Taotlus

E-kiri.eml

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

www.medipro.ee

www.jalgadehooldus.ee

FungiCheck kataloog.pdf

Usaldusväärne dermatofüütide testikomplekt

Revolutsiooniline

innovatsioon

www.medipro.eeHFL laboratories

2 3

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HFL Professionals

FungiCheck HFL FungiCheck testikomplekt on suur läbimurre dermatofüütide olemasolu diagnoosimisel küüneproovides.

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

või kahe triibu näol.

FungiChecki kasutamine praksises võiks saada rutiiniks, sest niiviisi pakute õige diagnoosi abil parimat hoolitsust.

Ja nii tõstate ka oma kabineti poolt pakutud hoolitsuse taset.

FungiCheck sobib kasutamiseks esmalt just uutel klientidel, kes teatavad küüne muutustest või traumast.

Pidage meeles, et umbes 50% kõigist küünetraumast teatanud klientidel on seennakkus ja 50% mitte. (3)

Oluline on seda kliendile vastuvõtul toonitada. Parima jalahooldusplaani loomise eelduseks on FungiCheck

testi läbiviimine.

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

on vale. (2, 5)

FungiCheck on saadaval ainult pediküürijatele, jalahooldusspetsialistidele,

jalaravispetsialistidele, arstidele ja dermatoloogidele.

FungiCheck eelised

Aeg Praegusel ajal kasutatavad laboratoorsed meetodid on mikroskoopiline meetod, PCR või kultiveerimismeetod. Mikroskoopiline meetod nõuab valdkonnapõhiseid teadmisi ja kogemusi ning mõned seened võivad jääda tähelepanuta. Nende meetodite õnnestumise määr on 50-73%. (7-8) Mõlemad meetodid on aeganõudvad, sest neid tehakse laboris.

Ravimid Erinevalt tavapärastest meetoditest saab testikomplekti FungiCheck kasutada ka klientidel, kes juba kasutavad seenevastaseid ravimeid või pealekantavaid tooteid. FungiCheck testi saab läbi viia pealekantavate toodete või seenevastaste ravimite (asoolide) kasutamise ajal. Testi tulemust see ei mõjuta. Negatiivse tulemuse korral on olemas korralikud alusandmed, kas suunata oma klient perearsti juurde või mitte. Positiivse tulemuse korral saab klient jätkata onühhomükoosi raviga. (4)

Kursus/koolitus Mõni meetod eeldab kursusel osalemist ja sageli on vaja seda igal aastal korrata. FungiCheck ei eelda kursuse läbimist. Iga testikarbiga kaasnev üksikasjalik kasutusjuhend annab teile korralikke juhiseid. Kasutusjuhendis viidatakse ka steriilsetele töövõtetele, kui soovite selle osa oma praktikas veel kord üle vaadata. Lisaks on saadaval tasuta tootekoolitused, kui soovite avardada oma teadmisi FungiChecki ja seennakkuste kohta.

Miks just Fungicheck? Tagab parima hoolitsuse tänu täpsele diagnostikale. FungiCheck kasutamisel saate olla kindel, et Teie kliendid saavad õige jalahooldusplaani ja tooted.

4 5

Onühhomükoos Rahvusvahelise inimeste ja loomade mükoloogiaühingu (ISHAM) andmetel on onühhomükoos küünte invasiivne seennakkus - olenemata algpõhjusest. See haigus esineb sõrme- või varbaküüntel ja põhjustab umbes 50% kõigist küünte anomaaliatest. (1)

Onühhomükoosi põhjustajad on dermatofüüdid ja mitte-dermatofüüdid (NDM-id). Mõistet Tinea unguium kasutatakse dermatofüütide põhjustatud onühhomükoosi korral; siiski on Tinea unguium ja onühhomükoos sageli kasutusel sünonüümina.

Üldiselt kasutatakse mõistet onühhomükoos kõigi küüneseente puhul, mille on põhjustanud kas dermatofüüdid või NDM-id. (1,3)

Uuringud näitavad, et (3) 91% küüneseentest on põhjustanud dermatofüüdid, samas kui mitte- dermatofüüdid põhjustavad umbes 9% kogu maailma onühhomükoosist. Euroopas tõuseb see protsent isegi 94% peale.

