Construction of EF-G knockdown strain of Mycobacterium smegmatis and drug resistance analysis / 生物工程学报

As the only translational factor that plays a critical role in two translational processes (elongation and ribosome regeneration), GTPase elongation factor G (EF-G) is a potential target for antimicrobial agents. Both Mycobacterium smegmatis and Mycobacterium tuberculosis have two EF-G homologous coding genes, MsmEFG1 (MSMEG_1400) and MsmEFG2 (MSMEG_6535), fusA1 (Rv0684) and fusA2 (Rv0120c), respectively. MsmEFG1 (MSMEG_1400) and fusA1 (Rv0684) were identified as essential genes for bacterial growth by gene mutation library and bioinformatic analysis. To investigate the biological function and characteristics of EF-G in mycobacterium, two induced EF-G knockdown strains (Msm-ΔEFG1(KD) and Msm-ΔEFG2(KD)) from Mycobacterium smegmatis were constructed by clustered regularly interspaced short palindromic repeats interference (CRISPRi) technique. EF-G2 knockdown had no effect on bacterial growth, while EF-G1 knockdown significantly retarded the growth of mycobacterium, weakened the film-forming ability, changed the colony morphology, and increased the length of mycobacterium. It was speculated that EF-G might be involved in the division of bacteria. Minimal inhibitory concentration assay showed that inhibition of EF-G1 expression enhanced the sensitivity of mycobacterium to rifampicin, isoniazid, erythromycin, fucidic acid, capreomycin and other antibacterial agents, suggesting that EF-G1 might be a potential target for screening anti-tuberculosis drugs in the future.

Clinical outcomes and molecular characterization of drug-resistant tuberculosis in pre- and extensively drug-resistant disease based on line probe assays

ABSTRACT Multidrug-resistant tuberculosis (MDR-TB) represents a significant impact in transmission, outcome, and health costs. The World Health Organization recommends implementation of rapid diagnostic methods for multidrug-resistance detection. This study was performed to evaluate the frequency of pre- and extensively drug resistant tuberculosis (pre-XDR-TB and XDR-TB) among MDR-TB patients, the pattern of resistance mutations for fluoroquinolones and the clinical outcome. Adult patients followed at a Brazilian regional reference center for TB, from January 2013 to June 2019 were included. Stored Mycobacterium tuberculosis (Mtb) cultures were recovered, the DNA was extracted, and the susceptibility test was performed using the line probe assay for second line antimycobacterial drugs, Genotype MTBDRsl version 2.0 (Hain Lifescience, CmbH, Germany). Among 33 MDR-TB included patients, we diagnosed XDR-TB or pre-XDR in five (15%) cases. Of these, mutations related to fluoroquinolones resistance were observed in four Mtb isolates, including one who had no phenotypic resistance profile. In two other patients with phenotypic resistance to ofloxacin, genotypic resistance was not found. Case fatality rate was 60% in pre/XDR-TB group, compared to 3.6% in the remaining of patients. This study observed few cases of pre-XDR and XDR-TB among a MDR-TB cohort. Phenotypic and genotypic assays presented good agreement. Clinical outcome was more favorable for patients with susceptibility to fluoroquinolones and injectable drugs.

Drug-Resistant Mycobacterium tuberculosis Isolates from New and Previously Treated TB Patients in China, 2017-2019

Abstract INTRODUCTION: Mycobacterium tuberculosis (MTB) is a causative agent of tuberculosis (TB) that causes death worldwide. METHODS: MTB was subjected to phenotypic drug-susceptibility tests (DST), and drug-resistant genes were sequenced. RESULTS: Previously treated patients were more likely to have positive smear results and exhibit drug resistance. New patients were more likely to be mono SM-resistant and less likely to be INH- and RIF-resistant. The most common mutations were katG (S315T), rpoB (S450L), rpsL (K43R), and embB (M306V). CONCLUSIONS: The proportion of mono-SM-resistant TB among new patients was higher.

