Transcriptomic and proteomic analyses of Mycobacterium tuberculosis strains isolated from tuberculous meningitis patients.

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.

Active Case Finding for Tuberculosis among Health Care Workers in a Teaching Hospital, Puducherry, India.

Background: Health care workers (HCWs) are at risk of acquiring tuberculosis (TB) infection and disease due to occupational exposure. But there are no national guidelines on routine screening for TB (active case finding (ACF)) among HCWs and understand its implementation and feasibility. Methods: This study was conducted among HCWs in a teaching hospital in India. We used symptom screening to identify those with presumptive TB and were further evaluated for diagnosis of TB. Results: A total of 1,001 HCWs were screened over a period of 18 months. In our study, 51 (5.1%) HCWs were found to have presumptive TB and on further evaluation, 5 (0.5%) of these patients were diagnosed with active TB. The number needed to screen (NNS) for one active TB among the HCWs was 200. Alcohol use was significantly associated with both presumptive TB (P = 0.037) and active TB (P = 0.035) among HCWs, and exposure to active TB patients (P = 0.014) in the family and workplace and increased frequency of exposures (P = <0.001) were associated with presumptive TB. Conclusion: ACF for TB among HCWs had a good yield in our study. ACF utilizing routine national TB program guidelines is feasible to be implemented among HCWs to aid in the early diagnosis and treatment of TB in this high-risk group.

Quantitative Real-Time Polymerase Chain Reaction for Detection of Mycobacterium leprae DNA in Tissue Specimens from Patients with Leprosy.

In leprosy, early diagnosis is crucial to prevent transmission and onset of disabilities of the disease. The purpose of this study was to determine usefulness of quantitative real-time polymerase chain reaction (PCR) in clinically diagnosed cases of leprosy. Thirty-two leprosy cases were included. The real-time PCR was performed using commercial kit targeting Mycobacterium leprae-specific insertion sequence element. The slit skin smear was positive in two (22.2%) borderline tuberculoid (BT) patients, five (83.3%) borderline lepromatous (BL) patients, and seven (50%) lepromatous leprosy (LL). The positivity of quantitative real-time PCR in BT, BL, LL, and pure neuritic leprosy were 77.8%, 83.3%, 100%, and 33.3%, respectively. Using histopathology as the gold standard, sensitivity of quantitative real-time PCR was 93.1%, and specificity was 100%. The DNA load was higher in LL (3,854.29/106 cells), followed by BL (140.37/106 cells), and BT (2.69/106 cells). Because of the high sensitivity and specificity of real-time PCR, our study strongly suggests the use of real-time PCR as a diagnostic tool for leprosy.

Diagnostic performance of real time PCR for the detection of Mycobacterium tuberculosis in cerebrospinal fluid samples.

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.

Development of prognostic scoring system for predicting 1-year mortality among pulmonary tuberculosis patients in South India.

BACKGROUND: Development of a prediction model using baseline characteristics of tuberculosis (TB) patients at the time of diagnosis will aid us in early identification of the high-risk groups and devise pertinent strategies accordingly. Hence, we did this study to develop a prognostic-scoring model for predicting the death among newly diagnosed drug sensitive pulmonary TB patients in South India. METHODS: We undertook a longitudinal analysis of cohort data under the Regional Prospective Observational Research for Tuberculosis India consortium. Multivariable cox regression using the stepwise backward elimination procedure was used to select variables for the model building and the nomogram-scoring system was developed with the final selected model. RESULTS: In total, 54 (4.6%) out of the 1181 patients had died during the 1-year follow-up period. The TB mortality rate was 0.20 per 1000 person-days. Eight variables (age, gender, functional limitation, anemia, leukopenia, thrombocytopenia, diabetes, neutrophil-lymphocyte ratio) were selected and a nomogram was built using these variables. The discriminatory power was 0.81 (95% confidence interval: 0.75-0.86) and this model was well-calibrated. Decision curve analysis showed that the model is beneficial at a threshold probability ~15-65%. CONCLUSIONS: This scoring system could help the clinicians and policy makers to devise targeted interventions and in turn reduce the TB mortality in India.

Corrigendum: Malnutrition leads to increased inflammation and expression of tuberculosis risk signatures in recently exposed household contacts of pulmonary tuberculosis.