Dermatofüüdid ja mitte-dermatofüüdid Dermatofüüdid on seened, mis vajavad kasvamiseks keratiini. Need seened võivad põhjustada naha, juuste ja küünte nakkust. Dermatofütoosi nimetatakse “Tinea” nakkusteks ja seetõttu võib see pärineda väljastpoolt.

Dermatofüüdid on inimestel enim vaevusi põhjustavad seened. (1, 3) Need on niitjad seened, mis suudavad lagundada keratiini ja kasutada seda toitainena. Neid leidub kõikjal maailmas. Dermatofüüte võib jagada järgmisse kolme seeneperekonda: Trichophyton, Epidermophyton ja Microsporum.

Varbaküüntel on kõige levinumad liigid Trichophyton Rubrum (70%) ja Trichophyton Mentagrophytes (20%). Perekond Epidermophyton sisaldab ainult ühte liiki: 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.

Uuringud näitavad, et enamiku küüneseentest on põhjustanud dermatofüüdid; pärmseened ja NDM-id põhjustavad umbes 9% kogu maailma onühhomükoosist.(3) Uuringud näitavad, et sellistes riikides nagu Iraan ja Kolumbia tekib NDM-ide põhjustatud küüneseen veidi suurema tõenäosusega. (6)

FungiCheck testriba piirdub dermatofüüdite põhjustatud küüneseene testimisega küüntes, sest need teevad pesa väljastpoolt ja asuvad seetõttu otse küünes. (4, 7)

Dermatofüüdid

6 7

Usaldusväärsus FungiCheck on teaduslikult ja kliiniliselt testitud testikomplekt. Uuringud näitavad, et FungiCheck, mille usaldusväärsus on 97%, on kõige usaldusväärsem meetod küüneproovist dermatofüütide tuvastamiseks.

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 - M. flavorosea (4, 7)

Tulemus Testi tulemuste põhjal saate koostada oma kliendile parima jalahooldusplaani või suunata kliendi ka perearsti juurde.

Roosa joon näitab testi õnnestumist. On väga oluline meeles pidada, et see joon peab alati nähtav olema, sest see on kontrolljoon. Teine violetne/hall joon näitab, kas test on positiivne. Järeldus: kaks joont on positiivsed ja 1 joon on negatiivne.

Dermatofüütide põhjustatud onühhomükoosi saab ravida professionaal ja see pole inimestele ohtlik. Kui tulemus on positiivne, võite alati saata perearstile kirja testi tulemustega, et ta saaks selle patsienditoimikusse lisada.

KUI test on negatiivne, on vägagi tõenäoline (50%), et kliendil on muu küüne anomaalia. Visuaalselt võivad muud küüneprobleemid (nt hüperkeratoos, onühholüüs, psoriaas või kollaste küünte sündroom) näha välja justkui seennakkus.

FungiCheck annab perearsti juurde suunamiseks korralikud alusandmed kui kliendil on nt mitu meditsiinilist kaebust või krooniline või eluohtlik haigus. Andke kliendile hermeetiline testiriba ja FungiCheck infolehe koopia, mille ta saab perearsti juurde kaasa võtta. Kui esineb kahtlus Candida osas ning klient põeb eluohtlikku haigust nagu nt vähk või HIV, peaks ta kindlasti perearsti juurde suunama.

Dermatofüütia või küünetrauma korral saab oma kliendile soovitada parimat jalahoolduslahendust, pakkudes seenevastast toodet.

See tagab olukorra, et olete soovitanud õige toote ning pakub paremaid võimalusi kasutatud toote tulemuste hindamiseks. Klient saab olla kindel, et ta ei raiska raha ebavajalikele toodetele.

See hoiab ära ebaeetilised otsused ja tekitab usalduse.

