The Use of Adhesive Tapes to Transfer Skin-scrapings for Sequential Laboratory Diagnosis of Dermatophytosis

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1 Med. Mycol. J. Med. Mycol. J. Vol. 57(No. 1), 2016 E 9 Vol. 57E, E 9 E 13, 2016 ISSN Original Article The Use of Adhesive Tapes to Transfer Skin-scrapings for Sequential Laboratory Diagnosis of Dermatophytosis Supapat Bunyaratavej, Penvadee Pattanaprichakul, Charussri Leeyaphan, Chuda Rujitharanawong, Chanai Muanprasart, Lalita Matthapan and Sumanas Bunyaratavej Department of Dermatology, Faculty of Medicine Siriraj Hospital, Mahidol University ABSTRACT Background: Dermatophytosis usually causes a skin problem, which requires both clinical findings and laboratory investigations for diagnosis. Whereas, fungal culture is considered as the gold standard procedure, it is more difficult to perform compared to potassium hydroxide(koh)examination. Objective: To evaluate the efficacy of KOH and fungal culture examinations from skin-scraping specimens, which were kept in transparent adhesive tapes at different time intervals. Methods: Skinscraping specimens were collected from clinically suspected cases by conventional technique using scalpel blades and kept using transparent adhesive tapes. KOH(10%)preparation and fungal cultures were then performed by highly experienced technicians at different time intervals: day 0(the day of specimen collection, which was set as the standard reference point), day 3 or 4, day 7, day 14, and day 28. Thereafter, the yield for detection of fungal elements by KOH examination and positive fungal cultures from specimens stored in adhesive tape was determined at different time intervals and compared to the results from the standard reference point. Results: The sensitivity of KOH examinations from 162 patients at different time intervals showed no significant difference when compared to the standard reference point at day 0. In contrast, the sensitivity of fungal culture progressively decreased as the storage period was prolonged. Conclusions: KOH examination of skin-scrapings from the adhesive tape storage technique yield good results even when specimens have been kept up to one month. On the other hand, the sensitivity of fungal cultures gradually declines as storage time is extended. Key words:dermatophytosis, laboratory examination, adhesive tape Introduction Dermatophytosis is the most common superficial fungal skin disease 1). Confirming diagnosis of dermatophytosis requires laboratory procedures such as skin-scraping for potassium hydroxide (KOH)examination and fungal cultures. There are only a few qualified laboratories in Thailand, however, that are capable of performing fungal culture and identification. The adhesive tape technique has been used for collecting specimens since 1953 for the reason that it helps to reduce spreading of scales 2). However, there is no scientific data to confirm the effectiveness of this method. This study, therefore, was designed to evaluate the effectiveness of using skin-scraping specimens that had been stored using adhesive tape technique for different time intervals. Methods The study included outpatients who were clinically suspected of glabrous skin dermatophytosis at the Department of Dermatology, Siriraj Address for correspondence : Samanas Bunyaratavej Department of Dermatology, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Wanglang Road, Bangkok, Bangkoknoi 10700, Thailand Received : 3, August 2015, Accepted: 5, November 2015 consultskin@yahoo.com

