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Lack of sensitive diagnostic tests impairs the understanding of the epidemiology of histoplasmosis, a disease whose burden is estimated to be largely underrated. Broad-range PCRs have been applied to identify fungal agents from pathology blocks, but sensitivity is variable. In this study, we compared the results of a specific <i>Histoplasma</i> qPCR (<i>H</i>. qPCR) with the results of a broad-range qPCR (28S qPCR) on formalin-fixed, paraffin-embedded (FFPE) tissue specimens from patients with proven fungal infections (<i>n</i> = 67), histologically suggestive of histoplasmosis (<i>n</i> = 36) and other mycoses (<i>n</i> = 31). The clinical sensitivity for histoplasmosis of the <i>H</i>. qPCR and the 28S qPCR was 94% and 48.5%, respectively. Samples suggestive for other fungal infections were negative with the <i>H</i>. qPCR. The 28S qPCR did not amplify DNA of <i>Histoplasma</i> in FFPE in these samples, but could amplify DNA of <i>Emergomyces</i> (<i>n</i> = 1) and <i>Paracoccidioides</i> (<i>n</i> = 2) in three samples suggestive for histoplasmosis but negative in the <i>H.</i> qPCR. In conclusion, amplification of <i>Histoplasma</i> DNA from FFPE samples is more sensitive with the <i>H.</i> qPCR than with the 28S qPCR. However, the 28S qPCR identified DNA of other fungi in <i>H</i>. qPCR-negative samples presenting like histoplasmosis, suggesting that the combination of both assays may improve the diagnosis.