Molecular Detection of Histoplasma capsulatum var. capsulatum in Human Clinical Samples

Abstract

We developed a seminested PCR for the diagnosis of histoplasmosis that amplifies a portion of the Histoplasma capsulatum H antigen gene. This assay is highly sensitive and specific, being able to detect genomic material corresponding to less than 10 yeast cells without cross-reaction against other bacterial or fungal pathogens.

Most Histoplasma infections are subclinical, frequently being limited by the host immune response. However, if the immune system fails to control the infection, this fungus can lead to acute or chronic lung infection or disseminated histoplasmosis (9).

Relying on the sole basis of the clinical symptoms, it is not possible to confirm a diagnosis of pulmonary histoplasmosis, since the clinical picture of histoplasmosis strongly mimics those of tuberculosis and some other lung diseases (14). Microscopic identification of histoplasma in May-Grünwald-Giemsa-stained slides is an acceptable approach, but an experienced operator is needed to obtain reliable results. The fungus can be cultured from different sources such as blood, bone marrow, respiratory secretions, or localized skin lesions in more than 85% of the cases (11, 12, 17, 20). Cultures from bone marrow yield the highest frequency of positive isolations (70 to 90%), while respiratory specimens give positive results less frequently (50 to 90%). A consistent identification of the cultured fungus usually takes between 2 and 6 weeks, introducing undesirable delay in the diagnosis and therapy. Serological methods are faster than culture, but they can lead to false-positive results, since the titer of specific antibodies against Histoplasma remains high for months or even years after the primary infection (1, 6, 7). In addition, cross-reactivity against Paracoccidioides brasiliensis can give false positive results (13). On the other hand, false-negative results due to low antibody titers can be observed in immunocompromised patients with active infection (18).

Another reliable alternative for diagnosing disseminated histoplasmosis is the detection of H. capsulatum antigen in body fluids (3-5, 15, 16, 21). Although this method is more sensitive than serum antibody detection assays, cross-reactivity against other pathogenic fungi capable of sharing infection sites with H. capsulatum remains a problem to be solved (8, 19).

Design of the seminested PCR assay.

The complete sequence of H. capsulatum H antigen gene (GenBank accession no. U20346) coding for β-glucosidase was used to design specific oligonucleotides by computational methods. Three oligonucleotides, Hc1, Hc2, and Hc3, placed at the fifth exon were chosen for their ability to differentiate the sequence of H. capsulatum from the sequences of other fungal β-glucosidases in the databases. The outer primers were Hc2 (5′-GCGGGGTTGGCTCTGCTCT-3′) and Hc3 (5′-TTGGAAACCCCGGGCTTG-3′), which produced a 439-bp fragment. For the seminested amplification reaction, Hc2 was used with the inner primer Hc1 (5′-TCATAGTAGGCTGTTCACCCCCG-3′), yielding a product of 330 bp. The first PCR was performed with 2 μl of purified DNA in a final volume of 50 μl (200 μM each deoxynucleoside triphosphate [dNTP], 0.4 μM each primer, 2 mM MgCl, 10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1 U of Taq polymerase). The conditions for seminested PCR were similar to those of the first reaction, except the Hc3 primer was replaced with Hc1, and 1 μl of the first reaction product was used as a template. In both cases, PCR amplification conditions were 96°C for 6 min; 35 cycles of 94°C for 1 min, 59°C for 1 min, and 72°C for 1 min; and a final extension at 72°C for 10 min. The sensitivity of the reaction was assayed by sequential dilution of spectrophotometrically dosed H. capsulatum DNA (2). As can be observed in Fig. 1, a 439-bp product was obtained after the first reaction only when quantities of DNA equivalent to ≥2.5 × 10⁵ genome copies were used. After the seminested reaction, the expected 330-bp fragment was obtained even when less than 10 genome copies were loaded in the first PCR tube.

FIG. 1.

Sensitivity of seminested PCR assay. (A) Schematic representation of primers location at the H. capsulatum H antigen (Ag) gene. Primer sequences are described in the text. (B) Amplification products of both the first reaction and seminested PCR, analyzed by ethidium bromide-stained agarose gel electrophoresis. The amounts of DNA (expressed as genome equivalents) loaded in the first reaction were 5 × 10⁶ (lane 1), 2.5 × 10⁵ (lane 2), 5 × 10⁴ (lane 3), 5 × 10³ (lane 4), 5 × 10² (lane 5), 5 × 10¹ (lane 6), and 5 (lane 7).

To assess seminested PCR specificity, we tested the assay with DNA from several fungal and bacterial pathogens usually associated with respiratory diseases. No amplification product was obtained when DNAs from Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Geotrichum sp., Cryptococcus neoformans, Candida albicans, Trichosporon sp., P. brasiliensis, Nocardia asteroides, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella sp., Streptococcus pneumoniae, Mycobacterium bovis, and M. tuberculosis were tested. DNA quality and absence of inhibitors were tested as described below.

