Aplicación de PCR a partir de cultivo en la identificación de subespecies del complejo Mycobacterium Tuberculosis / The use of culture-based PCR for the identification of subspecies of the mycobacterium tuberculosis complex

Los métodos diagnósticos clásicos de tuberculosis (TB) se basan en la utilización de baciloscopía y cultivo. La identificación del agente etiológico desde la positivización del cultivo requiere entre 10 y 15 días, mientras que el empleo de la reacción en cadena de la polimerasa (PCR) disminuye el tiempo a 24 h, lo que permite no solo identificar las subespecies del complejo Mycobacterium tuberculosis (CMTB) sino también diferenciarlas de otras especies ambientales clínicamente importantes (MOTT) facilitando el diagnóstico y tratamiento. El objetivo del presente trabajo fue determinar la utilidad de la PCR en la identificación temprana de las micobacterias pertenecientes al CMTB, a partir de cultivos positivos, de pacientes con sospecha de TB, atendidos en un hospital pediátrico de alta complejidad, durante un período de cuatro años. A cada muestra, se le realizó baciloscopía y cultivo en medio líquido. A los cultivos positivos, una inmunocromatografía lateral (TBIDR) y luego PCR. El 4,6% del total de muestras (510/11.162) pertenecientes a 198 pacientes presentó cultivos positivos. Cuatrocientos veintiseis (84%) correspondieron a muestras respiratorias. El rendimiento de la baciloscopía directa fue del 41% (194/470). Cuatrocientos treinta y ocho (86%) resultaron M. tuberculosis, 21 (4%) Mycobacterium bovis, 7 (1%), M. bovis-BCG y 44 (9%) MOTT. La utilización de medios de cultivos líquidos junto con el empleo de PCR favorecen una rápida orientación microbiológica y constituye una estrategia útil para optimizar el manejo clínico de estas infecciones, desde el punto de vista terapéutico y epidemiológico, especialmente en pediatría (AU)

Classical diagnostic methods for tuberculosis (TB) are based on the use of smear microscopy and culture. The identification of the etiological agent from positive culture requires 10 to 15 days, while the use of the polymerase chain reaction (PCR) reduces the time to 24 h, which allows not only to identify the subspecies of the Mycobacterium tuberculosis complex (MTC) but also to differentiate them from clinically important environmental mycobacteria other than tuberculosis (MOTT), facilitating diagnosis and treatment. The aim of this study was to determine the usefulness of PCR in the early identification of mycobacteria belonging to the MTC, from positive cultures of patients with suspected TB seen in a pediatric tertiary hospital over a 4-year period. For each sample, smear microscopy and culture in liquid medium was performed. Positive cultures were subjected to lateral immunochromatography (TBIDR) and then PCR. Of the total number of samples (510/11,162) belonging to 198 patients, 4.6% showed positive cultures; 426 (84%) were respiratory samples. The direct smear microscopy yield was 41% (194/470). Overall, 438 (86%) were found to be M. tuberculosis, 21 (4%) Mycobacterium bovis, 7 (1%), M. bovis-BCG, and 44 (9%) MOTT. The use of liquid culture media together with the use of PCR favors a rapid microbiological orientation and is a useful strategy to optimize the clinical management of these infections, from a therapeutic and epidemiological point of view, especially in children (AU)

Pericarditis purulenta por mycobacterium tuberculosis y streptococcus pneumoniae: revisión del tema, a propósito de un caso / Purulent pericarditis due to mycobacterium tuberculosis and streptococcus pneumoniae: case report and review

La pericarditis purulenta es una patología poco frecuente pero que conlleva alta mortalidad. En la era pre antibióticos, se observaba en pacientes con neumonía complicada y las cocáceas gram positivas eran los gérmenes frecuentemente involucrados. Por otro lado, la pericarditis tuberculosa representa el 1% del total de casos de tuberculosis, aunque es frecuente zonas endémicas, principalmente asociada a la infección por el virus de la inmunodeficiencia humana (VIH). Presentamos el caso de un paciente de 19 años, en situación calle, infectado con VIH, con diagnóstico de pericarditis purulenta, donde se demostró la co-infección de Mycobacterium tuberculosis y Streptecoccus pneumoniae en el pericardio. La pericarditis purulenta polimicrobiana es poco frecuente y la co-infección por los gérmenes mencionados es anecdótica. A pesar del tratamiento antimicrobiano, el aseo quirúrgico, los esteroides y la fibrinolisis intrapericárdica, esta patología tiene un pronóstico ominoso, en parte, debido a la condición basal de los enfermos que la padecen.

