Development of Specific Sequence-Characterized Amplified RegionMarkers for Detecting Histoplasma capsulatum in Clinical andEnvironmental Samples

María Guadalupe Frías De León,a Gabina Arenas López,b Maria Lucia Taylor,a Gustavo Acosta Altamirano,c andMaría del Rocío Reyes-Montesa

Departamentos de Microbiología-Parasitologíaa and Fisiología,b Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, Mexico, and HospitalJuárez de México, Mexico City, Mexicoc

Sequence-characterized amplified region (SCAR) markers, generated by randomly amplified polymorphic DNA (RAPD)-PCR,were developed to detect Histoplasma capsulatum selectively in clinical and environmental samples. A 1,200-bp RAPD-PCR-specific band produced with the 1281-1283 primers was cloned, sequenced, and used to design two SCAR markers, 1281-1283220

and 1281-1283230. The specificity of these markers was confirmed by Southern hybridization. To evaluate the relevance of theSCAR markers for the diagnosis of histoplasmosis, another molecular marker (M antigen probe) was used for comparison. Tovalidate 1281-1283220 and 1281-1283230 as new tools for the identification of H. capsulatum, the specificity and sensitivity ofthese markers were assessed for the detection of the pathogen in 36 clinical (17 humans, as well as 9 experimentally and 10 natu-rally infected nonhuman mammals) and 20 environmental (10 contaminated soil and 10 guano) samples. Although the twoSCAR markers and the M antigen probe identified H. capsulatum isolates from different geographic origins in America, the1281-1283220 SCAR marker was the most specific and detected the pathogen in all samples tested. In contrast, the 1281-1283230

SCAR marker and the M antigen probe also amplified DNA from Aspergillus niger and Cryptococcus neoformans, respectively.Both SCAR markers detected as little as 0.001 ng of H. capsulatum DNA, while the M antigen probe detected 0.5 ng of fungalDNA. The SCAR markers revealed the fungal presence better than the M antigen probe in contaminated soil and guano samples.Based on our results, the 1281-1283220 marker can be used to detect and identify H. capsulatum in samples from differentsources.

Histoplasmosis is a widespread respiratory infection caused bythe fungus Histoplasma capsulatum (2). The disease is en-

demic in North America, occurring mainly in the Ohio and Mis-sissippi river valleys, and frequent outbreaks occur in severalcountries of Latin America. Histoplasmosis has been registered inevery state of Mexico, and it has demonstrated a variable preva-lence in areas of endemicity (14, 31, 47). High lethality has beenrecorded in several Mexican outbreaks (50). The presence of fun-gal propagules in urban areas has been documented and may beimportant to explain clinical cases not associated with any expo-sure to high-risk infection sites (46).

Infection with the etiologic agent, the dimorphic fungus H.capsulatum, is initiated by inhalation of aerosolized microconidiaand mycelial fragments that convert into the virulent yeast phasein the parasitized host. The yeast cells proliferate within the hostphagocytes, mainly dendritic cells and macrophages. Usually, theactivation of cell-mediated immunity inhibits the yeast’s intracel-lular multiplication (10).

A wide variety of tests are used in the laboratory for the diag-nosis of histoplasmosis; however, several of them have particularlimitations (13, 41, 51–53). The diagnosis of this mycosis regularlyrequires histologic examination and/or fungal culture from clini-cal specimens such as blood, bone marrow, or bronchoalveolarlavage. Isolation of the pathogen requires 3 weeks for fungalgrowth, which delays and complicates an accurate diagnosis. Fur-thermore, identification of the organism can only be performed inbiosafety level 3 laboratories (41). In addition, confirmatory testsare needed for organisms suspected to be H. capsulatum, because

some saprobic microorganisms mimic the morphological moldphase of this fungus.

