Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients / Comparação de diferentes modalidades de diagnóstico para isolamento de Mycobacterium tuberculosis entre pacientes com suspeita de linfadenite tuberculosa

Abstract Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.

Resumo A tuberculose é uma doença transmissível com altas taxas de morbimortalidade nos países em desenvolvimento. O objetivo principal do estudo é comparar métodos convencionais, como cultura de bacilo álcool-ácido resistente (BAAR) e microscopia, com métodos de diagnóstico rápido. O objetivo secundário é comparar os achados histopatológicos e microbiológicos em pacientes com suspeita de linfadenite tubercular. Um total de 111 amostras (agosto de 2018 a setembro de 2019) de gânglios linfáticos foi processado ​​para microscopia de AFB, culturas de AFB, teste de susceptibilidade a drogas (DST), histopatologia e Xpert Mycobacterium tuberculosis (MTB)/ensaios de resistência à rifampicina (RIF). Das 111 amostras de linfonodos, 6 (5,4%) foram positivas para baciloscopia de AFB, 84 (75,6%) foram positivas para cultura de AFB, 80 (70,7%) foram positivas para o GeneXpert e 102 (91,8%) foram indicativas de tuberculose para estudos histopatológicos. A positividade da cultura do tubo indicador de crescimento de micobactérias (MGIT) foi 84 (75,6%), maior que a cultura sólida de Lowenstein-Jensen (LJ), 74 (66,6%). As culturas positivas foram submetidas a DST fenotípico. Dois casos eram multirresistentes (MDR) ao DST, enquanto três casos eram resistentes à rifampicina no GeneXpert. A sensibilidade do GeneXpert foi 62% contra o método convencional de cultura AFB. O fraco desempenho dos métodos convencionais de diagnóstico de linfadenite requer metodologia de diagnóstico precoce e precisa. O teste Xpert MTB/RIF pode ajudar no tratamento de casos de tuberculose multirresistente. No entanto, métodos rápidos e convencionais devem ser usados ​​para o isolamento completo do Mycobacterium tuberculosis.

Tuberculosis con resistencia a rifampicina en Chile / Tuberculosis with resistance to rifampicin in Chile

Resumen El éxito de los tratamientos acortados de la tuberculosis se debe a la asociación de fármacos bactericidas y esterilizantes, principalmente Rifampicina e Isoniazida. Cuando la Rifampicina no puede ser utilizada por resistencia, los tratamientos son más prolongados y el éxito en la curación se reduce. La resistencia a Rifampicina frecuentemente se acompaña de resistencia a Isoniazida (Multidrogoresistencia o MDR). La OMS informa que en 2019 se diagnosticaron sólo el 44% de los casos estimados de tuberculosis con resistencia a Rifampicina (se proyectaba 465.000 casos) y se trató sólo al 38% de los casos estimados, quedando una gran proporción de casos sin diagnosticar y sin tratar. En Chile la vigilancia de la susceptibilidad a fármacos de primera línea en cepas de Mycobacterium tuberculosis se efectúa mediante biología molecular desde 2014, observándose un progresivo incremento de casos resistentes a Rifampicina desde 1% (23 casos) para ese año hasta 2,2% (65 casos) en 2019. La mayoría de casos de resistencia a Rifampicina corresponden a resistencia inicial. En casos con resistencia a Rifampicina realizamos estudio de susceptibilidad a fármacos de segunda línea en el laboratorio de referencia nacional. La terapia de TB-MDR tradicional tiene baja eficacia, con abandonos frecuentes por su largo tiempo de terapia y toxicidad. Nuevos tratamientos sin inyectables y el uso de Clofazimina, Fluorquinolonas, Linezolid y Bedaquilina tienen una mejor tasa de curación. Recientemente, el Programa de Control de la Tuberculosis de Chile dispone de esta terapia más eficaz y de menor duración por vía oral con estos fármacos.