[This corrects the article DOI: 10.3389/fimmu.2022.1011166.].

Malnutrition leads to increased inflammation and expression of tuberculosis risk signatures in recently exposed household contacts of pulmonary tuberculosis.

Background: Most individuals exposed to Mycobacterium tuberculosis (Mtb) develop latent tuberculosis infection (LTBI) and remain at risk for progressing to active tuberculosis disease (TB). Malnutrition is an important risk factor driving progression from LTBI to TB. However, the performance of blood-based TB risk signatures in malnourished individuals with LTBI remains unexplored. The aim of this study was to determine if malnourished and control individuals had differences in gene expression, immune pathways and TB risk signatures. Methods: We utilized data from 50 tuberculin skin test positive household contacts of persons with TB - 18 malnourished participants (body mass index [BMI] < 18.5 kg/m2) and 32 controls (individuals with BMI ≥ 18.5 kg/m2). Whole blood RNA-sequencing was conducted to identify differentially expressed genes (DEGs). Ingenuity Pathway Analysis was applied to the DEGs to identify top canonical pathways and gene regulators. Gene enrichment methods were then employed to score the performance of published gene signatures associated with progression from LTBI to TB. Results: Malnourished individuals had increased activation of inflammatory pathways, including pathways involved in neutrophil activation, T-cell activation and proinflammatory IL-1 and IL-6 cytokine signaling. Consistent with known association of inflammatory pathway activation with progression to TB disease, we found significantly increased expression of the RISK4 (area under the curve [AUC] = 0.734) and PREDICT29 (AUC = 0.736) progression signatures in malnourished individuals. Conclusion: Malnourished individuals display a peripheral immune response profile reflective of increased inflammation and a concomitant increased expression of risk signatures predicting progression to TB. With validation in prospective clinical cohorts, TB risk biomarkers have the potential to identify malnourished LTBI for targeted therapy.

Evaluation of Host Protein Biomarkers by ELISA From Whole Lysed Peripheral Blood for Development of Diagnostic Tests for Active Tuberculosis.

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.

Effect of Neutralization of Gastric Aspirate on culture yield of mycobacterium tuberculosis in children with pulmonary tuberculosis.

INTRODUCTION: Conventionally gastric aspirates are neutralized with sodium bicarbonate to improve the culture yield of MTB. However, only limited data is there to support this practice. The aim of this study was to compare the contamination rate, culture yield and time to detection of Mycobacterium tuberculosis (MTB) in neutralized and non-neutralized gastric aspirate samples and report the drug resistance. MATERIALS AND METHODS: A total of 336 neutralized and non-neutralized gastric aspirate samples were simultaneously cultured by both LJ culture and MGIT 960 to compare the difference in isolation rate, time to detection and contamination rate. First line drug susceptibility testing was performed using MGIT 960 SIRE kit. RESULTS: MTB was isolated from 8.6% (29/336) of GA samples by one or more of the culture methods. The isolation rate of MTB from neutralized and non-neutralized GA samples by combined LJ and MGIT 960 culture was 7.1% (24/336) and 6.8% (23/336), respectively. Both of them detected 18 MTB isolates in common. However, the neutralized and non-neutralized GA samples detected additional 6 and 5 MTB isolates, respectively. The mean time to detection of MTB were similar. In MGIT 960 culture, contamination rate of non-neutralized samples (17%) was significantly lower when compared to neutralized samples (21.1%) (P = 0.044). Drug susceptibility testing of MTB isolates revealed that, out of 26 isolates, 2 were resistant to ethambutol, one each was resistant to isoniazid and rifampicin. CONCLUSION: The findings of this study suggest that non-neutralized samples should be routinely processed in addition to the neutralized samples for optimum isolation of MTB from gastric aspirate samples.

Pulmonary cryptosporidiosis in a case of adenocarcinoma of stomach: A rare case report.