Purulent pericarditis is a rare disease with a high mortality rate. In the pre-antibiotic era it was observed as a complication in patients with pneumonia. Gram-positive coccaceae were the most commonly implicated bacteria. Tuberculous pericarditis represents 1% of all tuberculosis (TBC) cases, although it is common in endemic areas, associated with human immunodeficiency virus (HIV) infection. We present the case of a 19-year-old homeless, admitted with HIV and malnutrition, diagnosed with purulent pericarditis. Mycobacterium tuberculosis and Streptococcus pneumoniae were found as a cause of purulent pericarditis. Polymicrobial purulent pericarditis is a rare condition and co-infection with the bacteria previously mentioned is merely anecdotal. Despite antimicrobial treatment, surgical management, steroids, and intrapericardial fibrinolysis, this pathology has an ominous prognosis, due in part to the pre-existing condition of these patients.

El dilema en los laboratorios de Koch, ¿Cultivar o no Cultivar? / The Dilemma in Koch's Laboratories, To Cultivate or Not to Cultivate?

La nueva norma técnica para el control y la eliminación de la tuberculosis es un gran avance para el diagnóstico de este microorganismo en Chile. Actualmente la principal técnica microbiológica para el diagnóstico de laboratorio es la biología molecular, que reduce el tiempo del resultado a tan solo un par de horas. La normativa actual indica que en el paciente caso presuntivo de tuberculosis (CPT) la técnica exclusiva a realizar es Biología molecular. La literatura indica que la detección a través de amplificación de material genético de la micobacteria tiene un límite de detección de 15,6 UFC/ ml, por tanto, todas las muestras bajo ese límite umbral potencialmente podrían no ser diagnosticadas bajo esta estructura emanada por el ministerio de Salud en Chile. Nuestra recomendación es continuar con el estudio de cultivo en medios líquidos o sólidos para todas las muestras hasta obtener literatura que avale lo contrario

The new technical standard for the control and elimination of tuberculosis in Chile is a great advance for the diagnosis of this microorganism. Currently the main microbiological technique for laboratory diagnosis is PCR, which reduces the time to result to just a couple of hours. The current regulations indicate that in the patient with a presumptive case of tuberculosis (CPT) t he exclusive technique to be performed is PCR. The literature indicates that the detection through amplification of genetic material of the mycobacterium has a detection limit of 15.6 CFU/ml, therefore, all samples under this threshold limit could potentially not be diagnosed under this structure emanated by the Ministry of Health in Chile. Our recommendation is to continue with the study of culture in liquid or solid media for all samples until literature confirms otherwise

Diversidad genética de Mycobacterium tuberculosis circulando en la jurisdicción V de Jalapa, Veracruz, México / Genetic diversity of Mycobacterium tuberculosis circulating in the jurisdiction V from Jalapa, Veracruz, Mexico

ANTECEDENTES: El estado de Veracruz se ubica en el sureste de México y presenta una alta prevalencia de tuberculosis (TBC) y drogo resistencia. Sin embargo, la composición de los genotipos circulantes es poco conocida. OBJETIVO: Caracterizar la diversidad genética de la TBC en la jurisdicción sanitaria V del estado de Veracruz. MÉTODOS: Estudio transversal realizado en aislados clínicos de pacientes con TBC residentes de la jurisdicción V. Se determinó la sensibilidad a medicamentos de primera línea. La genotipificación se realizó mediante espoligotipificación y MIRU-VNTR 15 loci. RESULTADOS: Entre los 74 aislados analizados se observó resistencia a un fármaco en 44 (59%) aislados. Linaje L4 (EuroAmericano) se presentó en 73 aislados. Se identificaron cinco sublinajes; H (40%), T (22%), LAM (16%), X (13%) y U (7%). El 32% de los aislados se agrupó mediante su espoligotipo y 40% en 10 complejos clonales. CONCLUSIONES: Es la primera descripción sobre la estructura genética de TBC en la región central de Veracruz. La diversidad de genotipos podría contribuir a su dispersión en la región. Esta información será útil para el desarrollo de intervenciones y reducir el impacto de TBC en la población.

