RESUMO
Candida tropicalis arises as one of the predominant non-Candida albicans Candida (NCAC) species causing invasive candidiasis in Asian countries. A rise in reports of C. tropicalis with a parallel increase in fluconazole resistance has also been observed. The genes and underlying pathways associated with azole antifungal resistance in C. tropicalis is still not properly understood. The RT-qPCR is the most promising approach for expression analysis of target genes to understand the mechanisms of resistance. The reliability and reproducibility of this technique depend on the selection of suitable reference genes for the normalization in expression study. The present study investigated the expression stability levels of ten genes including ACT1, EF1, GAPDH, PGK1, RDN5.8, RDN18, RDN28, SDHA, TUB1, and UBC13 for their suitability in fluconazole treated/untreated C. tropicalis. The stability levels of these genes were examined by the ∆∆CT, ΔCT, Pfaffl methods and five independent software including hkgFinder, geNorm, NormFinder, BestKeeper, and RefFinder software. We report, the EF1 and ACT1 were the most stable reference genes for normalization and can be used for the gene expression analysis in C. tropicalis. To the best of our knowledge, our study is the first to select and validate the reference genes in C. tropicalis for RT-qPCR based expression analysis.
Assuntos
Candida tropicalis/efeitos dos fármacos , Candidíase Invasiva/tratamento farmacológico , Farmacorresistência Fúngica/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos , Actinas/genética , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Candida tropicalis/genética , Candidíase Invasiva/microbiologia , Estudos de Viabilidade , Fluconazol/farmacologia , Fluconazol/uso terapêutico , Proteínas Fúngicas/genética , Genes Essenciais , Humanos , Índia , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
BACKGROUND: There is limited access to radiology facilities in most parts of India leading to significant under diagnosis and underreporting of smear negative clinically diagnosed tuberculosis (CDTB). Public Private Partnership (PPP) has a lot to contribute towards addressing this gap through providing access to chest x-ray (CXR) in far-off locations. METHOD: Mobile vans equipped with digital CXR equipment and support staff were provided by a Corporate Hospital working closely with government, with scheduled visits to government peripheral health institutes. Patients received upfront CXR and sputum microscopy along with GeneXpert in accordance with the revised TB diagnostic algorithm prescribed by the national program, Following a successful pilot in 2014 in district Rewari, "TB free Haryana" was launched in November 2015 with a phased roll out in 16 districts by 2018. RESULTS: The pilot initiative in 2014 confirmed practical and clinical feasibility and revealed a high rate (30% of people screened) of CDTB i.e. symptomatic cases with radiologic abnormalities compatible with Pulmonary TB. In the first year (2016), 5 districts were covered and a total of 3340 CXRs were carried out. There was an increase in the case notification rates of new CDTB (smear negative) in 2016 compared to 2015 (p = 0.036); yielding an additional 180 cases and an 11.67% increase in case detection. Scale up to a total of 13 and 16 districts took place successfully in 2017 and 2018 respectively; with 6268 CXRs and 8021 CXRs done in the respective years. CONCLUSION: PPP can involve Corporate Hospitals to improve the existing diagnostic infrastructure and provides access to CXR in a not-for-profit sustainable collaboration, with scale-up to the state level; and potential to replicate this initiative in other states.