Külgvool põhinev immunoanalüüs, mis on mõeldud küüntes dermatofüütidest saadud antigeenide tuvastamiseks, kasutades nitrotselluloosmembraanile immobiliseeritud dermatofüütidevastast

antikeha, mis reageerib kokkupuutel dermatofüütidega

Mikroskoopiline meetod

Kultiveerimismeetod FungiCheckPCR

73%50%60% 97%

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*

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

“Lõpuks ometi on loodud usaldusväärne ja kiire test, mida saab kliinikus kasutada väga levinud küüneinfektsiooni täpseks diagnoosimiseks”

Dr. Ivan Bristow, Dermatologist & Podiatrist, Southampton UK

Medipro OÜ Pirni 7/1-36, Tallinn

Tel: +372 5915 1408 [email protected] www.medipro.ee

11 02

00 2/

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Teaduslik uuring_FungiCheck - Dermatophyte test strip - Clinical Study - Journal of Dermatology (2).pdf

ORIGINAL ARTICLE

Clinical study of Dermatophyte Test Strip, an immunochromatographic method, to detect tinea unguium dermatophytes

Yuichiro TSUNEMI,1 Masataro HIRUMA2

1Department 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

Tinea unguium is a nail disease caused by dermatophyte infec-

tion of the nail plate.1,2 Its prevalence in advanced countries is

considered as at least 10% of the population,2 and in Japan it

is estimated at approximately 10%.1 The incidence increases

with aging in all countries and it is therefore predicted to

increase in number in association with the aging of society in

the future.2 Patients with tinea unguium pose a risk of becom-

ing the source of infection of other people.3 Tinea unguium is

particularly considered as a predictor of diabetic foot syn-

drome.3 Thus, tinea unguium does not only cause problems

with nail appearance, but it also seriously damages patients’

quality of life. It should therefore be treated actively as far as

possible.4

Because many other diseases have similar symptoms as

tinea unguium,5 differentiation is not easy, and a definitive

diagnosis by mycological examination is necessary before

treatment is started. Dermatophytes are classified into three

genera, namely, Trichophyton (T), Microsporum (M) and Epider- mophyton (E).6,7 T. rubrum accounts for the primary cause of

tinea unguium, followed by T. mentagrophytes.7,8

The diagnostic tests for tinea unguium include direct micro-

scopy with potassium hydroxide (KOH), fungal culture, periodic

acid-Schiff staining (PAS), fluorescence staining, confocal

microscopy and molecular biological methods such as poly-

merase chain reaction (PCR).9 Although direct microscopy with

KOH and fungal culture have been the gold standard for the

diagnosis of tinea unguium,7,9 these tests sometimes are a

burden in the clinical setting, because direct microscopy

requires experience to identify the fungal elements, and disso-

lution of nails is time-consuming. It takes 2–3 weeks to obtain

results using fungal culture, and the detection rate is lower

than with direct microscopy.10 Therefore, the development of

an easy and quick test to diagnose tinea unguium accurately is

in high demand.

The recently developed Dermatophyte Test Strip detects

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.

doi: 10.1111/1346-8138.13348 Journal of Dermatology 2016; : 1–7

using monoclonal antibodies that react specifically with the

polysaccharide present in the cell wall of dermatophytes.11–14

This antibody was found to react specifically with seven der-

matophytes (T. rubrum, T. mentagrophytes, T. violaceum,

T. tonsurans, M. gypseum, M. canis and E. floccosum).12,13

There have been a few preliminary reports on screening results

of dermatmycoses using a Dermatophyte Test Strip.15–17 Higa-

shi and his colleagues have collected 88 specimens from skin

and 72 from nails, examined them using direct microscopy and

Dermatophyte Test Strip, and compared the results.15

In this study, tinea unguium-suspected specimens were

subjected both to testing with the Dermatophyte Test Strip as

well as the gold standard of direct microscopy with KOH to

detect dermatophytes. Specimens that showed inconsistent

results between the two methods were subjected to PCR to

further confirm the presence or absence of dermatophytes.

The detection capacity of the Dermatophyte Test Strip was

then investigated for all specimens by comparing the results of

medical experts’ comprehensive evaluations based on the

results of direct microscopy and PCR.

METHODS

A multicenter, single-arm, comparative clinical study was con-

ducted to evaluate the efficacy of the Dermatophyte Test Strip

(JNC Corporation, Tokyo, Japan) to detect dermatophytes in

tinea unguium-suspected specimens in comparison with direct

microscopic examination. The study was conducted at 11

study sites (Table S1) from June to August 2014.