Although several immunological and molecular methods foridentifying H. capsulatum have been reported (4, 5, 16, 19, 22, 26,27, 30, 33, 38, 40, 42, 43, 49), some of them have distinct limita-tions, such as low sensitivity and specificity (12). Among the mo-lecular methods used for diagnosis, some fail to detect the fungalpresence due to the high genetic variability of H. capsulatum. Forthese reasons, a specific fungal marker, such as a sequence charac-terized amplified region (SCAR), could solve critical problems inhistoplasmosis diagnosis (1, 8, 23, 24, 39).

In the present study, a randomly amplified polymorphic DNA(RAPD)-PCR method was used to screen polymorphic DNAbands in order to select a marker capable of distinguishing H.capsulatum from related pathogenic microorganisms. A selectedRAPD-PCR band was converted to a SCAR marker with the aim ofdeveloping species-specific and sensitive PCR for H. capsulatum inclinical and environmental samples to allow for a fast histoplas-mosis diagnosis.

Received 8 August 2011 Returned for modification 9 September 2011Accepted 13 December 2011

Published ahead of print 21 December 2011

Address correspondence to Maria del Rocío Reyes-Montes, remoa@unam.mx.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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MATERIALS AND METHODSH. capsulatum. Forty isolates of H. capsulatum from different sources andgeographic origins were selected for the present study. Twenty-four iso-lates were from Mexico: EH-53, EH-316, EH-323-EH-328, EH-355-EH-357, and EH-359 human clinical isolates; EH-372-EH-377, EH-383H,EH-384H, EH-391, EH-393, and EH-408H from infected bats; and EH-554B from compost. Three isolates were from Guatemala: the Cepa 3clinical isolate and two isolates from bird guano (L-100-91 and Cepa 2),provided by the Facultad de Ciencias Químicas, Guatemala; four clinicalisolates (LA, Gli, DS, and RG) were from Colombia, provided by theCorporación para Investigaciones Biológicas, Colombia; six clinical iso-lates (951539, 01559, 01733 to 01735, and 01737) were from Argentina,provided by the Instituto Nacional de Enfermedades Infecciosas, ANLIS“Dr. Carlos G. Malbrán,” Argentina; the G-186B human reference strainwas from Panama from the American Type Culture Collection (ATCC);and the G-217B and Downs human reference strains were from theUnited States, also from the ATCC. The isolates and reference strainsstudied were deposited in the Histoplasma capsulatum Culture Collectionof the Laboratorio de Inmunología de Hongos, Departamento deMicrobiología-Parasitología, Facultad de Medicina, Universidad Nacio-nal Autónoma de México (UNAM). This collection can be accessed online(http://www.histoplas-mex.unam.mx), and it is registered in the databaseof the World Data Centre for Microorganisms of the World Federationfor Culture Collections under the acronym and number LIH-UNAMWDCM817.

The fungal isolates and strains were maintained in brain heart infusionagar (Bioxón; Becton Dickinson, Mexico City, Mexico) at 28°C and pre-served in mycobiotic agar (Bioxón) with sterile mineral oil at 4°C.

Tissue samples. A total of 17 samples from different tissues obtainedfrom patients with presumptive histoplasmosis were analyzed. Nine sam-ples from different organs obtained from mice and bats experimentallyinfected with H. capsulatum, and 10 tissue samples from naturally infectedanimals— one of a snow leopard (Uncia uncia), two of maras (Dolichotispatagonum), and seven of wallabies (Macropus rufogriseus)—were alsoprocessed. Blood samples from healthy human volunteers and tissue sam-ples from uninfected bats and mice were used as negative controls.

Soil and guano samples. We analyzed 10 soil samples (free of bird or batguano) that were experimentally contaminated with H. capsulatum myce-lium and 10 samples of bat or bird guano collected in epidemic sites fromMexico (Oaxaca, Guerrero, Morelos, Sinaloa, Nuevo León, and Puebla).Guano samples were taken from the Guano Collection of the Laboratorio deInmunología de Hongos, Departamento de Microbiología-Parasitología,Facultad de Medicina, UNAM. As negative controls, some soil samples werecollected from sites that, by mycological and molecular procedures, did notreveal fungal presence. The molecular procedure used was a nested PCR assayusing the Hcp100 gene fragment (4), with minor modifications (48).

