Correlation of Cyclic Threshold Values Generated by GeneXpert Ultra MTB/RIF and Fluorescence Microscopy to Predict Mycobacterial Burden in Suspected Cases of Pulmonary Tuberculosis.

BACKGROUND: Smear microscopy for acid-fast bacilli visualization is important to assess the infectivity rate in patients with pulmonary tuberculosis (PTB), but it has limited sensitivity; hence, it is important to find an alternative strategy. The aim of our study was to compare the fluorescence microscopy grading by Auramine O phenol staining technique of respiratory samples with the cyclic threshold (Ct) values of GeneXpert Ultra (Mycobacterium tuberculosis/rifampicin [MTB/RIF]) and assess the diagnostic efficacy of GeneXpert Ultra (MTB/RIF) compared to microscopy in suspected cases of PTB. METHODS: The study was conducted in the Mycobacteriology Laboratory, Department of Microbiology, in Kasturba Hospital, Manipal. The study was a prospective, single-centered, cross-sectional study. Four hundred and fifty-two respiratory samples were included in the study. An optimal Ct cutoff value for ruling smear-positivity and smear-negativity and the mean Ct cutoff value were calculated. Clinical and radiological data from the requisition forms were assessed. IBM SPSS statistics software version 22 was used. The correlation between GeneXpert Ultra (MTB/RIF) Ct values and smear status was calculated by polychoric correlation. The extended McNemar's test was used to find the association between the variables. RESULTS: GeneXpert Ultra (MTB/RIF) yielded a higher positivity rate of 22.2% compared to smear microscopy 17.2%. Ct value and smear grading yielded a positive correlation (P = 0.8681; P < 0.05). GeneXpert Ultra (MTB/RIF) yielded nontuberculous mycobacteria in five undetected cases and speciated as Mycobacterium abscessus complex. CONCLUSIONS: Our study confirms the GeneXpert Ultra (MTB/RIF) Ct value levels as a predictor of smear positivity.

Tuberculosis screening for pediatric household contacts in India: Time to adapt newer strategies under the National TB Elimination Programme!

INTRODUCTION: The study aimed to evaluate the effectiveness of screening pediatric household contacts (under the age of 15 years) for tuberculosis (TB) in India through verbal screening, tuberculin skin testing, and chest radiography at intervals of 0, 3, 6, 9, and 12 months. The study also aimed to determine the proportion of contacts who tested positive for TB and to describe the challenges in implementing regular follow-up. Current National TB Elimination Programme (NTEP) guidelines only require verbal screening for contacts under 6 years old at TB treatment initiation. The study aimed to fill this knowledge gap and provide valuable insights for improving TB screening in pediatric household contacts in India. METHODS: The study was conducted in two districts of Karnataka, India from 2021 to 2022, and utilized a cohort study design to enroll contacts of index tuberculosis (TB) cases diagnosed under the National TB Elimination Programme (NTEP). Participants were followed up at regular intervals for one year to evaluate the effectiveness of TB screening in pediatric household contacts. RESULTS: In this study, 686 pediatric household contacts were enrolled and screened for tuberculosis (TB) using verbal symptom screening, tuberculin skin testing (TST), and chest radiography. Projected figures estimated that 0.8%, 42%, and 4% of contacts would test positive for symptomatic screening, TST, and chest radiography, respectively. TB cases were detected in 2.91% (1.84-4.38) of contacts, with females above 6 years of age having a 22% higher risk of contracting the infection than males above 6 to < 15 years. However, not all cases were subjected to TST and chest radiography. The primary reason for not investigating child contact for TB was their reported healthy or asymptomatic status. CONCLUSION: The implementation of regular screening intervals for tuberculin skin test (TST) and chest radiography, along with verbal screening, among pediatric household contacts under the age of 15 years seems to be beneficial for the National TB Elimination Programme (NTEP), despite the challenges faced during implementation. Innovative strategies should be explored by NTEP to ensure effective implementation.

Lineage classification and antitubercular drug resistance surveillance of Mycobacterium tuberculosis by whole-genome sequencing in Southern India.

