Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients

Abstract Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.

Resumo A tuberculose é uma doença transmissível com altas taxas de morbimortalidade nos países em desenvolvimento. O objetivo principal do estudo é comparar métodos convencionais, como cultura de bacilo álcool-ácido resistente (BAAR) e microscopia, com métodos de diagnóstico rápido. O objetivo secundário é comparar os achados histopatológicos e microbiológicos em pacientes com suspeita de linfadenite tubercular. Um total de 111 amostras (agosto de 2018 a setembro de 2019) de gânglios linfáticos foi processado para microscopia de AFB, culturas de AFB, teste de susceptibilidade a drogas (DST), histopatologia e Xpert Mycobacterium tuberculosis (MTB)/ensaios de resistência à rifampicina (RIF). Das 111 amostras de linfonodos, 6 (5,4%) foram positivas para baciloscopia de AFB, 84 (75,6%) foram positivas para cultura de AFB, 80 (70,7%) foram positivas para o GeneXpert e 102 (91,8%) foram indicativas de tuberculose para estudos histopatológicos. A positividade da cultura do tubo indicador de crescimento de micobactérias (MGIT) foi 84 (75,6%), maior que a cultura sólida de Lowenstein-Jensen (LJ), 74 (66,6%). As culturas positivas foram submetidas a DST fenotípico. Dois casos eram multirresistentes (MDR) ao DST, enquanto três casos eram resistentes à rifampicina no GeneXpert. A sensibilidade do GeneXpert foi 62% contra o método convencional de cultura AFB. O fraco desempenho dos métodos convencionais de diagnóstico de linfadenite requer metodologia de diagnóstico precoce e precisa. O teste Xpert MTB/RIF pode ajudar no tratamento de casos de tuberculose multirresistente. No entanto, métodos rápidos e convencionais devem ser usados para o isolamento completo do Mycobacterium tuberculosis.

Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients / Comparação de diferentes modalidades de diagnóstico para isolamento de Mycobacterium tuberculosis entre pacientes com suspeita de linfadenite tuberculosa

Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.

A tuberculose é uma doença transmissível com altas taxas de morbimortalidade nos países em desenvolvimento. O objetivo principal do estudo é comparar métodos convencionais, como cultura de bacilo álcool-ácido resistente (BAAR) e microscopia, com métodos de diagnóstico rápido. O objetivo secundário é comparar os achados histopatológicos e microbiológicos em pacientes com suspeita de linfadenite tubercular. Um total de 111 amostras (agosto de 2018 a setembro de 2019) de gânglios linfáticos foi processado para microscopia de AFB, culturas de AFB, teste de susceptibilidade a drogas (DST), histopatologia e Xpert Mycobacterium tuberculosis (MTB)/ensaios de resistência à rifampicina (RIF). Das 111 amostras de linfonodos, 6 (5,4%) foram positivas para baciloscopia de AFB, 84 (75,6%) foram positivas para cultura de AFB, 80 (70,7%) foram positivas para o GeneXpert e 102 (91,8%) foram indicativas de tuberculose para estudos histopatológicos. A positividade da cultura do tubo indicador de crescimento de micobactérias (MGIT) foi 84 (75,6%), maior que a cultura sólida de Lowenstein-Jensen (LJ), 74 (66,6%). As culturas positivas foram submetidas a DST fenotípico. Dois casos eram multirresistentes (MDR) ao DST, enquanto três casos eram resistentes à rifampicina no GeneXpert. A sensibilidade do GeneXpert foi 62% contra o método convencional de cultura AFB. O fraco desempenho dos métodos convencionais de diagnóstico de linfadenite requer metodologia de diagnóstico precoce e precisa. O teste Xpert MTB/RIF pode ajudar no tratamento de casos de tuberculose multirresistente. No entanto, métodos rápidos e convencionais devem ser usados para o isolamento completo do Mycobacterium tuberculosis.

Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients / Comparação de diferentes modalidades de diagnóstico para isolamento de Mycobacterium tuberculosis entre pacientes com suspeita de linfadenite tuberculosa

Abstract Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.