DNA isolation. Mycelium cultures of H. capsulatum isolates andstrains were grown at 28°C in glucose yeast-extract medium with shaking,and the cultures were processed for DNA extraction as described else-where (35). The DNA from each mycelium culture of Coccidioides immi-tis, C. posadasii, Paracoccidioides brasiliensis, Blastomyces dermatitidis,Aspergillus fumigatus, A. niger, Candida albicans, Sporothrix schenckii,Chrysosporium carmichaelli, and Malbranchea sp. was extracted undersimilar conditions. The DNA from Cryptococcus neoformans and myce-lium cultures of C. carmichaelli and Malbranchea sp. were kindly providedby Laura Rosio Castañón (Facultad de Medicina, UNAM, Mexico), B.dermatitidis was kindly provided by Alejandro Bonifaz (Hospital General,Mexico), and Mycobacterium tuberculosis was kindly provided by MiriamBobadilla del Valle (Instituto Nacional de Ciencias Médicas y NutriciónSalvador Zubirán, Mexico). DNA from all of the fungi tested, as well as M.tuberculosis, was used to check the specificity of the H. capsulatum molec-ular markers studied. The concentration of each DNA sample was quan-tified fluorometrically and checked against standard lambda phage DNAconcentrations by electrophoresis in 0.8% agarose gels with ethidium bro-mide staining (10 �g/ml). Finally, the DNA was stored at 4°C.

RAPD-PCR for the selection of the H. capsulatum SCAR markers.Three primers were tested: 1281 (5=-AACGCGCAAC-3=), 1283 (5=-GCGATCCCCA-3=), and 1253 (5=-GTTTCCGCCC-3=), all of which were allsupplied by Operon Technologies, Inc. (Alameda, CA). The 1281 and1283 primers were assessed in the pairwise combination, according to thetwo-primer RAPD-PCR assay (18), while the 1253 primer was used singly(48). Each RAPD-PCR assay was performed twice to ensure reproducibil-ity. To select the best band for generating the SCAR markers, the RAPDpolymorphic patterns were compared to identify a common and repro-ducible band in the isolates from Mexico, Guatemala, Colombia, andArgentina, as well as in the reference strains from Panama and the UnitedStates (see details in Fig. 1).

Cloning, hybridization, and sequencing of a selected RAPD band. Aselected RAPD-PCR-specific band was purified with a QIAquick gel ex-traction kit (Qiagen, Inc., Valencia, CA), reamplified using the 1281 and1283 primers on the two-primer RAPD-PCR assay (18), and cloned intothe pGEM-T Easy Vector (Promega, Madison, WI). The plasmid harbor-ing insert-DNA with the expected molecular size was extracted, and thisresulting insert (the SCAR marker) was used for Southern hybridizationassays to confirm the presence of the selected marker in all of the H.capsulatum isolates and strains studied. Prehybridization and hybridiza-tion were performed according to the method of Sambrook et al. (37) at45°C. The hybridized bands were visualized by a colorimetric methodusing NBT/BCIP stock solution (Roche Molecular Biochemicals, Mann-heim, Germany). Each SCAR marker was sequenced at the Unidad deBiología Molecular, Instituto de Fisiología Celular, UNAM, using an ABIPrism 3100 automated DNA sequencer (Applied Biosystems, Inc., FosterCity, CA). The sequence alignments of each SCAR marker were analyzedby the BLAST algorithm (3) to check similarities among all fungal se-quences deposited in the GenBank database. All of these procedures aredetailed in Fig. 1.

Design of the primers for the SCAR markers. A pair of primers 20nucleotides long was designed based on the sequence of each SCAR, usingthe program Primer3 Input (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The primers were synthesized by Sigma-Genosys(The Woodlands, TX) (Fig. 1).