Factors associated with tuberculosis and multidrug-resistant tuberculosis in patients treated at a tertiary referral hospital in the state of Minas Gerais, Brazil / Fatores associados à tuberculose e à tuberculose multirresistente em pacientes atendidos em um hospital de referência terciária em Minas Gerais, Brasil

ABSTRACT Objective: To evaluate the risk factors for the development of tuberculosis and multidrug-resistant tuberculosis (MDR-TB) in patients treated at a tertiary referral hospital. Methods: This was a cross-sectional study based on data obtained from patients treated at the Júlia Kubitschek Hospital, located in the city of Belo Horizonte, Brazil, between October of 2012 and October of 2014. We evaluated sociodemographic, behavioral, clinical, and radiological variables. The outcome considered to identify associations between tuberculosis and the explanatory variables was the treatment prescribed. To evaluate the associations between MDR-TB and the same explanatory variables, the change in MDR-TB treatment was considered. Results: The factors associated with tuberculosis were alcoholism, comorbidities, pulmonary cavitations, and a radiological pattern suggestive of tuberculosis. Cavitation and previous treatment for tuberculosis were associated with MDR-TB. Conclusions: Despite the significant progress made in the fight against tuberculosis, there is a need for coordinated actions that include social protection measures and patient support.

RESUMO Objetivo: Avaliar os fatores de risco de pacientes atendidos em um hospital de referência terciária para o desenvolvimento de tuberculose e tuberculose multirresistente (TBMR). Métodos: Estudo transversal baseado em dados obtidos de pacientes atendidos no Hospital Júlia Kubitschek, na cidade de Belo Horizonte (MG), entre outubro de 2012 e outubro de 2014. As variáveis utilizadas foram agrupadas em características sociodemográficas, comportamentais, clínicas e radiológicas. O desfecho considerado para verificar associações entre tuberculose e variáveis explicativas foi o tratamento prescrito para tuberculose. Para avaliar a associação entre a tuberculose resistente e as mesmas variáveis explicativas considerou-se a mudança de tratamento para TBMR. Resultados: Alcoolismo, padrão radiológico sugestivo de tuberculose, presença de comorbidades e presença de cavitações pulmonares foram fatores associados à tuberculose. A TBMR foi associada a tratamento prévio para tuberculose e presença de cavitações. Conclusões: Apesar dos importantes progressos na luta contra a tuberculose, é necessário um conjunto de ações articuladas que incluam medidas de proteção social e suporte aos pacientes.

Rapid detection of Mycobacterium tuberculosis DNA and genetic markers for Isoniazid resistance in Ziehl-Neelsen stained slides

BACKGROUND Early diagnosis of tuberculosis (TB) and identification of strains of Mycobacterium tuberculosis resistant to anti-TB drugs are considered the main factors for disease control. OBJECTIVES To standardise a real-time polymerase chain reaction (qPCR) assay technique and apply it to identify mutations involved in M. tuberculosis resistance to Isoniazid (INH) directly in Ziehl-Neelsen (ZN) stained slides. METHODS Were analysed 55 independent DNA samples extracted from clinical isolates of M. tuberculosis by sequencing. For application in TB diagnosis resistance, 59 ZN-stained slides were used. The sensitivity, specificity and Kappa index, with a 95% confidence interval (CI95%), were determined. FINDINGS The agreement between the tests was, for the katG target, the Kappa index of 0.89 (CI95%: 0.7-1.0). The sensitivity and specificity were 97.6% (CI95%: 87.7-99.9) and 91.7% (CI95%: 61.5-99.5), respectively. For inhA, the Kappa index was 0.92 (CI95%: 0.8-1.0), the sensitivity and specificity were 94.4% (CI95%: 72.7-99.8) and 97.3% (CI95%: 85.8-99.9), respectively. The use of ZN-stained slides for drug-resistant TB detection showed significant results when compared to other standard tests for drug resistance. MAIN CONCLUSIONS qPCR genotyping proved to be an efficient method to detect genes that confer M. tuberculosis resistance to INH. Thus, qPCR genotyping may be an alternative instead of sequencing.

