Abstract
The major diagnostic antigens of Histoplasma capsulatum var. capsulatum are the H and M antigens, pluripotent glycoproteins that elicit both humoral and T-cell-mediated immune responses. The gene encoding the M antigen has previously been sequenced, and its sequence has significant overall homology to those of the genes for fungal catalases. Regions of the M-antigen gene with little or no homology were used to design four oligonucleotide sequences for application in the PCR detection and identification of H. capsulatum var. capsulatum. The PCR correctly identified the 31 H. capsulatum var. capsulatum strains isolated from human, animal, and soil specimens and 1 H. capsulatum var. duboisii isolate. PCR products of 111 and 279 bp were amplified with primers Msp1F-Msp1R and Msp2F-Msp2R, respectively. No amplification product was obtained from DNA extracted from an H. capsulatum var. farciminosum isolate. The specificity of the PCR with the M-antigen-derived primers was confirmed by the total absence of amplification products when genomic DNA from Paracoccidioides brasiliensis, Candida spp., Sporothrix schenckii, Cryptococcus neoformans, Blastomyces dermatitidis, Coccidioides immitis, Aspergillus niger, and Aspergillus fumigatus were applied in the reaction. This rapid, sensitive, and specific assay provides a way to identify typical and atypical isolates of H. capsulatum var. capsulatum.
Histoplasmosis, a systemic fungal disease caused by Histoplasma capsulatum var. capsulatum (15), is an important health problem worldwide. Although the majority of cases present as a mild to moderate flu-like disease requiring only supportive therapy, approximately 5% of patients develop a more serious pulmonary and extrapulmonary disease that can be life-threatening if diagnosis is delayed or if treatment is not initiated rapidly. Histoplasmosis is most prevalent in the midwestern states of the United States (27-30), although smaller regions of endemicity are scattered throughout most of Latin America (17, 22, 24). The disease is one of the most common systemic mycoses in Brazil, where epidemiological surveys carried out by use of the histoplasmin skin test indicate that this mycosis is endemic in all areas surveyed (9). These data suggest that the numbers of cases of histoplasmosis in Brazil may be underestimated and that the areas of endemicity are more widespread than previously thought.
The diagnosis of histoplasmosis is based on the results of clinical evaluation, culture of the organism, and various laboratory tests. Isolation and culture of H. capsulatum var. capsulatum provide a definitive diagnosis (15, 30), although complement fixation and precipitin tests that detect antibodies against the major serodiagnostic marker (the M antigen) play a significant role in the identification of infection (10, 11, 18, 33-36). The Histoplasma urine antigen test is also useful, particularly in cases of disseminated disease (31). However, serodiagnostic methods have limitations associated with false positivity, which arises from the presence of cross-reactive epitopes shared with other fungal pathogens.
At present, our group is focusing attention on the M glycoprotein because it is an immunodominant antigen that is responsible for the first precipitins to arise in acute-phase histoplasmosis; it is also commonly present in all phases of disease (11, 18, 19). The purification and evaluation of this molecule as a possible tool in the diagnosis of histoplasmosis were reported previously (33-35), together with the cloning, characterization, and expression of the gene encoding the M antigen (36). It was found that the sequence of the gene for the M antigen demonstrated significant homology to those of genes for other fungal catalases (8, 10, 23, 36); this discovery in turn raised the possibility that specific peptide sequences of the M-protein gene could be mapped by computational analysis. In the present study, four oligonucleotide primers whose sequences were based on regions of the M gene with little or no homology with the genes for other fungal catalases were designed for use in a PCR for the detection and identification of H. capsulatum var. capsulatum.
MATERIALS AND METHODS
Microorganisms.
Thirty-one H. capsulatum var. capsulatum isolates were included in this study. Eighteen H. capsulatum var. capsulatum strains were obtained from the Instituto de Pesquisa Clínica Evandro Chagas, Fundação Oswaldo Cruz; Hospital Universitário Pedro Ernesto, Universidade do Estado do Rio de Janeiro; and the Laboratório LÂmina, Rio de Janeiro, Brazil. The other four Brazilian isolates were kindly donated by Zoilo Camargo, Luzinete A. de Queiroz, Marilene R. Chang, and Maria do Rosário Rodrigues Silva. Fungal identification was done by conventional mycological methods, including morphology and the exoantigen test.
DNA isolation.