Whole-genome sequencing has created a revolution in tuberculosis management by providing a comprehensive picture of the various genetic polymorphisms with unprecedented accuracy. Studies mapping genomic heterogeneity in clinical isolates of Mycobacterium tuberculosis using a whole-genome sequencing approach from high tuberculosis burden countries are underrepresented. We report whole-genome sequencing results of 242 clinical isolates of culture-confirmed M. tuberculosis isolates from tuberculosis patients referred to a tertiary care hospital in Southern India. Phylogenetic analysis revealed that the isolates in our study belonged to five different lineages, with Indo-Oceanic (lineage 1, n = 122) and East-African Indian (lineage 3, n = 80) being the most prevalent. We report several mutations in genes conferring resistance to first and second line antitubercular drugs including the genes rpoB, katG, ahpC, inhA, fabG1, embB, pncA, rpsL, rrs, and gyrA. The majority of these mutations were identified in relatively high proportions in lineage 1. Our study highlights the utility of whole-genome sequencing as a potential supplemental tool to the existing genotypic and phenotypic methods, in providing expedited comprehensive surveillance of mutations that may be associated with antitubercular drug resistance as well as lineage characterization of M. tuberculosis isolates. Further larger-scale whole-genome datasets with linked minimum inhibition concentration testing are imperative for resolving the discrepancies between whole-genome sequencing and phenotypic drug sensitivity testing results and quantifying the level of the resistance associated with the mutations for optimization of antitubercular drug and precise dose selection in clinics. IMPORTANCE Studies mapping genetic heterogeneity of clinical isolates of M. tuberculosis for determining their strain lineage and drug resistance by whole-genome sequencing are limited in high tuberculosis burden settings. We carried out whole-genome sequencing of 242 M. tuberculosis isolates from drug-sensitive and drug-resistant tuberculosis patients, identified and collected as part of the TB Portals Program, to have a comprehensive insight into the genetic diversity of M. tuberculosis in Southern India. We report several genetic variations in M. tuberculosis that may confer resistance to antitubercular drugs. Further wide-scale efforts are required to fully characterize M. tuberculosis genetic diversity at a population level in high tuberculosis burden settings for providing precise tuberculosis treatment.

Crystal violet decolorization assay: a simplified colorimetric test for the rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates.

The increased prevalence of multi-drug resistant M. tuberculosis is quite possibly the direst and most difficult task for the early diagnosis and treatment. A rapid, reliable, and inexpensive diagnostic method is the need of the hour. The current study on crystal violet decolorization assay explores the possibility to develop a rapid and simple detection method to detect multi-drug-resistant tuberculosis isolates by comparing the results with the traditional liquid culture drug susceptibility testing method based on their sensitivity, specificity, positive predictive value, and negative predictive value. 70 isolates were used for the study and were detected as multi-drug resistant, mono drug-resistant, and sensitive by using crystal violet decolourization assay and further compared with the results of DST and using H37Rv as the standard control strain. The sensitivity, specificity, positive predictive value, and negative predictive value of crystal violet decolorization assay (Rifampicin: 100%, 94.60%, 100% and 82.40%; isoniazid: 100%, 94.10%, 100%, 86.40%) are calculated and the percentage were compared with the conventional liquid culture drug susceptibility testing for M. tuberculosis using rifampicin and isoniazid. Crystal violet decolourization assay is rapid, reproducible, and doesn't require any highly experienced personal or sophisticated laboratory instruments for interpretation. This assay is found to be nearly as reliable as conventional liquid culture drug susceptibility testing and may thus be of great help in phenotypic confirmation of multi-drug resistant tuberculosis by providing results more rapidly.

Etiology and Clinical Profile of Patients with a Tree-in-bud Appearance on High-resolution Computed Tomogram of the Thorax.

