Diagnostic value of a nested polymerase chain reaction for diagnosing cutaneous sporotrichosis from paraffin‐embedded skin tissue

Abstract

Summary Background The gold standard for diagnosis of cutaneous sporotrichosis involves the isolation of the fungus, Sporothrix, by a culture test. Generally, the sampling for the culture test is performed at the same time as skin biopsy under local anaesthesia. However, the culture test may occasionally return a false negative result. Objective The aim of our study was to investigate the diagnostic value of a molecular method for diagnosing cutaneous sporotrichosis from formalin‐fixed and paraffin‐embedded (FFPE) tissues. Methods Over a 30‐year period, we collected 52 cases of cutaneous sporotrichosis from biopsied specimens that had been positively diagnosed by a culture test. A nested PCR specific for Sporothrix detection was applied using FFPE tissue as template. The results were compared with control samples from 79 patients diagnosed with other cutaneous diseases according to histopathological, clinical findings and a cutler test. Results Of the 52 patients who were tested positive on the culture test, all cutaneous diseases were detected by PCR. Of the 59 patients in the control group, 58 tested negative by PCR. Under our conditions, the calculated sensitivity of this method was 100%, the specificity was 98.7% and the kappa coefficient was 0.984 (95% CI: 0.953‐1.000). Conclusions The specific PCR assay used appears to be a useful tool for the prompt and accurate diagnosis of sporotrichosis. Using this method, it would be possible to diagnose cutaneous sporotrichosis for patients who were suspected of cutaneous sporotrichosis but tested negative on culturing, and for pathologically suspected cutaneous sporotrichosis patients for whom the culture test was not undertaken.

Conjunctival sporotrichosis from cat to human: Case report

“…Sensitivity and specificity of PCR were as high as 100% and 98.7%, respectively. 13 In our case, histopathological review from cat wound (demonstrated yeast-like organism), history of cat contact and location of cat's wound which developed on its nose brought us recognition of mycoses that grew at subcutaneous level. Sporothrix schenckii was detected from PCR performing.…”

Nested PCR for the Diagnosis of Feline Sporotrichosis From Formalin-Fixed and Paraffin-Embedded Samples Using Different DNA Extraction Protocols

“…In this work, the nested PCR methodology performed was similar to that described by Hu et al (40) based on amplification of the 18S region of ribosomal RNA (ITS), using specific primers for Sporothrix sp.. A semi-nested PCR methodology was developed for the diagnosis of human sporotrichosis in tissues embedded in paraffin with high sensitivity and specificity, but few cases were analyzed and there was a lack of details that make its reproducibility difficult (49). Also, the authors used the same chemical DNA extraction kit but it was not reported how the paraffin blocks were sectioned (49). Hu et al (40) described a nested PCR assay for the detection of S. schenckii…”

“…A semi-nested PCR methodology was developed for the diagnosis of human sporotrichosis in tissues embedded in paraffin with high sensitivity and specificity, but few cases were analyzed and there was a lack of details that make its reproducibility difficult ( 49 ). Also, the authors used the same chemical DNA extraction kit but it was not reported how the paraffin blocks were sectioned ( 49 ). Hu et al ( 40 ) described a nested PCR assay for the detection of S. schenckii for diagnosing cutaneous sporotrichosis in frozen skin tissues ( 40 ).…”

“…Hu et al ( 40 ) described a nested PCR assay for the detection of S. schenckii for diagnosing cutaneous sporotrichosis in frozen skin tissues ( 40 ). Based on them, Hayashi et al ( 49 ) designed new primers that increased the sensitivity and specificity (100% and 98.7%, respectively) of their semi-nested PCR assay. In the present study, we used the same Sporothrix sp.…”

“…Prior to this study, the detection of Sporothrix sp. DNA by nested PCR in FFPE tissue samples was investigated only once, but using human samples ( 49 , 50 ). There is still no standardized and reproducible nested PCR methodology for the definitive diagnosis of animal sporotrichosis using FFPE tissues.…”