Evaluation of drug susceptibility profile of Mycobacterium tuberculosis Lineage 1 from Brazil based on whole genome sequencing and phenotypic methods

BACKGROUND The evaluation of procedures for drug susceptibility prediction of Mycobacterium tuberculosis based on genomic data against the conventional reference method test based on culture is realistic considering the scenario of growing number of tools proposals based on whole-genome sequences (WGS). OBJECTIVES This study aimed to evaluate drug susceptibility testing (DST) outcome based on WGS tools and the phenotypic methods performed on isolates of M. tuberculosis Lineage 1 from the state of Pará, Brazil, generally associated with low levels of drug resistance. METHODOLOGY Culture based DST was performed using the Proportion Method in Löwenstein-Jensen medium on 71 isolates that had been submitted to WGS. We analysed the seven main genome sequence-based tools for resistance and lineage prediction applied to M. tuberculosis and for comparison evaluation we have used the Kappa concordance test. FINDINGS When comparing the WGS-based tools against the DST, we observed the highest level of agreement using TB-profiler. Among the tools, TB-profiler, KvarQ and Mykrobe were those which identified the largest number of TB-MDR cases. Comparing the four most sensitive tools regarding resistance prediction, agreement was observed for 43 genomes. MAIN CONCLUSIONS Drug resistance profiling using next-generation sequencing offers rapid assessment of resistance-associated mutations, therefore facilitating rapid access to effective treatment.

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.

Genomic analysis of Latin American-Mediterranean family of Mycobacterium tuberculosis clinical strains from Kazakhstan

The human-adapted strains of the Mycobacterium tuberculosis complex (MTBC) comprise seven phylogenetic lineages originally associated with their geographical distribution. Here, we report the genomes of three drug-resistant clinical isolates of the Latin American-Mediterranean (LAM) family collected in Kazakhstan. We utilised whole-genome sequencing to study the distribution and drug resistance of these isolates. Phylogenetic analysis grouped the genomes described in this study with the sequences from Russia, Uzbekistan, and Kazakhstan belonging to the LAM family. One isolate has acquired extensive drug resistance to seven antituberculosis drugs. Our results suggest at least two multi-drug resistant (MDR)/extensively drug-resistant (XDR)-associated genotypes of the LAM family circulate in Kazakhstan.

Synthesis, characterization, and in vitro evaluation of gamma radiationinduced PEGylated isoniazid

Background: The search for innovative anti-tubercular agents has received increasing attention in tuberculosis chemotherapy because Mycobacterium tuberculosis infection has steadily increased over the years. This underlines the necessity for new methods of preparation for polymer-drug adducts to treat this important infectious disease. The use of poly(ethylene glycol)(PEG) is an alternative producing anti-tubercular derivatives. However, it is not yet known whether PEGylated isonicotinylhydrazide conjugates obtained by direct links with PEG are useful for therapeutic applications. Results: Here, we synthesized a PEGylated isoniazid (PEG-g-INH or PEG­INH) by gamma radiation-induced polymerization, for the first time. The new prodrugs were characterized using Raman and UV/Vis spectrometry. The mechanism of PEGylated INH synthesis was proposed. The in vitro evaluation of a PEGylated isonicotinylhydrazide macromolecular prodrug was also carried out. The results indicated that PEG­INH inhibited the bacterial growth above 95% as compared with INH, which showed a lower value (80%) at a concentration of 0.25 µM. Similar trends are observed for 0.1, 1, and 5 µM. Conclusions: In summary, the research suggests that it is possible to covalently attach the PEG onto INH by the proposed method and to obtain a slow-acting isoniazid derivative with little toxicity in vitro and higher antimycobacterial potency than the neat drug.

