The past, present and future of tuberculosis treatment / 浙江大学学报·医学版

Tuberculosis (TB) is an ancient infectious disease. Before the availability of effective drug therapy, it had high morbidity and mortality. In the past 100 years, the discovery of revolutionary anti-TB drugs such as streptomycin, isoniazid, pyrazinamide, ethambutol and rifampicin, along with drug combination treatment, has greatly improved TB control globally. As anti-TB drugs were widely used, multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis emerged due to acquired genetic mutations, and this now presents a major problem for effective treatment. Genes associated with drug resistance have been identified, including katG mutations in isoniazid resistance, rpoB mutations in rifampin resistance, pncA mutations in pyrazinamide resistance, and gyrA mutations in quinolone resistance. The major mechanisms of drug resistance include loss of enzyme activity in prodrug activation, drug target alteration, overexpression of drug target, and overexpression of the efflux pump. During the disease process, Mycobacterium tuberculosis may reside in different microenvironments where it is expose to acidic pH, low oxygen, reactive oxygen species and anti-TB drugs, which can facilitate the development of non-replicating persisters and promote bacterial survival. The mechanisms of persister formation may include toxin-antitoxin (TA) modules, DNA protection and repair, protein degradation such as trans-translation, efflux, and altered metabolism. In recent years, the use of new anti-TB drugs, repurposed drugs, and their drug combinations has greatly improved treatment outcomes in patients with both drug-susceptible TB and MDR/XDR-TB. The importance of developing more effective drugs targeting persisters of Mycobacterium tuberculosis is emphasized. In addition, host-directed therapeutics using both conventional drugs and herbal medicines for more effective TB treatment should also be explored. In this article, we review historical aspects of the research on anti-TB drugs and discuss the current understanding and treatments of drug resistant and persistent tuberculosis to inform future therapeutic development.

Identification of a predominant genotype of Mycobacterium tuberculosis in Brazilian indigenous population

After nearly a century of vaccination and six decades of drug therapy, tuberculosis (TB) kills more people annually than any other infectious disease. Substantial challenges to disease eradication remain among vulnerable and underserved populations. The Guarani-Kaiowá people are an indigenous population in Paraguay and the Brazilian state of Mato Grosso do Sul. This community, marginalized in Brazilian society, experiences severe poverty. Like other South American indigenous populations, their TB prevalence is high, but the disease has remained largely unstudied in their communities. Herein, Mycobacterium tuberculosis isolates from local clinics were whole genome sequenced, and a population genetic framework was generated. Phylogenetics show M. tuberculosis isolates in the Guarani-Kaiowá people cluster away from selected reference strains, suggesting divergence. Most cluster in a single group, further characterized as M. tuberculosis sublineage 4.3.3. Closer analysis of SNPs showed numerous variants across the genome, including in drug resistance-associated genes, and with many unique changes fixed in each group. We report that local M. tuberculosis strains have acquired unique polymorphisms in the Guarani-Kaiowá people, and drug resistance characterization is urgently needed to inform public health to ensure proper care and avoid further evolution and spread of drug-resistant TB

Frequency of first and second-line drug resistance-associated mutations among resistant Mycobacterium tuberculosis clinical isolates from São Paulo, Brazil

BACKGROUND Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis, and the number of new cases of multidrug resistant TB (MDR-TB), pre extensively drug-resistant TB (pre-XDR-TB) and extensively drug-resistant TB (XDR-TB) has increased considerably worldwide. OBJECTIVES Herein, using 156 M. tuberculosis isolates from 106 patients previously classified as MDR or pre-XDR or XDR isolates, we investigated the genetic mutation profiles associated with phenotypic resistances in patients with MDR-TB, pre-XDR-TB and XDR-TB, treatment outcomes and resistance evolution. METHODS Molecular analyses were performed by partial sequencing of the rpoB, katG, gyrA, gyrB, rrs genes and analysis of the fabG-inhA promoter region. Clinical, epidemiologic and demographic data were obtained from the TB Notification database system of São Paulo (TB-WEB) and the Information System for Special Tuberculosis Treatments (SITE-TB). FINDINGS Drug resistance was attributed to previously known mutations and a novel Asp449Val mutation in gyrB was observed in four isolates from the same patient. Ten patients had more than one isolate evaluated and eight of these patients displayed resistance progression. MAIN CONCLUSIONS The present study is the first to report the frequency of mutations related to second-line drug resistance in MDR-TB, pre-XDR-TB and XDR-TB isolates. The results could lead to the improvement of available technologies for the rapid detection of drug resistant TB.

