RESUMO
BACKGROUND: Phenotypic drug susceptibility testing (pDST) for Mycobacterium tuberculosis can take up to 8 weeks, while conventional molecular tests identify a limited set of resistance mutations. Targeted next-generation sequencing (tNGS) offers rapid results for predicting comprehensive drug resistance, and this study sought to explore its operational feasibility within a public health laboratory in Mumbai, India. METHODS: Pulmonary samples from consenting patients testing Xpert MTB-positive were tested for drug resistance by conventional methods and using tNGS. Laboratory operational and logistical implementation experiences from study team members are shared below. RESULTS: Of the total number of patients tested, 70% (113/161) had no history of previous TB or treatment; however, 88.2% (n = 142) had rifampicin-resistant/multidrug-resistant TB (RR/MDR-TB). There was a high concordance between resistance predictions of tNGS and pDST for most drugs, with tNGS more accurately identifying resistance overall. tNGS was integrated and adapted into the laboratory workflow; however, batching samples caused significantly longer result turnaround time, fastest at 24 days. Manual DNA extraction caused inefficiencies; thus protocol optimisations were performed. Technical expertise was required for analysis of uncharacterised mutations and interpretation of report templates. tNGS cost per sample was US$230, while for pDST this was US$119. CONCLUSIONS: Implementation of tNGS is feasible in reference laboratories. It can rapidly identify drug resistance and should be considered as a potential alternative to pDST.
CONTEXTE: Les tests phénotypiques de sensibilité aux médicaments (pDST) pour Mycobacterium tuberculosis peuvent prendre jusqu'à 8 semaines, tandis que les tests moléculaires conventionnels identifient un ensemble limité de mutations de résistance. Le séquençage ciblé de la prochaine génération (tNGS) offre des résultats rapides pour prédire la résistance globale aux médicaments, et cette étude avait pour objectif d'explorer sa faisabilité opérationnelle au sein d'un laboratoire de santé publique à Mumbai, en Inde. MÉTHODES: Des échantillons pulmonaires de patients consentants testés positifs au Xpert MTB ont été testés pour la résistance aux médicaments par des méthodes conventionnelles et en utilisant le tNGS. Les expériences des membres de l'équipe de l'étude en matière de fonctionnement du laboratoire et de mise en Åuvre logistique sont présentées ci-dessous. RÉSULTATS: Sur le nombre total de patients testés, 70% (113/161) n'avaient pas d'antécédents de TB ou de traitement ; cependant, 88,2% (n = 142) présentaient une TB résistante à la rifampicine/multirésistante aux médicaments (RR/MDR-TB). La concordance entre les prédictions de résistance de la tNGS et de la pDST était élevée pour la plupart des médicaments, la tNGS identifiant globalement la résistance avec plus de précision. La tNGS a été intégrée et adaptée au flux de travail du laboratoire ; toutefois, la mise en lots des échantillons a entraîné un délai d'obtention des résultats beaucoup plus long, le plus rapide étant de 24 jours. L'extraction manuelle de l'ADN a été source d'inefficacité ; le protocole a donc été optimisé. L'analyse des mutations non caractérisées et l'interprétation des modèles de rapport ont nécessité une expertise technique. Le coût du tNGS par échantillon s'élevait à US$230, contre US$119 pour le pDST. CONCLUSIONS: La mise en Åuvre de la tNGS est possible dans les laboratoires de référence. Elle permet d'identifier rapidement la résistance aux médicaments et devrait être considérée comme une alternative potentielle à la pDST.