Resumo A tuberculose é uma doença transmissível com altas taxas de morbimortalidade nos países em desenvolvimento. O objetivo principal do estudo é comparar métodos convencionais, como cultura de bacilo álcool-ácido resistente (BAAR) e microscopia, com métodos de diagnóstico rápido. O objetivo secundário é comparar os achados histopatológicos e microbiológicos em pacientes com suspeita de linfadenite tubercular. Um total de 111 amostras (agosto de 2018 a setembro de 2019) de gânglios linfáticos foi processado ​​para microscopia de AFB, culturas de AFB, teste de susceptibilidade a drogas (DST), histopatologia e Xpert Mycobacterium tuberculosis (MTB)/ensaios de resistência à rifampicina (RIF). Das 111 amostras de linfonodos, 6 (5,4%) foram positivas para baciloscopia de AFB, 84 (75,6%) foram positivas para cultura de AFB, 80 (70,7%) foram positivas para o GeneXpert e 102 (91,8%) foram indicativas de tuberculose para estudos histopatológicos. A positividade da cultura do tubo indicador de crescimento de micobactérias (MGIT) foi 84 (75,6%), maior que a cultura sólida de Lowenstein-Jensen (LJ), 74 (66,6%). As culturas positivas foram submetidas a DST fenotípico. Dois casos eram multirresistentes (MDR) ao DST, enquanto três casos eram resistentes à rifampicina no GeneXpert. A sensibilidade do GeneXpert foi 62% contra o método convencional de cultura AFB. O fraco desempenho dos métodos convencionais de diagnóstico de linfadenite requer metodologia de diagnóstico precoce e precisa. O teste Xpert MTB/RIF pode ajudar no tratamento de casos de tuberculose multirresistente. No entanto, métodos rápidos e convencionais devem ser usados ​​para o isolamento completo do Mycobacterium tuberculosis.

Diagnostic value of polymerase chain reaction targeting insertion sequence IS1081 for the diagnosis of pediatric tuberculosis

Background: Aim of this study was to evaluate the efficacy of PCR targeting IS1081in diagnosis of pediatric tuberculosis and compare the results with MGIT culture.Methods: This prospective study was conducted in the department of pediatrics, S.N. medical college, Agra. 100 subjects (28 pulmonary 72 extra pulmonary) were registered in study. The specimens obtained from these cases were subjected to Ziehl'Neelsen staining (ZN), MGIT 960 TB culture and PCR targeting insertion sequence IS1081. Sensitivity, specificity, PPV and NPV of PCR were calculated in pulmonary and extra pulmonary specimens. The results of PCR IS1081 were compared to MGIT culture.Results: Microscopy with ZN staining was positive in 12 (12%) samples. MGIT culture was positive in 44% samples with maximum positivity in sputum (70%). PCR IS1081 has shown 93.3% sensitivity in pulmonary tuberculosis, while PCR IS1081 has shown 93.1% sensitivity in extra pulmonary tuberculosis.' In diagnosis of childhood tuberculosis PCR IS1081 was found to be statistically significant (p value <0.05) as compared with MGIT culture. Result was statistically significant (p value <0.05) in CSF samples only.Conclusions: The study concluded that the PCR targeting sequence IS1081 technique is the most sensitive technique for a quick identification of MTB in pulmonary and extra pulmonary tuberculosis.

Bronchoscopic Analysis in Sputum Smear Negative Pulmonary Tuberculosis Patients A Hospital based Study

Introduction: India has the highest burden of TB cases inthe world, majority of them are pulmonary tuberculosis.The method of choice for diagnosis of PTB is microscopicexamination of AFB by sputum smear. However, 30 to 50%of patients with pulmonary tuberculosis can have negativesputum report or may not produce sputum. Flexible fibreopticbronchoscopy can provide excellent material for diagnosis forpatients with suspected sputum smear negative pulmonarytuberculosis. Study aimed to evaluate the role of fiberoptic bronchoscopy in sputum smear negative pulmonorytuberculosis.Material and methods: Forty suspected cases of pulmonaryTB with clinical and radiological evidence of tb and sputumsmear negative on 2 occasions were selected for thisprospective nonrandomised observational study. Detailedexamination of the bronchial tree was done and specimensincluding bronchial aspirate and lavage was collected andsend for investigations. Post bronchoscopy sputum (PBS) wasalso collected and sent for smear microscopy.Results: In our study of 40 patients, tuberculosis wasconfirmed in 13 (32.50%) by smear examination of AFB inBroncho alveolar fluid and by post bronchoscopy sputumsmear examination in 3/40 (7.5%) cases. A definitive diagnosisof tuberculosis was possible in 23 (57.5%) of the 40 patientsby AFB culture by BACTEC MGIT960.Conclusion: Fibreoptic bronchoscopy with post bronchoscopysputum,BAL and BAL AFB culture is a useful tool fordiagnosis and can thereby prompt treatment of sputum smearnegative pulmonary tuberculosis patients.

