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.

A Prospective Study of Fungal Colonization and Invasive Fungal Disease in Long-Term Mechanically Ventilated Patients in a Respiratory Intensive Care Unit.

BACKGROUND: Long-term mechanical ventilation in an Intensive Care Unit (ICU) exposes the patient to fungal colonization and invasive fungal disease due to the presence of indwelling catheters, administration of broad-spectrum antibiotics, and intravenous corticosteroids. A study is hence required to study the risk factors and incidence of fungal infection in these patients. METHODS: A prospective observational study was carried out in the respiratory ICU of a tertiary care hospital for a period of approximately 1 year in which patients on mechanical ventilation (>7 days) were enrolled. Blood, urine, and endotracheal aspirate (ETA) of these patients were sent for fungal culture on day 1 and day 7 of mechanical ventilation. Fiberoptic bronchoscopy was done on day 7 and bronchoalveolar lavage along with transbronchial lung biopsy (TBLB) were sent for fungal culture. RESULTS: During 7 days of ventilation, there was a statistically significant increase in the proportion of culture-positive ETA and urine samples. Overall, Candida albicans emerged as the most common colonizer. Blood candidemia was seen in 10% of patients on day 7 of mechanical ventilation. Fungal invasion of the lung, as evidenced by fungal culture-positive TBLB specimens, was seen in 17% of patients. Diabetes was found to be a statistically significant risk factor for respiratory and urinary tract colonization as well as invasive fungal disease. CONCLUSION: Long-term mechanical ventilation (>7 days) is strongly associated with fungal colonization of the respiratory tract and urinary tract. Appropriate prophylactic antifungals may be given and infection control practices to be observed to ensure minimum colonization and therefore infection in such settings.

Sequence Analysis of Fluoroquinolone Resistance-Associated Genes gyrA and gyrB in Clinical Mycobacterium tuberculosis Isolates from Patients Suspected of Having Multidrug-Resistant Tuberculosis in New Delhi, India.

Fluoroquinolones (FQs) are broad-spectrum antibiotics recommended for the treatment of multidrug-resistant tuberculosis (MDR-TB) patients. FQ resistance, caused by mutations in the gyrA and gyrB genes of Mycobacterium tuberculosis, is increasingly reported worldwide; however, information on mutations occurring in strains from the Indian subcontinent is scarce. Hence, in this study, we aimed to characterize mutations in the gyrA and gyrB genes of acid-fast bacillus (AFB) smear-positive sediments or of M. tuberculosis isolates from AFB smear-negative samples from patients in India suspected of having MDR-TB. A total of 152 samples from patients suspected of having MDR-TB were included in the study. One hundred forty-six strains detected in these samples were characterized by sequencing of the gyrA and gyrB genes. The extracted DNA was subjected to successive amplifications using a nested PCR protocol, followed by sequencing. A total of 27 mutations were observed in the gyrA genes of 25 strains, while no mutations were observed in the gyrB genes. The most common mutations occurred at amino acid position 94 (13/27 [48.1%]); of these, the D94G mutation was the most prevalent. The gyrA mutations were significantly associated with patients with rifampin (RIF)-resistant TB. Heterozygosity was seen in 4/27 (14.8%) mutations, suggesting the occurrence of mixed populations with different antimicrobial susceptibilities. A high rate of FQ-resistant mutations (17.1%) was obtained among the isolates of TB patients suspected of having MDR-TB. These observations emphasize the need for accurate and rapid molecular tests for the detection of FQ-resistant mutations at the time of MDR-TB diagnosis.

Detection of multi-drug resistance & characterization of mutations in Mycobacterium tuberculosis isolates from North- Eastern States of India using GenoType MTBDRplus assay.

BACKGROUND & OBJECTIVES: Information on drug resistance tuberculosis is sparse from North-East (N-E) States of India. We undertook this study to detect multi-drug resistant tuberculosis (MDR-TB) among MDR-TB suspects, and common mutations among MDR-TB cases using GenoType MTBDRplus. METHODS: All MDR suspect patients deposited sputum samples to peripheral designated microscopy centres (DMC) in North-East States. The district TB officers (DTOs) facilitated the transport of samples collected during January 2012 to August 2012 to our laboratory. The line probe assay to detect common mutations in the rpoB gene for rifampicin (RIF) and katG and inhA genes for isoniazid (INH), respectively was performed on 339 samples or cultures. RESULTS: A total of 553 sputum samples from MDR suspects were received of which, 181 (32.7%) isolates were found to be multi-drug resistant. Missing WT8 along with mutation in codon S531L was commonest pattern for rifampicin resistant isolates (65.1%) and missing WT along with mutations in codon S315T1 of katG gene was commonest pattern for isoniazid resistant isolates (86.2%). Average turn-around time for dispatch of LPA result to these States from cultures and samples was 23.4 and 5.2 days, respectively. INTERPRETATIONS & CONCLUSIONS: The MDR-TB among MDR-TB suspects in North-Eastern States of India was found to be 32.7 per cent. The common mutations obtained for RIF and INH in the region were mostly similar to those reported earlier.

