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.

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.

Evaluación del método de Petroff modificado con solución salina para el diagnóstico de micobacterias en el sistema Bact/Alert 3D / Assessment of Petroff method modified with saline solution for the diagnosis of mycobacteria in Bact/Alert 3D system

Introducción: Existen diferentes métodos de descontaminación de muestras pulmonares para el diagnóstico de micobacterias. El Programa Nacional de Control de Tuberculosis recomienda el método de Petroff modificado con solución salina, pero no existen evidencias documentadas que avalen este método. Objetivo: Evaluar el método de Petroff modificado con solución salina para el diagnóstico de micobacterias en el sistema Bact/Alert 3D. Métodos: Se realizó un estudio observacional analítico de pruebas diagnósticas utilizando 100 muestras pulmonares recibidas en el Laboratorio Nacional de Referencia e Investigaciones de Tuberculosis, Lepra y Micobacterias del Instituto Pedro Kourí, abril 2016 enero 2017. La muestra se dividió en 3 alícuotas y se descontaminaron mediante 3 métodos; luego se inocularon en los medios de cultivo sólido y líquido. Se compararon los resultados del cultivo en cuanto: tiempo de detección de crecimiento, tasa de contaminación, por ciento de positividad, además se calcularon indicadores de desempeño. Resultados: Al comparar el método Petroff modificado con solución salina con el Petroff modificado con solución fosfato en Löwenstein Jensen, el tiempo de detección de crecimiento, por ciento de positividad y la tasa de contaminación se comportaron de forma similar y la sensibilidad (93,75 por ciento), concordancia (96,47 por ciento) e índice de Youden (0,91) fueron elevadas. Al compararlo el Petroff modificado con solución salina con el N-Acetil-L-Cisteína, las variables no mostraron diferencias significativas y los Indicadores de Desempeño se comportaron por encima del 93 por ciento, para el medio sólido y líquido. Conclusiones: Los resultados avalan la continuidad del uso del Petroff modificado con solución salina como método de descontaminación de las muestras pulmonares en la red de laboratorios de Cuba y como alternativa en el pretratamiento de las muestras para el medio líquido (Bact/Alert 3D), además constituye un soporte para el Programa Nacional de Control de Tuberculosis(AU)

Introduction: There are different decontamination methods of pulmonary samples for the diagnosis of mycobacteria. The National Program for the Control of Tuberculosis recommends Petroff method modified with saline solution; but there are not documented evidences that endorse it. Objective: Assess Petroff method modified with saline solution for the diagnosis of mycobacteria in Bact / Alert 3D system. Methods: An observational analytic study of diagnostic tests was conducted; there were used 100 pulmonary samples received in the National Laboratory of References and Researches of Tuberculosis, Leprosy and Mycobacteria of Pedro Kourí Institute, from April 2016 to January 2017. The sample was divided in 3 aliquots and those were decontaminated using 3 methods; then, they were inoculated in the solid and liquid culture means. Cultures´ results were compared according to: growth's detection time, contamination rate, percent of positivity; in addition, performance indicators were calculated. Results: When comparing Petroff method modified with saline solution with Petroff method modified with phosphate solution in Löwenstein Jensen, the growth's detection time, the percent of positivity and the rate of contamination behaved similarly, and sensitivity (93,75percent), concordance (96,47percent) and Youden´s index (0,91) were high. When the Petroff method modified with saline solution was compared with N-Acetil-L- Cisteina, the variables did not show significative differences and the behaviour indicators were over 93percent for the solid and liquid mean. Conclusions: The results endorse the continuity of the use of Petroff method modified with saline solution as a decontamination method of pulmonary samples in the network of Cuban laboratories and as alternative to the pre-treatment of the samples for the liquid mean (Bact/Alert 3D); it also constitutes a support for the National Program for the Control of Tuberculosis(AU)

Nuevas herramientas para el diagnóstico de la tuberculosis / New tools for tuberculosis diagnosis