Evaluation of modified petroff’s method and manual MGIT culture system to facilitate detection of acid-fast bacilli in smear-negative presumptive cases of pulmonary tuberculosis

Background: Tuberculosis is a major public health problem in India. According to RNTCP guidelines, all efforts are to be made to diagnose tuberculosis on the basis of detection of AFB/ MTB in the clinical specimen; hence all those cases where AFB/ MTB have not been detected are presumptive cases of tuberculosis. In this background, the present study aims to detect AFB/ MTB in the sputum of clinico-radiologically presumptive cases of pulmonary tuberculosis using direct ZN microscopy, Modified Petroff’s method followed by ZN microscopy & MGIT liquid culture method. Materials and Methods: A total of 94 sputum samples which were negative for AFB by direct ZN microscopy at the DMC of the institute, were subjected to Modified Petroff’s method followed by microscopy & MGIT liquid culture test to find out any additional yield of bacteriologically confirmed disease. Result: Out of 94 specimens, 20 (21.28%) were positive for AFB on ZN microscopy post Modified Petroff’s method. Among all the specimens, 30 (31.91%) were found to be positive by MGIT liquid culture method with average time to detection growth (TTD) around 16.73 days (2-38 days). Conclusion: Modified Petroff’s method followed by ZN microscopy & MGIT test for mycobacterial culture can improve case detection.

Evaluation of GeneXpert MTB/RIF and BACTEC-MGIT 960 for the detection of tuberculosis among pneumoconiosis-associated tuberculosis patients / 中华劳动卫生职业病杂志

Objective@#To evaluate the performence of GeneXpert MTB/RIF and BACTEC-MGIT 960 on detecting Mycobacterium tuberculosis and rifampicin resistance for pneumoconiosis-associated tuberculosis patients.@*Methods@#The recruited 133 suspected active pneumoconiosis-associated tuberculosis hospitalized cases, morning sputum samples were collected to do modified L-J culture, conventional proportion method drug susceptibility test, GeneXpert MTB/RIF and BACTEC-MGIT 960. Analyze the sensitivity and specificity of the 133 sputum from patients, the positive rates of patients with tuberculosis in GeneXpert MTB/RIF test, BACTEC-MGIT 960 and modified L-J culture were 37.59%, 34.59% and 30.08% respectively. There was no significant difference among the three tests respectively (P>0.05) . According to the modified L-J culture, the sensitivity of GeneXpert MTB/RIF and BACTEC-MGIT 960 in detecting tuberculosis were 92.5% and 95.0% respectively, and specificity in rifampicin resistance were 86.0% and 91.4% respectively. There was no significant difference between GeneXpert MTB/RIF and BACTEC-MGIT 960 (P>0.05) . According to conventional proportion method drug susceptibility test, the sensitivity of GeneXpert MTB/RIF and BACTEC-MGIT 960 in detecting rifampicin resistance were 90.0% and 100%, and specificity were 92.6% and 96.4%. There was no significant difference between GeneXpert MTB/RIF and BACTEC-MGIT 960 (P>0.05) .@*Conclusion@#The GeneXpert MTB/RIF has good performence of detecting tuberculosis and rifampicin resistance. It has good application value among pneumoconiosis-associated tuberculosis patients.

Evaluation of different laboratory methods for auxiliary diagnosis of pulmonary tuberculosis / 临床检验杂志

Objective@#To evaluate the effectiveness of different laboratory methods for the supplementary diagnosis of pulmonary tuberculosis（PTB）and provide reference data for the early diagnosis of PTB. @*Methods@#A total of 298 suspected PTB patients, who were diagnosed and treated in the outpatient department of Shanghai Tongren Hospital from January 2016 to December 2018, were divided into 3 groups: active PTB (138 cases)，inactive PTB (43 cases) and non-PTB (117 cases) group. Sputum acid-fast staining, MGIT liquid culture system and Xpert MTB/RIF test were performed to detect the sputum specimens. The sensitivity and specificity were compared by Chi-square test. @*Results@#The three methods showed certain significance for distinguishing active PTB, inactive PTB combined with non-PTB (χ 2 values were 89.08, 138.94 and 137.12 respectively, all P＜0.01). There was no significant difference for the positive rate of the three methods between inactive PTB and non-PTB. The sensitivities of acid-fast staining, MGIT liquid culture, Xpert MTB/RIF test and the combination of three methods in the diagnosis of active PTB were 45.7% (63/138), 63.8% (88/138), 65.4% (87/133) and 78.2% (104/133) respectively. The sensitivities of MGIT culture and Xpert MTB/RIF test were significantly higher than that of acid-fast staining (χ 2 was 35.79 and 11.26 respectively，all P＜0.01). There was no significant difference for the sensitivities between MGIT liquid culture and Xpert MTB/RIF test（χ 2 was 29.87, P＞0.05) . The sensitivity of combined detection was higher than that of single detection（χ 2 was 30.84, 64.62, 70.14, respectively, all P＜0.01）. The diagnostic specificities of the three methods and their combination were 99.1%（116/117), 98.3%（115/116）, 99.1%（113/114) and 97.3%（110/113）respectively. There was no significant difference for the specificities of the three methods. @*Conclusion@#High sensitivities of MGIT liquid culture and Xpert MTB/RIF test were shown in PTB diagnosis. Combined detection of the three methods may improve the sensitivity of detection.