Evaluation of AAICare®-TB sequence analysis tool for accurate diagnosis of drug-resistant tuberculosis: A comparative study with TB-Profiler and Mykrobe.

A rapid and comprehensive drug susceptibility test is essential for eliminating drug resistant tuberculosis. Next generation sequencing (NGS) based susceptibility testing is being explored as a potential substitute for the conventional phenotypic and genotypic testing methods. However, the adoption of NGS based genotypic susceptibility testing depends on the availability of simple, accurate and efficient analysis tools. This preliminary study aimed to evaluate the performance of a Mycobacterium tuberculosis (Mtb) genome analysis pipeline, AAICare®-TB, for susceptibility prediction, in comparison to two widely used gDST prediction tools, TB-Profiler and Mykrobe. This study was performed in a National Reference Laboratory in India on presumptive drug-resistant tuberculosis (DR-TB) isolates. Whole genome sequences of the 120 cultured isolates were obtained through Illumina sequencing on a MiSeq platform. Raw sequences were simultaneously analysed using the three tools. Susceptibility prediction reports thus generated, were compared to estimate the total concordance and discordance. WHO mutation catalogue (1st edition, 2021) was used as the reference standard for categorizing the mutations. In this study, AAICare®-TB was able to predict drug resistance status for First Line (Streptomycin, Isoniazid, Rifampicin, Ethambutol and Pyrazinamide) and Second Line drugs (Fluoroquinolones, Second Line Injectables and Ethionamide) in 93 samples along with lineage and hetero-resistance as per the WHO guidelines.

Evaluation of Diagnostic Performance of Line Probe Assay on Smear-Negative Samples of Pulmonary Tuberculosis.

BACKGROUND: This study aims to compare the performance of line probe assay (LPA) on smear-negative samples with that of smear-positive samples for diagnosing pulmonary tuberculosis (PTB) and first-line drug sensitivity testing (FL DST). METHODS: A total of 196 sputum samples including both smear-positive (112) and negative (84) samples of patients suspected of PTB were subjected to LPA for TB detection and FL DST. TB culture followed by MPT 64 Ag was done and conventional FL DST was performed on all culture-positive isolates. Results of LPA on smear-negative were compared with smear-positive samples. RESULTS: The LPA confirmed the diagnosis of PTB in 104/112 smear-positive cases but in only 36/84 smear-negative cases. The assay had 47.36%, 72.72%, and 88.88% sensitivity and 86.96%, 95.23%, and 95.65% specificity in smear-negative cases compared to 89.09%, 95.83%, and 98.07% sensitivity and 100%, 98.36%, and 98.24% specificity in smear-positive cases for detecting Mycobacterium tuberculosis (MTB), rifampicin (RMP) resistance, and isoniazid (INH) resistance, respectively. CONCLUSION: LPA performance was better on smear-positive than smear-negative sputum samples. Further larger studies are needed to justify the use of LPA on smear-negative pulmonary samples for diagnosis.

Incidence and Patterns of Drug Resistance in Patients with Spinal Tuberculosis: a Prospective, Single-Center Study from a Tuberculosis-Endemic Country.