The high success rate of shortened Tuberculosis (TB) treatments has been achieved by the association of bactericidal and sterilizing drugs. The main drugs are Rifampicin and Isoniazide. When Rifampicin cannot be used by resistance, the treatment is prolonged and success in healing is significantly reduced. Resistance to Rifampicin is often accompanied by resistance to Isoniazide (Multidrug resistance or MDR). WHO reports that only 44% of estimated TB cases with resistance to Rifampicin were diagnosed in 2019 (465,000 cases projected) and only 38% of estimated cases were treated, with a large proportion of cases remaining undiagnosed and untreated. In Chile, monitoring of susceptibility to first-line drugs is conducted in strains of Mycobacterium tuberculosis by molecular biology since 2014, observing a progressive increase in cases with Rifampicin resistance from 1% for that year (23 cases) to 2.2% in 2019 (65 cases). Most cases of resistance to Rifampicin correspond to cases of initial resistance. In cases with resistance to Rifampicin we carry out susceptibility study to second-line drugs in a national reference laboratory. MDR-TB therapy has low efficacy, with frequent abandonments for its long therapy time and toxicity. New non-injectable treatments and use of Clofazimine, Fluorquinolones, Linezolid and Bedaquiline are achieving a better cure rate. Recently, Chile's TB Control Program has this most effective and shorter-lasting oral therapy with these drugs.

Development of molecular markers of Mycobacterium tuberculosis rifampicin resistance gene rpoB by PARMS technology / 生物工程学报

The purpose of this study is to provide a simple and reliable genetic typing approach for molecular drug susceptibility test of Mycobacterium tuberculosis, through the developing of fluorescence molecular marker of rifampicin resistance gene rpoB. Eleven fluorescent molecular markers of the rpoB gene were established by using the sequence difference between the amino acid positions 531, 526, 516, 511 and 513 of rpoB gene of rifampicin-resistant strains and the alleles of rifampicin-sensitive strains, combined with the PARMS technique (Penta-primer amplification refractory mutation system). We used 104 clinical isolates of Mycobacterium tuberculosis to validate this marker and it was verified by sequencing as 100% correct. These samples were also tested with proportional drug sensitivity test. The coincidence rate was 94.23%. The molecular markers had high reliability for genotyping of rpoB gene. It can also detect low-concentration drug-resistant samples (511/533 unit point mutations) whose phenotypic susceptibility cannot be detected. The eleven sets of fluorescent molecular markers could cover 92%-96% of rpoB gene mutation types of rifampicin-resistant strains, and provide new idea for rapid detection of rifampin-resistant Mycobacterium tuberculosis.

Diagnostic characteristics of Xpert MTB/RIF assay for the diagnosis of tuberculous meningitis and rifampicin resistance in Southern Brazil / Características diagnósticas do Xpert MTB/RIF para o diagnóstico de meningite tuberculosa e resistência à rifampicina no Sul do Brasil

ABSTRACT Background: The timely diagnosis of tuberculous meningitis (TBM) is challenging. Molecular diagnostic tools are necessary for TBM, particularly in low- and middle-income countries. Objectives: We aimed to calculate the diagnostics characteristics of Xpert MTB/RIF for the detection of Mycobacterium tuberculosis in the cerebrospinal fluid (CSF) and the frequency of rifampicin (RIF)-resistance in the CSF samples. Methods: A total of 313 consecutive CSF samples were studied and categorized into TBM definite, probable, possible, or not TBM cases based on the clinical, laboratory, and imaging data. Results: For the definite TBM cases (n=7), the sensitivity, specificity, efficiency, and positive likelihood ratio were 100, 97, 97, and 38%, respectively. However, for the TBM definite associated with the probable cases (n=24), the sensitivity decreased to 46%. All CSF samples that were Xpert MTB/RIF-positive were RIF susceptible. Conclusion: Xpert MTB/RIF showed high discriminating value among the microbiology-proven TBM cases, although the values for the probable and possible TBM cases were reduced. Xpert MTB/RIF contributes significantly to the diagnosis of TBM, mainly when coupled with the conventional microbiological tests and clinical algorithms.

