RESUMO
Drug-resistance due to AmpC ß-lactamases represents a growing problem worldwide. In this study, a previously collected sample of 108 cefoxitin-resistant clinical isolates was assessed for AmpC ß-lactamase production through routine phenotypic testing and double-disc cefoxitin/cloxcallin (DD-CC), cefoxitin/phenylboronic acid (CDT-PBA) and AmpC disc tests. The same isolates were characterized by a novel multiplex polymerase chain reaction molecular assay to detect the presence of blaACT, blaDHA, blaCIT, blaFOX, blaMIR and blaMOX. By phenotypic analysis, 56%, 55% and 48â% were detected as being AmpC ß-lactamase producers by the CDT-PBA, DD-CC and AmpC disc tests, respectively. By molecular analysis, 57ââ% were determined to be AmpC ß-lactamase producers, including 34â% blaFOX, 8â% blaCIT and 1.6â% blaDHAas mono-AmpC producers. The production of multiple AmpC molecular types was common, including 30â% with both blaCIT+FOX and 1.6â% each of blaCIT+DHA, blaACT+MIR, blaACT+FOX, blaACT+DHA and blaMIR+FOX. Molecular characterization of AmpC would help detect the prevalence of AmpC ß-lactamase producers, facilitate proper patient management and implement infection control practices.
Assuntos
Proteínas de Bactérias/genética , Enterobacteriaceae/genética , Reação em Cadeia da Polimerase Multiplex/métodos , beta-Lactamases/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Cefoxitina/farmacologia , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/isolamento & purificação , Enterobacteriaceae/metabolismo , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Genótipo , Humanos , Índia/epidemiologia , Testes de Sensibilidade Microbiana , Fenótipo , Sensibilidade e Especificidade , Atenção Terciária à Saúde , Resistência beta-Lactâmica/efeitos dos fármacos , Resistência beta-Lactâmica/genética , beta-Lactamases/biossínteseRESUMO
Reliable molecular diagnostics, which detect specific mutations associated with drug resistance, are promising technologies for the rapid identification and monitoring of drug resistance in Mycobacterium tuberculosis isolates. Pyrosequencing (PSQ) has the ability to detect mutations associated with first- and second-line anti-tuberculosis (TB) drugs, with the additional advantage of being rapidly adaptable for the identification of new mutations. The aim of this project was to evaluate the performance of PSQ in predicting phenotypic drug resistance in multidrug- and extensively drug-resistant tuberculosis (M/XDR-TB) clinical isolates from India, South Africa, Moldova, and the Philippines. A total of 187 archived isolates were run through a PSQ assay in order to identify M. tuberculosis (via the IS6110 marker), and to detect mutations associated with M/XDR-TB within small stretches of nucleotides in selected loci. The molecular targets included katG, the inhA promoter and the ahpC-oxyR intergenic region for isoniazid (INH) resistance; the rpoB core region for rifampin (RIF) resistance; gyrA for fluoroquinolone (FQ) resistance; and rrs for amikacin (AMK), capreomycin (CAP), and kanamycin (KAN) resistance. PSQ data were compared to phenotypic mycobacterial growth indicator tube (MGIT) 960 drug susceptibility testing results for performance analysis. The PSQ assay illustrated good sensitivity for the detection of resistance to INH (94%), RIF (96%), FQ (93%), AMK (84%), CAP (88%), and KAN (68%). The specificities of the assay were 96% for INH, 100% for RIF, FQ, AMK, and KAN, and 97% for CAP. PSQ is a highly efficient diagnostic tool that reveals specific nucleotide changes associated with resistance to the first- and second-line anti-TB drug medications. This methodology has the potential to be linked to mutation-specific clinical interpretation algorithms for rapid treatment decisions.
