Trends in antibiotic resistance among major bacterial pathogens isolated from blood cultures tested at a large private laboratory network in India, 2008-2014.

OBJECTIVE: There have been no long-term studies on trends in antibiotic resistance (ABR) on a national scale in India. Using a private laboratory network, the ABR patterns of organisms most commonly associated with bacteremia, obtained from patients across India between 2008 and 2014, were examined. METHODS: A retrospective study of patient blood cultures collected over a 7-year period (January 1, 2008-December 31, 2014) was conducted. Data on the microorganism(s) identified and their antimicrobial susceptibility were obtained from SRL Diagnostics (Mumbai, India). RESULTS: Of 135268 blood cultures, 18695 (14%) had at least one identified pathogen. In addition to continual high rates of methicillin-resistant Staphylococcus aureus (MRSA; approximately 44.2%), high resistance to nalidixic acid among Salmonella Typhi (98%) was observed, and carbapenem resistance increased in both Escherichia coli (7.8% to 11.5%; p=0.332) and Klebsiella pneumoniae (41.5% to 56.6%; p<0.001). Carbapenem resistance was also stable and high for both Acinetobacter species (approximately 69.6%) and Pseudomonas aeruginosa (approximately 49%). Resistance was also detected to colistin in the Gram-negatives and to vancomycin and linezolid in S. aureus. CONCLUSION: Increasing resistance to antibiotics of last-resort, particularly among Gram-negatives, suggests an urgent need for new antibiotics and improved antimicrobial stewardship programs in India.

Immunochromatographic assays for detection of Mycobacterium tuberculosis: what is the perfect time to test?

In this study, we aimed to correlate the analytical performance of SD BIOLINE TB Ag MPT64 Rapid Test kit (MPT64 assay) with the mycobacterial growth unit (GU) reported by the BACTEC MGIT 960 (MGIT 960) instrument. A total of 394 culture isolates reported positive by MGIT 960 were processed daily (until 'day 4') with the MPT64 assay until a positive MPT64 result was obtained and their GU values were noted daily before MPT64 testing. Based on this correlation of MPT64 positivity and corresponding GU values, a GU cut-off was determined. In the validation phase, with the experimentally determined GU cut-off value, 99.1% (576/581) of culture isolates were correctly identified as MTB within 2 days from instrument positivity. All results were available using a single-MPT64 assay strip, making the assay cost-effective. Thus, systematic implementation of the MPT64 assay proved to be cost-effective in a high-throughput laboratory without any delay in patient reporting.

Tuberculosis: laboratory diagnosis.

Delay in laboratory diagnosis of tuberculosis (TB) is a major obstacle in TB control programs. There is an imperative need for scale-up of peripheral health care laboratories with conventional and molecular technologies for rapid and reliable diagnosis of TB. A cost-effective diagnostic algorithm for rapid diagnosis of TB should be implemented and followed, thereby reducing cost burden on patients.

Evaluation of 24-locus MIRU-VNTR in extrapulmonary specimens: study from a tertiary centre in Mumbai.

Genotyping of Mycobacterium tuberculosis isolates is a useful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. There is a lack of information on the discriminatory power of standard 24-locus mycobacterial interspersed repetitive unit (MIRU) - variable number tandem repeats (VNTR) in India, which has the highest tuberculosis (TB) burden worldwide. Therefore, we assessed its utility on 69 M. tuberculosis (MTB) isolates from patients with extrapulmonary tuberculosis, in comparison to standard insertion sequence (IS) 6110-Restriction fragment length polymorphism (RFLP) fingerprinting and spoligotyping. IS6110-RFLP (HGDI, 0.9987) identified a single cluster of 3 (4.3%) single-copy IS6110 isolates. Spoligotyping showed 69.5% clustering (HGDI, 0.8857). In contrast, MIRU-VNTR analysis identified 69 (100%) unique strains (HGDI, 1.0000). Within the study limits, this observed high discriminatory power suggests that 24-locus MIRU-VNTR genotyping could potentially be used to study long-term transmission of MTB infection in Mumbai. Moreover, high congruence between the MIRU-VNTR-based and spoligotyping-based strain groupings suggests that CAS, EAI and Beijing are the predominant strain lineages in the Mumbai TB patient population. The Beijing lineage isolates were found to be more significantly associated with multi-drug resistance (p < 0.01) than CAS and EAI lineages.

Multiplex allele specific PCR for rapid detection of extensively drug resistant tuberculosis.