PCR conditions for SCAR markers. The PCR was performed in a25-�l reaction mixture containing 10 ng of genomic DNA, 2.5 mMMgCl2, 200 �M deoxynucleoside triphosphates (Applied Biosystems),0.001 nmol of each SCAR primer (forward and reverse), and 1 U of TaqDNA polymerase (Applied Biosystems) in 1� PCR buffer (Applied Bio-systems). The amplification conditions were as follows: one cycle at 94°Cfor 5 min; 30 cycles at 94°C for 30 s, 55°C for 30 s, 72°C for 1 min; and afinal extension cycle at 72°C for 5 min. In all of the PCR assays, 2 �l ofMilli-Q water was processed as the negative control. The amplified prod-ucts were resolved by electrophoresis in 1.5% agarose gels in 0.5� Tris-borate-EDTA buffer at 100 V. The products were sequenced as describedabove and deposited in the GenBank database.

(i) Sensitivity of the SCAR markers with H. capsulatum DNA. Thesensitivity of each marker was determined by standardized PCRs using arange of 5 to 0.001 ng of the EH-53 H. capsulatum DNA as the template.The assays were repeated, and sensitivity was defined as the smallestamount of DNA template necessary to give a visible product. The M an-tigen probe (27) was used to compare the sensitivity of the SCAR markers.The PCR using the M antigen probe was modified from the original de-scription using 30 ng/�l of DNA template, and the following PCR pro-gram: 1 cycle at 95°C for 3 min; 35 cycles at 95°C for 1 min, 60°C for 1 min,72°C for 1 min; and a final extension cycle at 72°C for 5 min.

(ii) Sensitivity of the SCAR markers with soil samples. Mycelial bio-mass of the EH-375 H. capsulatum isolate was used to contaminate soilsamples, which were processed according to the Reid and Schafer method(33) with minor modifications. Briefly, 1 g of biomass was prepared bybreaking the mycelium with a mortar and pestle and adding 9 ml of 150mM phosphate-buffered saline (pH 7.4). This mycelial suspension (1:10[wt/vol]) was diluted (1:15, 1:20, 1:30, 1:50, 1:70, 1:100, 1:200, 1:300, and

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1:500), and 1 ml of each dilution was plated in duplicate on mycobioticagar (Bioxón). The plates were incubated at 28°C for 5 days, and the H.capsulatum CFU were determined. Afterward, the CFU average for eachdilution was estimated. In addition, soil samples (100 mg each) from anonepidemic histoplasmosis area were distributed into several 2-ml Ep-pendorf tubes and then contaminated with 10 �l of each mycelium dilu-tion. Whole DNA was extracted from each contaminated soil sample,using the FastDNA SPIN kit (Qbiogene, Inc., Irvine, CA). Thereafter, PCRassays with SCAR markers and with the M antigen probe were performed.The data from the PCRs were compared to the CFU values obtained fromeach contaminated soil sample.

Specificity of the SCAR markers. The specificity of the SCAR markersand the M antigen probe was tested by PCR of the genomic DNA from allH. capsulatum isolates and strains used in the RAPD screening. In addi-tion, DNA from the pathogenic and nonpathogenic fungi C. immitis, C.posadasii, P. brasiliensis, B. dermatitidis, A. fumigatus, A. niger, C. neofor-mans, C. albicans, S. schenckii, C. carmichaelii, and Malbranchea sp. wereassayed, together with M. tuberculosis DNA, and the data were compared.

Usefulness of SCAR markers to detect H. capsulatum infection inclinical samples. A DNeasy tissue kit (Qiagen) was used to extract DNAfrom fresh or paraffin-embedded tissues from human clinical samples andfrom naturally or experimentally infected mammals (bat, mouse, leopard,wallaby, and mara). A DNA sample (6 �l) of each tissue was used for PCRassays with the SCAR markers and the M antigen probe, as describedabove. The data from the SCAR and M antigen probe markers were com-pared.

Usefulness of SCAR markers to detect H. capsulatum in guano sam-ples. The FastDNA SPIN kit (Qbiogene), was used to extract DNA from10 guano samples collected from different sites in Mexico that were cata-logued as having a high risk of histoplasmosis epidemic infection. For thePCR, 6 �l of DNA was added to the reaction mixture to visualize the bandscorresponding to the SCAR markers and M antigen probe. The data fromthe SCAR and M antigen probe markers were compared.

