam.mx, and it is registered in the database of the World Data Centre 91
for Microorganisms of the World Federation for Culture Collections under the acronym and 92
number LIH-UNAM WDCM817. 93
The fungal isolates and strains were maintained in brain heart infusion agar (Bioxón, Becton-94
Dickinson, Mexico City) at 28οC and preserved in mycobiotic agar (Bioxón) with sterile mineral 95
oil at 4οC. 96
Tissue samples. A total of 17 samples from different tissues obtained from patients with 97
presumptive histoplasmosis were analyzed. Nine samples from different organs obtained from 98
mice and bats experimentally infected with H. capsulatum and 10 tissue samples from naturally 99
infected animals, one of a snow leopard (Uncia uncia), two of maras (Dolichotis patagonum), 100
and seven of wallabies (Macropus rufogriseus), were also processed. Blood samples from healthy 101
human volunteers and tissue samples from uninfected bats and mice were used as negative 102
controls. 103
Soil and guano samples. We analyzed 10 soil samples (free of bird or bat guano) that were 104
experimentally contaminated with H. capsulatum mycelium and 10 samples of bat or bird guano 105
collected in epidemic sites from MX (Oaxaca, Guerrero, Morelos, Sinaloa, Nuevo León, and 106
Puebla). Guano samples were taken from the Guano Collection of the Laboratorio de 107
Inmunología de Hongos, Departamento de Microbiología-Parasitología, Facultad de Medicina, 108
UNAM. As negative controls, some soil samples were collected from sites that, by mycological 109
and molecular procedures, did not reveal fungal presence. The molecular procedure used was a 110
nested PCR assay using the Hcp100 gene fragment (4), with minor modifications (48). 111
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DNA isolation. Mycelium cultures of H. capsulatum isolates and strains were grown at 28οC in 112
glucose yeast-extract medium with shaking, and the cultures were processed for DNA extraction 113
as described elsewhere (35). The DNA from each mycelium culture of Coccidioides immitis, C. 114
posadasii, Paracoccidioides brasiliensis, Blastomyces dermatitidis, Aspergillus fumigatus, A. 115
niger, Candida albicans, Sporothrix schenckii, Chrysosporium carmichaelli, and Malbranchea 116
sp. was extracted under similar conditions. The DNA from Cryptococcus neoformans and 117
mycelium cultures of C. carmichaelli and Malbranchea sp. were kind gifts from Laura Rosio 118
Castañón (Facultad de Medicina, UNAM, MX), B. dermatitidis was a kind gift from Alejandro 119
Bonifaz (Hospital General, MX), and Mycobacterium tuberculosis was a kind gift from Miriam 120
Bobadilla del Valle (Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, MX). 121
DNA from all the fungi tested, as well as M. tuberculosis was used to check the specificity of the 122
H. capsulatum molecular markers studied. The concentration of each DNA sample was 123
quantified fluorometrically and checked against standard lambda phage DNA concentrations by 124
electrophoresis in 0.8% agarose gels with ethidium bromide staining (10 µg/ml). Finally, the 125
DNA was stored at 4ºC. 126
RAPD-PCR for the selection of the H. capsulatum SCAR markers. Three primers were 127
tested, 1281 (5’-AACGCGCAAC-3’), 1283 (5’-GCGATCCCCA-3’), and 1253 (5’-128
GTTTCCGCCC-3’), which were all supplied by Operon Technologies Inc. (Alameda, CA). The 129
1281 and 1283 primers were assessed in the pairwise combination, according to the two-primer 130
RAPD-PCR assay (18), while the 1253 primer was used singly (48). Each RAPD-PCR assay was 131
performed twice to ensure reproducibility. To select the best band for generating the SCAR 132
markers, the RAPD polymorphic patterns were compared to identify a common and reproducible 133
band in the isolates from MX, GT, CO, and AR, as well as in the reference strains from PA and 134
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the USA (see details in Fig. 1). 135
Cloning, hybridization, and sequencing of a selected RAPD band. A selected RAPD-PCR-136
specific band was purified with a QIAquick Gel extraction kit (Qiagen, Inc., Valencia, CA), 137
reamplified using the 1281-1283 primers on the two-primer RAPD-PCR assay (18), and cloned 138
into the pGEM®-T Easy Vector (Promega, Madison, WI). The plasmid harboring insert-DNA 139
with the expected molecular size was extracted, and this resulting insert (the SCAR marker) was 140
used for Southern hybridization assays, to confirm the presence of the selected marker in all of 141
the H. capsulatum isolates and strains studied. Prehybridization and hybridization were 142
performed according to Sambrook et al. (37), at 45°C. The hybridized bands were visualized by a 143
colorimetric method using NBT/BCIP stock solution (Roche Molecular Biochemicals, 144
Mannheim, Germany). Each SCAR marker was sequenced at the Unidad de Biología Molecular, 145
Instituto de Fisiología Celular, UNAM, using an ABI Prism 3100 automated DNA sequencer 146