Desempenho da cultura líquida MGIT após implementação em uma rede de laboratórios públicos do estado de São Paulo / Performance of liquid culture MGIT after implementation in a network of public laboratories of Sao Paulo state

A OMS, em 2007, recomendou a implementação da cultura líquida para o diagnóstico da tuberculose (TB) e teste de sensibilidade para países de baixa e média renda. Neste estudo foi avaliado o desempenho da cultura líquida MGIT em condição de rotina após dois anosde implantação em uma rede de laboratórios públicos. Foi efetuada análise retrospectiva de dados da cultura líquida, realizadas em dez laboratórios regionais do Instituto Adolfo Lutz, de janeiro a março de 2010. Foram incluídas amostras submetidas a baciloscopia, cultura líquida MGIT automatizada ou manual e identificação presuntiva do complexo Mycobacterium tuberculosis (CMTB). Foram detectadas 1.159 culturas positivas. Destas, 113 (9,7%) contaminaram, e 1.046 foram analisadas, sendo 850 (81,3%) CMTB, 116 (11,1%) micobactérias não tuberculosas e 6 (0,6%) Nocardia sp A taxa de contaminação foi de 2,2% e o acréscimo da cultura para o diagnóstico da TB foi de 29,9%. A média do tempo de detecção da cultura foi de 14,7 dias (DP+/- 11,7 dias). A acurácia da identificação presuntiva foi de 91,3%. A cultura líquida MGIT demonstrou ser excelente alternativa para efetuar diagnóstico da TB e das micobacterioses, em razão da rapidez possibilitando uma intervenção rápida e eficaz no tratamento.

In 2007, WHO recommended the implementation of liquid culture for tuberculosis (TB) diagnosis and drug-susceptibility test in low and middle-income countries. This study evaluated the performance of MGIT culture in routine condition after two years of its implementation in a public laboratories network.This is a retrospective study, which analyzed the data on the liquid culture performed in ten regional laboratories of the Institute Adolfo Lutz, from January to March 2010. The data included clinical samples submitted to microscopy, automated or manual MGIT culture and presumptive M. tuberculosis complex (MTBC) identification by analyzing the cord formation. Culture was positive in 1,159 samples. Of these, 113 (9.7%) contaminated, and 1,046 were analyzed, of which 850 (81.3%) were identified as MTBC, 116 (11.1%) as non-tuberculous mycobacteria and 6 (0.6%) as Nocardia sp. Contamination rate was 2.2% and the contribution of culture to the TB diagnosis was 29.9%. The detection mean time was 14.7 days (SD+/-11.7 days). The accuracy of the presumptive identification of MTBC was 91.3%. MGIT liquid culture demonstrated to be an excellent alternative for diagnosing TB and mycobacterioses, because of the rapidity of diagnosis, thus allowing an immediate and effective treatment.

Desempenho da cultura líquida MGIT após implementação em uma rede de laboratórios públicos do estado de São Paulo / Performance of liquid culture MGIT after implementation in a network of public laboratories of Sao Paulo state

A OMS, em 2007, recomendou a implementação da cultura líquida para o diagnóstico da tuberculose (TB) e teste de sensibilidade para países de baixa e média renda. Neste estudo foi avaliado odesempenho da cultura líquida MGIT em condição de rotina após dois anos de implantação em uma rede de laboratórios públicos. Foi efetuada análise retrospectiva de dados da cultura líquida,realizadas em dez laboratórios regionais do Instituto Adolfo Lutz, de janeiro a março de 2010. Foram incluídas amostras submetidas a baciloscopia, cultura líquida MGIT automatizada ou manual eidentificação presuntiva do complexo Mycobacterium tuberculosis (CMTB). Foram detectadas 1.159 culturas positivas. Destas, 113 (9,7%) contaminaram, e 1.046 foram analisadas, sendo 850 (81,3%) CMTB, 116 (11,1%) micobactérias não tuberculosas e 6 (0,6%) Nocardia sp. A taxa de contaminação foi de 2,2% e o acréscimo da cultura para o diagnóstico da TB foi de 29,9%. A média do tempo de detecção da cultura foi de 14,7 dias (DP+/- 11,7 dias). A acurácia da identificação presuntiva foide 91,3%. A cultura líquida MGIT demonstrou ser excelente alternativa para efetuar diagnóstico da TB e das micobacterioses, em razão da rapidez possibilitando uma intervenção rápida e eficaz no tratamento.