BACKGROUND: The state of Veracruz is placed in southeastern Mexico and has a high prevalence of tuberculosis (TB) and drug resistance. Nevertheless, the composition of circulating genotypes in the central region of the state is partially known. AIM: To characterize the genetic diversity of TB in the sanitary jurisdiction V of the state of Veracruz. METHODS: A cross-sectional study was conducted among clinical isolates from patients with TB living in the jurisdiction V, in Jalapa Ver., Mexico. Sensitivity to first-line drugs was determined, and genotyping was performed by spoligotyping and MIRU-VNTR 15 loci. RESULTS: Among the 74 isolates analyzed, resistance to one drug was observed in 44 isolates. L4 (EuroAmerican) was the major lineage identified. Five sublineages were the most abundant; H (40%), T (22%), LAM (16%), X (13%) and U (7%). Only 32% of the isolates were clustered by spoligotype and 40% were placed in ten clonal complexes. CONCLUSIONS: This is the first description of the genetic structure of TB in the central region of Veracruz. The diversity of genotypes could contribute to its dispersion. This information will be useful for the development of interventions to reduce the impact of TB in the population.

Biomarcadores en tuberculosis / Biomarkers in tuberculosis

Resumen Los métodos diagnósticos clásicos para la tuberculosis son de baja sensibilidad o son muy lentos en la obtención de resultados (baciloscopía, cultivo de Koch). De ahí nace la necesidad de nuevos métodos diagnósticos para esta enfermedad. Los biomarcadores surgen como una opción a esta problemática, con un buen rendimiento diagnóstico, costo y accesibilidad. Ellos permiten identificar la respuesta inflamatoria y/o metabólica del huésped, extrapolando la presencia de Mycobacterium tuberculosis; o identifican moléculas propias del patógeno. En la presente revisión se describen biomarcadores que presentan un buen rendimiento diagnóstico basados en metodologías de investigación de alto nivel (estudio de cohortes, prospectivos, muestreo consecutivo o aleatorizado, comparación de rendimiento diagnóstico frente a cultivo). Es necesario el desarrollo de estas nuevas técnicas con el fin de realizar el diagnóstico precoz de la enfermedad y lograr así su tan ansiada eliminación.

The classical laboratory diagnostic methods for tuberculosis have a low sensitivity or take a long time to know their results. New methods are underway. Biomarkers are a good option to improve our diagnostic approach to this disease. They have good performance, low cost and accessibility. They identify a patient's inflammatory or metabolic response to Mycobacterium Tuberculosis or identifies molecules that are typical of the pathogen. In this paper we sum up the biomarkers with a good diag-nostic performance described in well design investigations. Early diagnosis with these new techniques should contribute to the elimination of the disease.

Tuberculosis meníngea: reporte de casos años 2005-2017 / Meningeal tuberculosis: cases report years 2005-2017

INTRODUCCIÓN: La tuberculosis (TBC) continúa siendo un problema de salud pública mundial; su forma meníngea conlleva mayor letalidad y secuelas, en particular si se asocia a la infección por VIH/SIDA. OBJETIVO: Describir las características demográficas, presentación clínica, laboratorio y de las imágenes de los pacientes con TBC meníngea (aislamiento de Mycobacterium tuberculosis en LCR), analizando diferencias entre pacientes con y sin infección por VIH/SIDA. PACIENTES Y MÉTODOS: Estudio observacional y descriptivo, retrospectivo, de una serie de casos atendidos en el Hospital Dr. Alejandro Posadas de Buenos Aires, Argentina, desde enero de 2005 hasta diciembre de 2017. RESULTADOS: Se analizaron 36 pacientes, 22 de ellos mujeres, con una mediana de edad de 36,5 años. Veintidós pacientes presentaron co-infección por VIH, todos en estadio SIDA. El tiempo de inicio de síntomas tuvo una mediana 11 días. predominando fiebre, estado de conciencia alterado y cefalea. En el LCR se hallaron linfocitosis, hipoglucorraquia, hiperproteinorraquia y ácido láctico elevado. Se realizó tomografía computada de encéfalo a 34 pacientes, 16 sin alteraciones. En otros 16 se realizó resonancia magnética (RM) cerebral, 9 presentaban trastornos vasculares. La RM fue más sensible para identificar refuerzo meníngeo, trastornos de tipo vasculares, y lesiones de tipo granulomatosas. La mediana de inicio de tratamiento fue de 1 día, con 72,2% recibiendo co-adyuvancia con corticosteroides. La mortalidad observada fue de 27,7% y secuelas hubo en 36,1%. Sólo 5 pacientes requirieron intervención neuro-quirúrgica. CONCLUSIÓN: Siendo la TBC meníngea una afección de alta morbimortalidad, es imperioso asegurar un diagnóstico temprano en su evolución mediante la incorporación de la biología molecular e imagenología (RM) al amplio uso clínico.

