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Journal of Clinical Microbiology, April 2003, p. 1414-1418, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1414-1418.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Detection of Sporothrix schenckii in Clinical Samples by a Nested PCR Assay

Sindy Hu,1 Wen-Hung Chung,1 Shuen-Iu Hung,2 Hsin-Chun Ho,1 Zen-Whe Wang,1 Chien-Hsun Chen,1 Shu-Chuan Lu,3 Tseng-tong Kuo,3 and Hong-Shang Hong1*

Department of Dermatology,1 Department of Pathology, Chang Gung Memorial Hospital,3 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China2

Received 14 June 2002/ Returned for modification 23 September 2002/ Accepted 9 January 2003

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.

* Corresponding author. Mailing address: Department of Dermatology, Chang Gung Memorial Hospital, No. 199, Tung Hwa North Rd., Taipei 100, Taiwan, Republic of China. Phone: 886-2-2713-5211, ext. 3397. Fax: 886-2-2719-1623. E-mail: wenhungchung{at}yahoo.com.

Journal of Clinical Microbiology, April 2003, p. 1414-1418, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1414-1418.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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