The detection of dermatophytes using the Dermatophyte

Test Strip was performed at Yokohama Research Center of

JNC Corporation. Direct microscopic examination was per-

formed by Professor Shinichi Watanabe of Teikyo University

School of Medicine. PCR was performed by Professor Takashi

Sugita of Meiji Pharmaceutical University. This study was per-

formed in compliance with Good Clinical Practice based on the

Declaration of Helsinki and other applicable regulations. The

institutional review board of each study site reviewed and

approved the study protocol. Prior to the start of screening

procedures, signed informed consent was obtained from each

subject. The present study was monitored and audited by EPS

Associates Co., Ltd.

Study design Within 4 weeks after obtaining signed consent, the investigator

or subinvestigator confirmed the eligibility of the subject, col-

lected the baseline characteristic information (date of birth, age

and sex) and examined and interviewed the subject (main rea-

son for visit, disease or major complaint, medical history and

complications). A nail specimen was collected from each sub-

ject within 1 week after confirming the eligibility of a subject,

and the specimen was sent to the central laboratory where all

specimens were subjected to dermatophyte detection tests

with the Dermatophyte Test Strip and direct microscopy.18

Specimens that showed inconsistent results between the two

methods were subjected to PCR to judge the presence or

absence of tinea unguium.19–22

This trial was registered with the University Hospital Medical

Information Network Clinical Trials Registry (no. UMIN

000016760).

Subjects Male and female subjects aged 20 years or more who had a

nail lesion suspected of tinea unguium, and who provided

signed informed consent were included in the study. Subjects

were excluded from the study if any of the following applied: (i)

history of oral antifungal agent use within 6 months or of topi-

cal antifungal agent use within 1 month; (ii) participation in

another clinical trial or other clinical studies; and (iii) participa-

tion determined to be inappropriate by the study investigator

or subinvestigator.

Primary end-point The primary end-points were the concordance rate (positive

concordance rate, negative concordance rate and overall con-

cordance rate) and the inconsistent rate of detecting dermato-

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-

hensive evaluations based on the results of direct microscopy,

PCR, the clinical picture, underlying disease information and

specimen collection site.

Test specimen collection Specimens were collected by the dermatologists listed in

Table S1. Dermatologists at the 11 study sites selected

patients with suspected tinea unguium by visual examina-

tion and collected specimens from those patients. A speci-

men of approximately 20 mg was collected from a

suspected tinea unguium nail lesion in accordance with the

guideline.23 The lesions were recorded by taking pictures

and sketches.

The collected nail was sandwiched between autoclaved alu-

minum plates and crushed into small pieces by using a ham-

mer. The pieces were transferred to a test tube, further cut

and crushed using a pair of dissecting scissors and divided

into three portions after mixing for direct microscopy, the Der-

matophyte Test Strip and PCR, respectively. Because nine

specimens were extremely small, these specimens were only

cut into two pieces, forfeiting the piece for PCR.

Dermatophyte Test Strip Monoclonal antibodies that react specifically with the polysac-

charide present in the cell wall of dermatophytes12,13 are lin-

early immobilized on the test strip. The polysaccharides in the

suspected specimens are extracted using the extraction solu-

tion.14,24 In short, a nail specimen was cut into smaller pieces

with a nail clipper or nipper. These pieces were then added to

0.25 mL of extraction solution, and the mixture was stirred and

lightly ground with a plastic rod, and then left at rest for 1 min.

The test strip was then added. A positive judgment was made

if a dark purple line appeared on the test strip after 5–30 min

and a negative judgment if it was not visible within this time

limit (Fig. 1).

Y. Tsunemi and M. Hiruma

PCR and sequencing Nail specimen was weighed and subjected to DNA extrac-

tion. The nail specimen and crusher were placed in a 2-mL

test tube and liquid nitrogen was added. The nail specimen

was crushed using the Automill (TK-AM5-S; Tokken, Chiba,

Japan) at 1220 rpm for 30 s, and heated for 15 min at

100°C in the presence of lysing solution (100 mmol/L Tris-

HCl [pH 7.0], 1 mmol/L ethylenediaminetetraacetic acid

[EDTA] [pH 7.0], 0.1% sodium dodecylsulfate). DNA was

extracted by the standard method, dried and reconstituted in

50 lL Tris-EDTA buffer.

