Search

Antibiotics like colistin can save patients infected with carbapenem-resistant Pseudomonas aeruginosa. However, patients can succumb to such infections even if they undergo colistin therapy. This prompted us to investigate the probable antimicrobial resistance mechanisms and virulence determinants involved in colistin- and carbapenem-resistant P. aeruginosa (CCRPA). Of the 448 P. aeruginosa clinical strains, 19 isolates were resistant to both colistin and carbapenem. Carbapenemases and efflux pump encoding genes were assessed by multiplex PCR and qPCR, respectively. blaVIM was detected among six CCRPA isolates and blaIMP in one strain. The expression levels of pmrA and phoP, as well as pmrB genes and their association with colistin resistance, were assessed by qPCR and semi-quantitate PCR, respectively. pmrA and phoP genes were significantly enhanced in three and nine CCRPA isolates, respectively. We also phenotypically evaluated biofilms, pyocyanin, and alginate production among CCRPA strains. Alginate production was observed in 15 isolates, followed by biofilm (n = 8) and pyocyanin (n = 5). Our results highlighted the coexistence of colistin and carbapenem resistance and biofilm formation among clinical isolates of CCRPA. Further studies are required to trace the source and the origin of colistin and carbapenem resistance in this specific environment.

Background and objectives: Non tuberculous mycobacteria (NTM) is an emerging opportunistic pathogen increasing globally and indistinguishable from tuberculosis (TB), which remains a challenge particularly in developing countries. This study aimed to identify the prevalence and diversity of NTM among both pulmonary TB (PTB) and extrpulmonary TB (EPTB) clinical isolates from south India. Methodology: A total of 7633 specimens from TB suspects (PTB, n = 4327 and EPTB, n = 3306) were collected during the study period (July 2018–March 2020) in a tertiary care hospital. The study specimens were subjected to Ziehl Neelsen (ZN) staining and Auramine phenol (AP) staining followed by Lowenstein–Jensen (LJ) and mycobacteria growth indicator tube (MGIT) culture. The MPT64 immunochromatographic test (ICT) was performed among mycobacterial cultures and ICT negative isolates were subjected to Line Probe Assay (LPA). In addition, 53 (PTB, 48 and EPTB, 5) NTM MGIT positive cultures were collected from Intermediate Reference Laboratory (IRL), Puducherry and subjected to LPA for speciation. Results: Of the 7633 TB suspects, 0.6% were diagnosed as NTM diseases and 5.5% with Mycobacterium tuberculosis (MTBC). NTM infection was observed among 0.7% (31/4327) of PTB and 0.4% (14/3306) of EPTB. MTBC was detected among 6.1% (264/4327) of PTB and 4.6% (153/3306) of EPTB. Among 98 NTM cultures, 80.6% of isolates were recovered from PTB and 19.4% from EPTB specimens. Among pulmonary specimens, Mycobacterium intracellulare (26.6%), Mycobacterium abscessus (17.7%) and Mycobacterium kansasii (12.7%) were the most frequently detected species, while Mycobacterium intracellulare (21.1%), Mycobacterium scrofulaceum (15.8%) and Mycobacterium fortuitum (10.5%) were common in extrapulmonary specimens. Conclusion: The frequency of NTM infection among TB suspects was low at a South Indian tertiary care hospital. The most predominant NTM species isolated from both pulmonary and extrapulmonary specimens was M. intracellulare.

Background: Studying DNA methylation changes in disease condition provides the basis of disease pathogenesis or host immune response to infection. Evidences suggest that pathogen-mediated DNA methylation changes influences the expression pattern of genes contributing to disease condition to avert host immune response. Hence, we attempted to study the association between tubercle bacilli-mediated global DNA methylation changes in children with tuberculosis (TB) disease and healthy controls. Methods: Forty-three children diagnosed with TB and 33 healthy children were enrolled in this study. ELISA-based global DNA methylation quantification was performed to measure the changes in percentage of global genomic DNA methylation level. Results: Highly significant difference in global DNA methylation level was found between cases and controls and median global DNA methylation level was 6.25% (interquartile range [IQR] 2.5%–10%) in cases and 25% (IQR, 12.5%–25%) in controls (P < 0.001). Significant difference was found in GeneXpert-positive cases (P < 0.01). Receiver operating curve analysis showed that area under curve 0.81 for the total study population and 0.76 for GeneXpert-positive cases. Conclusion: The results show the significant difference in global DNA methylation level in children with TB disease that can serve as a potential biomarker in early diagnosis of TB disease. Measuring global DNA methylation level, however, not an accurate or sensitive diagnostic method but evaluating active demethylation at genome-wide level can be used to monitor disease progression and treatment efficacy.