2 E 10 Medical Mycology Journal Volume 57, Number 1, 2016 Table 1.Number of specimens collected from each site Hospital, from August 2010 to February Onychomycosis, inadequate specimens, and follow-up cases were excluded from the study. Data collection was carried out using the doubleblind coded technique. This study was approved by the institutional Ethics Committee. Skin scrapings were performed to collect equally adequate amounts of each specimen. The specimens were used for 10% KOH examination and for fungal culture on Sabouraud dextrose agar at day 0, which was set as the standard reference point; the rest of the scales were divided into 4 parts to perform KOH examination and fungal culture on day 3 or day 4, day 7, day 14, and day 28. All specimens were stored using adhesive tape technique, wherein a transparent vinyl tape was used to attach the scales on pieces of business-card-sized, transparent plastic sheets, which were then kept in envelopes until the day the procedures were performed. This technique would represent a real investigative procedure and duration when specimens were sent from one hospital to laboratory via postal service at different time intervals regarding the distance. In order to analyze sensitivity and specificity of KOH examination and fungal culture, day 0 was set as the standard reference point in comparing results at different time intervals. Positive culture results for Trichophyton spp., Epidermophyton spp., and Microsporum spp. were recorded as positive dermatophyte infection. Dermatophytosis was confirmed through fungal cultures performed on specimens stored for at least one day(day 0, 3 or 4, 7, 14, or 28). The manuscript has been already corrected. Fungi identified as belonging to other genera were considered as non-dermatophyte molds or contaminations. Results One hundred and sixty-two patients, who were clinically suspected of having skin dermatophytosis, were enrolled in the study during a six-month period. Fifty-four percent of the subjects were male, with mean age at 52 years(sd = 16.5 years). Most of the specimens were collected from the feet(table 1). This study was designed to compare the effectiveness of KOH examination with the gold standard method, i.e., fungal culture of specimens collected at day 0. The accuracy of KOH examination at day 0 based on the results of fungal culture at day 0 was 98.14%, while the sensitivity, specificity, positive predictive value, and negative predictive value were 99. 2%, 95. 5%, 98. 3% and 97. 7%, respectively. In addition, results of KOH examination at day 3 or 4, 7, 14, 28 were also highly correlated with the results of fungal culture at day 0, as shown in Table 2. For KOH examination, the sensitivity, specificity, positive predictive value, and negative predictive value at the different time intervals(day 3 or 4, 7, 14, 28)compared with KOH examination at day 0 were particularly high(table 2). On the other hand, the analysis of fungal cultures performed at different time intervals(day 3 or 4, 7, 14, 28)based on the results of fungal culture at day 0 showed that the sensitivity, and negative predictive value of fungal culture decreased through time. On the other hand, the specificity and the positive predictive value remained very high over 90%(Table 2). Interestingly, among the 93 dermatophyteinfected patients, results showed that the number of positive dermatophyte fungal culture examinations decreased significantly through time(p < 0.05)(Fig. 1). Discussion Location 1. Feet 2. Body 3. Groin 4. Hands 5. Face 6. Head Number(%) 69(42.6%) 55(34.0%) 20(12.4%) 11(6.8%) 6(3.7%) 1(0.6%) The use of adhesive tapes was introduced in mycological investigations in ). An adhesive tape technique was developed to improve patient s cooperation in the collection of specimens, particularly in children, who were generally afraid of scalpel blades. The technique was initially used to investigate tinea versicolor, but subsequent studies also showed some benefit in dermatophytosis 3 5). In this study, we did not use adhesive tape to replace scalpel blades, as in most previous studies about adhesive tape technique, but we

3 Med. Mycol. J. Vol. 57(No. 1), 2016 E 11 Table 2.Comparison between KOH examination and fungal culture at different time intervals (Day 0, 3 or 4, 7, 14, 28) Accuracy(%) Sensitivity(%) Specificity(%) PPV(%) NPV(%) KOH examination at different intervals compared with fungal culture at day 0 Day KOH examination at different intervals compared with KOH examination at day Fungal culture at different intervals compared with fungal culture at day * The number of positive dermatophyte fungal culture specimens is presented as percentage. Fig. 1.Number of positive dermatophyte fungal culture specimens at different time intervals(day 0, 3 or 4, 7, 14, 28)in 93 cases that were definitely diagnosed as true dermatophyte infection. used it to attach scales on transparent plastic sheet to prevent spreading inside the envelope. Our results showed the excellent sensitivity and accuracy of KOH examination. Although specimens were kept for up to almost one month, the sensitivity and accuracy remained higher than 90%. On the other hand, results showed that the sensitivity and accuracy of fungal culture gradually decreased as storage time was extended, and were less than 50% when specimens were kept using the adhesive tape technique for one month. Moreover, species identification of fungal cultures from prolonged-storage specimens was more difficult than in cultures performed on day 0. Although Knudsen reported that microscopic and macroscopic identification of fungal species were not affected by the vinyl adhesive tape method, the yield of fungal cultures still decreased in correlation with prolonged storage duration, similar to our results 6). The decrease in sensitivity of fungal culture may be explained by the fact that adhesive tape creates an anaerobic environment that interferes with fungal growth. Moreover, lack of keratin, which is considered as a nutrient source of dermatophytes, and a dry environment, can also precipitate fungal death. Scientific data, however, show that there is no substance in the adhesive tape that can inhibit fungal viability 7, 8). Although initial and sequential KOH preparations revealed positive branching hyphae, subsequent results of fungal culture yielded nondermatophyte molds or contaminations. This could be due to the fact that most of the