BACKGROUND. Tuberculosis (TB) continues to be a global public health problem; its meningeal form leads to greater lethality and sequelae, particularly if it is associated with HIV / AIDS infection. AIM: To describe the demographic characteristics, clinical presentation, laboratory and images of patients with meningeal TB (isolation of Mycobacterium tuberculosis in CSF), analyzing differences between HIV and non-HIV patients. METHODS: We performed an observational and descriptive study, with retrospective analysis of patients attending at the Dr. Alejandro Posadas Hospital, Buenos Aires, since January 2005 to December 2017. RESULTS: Thirty-six patients were analyzed, with 22 women with a median age of 36.5 years. Twenty two patients had HIV coinfection, all in the AIDS stage. The symptom onset time was median 11 days. The predominant ones were fever, altered consciousness and headache. In the cerebrospinal fluid were lymphocitosis, hypoglycorrhachia, hyperproteinorrhachia and high lactic acid, according to previously described findings. Of 34 patients who underwent brain scan, 16 patients had no significant pathological findings. MRI was performed in 16 patients, 9 had vascular disorders. Brain MRI was more sensitive to identify meningeal reinforcement than computerized tomography, vascular disorders, and granulomatous lesions. The median onset of treatment was 1 day, with 72.2% of the total receiving coadjuvants with corticosteroids. Mortality of 27.7% and sequelae in 36.1% were observed. Only 5 patients required neurosurgical intervention. CONCLUSION: Since meningeal TB is a disease with high morbidity and mortality, it is imperative to ensure an early diagnosis in its evolution by incorporating molecular biology and imaging (MRI) into broad clinical use.

Molecular epidemiology of Mycobacterium tuberculosis in Brazil before the whole genome sequencing era: a literature review

Molecular-typing can help in unraveling epidemiological scenarios and improvement for disease control strategies. A literature review of Mycobacterium tuberculosis transmission in Brazil through genotyping on 56 studies published from 1996-2019 was performed. The clustering rate for mycobacterial interspersed repetitive units - variable tandem repeats (MIRU-VNTR) of 1,613 isolates were: 73%, 33% and 28% based on 12, 15 and 24-loci, respectively; while for RFLP-IS6110 were: 84% among prison population in Rio de Janeiro, 69% among multidrug-resistant isolates in Rio Grande do Sul, and 56.2% in general population in São Paulo. These findings could improve tuberculosis (TB) surveillance and set up a solid basis to build a database of Mycobacterium genomes.

Escrofuloderma, una manifestación extrapulmonar de tuberculosis: a propósito de un caso y actualización de esquema de tratamiento en Chile / Scrofuloderma, a manifestation extrapulmonary tuberculosis: about a case and treatment scheme update in Chile

La tuberculosis (TBC) es una enfermedad infecciosa causada por organismos del complejo Mycobacterium tuberculosis. Las presentaciones extrapulmonares constituyen hasta el 25% de los casos de TBC reportados en nuestro país. La TBC cutánea es una manifestación extrapulmonar rara que representa el 1-2% de los casos, siendo el escrofuloderma y el lupus vulgar las formas clínicas más comunes. El escrofuloderma es una manifestación endógena de la infección, como resultado de la extensión contigua a la piel suprayacente desde estructuras adyacentes. La biopsia de piel asociada a técnicas moleculares y cultivo de micobacterias constituyen el gold standard diagnóstico de la TBC cutánea. El tratamiento de la TBC cutánea sigue las mismas recomendaciones que para otras formas de TBC. Presentamos el caso de un paciente con escrofuloderma.

