Recovery rates of mycobacterium from suspected extra-pulmonary tuberculosis patients using liquid culture at a tertiary referral centre of India.

Tuberculosis still remains a serious public health problem in developing countries. Rapid isolation of mycobacteria is critical for accurate diagnosis and management of tuberculosis. In the present study BACTEC MGIT 960 system was evaluated against Lowenstein Jensen (LJ) medium for isolation of mycobacteria from different extra-pulmonary specimens (N = 371). The samples were processed using NaOH-NALC method and inoculated in BACTEC MGIT and on LJ medium. The BACTEC MGIT 960 system detected 93 (25.06%) samples positive for acid fast bacilli and by LJ only 38 samples (10.24%) was positive. Furthermore, total 99 (26.68%) samples were detected positive by both the culture methods. The mean turnaround time to detection of mycobacteria by MGIT 960 were significantly less (12.4 days) as compared with LJ (22.76 days). In conclusion, BACTEC MGIT 960 system is more sensitive and rapid culture system for isolation of mycobacteria. However LJ culture method also suggested to further increase the detection rate of EPTB cases.

Non-tuberculous mycobacterial infections in geriatric patients-A neglected and emerging problem.

The diseases caused by Non-tuberculous mycobacteria (NTM) has increased steadily in the last two decades. Increase in incidence of NTM infections are being reported in elderly people as they are more susceptible and often experiencing high morbidity. There is prediction that NTM infections will further rise because of expected increase in elderly population by 2050. Given the importance of NTM infection in the elderly, the interest in studying NTM characteristics in the aged population is increasing. In this review, we summarize the characteristics of NTM infection among elderly patients. We focus on epidemiology, clinical presentation, and treatment options of NTM in this age group. We highlight the differences in the diagnosis and treatment between rapid and slow growing mycobacterial infections. The current recommendations for treatment of NTM have been discussed. Finally, we have reviewed the prognosis of NTM disease in elderly patients.

Laboratory Cost Analysis of Conventional and Newer Molecular Tests for Diagnosis of Presumptive Multidrug-Resistant Tuberculosis Patients.

Introduction: Tuberculosis (TB) remains a deadliest infectious disease. Lack of rapid test with low cost is one of the important challenges to eradicate the TB. The objective of the study was to analyze the laboratory costs of conventional and newer molecular tests, for diagnosis of presumptive multidrug-resistant TB (MDR-TB) patients. Methods: A detailed laboratory cost of various conventional tests (Ziehl - Neelsen [ZN] microscopy, light-emitting diode-fluorescent microscopy [LED-FM], culture and drug susceptibility testing [DST] using solid Lowenstein-Jensen media and liquid media [BACTEC MGIT 960]) was compared with rapid methods (GenoType MTBDRplus line probe assay [LPA] and GeneXpert MTB/RIF assay). Laboratory cost was also calculated in terms of cost per TB and MDR-TB case detected by using different diagnostic scenarios. Results: Cost per test for ZN microscopy, LED-FM, LPA, GeneXpert MTB/RIF assay, solid culture plus DST, liquid culture plus DST was found as $2.5 (INR 156.8), $2.0 (INR128.9), $18.6 (INR1210), $13.8 (INR 895.2), $21.5 (INR 1396.6), and $29.1 (INR 1888.2), respectively. The laboratory cost for detecting TB and MDR-TB by diagnostic scenarios involving molecular DST was found to be less as compared to involving only conventional liquid culture-based test. Conclusions: The implementation of rapid molecular tests with selective use of liquid culture-based DST may be less in cost as compared to the use of culture-based DST alone, at high burden reference TB laboratory.

Multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis strains in geriatrics: An analysis and its implications in tuberculosis control.