We read with great interest the article "the deadly duo of hypertension and diabetes in India: further affirmation from a new epidemiological study" by Metri et al.1 They rightly pointed out that the prevalence of hypertension in Indian patients with type 2 diabetes patients is high and therefore early screening and management of hypertension should be included in the treatment of patients with type 2 diabetes. We wish to share our study findings on the prevalence of hypertension in newly onset diabetes mellitus (DM). We find that the prevalence of hypertension in all males and females with DM was 44.59, 44.34, and 45.16%, respectively.2.

Tubercular lymphadenitis in the 21st century: A 5-Year single-center retrospective study from South India.

Background: Tubercular lymphadenitis (TBLN) remains the most frequent manifestation for extrapulmonary TB despite advancements in diagnostics and management over the years. Our study intends to explore five-year trend of TBLN in a tertiary care centre from south India, and aims to study clinico-demographic and diagnostic factors in the management of TBLN. Methods: All the adult patients (≥18 years) diagnosed and confirmed for TB lymphadenitis between January 2015 to December 2019 were retrospectively evaluated. Demographic factors, clinical manifestations, and different diagnostic approaches used in the management of TBLN were analysed using SPSS ver. 16. Results: A total of 164 patients with confirmed TBLN were included. Patients aged 18-45 years were the most affected (63.41%) with female dominancy. The most affected lymph nodes were cervical lymph nodes (84.1%) presenting with single palpable enlarged lymph node (80.5%). Majority (78.7%) of the lymph nodes were non-matted and 68.9% of enlarged lymph nodes were >3cm size. Excisional biopsy was performed for the majority of the patients 99 (60.4%) and 60.4% of the cases were managed with a combination of surgical excision and anti-tubercular treatment (ATT). Conclusions: The declining trend of TBLN observed in this study highlights the outcome of good public health policies; however, young females and high-risk groups like HIV infected or AIDS (affected more in the study) demand further attention. Overall, the advanced diagnostic tools along with surgical management and ATT can lead us to earlier diagnosis and successful treatment outcomes.

Challenges Perceived by Health Care Providers for Implementation of Contact Screening and Isoniazid Chemoprophylaxis in Karnataka, India.

Background: In India, challenges in pediatric TB contact screening and chemoprophylaxis initiation are still underexplored. Elucidating these challenges will help in better implementation of the programme at the grass-roots level thereby helping in early detection of pediatric cases and timely initiation of preventive therapy. This study aimed at exploring the challenges faced by the health care provider in contact screening and chemoprophylaxis initiation implementation of the pediatric household contacts. Methods: A qualitative study was conducted in the districts of Bengaluru and Udupi and in-depth interviews of key participants were adopted to explore the challenges. Qualitative data analysis was done after developing transcripts by generating themes and codes. Results: The key challenges were identified as stigma towards the disease, migrant patients with changing address, difficulty in sample collection, anxiety among parents due to long duration of the prophylactic treatment and adherence to IPT is not well documented, inadequate transportation from rural areas, and the ongoing COVID-19 pandemic. Conclusions: It is important for the National TB programme to address these challenges efficiently and effectively. Innovative solutions, feasible engagements, and massive efforts are to be taken by the programme to improve contact screening and isoniazid chemoprophylaxis implementation.

Detection of Biofilm Production and Its Impact on Antibiotic Resistance Profile of Bacterial Isolates from Chronic Wound Infections.

BACKGROUND: Microorganisms are known to be involved in the formation of biofilm. These biofilms are often seen in chronic wound infections, surgical site infections, implants etc., These are capable of causing recalcitrant infections and most of them are also known to possess high antibiotic resistance. OBJECTIVES: This study was conducted to detect the biofilm formation in bacterial isolates from chronic wound infections. MATERIALS AND METHODS: In the present study, ninety two isolates from chronic wound infections were identified by MALDI-TOF-MS (bioMerieux) and VITEK-2-MS (bioMerieux). These isolates were further screened for biofilm formation by three methods i. e., Tissue Culture Plate method (TCP), Tube Method (TM) and Congo Red Agar (CRA) method. Impact of biofilm production was correlated with the antibiotic resistant pattern. STATISTICAL ANALYSIS: Statistical analysis was done for all three methods considering TCP as Gold Standard and parameters like senitivity and specificity of TM i.e. 47.2 and 100% respectively. RESULTS: Out of 92 isolates, biofilm formation was seen in 72 isolates (78.2%) by TCP method. 64 isolates were strong biofilm producers, 8 isolates were moderate biofilm producers and 20 isolates were nonbiofilm producing. High prevalence of biofilm formation was seen in nonhealing ulcers infected with Staphylococcus aureus followed by Klebsiella pneumoniae. CONCLUSION: Among three screening methods used for detection of biofilm production, TCP method is considered to be a standard and most reliable for screening of biofilm formation in comparison to TM and CRA.

