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AIMS: The use of metagenomics for pathogen identification in clinical practice has been limited. Here we describe a workflow to encourage the clinical utility and potential of NGS for the screening of bacteria, fungi, and antimicrobial resistance genes (ARGs). METHODS AND RESULTS: The method includes target enrichment, long-read sequencing, and automated bioinformatics. Evaluation of several tools and databases was undertaken across standard organisms (n = 12), clinical isolates (n = 114), and blood samples from patients with suspected bloodstream infections (n = 33). The strategy used could offset the presence of host background DNA, error rates of long-read sequencing, and provide accurate and reproducible detection of pathogens. Eleven targets could be successfully tested in a single assay. Organisms could be confidently identified considering ≥60% of best hits of a BLAST-based threshold of e-value 0.001 and a percent identity of >80%. For ARGs, reads with percent identity of >90% and >60% overlap of the complete gene could be confidently annotated. A kappa of 0.83 was observed compared to standard diagnostic methods. Thus, a workflow for the direct-from-sample, on-site sequencing combined with automated genomics was demonstrated to be reproducible. CONCLUSION: NGS-based technologies overcome several limitations of current day diagnostics. Highly sensitive and comprehensive methods of pathogen screening are the need of the hour. We developed a framework for reliable, on-site, screening of pathogens.
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Sequenciamento por Nanoporos , Humanos , Bactérias/genética , Fungos/genética , Biologia Computacional , Genômica , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
BACKGROUND: Monitoring of hepatitis B virus (HBV) viral load has become an essential phase in the treatment of HBV. There are many commercial assays available for HBV viral load quantification. In this study, we have evaluated the performance characteristics of Amplisure® HBV Kit in comparison with the Qiagen artus HBV QS-RGQ kit for HBV DNA quantitation. METHODS: Comparison of 2 methods was carried out on 200 clinical samples, 150 HBV DNA positive and 50 HBV DNA negative, by a reference method. Results obtained with Amplisure® HBV Kit (Amplisure HBV) were compared using the Qiagen artus HBV QS-RGQ assay results as the comparator method. RESULT: The overall performance of the Amplisure HBV compared with the comparator method shows positive and negative clinical agreement of 100 and 76%, respectively. Among the 12 qualitative discrepant samples, all positive with Amplisure HBV were sequenced and 10 were below comparator method's LOD. For 5 weak positives (-0.22 to 0.98 log IU/mL), the sequencing failed. The 7 other positives (0.48 to 1.89 log IU/mL) were confirmed positive by sequencing. Quantitative comparison gave an r2 of 0.967 with a mean log difference of 0.09 log10 IU/mL. CONCLUSION: This study shows that Amplisure® HBV Quantitative Kit shows comparable performance with artus HBV QS-RGQ assays and can be useful in management and therapeutic monitoring of HBV in a clinical practice.
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DNA Viral , Vírus da Hepatite B , DNA Viral/genética , Vírus da Hepatite B/genética , Carga ViralRESUMO
Treatment decisions for tuberculosis (TB) in the absence of full drug-susceptibility data can result in amplifying resistance and may compromise treatment outcomes. Genomics of Mycobacterium tuberculosis (M.tb) from clinical samples enables detection of drug resistance to multiple drugs. We performed whole-genome sequencing (WGS) for 600 clinical samples from patients with tuberculosis to identify the drug-resistance profile and mutation spectrum. We documented the reasons reported by clinicians for referral. WGS identified a high proportion (51%) of pre-extensively drug-resistant (pre-XDR) cases followed by multidrug-resistant tuberculosis (MDR-TB) (15.5%). This correlates with the primary reason for referral, as non-response to the first-line treatment (67%) and treatment failure or rifampicin resistance (14%). Multivariate analysis indicated that all young age groups (P < 0.05), male gender (P < 0.05), and Beijing strain (P < 0.01) were significant independent predictors of MDR-TB or MDR-TB+ [pre-extensively