RESULTSSelection of the H. capsulatum SCAR markers. The RAPD-PCRassays, both the two-primer (primers 1281 and 1283) and thesingle-primer (primer 1253) reactions, resolved polymorphicbands in all of the H. capsulatum isolates and reference strainsanalyzed. The polymorphic patterns were reproducible in re-peated agarose gel electrophoresis with high resolution. Usually,the 1281/1283 primer combination generated a band pattern be-tween 250 and 1,470 bp, whereas the 1253 primer showed a poly-morphic band pattern between 158 and 1,200 bp. Irrespective ofthe RAPD primers used, it was possible to detect a common bandof 1,200-bp in all isolates and strains studied, except for the iso-lates from Argentina, as observed in the RAPD assays (data notshown). This 1,200-bp band was named Hc1200, and its reampli-fication using the 1281/1283 primers yielded two bands of 900 and800 bp, named Hc900 and Hc800, respectively. Due to these unex-pected double bands, the reamplification conditions were modi-fied by varying the MgCl2 concentrations, increasing the anneal-ing temperature, and reducing the number of cycles. However, thereamplified product under these modified conditions showed thesame two bands. Next, the two bands were successfully cloned intothe pGEM-T Easy vector, and the two generated inserts werechecked by both PCR and restriction digestion analyses. The in-serts showed the expected molecular sizes of 900 and 800 bp, iden-tifying both insert-DNAs as putative H. capsulatum markers,which were named 1281-1283900 and 1281-1283800, respectively.

Southern hybridization. Hybridization assays were per-formed to corroborate the recognition by the 1281-1283900 and1281-1283800 markers of all H. capsulatum isolates and strainsstudied. The markers were used as probes in Southern blotting

FIG 1 Schematic representation of the methodology for obtaining the 1281-1283220 and 1281-1283230 SCAR markers.

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against the molecular patterns generated by the 1281-1283 prim-ers in the two-primer RAPD-PCR assays of all of the isolates andstrains of H. capsulatum studied. Hybridizations were detectedonly in the common band of 1,200-bp in this RAPD pattern. Theseresults were consistent even at low hybridization temperatures,with 38°C used for 1281-1283900 and 39°C used for 1281-1283800

(data not shown).BLAST analysis of the insert-DNA sequences. The sequences

of the 1281-1283900 and 1281-1283800 markers were comparedbetween themselves and among all fungal sequences deposited inGenBank. This analysis revealed that 1281-1283900 and1281-1283800 correspond to different segments of the 1,200-bp bandand share similarity with other gene regions from Saccharomycescerevisiae, Schizosaccharomyces pombe, Neurospora crassa, severalspecies of Aspergillus (A. awamori, A. kawachii, A. niger, A. orizae,A. flavus, and A. shirousamii), Botrytis cinerea, Giberella zeae, andPneumocystis carinii. Similar sequences shared with these fungiwere trimmed to generate the first two SCAR markers: the 300-bp1281-1283300 and the 440-bp 1281-1283400 from the 1281-1283800

and 1281-1283900 markers, respectively.Generation of primers for the SCAR markers. Based on the

1281-1283300 and 1281-1283400 SCAR sequences, two sets ofSCAR primers for H. capsulatum were designed. For the SCAR1281-1283300, the 1281-1283220F (5=-CATTGTTGGAGGAACCTGCT-3=) and 1281-1283220R (5=-GAGCTGCAGGATGTTTGTTG-3=) primers delimit a fragment of 220 bp. For the SCAR 1281-1283400, the 1281-1283230F (5=-GGAGCCATGACGTTAAATGG-3=) and 1281-1283230R (5=-TATTGCCAATGGGTTTGTCA-3=)primers delimit a fragment of 230 bp. Sequences of 220 and 230 bpwere deposited in GenBank under accession numbers JN089378and JN089379, respectively. Both sequences define the new SCARmarkers, 1281-1283220 and 1281-1283230.