PURPOSE: Detection of carbapenemases among Gram-negative bacteria (GNB) is important for both clinicians and infection control practitioners. The Clinical and Laboratory Standards Institute recommends Carba NP (CNP) as confirmatory test for carbapenemase production. The reagents required for CNP test are costly and hence the test cannot be performed on a routine basis. The present study evaluates modifications of CNP test for rapid detection of carbapenemases among GNB. MATERIALS AND METHODS: The GNB were screened for carbapenemase production using CNP, CarbAcineto NP (CANP), and modified CNP (mCNP) test. A multiplex polymerase chain reaction (PCR) was performed on all the carbapenem-resistant bacteria for carbapenemase genes. The results of three phenotypic tests were compared with PCR. RESULTS: A total of 765 gram negative bacteria were screened for carbapenem resistance. Carbapenem resistance was found in 144 GNB. The metallo-β-lactamases were most common carbapenemases followed by OXA-48-like enzymes. The CANP test was most sensitive (80.6%) for carbapenemases detection. The mCNP test was 62.1% sensitive for detection of carbapenemases. The mCNP, CNP, and CANP tests were equally sensitive (95%) for detection of NDM enzymes among Enterobacteriaceae. The mCNP test had poor sensitivity for detection of OXA-48-like enzymes. CONCLUSION: The mCNP test was rapid, cost-effective, and easily adoptable on routine basis. The early detection of carbapenemases using mCNP test will help in preventing the spread of multidrug-resistant organisms in the hospital settings.

Competing Interests: KEK has a patent (WO2015170108A1) on biomarkers and combinations thereof for diagnosing tuberculosis. All other authors declare no competing interests.

Introduction: Mycobacterium tuberculosis has been extensively studied for mutations leading to drug resistance. Pyrazinamide is a drug acting on the semi-dormant bacteria that is responsible for relapse of tuberculosis. This drug helped reduce the treatment duration of tuberculosis from nine to six months. However, this drug is not being screened for resistance along with Rifampicin and Isoniazid., Aims and Objectives: This study aimed to estimate the proportion of pncA gene mutation among tuberculosis patients and its association between treatment outcomes, clinical characteristics, and phenotypic drug resistance., Method: ology: A total of 154 samples included 73 drug-resistant and 81 drug-susceptible isolates. The isolates were subjected to DNA extraction and amplification using conventional PCR. The PCR product was sequenced by the Sanger sequencing method, and phenotypic drug susceptibility testing was done using the broth dilution method. The association of this gene with the treatment outcome was done by following up with the patients till the end of the regimen., Results: None of the drug susceptible tuberculosis patients showed significant non-synonymous mutations. Among the drug-resistant TB patients, seven unique significant mutations out of 73 isolates (9.6%) were distributed among Isoniazid-resistant tuberculosis and Multi-Drug Resistant Tuberculosis isolates. No association was found between the mutations and the clinical characteristics of the subjects harboring these isolates., Conclusion: This study estimated seven unique mutations in drug-resistant tuberculosis and none in drug-sensitive tuberculosis. Isolates harboring was not significantly associated with the participant's treatment outcome and other clinical characteristics. The pyrazinamide resistance testing by the phenotypic and genotypic methods was found to be in concordance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.)

Background: Tuberculous meningitis (TBM) is caused by the dissemination of Mycobacterium tuberculosis (MTB) from the primary site of infection to the central nervous system. However, the bacterial factors associated with the pathogenesis of TBM remain unclear. This study employed transcriptomic and proteomic methods to comprehensively analyze the changes in genes and proteins and their associated pathways in MTB strains isolated from cerebrospinal fluid (CSF) of TBM and sputum of pulmonary TB (PTB) cases., Methodology: Five MTB strains were subjected to OMICs (transcriptomic and proteomic) analysis. Among five MTB strains, two were isolated from CSF and sputum samples of the same patient with PTB and TBM infections, one from the sputum of a different PTB patient, and a strain obtained from the CSF of another TBM patient. H37Rv was used as a reference strain. The reliability of transcriptomic results was validated by real time polymerase chain reaction with selected genes from 100 MTB isolates (CSF, 50 and sputum, 50)., Results: The transcriptomic study revealed that overlapping differentially expressed genes of MTB strains isolated from TBM patients showed featured enrichment in benzoate degradation, lysine degradation, tryptophan metabolism, fatty acid degradation, ATP binding cassette transporters, microbial metabolism in diverse environments, biosynthesis of antibiotics, and metabolic pathways. Eleven genes were upregulated, and four were downregulated in MTB strains isolated from TBM compared to PTB. From proteomic analysis, we identified three candidate proteins belonging to plasminogen binding proteins (PBP) (enolase, dnaK, and isocitrate lyase 1) that were significantly upregulated in MTB strains isolated from TBM., Conclusion: Overall, the transcriptomic and proteomic analyses provided an important base for understanding the unique feature of TBM pathogenesis. To the best of our knowledge, this is the first report highlighting the importance of PBPs on TBM pathogenesis., Competing Interests: None

