PCR multiplex en la detección de Tinea unguium en pacientes de Caracas – Venezuela // PCR multiplex in the detection of Tinea unguiumin patients from Caracas – Venezuela
Abstract
Resumen
LaTinea unguium(T. unguium) es una micosis superficial producida por dermatofitos que infectan la lámina ungueal de manos y pies. Afecta a más del 10% de la población mundial y su prevalencia viene aumentando en los últimos años. Los métodos convencionales para su diagnóstico, basados en el examen directo y el cultivo micológico, presentan baja especificidad y sensibilidad respectivamente. El reciente avance de técnicas de secuenciación del ADN, ha permitido desarrollar varios métodos moleculares para la identificación directa y más precisa de dermatofitos a partir de muestras clínicas, con resultados rápidos en 24-48 horas. El objetivo de este trabajo fue comparar la efectividad de la PCR multiplex con los métodos tradicionales para el diagnóstico de dermatofitos. Se evaluaron un total de 49 pacientes con diagnóstico clínico de T. unguiumy 10 muestras de individuos sanos. Se aplicaron las técnicas convencionales y la PCR multiplex para detectar secuencias comunes de los dermatofitos y secuencias específicas para Trichophyton (T.) rubrum yT. mentagrophytes. La PCR multiplex fue positiva para dermatofitos en 19 pacientes, superó el cultivo micológico por 11 muestras, detectando secuencias del T. mentagrophytes y otros dermatofitos,que fueron negativos al cultivo micológico. Se obtuvo una sensibilidad del 97% y especificidad del 73,2%. Los resultados obtenidos en este trabajo demuestran que la PCR multiplex puede detectar especies que no son aisladas en cultivo, logrando mejorar el diagnóstico y la toma de decisiones para el tratamiento en las infecciones fúngicas.
Abstract
Tinea unguiumis a superficial fungal infection due to dermatophytes that affects the ungueal plate of fingers and toes. It affects more than 10% of the population throughout the world with an increasing prevalence in the last years. Conventional diagnostic methods such as direct examination and mycological and fungal culture are of low specificity and sensibility respectively. Recent advances in DNA sequencing have allowed direct identification of dermatophytes with quick results in 24-48 hours. The aim of this study was to compare the effectiveness of PCR multiplex vstraditional methods in the diagnosis of dermatophyte infection of the ungueal plate, and to determine future parameters for its standardization. Forty-nine patients with diagnosis of tinea unguium and 10 samples of healthy control individuals were evaluated. Conventional techniques and PCR multiplex were applied to all samples to identify common DNA sequences of dermatophytes and specific sequences of T. rubrumand T. mentagrophytes. PCRm was positive for dermatophytes in 19 patients, overcoming the mycological culture by 11 samples, detecting the sequence of T. mentagrophytesand other dermatophytes, which were negative to the mycological culture. A high sensitivity of 97% and a specificity of 73.2% were obtained. The results obtained in this work show that multiplex PCR can detect species that are not isolated in culture, achieving better diagnosis and decision making for treatment in fungal infections.
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References
López-Estebaranz JL, Sopena-Barona J. Dermatofitosis cutáneas. Etiología, epidemiología y manifestaciones clínicas. Med Clin 2006; 126(1):14-19.
Manzano-Gayosso P. Las micosis superficiales: su relevancia médica y socioeconómica. Gac Med Méx 2008; 144(2):123-124.
Hiok-Hee T.Superficial fungal infections seen at the National Skin Centre, Singapore 2005; 46:77-80.
Arenas R.Dermatofitosis en México. Rev Iberoam Micol 2002; 19 (2):63-67.
Martínez Méndez D, Hernández Valles R, Alvarado P y Mendoza M. Las micosis en Venezuela: casuística de los Grupos de Trabajo en Micología (1984-2010). Rev Iberoam Micol 2013; 30(1):39–46; doi: 10.1016/j.riam.2012.10.001.
Lemus-Espinoza D, Maniscalchi MT, Villarroel O, Bónoli SB, Wahab F, García O. Superficial mycoses in patients from Anzoátegui State, Venezuela, periodo 2002-2012. Invest Clin 2014; 55(4):311-320.
Elewski B, Hazen P. The superficial mycoses and the dermatophytes. Continuing Medical Education, J Am Acad Dermatol 1989; 21(4 Pt 1):655-673.
Petrini B, Von Rosen L.Optimal dermatophyte diagnosis requires both microscopy and culture. Lakartidningen 2002; 99(41):4084.
