Pathology and diagnosis of Mycobacterium bovis in naturally infected dromedary camels (Camelus dromedarius) in India.

The present study investigated the pathological features of tuberculosis (TB) caused by Mycobacterium bovis and its diagnosis in naturally infected dromedary camels from an organized farm in India. During the period of the 5-year study, a total of 18 (19.56 %) camels out of 92 examined showed gross lesions compatible with TB at post-mortem. The clinical signs and pathological lesions in these camels were studied, and the efficacy of different diagnostic tests was also assessed. On the basis of occurrence and distribution of gross TB lesions, the infected camels revealed two different lesional patterns as pulmonary (n = 15) and disseminated (n = 3) form. The histopathology of affected organs revealed typical granulomatous lesions wherein the giant cells and acid-fast bacilli were occasionally observed in pulmonary form whereas they frequently observed in disseminated form. The single intradermal tuberculin test (SIDT) detected TB in 10 (55.55 %) whereas the Ziehl-Neelsen (ZN) stain and IS6110 PCR from tissue lesions detected 13 (72.22 %) and 18 (100 %) of the infected camels, respectively. The study suggests that pulmonary form of the TB is more common in camels indicating respiratory route as the major source of exposure in camel herds. Moreover, very low sensitivity of SIDT was observed which highlights the difficulty for confirmation of TB in live camels.

In-house PCR with DNA extracted directly from positive slides to confirm or exclude the diagnosis of tuberculosis: focus on biosafety.

The possibility to obtain DNA from smears is a valuable alternative to remedy the lack of samples when they are totally used for bacilloscopy; this technique solves the biosafety problem related to a possible accident with the transportation of flasks containing potentially transmissible clinical samples. Hence, the purpose of this study was to utilize the insertion sequence IS6110 for amplification of DNA from a smear-positive sample for tuberculosis (TB) diagnosis. Among the 52 positive bacilloscopies, sensitivity, specificity, positive predictive value and negative predictive value were 52.3%, 100%, 100% and 89.7%, respectively whereas accuracy was 90.7%. The IS6110-based PCR for TB diagnosis developed in DNA extracted from a positive smear is a fast, simple, specific, and safe method.

Comparison of Protein B Polymerase Chain Reaction (PCR) With IS6110 PCR for Diagnosis of Tuberculous Meningitis Patients.

Purpose Tuberculous meningitis (TBM) is a diagnostic challenge. With the conventional staining and culture techniques being too insensitive and time-consuming, and the commercial detection systems being costly, polymerase chain reaction (PCR) seems lucrative for routine laboratories. The purpose of this study was to evaluate the diagnostic potential of protein b antigen polymerase chain reaction (Pab PCR) for TBM, in comparison to IS6110. Another purpose was to compute a cut-off at which adenosine deaminase (ADA) could diagnose TBM. Material and methods This is a prospective case-control study to measure the diagnostic accuracy of PCR, BACTEC culture, Lowenstein-Jensen (LJ) culture, ADA, and acid-fast bacilli (AFB) smear tests in TBM. CSF from 50 TBM patients (10 confirmed, 40 clinically suspected) and 40 controls was subjected to Pab PCR and IS6110 PCR, and performance was compared against culture and composite reference standards. Results The overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of Pab PCR in diagnosing TBM were 82%, 100%, 100%, and 81.63%, respectively, and that of IS6110 PCR were 74%, 100%, 100%, and 75.47%, respectively. Both PCRs outperformed culture (p<0.001). Though performance of both PCRs was comparable (p=0.335) with excellent agreement (k=0.86), Pab PCR detected four additional cases, one culture-positive and three culture-negative clinically suspected. ADA of 6.5 IU/L was able to differentiate between TBM and non-TBM with 86% sensitivity and 63% specificity. Conclusions Molecular tools such as PCR have the potential to increase the clinician's ability to diagnose tuberculous meningitis. Pab PCR is a rapid and reliable method for diagnosing TBM in routine microbiology laboratories.

Evaluation and comparison of molecular and conventional diagnostic modalities for detecting pulmonary tuberculosis in bronchoalveolar lavage fluid.

