Vithal Prasad Myneedu - Academia.edu (original) (raw)

Papers by Vithal Prasad Myneedu

The Lancet Infectious Diseases, 2021

doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by pee... more doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

SSRN Electronic Journal, 2020

JOURNAL OF CLINICAL AND DIAGNOSTIC RESEARCH, 2020

Introduction: Lowenstein Jensen (LJ) culture contamination is one of the most frequent problems e... more Introduction: Lowenstein Jensen (LJ) culture contamination is one of the most frequent problems encountered during Mycobacterium tuberculosis (M. tuberculosis) culture. Contaminated cultures are repeated at an additional cost and thus hinder diagnosis. This problem is of more significant concern in Extrapulmonary (EP) samples which have very fewer bacilli loads. Unfortunately, our current contaminant knowledge from past studies is minimal, which is entirely based on pulmonary samples, and not on EP samples. Development of newer culture methods will remain incomplete unless we have a good knowledge about contaminant profiles from both types of samples. Aim: To isolate and identify bacterial and fungal contaminants growing on LJ media during M. tuberculosis complex culture in both pulmonary and EP samples. Materials and Methods: LJ media pairs (5074) were inoculated, of which 2030 were inoculated with pulmonary samples and 3044 with EP samples. Mycobacterial, non-Mycobacterial and fungal growth were differentiated based on characteristics like colony morphology (on Chocolate agar, blood agar, Mackonkey Agar and Sabouraud's Dextrose Agar), staining (Gram stain and Ziehl Neelsen) and biochemical reactions (Indole, Urea, Citrate and Triple Sugar Iron). Statistical analysis was done using SPSS version 20 (IBM ® , New York, NY, USA) and Chi-square test was performed. Results: Overall Contamination Rate (CR) was 2.2%. Individually, CR was 2.9% (60/2030) in pulmonary samples and 1.7% (52/3044) in EP samples (p<0.05). Of the total 303 organisms isolated in the study, 87.8% (266/303) were of bacterial origin and remaining 12.2% (37/303) were fungal. Bacteria isolated belonged to 12 different genera amongst which aerobic spore bearers (Bacillus spp) were the most common. All the 37 fungal isolates were moulds, of which 22 were Aspergillus spp (A. flavus, A. fumigatus and A. niger). Bacterial contaminants were more in pulmonary samples, whereas fungal in EP samples (p<0.05). All the contaminants were breakthrough as none grew as mixture along with acid-fast organisms. Conclusion: Breakthrough nature of contaminants indicates that they probably act by completely inhibiting the growth of any acid-fast bacilli present in the sample. This observation becomes quite relevant in EP samples wherein M. tuberculosis bacilli load is already very less. This M. tuberculosis growth masking can thus decrease the overall sensitivity of LJ culture.

Journal of Infectious Diseases, 1996

Infection Prevention in Practice, 2020

Background: Everyday, tuberculosis hospitals collect enormous amount of sputum containing viable ... more Background: Everyday, tuberculosis hospitals collect enormous amount of sputum containing viable Mycobacterium tuberculosis bacilli, the disposal of which is a challenging task. Chemical (5% phenol) and physical (autoclaving) disinfection methods involve cost, space and cause further environmental degradation. Over the years, use of microwave for sterilisation of biomedical waste has become widespread. However, its efficacy to sterilise large volume of M. tuberculosis positive sputum has never been investigated. Aim: To evaluate the effectiveness of microwave in sterilising large volumes of M. tuberculosis positive sputum samples. Methods: 226 sputum samples positive for M. tuberculosis were checked by Ziehl-Neelsen staining and liquid culture (MGIT Ô) both before and after microwaving. c 2 test was performed, and p-value <0.05 was considered significant. Findings: Before microwaving, samples containing acid fast bacilli (AFB) and live M. tuberculosis bacilli were 93.8% and 95% (z94.7%) respectively; which came down to 14.2% (32) and <1% (z0.9%) in post microwave. In the 32 post-microwave AFB positive samples, bacilli appeared apoptotic, decreased in size, fragmented, loosely arranged and were easily missed as stain artefacts. Their beaded appearance was not appreciable. Background pus cells were of smaller size, did not take up methylene blue stain properly, and multilobed nuclear material was missing. Conclusion: The study shows efficacy of microwave as an alternative sterilisation method for large volume sputum samples containing M. tuberculosis bacilli. Microwave can become an effective sterilisation method, especially for isolated tuberculosis care centres in countries which struggle for disposal of sputum, the biomedical waste.