Fungal DNA was amplified using the following primer set

that amplified the internal transcribed spacer regions: primer

IntF (50-AACTTGGTCATTTAGAGGAA-30) and NL4 (50-GGT

CCGTGTTTCAAGACGG-30),19,20 followed by another round of

PCR using the IntF primer and a nested primer ITS4R

(50-TCCTCCGCTTATTGATATGC-30).21 Sequences of PCR

products were confirmed by the direct sequencing using the

IntF and ITS4R primers.

Statistical analyses The planned number of subjects enrolled in this study was

200. The safety analysis set included subjects who provided

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-

jects completed the study. Subjects’ age (mean standard

deviation) was 64.0 16.1. This study included 96 males

(43.2%) and 126 females (56.8%). Specimens collected from

all the subjects were tested for the presence of dermatophytes

using the Dermatophyte Test Strip and direct microscopy, and

all subjects were therefore included in the efficacy and safety

analyses (ITT, n = 222).

The 45 specimens, for which the Dermatophyte Test Strip

and direct microscopy results differed, had to be subjected to

PCR to further confirm the presence or absence of dermato-

phytes. However, 40 specimens were subjected to the PCR

test because no piece for the PCR test was left in five

specimens.

Detection of dermatophytes Using the Dermatophyte Test Strip, dermatophytes were

detected in 201 of 222 (90.5%) specimens but were absent

in 21 of 222 (9.5%) specimens (Table 1). Dermatophytes

were detected in 170 of 222 (76.6%) specimens but were

absent in 52 of 222 (23.4%) by direct microscopy. Of the

Figure 1. Appearance of the Dermatophyte Test Strip. The

control line (CL) should always appear after the assay. If it

does not appear, the test is invalid. A positive judgment is made if the test line (TL) is visible in 5–30 min after the assay,

and negative if it is not visible.

Table 1. Results of the Dermatophyte Test Strip and direct microscopy (n = 222)

Dermatophyte detection

Dermatophyte

Test Strip

Direct

microscopy

n Ratio (%) n Ratio (%)

Positive 201 90.5 170 76.6 Negative 21 9.5 52 23.4

Dermatophyte Test Strip for tinea unguium

170 specimens, 163 were positive and seven were negative

with the Dermatophyte Test Strip. Of the 222 specimens,

177 showed matched test results between the test strip and

direct microscopy, and 45 specimens showed inconsistent

results. Of the 177 specimens, 163 were positive with the

Dermatophyte Test Strip and direct microscopy, and 14 were

negative with the Dermatophyte Test Strip and direct micro-

scopy. Of the 45 specimens that showed inconsistent results

between the Dermatophyte Test Strip and direct microscopy,

PCR could further clarify the results of 40 specimens

(Tables 2, 3). Of the 40 specimens, 35 were positive with

the Dermatophyte Test Strip and negative with direct micro-

scopy, and five were negative with the Dermatophyte Test

Strip and positive with direct microscopy. Of the 35 speci-

mens that were positive with the Dermatophyte Test Strip,

33 were positive with PCR. Of the five specimens that were

negative with the Dermatophyte Test Strip, four were positive

with PCR (Table 2). Of the 37 positive specimens, 23 speci-

mens were positive for T. rubrum and 14 specimens for

T. mentagrophytes complex. Of the 40 specimens subjected

to PCR, 34 (85.0%) and six (15.0%) specimens showed the

same results as the Dermatophyte Test Strip and direct

microscopy, respectively (Table 2). Concerning the five speci-

mens that were not left to be subjected to PCR, the results

of direct microscopy were used to analyze the efficacy end-

point in the ITT population. In the PP population, the five

specimens that were not subjected to PCR were excluded

from analysis as undeterminable due to protocol deviations,

and the concordance and inconsistent rates between the

results of the final determination and the Dermatophyte Test

Strip were analyzed (n = 217).