In 2007, WHO recommended the implementation of liquid culture for tuberculosis (TB) diagnosis anddrug-susceptibility test in low and middle-income countries. This study evaluated the performanceof MGIT culture in routine condition after two years of its implementation in a public laboratoriesnetwork. This is a retrospective study, which analyzed the data on the liquid culture performed in ten regional laboratories of the Institute Adolfo Lutz, from January to March 2010. The data included clinical samples submitted to microscopy, automated or manual MGIT culture and presumptive M. tuberculosis complex (MTBC) identification by analyzing the cord formation. Culture waspositive in 1,159 samples. Of these, 113 (9.7%) contaminated, and 1,046 were analyzed, of which 850 (81.3%) were identified as MTBC, 116 (11.1%) as non-tuberculous mycobacteria and 6 (0.6%)as Nocardia sp. Contamination rate was 2.2% and the contribution of culture to the TB diagnosis was 29.9%. The detection mean time was 14.7 days (SD+/-11.7 days). The accuracy of the presumptive identification of MTBC was 91.3%. MGIT liquid culture demonstrated to be an excellent alternative for diagnosing TB and mycobacterioses, because of the rapidity of diagnosis, thus allowing an immediate and effective treatment.

Rapid culture-based detection of living mycobacteria using microchannel electrical impedance spectroscopy (m-EIS)

BACKGROUND: Multiple techniques exist for detecting Mycobacteria, each having its own advantages and drawbacks. Among them, automated culture-based systems like the BACTEC-MGIT™ are popular because they are inexpensive, reliable and highly accurate. However, they have a relatively long "time-to-detection" (TTD). Hence, a method that retains the reliability and low-cost of the MGIT system, while reducing TTD would be highly desirable. METHODS: Living bacterial cells possess a membrane potential, on account of which they store charge when subjected to an AC-field. This charge storage (bulk capacitance) can be estimated using impedance measurements at multiple frequencies. An increase in the number of living cells during culture is reflected in an increase in bulk capacitance, and this forms the basis of our detection. M. bovis BCG and M. smegmatis suspensions with differing initial loads are cultured in MGIT media supplemented with OADC and Middlebrook 7H9 media respectively, electrical "scans" taken at regular intervals and the bulk capacitance estimated from the scans. Bulk capacitance estimates at later time-points are statistically compared to the suspension's baseline value. A statistically significant increase is assumed to indicate the presence of proliferating mycobacteria. RESULTS: Our TTDs were 60 and 36 h for M. bovis BCG and 20 and 9 h for M. smegmatis with initial loads of 1000 CFU/ml and 100,000 CFU/ml respectively. The corresponding TTDs for the commercial BACTEC MGIT 960 system were 131 and 84.6 h for M. bovis BCG and 41.7 and 12 h for M smegmatis, respectively. CONCLUSION: Our culture-based detection method using multi-frequency impedance measurements is capable of detecting mycobacteria faster than current commercial systems.

Evaluation of anti-tubercular activity of linolenic acid and conjugated-linoleic acid as effective inhibitors against Mycobacterium tuberculosis

OBJECTIVE@#To evaluate a new pharmacological activity/effect of linolenic acid (α- and γ-form) and conjugated-linoleic acid (CLA) causing antibacterial activity against Mycobacterium tuberculosis (Mtb).@*METHODS@#The anti-Mtb activity/effect of linolenic acid and CLA were determined using different anti-Mtb indicator methods such as resazurin microtiter assay (REMA) and MGIT 960 system assay. The Mtb was incubated with various concentrations (12.5-200 μg/mL) of the compounds and anti-Mtb first-line drugs for 5 d in the REMA, and for 3 wk in MGIT 960 system assay.@*RESULTS@#Linolenic acid and CLA obviously indicated their anti-Mtb activity/effect by strongly inhibiting the growth/proliferation of Mtb in a dose-dependent manner in the REMA and the MGIT 960 system assay. Interestingly, linolenic acid and CLA consistently induced anti-Mtb activity/effect by effectively inhibiting the growth/proliferation of Mtb in MGIT 960 system for 21 d with a single treatment, and their minimum inhibitory concentrations were measured as 200 μg/mL respectively.@*CONCLUSIONS@#These results demonstrate that linolenic acid and CLA not only have effective anti-Mtb activity/properties, but also induce the selective-anti-Mtb effects by strongly inhibiting and blocking the growth/proliferation of Mtb through a new pharmacological activity/action. Therefore, this study provides novel perspectives for the effective use of them and the potential that can be used as potent anti-Mtb candidate drugs, as well as suggests the advantage of reducing the cost and/or time for developing a new/substantive drug by effectively repurposing the existing drugs or compounds as one of new strategies for the global challenge of tuberculosis.