Objective: This study aimed to analyze the trends of tuberculosis (TB) disease, drugs susceptibility patterns in geriatric TB over a period of three years (from 2010 to 2012). Materials & methods: In this study, laboratory data on diagnosis of geriatric tuberculosis suspected patients (age ≥60 years) was analyzed retrospectively at National Reference Laboratory (NRL). Results: Among 12,140 geriatric TB suspects, 1621 (13%) were acid-fast bacillus (AFB) smear-positive and 10,519 (87%) were smear-negative. Analysis of 915 culture results showed 470 (51%) as positive for Mycobacterium tuberculosis complex (MTBC), 63 (7%) contaminated and 36 (4%) identified as mycobacteria other than tuberculosis (MOTT). A total 210/470 (45%) were multidrug-resistant TB (MDR-TB) strains. Among the mono-resistant strains, isoniazid mono-resistant was found more frequently (134/470, 28%) whereas, it was least among rifampicin mono-resistant 5/470 (1%). The second-line drug susceptibility testing (DST) results showed 7% (17/240) extensively drug-resistant TB (XDR-TB) strains. Most common second line mono-resistant strain was observed with ofloxacin, 16% (38/240). Conclusion: This study shows high number of MDR/XDR geriatric TB patients at tertiary care TB hospital. The study highlighted the need of separate line of early identification, diagnosis and treatment of geriatric TB patients. However, further study with improved sample size may needed to confirm the findings.

Direct Molecular Detection of Drug-Resistant Tuberculosis from Transported Bio-Safe Dried Sputum on Filter-Paper.

In 2019, amongst half a million new rifampicin-resistant tuberculosis (TB) cases, 78% were multi drug-resistant TB (MDR-TB). Access to rapid and Universal-Drug susceptibility testing (DST) to patients in remote areas is a major challenge to combat drug-resistant TB. To overcome this challenge, we had recently reported the development of 'TB Concentration & Transport kit' for bio-safe ambient temperature transport of dried sputum on filter-paper (Trans-Filter). The present study was conducted to evaluate the utility of DNA extracted from sputum on Trans-Filter in a Multiplex PCR-based sequencing assay (Mol-DSTseq) for diagnosing drug-resistant TB. The developed Mol-DSTseq assays were standardized on Mycobacterium tuberculosis clinical isolates (n = 98) and further validated on DNA extracted from sputum on Trans-Filter (n = 100). Using phenotypic DST as gold standard, the Mol-DSTseq assay showed 100% (95% Confidence Interval [CI] 79.4-100%) and 73.3% (95% CI 54.1-87.7%) sensitivity for detecting rifampicin and isoniazid resistance with a specificity of 85.1% (95% CI 66.2-95.8%) and 100% (95% CI:82.3-100%), respectively. For fluoroquinolones and aminoglycosides, the Mol-DSTseq assay showed a sensitivity of 78.5% (95% CI 49.2-95.3%) and 66.6% (95% CI 9.4-99.1%) with a specificity of 88.2% (95% CI 72.5-96.7%) and 100% (95% CI 93.1-100%), respectively. The Mol-DSTseq assays exhibited a high concordance of ~ 83-96% (κ value: 0.65-0.81) with phenotypic DST for all drugs. In conclusion, the 'TB Concentration and Transport kit' was compatible with Mol-DSTseq assays and has the potential to provide 'Universal-DST' to patients residing in distant areas in high burden countries, like India for early initiation of anti-tubercular treatment.

Compatibility of a novel filter paper-based bio-safe sputum transport kit with line probe assay for diagnosing drug-resistant tuberculosis: a single-site evaluation study.

