Taotlus

Saatja: [email protected] Saaja: [email protected] Teema: FungiCheck kiirtest Kuupäev: 2025-02-20 11:44 Tähelepanu! Tegemist on väljastpoolt asutust saabunud kirjaga. Tundmatu saatja korral palume linke ja faile mitte avada. Lugupeetud Terviseamet Pöördume Teie poole sooviga uurida, kas FungiCheck kiirtest dermatofüütide tuvastamiseks küüneproovis võiks olla aktsepteeritav perearstide seas kui legitiimne diagnostiline vahend nagu nt Covid19 testid. Oleme lisatud manusena edastanud teadusartikli, mis toetab testi usaldusväärsust ja tõhusust. Lisasin kaasa ka informatiivse kataloogi kiirtesti kohta. FungiCheck kiirtest on murranguline lahendus dermatofüütide tuvastamisel küüneproovides, pakkudes täpseid ja usaldusväärseid tulemusi vaid 10 minutiga. Testi lihtsus ja kiirus võimaldavad selle kasutamist jalaravikabinettides, podoloogide juures, praktilises tervishoiukeskkonnas, sealhulgas perearstide praksistes, kus küüneprobleemidega patsiendid sageli esmalt abi otsivad. FungiChecki eelised: Kiirus ja täpsus: Tulemus 10 minutiga, mis välistab pika ooteaja laboratoorsetele testidele. Usaldusväärsus: Kliinilised uuringud kinnitavad 97% õnnestumise määra. Lihtsus ja mugavus: Testi saab kasutada ka ilma erikoolituseta, järgides detailset kasutusjuhendit. Sobivus ravimite kasutajatele: FungiChecki saab kasutada ka patsientidel, kes juba kasutavad seenevastaseid ravimeid. Positiivne tulemus võimaldab suunata patsiendi ravi saamiseks arsti juurde, negatiivne tulemus aitab välistada dermatofüütide esinemise ja keskenduda teistele võimalikele küüneprobleemidele. Arvestades küüneprobleemide laialdast esinemist ja sagedast segiajamist teiste haigusseisunditega, on oluline, et perearstid omaksid ja tunnustaksid kiiret ja täpset diagnostikavahendit, mis vähendaks ebavajalikke ravikuure ning tagaks õigeaegse diagnoosi. Palume Teil hinnata FungiCheck testi sobivust ning anda tagasisidet selle võimaliku kasutuselevõtu kohta Eesti tervishoiusüsteemis. Millised tingimused peavad selleks olema täidetud ja mis sammud tuleks astuda? Ootame Teie vastust ja oleme avatud edasisele arutelule. Lugupidamisega/Best Regards Triin Uuk Müügijuht/Sales manager Medipro OÜ +372 59151408 <https://www.facebook.com/jalgadehooldusekabinet> <https://www.instagram.com/jalgadehooldus> www.medipro.ee <http://www.medipro.ee/> www.jalgadehooldus.ee <http://www.jalgadehooldus.ee> Usaldusväärne dermatofüütide testikomplekt Revolutsiooniline innovatsioon HFL laboratories www.medipro.ee  FungiCheck Miks just Fungicheck? HFL FungiCheck testikomplekt on suur läbimurre dermatofüütide olemasolu diagnoosimisel küüneproovides. Tagab parima hoolitsuse tänu täpsele diagnostikale. FungiCheck kasutamisel saate olla kindel, et Teie kliendid saavad õige jalahooldusplaani ja tooted. See on lihtne test, mida saab läbi viia Teie enda praksises andes Teile usaldusväärse ja täpse tulemuse vaid 10 minutiga. Võtke küüneproov, asetage see katsutisse. Lisage sinna vajaminev kogus pakendis kaasas olevat vedelikku ja segage. Viimaseks sammuks on testriba asetamine katsutisse, mille järel võib varsti näha vastust ühe FungiCheck eelised või kahe triibu näol. Aeg Praegusel ajal kasutatavad laboratoorsed meetodid on mikroskoopiline meetod, PCR või kultiveerimismeetod. Mikroskoopiline meetod nõuab valdkonnapõhiseid teadmisi ja FungiChecki kasutamine praksises võiks saada rutiiniks, sest niiviisi pakute õige diagnoosi abil parimat hoolitsust. kogemusi ning mõned seened võivad jääda tähelepanuta. Nende meetodite õnnestumise Ja nii tõstate ka oma kabineti poolt pakutud hoolitsuse taset. määr on 50-73%. (7-8) Mõlemad meetodid on aeganõudvad, sest neid tehakse laboris. FungiCheck sobib kasutamiseks esmalt just uutel klientidel, kes teatavad küüne muutustest või traumast. Ravimid Erinevalt tavapärastest meetoditest saab testikomplekti FungiCheck kasutada ka klientidel, Pidage meeles, et umbes 50% kõigist küünetraumast teatanud klientidel on seennakkus ja 50% mitte. (3) kes juba kasutavad seenevastaseid ravimeid või pealekantavaid tooteid. FungiCheck testi Oluline on seda kliendile vastuvõtul toonitada. Parima jalahooldusplaani loomise eelduseks on FungiCheck saab läbi viia pealekantavate toodete või seenevastaste ravimite (asoolide) kasutamise ajal. Testi tulemust see ei mõjuta. Negatiivse tulemuse korral on olemas korralikud alusandmed, testi läbiviimine. kas suunata oma klient perearsti juurde või mitte. Positiivse tulemuse korral saab klient jätkata onühhomükoosi raviga. (4) Ärge unustage ka seda, et kui Te ei kasuta diagnostilist testi, siis põhineb diagnoos kindlustunde asemel alati aimdusel. Peaksite teadma seda, et uuringud on näidanud, et iga kolmas visuaalne küüneseene diagnoos Kursus/koolitus Mõni meetod eeldab kursusel osalemist ja sageli on vaja seda igal aastal korrata. on vale. (2, 5) FungiCheck ei eelda kursuse läbimist. Iga testikarbiga kaasnev üksikasjalik kasutusjuhend annab teile korralikke juhiseid. Kasutusjuhendis viidatakse ka steriilsetele töövõtetele, FungiCheck on saadaval ainult pediküürijatele, jalahooldusspetsialistidele, kui soovite selle osa oma praktikas veel kord üle vaadata. Lisaks on saadaval tasuta tootekoolitused, kui soovite avardada oma teadmisi FungiChecki ja seennakkuste kohta. jalaravispetsialistidele, arstidele ja dermatoloogidele. Wordt lid van onze besloten facebookpagina en deel uw