Introducción: en las últimas décadas se han desarrollado nuevas herramientas para disminuir el tiempo de diagnóstico de las infecciones por Mycobacterium tuberculosis. Objetivo: introducir nuevas herramientas para la identificación de M. tuberculosis y comparar los resultados con el cultivo en Löwenstein Jensen. Métodos: se estudiaron 1 368 muestras recibidas en el Laboratorio Nacional de Referencia de Tuberculosis del Instituto de Medicina Tropical Pedro Kourí, de agosto 2010 - agosto 2014. Las muestras después de procesadas fueron inoculadas en paralelo en Löwenstein Jensen y en BacT ALERT. Los resultados se ana­lizaron y compararon con relación al total de aislamientos, tiempo de detección de crecimiento y tasa de contaminación, se calcularon además los indicadores de desempeño del BacT ALERT. Resultados: por Bact/ ALERT se identificó Mycobacterium tuberculosis en 126 (98,5 por ciento) muestras y 116 (88,5 por ciento) por el Löwenstein Jensen. El tiempo de detección de crecimiento de Mycobacterium tuberculosis por el BacT/ ALERT fue de 16,6 días, dos veces menor que el obtenido por el Löwenstein Jensen (35,5 días). La tasa de contaminación por el Bact/ ALERT y Löwenstein Jensen fue de 11 por ciento y 7,8 por ciento, respectivamente. La sensibilidad, especificidad e índice de Youden fue de 99,1 por ciento, 99,0 por ciento y 0,98, respectivamente; y el índice de validez del 99 por ciento. Conclusiones: el sistema Bact/ ALERT resultó un método útil porque acortó significativamente el tiempo de diagnóstico de la tuberculosis permitiendo comenzar el tratamiento de forma oportuna, sobre todo en pacientes con baciloscopia negativa. El uso combinado del Löwenstein Jensen y medio líquido aseguró la recuperación del total de cepas de M. tuberculosis(AU)

Background: in recent decades, new tools have been developed to decrease the time of diagnosis of infections with Mycobacterium tuberculosis. Objective: to introduce the new tools for identification of M. tuberculosis and compare the results with culture in Löwenstein Jensen. Methods: 1 368 samples received at the National Tuberculosis Reference Laboratory of the IPK from August 2010 to August 2014. After samples processed were inoculated in parallel on Löwenstein Jensen and BacT ALERT. The results obtained are analyzed and compared with respect to the total isolates, time detection of growth and contamination rate, the performance indicators of BacT ALERT also calculated. Results: by Bact / ALERT were identified Mycobacterium tuberculosis in 126 (98.5 percent) samples and 116 (88.5 percent) by Löwenstein Jensen. The time detection of growth of Mycobacterium tuberculosis by BacT / ALERT was 16.6 days, two times lower than that obtained by the Löwenstein Jensen (35.5 days). The contamination rate by Bact / ALERT and Löwenstein Jensen was 11 percent and 7.8 percent, respectively. The sensitivity, specificity and Youden index was 99.1 percent, 99.0 percent and 0.98, respectively; and the validity index was 99 percent. Conclusions: bact / ALERT system proved a useful method because it significantly shortened the time of tuberculosis diagnosis and start allowing treatment in a timely manner, especially in smear-negative patients. The combined use of liquid medium and Löwenstein Jensen assured recovery of all strains of Mycobacterium tuberculosis(AU)

Tuberculosis Burden -In Non-HIV Patients at a Tertiary Care Hospital in Hyderabad Telangana State.

Introduction: The revised national tuberculosis control program of India has been able to reduce the disease burden significantly. Despite; TB continues to affect 40% of our population. To achieve the desired goal of NSP {national strategic plan} 2012-2017 we need to have a focused approach on the disease prevalence and the most vulnerable. Aim: To estimate the burden of tuberculosis both pulmonary and extra pulmonary in non HIV patients at a tertiary care hospital in Hyderabad Telangana state South India. Materials and Methods: Over a period of two years from Jan.2013- Jan.2015 a total of two hundred and twenty six specimens, seventy eight from pulmonary and one hundred forty eight from extra pulmonary tuberculosis suspects were processed by various methods to achieve diagnosis. Results: Microbiologically the disease was observed in 25% of the studied subjects. Pulmonary tuberculosis accounted for 10% and extra pulmonary in 15%. Overall smear by ZN stain was positive in 17% and culture in 20%. Cytology could detect disease in 67% of the suspects. Biochemical findings were insignificant. Drug resistance was noted in 4.4% of the cases. Drug resistance and MDR tuberculosis was more common in pulmonary form than in extra pulmonary. Females dominated both in the suspect’s and culture confirmed cases as 53% & 76% respectively. The most affected age group for extra pulmonary disease remained as 6-35 years for both the sexes. In case of pulmonary tuberculosis it was noted as 36-50 years for men. Conclusion: TB affects one third of the suspects. In the present study EPTB has exceeded PTB in the proportion of the laboratory confirmed suspects of tuberculosis. Both the forms of disease are more common in females and young age which needs to be prioritized in the control program to achieve the desired target of NSP.