RESUMO Introdução: O diagnóstico da meningite tuberculosa (TBM) é desafiador. Ferramentas de diagnóstico molecular são necessárias para esse diagnóstico, particularmente em países de baixa e média renda. Objetivos: Calcular as características diagnósticas do Xpert MTB/RIF para a detecção de Mycobacterium tuberculosis no líquido cefalorraquidiano (LCR) e a frequência de resistência à rifampicina (RIF) nas amostras do LCR. Métodos: Um total de 313 amostras consecutivas de LCR foram estudadas e categorizadas em casos de TBM definida, provável, possível ou não TBM, com base nos dados clínicos, laboratoriais e de imagem. Resultados: Para os casos definidos de TBM (n=7), sensibilidade, especificidade, eficiência e razão de verossimilhança positiva foram de 100, 97, 97 e 38%, respectivamente. No entanto, para os casos de TBM definidos associados aos prováveis (n=24), a sensibilidade diminuiu para 46%. Todas as amostras de LCR que foram positivas para Xpert MTB/RIF foram suscetíveis a RIF. Conclusão: O Xpert MTB/RIF mostrou alto valor discriminante entre os casos TBM comprovados por microbiologia, porém o valor nos casos prováveis e possíveis de TBM foram reduzidos. O Xpert MTB/RIF contribui significativamente para o diagnóstico de TBM, principalmente quando associado aos testes microbiológicos convencionais e algoritmos clínicos.

Accuracy of Xpert® MTB/RIF for the detection of tuberculosis and rifampicin-resistance tuberculosis in China: A systematic review and meta-analysis / 北京大学学报(医学版)

OBJECTIVE@#To systematically review the diagnostic accuracy of Xpert® Mycobacterium tuberculosis/rifampicin (Xpert® MTB/RIF) for the detection of active tuberculosis (TB) and rifampicin-resistance TB in Chinese patients.@*METHODS@#Four Chinese databases (SinoMed, CNKI, WanFang database, and VIP) and three English databases (PubMed, Embase, and The Cochrane Library) were searched from January 1, 2000 to September 15, 2017, to identify diagnostic tests about the accuracy of Xpert® MTB/RIF in Chinese patients. Two investigators screened the articles and extracted the information independently, and then the quality of each included study was evaluated by Quality Assessment of Diagnostic Accuracy Studies (QUADAS)-2. Bivariate random-effects meta-analysis was conducted to pool the sensitivity and specificity. In addition, subgroup analyses were performed based on patient type (TB patient and TB suspected patient), sample type (sputum, bronchoalveolar lavage fluid and others). All statistical analyses were conducted with Stata version 13.0.@*RESULTS@#A total of 47 articles were included in this systematic review. Most of them (38 articles) were in Chinese and only 9 articles were in English. All the articles were published during 2014 to 2017, and the sample size ranged from 31 to 3 151. Forty articles including 42 comparisons about TB were finally included with the pooled sensitivity of 0.94 (95%CI: 0.92, 0.95) and the pooled specificity of 0.87 (95%CI: 0.84, 0.91). Subgroup analysis showed that different patient and specimen types had no significant differences on sensitivity, but the specificity of sputum group was higher than that of bronchoalveolar lavage fluid. As for the detection of rifampicin-resistant TB, 33 articles (38 comparisons) were analyzed, the pooled sensitivity and specificity were 0.92 (95%CI: 0.89, 0.94) and 0.98 (95%CI: 0.97, 0.99) respectively. There were no significant differences between the patient and specimen in the subgroup analyses. The Deeks funnel plot showed a possible publication bias for detecting active tuberculosis (P=0.08) and no publication bias for rifampicin-resistant TB (P=0.24). The likelihood ratio scatter gram showed that in clinical applications, Xpert® MTB/RIF had a good diagnostic ability for detecting active tuberculosis, and it had good clinical diagnostic value in detecting rifampicin-resistant TB.@*CONCLUSION@#Xpert® MTB/RIF has good sensitivity and specificity in detecting TB and rifampicin-resistant TB in Chinese people. In particular, it has good clinical value in diagnosing rifampicin-resistance TB.