(Applied Biosystems Inc. Foster City, CA). The sequence alignments of each SCAR marker were 147
analyzed by the BLAST algorithm (3) to check similarities among all fungal sequences deposited 148
in the GenBank database. 149
All the aforementioned procedures are detailed in Fig. 1. 150
Design of the primers for the SCAR markers. A pair of primers 20 nucleotides long was 151
designed based on the sequence of each SCAR, using the program Primer3 Input 152
(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The primers were synthesized by 153
Sigma-Genosys (The Woodlands, TX) (Fig. 1). 154
PCR conditions for SCAR markers. The PCR was performed in a 25-µl reaction mixture 155
containing: 10 ng genomic DNA; 2.5 mM MgCl2; 200 µM of dNTPs (Applied Biosystems); 156
0.001 nmol of each SCAR primer (forward and reverse); and 1 U Taq DNA polymerase (Applied 157
Biosystems) in 1X PCR buffer (Applied Biosystems). The amplification conditions were as 158
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follows: one cycle at 94°C for 5 min; 30 cycles at 94°C for 30 s, 55°C for 30 s, 72°C for 1 min; 159
and a final extension cycle at 72°C for 5 min. In all the PCR assays, 2 μl Milli-Q water were 160
processed as the negative control. The amplified products were resolved by electrophoresis in 161
1.5% agarose gels in 0.5X Tris-borate-EDTA buffer at 100 V. The products were sequenced as 162
described above and deposited in the GenBank database. 163
Sensitivity of the SCAR markers. a) With H. capsulatum DNA. The sensitivity of each 164
marker was determined by standardized PCR reactions using a range of 5 to 0.001 ng of the EH-165
53 H. capsulatum DNA as the template. The assays were repeated, and sensitivity was defined as 166
the smallest amount of DNA template necessary to give a visible product. The M antigen probe 167
(27) was used to compare the sensitivity of the SCAR markers. The PCR reaction using the M 168
antigen probe was modified from the original description using 30 ng/µl of DNA template and 169
the following PCR program: 1 cycle at 95°C for 3 min; 35 cycles at 95°C for 1 min, 60°C for 1 170
min, 72°C for 1 min; and a final extension cycle at 72°C for 5 min. b) With soil samples. 171
Mycelial biomass of the EH-375 H. capsulatum isolate was used to contaminate soil samples, 172
which were processed according to the Reid and Schafer method (33) with minor modifications. 173
Briefly, 1 g of biomass was prepared by breaking the mycelium with a mortar and pestle and 174
adding 9 ml of 150 mM PBS (pH 7.4). This mycelial suspension (1:10 w/v) was diluted (1:15, 175
1:20, 1:30, 1:50, 1:70, 1:100, 1:200, 1:300, and 1:500), and 1 ml of each dilution was plated in 176
duplicate on mycobiotic agar (Bioxón). The plates were incubated at 28°C for 5 days, and the H. 177
capsulatum CFU were determined. Afterwards, the CFU average for each dilution was estimated. 178
In addition, soil samples (100 mg each) from a non-epidemic histoplasmosis area were 179
distributed into several 2-ml Eppendorff tubes and then contaminated with 10 µl of each 180
mycelium dilution. Whole DNA was extracted from each contaminated soil sample, using the 181
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FastDNA® SPIN kit (Qbiogene Inc., Irvine, CA). Thereafter, PCR assays with SCAR markers 182
and with the M antigen probe were performed. Data from the PCRs were compared to the CFU 183
values obtained from each contaminated soil sample. 184
Specificity of the SCAR markers. The specificity of the SCAR markers and the M antigen 185
probe was tested by PCR of the genomic DNA from all H. capsulatum isolates and strains used in 186
the RAPD screening. In addition, DNA from the pathogenic and non-pathogenic fungi C. 187
immitis, C. posadasii, P. brasiliensis, B. dermatitidis, A. fumigatus, A. niger, C. neoformans, C. 188
albicans, S. schenckii, C. carmichaelii, and Malbranchea sp. were assayed, together with M. 189
tuberculosis DNA, and the data were compared. 190
Usefulness of SCAR markers to detect H. capsulatum infection in clinical samples. A 191
DNAeasy® tissue kit (Qiagen) was used to extract DNA from fresh or paraffin-embedded tissues 192
from human clinical samples and from naturally or experimentally infected mammals (bat, 193
mouse, leopard, wallaby, and mara). A DNA sample (6 µl) of each tissue, was used for PCR 194
assays with the SCAR markers and the M antigen probe, as described above. The data from the 195
SCAR and M antigen probe markers were compared. 196
Usefulness of SCAR markers to detect H. capsulatum in guano samples. The FastDNA® 197
SPIN kit (Qbiogene), was used to extract DNA from 10 guano samples collected from different 198
sites in MX that were catalogued as having a high risk of histoplasmosis epidemic infection. For 199
the PCR, 6 µl of DNA was added to the reaction mixture to visualize the bands corresponding to 200
