New insights on tuberculosis transmission dynamics and drug susceptibility profiles among the prison population in Southern Brazil based on whole-genome sequencing

ABSTRACT Background: The rate of tuberculosis (TB) infection among the prison population (PP) in Brazil is 28 times higher than that in the general population, and prison environment favors the spread of TB. Objective: To describe TB transmission dynamics and drug resistance profiles in PP using whole-genome sequencing (WGS). Methods: This was a retrospective study of Mycobacterium tuberculosis cultivated from people incarcerated in 55 prisons between 2016 and 2019; only one isolate per prisoner was included. Information about movement from one prison to another was tracked. Clinical information was collected, and WGS was performed on isolates obtained at the time of TB diagnosis. Results: Among 134 prisoners included in the study, we detected 16 clusters with a total of 58 (43%) cases of M. tuberculosis. Clusters ranged from two to seven isolates with five or fewer single nucleotide polymorphism (SNP) differences, suggesting a recent transmission. Six (4.4%) isolates were resistant to at least one anti-TB drug. Two of these clustered together and showed resistance to rifampicin, isoniazid, and fluoroquinolones, with 100% concordance between WGS and phenotypic drug-susceptibility testing. Prisoners with clustered isolates had a high amount of movement between prisons (two to eight moves) during the study period. Conclusions: WGS demonstrated the recent transmission of TB within prisons in Brazil. The high movement among prisoners seems to be related to the transmission of the same M. tuberculosis strain within the prison system. Screening for TB before and after the movement of prisoners using rapid molecular tests could play a role in reducing transmission.

GenoType MTBDRsl for detection of second-line drugs and ethambutol resistance in multidrug-resistant Mycobacterium tuberculosis isolates at a high-throughput laboratory

We assessed the performance of MTBDRsl for detection of resistance to fluoroquinolones, aminoglycosides/cyclic peptides, and ethambutol compared to BACTEC MGIT 960 by subjecting simultaneously to both tests 385 phenotypically multidrug-resistant-Mycobacterium tuberculosis isolates from Sao Paulo, Brazil. Discordances were resolved by Sanger sequencing. MTBDRsl correctly detected 99.7% of the multidrug-resistant isolates, 87.8% of the pre-XDR, and 73.9% of the XDR. The assay showed sensitivity of 86.4%, 100%, 85.2% and 76.4% for fluoroquinolones, amikacin/kanamycin, capreomycin and ethambutol, respectively. Specificity was 100% for fluoroquinolones and aminoglycosides/cyclic peptides, and 93.6% for ethambutol. Most fluoroquinolone-discordances were due to mutations in genome regions not targeted by the MTBDRsl v. 1.0: gyrA_H70R and gyrB_R446C, D461N, D449V, and N488D. Capreomycin-resistant isolates with wild-type rrs results on MTBDRsl presented tlyA mutations. MTBDRsl presented good performance for detecting resistance to second-line drugs and ethambutol in clinical isolates. In our setting, multidrug-resistant. isolates presented mutations not targeted by the molecular assay.

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.

Transmission and prevalence of drug-resistant tuberculosis in a Brazilian setting under a directly observed therapy short-course strategy

Abstract INTRODUCTION: We aimed to estimate the prevalence and transmission of drug-resistant tuberculosis in a high-burden Brazilian setting under directly observed therapy short-course strategy. METHODS: Isolates of culture-confirmed pulmonary tuberculosis patients from Guarulhos, Brazil, diagnosed in October 2007-2011 were subjected to drug susceptibility and IS6110-restriction fragment length polymorphism testing. RESULTS: The overall resistance prevalence was 11.5% and the multi-drug resistance rate was 4.2%. Twenty-six (43.3%) of 60 drug-resistant isolates were clustered. Epidemiological relationships were identified in 11 (42.3%) patients; 30.8% of the cases were transmitted in households. CONCLUSIONS: Drug-resistant tuberculosis was relatively low and transmitted in households and the community.