Effective tuberculosis (TB) control is hindered by lack of rapid diagnostic tests for detection of drug-resistant TB (DR-TB). Use of molecular tools for rapid detection of multi-and extensively- DR-TB, could facilitate early initiation of appropriate anti-tubercular treatment (ATT) regimen thereby interrupting transmission. Understanding the urgent situation, we standardized and evaluated 4 individual multiplex allele specific PCR (MAS-PCR) assays on 450 sputum specimens for Mycobacterium tuberculosis (MTB) detection and determination of drug resistance by targeting katG315, rpoB531, gyrA 94, rrs 1401 codon mutations for determination of resistance to Isoniazid (INH), Rifampicin (RIF), Fluoroquinolones (FQ) and Aminoglycosides (AG) respectively. Using a single sputum specimen, MAS-PCR correctly identified 97.2% (281/289; 95% CI:95-99) culture positive patients as MTB positive, 100% (271/271; 95% CI:99-100) for smear positive cases and 55.5% (10/18; 95% CI:34-75) for smear negative cases; and correctly identified 93.6% (104/111; 95% CI:87-97) of culture negative patients. Individual MAS-PCR assays reported variable diagnostic accuracy for determination of drug resistance. On comparison with phenotypic drug susceptibility testing, MAS-PCR assays correctly identified 89.2% (191/214; 95% CI:84-93), 94.9% (187/197; 95% CI:91-97), 72.5% (98/135; 95% CI:65-79) and 92.3% (24/26; 95% CI:75-99) of INH resistant, RIF resistant, FQ resistant and AG resistant specimens respectively; and correctly identified 94% (63/67; 95% CI:85-98), 86.9% (73/84; 95% CI:78-93), 93.1% (136/146; 95% CI:88-96) and 99.2% (253/255; 95% CI:97-100) of INH sensitive, RIF sensitive, FQ sensitive and AG sensitive specimens respectively. Thus, use of MAS-PCR assays for rapid detection of DR-TB is recommended, enabling early initiation of appropriate ATT.

Determination of risk factors for isoniazid monoresistance and multidrug-resistant tuberculosis in treatment failure patients.

India has a high number of drug-resistant tuberculosis cases. Patient records were screened to determine risk factors possibly associated with monoresistance and multidrug-resistant tuberculosis (MDR-TB) in comparison with patients with susceptible TB. We retrospectively screened and selected 250 patient records from a tertiary care centre, in which detailed clinical histories were provided. Of the 250 patients, 10 had isoniazid monoresistant TB, 184 patients had MDR-TB, and 56 patients had TB susceptible to first-line drugs. Binary regression analysis revealed that previous treatment with a fluoroquinolone and an injectable other than streptomycin (odds ratio (OR) 3.889, 95% confidence interval (CI) 1.828-8.272) was associated with MDR-TB. Previous history of TB (OR 0.697, 95% CI 0.363-1.338) and the presence of cavities on radiographs (OR 0.371, 95% CI 0.160-0.862) did not show any association. None of these reported risk factors were associated with isoniazid monoresistance. In conclusion, a history of previous treatment with a fluoroquinolone and an injectable other than streptomycin was found to be a risk factor for MDR-TB.

Clonal population of Mycobacterium tuberculosis strains reside within multiple lung cavities.

BACKGROUND: Unsuccessful treatment outcomes among patients with multi-/extensively-drug resistant tuberculosis (TB) have hampered efforts involved in eradicating this disease. In order to better understand the etiology of this disease, we aimed to determine whether single or multiple strains of Mycobacterium tuberculosis (MTB) are localized within lung cavities of patients suffering from chronic progressive TB. METHODOLOGY/FINDINGS: Multiple cavity isolates from lung of 5 patients who had undergone pulmonary resection surgery were analyzed on the basis of their drug susceptibility profile, and genotyped by spoligotyping and 24-loci MIRU-VNTR. The patients past history including treatment was studied. Three of the 5 patients had extensive drug resistant TB. Heteroresistance was also reported within different cavity isolates of the lung. Both genotyping methods reported the presence of clonal population of MTB strain within different cavities of the each patient, even those reporting heteroresistance. Four of the 5 patients were infected with a population of the Beijing genotype. Post-surgery they were prescribed a drug regimen consisting of cycloserine, a fluoroquinolone and an injectable drug. A 6 month post-surgery follow-up reported only 2 patients with positive clinical outcome, showing sputum conversion. CONCLUSION: Identical spoligotype patterns and MIRU-VNTR profiles between multiple cavities of each patient, characterize the presence of clonal population of MTB strains (and absence of multiple MTB infection).

A multisite assessment of the quantitative capabilities of the Xpert MTB/RIF assay.