Purpose: We aimed this study to standardize real time - polymerase chain reaction (RT-PCR) for the detection of Mycobacterium tuberculosis (Mtb) in cerebrospinal fluid (CSF) samples and compare its diagnostic performance with GeneXpert (Xpert), Mycobacteria Growth Indicator Tube (MGIT) and Multiplex PCR (MPCR) for tuberculous meningitis (TBM)., Methodology: A total of 217 CSF samples were obtained from patients with suspected TBM during the study period between January 2019 and December 2021. The optimal cycle threshold (CT) of RT-PCR was determined by comparing different gene targets of Mtb (IS6110, 16SrRNA, HSP65 and Ag85B). Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) was determined for RT-PCR, Xpert, MGIT960 and MPCR. Diagnostic accuracy of these assays was compared by using clinical diagnosis as reference standard., Results: IS6110RT-PCR was found to be highly sensitive as compared to other gene targets. Sensitivities of IS6110RT-PCR, MPCR, Xpert and MGIT against a reference standard of definite, probable and possible TBM were 36.7%, 21.1%, 16.7% and 6.7%, respectively; specificities were 97.6%, 100%, 100% and 100%, respectively. Xpert, RT-PCR, MPCR and MGIT960 detected 6.91% (n = 15), 5.99% (n = 13), 5.99% (n = 13) and 2.76% (n = 6) of definite TBM, respectively. RT-PCR detected 6.45% (n = 14) and 2.76% (n = 6) of possible TBM and probable TBM, respectively and MPCR detected 1.38% (n = 3) of possible and probable TBM each., Conclusion: IS6110RT-PCR is highly sensitive for primary screening of suspected TB cases, which may help clinicians to start appropriate patient's treatment with clinical suspicion of TBM., Competing Interests: Conflict of interest None., (Copyright © 2023 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

Tuberculosis (TB) remains a significant global health crisis and the number one cause of death for an infectious disease. The health consequences in high-burden countries are significant. Barriers to TB control and eradication are in part caused by difficulties in diagnosis. Improvements in diagnosis are required for organisations like the World Health Organisation (WHO) to meet their ambitious target of reducing the incidence of TB by 50% by the year 2025, which has become hard to reach due to the COVID-19 pandemic. Development of new tests for TB are key priorities of the WHO, as defined in their 2014 report for target product profiles (TPPs). Rapid triage and biomarker-based confirmatory tests would greatly enhance the diagnostic capability for identifying and diagnosing TB-infected individuals. Protein-based test methods e.g. lateral flow devices (LFDs) have a significant advantage over other technologies with regard to assay turnaround time (minutes as opposed to hours) field-ability, ease of use by relatively untrained staff and without the need for supporting laboratory infrastructure. Here we evaluate the diagnostic performance of nine biomarkers from our previously published biomarker qPCR validation study; CALCOCO2, CD274, CD52, GBP1, IFIT3, IFITM3, SAMD9L, SNX10 and TMEM49, as protein targets assayed by ELISA. This preliminary evaluation study was conducted to quantify the level of biomarker protein expression across latent, extra-pulmonary or pulmonary TB groups and negative controls, collected across the UK and India, in whole lysed blood samples (WLB). We also investigated associative correlations between the biomarkers and assessed their suitability for ongoing diagnostic test development, using receiver operating characteristic/area under the curve (ROC) analyses, singly and in panel combinations. The top performing single biomarkers for pulmonary TB versus controls were CALCOCO2, SAMD9L, GBP1, IFITM3, IFIT3 and SNX10. TMEM49 was also significantly differentially expressed but downregulated in TB groups. CD52 expression was not highly differentially expressed across most of the groups but may provide additional patient stratification information and some limited use for incipient latent TB infection. These show therefore great potential for diagnostic test development either in minimal configuration panels for rapid triage or more complex formulations to capture the diversity of disease presentations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Garlant, Ellappan, Hewitt, Perumal, Pekeleke, Wand, Southern, Kumar, Belgode, Abubakar, Sinha, Vasan, Joseph and Kempsell.)