Abandono del tratamiento en tuberculosis multirresistente: factores asociados en una región con alta carga de la enfermedad en Perú / Abandonment of therapy in multidrug-resistant tuberculosis: Associated factors in a region with a high burden of the disease in Perú

Resumen Introducción. En la tuberculosis multirresistente, el abandono del tratamiento constituye un grave problema de salud pública que afecta la calidad de vida de los pacientes, sus familias y la comunidad. El enfrentarlo supone una carga para los sistemas sanitarios debido a que provoca fuentes de transmisión libre en la comunidad e incrementa la prevalencia y la mortalidad. De ahí, la necesidad de investigar los factores asociados con esta situación. Objetivo. Determinar los factores de riesgo asociados con el abandono del tratamiento en pacientes con tuberculosis multirresistente en la región de Callao (Perú). Materiales y métodos. Se hizo un estudio analítico de casos y controles (80 casos y 180 controles) en tratamiento entre el 1° enero del 2010 y el 31 diciembre del 2012. Los factores se determinaron mediante regresión logística, y se calcularon los odds ratios (OR) y los intervalos de confianza (IC) del 95 %. Resultados. En el análisis multivariado se determinaron los siguientes factores de riesgo: no tener conocimiento de la enfermedad (OR=23,10; IC95%: 3,6-36,79; p=0,002); no creer en la curación (OR=117,34; IC95%: 13,57-124,6; p=0,000); no tener apoyo social (OR=19,16; IC95%: 1,32-27,77; p=0,030); no considerar adecuado el horario de atención (OR=78,13; IC95%: 4,84-125,97; p=0,002), y no recibir los resultados de laboratorio (OR=46,13; IC95%: 2,85-74,77; p=0,007). Conclusión. Los servicios de salud deben esforzarse en la determinación precoz de las condiciones que podrían convertirse en factores de riesgo, lo cual ayudaría a implementar preventivamente intervenciones efectivas, rápidas y de alto impacto.

Abstract Introduction: In the context of multidrug-resistant tuberculosis, abandonment of therapy represents a serious public health problem that affects the quality of life of patients, families, and communities. Managing this phenomenon places a burden on health systems since it causes free sources of transmission in the community, thereby increasing prevalence and mortality. Thus, there is a need to study factors associated with this problem. Objective: This study sought to identify risk factors associated with the abandonment of therapy by patients with multidrug-resistant tuberculosis in the Peruvian region of Callao. Materials and methods: We conducted an analytical case-control study (cases=80; controls=180) in patients under treatment from January 1st, 2010, to December 31, 2012. Risk factors were identified using logistic regression; odds ratios (OR) and 95% confidence intervals (CI) were calculated. Results: The multivariate analysis identified the following risk factors: Being unaware of the disease (OR=23.10; 95% CI 3.6-36.79; p=0.002); not believing in healing (OR=117.34; 95% CI 13.57-124.6; p=0.000); not having social support (OR=19.16; 95% CI 1.32-27.77; p=0.030); considering the hours of attention to be inadequate (OR=78.13; 95% CI 4.84-125.97; p=0.002), and not receiving laboratory reports (OR=46.13; 95% CI 2.85-74.77; p=0.007). Conclusion: Health services must focus on the early detection of conditions that may represent risk factors to proactively implement effective, rapid and high-impact interventions.