A single colony of yeast-phase H. capsulatum var. capsulatum was grown at 37°C in the citrate broth of Pine et al. (18) in a gyratory shaker at 120 rpm for 3 days. The yeast cells were harvested and washed three times in sterile distilled water by centrifugation at 2,000 × g. Genomic DNA was extracted from H. capsulatum var. capsulatum yeast cells with the Puregene DNA isolation kit (Gentra Systems, Inc. Minneapolis, Minn.). For atypical strains, which were not able to convert to the yeast phase, the DNA was extracted from fungal hyphae by a previously described method (7). DNA quantification was done in a Gene Quant pro RNA/DNA calculator (Amersham Pharmacia Biotech, Cambridge, United Kingdom).
Other DNA samples were kindly provided by Brent Lasker, Centers for Disease Control and Prevention, Atlanta, Ga. (H. capsulatum var. capsulatum isolates from the United States, H. capsulatum var. duboisii, H. capsulatum var. farciminosum, and Blastomyces dermatitidis); Rosaní S. Reis, IPEC-FIOCRUZ (Sporothrix schenckii); Patrícia M. S. Tavares, IPEC-FIOCRUZ (Candida spp.); Márcia dos S. Lazéra, IPEC-FIOCRUZ (Coccidioides immitis); Marília Nishikawa, INCQS-FIOCRUZ (Aspergillus spp.); and Maria José Mendes-Giannini (Cryptococcus neoformans).
Primer design.
Oligonucleotide primers were designed on the basis of the nucleotide sequence of the M antigen from H. capsulatum var. capsulatum deposited in GenBank (accession no. AFO26268) after comparison with the sequences of other catalases from eukaryotes. Alignments were made with the FASTA program (version 3). Table 1 includes the primer sequences, the positions of the primers in the open reading frame of the gene, and the predicted sizes of the amplified products.
TABLE 1.
PCR primer pairs selected from recombinant M protein
| PCR primer | Nucleotide sequence (5′-3′) | Complementary positions | Amplicon size (bp) |
|---|---|---|---|
| Pair 1 | |||
| Msp1F | ACA AGA GAC GAC GGT AGC TTC ACG | 2663-2686 | 111 |
| Msp1R | GCG TTG GGG ATC AAG CGA TGA GCC | 2750-2773 | |
| Pair 2 | |||
| Msp2F | CGG GCC GCG TTT AAC AGC GCC | 2702-2722 | 279 |
| Msp2R | ACC AGC GGC CAT AAG GAC GTC | 2960-2980 |
PCR assay.
PCR was performed with 100 ng of extracted DNA amplified in a 25-μl reaction volume consisting of PCR buffer (10 mM Tris-HCl, 50 mM KCl), 1.5 mM MgCl2, 200 μM deoxynucleoside triphosphates, 2.5 U of Taq DNA polymerase (Gibco BRL, Grand Island, N.Y.), and 20 pmol of each primer. Thirty-five cycles of repeated denaturation, primer annealing, and enzymatic chain extension were performed in a 2400 Thermal Cycler (Perkin-Elmer Applied Biosystems, Foster City, Calif.). The amplification program was carried out as follows: 95°C for 5 min, 1 min at 95°C, 1 min at 70°C, and 1 min at 72°C, followed by a single terminal extension at 72°C for 5 min. An internal PCR control was used in all amplifications to verify the efficiency of the test and to ensure that PCR inhibition was absent. The universal fungal primers ITS1 and ITS4, derived from highly conserved regions of the fungal rRNA gene, were used for this purpose (32).
PCR was carried out with serially diluted DNA extracted from H. capsulatum var. capsulatum strain F-182 (100 ng to 1 pg) to determine the analytical sensitivity of the test.
Electrophoresis.
The PCR products were electrophoresed through 1% agarose (Sigma Chemical Co., St. Louis, Mo.) dissolved in Tris-borate-EDTA buffer (0.1 M Tris, 0.09 M boric acid, 0.001 M EDTA [pH 8.4]). Electrophoresis was conducted at 80 V for 60 min, with 5 μl of each PCR amplicon plus 1 μl of tracking dye added to each well; the bands were visualized with a UV transilluminator after ethidium bromide staining.
Nucleotide sequence analysis.
The products amplified by PCR were purified with the QIAquick PCR purification kit (Qiagen Inc., Valencia, Calif.). The nucleotide sequences were determined with an automated sequencer (Perkin-Elmer Applied Biosystems) according to the instructions of the manufacturer.