CONTEXT: In cases of sputum smear-negative and sputum-scarce (SSN/SC) pulmonary tuberculosis (PTB), bronchoalveolar lavage (BAL) fluid may be helpful in establishing diagnosis. No specific recommendations for BAL samples have yet been formulated due to limited literature. AIMS: 1. To find a sensitive and specific protocol for same-day diagnosis of PTB using BAL in SSN/SC clinically suspected patients. 2. To evaluate the need to routinely perform MGIT for all BAL samples. SETTINGS AND DESIGN: Prospective observational study design in a tertiary care hospital in New Delhi. METHODS AND MATERIAL: Fibreoptic bronchoscopy was performed and BAL collected from 175 clinically suspected SSN/SC PTB patients. BAL samples were subjected to: ZN Stain, Xpert MTB/RIF CBNAAT, BACTEC MGIT 960 liquid culture and M. tuberculosis complex DNA Real time PCR. The results of the various diagnostic tests were analysed using a) MGIT as gold standard and b) a composite reference standard (CRS) for a final diagnosis of PTB. STATISTICAL ANALYSIS USED: Microsoft Excel 2016 and SPSS version 21.0 were used. Sensitivity, specificity and predictive values were calculated and compared using McNemar test. A p value of <0.05 was considered statistically significant. RESULTS: 34 Cases had a final diagnosis of TB as per the CRS. Using CRS, MGIT had a sensitivity of 50.0% (32.4%-67.6%). There was no statistically significant difference between sensitivities of CBNAAT and PCR; both were more sensitive than ZN stain. Sensitivity and specificity of CBNAAT was 79.4% (62.1%-91.3%) and 100.0% (97.4%-100.0%) respectively. The preferred protocol for the hospital is CBNAAT and ZN stain. There was no statistically significant difference in sensitivity by adding PCR or MGIT to this protocol. CONCLUSIONS: We found it a good strategy to perform CBNAAT and ZN stain on BAL fluid for accurate and same-day PTB diagnosis. CBNAAT is useful for ruling PTB in even when BAL cultures are negative. It is prudent to continue to routinely perform MGIT for all BAL samples.

Detection of Mycobacterium tuberculosis in paucibacillary sputum: performances of the Xpert MTB/RIF ultra compared to the Xpert MTB/RIF, and IS6110 PCR.

The Xpert MTB/RIF Ultra assay has recently been launched to improve the detection of smear negative disease. This retrospective study compares the sensitivity of Xpert MTB/RIF Ultra with that of Xpert MTB/RIF tests and IS6110 real-time PCR in sputum. Diagnostic performance of three molecular tests was evaluated using 48 culture-positive clinical respiratory specimens diluted to obtain paucibacillary sputum specimens. Xpert MTB/RIF Ultra had the highest sensitivity of 100% compared to 42% (P < 0.001) for Xpert MTB/RIF and 64.5% (P = 0.02) for IS6110-PCR. All "very low" or "low" positive specimens using Xpert MTB/RIF Ultra were tested positive using IS6110-PCR, but 35.4% were found negative using Xpert MTB/RIF. Xpert MTB/RIF Ultra is more sensitive than the two other tests for sputum with a low bacterial load. Adding detection of IS6110 and IS1081 to rpoB, is a key evolution of the assay and improves the detection of Mycobacterium tuberculosis' DNA in paucibacillary sputum.

Role of PCR method using IS6110 primer in detecting Mycobacterium tuberculosis among the clinically diagnosed childhood tuberculosis patients at an urban hospital in Dhaka, Bangladesh.

OBJECTIVE: Better methods are needed for the accurate detection of child tuberculosis (TB). This study compared different laboratory tests and evaluated IS6110 PCR for the detection of Mycobacterium tuberculosis (MTB) among clinically diagnosed child TB patients. METHODS: A total of 102 paediatric patients (<15 years old) with clinically diagnosed TB were enrolled in this study. The patients were admitted to the icddr,b hospital in Dhaka between 2003 and 2005. Sputum/gastric lavage samples were collected for smear microscopy, culture (solid/Lowenstein-Jensen medium and liquid/MGIT), and IS6110 PCR testing. The sensitivity, specificity, and positive and negative predictive values (PPV, NPV) of smear microscopy and PCR were compared to the two culture methods. RESULTS: Three patients were positive on smear microscopy (2.9%). MTB was detected by conventional culture in 15.7% (16/102), liquid culture in 14% (14/100), and IS6110 PCR in 61.8% (63/102). PCR detected an additional 45 patients who were undetected with the three other tests. Compared to conventional and liquid culture, respectively, smear microscopy showed sensitivity of 18.8% and 21.4%, specificity of 100% individually, PPV of 100% individually, and NPV of 86.9% and 88.7%, whereas PCR had sensitivity of 87.5% and 92.9%, specificity of 43% individually, PPV of 22.2% and 21%, and NPV of 94.9% and 97.4%. CONCLUSIONS: PCR can be useful compared to smear microscopy and culture methods and is applicable as a rapid screening test for child TB. A larger scale study is required to determine its diagnostic efficacy in improving the detection of child TB in the presence and absence of severe malnutrition.

Diagnosis of tuberculous meningitis: Current scenario from a Tertiary Neurocare Centre in India.