Comparison of the results between the Dermatophyte Test Strip and direct microscopy Of the 201 specimens that showed a positive result with the

Dermatophyte Test Strip, 163 specimens were also positive

with direct microscopy. The concordance rate was 81.1%

(95% confidence interval [CI], 75.4–86.8) (Tables 3, 4). Of the

21 specimens that showed a negative result with the Dermato-

phyte Test Strip, 14 specimens also showed a negative result

with direct microscopy. The concordance rate was 66.7%

(95% CI, 44.1–89.2). Overall, the number of specimens with

consistent results between the Dermatophyte Test Strip and

direct microscopy, either positive or negative, was 177 of 222

specimens. The concordance rate was 79.7% (95% CI, 74.2–

85.2; j-coefficient, 0.29). The number of inconsistent speci-

mens between the two methods was 45 of 222 specimens

(20.3%; 95% CI, 14.8–25.8).

Comparison of the results between the Dermatophyte Test Strip, direct microscopy adjusted by PCR, and final determination Of the 201 specimens that showed a positive result with the Der-

matophyte Test Strip, 196 specimens also showed a positive

result for the combined results of direct microscopy and PCR

that resulted in a concordance rate of 97.5% (95% CI, 95.1–

99.9) in the ITT population (Tables 3, 4). Of the 21 specimens

that showed a negative result with the Dermatophyte Test Strip,

15 also showed a negative result with the combined results of

direct microscopy and PCR that resulted in a concordance rate

of 71.4% (95% CI, 49.7–93.1). Overall, the number of specimens

that showed consistent results between the Dermatophyte Test

Strip and direct microscopy adjusted by PCR, either positive or

negative, was 211 of 222 specimens, which resulted in a concor-

dance rate of 95.0% (95% CI, 92.0–98.1; j-coefficient, 0.70).

The number of inconsistent specimens between the two meth-

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

Dermatophyte Test Strip, 196 specimens also showed a posi-

tive result with the final determination, which resulted in a con-

cordance rate of 99.0% (95% CI, 97.3–100.0) in the PP

population (Tables 3, 4). Of the 19 specimens that showed a

negative result with the Dermatophyte Test Strip, 15 specimens

also showed a negative result with the final determination,

Table 2. Results of PCR

Inconsistent (Dermatophyte Test Strip/direct microscopy) PCR n Ratio (%)

Positive/negative Positive 33 82.5

Negative 2 5.0 Negative/positive Positive 4 10.0

Negative 1 2.5

Total 40 100.0

PCR, polymerase chain reaction.

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.

Y. Tsunemi and M. Hiruma

which resulted in a concordance rate of 78.9% (95% CI, 58.0–

99.9). Overall, the number of specimens for which the results

of the Dermatophyte Test Strip and the final determination

were consistent, either positive or negative, was 211 of 217

specimens, which resulted in a concordance rate of 97.2%

(95% CI, 94.8–99.7; j-coefficient, 0.82). The number of incon-

sistent specimens between the two methods was six of 217

specimens (2.8%; 95% CI, 0.4–5.2).

No adverse events were observed in any of the 222 subjects

throughout the study.

DISCUSSION

The clinical presentation of tinea unguium includes clouding,

thickening and deformation of the nail plate, and subungual

hyperkeratosis. The diagnosis of tinea unguium is sometimes

challenging because it mimics many diseases including psoria-

sis, lichen planus and onychodystrophy.5 Therefore, to distin-

guish tinea unguium from other diseases, mycological

examination is essential.11 Direct microscopy and fungal cul-

ture are recommended in the guidelines.23,26,27 However, there

are disadvantages to direct microscopy; for example, detection

sensitivity is likely to be affected by the skill and experience

level of the person who performs the test. Fungal culture, on

the other hand, takes 2–3 weeks to identify the pathogenic

fungi and also has low sensitivity (40–77%).10,28 Recent devel-

opments in molecular biological methods offer highly sensitive

and reliable methods such as PCR, which detects dermato-

phytes directly in the affected lesions, but these methods are

not practical in the clinical setting or on a commercial basis,

because dedicated equipment and special skills are required.

Therefore, there has been demand for the development of a

quick, reliable and practical detection method.