the SCAR markers and M antigen probe. The data from the SCAR and M antigen probe markers 201
were compared. 202
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RESULTS 204
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Selection of the H. capsulatum SCAR markers. The RAPD-PCR assays, both the two-primer 205
(1281-1283) and the single primer (1253) reactions, resolved polymorphic bands in all of the H. 206
capsulatum isolates and reference strains analyzed. The polymorphic patterns were reproducible 207
in repeated agarose gel electrophoresis with high resolution. Usually, the 1281-1283 primer 208
combination generated a band pattern between 250 and 1470 bp, whereas the 1253 primer 209
showed a polymorphic band pattern between 158 and 1200 bp. Irrespective of the RAPD primers 210
used, it was possible to detect a common band of 1200-bp in all isolates and strains studied, 211
except for the isolates from AR, as observed in the RAPD assays (data not shown). This 1200-bp 212
band was named Hc1200, and its reamplification using the 1281-1283 primers yielded two bands 213
of 900 and 800 bp, named Hc900 and Hc800, respectively. Due to these unexpected double bands, 214
the reamplification conditions were modified by varying the MgCl2 concentrations, increasing the 215
annealing temperature, and reducing the number of cycles. However, the reamplified product 216
under these modified conditions showed the same two bands. Next, the two bands were 217
successfully cloned into the pGEM®-T Easy Vector, and the two generated inserts were checked 218
by both PCR and restriction digestion analyses. The inserts showed the expected molecular sizes 219
of 900 and 800 bp, identifying both insert-DNAs as putative H. capsulatum markers, which were 220
named 1281-1283900 and 1281-1283800, respectively. 221
Southern hybridization. Hybridization assays were performed to corroborate the recognition 222
by the 1281-1283900 and 1281-1283800 markers of all H. capsulatum isolates and strains studied. 223
The markers were used as probes in Southern blotting against the molecular patterns generated by 224
the 1281-1283 primers in the two-primer RAPD-PCR assays of all the isolates and strains of H. 225
capsulatum studied. Hybridizations were detected only in the common band of 1200-bp in this 226
RAPD pattern. These results were consistent even at low hybridization temperatures, with 38°C 227
used for 1281-1283900 and 39°C for 1281-1283800 (data not shown). 228
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BLAST analysis of the insert-DNA sequences. The sequences of the 1281-1283900 and 1281-229
1283800 markers were compared between themselves and among all fungal sequences deposited 230
in GenBank. This analysis revealed that 1281-1283900 and 1281-1283800 correspond to different 231
segments of the 1200-bp band and share similarity with other gene regions from Saccharomyces 232
cerevisiae, Schizosaccharomyces pombe, Neurospora crassa, several species of Aspergillus (A. 233
awamori, A. kawachii, A. niger, A. orizae, A. flavus, and A. shirousamii), Botrytis cinerea, 234
Giberella zeae, and Pneumocystis carinii. Similar sequences shared with these fungi were 235
trimmed to generate the first two SCAR markers, the 300-bp 1281-1283300 of and the 440-bp 236
1281-1283400 from the 1281-1283800 and 1281-1283900 markers, respectively. 237
Generation of primers for the SCAR markers. Based on the 1281-1283300 and 1281-1283400 238
SCAR sequences, two sets of SCAR primers for H. capsulatum were designed. For the SCAR 239
1281-1283300, the 1281-1283220F (5´-cattgttggaggaacctgct-3´) and 1281-1283220R (5´-240
gagctgcaggatgtttgttg-3´) primers delimit a fragment of 220-bp. For the SCAR 1281-1283400, the 241
1281-1283230F (5´-ggagccatgacgttaaatgg-3´) and 1281-1283230R (5´-tattgccaatgggtttgtca-3´) 242
primers delimit a fragment of 230-bp. Sequences of 220 and 230 bp were deposited in GenBank, 243
with the respective accession numbers JN089378 and JN089379. Both sequences define the new 244
SCAR markers, 1281-1283220 and 1281-1283230. 245
According to the BLAST algorithm search, the 1281-1283220 SCAR marker corresponds to the 246
mRNA of a hypothetical protein of an Ajellomyces capsulatus NAm class 1 strain, whereas the 247
1281-1283230 SCAR marker corresponds to a partial mRNA of an alpha-amylase A precursor of 248
the same A. capsulatus strain. 249
Sensitivity of the SCAR markers and M antigen probe. After testing different dilutions of an 250
H. capsulatum DNA sample, the 1281-1283220 and 1281-1283230 SCAR markers were found to 251
have greater sensitivity (0.001 ng/µl (Fig. 2a and b) than the M antigen probe (0.5 ng/µl) (Fig. 252
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2c). The sensitivity of the SCAR markers was confirmed in soil samples contaminated with H. 253
capsulatum mycelium, in which 20 CFU/g-soil, corresponding to a mycelium dilution of 1:500, 254
