Mutation detection and minimum inhibitory concentration determination against linezolid and clofazimine in confirmed XDR-TB clinical isolates.

BACKGROUND: The emergence of multidrug-resistant tuberculosis (MDR-TB) has complicated the situation due to the decline in potency of second-line anti-tubercular drugs. This limits the treatment option for extensively drug-resistant tuberculosis (XDR-TB). The aim of this study was to determine and compare the minimum inhibitory concentration (MIC) by agar dilution and resazurin microtiter assay (REMA) along with the detection of mutations against linezolid and clofazimine in confirmed XDR-TB clinical isolates. RESULTS: A total of 169 isolates were found positive for Mycobacterium tuberculosis complex (MTBC). The MIC was determined by agar dilution and REMA methods. The isolates which showed non-susceptibility were further subjected to mutation detection by targeting rplC gene (linezolid) and Rv0678 gene (clofazimine). The MIC for linezolid ranged from 0.125 µg/ml to > 2 µg/ml and for clofazimine from 0.25 µg/ml to > 4 µg/ml. The MIC50 and MIC90 for linezolid were 0.5 µg/ml and 1 µg/ml respectively while for clofazimine both were 1 µg/ml. The essential and categorical agreement for linezolid was 97.63% and 95.26% and for clofazimine, both were 100%. The sequencing result of the rplC gene revealed a point mutation at position 460 bp, where thymine (T) was substituted for cytosine (C) while seven mutations were noted between 46 to 220 bp in Rv0678 gene. CONCLUSION: REMA method has been found to be more suitable in comparison to the agar dilution method due to lesser turnaround time. Mutations in rplC and Rv0678 genes were reasons for drug resistance against linezolid and clofazimine respectively.

Direct detection of resistance to fluoroquinolones/SLIDs in sputum specimen by GenoType MTBDRsl v.2.0 assay A study from Eastern Uttar Pradesh, India.

BACKGROUND: According to World Health Organization (WHO), drug-resistant tuberculosis (DR-TB) is a major contributor to antimicrobial resistance globally and continues to be a public health threat. Annually, about half a million people fall ill with DR-TB globally. The gradual increase in resistance to fluoroquinolones (FQs) and second-line injectable drugs (SLIDs), poses a serious threat to effective TB control and adequate patient management. Therefore, WHO suggests the use of GenoType MTBDRsl v.2.0 assay for detection of multiple mutations associated with FQs and SLIDs. Hence, the study was conducted to determine the prevalence of resistance to FQs and SLIDs by comparing direct GenoType MTBDRsl v.2.0 assay with phenotypic drug susceptibility testing (DST). METHODS: The study was conducted on 1320 smear positive sputum samples from a total of 2536 RR-TB, confirmed by GeneXpert MTB/RIF. The smear positive specimens were decontaminated, and DNA extraction was performed. Furthermore, the extracted DNA was used for GenoType MTBDRsl v.2.0 assay. While 20% of the decontaminated specimens were inoculated in Mycobacterium growth indicator tube (MGIT) for drug susceptibility testing (DST). RESULTS: Out of 1320 smear positive sputum samples, 1178 were identified as Mycobacterium tuberculosis complex (MTBC) and remaining were negative by GenoType MTBDRsl v.2.0 assay. Of the 1178 MTBC positive, 26.6% were sensitive to both FQs and SLIDs, whereas 57.3% were only FQs resistant and 15.9% were resistant to both FQs and SLIDs. Further DST of 225 isolates by liquid culture showed that 17% were sensitive to both FQs and SLIDs, 61.3% were only FQs resistant and 21.3% were resistant to both. The specificity for FQs and SLIDs was 92.31% and 100% whereas sensitivity was 100% respectively by GenoType MTBDRsl v.2.0 assay in direct sputum samples. CONCLUSIONS: Our study clearly suggests that GenoType MTBDRsl v.2.0 assay is a reliable test for the rapid detection of resistance to second-line drugs after confirmation by GeneXpert MTB/RIF assay for RR-TB. Though, high rate FQ (ofloxacin) resistance was seen in our setting, moxifloxacin could be used as treatment option owing to very low resistance.