Characterization of clinical isolates of Mycobacterium tuberculosis from indigenous peoples of Colombia / Caracterización de aislamientos clínicos de Mycobacterium tuberculosis de pueblos indígenas de Colombia

ABSTRACT Introduction: Tuberculosis continues to be a public health priority. Indigenous peoples are vulnerable groups with cultural determinants that increase the risk of the disease. Objective: To determine molecular epidemiology and phenotypical features and of Mycobacterium tuberculosis isolates from indigenous people in Colombia during the period from 2009 to 2014. Materials and methods: We conducted an analytical observational study; we analyzed 234 isolates to determine their patterns of sensitivity to antituberculosis drugs and their molecular structures by spoligotyping. Results: The isolates came from 41 indigenous groups, predominantly the Wayúu (13.10%) and Emberá Chamí (11.35%). We found 102 spoligotypes distributed among seven genetic families (37.2% LAM, 15.8% Haarlem, 8.1% T, 3.4% U, 2.6% S, 2.1% X, and 0.9%, Beijing). The association analysis showed that the non-clustered isolates were related to prior treatment, relapse, orphan spoligotypes, and the Beijing family. The H family presented an association with the Arhuaco and Camëntŝá indigenous groups, the U family was associated with the Wounaan group, and the T family was associated with the Motilón Barí group. Conclusions: This is the first national study on M. tuberculosis characterization in indigenous groups. The study evidenced that diagnosis in indigenous people is late. We described 53% of orphan patterns that could be typical of the Colombian indigenous population. The high percentage of grouping by spoligotyping (62%) could indicate cases of active transmission, a situation that should be corroborated using a second genotyping marker. A new Beijing spoligotype (Beijing-like SIT 406) was identified in Colombia.

RESUMEN Introducción. La tuberculosis es prioridad de salud pública. Los pueblos indígenas son vulnerables debido a los factores culturales determinantes que aumentan el riesgo de tuberculosis. Objetivo. Determinar la epidemiologia molecular y las características fenotípicas de los aislamientos de Mycobacterium tuberculosis de pueblos indígenas de Colombia entre 2009 y 2014. Materiales y métodos. Se hizo un estudio observacional analítico; se analizaron 234 aislamientos para determinar la sensibilidad a los fármacos antituberculosos y la estructura molecular usando spoligotyping. La información epidemiológica se recolectó utilizando el formato único de vigilancia de micobacterias. Resultados. Los aislamientos provenían de 41 grupos indígenas, principalmente los wayúu (13,10 %) y emberá chamí (11,35 %). Se encontraron 102 genotipos distribuidos en siete familias genéticas (37,2 %, LAM; 15,8 %, Haarlem; 8,1 %, T; 3,4 %, U; 2,6 %, S; 2,1 %, X, y 0,9%, Beijing). El análisis de asociación mostró que los aislamientos no agrupados se asociaron con el tratamiento previo, las recaídas, los genotipos huérfanos y la familia Beijing. La familia H presentó una asociación con los grupos indígenas arhuaco y camëntŝá, la familia U se asoció con el grupo wounaan y la familia T con el grupo motilón barí. Conclusiones. Este es el primer estudio nacional de caracterización de M. tuberculosis en grupos indígenas. Se evidenció que el diagnóstico en indígenas es tardío, y que 53 % de los patrones huérfanos podrían ser típicos de la población indígena colombiana. El alto porcentaje de agrupamiento por spoligotyping (62%) podría indicar casos de transmisión activa, una situación que debe ser corroborada usando un segundo marcador de genotipificación. Se identificó un nuevo genotipo (Beijing-like SIT 406) en Colombia.

Perfil dos municípios brasileiros, a presença de tuberculose e sua forma drogarresistente / Brazilian cities profile, the occurence of tuberculosis an its drug-resistant form

Resumo A tuberculose tem estreita relação com as condições de vida das pessoas. O presente estudo classifica os municípios brasileiros de acordo com a presença ou ausência de casos de tuberculose (TB) e sua forma drogarresistente (TB-DR), e os descreve quanto às condições de saúde da população, os indicadores de controle da tuberculose, perfil demográfico e socioeconômico. Em 2014, 327 municípios registraram casos de TB-DR. Regiões integradas de desenvolvimento ou regiões metropolitanas concentraram 80,1% dos casos de TB-DR do País. Os municípios com casos de TB-DR tiveram piores indicadores de desfecho da TB, mas maiores percentuais de realização de exame de cultura, além de mais habitantes e melhores indicadores socioeconômicos. Os 3.644 municípios com casos de TB, mas sem casos de TB-DR, tiveram os piores indicadores socioeconômicos entre os três grupos. Os 1.594 municípios sem casos de TB tiveram as menores taxas de desemprego e de detecção de AIDS, e maior cobertura de atenção básica. Os diferentes perfis encontrados no estudo podem dar suporte à lapidação de estratégias nacionais de controle da doença e sua forma drogarresistente no País.