RATIONALE: The Xpert MTB/RIF is an automated molecular test for Mycobacterium tuberculosis that estimates bacterial burden by measuring the threshold-cycle (Ct) of its M. tuberculosis-specific real-time polymerase chain reaction. Bacterial burden is an important biomarker for disease severity, infection control risk, and response to therapy. OBJECTIVES: Evaluate bacterial load quantitation by Xpert MTB/RIF compared with conventional quantitative methods. METHODS: Xpert MTB/RIF results were compared with smear-microscopy, semiquantiative solid culture, and time-to-detection in liquid culture for 741 patients and 2,008 samples tested in a multisite clinical trial. An internal control real-time polymerase chain reaction was evaluated for its ability to identify inaccurate quantitative Xpert MTB/RIF results. MEASUREMENTS AND MAIN RESULTS: Assays with an internal control Ct greater than 34 were likely to be inaccurately quantitated; this represented 15% of M. tuberculosis-positive tests. Excluding these, decreasing M. tuberculosis Ct was associated with increasing smear microscopy grade for smears of concentrated sputum pellets (r(s) = -0.77) and directly from sputum (r(s) =-0.71). A Ct cutoff of approximately 27.7 best predicted smear-positive status. The association between M. tuberculosis Ct and time-to-detection in liquid culture (r(s) = 0.68) and semiquantitative colony counts (r(s) = -0.56) was weaker than smear. Tests of paired same-patient sputum showed that high viscosity sputum samples contained ×32 more M. tuberculosis than nonviscous samples. Comparisons between the grade of the acid-fast bacilli smear and Xpert MTB/RIF quantitative data across study sites enabled us to identify a site outlier in microscopy. CONCLUSIONS: Xpert MTB/RIF quantitation offers a new, standardized approach to measuring bacterial burden in the sputum of patients with tuberculosis.

Xpert MTB/RIF: a new pillar in diagnosis of extrapulmonary tuberculosis?

Approximately 10 to 15% of tuberculosis (TB) cases in India are estimated to have extrapulmonary disease, and due to a lack of diagnostic means, they often remain untreated. The early detection of Mycobacterium tuberculosis and multidrug resistance is a priority in TB diagnosis to improve the successful treatment rate of TB and reduce transmission. The Xpert MTB/RIF (Xpert) test, recently endorsed by the World Health Organization for the detection of pulmonary TB, was evaluated to test its utility in 547 patients with suspected extrapulmonary tuberculosis. Five hundred forty-seven extrapulmonary specimens were split and processed simultaneously for both culture (solid and liquid) and Xpert testing. For culture, the sensitivity was low, 53% (150/283 specimens). Xpert sensitivity and specificity results were assessed in comparison to a composite reference standard made up of smear and culture results and clinical, radiological, and histological findings. The sensitivity of the Xpert assay was 81% (228/283 specimens) (64% [89/138] for smear-negative cases and 96% [139/145] for smear-positive cases), with a specificity of 99.6%. The sensitivity was found to be high for the majority of specimen types (63 to 100%) except for cerebrospinal fluid, the sensitivity of which was 29% (2/7 specimens). The Xpert test correctly identified 98% of phenotypic rifampin (RIF)-resistant cases and 94% of phenotypic RIF-susceptible cases. Sequencing of the 6 discrepant samples resolved 3 of them, resulting in an increased specificity of 98%. In conclusion, the results of this study suggest that the Xpert test also shows good potential for the diagnosis of extrapulmonary TB and that its ease of use makes it applicable for countries where TB is endemic.

Fading of auramine-stained mycobacterial smears and implications for external quality assurance.

Light-emitting diode fluorescence microscopy is being scaled up for tuberculosis control, but fading of auramine-stained slides could compromise external quality assurance. We stored auramine-stained slides and reexamined them over time. Slides stored in all environments faded quickly, with significant changes in the proportion of positive slides in as little as 1 week.

Insilico analysis of homocamptothecin (hCPT) analogues for anti-tumour activity.

Cancer being a leading cause of death, the development of anti-cancer drugs like Camptothecin (CPT) has been promoted. CPT has lactone ring instability and lacks lipophilicity resulting in drug efflux. Owing to these limitations, homocamptothecin (hCPT), a CPT analogue was developed, which due to seven membered beta-hydroxylactone ring has better lipophilicity leading to reduced drug efflux. Analogues of hCPT were designed and docked into catalytic site of 1t8i (PDB id) protein (top-I). The docking energies and formation of hydrogen-bonds between the analogue and protein were compared with the original hCPT. Further, ADME properties, LogP and IC50 values were determined computationally.