Characterization of mycobacterium isolates from pulmomary tuberculosis suspected cases visiting Tuberculosis Reference Laboratory at Ethiopian Health and Nutrition Research Institute, Addis Ababa Ethiopia: A cross sectional study

Objective: To characterize mycobacterium isolates from pulmomary tuberculosis suspected cases visiting National Tuberculosis Reference Laboratory at Ethiopian Health and Nutrition Research Institute, for diagnosis of pulmonary tuberculosis from January 4 to February 22, 2010 with total samples of 263. Methods: Sputum specimens were collected and processed; the deposits were cultured. For culturing Lowenstein Jensen medium (LJ) and Mycobacteria Growth Indicator Tube (BACTEC MGIT 960) were used. Capilia Neo was used for detecting NTM isolates from isolates of BACTEC MGIT 960. In Armauer Hansen Research Institute, Addis Ababa Ethiopia, Deletion typing PCR method for species identification (from confirmed Mycobacterium tuberculosis complex (MTBC) isolates by Capilia Neo) was done. Results: Out of 263 enrolled in the study, 124 and 117 of them were positive for mycobacterium growth by BACTEC MGIT 960 and LJ culture method, respectively. From BACTEC MGIT 960 positive media of 124 isolates, 117 were randomly taken to perform Capilia TB Neo test. From these 7 (6%) of them were found to be NTM and 110 (94%) were MTBC. From these 110 MTBC isolates, 81 of them were randomly taken and run by the deletion typing RD9 PCR method of molecular technique. Out of these 78 (96.3%) were found to be species of Mycobacterium tuberculosis and 3 (3.7%) were found to be not in the MTBC. Regarding the types of methods of culture media, Mycobacteria Growth Indicator Tube (BACTEC MGIT 960) method was found to have excellent agreement (with kappa value of 0.78) with the routine method of LJ. Conclusions: Pulmonary tuberculosis suspected cases visiting the National Tuberculosis Reference Laboratory at EHNRI that were confirmed to be pulmonary tuberculosis are caused by the species of Mycobacterium tuberculosis, hence treatment regimen including pyrazinamide can be applied to the patients as the first choice in the study area in Addis Ababa, Ethiopia. There is indication of the presence of NTM in patients visiting the tuberculosis reference laboratory and this is important because NTM is known to cause pulmonary disease similar with sign and symptom of pulmonary tuberculosis but different in treatment. BACTEC MGIT 960 has excellent agreement with LJ media but it has high tendency of having high contamination rate unless a better decontamination method is designed.

Characterization of mycobacterium isolates from pulmomary tuberculosis suspected cases visiting Tuberculosis Reference Laboratory at Ethiopian Health and Nutrition Research Institute, Addis Ababa Ethiopia: a cross sectional study

