RESUMO
The gold-standard method for dermatophyte identification involves direct microscopy and culture, which have inherent shortcomings. Only few molecular methods have been standardised for routine clinical work. This study aimed to develop and test a platform for identifying the most common dermatophytes in Israel using multiplex real-time polymerase chain reaction (RT-PCR). Specific primers were designed for the multiplex system (LightCycler 480) according to known cultures and validated by reference isolates. The dermatophyte detection rate was compared to smear and culture in 223 clinical samples obtained from a tertiary medical centre. Inconsistencies between methods were evaluated by sequencing. The RT-PCR was further evaluated in 200 community-based samples obtained from a health maintenance organisation and 103 military-personnel-based samples analysed at a central laboratory. In hospital-based clinical samples, complete concordance between methods was observed in 190 samples (85%; Kappa = 0.69). In most cases of non-concordance, sequencing was consistent with RT-PCR results. RT-PCR correctly identified all smear- and culture-positive cases in community and military-personnel samples. The results were available within 4 hours. The multiplex RT-PCR platform is a rapid and efficient method for identifying dermatophyte species in clinical samples and may serve as a first step in the diagnostic algorithm of superficial fungal infections.
Assuntos
Arthrodermataceae/isolamento & purificação , Dermatomicoses/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Arthrodermataceae/genética , Criança , Pré-Escolar , Primers do DNA/genética , Feminino , Humanos , Lactente , Israel , Masculino , Técnicas Microbiológicas/métodos , Pessoa de Meia-Idade , Fatores de Tempo , Adulto JovemRESUMO
MicroRNAs (miRNAs) are short non-coding RNAs that postranscriptionally regulate viral and host gene expression. Reliable and simple assays for detecting and analyzing miRNAs during viral infections are critical for clinical and research purposes. A highly sensitive quantitative real-time PCR (qPCR) assay was developed. This approach, using a generic hydrolysis probe, detects and quantifies miRNAs in cell culture and in clinical samples obtained from patients. The assay is based on preparation of cDNA libraries by polyadenylation of total RNA and reverse transcription, followed by detection of specific miRNAs in the cDNA library by qPCR. The qPCR test was highly sensitive and specific, distinguishing between miRNAs that differ by as little as a single nucleotide with remarkable reproducibility. When applied to clinical samples the assay could detect the differential expression of EBV encoded miRNAs in peripheral blood cells of healthy EBV carriers and patients with acute EBV infection, which makes it a powerful tool for the study of differential expression of miRNAs in health and during viral infections.