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.

Accuracy of a rapid molecular test for tuberculosis in sputum samples, bronchoalveolar lavage fluid, and tracheal aspirate obtained from patients with suspected pulmonary tuberculosis at a tertiary referral hospital / Acurácia do teste rápido molecular para tuberculose em amostras de escarro, lavado broncoalveolar e aspirado traqueal obtidos de pacientes com suspeita de tuberculose pulmonar em um hospital de referência terciária

ABSTRACT Tuberculosis continues to be a major public health problem worldwide. The aim of the present study was to evaluate the accuracy of the Xpert MTB/RIF rapid molecular test for tuberculosis, using pulmonary samples obtained from patients treated at the Júlia Kubitschek Hospital, which is operated by the Hospital Foundation of the State of Minas Gerais, in the city of Belo Horizonte, Brazil. This was a retrospective study comparing the Xpert MTB/RIF test results with those of standard culture for Mycobacterium tuberculosis and phenotypic susceptibility tests. Although the Xpert MTB/RIF test showed high accuracy for the detection of M. tuberculosis and its resistance to rifampin, attention must be given to the clinical status of the patient, in relation to the test results, as well as to the limitations of molecular tests.

RESUMO A tuberculose permanece como um grave problema de saúde pública. O objetivo deste estudo foi avaliar a acurácia do teste rápido molecular Xpert MTB/RIF em amostras pulmonares no Hospital Júlia Kubitschek, Fundação Hospitalar do Estado de Minas Gerais, localizado em Belo Horizonte (MG). Trata-se de um estudo descritivo retrospectivo, considerando-se como método padrão a cultura para o bacilo da tuberculose e o teste de sensibilidade fenotípico. O teste Xpert MTB/RIF apresentou ótima acurácia para a detecção da tuberculose e resistência à rifampicina, mas é necessária a atenção a dados clínicos do paciente em relação ao resultado do exame e às limitações dos testes moleculares.

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.

Evaluation of the commercial kit SIRE nitratase for detecting resistant mycobacterium tuberculosis in Brazil

Abstract INTRODUCTION: This study aimed to evaluate a new commercial kit, Kit SIRE Nitratase-PlastLabor, for testing the drug susceptibility of clinical Mycobacterium tuberculosis isolates. METHODS: The accuracy of the Kit SIRE Nitratase was evaluated by examining the susceptibility (streptomycin, isoniazid, rifampicin, and ethambutol) of 40 M. tuberculosis isolates, using the proportion method with Lowenstein-Jensen medium or the BACTEC MGIT 960 system. RESULTS: The detection accuracy for streptomycin, isoniazid, rifampicin, and ethambutol was 95%, 97.5%, 100%, and 80%, respectively. CONCLUSIONS: The exceptional accuracy demonstrated by Kit SIRE Nitratase for isoniazid and rifampicin makes the kit an attractive option for screening M. tuberculosis strain resistance.

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.

Effects of the encapsulation of usnic acid into liposomes and interactions with antituberculous agents against multidrug-resistant tuberculosis clinical isolates

Mycobacterium tuberculosis (Mtb) has acquired resistance and consequently the antibiotic therapeutic options available against this microorganism are limited. In this scenario, the use of usnic acid (UA), a natural compound, encapsulated into liposomes is proposed as a new approach in multidrug-resistant tuberculosis (MDR-TB) therapy. Thus the aim of this study was to evaluate the effect of the encapsulation of UA into liposomes, as well as its combination with antituberculous agents such as rifampicin (RIF) and isoniazid (INH) against MDR-TB clinical isolates. The in vitro antimycobacterial activity of UA-loaded liposomes (UA-Lipo) against MDR-TB was assessed by the microdilution method. The in vitro interaction of UA with antituberculous agents was carried out using checkerboard method. Minimal inhibitory concentration values were 31.25 and 0.98 µg/mL for UA and UA-Lipo, respectively. The results exhibited a synergistic interaction between RIF and UA [fractional inhibitory concentration index (FICI) = 0.31] or UA-Lipo (FICI = 0.28). Regarding INH, the combination of UA or UA-Lipo revealed no marked effect (FICI = 1.30-2.50). The UA-Lipo may be used as a dosage form to improve the antimycobacterial activity of RIF, a first-line drug for the treatment of infections caused by Mtb.