BackgroundThe WHO End TB Strategy requires universal drug susceptibility testing and treatment of... more BackgroundThe WHO End TB Strategy requires universal drug susceptibility testing and treatment of all people with tuberculosis. However, available second-line diagnostic tools are cumbersome and require sophisticated laboratory infrastructure, and ultimately less than half of those with drug-resistant tuberculosis receive appropriate treatment. Xpert MTB/XDR was developed to help overcome these limitations.MethodsWe assessed the diagnostic accuracy of sputum-based Xpert MTB/XDR for isoniazid, fluoroquinolone, ethionamide and second-line injectable resistance detection in adults with an Xpert MTB/RIF or Ultra Mycobacterium tuberculosis-positive result against a composite reference standard of phenotypic drug-susceptibility testing and whole genome sequencing (NCT03728725). Participants with pulmonary tuberculosis symptoms and ≥1 risk factor for drug resistance were consecutively enrolled between four clinical sites in India, Moldova and South Africa.FindingsBetween 31 July 2019 and 2...

Immunology and Infectious Diseases

Background & Objectives: Globally, tuberculosis (TB) still remains a major public health problem.... more Background & Objectives: Globally, tuberculosis (TB) still remains a major public health problem. India is a high TB burden country contributing to 26 per cent of global TB burden. Pulmonary tuberculosis (PTB) cases are more common (~ 90% of cases) while extra pulmonary tuberculosis (EPTB) constitutes around 10 to 20% of all tuberculosis cases in India. The diagnosis of the EPTB cases is difficult because of few bacilli and consequently is associated with low sensitivity of Zhiel-Neelson (ZN) smear and culture on LJ media. The present study evaluates the utility of PCR for the detection of M. tuberculosis in paucibacillary extra pulmonary and pulmonary tuberculosis samples. Methods: A total of 561 samples (553 EPTB & 8 PTB cases) were collected from the extra pulmonary and pulmonary tuberculosis patients which were processed for ZN smear, culture on LJ media and conventional PCR using two gene targets (IS6110 and MPB64). Results: The PCR positivity of IS6110 and MPB64 gene targets was found to be 91.3% (N=63/69) and 89.9% (N= 62/69) in majority of smear negative & culture positive (as a gold standard) extra pulmonary cases, respectively. However the PCR positivity was observed 100% in smear positive, culture positive Line probe assay tested MDR PTB cases (true positive controls; N=34). Further the PCR specificity was determined >95% (true negative healthy controls; N=26). The positivity of M. tuberculosis by IS6110 & MPB 64 gene targets was found to be range of 88% to 100% in various clinical paucibacillary extra pulmonary samples i.e. pleural fluid, ascitic fluid, lymph node, pus, CSF and others. Our data on 64 samples (non respiratory, n=63 & respiratory samples, n=1) revealed 40.6% positivity by Cobas TaqMan Real Time PCR (utilizing 16S rRNA probe; Roche, USA). Interpretation & Conclusion: Our data revealed that utility of both PCR and Real Time PCR in rapid diagnosis of M. tuberculosis in paucibacillary extra pulmonary tuberculosis samples in Indian scenario.

Current Microbiology, 2022

In 2019, amongst half a million new rifampicin-resistant tuberculosis (TB) cases, 78% were multi ... more 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.

Background: Silent presence of non-tuberculous mycobacterium (NTM) has been observed since the la... more Background: Silent presence of non-tuberculous mycobacterium (NTM) has been observed since the last 100 years, but now the increasing incidence of NTM is of great concern for clinical microbiologists as well as clinicians. Although many advanced efforts are being made for identification and control of Mycobacterium tuberculosis, still the silently growing menace of non-tuberculous mycobacteria is receiving negligible attention. Objectives: This study was aimed to find NTMs in positive cultures and identify them up to species level. Material & Methods: During the study period, i.e. from January 2009 to June 2011, a total of 4104 positive cultures were subjected to species identification by different morphological and biochemical tests. All the tests for identification were performed as per standard procedure along with the standard strains of NTM provided by JALMA, Agra. Results: The identification of positive cultures showed 4044/15581 (25.95%) Mycobacterium tuberculosis complex and...