Analysis of anti-tuberculosis drug resistance and sociodemographic and clinical aspects of patients admitted in a referral hospital / Análise da resistência a medicamentos antituberculose e aspectos sociodemográficos e clínicos de pacientes atendidos em hospital referência

ABSTRACT Objective To determine the occurrence of anti-tuberculosis drug resistance and its association with sociodemographic and clinical characteristics of patients in a referral hospital. Methods This was a cross-sectional study based on data from patients who had mycobacterial culture identified and defined antimicrobials sensitivity profile (June 2014 to February 2016). The descriptive statistical analysis and Fisher's exact test were used to compare proportions. Results The study included 104 patients who had positive results for Mycobacterium tuberculosis . Bacilloscopy had high positivity (93.3%). A total of 15 patients (14.4%) had resistant strains and six (5.6%) multidrug-resistant. The sociodemographic and clinical characteristics were not related with resistance. Conclusion This study contributed to further the understandings about the tuberculosis patients' profile, the study also served as a tool for development of specific public policies. Patients diagnosed with resistant tuberculosis must be under greater supervision.

RESUMO Objetivo Verificar a ocorrência de resistência a fármacos antituberculose e a associação com características sociodemográficas e clínicas de pacientes de um hospital referência. Métodos Estudo transversal, com dados de pacientes que tiveram a cultura de micobactérias identificada e o respectivo perfil de sensibilidade aos antimicrobianos definido (junho de 2014 a fevereiro de 2016). Foram realizados a análise estatística descritiva e o teste exato de Fisher, para comparação de proporções. Resultados O estudo envolveu 104 pacientes, e todos tiveram resultados para Mycobacterium tuberculosis . A baciloscopia atingiu alta positividade (93,3%), e 15 pacientes (14,4%) apresentaram linhagens resistentes, sendo 6 (5,6%) multirresistentes. As características sociodemográficas e clínicas não foram associadas à resistência. Conclusão A pesquisa permitiu conhecer melhor o perfil dos pacientes com tuberculose e constitui ferramenta para elaboração de políticas públicas específicas. Os pacientes diagnosticados com tuberculose resistente devem ser submetidos à maior supervisão.

Detection of Drug Resistant Mycobacterium Tuberculosis Strains Using Kit SIRE Nitratase®: a Multicenter Study

Abstract (1) Background: The Commercial Kit SIRE Nitratase® PlastLabor, is a drug susceptibility test kit used to detect Mycobacterium tuberculosis resistance to first-line TB treatment drugs. The present study aimed at evaluating its performance in a multicenter study. (2) Methods: To determine its accuracy, the proportion methods in Lowenstein Jensen medium or the BACTECTMMGITTM960 system was used as a gold standard. (3) Results: The study revealed that the respective accuracies of the kit with 190 M. tuberculosis clinical isolates, using the proportion methods in Lowenstein Jensen medium or BACTECTMMGITTM960 system as a gold standard, were 93.9% and 94.6%, 96.9% and 94.6%, 98.0% and 97.8%, and 98.0% and 98.9%, for streptomycin, isoniazid, rifampicin, and ethambutol, respectively. (4) Conclusion: Thus, the kit can rapidly screen resistance to streptomycin, isoniazid, rifampicin, and ethambutol. Additionally, it does not require sophisticated equipment; hence, it can be easily used in the laboratories of low and middle income countries.

Mutaciones que confieren resistencia a fármacos antituberculosis de primera línea en Perú: una revisión sistemática de la literatura / Mutations conferring resistance to first-line anti-tuberculosis drugs in Peru: a systematic review of the literature

RESUMEN Objetivos. Sistematizar la información disponible referente a las mutaciones que confieren resistencia a los fármacos antituberculosis de primera línea. Materiales y métodos. Se realizó una revisión sistemática de la literatura científica para identificar artículos que reportaron mutaciones que confieren resistencia a fármacos antituberculosis de primera línea. Esta búsqueda hizo énfasis en la resistencia a los fármacos de isoniazida y rifampicina en cepas de M. tuberculosis de pacientes peruanos. La búsqueda fue realizada en PubMed y LILACS (Literatura Latinoamericana y del Caribe en Ciencias de la Salud). Resultados. Se incluyeron 14 artículos de los cuales tres reportaron mutaciones asociadas con resistencia a isoniazida, seis a rifampicina, ocho a pirazinamida y uno a etambutol. Todas las mutaciones a isoniazida o rifampicina fueron identificadas directa o indirectamente mediante la prueba de diagnóstico molecular GenoType MTBDRplus® v2.0. La mayor variabilidad de mutaciones fue determinada en la resistencia a pirazinamida. Conclusiones. Existe una gran variabilidad de mutaciones asociadas con resistencia a fármacos antituberculosis que han sido reportadas en Perú, y se sistematizan en el presente reporte. Estas mutaciones deben de ser tomadas en cuenta para el desarrollo de dispositivos diagnósticos o selección de pruebas diagnósticas a ser aplicadas en nuestro país.

