Drug resistance in tuberculosis- resurvey in Wardha district, India after implementation of revised national TB control program

Level of drug resistance among new TB patients indicates the efficacy of any control programme. A drug resistance survey, as a part of global study in new pulmonary tuberculosis (NPTB) patients, was conducted in Wardha district, India under WHO /IUTLD in 2001, before the implementation of the Revised National Tuberculosis Control Programme (RNTCP) which was implemented in the district in 2002. The present study was conducted in Wardha district in 2014-2015, thirteen years after the implementation of RNTCP, and the drug resistance in isolates from NPTB patients was compared to the results of 2001 survey. The methodology used was same in both the surveys. : In addition to 132 isolates from Wardha, the study also included 112 isolates from adjoining city, Nagpur and total of 244 sputum isolates were subjected to drug sensitivity by standard 1% proportion method on Lowenstein Jensen’s medium. In addition molecular typing of resistant strains was done.: In Wardha, compared to 2001 survey, overall susceptibility to first line drugs washigher (94.7% vs 80.2%); and resistance to streptomycin (3% vs 7.6%) and isoniazid (2.2% vs 15.2%) were significantly lower (p≤ 0.05). MDR was 0.75% against 0.50% in the earlier study but the difference was statistically not significant. Only two MDR isolate were recovered, of which only one was from Wardha.After the implementation of RNTCP in Wardha District, drug resistancein new PTB patients has shown a decline and MDR continues to be low reflecting upon the efficiency of the program.

Evaluating the Frequency of Resistance to Pyrazinamide Among Drug-resistant Strains of Mycobacterium tuberculosis in Isfahan, Iran

Background: Pyrazinamide is one of the most important first-line medications for the treatment of tuberculosis and an alternative intake for MDR-TB and XDR-TB patients. Objectives: The purpose of this study was to evaluate resistance to pyrazinamide in the isolates resistant to the Mycobacterium tuberculosis drug in patients in the city of Isfahan. Methods: In this study, the drug susceptibility test was performed with pyrazinamide using the proportion method and PZA assay on 47 isolates resistant to Mycobacterium tuberculosis. Then, the mutations of the pncA and rpsA genes of the isolates resistant to pyrazinamide were evaluated by the sequencing method. Results: According to the proportion method, 19 cases were resistant to pyrazinamide, 16 of which had mutations in their pncA and rpsA genes. Besides, five new mutations were recorded, and three isolates lacked mutations in the mentioned genes. Conclusions: Pyrazinamide resistance is high in MDR-TB and INH mono-resistant isolates. Therefore, evaluating the susceptibility to pyrazinamide in patients with MDR-TB before the initiation of treatment with pyrazinamide is considered essential.

Effect of Piper nigrum (Linn.) seeds extract and second line anti-tuberculosis drugs on a few Mycobacterium tuberculosis strains

Piper nigrum (Linn.) belonging to the family Piperaceae have been reported for its multitudinous medicinal values. The present study was undertaken to examine the direct effect of Ethionamide (ETH), Para amino salicylic acid (PAS), ethanolic extracts of P. nigrum on Mycobacterium tuberculosis (MTB) strain H37Rv and Multi drug-resistant (MDR)-strains-12, 19 and 21. The proportion method was used to detect the anti-mycobacterial drug susceptibility testing for mycobacteria using Lowenstein Jensen (LJ) medium. It was found that P. nigrum does not interfere with single or in the combination of both ETH and PAS showing the bioenhancer activity. In vitro study of ethanolic extract of P. nigrum observed that the extract inhibited the growth of H37Rv strains and MDR strains-12, MDR strains 19, and MDR strains 21. The present results will pave new avenues to find a new medicine that possesses P. nigrum alone or in combination with drugs to combat MDR-strains controlling tuberculosis.

The simple direct slide method is comparable to indirect Lowenstein Jensen proportion culture for detecting rifampicin resistant tuberculosis

Introduction. Drug resistant tuberculosis remains a worldwide problem that requires prompt diagnosis. Hypothesis/Gap statement. The WHO recommended direct, rapid Xpert MTB/RIF is prohibitively costly, therefore, there is a need to validate a rapid, affordable DST for use in low- and middle-income settings. Aim. The technical performance and time to results of a simple, direct microscopy-based slide DST (SDST) assay for diagnosis of rifampicin-resistant TB was evaluated in Uganda. Methodology. Sputum samples from 122 smear-positive re-treatment TB patients presenting to the TB treatment centre at Uganda’s National Referral Hospital, Mulago, Kampala, Uganda were examined. The sputum samples were tested by the direct SDST which was compared to the indirect Lowenstein Jensen Proportion Method (LJDST) method as the gold standard. The time to results was defined as the time from DST setting to results interpretation. The results were further analysed for sensitivity and specificity as well as agreement between LJDST and SDST for rifampicin resistance determination. Results. A total of 117 smear positive sputum samples with valid results for both tests were compared. The median time to results for SDST was 14 days with an interquartile range (IQR) of 10–14 days compared to 60 days with IQR of 60–75 days for LJDST. The number for rifampicin resistance by the gold standard LJDST was 26. The SDST had a sensitivity of 96 % (95 %; CI 81–99 %) and a specificity of 97.8 % (95 %; CI 93–100 %). The Positive Predictive and Negative Predictive values for SDST were 92.3 % (95 %; CI 76.8–99 %) and 98.9 % (95 %; CI 94–100 %), respectively. The kappa agreement between SDST and LJDST was 92.3 %. Conclusion. The SDST was found to be a rapid and accurate direct test for the detection of rifampicin resistance among retreatment TB cases in low-income settings.