Evaluation of anti-tubercular activity of linolenic acid and conjugated-linoleic acid as effective inhibitors against Mycobacterium tuberculosis

Objective: To evaluate a new pharmacological activity/effect of linolenic acid (α- and γ-form) and conjugated-linoleic acid (CLA) causing antibacterial activity against Mycobacterium tuberculosis (Mtb). Methods: The anti-Mtb activity/effect of linolenic acid and CLA were determined using different anti-Mtb indicator methods such as resazurin microtiter assay (REMA) and MGIT 960 system assay. The Mtb was incubated with various concentrations (12.5-200 μg/mL) of the compounds and anti-Mtb first-line drugs for 5 d in the REMA, and for 3 wk in MGIT 960 system assay. Results: Linolenic acid and CLA obviously indicated their anti-Mtb activity/effect by strongly inhibiting the growth/proliferation of Mtb in a dose-dependent manner in the REMA and the MGIT 960 system assay. Interestingly, linolenic acid and CLA consistently induced anti-Mtb activity/effect by effectively inhibiting the growth/proliferation of Mtb in MGIT 960 system for 21 d with a single treatment, and their minimum inhibitory concentrations were measured as 200 μg/mL respectively. Conclusions: These results demonstrate that linolenic acid and CLA not only have effective anti-Mtb activity/properties, but also induce the selective-anti-Mtb effects by strongly inhibiting and blocking the growth/proliferation of Mtb through a new pharmacological activity/action. Therefore, this study provides novel perspectives for the effective use of them and the potential that can be used as potent anti-Mtb candidate drugs, as well as suggests the advantage of reducing the cost and/or time for developing a new/substantive drug by effectively repurposing the existing drugs or compounds as one of new strategies for the global challenge of tuberculosis.

The anti-tubercular activity of Melia azedarach L. and Lobelia chinensis Lour. and their potential as effective anti-Mycobacterium tuberculosis candidate agents

Objective To evaluate the anti-mycobacterial activity of Melia azedarach L. (M. azedarach) and Lobelia chinensis Lour. (L. chinensis) extracts against the growth of Mycobacterium tuberculosis (M. tuberculosis). Methods The anti-M. tuberculosis activity of M. azedarach and L. chinensis extracts were evaluated using different indicator methods such as resazurin microtiter assay (REMA) and mycobacteria growth indicator tube (MGIT) 960 system assay. The M. tuberculosis was incubated with various concentrations (50–800 μg/mL) of the extracts for 5 days in the REMA, and for 4 weeks in MGIT 960 system assay. Results M. azedarach and L. chinensis extracts showed their anti-M. tuberculosis activity by strongly inhibiting the growth of M. tuberculosis in a concentration-dependent manner in the REMA and the MGIT 960 system assay. Particularly, the methanol extract of M. azedarach and n-hexane extract of L. chinensis consistently exhibited their effects by effectively inhibiting the growth of M. tuberculosis in MGIT 960 system for 4 weeks with a single-treatment, indicating higher anti-M. tuberculosis activity than other extracts, and their minimum inhibitory concentrations were measured as 400 μg/mL and 800 μg/mL, respectively. Conclusions These results demonstrate that M. azedarach and L. chinensis extracts not only have unique anti-M. tuberculosis activity, but also induce the selective anti-M. tuberculosis effects by consistently inhibiting or blocking the growth of M. tuberculosis through a new pharmacological action. Therefore, this study suggests the potential of them as effective candidate agents of next-generation for developing a new anti-tuberculosis drug, as well as the advantage for utilizing traditional medicinal plants as one of effective strategies against tuberculosis.

