Evaluation of kit-based loop-mediated isothermal amplification (TB-LAMP) assay in the diagnosis of tubercular lymphadenitis.

Background: The rapid and accurate diagnosis of tubercular lymphadenitis remains a challenging task today. The World Health Organization (WHO) endorsed the LoopAMP MTBC kit (TB-LAMP) as a replacement for sputum smear microscopy in the diagnosis of pulmonary tuberculosis (PTB). However, no prospective diagnostic accuracy study of TB-LAMP for tubercular lymphadenitis in adults has been performed yet. The current study evaluated the diagnostic performance of TB-LAMP in tubercular lymphadenitis (LNTB). Methods: In a prospective observational study conducted at a tertiary care hospital in India, 90 subjects (age >18 years) suspected of LNTB were recruited consecutively and followed up for 6 months between January 2019 and December 2020. Samples were processed for microscopy, culture, GeneXpert, histopathology and TB-LAMP. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of TB-LAMP against the composite reference standard (CRS) and culture were determined. Results: TB-LAMP showed a sensitivity of 83.78 % (95 % CI, 73.76-90.47) and a specificity of 81.25 % (95 % CI, 56.99-93.41), respectively, against the CRS. The PPV and NPV were 95.38 % (95 % CI, 87.29-98.42) and 52.00 % (95 % CI, 33.50-69.97), respectively. TB-LAMP showed a sensitivity of 88.89 % (95 % CI, 71.94-96.15) and a specificity of 36.17 % (95 % CI, 23.97-50.46), respectively, against culture. The PPV and NPV were 44.44 % (95 % CI, 32-57.62) and 85 % (95 % CI, 63.96-94.76), respectively. Conclusion: TB-LAMP can be used instead of conventional microscopy for the diagnosis of TB in lymph node specimens at primary healthcare centres. It provides rapid and cost-effective diagnosis of LNTB in resource-limited settings due to good sensitivity and NPV.

Prevention of relapse in drug sensitive pulmonary tuberculosis patients with and without vitamin D3 supplementation: A double blinded randomized control clinical trial.

BACKGROUND: The immunomodulatory effects of vitamin D are widely recognized and a few studies have been conducted to determine its utility in the treatment of tuberculosis, with mixed results. This study was conducted to see if vitamin D supplementation in patients with active pulmonary tuberculosis (PTB) in the Indian population contributed to sputum smear and culture conversion as well as the prevention of relapse. METHODS: This randomized double-blind placebo-controlled trial was conducted in three sites in India. HIV negative participants aged 15-60 years with sputum smear positive PTB were recruited according to the Revised National Tuberculosis Control Program guidelines and were randomly assigned (1:1) to receive standard anti-tubercular treatment (ATT) with either supplemental dose of oral vitamin D3 (60,000 IU/sachet weekly for first two months, fortnightly for next four months followed by monthly for the next 18 months) or placebo with same schedule. The primary outcome was relapse of PTB and secondary outcomes were time to conversion of sputum smear and sputum culture. RESULTS: A total of 846 participants were enrolled between February 1, 2017 to February 27, 2021, and randomly assigned to receive either 60,000 IU vitamin D3 (n = 424) or placebo (n = 422) along with standard ATT. Among the 697 who were cured of PTB, relapse occurred in 14 participants from the vitamin D group and 19 participants from the placebo group (hazard risk ratio 0.68, 95%CI 0.34 to 1.37, log rank p value 0.29). Similarly, no statistically significant difference was seen in time to sputum smear and sputum culture conversion between both groups. Five patients died each in vitamin D and placebo groups, but none of the deaths were attributable to the study intervention. Serum levels of vitamin D were significantly raised in the vitamin D group as compared to the placebo group, with other blood parameters not showing any significant difference between groups. CONCLUSIONS: The study reveals that vitamin D supplementation does not seem to have any beneficial effect in the treatment of PTB in terms to the prevention of relapse and time to sputum smear and culture conversion. TRIAL REGISTRATION: CTRI/2021/02/030977 (ICMR, Clinical trial registry-India).

