Genetic diversity of Mycobacterium avium sp. paratuberculosis by mycobacterial interspersed repetitive Unit-Variable number tandem repeat and multi-locus short-sequence repeat one-sentence summary: Genetic diversity of Mycobacterium avium sp. paratuberculosis isolates.

Background: Paratuberculosis is an enteric disease caused by Mycobacterium avium sp. paratuberculosis (MAP) that affects mainly ruminant producing losses to the livestock industry. Many molecular epidemiological methods have been used to discriminate MAP isolates. Method: The aim of this study was to describe the genetic diversity of the Argentinean MAP isolates using a combination of two molecular systems, the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) ("automated and "non-automated") and the multi-locus short-sequence repeat (MLSSR) system. Results: Thirty-two isolates were identified as MAP of C type by IS900 polymerase chain reaction (PCA) and IS1311 PCA-restriction enzyme analysis. The main patterns found by both MIRU-VNTR systems were INMV1 (54.5%), INMV2 (24.2%) and INMV11 (9.1%). The INMV5, INMV8 and INMV16 were represented with one isolate each (3.0%). Only 4 MIRU-VNTR loci were polymorphic. Conclusion: Those isolates sharing the same INMV patterns were analyzed by MLSSR, being locus 2 the most polymorphic one showing isolates with 9, 10, 11, and more than 11 "G" repeats. Besides, the global discriminatory power among isolates could be increased using both techniques. Based on these results, a short version of the "automated" MIRU-VNTR could be used as a screening tool to group isolates genetically related and subsequently perform the SSR using locus 2 on those isolates sharing the same INMV pattern.

Surveillance and characterization of drug-resistant Mycobacterium tuberculosis isolated in a reference hospital from Argentina during 8 years' period.

Background: Argentina is considered a country with a middle tuberculosis (TB) incidence. However, according to the last national epidemiological report released in 2018, since 2013, the trends are steadily increasing. The aims of this study were to determine the drug-resistance (DR), multi-DR and extensively DR (MDR/XDR-TB), and rifampicin resistance (RIF-R) burden as a part of the local TB diagnosis (June 2010-August 2018); to detect the mutations associated to isoniazid (INH) and RIF-R and their geographical distribution; and to analyze the lineage relationship among the genetic patterns of the isolates circulating in the community. Methods: Respiratory and extrapulmonary specimens were processed by Ziehl-Neelsen stain and cultured on specific media. Drug-susceptibility testing of isolates was performed by the MGIT 960 and a colorimetric micro-method. Mutations conferring DR were detected by Genotype and DNA sequencing. Results: The study showed a DR-TB prevalence of approximately 20% of the isolated strains, while M/XDR-TB-and particularly RIF-R-affected more than 5.0% of the total amount of cases. DR geographical distribution revealed isolates carrying mutations in the inhA gene promoter region only constrained to three districts where it was also registered two same family relatives' cases with the infrequent rpoB S522 L/Q mutation. The fact that most DR/MDR-TB isolates were not grouped in genetic clusters suggested that these cases may mostly have occurred due to endogenous reactivation rather than recently transmission. Conclusion: According to the obtained results, it would be convenient, in highly MDR-TB suspected individuals, to confirm phenotypically, the INH and RIF susceptibility detected by molecular tests.

String test: A new tool for tuberculosis diagnosis and drug-resistance detection in children.

Background: There is a critical need to improve the diagnostic accuracy of tuberculosis (TB) in children. Several techniques have been developed to improve the quality of sputum samples; however, these procedures are very unpleasant and invasive and require hospitalization and trained personnel. This study aims to explore the potential use of a new and noninvasive tool, "string test," for TB diagnosis in children and in adults not able to render sputum samples and at risk of developing multidrug-resistant TB (MDR-TB). Methods: Children with clinical suspicion of TB attending the pediatric consultation at the Cetrangolo or Cordero Hospitals and adults suspected of MDR-TB and unable to produce sputum attending the Infectious Disease Unit of Cetrangolo Hospital were included in this study. Subjects and Methods: The "string test" is a string that is swallowed by the patients and exposed to gastrointestinal secretions that were late analyzed for TB diagnosis and drug-resistance detection by GenoType MTBDRplus. MedCalc software was used to perform statistical analysis. Results: This technique could be applied on 62.1% of selected children. About 11 (30.6%) children were diagnosed as TB cases, 8 (22.2%) from gastric aspirate and using the "string test." Six out of 19 adults were also diagnosed. Genotype directly on the string specimen detected two MDR-TB in adults and two isoniazid-resistant cases before obtaining the isolate. Conclusion: This test was safe, cheap, and easily implemented without requiring hospitalization. This research could represent a significant step forward to diagnose and rapidly detect drug-resistant TB in children.

Evaluation of Mycobacterium tuberculosis cross-resistance to isoniazid, rifampicin and levofloxacin with their respective structural analogs.