Assuntos
Antituberculosos/uso terapêutico , Farmacorresistência Bacteriana Múltipla/genética , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Proteínas de Bactérias/genética , Sequência de Bases , Catalase/genética , DNA Girase/genética , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Humanos , Isoniazida/uso terapêutico , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Oxirredutases/genética , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNARESUMO
PURPOSE: Tuberculous meningitis (TBM) is the most severe form of tuberculosis (TB). Difficulty in diagnosing the condition along with other factors, increases its potential for high morbidity and mortality. Targeted Next Generation Sequencing (tNGS) generates high quality sequence read depths, enabling the identification of low-frequency alleles linked to Drug resistance (DR). The paucibacillary nature of tuberculous meningitis is a challenge for making a definitive diagnosis. METHODS: tNGS was performed on 20 cerebrospinal fluid (CSF) samples where, MGIT has shown Positive MTB Cultures. We simultaneously performed pyrosequencing (PSQ) and phenotypic Drug susceptibility testing (pDST) for these 20 samples. RESULTS: Sequencing results (from tNGS and PSQ) were compared with reference standards i.e. pDST. tNGS detected MTB in 7/20 (35%) CSF samples whereas, PSQ detected MTB in 17/20 (85%). CONCLUSION: Although tNGS has ability to detect minority variants along with detection of additional targets than PSQ, PSQ remains the diagnostic choice in our tertiary lab.
RESUMO
We screened and spoligotyped 150 consecutive phenotypically confirmed extensively drug-resistant Mycobacterium tuberculosis (XDR-TB) isolates (January 2008 to March 2009) for rifampin, isoniazid, fluoroquinolone, and aminoglycoside resistance targeting rpoB, inhA, katG, gyrA, gyrB, and rrs. Mutations predominant among XDR-TB were S315T (katG) (100% of isolates), S531L (rpoB) (97% of isolates), D94G (gyrA) (53% of isolates), and A1401G (rrs) (71% of isolates). Spoligotyping revealed 62% of the isolates to be Beijing.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana Múltipla , Tuberculose Extensivamente Resistente a Medicamentos/diagnóstico , Mutação de Sentido Incorreto , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Análise por Conglomerados , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Genótipo , Humanos , Índia , Tipagem Molecular , Mycobacterium tuberculosis/isolamento & purificaçãoRESUMO
Drug resistance in tuberculosis (TB) is a matter of grave concern for TB control programs, as there is currently no cure for some extensively drug-resistant (XDR) strains. There is concern that this resistance could transmit, stressing the need for additional control measures, rapid diagnostic methods, and newer drugs for treatment. We developed an in-house assay that can rapidly detect resistance to drugs involved in the definition of XDR-TB directly from smear-positive specimens. Two hundred fifteen phenotypically XDR-TB isolates and 50 pansusceptible isolates were analyzed using a reverse line blot hybridization (RLBH) assay. The assay was also successfully applied to 73 smear-positive clinical specimens. The RLBH assay exhibited good sensitivity for the detection of resistance to isoniazid (99%), rifampin (99%), fluoroquinolones (95.3%), and second-line aminoglycosides (94.8%). The results from application of this assay on direct smear-positive clinical specimens revealed 93% concordance with the phenotypic drug susceptibility test (DST) results for the above-mentioned drugs. The time to accurate DST results was significantly reduced from weeks to 3 days. This molecular assay is a highly accurate tool for screening for XDR-TB, which achieves a substantial reduction in diagnostic delays.
Assuntos
Técnicas de Laboratório Clínico/métodos , Farmacorresistência Bacteriana Múltipla , Tuberculose Extensivamente Resistente a Medicamentos/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Antituberculosos/farmacologia , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Humanos , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/efeitos dos fármacos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: We evaluated the performance of pyrosequencing, a genotypic test which detects TB and XDR-defining mutations within 6 h, directly on CSF samples for diagnosing TB meningitis(TBM). METHODS: This retrospective, diagnostic accuracy study was conducted in Hinduja hospital, Mumbai from May-2017 to May-2019. 107 consecutive patients with physician-suspected TBM for whom CSF pyrosequencing was requested were screened. Seven patients with incomplete data were excluded. Diagnostic accuracy of pyrosequencing was compared with Xpert MTB/Rif and TBMGIT (TB Mycobacterial Growth Indicator Tube) culture against the uniform case definition of definite or probable TBM. Susceptibility concordance rate of pyrosequencing with TBMGIT culture and Xpert MTB/Rif was determined. RESULTS: The study cohort comprised of 100 patients[Definite(n = 33), Probable(n = 20), Possible(n = 30), Alternative(n = 17)] with 50% males[median age(years):38(Range:2-87)]. Against the uniform case definition, pyrosequencing had 98·11%(95%CI 89·93-99·95; n = 52/53) sensitivity and 97·79%(86·31-99·67; n = 44/45) negative predictive value(NPV) compared with 43.39%(29·83-57·72; n = 23/53,p < 0.0001) sensitivity and 61.04%(55·31-66·48; n = 47/77) NPV for Xpert MTB/Rif and 45·28%(31·56-59·55; n = 24/53,p < 0.0001) sensitivity and 61·84%(55·92-67·43; n = 47/76) NPV for TBMGIT culture. Susceptibility concordance rate of pyrosequencing with phenotypic Drug Susceptibility Testing was 91.3%(n = 21/23) and with Xpert MTB/Rif was 95·45%(n = 21/22). CONCLUSION: CSF pyrosequencing is significantly more sensitive than Xpert MTB/Rif and TBMGIT culture for diagnosing TBM. Additionally, it facilitates early therapeutic decision-making by providing information on XDR-defining mutations.