Evaluation of genotype MTBDRplus V2 and genotype MTBDRsl V2 for the diagnosis of extrapulmonary tuberculosis in India.

Microbiological diagnosis of extra-pulmonary tuberculosis (EPTB) has been one of the most difficult aspects of tuberculosis (TB) management. Availability of better imaging and diagnostic modalities has led to an increase in the number of diagnosed cases. The current upsurge in multidrug-resistant tuberculosis warrants routine testing of EPTB samples for resistance at baseline with shorter turn-around time. A total of 369 EPTB specimens were subjected to Ziehl-Neelsen (ZN) stain, liquid culture (LC) with phenotypic drug susceptibility testing, MTBDRplus V.2 and MTBDRsl V.2. The molecular categorisation of resistant specimens was further reconfirmed with sequencing. The sensitivity and specificity of MTBDRplus V.2 to detect Mycobacterium tuberculosis (MTB) when compared to ZN stain was 97.9% and 89.2%, respectively while it was 73.4% and 83.8%, respectively when compared to LC. Similarly, for MTBDRsl V.2, the sensitivity and specificity for detection of MTB when compared with ZN was 95.6% and 91.9%, respectively and 75% and 89.2%, respectively when compared to LC. In smear-positive specimens, 94% (141/150) and 86% (129/150) valid results were observed in MTBDRplus V.2 and MTBDRsl V.2, respectively. The utilisation of both MTBDRplus V.2 and MTBDRsl V.2 for the diagnosis of smear-positive EPTB specimens would be useful in programmatic management of TB in high-burden settings.

Nontuberculous mycobacteria: A report of eighteen cases from a tertiary care center in India.

CONTEXT: Nontuberculous mycobacteria (NTM) are ubiquitous mycobacteria present in environment and generally affect patients with either structural lung disease or immunosuppression and commonly involve lungs, lymph node, or skin. MATERIALS AND METHODS: Between July 2016 and February 2019, 18 cases of NTM were diagnosed and their relevant clinical, diagnostic, and treatment details were recorded after taking informed consent. RESULTS: We report 18 cases of NTM involving lungs (n = 11), skin and soft tissue (n = 3), joint (n = 2), genitourinary (n = 1), and central nervous system (n = 1). History of immunosuppression was present in two patients, whereas history of some form of intervention was seen in six patients. Mycobacterium fortuitum group (n = 5) was the most commonly isolated organism, followed by Mycobacterium avium complex (n = 4), Mycobacterium abscessus (n = 3), Mycobacterium kansasii (n = 2), and Mycobacterium chelonae (n = 1). In two patients, M. chelonae and M. abscessus were isolated in succession. Of these 18 patients, clinical response was present in 15 of the patients. Diagnosis and treatment of NTM in resource limited settings is extremely challenging. CONCLUSION: Most of the patients with NTM are misdiagnosed and are treated as tuberculosis in India, sometimes with a multidrug resistance regimen, which results in significant morbidity and mortality. We present these cases to shed some light on the epidemiology of NTM in this part of India.

High degree of fluoroquinolone resistance among extrapulmonary tuberculosis patients at a tertiary care center in North India.