The emergence of drug-resistant, multidrug-resistant and extensively drug-resistant tuberculosis (TB) is of major public health concern in several countries. In this study, the pharmacodynamic relationships among the structural analogs of antibiotics belonging to the same family were taken into consideration. The aim of this study was to compare the susceptibility of Mycobacterium tuberculosis to isoniazid (INH), rifampicin and levofloxacin (LX) to their respective structural analogs, which are frequently used as second-line agents. The microplate colorimetric method was used to determine the MIC to INH, ethionamide (ETH), rifampicin, rifabutin, LX and moxifloxacin (MOX) in clinical isolates previously shown to be drug resistant. Mutations conferring drug resistance were detected by GenoType MTBDR plus and DNA sequencing. INH and ETH cross-resistance was found in 95.12% (39/41) of the INH-resistant isolates harboring a mutation in inhAP or inhA open reading frame, but rifabutin cross-resistance was observed in 90.0% (63/70) of the clinical isolates originally shown to be resistant to rifampicin. Isolates with high LX-resistance levels also showed high MIC to MOX. Fluoroquinolone cross-resistance was verified in isolates containing the gyrA94 and the gyrA90 mutation. In general, isolates with high INH, rifampicin and LX-resistance levels also displayed high MIC values for their structural analogs. These findings suggest the need to test in vitro the second-line drugs before their incorporation in the therapeutic schemes.

Meta-análisis: diagnóstico de tuberculosis resistente por técnicas no comerciales de amplificación / Meta-analysis: drug-resistant tuberculosis diagnosis by non commercial amplification techniques / Meta-análise: diagnóstico de tuberculose resistente por técnicas não comerciais de amplificação

La emergencia de tuberculosis (TB) multidrogo y extensivamente-resistente reactivó la necesidad de contar con métodos rápidos para detectar resistencia a isoniacida (INH) y rifampicina (RIF). Por tal motivo, los objetivos de este trabajo fueron evaluar, mediante meta-análisis, la exactitud global y la posible utilidad de métodos caseros basados en PCR para la detección rápida de resistencia a INH y RIF en aislamientos clínicos de Mycobacterium tuberculosis. La búsqueda bibliográfica incluyó Medline/PubMed, BioMedLib. Para estimar la variabilidad entre los resultados de los estudios y el grado de exactitud diagnóstica de los métodos utilizados se realizaron gráficos "forest plot" y curvas SROC (summary receiver operating characteristic) mediante el software Meta-DiSc. Fueron seleccionados 15 estudios, conteniendo 1311 aislamientos resistentes a INH y 953 a RIF. Para la detección de resistencia a INH la sensibilidad y especificidad globales fueron: 84,0% y 96,0% respectivamente, mientras que para la detección de resistencia a RIF esos valores fueron 92,0% y 97,0%. Además, estos métodos mostraron alta exactitud diagnóstica, con áreas bajo la curva SROC>0,9. La alta sensibilidad y especificidad obtenidas con métodos moleculares caseros sugieren que algunos de ellos podrían ser aplicados para el diagnóstico rápido de resistencia a partir del aislamiento de M. tuberculosis.

Due to the emergency of multidrug and extensively-drug resistant tuberculosis, molecular methods for a rapid detection of isoniazid (INH) and rifampicin (RIF) resistance are urgently needed. For that reason, the objectivesof this study were to asses through a meta-analysis the global accuracy and the utility of the home-made molecular methods based in PCR for INH and RIF resistance rapid detection from Mycobacterium tuberculosis clinical isolates. The articles were searched using Medline/PubMed, BioMedLib. The variability among different studies results and the diagnostic accuracy of the used methods were estimated by forest plot and summary receiver operating characteristic (SROC) curves performed with software Meta-DiSc. Fifteen studies were chosed: 1311 containing INH resistant and 953 RIF resistant isolates. The pooled sensitivity and specificity for INH resistance detection was 84.0% and 96.0% respectively, while 92.0% and 97.0% were the pooled values for RIF resistance detection. Besides, these methods showed a high diagnostic accuracy, with the area under the SROC curve >0.9. Due to the high sensitivity and specificity obtained with the home-made molecular methods, some of these tests could be applied for a rapid detection of M. tuberculosis drug resistance in clinical practice.

A emergência de tuberculose (TB) multidrogas e extensivamente-resistente reativou a necessidade de contar com métodos rápidos para detectar resistência à isoniazida (INH) e rifampicina (RIF). Por isso, o objetivo deste trabalho foi a avaliação através da meta-análise, da exatidão global e da possível utilidade de métodos caseiros baseados em PCR para detectar rapidamente a resistência a INH e RIF em isolamentos clínicos de Mycobacterium tuberculosis. A pesquisa bibliográfica incluiu Medline/PubMed, Bio MedLib. Para estimar a variabilidade entre os resultados dos estudos e o grau de exatidão diagnóstica dos métodos utilizados, foram realizados gráficos "forest plot" e curvas SROC (summary receiver operating characteristic) com o software Meta-Disc. Foram selecionados 15 estudos, contendo 1311 isolamentos resistentes a INH e 953 a RIF. Para a detecção de resistência a INH, a sensibilidade e especificidade globais foram 84,0% e 96,0% respectivamente, enquanto que para a detecção de resistência a RIF esses valores foram de 92,0% e 97,0%. Alem disso, os mesmos métodos mostraram elevada exatidão diagnóstica, com áreas inferiores à curva SROC>0,9. A elevada sensibilidade e especificidade obtida através de métodos moleculares caseiros sugere que alguns deles poderiam ser aplicados para o diagnóstico rápido de resistência a partir do isolamento de M. tuberculosis.