ABSTRACT Objective. To systematize available information regarding mutations that confer resistance to first-line anti-tuberculosis drugs. Materials and Methods. A systematic review of the scientific literature was conducted to identify articles that reported mutations conferring resistance to first-line anti-tuberculosis drugs. This search emphasized resistance to isoniazid and rifampicin drugs in M. tuberculosis strains of Peruvian patients. The search was performed on PubMed and LILACS (Latin American and Caribbean Health Sciences Literature). Results. Fourteen (14) articles were included, of which three reported mutations associated with resistance to isoniazid, six to rifampicin, eight to pyrazinamide and one to ethambutol. All mutations to isoniazid or rifampicin were identified directly or indirectly by the molecular diagnostic test GenoType MTBDRplus® v2.0. The greatest variability of mutations was determined in resistance to pyrazinamide. Conclusions. There is a great variability of mutations associated with resistance to anti-tuberculosis drugs that have been reported in Peru, and they are systematized in this report. These mutations must be taken into account for the development of diagnostic devices or selection of diagnostic tests to be applied in our country.

Vigilancia por el laboratorio de la tuberculosis resistente en menores de 15 años, Colombia, 2010 a 2015 / Resistant tuberculosis in children under 15 years of age, Colombia 2010-2015

Resumen Introducción. La tuberculosis en los niños es un reflejo de transmisión reciente en la comunidad. Se estima que en el mundo cada año un millón de niños enferma por esta causa; en Colombia se notificaron 291 casos en el 2015. Objetivo. Actualizar la información obtenida de las actividades de vigilancia por el laboratorio de la farmacorresistencia del bacilo Mycobacterium tuberculosis en menores de 15 años en Colombia entre el 2010 y el 2015. Materiales y métodos. Se llevó a cabo un estudio retrospectivo de corte transversal. Se estudiaron las variables de procedencia, sexo, edad, tipo de tuberculosis y estado de HIV en los casos sensibles y resistentes. Estos se clasificaron como caso nuevo sin tratamiento o caso previamente tratado para analizar el perfil de resistencia a fármacos de primera y segunda línea. Resultados. De los 3.440 casos notificados, en el 16,4 % se practicó la prueba de sensibilidad. El 50,6 % eran mujeres, la forma pulmonar se presentó en el 70,6 % y el 1,4 % presentó coinfección de tuberculosis y HIV. Se estudiaron 565 casos, de los cuales 503 (89,0 %) eran nuevos: el 3,9 % con tuberculosis multirresistente y el 9,5 % con resistencia global. Los previamente tratados fueron 62 (10,9 %), 4,8 % con multirresistencia y 19,3 % con resistencia global. No se evidenciaron diferencias estadísticamente significativas en los años estudiados. La proporción de tuberculosis extremadamente resistente en los casos nuevos evaluados fue de 9,0 %. Conclusiones. Es necesario que el Ministerio de Salud y Protección Social y el Instituto Nacional de Salud promuevan el uso de pruebas diagnósticas rápidas y muy sensibles, como las moleculares recomendadas por la Organización Mundial de la Salud.

Abstract Introduction: Tuberculosis in children is a recent transmission reflection in the community. It is estimated that every year one million children get sick in the world because of this. In Colombia, 291 cases were notified in 2015. Objective: To update the information obtained from the surveillance activities of the drug-resistance laboratory in children younger than 15 years of age in Colombia between 2010 and 2015. Materials and methods: This was a cross-sectional retrospective study. We studied the variables of origin, gender, age, type of tuberculosis, and HIV status in sensitive and resistant cases. We classified them according to their treatment background between new and previously treated to analyze their first and second line drug resistance profile. Results: From the notified cases, 16.4 % had a sensitivity test. 50.6 % were women, the pulmonary form was present in 70.6% cases, and 1.4 % presented with tuberculosis/HIV coinfection. We studied 565 cases, from which 503 (89.1 %) were new, presenting with multidrug-resistant tuberculosis, and a global resistance of 3.9 % and 9.5 %, respectively. From them, 62 had been previously treated (10.9 %), with 4.8 % and 19.3 % multidrug resistance and global resistance, respectively. There was no evidence of statistically significant differences during the studied years. Extremely resistant tuberculosis in new cases was 9.0 %. Conclusions: It is necessary for the Ministerio de Salud y Protección Social and the Instituto Nacional de Salud to promote the use of faster and more sensitive diagnostic tests such as the molecular ones recommended by the World Health Organization.

Speeding up the diagnosis of multidrug-resistant tuberculosis in a high-burden region with the use of a commercial line probe assay / Agilizando o diagnóstico da tuberculose multirresistente em uma região endêmica com o uso de um teste comercial de sondas em linha

ABSTRACT Objective: To evaluate the rapid diagnosis of multidrug-resistant tuberculosis, by using a commercial line probe assay for rifampicin and isoniazid detection (LPA-plus), in the routine workflow of a tuberculosis reference laboratory. Methods: The LPA-plus was prospectively evaluated on 341 isolates concurrently submitted to the automated liquid drug susceptibility testing system. Results: Among 303 phenotypically valid results, none was genotypically rifampicin false-susceptible (13/13; 100% sensitivity). Two rifampicin-susceptible isolates harboured rpoB mutations (288/290; 99.3% specificity) which, however, were non-resistance-conferring mutations. LPA-plus missed three isoniazid-resistant isolates (23/26; 88.5% sensitivity) and detected all isoniazid-susceptible isolates (277/277; 100% specificity). Among the 38 (11%) invalid phenotypic results, LPA-plus identified 31 rifampicin- and isoniazid-susceptible isolates, one isoniazid-resistant and six as non-Mycobacterium tuberculosis complex. Conclusions: LPA-plus showed excellent agreement (≥91%) and accuracy (≥99%). Implementing LPA-plus in our setting can speed up the diagnosis of multidrug-resistant tuberculosis, yield a significantly higher number of valid results than phenotypic drug susceptibility testing and provide further information on the drug-resistance level.