ervaring HFL Professionals 2 3  Onühhomükoos Dermatofüüdid ja mitte-dermatofüüdid Rahvusvahelise inimeste ja loomade mükoloogiaühingu (ISHAM) andmetel on onühhomükoos küünte invasiivne Dermatofüüdid on seened, mis vajavad kasvamiseks keratiini. Need seened võivad põhjustada naha, seennakkus - olenemata algpõhjusest. See haigus esineb sõrme- või varbaküüntel ja põhjustab umbes 50% kõigist juuste ja küünte nakkust. Dermatofütoosi nimetatakse “Tinea” nakkusteks ja seetõttu võib see pärineda küünte anomaaliatest. (1) väljastpoolt. Onühhomükoosi põhjustajad on dermatofüüdid ja Uuringud näitavad, et (3) 91% küüneseentest on Dermatofüüdid on inimestel enim vaevusi põhjustavad Uuringud näitavad, et enamiku küüneseentest on mitte-dermatofüüdid (NDM-id). Mõistet Tinea unguium põhjustanud dermatofüüdid, samas kui mitte- seened. (1, 3) Need on niitjad seened, mis suudavad põhjustanud dermatofüüdid; pärmseened ja NDM-id kasutatakse dermatofüütide põhjustatud onühhomükoosi dermatofüüdid põhjustavad umbes 9% kogu maailma lagundada keratiini ja kasutada seda toitainena. Neid põhjustavad umbes 9% kogu maailma onühhomükoosist.(3) korral; siiski on Tinea unguium ja onühhomükoos sageli onühhomükoosist. Euroopas tõuseb see protsent isegi leidub kõikjal maailmas. Dermatofüüte võib jagada Uuringud näitavad, et sellistes riikides nagu Iraan ja kasutusel sünonüümina. 94% peale. järgmisse kolme seeneperekonda: Trichophyton, Kolumbia tekib NDM-ide põhjustatud küüneseen veidi Epidermophyton ja Microsporum. suurema tõenäosusega. (6) Üldiselt kasutatakse mõistet onühhomükoos kõigi küüneseente puhul, mille on põhjustanud kas Varbaküüntel on kõige levinumad liigid Trichophyton FungiCheck testriba piirdub dermatofüüdite põhjustatud dermatofüüdid või NDM-id. (1,3) Rubrum (70%) ja Trichophyton Mentagrophytes (20%). küüneseene testimisega küüntes, sest need teevad pesa Perekond Epidermophyton sisaldab ainult ühte liiki: väljastpoolt ja asuvad seetõttu otse küünes. (4, 7) E. floccosum (5%). Mitte-dermatofüütset onühhomükoosi (NDO) põhjustavad hüaliin ja tumeda pigmendiga niitjad seened, mida tavaliselt leidub saprofüütidena või taime patogeenidena (taimed ja toit). Erinevalt dermatofüütidest ei ela nad tavaliselt keratiinist. Nad elavad peremeeskoe keratiniseerimata rakkudevahelisel sideainel ja peavad ära kasutama varasemat keratiini hävinemist nt dermatofüütide, trauma või muude küünehaiguste tõttu. Seetõttu peetakse neid mõnikord küüneplaadi sekundaarseteks vallutajateks. Dermatofüüdid 4 5  Usaldusväärsus Tulemus FungiCheck on teaduslikult ja kliiniliselt testitud testikomplekt. Uuringud näitavad, et FungiCheck, Testi tulemuste põhjal saate koostada oma kliendile parima jalahooldusplaani või suunata kliendi ka perearsti juurde. mille usaldusväärsus on 97%, on kõige usaldusväärsem meetod küüneproovist dermatofüütide tuvastamiseks. Roosa joon näitab testi õnnestumist. On väga oluline FungiCheck annab perearsti juurde suunamiseks korralikud meeles pidada, et see joon peab alati nähtav olema, alusandmed kui kliendil on nt mitu meditsiinilist kaebust või sest see on kontrolljoon. Teine violetne/hall joon näitab, krooniline või eluohtlik haigus. kas test on positiivne. Järeldus: kaks joont on positiivsed ja Andke kliendile hermeetiline testiriba ja FungiCheck infolehe 1 joon on negatiivne. koopia, mille ta saab perearsti juurde kaasa võtta. Kui esineb kahtlus Candida osas ning klient põeb eluohtlikku haigust Dermatofüütide põhjustatud onühhomükoosi saab nagu nt vähk või HIV, peaks ta kindlasti perearsti juurde ravida professionaal ja see pole inimestele ohtlik. suunama. 60% 50% 73% 97% Kui tulemus on positiivne, võite alati saata perearstile kirja testi tulemustega, et ta saaks selle patsienditoimikusse Dermatofüütia või küünetrauma korral saab oma kliendile lisada. soovitada parimat jalahoolduslahendust, pakkudes seenevastast toodet. KUI test on negatiivne, on vägagi tõenäoline (50%), et Mikroskoopiline Kultiveerimismeetod PCR FungiCheck kliendil on muu küüne anomaalia. Visuaalselt võivad See tagab olukorra, et olete soovitanud õige toote ning meetod muud küüneprobleemid (nt hüperkeratoos, onühholüüs, pakub paremaid võimalusi kasutatud toote tulemuste psoriaas või kollaste küünte sündroom) näha välja justkui hindamiseks. Klient saab olla kindel, et ta ei raiska raha seennakkus. ebavajalikele toodetele. See hoiab ära ebaeetilised otsused ja tekitab usalduse. FungiCheck testikomplekt testib küüneproovi kõigi dermatofüütide suhtes Trichophyton Mentagrophytes - Trichophyton Rubrum - Trichophyton Tonsurans - Trichophyton Violaceum - Trichophyton Verrucosum - Microsporum Gypseum - Microsporum Canis - Epidermophyton Floccosum - Aspergillus Flavus - Aspergillus Fumigatus - Aspergillus Niger - Aspergillus Terreus - Neosartorya Fischeri - Paecilomyces Lilacinus - Penicillium Griseofulvum - Veronaea Botryosa - Fusarium Solani - Exophiala Dermatitidis (M-Y form) - Exophiala Dermatitidis (G form) - Exophiala Spinifera - Hortaea Werneckii - Malbranchea Circinata - Külgvool põhinev immunoanalüüs, mis on mõeldud küüntes dermatofüütidest saadud antigeenide M. flavorosea (4, 7) tuvastamiseks, kasutades nitrotselluloosmembraanile immobiliseeritud dermatofüütidevastast antikeha, mis reageerib kokkupuutel dermatofüütidega 6 7  “Lõpuks ometi on loodud usaldusväärne ja kiire test, mida saab kliinikus kasutada väga levinud küüneinfektsiooni täpseks diagnoosimiseks” Dr. Ivan Bristow, Dermatologist & Podiatrist, Southampton UK 1 Boni E. Elewski* Onychomycosis: Pathogenesis, Diagnosis, and Management - Clin Microbiol Rev. 1998 Jul; 11(3): 415–429. 