Determinação do nível de resistência a isoniazida em cepas de Mycobacterium tuberculosis e correlação com mutações nos genes que conferem resistência a este fármaco: katG e inhA / Determination of the level of resistance to isoniazid in strains of Mycobacterium tuberculosis and correlation with mutations in genes that confer resistance to this drug: katG and inhA

A tuberculose (TB) resistente aos fármacos é um problema mundial. As mutações mais frequentes associadas à resistência à isoniazida em Mycobacterium tuberculosis ocorrem no códon 315 do gene katG, causando níveis moderados a altos de resistência, e na região promotora do gene inhA, associadas a baixos níveis de resistência. Mutações no gene rpoB associam-se à resistência à rifampicina. Este estudo analisou mutações nos genes katG, inhA e rpoB e níveis fenotípicos de resistência à isoniazida em isolados de M. tuberculosis de pacientes com TB resistente do estado de São Paulo, no período de outubro/2008 a março/2009 e no ano de 2016. Dos 374 isolados analisados, 165 (44%) eram monorresistentes à isoniazida, 47 (13%) polirresistentes (resistentes à isoniazida+fármaco(s) de 1ª linha exceto rifampicina), 122 (33%) multirresistentes (MR), 24 (6%) pré-extensivamente resistentes (pré-XDR) e 16 (4%) XDR. Entre os isolados com a mutação Ser315Thr no katG, 99,4% apresentaram resistência intermediária ou alta à isoniazida, enquanto 73,7% dos isolados com a mutação C-15T no inhA apresentaram baixa resistência. Dos isolados com mutações no katG e inhA, 66,6% mostraram resistência alta e 32,4% resistência intermediária. Observou-se que 58,8% dos isolados tipados estavam em cluster e 43,7% deles pertenciam...(AU)

Drug-resistant tuberculosis (TB) is a problem worldwide. The most frequent mutations associated with isoniazid resistance in Mycobacterium tuberculosis isolates occur in codon 315 of the katG gene, which have been associated with moderate- to high-levels of resistance, and in the promoter region of the inhA gene, causing low-level isoniazid resistance. In its turn, the rpoB gene is associated with resistance to rifampicin. This study analyzed mutations in katG, inhA and rpoB genes and phenotypic levels of isoniazid resistance in M. tuberculosis isolates from drug-resistant TB patients from the state of São Paulo, in the period of October 2008 to March 2009 and during the year of 2016. Of the 374 M. tuberculosis isolates analyzed 165 (44%) were monoresistant to isoniazid, 47 (13%) polydrug-resistant (resistant to isoniazid+1st line drug(s) except rifampicin), 122 (33%) multidrug-resistant (MDR), 24 (6%) pre-extensively drug-resistant (pre-XDR) and 16 (4%) XDR. Among isolates with the katG Ser315Thr mutation, 99.4% had intermediate or high-level isoniazid resistance, while 73.7% of the isolates with the inhA C15T mutation had low-level resistance. Among isolates with mutations in katG and inhA, 66.6% had high-level resistance and 32.4% had intermediate resistance. It was observed that 58.8% of the isolates submitted to typing were clustered, and 43.7% of them belonged to the five most prevalent clusters: SP5, SP2c, SP12, SP5ac and SP1i. Isolates with katG 315 mutations were more frequently clustered when compared to isolates with other katG mutations or with no mutations (p= 0.002 and 0.01, respectively). Isolates with the inhA C-15T mutation were more frequently clustered when compared to isolates with katG 315 mutations (p= 0.006). Statistically significant differences were found between cure rates of isoniazidmonoresistant TB patients and MDR-TB patients (63.4% vs 47.1%, p= 0.02), as well as between isoniazid-monoresistant TB patients and XDR-TB patients (63.4% vs 12.5%, p= 0.01). Patients with polydrug-resistant TB also had higher cure rates than patients with MDR-TB (70% vs 47.1%, p= 0.02) and XDR-TB (12.5%, p= 0.004). The present study showed that katG mutations are associated with higher levels of isoniazid resistance and inhA mutations are found in isolates with lower resistance levels. In addition, drugresistant TB was transmitted among patients from the state of São Paulo, whose cure rates were relatively low. (AU)

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.

Performance of an in-house real-time polymerase chain reaction for identification of Mycobacterium tuberculosis isolates in laboratory routine diagnosis from a high burden setting

Abstract Brazil is one of the high burden countries for tuberculosis, and a rapid diagnosis is essential for effective control of the disease. In the present study, an in-house real-time polymerase chain reaction (PCR) assay targeting the mpt64 gene for identification of Mycobacterium tuberculosis complex isolates was evaluated under routine diagnosis conditions in a reference laboratory. From May 2011 to July 2012, 1,520 isolates of mycobacteria were prospectively submitted for phenotypic and/or PRA-hsp65 identification and to real-time PCR. The mpt64 real-time PCR showed 99.7% sensitivity and 96% specificity and detected 79.4% of the cases missed by phenotypic and PRA-hsp65 identification. The in-house real-time PCR assay showed high sensitivity and specificity and was successfully implemented in the routine diagnosis of tuberculosis in a reference laboratory from a high burden setting.