Meta-análisis: diagnóstico de tuberculosis resistente por técnicas no comerciales de amplificación / Meta-analysis: drug-resistant tuberculosis diagnosis by non commercial amplification techniques / Meta-análise: diagnóstico de tuberculose resistente por técnicas nÒo comerciais de amplificaþÒo

La emergencia de tuberculosis (TB) multidrogo y extensivamente-resistente reactivó la necesidad de contar con métodos rápidos para detectar resistencia a isoniacida (INH) y rifampicina (RIF). Por tal motivo, los objetivos de este trabajo fueron evaluar, mediante meta-análisis, la exactitud global y la posible utilidad de métodos caseros basados en PCR para la detección rápida de resistencia a INH y RIF en aislamientos clínicos de Mycobacterium tuberculosis. La búsqueda bibliográfica incluyó Medline/PubMed, BioMedLib. Para estimar la variabilidad entre los resultados de los estudios y el grado de exactitud diagnóstica de los métodos utilizados se realizaron gráficos "forest plot" y curvas SROC (summary receiver operating characteristic) mediante el software Meta-DiSc. Fueron seleccionados 15 estudios, conteniendo 1311 aislamientos resistentes a INH y 953 a RIF. Para la detección de resistencia a INH la sensibilidad y especificidad globales fueron: 84,0% y 96,0% respectivamente, mientras que para la detección de resistencia a RIF esos valores fueron 92,0% y 97,0%. Además, estos métodos mostraron alta exactitud diagnóstica, con áreas bajo la curva SROC>0,9. La alta sensibilidad y especificidad obtenidas con métodos moleculares caseros sugieren que algunos de ellos podrían ser aplicados para el diagnóstico rápido de resistencia a partir del aislamiento de M. tuberculosis.(AU)

Due to the emergency of multidrug and extensively-drug resistant tuberculosis, molecular methods for a rapid detection of isoniazid (INH) and rifampicin (RIF) resistance are urgently needed. For that reason, the objectivesof this study were to asses through a meta-analysis the global accuracy and the utility of the home-made molecular methods based in PCR for INH and RIF resistance rapid detection from Mycobacterium tuberculosis clinical isolates. The articles were searched using Medline/PubMed, BioMedLib. The variability among different studies results and the diagnostic accuracy of the used methods were estimated by forest plot and summary receiver operating characteristic (SROC) curves performed with software Meta-DiSc. Fifteen studies were chosed: 1311 containing INH resistant and 953 RIF resistant isolates. The pooled sensitivity and specificity for INH resistance detection was 84.0% and 96.0% respectively, while 92.0% and 97.0% were the pooled values for RIF resistance detection. Besides, these methods showed a high diagnostic accuracy, with the area under the SROC curve >0.9. Due to the high sensitivity and specificity obtained with the home-made molecular methods, some of these tests could be applied for a rapid detection of M. tuberculosis drug resistance in clinical practice.(AU)

A emergÛncia de tuberculose (TB) multidrogas e extensivamente-resistente reativou a necessidade de contar com métodos rápidos para detectar resistÛncia O isoniazida (INH) e rifampicina (RIF). Por isso, o objetivo deste trabalho foi a avaliaþÒo através da meta-análise, da exatidÒo global e da possível utilidade de métodos caseiros baseados em PCR para detectar rapidamente a resistÛncia a INH e RIF em isolamentos clínicos de Mycobacterium tuberculosis. A pesquisa bibliográfica incluiu Medline/PubMed, Bio MedLib. Para estimar a variabilidade entre os resultados dos estudos e o grau de exatidÒo diagnóstica dos métodos utilizados, foram realizados gráficos "forest plot" e curvas SROC (summary receiver operating characteristic) com o software Meta-Disc. Foram selecionados 15 estudos, contendo 1311 isolamentos resistentes a INH e 953 a RIF. Para a detecþÒo de resistÛncia a INH, a sensibilidade e especificidade globais foram 84,0% e 96,0% respectivamente, enquanto que para a detecþÒo de resistÛncia a RIF esses valores foram de 92,0% e 97,0%. Alem disso, os mesmos métodos mostraram elevada exatidÒo diagnóstica, com áreas inferiores O curva SROC>0,9. A elevada sensibilidade e especificidade obtida através de métodos moleculares caseiros sugere que alguns deles poderiam ser aplicados para o diagnóstico rápido de resistÛncia a partir do isolamento de M. tuberculosis.(AU)