Detection of clinically important non tuberculous mycobacteria (NTM) from pulmonary samples through one-step multiplex PCR assay.

BACKGROUND: The burden of non-tuberculous mycobacterial (NTM) disease is increasing worldwide but still its diagnosis is delayed and it is mistaken as multidrug-resistant tuberculosis (MDR-TB).The present study was performed to develop a multiplex PCR assay for detection and identification of clinically most common NTM to the species level from pulmonary samples. RESULTS: Out of 50 isolates, 26 were identified as Mycobacterium kansasii (MK), 20 were identified as Mycobacterium abscessus (MA) and 4 were identified as Mycobacterium avium complex (MAC) through multiplex PCR and further confirmed by sequencing. CONCLUSION: Our study showed that multiplex PCR assay is a simple, convenient, and reliable technique for detection and differential identification of major NTM species.

Detection of mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains inhibiting wild type probe hybridization in the MTBDR plus assay by DNA sequencing directly from clinical specimens.

BACKGROUND: The potential of genetic testing for rapid and accurate diagnosis of drug-resistant Mycobacterium tuberculosis strains is vital for efficient treatment and reduction in dissemination. MTBDR plus assays rapidly detect mutations related to drug resistance and wild type sequences allied with susceptibility. Although these methods are promising, the examination of molecular level performance is essential for improved assay result interpretation and continued diagnostic development. Therefore this study aimed to determine novel mutations that were inhibiting wild type probe hybridization in the Line probe assay by DNA sequencing. Using data collected from Line Probe assay (GenoType MTBDRplus assay) the contribution of absent wild type probe hybridization to the detection of rifampicin resistance was assessed via comparison to a reference standard method i.e. DNA sequencing. RESULTS: Sequence analysis of the rpoB gene of 47 MTB resistant strains from clinical specimens showed that 37 had a single mutation, 9 had double mutations and one had triple mutations in the ropB gene. CONCLUSIONS: The absence of wild type probe hybridization without mutation probe hybridization was mainly the result of the failure of mutation probe hybridization and the result of the novel or rare mutations. Additional probes are necessary to be included in the Line probe assay to improve the detection of rifampicin-resistant Mycobacterium tuberculosis strains.

Detection of Beijing strains of MDR M. tuberculosis and their association with drug resistance mutations in katG, rpoB, and embB genes.

BACKGROUND: Molecular epidemiological studies of Mycobacterium tuberculosis (MTB) are the core of current research to find out the association of the M. tuberculosis genotypes with its outbreak and transmission. The high prevalence of the Beijing genotype strain among multidrug resistance (MDR) TB has already been reported in various studies around India. The overall objective of this study was to detect the prevalence of Beijing genotype strains of MDR M. tuberculosis and their association with the clinical characteristics of TB patients. METHODS: In this study 381 M. tuberculosis clinical isolates were obtained from sputum samples from 2008 to 2014. The multiplex-PCR and Spoligotyping (n = 131) methods were used to investigate the prevalence of the Beijing genotype strain by targeting the Rv2820 gene and their association with drug resistance and clinical characteristics of TB patients. The drug susceptibility testing of first-line anti-TB drugs was performed by using the proportion method and MGIT960. A collection of isolates having Beijing and non-Beijing strains were also characterized to see if Beijing genotype strains had a higher rate of mutations at codons 516, 526 and 531 of the 81-bp region of the rpoB gene, codon 315 of the katG gene, and codon 306 of the embB gene. RESULTS: The sensitivities and specificities of multiplex-PCR assay compared to that of standard Spoligotyping was detected to be 100%. Further, we observe that the multi drug-resistance was significantly associated with Beijing genotype strains (p = 0.03) and a strong correlation between Beijing genotype strains and specific resistance mutations at the katG315, rpoB531, and embB306 codons (p = < 0.0001, < 0.0001 & 0.0014 respectively) was also found. CONCLUSIONS: This rapid, simple, and cost-effective multiplex PCR assay can effectively be used for monitoring the prevalence of Beijing genotype strains in low resource settings. Findings of this study may provide a scientific basis for the development of new diagnostic tools for detection and effective management of DR-TB in countries with a higher incidence rate of Beijing genotype strains.