drug-resistant tuberculosis (XDR-TB) and XDR-TB]. Ser315Thr (72.5%) in the inhA gene and Ser450Leu in the rpoB gene (65.5%) were the most prevalent mutations, as were resistance-conferring mutations to pyrazinamide (41%) and streptomycin (61.33%). Mutations outside the rifampicin resistance-determining region (RRDR), Ile491Phe and Val170Phe, were seen in 1.3% of cases; disputed mutations in rpoB (Asp435Tyr, His445Asn, His445Leu, and Leu430Pro) were seen in 6% of cases, and mutations to newer drugs such as bedaquiline and linezolid in 1.0% and 7.5% of cases, respectively. This study on clinical samples highlights that there is a high proportion of pre-XDR cases and emerging resistance to newer drugs; ongoing transmission of these strains can cause serious threat to public health; and whole-genome sequencing can effectively identify and support precision medicine for TB. IMPORTANCE: The current study is based on real-world data on the TB drug-resistance profile by whole-genome sequencing of 600 clinical samples from patients with TB in India. This study indicates the clinicians' reasons for sending samples for WGS, which is for difficult-to-treat cases and/or relapse and treatment failure. The study reports a significant proportion of cases with pre-XDR-TB strains that warrant policy makers' attention. It reflects the current iterative nature of the diagnostic tests under programmatic conditions that leads to delays in appropriate diagnosis and empirical treatment. India had an estimated burden of 2.95 million TB cases in 2020 and 135,000 multidrug-resistant cases. However, WGS profiles of M.tb from India remains disproportionately poorly represented. This study adds a significant body of data on the mutation profiles seen in M.tb isolated from patients with TB in India, mutations outside the RRDR, disputed mutations, and resistance-conferring mutations to newer drugs such as bedaquiline and linezolid.
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Antituberculosos , RNA Polimerases Dirigidas por DNA , Farmacorresistência Bacteriana Múltipla , Tuberculose Extensivamente Resistente a Medicamentos , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis , Oxirredutases , Tuberculose Resistente a Múltiplos Medicamentos , Sequenciamento Completo do Genoma , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Índia/epidemiologia , Masculino , Feminino , Adulto , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Pessoa de Meia-Idade , Farmacorresistência Bacteriana Múltipla/genética , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Proteínas de Bactérias/genética , Adulto Jovem , Adolescente , Idoso , Rifampina/farmacologia , Rifampina/uso terapêuticoRESUMO
Robust efforts are essential to sustain and increase the advancements made in battling TB, as well as to tackle persistent issues that have caused the fight against the disease to be uneven. The End TB Strategy proposes that new technologies are to be developed by 2025 to encourage a quick growth in TB occurrence diminishment. This calls for a cross-sectoral focus on creating and distributing suitable medical and programmatic modernizations in a fair manner. However, many difficulties and differences still exist in the realms of research and development regarding vaccines, drugs, technical advances, and services related to TB. Therefore, priority needs to be given to overcoming these difficulties and discrepancies for a better future. On World TB Day 2023, SEAR Union, TB Alliance, the National Institute of Advanced Studies (NIAS) and Open Source Pharma Foundation (OSPF) gathered to discuss an important topic under the heading: "YES, WE HAVE THE POWER TO END TB!" With a commitment to putting the patient first and increasing their collective efforts, the organizations recognized that it is possible to make this goal a reality. The organizations involved in the discussion have declared their commitment to engaging in collaborative efforts to end TB globally. They advocate for strengthening access to TB services, controlling and preventing TB, improving surveillance and drug resistance management, and investing in research and development. Furthermore, they recognize the importance of reducing stigma and integrating patient voices in this endeavour. This Round Table serves as a framework to build on and ensure that the goal of ending TB is achievable.