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.

Isoniazid and rifampin drug susceptibility testing: application of 2,3,5-triphenyl tetrazolium chloride assay and microscopic-observation drug-susceptibility assay directly on Ziehl-Neelsen smear positive sputum specimens

Abstract The current study was aimed to evaluate the performance of direct 2,3,5-triphenyl tetrazolium chloride assay and direct microscopic observation drug susceptibility assay with indirect Löwenstein-Jensen proportion method directly on Ziehl-Neelsen smear positive sputum specimens. Methods Direct acid fast bacilli smear positive sputum specimens (n = 264) were subjected to isoniazid and rifampicin drug susceptibility testing by direct 2,3,5-triphenyl tetrazolium chloride assay, direct microscopic observation drug susceptibility assay, and the performances were compared with indirect Löwenstein-Jensen proportion method. Results The direct 2,3,5-triphenyl tetrazolium chloride assay demonstrated an overall sensitivity, specificity, positive predictive value, and negative predictive value of 99.2%, 82.4%, 99.2%, and 88.5%, respectively, for the detection of isoniazid and rifampicin resistant Mycobacterium tuberculosis isolates when compared to indirect Löwenstein-Jensen proportion method. Likewise, the overall sensitivity, specificity, positive predictive value and negative predictive value of direct microscopic observation drug susceptibility assay were 98.8%, 82.4%, 99.2%, and 78.2%, respectively. Conclusion The direct 2,3,5-triphenyl tetrazolium chloride assay was found to be an economical alternative method for the rapid and accurate detection of isoniazid and rifampicin resistance from direct acid fast bacilli smear positive sputum specimens.

A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis

Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in therpoB, katG, inhA,ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 forrpoB, katG, inhA,ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours.

Role of a GenoType MTBDRplus line probe assay in early detection of multidrug-resistant tuberculosis at a Brazilian reference center

Resistance to Mycobacterium tuberculosis is a reality worldwide, and its diagnosis continues to be difficult and time consuming. To face this challenge, the World Health Organization has recommended the use of rapid molecular tests. We evaluated the routine use (once a week) of a line probe assay (Genotype MTBDRplus) for early diagnosis of resistance and for assessment of the main related risk factors over 2 years. A total of 170 samples were tested: 15 (8.8%) were resistant, and multidrug resistance was detected in 10 (5.9%). The sensitivity profile took 3 weeks (2 weeks for culture and 1 week for rapid testing). Previous treatment for tuberculosis and the persistence of positive acid-fast smears after 4 months of supervised treatment were the major risk factors observed. The use of molecular tests enabled early diagnosis of drug-resistant bacilli and led to appropriate treatment of the disease. This information has the potential to interrupt the transmission chain of resistant M. tuberculosis.

Xpert MTB/RIF test performance assay in respiratory samples at real work settings in a developing country / Rendimiento de la prueba Xpert MTB/RIF en muestras respiratorias en el escenario real de trabajo en un país en desarrollo