2 Tsunemi Y, Takehara K, Oe M, Sanada H, Kawashima M. Diagnostic accuracy of tinea unguium based on clinical observation. The Journal of Dermatology. 2015;42(2):221-2. 3 Onychomycosis: A Review_J Fungi (Basel). 2015 Jun; 1(1): 30–43.Published online 2015 Mar 27. doi: 10.3390/jof1010030 4 J Dermatol. 2016 Dec;43(12):1417-1423. doi: 10.1111/1346-8138.13348. Epub 2016 Mar 19.Clinical study of Dermatophyte Test Strip, an immunochromatographic method, to detect tinea unguium dermatophytes. 5 Evaluation of clinicomycological aspects of onychomycosis_ Indian J Dermatol. 2008; 53(4): 174–178. doi: 10.4103/0019-5154.44788 6 Jundishapur J Microbiol. 2016 Jul 26;9(8):e40543. eCollection 2016 Aug. 7 Screening for tinea unguium by Dermatophyte Test Strip* 1102002/2501/EST.2e Y. Tsunemi,1 K. Takehara,2 Y. Miura,2 G. Nakagami,2 H. Sanada2 and M. Kawashima1 8 Monica A. Lawry, MD; Eckart Haneke, MD, PhD; Katherine Strobeck, MD; et al - Methods for Diagnosing Onychomycosis. Arch Dermatol. 2000;136(9):1112-1116. doi:10.1001/archderm.136.9.1112 Medipro OÜ Pirni 7/1-36, Tallinn Tel: +372 5915 1408 [email protected] www.medipro.ee doi: 10.1111/1346-8138.13348 Journal of Dermatology 2016;

: 1–7 ORIGINAL ARTICLE Clinical study of Dermatophyte Test Strip, an immunochromatographic method, to detect tinea unguium dermatophytes Yuichiro TSUNEMI,1 Masataro HIRUMA2 1 Department of Dermatology, Tokyo Women’s Medical University, 2Ochanomizu Institute for Medical Mycology and Allergology, Tokyo, Japan ABSTRACT The Dermatophyte Test Strip visualizes mycotic antigens by immunochromatography. It allows easy and fast detection of dermatophytes. A multicenter, single-arm, comparative clinical study was designed to evaluate the capacity of Dermatophyte Test Strip to detect dermatophytes in suspected tinea unguium specimens in compar- ison with direct microscopy and polymerase chain reaction (PCR). Signed consent was obtained from 222 sub- jects and all subjects completed the study. With the Dermatophyte Test Strip, dermatophytes were detected in 201 of 222 (90.5%) specimens but not in 21 of 222 (9.5%) specimens. With direct microscopy, dermatophytes were detected in 170 of 222 (76.6%) specimens but not in 52 of 222 (23.4%). Of the 45 specimens that showed inconsis- tent results between the two methods, PCR gave further results for 40 specimens, of which 37 (92.5%) specimens were positive and three (7.5%) were negative for dermatophytes. The positive concordance rate, negative concor- dance rate and overall concordance rate between the Dermatophyte Test Strip and direct microscopy were 81.1%, 66.7% and 79.7%, respectively. When inconsistent results were corrected using the results of PCR, these rates were 97.5%, 71.4% and 95.0%, respectively. When five specimens that could not be tested by PCR because no piece for the PCR test was left were excluded from analysis, these rates were 99.0%, 78.9% and 97.2%, respectively. The present results indicate good detection capacity of the Dermatophyte Test Strip. The Dermato- phyte Test Strip provides a reliable, convenient and quick method to test for tinea unguium. Key words: dermatophytes, immunochromatography, immunological diagnosis, tinea unguium, Trichophyton. INTRODUCTION genera, namely, Trichophyton (T), Microsporum (M) and Epider- mophyton (E).6,7 T. rubrum accounts for the primary cause of Tinea unguium is a nail disease caused by dermatophyte infec- tinea unguium, followed by T. mentagrophytes.7,8 tion of the nail plate.1,2 Its prevalence in advanced countries is The diagnostic tests for tinea unguium include direct micro- considered as at least 10% of the population,2 and in Japan it scopy with potassium hydroxide (KOH), fungal culture, periodic is estimated at approximately 10%.1 The incidence increases acid-Schiff staining (PAS), fluorescence staining, confocal with aging in all countries and it is therefore predicted to microscopy and molecular biological methods such as poly- increase in number in association with the aging of society in merase chain reaction (PCR).9 Although direct microscopy with the future.2 Patients with tinea unguium pose a risk of becom- KOH and fungal culture have been the gold standard for the ing the source of infection of other people.3 Tinea unguium is diagnosis of tinea unguium,7,9 these tests sometimes are a particularly considered as a predictor of diabetic foot syn- burden in the clinical setting, because direct microscopy drome.3 Thus, tinea unguium does not only cause problems requires experience to identify the fungal elements, and disso- with nail appearance, but it also seriously damages patients’ lution of nails is time-consuming. It takes 2–3 weeks to obtain quality of life. It should therefore be treated actively as far as results using fungal culture, and the detection rate is lower possible.4 than with direct microscopy.10 Therefore, the development of Because many other diseases have similar symptoms as an easy and quick test to diagnose tinea unguium accurately is tinea unguium,5 differentiation is not easy, and a definitive in high demand. diagnosis by