In this study, the detection capacity of the Dermatophyte

Test Strip was evaluated by comparing the results of the

Dermatophyte Test Strip and direct microscopy using nail

specimens obtained from subjects with suspected tinea

unguium. The specimens that showed inconsistent results

between the two methods were further subjected to PCR, the

results of which were then added to the comparison in the ITT

population. Finally, the results of medical experts’ comprehen-

sive evaluations based on the results of direct microscopy,

PCR, the clinical picture, underlying disease information and

specimen collection site were utilized as the final determina-

tion, and PP analysis was carried out.

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

microscopy which is a current standard test in daily dermatologi-

cal practice. However, because direct microscopy is not as

accurate as PCR, PCR was supplementally used to obtain more

accurate data to assess the accuracy of the Dermatophyte Test

Strip. It would be of interest to compare the results of the Der-

matophyte Test Strip and those of PCR in a future study.

The present results of direct microscopy showed that 170

and 52 subjects, respectively, were positive and negative

(Table 3). When the results were corrected by the PCR results,

the numbers turned to 202 and 20 subjects, respectively. The

cause of the false-negative results by direct microscopy is

considered to be that the morphological fungal element was

not preserved although the DNA was intact because most of

the specimens were collected from the tip of the nail. When

the results were further analyzed in the PP population, in which

five specimens could not be subjected to PCR, the concor-

dance rates increased with j-coefficient of 0.82 (Table 3).

These results indicate that the Dermatophyte Test Strip is

practical and useful as a detection method that can be imple-

mented in the actual clinical setting.

In a previous study, the sensitivity, specificity and concor-

dance rates of the same test strip have been evaluated by direct

microscopy as the standard.11 In this previous study, Tsunemi

and his colleagues have shown that the sensitivity, specificity,

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.

Dermatophyte Test Strip for tinea unguium

positive concordance rate, negative concordance rate and over-

all concordance rate were 97.8%, 78.4%, 84.8%, 96.7% and

89.1%, respectively. Because the sensitivity of the test strip and

negative concordance rate between the two methods were high,

the test strip was considered to be useful in screening for tinea

unguium. However, in the present study, the results were some-

what different (Tables 3, 4); the positive and overall concordance

rates between the previous study and the present study appear

to be comparable, but the negative concordance rate differs by

30%. The difference in the negative concordance rate between

the present and previous studies appeared to be increased

because the number of subjects without tinea unguium in this

study was extremely small. In addition, the cases with extremely

small specimens had negative results with the Dermatophyte

Test Strip in the present study (data not shown).

Both the previous and present studies indicate that the Der-

matophyte Test Strip can quickly provide accurate results. The

Dermatophyte Test Strip is also convenient and practical in the

actual clinical setting because it is provided as a ready-to-use

kit. Although direct microscopy is the gold standard for the

diagnosis of tinea unguium, it has disadvantages; for example,

it takes time to dissolve the nail specimens and it requires skill

to identify the fungal elements. The Dermatophyte Test Strip is

a useful method that overcomes these disadvantages. It should

however be noted that the selection of the sampling site and

method of sampling are critical for any test methods. Special-

ists with sufficient experience should collect the specimens in

accordance with the method recommended in the guideline.23

In conclusion, the present results showed that the Dermato-

phyte Test Strip has high detection capacity and provides a reli-

able, convenient and quick test for the detection of tinea

unguium.

ACKNOWLEDGMENTS: We thank all patients, physicians

and medical staff who supported this study. We also thank Professor

Shinichi Watanabe of Teikyo University School of Medicine for perform-

ing direct microscopy and Professor Takashi Sugita at Meiji Pharma-

ceutical University for performing PCR. The authors retained editorial

control over the content. Editorial assistance in the preparation of this

manuscript was provided by WysiWyg Co., Ltd, and financial support

by JNC Corporation.

CONFLICT OF INTEREST: This clinical performance study

was supported by JNC Corporation. Y. T. and M. H. served as consul-

tants to JNC Corporation.

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SUPPORTING INFORMATION

Additional Supporting Information may be found in the online

version of this article:

Table S1. Study investigators and institutions.