Background: The treatment of drug-resistant tuberculosis (TB) involves various regimens. Among them, the most promising antibiotic regimens are fluoroquinolone (FQ) drugs. Drug susceptibility testing (DST) for FQs is not included as routine assessment for baseline TB diagnosis. Limited resources are available about FQ resistance among extrapulmonary TB (EPTB) cases. Methods: A total of 447 culture-positive specimens were subjected to DST for first-line anti-TB drugs (FLDs) and second-line anti-TB drugs. DST was performed using automated mycobacterium growth indicator tube-960 liquid culture techniques. The study was carried out during the period of April 2016 to March 2017. In addition, DST of FQs was also performed in FLD-sensitive strains. Results: Mycobacterium tuberculosis was isolated from 447 specimens. Of the 447 culture-positive EPTB specimens, 54 were rifampicin-resistant (RR)/multidrug-resistant TB (MDR-TB) isolates, 45 isolates were resistant to any drug, and the remaining 348 were sensitive to FLDs. Monoresistance of FQs was observed in 20.4% (11/54) among RR/MDR-TB isolates and 4.3% (15/348) among FLD-sensitive isolates. Conclusion: The high degree of FQ resistance observed in EPTB specimens among drug-sensitive and MDR-TB isolates is alarming. This study reflects the need to expand culture and DST for EPTB cases and include FQs within first-line DST in such settings. Furthermore, there should be a rational use of FQs for the treatment of other diseases.

Diagnostic Evaluation of Non-Interpretable Results Associated with rpoB Gene in Genotype MTBDRplus Ver 2.0.

BACKGROUND: Line probe assay (LPA) is standard diagnostic tool to detect multidrug resistant tuberculosis. Noninterpretable (NI) results in LPA (complete missing or light wild-type 3 and 8 bands with no mutation band in rpoB gene region) poses a diagnostic challenge. METHODS: Sputum samples obtained between October 2016 and July 2017 at the Intermediate Reference Laboratory, All India Institute of Medical Sciences Hospital, New Delhi, India were screened. Smear-positive and smear-negative culturepositive specimens were subjected to LPA Genotype MTBDRplus Ver 2.0. Smear-negative with culture-negative and culture contamination were excluded. LPA NI samples were subjected to phenotypic drug susceptibility testing (pDST) using MGIT-960 and sequencing. RESULTS: A total of 1,614 sputum specimens were screened and 1,340 were included for the study (smear-positive [n=1,188] and smear-negative culture-positive [n=152]). LPA demonstrated 1,306 (97.5%) valid results with TUB (Mycobacterium tuberculosis) band, 24 (1.8%) NI, three (0.2%) valid results without TUB band, and seven (0.5%) invalid results. Among the NI results, 22 isolates (91.7%) were found to be rifampicin (RIF) resistant and two (8.3%) were RIF sensitive in the pDST. Sequencing revealed that rpoB mutations were noted in all 22 cases with RIF resistance, whereas the remaining two cases had wild-type strains. Of the 22 cases with rpoB mutations, the most frequent mutation was S531W (n=10, 45.5%), followed by S531F (n=6, 27.2%), L530P (n=2, 9.1%), A532V (n=2, 9.1%), and L533P (n=2, 9.1%). CONCLUSION: The present study showed that the results of the Genotype MTBDRplus assay were NI in a small proportion of isolates. pDST and rpoB sequencing were useful in elucidating the cause and clinical meaning of the NI results.

Choosing the therapy for neurological infection with rapidly growing mycobacteria.

The management of neurological infections due to non-tubercular mycobacteria is extremely challenging because of scarce literature, issues with penetration, lack of easily available susceptibility platforms and adverse effects associated with long term therapy. We report a case of a young girl with neurological infection due to rapidly growing mycobacteria to discuss the factors that should be considered while choosing the therapy for such rare and persistent infections.

Persistent Laparoscopic Port-site Discharging Sinus: A Rare Case of Mycobacterium senegalense Infection.

Laparoscopic port-site infections, though infrequent, undermine the advantages provided by minimally invasive surgeries. Persistent nonhealing discharging sinuses, not responding to conventional antibiotic therapy, pose diagnostic and therapeutic challenges. Sizeable number of these infections is caused by rapidly growing nontuberculous mycobacteria (NTM), and diagnosing these requires a high index of suspicion. We present a case of a nonhealing laparoscopic cholecystectomy umbilical port-site infection caused by Mycobacterium senegalense, a rare NTM. The patient recovered completely after 6 months of combination therapy with clarithromycin, trimethoprim-sulfamethoxazole, and levofloxacin.