BACKGROUND: Near-patient access to appropriate tests is a major obstacle for the efficient diagnosis of tuberculosis (TB) and associated drug resistance. METHODS: We recently developed the "TB Concentration & Transport" kit for bio-safe, ambient-temperature transportation of dried sputum on Trans-Filter, and the "TB DNA Extraction" kit for DNA extraction from Trans-Filter for determining drug resistance by DNA sequencing. In the present study, we evaluated the compatibility of Kit-extracted DNA with Hain's line probe assays (LPAs), which are endorsed by National TB programmes for the detection of drug resistance in sputum collected from presumptive multidrug-resistant TB patients (n=207). RESULTS: Trans-Filter-extracted DNA was seamlessly integrated with the LPA protocol (Kit-LPA). The sensitivity of Kit-LPA for determining drug resistance was 83.3% for rifampicin (95% CI 52-98%), 77.7% for isoniazid (95% CI 52-94%), 85.7% for fluoroquinolones (95% CI 42-100%) and 66.6% for aminoglycosides (95% CI 9-99%), with a specificity range of 93.7% (95% CI 87-97) to 99.1% (95% CI 95-100) using phenotypic drug susceptibility testing (DST) as a reference standard. A high degree of concordance was noted between results obtained from Kit-LPA and LPA (99% to 100% (κ value: 0.83-1.0)). CONCLUSIONS: This study demonstrates successful integration of our developed kits with LPA. The adoption of these kits across Designated Microscopy Centres in India can potentially overcome the existing challenge of transporting infectious sputum at controlled temperature to centralised testing laboratories and can provide rapid near-patient cost-effective "Universal DST" services to TB subjects residing in remote areas.

Evaluation of 'TBDetect' sputum microscopy kit for improved detection of Mycobacterium tuberculosis: a multi-centric validation study.

OBJECTIVES: The present study aimed to evaluate the performance of the 'TBDetect' kit-based bio-safe fluorescent microscopy filter (BioFM-Filter) microscopy in comparison with direct smear microscopy and culture for the detection of pulmonary tuberculosis (TB) in a multi-centric setting in India. METHODS: The TBDetect kit enables sputum concentration through filtration using the BioFM-Filter for improved and bio-safe smear microscopy. We evaluated the performance of the TBDetect kit in a six-site multi-centric validation study on sputum collected from 2086 presumptive TB patients. RESULTS: The combined positivity of TBDetect microscopy performed on these sputum samples was 20% (n = 417/2086) vs 16.1% of light-emitting diode fluorescence microscopy (LED-FM, n = 337/2086) and 16% of Ziehl Neelsen (ZN) smear microscopy (n = 333/2086). The increment in positivity of TBDetect over both LED-FM and ZN smears was significant (p < 0.001). The overall sensitivity of TBDetect for six sites was ~55% (202/367, 95% confidence interval (CI): 50, 60%) vs 52% (191/367, 95% CI: 47, 57%) for LED-FM (p 0.14) and 50.9% (187/367, 95% CI: 46, 56%) for ZN smear (p < 0.05), using Mycobacterium Growth Indicator Tube culture (MGIT, n = 1949, culture positive, n = 367) as the reference standard. A bio-safety evaluation at six sites confirmed efficient sputum disinfection by TBDetect; 99.95% samples (1873/1874) were sterile after 42 days of incubation. Scientists and technicians at the study sites indicated the ease of use and convenience of TBDetect microscopy during feedback. CONCLUSIONS: TBDetect added value to the smear microscopy test due to its improved performance, convenience and user safety. These findings indicate that equipment-free TBDetect technology has the potential to improve TB diagnosis in basic laboratory settings by leveraging on the existing nationwide network of designated microscopy centres and primary healthcare centres.

Amplification of Hsp 65 gene and usage of restriction endonuclease for identification of non tuberculous rapid grower mycobacterium.