Assuntos
Líquido Cefalorraquidiano/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Meníngea/líquido cefalorraquidiano , Tuberculose Resistente a Múltiplos Medicamentos/líquido cefalorraquidiano , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Líquido Cefalorraquidiano/microbiologia , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Reprodutibilidade dos Testes , Estudos Retrospectivos , Tuberculose Meníngea/diagnóstico , Tuberculose Meníngea/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Adulto JovemRESUMO
BACKGROUND: Timely drug resistance detection is essential to global tuberculosis management. Unfortunately, rapid molecular tests assess resistance to only a few drugs, with culture required for comprehensive susceptibility test results. METHODS: We evaluated targeted next generation sequencing (tNGS) for tuberculosis on 40 uncultured sputum samples. Resistance profiles from tNGS were compared with profiles from Xpert MTB/RIF, line probe assay (LPA), pyrosequencing (PSQ), and phenotypic testing. Concordance, sensitivity, specificity, and overall test agreement were compared across assays. RESULTS: tNGS provided results for 39 of 40 samples (97.5%) with faster turnaround than phenotypic testing (median 3 vs. 21 days, p = 0.0068). Most samples were isoniazid and rifampin resistant (N = 31, 79.5%), 21 (53.8%) were fluoroquinolone resistant, and 3 (7.7%) were also resistant to Kanamycin. Half were of the Beijing lineage (N = 20, 51.3%). tNGS from uncultured sputum identified all resistance to isoniazid, rifampin, fluoroquinolones, and second-line injectable drugs that was identified by other methods. Agreement between tNGS and existing assays was excellent for isoniazid, rifampin, and SLDs, very good for levofloxacin, and good for moxifloxacin. CONCLUSION: tNGS can rapidly identify tuberculosis, lineage, and drug resistance with faster turnaround than phenotypic testing. tNGS is a potential alternative to phenotypic testing in high-burden settings.
Assuntos
Técnicas Bacteriológicas , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Sequenciamento de Nucleotídeos em Larga Escala , Pulmão/microbiologia , Mycobacterium tuberculosis/genética , Tuberculose Pulmonar/microbiologia , Adolescente , Adulto , Antituberculosos/uso terapêutico , Estudos de Viabilidade , Feminino , Genótipo , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Fenótipo , Valor Preditivo dos Testes , Escarro/microbiologia , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/tratamento farmacológico , Adulto JovemRESUMO
BACKGROUND: In the era of increasing drug resistance in pulmonary tuberculosis (TB), it is prudent to assess causes of poor response to anti tubercular therapy (ATT) and drug sensitivity pattern (DSP) in osteoarticular TB. MATERIALS AND METHODS: As a part of Bombay Orthopaedic society's research project, members were asked to refer non responders to ATT to our institute. Cases were enrolled from October 2010 to March 2014. Deep tissue samples were obtained in all but five cases and subjected to a battery of tests including histopathology (HPE) and TB culture and sensitivity. The DSP was compared with the study performed by the principle author from 2004 to 2007 and published in 2009. RESULTS: 39 male and 50 female patients with a mean age of 24.85 years (2-66) were included and classified in four groups after results. (1) Culture and HPE positive-36. 24 had MDR and three XDR TB. Primary resistance to even second line drugs and deterioration of DSP since last study was noted, (2) culture negative and HPE positive-21. The cause of poor response was surgical in more than half cases, (3) non representative samples or lost to follow-up-15, (4) TB mimics-16. CONCLUSION: There is increasing incidence of primary resistance to second line drugs, primary resistance in children and worsening of resistance patterns as compared to older studies. ATT initiation is a fateful decision and every attempt should be made to rule out TB mimics and establish DSP before initiation.