Mutation in atpE and Rv0678 genes associated with bedaquline resistance among drug-resistant tuberculosis patients: A pilot study from a high-burden setting in Northern India.

Background: Mutations in atpE gene or transcriptional repressor Rv0678 gene associated with inhibition of adenosine 5'-triphosphate synthase and upregulation of efflux pumps, respectively, may potentially lead to in vitro resistance to bedaquiline. This is the first study from India, which looks at mutations associated with this novel drug. Methods: In 2019 (January to June), a total of 68 laboratory-confirmed pre-extensively drug-resistant tuberculosis (XDR-TB) (fluoroquinolone resistant [n = 52] and second-line injectables resistant [n = 12]) and 4 × DR-TB culture specimens were included. All specimens were evaluated for genetic analysis using predesigned primers of atpE and Rv0678 genes. Results: Among the pre-XDR-TB isolates (n = 64), there were no mutations found in either atpE or Rv0678. However, among the XDR-TB isolates (n = 4), one specimen (25%) was found to be associated with a mutation in atpE gene at position 49, resulting in the amino acid leucine replaced by proline (L-49-P). No mutations were observed with the Rv0678 gene. Conclusion: In this study, genetic analysis showed that only one-fourth XDR-TB isolates had a mutation in the atpE gene; there were no other mutations found in the Rv0678 gene. To the best of our knowledge, this novel mutation (L-49-P) in atpE gene is being reported for the first time in northern India.

High degree of fluoroquinolone resistance among pulmonary tuberculosis patients in New Delhi, India.

BACKGROUND & OBJECTIVES: : The fluoroquinolones (FQs) group of antibiotics is the backbone drugs for the management of drug-resistant tuberculosis (TB). In routine clinical practice, drug susceptibility testing (DST) for FQs is not performed, and the patients are empirically treated. A limited information exists regarding FQs resistance among pulmonary TB cases. The present study was conducted to determine the FQs resistance among drug sensitive and drug-resistant pulmonary TB patients in a tertiary care centre in north India. METHODS: : A total of 1619 sputum/smear-positive specimens of pulmonary TB patients were subjected to DST for first-line drugs (FLDs) and second-line drugs. In addition, FQs DST was also performed using automated Mycobacterial Growth Indicator Tube-960 liquid culture technique. The immuno-chromatographic assay was performed to distinguish Mycobacterium tuberculosis complex (MTBC) from non-MTBC. RESULTS: : Mycobacterium tuberculosis (Mtb) was isolated in 1499 sputum specimens; 1099 culture specimens were sensitive to FLDs, 249 grew as multidrug-resistant (MDR) Mtb and the remaining 151 isolates revealed any drug resistance to FLDs. While FQs monoresistance among the FLD sensitive isolates was 3.1 per cent (35/1099), 27.3 per cent (68/249) among MDR Mtb isolates had additional FQs resistance. INTERPRETATION & CONCLUSIONS: : FQs resistance among drug sensitive and MDR Mtb isolates was high in Delhi, India. Based on these findings, it is recommended that the DST for FQs should be routinely performed to avoid further amplification of drug resistance.

A prospective study of non-tuberculous mycobacterial disease among tuberculosis suspects at a tertiary care centre in north India.