mycological examination is necessary before The recently developed Dermatophyte Test Strip detects treatment is started. Dermatophytes are classified into three dermatophytes easily and rapidly by immunochromatography Correspondence: Yuichiro Tsunemi, M.D., Ph.D., Department of Dermatology, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Email: [email protected] Received 15 October 2015; accepted 31 January 2016. © 2016 Japanese Dermatological Association 1 Y. Tsunemi and M. Hiruma using monoclonal antibodies that react specifically with the This trial was registered with the University Hospital Medical polysaccharide present in the cell wall of dermatophytes.11–14 Information Network Clinical Trials Registry (no. UMIN This antibody was found to react specifically with seven der- 000016760). matophytes (T. rubrum, T. mentagrophytes, T. violaceum, T. tonsurans, M. gypseum, M. canis and E. floccosum).12,13 Subjects There have been a few preliminary reports on screening results Male and female subjects aged 20 years or more who had a of dermatmycoses using a Dermatophyte Test Strip.15–17 Higa- nail lesion suspected of tinea unguium, and who provided shi and his colleagues have collected 88 specimens from skin signed informed consent were included in the study. Subjects and 72 from nails, examined them using direct microscopy and were excluded from the study if any of the following applied: (i) Dermatophyte Test Strip, and compared the results.15 history of oral antifungal agent use within 6 months or of topi- In this study, tinea unguium-suspected specimens were cal antifungal agent use within 1 month; (ii) participation in subjected both to testing with the Dermatophyte Test Strip as another clinical trial or other clinical studies; and (iii) participa- well as the gold standard of direct microscopy with KOH to tion determined to be inappropriate by the study investigator detect dermatophytes. Specimens that showed inconsistent or subinvestigator. results between the two methods were subjected to PCR to further confirm the presence or absence of dermatophytes. Primary end-point The detection capacity of the Dermatophyte Test Strip was The primary end-points were the concordance rate (positive then investigated for all specimens by comparing the results of concordance rate, negative concordance rate and overall con- medical experts’ comprehensive evaluations based on the cordance rate) and the inconsistent rate of detecting dermato- results of direct microscopy and PCR. phytes in the suspected specimens between the Dermatophyte Test Strip and direct microscopy or the adjusted results based on PCR and final determination by medical experts’ compre- METHODS hensive evaluations based on the results of direct microscopy, A multicenter, single-arm, comparative clinical study was con- PCR, the clinical picture, underlying disease information and ducted to evaluate the efficacy of the Dermatophyte Test Strip specimen collection site. (JNC Corporation, Tokyo, Japan) to detect dermatophytes in tinea unguium-suspected specimens in comparison with direct Test specimen collection microscopic examination. The study was conducted at 11 Specimens were collected by the dermatologists listed in study sites (Table S1) from June to August 2014. Table S1. Dermatologists at the 11 study sites selected The detection of dermatophytes using the Dermatophyte patients with suspected tinea unguium by visual examina- Test Strip was performed at Yokohama Research Center of tion and collected specimens from those patients. A speci- JNC Corporation. Direct microscopic examination was per- men of approximately 20 mg was collected from a formed by Professor Shinichi Watanabe of Teikyo University suspected tinea unguium nail lesion in accordance with the School of Medicine. PCR was performed by Professor Takashi guideline.23 The lesions were recorded by taking pictures Sugita of Meiji Pharmaceutical University. This study was per- and sketches. formed in compliance with Good Clinical Practice based on the The collected nail was sandwiched between autoclaved alu- Declaration of Helsinki and other applicable regulations. The minum plates and crushed into small pieces by using a ham- institutional review board of each study site reviewed and mer. The pieces were transferred to a test tube, further cut approved the study protocol. Prior to the start of screening and crushed using a pair of dissecting scissors and divided procedures, signed informed consent was obtained from each into three portions after mixing for direct microscopy, the Der- subject. The present study was monitored and audited by EPS matophyte Test Strip and PCR, respectively. Because nine Associates Co., Ltd. specimens were extremely small, these specimens were only cut into two pieces, forfeiting the piece for PCR. Study design Within 4 weeks after obtaining signed consent, the investigator Dermatophyte Test Strip or subinvestigator confirmed the eligibility of the subject, col- Monoclonal antibodies that react specifically with the polysac- lected the baseline characteristic information (date of birth, age charide present in the cell wall of dermatophytes12,13 are lin- and sex) and examined and interviewed the subject (main rea- early immobilized on the test strip. The polysaccharides in the son for visit, disease or