The diagnosis of histoplasmosis has historically been based on the findings of culture and microscopy, which lack sensitivity, require interpretive expertise, and in particular, fail to provide the timely diagnosis required for the institution of treatment of patients infected with both HIV and histoplasma (9
). The advent of DNA-based diagnosis has resulted in the development of a number of molecular tests that employ different DNA targets (2
). The choice of rRNA gene targets can contribute to an unacceptable rate of false-positive results, particularly when taxonomically close relatives cross-react with Histoplasma
). To circumvent this problem, Bialek et al. developed a nested PCR assay using the Hc100 gene, which they determined was specific for H. capsulatum
). They recognized the need for this assay to be validated further with patient samples collected from a region where Histoplasma
is endemic. This challenge was first addressed by Maubon et al. (22
), who tested 40 samples from 27 patients in French Guiana, South America, and found that all of the cultures positive for H. capsulatum
= 15) were also positive by the Hc100 PCR. The study we report here was designed to validate the Hc100 PCR assay for the diagnosis of histoplasmosis with samples from the largest study population evaluated to date: 146 clinical samples from 135 patients in Medellín, Colombia, suspected of having histoplasmosis. We obtained a sensitivity of 100% and a specificity 92.4% compared to the results for the negative controls and a specificity of 95.2% compared to the results for patients with other infectious diseases.
We also provide valuable information on the representativeness and reliability of the Hc100 gene as an indicator of H. capsulatum by verifying the presence of this gene in a series of H. capsulatum isolates from North, Central, and South America and from Africa, as well as by determining the absence of this target in the closely related fungi Paracoccidioides brasiliensis and Blastomyces dermatitidis, as well as Aspergillus, Cryptococcus, Candida, Coccidioides, and Mycobacterium species. These studies verified that clinical samples from patients with infections due to other respiratory pathogens would not be expected to display cross-reactivity with the Hc100 gene target.
We found that the Hc100 nested PCR assay was positive for 13 of the clinical samples from patients with respiratory symptoms who were negative for H. capsulatum
by culture. Histoplasmosis is endemic in Colombia, and judging from the high rate of histoplasmin skin test positivity for healthy adults (22%), it can be speculated that such patients were infected with H. capsulatum
). If this value is reflected in the total number of inhabitants of the country, the number of infected persons would be close to 6 million. It is therefore possible to find subclinical histoplasmosis in both symptomatic patients and asymptomatic persons who remain healthy. A test as sensitive as the one that is being implemented could well detect these subclinical infections in patients who were not culture positive for H. capsulatum.
These positive results may well occur for persons with latent histoplasmosis, in a manner similar to that for tuberculosis (17
When we analyzed the 60 samples from patients with proven respiratory infections other than histoplasmosis, we found that one patient with established cryptococcosis and two patients with diagnoses of esophageal and pleural candidiasis, respectively, were positive by the Hc100 nested PCR. All amplified products were confirmed by sequence analysis, and they showed over 98% identity with the gene coding for the H. capsulatum
-specific 100-kDa protein. The findings suggest that the patients mentioned above may also have concomitantly had histoplasmosis that had not been previously detected by conventional tests. It is known that the identification of microorganisms by isolation in culture, especially if the microorganism is H. capsulatum
, has its limitations. Culture methods can produce false-negative results for about 20% and 50% of patients with disseminated pulmonary histoplasmosis and chronic pulmonary histoplasmosis, respectively (28
). Concerning the cryptococcosis patient whose sample was positive by the Hc100 nested PCR, the clinical record showed that he had AIDS and a low CD4+
lymphocyte count (77 cells per microliter). Thus, in this group of patients, one may find nonapparent coinfections with AIDS-associated microorganisms (1
). For the patients with candidiasis, we were, unfortunately, unable to access their medical records to obtain detailed data that could have revealed an explanation for their positive reactions.
In addition, we confirmed the detection limits of the test and showed that it is highly sensitive, detecting approximately either 10 yeast cells per reaction or H. capsulatum
DNA at concentrations as low as 20 fg, suggesting that the Hc100 nested PCR reaches an analytical sensitivity similar to the analytical sensitivities reported in other studies (3
Histoplasmosis is not a mandatory reportable disease; thus, its prevalence is difficult to calculate. We determined the positive and negative likelihood ratios and obtained values of 25 and 0, respectively. These values indicate that a positive result by the Hc100 nested PCR is about 25 times more probable for a patient with histoplasmosis than for an individual without histoplasmosis.
In conclusion, the Hc100 nested PCR is a promising diagnostic tool that can be implemented to detect H. capsulatum DNA in a variety of clinical samples. Thus, this molecular test appears to be much more sensitive than culture, with the latter being considered the gold standard. This method should be valuable in areas where H. capsulatum is endemic.