RESUMO Objetivo: Avaliar o diagnóstico rápido de tuberculose multirresistente, utilizando um teste comercial de sondas em linha (LPA-plus), na rotina de um laboratório de referência de tuberculose. Métodos: O teste LPA-plus foi avaliado prospectivamente em 341 isolados simultaneamente submetidos ao teste de suscetibilidade aos antimicrobianos em meio líquido, pelo sistema automatizado. Resultados: Entre os 303 resultados fenotipicamente válidos, nenhum foi genotipicamente falso suscetível à rifampicina (13/13; 100% de sensibilidade). Dois isolados sensíveis à rifampicina apresentavam mutações no gene rpoB (288/290; especificidade de 99,3%), as quais, no entanto, não são associadas à resistência a rifampicina. O LPA-plus não identificou resistência à isoniazida em três isolados fenotipicamente resistentes (23/26; 88,5% de sensibilidade) e detectou todos os isolados sensíveis à isoniazida (277/277; especificidade de 100%). Entre os 38 (11%) resultados fenotípicos inválidos, o LPA-plus identificou 31 isolados sensíveis à rifampicina e à isoniazida, um resistente à isoniazida e seis como micobactérias não tuberculosas. Conclusões: O LPA-plus mostrou excelente concordância (≥91%) e acurácia (≥99%). Sua implementação pode acelerar o diagnóstico da tuberculose multirresistente, produzir número significativamente maior de resultados válidos do que o teste fenotípico de suscetibilidade aos antimicrobianos e fornecer informações adicionais sobre o nível de resistência aos fármacos.

Mycobacterium tuberculosis spoligotypes and drug-resistant characterization from Beira compared to genotypes circulating in Mozambique

Abstract INTRODUCTION Mozambique is one of three countries with high prevalence of tuberculosis (TB), TB/human immunodeficiency virus coinfection, and multidrug-resistant TB. We aimed to describe Mycobacterium tuberculosis spoligotypes circulating among drug resistant (DR) strains from Beira, Mozambique comparing them with genotypes in the country. METHODS: We performed spoligotyping of 79 M. tuberculosis suspected of DR-TB compared all spoligotype patterns published on the international database and PubMed. RESULTS: Both in Beira and Mozambique (n=578), the main clades were Latin-American-Mediterranean, East-African-Indian, Beijing and T, with no extensively DR TB cases. CONCLUSIONS: Beira and Mozambique share the same population genetic structure of M. tuberculosis.

Diagnóstico molecular de tuberculosis multidrogorresistente en muestras de esputo mediante el análisis de curvas de melting / Molecular diagnosis of multidrug-resistant tuberculosis in sputum samples by melting curve analysis

RESUMEN Objetivos. Analizar curvas de melting para el diagnóstico de tuberculosis multidrogorresistente a partir de muestras de esputo. Materiales y métodos. Se colectaron muestras de esputo (n = 250) de pacientes con sospecha clínica de tuberculosis pulmonar según resultado de baciloscopia y cultivados en medio sólido Lowenstein Jensen. Según el método de referencia se trabajó con 124 muestras sensibles a rifampicina e isoniacida, 24 resistentes a rifampicina, 33 resistentes a isoniacida y 69 multidrogorresistentes. Se evaluó por PCR en tiempo real y luego por las curvas de melting, se utilizó el gen rpoB como biomarcador de resistencia a rifampicina, y el gen katG y región promotora inhA como biomarcadores de resistencia a isoniacida. La cepa H37Rv fue considerada como control sensible a drogas. Se compararon los resultados del método de referencia y los resultados del análisis de curvas de melting para evaluar los parámetros de sensibilidad, especificidad, valor predictivo positivo y valor predictivo negativo. Resultados. La resistencia a rifampicina mostró una sensibilidad de 90,3 %, especificidad de 90,4 %, valor predictivo positivo de 84,8 % y valor predictivo negativo de 94,0 %. La resistencia a isoniacida mostró una sensibilidad de 90,2 %, especificidad de 93,9 %, valor predictivo positivo de 91,1 % y valor predictivo negativo de 93,3 %. La detección de tuberculosis multidrogorresistente mostró valores de 89,9 %, 90,6 %, 78,5 % y 95,9 % para sensibilidad, especificidad, valor predictivo positivo y valor predictivo negativo, respectivamente. Conclusiones. El análisis de curvas de melting mostró ser seguro y confiable para ser utilizado en el diagnóstico rápido de tuberculosis multidrogorresistente en muestras de esputo.