Assuntos
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
The development and rollout of the Xpert® Mycobacterium tuberculosis/rifampicin assay for the GeneXpert platform is considered an important breakthrough in the fight against tuberculosis. Xpert though robust is known to have issues that occur with very low load of tuberculosis detection, wherein it is recommended to confirm resistance if resistance is not suspected using another genotypic test.
RESUMO
Tubercular meningitis (TBM) is a serious form of tuberculosis (TB). The diagnosis of TBM & susceptibility/resistance is difficult as TB MGIT culture lacks sensitivity & timeliness. Timely and accurate diagnosis of the TBM is the need of the hour for initiation of appropriate therapy. We have exploited pyrosequencing to detect TB and associated Multi/extensively drug resistant (MDR/ XDR-TB) directly from Cerebrospinal Fluid (CSF) specimens.
Assuntos
Técnicas Bacteriológicas , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/genética , Tuberculose Extensivamente Resistente a Medicamentos/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala , Mycobacterium tuberculosis/genética , Análise de Sequência de DNA/métodos , Tuberculose Meníngea/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Adolescente , Adulto , Antituberculosos/uso terapêutico , Líquido Cefalorraquidiano/microbiologia , DNA Bacteriano/líquido cefalorraquidiano , Tuberculose Extensivamente Resistente a Medicamentos/líquido cefalorraquidiano , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Fenótipo , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Fatores de Tempo , Tuberculose Meníngea/líquido cefalorraquidiano , Tuberculose Meníngea/tratamento farmacológico , Tuberculose Meníngea/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/líquido cefalorraquidiano , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Fluxo de Trabalho , Adulto JovemRESUMO
Delayed diagnosis of drug resistance has been a major obstacle to proper management and control of drug-resistant tuberculosis (TB). Expanded access to rapid molecular diagnostics such as Xpert MTB/RIF has been helpful, but has generated confusion about how to interpret genotype-phenotype discordance. Optimal management is not clearly defined for patients with rifampin resistance by Xpert MTB/RIF but rifampin susceptibility by phenotypic testing. To resolve this discrepancy, we performed pyrosequencing of discordant isolates identified at a reference laboratory over a 6-month period. We present here strategies to address genotype-phenotype discordance using sequencing.
RESUMO
MGIT 960 drug susceptibility testing (DST) for Mycobacterium tuberculosis was compared for performance and speed with pyrosequencing (PSQ). Pulmonary samples (nâ¯=â¯100), from GeneXpert/MTB/Rifampicin-resistant patients receiving second-line treatment for 1-3 months, were subjected to DST and PSQ for seven drugs (isoniazid, rifampicin, kanamycin, amikacin, capreomycin, moxifloxacin, and ofloxacin). The mean time-to-result was 35 and two days for DST and PSQ, respectively. Average concordancy was 92.7%. Theoretically, PSQ showed substantial incremental value over the commercial Genotype MTBDRplus/sl. Mutations not considered in commercial molecular tests were observed by PSQ. Our findings corroborated the association between S315T (katG region) and S531L (rpoB region) and phenotypic resistance. PSQ is more rapid, can be performed from the sample, provides information about all known mutations simultaneously, allows extensive post-processing analyses, and is open to the inclusion of new mutations. It indicates the exact mutation conferring resistance to the particular drug, unlike the qualitative DST.
Assuntos
Antituberculosos/farmacologia , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/genética , Estudos de Viabilidade , Genótipo , Humanos , Testes de Sensibilidade Microbiana/métodos , Mutação , Mycobacterium tuberculosis/genética , Fenótipo , Saúde Pública , Análise de Sequência de DNA/métodosAssuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana Múltipla , Tuberculose Extensivamente Resistente a Medicamentos/diagnóstico , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Adulto , Antituberculosos/administração & dosagem , DNA Bacteriano/genética , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Feminino , Genótipo , Humanos , Índia , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Adulto JovemRESUMO
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.
Assuntos
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/microbiologiaRESUMO
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).
Assuntos
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
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.
Assuntos
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
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.