Detection ofHistoplasma capsulatumfrom clinical specimens by cycling probe-based real-time PCR and nested real-time PCR

Abstract

We developed new cycling probe-based real-time PCR and nested real-time PCR assays for the detection of Histoplasma capsulatum that were designed to detect the gene encoding N-acetylated α-linked acidic dipeptidase (NAALADase), which we previously identified as an H. capsulatum antigen reacting with sera from patients with histoplasmosis. Both assays specifically detected the DNAs of all H. capsulatum strains but not those of other fungi or human DNA. The limited of detection (LOD) of the real-time PCR assay was 10 DNA copies when using 10-fold serial dilutions of the standard plasmid DNA and 50 DNA copies when using human serum spiked with standard plasmid DNA. The nested real-time PCR improved the LOD to 5 DNA copies when using human serum spiked with standard plasmid DNA, which represents a 10-fold higher than that observed with the real-time PCR assay. To assess the ability of the two assays to diagnose histoplasmosis, we analyzed a small number of clinical specimens collected from five patients with histoplasmosis, such as sera (n = 4), formalin-fixed paraffin-embedded (FFPE) tissue (n = 4), and bronchoalveolar lavage fluid (BALF) (n = 1). Although clinical sensitivity of the real-time PCR assay was insufficiently sensitive (33%), the nested real-time PCR assay increased the clinical sensitivity (77%), suggesting it has a potential to be a useful method for detecting H. capsulatum DNA in clinical specimens.

Laboratory Diagnostics for Histoplasmosis

“…Laboratory-developed PCR assays using a variety of molecular targets have been developed (Table 4). Compared to culture and a criterion-based or clinical diagnosis of histoplasmosis, the sensitivity of molecular assays in published studies has ranged between 67 and 100% (30-35) and 33 and 87% (36,37), respectively. A fluorescence in situ hybridization (FISH) technique that successfully detects H. capsulatum rRNA in blood cultures may circumvent the need for colony growth to obtain a definitive and timely diagnosis (35).…”

Identification of Histoplasma causing an unexplained disease cluster in Matthews Ridge, Guyana

“…Unfortunately, commercial kits to detect Histoplasma antigens based on enzyme immunoassay are still not available in China. PCR, nested PCR and real-time PCR using Histoplasma specific primers were reported to distinguish cultured Histoplasma from other pathogenic fungi [14]. There are few reports on the detection of capsular Histoplasma by high-throughput sequencing.…”

“…Real-time PCR was performed on all samples from the 12 patients to screen for Influenza A virus (in-house developed), Coronavirus (in-house developed), Black creek canal virus (in-house developed), Andes virus (inhouse developed), Guanarito virus (in-house developed), Sabia virus (inhouse developed), Nipah virus (in-house developed), Flavivirus (in-house developed), Leptospira [12,13], and Histoplasma [14,15], according to the cited publications or in-house protocols.…”

Timely Diagnosis of Histoplasmosis in Non-endemic Countries: A Laboratory Challenge

“…The results of this work led the authors to conclude that multicopy targets were the best option when designing an assay as they provide and increase in sensitivity without decreasing specificity; real time PCR proved to be more advantageous than conventional PCR. In contrast, one study limited to a small number of samples from a single laboratory showed a better sensitivity of nested PCR assays as compared to designs based on cycling-probe real-time PCR (Muraosa et al, 2016). Such differences highlight the need of collaborative networks to assess the diagnostic yield of different molecular assay designs for the diagnosis of histoplasmosis, particularly in areas of low prevalence.…”