ABSTRACT Objectives. To analyze melting curves for the diagnosis of multidrug-resistant tuberculosis from sputum samples. Materials and Methods. Sputum samples (n = 250) were collected from patients with clinical suspicion of pulmonary tuberculosis as a result of bacilloscopy and cultured in solid medium Lowenstein Jensen. According to the reference method, 124 samples sensitive to rifampicin and isoniazid, 24 resistant to rifampicin, 33 resistant to isoniazid, and 69 multidrug-resistant were used. It was evaluated by real-time PCR and then by melting curves, the rpoB gene was used as a biomarker of rifampicin resistance, and the katG gene and inhA promoter region were used as biomarkers of isoniazid resistance. The H37Rv strain was considered a drug-sensitive control. The results of the reference method and the results of the melting curve analysis were compared to evaluate the parameters of sensitivity, specificity, positive predictive value and negative predictive value. Results. Rifampicin resistance showed a sensitivity of 90.3%, specificity of 90.4%, positive predictive value of 84.8% and negative predictive value of 94.0%. Isoniazid resistance showed a sensitivity of 90.2%, specificity of 93.9%, positive predictive value of 91.1% and negative predictive value of 93.3%. The detection of multidrug-resistant tuberculosis showed values of 89.9%, 90.6%, 78.5% and 95.9% for sensitivity, specificity, positive predictive value and negative predictive value, respectively. Conclusions. The melting curve analysis showed to be safe and reliable to be used in the rapid diagnosis of multidrug-resistant tuberculosis in sputum samples.

Predictors of mortality in multidrug-resistant tuberculosis patients from Brazilian reference centers, 2005 to 2012

ABSTRACT Objectives To determine the main predictors of death in multidrug-resistant (MDRTB) patients from Brazil. Design Retrospective cohort study, a survival analysis of patients treated between 2005 and 2012. Results Of 3802 individuals included in study, 64.7% were men, mean age was 39 (1-93) years, and 70.3% had bilateral pulmonary disease. Prevalence of human immunodeficiency virus (HIV) was 8.3%. There were 479 (12.6%) deaths. Median survival time was 1452 days (4 years). Factors associated with increased risk of death were age greater than or equal to 60 years (hazard rate [HR] = 1.6, confidence interval [CI] = 1.15-2.2), HIV co-infection (HR = 1.46; CI = 1.05-1.96), XDR resistance pattern (HR = 1.74, CI = 1.05-2.9), beginning of treatment after failure (HR = 1.72, CI = 1.27-2.32), drug abuse (HR = 1.64, CI = 1.22-2.2), resistance to ethambutol (HR = 1.30, CI = 1.06-1.6) or streptomycin (HR = 1.24, CI = 1.01-1.51). Mainly protective factors were presence of only pulmonary disease (HR = 0.57, CI = 0.35-0.92), moxifloxacin use (HR = 0.44, CI = 0.25-0.80), and levofloxacin use (HR = 0.75; CI = 0.60-0.94). Conclusion A more comprehensive approach is needed to manage MDRTB, addressing early diagnostic, improving adhesion, and comorbidities, mainly HIV infection and drug abuse. The latest generation quinolones have an important effect in improving survival in MDRTB.

The role of the Xpert MTB/RIF assay among adolescents suspected of pulmonary tuberculosis in Rio de Janeiro, Brazil

Abstract INTRODUCTION The teste rápido molecular para tuberculose (TRM-TB) was introduced in 2014 in Brazil for tuberculosis screening. However, its role in adolescents in Brazil has not been studied. METHODS A descriptive study of adolescents with suspected tuberculosis using National Laboratory software. RESULTS Of 852 (15.4%) suspected cases, 131 were positive by TRM-TB and 2% were resistant to rifampicin. Among TRM-TB-positive cases, 105 (91.4%) were culture-positive. Sixty-four of 96 samples were sensitive to rifampicin by TRM-TB; 11 were resistant to other drugs by drug sensitivity test (DST). CONCLUSIONS Among suspected cases, 16% were diagnosed by TRM-TB, of which 17% were drug-resistant by DST.

Combination of commercially available molecular assays and culture based methods in diagnosis of tuberculosis and drug resistant tuberculosis

ABSTRACT Early diagnosis of tuberculosis is of major clinical importance. Among 4733 clinical specimens collected from 3363 patients and subjected to Ziehl-Neelsen microscopy, 4109 were inoculated onto Löwenstein-Jensen slants and 3139 in Bactec/9000MB. Polymerase Chain Reaction (PCR) was performed in 3139 specimens, whereas, a genotypic assay was directly applied in 93 Mycobacterium tuberculosis complex PCR-positive for isoniazid and rifampicin resistance detection specimens (GenoType MTBDRplus). Recovered M. tuberculosis isolates (64) as well as, 21 more sent from Regional Hospitals were tested for antimycobacterial resistance with a phenotypic (manual MGIT-SIRE) and a genotypic assay (GenoType MTBDRplus). PCR in the clinical specimens showed excellent specificity (97.4%) and accuracy (96.8%), good sensitivity (70.4%), but low positive predictive value (40.3%). MGIT-SIRE performed to M. tuberculosis did not confer a reliable result in 16 isolates. Of the remaining 69 isolates, 15 were resistant to streptomycin, seven to isoniazid, seven to ethambutol and five to rifampicin. GenoType MTBDRplus correctly detected isoniazid (seven) and rifampicin-resistant M. tuberculosis strains (five), showing an excellent performance overall (100%). Susceptibility results by the molecular assay applied directly to clinical specimens were identical to those obtained from recovered isolates of the corresponding patients. Combining molecular and conventional methods greatly contribute to early diagnosis and accurate susceptibility testing of tuberculosis.