Tuberculosis (TB) is an infectious disease caused by organisms of the Mycobacterium tuberculosis complex. Extrapulmonary presentations may constitutes up to 25% of TB cases. Reported in our country Cutaneous tuberculosis is a rare extrapulmonary manifestation that represents 1-2% of cases, with scrofuloderma and lupus vulgaris being the most common clinical forms. Scrofuloderma is an endogenous manifestation of the infection, because of contiguous extension to the overlying skin from adjacent structures. Skin biopsy associated with molecular techniques and mycobacterial culture constitute the gold standard for diagnosis of cutaneous TB. The treatment of cutaneous TB follows the same recommendations as for other forms of TB. We present the case of a patient with scrofuloderma.

Formulation and Validation of Recombinant Antigens CFP10 and ESAT6 for Tuberculosis Diagnosis

Abstract The rapid and accurate diagnosis of tuberculosis (TB), especially considering limited resources, is still a challenge. Development of new methodologies and tests are needed to overcome several disadvantages of the available standard tests. We evaluated the diagnostic potential of two antigens specific for Mycobacterium tuberculosis, the CFP10 and ESAT6 recombinant proteins, and developed stable formulations thereof. Sensitivity and specificity of the delayed-type hypersensitivity (DTH) skin testing and the induction of gamma interferon production (IFN-γ) by lymphocytes, as a non-invasive test, were evaluated using the CFP10 and ESAT6 protein formulations. The recombinant proteins produced by our group presented a high DTH response and the ability to differentiate between tuberculosis infection, BCG vaccination, and the contact with non-tuberculous mycobacteria (NTM). The production of IFN-γ by stimulation with individual and combined proteins was detected in a panel of 40 individuals and showed a specificity of 100% and a sensitivity of 90% when the two proteins were used together. Lyophilized formulations were stable under all conditions, while soluble formulations were stable under freezing at -20 ºC and -80 ºC. The proposed formulations containing the ESAT6 and CFP10 recombinant antigens constitute satisfactory tools for TB testing, suitable to be developed and implemented in a large-scale trial.

Evaluation of Multidrug Resistant Loop-mediated Isothermal Amplification Assay for Detecting the Drug Resistance of / 生物医学与环境科学（英文）

Objective@#To evaluate multidrug resistant loop-mediated isothermal amplification (MDR-LAMP) assay for the early diagnosis of multidrug-resistant tuberculosis and to compare the mutation patterns associated with the @*Methods@#MDR-LAMP assay was evaluated using 100 @*Results@#The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MDR-LAMP were 85.5%, 93.6%, 96.7%, and 74.4% for the detection of resistance to isoniazid and rifampicin, respectively, and 80.5%, 92.3%, 98.6%, and 41.4% for the detection of @*Conclusion@#MDR-LAMP is a rapid and accessible assay for the laboratory identification of rifampicin and isoniazid resistance of

Endometritis granulomatosa por tuberculosis en mujer postmenopaúsica / Granulomatous tuberculosis endometritis in a posmenopausal woman

INTRODUCCIÓN: La tuberculosis (TBC) genital es una infección relativamente poco frecuente en la mujer. Afecta principalmente a mujeres menores de 40 años, y el motivo de consulta más usual es la esterilidad, de ahí la importancia de su diagnóstico precoz. CASO CLÍNICO clínico: Se presenta el caso de una paciente con dolor pélvico crónico que acude a nuestras consultas para valoración. Durante el estudio se toma biopsia dirigida de la cavidad endometrial diagnosticándose la presencia de granulomas no necrotizantes. Posteriormente se realiza un cultivo microbiológico que resulta positivo para micobacterias y se determina el DNA, mediante reacción en cadena de la polimerasa, de mycobacterium tuberculosis, como causante del cuadro. DISCUSIÓN: El diagnóstico definitivo de TBC requiere el aislamiento en cultivo del bacilo de Koch, aunque en los casos de TBC genital, al ser una entidad paucibacilar, puede no resultar positivo. En éste caso, sería suficiente el diagnóstico de presunción basado en la sospecha clínica y el hallazgo histológico de granulomas. CONCLUSIÓN: La tuberculosis genital es una entidad poco frecuente en nuestro medio, aunque es una causa importante de infertilidad femenina y su predominio generalmente se subestima debido a la naturaleza paucisintomática de la misma. El diagnóstico temprano y el tratamiento multidisciplinar son fundamentales.