Abstract Tuberculosis is closely related to living conditions. This study classifies Brazilian municipalities according to the occurrence of tuberculosis (TB) and drug-resistant TB (DR-TB) cases and describes them with regard to the population's health conditions, tuberculosis control indicators, demographic and socioeconomic profile. In 2014, 327 municipalities reported DR-TB cases. Integrated regions of development or metropolitan regions accounted for 80.1% of national DR-TB cases. Municipalities with DR-TB cases had worse TB outcome indicators, but higher culture test percentages, and more inhabitants and better socioeconomic indicators. The 3,644 municipalities with TB cases, but without DR-TB cases, had the worst socioeconomic indicators among the three groups. The 1,594 municipalities without TB cases had the lowest rates of unemployment and AIDS detection and greater coverage of primary healthcare. The different profiles found in the study can sustain improved national interventions for TB and drug-resistant TB control in Brazil.

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.

Tuberculosis por Mycobacterium africanum multirresistente: Caso clinico pediatrico / Tuberculosis by multiresistant Mycobacterium africanum: Pediatric clinical case

La tuberculosis, considerada desde 2003 por la Organización Mundial de la Salud una emergencia global de salud, provoca una mortalidad anual de alrededor de 2 millones de personas, fundamentalmente, en países en vías de desarrollo. En la población pediátrica española, la incidencia es de 5 casos/100 000 niños de entre 5 y 14 años y 13 casos/100 000 niños de entre 0 y 4 años. La infección se transmite por vía respiratoria por enfermos bacilíferos. Los niños eliminan escasos bacilos en secreciones respiratorias y no suelen transmitir la infección. En España, el porcentaje de resistencias a isoniazida en la población general es de 5% y es superior en la población inmigrante, lo cual es importante tener en cuenta para el tratamiento de los casos. Se presenta un caso de tuberculosis por Mycobacterium africanum multirresistente al tratamiento, con evolución satisfactoria posterior a la terapia múltiple.

Tuberculosis, considered since 2003 by the World Health Organization a global health emergency, causes annual mortality of approximately 2 million people, mainly in developing countries. In the Spanish pediatric population, the incidence is 5 cases/100 000 children between 5 and 14 years and 13 cases/100 000 children between 0 and 4 years. The infection is transmitted through the respiratory tract by baciliferous patients. Children eliminate few bacilli in respiratory secretions and do not usually transmit the infection. In Spain, the resistance to isoniazid in the general population is 5%, being higher in the immigrant population, which is important to take into account for the treatment of cases. A case of tuberculosis due to Mycobacterium africanum multiresistant to treatment is presented, with satisfactory evolution after multiple therapy.

In vitro anti-tuberculosis activity of azole drugs against Mycobacterium tuberculosis clinical isolates / Actividad in vitro de azoles contra aislamientos clínicos de Mycobacterium tuberculosis