Deep learning, computer-aided radiography reading for tuberculosis: a diagnostic accuracy study from a tertiary hospital in India.

In general, chest radiographs (CXR) have high sensitivity and moderate specificity for active pulmonary tuberculosis (PTB) screening when interpreted by human readers. However, they are challenging to scale due to hardware costs and the dearth of professionals available to interpret CXR in low-resource, high PTB burden settings. Recently, several computer-aided detection (CAD) programs have been developed to facilitate automated CXR interpretation. We conducted a retrospective case-control study to assess the diagnostic accuracy of a CAD software (qXR, Qure.ai, Mumbai, India) using microbiologically-confirmed PTB as the reference standard. To assess overall accuracy of qXR, receiver operating characteristic (ROC) analysis was used to determine the area under the curve (AUC), along with 95% confidence intervals (CI). Kappa coefficients, and associated 95% CI, were used to investigate inter-rater reliability of the radiologists for detection of specific chest abnormalities. In total, 317 cases and 612 controls were included in the analysis. The AUC for qXR for the detection of microbiologically-confirmed PTB was 0.81 (95% CI: 0.78, 0.84). Using the threshold that maximized sensitivity and specificity of qXR simultaneously, the software achieved a sensitivity and specificity of 71% (95% CI: 66%, 76%) and 80% (95% CI: 77%, 83%), respectively. The sensitivity and specificity of radiologists for the detection of microbiologically-confirmed PTB was 56% (95% CI: 50%, 62%) and 80% (95% CI: 77%, 83%), respectively. For detection of key PTB-related abnormalities 'pleural effusion' and 'cavity', qXR achieved an AUC of 0.94 (95% CI: 0.92, 0.96) and 0.84 (95% CI: 0.82, 0.87), respectively. For the other abnormalities, the AUC ranged from 0.75 (95% CI: 0.70, 0.80) to 0.94 (95% CI: 0.91, 0.96). The controls had a high prevalence of other lung diseases which can cause radiological manifestations similar to PTB (e.g., 26% had pneumonia, 15% had lung malignancy, etc.). In a tertiary hospital in India, qXR demonstrated moderate sensitivity and specificity for the detection of PTB. There is likely a larger role for CAD software as a triage test for PTB at the primary care level in settings where access to radiologists in limited. Larger prospective studies that can better assess heterogeneity in important subgroups are needed.

Whole-genome sequencing reveals genetic signature of bedaquiline resistance in a clinical isolate of Mycobacterium tuberculosis.

OBJECTIVES: The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis strains has opened up new challenges for tuberculosis (TB) control in India. This study examined molecular markers of resistance to bedaquiline, a new antituberculous drug with the potential to dramatically improve MDR-TB treatment outcomes and to reduce mortality. METHODS: A clinical M. tuberculosis isolate with a MDR-TB profile was subjected to whole-genome sequencing using an Illumina NextSeq500 platform, followed by genome-wide sequence analysis. RESULTS: A mutation in the Rv0678 coding region associated with in vitro bedaquiline resistance was identified. The strain represented the Delhi/CAS lineage. CONCLUSIONS: This first report of a potentially bedaquiline-resistant M. tuberculosis strain in India highlights the role of genome-wide sequence analysis of isolates from TB cases with a history of treatment in countries with a high burden of MDR-TB and XDR-TB.