Tuberculosis (TB) remains a major global threat and diagnosis of active TB ((ATB) both extra-pulmonary (EPTB), pulmonary (PTB)) and latent TB (LTBI) infection remains challenging, particularly in high-burden countries which still rely heavily on conventional methods. Although molecular diagnostic methods are available, e.g., Cepheid GeneXpert, they are not universally available in all high TB burden countries. There is intense focus on immune biomarkers for use in TB diagnosis, which could provide alternative low-cost, rapid diagnostic solutions. In our previous gene expression studies, we identified peripheral blood leukocyte (PBL) mRNA biomarkers in a non-human primate TB aerosol-challenge model. Here, we describe a study to further validate select mRNA biomarkers from this prior study in new cohorts of patients and controls, as a prerequisite for further development. Whole blood mRNA was purified from ATB patients recruited in the UK and India, LTBI and two groups of controls from the UK (i) a low TB incidence region (CNTRLA) and (ii) individuals variably-domiciled in the UK and Asia ((CNTRLB), the latter TB high incidence regions). Seventy-two mRNA biomarker gene targets were analyzed by qPCR using the Roche Lightcycler 480 qPCR platform and data analyzed using GeneSpring™ 14.9 bioinformatics software. Differential expression of fifty-three biomarkers was confirmed between MTB infected, LTBI groups and controls, seventeen of which were significant using analysis of variance (ANOVA): CALCOCO2, CD52, GBP1, GBP2, GBP5, HLA-B, IFIT3, IFITM3, IRF1, LOC400759 (GBP1P1), NCF1C, PF4V1, SAMD9L, S100A11, TAF10, TAPBP, and TRIM25. These were analyzed using receiver operating characteristic (ROC) curve analysis. Single biomarkers and biomarker combinations were further assessed using simple arithmetic algorithms. Minimal combination biomarker panels were delineated for primary diagnosis of ATB (both PTB and EPTB), LTBI and identifying LTBI individuals at high risk of progression which showed good performance characteristics. These were assessed for suitability for progression against the standards for new TB diagnostic tests delineated in the published World Health Organization (WHO) technology product profiles (TPPs)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Perumal, Abdullatif, Garlant, Honeyborne, Lipman, McHugh, Southern, Breen, Santis, Ellappan, Kumar, Belgode, Abubakar, Sinha, Vasan, Joseph and Kempsell.)

Tuberculous meningitis (TBM) is the most severe form of Mycobacterium tuberculosis (Mtb) infection in humans and is a public health concern worldwide. We evaluated the performance of GeneXpert MTB/RIF (GeneXpert) for the diagnosis of TBM. In addition, genetic diversity and drug susceptibility profiling of Mtb strains isolated from TBM patients were also investigated. A total of 293 TBM suspected cerebrospinal fluid (CSF) samples were collected and subjected to GeneXpert and Mycobacterial Growth Indicator Tube (MGIT 960) culture, respectively. Sensitivity and specificity of GeneXpert was 72.7% and 98.5%, respectively by using MGIT 960 as a gold standard (GeneXpert (n = 20, 6.8%) vs MGIT 960 (n = 22, 7.5%)). All Mtb positive cultures were subjected to 24-locus Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR) typing, Line probe assay (LPA) and MGIT 960- Drug Susceptibility Testing (DST). The rpoB gene was amplified and sequenced for selected isolates. Among our TBM patients, East African Indian (EAI) lineage (n = 16, 72.7%) was most predominant followed by Beijing (n = 3, 13.6%), S-family (n = 2, 9.1%) and Delhi/CAS (n = 1, 4.5%). Three Mtb strains were found to be Isoniazid (INH) resistant by MGIT 960; however LPA revealed that two strains were INH resistant and one strain was multi drug resistant (MDR) (Resistant to Isoniazid and Rifampicin (RIF)). We identified rifampicin resistant isolate with the mutation D516F in rifampicin resistance-determining region (RRDR) and observed discordant results between LPA, GeneXpert and MGIT 960. In addition, GeneXpert showing false RIF resistance was identified (no mutation in RRDR). We conclude that GeneXpert is useful for the diagnostic confirmation of TBM; however a GeneXpert negative sample should be subjected to MGIT 960 culture or LPA to rule out TBM. EAI lineage was the most predominant among TBM patients in South India and associated with drug resistance. The discordance between GeneXpert, MGIT 960 and LPA with respect to rifampicin resistance has to be ruled out to avoid TB treatment failure or relapse., Competing Interests: The authors have declared that no competing interests exist.

