The efficacy of a nested PCR in detecting cytochrome c oxidase subunit 1 gene of Sarcoptes scabiei var. hominis for diagnosing scabies J.E. Hahm, C.W. Kim, S.S. Kim Department of Dermatology, Kangdong Sacred Heart Hospital, Hallym University College of Medicine, Seoul, Korea British Journal of Dermatology. DOI: 10.111/bjd.16657

Chul Woo Kim, Lead author Associate Professor of Department of Dermatology, Kangdong Sacred Heart Hospital, Hallym University College of Medicine, Seoul, Korea M.D. in Hallym University College of Medicine M.S. in Hallym University College of Medicine Visiting Associated Professor, Department of Dermatology, Columbia University Medical Center, USA

Introduction What’s already known? Previous studies have demonstrated the usefulness of conventional PCR in diagnosing scabies but the positive diagnosis rate was too low to obtain satisfactory results. Those researches comparing PCR for scabies diagnosis against microscopy detection did not include dermoscopy; therefore, it is possible that there were high false-negative rates by microscopy and the effectiveness of a PCR assay was overstated.

Objective A widespread scabies infestation, associated to long-term residence in nursing homes, is becoming a serious issue in developed countries. Mineral oil examination is regarded as the gold standard in diagnosing scabies, but the sensitivity of this method is generally low—approximately 50%. Molecular tests may contribute to enhance the sensitivity of current tests for laboratory diagnosis of human scabies. In this study, we developed new primers for a nested PCR for the cytochrome c oxidase subunit 1 (cox1) gene of Sarcoptes scabiei var. hominis to increase the sensitivity of a previously developed conventional PCR.

Methods 1. Study subjects and samples Clinically suspected scabies patients were examined with a dermoscope on body areas where burrows are frequently found. It helps in identifying triangular structures corresponding to the anterior section of the mite and the scrapings of them were analyzed for the presence of mites or eggs under the microscope. Skin scrapings were collected from both microscopy-positive and negative patients for PCR. Microscopy-positive : diagnosed with definite scabies when at least one mite or egg was seen under the microscope Microscopy-negative : those with suspected signs of scabies, but whose skin scrapings turned up negative

Methods 2. DNA extraction from samples 3. Nested PCR amplification To increase sensitivity, new primers were designed for a nested PCR, in addition to the existing primers designed by Wong et al. that amplify the 262-bp fragment of the cox1 gene of S. scabiei. The PCR program for each primer pair is presented in Table 2. Target gene Primer name Round of nested PCR PCR programs for each primer cox1 nest-scab-F2 First 95 ℃ for 10 min; 40 cycles of 95 ℃ for 1 min, 53.3 ℃ for 1 mi n, and 72 ℃ for 1 min; a final extension at 72 ℃ for 10 min nest-scab-R1 scabF1-1 Second 95 ℃ for 10 min; 40 cycles of 95 ℃ for 1 min, 51.3 ℃ for 1 mi n, and 72 ℃ for 1 min; a final extension at 72 ℃ for 10 min scabR2-3 Table 2(part). The PCR programs used in this study

Methods 4. Electrophoresis 5. Sequencing Positive controls scrapings from microscopically confirmed scabies patients Negative controls scrapings from patients with other dermatological conditions DNA extracted from house dust mites (D. pteronyssinus, D. farinae) pure cultures of dermatophytes (M. canis, T. rubrum) DNA extracted from specimens infected with D. folliculorum 5. Sequencing Sequenced with an ABI PRISM 3730XL DNA analyzer (Applied Biosystems, Foster city, USA) according to manufacturer’s instructions

No. of samples with the following microscopy result Results Of the total 63 samples, 28 were microscopy-positive and 35 were negative with no differences in sex and age between the two groups. cox1 nested PCR result No. of samples with the following microscopy result Sensitivity of the microscopya P valueb Positive Negative Total 28 9 37 75.68% 0.004 26 35 63 aSensitivity was calculated by postulating that the cox1 nested PCR has a sensitivity of 100%. bDifference between the two methods was statistically significant (P < 0.05). Table 4. Results of microscopy and cox1 nested PCR in diagnosing scabies

Results FIG2. PCR amplification of negative controls and scabies cases. (A) cox1 gene of scabies mites using nested PCR. (B) Human β-globin gene. (C) EF-1α gene of Dermatophagoides spp. Lanes: M, molecular marker; 1-3. Atopic dermatitis; 4-5. Allergic contact dermatitis; 6. Seborrheic dermatitis; 7. Xerosis cutis; 8. Psoriasis; 9. Onychomycosis; 10. Microsporum canis; 11. Trichophyton rubrum; 12. Tinea versicolor; 13. Demodecidosis; 14. Dermatophagoides farinae; 15. Dermatophagoides pteronyssinus; 16-19. Microscopy-positive scabies patients.

Discussion At present, the confirmative diagnostic method for scabies is observing eggs or mites collected from skin scrapings under a microscope. However, microscopic detection rates vary widely, ranging from 10 to 70%. Therefore, it is necessary to improve the detection technique or develop a new reliable method. To the best of our knowledge, this is the first study using a nested PCR of the cox1 gene to diagnose scabies infestations. The detection rate of scabies by nested PCR in microscopy-negative patients was higher than by conventional PCR tested by Wong et al. (14.5% of 83 samples).

Discussion Previous studies comparing PCR for scabies diagnosis against microscopy detection, did not include dermoscopy; therefore, it is possible that the sensitivity of the PCR assays was overestimated due to the high false-negative rates by microscopy. Even though false-negatives were minimized in this study owing to the inclusion of dermoscopy, the microscopy results were significantly inferior to that of the cox1 nested PCR. If the cox1 nested PCR was considered as the ‘diagnostic gold standard’, then the sensitivity of microscopy detection in this study is 75.68% (95% CI, 58.80–88.23), which is higher than the 58.6% reported by Wong et al.

Discussion PCR testing has the advantage of high sensitivities, and no long-term training requirement. Of note, diagnosis by PCR yielded positive results for some patients with scabies symptoms yet whose microscopy results were negative. This technique can therefore be employed as an adjunct method for scabies diagnosis, especially in suspected but microscopy-negative cases, outbreak investigations, or environmental sampling when large quantities of specimens are collected.

Conclusions What does this study add? The detection rate of scabies by nested PCR in microscopy-negative patients (25.7%, 9 of 35 samples) was higher than by conventional PCR (14.5%, 12 of 83 samples). Even though false-negatives were minimized in this study owing to the inclusion of dermoscopy, the microscopy sensitivity was 75.68% (95% CI, 58.80 to 88.23), which was significantly inferior to that of the cox1 nested PCR (P = 0.004).

Research Team

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