major complaint, medical history and suspected specimens are extracted using the extraction solu- complications). A nail specimen was collected from each sub- tion.14,24 In short, a nail specimen was cut into smaller pieces ject within 1 week after confirming the eligibility of a subject, with a nail clipper or nipper. These pieces were then added to and the specimen was sent to the central laboratory where all 0.25 mL of extraction solution, and the mixture was stirred and specimens were subjected to dermatophyte detection tests lightly ground with a plastic rod, and then left at rest for 1 min. with the Dermatophyte Test Strip and direct microscopy.18 The test strip was then added. A positive judgment was made Specimens that showed inconsistent results between the two if a dark purple line appeared on the test strip after 5–30 min methods were subjected to PCR to judge the presence or and a negative judgment if it was not visible within this time absence of tinea unguium.19–22 limit (Fig. 1). 2 © 2016 Japanese Dermatological Association  Dermatophyte Test Strip for tinea unguium signed consent and test specimens. Subjects whose speci- mens were subjected both to the Dermatophyte Test Strip detection and direct microscopy were included in the efficacy analysis set (intention to treat [ITT]). To evaluate the endpoints, the concordance rate (positive concordance rate, negative concordance rate and overall con- cordance rate) and the inconsistent rate of the detection of dermatophytes in the suspected specimens between the Der- matophyte Test Strip test and direct microscopy or adjusted results based on PCR (ITT) were obtained. The results of the medical experts’ comprehensive evaluations based on the results of direct microscopy, PCR, the clinical picture, underly- ing disease information and specimen collection site were uti- lized as the final determination (per protocol [PP]). The simple j-coefficient was used to measure the level of agreement between the two methods.25 Any adverse events that occurred after obtaining the signed consent until the day when the test specimens were collected were recorded by the investigators or subinvestigators. Adverse events were coded using the Medical Dictionary for Regulatory Activities, version 17.0. All statistical analyses and data management were con- ducted by L Data Science (Tokyo, Japan). RESULTS Signed consent was obtained from 222 subjects, and all sub- Figure 1. Appearance of the Dermatophyte Test Strip. The jects completed the study. Subjects’ age (mean  standard control line (CL) should always appear after the assay. If it deviation) was 64.0  16.1. This study included 96 males does not appear, the test is invalid. A positive judgment is (43.2%) and 126 females (56.8%). Specimens collected from made if the test line (TL) is visible in 5–30 min after the assay, all the subjects were tested for the presence of dermatophytes and negative if it is not visible. using the Dermatophyte Test Strip and direct microscopy, and all subjects were therefore included in the efficacy and safety analyses (ITT, n = 222). PCR and sequencing The 45 specimens, for which the Dermatophyte Test Strip Nail specimen was weighed and subjected to DNA extrac- and direct microscopy results differed, had to be subjected to tion. The nail specimen and crusher were placed in a 2-mL PCR to further confirm the presence or absence of dermato- test tube and liquid nitrogen was added. The nail specimen phytes. However, 40 specimens were subjected to the PCR was crushed using the Automill (TK-AM5-S; Tokken, Chiba, test because no piece for the PCR test was left in five Japan) at 1220 rpm for 30 s, and heated for 15 min at specimens. 100°C in the presence of lysing solution (100 mmol/L Tris- HCl [pH 7.0], 1 mmol/L ethylenediaminetetraacetic acid Detection of dermatophytes [EDTA] [pH 7.0], 0.1% sodium dodecylsulfate). DNA was Using the Dermatophyte Test Strip, dermatophytes were extracted by the standard method, dried and reconstituted in detected in 201 of 222 (90.5%) specimens but were absent 50 lL Tris-EDTA buffer. in 21 of 222 (9.5%) specimens (Table 1). Dermatophytes Fungal DNA was amplified using the following primer set were detected in 170 of 222 (76.6%) specimens but were that amplified the internal transcribed spacer regions: primer absent in 52 of 222 (23.4%) by direct microscopy. Of the IntF (50 -AACTTGGTCATTTAGAGGAA-30 ) and NL4 (50 -GGT CCGTGTTTCAAGACGG-30 ),19,20 followed by another round of Table 1. Results of the Dermatophyte Test Strip and direct PCR using the IntF primer and a nested primer ITS4R microscopy (n = 222) (50 -TCCTCCGCTTATTGATATGC-30 ).21 Sequences of PCR products were confirmed by the direct sequencing using the Dermatophyte Direct IntF and ITS4R primers. Test Strip microscopy Dermatophyte detection n Ratio (%) n Ratio (%) Statistical analyses Positive 201 90.5 170 76.6 The planned number of subjects enrolled in this study was Negative 21 9.5 52 23.4 200. The safety analysis set included subjects who provided © 2016 Japanese Dermatological Association 3 Y. Tsunemi and M. Hiruma 170 specimens, 163 were positive and seven were negative Comparison of the results between the with the Dermatophyte Test Strip. Of the 222 specimens, Dermatophyte Test Strip and direct microscopy 177 showed matched test results between