Correlation between the BACTEC MGIT 960 culture system with Genotype MTBDRplus and TB-SPRINT in multidrug resistant Mycobacterium tuberculosis clinical isolates from Brazil

BACKGROUND The accurate detection of multidrug-resistant tuberculosis (MDR-TB) is critical for the application of appropriate patient treatment and prevention of transmission of drug-resistant Mycobacterium tuberculosis isolates. The goal of this study was to evaluate the correlation between phenotypic and molecular techniques for drug-resistant tuberculosis diagnostics. Molecular techniques used were the line probe assay genotype MTBDRplus and the recently described tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT) bead-based assay. Conventional drug susceptibility testing (DST) was done on a BACTECTM MGIT 960 TB. METHOD We studied 80 M. tuberculosis complex (MTC) clinical isolates from Minas Gerais state, of which conventional DST had classified 60 isolates as MDR and 20 as drug susceptible. FINDINGS Among the 60 MDR-TB isolates with MGIT as a reference, sensitivity, specificity, accuracy, and kappa for rifampicin (RIF) resistance using TB-SPRINT and MTBDRplus, were 96.7% versus 93.3%, 100.0% versus 100.0%, 97.5% versus 95.0% and 0.94 versus 0.88, respectively. Similarly, the sensitivity, specificity, accuracy, and kappa for isoniazid (INH) resistance were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. Finally, the sensitivity, specificity, accuracy, and kappa for MDR-TB were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. MAIN CONCLUSIONS Both methods exhibited a good correlation with the conventional DST. We suggest estimating the cost-effectiveness of MTBDRplus and TB-SPRINT in Brazil.

Precisión diagnóstica de tres pruebas moleculares para detectar la tuberculosis multirresistente / Diagnostic accuracy of three technologies for the diagnosis of multi-drug resistant tuberculosis

Resumen Introducción. La tuberculosis multirresistente (TB-MDR) y la extremadamente resistente (TB-XDR) constituyen un problema de salud pública a nivel mundial. Su detección oportuna permitiría reducir la carga de la enfermedad y su impacto económico en los sistemas de salud. Objetivo. Revisar sistemáticamente la información relacionada con la precisión diagnóstica de tres pruebas moleculares para detectar la tuberculosis multirresistente y la extremadamente resistente. Materiales y métodos. Se hizo una revisión sistemática de la literatura, según los lineamientos de Cochrane, de los estudios en población inmunocompetente relacionados con la precisión diagnóstica de tres pruebas moleculares para detectar la tuberculosis multirresistente y la extremadamente resistente. La búsqueda de los estudios publicados a partir del 2007 se hizo en Medline y Embase. La precisión diagnóstica de las pruebas se estableció con base en los valores máximos y mínimos de sensibilidad y especificidad, y en los valores predictivos positivos y negativos. Resultados. Se detectaron ocho estudios sobre la precisión diagnóstica de la prueba GeneXpert MTB/RIF(r), 12 sobre la de GenoType MTBDRplus(r) y 13 sobre la de GenoType MTBDRsl(r). La especificidad de GeneXpert MTB/RIF(r) osciló entre 91 y 100 % y su sensibilidad, entre 33,3 y 100 %. La sensibilidad de GenoType MTBDRplus(r) varió entre 82 y 100 %, en tanto que la sensibilidad y la especificidad de GenoType(r) MTBDRsl fluctuaron entre 56 y 100 % y 21 y 100 %, respectivamente. Conclusión. Según los estudios consultados, los tres métodos de diagnóstico evaluados presentabanuna adecuada eficacia diagnóstica para detectar la tuberculosis multirresistente y la extremadamente resistente.