Background: There is paucity of data on incidence and pattern of drug resistance in spinal TB. This prospective observational study was conducted to document the incidence and drug-resistance pattern among primary and presumptive resistant cases. Methods: 59 consecutive cases diagnosed clinico-radiologically (imaging) were grouped into Group A (n = 51, primary cases) and Group B (n = 8, presumptive resistant cases) based on pre-defined criteria (INDEX-TB guidelines). Tissue samples obtained percutaneously (37.29%, 22/59) and on surgery (62.71%, 37/59) were subjected to genotypic DST (CBNAAT, LPA) and phenotypic DST (BACTEC MGIT 960 culture and sensitivity using fixed critical concentration of drugs). Results: Etiological diagnosis was ascertained in all. 13/51 (25.49%) in Group A, while 3/8 (37.5%) in Group B and 16/59 (27.12%) overall demonstrated drug resistance. 12/16 (75%) had no prior history of ATT intake. 4 demonstrated INH (Isoniazid) mono-resistance. 12 polydrug resistance demonstrated: 5MDR, 3pre-XDR, while RIF + FQ (fluoroquinolones), FQ + Lz (linezolid), only SLID (second-line injectable drugs), and only FQ resistance observed in 1 case each. Isolated RIF (Rifampicin) resistance and XDR pattern were not observed. Overall frequency of RIF resistance was 16.4% (9/55) and INH was 25% (12/48) with low-(n-2) and high-level INH resistance (n-10). Among second-line drugs, FQ resistance was more than SLID resistance and within FQ, levofloxacin resistance was more frequent than moxifloxacin. MGIT demonstrated positive growth in 16/59 samples, out of which 1 sample was positive for nontuberculous mycobacteria (M. chelonae) but on genotypic testing demonstrated MTB resistant to RIF and FQ. Conclusion: This is the first report on incidence and drug-resistant pattern in culture-positive/negative cases. High (25.49%) primary drug resistance is worrisome. This being the first study in  spinal TB cases which document prevalent drug-resistant pattern as evaluated for consecutive culture-positive/negative cases. The tissue obtained must be submitted for AFB culture and molecular tests to ascertain drug resistance in culture-positive/negative cases. However, in the presence of insufficient tissue sample histology and CBNAAT can ascertain etiological diagnosis in 100% cases. INH resistance is more than RIF with isolated RIF resistance unreported.

Chronic pulmonary aspergillosis in a tertiary tuberculosis institute: A common entity missed commonly.

The disease chronic pulmonary aspergillosis (CPA), which has 3 million cases globally, has a substantial impact on global health. The morbidity and mortality it cause are also rather severe. Patients with modest immune suppression or those with underlying structural and chronic lung illnesses are more likely to develop this condition. CPA pose a diagnostic and management challenge to clinicians. The condition causes patients to have persistent respiratory difficulties, which lowers their quality of life, and the therapy is lengthy and offers few choices. Particularly in a nation like India, where tuberculosis (TB) is prevalent and patients exhibit identical signs and symptoms, a strong index of suspicion is required. Treated pulmonary TB patients, presenting with symptoms or chest x-ray abnormalities, especially those with presence of cavity are also more prone to develop CPA. The constellation of symptoms together with presence of microbiological criteria and suggestive radiology can help to reach at the diagnosis. The field of mycology has made major developments, but there is still much to understand about this illness and to establish timely diagnoses and make the best use of the existing treatment choices. The burden of CPA in patients with treated TB is highlighted in this article along with the most recent research and clinical guidelines.

Evaluation of Mycobacterium tuberculosis derived cell-free DNA using pleural fluid and paired plasma samples for the diagnosis of pleural tuberculosis.

Pleural tuberculosis (pTB) is a grave clinical challenge. A novel cell-free M. tuberculosis DNA (cfM.tb-DNA) probe-based-qPCR assay was developed for the diagnosis of pTB. Total cell-free DNA was extracted from pleural fluid (PF) and paired plasma samples and cfM.tb-DNA was quantified by probe-based qPCR targeting devR (109-bp) gene of M. tuberculosis in patients with pleural effusion. Patient categorization was done using 'Composite-Reference-Standard' formulated for the study. Assay cut-offs were determined from samples in the 'Development set' (n = 17; 'Definite & Probable' pTB; n = 9 and 'Non-TB'; n = 8) by ROC-curve analysis and applied to 'Validation set' (n = 112; 'Definite' pTB; n = 8, 'Probable' pTB; n = 34, 'Possible' pTB; n = 28 and 'Non-TB'; n = 42). cfM.tb-DNA qPCR had a sensitivity of 62.5% (95%CI; 24.4,91.4) in 'Definite' pTB category and 59.5% (95%CI; 43.2,74.3) in 'Definite & Probable' pTB category with 95.2% (95%CI; 83.8,99.4) specificity using PF. In plasma (n = 85), the assay had a sub-optimal sensitivity of 7.6% (95%CI; 0.95,25.1) with 88.2% (95%CI; 72.5,96.7) specificity in 'Definite & Probable' pTB group. Xpert MTB/RIF assay detected only six-samples in the 'Validation set'. Logistic regression analysis indicated that PF-cfM.tb-DNA qPCR provided incremental advantage over existing pTB diagnostic algorithms. To the best of our knowledge, this is the first report describing the utility of cfM.tb-DNA for pTB diagnosis in India.