Immunology and Infectious Diseases, 2013

The Indian journal of chest diseases & allied sciences, 2007

Setting. Tertiary referral hospital in Delhi, India with a well established directly observed tre... more Setting. Tertiary referral hospital in Delhi, India with a well established directly observed treatment, short-course (DOTS) infastructure. Objective. The study aimed to evaluate pilot project of DOTS-Plus strategy in India using a standardised treatment regimen (STR). Methods. Retrospective analysis of the records of 66 patients with multidrug-resistant tuberculosis (MDR-TB) who failed on short-course chemotherapy and were treated with a fully supervised STR containing kanamycin, pyrazinamide (both for initial intensive phase of 6-9 months), ofloxacin, ethionamide and cycloserine was done. Continuation phase drugs were given for at least 18 months after conversion to a negative culture. Thirteen patients required intensive phase of more than six months (mean duration of 7.4 months). Clinical and bacteriological progress was monitored at regular intervals. Results. Of the 66 patients included for analysis, 53 (80.9%) became culture-negative, 77.3% of these within three months. Four ...

ERJ Open Research, 2021

BackgroundNear-patient access to appropriate tests is a major obstacle for the efficient diagnosi... more BackgroundNear-patient access to appropriate tests is a major obstacle for the efficient diagnosis of Tuberculosis (TB) and associated drug resistance.MethodsWe 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 Multi-drug resistant TB patients (n=207).ResultsTrans-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% Confidence Interval [CI]: 52, 98%), 77.7% for isoniazid (95% CI: 52, 94%), 85.7% for fluoroquinolones (95% CI: 42, 100...

Clinical Microbiology and Infection, 2021

OBJECTIVES The present study aimed to evaluate the performance of 'TBDetect' kit-based Bi... more OBJECTIVES The present study aimed to evaluate the performance of 'TBDetect' kit-based Bio-safe fluorescent microscopy filter (BioFM-Filter) microscopy in comparison to 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 BioFM-Filter for improved and bio-safe smear microscopy. We evaluated the performance of 'TBDetect' kit in a 6-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 smear was significant (p<0.001). The overall sensitivity of 'TBDetect' for 6 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 6 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 (DMCs) and Primary Healthcare Centres (PHCs).

Diagnostic Microbiology and Infectious Disease, 2020

In GenoType MTBDRplus assay [line probe assay (LPA)], when Mycobacterium tuberculosis (M. tubercu... more 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.

PLOS ONE, 2019

India has the highest burden of Tuberculosis (TB) and multidrug-resistant TB (MDR-TB) worldwide. ... more India has the highest burden of Tuberculosis (TB) and multidrug-resistant TB (MDR-TB) worldwide. Innovative technology is the need of the hour to identify these cases that remain either undiagnosed or inadequately diagnosed due to the unavailability of appropriate tools at primary healthcare settings. We developed and evaluated 3 kits, namely 'TB Detect' (containing BioFM-Filter device), 'TB Concentration and Transport' (containing Trans-Filter device) and 'TB DNA Extraction' kits. These kits enable bio-safe equipment-free concentration of sputum on filters and improved fluorescence microscopy at primary healthcare centres, ambient temperature transport of dried inactivated sputum filters to central laboratories and molecular detection of drug resistance by PCR and DNA sequencing (Mol-DST). In a 2site evaluation (n = 1190 sputum specimens) on presumptive TB patients, BioFM-Filter smear exhibited a significant increase in positivity of 7% and 4% over ZN smear and LED-FM smear (p<0.05), respectively and an increment in smear grade status (1+ or 2+ to 3+) of 16% over ZN smear and 20% over LED-FM smear. The sensitivity of Mol-DST in presumptive MDR-TB and XDR-TB cases (n = 148) was 90% for Rifampicin (95% confidence interval [CI], 78-96%), 84% for Isoniazid (95% CI, 72-92%), 83% for Fluoroquinolones (95% CI, 66-93%) and 75% for Aminoglycosides (95% CI, 35-97%), using phenotypic DST as the reference standard. Test specificity was 88-93% and concordance was~89-92% (κ value 0.8-0.9). The patient-friendly kits described here address several of the existing challenges and are designed to provide 'Universal Access' to rapid TB diagnosis, including drug-resistant disease. Their utility was demonstrated by application to sputum at 2 sites in India. Our

ACS infectious diseases, Jan 25, 2018

Pulmonary tuberculosis is the most common manifestation of tuberculosis, and to this day, sputum ... more Pulmonary tuberculosis is the most common manifestation of tuberculosis, and to this day, sputum smear microscopy remains the most widely used diagnostic test in resource-limited settings despite its suboptimal sensitivity. Here we report the development of two DNA aptamer-based diagnostic tests, namely aptamer linked immobilized sorbent assay (Aptamer ALISA) and electrochemical sensor (ECS), for the direct detection of a TB biomarker HspX in sputum. First we compared the performance of Aptamer ALISA with anti-HspX polyclonal antibody-based enzyme linked immunosorbent assay (Antibody ELISA) in a blinded study of 314 sputum specimens. Aptamer ALISA displayed a high sensitivity of 94.1% (95% CI 86.8-98%) as compared to 68.2% sensitivity (95% CI 57.2-77.9%) of Antibody ELISA ( p-value < 0.05) using culture as the reference standard without compromising test specificity of 100%. Out of nine smear-negative culture-positive samples, six were positive by Aptamer ALISA and only two were ...

