downloaded from juárez de méxico, mexi co city, 07760, mexico · 22 1281-1283 primers was cloned,...

1

JCM05271-11 version 2 1

Development of specific SCAR markers for detecting Histoplasma capsulatum in clinical 2

and environmental samples 3

4

María Guadalupe Frías De León1, Gabina Arenas López2, Maria Lucia Taylor1, Gustavo Acosta 5

Altamirano3, María del Rocío Reyes-Montes1* 6

7

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

11

Running title: Specific-SCAR markers for H. capsulatum detection 12

13

*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

17

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

on June 21, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

http://jcm.asm.org/

2

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

38

Keywords: Histoplasma capsulatum; RAPD-PCR; SCAR-markers; molecular diagnosis. 39

40


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

3

41

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

4

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

74

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

5

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

6

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

7

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

8

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

9

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

203

RESULTS 204


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

10

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

11

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

12

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

13

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

14

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

15

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

16

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

17

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

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


.asm.org/

Dow

nloaded from

http://jcm.asm.org/


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

downloaded from juárez de méxico, mexi co city, 07760, mexico · 22 1281-1283 primers was cloned,...

Documents