Elewski B.Onychomycosis: pathogenesis, diagnosis, and management. Clin Microbiol Rev 1998; 11(3):415–429.
Nenoff P, Krüger C, Schaller J, GinterHanselmayer G, Schulte-Beerbühl R, Tietz HJ. Mycology - an update part 2: dermatomycoses: clinical picture and diagnostics. J Dtsch Dermatol Ges 2014;12(9):749-77. doi: 10.1111/ddg.12420.
Ovrén E, Berglund L, Nordlind K, Rollman O. Dermatophytosis: fluorostaining enhances speed and sensitivity in direct microscopy of skin, nail and hair specimens from dermatology outpatients. Mycoses 2016; 59(7):436-441. doi: 10.1111/myc.12491.
Hayette MP, Seidel L, Adjetey C, Darfouf R, Wéry M, Boreux R1, Sacheli R, Melin P, Arrese J. Clinical evaluation of the DermaGenius® Nail real-time PCR assay for the detection of dermatophytes and Candida albicans in nails. Med Mycol 2019; 1;57(3):277-283. doi: 10.1093/mmy/myy020.
Sherman S, Goshen M, Treigerman O, Ben-Zion K, Carp MJ, Maisler N, Ehrenreich IB, Kimchi A, Lifshitz S, Smollan G, Davidovici B, David M, Hodak E, Segal R. Evaluation of multiplex real-time PCR for identifying dermatophytes in clinical samples-A multicentre study. Mycoses 2018;61(2):119-126. doi: 10.1111/myc.12713.
Pankewitz F, Nenoff P, Uhrlaß S, Bezold G, Winter I, Gräser Y.Development of a novel polymerase chain reaction-enzyme-linked immunosorbent assay for the diagnosis of Trichophyton rubrumonychomycosis. Br J Dermatol 2013; 168(6):1236-42. doi: 10.1111/bjd.12221.
Ding J, Li J, Liu Z, Tan Z.Clinical identification of common species of dermatophytes by PCR and PCR-RFLP. J Huazhong Univ Sci Technolog Med Sci 2004; 24(6):642-4.
Dhib I, Fathallah A, Yaacoub A, Hadj Slama F, Said MB, Zemni R.Multiplex PCR assay for the detection of common dermatophyte nail infections. Mycoses 2014; 57(1):19-26.
Brito Gamboa A, Mendoza M, Fernández A, Díaz E. Detection of Candida dubliniensis in patients with candidiasis in Caracas, Venezuela. Rev Iberoam Micol 2006; 23(2):81-84.
Hay R.The future of onychomycosis therapy may involve a combination of approaches. Br J Dermatol 2001;145 Suppl 60:3-8.
Westerberg DP, Voyack MJ.Onychomycosis: Current trends in diagnosis and treatment. Am Fam Physician 2013; 88(11):762-70.
Lipner SR, Scher RK.Onychomycosis – a small step for quality of care. Curr Med Res Opin 2016; 32(5):865-7. doi: 10.1185/03007995.2016.1147026.
Shemer A, Trau H, Davidovici B, Grunwald MH, Amichai B.Collection of fungi samples from nails: comparative study of curettage and drilling techniques. J Eur Acad Dermatol Venereol 2008; 22(2):182-5. doi: 10.1111/j.1468-3083.2007.02361.x.
Shemer A, Trau H, Davidovici B, Grunwald MH, Amichai B. Nail sampling in onychomycosis: comparative study of curettage from three sites of the infected nail. J Dtsch Dermatol Ges 2007;5(12):1108-11.
Han HW, Hsu MM, Choi JS, Hsu CK, Hsieh HY, Li HC, Chang HC, Chang TC.Rapid detection of dermatophytes and Candida albicansin onychomycosis specimens by an oligonucleotide array. BMC Infect Dis 2014 Nov 7;14:581. doi: 10.1186/s12879-014-0581-5.
Bhatia VK, Sharma PC.Epidemiological studies on Dermatophytosis in human patients in Himachal Pradesh, India. SpringerPlus 2014; 3:134. doi: 10.1186/2193-1801-3-134.
Baranová Z, Kampe T, Dorko E, Rimárová K. Epidemiological and clinical aspects of dermatophytoses in Eastern Slovakia: a retrospective three-year study. Cent Eur J Public Health. 2018; 26 Suppl:S72-S75. doi: 10.21101/cejph.a5279.