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Tuberculose , Humanos , Tuberculose/epidemiologia , Tuberculose/prevenção & controleRESUMO
SARS-CoV-2 polymerase chain reaction (PCR) tests generally report only binary (positive or negative) outcomes. Quantitative PCR tests can provide epidemiological information on viral transmission patterns in populations. SARS-CoV-2 transmission patterns during India's SARS-CoV-2 viral waves remain largely undocumented. We analyzed 2.7 million real-time PCR testing records collected in Mumbai, a bellwether for other Indian cities. We used the inverse of cycle threshold (Ct) values to determine the community-level viral load. We quantified wave-specific differences by age, sex, and slum population density. Overall, PCR positivity was 3.4% during non-outbreak periods, rising to 23.2% and 42.8% during the original (June-November 2020) and Omicron waves (January 2022), respectively, but was a surprisingly low 9.9% during the Delta wave (March-June 2021; which had the largest increase in COVID deaths). The community-level median Ct values fell and rose ~7-14 days prior to PCR positivity rates. Viral loads were four-fold higher during the Delta and Omicron waves than during non-outbreak months. The Delta wave had high viral loads at older ages, in women, and in areas of higher slum density. During the Omicron wave, differences in viral load by sex and slum density had disappeared, but older adults continued to show a higher viral load. Mumbai's viral waves had markedly high viral loads representing an early signal of the pandemic trajectory. Ct values are practicable monitoring tools.
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Tuberculosis (TB) is a preventable, treatable, and curable disease. However, in 2020, 9â9 million people were estimated to have developed tuberculosis, and 1.5 million people were estimated to have died from it. Whereas in India, 2.6 million were diagnosed with TB and 436,000 succumbed to TB in 2019. India (26%) is the major contributor to the global drop in TB cases. The COVID-19 pandemic has substantially reduced access to services for the diagnosis and treatment of TB, resulting in an increase in deaths and a reversal in global progress. [1] Presently, TB incidence is falling at a rate of 2% per year, obstructed mainly by the rearing pandemic of drug-resistant tuberculosis (DRTB). Particularly concerning is multi-drug resistant TB (MDRTB), defined as resistance towards isoniazid (INH) and rifampicin (RIF). [2] The World Health Organization (WHO) targeted to reduce worldwide TB incidence by 90% until 2035. (1) Early initiation of effective treatment based on susceptibility patterns of the Mycobacterium tuberculosis complex (MTBC) is considered key to successful TB control in countries with high DRTB incidence. Worldwide MDRTB treatment outcomes are poor, with cure rates less than 60% (2) due to the lack of comprehensive Drug Susceptibility Testing (DST) in most high MDRTB burden countries. This is leading to the inadequate anti-TB activity of the provided regimens (3-5), unlike regimens advised for DST assure optimal results. (6) In addition to resistances to the established regimens, the resistance to the newer DRTB drugs is increasing. On World TB Day 2022, Academy of Advanced Medical Education, Thyrocare Technologies Limited and HyastackAnalytics - IITB along with expert pulmonologist and renowned physicians from India convened for an advisory board meeting in Delhi on 20th March 2022 to discuss the role of Whole Genome Sequencing (WGS) in the diagnosis and management of TB. Objectives and specific topics relating to WGS in MDRTB were discussed, each expert shared their views, which led to a group discussion with a commitment to putting the patient first, and increasing their collective efforts, the organizations recognized that it is possible to make this goal a reality. The organizations involved in the discussion have declared their commitment to engaging in collaborative efforts to tackle DRTB detection efficiently. They advocate for strengthening access to WGS TB services, controlling and preventing TB, improving surveillance and drug resistance management, and investing in research and development. This Round Table serves as a framework to build on and ensure that the goal of ending TB is achievable with WGS services wherever needed. Post discussion, a uniform consensus was said to be arrived if more than 80% board members agreed to the statement. The present paper is the outcome of aspects presented and discussed in the advisory board meeting.