Antimycobacterial susceptibility evaluation of rifampicin and isoniazid benz-hydrazone in biodegradable polymeric nanoparticles against Mycobacterium tuberculosis H37Rv strain.

INTRODUCTION: Tuberculosis (TB) is the single largest infectious disease which requires a prolonged treatment regime with multiple drugs. The present treatment for TB includes frequent administration of a combination of four drugs for a duration of 6 months. This leads to patient's noncompliance, in addition to developing drug-resistant strains which makes treatment more difficult. The formulation of drugs with biodegradable polymeric nanoparticles (NPs) promises to overcome this problem. MATERIALS AND METHODS: In this study, we focus on two important drugs used for TB treatment - rifampicin (RIF) and isoniazid (INH) - and report a detailed study of RIF-loaded poly lactic-co-glycolic acid (PLGA) NPs and INH modified as INH benz-hydrazone (IH2) which gives the same therapeutic effect as INH but is more stable and enhances the drug loading in PLGA NPs by 15-fold compared to INH. The optimized formulation was characterized using particle size analyzer, scanning electron microscopy and transmission electron microscopy. The drug release from NPs and stability of drug were tested in different pH conditions. RESULTS: It was found that RIF and IH2 loaded in NPs release in a slow and sustained manner over a period of 1 month and they are more stable in NPs formulation compared to the free form. RIF- and IH2-loaded NPs were tested for antimicrobial susceptibility against Mycobacterium tuberculosis H37Rv strain. RIF loaded in PLGA NPs consistently inhibited the growth at 70% of the minimum inhibitory concentration (MIC) of pure RIF (MIC level 1 µg/mL), and pure IH2 and IH2-loaded NPs showed inhibition at MIC equivalent to the MIC of INH (0.1 µg/mL). CONCLUSION: These results show that NP formulations will improve the efficacy of drug delivery for TB treatment.

Genetic diversity of Mycobacterium tuberculosis in south coastal Karnataka, India, using spoligotyping.

Background & objectives: Despite high occurrence of tuberculosis in India very little information is available about the genetic diversity of Mycobacterium tuberculosis isolates prevailing in coastal Karnataka, India. Thus, the present study was undertaken to explore the genetic biodiversity of M. tuberculosis isolates prevailing in south coastal region of Karnataka (Udupi District), India. Methods: A total of 111 Mycobacterial isolates were cultured in Lowenstein Jensen (LJ) medium and after obtaining growth, DNA was extracted and spoligotyping was performed. SITVIT WEB database was used to locate families of spoligotypes. Results: On analyzing the hybridization results of all 111 isolates on SITVIT WEB database 57 (51.35%) isolates were clustered into 11 Spoligotype International Types (SIT). The largest cluster of 14 (12.61%) isolates was SIT-48 (EAI1-SOM), followed by SIT-1942 (CAS1-Delhi) with 11 isolates (9.9%) and SIT-11 with seven (6.30%). Moreover, 23 isolates (20.72%) had unique spoligotypes and 31 (27.92%) were orphans. Spotclust analysis revealed that majority (67%) of orphan isolates were variants of CAS (37%) and EAI-5 (34%). Interpretation & conclusions: The present study revealed high biodiversity among the circulating isolates of M. tuberculosis in this region with the presence of mixed genotypes earlier reported from north and south India along with certain new genotypes with unique SITs. The study highlights the need for further longitudinal studies to explore the genetic diversity and to understand the transmission dynamics of prevailing isolates.

Genotypic detection of fluoroquinolone resistance in drug-resistant Mycobacterium tuberculosis at a tertiary care centre in south Coastal Karnataka, India.