INTRODUCTION: Genital tuberculosis (TB) is a relatively rare afection in women. It mainly affects women younger than 40 years, and the most frequent reason for consultation is sterility, therefore early diagnosis is important. CLINICAL CASE: We presented the case of a patient with chronic pelvic pain who comes to our consultations. During the study, we take an endometrial biopsy diagnosing the presence of non-necrotizing granulomas. Finally, we determined the mycobacterium tuberculosis DNA through the polymerase chain reaction and positive microbiological culture, as the cause of pathology. DISCUSSION: The definitive diagnosis of TB requires the isolation in culture of the Koch bacillus, although in genital TB cases, as it is a paucibacillary entity, it may not be positive. In this case, the presumptive diagnosis based on clinical suspicion and the histological granulomas would be enough. CONCLUSIONS: Genital tuberculosis is a rare entity in our environment, although it is an important cause of female infertility and its prevalence is generally underestimated due to its paucisymptomatic nature. Early diagnosis and multidisciplinary treatment are essential.

Disseminated tuberculosis associated with reactive arthritis of Poncet in an immunocompetent patient

Abstract Cutaneous tuberculosis is a rare extrapulmonary manifestation of tuberculosis which, like disseminated tuberculosis, commonly occurs in immunocompromised patients. Poncet reactive arthritis is a seronegative arthritis affecting patients with extrapulmonary tuberculosis, which is uncommon even in endemic countries. We report a previously healthy 23-year-old male patient with watery diarrhea associated with erythematous ulcers on the lower limbs and oligoarthritis of the hands. Histopathological examination of the skin showed epithelioid granulomatous process with palisade granulomas and central caseous necrosis. AFB screening by Ziehl-Neelsen staining showed intact bacilli, the culture was positive for Mycobacterium tuberculosis, and colonoscopy revealed multiple shallow ulcers. Disseminated tuberculosis associated with reactive Poncet arthritis was diagnosed, with an improvement of the clinical and skin condition after appropriate treatment.

Resistencia cruzada entre isoniacida y etionamida y su alta correlación con la mutación C-15T en aislamientos de Mycobacterium tuberculosis de Perú / Cross-resistance between isoniazid and ethionamide and its strong association with mutation C-15T in Mycobacterium tuberculosis isolates from Peru