Genotype analysis of ofloxacin-resistant multidrug-resistant Mycobacterium tuberculosis isolates in a multicentered study from India.

Background & objectives: Drug resistance surveillance offers useful information on trends of drug resistance and the efficacy of control measures. Studies and reports of drug-resistant mutations and phenotypic assays thus become important. This study was conducted to investigate the molecular characteristics of ofloxacin (OFX)-resistant, multidrug-resistant tuberculosis (MDR-TB) isolates from different geographical regions of India and their association with strains of different genotypes. Further, the nitrate reductase assay (NRA) was tested against Mycobacteria Growth Indicator Tube(MGIT) for the determination of OFX resistance as an alternative and cost-effective method. Methods: A total of 116 Mycobacterium tuberculosis isolates were used to assess the mutations in the gyrA, gyrB genes and resistance levels to OFX. Mutational analysis in gyrA and gyrB genes and genotype analysis of M. tuberculosis isolates was done by gene-specific polymerase chain reaction (PCR) followed by DNA sequencing and spoligotyping, respectively. Results: Three (6.25%), 12 (44.44%) and 12 (29.27%) MDR-TB isolates from western, northern and southern India, respectively, were found to be OFX-resistant MDR-TB isolates. OFX resistance was observed to be significantly higher in MDR-TB cases for all study regions. Beijing genotypes from northern India were observed to be associated with OFX-resistant MDR-TB cases (P <0.05). Among 35 (30.15%) phenotypically OFX-resistant isolates, 22 (62.86%) had mutations in the gyrA gene and two (5.71%) isolates had mutations in the gyrB gene. Interpretation & conclusions: These results caution against the PCR-based prediction of OFX resistance patterns and highlight the need for searching other genetic loci for the detection of mutations conferring resistance to OFX in M. tuberculosis. Our study also showed the usefulness of NRA as an alternative method to detect OFX resistance.

Mutation in MPT64 gene influencing diagnostic accuracy of SD Bioline assay (capilia).

BACKGROUND: Success of India's TB control program relies on rapid case detection, monitoring, care and treatment of drug resistance. Patients on multidrug resistance (MDR) treatment are monitored by follow up cultures. Discordant results (culture and smear positive while capilia negative) are usually declared negative Mycobacterium tuberculosis complex (MTBC). This study was designed to understand the possible causes of discordant results. METHODS: The capilia kit was evaluated to test its utility among 4737 follow up MDR patients enrolled during a period of 1 year. A total of 889 were liquid culture positive, 3375 were negative and 473 were contaminated. Of the 889 cultures positive, 829 were found positive by ZN smear, capilia test and MTBDR plus assay. The cultures which gave a positive result on Mycobacterium Growth Indicator Tube 960 (MGIT 960) and ZN smear but were negative on capilia test with no growth on Brain Heart Infusion agar (BHI) were included in this study. The conflicting results of capilia were compared with other molecular techniques; MTBDR plus assay and DNA sequence analysis of MPT64 gene. RESULTS: Out of 889 culture positive, 60 (6.7%) were found positive on liquid culture and ZN smear but were negative on capilia. Of these 60 cultures, 10 (16.7%) were found positive by both MTBDR plus assay and PCR. The sequencing analysis revealed that all of the capilia negative isolates had mutations within the MPT64 gene. CONCLUSION: Re-evaluation of culture positive but capilia negative isolates should be done before declaring them as Mycobacterium other than tuberculosis (MOTT) because such cases can act as chronic carriers of TB in the population which can lead to the rise of this lethal disease.

Rapid detection of drug-resistant Mycobacterium tuberculosis directly from clinical specimens using allele-specific polymerase chain reaction assay.