the test strip and Of the 201 specimens that showed a positive result with the direct microscopy, and 45 specimens showed inconsistent Dermatophyte Test Strip, 163 specimens were also positive results. Of the 177 specimens, 163 were positive with the with direct microscopy. The concordance rate was 81.1% Dermatophyte Test Strip and direct microscopy, and 14 were (95% confidence interval [CI], 75.4–86.8) (Tables 3, 4). Of the negative with the Dermatophyte Test Strip and direct micro- 21 specimens that showed a negative result with the Dermato- scopy. Of the 45 specimens that showed inconsistent results phyte Test Strip, 14 specimens also showed a negative result between the Dermatophyte Test Strip and direct microscopy, with direct microscopy. The concordance rate was 66.7% PCR could further clarify the results of 40 specimens (95% CI, 44.1–89.2). Overall, the number of specimens with (Tables 2, 3). Of the 40 specimens, 35 were positive with consistent results between the Dermatophyte Test Strip and the Dermatophyte Test Strip and negative with direct micro- direct microscopy, either positive or negative, was 177 of 222 scopy, and five were negative with the Dermatophyte Test specimens. The concordance rate was 79.7% (95% CI, 74.2– Strip and positive with direct microscopy. Of the 35 speci- 85.2; j-coefficient, 0.29). The number of inconsistent speci- mens that were positive with the Dermatophyte Test Strip, mens between the two methods was 45 of 222 specimens 33 were positive with PCR. Of the five specimens that were (20.3%; 95% CI, 14.8–25.8). negative with the Dermatophyte Test Strip, four were positive with PCR (Table 2). Of the 37 positive specimens, 23 speci- Comparison of the results between the mens were positive for T. rubrum and 14 specimens for Dermatophyte Test Strip, direct microscopy T. mentagrophytes complex. Of the 40 specimens subjected adjusted by PCR, and final determination to PCR, 34 (85.0%) and six (15.0%) specimens showed the Of the 201 specimens that showed a positive result with the Der- same results as the Dermatophyte Test Strip and direct matophyte Test Strip, 196 specimens also showed a positive microscopy, respectively (Table 2). Concerning the five speci- result for the combined results of direct microscopy and PCR mens that were not left to be subjected to PCR, the results that resulted in a concordance rate of 97.5% (95% CI, 95.1– of direct microscopy were used to analyze the efficacy end- 99.9) in the ITT population (Tables 3, 4). Of the 21 specimens point in the ITT population. In the PP population, the five that showed a negative result with the Dermatophyte Test Strip, specimens that were not subjected to PCR were excluded 15 also showed a negative result with the combined results of from analysis as undeterminable due to protocol deviations, direct microscopy and PCR that resulted in a concordance rate and the concordance and inconsistent rates between the of 71.4% (95% CI, 49.7–93.1). Overall, the number of specimens results of the final determination and the Dermatophyte Test that showed consistent results between the Dermatophyte Test Strip were analyzed (n = 217). Strip and direct microscopy adjusted by PCR, either positive or negative, was 211 of 222 specimens, which resulted in a concor- Table 2. Results of PCR dance rate of 95.0% (95% CI, 92.0–98.1; j-coefficient, 0.70). Inconsistent (Dermatophyte The number of inconsistent specimens between the two meth- Test Strip/direct microscopy) PCR n Ratio (%) ods was 11 of 222 specimens (5.0%; 95% CI, 1.9–8.0). Of the 198 specimens that showed a positive result with the Positive/negative Positive 33 82.5 Dermatophyte Test Strip, 196 specimens also showed a posi- Negative 2 5.0 Negative/positive Positive 4 10.0 tive result with the final determination, which resulted in a con- Negative 1 2.5 cordance rate of 99.0% (95% CI, 97.3–100.0) in the PP Total 40 100.0 population (Tables 3, 4). Of the 19 specimens that showed a negative result with the Dermatophyte Test Strip, 15 specimens PCR, polymerase chain reaction. also showed a negative result with the final determination, Table 3. Comparison of the results of the Dermatophyte Test Strip with those of direct microscopy, direct microscopy adjusted by PCR, and final determination Direct microscopy Direct microscopy/PCR Final determination† Positive Negative Total Positive Negative Total Positive Negative Total Population (n) ITT (222) ITT (222) PP (217) Dermatophyte Test Strip Positive 163 38 201 196 5 201 196 2 198 Negative 7 14 21 6 15 21 4 15 19 Total 170 52 222 202 20 222 200 17 217 j-coefficient 0.29 0.70 0.82 † Determination based on results of direct microscopy, PCR and evaluation by specialists. ITT, intention to treat; PP, per protocol; PCR, polymerase chain reaction. 