was detected (Fig. 3a and b). In contrast, the M antigen probe detected 640 CFU/g-soil, 255
corresponding to a mycelium dilution of 1:30 (Fig. 3c). A negative soil sample control tested by a 256
nested PCR assay with the Hcp100 marker never amplified the H. capsulatum-specific product 257
that reveals the fungal presence in this sample. 258
Specificity of the SCAR markers and the M antigen probe. The 1281-1283220 and 1281-259
1283230 SCAR markers each amplified a unique band in all the isolates and strains of H. 260
capsulatum tested, with the expected molecular sizes of 220 and 230 bp, respectively, 261
irrespective of their geographic origin. Although none of the H. capsulatum isolates from AR 262
showed the Hc1200 RAPD marker, the 1281-1283220 and 1281-1283230 SCAR markers detected H. 263
capsulatum in the Argentinean samples. Non-specific bands were not detected under any of the 264
PCR conditions used. 265
To assess the PCR specificity of the 1281-1283220 and 1281-1283230 SCAR markers, the DNA 266
from several fungal species and M. tuberculosis was tested. No amplification was observed with 267
the 1281-1283220 SCAR for C. immitis, C. posadasii, P. brasiliensis, B. dermatitidis, A. 268
fumigatus, A. niger, C. neoformans, C. albicans, S. schenckii, C. carmichaelii, Malbranchea sp. 269
and M. tuberculosis (Fig. 4a). However, the 1281-1283230 SCAR amplified DNA from A. niger, 270
despite the high-stringency conditions of the PCR (Fig. 4b). In addition, the M antigen probe 271
amplified its characteristic 279-bp band from DNA samples of C. neoformans (Fig. 4c). 272
Usefulness of SCAR markers and M antigen probe to detect H. capsulatum infection in 273
human and animal tissues. Concerning the specificity of the SCAR markers and the M antigen 274
probe for clinical samples, the analysis of 17 biological specimens from patients with clinical 275
symptoms presumptive of histoplasmosis showed that only seven specimens were positive with 276
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the 1281-1283220 and 1281-1283230 SCAR markers and with the M antigen probe, revealing in 277
each case a single band with the expected molecular size. These results were consistent with the 278
histopathology findings for these patients because, in all these cases, yeast-like H. capsulatum 279
cells were observed. In contrast, the M antigen probe was positive in a sample from a patient with 280
an initial presumptive cryptococcosis diagnosis that was confirmed by C. neoformans isolation, 281
whereas the SCAR markers were always negative for this patient samples. The SCAR markers 282
identified H. capsulatum in nine tissue samples from mice experimentally infected with this 283
fungus, whereas the M antigen probe identified only seven samples. All three markers amplified 284
their corresponding bands in three samples from wild mammals that were naturally infected in 285
their shelters or in captivity conditions (bat, mara, and leopard). However, of seven samples from 286
a captive wallaby (liver, lungs, lymph node, pancreas, kidney, intestine, and gastric mucosa), 287
three (liver, lungs, and lymph node) were positive with the two SCAR markers and only two 288
(liver and lungs) with the M antigen probe. 289
Usefulness of SCAR markers to detect H. capsulatum in guano samples. Out of 10 samples 290
of guano collected in different epidemic sites of MX, four were positive with the SCAR markers 291
(two from Morelos, one from Guerrero and one from Oaxaca) (Fig. 5a and b), and only two were 292
positive with the M antigen probe (Morelos and Guerrero) (Fig. 5c). 293
294
DISCUSSION 295
Diverse molecular markers for H. capsulatum identification for diagnostic and epidemiologic 296
purposes have been reported by several authors (7, 25, 26, 32, 33, 38, 42). However, most of 297
these markers have low sensitivity and specificity, as well as poor reproducibility. Some of these 298
markers present different types of limitations associated with complicated methodologies that 299
involve high costs. A small number of markers have been obtained from ribosomal genes, whose 300
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conserved nature within the fungi kingdom can lead to non-specific results among various fungal 301
species (16, 25, 26, 33, 38, 43, 49). Commercially available probes used for diagnostic tests are 302
not a panacea, because such probes have shown non-specific results (6). 303
Markers designed from specific genes of H. capsulatum (4, 5, 26) are the most noteworthy for 304
fungal identification in a wide spectrum of clinical samples and infectious sources, due to their 305
apparent specificity. Nevertheless, such markers have not been extensively evaluated with fungal 306
isolates from different geographic regions or validated by comparison with other markers to 307
ensure their efficacy as a diagnostic tool. 308
A nested PCR assay, using a highly specific and sensitive 210-bp amplification product from a 309