Background & objectives: The burden of non-tuberculous mycobacterial (NTM) disease is increasing worldwide. The disease shares clinicoradiological features with tuberculosis (TB), Nocardia and several fungal diseases, and its diagnosis is frequently delayed. The present study was performed to determine the frequency of NTM disease among TB suspects in a tertiary care centre in north India. Methods: In this prospective study, mycobacterial culture isolates from pulmonary and extrapulmonary specimens among TB suspects were tested with immunochromatographic assay (ICA). All ICA-negative isolates were considered as NTM suspects and further subjected to 16S-23S rRNA internal transcribed spacer gene sequencing for confirmation and species identification. Patients with active disease were treated with drug regimen as per the identified NTM species. Follow up of patients was done to determine clinical, radiological and microbiological outcomes. Results: Of the 5409 TB suspects, 42 (0.77%) were diagnosed with NTM disease. Patients with active disease consenting for treatment were treated and followed up. Thirty four patients had NTM pulmonary disease (NTM-PD) and the remaining eight had extrapulmonary NTM (EP-NTM) disease. Mycobacterium intracellulare and M. abscessus, respectively, were most frequently isolated from NTM-PD and EP-NTM patients. Fifteen NTM-PD and seven EP-NTM patients successfully completed the treatment. Ten patients died due to unrelated causes, five were lost to follow up and another four declined the treatment. Interpretation & conclusions: Our study showed that the frequency of NTM disease was low among TB suspects at a large tertiary care centre in north India and this finding was similar to other Indian studies. More studies need to be done in other parts of the country to know the geographical variation in NTM disease, if any.

Impact of nicotine replacement therapy as an adjunct to anti-tuberculosis treatment and behaviour change counselling in newly diagnosed pulmonary tuberculosis patients: an open-label, randomised controlled trial.

We evaluated the impact of intensive smoking cessation activities as an adjunct to anti-tuberculosis treatment on patient-related treatment outcomes. In this open-label, randomised controlled trial, self-reporting smokers with pulmonary tuberculosis who initiated standard anti-tuberculosis treatment were randomised to either nicotine replacement therapy and behaviour change counselling (n = 400) or counselling alone (n = 400) provided at baseline and two follow-up visits. The primary outcomes were change in TBscore at 24-weeks and culture conversion at 8-weeks. Biochemical smoking quit rates defined as serum cotinine levels <10 ng/mL and/or exhaled carbon monoxide levels <6 ppm (47·8% vs 32·4%, p-value =< 0·001) and self-reported quit rates (69.3% vs 38·7%, p-value =< 0·001) were significantly higher in the intervention arm at 24-weeks. Though the TBscores at 24 weeks (95% CI) were lower in the intervention arm [2·07 (1·98, 2·17) versus 2.12 (2·02, 2·21)], the difference was not clinically meaningful. Patients in the control arm required treatment extension more often than intervention arm (6·4% vs 2·6%, p-value = 0·02). Combining nicotine replacement therapy with behaviour change counselling resulted in significantly higher quit rates and lower cotinine levels, however, impact on patient-related (TBscore) or microbiological outcomes (culture conversion) were not seen.

Diagnostic utility of a line probe assay for multidrug resistant-TB in smear-negative pulmonary tuberculosis.

OBJECTIVE: To evaluate the performance of Genotype MTBDRplus VER 2.0 in the diagnosis of Mycobacterium tuberculosis (MTB) in sputum smear-negative pulmonary TB cases. METHODS: A total of 572 Ziehl-Neelsen sputum smear-negative samples were selected and subjected to line probe assay (Genotype MTBDRplus VER 2.0), and culture in mycobacterial growth indicator tube (MGIT-960). Immunochromatographic test was used to confirm the MTB-complex (MTBC) in culture-positive samples and phenotypic drug-susceptibility testing was done using MGIT-960. RESULTS: The line probe assay was able to diagnose MTBC in 38.2% (213/558) of specimens after excluding 14 nontuberculous mycobacteria. Sensitivity and specificity of the assay were 68.4% and 89.3% respectively, considering MGIT-960 culture as gold standard after excluding contaminated and invalid results. On comparing with composite reference standard, the assay had 71.5% sensitivity and 100% specificity in the diagnosis of tuberculosis. The sensitivity and specificity for detecting resistance to rifampicin (RMP) were 100% and 99.24% respectively and for resistance to isoniazid (INH) were 97.62% and 98.55%, respectively. CONCLUSION: Genotype MTBDRplus VER 2.0 is a rapid and precise diagnostic tool for detection of MTB in sputum smear-negative samples. It also facilitates accurate diagnosis of RMP and INH resistance within turn around-time.