Cryptosporidium species are commonly known to cause chronic intractable diarrhea in patients suffering from human immunodeficiency virus (HIV)-acquired immunodeficiency syndrome, however extra-intestinal presentations have been rarely reported. Hereby, we report a rare case of isolated pulmonary cryptosporidiosis in a 75-year-old HIV-negative patient with metastatic carcinoma of the stomach who was managed conservatively with hemostatic radiotherapy for palliative care. The patient had presented with cough with expectoration for 2 months. Sputum microscopic examination was suggestive of pulmonary cryptosporidiosis. There was no evidence of intestinal cryptosporidiosis. Therapy for pulmonary cryptosporidiosis was started with tablet nitazoxanide. The patient succumbed to the disease few days later following discharge. Although rare, patients with disseminated gastrointestinal malignancy can potentially have isolated pulmonary cryptosporidiosis.

Evaluation of real time polymerase chain reaction targeting mpb64 gene for diagnosis of extrapulmonary tuberculosis.

BACKGROUND: Paucibacillary nature of extrapulmonary tuberculosis (EPTB) has paved way for molecular methods increasingly being used for diagnosis. We undertook a study for evaluation of sensitivity and specificity of real-time polymerase chain reaction (RT-PCR) targeting mpb64 gene for diagnosis of EPTB. METHODS: A total of 152 clinical samples from suspected cases of EPTB were included in this study. All samples were extracted using spin column based commercial DNA extraction kit and were subjected to RT-PCR targeting mpb64 and IS6110. Smear and culture was also done for samples whenever quantity was sufficient. Cytology report was noted from hospital information system. Receiver operating characteristic (ROC) curve analysis was done for determining cut-off Ct value for mpb64 RT-PCR. Melt curve analysis was done for samples whose cycle threshold (Ct) value was more than 37. The sensitivity and specificity of the mpb64 RT-PCR was calculated using a composite gold standard i.e., positive for one or more of the following: microscopy (including fine needle aspiration cytology (FNAC), acid-fast bacilli positivity), culture and IS6110 RT-PCR. RESULTS: Out of the 152 samples, 72 (47.4%) were positive for tuberculosis by composite gold standard. Samples consisted of ascitic fluid (12), CSF (35), pus (23), lymph node aspirate (35), pleural fluid (37), synovial fluid (4), urine (1), pericardial fluid (1) and tissue bits (4). Microscopy (AFB smear including lymph node aspirate) was done for 124 samples of which 43 (34.7%) were positive. Culture results were available for 79 samples, 25 (31.6%) of which were positive and 42 (27.6%) of the 152 samples were positive by IS6110 PCR. Based on ROC and melt curve analysis, mpb64 RT-PCR was able to detect 38 (52.8%) of the 72 positive samples. In comparison to IS6110 RT PCR, 4 additional cases were detected by mpb64 RT-PCR. Compared to composite gold standard mpb64 showed overall sensitivity of 52.8%. CONCLUSION: The mpb64 RT-PCR is highly specific or MTB and can be used as a supplemental test for diagnosis of EPTB along with other diagnostic tests. However the overall sensitivity of mpb64 RT-PCR is too low to be used as an independent test for diagnosis of EPTB. Combining the results of IS6110 RT PCR and mpb64 RT PCR improved the overall sensitivity and hence mpb64 can be used as an additional target for diagnosis of EPTB.

Prevalence and speciation of non-tuberculous mycobacteria among pulmonary and extrapulmonary tuberculosis suspects in South India.

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.

Prevalence and Time Trend of SARS-CoV-2 Infection in Puducherry, India, August-October 2020.

We conducted 3 population-based cross-sectional surveys, at 1-month intervals, to estimate the prevalence and time-trend of severe acute respiratory syndrome coronavirus 2 infection in Puducherry, India. Seropositivity rate increased from 4.9% to 34.5% over 2 months and was 20-fold higher than the number of diagnosed cases of infection.

Molecular diagnosis, genetic diversity and drug sensitivity patterns of Mycobacterium tuberculosis strains isolated from tuberculous meningitis patients at a tertiary care hospital in South India.

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.

Evaluation of factors influencing Mycobacterium tuberculosis complex recovery and contamination rates in MGIT960.

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.

Diagnostic performance of real time PCR and MALDI-TOF in the detection of nontuberculous mycobacteria from clinical isolates.