South African physical sciences teachers’ use of formulae and proportion when answering reaction-based stoichiometry calculation questions

Stoichiometry calculation competence tends to be particularly poor in the developing world, even among teachers. Various methods, including the unit factor method, have the potential to be effective in developing such competence. To evaluate the likelihood of such a generic proportion method, which downplays traditional formula usage, succeeding in a particular context, it is necessary first to understand teachers’ existing competence with proportion and the extent to which their calculation success relies on the explicit provision of and substitution into formulae in their written solutions. This quantitative survey study of 171 South African Physical Sciences teachers’ use of formulae and proportion found that most teachers failed to recognise the need to use proportion for some of the four reaction-based stoichiometry calculation questions used. Provision of and substitution into a formula in the written solution was found to be valuable in helping participants who underutilised proportion to obtain process marks, but to be largely irrelevant to obtaining the correct answer. The findings suggest that professional development interventions in similar contexts should focus on proportion within stoichiometry, particularly on recognition of its relevance to reaction-based questions. While a generic proportion method is well suited to this purpose, questions are raised concerning the likelihood that teachers would accept such a method in a context where process marks favour traditional formats of formula provision and substitution and where process mark attainment is highly valued. The findings also point to the need for teacher education to address competencies other than proportion.

QUANTITATIVE ANALYSIS OF GYRA AND GYRB KNOWN MUTATIONS IN DRUG-RESISTANT MYCOBACTERIUM TUBERCULOSIS STRAINS TREATED WITH OFLOXACIN

Objective: The aim of the study is to measure the minimum inhibitory concentrations (MICs) of ofloxacin antibiotic from gyrA and gyrB mutations present in fluoroquinolones (FQs) resistant strains of Mycobacterium tuberculosis (MTB) and to further concentrate the potential association between gene mutations and phenotypic resistance based on their MICs. Methods: Different levels of ofloxacin MICs levels were detected in 31 archived multi drug-resistant MTB isolates showing gyrA mutations in codon at A90V, S91P, D94A, D94N/Y, D94 G, and D94H and two gyrB probes (N538D and E540V). The MIC determinations were made using the 1% proportion method. Results: Genotypic assay detected 32 mutations in the gyrA (n=29) and gyrB (n=3) genes among the 31 FQs resistant isolates. Most frequently seen in gyrA mutations at codon D94G (n=16; 50%), these mutations had a clearly elevated MIC level from 2 to 16 μg/ml, that was phenotypically resistant. The A90V mutation region consistently exhibited the lowest levels of ofloxacin resistance, with three out of eight (37.50%) of these isolates had a MIC of <2 μg/ml. In addition, a further strain of S91P mutations for MIC was determined to be less than the critical concentration (CC). These low levels of resistance have been detected in a phenotypic manner which is noticeable in the study. Furthermore, fewer mutations in codons at D94A, D94N/Y were identified. Three wild-type absent isolates from gyrB QRDR were identified and the MIC of those strains for ofloxacin was lower than the critical cutoff value. Conclusion: Based on the results, it is shown that different resistance mutations were associated with different levels of MICs and the current concentration for MGIT will be lowered from 2 μg/ml to 1 μg/ml for the ofloxacin drug.

Detection of Multi-Drug Resistant Tuberculosis Using the Genotype MTBDR DNA Strip Assay and Lowenstein Jensen Proportion Method

Pulmonary tuberculosis (TB) remains a significant public health issue in low-middle income countries. Accurate and timely diagnosis is key to effective management. Diagnosing Multi Drug Resistance Tuberculosis (MDR-TB) is mostly done with phenotypic Lowenstein Jenson (LJ) proportion method with long turn around which delays treatment. The genotypic MTBDR plus was introduced by World Health Organisation (W.H.O) for the same purpose hence, this study aims to detect MDR-TB using both two methods. Sputum samples were collected from cases of pulmonary TB diagnosed with Genexpert and Ziehl Neelsen stain. Positive samples were subjected to MTBDR plus and the LJ proportion method with the LJ method considered gold standard. Chi square analysis was used to evaluate the Sensitivity, Specificity, Positive Predictive value (PPV), Negative Predictive value (NPV), of the MTBDR plus method compared to the LJ Proportion method. Kappa values were also estimated as a measure of agreement between the two methods. In evaluating the performance of MTBDR plus compared to the LJ proportion method, the sensitivity, specificity, PPV and NPV for the detection of rifampicin and isoniazid was 93.7%, 90.2%, 92.5% ,91.7% and 88.5%, 95.5%,92.0% and 93.3% respectively, while evaluation MDR-TB was 74.5%, 94.4%, 88.4% and 86.6% with a Kappa value of 0.85,0.84 and 0.71 for Isoniazid, Rifampicin and MDR-TB which indicate almost perfect agreement for both rifampicin and isoniazid and substantial agreement for Multi Drug Resistance (MDR). Compared to LJ proportion method, MTBDRplus performed well in the detection of drug resistance to rifampicin, isoniazid and MDR-TB, hence, is a rapid and efficient tool for the diagnosis and initiation of treatment for MDR-TB.