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Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Tuberculose , Humanos , Antituberculosos/uso terapêutico , Antituberculosos/farmacologia , Testes de Sensibilidade Microbiana , Pandemias , Mycobacterium tuberculosis/genética , Tuberculose/diagnóstico , Tuberculose/tratamento farmacológico , Tuberculose/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Genômica , Sequenciamento Completo do GenomaAssuntos
Antituberculosos/uso terapêutico , Farmacorresistência Bacteriana , Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Pulmonar/tratamento farmacológico , Estudos de Coortes , Controle de Doenças Transmissíveis , Genótipo , Humanos , Índia , Infectologia/métodos , Linezolida/uso terapêutico , Testes de Sensibilidade Microbiana , Valor Preditivo dos Testes , RifampinaRESUMO
Introduction: The mechanistic details of first line drug (FLD) resistance have been thoroughly explored but the genetic resistance mechanisms of second line injectables, which form the backbone of the combinatorial drug resistant tuberculosis therapy, are partially identified. This study aims to highlight the genetic and spoligotypic differences in the second line drug (SLD) resistant and sensitive Mycobacterium tuberculosis (Mtb) clinical isolates from Mumbai (Western India) and Lucknow (Northern India). Methods: The rrs, eis, whiB7, tlyA, gyrA and gyrB target loci were screened in 126 isolates and spoligotyped. Results: The novel mutations were observed in whiB7 loci (A43T, C44A, C47A, G48T, G59A and T152G in 5'-UTR; A42C, C253T and T270G in gene), tlyA (+CG200, G165A, C415G, and +G543) and gyrB (+G1359 and +A1429). Altogether, the rrs, eis, and whiB7 loci harbored mutations in ~86% and ~47% kanamycin resistant isolates from Mumbai and Lucknow, respectively. Mumbai strains displayed higher prevalence of mutations in gyrA (~85%) and gyrB loci (~13%) as compared to those from Lucknow (~69% and ~3.0%, respectively). Further, spoligotyping revealed that Beijing lineage is distributed equally amongst the drug resistant strains of Mumbai and Lucknow, but EAI-5 is existed at a higher level only in Mumbai. The lineages Manu2, CAS1-Delhi and T1 are more prevalent in Lucknow. Conclusion: Besides identifying novel mutations in whiB7, tlyA and gyrB target loci, our analyses unveiled a potential polymorphic and phylogeographical demarcation among two distinct regions.
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INTRODUCTION: cytomegalovirus (CMV) infection has been reported to be associated with onset/exacerbation of systemic lupus erythematosus (SLE). In an attempt to verify this, we studied CMV infection in SLE patients. METHODS: forty-two SLE patients were studied at 3-time points; disease onset/flare, at peak of immunosuppression (at 6 weeks) and at low doses of immunosuppression (at 6 months). We studied healthy blood donors as controls, only once. Clinical assessment and SLE Disease Activity Index scoring were done at each visit. RT-PCR and ELISA were performed to detect CMV viral-load and anti-CMV antibodies (Ab) respectively. RESULTS: nine of 106 patients had detectable viral-load (145-50,000 copies/ml). Of these nine, three patients had significant viral-load, 6 patients had low viral-loads of doubtful clinical significance. None of the patients developed CMV disease. Six of 42 cases were positive for IgM Abs. All controls were negative for CMV DNA as well as CMV IgM Abs. All samples from patients and controls were positive for CMV IgG Ab indicating widespread prevalence. CONCLUSION: significantly, a higher seroprevalence of CMV IgM Abs against CMV observed in SLE patients when compared to controls, indicating possible reactivation due to immune modulation.
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Infecções por Citomegalovirus/epidemiologia , Imunossupressores/administração & dosagem , Lúpus Eritematoso Sistêmico/complicações , Adulto , Anticorpos Antivirais/sangue , Estudos de Casos e Controles , Infecções por Citomegalovirus/diagnóstico , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/imunologia , Índia , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estudos Soroepidemiológicos , Fatores de Tempo , Carga Viral , Adulto JovemRESUMO
Over the past decades, drug-resistant Mycobacterium tuberculosis strains have presented a significant challenge, with inadequate diagnosis of tuberculosis (TB) cases. Here, we report the draft whole-genome sequence of drug-resistant M. tuberculosis strain S7, which was isolated from a patient from Tripura, India, who was diagnosed with pulmonary TB.