OBJECTIVES: This study aimed to characterise mutations associated with fluoroquinolone resistance in drug-resistant Mycobacterium tuberculosis clinical isolates at a tertiary care centre in south Coastal Karnataka, India. METHODS: DNA from 50 M. tuberculosis clinical isolates was extracted and the gyrA and gyrB genes were amplified. Purified amplicons of gyrA and gyrB were sequenced and extended-sequencing PCR products were analysed. Analysis of mutations in gyrA and gyrB was done using the MUBII-TB-DB database. Statistical analysis was performed using SPSS v.22 and data were compared by χ2 test. A P-value of <0.05 was considered statistically significant. RESULTS: Mutations conferring resistance to fluoroquinolones were observed in 9 isolates (18%). The gyrA A281G (D94G) mutation was observed in 3 isolates (6%), whereas mutations G280T (D94Y) and A281C (D94A) were each observed in 1 isolate (2%). Mutation G1498A (D500N) in gyrB alone was observed in 2 isolates (4%). Two isolates (4%) had mutations both in gyrA and gyrB; gyrA mutation T271C (S91P) was observed in one isolate, whereas the other isolate had gyrA C269G (A90G), but both isolates had a common G1498A (D500N) gyrB mutation. G284C mutation conferring S95T polymorphism (not associated with fluoroquinolone resistance) was observed in 39/50 isolates (78%). CONCLUSION: gyrA mutations at codons 94, 91 and 90 and gyrB mutation G1498A (D500N) were the most common mutations associated with fluoroquinolone resistance in clinical isolates in this study. Future studies including a larger number of samples are desirable to fully explore the true extent of fluoroquinolone resistance and mutations associated with them.

Rapid detection of multidrug resistant tuberculosis in respiratory specimens at a tertiary care centre in south coastal Karnataka using Genotype MTBDR plus assay.

BACKGROUND AND OBJECTIVES: Despite high prevalence of MDR-TB in India very limited information about MDR-TB and mutation patterns in rpoB, inhA and katG genes among MDR-isolates of Mycobacterium tuberculosis in south coastal Karnataka region is available; thus present study is an attempt to explore the extent of MDR-TB and mutation patterns prevalent among clinical isolates in this region using GenoType MTBDR plus assay. MATERIALS AND METHODS: A total of 256 sputum samples from Pulmonary TB patients suspected of MDR-TB were tested by GenoType MTBDR plus as per manufacturer's guidelines for detection of mutations conferring resistance to rifampicin and isoniazid. The results of GenoType MTBDR plus were recorded and analysed using SPSS version 22. For all analyses, a p value <0.05 was considered statistically significant. RESULTS: Fifty (19.53%) isolates were found MDR, 32 (12.50%) isolates were found mono-resistant to isoniazid and 15 (5.86%) isolates were found mono-resistant to rifampicin. Eleven isolates (4.3%) were found NTM. Mutation in codon S531L, S315T1 and C15T were most common in rpoB, katG and inhA genes respectively. Unknown mutations were found in 50.77% (33/65), 3.66% (3/82) and 26.83% (22/82) isolates for rpoB, katG and inhA genes respectively. Hetero-resistance in MDR, rifampicin monoresistant and isoniazid monoresistant isolates was found to be 26% (13/50), 20% (3/15) and 34.37% (11/32) respectively. CONCLUSION: Mutation in codon S531L, S315T1 and C15T were most common mutations associated with MDR-TB. Further high number of isolates showed mutations in unknown regions and hetero-resistance thus more elaborate studies based on sequencing are desirable in this region.

Mycobacterium fortuitum Infection at Umbilical Hernioplasty Site.

Non Tuberculous Mycobacteria (NTM) are a group of rapidly growing mycobacteria and are generally considered to be of low virulence. Of late, there has been an increase in incidence of infections due to these organisms. Among them, Mycobacterium fortuitum, M. chelonae and M. abscessus are the common species which have been identified. Though they are occasionally implicated in pulmonary infections, NTM are very commonly associated with cutaneous infections, especially surgical site infections. Identification of NTM infection at such sites should be suspected when there is delayed healing of the wound. Histopathological Examination (HPE) of the wound site may reveal a classical picture of granulomas, epithelioid cells and giant cells which may lead to a suspicion of tuberculosis. It is important to perform mycobacterial culture and sensitivity testing of the wound tissue as this helps to differentiate tuberculous and non tuberculous infections. Here, we present a case of a patient who underwent mesh hernioplasty for umbilical hernia and was diagnosed with M. fortuitum infection at the site of umbilical hernioplasty.

