In vitro anti-tuberculosis activity of azole drugs against Mycobacterium tuberculosis clinical isolates

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.Inst. de BiotecnologíaFil: Imperiale, Belen Rocio. Hospital Dr. Antonio A. Cetrángolo. Laboratorio de Referencia del Programa de Control de la Tuberculosis de la provincia de Buenos Aires; ArgentinaFil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Morcillo, Nora. Hospital Dr. Antonio A. Cetrángolo. Laboratorio de Referencia del Programa de Control de la Tuberculosis de la provincia de Buenos Aires; Argentin

Rifampin-Isoniazid Oligonucleotide Typing: an Alternative Format for Rapid Detection of Multidrug-Resistant Mycobacterium tuberculosis

Fil: Hernandez-Neuta, Iván. Corporacion CorpoGen; Colombia.Fil: Varela, Andres. Corporacion CorpoGen; Colombia.Fil: Martin, Anandi. Institute of Tropical Medicine. Mycobacteriology Unit; Bélgica.Fil: von Groll, Andrea. Institute of Tropical Medicine. Mycobacteriology Unit; Bélgica.Fil: Jureen, Pontus. Swedish Institute for Infectious Disease Control; Suecia.Fil: López, Beatriz. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Imperiale, Belen. Hospital Dr. Cetrángolo; Argentina.Fil: Skenders, Girts. State Agency of Tuberculosis and Lung Diseases; Letonia.Fil: Ritacco, Viviana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Hoffner, Sven. Swedish Institute for Infectious Disease Control; Suecia.Fil: Morcillo, Nora. Hospital Dr. Cetrángolo; Argentina.Fil: Palomino, Juan Carlos. Institute of Tropical Medicine. Mycobacteriology Unit; Bélgica.Fil: Del Portillo, Patricia. Corporacion CorpoGen; Colombia.on of rifampin and isoniazid resistance in clinical isolates of Mycobacterium tuberculosis was based on the same amplification/reverse hybridization principle of the widely used spoligotyping. The test involved probing nine DNA regions that are targets of common drug resistance-associated mutations in the genes rpoB, katG, and inhA. Addition of quaternary amine tetramethyl ammonium chloride to the hybridization buffer promoted multiple hybrid formations at a single annealing temperature irrespective of the different GC contents of probes. The assay was standardized using 20 well-documented strains from the Institute of Tropical Medicine (Belgium) and evaluated blindly in a central laboratory with 100 DNA samples that were obtained from cultured clinical isolates and shipped dried from three other countries. Compared with drug susceptibility testing, both sensitivity and specificity for rifampin resistance detection were 93.0% while for isoniazid the values were 87.7% and 97.7%, respectively. Compared with sequencing and GenoType MTBDRplus methods, sensitivity and specificity reached 96.4% and 95.5% for rifampin and 92.7% and 100% for isoniazid. Altogether, 40/45 (89%) multidrug-resistant isolates were correctly identified. Advantages of this in-house development include versatility, capacity to run up to 41 samples by triplicate in a single run, and reuse of the membrane at least 10 times. These features substantially reduce cost per reaction and make the assay an attractive tool for use in reference laboratories of countries that have a high burden of multidrug-resistant tuberculosis but that cannot afford expensive commercial tests because of limited resources

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.Instituto de BiotecnologíaFil: Moyano, Roberto Damian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Moyano, Roberto Damian. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Imperiale, Belen Rocio. Academia Nacional de Medicina. Instituto de Medicina Experimental (IMEX); ArgentinaFil: Imperiale, Belen Rocio. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Romero, Magalí. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Centro de Diagnóstico e Investigación Veterinaria (CEDIVE); ArgentinaFil: Santangelo, María De La Paz. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Santangelo, María De La Paz. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alvarado Pinedo, Maria Fiorella. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Centro de Diagnóstico e Investigación Veterinaria (CEDIVE); ArgentinaFil: Travería, Gabriel. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Centro de Diagnóstico e Investigación Veterinaria (CEDIVE); ArgentinaFil: Romano, Maria Isabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Romano, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

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

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.Instituto de BiotecnologíaFil: Imperiale, Belen Rocio. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Di Giulio, Angela Beatríz. Hospital Dr. Petrona V. de Cordero. Laboratorio de Micobacterias; ArgentinaFil: Mancino, María Belén. Hospital Dr. Antonio A. Cetrángolo. Laboratorio de Referencia del Programa de Control de la Tuberculosis de la provincia de Buenos Aires; ArgentinaFil: Zumarraga, Martin Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Morcillo, Nora. Hospital Dr. Antonio A. Cetrángolo. Laboratorio de Referencia del Programa de Control de la Tuberculosis de la provincia de Buenos Aires; Argentin