Diagnostic utility of GenoType MTBDRsl assay for the detection of moxifloxacin-resistant mycobacterium tuberculosis, as compared to phenotypic method and whole-genome sequencing.

Background: Recently, moxifloxacin (MFX)-resistant results of Mycobacterium tuberculosis (Mtb) obtained by GenoType MTBDRsl (second-line line probe assay [SL-LPA]) have been stratified to determine their resistance level; however, its accuracy has not been well studied. Therefore, the study aimed to evaluate the diagnostic accuracy of SL-LPA, with phenotypic drug susceptibility testing (pDST) and whole-genome sequencing (WGS) for the detection of MFX-resistant Mtb and their resistance level. Methods: A total of 111 sputum samples were subjected to SL-LPA according to the diagnostic algorithm of the National Tuberculosis Elimination Program. Results were compared with pDST of MFX (at critical concentration [CC, 0.25 µg/ml] and clinical breakpoint [CB, 1.0 µg/ml] using BACTEC mycobacterial growth indicator tube-960), and WGS. Results: At CC, SL-LPA and pDST yielded concordant results of MFX for 104 of 111 (94%). However, at CB, 23 of 30 (77%) isolates carrying gyrA mutation known to confer low-level resistance to MFX were scored as susceptible by pDST. Among 46 Mtb isolates carrying gyrA mutations known to confer high-level resistance to MFX, 36 (78%) isolates yielded concordant results, while 10 (22%) isolates were scored as susceptible at CB by pDST. WGS identified gyrA mutations in all isolates suggested by SL-LPA. Conclusion: It is concluded that the stratification of MFX-resistant results by SL-LPA/genotypic method is not very well correlated with pDST (at CB), and hence, pDST may not be completely replaced by SL-LPA. gyrA D94G and gyrAA90V are the most prevalent mutations in MFX-resistant Mtb.

Detection of multidrug and extensively drug-resistance and mutation pattern in geriatric patients from North Indian referral institute.

INTRODUCTION: Geriatric population are predisposed to reactivation to tuberculosis (TB) and multi-drug resistance (MDR) due to deteriorated immune system. Limited data is available in this population hence present study is undertaken to study drug resistance and associated mutations among geriatric presumptive DR-TB patients by genotypic methods METHODS: From October 2011 to December 2018, demographic characteristics of enrolled patients was collected. Smear-positive processed sputum samples were subjected directly while cultures positive for Mycobacterium Tuberculosis (MTB) from smear-negative pulmonary and all extra-pulmonary samples were subjected to LPA. The LPA used were Genotype MTBDR plus (1st line LPA) for detection of susceptibility to rifampicin (RIF) and isoniazid (INH) and Genotype MTBDR sl (2nd line LPA), for susceptibility to fluoroquinolones (FQ) and aminoglycosides (AG). RESULTS: Total of 2041 samples were received from presumptive MDR-TB patients above 60 years of age during study period, of which 1406; 68.9% were within 60-70 year followed by 495; 24.3% within 71-80 year and 140; 6.9% more than 80 years. Total of 1055 MTB were detected, of which those diagnosed as RIF resistant were 117/1055; 11.2% including 89/1055; 8.5% MDR-TB and resistance to INH was in 84/1055; 8%. Total 67, 2nd line LPA gave valid results, of which 19/67 (28.4%) isolates were resistant to only FQ, and one isolate was resistant to AG. CONCLUSION: Study finding highlights need for dedicated efforts for diagnosis, and treatment of geriatric tuberculosis. Suitable intervention at programmatic country level at country will help in strengthening tuberculosis control strategies in this population.

Study of spectrum of fungal isolates in the sputum of newly diagnosed pulmonary tuberculosis patients.

