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BACKGROUND Low sensitivity of the polymerase chain reaction (PCR) assay for diagnosing scabies has been pointed out due to the difficulty in obtaining tissue containing S. scabiei DNA. OBJECTIVES To evaluate nested real-time quantitative PCR (nRT-qPCR) with non-expert dependent standardized cotton swab sampling (CSW) as a tool for diagnosing scabies. METHODS All patients underwent dermoscopic and microscopic examination (MS) with scraped samples (Sc). Patient samples were acquired with a single, dry swab rubbed across the flexor areas of both wrists as well as the 8 interdigital spaces and on any suspected scabies lesions. nRT-qPCRs were performed with scraped and CSW samples. RESULTS Out of 125 patients with suspected scabies, 120 patients were sampled, and 57 were positive (MS positive n=53, nRT-qPCR with Sc positive n=52, nRT-qPCR with CSW positive n=46) and 63 were negative for scabies. The sensitivities of these tests were 93.0%, 91.2% and 80.7%, respectively, which were not different statistically (p > 0.05). However, upon subsequent monitoring after treatment, the sensitivity of nRT-qPCR with CSW was only 36.6%, which was significantly lower than 83.0% for MS and 92.7% for nRT-qPCR with Sc (p = 0.00, respectively). The obtained sequences showed 97% to 100% homology with scabies sequences deposited in GenBank. CONCLUSIONS CSW with nRT-qPCR shows sensitivity close to microscopic examination with scraping performed by experts as diagnosing scabies in an outpatient setting, but not for post-treatment monitoring. CSW with nRT-qPCR may be useful for physicians unfamiliar with a traditional diagnostic method, and for screening an outbreak in community facilities.