To evaluate the performance of TrueNAT (RT Micro PCR device) assay in comparison with GeneXpert on sputum samples from pulmonary cases of tuberculosis. 274 samples were processed to detect MTB by ZN smear examination, MGIT culture and molecular methods that included RT-PCR (ABI 7500 & TrueNAT) and GeneXpert for case detection of TB. The overall performance of the test with MGIT(Mycobacterium Growth Indicator Tube) culture as gold standard, sensitivity of smear, RT PCR/TrueNAT and Genexpert was 61.5% (CI:53.3–69.3%), 94.7% (CI:89.8–97.6%) & 96.0% (CI: 91.5–98.5%), respectively. Amongst the S+ (108) samples, RT-PCR/TrueNAT and GeneXpert showed a sensitivity of 99% (CI:94.9%–99.8%) and 100% (98.6%–100.0%), respectively. High concordance was observed between GeneXpert and TrueNAT for case detection of TB. The GeneXpert MTB/RIF test was independent on the user's skills. It has a short turn-around time and simultaneously detects RIF resistance with M. tuberculosis in less than 3 h. The TrueNAT MTB has good sensitivity and specificity in case detection with hands on time of less than 3 h as well as fits the requirements in resourcelimited health care settings. Larger, multi-site studies are required to obtain better estimates of the performance of TrueNAT MTB.

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The current study was undertaken to characterize the RRDR rpoB gene mutations among the rifampicin-resistant Mycobacterium tuberculosis (MTB) isolates from Pakistan. Rifampicin mutation patterns were analyzed by using PCR followed by rpoB gene sequencing. Among the 1080 referred TB cases, 63 (6%) were resistant against at least one first-line TB drug. Out of these 63 resistant isolates, 24 isolates (38%) were found to be resistant to isoniazid and rifampicin. Sequence analysis of multidrug-resistant tuberculosis (MDR-TB) isolates detected a single mutation in the RRDR region of the rpoB gene at codon 531, 516, 512, 528 and 533; however, 5 MDR-TB isolates lack any mutation in the RRDR region. A double mutation was observed in 1 MDR-TB isolate at codon 512 and 516 which are reported for the first time from Pakistan. Moreover, in 1 isolate a novel silent mutation was observed at codon 528. Further studies about these mutations may be helpful in the development of diagnostic tools for the detection of MTB in a high TB endemic area like Pakistan.

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This study explored the genetic diversity of Mycobacterium tuberculosis isolates in Iraq by spoligotyping and 15-locus-based mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) methods. Initially, 270 isolates from 134 patients were collected and then 134 non-duplicating isolates (1 isolate/patient) were subjected to the study analyses, 70 isolates were found to be multidrug resistant (MDR) upon testing by proportion method on Löwenstein–Jensen medium. Spoligotyping yielded 39 patterns; 111/134 (82.2%) isolates being grouped in 16 clusters vs. 23/134 (17.2%) isolates being unique. SIT1144/T1 represented the largest cluster (n= 20, 14.9%), followed by SIT25/CAS1_Delhi (n= 19, 14.2), SIT22/CAS1_Delhi (n= 12, 9%); the other clusters ranged from 2 to 8 isolates. The SIT1144 is not reported in neighboring countries and only 4 isolates were reported worldwide (2 in USA, 1 in Venezuela, and 1 in Greece). This study reported 4 isolates belonging to SIT41/Turkey family, and thus it seems that this family is not exclusive to Turkey as previously thought. CAS lineage was predominant in this study (42.5%), followed by ill-defined T (29.9%). Highly diverse MIRU-VNTR genotypes were displayed; 100 distinct MIRU-VNTR genotypes were detected (8 clusters with 2–8 strains/cluster and 92 unique). The clustering rate was 18.03%. The discriminatory efficiency of MIRU-VNTR was high (Hunter-Gaston discriminatory index [HGDI]= 0.992); it was higher than that of spoligotyping (HGDI; 0.930). However, the highest discriminatory power was provided by spoligotyping and MIRUs together. Owing to the low clustering rate by MIRU-VNTR, these results suggest that drug-resistance TB in Iraq is due to acquired resistance as opposed to transmission. Conclusion: Iraq is specific in having its own most predominant lineage (SIT1144/T1) which is not found among neighboring countries. The 15-locus MIRU-VNTR can be useful in discriminating M. tuberculosis isolates in Iraq.

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Objective: The study sought to assess the extent to which healthcare workers (HCWs) adhere to the National Tuberculosis Program (NTP) guidelines for the diagnosis of smear negative tuberculosis in Nigeria. Method: This was a cross-sectional retrospective desk analysis of case files of 280 smear negative pulmonary TB in six States in southern Nigeria. Results: About 93% of the 280 patients had their first set of sputum smear microscopy tests done, but only 3.6% had the second set of diagnostic tests as prescribed by the NTP guidelines. Only 45.7% (128/280) received broad spectrum antibiotics after their first smear microscopy. 98% had a chest X-ray done, while 93.6% (262/280) had HIV counseling and testing (HCT), out of which 45.0% were HIV positive. Overall, only 2 patients (0.7%) were diagnosed in strict compliance with the NTP guidelines. There was no significant difference in the pattern of diagnosis of smear negative TB cases and smear positive TB cases. Conclusion: The adherence of HCWs to the NTP guidelines for diagnosis of smear negative TB is apparently sub-optimal and needs improvement.