BACKGROUND: Tuberculous meningitis (TBM) is a condition that is caused by Mycobacterium tuberculosis complex and is difficult to diagnose due to the nonspecificity of the presentations. The study analyzed the different modes of diagnosis available in a developing country set up over a period of five years to understand the diagnostic values of the current conventional and automated methods of diagnosis of TBM among the patients suspected with chronic meningitis. METHODS: A total of 10,281 cerebrospinal fluid samples (CSF) were collected from suspected chronic meningitis patients, of which 790 samples were from individuals who had clinically suspected TBM. The samples were subjected to CSF cytology and staining, culturing, immunological tests, molecular techniques, and methods for detection of drug resistance. RESULTS: The TBM patients were predominantly male, with a mean age of 21-40 years. CSF pleocytosis and lymphocytic predominance were noted. Culture had 40.13% positivity among clinically suspected TBM patients. The multidrug-resistant M. tuberculosis (MDR-TB) constituted 3.14% of the total clinical isolates. With IS6110 PCR, a specificity of 92.86% and sensitivity of 100% are seen with an assay threshold of 30pg/ml. Line probe assay (LPA) using culture isolates had a sensitivity of 97.67% and a specificity of 100%. Direct CSF LPA showed a sensitivity of 96.15% and a specificity of 100%. CONCLUSIONS: A combination of techniques that involved culture, cytology, and DNA amplification methods was found to be promising in specific, accurate, and rapid detection of M. tuberculosis in the CSF samples from patients.

Noninvasive Tuberculosis Screening in Free-Living Primate Populations in Gombe National Park, Tanzania.

Recent advances in noninvasive detection methods for mycobacterial infection in primates create new opportunities for exploring the epidemiology of tuberculosis in free-living species. Chimpanzees (Pan troglodytes schweinfurthii) and baboons (Papio anubis) in Gombe National Park, Tanzania, were screened for infection with pathogens of the Mycobacterium tuberculosis Complex using Fecal IS6110 PCR; none was positive. This study demonstrates the feasibility of large-scale mycobacterial screening in wild primates.

Upconversion nanoparticle-based Förster resonance energy transfer for detecting the IS6110 sequence of Mycobacterium tuberculosis complex in sputum.

Upconversion nanoparticles (UCNPs), which are excited at near-infrared wavelength (980 nm), emit high-energy photons. Since UCNPs display a high signal-to-noise ratio and no photobleaching, they are extremely useful for diagnostic application. In this study, we applied UCNPs for detecting the IS6110 sequence of the Mycobacterium tuberculosis complex (MTBC) and evaluated the feasibility of the system for use in molecular diagnostics. Using biotinylated primers, IS6110 DNA PCR was performed and the PCR amplicon was then mixed with streptavidin-conjugated UCNPs, followed by intercalation with SYTOX Orange dye. Fluorescence detection for the Förster resonance energy transfer (FRET) of the UCNPs (UCNP-FRET) was then performed. The estimated lowest detection by UCNP-FRET was 10(2) copies/µL of IS6110 DNA (157 bp). The kappa agreement of the UCNP-FRET assay with conventional PCR was 0.8464 (95% confidence interval, 0.7442-0.9486) and false-negative results were reduced. Our results demonstrated the successful implementation of the UCNP-FRET system in detecting the IS6110 sequence of the MTBC and its potential application for molecular diagnostics.

Comparison of LCD array and IS6110-PCR with conventional techniques for detection of Mycobacterium bovis isolated from Egyptian cattle and Buffaloes.

Bovine tuberculosis is a chronic bacterial and major infectious disease of cattle and buffaloes caused by Mycobacterium bovis. Rapid diagnosis of bovine tuberculosis is considered one of the cornerstones for worldwide control as it permits early epidemiological and therapeutic interventions. Therefore, this study was designed to evaluate conventional techniques (tuberculin test, Ziehl Neelsen staining and culturing) in comparison with proven molecular laboratory techniques (LCD array and IS6110 PCR) for identification of Bovine tuberculosis. A total of 902 Egyptian animals (480 buffaloes and 422 cattle) were examined by tuberculin test, and the positive reactors were slaughtered. Tissue samples were collected for staining as well as culturing. Moreover, LCD array and PCR using IS6110 on DNA extracted from tissue and culture samples were carried out for molecular identification of M. bovis. According to the results, the tuberculin positive cases for cattle and buffaloes were 2.14% (9 cases) and 5.62% (27 cases), respectively. After post-mortem examination, the prevalence of tuberculin positive cases with visible lesions was 88.9% for cattle and 14.8% for buffaloes. Alternatively, these percentages were 11.1% and 85.2% for cattle and buffalo carcasses with non-visible lesions. The percentage of cattle and buffaloes showing positive culture was 88.9% and 62.9%, respectively. This percentage was 69.5% after staining with Ziehl Neelsen. In contrast, LCD array and IS6110 were 100%, confirming the isolation results. In conclusion, LCD array depending on 16S RNA and DNA hybridization with specific probes for detection of M. bovis are rapid, sensitive and labor-saving when combined with IS6110-PCR.