Use of the seminested PCR assay in clinical samples.

Table 1 shows the results of a retrospective assay performed on 30 samples obtained from regional public health centers. All patients lived in an area of endemicity surrounding the city of Rosario, Argentina. Samples were submitted to microscopic observation (May-Grunwald-Giemsa staining); cultured in Mycosel, Sabouraud, Sabouraud-chloramphenicol (Chloromycetin), and V₈ agars at 28°C and brain heart infusion agar plus blood at 37°C; and analyzed by PCR. DNA was extracted from 500 μl of blood samples as follows. Cellular pellets were recovered by centrifugation (10 min at 10,000 × g), washed twice with 500 μl of sterile distilled water, resuspended in 500 μl of phosphate-buffered saline (PBS) containing 3.5 mg of zymosan per ml (Sigma, St. Louis, Mo.), and incubated at 37°C for 2 h. Then, sodium dodecyl sulfate (SDS) and proteinase K were added to final concentrations of 0.1% (wt/vol) and 100 μg/ml, respectively. After a 30-min incubation at 65°C, samples were boiled for 10 min, cooled and extracted with phenol-chloroform, precipitated with ethanol, and resuspended in 10 μl of TE (10 mM Tris [pH 7.5], 1 mM EDTA). Tissue samples (100 to 300 mg) were incubated overnight at 37°C with 500 μl of a solution containing 100 mM NaCl, 10 mM Tris-HCl (pH 8), 25 mM EDTA, 0.5% (wt/vol) SDS, and 100 μg of proteinase K per ml, extracted with phenol-chloroform, and precipitated in alcohol. Samples from mucocutaneous lesions were recovered by scraping them off with a sterile scalpel blade in the presence of PBS and then were centrifuged, resuspended in PBS-zymosan, and processed as described above. In parallel to each PCR assay, inhibitory and cross-contamination controls were performed with either 0.33 pg of H. capsulatum DNA or 2 μl of water, respectively. DNA quality was tested by a random amplification of polymorphic DNA (RAPD) assay, performed either with the pulmonary pathogen DNA samples or samples with a unique degenerate primer, P_S20 (5′-GCTGCAGCYTCRTCNGGRTT-3′), as previously described (10).

TABLE 1.

Results of 30 samples analyzed by staining, culture, and PCR assay

Sample no.	Sample	Result by:
		MGGa staining	Culture	PCR
1	Biopsy	Negative	Negative	Negative
2	Biopsy	H. capsulatum	H. capsulatum	Positive
3	Blood	Negative	Negative	Negative
4	Blood	Negative	Candida spp. and Cryptococcus neoformans	Negative
5	Blood	Negative	Negative	Negative
6	Blood	Negative	Negative	Negative
7	Blood	Negative	Negative	Negative
8	Blood	Negative	Negative	Negative
9	Blood	Negative	Negative	Negative
10	Blood	Negative	Negative	Negative
11	Blood	Negative	C. neoformans	Negative
12	Blood	Negative	Negative	Negative
13	Blood	Negative	Negative	Negative
14	Blood	Negative	Negative	Negative
15	Blood	Negative	Negative	Negative
16	Blood	Negative	Negative	Negative
17	Blood	Negative	Negative	Negative
18	Blood	Negative	Negative	Negative
19	Blood	Negative	Negative	Negative
20	Blood	Negative	Negative	Negative
21	Blood	Negative	Negative	Negative
22	Blood	Negative	Negative	Negative
23	Blood	Negative	Negative	Positive
24	Blood	Negative	Negative	Positive
25	Blood	Negative	H. capsulatum	Positive
26	Blood	Negative	H. capsulatum	Positive
27	Scraping	Negative	Negative	Negative
28	Scraping	H. capsulatum	H. capsulatum	Positive
29	Scraping	H. capsulatum	H. capsulatum	Positive
30	Scraping	H. capsulatum	H. capsulatum	Positive

^aMCG, May-Grünwald-Giemsa.

Four of the 24 blood samples were positive for PCR, although 2 of them have been considered negative by other methods. From the two culture-negative PCR-positive samples, one corresponded to a patient with confirmed clinical histoplasmosis, and the second was lost to follow-up. A complete agreement between the three methods was found in four other skin samples obtained from AIDS patients with cutaneous manifestations. Similar results were found when two biopsy specimens were analyzed. All negative samples were tested for the presence of PCR inhibitors in parallel reactions as mentioned above. Even considering that the number of samples analyzed was quite limited, these results are promising and lead us to propose PCR as a useful additional tool for the diagnosis of H. capsulatum.