Background & objectives: Rapid detection of drug resistance in Mycobacterium tuberculosis (MTB) is essential for the efficient control of tuberculosis. Hence, in this study a nested-allele-specific (NAS) PCR, nested multiple allele-specific PCR (NMAS-PCR) and multiple allele-specific (MAS) PCR assays were evaluated that enabled detection of the most common mutations responsible for isoniazid (INH) and rifampicin (RIF) resistance in MTB isolates directly from clinical specimens. Methods: Six pairs of primers, mutated and wild type, were used for the six targets such as codon 516, 526 and 531 of rpoB, codon 315 of katG and C15-T substitution in the promoter region of mabA-inhA using allele-specific (AS) PCR assays (NAS-PCR, NMAS-PCR and MAS-PCR). The performance of AS PCR method was compared with phenotypic drug susceptibility testing (DST). Results: The usefulness of AS PCR assays was evaluated with 391 clinical specimens (251 Acid fast bacilli smear positive and MTB culture positive; 93 smear negative and MTB culture positive; 47 smear positive and MTB culture negative) and 344 MTB culture positive isolates. With culture-based phenotypic DST as a reference standard, the sensitivity and specificity of the NAS-PCR, NMAS-PCR and MAS-PCR assay for drug resistance-related genetic mutation detection were 98.6 and 97.8 per cent for INH, 97.5 and 97.9 per cent for RIF and 98.9 and 100 per cent for multidrug resistance (MDR). Interpretation & conclusions: The performance of AS PCR assays showed that those could be less expensive and technically executable methods for rapid detection of MDR-TB directly from clinical specimens.

Evaluation of Xpert MTB/RIF Assay for Diagnosis of Tuberculosis in Children.

Introduction: Childhood tuberculosis (TB) is now a global priority. With the advent of Xpert MTB/RIF, more TB cases in children are being reported. This study was undertaken to evaluate the performance of Xpert in diagnosis of pulmonary and extra-pulmonary TB in children. Methods: Specimens from 171 suspected TB cases in children aged <15 years were tested with Xpert, culture and smear microscopy in the Department of Microbiology, Institute of Medical Sciences, India. Results: The specimens included 106 gastric aspirates, 51 cerebrospinal fluids, 8 induced sputum and 6 lymph node aspirates. Xpert detected Mycobacterium tuberculosis in 19 cases (14 pulmonary and 5 extra-pulmonary), 7 of which were rifampicin-resistant. Sensitivity, specificity, positive predictive value and negative predictive value of Xpert compared with culture were 88.89, 98.04, 84.21 and 98.68%, respectively. The sensitivity was 100% in children aged 1-5 years and 6-10 years and in gastric aspirates. Conclusion: Xpert is an efficient diagnostic tool in childhood tuberculosis.

PknB remains an essential and a conserved target for drug development in susceptible and MDR strains of M. Tuberculosis.

BACKGROUND: The Mycobacterium tuberculosis (M.tb) protein kinase B (PknB) which is now proved to be essential for the growth and survival of M.tb, is a transmembrane protein with a potential to be a good drug target. However it is not known if this target remains conserved in otherwise resistant isolates from clinical origin. The present study describes the conservation analysis of sequences covering the inhibitor binding domain of PknB to assess if it remains conserved in susceptible and resistant clinical strains of mycobacteria picked from three different geographical areas of India. METHODS: A total of 116 isolates from North, South and West India were used in the study with a variable profile of their susceptibilities towards streptomycin, isoniazid, rifampicin, ethambutol and ofloxacin. Isolates were also spoligotyped in order to find if the conservation pattern of pknB gene remain consistent or differ with different spoligotypes. The impact of variation as found in the study was analyzed using Molecular dynamics simulations. RESULTS: The sequencing results with 115/116 isolates revealed the conserved nature of pknB sequences irrespective of their susceptibility status and spoligotypes. The only variation found was in one strains wherein pnkB sequence had G to A mutation at 664 position translating into a change of amino acid, Valine to Isoleucine. After analyzing the impact of this sequence variation using Molecular dynamics simulations, it was observed that the variation is causing no significant change in protein structure or the inhibitor binding. CONCLUSIONS: Hence, the study endorses that PknB is an ideal target for drug development and there is no pre-existing or induced resistance with respect to the sequences involved in inhibitor binding. Also if the mutation that we are reporting for the first time is found again in subsequent work, it should be checked with phenotypic profile before drawing the conclusion that it would affect the activity in any way. Bioinformatics analysis in our study says that it has no significant effect on the binding and hence the activity of the protein.