The anti-tubercular activity of Melia azedarach L. and Lobelia chinensis Lour. and their potential as effective anti-Mycobacterium tuberculosis candidate agents

Objective: To evaluate the anti-mycobacterial activity of Melia azedarach L. (M. azedarach) and Lobelia chinensis Lour. (L. chinensis) extracts against the growth of Mycobacterium tuberculosis (M. tuberculosis). Methods: The anti-M. tuberculosis activity of M. azedarach and L. chinensis extracts were evaluated using different indicator methods such as resazurin microtiter assay (REMA) and mycobacteria growth indicator tube (MGIT) 960 system assay. The M. tuberculosis was incubated with various concentrations (50–800 mg/mL) of the ex-tracts for 5 days in the REMA, and for 4 weeks in MGIT 960 system assay. Results: M. azedarach and L. chinensis extracts showed their anti-M. tuberculosis ac-tivity by strongly inhibiting the growth of M. tuberculosis in a concentration-dependent manner in the REMA and the MGIT 960 system assay. Particularly, the methanol extract of M. azedarach and n-hexane extract of L. chinensis consistently exhibited their effects by effectively inhibiting the growth of M. tuberculosis in MGIT 960 system for 4 weeks with a single-treatment, indicating higher anti-M. tuberculosis activity than other extracts, and their minimum inhibitory concentrations were measured as 400 mg/mL and 800 mg/mL, respectively. Conclusions: These results demonstrate that M. azedarach and L. chinensis extracts not only have unique anti-M. tuberculosis activity, but also induce the selective anti-M. tuberculosis effects by consistently inhibiting or blocking the growth of M. tuberculosis through a new pharmacological action. Therefore, this study suggests the potential of them as effective candidate agents of next-generation for developing a new anti-tuberculosis drug, as well as the advantage for utilizing traditional medicinal plants as one of effective strategies against tuberculosis.

Effectiveness of Tuberculosis Smear Microscopy in Laboratory Diagnosis of Mycobacterium tuberculosis in Resource Constrained Settings.

Aim: To determine the correlation of accuracy of direct smear microscopy compared with BACTEC MGIT 960. Design: The study prospectively compare direct smear microscopy with BACTEC MGIT 960 using the reference standard, Lowenstein Jensen culture. Place and Duration: The study was conducted in Zankli Medical Centre, Abuja, between November 2004 and July 2005. Methodology: 340 suspected patients for Mycobacterium tuberculosis referred from direct observation therapy clinics located in six different government owned health facilities were referred to our facility. These patients; male (192) and female (148) were between the age of 10 and 64 years old. Three sputa samples were collected over two consecutive days and direct smear microscopy and culture were performed on these samples. Results: When compared with the reference standard, BACTEC MGIT 960 has a sensitivity and specificity of 100.0% and 56.4% respectively, and a negative predictive value of 100.0%; indicating the proportion of AFB negative participants were actually not infected with M. tuberculosis when tested with BACTEC MGIT 960. The sensitivity of direct microscopy was significantly lower than BACTEC MGIT 960 (84.9% versus 100%, p<0.001) and the specificity was significantly higher (96.6% versus 56.4%, p<0.001). Conclusions: For the purpose of effectiveness of tuberculosis program in developing countries, direct smear microscopy may still be relevant in the diagnosis of Mycobacterium tuberculosis.

A novel low-cost method for Mycobacterium avium subsp. paratuberculosis DNA extraction from an automated broth culture system for real-time PCR analysis

PCR is a highly accurate technique for confirming the presence of Mycobacterium avium subsp. paratuberculosis (Map) in broth culture. In this study, a simple, efficient, and low-cost method of harvesting DNA from Map cultured in liquid medium was developed. The proposed protocol (Universidad Austral de Chile [UACH]) was evaluated by comparing its performance to that of two traditional techniques (a QIAamp DNA Stool Mini Kit and cethyltrimethylammonium bromide [CTAB] method). The results were statistically assessed by agreement analysis for which differences in the number of cycles to positive (CP) were compared by Student's t-test for paired samples and regression analysis. Twelve out of 104 fecal pools cultured were positive. The final PCR results for 11 samples analyzed with the QIAamp and UACH methods or ones examined with the QIAamp and CTAB methods were in agreement. Complete (100%) agreement was observed between data from the CTAB and UACH methods. CP values for the UACH and CTAB techniques were not significantly different, while the UACH method yielded significantly lower CP values compared to the QIAamp kit. The proposed extraction method combines reliability and efficiency with simplicity and lower cost.

Detection of Mycobacterium in fine-needle aspiration biopsy specimens from children with tuberculous lymphadenitis / 中华微生物学和免疫学杂志