RESUMO
PURPOSE: The purpose of this study was to investigate New Delhi metallo-ß-lactamase (NDM) production among Gram-negative bacilli. METHODOLOGY: Antibiogram-resistotyping and detection of New Delhi metallo-ß-lactamase (NDM) in clinical isolates of Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa and comparative evaluation of the diagnostic performance of three phenotypic methods for NDM detection, with PCR considered as the gold standard, were performed. Minimum inhibitory concentration (MIC) of antibiotics against NDM-positive strains using E-tests and clonal relationship analysis using enterobacterial repetitive intergenic consensus (ERIC)-PCR in these strains were determined. RESULTS: The most effective antibiotics against strains of the species K. pneumoniae were Colistin, Chloramphenicol and Tigecycline; against P. aeruginosa were Fosfomycin and Polymyxins, and against A. baumannii were Polymyxins, Ampicillin/Sulbactam and Minocycline. Overall, 66, 31 and 40 different resistotypes were observed among K. pneumoniae, A. baumannii and P. aeruginosa strains, respectively. The blaNDM-1 gene was detected in 28 (8.5â%) strains of the bacteria investigated. The sensitivities and specificities of the Meropenem-EDTA combined disk test, the meropenem-dipicolinic acid combined disk test and the modified Hodge test methods for NDM detection were 96.43, 55.15; 96.43, 54.85; and 89.29, 35.15, respectively. Additionally, in spite of the low positive predictive values of these tests, their negative predictive values were high. ERIC-PCR results revealed two main clusters in NDM-positive strains of each of the species P. aeruginosa and A. baumannii, and ten main clusters in K. pneumoniae. In all the NDM-positive strains maximum MIC rates (>256) were observed for all beta-lactam antibiotics. CONCLUSION: There were high levels of antibiotic resistance and a high frequency of multi-drug resistance and extensive-drug resistance profiles, as well as highly prevalent blaNDM-1 genes in the bacteria investigated.
Assuntos
Acinetobacter baumannii/enzimologia , Antibacterianos/farmacologia , Klebsiella pneumoniae/enzimologia , Pseudomonas aeruginosa/enzimologia , beta-Lactamases/genética , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Carbapenêmicos/farmacologia , Análise por Conglomerados , Farmacorresistência Bacteriana , Genótipo , Humanos , Irã (Geográfico) , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Meropeném , Testes de Sensibilidade Microbiana , Fenótipo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Tienamicinas/farmacologiaRESUMO
Abstract Candida albicans is the etiological agent most frequently associated with oral candidiasis in human immunodeficiency virus (HIV) carriers. Strain typification is important to disease epidemiology, particularly with simple, low-cost methodologies such as resistotyping. The present study was designed to use resistotyping to identify possible phenotypic differences between C. albicans strains isolated from the oral cavity of HIV+ and HIV-seronegative patients. Analyses were run using resistotyping (boric acid, cetrimide, sodium periodate, sodium selenite and silver nitrate) to identify phenotypical differences between C. albicans. Descriptive statistics was performed. Of the 149 clones isolated from HIV+ patients the most frequent (47.0%) resistotype was ABCDE. The most frequent resistotype (64.8%) in the 74 clones from HIV-seronegative patients was --CDE. Phenotypic differences were identified between the strains isolated from each group. HIV+ patients exhibited greater strain diversity. Although it has limitations, resistotyping effectively identified differences between C. albicans strains.
Resumen Candida albicans es el agente etiológico más frecuentemente asociado con la candidiasis oral en portadores del virus de la inmunodeficiencia humana (VIH). La tipificación de la cepas es importante para conocer la epidemiología de la enfermedad, particularmente con metodologías simples y de bajo costo, como la resistotipificación. El presente estudio fue diseñado para identificar posibles diferencias fenotípicas por el método de resistotipificación entre cepas de C. albicans aisladas de la cavidad oral de pacientes VIH+ y seronegativos. Se realizó estadística descriptiva. Los análisis se realizaron utilizando resistotipificación (ácido bórico, cetrimida, peryodato de sodio, selenito de sodio y nitrato de plata) para identificar diferencias fenotípicas entre C. albicans. De las 149 clonas aisladas de pacientes VIH+, el resistotipo más frecuente (47.0%) fue ABCDE. El resistotipo más frecuente (64.8%) en las 74 clonas de pacientes seronegativos al VIH fue --CDE. Se identificaron diferencias fenotípicas entre las cepas aisladas de cada grupo. Los pacientes VIH + exhibieron una mayor diversidad de cepas. Aunque tiene limitaciones, la resistotipificación identificó de manera efectiva las diferencias entre las cepas de C. albicans.