RESULTS
The PCR system with the two specific primer pairs was specific for H. capsulatum var. capsulatum regardless of where the fungus was isolated (Table 2). Primer set Msp1F-Msp1R and primer set Msp2F-Msp2R amplified the predicted products of 111 and 279 bp, respectively, from all DNA templates obtained from 31 H. capsulatum var. capsulatum isolates studied, even those that had atypical morphologies and/or that were exoantigen negative (strains 16070, 90628, and 90652). The test displayed 100% sensitivity (Fig. 1), and it was able to amplify the predicted amplicons from as little as 1 ng of DNA (results not shown). Nucleotide sequence analysis of the amplicons revealed the corrected sequences of the H. capsulatum var. capsulatum M-antigen gene. The amplification of the rRNA gene produced a 600-bp amplicon for all fungi tested. These results demonstrate that the specific DNA targets were suitably amplified and ensured that PCR inhibition was absent.
TABLE 2.
H. capsulatum isolates used to evaluate PCR sensitivity
| Strain | Variety | Source | Origina |
|---|---|---|---|
| D-293 | H. capsulatum var. duboisii | Human | Africa |
| Hcf | H. capsulatum var. farciminosum | Unknown | Unknown |
| Downs | H. capsulatum var. capsulatum | Human | United States |
| G-222B | H. capsulatum var. capsulatum | Human | United States |
| G-217 | H. capsulatum var. capsulatum | Human | United States |
| G-186B | H. capsulatum var. capsulatum | Human | United States |
| G-184B | H. capsulatum var. capsulatum | Human | United States |
| FLS-1 | H. capsulatum var. capsulatum | Soil | United States |
| G-117B | H. capsulatum var. capsulatum | Human | United States |
| F-182 | H. capsulatum var. capsulatum | Human | United States |
| CADAM | H. capsulatum var. capsulatum | Dog | Campo Grande, RJ, Brazil |
| RS-1 | H. capsulatum var. capsulatum | Rat | Ilha Grande, RJ, Brazil |
| RS-9 | H. capsulatum var. capsulatum | Rat | Ilha Grande, RJ, Brazil |
| RS-36 | H. capsulatum var. capsulatum | Rat | Ilha Grande, RJ, Brazil |
| TI-14 | H. capsulatum var. capsulatum | Soil | Tinguá, RJ, Brazil |
| TI-05 | H. capsulatum var. capsulatum | Soil | Tinguá, RJ, Brazil |
| RPS-45 | H. capsulatum var. capsulatum | Soil | Rio da Prata, RJ, Brazil |
| RPS-86 | H. capsulatum var. capsulatum | Soil | Rio da Prata, RJ, Brazil |
| AC-2 | H. capsulatum var. capsulatum | Soil | Niterói, Brazil |
| 4959 | H. capsulatum var. capsulatum | Human | Rio de Janeiro, RJ, Brazil |
| 3669 | H. capsulatum var. capsulatum | Human | Rio de Janeiro, RJ, Brazil |
| 3356 | H. capsulatum var. capsulatum | Human | Rio de Janeiro, RJ, Brazil |
| 3237 | H. capsulatum var. capsulatum | Human | Rio de Janeiro, RJ, Brazil |
| 4631 | H. capsulatum var. capsulatum | Human | Rio de Janeiro, RJ, Brazil |
| 3612 | H. capsulatum var. capsulatum | Human | Rio de Janeiro, RJ, Brazil |
| 16070 | H. capsulatum var. capsulatum | Human | Rio de Janeiro, RJ, Brazil |
| 90628 | H. capsulatum var. capsulatum | Human | Rio de Janeiro, RJ, Brazil |
| 90652 | H. capsulatum var. capsulatum | Human | Rio de Janeiro, RJ, Brazil |
| 9414 | H. capsulatum var. capsulatum | Human | Rio de Janeiro, RJ, Brazil |
| SP-49 | H. capsulatum var. capsulatum | Human | São Paulo, SP, Brazil |
| RE-5646 | H. capsulatum var. capsulatum | Human | Recife, PE, Brazil |
| BG-365 | H. capsulatum var. capsulatum | Human | Campo Grande, MS, Brazil |
| GO-764 | H. capsulatum var. capsulatum | Human | GoiÂnia, GO, Brazil |
RJ, Rio de Janeiro; SP, Sao Panlo; PE, Pernambuco; MS, Mato Grosso do Sul; GO, Goiás.
FIG. 1.
Sensitivity and specificity of the PCR assay with DNA from various pathogenic fungi by using primers specific for H. capsulatum and based on the M-antigen gene sequence. (A) Msp1F-Msp1R primer pair; (B) Msp2F-Msp2R primer pair. Lanes: M, DNA molecular weight marker (marker IX; 72 to 1,356 bp; the numbers on both sides of the gel are in base pairs); 1, H. capsulatum var. capsulatum from a human (strain 4959); 2, H. capsulatum var. capsulatum from an animal (strain RS-1); 3, H. capsulatum var. capsulatum from soil (strain Ti-14); 4, H. capsulatum var. duboisii; 5, H. capsulatum var. farciminosum; 6, P. brasiliensis; 7, C. neoformans; 8, S. schenckii; 9, B. dermatitidis; 10, A. fumigatus; 11, A. niger; 12, C. albicans; 13, C. immitis.
