Detection of Sporothrix schenckii in clinical samples by a nested PCR assay

Abstract

Cutaneous sporotrichosis is a chronic granulomatous fungal infection caused by Sporothrix schenckii with worldwide distribution. Its traditional diagnosis is time-consuming and difficult to differentiate from that of a clinical sporotrichoid lesion caused by various pathogens. In this study, a nested PCR assay for the detection of S. schenckii was evaluated by using a sequence of 18S rRNA gene as a target. For the examination of specificity and sensitivity, five clinical isolates with 1 ATCC 10213 strain of S. schenckii, 10 strains of clinical common fungi, 3 strains of Mycobacterium spp., Staphylococcus aureus, and normal human skin tissue were used. The expected fragment was amplified from six S. schenckii isolates in the first round and nested PCR but not from other microorganisms and human DNA. Their sequences were 100% identical to the S. schenckii 18S rRNA gene sequence deposited in GenBank. A detection limit of 40 fg of S. schenckii DNA extract was determined with ethidium bromide staining. Serial dilution studies demonstrated that the nested PCR could detect a DNA amount of 1 CFU of S. schenckii in tissue samples. We further investigated the nested PCR assay for the detection of S. schenckii from the tail tissues of 5 experimentally infected mice and from the clinical biopsy specimens of 12 patients with sporotrichosis confirmed by culture or histochemical staining. The nested PCR assay was positive in all 5 infected mice and in 11 of the 12 clinical specimens. The high sensitivity and specificity of this nested PCR indicate that the assay can provide rapid diagnosis with sufficient accuracy to be clinically useful for patients with sporotrichosis.

FIG. 1.

Specificity of the nested PCR assay. (A) DNA extracted from common fungi. (B) DNA extracted from bacteria, mycobacteria, and normal human tissue. Lanes: M, molecular size marker (100-bp ladder [Promega]); 1, distilled water; 2, S. schenckii; 3, C. albicans; 4, C. glabrata; 5, C. parapsilosis; 6, C. tropicalis; 7, T. asahii; 8, T. rubrum; 9, Fonsecaea spp.; 10, Cladosporum spp.; 11, Penicillium spp.; 12, A. flavus; 13, S. schenckii; 14, M. tuberculosis; 15, M. marinum; 16, M. chelonae; 17, S. aureus; 18, normal human skin.

FIG. 2.

Sensitivity of the PCR assay with DNA from S. schenckii. (A) First-round PCR assay. (B) Nested PCR assay. Lanes: M, molecular size marker (100-bp ladder [Promega]); 1, distilled water; 2, 4 × 10⁻¹¹ g of S. schenckii DNA; 3, 4 × 10⁻¹² g of S. schenckii DNA; 4, 4 × 10⁻¹³ g of S. schenckii DNA; 5, 4 × 10⁻¹⁴ g of S. schenckii DNA; 6, 4 × 10⁻¹⁵ g of S. schenckii DNA; 7, 4 × 10⁻¹⁶ g of S. schenckii DNA.

FIG. 3.

Detection of S. schenckii in DNA extracts of 12 clinical samples by nested PCR assay. Lanes: M, molecular size marker (100-bp ladder [Promega]); 1, positive control of S. schenckii; 2, distilled water; 3 to 14, patient no. 1 to 12, respectively. Lane 8 (patient no. 6) showed a negative result.