According to the BLAST algorithm search, the 1281-1283220

SCAR marker corresponds to the mRNA of a hypothetical proteinof an Ajellomyces capsulatus North American (NAm) class 1 strain,whereas the 1281-1283230 SCAR marker corresponds to a partialmRNA of an alpha-amylase A precursor of the same A. capsulatusstrain.

Sensitivity of the SCAR markers and M antigen probe. Aftertesting different dilutions of an H. capsulatum DNA sample, the1281-1283220 and 1281-1283230 SCAR markers were found to have

greater sensitivity (0.001 ng/�l) (Fig. 2a and b) than the M antigenprobe (0.5 ng/�l) (Fig. 2c). The sensitivity of the SCAR markerswas confirmed in soil samples contaminated with H. capsulatummycelium, in which 20 CFU/g of soil, corresponding to a myce-lium dilution of 1:500, was detected (Fig. 3a and b). In contrast,the M antigen probe detected 640 CFU/g of soil, corresponding toa mycelium dilution of 1:30 (Fig. 3c). A negative soil sample con-trol tested by a nested PCR assay with the Hcp100 marker neveramplified the H. capsulatum-specific product that reveals the fun-gal presence in this sample.

Specificity of the SCAR markers and the M antigen probe.The 1281-1283220 and 1281-1283230 SCAR markers each ampli-fied a unique band in all of the isolates and strains of H. capsula-tum tested, with the expected molecular sizes of 220 and 230 bp,respectively, irrespective of their geographic origin. Althoughnone of the H. capsulatum isolates from Argentina showed theHc1200 RAPD marker, the 1281-1283220 and 1281-1283230 SCARmarkers detected H. capsulatum in the Argentinean samples.Nonspecific bands were not detected under any of the PCR con-ditions used.

To assess the PCR specificity of the 1281-1283220 and 1281-1283230 SCAR markers, the DNA from several fungal species andM. tuberculosis was tested. No amplification was observed with the1281-1283220 SCAR for C. immitis, C. posadasii, P. brasiliensis, B.dermatitidis, A. fumigatus, A. niger, C. neoformans, C. albicans, S.schenckii, C. carmichaelii, Malbranchea sp., and M. tuberculosis(Fig. 4a). However, the 1281-1283230 SCAR amplified DNA fromA. niger, despite the high-stringency conditions of the PCR (Fig.4b). In addition, the M antigen probe amplified its characteristic279-bp band from DNA samples of C. neoformans (Fig. 4c).

Usefulness of SCAR markers and M antigen probe to detectH. capsulatum infection in human and animal tissues. Concern-ing the specificity of the SCAR markers and the M antigen probefor clinical samples, the analysis of 17 biological specimens frompatients with clinical symptoms presumptive of histoplasmosisshowed that only seven specimens were positive with the 1281-1283220 and 1281-1283230 SCAR markers and with the M antigen

FIG 2 (a to c) Sensitivity of the SCAR markers and M antigen probe evaluatedwith different concentrations of H. capsulatum genomic DNA. The markerswere processed by PCR as described in Materials and Methods. (a) PCR withthe 1281-1283220 SCAR marker. (b) PCR with the 1281-1283230 SCAR marker.(c) PCR with the M antigen probe. C (�), negative control; M, molecular sizemarker. FIG 3 (a to c) Correlations between the sensitivity of the molecular markers

studied and the CFU of soil samples contaminated with H. capsulatum myce-lium dilutions. The two SCAR markers and the M antigen probe were pro-cessed by PCR as described in Materials and Methods. (a) PCR with the 1281-1283220 SCAR marker. (b) PCR with the 1281-1283230 SCAR marker. (c) PCRwith the M antigen probe. C (�), negative control; M, molecular size marker.