Bonifaz A.Micología Médica Básica. Tercera edición. México: Mc Graw Hill; 2010.
Eba M, Njunda AL, Mouliom RN, Kwenti ET, Fuh AN, Nchanji GT, Atashili J. Onychomycosis in diabetic patients in Fako Division of Cameroon: prevalence, causative agents, associated factors and antifungal sensitivity patterns. BMC Res Notes 2016; 22;9(1):494.
Gulcan A, Gulcan E, Oksuz S, Sahin I, Kaya D. Prevalence of toenail onychomycosis in patients with type 2 diabetes mellitus and evaluation of risk factors. J Am Podiatr Med Assoc 2011;101(1):49-54.
Angulo A, Bravo N, Falco A, Pulido A, Rivera Z, Cavallera E.Dermatofitosis por Trichophyton rubrum. Experiencia de 10 Años en el Departamento de Micología del Instituto de Biomedicina. Dermatol Venez 2008; 46 (4): 12-17.
Rios-Yuil JM, Bonifaz A, Arenas R, Araiza J, Fernández R, Mercadillo-Pérez P, Ponce-Olivera R. Mycological studies of nail samples obtained by curettage vs. vertical perforation of the nail plate. J Eur Acad Dermatol Venereol 2013; 27(9):1189-1190. doi: 10.1111/jdv.12035.
Prieto R, Janeiro J, Fiaño L, ibáñez L, Alfonso A, González C. Diagnóstico de infecciones por dermatofitos en uñas con detección rápida específica de Trichophyton rubrum. Rev Inter de Ciencias Podol 2011; 5 (2):9-16.
Nazar JR, Gerosa PE, Díaz OA.Onicomicosis: epidemiología, agentes causales y evaluación de los métodos diagnósticos de laboratorio. Rev Argent Microbiol 2012; 44(1): 21-25.
Pérez JE, Cárdenas C, Hoyos AM.Características clínicas, epidemiológicas y microbiológicas de la onicomicosis en un laboratorio de referencia, Manizales (Caldas), 2009. Infect 2011; 15(3):168-176.
Yue X, Li Q, Wang H, Sun Y, Wang A, Zhang Q, Zhang C. An ultrastructural study of Trichophyton rubruminduced onychomycosis. BMC Infect Dis 2015; 17;15:532. doi: 10.1186/s12879-015-1240-1.
Spiliopoulou A, Bartzavali C, Jelastopulu E, Anastassiou ED, Christofidou M. Evaluation of a commercial PCR test for the diagnosis of dermatophyte nail infections. J Med Microbiol 2015; 64(Pt 1):25-31. doi: 10.1099/jmm.0.079962-0.
Ignjatović VA, Stevanović MT, Djurdjević VS, Petrović MM, Stanucević MP, Džamić AM, Čalovski I. Onychomycosis - Sampling, as Efficiant Part of Hospital Pharmacology. Hospital Pharmacology 2014; 1(3):130-137.
Petinataud D, Berger S, Ferdynus C, Debourgogne A, Contet-Audonneau N.Optimising the diagnostic strategy for onychomycosis from sample collection to fungal identification evaluation of a diagnostic kit for real-time PCR. Mycoses 2016; 59(5):304-311.
Kondori N, Abrahamsson AL, Ataollahy N, Wenneras C. Comparison of a new commercial test, Dermatophyte-PCR kit, with conventional methods for rapid detection and identification of Trichophyton rubrumin nail specimens. Med Mycol 2010; 48(7):1005-1008. doi: 10.3109/13693781003743130.
Mehlig L1, Garve C, Ritschel A, Zeiler A, Brabetz W, Weber C, Bauer A.Clinical evaluation of a novel commercial multiplex-based PCR diagnostic test for differential diagnosis of dermatomycoses. Mycoses 2014;57(1):27-34. doi: 10.1111/myc.12097.
Verrier J, Pronina M, Peter C, Bontems O, Fratti M, Salamin K, Schürch S, Gindro K, Wolfender JL, Harshman K, Monod M. Identification of infectious agents in onychomycoses by PCR-terminal restriction fragment length polymorphism.J Clin Microbiol 2012;50(3):553-61. doi: 10.1128/JCM.05164-11.
Wisselink GJ, van Zanten E, KooistraSmid AM.Trapped in keratin; a comparison of dermatophyte detection in nail, skin and hair samples directly from clinical samples using culture and real-time PCR. J Microbiol Methods 2011;85(1):62-66. doi: 10.1016/j.mimet.2011.01.023.