gene coding for a co-activator protein (Hcp100), has been successfully used for H. capsulatum 310
diagnosis. This approach was first described by Bialek et al. (4) and further validated by Maubon 311
et al. (28) and more recently by Muñoz et al. (29) in human clinical samples. The Hcp100 marker 312
has also been used to detect H. capsulatum infection in tissue samples from two captive snow 313
leopards (11). The same marker was also used to detect H. capsulatum in contaminated compost, 314
which is a frequent source of fungal infection (48). The Hcp100 marker has also been 315
successfully employed to identify H. capsulatum isolates from two captive infected maras (36). 316
For histoplasmosis diagnosis, another molecular marker (H-antigen) has been proposed, using a 317
semi-nested PCR (5). Despite the high sensitivity and specificity of the Hcp100 and H-antigen 318
markers, the nested and semi-nested PCR employed with these markers generates additional 319
problems and needs to be carefully controlled to avoid non-specific amplifications, which lead to 320
misinterpretation (4, 5). 321
Other molecular markers, designed to be used in methods such as PCR-EIA, real-time PCR, 322
and Southern blot, have shown good results (7, 22, 25, 43). However, their usefulness is 323
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restricted due to their high cost and complexity together with the lack of standardized protocols to 324
perform them. 325
Because of all the inconveniences mentioned, it is necessary to establish more specific and 326
sensitive H. capsulatum identification markers with broad spectra of recognition (12), as there is 327
great genetic diversity among the fungal isolates from different sources and geographic origins 328
(9, 20, 21, 34, 35, 44-46). SCAR markers are excellent candidates for this role, given that their 329
sequences have proven to be useful in the construction of genomic libraries, in biological control 330
by monitoring fungal strains in the environment, in breeding programs, and in the development of 331
sensitive assays that associate the clinical form of the disease with the fungal burden. 332
Furthermore, the SCAR markers are able to discriminate a specific DNA amplification in a 333
sample containing a mixture of fungi (1). 334
During the process of the Hc1200 band purification, which included a reamplification step by 335
PCR, the Hc1200 band was lost, and two new bands of 800 and 900 bp appeared instead, despite 336
the variations in the MgCl2 concentration and annealing temperature that were performed. This 337
phenomenon is not rare and has been reported elsewhere (17). Also, the resulting clones, 1281-338
1283900 and 1281-1283800, hybridized with only the 1200-bp band from the RAPD polymorphic 339
patterns generated by all H. capsulatum isolates and strains tested, confirming that the 800- and 340
900-bp bands were generated from the Hc1200 band. Hence, the presence of a single hybridized 341
band in the Southern blot for each marker, suggests that these two hybridized DNA regions are 342
unique or single-copy in the genome of the pathogen. To ensure the usefulness of the SCAR 343
markers, it was necessary to evaluate their specificity and sensitivity in comparison with the M 344
antigen probe, which has been reported to be highly sensitive (0.001 ng) and unable to cross-react 345
with other fungi related to H. capsulatum (27). Hence, we tested the specificity of each SCAR 346
marker with H. capsulatum isolates from different geographic areas, with other microorganisms, 347
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and with environmental samples. The 1281-1283220 SCAR was the most sensitive (with clinical 348
and environmental samples) and 100% specific, in contrast to the 1281-1283230 SCAR, which 349
amplified the genetic material of A. niger and the M antigen probe, which recognized a clinical 350
sample containing C. neoformans. Considering that these last two markers were designed from 351
sequences that showed no similarity with any others deposited in GenBank, their non-specific 352
recognitions might be explained by microorganisms that remain without known sequences and 353
that were not considered for the design of these markers (6). 354
Based on the results of the 1281-1283220 SCAR, it is an ideal candidate for the identification of 355
H. capsulatum in clinical samples and in different infection sources. Hence, this marker is useful 356
for the detection of H. capsulatum in soil contaminated with bird or bat guano, as fungal culture 357
isolation from infected organs in mice after intraperitoneal injection of soil suspensions has a 358
very slow development. Furthermore, other methodologies such as specific antibody and antigen 359
detection, as well as histopathologic observations are less sensitive than PCR-based methods. 360