Abstract Introduction: Multi-drug resistant (MDR-TB) and extensively drug-resistant (XDR-TB) tuberculoses are a global public health problem. Their timely detection might reduce the burden of the disease and the economic impact on health systems worldwide. Objective: To conduct a literature review of the diagnostic accuracy of three molecular tests to detect multi-drug resistant and extensively drug-resistant tuberculoses. Materials and methods: A systematic literature review following Cochrane methodology was carried out to study the diagnostic accuracy of three molecular tests to detect MDR-TB and XDR-TB in previous studies among immunocompetent population. Articles indexed in Medline and Embase were reviewed starting in 2007. Diagnostic accuracy was reported by sensitivity, specificity, and positive and negative predictive values of each test. Results: In total, 8, 12 and 13 studies were included to assess the diagnostic accuracy of GeneXpert MTB/RIF(r), GenoType MTBDRplus (r) and GenoType MTBDRsl (r), respectively. The specificity of GeneXpert MTB/RIF(r) ranged between 91 and 100%, and its sensitivity between 33.3 and 100%. The sensitivity of GenoType(r) MTBDRplus (r) ranged between 88 and 100%. The sensitivity and specificity of GenoType MTBDRsl (r) to evaluate drug resistance ranged between 56 and 100% and 21 and 100%, respectively. Conclusion: The three diagnostic tests evaluated have shown an adequate diagnostic accuracy to detect MDR and XDR tuberculoses.

Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus

BACKGROUND To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed. OBJECTIVE To evaluate the performance of multiplex ligaton-dependent probe amplification (MLPA) against Genotype® MTBDRplus to detect resistance to isoniazid (INHr) and rifampicin (RIFr). METHOD 96 culture isolates characterised for identification, drug susceptibility testing (DST) and sequencing of rpoB, katG, and inhA genes were evaluated by the MLPA and Genotype®MTBDRplus assays. RESULTS With sequencing as a reference standard, sensitivity (SE) to detect INHr was 92.8% and 85.7%, and specificity (SP) was 100% and 97.5%, for MLPA and Genotype®MTBDRplus, respectively. In relation to RIFr, SE was 87.5% and 100%, and SP was 100% and 98.8%, respectively. Kappa value was identical between Genotype®MTBDRplus and MLPA compared with the standard DST and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)]. CONCLUSION Compared to Genotype®MTBDRplus, MLPA showed similar sensitivity to detect INH and RIF resistance. The results obtained by the MLPA and Genotype®MTBDRplus assays indicate that both molecular tests can be used for the rapid detection of drug-resistant TB with high accuracy. MLPA has the added value of providing information on the circulating M. tuberculosis lineages.

Descripción de las mutaciones de Mycobacterium tuberculosis que confieren resistencia a rifampicina e isoniacida detectadas mediante GenoType(r) MTBDR plus V.2 en Colombia / Description of Mycobacterium tuberculosis mutations conferring resistance to rifampicin and isoniazid detected by GenoType(r) MTBDR plus V.2 in Colombia

Resumen Introducción: La metodología de GenoType(r) MTBDRplus V.2 es una técnica molecular aprobada por la Organización Mundial de la Salud y la Organización Panamericana de la Salud para la detección de las mutaciones en el gen rpoβ del complejo Mycobacterium tuberculosis, las cuales confieren resistencia a la rifampicina, y las de los genes katG e inhA que la confieren frente a la isoniacida. Debido a la variación genética en las cepas circulantes a nivel mundial, los programas nacionales de control de la tuberculosis deben comprobar el desempeño de los nuevos métodos de diagnóstico para su aplicación como prueba rápida. Objetivo: Describir las mutaciones detectadas mediante la técnica GenoType(r) MTBDRplus V.2 en muestras pulmonares y aislamientos de M. tuberculosis procesados en el Laboratorio Nacional de Referencia del Instituto Nacional de Salud durante el 2014. Materiales y métodos: Se hizo un estudio retrospectivo descriptivo que determinó la expresión de los genes inhA, KatG y rpoβ responsables de la resistencia a isoniacida y rifampicina, utilizando la técnica GenoType(r) MTBDRplus V.2 en 837 muestras y aislamientos de casos de tuberculosis. Resultados. Se obtuvieron 689 resultados de pruebas: 581(84,3 %) sensibles, 58 (8,4 %) resistentes y 50 (7,2 %) multirresistentes. Se detectaron diversas mutaciones en el gen rpoβ, de las cuales la más frecuente fue la Ser531Leu (36,6 %), seguida por la Asp516Val (21,6 %), en tanto que en el gen katG la más frecuente fue la Ser315Thr1 (91,9 %). Conclusiones: Se detectaron varias mutaciones en los casos resistentes reportados en el país, con frecuencias similares a las reportadas en otros países de la región de América del Sur.

Abstract Introduction: The GenoType(r)MTBDRplusV.2 assay is a molecular technique endorsed by the World Health Organization and the Pan American Health Organization that allows for the identification of the Mycobacterium tuberculosis complex and the detection of mutations in the rpoβ gene for rifampicin resistance, and katG and inhA genes for isoniazid resistance. Due to the genetic variability in the circulating strains around the world, the national tuberculosis control programs should assess the performance of these new diagnostic technologies and their use under program conditions as rapid tests. Objective: To describe the mutations identified by the GenoType(r)MTBDRplusV.2 assay in pulmonary samples and Mycobacterium tuberculosis isolates in the Laboratorio Nacional de Referencia of the Instituto Nacional de Salud in 2014. Materials and methods. We conducted a retrospective, descriptive study to detect the expression of inhA, KatG and rpoβ genes, responsible for resistence against isoniazid and rifampicin using the GenoType(r) MTBDRplus V.2 assay in 837 samples and isolates from tuberculosis cases. Results: Several mutations in the rpoβ gene were identified. Ser531Leu was the most frequent (36.6%) followed by Asp516Val (21.6%), while Ser315Thr1 was the most frequent mutation in the katG gene (91.9%). Conclusions: We were able to identify different mutations present in MDR-TB strains in the country, with frequencies similar to those reported in other countries in the South American region.