Tuberculosis is a state of immunosupression which exposes the patients to further opportunistic pathogens like fungus. Methods: 102 newly diagnosed sputum positive pulmonary tuberculosis cases were enrolled. Significant fungal isolates were seen in 31/102 (30.4%) patients. Aspergillus spp. were isolated in 13/31(41.9%) of the positive fungal cultures while Candida spp. were isolated in 15/31 (48.4%). Low body mass index, duration of symptoms, haemoptysis, severity of radiological features and IgG Aspergillus antibodies were independent risk factors for positive fungal culture. Significant proportion of patients with PTB have fungal colonisation of their airways which can lead to poor clinical outcomes. Few easily ascertained clinical parameters can help the clinician to determine patients who are at a higher risk of fungal colonisation.

SARS-CoV-2 Mutations and Their Impact on Diagnostics, Therapeutics and Vaccines.

With the high rate of COVID-19 infections worldwide, the emergence of SARS-CoV-2 variants was inevitable. Several mutations have been identified in the SARS-CoV-2 genome, with the spike protein as one of the mutational hot spots. Specific amino acid substitutions such as D614G and N501Y were found to alter the transmissibility and virulence of the virus. The WHO has classified the variants identified with fitness-enhancing mutations as variants of concern (VOC), variants of interest (VOI) or variants under monitoring (VUM). The VOCs pose an imminent threat as they exhibit higher transmissibility, disease severity and ability to evade vaccine-induced and natural immunity. Here we review the mutational landscape on the SARS-CoV-2 structural and non-structural proteins and their impact on diagnostics, therapeutics and vaccines. We also look at the effectiveness of approved vaccines, antibody therapy and convalescent plasma on the currently prevalent VOCs, which are B.1.17, B.1.351, P.1, B.1.617.2 and B.1.1.529. We further discuss the possible factors influencing mutation rates and future directions.

Downregulation of Lipid Phosphate Phosphatase 3 Correlates With Tumor-Infiltrating Immune Cells in Oral Cancer.

Background Sphingosine-1-phosphate (S1P) is a potent oncogenic lipid. Intracellular levels of S1P are tightly regulated by eight S1P-metabolizing enzymes. S1P synthesis is catalyzed by two sphingosine kinases, i.e., sphingosine kinase 1 (SphK1) and sphingosine kinase 2 (SphK2). Five lipid phosphatases (two S1P phosphatases and lipid phosphate phosphatases (LPPs) 1, 2, and 3) reversibly convert S1P back to sphingosine. Previously, we have determined the mRNA expression profile of eight S1P-metabolizing enzymes in tumor tissues and adjacent normal tissues from oral squamous cell carcinoma (OSCC) patients. Except for SphK1, the role of S1P-metabolizing enzymes in OSCC has been poorly studied. Methods We have determined the protein expression of four S1P-metabolizing enzymes (SphK1, SphK2, sphingosine-1-phosphate phosphatase 1 (SGPP1), and lipid phosphate phosphatase 3 (LPP3)) by immunohistochemistry (IHC) in tumor tissues of 46 OSCC patients. Six subjects with non-dysplastic oral mucosa were also included in the study. The immunoreactivity score (IRS) was calculated for each protein in every subject. Further, we determined the associations of expression of S1P-metabolizing enzymes with clinicopathological features of OSCC patients. Results We demonstrate the low IRS for SphK2 and LPP3 in OSCC tumors. Importantly, expression of SphK2 and LPP3 was downregulated in malignant epithelial cells compared to non-malignant mucosa. Further, LPP3 expression negatively correlated with tumor­node­metastasis (TNM) staging of patients (r = -0.307, p = 0.043). Importantly, expression of LPP3 in tumors was found to be an independent predictor of perinodal extension (b = -0.440, p = 0.009), lymphovascular invasion (b = -0.614, p < 0.001), lymph node ratio (b = 0.336, p = 0.039), and TNM staging (b = -0.364, p = 0.030). Conclusion Taken together, our data show that expression of SphK2 and LPP3 is decreased compared to normal mucosa. Thus, the S1P signaling pathway could represent a potential therapeutic target.

Correlation of inhA mutations and ethionamide susceptibility: Experience from national reference center for tuberculosis.