Moreover, these methodologies present a critical disadvantage in relation to molecular methods, 361
in that they cannot be directly applied to environmental samples. 362
The use of the 1281-1283220 SCAR marker will contribute to diagnosis and the knowledge of 363
the distribution of endemic and epidemic histoplasmosis in different countries of the Americas 364
and to the definition of areas of high risk of infection in the environment. 365
366
ACKNOWLEDGMENTS 367
This research was supported by a grant of Dirección General de Asuntos del Personal 368
Académico (DGAPA) DGAPA-UNAM- IN219703-2. M. G. Frías De León thanks the Biological 369
Science Graduate Program of UNAM and CONACyT for a scholarship (Ref. No.172552). 370
The authors thank Ingrid Mascher for editorial assistance. 371
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372
REFERENCES 373
1. Abbasi, P. A., S. A. Miller, T. Meulia, H. A. Hoiting, and J. Kim. 1999. Precise 374
detection and tracing of Trichoderma hamatum 382 in compost-amended potting mixes using 375
molecular markers. Appl. Environ. Microbiol. 65:5421-5426. 376
2. Ajello, L. 1971. Distribution of Histoplasma capsulatum in the United States, p. 103–122. 377
In L. Ajello, E. W. Chick, and M. L. Furcolow (ed.), Histoplasmosis: Proceedings of the Second 378
National Conference. Charles C Thomas Publishers, Springfield, ILL. 379
3. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. 380
J. Lipman. 1997. Gapped BLAST and PSI-BLAST: A new generation of protein data base 381
search programs. Nucleic Acids Res. 25:3389-3402. 382
4. Bialek, R., A. Feucht, C. Aepinus, G. Just-Nubling, V. J. Robetson, J. Knobloch, and 383
R. Hohle. 2002. Evaluation of two nested PCR assays for detection of Histoplasma capsulatum 384
DNA in human tissue. J. Clin. Microbiol. 40:1642-1647. 385
5. Bracca, A., M. E. Tosello, J. E. Girardini, S. L. Amigot, C. Gomez, and E. Serra. 386
2003. Molecular detection of Histoplasma capsulatum var. capsulatum in human clinical 387
samples. J. Clin. Microbiol. 41:1753-1755. 388
6. Brandt, M. E., D. Gaunt, N. Iqbal, S. McClinton, S. Hambleton, and L. Sigler. 2005. 389
False-positive Histoplasma capsulatum Gen-Probe chemiluminescent test result caused by a 390
Chrysosporium species. J. Clin. Microbiol. 43:1456-1458. 391
7. Buitrago, M. J., J. Berenguer, E. Mellado, J. L. Rodríguez-Tudela, and M. Cuenca-392
Estrella. 2006. Detection of imported histoplasmosis in serum of HIV-infected patients using a 393
real-time PCR-based assay. Eur. J. Clin. Microbiol. 25:665-668. 394
8. Castrillo, L. A., J. D. Vandenberg, and S. P. Wraight. 2003. Strain-specific detection 395
on June 21, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
18
of introduced Beauveria bassiana in agricultural fields by use of sequence-characterized 396
amplified region markers. J. Invertebr. Pathol. 82:75-83. 397
9. Chávez-Tapia, C. B., R. Vargas-Yáñez, G. Rodríguez-Arellanes, G. R. Peña-398
Sandoval, J. J. Flores-Estrada, M. R. Reyes-Montes, and M. L. Taylor. 1998. I. El 399
murciélago como reservorio y responsable de la dispersión de Histoplasma capsulatum en la 400
naturaleza. II. Papel de los marcadores moleculares del hongo aislado de murciélagos infectados. 401
Rev. Inst. Nal. Enf. Resp. Méx. 11:187-191. 402
10. Deepe, G. S., Jr. 2000. Histoplasma capsulatum, p. 2718–2732. In G. L. Mandell, J. E. 403
Bennett, and R. Dolin (ed.), Principles and practice of infectious diseases, 5th ed., vol. 2. 404
Churchill Livingstone, Philadelphia, PA. 405
11. Espinosa-Avilés, D., M. L. Taylor, M. R. Reyes-Montes, A. Pérez-Torres. 2008. 406
Molecular findings of disseminated histoplasmosis in two captive snow leopards (Uncia uncia). 407
J. Zoo Wildl. Med. 39:450-454. 408
12. Frías De León, M. G., M. L. Taylor, A. Hernández-Ramírez, and M. R. Reyes-409
Montes. 2007. Utilidad de las técnicas moleculares en el diagnóstico de la histoplasmosis. Rev. 410
Mex. Micol. 25:83-90. 411
13. Gomez, B., J. Figueroa, A. Hamilton, and B. Ortiz. 1997. Development of a novel 412
antigen detection test for histoplasmosis. J. Clin. Microbiol. 35:2618-2622. 413
14. González-Ochoa, A., and D. Félix. 1971. Distribución geográfica de la reactividad 414
cutánea a la histoplasmina en México. Rev. Invest. Salud Publ. Méx. 31:74-77. 415
15. Güssow, D., and T. Clackson. 1989. Direct clone characterization from plaques and 416
colonies by the polymerase reaction. Nucleic Acids Res. 17:4000. 417
16. Haynes, K., T. Westerneng, J. Fell, and W. Moens. 1995. Rapid detection and 418
identification of pathogenic fungi by polymerase chain reaction amplification of large subunit 419
on June 21, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
19
ribosomal DNA. J. Med. Vet. Mycol. 33:319-325. 420
17. Hernández, P., A. Marín, and G. Dorado. 1999. Development of SCAR by direct 421
sequencing of RAPD products: a practical tool for the introgression and marker-assisted selection 422
of wheat. Mol. Breeding 5:245-253. 423