Resumen Diversos estudios han evidenciado una resistencia cruzada entre isoniacida y etionamida, 2 de los fármacos utilizados en el tratamiento de la tuberculosis multirresistente.El objetivo del presente estudio fue determinar la resistencia cruzada entre ambos fármacos en aislados de Mycobacterium tuberculosis obtenidos en un hospital de Lima (Perú), conalta proporción de pacientes con tuberculosis. Se calculó la frecuencia de mutaciones asociadas con la resistencia a la isoniacida (INH) evaluando el gen katG y la región promotorainhA mediante la prueba molecular Genotype MTBDRplus v2.0. El método gold standard conocido como agar proporciones en placa (APP) permitió la identificación de resistencia a INH yetionamida. De 107 aislamientos resistentes a INH, 54 fueron multirresistentes (identificadosmediante la prueba Genotype MTBDRplus) y 49 (es decir, el 45,8% del total) también fueronresistentes a etionamida por el método APP. En los aislamientos resistentes a INH, se encontraron mutaciones en el gen katG en el 50,5% (54/107); en la región promotora inhA en el23,3% (25/107), y un 14,0% (15/107) presentaron mutaciones en ambos. Un 12,1% (13/107)fueron resistentes a INH por ausencia de banda wild type y banda de mutación. La mutaciónC-15T en la región promotora inhA presentó una fuerte asociación con la resistencia a etionamida y alcanzó el 73,4% (36/49) de los aislamientos resistentes a dicho fármaco. Los resultadosdel presente estudio sugieren que la identificación de mutaciones relacionadas con resistenciaa INH, sobre todo en la región promotora inhA, podría ser de gran utilidad para identificarla resistencia cruzada a etionamida y mejorar el tratamiento de las personas afectadas portuberculosis.© 2019 Asociacion Argentina de Microbiolog´ía. Publicado por Elsevier Espana, S.L.U. Este es unart´ículo Open Access bajo la licencia CC BY-NC-ND (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Abstract Several studies have shown cross-resistance between isoniazid and ethionamide, 2of the drugs used in the treatment of multidrug-resistant tuberculosis. The objective of this study was to determine the cross-resistance between both drugs in Mycobacterium tuberculosis isolates from a hospital with high incidence of tuberculosis in Lima, Peru. The frequency of mutations to isoniazid in the katG gene and the inhA promoter region was identified by the Genotype MTBDRplus v2.0 molecular test. The gold standard Agar Proportion method (APM) allowed todetect resistance to isoniazid and ethionamide. Of 107 isoniazid-resistant isolates (54 multidrug-resistant isolates identified by the Genotype MTBDRplus test, 45.8% (49/107) were also resistant to ethionamide by the APM. Mutations were found in the katG gene in 50.5% (54/107), in the promoter region inhA in 23.3% (25/107) and 14.0% (15/107) that share both mutations in the resistant isolates to INH. The absence of the wild type and mutation bands indicated that 12.1% (13/107) of the isolates were resistant to INH. The mutation C-15T in the inhA promoter region showed a strong association with resistance to ethionamide in 73.4% (36/49) of the isolates analyzed. The results of the present study suggest that the identification of mutations related to resistance to isoniazid, especially in the inhA promoter region, could be very useful to identify cross-resistance to ethionamide and improve the treatment of individuals suffering from this disease.

The clinical and molecular diagnosis of childhood and adolescent pulmonary tuberculosis in referral centers

Abstract INTRODUCTION: The diagnostic accuracy of Xpert MTB/RIF (Xpert) in pulmonary tuberculosis (PTB) in children is lower than in adults. In Brazil, the diagnosis of PTB is based on a diagnostic score system (DSS). This study aims to study the role of Xpert in children and adolescents with PTB symptoms. METHODS: A cross-sectional study was conducted in 3 referral centers to TB. Children and adolescents (0-19 years old) whose respiratory samples were submitted to Xpert were included. Statistical analysis (bivariate and logistic regression) to assess the simultaneous influence of TB-related variables on the occurrence of Xpert detectable in TB cases was done. To evaluate the agreement or disagreement between Xpert results with acid-fast bacillus (AFB) and cultures, κ method was used (significancy level of 5%). RESULTS: Eighty-eight patients were included in the study and PTB occurred in 43 patients (49%) and Xpert was detectable in 21 patients (24%). Adolescents and positive culture results were independent predictive variables of Xpert positivity. DSS sensitivity compared with the final diagnosis of TB was 100% (95% CI, 88.1-100%), specificity was 97.2% (95% CI, 85.5-99.9%). The accuracy of the method was 98.5% (95% CI, 91.7-99.9%). CONCLUSIONS: Xpert contributed to diagnosis in 9% of patients with AFB and in culture negative cases. DSS indicated relevance for this diagnostic approach of intrathoracic TB (ITB) in reference centers for presenting data both with high sensitivity and specificity.

Rapid detection of Mycobacterium tuberculosis in children using blood and urine specimens

Abstract INTRODUCTION: Laboratory and clinical features of childhood tuberculosis (TB) are non-specific and establishing an accurate diagnosis remains a challenge. This study evaluated a Single tube nested-PCR (STNPCR) to detect genomic DNA of Mycobacterium tuberculosis complex in blood and urine. METHODS: Biological samples were obtained from children (<15 years old) with clinical suspicion of pulmonary and extrapulmonary TB at public hospitals in Recife-Pernambuco, Brazil. Cultures yielded negative results in a majority of childhood TB cases, which are generally paucibacillary. A set of clinical, epidemiological, radiological, and laboratory criteria with evident clinical improvement after anti-TB treatment were frequently used to define childhood TB cases. RESULTS: Ninety children with clinical suspicion were enrolled in this study (44 with TB and 46 without TB). The pulmonary TB group had 20 confirmed cases and 46 negative controls, while the extrapulmonary TB group had 24 confirmed cases. The STNPCR showed sensitivities to pulmonary and extrapulmonary TB of 47.4% and 52.2% (blood) and 38.8% and 20% (urine), respectively. Considering the low performance of STNPCR on separate samples, we decided to perform a combined analysis (parallel sensitivity analysis) of the results from blood and urine samples. The parallel sensitivity increased to 65% in blood and 62.5% in urine. The specificity in both samples ranged from 93.5-97.8%. CONCLUSIONS: Although STNPCR showed moderate sensitivity, the specificity is high; therefore, the test can be used as an auxiliary tool to diagnose TB in children. It is a rapid test that demonstrated better performance than other diagnostic tests in paucibacillary samples as it does in childhood tuberculosis.