When DNA from H. capsulatum var. duboisii was used as the template, identical PCR products were amplified with both primer sets. However, no DNA fragments were obtained when H. capsulatum var. farciminosum was used in the reaction mixture (Fig. 1).
For specificity testing, 31 strains of 12 fungal species were examined (Table 3). No target DNA products were amplified when DNAs from the phylogenetically related pathogens Paracoccidioides brasiliensis, C. immitis, and B. dermatitidis or the unrelated fungi Candida albicans, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida glabrata, S. schenckii, C. neoformans, Aspergillus niger, and Aspergillus fumigatus were tested with primers Msp1F-Msp1R and Msp2F-Msp2R.
TABLE 3.
Fungal species used to evaluate PCR specificity
| Strain identification | Species | Source | Origin |
|---|---|---|---|
| 16851 | Paracoccidioides brasiliensis | Human | Brazil |
| Pb 339 | Paracoccidioides brasiliensis | Human | Brazil |
| Pb 2 | Paracoccidioides brasiliensis | Human | Brazil |
| Pb 18 | Paracoccidioides brasiliensis | Human | Brazil |
| Pb 192 | Paracoccidioides brasiliensis | Human | Brazil |
| 164 | Paracoccidioides brasiliensis | Human | Colombia |
| 165 | Paracoccidioides brasiliensis | Human | Colombia |
| 27 | Cryptococcus neoformans | Human | Brazil |
| 37 | Cryptococcus neoformans | Human | Brazil |
| 45 | Cryptococcus neoformans | Human | Brazil |
| 540 | Cryptococcus neoformans | Human | Brazil |
| 15242 | Cryptococcus neoformans | Human | Brazil |
| 48 | Sporothrix schenckii | Cat | Brazil |
| 142 | Sporothrix schenckii | Cat | Brazil |
| 15677 | Sporothrix schenckii | Human | Brazil |
| 16415 | Sporothrix schenckii | Human | Brazil |
| 16459 | Sporothrix schenckii | Human | Brazil |
| 17500 | Sporothrix schenckii | Human | Brazil |
| 46-6776 | Coccidioides immitis | Soil | Brazil |
| 47-19 | Coccidioides immitis | Rat | Brazil |
| 48-19 | Coccidioides immitis | Rat | Brazil |
| Bd 3-4 | Blastomyces dermatitidis | Human | United States |
| ATCC 16913 | Aspergillus fumigatus | Human | United States |
| ATCC 1004 | Aspergillus niger | Unknown | United States |
| ATCC 16404 | Aspergillus niger | Fruits | United States |
| ATCC 9642 | Aspergillus niger | Environment | Australia |
| 121 | Candida albicans | Human | Brazil |
| 80848 | Candida parapsilosis | Human | Brazil |
| 22 | Candida tropicalis | Human | Brazil |
| 30 | Candida krusei | Human | Brazil |
| 78752 | Candida glabrata | Human | Brazil |
DISCUSSION
The detection of H. capsulatum infections is still largely dependent on culture of the organism from material taken from the patient; once the organism is in culture, precise identification relies on visualization of the typical morphology and the demonstration of dimorphism. Identification of the fungal species grown in cultures of clinical specimens is essential for appropriate clinical decision making and the monitoring of antifungal therapy. Unfortunately, saprophobic fungi such as members of the genera Chrysosporium, Corynascus, Renispora, and Sepedonium produce structures which resemble the tuberculated macroconidia of H. capsulatum var. capsulatum, making visual identification difficult. In addition, many laboratories, particularly those in developing countries, do not have direct access to trained mycologists capable of confirming a visual identification. The use of thermal dimorphism for identification may also be problematic since conversion to the yeast phase in vitro is strain dependent, and the process may take up to 4 weeks (30).