Differentiation of Mycobacterium tuberculosis complex from non-tubercular mycobacteria by nested multiplex PCR targeting IS6110, MTP40 and 32kD alpha antigen encoding gene fragments.

BACKGROUND: Control of the global burden of tuberculosis is obstructed due to lack of simple, rapid and cost effective diagnostic techniques that can be used in resource poor-settings. To facilitate the early diagnosis of TB directly from clinical specimens, we have standardized and validated the use of nested multiplex PCR, targeting gene fragments IS6110, MTP40 and 32kD α-antigen encoding genes specific for Mycobacterium tuberculosis complex and non-tubercular mycobacteria (NTM), in comparison to smear microscopy, solid culture and single step multiplex PCR. The results were evaluated in comparison to a composite reference standard (CRS) comprising of microbiological results (smear and culture), clinical, radiological and cytopathological findings, clinical treatment and response to anti-tubercular therapy. METHODS: The nested multiplex PCR (nMPCR) assay was evaluated to test its utility in 600 (535 pulmonary and 65 extra-pulmonary specimens) clinically suspected TB cases. All specimens were processed for smear, culture, single step multiplex PCR and nested multiplex PCR testing. RESULTS: Out of 535 screened pulmonary and 65 extra-pulmonary specimens, 329 (61.5%) and 19 (29.2%) cases were culture positive for M. tuberculosis. Based on CRS, 450 patients had "clinical TB" (definitive-TB, probable-TB and possible-TB). Remaining 150 were confirmed "non-TB" cases. For culture, the sensitivity was low, 79.3% for pulmonary and 54.3% for extra-pulmonary cases. The sensitivity and specificity results for nMPCR test were evaluated taken composite reference standard as a gold standard. The sensitivity of the nMPCR assay was 97.1% for pulmonary and 91.4% for extra-pulmonary TB cases with specificity of 100% and 93.3% respectively. CONCLUSION: Nested multiplex PCR using three gene primers is a rapid, reliable and highly sensitive and specific diagnostic technique for the detection and differentiation of M. tuberculosis complex from NTM genome and will be useful in diagnosing paucibacillary samples. Nested multiplex PCR assay was found to be better than single step multiplex PCR for assessing the diagnosis of TB.

Risk factors associated with fluoroquinolone-resistant enterococcal urinary tract infections in a tertiary care university hospital in north India.

BACKGROUND & OBJECTIVES: Fluoroquinolone resistance in both Gram-positive and Gram-negative bacteria has increased with the widespread use of fluoroquinolones. Fluoroquinolone resistance in Gram-negative bacilli has been widely studied, though staphylococci and enterococci are also notably resistant. Enterococci being the second most common cause of healthcare-associated urinary tract infections (UTIs) fluoroquinolones are often the drug of choice. This study was undertaken to assess the risk factors associated with fluoroquinolone-resistant enterococcal UTI in a tertiary level health facility in north India. METHODS: A total of 365 patients with UTI caused by enterococci were studied over a period of two years. Patients with ciprofloxacin-resistant and susceptible UTI were considered as cases and controls, respectively. Resistance profile of the isolates against common antibiotics was studied by minimum inhibitory concentration (MIC) determination. Mechanisms for fluoroquinolone resistance was studied by efflux pump inhibitor activity and multiplex PCR targeting the qnr genes. RESULTS: A total of 204 (55.89%) cases and 161 (44.1%) controls were identified. The fluoroquinolone-resistant isolates were significantly resistant to ampicillin, high strength aminoglycosides and vancomycin. The majority (78%) of the resistant isolates showed efflux pump activity. Treatment in indoor locations, presence of urinary catheters and pregnancy along with recent exposure to antibiotics especially fluoroquinolones, third generation cephalosporins and piperacillin-tazobactam were identified as independent risk factors. INTERPRETATION & CONCLUSIONS: Our results showed that fluoroquinolone resistance in enterococcal UTI was largely associated with indoor usage of antibiotics and use of indwelling devices. Knowledge of risk factors is important to curb this emergence of resistance.