OBJECTIVE@#To characterize mycobacterium isolates from pulmomary tuberculosis suspected cases visiting National Tuberculosis Reference Laboratory at Ethiopian Health and Nutrition Research Institute, for diagnosis of pulmonary tuberculosis from January 4 to February 22, 2010 with total samples of 263.@*METHODS@#Sputum specimens were collected and processed; the deposits were cultured. For culturing Lowenstein Jensen medium (LJ) and Mycobacteria Growth Indicator Tube (BACTEC MGIT 960) were used. Capilia Neo was used for detecting NTM isolates from isolates of BACTEC MGIT 960. In Armauer Hansen Research Institute, Addis Ababa Ethiopia, Deletion typing PCR method for species identification (from confirmed Mycobacterium tuberculosis complex (MTBC) isolates by Capilia Neo) was done.@*RESULTS@#Out of 263 enrolled in the study, 124 and 117 of them were positive for mycobacterium growth by BACTEC MGIT 960 and LJ culture method, respectively. From BACTEC MGIT 960 positive media of 124 isolates, 117 were randomly taken to perform Capilia TB Neo test. From these 7 (6%) of them were found to be NTM and 110 (94%) were MTBC. From these 110 MTBC isolates, 81 of them were randomly taken and run by the deletion typing RD9 PCR method of molecular technique. Out of these 78 (96.3%) were found to be species of Mycobacterium tuberculosis and 3 (3.7%) were found to be not in the MTBC. Regarding the types of methods of culture media, Mycobacteria Growth Indicator Tube (BACTEC MGIT 960) method was found to have excellent agreement (with kappa value of 0.78) with the routine method of LJ.@*CONCLUSIONS@#Pulmonary tuberculosis suspected cases visiting the National Tuberculosis Reference Laboratory at EHNRI that were confirmed to be pulmonary tuberculosis are caused by the species of Mycobacterium tuberculosis, hence treatment regimen including pyrazinamide can be applied to the patients as the first choice in the study area in Addis Ababa, Ethiopia. There is indication of the presence of NTM in patients visiting the tuberculosis reference laboratory and this is important because NTM is known to cause pulmonary disease similar with sign and symptom of pulmonary tuberculosis but different in treatment. BACTEC MGIT 960 has excellent agreement with LJ media but it has high tendency of having high contamination rate unless a better decontamination method is designed.

To Assess the Burden of and Trends in Extra Pulmonary Tuberculosis at a Tertiary Care Hospital in India.

Introduction: Extra pulmonary form of tuberculosis is an important public health disease which cannot be ignored because of its low transmissibility. Data on the exact burden of the disease in developing countries is scarce. Aim: To assess the burden of the disease in a tertiary care hospital of India. To study the clinical trends in the disease, and the utility of various diagnostic modalities for its diagnosis. To identify the Mycobacterial species and perform drug susceptibility test. Materials and Methods: A cross sectional study was carried out for a period of two years. A total of one hundred and forty seven samples were tested for extrapulmonary tuberculosis using a combination of bacteriological, cytological, histological and biochemical techniques to achieve proper diagnosis of the disease. Results: Young adults and females predominated in the study group and positive cases. Microbiologically, 26% of the specimens were positive. Eighteen percent of them were found to be culture positive for M. tuberculosis. Smear by Ziehl Neelsen stain was positive in 9%. A combination of culture media both solid and liquid maximized the yield of Mycobacteria. Lymph node tuberculosis was found to be the predominant type followed by others. Fifteen percent of the strains were found to be resistant to the first line drugs used in treatment of tuberculosis. Cytology and biochemical findings were found to be less specific in diagnosis of extrapulmonary tuberculosis. Conclusion: Extrapulmonary form of tuberculosis is seen in significant number of the suspects. Hence, attention should be paid towards its proper and early diagnosis followed by rational management, as if neglected may lead to associated complications and sequalae.

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 .

Occurrence of Non-Tuberculous Mycobacterium in clinical samples - a potential pathogen.

Background: Silent presence of non-tuberculous mycobacterium (NTM) has been observed since the last 100 years, but now the increasing incidence of NTM is of great concern for clinical microbiologists as well as clinicians. Although many advanced efforts are being made for identification and control of Mycobacterium tuberculosis, still the silently growing menace of non-tuberculous mycobacteria is receiving negligible attention. Objectives: This study was aimed to find NTMs in positive cultures and identify them up to species level. Material & Methods: During the study period, i.e. from January 2009 to June 2011, a total of 4104 positive cultures were subjected to species identification by different morphological and biochemical tests. All the tests for identification were performed as per standard procedure along with the standard strains of NTM provided by JALMA, Agra. Results: The identification of positive cultures showed 4044/15581 (25.95%) Mycobacterium tuberculosis complex and 60/15581(0.38%) NTM. The mycobacterium species identification results showed that out of total 60 NTM, 21 different species of NTM were found and they belonged to all the four groups of runyon. The most common species identified in this study was M.simiae (07) followed by M.avium(06), M.gordonae(05), M.kansasii(05), M.fortuitum(05), M.chelonae(05), M.pheli(05), M.terrae(04), M.szulgai(02), M.vaccae(02), M.flavescens(02), M. trivale(02), M.malmoense(01), M.scrofulaceum(01), M.intracellulare(01), M.xenopi(01), M.ulcerans(01), M.tusciae(01), M.triplex(01), M.septicum(01), M.mucogenicum(01). Conclusion: The isolation of NTMs from different clinical samples indicated that they may be the causative agents for pulmonary and extra-pulmonary non-tuberculous diseases. Elaborate and focused studies are needed to differentiate NTMs amongst commensal/colonizer, pathogen and laboratory contaminants.