This report describes a one-step PCR assay for the detection and identification of DNA sequences from the gene encoding the M antigen of H. capsulatum var. capsulatum. This assay had a sensitivity and specificity of 100%. All H. capsulatum var. capsulatum isolates tested in the study, including those incompletely distinguished by morphological and exoantigen tests, were correctly identified. Thus, H. capsulatum var. capsulatum 90628 and 90652 did not convert at 37°C, did not yield macroconidia and microconidia after 2 months of cultivation, and did not produce exoantigen; however, both were PCR positive. The same was true of H. capsulatum var. capsulatum 16070, which also did not yield the characteristic morphology when it was grown at 25 and 37°C (although it was exoantigen positive). The presence of a unique band as a product of amplification has been considered a positive result for PCR with specific primers (1). A single band of 1.0 pg was amplified from target H. capsulatum var. capsulatum DNA with the specific primers Msp1F-Msp1R and Msp2F-Msp2R, and sequencing confirmed the identities of the predicted 111- and 279-bp products, so validating the sensitivity and specificity of this test. Both primer pairs were equally effective, which confirms that the approach taken was suitably robust. Certainly, the sensitivity of the test described in this report compares favorably with that of a previously disclosed assay, which had a sensitivity of 1.0 pg when primers Pm1 and Pm2 were used in a single PCR amplification for the rapid identification of Penicillium marneffei (26).
The source of the H. capsulatum var. capsulatum isolates, whether clinical or environmental, had no impact on the sensitivity of detection, which proves the utility of the primers selected for PCR assay. The specificity of this assay was demonstrated by the fact that no specific band was amplified when the DNAs of other pathogenic fungal species were used as the DNA template. Thus, for example, the absence of any amplification product from Candida species demonstrates that this one-step PCR test can be used to avoid the false detection of such potentially saprophytic fungi.
Recently, applications have been developed to apply nested PCR assays in the detection of H. capsulatum var. capsulatum in cultures of clinical and environmental isolates (4, 5, 6, 21). These studies were accomplished with either a single-copy gene (4) or ribosomal genes (21). A PCR assay targeting the rRNA genes has the ability to achieve a higher sensitivity. However, the conserved nature of rRNA genes may cause nonspecific amplification (3). Nested PCR has several inherent problems including contamination during the manipulation and nonspecific amplification. The latter was demonstrated by Bialek and coworkers (2, 6), who found that even the high stringency of their nested PCR could not prevent nonspecific amplification. Methods to detect multiple fungal species (20, 25) have included a PCR-enzyme immunoassay which could detect H. capsulatum var. capsulatum (16). However, that test requires the use of multiple heterologous probes, and in addition, the production of minor hybridization signals among taxonomically related fungi is a problem. The technological complexity of this methodology may make it unsuitable for use in less well equipped centers.
The system of DNA detection described here could be expanded to detect individual varieties within species of the genus Histoplasma. Thus, one band was also observed when the H. capsulatum var. duboisii template was evaluated by this test. Although this variety is distinct from H. capsulatum var. capsulatum in term of its epidemiology, clinical manifestations, and the morphology of the yeast phase, isolates of H. capsulatum var. duboisii can be sexually crossed with H. capsulatum var. capsulatum (13). These fungi are classified as a single biological species, in which the two varieties share a specific mate recognition system (14). Surprisingly, amplification did not occur when the DNA template consisted of H. capsulatum var. farciminosum DNA. The latter causes subcutaneous and ulcerated lesions of the skin only in horses and mules (15). The taxonomic status of H. capsulatum var. farciminosum is controversial at present. Kwon-Chung and Bennett (15) described it as a separate species, although more recently (1999), Kasuga et al. (12) have suggested that H. capsulatum var. farciminosum is taxonomically related to H. capsulatum var. capsulatum strains isolated in South America, although it is phenotypically and pathologically distinct. However, because we examined only one isolate of H. capsulatum var. farciminosum, we cannot contribute meaningfully to this taxonomic debate.
In conclusion, the PCR assay described in this report, which was developed with specific primers pairs whose sequences were based on the H. capsulatum var. capsulatum M-protein gene sequence, proved to be a sensitive and specific test for the identification of typical and atypical isolates of this fungal species. This methodology has obvious applications in the identification of H. capsulatum var. capsulatum strains cultured from clinical samples, and studies are in progress to determine its application to the diagnosis of histoplasmosis in a diagnostic laboratory setting. This study also represents the first step toward determining whether this test has applicability to the direct detection of H. capsulatum var. capsulatum in clinical samples such as blood or tissue; future work will be targeted toward this goal.
Acknowledgments
We thank the Mycotic Diagnosis Section staff for support on the isolation of H. capsulatum var. capsulatum; Andréa Pussenti Derossi and Rosane Orofino Costa for providing the H. capsulatum var. capsulatum isolates from the Hospital Universitário Pedro Ernesto, Universidade do Estado do Rio de Janeiro, and Laboratório Diagnostico das Americas, Rio de Janeiro, Brazil; Viviana Malirat and José Júnior F. Barros, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, for nucleotide sequence analysis.
The research was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico of Brazil and an International Collaborative grant from the Wellcome Trust of the United Kingdom.
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