Long-term outbreak of Klebsiella pneumoniae& third generation cephalosporin use in a neonatal intensive care unit in north India.

BACKGROUND & OBJECTIVES: The indiscriminate use of third generation cephalosporin has contributed to the emergence and widespread dissemination of extended spectrum ß lactamases (ESBL) genes in Klebsiella pneumoniae. This study was undertaken to elaborate the genetic behaviour of ESBL - producing K. pneumoniae isolates in the neonatal intensive care unit (NICU) of a tertiary care hospital in north India causing successive outbreaks in context with empirical third generation cephalosporin use. METHODS: Isolates of K. pneumoniae (43 from blood, 3 from pus and endotracheal tube, 4 from environment) causing successive outbreaks in the NICU of a tertiary care university hospital were studied for two years. Antimicrobial susceptibility testing was done by disc diffusion and minimum inhibitory concentration (MIC) determination by agar dilution methods. ESBL production was determined by phenotypic and genotypic methods. Clonal relatedness among the isolates was studied by enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). Genetic environment of these isolates was assessed by the presence of integrons and gene cassettes. Transformation experiments were done, and plasmids of these isolates were characterized by stability testing and incompatibility testing. Subsequently, a change in the ongoing antibiotic policy was adopted, and corresponding changes in the behaviour of these isolates studied. RESULTS: During the period from August 2011 to January 2013, 46 isolates of monoclonal ESBL K. pneumoniae were obtained from different neonates and four similar environmental isolates were studied. Multidrug-resistant ESBL isolates harboured both blaCTXM-15 and bla SHV-5. The dfr and aac-6 ' resistant genes were found in gene cassettes. A 50 kb plasmid belonging to IncFIIA group was detected in all the isolates which was transferable and stable. The emergence and regression of the outbreaks coincided with antibiotic usage in the NICU, with widespread empirical use of cefotaxime being responsible for their persistence in the environment. INTERPRETATION & CONCLUSIONS: The study indicates that empirical use of third generation cephalosporins may promote the emergence, persistence, and dissemination of resistant isolates in the hospital environment. Periodic review of antibiotic policy is necessary for rationalized use of antibiotics.

A study of Mycobacterium tuberculosis genotypic diversity & drug resistance mutations in Varanasi, north India.

BACKGROUND & OBJECTIVES: One-fifth of the world's new tuberculosis (TB) cases and two-thirds of cases in the South East Asian region occur in India. Molecular typing of Mycobacterium tuberculosis isolates has greatly facilitated to understand the transmission of TB. This study was aimed to investigate the molecular epidemiology of M. tuberculosis genotypes in Varanasi, north India, and their association with clinical presentation among patients with pulmonary TB. METHODS: M. tuberculosis isolates from 104 TB patients attending a tertiary referral hospital of north India were screened for susceptibility to isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and streptomycin (STR) by proportion method and multiplex-allele-specific-polymerase chain reaction (MAS-PCR). These were genotyped by spoligotyping. The spoligotype patterns were compared with those in the international SITVIT2 spoligotyping database. RESULTS: Eighty three of 104 isolates were distributed in 38 SITs, of which SIT3366 was newly created within the present study. The mass of ongoing transmission with MDR-TB isolates in Varanasi, northern India, was linked to Beijing genotype followed by the CAS1_Delhi lineage. HIV-seropositive patients had a significantly higher proportion of clustered isolates than HIV-seronegative patients and compared with the wild type(wt) isolates, the isolates with katG315Thr mutation were considerably more likely to be clustered. INTERPRETATION & CONCLUSIONS: This study gives an insight into the M. tuberculosis genetic biodiversity in Varanasi, north India, the predominant spoligotypes and their impact on disease transmission. In this region of north India, TB is caused by a wide diversity of spoligotypes with predominance of four genotype lineages: Beijing, CAS, EAI and T. The Beijing genotype was the most frequent single spoligotype and strongly associated with multi drug resistant (MDR)-TB isolates. These findings may have important implications for control and prevention of TB in north India.