Use of fluorescein diacetate (FDA) staining to detect viable Mycobacterium tuberculosis.

Objective: Drug resistant tuberculosis has long been a common problem prevailing in developing countries including Bangladesh. Present study focused on the rapid identification of live Mycobacterium tuberculosis among treatment failure cases. Materials and Methods: Sputum samples from a total of 100 category-I and category-II treatment failure cases, assumed as multidrug resistant tuberculosis, were studied through fluorescein diacetate (FDA) staining under light emitting diode (LED) fluorescence microscope. Considering culture method as gold standard, we also compared the results of FDA staining with that of auramine O staining. Results: A total of 85% acid-fast bacilli were detected by FDA staining, 82% by auramine O staining and a total of 85% isolates were detected in Lowenstein-Jensen (LJ) culture. The sensitivity of FDA staining (96.47%) was estimated to be slightly higher than that of auramine O staining (91.76%). Moreover, 76.47% cases were detected as multidrug resistant tuberculosis (MDR-TB). Conclusion: Taken together, FDA staining method has been proposed to be appropriate for the rapid diagnosis of drug resistant tuberculosis.

Rapid culture diagnosis of tuberculous lymphadenitis from a tertiary care centre in an endemic nation: Potential and pitfalls.

In spite of low sensitivity and specificity, standard diagnostic algorithm recommends fine needle aspiration cytology (FNAC) and direct microscopic screening for acid-fast bacilli (AFB) for the routine diagnosis of tuberculous lymphadenopathy (LNTB). In this study, the diagnostic utility of liquid broth based automated culture (BacT/ALERT 3D) technique was assessed in comparison with conventional techniques in 89 clinically suspected tubercular lymphadenitis patients. 60% (n = 53) were positive by FNAC and 38.4% (n = 34) demonstrated AFB in smear examination. BacT/ALERT yielded isolation in 43.1% (n = 38) aspirates, confirming tubercular aetiology. We also found six paediatric culture-positive cases which showed negative outcome by both FNAC and smear. Thus, we conclude that culture by BacT/ALERT, may be used for faster yield of Mycobacteria in LNTB, especially in children. Additionally, this could also be used as a platform for further differentiation of Mycobacterium tuberculosis from non-tuberculous mycobacteria (NTM) infection and for testing of anti-tubercular chemotherapeutic agents whenever drug resistance is suspected.

Exploitation of Culture Medium for Mycobacterium tuberculosis

The culture media for Mycobacterium tuberculosis (MTB) were contrived from both egg-based and agar based ingredients. In 1903, Dorset introduced the first egg-based medium. It was followed by the invention of Lowenstein-Jensen and Ogawa media that contain whole eggs as nutrient and malachite green to inhibit the growth of contaminants. These media have been used worldwide for their usefulness and inexpensiveness. However they have a fundamental disadvantage that the cultivation time for mycobacterial growth takes more than 4 weeks. In 1947, Dubos introduced the first agar medium and followed by the invention of the 7H10 by Middlebrook and Cohn. A powder base of these media contains agar, combination of seven salts, L-glutamic acid, pyridoxine, biotin, and malachite green. This requires the addition of the oleic acid, albumin, dextrose, and catalase (OADC) growth supplement. BACTEC MGIT960 has recently been introduced for rapid cultivation of MTB, which is fully automated liquid culture system with modified 7H9 broth. Agar-based medium developed by Middlebrook has a number of advantages over egg-based medium. One of them is transparency, which enables earlier detection of growing colonies. The major disadvantages of agar media are the high cost of OADC and the need for a CO2 incubator. In conclusion, there is a need for a new agar medium, which can be produced at a lower cost and earlier growth detection. In this review, we introduced the growth promoting factors which can be used as an alternative new growth supplement, cAMP and resuscitation-promoting factor (Rpf). These factors may abrogate a lag in adaption of the bacilli in culture media.