BACKGROUND: The rapid grower mycobacteria have emerged as significant group of human pathogen amongst the Runyon group IV organisms that are capable of causing infection in both the healthy and immunocompromised hosts. Study aimed to identification of species amongst rapid grower non tuberculous mycobacterial isolates by polymerase chain reaction - restriction enzyme analysis (PRA). Analysis and comparison of results with standard biochemical tests. METHODS: Rapid grower non tuberculous mycobacteria had been collected from liquid culture section during the study period. All isolates were identified by conventional biochemical tests. A 441bp fragment of hsp65 genes was amplified and digested by two restriction enzymes, BstEII and HaeIII. Digested products were analyzed using polyacrilamid gel electrophoresis (PAGE). RESULTS: During study, 121 rapid grower mycobacterial isolates were subjected for species identification. Isolates were obtained from pulmonary samples (72) and extrapulmonary samples (49). In the PRA test 8 different types of rapid grower mycobacteria were identified after analyzing the fragments generated through restriction enzymes. Mycobacterium chelonae (57/121) was the most common isolate in pulmonary and extrapulmonary samples. Mycobacterium fortuitum (42), Mycobacterium abscessus (11), Mycobacterium immunogen (06), Mycobacterium peregrinum (02), Mycobacterium smegmatis (01), Mycobacterium wolinskyi (01), Mycobacterium goodii (01) were identified as other species of rapid grower non tuberculous mycobacteria. CONCLUSION: PRA is a rapid and accurate system for the identification of species of non tuberculous mycobacteria. Results of PRA and biochemical tests are concordant up to 98%.

Isoniazid resistance among rifampicin-susceptible Mycobacterium tuberculosis isolates from tuberculosis patients.

OBJECTIVE/BACKGROUND: With the introduction of novel molecular techniques that rely on rifampicin (RIF) susceptibility, resistance to isoniazid (INH) or other first-line drugs remains undetected. Such patients are prescribed first-line antituberculosis therapy and are on RIF monodrug therapy during the continuation phase, which may lead to therapeutic failure and emergence of multidrug resistance. We aimed to study INH resistance among RIF-susceptible Mycobacterium tuberculosis (MTB) isolates from retreatment patients. METHODS: The Drug Susceptibility Testing data for four first-line drugs (streptomycin [SM], INH, RIF, and ethambutol [EMB]) using BACTEC MGIT 960 (Becton Dickinson, Franklin 124 Lakes, NJ ,USA) and for two drugs (INH and RIF) using line probe assay was analyzed retrospectively at the Department of Microbiology, National Institute of Tuberculosis and Respiratory Diseases (New Delhi, India). RESULTS: We analyzed 4910 drug susceptibility results performed using the BACTEC MGIT960 liquid culture system from 2009 to 2015. We found that 969 (19.7%) isolates were sensitive to all four first-line drugs, 3941 (80.3%) isolates were resistant to one or more drugs, and 3041 (61.9%) isolates were resistant to both RIF and INH with or without resistance to any other drug (multidrug resistant). Monodrug resistance to SM and EMB was observed in 94 (1.9%) and 8 (0.16%) isolates, respectively. RIF resistance without INH resistance was observed in 22 (0.44%) isolates. There were 776 isolates sensitive to RIF, but resistant to INH. Among these, INH resistance with EMB and/or SM was observed in 367 (7.47%) isolates, whereas 409 (8.3%) isolates were resistant to INH alone. The results of line probe assay from 2012 to 2015 were also analyzed, and the resistance to INH alone among all isolates with valid results was found to be 9.32% (1462/15,676). More than 75% of these isolates harbor mutations in the kat G gene associated with high-level resistance. CONCLUSION: INH resistance among RIF-susceptible isolates was present in 10-15% of the total cases. Among these cases, the use of RIF susceptibility alone will fail to detect INH resistance. Since higher rates of failure, relapse, or acquired resistance are linked with INH resistance, rollout of techniques focusing on RIF resistance must, therefore, be accompanied by strict monitoring for better management of patients.

Utility of MPT64 Antigen Detection for Rapid Confirmation of Mycobacterium tuberculosis Complex.