PloS one, 2018

There is lack of information on the proportion of new smear-positive pulmonary tuberculosis (PTB)... more There is lack of information on the proportion of new smear-positive pulmonary tuberculosis (PTB) patients treated with a 6-month thrice-weekly regimen under Revised National Tuberculosis Control Programme (RNTCP) who develop recurrent TB after successful treatment outcome. To estimate TB recurrence among newly diagnosed PTB patients who have successfully completed treatment and to document endogenous reactivation or re-infection. Risk factors for unfavourable outcomes to treatment and TB recurrence were determined. Adult (aged ≥ 18 yrs) new smear positive PTB patients initiated on treatment under RNTCP were enrolled from sites in Tamil Nadu, Karnataka, Delhi, Maharashtra, Madhya Pradesh and Kerala. Those declared "treatment success" at the end of treatment were followed up with 2 sputum examinations each at 3, 6 and 12 months after treatment completion. MIRU-VNTR genotyping was done to identify endogenous re-activation or exogenous re-infection at TB recurrence. TB recurr...

Journal of Epidemiology and Global Health, 2017

Data regarding prevalence of multi-drug resistant tuberculosis (MDR-TB) and associated common mut... more Data regarding prevalence of multi-drug resistant tuberculosis (MDR-TB) and associated common mutations is scarce from Punjab region. The study was designed to determine rate of MDR-TB among presumptive MDR-TB from Punjab and mutation patterns using GenoType MTBDRplus assay. Total of 812 consecutive sputum samples were received from January 2012 to July 2013, from 14 districts of Punjab at the National Reference Laboratory at New Delhi for diagnosis of MDR-TB as hand holding activity. Presumptive MDR-TB patients were identified on basis of criterion B defined by the programme. Smear positive and negatives patients were found to be 636/798 (79.7%) and 162/ 798 (20.3%) respectively. Total of 606 GenoType MTBDRplus tests were conducted and mutations in rpoB, kat G and inhA genes analyzed. Total of 94/606 (15.5%), 43/606 (7.1%) and 40/606 (6.6%) were found to be RIF and INH resistant, mono-RIF resistant and 40/606 (6.6%) mono-INH resistant respectively. Commonest known mutation for RIF in rpoB gene and INH in kat G gene was S531L (80/ 137; 58.4%) and S315T1 (119/134; 88.8%) respectively. Mutations in inhA were found in 21/134 (15.7%) strains. Average turnaround time (TAT) for dispatch of result toPunjab was 4.6 days. Prevalence of RIF resistance in Punjab was found to be 22.6%. Common mutations for RIF and INH were similar to that in other regions of country. GenoType MTBDRplus was found to be useful assay for rapid detection of MDR-TB, responsible for determining better management of MDR-TB patients under the programme.

Journal of Medical Science And clinical Research, 2017

The rapid grower mycobacteria have emerged as significant group of human pathogen amongst the Run... more 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. In this study, we analyzed and identified 121 rapidly growing mycobacterium isolated from clinical samples by polymerase chain reactionrestriction enzyme analysis (PRA) at a national reference laboratory. The results were analyzed and compared with standard biochemical test. In this study, 8 different types of rapid grower mycobacteria were identified by analyzing the fragment generated through restriction enzymes. More than 50% of the isolates were from the pulmonary samples sputum. In pulmonary as well as extrapulmonary samples the most common isolate was M. chelonae (57/121). All strains of M. chelonae were having the same band fragments size. The others species identified in this study were M.fortuitum (42), M. abscessus (11), M. immunogen (06), M. peregrinum (02), M. smegmatis (01), M. wolinskyi (01), M.goodii(01). The study showed that in pulmonary as well as extrapulmonary sample M. chelonae was the most common isolate. PCR-REA is a rapid accurate system with concordance of 119/121 (98.34%) when compared to standard biochemical tests for identification of clinically important species of rapidly growing mycobacterium.