Rapid genotypic detection of rpoB and katG gene mutations in Mycobacterium tuberculosis clinical isolates from Northern India as determined by MAS-PCR.

BACKGROUND: There is a growing need to develop rapid laboratory research methods to counter the menace of drug resistant tuberculosis (MDR-TB) cases worldwide especially in developing countries. The present study was undertaken to investigate the type and frequency of rpoB and katG mutations in rifampicin (RIF) and isoniazid (INH) resistant strains respectively of Mycobacterium tuberculosis (MTB) circulating in Northern India and to explore the utility of multiplex-allele-specific (MAS)-PCR assay for detection of drug-resistant MTB isolates in low resource set up. METHODS: Phenotypic and genotypic drug susceptibility testing (DST) was performed on 354 MTB isolates. RESULTS: Mutation in rpoB gene was found most frequently at codons 531, 526 and 516 (59.83%, 45.29% and 22.22%, respectively). Further, combinations of 2-3 point mutations were also observed in 19.66% of RIF-resistant MTB strains. The frequency of mutations in katG gene was found at codon 315 among 82.95% of the INH-resistant MTB isolates. MAS-PCR detected rpoB and katG mutations in phenotypically resistant isolates with sensitivities of 93% and 83% respectively. CONCLUSION: MAS-PCR assays can be used for rapid detection of drug-resistant TB strains in routine diagnostic practice, enabling early administration of appropriate treatment regimens to the affected patients.

Coagulase-Negative Staphylococci in Neonatal Blood: How Concerning?

Objective Coagulase-negative staphylococci (CoNS) are being implicated as one of the leading causes of bloodstream infection (BSI). To study the spectrum, prevalence, and antimicrobial susceptibility of CoNS causing BSI in neonates. Materials and Methods A cross-sectional study was done in level III neonatal intensive care unit (NICU). Blood samples in automated culture bottles were processed as per the standard technique. Previously validated methods were followed for the characterization of CoNS and for AST of standard antibiotics by Kirby Bauer disk diffusion and vancomycin by agar dilution. The prevalence of causative organisms and susceptibility of CoNS were statistically analyzed. Categorical variables were compared by chi-square or Fisher's exact probability tests. Result In total, 1,365 blood samples (1,365 neonates) were studied, of which 383 (28.05%) were positive and 982 (71.94%) were negative. Gram-positive organisms (GPC) predominated ( n = 238; 62.14%) ( p < 0.001) with 41.77% (160/383) S. aureus and 13.83% (53/383) CoNS. CoNS included S. epidermidis (19, 38%), S . haemolyticus (7, 14%), S. hominis (6, 12%), S. simulans (6,12%), S. capitis (5,10%), S. cohnii (4, 8%), S. warneri (1, 2%), and S. xylosus (1, 2%). The susceptibility to netilmicin, linezolid, and vancomycin was 100% ( p ≤ 0.001), and 54% ( n = 27) had vancomycin MIC of 0.125 µg/mL but methicillin-resistant CoNS (MRCoNS) was 70%. Methicillin-susceptible (MS) CoNS had lower MIC of vancomycin ( p < 0.05) than MRCoNS. Conclusion The spectrum of pathogens causing BSI in neonates is changing with predominance of GPC and among CoNS, S. epidermidis . Considerable proportion of MRCoNS with the emergence of MIC creep for vancomycin requires immediate attention.

Colonization with vancomycin-intermediate Staphylococcus aureus strains containing the vanA resistance gene in a tertiary-care center in north India.

A nasal carriage survey for methicillin-resistant Staphylococcus aureus (MRSA) in an intensive care unit detected four strains of MRSA with reduced susceptibility to vancomycin. The vanA gene was found in two of these vancomycin-intermediate Staphylococcus aureus (VISA) strains. The absence of selective vancomycin pressure might have resulted in reduced expression of the resistant gene.

Diagnosis of Genital Tuberculosis in Infertile Women by Using the Composite Reference Standard.