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Antituberculosos/farmacologia , Fluoroquinolonas/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Estudos de Coortes , Humanos , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/estatística & dados numéricosRESUMO
To prevent the spread of carbapenemases-producing Enterobacteriaceae (CPE) active surveillance, contact isolation and cohorting infected patients should be practiced. Rectal swabs for the Xpert MDRO-assay of 32 patients were included. 71.85% were positive for targets incorporated into the MDRO-assay; whereas 28% were phenotypically not CRE and Xpert negative (9.37% had different mechanism [bla OXA]). The assay identified 59.3%, 9.37% and 3.1% as bla NDM, bla NDM+VIM and bla VIM, respectively. The assay is a screening test that identifies CPE harbouring organism within an hour and can be installed at tertiary-care facilities to screen colonized patients.
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Proteínas de Bactérias/análise , Técnicas Bacteriológicas/métodos , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/enzimologia , Reação em Cadeia da Polimerase Multiplex/métodos , beta-Lactamases/análise , Proteínas de Bactérias/genética , Enterobacteriaceae/isolamento & purificação , Humanos , Programas de Rastreamento/métodos , Reto/microbiologia , beta-Lactamases/genéticaRESUMO
OBJECTIVE/BACKGROUND: The in vitro drug-susceptibility testing of Mycobacterium tuberculosis reports isolates as resistant or susceptible on the basis of single critical concentrations. It is evident that drug resistance in M. tuberculosis is quite heterogeneous, and involves low level, moderate level, and high level of drug-resistant phenotypes. Thus, the aim of our study was to correlate rrs (X52917) and eis (AF144099) promoter mutations, found in M. tuberculosis isolates, with corresponding minimum inhibitory concentrations of amikacin, kanamycin, and capreomycin. METHODS: Ninety M. tuberculosis clinical isolates were analyzed in this study. The minimum inhibitory concentrations were determined by MGIT 960 for 59 isolates with resistance-associated mutations in the rrs and eis promoter gene regions, and 31 isolates with wild-type sequences, as determined by the GenoType MTBDRsl (version 1) assay. RESULTS: The rrs A1401G mutation was identified in 48 isolates resistant to the second-line injectables. The eis promoter mutations C-14T (n=3), G-10C (n=3), G-10A (n=3), and C-12T (n=2) were found within 11 isolates with various resistance profiles to the second-line injectables. Thirty-one isolates had wild-type sequences for the rrs and eis promoter gene regions of interest, one of which was amikacin, kanamycin, and capreomycin resistant. The isolates with the rrs A1401G mutation had amikacin, kanamycin, and capreomycin minimum inhibitory concentrations of >40mg/L, >20mg/L, and 5-15mg/L, respectively. The isolates with eis promoter mutations had amikacin, kanamycin, and capreomycin minimum inhibitory concentrations of 0.25-1.0mg/L, 0.625-10mg/L, and 0.625-2.5mg/L, respectively. CONCLUSION: This study provides a preliminary basis for the prediction of phenotypic-resistance levels to the second-line injectables based upon the presence of genetic mutations associated with amikacin, kanamycin, and capreomycin resistance. The results suggest that isolates with eis promoter mutations have consistently lower resistance levels to amikacin, kanamycin, and capreomycin than isolates with the rrs A1401G mutation.
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Antibacterianos/farmacologia , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Regiões Promotoras Genéticas , RNA Ribossômico 16S/genética , Acetiltransferases , Amicacina/farmacologia , Capreomicina/farmacologia , Genes de RNAr , Genótipo , Humanos , Canamicina/farmacologia , Testes de Sensibilidade Microbiana , Mutação , Fenótipo , Análise de Sequência de DNA , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaAssuntos
COVID-19/virologia , SARS-CoV-2/genética , COVID-19/transmissão , Doenças Transmissíveis Importadas/transmissão , Doenças Transmissíveis Importadas/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Índia , Mutação , Saúde Pública , SARS-CoV-2/isolamento & purificação , África do Sul , TanzâniaRESUMO
This study correlates MICs of rifampicin (RIF) and isoniazid (INH) with GenoType MTBDRplus assay results for drug-resistant Mycobacterium tuberculosis (MTB) clinical isolates. MICs of RIF and INH were established for 84 and 90 isolates, respectively, testing 7 concentrations of each drug. Genotypic resistance to each drug was determined by GenoType MTBDRplus assay with 50 representative mutations confirmed by pyrosequencing, with mutations in the rpoB gene associated with RIF resistance and mutations in the katG and/or inhA genes associated with INH resistance. Based upon the correlation of MICs with specific genetic profiles, relative resistance levels were established for each isolate. Results indicate that MTB phenotypic resistance, currently based upon the testing of isolate susceptibility to a single drug concentration, may be more accurately profiled via quantitative MICs, and therefore, the correlation of molecular diagnostic results with specific MICs may allow for more optimal treatment of infections.