4 © 2016 Japanese Dermatological Association  Dermatophyte Test Strip for tinea unguium Table 4. Concordance and inconsistent rates in comparison with the detection results between the Dermatophyte Test Strip and direct microscopy, direct microscopy adjusted by PCR, and final determination Concordance rate (95% CI) Fisher’s exact test (95% CI) Comparison of Dermatophyte Test Strip with direct microscopy Positive concordance 81.1% (163/201) (75.4–86.8) (75.0–86.3) Negative concordance 66.7% (14/21) (44.1–89.2) (43.0–85.4) Overall concordance 79.7% (177/222) (74.2–85.2) (73.8–84.8) Inconsistent 20.3% (45/222) (14.8–25.8) (15.2–26.2) Comparison of Dermatophyte Test Strip with direct microscopy adjusted by PCR Positive concordance 97.5% (196/201) (95.1–99.9) (94.3–99.2) Negative concordance 71.4% (15/21) (49.7–93.1) (47.8–88.7) Overall concordance 95.0% (211/222) (92.0–98.1) (91.3–97.5) Inconsistent 5.0% (11/222) (1.9–8.0) (2.5–8.7) Comparison of Dermatophyte Test Strip with final determination Positive concordance 99.0% (196/198) (97.3–100.0) (96.4–99.9) Negative concordance 78.9% (15/19) (58.0–99.9) (54.4–94.0) Overall concordance 97.2% (211/217) (94.8–99.7) (94.1–99.0) Inconsistent 2.8% (6/217) (0.4–5.2) (1.0–5.9) CI, confidence interval; PCR, polymerase chain reaction. which resulted in a concordance rate of 78.9% (95% CI, 58.0– specimens obtained from subjects with suspected tinea 99.9). Overall, the number of specimens for which the results unguium. The specimens that showed inconsistent results of the Dermatophyte Test Strip and the final determination between the two methods were further subjected to PCR, the were consistent, either positive or negative, was 211 of 217 results of which were then added to the comparison in the ITT specimens, which resulted in a concordance rate of 97.2% population. Finally, the results of medical experts’ comprehen- (95% CI, 94.8–99.7; j-coefficient, 0.82). The number of incon- sive evaluations based on the results of direct microscopy, sistent specimens between the two methods was six of 217 PCR, the clinical picture, underlying disease information and specimens (2.8%; 95% CI, 0.4–5.2). specimen collection site were utilized as the final determina- No adverse events were observed in any of the 222 subjects tion, and PP analysis was carried out. throughout the study. The Dermatophyte Test Strip was developed for use in daily dermatological practice; therefore, in this study, the results of the Dermatophyte Test Strip were compared with those of direct DISCUSSION microscopy which is a current standard test in daily dermatologi- The clinical presentation of tinea unguium includes clouding, cal practice. However, because direct microscopy is not as thickening and deformation of the nail plate, and subungual accurate as PCR, PCR was supplementally used to obtain more hyperkeratosis. The diagnosis of tinea unguium is sometimes accurate data to assess the accuracy of the Dermatophyte Test challenging because it mimics many diseases including psoria- Strip. It would be of interest to compare the results of the Der- sis, lichen planus and onychodystrophy.5 Therefore, to distin- matophyte Test Strip and those of PCR in a future study. guish tinea unguium from other diseases, mycological The present results of direct microscopy showed that 170 examination is essential.11 Direct microscopy and fungal cul- and 52 subjects, respectively, were positive and negative ture are recommended in the guidelines.23,26,27 However, there (Table 3). When the results were corrected by the PCR results, are disadvantages to direct microscopy; for example, detection the numbers turned to 202 and 20 subjects, respectively. The sensitivity is likely to be affected by the skill and experience cause of the false-negative results by direct microscopy is level of the person who performs the test. Fungal culture, on considered to be that the morphological fungal element was the other hand, takes 2–3 weeks to identify the pathogenic not preserved although the DNA was intact because most of fungi and also has low sensitivity (40–77%).10,28 Recent devel- the specimens were collected from the tip of the nail. When opments in molecular biological methods offer highly sensitive the results were further analyzed in the PP population, in which and reliable methods such as PCR, which detects dermato- five specimens could not be subjected to PCR, the concor- phytes directly in the affected lesions, but these methods are dance rates increased with j-coefficient of 0.82 (Table 3). not practical in the clinical setting or on a commercial basis, These results indicate that the Dermatophyte Test Strip is because dedicated equipment and special skills are required. practical and useful as a detection method that can be imple- Therefore, there has been demand for the development of a mented in the actual clinical setting. quick, reliable and practical detection method. In a previous study, the sensitivity, specificity and concor- In this study, the detection capacity of the Dermatophyte dance rates of the same test strip have been evaluated by direct Test Strip was evaluated by comparing the results of the microscopy as the standard.11 In this previous study, Tsunemi Dermatophyte Test Strip and direct microscopy using nail and his colleagues have shown that the sensitivity, specificity, © 2016 Japanese Dermatological Association 5 Y. Tsunemi and M. Hiruma positive concordance rate, negative concordance rate and over- 4 Firooz A, Khamesipour A, Dowlati Y. Itraconazole pulse therapy all concordance rate were 97.8%, 78.4%, 84.8%, 96.7% and improves the quality of life of patients with toenail onychomycosis. J Dermatolog Treat 2003; 14: 95–98. 