Fitness of drug resistant Mycobacterium tuberculosis and the impact on the transmission among household contacts

There has been an on-going debate on whether the development of drug resistance in Mycobacterium tuberculosis reduces its relative fitness and its ability to cause disease. The aim of this study was to explore this relationship. For this purpose, we evaluated the in vitro growth of clinical isolates and the transmission of the strains within the patients' households. Clinical and epidemiological data from patients in households, drug-susceptibility and genetic patterns of the isolates were collected. BACTEC MGIT 960™ system with the Epicenter™ software was used to perform fitness experiments and calculate the relative fitness (RF) comparing with the H73Rv reference strain. From 39 households, 124 patients and 388 contacts were included. Concerning transmission, 20 Multi drug-resistant (MDR) and 16 drug sensitive (DS) index cases generated 23 and 28 secondary cases, respectively. An average RF drop of 16.7% was found for MDR strains, but only mutations in rpoB codons 531 were associated with reduced fitness. When the strains were transmitted, their RF tended to decrease, and strains with low RF were less frequently transmitted. Within the limitations of this study, the results showed that the decrease in RF was associated to a limited transmission among the households' contacts.Instituto de BiotecnologíaFil: Morcillo, Nora. Hospital Dr. Antonio A. Cetrángolo. Laboratorio de Referencia del Programa de Control de la Tuberculosis de la provincia de Buenos Aires; ArgentinaFil: Imperiale, Belen Rocio. Hospital Dr. Antonio A. Cetrángolo. Laboratorio de Referencia del Programa de Control de la Tuberculosis de la provincia de Buenos Aires; ArgentinaFil: Di Giulio, Angela Beatríz. Hospital Dr. Petrona V. de Cordero. Laboratorio de Micobacterias; ArgentinaFil: Zumarraga, Martin Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Takif, Howard. Instituto Venezolano de Investigaciones Científicas; VenezuelaFil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

Crystal violet decolorization assay for rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates: A multicenter study

Background: Effective control of tuberculosis is achieved by early diagnosis and drug susceptibility testing for initiation of appropriate treatment. The performance of crystal violet decolorization assay (CVDA) for susceptibility testing of Mycobacterium tuberculosis to isoniazid (INH) and rifampicin (RIF) was compared in a multicenter study. Methods: Seventy-two M. tuberculosis isolates were tested in two phases by CVDA. Results: In Phase I, the specificity, sensitivity, positive predictive value (PPV), negative predictive value (NPV), and agreement for INH were 100%, respectively. Specificity, sensitivity, PPV, NPV, and agreement for RIF were 98.2%, 100%, 94.1%, 100%, and 98.6%, respectively. In Phase II, specificity, sensitivity, PPV, NPV, and agreement were 98%, 100%, 95.4%, 100%, and 98.6% for INH, respectively. Specificity, sensitivity, PPV, NPV, and agreement for RIF were 96.3%, 88.2%, 88.2%, 96.3%, and 94.4%, respectively. Results in the study were obtained on average 10.9 ± 3.1 days in Phase I and 9.8 ± 2.2 days in Phase II. Conclusion: CVDA can be performed for drug susceptibility testing in developed and developing countries. In addition, further studies with larger sample size are needed for evaluation of this method

Genetic diversity of Mycobacterium avium complex strains isolated in Argentina by MIRU-VNTR