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Antituberculosos/farmacologia , Farmacorresistência Bacteriana Múltipla , Técnicas de Genotipagem/métodos , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Proteínas de Bactérias/genética , Catalase/genética , RNA Polimerases Dirigidas por DNA/genética , Genótipo , Humanos , Isoniazida/farmacologia , Mutação , Mycobacterium tuberculosis/isolamento & purificação , Oxirredutases/genética , Fenótipo , Rifampina/farmacologiaRESUMO
OBJECTIVE: To correlate gyrA mutations found on the Genotype MTBDRsl assay in Mycobacterium tuberculosis (MTB) isolates with Minimum Inhibitory Concentrations (MICs) to the fluoroquinolones compounds ofloxacin (OFX) and moxifloxacin (MXF). METHODS: MICs for OFX and MXF were ascertained for 93 archived clinical MTB isolates that showed gyrA mutations at Ala90Val, Ser91Pro, Asp94Ala, Asn/Tyr, Gly and His. Thirty fluoroquinolones susceptible isolates as determined by presence of all wild-type gyrA bands on the Genotype MTBDRsl assay were also included. RESULTS: gyrA mutations at Ala90Val (n = 25), Ser91Pro (n = 6), Asp94Ala (n = 4), Asp94Asn/Tyr (n = 13), Asp94Gly (n = 42) and Asp94His (n = 3) were observed. Isolates with mutations at Ala90Val or Ser91Pro had MIC90 of 4.0 µg/ml and 1.0 µg/ml for OFX and MXF, respectively, and isolates with mutations at Asp 94Ala, Asn/Tyr, Gly and His had MIC90 of 8.0 µg/ml, and 2.5 µg/ml for OFX and MXF, respectively. CONCLUSIONS: MTB MICs were found to be consistently lower for MXF than for OFX among isolates with the same gyrA mutation (e.g. Ala90Val). The majority of MTB isolates containing mutations at Asp94Ala, Asn/Tyr, Gly and His in gyrA were associated with a moderate level of resistance to MXF (MIC = 2.5 µg/ml), although 3 isolates with the mutations Asp94Asn/Tyr/Gly were associated with a high level of resistance to both fluoroquinolones (MXF MICs = 5.0-8.0 µg/ml, OFX MICs = ≥10.0 µg/ml).
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Antituberculosos/farmacologia , DNA Girase/genética , Fluoroquinolonas/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Ofloxacino/farmacologia , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana/genética , Farmacorresistência Bacteriana Múltipla/genética , Fluoroquinolonas/administração & dosagem , Genótipo , Humanos , Testes de Sensibilidade Microbiana/métodos , Moxifloxacina , Mutação , Mycobacterium tuberculosis/genética , Ofloxacino/administração & dosagemRESUMO
To evaluate the performance of TrueNAT (RT Micro PCR device) assay in comparison with GeneXpert on sputum samples from pulmonary cases of tuberculosis. 274 samples were processed to detect MTB by ZN smear examination, MGIT culture and molecular methods that included RT-PCR (ABI 7500 & TrueNAT) and GeneXpert for case detection of TB. The overall performance of the test with MGIT(Mycobacterium Growth Indicator Tube) culture as gold standard, sensitivity of smear, RT PCR/TrueNAT and Genexpert was 61.5% (CI:53.3-69.3%), 94.7% (CI:89.8-97.6%) & 96.0% (CI: 91.5-98.5%), respectively. Amongst the S+ (108) samples, RT-PCR/TrueNAT and GeneXpert showed a sensitivity of 99% (CI:94.9%-99.8%) and 100% (98.6%-100.0%), respectively. High concordance was observed between GeneXpert and TrueNAT for case detection of TB. The GeneXpert MTB/RIF test was independent on the user's skills. It has a short turn-around time and simultaneously detects RIF resistance with M. tuberculosis in less than 3h. The TrueNAT MTB has good sensitivity and specificity in case detection with hands on time of less than 3h as well as fits the requirements in resourcelimited health care settings. Larger, multi-site studies are required to obtain better estimates of the performance of TrueNAT MTB.