18. Hu, J., J. Van-Eysden, and C. F. Quiros. 1995. Generation of DNA-based markers in 424
specific genome regions by two-primer RAPD reactions. Genome Res. 4:346-351. 425
19. Huffnagle, K., and R. Gander. 1993. Evaluation of Gen-Probe´s Histoplasma 426
capsulatum and Cryptococcus neoformans Accuprobes. J. Clin. Microbiol. 31:419-421. 427
20. Kasuga, T., J. W. Taylor, and T. J. White. 1999. Phylogenetic relationships of varieties 428
and geographical groups of the human pathogenic fungus Histoplasma capsulatum Darling. J. 429
Clin. Microbiol. 37:653–663. 430
21. Kasuga, T., T. J. White, G. Koenig, J. McEwen, A. Restrepo, E. Castañeda, C. Da 431
Silva-Lacaz, E. M. Heins-Vaccari, R. S. De Freitas, R. M. Zancopé-Oliveira, Z. Qin, R. 432
Negroni, D. A. Carter, Y. Mikami, M. Tamura, M. L. Taylor, G. F. Miller, N. Poonwan, and 433
J. W. Taylor. 2003. Phylogeography of the fungal pathogen Histoplasma capsulatum. Mol. Ecol. 434
12:3383–3401. 435
22. Keath, E. J., E. D. Spitzer, A. A. Painter, S. J. Travis, G. S. Kobayashi, and G. 436
Medoff. 1989. DNA probe for the identification of Histoplasma capsulatum. J. Clin. Microbiol. 437
27:2369-2372. 438
23. Lecomte, P., J. P. Peros, D. Blancard, N. Bastien, and C. Délye. 2000. PCR assays that 439
identify the grapevine dieback fungus Eutypa lata. Appl. Environ. Microb. 66:4475–4480. 440
24. Li, K. N., D. I. Rouse, E. J. Eyestone, and T. L. German. 1999. The generation of 441
specific DNA primers using random amplified polymorphic DNA and its application to 442
Verticillium dahlie: Mycol. Res. 11:1361-1368. 443
on June 21, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
20
25. Lindsley, M. D., S. F. Hurst, J. I. Nauren, and C. J. Morrison. 2001. Rapid 444
identification of dimorphic and yeast-like fungal pathogens using specific DNA probes. J. Clin. 445
Microbiol. 39:3505-3511. 446
26. Martagon-Villamil, J., N. Shrestha, M. Sholtis, C. M. Isada, G. S. Hall, T. Bryne, B. 447
A. Lodge, L. B. Reller, and G. W. Procop. 2003. Identification of Histoplasma capsulatum 448
from culture extracts by real-time PCR. J. Clin. Microbiol. 41:1295-1298. 449
27. Matos-Guedes, H. L., A. Jefferson-Guimarães, M. de Medeiros Muniz, C. Vera 450
Pizzini, A. J. Hamilton, J. M. Peralta, G. S. Deepe, and R. M. Zancopé-Oliveira. 2003. PCR 451
assay for identification of Histoplasma capsulatum based on the nucleotide sequence of the M 452
antigen. J. Clin. Microbiol. 41:535-539. 453
28. Maubon, D., S. Simon, and C. Aznar. 2007. Histoplasmosis diagnosis using a 454
polymerase chain reaction method. Application on human samples in French Guiana, South 455
America. Diagnostic Microbiol. Infect. Dis. 58:441–444. 456
29. Muñoz, C., B. L. Gómez, A. Tobón, K. Arango, A. Restrepo, M.M. Correa, C. 457
Muskus, L. E. Cano, and A. González. 2010. Validation and clinical application of a molecular 458
method for identification of Histoplasma capsulatum in human specimens in Colombia, South 459
America. Clin. Vaccine Immunol. 17:62–67. 460
30. Padhye, A., G. Smith, P. Standard, D. McLaughlin, and L. Kaufman. 1994. 461
Comparative evaluation of chemioluminiscent DNA probe assays and exoantigen test for rapid 462
identification of Blastomyces dermatitidis and Coccidioides immitis. J. Clin. Microbiol. 32:867-463
870. 464
31. Pedroza-Serés, M., H. Quiroz-Mercado, J. Granados, and M. L. Taylor. 1994. The 465
syndrome of presumed ocular histoplasmosis in Mexico: A preliminary study. J. Med. Vet. 466
Mycol. 32:83-92. 467
on June 21, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
21
32. Pounder, J. I., D. Hansen, and G. L. Woods. 2006. Identification of Histoplasma 468
capsulatum, Blastomyces dermatitidis and Coccidioides species by repetitive-sequence-based 469
PCR. J. Clin. Microbiol. 44:2977-2982. 470
33. Reid, T. M., and M. P. Schafer. 1999. Direct detection of Histoplasma capsulatum in 471
soil suspensions by two-stage PCR. Mol. Cell. Probes 13:269-273. 472
34. Reyes-Montes, M. R., M. Bobadilla-del Valle, M. A. Martínez-Rivera, G. Rodríguez-473
Arellanes, E. Flores-Robles, J. Sifuentes-Osornio, and M. L. Taylor. 1998. Tipificación de 474
aislados clínicos de Histoplasma capsulatum por métodos fenotípicos y genotípicos. Rev. Inst. 475
Nal. Enf. Resp. Méx. 11:195-201. 476
35. Reyes-Montes, M. R., M. Bobadilla del Valle, M. A. Martínez-Rivera, G. Rodríguez-477
Arellanes, E. Maravilla, J. Sifuentes-Osornio, and M. L. Taylor. 1999. Relatedness analyses 478
of Histoplasma capsulatum isolates from Mexican patients with AIDS-associated histoplasmosis 479
by using histoplasmin electrophoretic profiles and randomly amplified polymorphic DNA 480
patterns. J. Clin. Microbiol. 37:1404-1408. 481
36. Reyes-Montes, M. R., G. Rodríguez-Arellanes, A. Pérez-Torres, A. G. Rosas-Rosas, 482
A. Parás-García, C. Juan-Sallés, and M. L. Taylor. 2009. Identification of histoplasmosis 483
infection source from two captive maras (Dolichotis patagonum) of the same colony by using 484