Mycobacterium avium sp. avium (MAA), M. avium sp. hominissuis (MAH), and M. avium sp. paratuberculosis (MAP) are the main members of the M. avium complex (MAC) causing diseases in several hosts. The aim of this study was to describe the genetic diversity of MAC isolated from different hosts. Twenty-six MAH and 61 MAP isolates were recovered from humans and cattle, respectively. GenoType CM® and IS1311-PCR were used to identify Mycobacterium species. The IS901-PCR was used to differentiate between MAH and MAA, while IS900-PCR was used to identify MAP. Genotyping was performed using a mycobacterial interspersed repetitive-unitvariable-number tandem-repeat (MIRU-VNTR) scheme (loci: 292, X3, 25, 47, 3, 7, 10, 32) and patterns (INMV) were assigned according to the MAC-INMV database (http://mac-inmv.tours. inra.fr/). Twenty-two (22/26, 84·6%) MAH isolates were genotyped and 16 were grouped into the following, INMV 92, INMV 121, INMV 97, INMV 103, INMV 50, and INMV 40. The loci X3 and 25 showed the largest diversity (D: 0·5844), and the global discriminatory index (Hunter and Gaston discriminatory index, HGDI) was 0·9300. MAP (100%) isolates were grouped into INMV 1, INMV 2, INMV 11, INMV 8, and INMV 5. The HGDI was 0·6984 and loci 292 and 7 had the largest D (0·6980 and 0·5050). MAH presented a higher D when compared with MAP. The MIRU-VNTR was a useful tool to describe the genetic diversity of both MAH and MAP as well as to identify six new MAH patterns that were conveniently reported to the MAC-INMV database. It was also demonstrated that, in the geographical region studied, human MAC cases were produced by MAH as there was no MAA found among the human clinical samples.Inst. de BiotecnologíaFil: Imperiale, Belen Rocio. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Moyano, Roberto Damian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Di Giulio, A.B. Hospital Dr. Petrona V. de Cordero. Laboratorio de Micobacterias; San Fernando, ArgentinaFil: Romero, M.A. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Centro de Diagnóstico e Investigación Veterinaria; ArgentinaFil: Alvarado Pinedo, Maria Fiorella. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Centro de Diagnóstico e Investigación Veterinaria; ArgentinaFil: Santangelo, María De La Paz. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Travería, G.E. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Centro de Diagnóstico e Investigación Veterinaria; ArgentinaFil: Morcillo, Nora S.Hospital Dr. Antonio A. Cetrángolo. Laboratorio de Referencia del Programa de Control de la Tuberculosis de la provincia de Buenos Aires; ArgentinaFil: Romano, Maria Isabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Relevancia clínica, diversidad y variabilidad genética de distintas especies del género Mycobacterium

Introducción: El término micobacterias no tuberculosas (MNT) incluye distintas especies ambientales capaces de enfermar humanosy/o animales incluso mediante una probable transmisión zoonótica Objetivos. Determinar: la importancia clínica de varias especies del género Mycobacterium y la diversidad genética del Complejo M. avium (MAC), la sensibilidad bacteriana in vitro yel éxito del tratamiento especifico. Materiales y Métodos: Recolección de datos clínicos, epidemiológicos y aislamientos en el periodo 2009-2016; identificación molecular de los aislamientos; determinación de la sensibilidad bacteriana in vitro y de la diversidad genética del MAC; evaluación del tratamiento. Resultados: Fueron diagnosticados 225 casos de micobacteriosis, con prevalencia estable ≈ 6% por año, y 22 especies recuperadas: 4 de rápido desarrollo aisladas de 66 pacientes y 18 de lento desarrollo. MAC fue aislado en 95 casos, 40 M. avium hominissuis, 51 M. intracellulare, 3 M. chimaera, 1 M. colombiense. Se observó mayor probabilidad de enfermar por M. intracellulare en pacientes tratados previamente por tuberculosis (TB). Los pacientes HIV+ tuvieron riesgo incrementado de enfermedad causada por M. avium hominissuis. Los aminoglucósidos, fluoroquinolonas y macrólidos fueron las drogas más activas frente a la mayoría de las MNT. Aproximadamente la mitad de los casos curaron. Conclusiones: M. intracellulare, M. aviumhominissuis con una gran variabilidad genética, y M. abscessus fueron los patógenos más frecuentemente hallados. Un hallazgo importante fue el de casos de enfermedad mixta TB+MNT. Estos pacientes requirieron una terapia con agregado de drogas de segunda línea al esquema terapéutico para TB habiendo curado la mayoría de ellos.Fil: Imperiale, Belen. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; ArgentinaFil: Di Giulio, Beatriz. Hospital Petrona V. de Cordero; ArgentinaFil: Moyano, Roberto Damian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; ArgentinaFil: Santangelo, María de la Paz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; ArgentinaFil: Tartara, Silvina. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; ArgentinaFil: Alonso, Viviana. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; ArgentinaFil: Sanjurjo, Myrian. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; ArgentinaFil: Garcia, Graciela. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; ArgentinaFil: Castellano, Patricia. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; ArgentinaFil: Romano, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; ArgentinaFil: Morcillo, Nora. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; Argentin