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Determination of lipid content of any biological sample is essential for various kinds of studies related to pathogenicity and drug development. Thus, reliable methods for the quantitative extraction of lipids are of critical importance. The mycobacterial cell wall is largely composed of lipids. Commonly used methods to extract lipids, such as the Bligh and Dyer method or the Folch method, yield a low amount of lipids when applied to mycobacterial cells. This study presents an efficient modification of Chandramauli's method, a less known method developed at this institute earlier that is able to yield a considerably higher concentration of mycobacterial lipids.

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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.

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It is often difficult to discern true mycobacterial infection from colonization due to Mycobacterium gordonae (M. gordonae) since this organism is ubiquitous and is commonly an innocuous saprophyte. This study reports a rare case of caseating hilar adenopathy and pulmonary disease caused by M. gordonae in a patient with chronic obstructive pulmonary disease (COPD) and rheumatoid arthritis (RA) on maintenance steroids and methotrexate. Pathologic exam and cultures of lymph node excision biopsy and bronchoalveolar lavage (BAL) confirmed the diagnosis. Triple antimycobacterial therapy with azithromycin, ethambutol and rifabutin was administered. The patient had significant clinical and radiologic improvement and follow-up cultures confirmed microbiologic cure. Mycobacterium gordonae can be a rare cause of significant pulmonary infection, and positive sputum or BAL cultures for M. gordonae should not be automatically discarded and considered as nonpathogenic contaminants or colonizing organisms, especially in immunocompromised hosts with comorbidities. A detailed review of the case and relevant literature is provided.

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Bifocal musculoskeletal tuberculosis is a rare presentation of the disease. The following report documents the case of a 28-year-old Moroccan man presenting simultaneously with a swelling of the palm of the hand and the sternoclavicular joint. The HIV serology and hepatitis (B, C) serology were negative, as well as the tuberculin skin test. The radiographs of both locations were normal. The histopathology confirmed the diagnosis. The antibacillary chemotherapy produces excellent results. It is therefore indispensable to bear in mind the possibility of such atypical presentations of tuberculosis when making a rapid and pertinent diagnosis and prescribing the appropriate treatment.

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This study compared the ambient air survival of two clinical strains of Mycobacterium tuberculosis that had caused significantly different numbers of cases in the same population. No difference in the survival ability between the two strains was found. However, a significant difference was observed in colony morphology between the two strains. These findings provide new insight into potential microbial determinants for the occurrence of large clusters of tuberculosis cases in a population.

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Objective: To assess the quality of week 8 sputum smear AFB microscopy performed by peripheral TB laboratories in Nigeria. Method: A cross-sectional review was performed of all week 8 tuberculosis sputum smear slides reported for the first quarter of 2009 by peripheral laboratories in five States of Nigeria. Each slide was reviewed by two independent external slide readers as external quality check and also crosschecked with fluorescent microscopy. Results: In Akwa Ibom, Anambra, Enugu, Kogi and Ogun States, a total of 415, 315, 231, 206 and 428week 8 slides respectively were studied (a grand total of 1595 slides studied). The wide range of conversion rates between the different States as reported by peripheral labs (83.8% in Anambra State to 98.1% in Kogi State) was also observed by the external quality check (68.4% in Kogi State to 88.0% in Akwa Ibom State). In all the States, the studied sputum conversion rates reported by the peripheral labs were significantly higher than values obtained from external quality check and fluorescent microscopy (p = 0.000). Conclusion/recommendation: There is a wide range of sputum conversion rates between States, but the conversion rate in each State is significantly higher than those of external quality check possibly indicating many false negative reports by peripheral labs. It is recommended that training and re-training of laboratory persons be continued. Internal and external quality checks should also continue to be practiced in the national TB program.

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Tuberculosis verrucosa cutis (TVC), also known as warty tuberculosis, anatomist's wart or prosector's wart is characterized by the presence of verrucous plaque-like lesions, resulting from direct inoculation of the causative organism into the skin of a previously infected patient. A 59-year-old man presented with a hyperpigmented plaque on the chest wall which closely mimicked a keloid. He was a case of sputum-positive pulmonary tuberculosis and had repeatedly been applying early morning saliva on the lesion as a part of the indigenous practices for quick healing. There was further progression of the lesion with discharge from several sites. A smear for acid fast bacilli was positive from the discharge and growth on Lowenstein Jensen medium revealed growth of Mycobacterium tuberculosis. Biopsy was compatible with TVC and the patient was started on 6 months anti-tubercular therapy. However, the plaque continued to persist with continuing discharge from multiple openings which necessitated surgical intervention, finally leading to near complete resolution of the plaque of TVC.

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