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probe, revealing in each case a single band with the expected mo-lecular size. These results were consistent with the histopathologyfindings for these patients because, in all of these cases, yeast-likeH. capsulatum cells were observed. In contrast, the M antigenprobe was positive in a sample from a patient with an initial pre-sumptive cryptococcosis diagnosis that was confirmed by C. neo-formans isolation, whereas the SCAR markers were always nega-tive for this patient’s samples. The SCAR markers identified H.capsulatum in nine tissue samples from mice experimentally in-fected with this fungus, whereas the M antigen probe identifiedonly seven samples. All three markers amplified their correspond-ing bands in three samples from wild mammals that were natu-rally infected in their shelters or in captivity conditions (bat, mara,and leopard). However, of seven samples from a captive wallaby(liver, lungs, lymph node, pancreas, kidney, intestine, and gastricmucosa), three (liver, lungs, and lymph node) were positive withthe two SCAR markers and only two (liver and lungs) were posi-tive with the M antigen probe.

Usefulness of SCAR markers to detect H. capsulatum inguano samples. Of 10 samples of guano collected in different ep-idemic sites of Mexico, four were positive with the SCAR markers(two from Morelos, one from Guerrero, and one from Oaxaca)(Fig. 5a and b), and only two were positive with the M antigenprobe (Morelos and Guerrero) (Fig. 5c).

DISCUSSION

Diverse molecular markers for H. capsulatum identification fordiagnostic and epidemiologic purposes have been reported by sev-eral authors (7, 25, 26, 32, 33, 38, 42). However, most of thesemarkers have low sensitivity and specificity, as well as poor repro-ducibility. Some of these markers present different types of limi-tations associated with complicated methodologies that involvehigh costs. A small number of markers have been obtained fromribosomal genes, whose conserved nature within the fungi king-

dom can lead to nonspecific results among various fungal species(16, 25, 26, 33, 38, 43, 49). Commercially available probes used fordiagnostic tests are not a panacea, because such probes haveshown nonspecific results (6).

Markers designed from specific genes of H. capsulatum (4, 5,26) are the most noteworthy for fungal identification in a widespectrum of clinical samples and infectious sources, due to theirapparent specificity. Nevertheless, such markers have not beenextensively evaluated with fungal isolates from different geo-graphic regions or validated by comparison with other markers toensure their efficacy as a diagnostic tool.

A nested PCR assay, using a highly specific and sensitive 210-bpamplification product from a gene coding for a coactivator pro-tein (Hcp100), has been successfully used for H. capsulatum diag-nosis. This approach was first described by Bialek et al. (4) andfurther validated by Maubon et al. (28) and more recently byMuñoz et al. (29) in human clinical samples. The Hcp100 markerhas also been used to detect H. capsulatum infection in tissue sam-ples from two captive snow leopards (11). The same marker wasalso used to detect H. capsulatum in contaminated compost,which is a frequent source of fungal infection (48). The Hcp100marker has also been successfully used to identify H. capsulatumisolates from two captive infected maras (36). For histoplasmosisdiagnosis, another molecular marker (H-antigen) has been pro-posed, using a seminested PCR (5). Despite the high sensitivityand specificity of the Hcp100 and H-antigen markers, the nestedand seminested PCR used with these markers generates additionalproblems and needs to be carefully controlled to avoid nonspecificamplifications, which lead to misinterpretation (4, 5).

Other molecular markers, designed to be used in methods suchas PCR-EIA, real-time PCR, and Southern blotting, have showngood results (7, 22, 25, 43). However, their usefulness is restricteddue to their high cost and complexity, together with the lack ofstandardized protocols to perform them.

Because of all of the inconveniences mentioned, it is necessaryto establish more specific and sensitive H. capsulatum identifica-tion markers with broad spectra of recognition (12), since there isgreat genetic diversity among the fungal isolates from differentsources and geographic origins (9, 20, 21, 34, 35, 44–46). SCAR

FIG 4 Specificity of the SCAR markers and M antigen probe. PCR assayswere performed with DNA from H. capsulatum and other pathogenic andnonpathogenic fungi (C. immitis, C. posadasii, P. brasiliensis, B. dermatiti-dis, A. fumigatus, A. niger, C. neoformans, C. albicans, S. schenckii, C. car-michaelli, and Malbranchea sp.), as well as from M. tuberculosis. The twoSCAR markers and the M antigen probe were processed by PCR as de-scribed in Materials and Methods. (a) PCR with the 1281-1283220 SCARmarker. (b) PCR with the 1281-1283230 SCAR marker. (c) PCR with the Mantigen probe. C (�), negative control; M, molecular size marker.