Background: Latent tuberculosis has been associated with the persistence of dormant Mycobacterium tuberculosis in the organism of infected individuals, who are reservoirs of the bacilli and the source for spreading the disease in the community. New active anti-TB drugs exerting their metabolic action at different stages and on latent/dormant bacilli are urgently required to avoid endogenous reactivations and to be part of treatments of multi- and extensively-drug resistant tuberculosis (M/XDR-TB). It was previously reported that azole drugs are active against M. tuberculosis. For that reason, the aims of this study were to determine the in vitro activity of azole drugs, imidazole (clotrimazole, CLO and econazole, ECO) and nitroimidazole (metronidazole, MZ and ipronidazole, IPZ), against a collection of MDR M. tuberculosis clinical isolates; and to analyze their potential use in both the LTB and the active forms of M/XDR-TB treatments. Methods: A total of 55 MDR M. tuberculosis isolates and H37Rv were included. MZ and IPZ activity against M. tuberculosis isolates were tested using anaerobic culture conditions. The activity of ECO and CLO was measured by the minimal inhibitory concentration (MIC) using a microdilution colorimetric method. Results: MZ and IPZ showed bacteriostatic activity against M. tuberculosis strains. MIC5o and MIC90 to ECO was 4.0 µg/ml, while MIC50 to CLO was 4.0 µg/ml and MIC90 was 8.0 µg/ml respectively. Conclusion: All azole compounds tested in the study showed inhibitory activity against MDR M. tuberculosis clinical isolates.

Introducción: La tuberculosis (TB) latente ha sido asociada a la persistencia de Mycobacterium tuberculosis durmientes en el organismo de las personas infectadas, las cuales constituyen un reservorio del bacilo y una fuente de diseminación de la enfermedad en la comunidad. Urge la necesidad de contar con nuevos fármacos antituberculosos con acción sobre el bacilo en estado latente/durmiente, a fin de evitar reactivaciones endógenas y para ser incluidas en el tratamiento de la TB multirresistente y extensivamente resistente (M/XDR-TB). Se ha reportado que los azoles son activos contra M. tuberculosis. Por esta razón, los objetivos del presente estudio fueron determinar la actividad in vitro sobre aislamientos clínicos de M/XDR-TB de distintos azoles, incluyendo los imidazoles econazol (ECO) y clotrimazol (CLO) y los 5-nitro-imidazoles ipronidazol (IPZ) y metronidazol (MZ), así como analizar su potencial uso contra las formas latente y activa de esta enfermedad. Métodos: Fueron incluidos 55 aislamientos clínicos de M. tuberculosis MDR y la cepa de referencia H37Rv. Se evaluó la actividad del MZ y el IPZ sobre los aislamientos en condiciones de cultivo anaeróbico, mientras que la actividad del ECO y el CLO fue estimada determinando la concentración inhibitoria mínima (CIM) mediante el método colorimétrico de microdilución en placa. Resultados: El MZ y el IPZ presentaron actividad bacteriostática frente a las cepas de M. tuberculosis. La CIM50 y CIM90 del ECO fue de 4 µg/ml, mientras que el CLO presentó una CIM50 de 4 µg/ml y una CIM90 de 8 µg/ml. Conclusión: Todos los compuestos azólicos evaluados presentaron actividad inhibitoria frente a aislamientos clínicos de M. tuberculosis.

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.

Natural Compounds from Mexican Medicinal Plants as Potential Drug Leads for Anti-Tuberculosis Drugs

ABSTRACT In Mexican Traditional Medicine 187 plant species are used in the treatment of respiratory conditions that may be associated with tuberculosis. In this contribution, we review the ethnobotany, chemistry and pharmacology of 63 species whose extracts have been assayed for antimycobacterial activity in vitro. Among these, the most potent is Aristolochia brevipes (MIC= 12.5 µg/mL), followed by Aristolochia taliscana, Citrus sinensis, Chrysactinia mexicana, Persea americana, and Olea europaea (MIC<64 µg/mL). Other potent extracts (inhibition > 95%, 50 µg/mL) include: Amphipterygium adstringens, Larrea divaricata, and Phoradendron robinsoni. Several active compounds have been identified, the most potent are: Licarin A (isolated from A. taliscana), and 9-amino-9-methoxy-3,4-dihydro-2H-benzo[h]-chromen-2-one (transformation product of 9-methoxytariacuripyrone isolated from Aristolochia brevipes), both with MIC= 3.125 µg/mL, that is 8-fold less potent than the reference drug Rifampicin (MIC= 0.5 µg/mL). Any of the compounds or extracts here reviewed has been studied in clinical trials or with animal models; however, these should be accomplished since several are active against strains resistant to common drugs.