Background: Tuberculosis (TB) is a major public health problem worldwide. Contamination rate and poor recovery of Mycobacterium tuberculosis complex (MTBC) in MGIT960 culture may affect the early diagnosis of TB. Evidence is needed to determine the factors associated with contamination rates and MTBC recovery in MGIT960. Hence, we undertook this study to compare the factors influencing MTBC culture positivity and contamination rates in MGIT960 in patients with Pulmonary tuberculosis (PTB)., Methods: A total of 849 sputum samples from newly diagnosed smear-positive TB cases enrolled into the Regional Prospective Observational Research for Tuberculosis India cohort between May 2014 to March 2017 were analyzed. Samples were inoculated into MGIT960 and positive cultures were examined for the presence of MTBC by immunochromatographic test for detection of MPT64 antigen., Results: Of the 849 cases, 811 (95.5%) were culture positive for MTBC, 23 (2.7%) were culture negative and 15 (1.8%) were contaminated. Salivary sputum showed significantly less culture yield compared to mucopurulent/blood stained samples (p = 0.021). Sputum from individuals <20 or ≥60 years showed lower culture yield of 93.9%, compared to those aged 20-59years (98.2%) (p = 0.002). Based on smear grading, culture isolation of MTBC by MGIT960 was 86.1%, 93.6% and 99.5% for negative, scanty and positive (1+/2+/3+) samples, respectively (p ≤ 0.0001). Sputum from HIV negative patients showed higher culture yield, compared to HIV positive patients (p ≤ 0.0001). Chest X-Ray revealed that patient with cavity showed higher culture isolation of MTBC compared to patients without cavity (p = 0.035). Contamination rates were higher in smear negatives (6.0%), compared to scanty (2.1%) and smear positives (1.1%) (p = 0.007). However, delay in transport of the specimen to the laboratory was the only independent factor significantly associated with increase in culture contamination., Conclusion: Our results highlight that extremes of age, smear negativity, HIV infection, sputum quality and cavitation significantly influence the culture yield of MTBC, whereas transport duration and smear grading affected the contamination rates in MGIT960. Hence, addressing these factors may improve the diagnostic performance of MGIT960., Competing Interests: Declaration of competing interest All authors have none to declare., (Copyright © 2020 Tuberculosis Association of India. All rights reserved.)

Objectives: This study aimed to identify the multiple drug resistance mechanisms and various virulence genes in high-level carbapenem-resistant Pseudomonas aeruginosa (CARPA) isolates collected from patients hospitalised in a tertiary care hospital in South India., Methods: A total of 156 CARPA isolates were included in the study. Multiplex PCR was optimised to detect carbapenemase and extended-spectrum β-lactamase (ESBL) genes. Semi-quantitative reverse transcription PCR (RT-PCR) was optimised to evaluate oprD deficiency. Efflux pump activity was determined by phenylalanine-arginine β-naphthylamide (PAβN) assay, and expression of mexA and mexC genes was evaluated by real-time quantitative PCR (qRT-PCR). Presence of the virulence genes exoS, algD, algU, lasR and rhlR were detected by qRT-PCR and plcH and lasB by RT-PCR., Results: Genes encoding carbapenemases and ESBLs were detected in 48.7% (bla VIM , 23.1%; bla NDM-1 , 17.3%; bla VIM +bla NDM-1 , 7.1%; and bla IMP , 1.3%) and 4% (bla VEB , 4.5%) of CARPA isolates, respectively. Loss of porin OprD was observed in nine isolates (5.8%), two of which harboured the bla VIM gene. Among efflux pump-positive isolates, mexA expression was significantly upregulated. algD expression was detected in 93% of CARPA isolates, followed by algU (89%), rhlR (84%), lasR (81%) and exoS (76%). The lasB and plcH genes were detected in 94% and 92% of isolates, respectively., Conclusions: Co-existence of multiple antimicrobial resistance mechanisms together with virulence genes in carbapenem-resistant isolates has become an alarming emerging threat. Continuous monitoring of multidrug-resistant pathogens is important for clinicians in order to determine therapeutic options against such infections., (Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)