Detection of Histoplasma DNA from Tissue Blocks by a Specific and a Broad-Range Real-Time PCR: Tools to Elucidate the Epidemiology of Histoplasmosis

Abstract

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 Histoplasma qPCR (H. 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 (n = 67), histologically suggestive of histoplasmosis (n = 36) and other mycoses (n = 31). The clinical sensitivity for histoplasmosis of the H. qPCR and the 28S qPCR was 94% and 48.5%, respectively. Samples suggestive for other fungal infections were negative with the H. qPCR. The 28S qPCR did not amplify DNA of Histoplasma in FFPE in these samples, but could amplify DNA of Emergomyces (n = 1) and Paracoccidioides (n = 2) in three samples suggestive for histoplasmosis but negative in the H. qPCR. In conclusion, amplification of Histoplasma DNA from FFPE samples is more sensitive with the H. qPCR than with the 28S qPCR. However, the 28S qPCR identified DNA of other fungi in H. qPCR-negative samples presenting like histoplasmosis, suggesting that the combination of both assays may improve the diagnosis.

Rapid Diagnosis of Central Nervous System Scedosporiosis by Specific Quantitative Polymerase Chain Reaction Applied to Formalin-Fixed, Paraffin-Embedded Tissue

“…A PCR targeting Fusarium would have been a logical next assay in hyalohyphomycosis but was not performed as cultures grew S. apiospermum. It has been demonstrated before that specific qPCR assays offer improved sensitivity compared to broad-range PCR and sequencing when applied to FFPE tissue in the context of mold infections and histoplasmosis [22,23]. The optimal molecular strategy may include both broad-range assays and specific assays selected for fungi, likely causing a given histopathologic result.…”

Animal Histoplasmosis in Europe: Review of the Literature and Molecular Typing of the Etiological Agents

“…This entailed the isolation of H. capsulatum , histopathology or direct microscopy revealing the typical morphological features of histoplasmosis (2–4 µm narrow-based budding yeast cells) [ 29 ]. In the case of a histopathological diagnosis, histoplasmosis was confirmed by specific H. capsulatum PCRs or panfungal PCRs followed by amplicon identification by Sanger sequencing or hybridization on a chip [ 30 , 31 ]. Data on host, living environment, type of infection and diagnosis were provided by referring veterinaries.…”

A multicentre external quality assessment: A first step to standardise PCR protocols for the diagnosis of histoplasmosis and coccidioidomycosis

“…8 On the other hand, histopathological observation of fungal structures in tissues requires skilled personnel, as several protozoa and fungi, especially other dimorphic fungi, may be challenging to differentiate from Histoplasma and Coccidioides. [8][9][10][11][12] In addition, the sensitivity of these conventional methods is moderate, ranging from 0 to around 75% depending on clinical manifestations and origin of the samples. 8 Besides these golden standards, specific antigen detection tests may be a good additional tool for the diagnosis of histoplasmosis and coccidioidomycosis; however, cross-reactions are described with other fungi, sensitivity is decreased in immunocompetent patients [13][14][15][16][17][18][19][20] and antigen detection tests are still not largely available.…”

“…Molecular diagnostic testing on clinical samples has shown its usefulness in diagnosing fungal infections in animals 26,27 and humans 12,[28][29][30][31][32][33][34][35] and detecting environmental niches. [36][37][38][39] Besides existing broad-range fungal PCRs that rely upon amplicon identification by sequencing, multiple specific in-house assays were developed during the past years to detect H. capsulatum and Coccidioides spp.…”

“…house PCR protocols for detecting H. capsulatum and Coccidioides spp. Techniques based on PCR are essential to achieve a rapid diagnosis of imported12,34,35,40,79,80 and autochthonous 26 endemic mycosis cases. Until today, only one inter-laboratory study compared seven different H. capsulatum PCR protocols in four laboratories from Latin America and one in Spain 81 showing that protocols based on qPCR were the most sensitive and reproducible.…”