molecular and immunologic assays. Rev. Argent. Microbiol. 41:102-104. 485
37. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Small-scale preparation of plasmid 486
DNA. Lysis by alkali, p. 1.25-1.28. In N. Ford, C. Nolan and M. Ferguson (ed.), Molecular 487
cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, New York, NY. 488
38. Sandhu, G. S., B. C. Kline, L. Stockman, and G. D. Roberts. 1995. Molecular probes 489
for diagnosis of fungal infections. J. Clin. Microbiol. 33:2913-2919. 490
39. Schilling, A. G., E. M. Moller, and H. H. Geiger. 1996. Polymerase chain reaction-491
on June 21, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
22
based assays for species specific detection of Fusarium culmorum, F. graminearum, and F. 492
avenaceum. Phytopathology 86:515–522. 493
40. Spitzer, E. D., B. A. Lasker, S. J. Travis, G. S. Kobayashi, and G. Medoff. 1989. Use 494
of mitochondrial and ribosomal DNA polymorphisms to classify clinical and soil isolates of 495
Histoplasma capsulatum. Infect. Immun. 57:1409-1412. 496
41. Stevens, D. A. 2002. Diagnosis of fungal infections: current status. J. Antimicrob. Chem 497
49 (Suppl. 1):11-19. 498
42. Stockman, L., K. A. Clark, J. M. Hunt, and G. Roberts. 1993. Evaluation of 499
commercially available acridinium ester-labeled chemiluminiscent DNA probes for culture 500
identification of Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans and 501
Histoplasma capsulatum. J. Clin. Microbiol. 31:845-850. 502
43. Tang, Y. W., H. Li, M. M. Durkin, S. E. Sefers, S. Meng, P. A. Connolly, C. Stratton, 503
and L. J. Wheat. 2006. Urine polymerase chain reaction is not as sensitive as urine antigen for 504
the diagnosis of disseminated histoplasmosis. Diagn. Microbiol. Infect. Dis. 53:46-51. 505
44. Taylor, M. L., C. B. Chávez–Tapia, and M. R. Reyes-Montes. 2000. Molecular typing 506
of Histoplasma capsulatum isolated from infected bats, captured in Mexico. Fungal Genet. Biol. 507
30:207-212. 508
45. Taylor, M. L., and M. R. Reyes-Montes. 2002. Nuevas aportaciones sobre la 509
epidemiología de la histoplasmosis en México: avances en el conocimiento de aspectos 510
inmunológicos y moleculares y de la distribución geográfica del agente etiológico. VITAE 511
Academia Biomédica Digital, CAIBO 10:1-3, 512
http://caibco.ucv.ve/vitae/VitaeDiez/Articulos/Micologia/Histoplasmosis/ArchivosHTML/Antece513
dentes.htm. 514
46. Taylor, M. L., M. R. Reyes-Montes, C. B. Chávez-Tapia, E. Curiel-Quesada, E. 515
on June 21, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
23
Duarte-Escalante, G. Rodríguez-Arellanes, G. R. Peña-Sandoval, and F. Valenzuela-Tovar. 516
2000. Ecology and molecular epidemiology findings of Histoplasma capsulatum, in México, p. 517
29-35. In M. Benedik (ed.), Research advances in microbiology. Global Research Network. 518
Kerala. 519
47. Taylor, M. L., M. R. Reyes-Montes, M. A. Martínez-Rivera, G. Rodríguez-Arellanes, 520
E. Duarte-Escalante, and J. J. Flores-Estrada. 1997. Histoplasmosis en México. Aportaciones 521
inmunológicas y moleculares sobre su epidemiología. Ciencia y Desarrollo. 23:58-63. 522
48. Taylor, M. L., G. M. Ruiz-Palacios, M. R. Reyes-Montes, G. Rodríguez-Arellanes, L. 523
E. Carreto-Binaghi, E. Duarte-Escalante, A. Hernández-Ramírez, A. Pérez, R. O. Suárez-524
Álvarez, Y. A. Roldán-Aragón, M. Romero-Martínez, J. H. Sahaza-Cardona, J. Sifuentes-525
Osornio, L. E. Soto-Ramírez, and G. R. Peña-Sandoval. 2005. Identification of the infectious 526
source of an unusual outbreak of histoplasmosis in a hotel in Acapulco, state of Guerrero, 527
Mexico. FEMS Immunol. Med. Microbiol. 45:435-441. 528
49. Ueda, Y., A. Sano, M. Tamura, T. Inomata, K. Kamei, K. Yokoyama, F. Kishi, J. Ito, 529
Y. Mikami, M. Miyaji, and K. Nishimura. 2003. Diagnosis of histoplasmosis by detection of 530
the internal transcribed spacer region of fungal rRNA gene from a paraffin-embedded skin 531
sample from a dog in Japan. Vet. Microbiol. 94:219-224. 532
50. Vaca-Marín, M. A., M. A. Martínez-Rivera, and J. J. Flores-Estrada. 1998. 533
Histoplasmosis en México, aspectos históricos y epidemiológicos. Rev. Inst. Nal. Enf. Resp. 534
Mex. 11:208-215. 535
51. Wheat, L. J., T. Garringer, E. Brizendine, and P. Connolly. 2002. Diagnosis of 536
histoplasmosis by antigen detection based upon experience at the histoplasmosis reference 537
laboratory. Diagn. Microbiol. Infect. Dis. 43:29-37. 538
on June 21, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
24
52. Wheat, L. J., R. Kohler, and R. Tewari. 1986. Diagnosis of disseminated 539
histoplasmosis by detection of Histoplasma capsulatum antigen in serum and urine specimens. N. 540
Engl. J. Med. 314:83-88. 541
53. Williams, B., M. Fojtasek, P. Connolly-Stringfield, and L. J. Wheat. 1994. Diagnosis 542
of histoplasmosis by antigen detection during an outbreak in Indianapolis. Arch. Pathol. Lab. 543
Med. 118:1205-1208. 544
on June 21, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from