Introduction: The Xpert MTB/RIF test detects DNA from Mycobacterium tuberculosis complex and susceptibility to rifampin. It has been evaluated repeatedly under "ideal" conditions including centrifugation of sputum and bronchoalveolar lavage, Ziehl Neelsen (ZN) and auramine/rhodamine staining, as well as with solid and liquid automated culture methods. Results from such evaluations cannot be extrapolated to low-income countries that do not routinely use all these processes. Objective: To assess the performance of the Xpert MTB/RIF test in respiratory samples under "real" conditions of work in a low-income country and its correlation with phenotypic susceptibility testing. Materials and methods: We conducted a cross-sectional study to assess the performance of the Xpert MTB/RIF test in =12 year-old patients with suspected pulmonary tuberculosis. In routine sample processing at the Hospital we do not use sputum centrifugation, staining with auramine/rhodamine or automated liquid culture. Results: We screened 152 patients of whom 108 were eligible for the study and 103 were included in the analysis; 34% of the samples were positive. The overall test sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were 91%, 92%, 83% and 96%, respectively. In ZN-negative samples the sensitivity, specificity, PPV and NPV were 87%, 91%, 68% and 97%, respectively. The results of sensitivity and resistance to rifampin were concordant with susceptibility testing using the multiple proportions method (kappa=1, p<0.0001). Conclusions: The Xpert MTB/RIF test overall performance was similar to the one achieved under ideal conditions. Its performance in ZN-negative samples was better under "real" conditions of work in a low-income country.

Introducción. La prueba Xpert MTB/RIF detecta el ADN del complejo Mycobacterium tuberculosis y la sensibilidad a rifampicina. La prueba ha sido evaluada en condiciones "ideales" que incluyen la centrifugación de esputo y el lavado broncoalveolar, la tinción de Ziehl Neelsen (ZN) y de auramina-rodamina y los métodos de cultivo sólido y de cultivo líquido automatizado. Los resultados de tales evaluaciones no pueden extrapolarse a países de bajos ingresos que no utilizan habitualmente todos estos procesos. Objetivo. Evaluar el rendimiento de la prueba Xpert MTB/RIF en muestras respiratorias bajo condiciones "reales" de trabajo y su correlación con las pruebas fenotípicas de sensibilidad. Materiales y métodos. Se llevó a cabo un estudio transversal para evaluar el rendimiento de la prueba Xpert MTB/RIF en pacientes =12 años con sospecha de tuberculosis pulmonar. En el procesamiento rutinario de muestras en el Hospital del estudio no se usa la centrifugación del esputo, la tinción con auramina-rodamina ni el cultivo líquido automatizado. Resultados. Se incluyeron 152 pacientes, de los cuales 108 eran elegibles y 103 se incluyeron en el análisis. El 34 % de las muestras fueron positivas; la sensibilidad de la prueba fue de 91 %, la especificidad de 92 %, el valor diagnóstico positivo de 83 % y el valor diagnóstico negativo global de 96 %. En las muestras negativas con Ziehl Neelsen, la sensibilidad fue de 87 %, la especificidad de 91 % y los valores diagnósticos positivo y negativo alcanzaron 68 y 97 %, respectivamente. Los resultados de sensibilidad o resistencia a la rifampicina concordaron con los de la prueba fenotípica de sensibilidad (valor de kappa=1, p<0,0001). Conclusiones. El rendimiento global de la prueba fue similar al obtenido bajo condiciones "ideales". En las muestras negativas con Ziehl Neelsen se obtuvo un mejor rendimiento en las condiciones "reales" de trabajo de un país de bajos ingresos.

Multiple antibiotic susceptibility of polyphosphate kinase mutants (ppk1 and ppk2) from Pseudomonas aeruginosa PAO1 as revealed by global phenotypic analysis

BACKGROUND: Pseudomonas aeruginosa is known to be a multidrug resistant opportunistic pathogen. Particularly, P. aeruginosa PAO1 polyphosphate kinase mutant (ppk1) is deficient in motility, quorum sensing, biofilm formation and virulence. FINDINGS: By using Phenotypic Microarrays (PM) we analyzed near 2000 phenotypes of P. aeruginosa PAO1 polyP kinase mutants (ppk1 and ppk2). We found that both ppk mutants shared most of the phenotypic changes and interestingly many of them related to susceptibility toward numerous and different type of antibiotics such as Ciprofloxacin, Chloramphenicol and Rifampicin. CONCLUSIONS: Combining the fact that ppk1 mutants have reduced virulence and are more susceptible to antibiotics, polyP synthesis and particularly PPK1, is a good target for the design of molecules with anti-virulence and anti-persistence properties.

In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA

Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.