Evaluation of crystal violet decolorization assay for minimal inhibitory concentration detection of primary antituberculosis drugs against Mycobacterium tuberculosis isolates

In this study we evaluated the crystal violet decolorization assay (CVDA) for detection of minimum inhibitory concentration (MIC) of antituberculosis drugs. 53 isolates were tested in this study and 13 of them were multidrug resistant (MDR) isolates. The antibiotics concentrations were 2-0.06 mg/L for isoniazid (INH) and rifampicin (RIF) and were 16-0.25 mg/L for streptomycin (STM) and ethambutol (EMB). Crystal violet (CV-25 mg/L) was added into the microwells on the seventh day of incubation and incubation was continued until decolorization. Decolorization of CV was the predictor of bacterial growth. Overall agreements for four drugs were detected as 98.1%, and the average time was detected as 9.5 ± 0.89 day after inoculation. One isolate for INH and two isolates for STM were determined resistant in the reference method, but susceptible by the CVDA. One isolate was susceptible to EMB by the reference method, but resistant by the CVDA. All results were concordant for RIF. This study shows that CVDA is a rapid, reliable and suitable for determination of MIC values of Mycobacterium tuberculosis. And it can be used easily especially in countries with limited-sources.

Detection of multidrug-resistant Mycobacterium tuberculosis strains isolated in Brazil using a multimarker genetic assay for katG and rpoB genes

Abstract Multidrug-resistant tuberculosis (MDRTB) is a serious world health problem that limits public actions to control tuberculosis, because the most used anti-tuberculosis first-line drugs fail to stop mycobacterium spread. Consequently, a quick detection through molecular diagnosis is essential to reduce morbidity and medical costs. Despite the availability of several molecular-based commercial-kits to diagnose multidrug-resistant tuberculosis, their diagnostic value might diverge worldwide since Mycobacterium tuberculosis genetic variability differs according to geographic location. Here, we studied the predictive value of four common mycobacterial mutations in strains isolated from endemic areas of Brazil. Mutations were found at the frequency of 41.9% for katG, 25.6% for inhA, and 69.8% for rpoB genes in multidrug-resistant strains. Multimarker analysis revealed that combination of only two mutations (“katG/S315T + rpoB/S531L”) was a better surrogate of multidrug-resistant tuberculosis than single-marker analysis (86% sensitivity vs. 62.8%). Prediction of multidrug-resistant tuberculosis was not improved by adding a third or fourth mutation in the model. Therefore, rather than using diagnostic kits detecting several mutations, we propose a simple dual-marker panel to detect multidrug-resistant tuberculosis, with 86% sensitivity and 100% specificity. In conclusion, this approach (previous genetic study + analysis of only prevalent markers) would considerably decrease the processing costs while retaining diagnostic accuracy.

Resistance patterns, prevalence, and predictors of fluoroquinolones resistance in multidrug resistant tuberculosis patients

Abstract Background Fluoroquinolones are the backbone of multidrug resistant tuberculosis treatment regimens. Despite the high burden of multidrug resistant tuberculosis in the country, little is known about drug resistance patterns, prevalence, and predictors of fluoroquinolones resistance among multidrug resistant tuberculosis patients from Pakistan. Objective To evaluate drug resistance patterns, prevalence, and predictors of fluoroquinolones resistance in multidrug resistant tuberculosis patients. Methods This was a cross-sectional study conducted at a programmatic management unit of drug resistant tuberculosis, Lady Reading Hospital Peshawar, Pakistan. Two hundred and forty-three newly diagnosed multidrug resistant tuberculosis patients consecutively enrolled for treatment at study site from January 1, 2012 to July 28, 2013 were included in the study. A standardized data collection form was used to collect patients’ socio-demographic, microbiological, and clinical data. SPSS 16 was used for data analysis. Results High degree of drug resistance (median 5 drugs, range 2–8) was observed. High proportion of patients was resistant to all five first-line anti-tuberculosis drugs (62.6%), and more than half were resistant to second line drugs (55.1%). The majority of the patients were ofloxacin resistant (52.7%). Upon multivariate analysis previous tuberculosis treatment at private (OR = 1.953, p = 0.034) and public private mix (OR = 2.824, p = 0.046) sectors were predictors of ofloxacin resistance. Conclusion The high degree of drug resistance observed, particularly to fluoroquinolones, is alarming. We recommend the adoption of more restrictive policies to control non-prescription sale of fluoroquinolones, its rational use by physicians, and training doctors in both private and public–private mix sectors to prevent further increase in fluoroquinolones resistant Mycobacterium tuberculosis strains.