Research article Evaluation of Genotype MTBDRplus Assay for identifying Multidrug Resistant Mycobacterium tuberculosis isolates in Nepal

Background and Objectives: Multidrug-resistant (MDR) Mycobacterium tuberculosis strains are serious threats to the control of tuberculosis and comprise an increasing public health problem. Rapid detection of such strains is quite critical in timely management of such issues. The study was performed with an objective to compare Genotype MTBDRplus reverse hybridization probe assay (Hain Lifescince, GmBH, Nehern, Germany) with culture based proportion method for rapidly identifying MDR-TB strains from suspected multi drug resistant cases, referred to GENETUP Kathmandu, Nepal. Methodology: A commercially available new Genotype MTBDRplus assay was evaluated for its ability to detect mutations in Mycobacterial isolates conferring resistance to rifampicin (RMP) and isoniazid (INH). A total of 64 MDR isolates (i.e., at least resistant to RMP and INH), 5 fully susceptible strains and 1 RMP sensitive strains by conventional proportion method were analyzed using Genotype MTBDRplus assay. MTBDRplus assay is designed to detect the mutations in the hot spot region of rpoB gene, katG and regulatory region of inhA gene. Results: The MTBDRplus assay detected 59 of 61 RMP resistant strains (96.72%) with mutations on 81-bp hot spot region of rpoB gene and 60 of 63 INH resistant strains (95.23%) with mutation in codon 315 katG and regulatory region of inhA. The sensitivity and specificity for the detection of RMP resistance were 96.72% and 100% respectively. While, value of the same two variables for INH resistance were 95.23% and 100%, respectively. Conclusions: The new Genotype MTBDRplus assay represents a rapid, reliable, upgraded tool with high sensitivity and specificity for the detection of INH and RMP resistance strains that can readily be included in a routine laboratory work for the early diagnosis and control of MDR-TB.

Multi Drug Resistant Pathogens Causing Urinary Tract Infections in Children at Kathmandu Model Hospital.

Introduction: Antibiotic resistance of urinary tract pathogens has increased globally. Updated knowledge of the antibiotic resistance patterns of uropathogens in the health institutes is important for the selection of an appropriate empirical antimicrobial therapy. The aim of this study was to evaluate the multi drug resistant urinary isolates in the children from 1 to15 years and evaluate the options for empiric antibiotic therapy. Materials and Methods: The study was conducted from December 2011 to May 2012 in the Bacteriology laboratory, Kathmandu Model Hospital. Urine samples received in the laboratory were processed for routine, culture and its sensitivity. The antimicrobial susceptibility of bacterial isolates was determined following Clinical and Laboratory Standard Institute (CLSI) recommended Kirby-Bauer Disc Diffusion method. Results: Of the total 372 urine samples received in the laboratory, 60 (16.13%) showed significant growth; of which 55.0 % (33/60) were MDR isolates. Escherichia coli were the predominant isolate from urine sample. Out of 49 Escherichia coli isolates, 27 (45.0%) were Multi drug resistant. Enterococcus faecalis (N=3) was the most predominant Gram positive isolate and 66.67 % (2/3) of this organism were multi drug resistant. Among the first line drugs used against gram negative isolates, nitrofurantoin was the most effective drug followed by quinolones, while among the second line drugs; meropenem was the most effective drug followed by chloramphenicol and amikacin, whereas; nitrofurantoin (100%) was the most effective drug for Gram positive isolates followed by norfloxacin and cefotaxime. Conclusion: High percentages of multi drug resistant uropathogens were revealed in children. Nitrofurantoin was found to be the most effective drug for gram positive, gram negative and multi drug resistant isolates.

Oxidative stress, antioxidant status and lipid profile in ischemic heart disease patients from western region of Nepal.

Disturbed lipid profile is one of the most important and potent risk factors in ischemic heart disease (IHD). In recent years, it has been demonstrated that raised oxidative stress promotes several undesirable pathways including the formation of oxidised LDL (O-LDL) and oxidized cholesterol which encourages cholesterol accumulation in arterial tissues. We, therefore, aimed to ascertain the redox balance by measuring oxidative stress (OS) and total antioxidant activity (TAA) along with lipid profile to determine their possible association with IHD. Our study group comprised of 28 confirmed cases of IHD. The inclusion criterion was history of chest pain, ischemic changes in the ECG and good left ventricular (LV) function. Patients with diabetes mellitus, poor LV function, previous infarct and valvular heart disease were excluded. Lipid profile, plasma thiobarbituric acid reactive substances (TBARS), plasma total antioxidant activity (TAA) and urinary TBARS were estimated in these patients by standard procedures and the values were compared with 30 age, sex and socioeconomically matched normal healthy control subjects. Body mass index (BMI) and waist/hip ratio (W/H ratio) was also noted in both the groups. Lipid profile and OS (TBARS levels) were significantly raised in IHD patients. Though statistically not significant but TAA tended to be lower and urinary TBARS levels tended to be higher in patients. BMI, W/H ratio, smoking and alcohol did not show discernible association with lipid profile, OS or TAA. OS is significantly raised in majority of IHD patients. The non association of BMI, W/H ratio, smoking and alcohol with lipid profile, OS and TAA suggest that there are other risk factors which primarily contribute to the initiation and progression of IHD.