FIG 5 Detection of H. capsulatum in guano samples, collected from differentepidemic sites in Mexico. The two SCAR markers and the M antigen probewere processed by PCR as described under Materials and Methods. (a) PCRwith the 1281-1283220 SCAR marker. (b) PCR with the 1281-1283230 SCARmarker. (c) PCR with the M antigen probe. OC, Oaxaca; NL, Nuevo León; GR,Guerrero; MS, Morelos; SL, Sinaloa; PL, Puebla; C (�), positive control; C(�), negative control; M, molecular size marker.

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markers are excellent candidates for this role, given that their se-quences have proven to be useful in the construction of genomiclibraries, in biological control by monitoring fungal strains in theenvironment, in breeding programs, and in the development ofsensitive assays that associate the clinical form of the disease withthe fungal burden. Furthermore, the SCAR markers are able todiscriminate a specific DNA amplification in a sample containinga mixture of fungi (1).

During the process of the Hc1200 band purification, which in-cluded a reamplification step by PCR, the Hc1200 band was lost,and two new bands of 800 and 900 bp appeared instead, despitethe variations in the MgCl2 concentration and annealing temper-ature that were performed. This phenomenon is not rare and hasbeen reported elsewhere (17). Also, the resulting clones, 1281-1283900 and 1281-1283800, hybridized with only the 1,200-bp bandfrom the RAPD polymorphic patterns generated by all H. capsu-latum isolates and strains tested, confirming that the 800- and900-bp bands were generated from the Hc1200 band. Hence, thepresence of a single hybridized band in the Southern blot for eachmarker suggests that these two hybridized DNA regions areunique or single-copy in the genome of the pathogen. To ensurethe usefulness of the SCAR markers, it was necessary to evaluatetheir specificity and sensitivity in comparison to the M antigenprobe, which has been reported to be highly sensitive (0.001 ng)and unable to cross-react with other fungi related to H. capsula-tum (27). Hence, we tested the specificity of each SCAR markerwith H. capsulatum isolates from different geographic areas, withother microorganisms, and with environmental samples. The1281-1283220 SCAR was the most sensitive (with clinical and en-vironmental samples) and 100% specific, in contrast to the 1281-1283230 SCAR, which amplified the genetic material of A. nigerand the M antigen probe, which recognized a clinical sample con-taining C. neoformans. Considering that these last two markerswere designed from sequences that showed no similarity with anyothers deposited in GenBank, their nonspecific recognitionsmight be explained by microorganisms that remain withoutknown sequences and that were not considered for the design ofthese markers (6).

Based on the results of the 1281-1283220 SCAR, it is an idealcandidate for the identification of H. capsulatum in clinical sam-ples and in different infection sources. Hence, this marker is usefulfor the detection of H. capsulatum in soil contaminated with birdor bat guano, since fungal culture isolation from infected organsin mice after intraperitoneal injection of soil suspensions has avery slow development. Furthermore, other methodologies suchas specific antibody and antigen detection, as well as histopatho-logic observations are less sensitive than PCR-based methods.Moreover, these methodologies present a critical disadvantage inrelation to molecular methods, in that they cannot be directlyapplied to environmental samples.

The use of the 1281-1283220 SCAR marker will contribute todiagnosis and the knowledge of the distribution of endemic andepidemic histoplasmosis in different countries of the Americasand to the definition of areas of high risk of infection in the envi-ronment.

ACKNOWLEDGMENTS

This research was supported by a grant of Dirección General de Asuntosdel Personal Académico (DGAPA-UNAM-IN219703-2). M.G.F.D.L

thanks the Biological Science Graduate Program of UNAM andCONACyT for a scholarship (reference no. 172552).

We thank Ingrid Mascher for editorial assistance.

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