This study aimed to evaluate the performance of real-time PCR (qPCR) and MALDI-TOF for accurate and timely detection of nontuberculous mycobacterium (NTM) from clinical isolates. We collected fifty NTM suspected Mycobacteria Growth Indicator Tube (MGIT) cultures and analysed the diagnostic performance of qPCR and VITEK MS using Line Probe Assay (LPA) GenoType CM (Common Mycobacteria) as gold standard. The qPCR assays targeting 16S rRNA, ITS and IS6110 genes were developed for the identification of NTM and Mycobacterium tuberculosis complex (MTBC). LPA GenoType CM, a PCR technique targeting 23S rRNA gene, followed by reverse hybridization and line probe technology identified 90% of Mycobacterium species including M. fortuitum (16%,n = 8), M. intracellulare (10%,n = 5), M. gordonae (10%,n = 5), M. xenopi (4%,n = 2), M. scrofulaceum (4%,n = 2), Mycobacterium additional species (AS) (32%,n = 16) and MTBC (14%,n = 7), qPCR detected 80% of Mycobacterium species (NTM, 66% (n = 33) and MTBC, 14% (n = 7)) and MALDI-TOF, 52% (M. fortuitum (12%,n = 6), M. intracellulare (10%, n = 5), M. simiae (8%,n = 4), M. gordonae (8%,n = 4), and MTBC (14%,n = 7)). Sensitivity of qPCR and MALDI-TOF was 88.9% and 57.8%, respectively with 100% specificity. The combination of qPCR and MALDI-TOF remains an appropriate test for timely diagnosis of Mycobacterium species. This may eventually assist to detect the cases that may have been missed by phenotypic tests and enhance the NTM diagnosis capability to improve effective patient management.

Effect of malnutrition on radiographic findings and mycobacterial burden in pulmonary tuberculosis.

BACKGROUND: The relationship between malnutrition and tuberculosis (TB) severity is understudied. We investigated the effect of malnutrition on radiographic findings and mycobacterial burden. METHODS: Subjects included newly diagnosed, smear-positive, culture-confirmed, pulmonary TB cases enrolled in the Regional Prospective Observational Research for TB (RePORT) cohort. Multivariate regression models were used to evaluate the relationship at start of treatment between body mass index (BMI) and chest radiograph (CXR) findings of cavitation and percentage of lung affected and mycobacterial growth indicator tube (MGIT) time to positive (TTP). Severe malnutrition was defined as BMI<16 kg/m2, moderate malnutrition as 16-18.4kg/m2, and "normal"/overweight as ≥18.5 kg/m2. RESULTS: Of 173 TB cases with chest x-ray data, 131 (76%) were male. The median age was 45 years (range 16-82); 42 (24%) had severe malnutrition and 58 (34%) moderate malnutrition. Median percentage of lung affected was 32% (range 0-95), and 132 (76%) had cavitation. Individuals with severe malnutrition had, on average, 11.1% [95% CI: 4.0-13.3] more lung affected, compared to those with normal BMI, controlling for diabetes and cavitation. In multivariable analyses, cases with severe malnutrition had a 4.6-fold [95% CI, 1.5-14.1] increased odds of cavitation compared to those with normal BMI, controlling for smoking. Median MGIT TTP was 194.5 hours. Neither severe (aRR 0.99; 95% CI, 0.9-1.2) nor moderate (aRR 0.97; 95% CI, 0.8-1.1) malnutrition was associated with MGIT TTP. CONCLUSION: We found that malnutrition was associated with increased extent of disease and cavitation on CXR. These findings may reflect the immunomodulatory effect of malnutrition on pulmonary pathology.

Assessment of global DNA methylation in children with tuberculosis disease.

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.

Unforeseeable presentation of Chryseobacterium indologenes infection in a paediatric patient.

BACKGROUND: We report for the first time a case of community acquired Chryseobacterium indologenes soft tissue infection in an immunocompetent patient. CASE PRESENTATION: A 11 year female child, from South-Asia of Indian origin presented with fever, pain and swelling in right leg for 3 days with no significant past history. Incision and drainage was done and pus was sent for culture and sensitivity. Radiological investigation showed subtle irregular soft tissue density. Pus culture grew multidrug resistant C. indologenes. CONCLUSION: Though of low pathogenicity, our case emphasises its unpredictable nature and the need to determine minimum inhibitory concentration breakpoints for therapy.