89.1%, respectively. Because the sensitivity of the test strip and 5 Allevato MA. Diseases mimicking onychomycosis. Clin Dermatol negative concordance rate between the two methods were high, 2010; 28: 164–177. the test strip was considered to be useful in screening for tinea 6 Hube B, Hay R, Brasch J, Veraldi S, Schaller M. Dermatomycoses unguium. However, in the present study, the results were some- and inflammation: the adaptive balance between growth, damage, and survival. J Mycol Med 2015; 25: e44–e58. what different (Tables 3, 4); the positive and overall concordance 7 Welsh O, Vera-Cabrera L, Welsh E. Onychomycosis. Clin Dermatol rates between the previous study and the present study appear 2010; 28: 151–159. to be comparable, but the negative concordance rate differs by 8 Faergemann J, Baran R. Epidemiology, clinical presentation and 30%. The difference in the negative concordance rate between diagnosis of onychomycosis. Br J Dermatol 2003; 149(Suppl 65): 1– the present and previous studies appeared to be increased 4. 9 Feuilhade de Chauvin M. New diagnostic techniques. J Eur Acad because the number of subjects without tinea unguium in this Dermatol Venereol 2005; 19(Suppl 1): 20–24. study was extremely small. In addition, the cases with extremely 10 Karimzadegan-Nia M, Mir-Amin-Mohammadi A, Bouzari N, small specimens had negative results with the Dermatophyte Firooz A. Comparison of direct smear, culture and histology for Test Strip in the present study (data not shown). the diagnosis of onychomycosis. Australas J Dermatol 2007; 48: 18–21. Both the previous and present studies indicate that the Der- 11 Tsunemi Y, Takehara K, Miura Y, Nakagami G, Sanada H, Kawa- matophyte Test Strip can quickly provide accurate results. The shima M. Screening for tinea unguium by Dermatophyte Test Strip. Dermatophyte Test Strip is also convenient and practical in the Br J Dermatol 2014; 170: 328–331. actual clinical setting because it is provided as a ready-to-use 12 Kajitani K, Noriki S, Ishida H, inventers; Toyobo Co., Ltd., assignee. kit. Although direct microscopy is the gold standard for the Monoclonal antibody against dermatophyte, hybridoma producing the antibody and method for producing the antibody. Japanese diagnosis of tinea unguium, it has disadvantages; for example, patent 4117542 July 16, 2008. it takes time to dissolve the nail specimens and it requires skill 13 Kajitani K, Noriki S, Ishida H, inventers; Toyobo Co., Ltd., assignee. to identify the fungal elements. The Dermatophyte Test Strip is Dermatophyte detection method, reagent for dermatophyte detec- a useful method that overcomes these disadvantages. It should tion, and antigenicity activation method. Japanese patent 4117563 July 16, 2008. however be noted that the selection of the sampling site and 14 Noriki S, inventer; University of Fukui, JNC Corporation, assignees. method of sampling are critical for any test methods. Special- Non-heating detection method for dermatophyte. European patent ists with sufficient experience should collect the specimens in 2009111 May 29, 2013. accordance with the method recommended in the guideline.23 15 Higashi Y, Miyoshi H, Takeda K et al. Evaluation of a newly-devel- In conclusion, the present results showed that the Dermato- oped immunochromatography strip test for diagnosing dermatophy- tosis. Int J Dermatol 2012; 51: 406–409. phyte Test Strip has high detection capacity and provides a reli- 16 Mochizuki T, Anzawa K, Sakata Y et al. A sampling method using able, convenient and quick test for the detection of tinea cotton swab is suitable for immunochromatography strip test for unguium. detection of dermatophyte antigens from human skin lesions. Rin- sho Kensa 2012; 56: 1503–1507. (in Japanese). 17 Tanabe H. Immunochromatography strip test of dermatophyto- ACKNOWLEDGMENTS: We thank all patients, physicians sis. Japanese J Clin Dermatol 2012; 66(Suppl): 71–75. (in and medical staff who supported this study. We also thank Professor Japanese). Shinichi Watanabe of Teikyo University School of Medicine for perform- 18 Elewski BE. Onychomycosis: pathogenesis, diagnosis, and manage- ing direct microscopy and Professor Takashi Sugita at Meiji Pharma- ment. Clin Microbiol Rev 1998; 11: 415–429. ceutical University for performing PCR. The authors retained editorial 19 Sugita T, lkeda R, Shinoda T. Diversity among strains of Cryptococ- control over the content. Editorial assistance in the preparation of this cus neoformans var. gattii as revealed by a sequence analysis of manuscript was provided by WysiWyg Co., Ltd, and financial support multiple genes and a chemotype analysis of capsular polysaccha- by JNC Corporation. ride. Microbiol Immunol 2001; 45: 757–768. 20 Kurtzman CP, Robnett CJ. Phylogenetic relationship among yeasts of the ‘Saccharomyces complex’ determined from multigene sequence analyses. FEMS Yeast Res 2003; 3: 417–432. 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British Association of Dermatologists’ guidelines for the management of onychomycosis 2014. Br J Dermatol 2014; 171: 937–958. SUPPORTING INFORMATION 27 Drake LA, Dinehart SM, Farmer ER et al. Guidelines of care for Additional Supporting Information may be found in the online superficial mycotic infections of the skin: onychomycosis. Guideli- nes/Outcomes Committee. American Academy of Dermatology. version of this article: J Am Acad Dermatol 1996; 34: 116–121. Table S1. Study investigators and institutions. © 2016 Japanese Dermatological Association 7