Objective To improve the diagnosis of tuberculosis ( TB) by analyzing Mycobacterium infection in fine-needle aspiration biopsy specimens from children with tuberculous lymphadenitis .Methods Fine-needle aspiration biopsy was performed on 269 children with tuberculous lymphadenitis diagnosed by Shanghai Public Health Clinical Center from January 2011 to September 2013 .The needle aspiration biopsy specimens were processed for acid-fast bacillus (AFB) smear test, mycobacterial culture and Mycobacterium identification ( p-nitrobenzoic acid inhibition test ) .Results Cytological diagnosis of tuberculous lymphade-nitis was made for 269 patients.The positive results by AFB smear test were detected in 63.19% of 269 specimens (n=170) and 40.15%(n=108) specimens were positive in mycobacterial culture .The differ-ence between the two tests were significant (P<0.01).The positive rate of Mycobacterium detected by using BACTEC MGIT 960 automated system and L?wenstein-Jensen culture method were 38 .66% ( n=104 ) and 28.99%(n=78), respectively, showing the significant difference between two tests (P<0.05).AFB smear test in combination with mycobacterial culture could precisely diagnose 70.63% of tuberculous lym-phadenitis in children.Of the 108 clinical isolates, 105 strains (97.2%) were Mycobacterium tuberculosis complex and the rest were non-tuberculous Mycobacterium strains (2.8%).Conclusion The positive rate by AFB smear test was significantly increased in fine needle aspiration biopsy specimens after a series of treatments including sample digestion , centrifugation and precipitation , but the positive rate of mycobacterial culture was reduced .Diagnostic accuracy could be significantly improved by using BACTEC MGIT 960 sys-tem.Mycobacterium tuberculosis complex was the predominant pathogenic bacterium in children with tubercu-lous lymphadenitis .

Diagnostic role of MGIT culture of BAL samples in sputum smearnegative pulmonary tuberculosis.

Background: In view of the diagnostic difficulties associated with sputum- negative pulmonary TB (PTB), we aimed at exploring if bronchoalveolar lavage (BAL) samples can be subjected to smear- microscopy and rapid mycobacterial culture (by Mycobacterial Growth Indicator Tube (MGIT) method) to achieve improved diagnosis of this condition. Methods: Patients presenting with clinico-radiological features suggestive of pulmonary tuberculosis and whose sputum smears were negative for acid- fast bacilli (AFB) or who could not expectorate sputum were prospectively enrolled in this study. BAL samples collected from them were subjected to smear- microscopy for AFB and micro-MGIT culture. BAL samples were also inoculated on Lowenstein- Jensen (LJ) slants. Results: A total of 105 patients (74 males) were recruited in the study, with a mean (±SD) age of 51 (± 15) years. The diagnosis of PTB was made in 52 patients on the basis of clinico- radiological presentation, with or without microbiological confirmation. Thirty- four patients (65.4 %) had microbiologically confirmed PTB. Of them, AFB were detected in 12 BAL samples, while culture- positivity was noted in 24 and 27 patients by the LJ and MGIT methods respectively. Intertest agreement between the LJ and MGIT methods was found to be significant (ê= 0.655; p= <0.001). However, the mean time to positivity was significantly lower for the MGIT method than for the LJ method (p= <0.001). Conclusion: Examination of BAL samples by smear- microscopy and micro-MGIT culture can, therefore, provide a rapid and definitive diagnosis of PTB in sputum- negative patients.

Usefulness of the BACTEC MGIT 960 System for Mycobacterial Culture and TB Ag MPT64 Immunochromatographic Assay to Identify Mycobacterium tuberculosis / 영남의대학술지

BACKGROUND: This study was conducted to evaluate the usefulness of the BACTEC MGIT (Mycobacterium Growth Indicator Tube) 960 system for mycobacteria culture and immunochromatographic assay to identify Mycobacterium tuberculosis (MTB) in positive MGIT culture. METHODS: Mycobacteria-culture-positive cases were retrospectively analyzed from December 2010 to July 2011. The detection rates and the recovery times of the mycobacteria between the Ogawa media and the MGIT were compared. An immunochromatographic assay (ICA) (SD BIO-LINE) was also performed in the positive MGIT culture for identification, and the results were compared with those of the Ogawa media in the Korea National Tuberculosis Association. RESULTS: Among the 261 patients (M:F, 168:93; mean age, 61.6+/-17.16 yrs), 450 specimens (sputa, 365; bronchial washing, 61; and pleural effusion, 24) were found positive with mycobacteria. Mycobacteria were grown both on the MGIT and Ogawa media in 310 cases (68.9%); only on the MGIT in 115 cases (22.6%); and only on the Ogawa media in 25 cases (5.5%) (p<0.05).The recovery time was 28.2+/-8.9 days in the Ogawa media and 11.1+/-5.8 days in the MGIT (p<0.05). Among the 127 cases from the positive MGIT culture, all 92 cases that were confirmed as MTB cases bythe Korea National Tuberculosis Association were identified as MTB by ICA, with 100% sensitivity. CONCLUSION: MGIT increases the detection rate and shortens the recovery time of mycobacteria in clinical respiratory specimens, and the TB Ag MPT64 kit using ICA is useful in identifying MTB in a positive MGIT culture.