Role of multiplex polymerase chain reaction in diagnosing tubercular meningitis.

Tuberculous meningitis (TBM) is one of the most serious manifestations of extrapulmonary tuberculosis. Timely and accurate diagnosis provides a favorable prognosis in patients with TBM. The study evaluated the use of multiplex polymerase chain reaction (PCR) in the diagnosis of TBM. A study was conducted on 74 patients clinically suspected with TBM. The cerebrospinal fluid (CSF) specimens were processed for smear microscopy, middle brook 7H9 culture, and multiplex PCR using primers directed against IS6110 gene and 38 kD protein for detection of Mycobacterium tuberculosis. The results were analyzed to assess the role of multiplex PCR in the diagnosis of TBM. A total of 26 (35.1%) patients were diagnosed with TBM. Microscopy was negative in all while culture was positive in two cases only. Comparing with clinical diagnosis and CSF adenosine deaminase levels of ≥10 U/L, multiplex PCR showed sensitivity, specificity, positive predictive value, and negative predictive value of 71.4%, 89.6%, 83.3%, and 81.2%, respectively, in the diagnosis of TBM.

Role of real-time PCR for detection of tuberculosis and drug resistance directly from clinical samples.

BACKGROUND: Only a few studies done earlier in India reveal the utility of real-time PCR in detecting drug resistance in cases of pulmonary tuberculosis. OBJECTIVES: The study was carried out to standardise real-time PCR (Quantitative real-time PCR, qPCR) targeting 16s RNA for the rapid detection of tuberculosis and its drug resistance from suspected TB patients. MATERIALS AND METHODS: Sputum samples from 100 clinically suspected tuberculosis patients, after processing were subjected to microscopy, MGIT culture and qPCR. qPCR targeted 16sRNA for detecting Mycobacterium tuberculosis complex, KatG and rpoB genes for detection of resistance to isoniazid and rifampicin respectively. 1% proportionate method and Line probe assay (Hain Lifesciences, Nehren, Germany) were used to confirm the MDR isolates. RESULTS: The study showed positivity of microscopy, culture and qPCR for M. tuberculosis as 37%, 44% and 46% respectively. Sensitivity of 100% and specificity of 96.5% in the detection of M. tuberculosis was observed for qPCR in comparison to culture. MDRTB was detected in 14 cases whereas monoresistance to rifampicin and isoniazid was detected in 1 and 3 samples respectively. CONCLUSION: Real-time PCR targeting 16sRNA, KatG and rpoB is a sensitive, specific, rapid and reliable technique to detect pulmonary tuberculosis and its MDR status directly from the sputum samples.

Study of drug resistance in pulmonary tuberculosis cases in south coastal Karnataka.

The present cross-sectional study was conducted for the first time from the Udupi district of coastal Karnataka to know the prevalence of drug resistance and comparative analysis of MDR and non-MDR cases of pulmonary tuberculosis. Details of 862 smear positive cases of pulmonary tuberculosis with age ⩾15 years from 12 designated microscopy centres of the Udupi district were studied. Initially 2 sputum samples trailed by one follow-up sample were collected from each patient and processed for culture and drug sensitivity on the Lowenstein-Jensen medium. A total resistance of 33.4% was observed that includes the mono-resistance of 22.5%, multidrug resistance (MDR) of 6.3% and extensive drug resistance (XDR) of 0.3%. Significant odds ratio (OR) was observed in category 2 cases (OR 3.9) for the development of MDR tuberculosis. A significant statistical association was observed using Fisher's exact test while comparing mortality rate (19.3% vs. 1.8%), treatment failure (8.8% vs. 3.8%) and cure rate (68.4% vs. 85.4%) between MDR and non-MDR cases (p<0.001). Category 2 patients are important risk factors for the development of MDR in pulmonary tuberculosis. Due to high mortality and low cure rate in MDR cases it is imperative to know the drug sensitivity report before institution of anti-tubercular treatment.