4 E 12 Medical Mycology Journal Volume 57, Number 1, 2016 Fungal culture Fig. 2.Comparison of the sensitivity and accuracy of KOH examination and fungal culture at different intervals compared with fungal culture at day 0. Fig. 3.Representative pictures showing the use of adhesive tapes to transport skin-scrapings for sequential laboratory diagnosis of dermatophytosis. specimens in this study were collected from the feet, in which non-dermatophyte molds or contaminations could be predominant. Applying the knowledge we gained from this study in clinical practice, we recommend that clinicians send the scraping specimens via regular postal mail to the laboratory for KOH examination any time between specimen collection up to a one-month interval, within which period the sensitivity and PPV remain high. On the contrary, if fungal culture is required, specimens should be delivered using the fastest method, such as express mail or overnight service, to yield reliable results. This study, however, has some limitations. First, dermatophyte onychomycosis was not included in this study due to its hard keratin. Due to the high prevalence and necessity of species identification for treatment of onychomycosis, further studies should be conducted to include onychomycosis. Second, results of KOH preparation and fungal culture depend highly on the individual experience of each technician. Different results, therefore, might be obtained if the study was performed elsewhere. We believe, however, that the decrease in the sensitivity of fungal culture and the consistently high sensitivity of KOH examination through prolonged storage should be the same for every laboratory. Lastly, due to the difficulty of identifying microorganisms to the species level, particularly when the specimens were stored for prolonged periods, there were inconsistencies in species identification for the same specimen. Nevertheless, in this study, we used only the number of dermatophytes and non-dermatophytes to calculate the results, as the appearance of dermatophytes is very different from non-dermatophyte molds. Hence, this limitation should not significantly affect the results.

5 Med. Mycol. J. Vol. 57(No. 1), 2016 E 13 Conclusion Our study revealed the potential benefit of storing specimens using adhesive tape and delivering them via mail to the laboratory for KOH examination and fungal culture. Because of the decreasing reliability of fungal culture results over time, specimens should be sent using the fastest delivery system, unless only KOH examination is required. Acknowledgement The authors would like to thank Assistant Professor Dr. Chulaluk Komoltri for her advice in the statistical analysis. References 1)Porto JA: The use of cellophane tape in the diagnosis of Tinea versicolor. J Invest Dermatol 21: , )Dvorák J, Hubálek Z, Otcenásek M: Survival of dermatophytes in human skin scales. Arch Dermatol 98: , )Miranda MF, Silva AJ: New uses of vinyl tape for reliable collection and diagnosis of common superficial mycoses. Skinmed 2: , )Milne LJ, Barnetson RS: Diagnosis of dermatophytoses using vinyl adhesive tape. Sabouraudia 12: , )Moghaddami M, Hakim SH: Evaluation of two methods, conventional scraping and stripping, for diagnosis of dermatophytosis. Iran J Public Health 24: 7-10, ) Knudsen EA: The survival of dermatophytes from tape strippings of skin. Sabouraudia 18: , )Barnetson RS, Milne LJ: Skin sampling for Candida with adhesive tape. Br J Dermatol 88: , )Knudsen EA: Growth on Sabouraud s agar of dermatophytes obtained by conventional scraping and by stripping with vinyl tape. Br J Dermatol 90: , 1974.