Detecção do Complexo Mycobacterium tuberculosis em escarro por PCR em tempo real / Detection of Mycobacterium tuberculosis complex in sputum by real-time PCR

A tuberculose é uma doença infecciosa de distribuição universal, constituindo um sério problema de saúde pública no Brasil e no mundo. Micobactérias que causam tuberculose em humanos ou animais possuem alta similaridade genética e, por este motivo, estão agrupadas dentro do Complexo Mycobacterium tuberculosis (CMT). A forma clínica mais comum é a tuberculose pulmonar. As técnicas tradicionais de diagnóstico laboratorial da tuberculose são a baciloscopia e a cultura. A baciloscopia, apesar de ser uma técnica rápida e de baixo custo, apresenta baixa sensibilidade e especificidade. A cultura é o padrão-ouro para o diagnóstico da tuberculose, porém consome muito tempo devido ao crescimento lento das espécies do CMT. Considerando que o diagnóstico rápido e acurado é essencial ao controle da tuberculose, o objetivo deste estudo foi avaliar o desempenho de uma metodologia de PCR em tempo real in house tendo como alvo o gene mpt64, específico para o CMT, diretamente em amostras de escarro de pacientes com suspeita de tuberculose, e comparar esta metodologia com os métodos fenotípicos de baciloscopia e cultura. Foram analisadas 715 amostras de escarro no período de agosto de 2012 a outubro de 2013. Estas amostras foram submetidas à baciloscopia, cultura, identificação de micobactérias por métodos bioquímicos e pelo método molecular de PRAhsp65, e PCR em tempo real. Com relação aos casos confirmados de tuberculose (n= 62 ou 8,7%), a baciloscopia apresentou sensibilidade de 82,3% (IC 95%, 71 – 89,8%). As técnicas de cultura e PCR em tempo real mostraram a mesma sensibilidade (90,3%; IC 95%, 80,5 – 95,5%). Os valores de especificidade foram 99,7% (IC 95%, 98,9 – 99,9%), 99,4% (IC 95%, 98,4 – 99,8%) e 98,6% (IC 95%, 97,4 – 99,3%) para a baciloscopia, cultura e PCR em tempo real, respectivamente. A metodologia de PCR em tempo real in house avaliada neste estudo apresentou sensibilidade igual à da cultura, que, apesar de demorada, continua sendo o padrão-ouro...

Tuberculosis is an infectious disease of global distribution, constituting a serious public health problem in Brazil and worldwide. Mycobacteria that cause tuberculosis in humans or animals have high genetic similarity, and are grouped within the Mycobacterium tuberculosis complex. The most common clinical presentation is pulmonary tuberculosis. Traditional techniques for the laboratory diagnosis of tuberculosis are smear microscopy and culture. The former, despite being a quick and inexpensive technique has low sensitivity and specificity. Culture is the gold standard in the diagnosis of tuberculosis, but very time consuming due to the slow growth of these microorganisms. Considering that the rapid and accurate diagnosis is essential for tuberculosis control, the aim of this study was to evaluate the performance of an in-house real-time PCR methodology targeting the mpt64 gene, specific for M. tuberculosis complex, applied directly in sputum samples of patients with suspected tuberculosis, and to compare this technique with the phenotypic methods of smear microscopy and culture. We analyzed 715 sputum samples from August 2012 to October 2013. These samples were submitted to smear microscopy, culture, identification of mycobacteria by biochemical methods and by the molecular method of PRAhsp65, and real-time PCR. With respect to confirmed tuberculosis cases (n=62 or 8.7%), the smear microscopy had a sensitivity of 82.3% (95% CI, 71 - 89.8%). Culture and real-time PCR showed the same sensitivity (90.3%; 95% CI, 80.5 - 95.5%). The specificity was 99.7% (95% CI, 98.9 - 99.9%), 99.4% (95% CI, 98.4 - 99.8%) and 98.6% (95% CI, 97.4 - 99.3%) for smear microscopy, culture and real-time PCR, respectively. The in-house real-time PCR targeting the mpt64 gene had a sensitivity equal to that of culture, which is still the gold standard, although being time consuming...