PCR interna con ADN extraído directamente de portaobjetos positivos para confirmar o excluir el diagnóstico de tuberculosis: enfoque en la bioseguridad / In-house PCR with DNA extracted directly from positive slides to confirm or exclude the diagnosis of tuberculosis: focus on biosafety

The possibility to obtain DNA from smears is a valuable alternative to remedy the lack of samples when they are totally used for bacilloscopy; this technique solves the biosafety problem related to a possible accident with the transportation of flasks containing potentially transmissible clinical samples. Hence, the purpose of this study was to utilize the insertion sequence IS6110 for amplification of DNA from a smear-positive sample for tuberculosis (TB) diagnosis. Among the 52 positive bacilloscopies, sensitivity, specificity, positive predictive value and negative predictive value were 52.3%, 100%, 100% and 89.7%, respectively whereas accuracy was 90.7%. The IS6110-based PCR for TB diagnosis developed in DNA extracted from a positive smear is a fast, simple, specific, and safe method

La posibilidad de obtener ADN a partir de frotis es una valiosa alternativa para remediar la falta de muestras cuando estas son totalmente utilizadas para la baciloscopia; esta opción soluciona, además, el problema de bioseguridad asociado a la posibilidad de accidente al transportar frascos que contienen muestras clínicas potencialmente infectivas. Por lo tanto, el propósito de este estudio fue utilizar para el diagnóstico de la tuberculosis la secuencia de inserción IS6110 para amplificación del ADN a partir de frotis que resultaron positivos por baciloscopia. Del análisis de 52 baciloscopias positivas surge que la sensibilidad, la especificidad, el valor predictivo positivo y el valor predictivo negativo de esta técnica fueron, respectivamente, del 52,3%, del 100%, del 100% y del 89,7%; y la precisión fue del 90,7%. La PCR IS6110 para el diagnóstico de tuberculosis, desarrollada con ADN extraído de frotis positivos, es un método rápido, simple, específico y seguro

Uso clínico de la reacción en cadena de la polimerasa e hibridización en el diagnóstico de tuberculosis / Clinical use of the polymerase chain reaction and hybridization in diagnosing tuberculosis

La tuberculosis (TBC) es una causa importante de morbimortalidad en nuestro país. Las herramientas de diagnóstico no son 100% eficientes y rápidas para ayudar al clínico en su decisión médica. Nuevas pruebas, como la reacción en cadena de la polimerasa (PCR), son necesarias. Evaluar la utilidad clínica de una PCR para IS6110, en muestras clínicas de pacientes con sospecha de tuberculosis pulmonar o extrapulmonar. Sesenta y una muestras de 46 pacientes del Hospital General del Este, Dr. Domingo Luciani de Caracas, Venezuela, fueron procesadas para la detección del M. Tuberculosis. Se practicó baciloscopia, cultivo en LJ y PCR e hibridación. Los pacientes se clasificaron como TBC positivos si tenían evidencia clínica o radiológica, y positivas algunas de estas pruebas: PPD, ZN, cultivo, biopsia positiva o respuesta favorable al tratamiento. Los TBC negativos con evidencia clínica o radiológica positiva y todas las pruebas negativas. La sensibilidad de la PCR sola fue del 75% y la especificidad del 61%. La PCR e hibridación juntas mostraron sensibilidad del 90% y especificidad del 57%. Los valores predictivos positivos y negativos fueron del 62% y 88%, respectivamente

Tuberculosis (TBC) remains a leading cause of morbidity and mortality in Venezuela. The diagnos tic tools used are not 100% specific, sensitive, efficient or fast enough to help clinicians take the right clinical decision. New tests, like polymerase chain reaction (PCR), are necessary. We evaluated the clinical usefulness of an IS6110 in-house PCR in clinical samples from patients suspected of having pulmonary or extrapulmonary TBC. Sixty one samples from 46 patients were processed for detection of M. tuberculosis by ZN stained smear examination, LJ medium culture, and PCR and hybridization assay. The patients were classified as TBC positive when they had clinical or radiological evidence plus any positive of the following tests: PPD, ZN, LJ culture, biopsy or anti-TBC treatment response and TBC negative when they had clinical or radiological evidence but all the applied tests used were negative. PCR sensibility was 75% and specificity was 61%, using PCR alone, but when PCR and hybridization were evaluated together the sensibility was 90% and specificity was 57%. The predictive positive and negative values were 62% and 88% respectively