Ethambutol resistance of indigenous Mycobacterium tuberculosis isolated from human patients

The present study was conducted to find out the ethambutol resistance pattern of indigenous isolates of Mycobacterium tuberculosis from Tuberculosis diagnosed human patients. A total of 172 specimens were collected from six different sources and comprised of 84.9 percent sputum, 10.5 percent pus and 4.7 percent bronchial washings. There were 70.9 percent males and 29.1 percent females with 84.30 percent pulmonary and 15.69 percent extra-pulmonary tuberculosis. The Mycobacterium tuberculosis isolates collected from primary culture were further studied to determine their pattern and level of resistance. The inoculums were prepared using 0.5 Mac Farland turbidity standards. Five different concentration of ethambutol were used in Lowenstein Jensen (LJ) medium i.e. 2μg/ml, 4μg/ml, 6μg/ml, 8μg/ml and 10μg/ml for sensitivity testing. Data showed 10 (5.8 percent) resistant and 162 (94.2 percent) sensitive Mycobacterium tuberculosis out of total 172 clinical isolates. The growth was not inhibited at 1st (2μg/ml) and 2nd (4μg/ml) drug levels, while growth of 50 percent isolates inhibited at 3rd level (6μg/ml), 30 percent inhibited at 4th level (8μg/ml) and 20 percent at 5th level (10μg/ml). The last three levels are above the therapeutic index and not recommended in actual clinical practice. It is thus conceivable to explore some other more effective chemotherapeutic agents, modify combinations or find more effective procedures to stop morbidity and mortality due to ethambutol resistant Mycobacterium tuberculosis.

Ovo desidratado para substituir ovo in natura na preparaçäo do meio Löwenstein-Jensen para cultivo de micobactérias / Dehydrated egg to replace in natura egg in the preparation of Löwenstein-Jensen medium for mycobacteria culture

Os métodos bacteriológicos säo extremamente importantes para diagnosticar tuberculose pulmonar. O meio de cultura mais comumente usado para isolar micobactérias é o Löwenstein-Jensen (LJ), meio sólido à base de ovos. Os ovos precisam sofrer uma limpeza prévia que consiste em escová-los com água e sabäo, com posterior descontaminaçäo por 30 minutos em álcool 70 por cento. O ovo "in natura" apresenta dois inconvenientes: esta limpeza, que é demorada e o sistema de compras e armazenamento, pois os ovos devem ser frescos. Com o propósito de simplificar este procedimento, este estudo comparou o meio preparado com ovos frescos e com ovos desidratados. Amostras clínicas foram precessadas para cultura e o inoculo foi semeado nos dois tipos de meios. Os resultados das culturas mostraram que das 150 amostras positivas pela baciloscopia 123 foram positivas no meio convencional e 24 foram negativas, enquanto que no meio teste, foram 121 positivas e 26 negativas, mostrando uma diferença que näo foi significativa. Contaminaçöes ocorreram igualmente nos dois meios (3 amostras). Ao comparar-se os dois meios a sensibilidade foi de 98 por cento e a especificidade de 100 por cento. Conclui-se que o ovo desidratado pode ser uma forma alternativa e vantajosa, no preparo do meio de Löwenstein-Jensen. (AU)

Bacteriological methods are the most important to diagnose pulmonary tuberculosis. The culture medium most used to isolate mycobacteria is Löwenstein-Jensen (LJ), which is a solid medium containing eggs, glycerol and asparagine as carbon and nitrogen source. The eggs used to prepare the medium need a previous cleaning, consisting of brushing with water and soap, and decontamination with 70% alcohol during 30 minutes. This procedure takes time; besides it is not so simple to organize the acquisition of fresch eggs. With the purpose of simplifying the whole procedure of making medium, we propose the replacement of "in natura" by dehydrated eggs. This study compares the performance of conventional LJ and LJ made with dehydrated eggs. Clinical specimens sent to the laboratory for tuberculosis diagnosis were decontaminated by Petroff method and inoculated in tubes of conventional LJ and the test LJ. It was used 200 clinical specimens, 150 were smear positive and 50 smear negative. Among the 150 samples smear positive 123 were positive in traditional LJ and 24 were culture negative, while in the test LJ 121 were positive and 26 were negative. The difference was not significant. Three cultures were contaminated in both media. Comparison of the two media showed a sensibility of 98% and specificity of 100% The conclusion is that the dehydrated egg could be na alternative way to make LJ medium, propitiating a simpler and more practical medium preparation. (AU)