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.

Caracterización de aislamientos clínicos de Mycobacterium tuberculosis obtenidos de individuos positivos para HIV en Colombia, 2012 / Characterization of clinical isolates of Mycobacterium tuberculosis from HIV positive individuals in Colombia, 2012

Resumen Introducción: Una tercera parte de los casos nuevos de tuberculosis se atribuye a la propagación del HIV. En 2012, se presentaron en Colombia 1.397 casos de tuberculosis concomitante con la infección por HIV, es decir, 11,8 % del total de notificados. El uso de las herramientas de epidemiología molecular contribuye a una mejor comprensión de la transmisión de la enfermedad. Objetivo: Caracterizar los aislamientos clínicos de Mycobacterium tuberculosis de individuos positivos para HIV recibidos en el Laboratorio Nacional de Referencia del Instituto Nacional de Salud. Materiales y métodos: Se hizo un estudio observacional descriptivo. Se estudiaron 63 aislamientos de individuos con tuberculosis e infección por HIV mediante pruebas de identificación, sensibilidad y genotipificación. Resultados: Dos de los casos nuevos (3,3 %) eran resistentes a rifampicina y uno (1,6 %) a isoniacida, en tanto que tres (5,0 %) lo eran a la isoniacida combinada con estreptomicina. Los casos previamente tratados fueron sensibles. No se evidenció multirresistencia. Hubo 20 (31,7 %) aislamientos de la familia genética LAM9, 8 (12,7 %) de la H1 y 7 (11,1 %) de la T1 . Diecinueve aislamientos correspondieron a patrones huérfanos. Se observó un único agrupamiento entre los aislamientos analizados. No se encontraron diferencias estadísticamente significativas entre la resistencia a fármacos y las familias genéticas. Conclusión: La resistencia encontrada demostró la transmisión de cepas resistentes a rifampicina e isoniacida. Las familias genéticas LAM9, T1 y H1 corresponden a las descritas en la población general. No se evidenció transmisión activa en los aislamientos estudiados. Se necesitan estudios más completos para conocer la situación real de la infección concomitante de tuberculosis y HIV en el país.

Abstract Introduction: One third of the increase in tuberculosis cases is attributed to the spread of HIV. In 2012, 1,397 HIV-associated tuberculosis cases were reported in Colombia, i.e., 11.8% of the total cases. Molecular epidemiology tools help to understand the transmission of tuberculosis. Objective: To characterize clinical isolates of Mycobacterium tuberculosis derived from HIV-infected individuals, received at the Laboratorio Nacional de Referencia in the Instituto Nacional de Salud. Materials and methods: This was a descriptive observational study. We analyzed 63 isolates of M. tuberculosis from HIV-infected individuals. Identification, drug susceptibility and genotyping assays were performed. Results: Of the new cases evaluated, three (5.0%) were resistant to isoniazid combined with streptomycin; two (3.3%) to rifampicin, and one (1.6%) to isoniazid. Previously treated cases were sensitive. No multidrug resistance was evident. Among the predominant genotypes, 20 isolates were (31.7%) LAM9, eight (12.7%), H1, and seven (11.1%), T1. Nineteen isolates corresponded to orphan patterns. One single grouping was observed among tested isolates. We found no statistically significant difference between the proportions of the antituberculous drug resistance and genotypes. Conclusion: We found resistant isolates to the most powerful drugs, rifampicin and isoniazid, among new cases, showing the transmission of resistant strains. Genetic families of M. tuberculosis LAM9, T1 and H1 correspond to those described in the general population. We detected no active transmission among studied isolates. More comprehensive studies are needed to assess the real situation of HIV associated tuberculosis in the country regarding sensitivity and transmission.