BACKGROUND: Rapid differentiation of the Mycobacterium tuberculosis complex (MTBC) and mycobacteria other than tuberculosis (MOTT) is crucial to facilitate early and effective treatment of the patients. Clinical presentation of MTBC and MOTT is not always very clear and routine conventional methods are time consuming. MATERIALS AND METHODS: In the present study, the MPT64 protein detection-based immunochomatographic test (SD Bioline Kit, Standard Diagnostics, Inc., Korea) was compared with the conventional biochemical method. RESULTS: The sensitivity, specificity, positive predictive, and negative predictive values of the SD AgMPT64 kit were found to be 100, 96.4, 98.72, and 100%, respectively. CONCLUSIONS: Our results have demonstrated that the SD bioline kit is a rapid, reliable method and it can be used in the Revised National Tuberculosis Control Program (RNTCP) of India, for the appropriate management of tuberculosis.

Identification of mycobacterial species by PCR restriction enzyme analysis of the hsp65 gene­an Indian experience.

Nowadays, nontuberculous mycobacteria (NTM) often cause pulmonary and extrapulmonary disease. Species identification of NTM determines the line of treatment and management of the disease. The routine diagnostic methods, i.e., smear microscopy and biochemical identification, of nontuberculous mycobacteria are tedious and time consuming and not all laboratories can perform these tests on a routine basis. A PCR targeting the hsp65 gene was implemented using standard strains and was applied to 109 clinical isolates. The PCR-amplified product was subjected to restriction enzyme analysis using BstEII and HaeIII. The results obtained were compared with that of biochemical tests. Of 109 NTM, 107 were identified to species level. PCR plus restriction enzyme analysis (PRA) identified 12 types of NTM. Common species identified were Mycobacterium chelonae (32), a rapid growing NTM, and Mycobacterium avium complex (21), among the slow growing NTM. PRA and biochemical identification showed 95.32% (102/107) concordant results. PRA is fast, cheap, and accurate for identification of potentially pathogenic NTM.

Study of opportunistic intestinal parasitic infections in human immunodeficiency virus/acquired immunodeficiency syndrome patients.

INTRODUCTION: Intestinal parasites predominantly coccidian parasites are a common cause for diarrhea in human immunodeficiency virus (HIV)-positive patients. MATERIALS AND METHODS: The study was conducted during January 2009-December 2010. A total of 1,088 stool samples from 544 seropositive HIV positive cases were examined microscopically for ova and cyst using wet mount preparations and stained smears. Out of 544 patients, 343 had prolonged diarrhea for more than 4 weeks, 57 had acute diarrhea of lesser than 7 days and 144 were asymptomatic cases who attended out-patient department; included in this study after taking consent from patients. Enteric pathogens were detected in 274 (50.36%) of the 544 patients. RESULTS AND CONCLUSIONS: The parasites identified were Cryptosporidium (135), Isospora belli (42), Cyclospora (12), Microsporidia (02), Entamoeba histolytica (49), Hookworm (34). Intestinal parasites in chronic diarrhea were significantly higher than the acute diarrhea (63.05% vs. 7.35%; P < 0.05). Parasitic pathogens were frequently associated with HIV-positive patients with diarrhea in Western India. Stools of all HIV-positive patients with diarrhea should thoroughly be investigated to identify etiologic agents for proper management. MATERIALS AND METHODS: The study was conducted during January 2009-December 2010. A total of 1,088 stool samples from 544 seropositive HIV positive cases were examined microscopically for ova and cyst using wet mount preparations and stained smears. Out of 544 patients, 343 had prolonged diarrhea for more than 4 weeks, 57 had acute diarrhea of lesser than 7 days and 144 were asymptomatic cases who attended out-patient department; included in this study after taking consent from patients. Enteric pathogens were detected in 274 (50.36%) of the 544 patients. RESULTS AND CONCLUSIONS: The parasites identified were Cryptosporidium (135), Isospora belli (42), Cyclospora (12), Microsporidia (02), Entamoeba histolytica (49), Hookworm (34). Intestinal parasites in chronic diarrhea were significantly higher than the acute diarrhea (63.05% vs. 7.35%; P > 0.05). Parasitic pathogens were frequently associated with HIV-positive patients with diarrhea in Western India. Stools of all HIV-positive patients with diarrhea should thoroughly be investigated to identify etiologic agents for proper management.