Female genital tuberculosis (FGTB) can be asymptomatic or even masquerade as other gynecological conditions. Conventional methods of FGTB diagnosis include various imaging, bacteriological, molecular, and pathological techniques that are only positive in a small percentage of patients, leaving many cases with undiagnosed condition. In the absence of a perfect diagnostic method, composite reference standards (CRSs) have been advocated in this diagnostic study. This study assesses the agreement between traditional diagnostic modalities using CRS and prevalent TB groups among different fallopian tube infertility manifestations. A total of 86 women with primary and secondary infertility were included in the study and subjected to bacteriological, pathological, and radiological examination for the diagnosis of FGTB. Results were evaluated statistically for concordance of the diagnostic tests to the CRS by sensitivity and specificity, while PPV and NPV were calculated for the performance of diagnostic tests of FGTB. We observed that 11.2% of women were found to be true positives by means of CRS. The positive findings by CRS were as follows: ultrasonography (13.9%), laparoscopy (14%), hysteroscopy (12%), GeneXpert (4.8%), culture (4.8%), polymerase chain reaction (4.8%), and histopathology (6.4%). GeneXpert and culture were found to have a perfect agreement with CRS. Hysterosalpingography, laparoscopy, and hysteroscopy have a fair agreement with CRS. Out of 43 women with tubal factor infertility, 6 women were found in the definitive TB group with mixed conditions of tubal manifestations. This study evaluates and demonstrates the reliability of the collective assessment of various diagnostic methods with CRS findings that help in identifying different TB groups of genital tuberculosis patients from all infertile patients by applying the criteria of CRS.

Evaluation of the performance of nitrate reductase assay for rapid drug-susceptibility testing of mycobacterium tuberculosis in north India.

The objective of the study was to evaluate the performance of nitrate reductase assay (NRA) as a rapid, reliable and inexpensive method for drug-susceptibility testing (DST) of Mycobacterium tuberculosis against first-line antitubercular drugs, such as rifampicin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB). In total, 286 isolates were subjected to test by proportion method (PM) and NRA. By comparing the results of NRA with those of the gold standard PM, sensitivities and specificities were 98.4%, 97%, 88.5%, and 94.2% and 100%, 100%, 94%, and 99% for RIF, INH, STR, and EMB respectively. The positive predictive values were 100%, 100%, 95%, and 98% for RIF, INH, STR, and EMB respectively. The negative values were 99%, 98%, 87%, and 96% for RIF, INH, STR, and EMB respectively. The median time of obtaining results was shorter using NRA (10 days) compared to PM (28 days). An excellent agreement was observed between the two phenotypic tests with the K values of 0.98, 0.97, 0.81, and 0.93 for RIF, INH, STR, and EMB respectively. The results demonstrated that NRA is suitable for the early determination of INH and RIF resistance and has the potential to be a useful tool for rapid drug-sensitivity test of M. tuberculosis in resource-constrained settings.

Antitubercular drug resistance in four healthcare facilities in North India.

Tuberculosis (TB) is a major public-health problem in India, having the highest number of incident and multidrug-resistant (MDR) TB cases. The study was carried out to appraise the prevalence of first-line anti-TB drug resistance in Mycobacterium tuberculosis (MTB) and its patterns among different types of TB patients from different settings in a province of North India. Of 3,704 clinical specimens, 345 (9.3%) were culture-positive, and drug-susceptibility testing was carried out for 301 MTB strains. A high level of primary and acquired drug resistance of MTB was observed in the region studied, with weighted mean of 10.5% and 28.08%, 12.81% and 29.72%, 17.12% and 29.94%, 11.97% and 27.84%, and 10.74% and 23.54% for rifampicin, isoniazid, streptomycin, ethambutol-resistant and MDR cases respectively. Drug resistance was significantly higher in pulmonary (p = 0.014) and acquired drug-resistant TB cases (p < 0.001). Any drug resistance (p = 0.002) and MDR TB were significantly (p = 0.009) associated with HIV-seropositive cases. An urgent plan is needed to continuously monitor the transmission trends of drug-resistant strains, especially MDR-TB strains, in the region.