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.

Analysis of anti-tuberculosis drug resistance and sociodemographic and clinical aspects of patients admitted in a referral hospital / Análise da resistência a medicamentos antituberculose e aspectos sociodemográficos e clínicos de pacientes atendidos em hospital referência

ABSTRACT Objective To determine the occurrence of anti-tuberculosis drug resistance and its association with sociodemographic and clinical characteristics of patients in a referral hospital. Methods This was a cross-sectional study based on data from patients who had mycobacterial culture identified and defined antimicrobials sensitivity profile (June 2014 to February 2016). The descriptive statistical analysis and Fisher's exact test were used to compare proportions. Results The study included 104 patients who had positive results for Mycobacterium tuberculosis . Bacilloscopy had high positivity (93.3%). A total of 15 patients (14.4%) had resistant strains and six (5.6%) multidrug-resistant. The sociodemographic and clinical characteristics were not related with resistance. Conclusion This study contributed to further the understandings about the tuberculosis patients' profile, the study also served as a tool for development of specific public policies. Patients diagnosed with resistant tuberculosis must be under greater supervision.

RESUMO Objetivo Verificar a ocorrência de resistência a fármacos antituberculose e a associação com características sociodemográficas e clínicas de pacientes de um hospital referência. Métodos Estudo transversal, com dados de pacientes que tiveram a cultura de micobactérias identificada e o respectivo perfil de sensibilidade aos antimicrobianos definido (junho de 2014 a fevereiro de 2016). Foram realizados a análise estatística descritiva e o teste exato de Fisher, para comparação de proporções. Resultados O estudo envolveu 104 pacientes, e todos tiveram resultados para Mycobacterium tuberculosis . A baciloscopia atingiu alta positividade (93,3%), e 15 pacientes (14,4%) apresentaram linhagens resistentes, sendo 6 (5,6%) multirresistentes. As características sociodemográficas e clínicas não foram associadas à resistência. Conclusão A pesquisa permitiu conhecer melhor o perfil dos pacientes com tuberculose e constitui ferramenta para elaboração de políticas públicas específicas. Os pacientes diagnosticados com tuberculose resistente devem ser submetidos à maior supervisão.

Genotype®MTBDRplus and Xpert®MTB/RIF in the diagnosis of tuberculosis and resistant tuberculosis: cost analysis in a tertiary referral hospital

Abstract INTRODUCTION: The present study sought to assess the mean and activity based cost (ABC) of the laboratory diagnosis for tuberculosis through the application of conventional and molecular techniques-Xpert®MTB/RIF and Genotype®MTBDRplus-in a tertiary referral hospital in Brazil. METHODS: The mean cost and ABC formed the basis for the cost analysis of the TB laboratory diagnosis. RESULTS: The mean cost and ABC were US$ 4.00 and US$ 3.24, respectively, for a bacilloscopy; US$ 6.73 and US$ 5.27 for a Lowenstein-Jensen (LJ) culture; US$ 105.42 and US$ 76.56 for a drug sensitivity test (DST)-proportions method (PM) in LJ; US$ 148.45 and US$ 136.80 for a DST-BACTECTM MGITTM 960 system; US$ 11.53 and US$ 9.89 for an Xpert®MTB/RIF; and US$ 84.21 and US$ 48.38 for a Genotype®MTBDRplus. CONCLUSIONS: The mean cost and ABC proved to be good decision-making parameters in the diagnosis of TB and MDR-TB. The effective implementation of algorithms will depend on the conditions at each location.