BACKGROUND: Detection of ethionamide (ETH) resistance is crucial as it is part of antitubercular regime. It is crucial to examine the role of inhA gene mutations as a surrogate marker for the detection of ETH resistance, in the Indian context. The present retrospective study was designed with this objective. SUBJECTS AND METHODS: The study was conducted in National Reference Laboratory within the tertiary care institute from January 1, 2018, to June 30, 2019, over 18 months duration. A total of 6612 sputum samples from presumptive multidrug-resistant tuberculosis (TB) patients were received from four districts of Delhi, outdoor and inpatients. Line probe assay (LPA) was performed for smear-positive or culture-positive samples for Mycobacterium tuberculosis. All isolates found to be INH resistant by LPA were cultured and phenotypic susceptibility to ETH was conducted for selected isolates as per the guidelines. RESULTS: A total of 246 isolates were analyzed, for which phenotypic susceptibility to ETH and mutations in inhA were available. ETH resistance was detected among 87/108 (80.5%) isolates with inhA mutation. Sensitivity and specificity of inhA mutation for detection of ETH resistance were 80.5% and 83.8%, respectively. No inhA mutation was detected in 29/116 (25%) ETH-resistant isolates in our study, whereas ETH was found to be phenotypically susceptible in spite of the presence of inhA mutation among 21/130 (16.1%) isolates. CONCLUSIONS: Mutations in inhA gene in LPA predict ETH resistance with fairly good sensitivity and specificity. However, it is imperative to perform phenotypic detection of ETH resistance at proper concentration, in addition to detecting inhA mutation.

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.

Genotypic characterization of 'inferred' rifampin mutations in GenoType MTBDRplus assay and its association with phenotypic susceptibility testing of Mycobacterium tuberculosis.

In GenoType MTBDRplus assay [line probe assay (LPA)], when Mycobacterium tuberculosis (M. tuberculosis) sample DNA fails to hybridize to at least 1 rpoB wild-type probe and any mutation probe, it is inferred as rifampin (RIF)-resistant. In this study, we sought to identify such 'inferred' mutations in M. tuberculosis isolates (n = 203) by rpoB gene sequencing and determined their association with phenotypic resistance. D516Y, H526N, L511P mutations were associated with both phenotypically sensitive (59%, n = 38/64) and resistant (23.7%, n = 33/139) antimicrobial susceptibility testing (AST) results, whereas S531W mutation was associated with only RIF-resistant isolates (33%, n = 46/139). These results demonstrated that, at standard drug concentrations, some 'inferred' mutations may be missed by RIF-AST (phenotypically sensitive). The use of LPA permits identification of these RIF-resistant isolates, and incorporation of additional mutation probes (e.g., S531W) could further increase LPA specificity. Further studies are needed to establish the significance of the type of 'inferred' mutation with clinical/treatment outcomes.

Emerging trends in microbiological diagnostics in children.

The targets of the WHO's End TB Strategy and the United Nations' (UN) Sustainable Development Goals (SDGs) have been expanded to"Find. Treat. All #EndTB" with universal access to TB diagnosis, treatment and care by 2022 in an effort to end the global TB epidemic. Trends to achieve the above targets in children have led to greater emphasis on the newer diagnostics paving way to microbiological confirmation and universal drug sensitivity in children.

Prevalence of allergic bronchopulmonary aspergillosis in asthmatic patients: A prospective institutional study.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is characterized by an allergic inflammatory response to colonization by Aspergillus species, most commonly Aspergillus fumigatus. AIM: To study the prevalence of ABPA in asthmatic patients presenting to our institute. MATERIALS AND METHODS: All consecutive asthma patients attending our allergy clinic Out Patient Department (OPD) over a period of 20 months were tested with skin prick test (SPT) for Aspergillus antigens and those who were found positive were further evaluated for ABPA using Greenberger's criteria. RESULTS: Seventy consecutive asthmatic patients were screened by SPT using Aspergillus antigens. Thirteen patients (18.57%) were found to be SPT positive, out of which nine patients (12.9%) were diagnosed as having ABPA using Greenberger's criteria. ABPA was common among 25-35 age group with no gender predilection. ABPA patients had longer duration of illness, predominantly mixed pattern in PFT, higher mean absolute eosinophil count (AEC) and serum total IgE compared to non-ABPA asthmatic patients. Specific IgE for A. fumigatus was positive in all ABPA patients and serum precipitins were positive in seven patients (77.58%). Chest X-ray abnormalities were seen in five patients (55.6%) and HRCT showed central bronchiectasis in eight patients (88.9%) with varying other radiological features. None were sputum fungal culture positive and five patients (55.6%) have been misdiagnosed as pulmonary tuberculosis in the past. CONCLUSION: The prevalence of ABPA is significantly higher in bronchial asthma patients presenting to tertiary care centers and hence awareness is required among physicians for early diagnosis and management of ABPA to achieve better asthma control and to avoid permanent lung damage.