A study of oxidative stress, antioxidant status and lipid profile in diabetic patient in the western region of Nepal.

AIMS AND OBJECTIVES: Diabetes mellitus (DM) is often termed as a disease of premature aging. Several studies have indicated lopsided redox balance due to pro oxidant environment as one of the important etiological factors. Some recent researches also indicate a causal relationship with oxidative stress (OS). So far, no study has been undertaken on this aspect in Nepali populations. We, therefore, aimed this maiden study in Nepali population to examine redox balance by measuring OS and antioxidant status along with lipid profile in 37 patients of DM type- 2 and 30 matched normal subjects. METHODOLOGY: Thirty seven patients of DM type-2 without any complications (mean age= 57.6+/- 10.6 years) and 30 normal subjects (mean age= 55.8 +/- 14.8 years) were included in this study. Body Mass Index (BMI) and Waist/Hip (W/H) ratio were measured. Fasting blood sample was collected for the analysis of total antioxidant activity (TAA), plasma and urinary thiobarbituric acid reactive substances (TBARS) and lipid profile by standard procedures in both the groups. The statistical analysis was done with SPSS 10 version. RESULTS: Total cholesterol, triglyceride, VLDL-cholesterol, LDL-cholesterol, plasma and urinary TBARS were significantly raised whereas, plasma TAA was significantly reduced in DM type-2 patients as compared to controls. The comparison of old and fresh cases revealed that though TAA was lower and PTBARS and UTBARS were higher in patients but did not attain the level of significance. W/H ratio is significantly higher in patients compared to normal subjects. But, no significant correlation of BMI and W/H with lipid profile is observed in both control and patients. CONCLUSION: Oxidative stress is raised in type 2 DM patients. This along with deranged lipid profile and decreased antioxidant status could be the risk factors in the development of complications associated with DM.

Cigarette smoke induced oxidative insult in local population of Pokhara.

OBJECTIVES: To assess the effect of cigarette smoking on lipid peroxidation induced oxidative stress, antioxidants, uric acid and blood sugar in normal subjects. METHODS: The study included 61 normal subjects with regular smoking habit and 57 never-smokers normal subjects matched in respect to socio-economic status, age and BMI. Information regarding smoking habit and other personal details were collected by oral questionnaire. Total antioxidant activity (TAA), reduced glutathione (GSH), alpha-tocopherol (alpha-T), ascorbic acid (AA), uric acid (UA), plasma and urinary thiobarbituric acid reactive substances (TBARS), fasting blood sugar (FBS) and urinary creatinine (Cr) were estimated by standard procedures in both the groups. Ferric Reducing Antioxidant Power (FRAP) procedure is used to estimate TAA which measures total dietary antioxidants. Statistical analysis was done with SPSS version 10. RESULTS: The mean pack years smoked by smokers was 14.4 +/- 15.8. The plasma TBARS level in smokers and never-smokers was 2.6 +/- 0.8 and 2.5 +/- 0.6 micromol/L respectively. The respective figure for urinary TBARS level was 4.6 +/- 2.7 and 3.7 +/- 1.4 micromol/gmCr. Smokers did not show any significant difference from never-smokers with respect to GSH, alpha-T, AA, plasma TBARS and FBS. However, the smokers had significantly lower levels of TAA (p<0.05) and raised level of urinary TBARS (p<0.05) and uric acid (p<0.01) as compared to never-smokers. CONCLUSION: Our study suggests that smoking induces mild lipid peroxidation but the body is able to compensate for it by removing its adducts. Importantly it also indicates enhanced oxidation of purines which are essential components of both DNA and RNA. Dietary antioxidants are consumed to scavenge free radicals (FR) and other reactive species (RS) in smoke. Female smokers are more prone to oxidative insult than male smokers. In summary RS present in smoke induce mild lipid peroxidation but are not the major contributors of redox imbalance in smoke induced toxicity in the selected subjects.