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BACKGROUND: Control of the global Tuberculosis (TB) burden is hindered by the lack of a simple and effective diagnostic test that can be utilized in resource-limited settings. METHODS: We evaluated the performance of Truenat MTB™, a chip-based nucleic acid amplification test in the detection of Mycobacterium tuberculosis (MTB) in clinical sputum specimens from 226 patients with suspected pulmonary tuberculosis (TB). The test involved sputum processing using Trueprep-MAG™ (nanoparticle-based protocol run on a battery-operated device) and real-time PCR performed on the Truelab Uno™ analyzer (handheld, battery-operated thermal cycler). Specimens were also examined for presence of MTB using smear microscopy, liquid culture and an in-house nested PCR protocol. Results were assessed in comparison to a composite reference standard (CRS) consisting of smear and culture results, clinical treatment and follow-up, and radiology findings. RESULTS: Based on the CRS, 191 patients had "Clinical-TB" (Definite and Probable-TB). Of which 154 patients are already on treatment, and 37 were treatment naïve cases. Remaining 35 were confirmed "Non-TB" cases which are treatment naïve cases. The Truenat MTB test was found to have sensitivity and specificity of 91.1% (CI: 86.1-94.7) and 100% (CI: 90.0-100) respectively, in comparison to 90.58% (CI: 85.5-94.3) and 91.43% (CI: 76.9-98.2) respectively for the in-house nested PCR protocol. CONCLUSION: This preliminary study shows that the Truenat MTB test allows detection of TB in approximately one hour and can be utilized in near-care settings to provide quick and accurate diagnosis.
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Técnicas e Procedimentos Diagnósticos/instrumentação , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium tuberculosis/fisiologia , Sistemas Automatizados de Assistência Junto ao Leito , Tuberculose Pulmonar/diagnóstico , Humanos , Mycobacterium tuberculosis/genética , Nanopartículas , Reação em Cadeia da Polimerase em Tempo Real , Escarro/microbiologia , Fatores de TempoRESUMO
BACKGROUND: The emergence of resistant tuberculosis (TB) is a major setback to the global control of the disease as the treatment of such resistance is complex and expensive. Use of direct detection of mutations by molecular methods could facilitate rapid diagnosis of resistance to offset diagnostic delays. We evaluated the performance of the Genotype MTBDRsl (Hain Life Sciences) for the detection of second line resistant TB directly from stored smear positive sputum sediments. METHODOLOGY/PRINCIPAL FINDINGS: The assay showed a diverse range of sensitivity and specificity, 91.26% [95% CI, 84-96] and 95.5% [95% CI, 87-99] for FQ (PPV â¼97% & NPV â¼ 87.67%), 56.19% [95%CI, 46-66] and 81% [95%CI, 66-91] for EMB (PPV â¼ 88.06% & NPV â¼ 43.21%) and 100% for SLD. Diagnostic accuracy for FQ, SLD and EMB was 94%, 100% and 63.51%, respectively. 1.17% (2/170) were heteroresistance strains, where the heteroresistance was linked to rrs gene. A varying rate of validity was observed 100% (170/170) for FQ, 94.11% (160/170) for EMB, 88.23% (150/170) for SLD. CONCLUSIONS/SIGNIFICANCE: Genotype MTBDRsl is simple, rapid, economical assay that can be used to detect commonly known resistance associated with Fluoroquinolone, second line injectable drugs and ethambutol. The assay detects the targeted resistance in less time as compared to phenotypic DST. But due to low NPV to FQ (88%) and EMB (43.21%), the assay results must be interpreted in coordination with the phenotypic DST.