Evaluation of nitrate reductase assay in 7H11 agar for diagnosis of multi-drug-resistant tuberculosis in eastern Nepal.

BACKGROUND: Emergence of multi-drug-resistant tuberculosis is a serious challenge for successful global tuberculosis control. Early diagnosis of drug-resistant tuberculosis by direct nitrate reductase assay (NRA) aids in appropriate treatment and reduction in disease transmission, particularly in countries with high tuberculosis burden. The aim of this study was to evaluate the performance of NRA for direct detection of resistance to rifampicin and isoniazid in Mycobacterium tuberculosis in laboratories with limited resources. METHODS: Fifty-eight new smear-positive sputum samples were processed as per the guidelines of revised national tuberculosis control program, India. The performance of NRA on middlebrook 7H11 agar was evaluated for detection of rifampicin and isoniazid resistance directly on smear-positive sputum specimens, and the results were compared with conventional proportion method. Sensitivity and specificity of the test were compared with the gold standard proportion method. Mc Nemar chi-square test was used to find out the significant difference between two methods. RESULTS: Direct NRA for detection of rifampicin resistance was 85.7% sensitive and 100% specific, whereas sensitivity and specificity of isoniazid resistance were 87.5% and 100%, respectively. Agreement between NRA and proportion method was 98% for both the drugs. The mean days of drug susceptibility testing results were 19.3 days for NRA and 72 days for conventional proportion method. The results of NRA were available in 21 days for 83% of the samples. CONCLUSIONS: Direct NRA on middlebrook 7H11 medium is a highly sensitive, reliable, and significantly faster method to perform drug susceptibility testing. It has the potential to be implemented for rapid detection of multi-drug-resistant tuberculosis against insufficient resources.

Bacteriological quality of bottled drinking water versus municipal tap water in Dharan municipality, Nepal.

BACKGROUND: Water-related diseases are of great concern in developing countries like Nepal. Every year, there are countless morbidity and mortality due to the consumption of unsafe drinking water. Recently, there have been increased uses of bottled drinking water in an assumption that the bottled water is safer than the tap water and its use will help to protect from water-related diseases. So, the main objective of this study was to analyze the bacteriological quality of bottled drinking water and that of municipal tap water. METHODS: A total of 100 samples (76 tap water and 24 bottled water) were analyzed for bacteriological quality and pH. The methods used were spread plate method for total plate count (TPC) and membrane filter method for total coliform count (TCC), fecal coliform count (FCC), and fecal streptococcal count (FSC). pH meter was used for measuring pH. RESULTS: One hundred percent of the tap water samples and 87.5 % of the bottled water samples were found to be contaminated with heterotrophic bacteria. Of the tap water samples, 55.3 % were positive for total coliforms, compared with 25 % of the bottled water. No bottled water samples were positive for fecal coliforms and fecal streptococci, in contrast to 21.1 % and 14.5 % of the tap water samples being contaminated with fecal coliforms and fecal streptococci, respectively. One hundred percent of the tap water samples and 54.2 % of the bottled water samples had pH in the acceptable range. CONCLUSIONS: All of the municipal tap water samples and most of the bottled drinking water samples distributed in Dharan municipality were found to be contaminated with one or more than one type of indicator organisms. On the basis of our findings, we may conclude that comparatively, the bottled drinking water may have been safer (than tap water) to drink.

Effect of urogenital cleaning with paper soap on bacterial contamination rate while collecting midstream urine specimens.

CONTEXT: Urinary tract infection (UTI) is one of the major health problems. Urine culture is considered as a gold standard method for the diagnosis of UTI. But, improper sample collection can lead to contamination with normal urogenital flora. Use of any portable disinfectant that can reduce contamination rate would be the significant help in urine culture interpretation. AIMS: To observe the effect of urogenital cleaning with paper soap on bacterial contamination rate while collecting specimens. MATERIALS AND METHODS: A cross-sectional comparative study was done in 600 patients aged 15-45 years, equally divided into three groups. The first group was given sterile container and instructed to collect midstream clean catch urine (MSU) after urogenital cleaning with provided piece of paper soap. The second group was given sterile container and strictly instructed to collect the MSU sample after urogenital cleansing by tap water only. The third group was given the sterile container and asked for midstream urine. Collected specimens were inoculated in CLED media, incubated aerobically for overnight at 37°C. Reporting of culture was done according to the guideline of American Society of Microbiology. RESULTS: The contamination rate in the three groups were 6.0%, 13.0%, and 27.5%, respectively (P value < 0.05), which was statistically significant. CONCLUSIONS: Contamination rate was significantly lower in group who provided urine specimen after urogenital cleaning with paper soap. Thus, cleaning the urogenital area may reduce the need of the repeat sample to rule out actual contamination and prevent from the unnecessary antibiotic treatment.

Heterologous expression of Aspen PTM3, a MADS box gene in cotton.

The PTM3 gene of Aspen was ectopically expressed in cotton to explore the opportunity to introduce desirable agronomic traits with the potential to improve yield and modify the duration of the parent cotton variety. Sixty-seven transgenic cotton lines expressing Aspen PTM3 (MADS box) gene were developed. The transgenic cotton lines expressing PTM3 gene showed earliness of 4-15 days variations in flowering and maturity. The transgenic lines were confirmed by kanamycin leaf paint assay, GUS assay and PCR. Among 67 transgenic lines, the event-10 showed profuse branching, event-24 showed abnormal growth and the remaining events exhibited single erect phenotype. In addition, the event-24 produced no flower and this might be due to the positional effect of PTM3 gene integration. Southern blot analysis performed for event-10, 24 and 48 showed distinct single copy integrations of PTM3 gene cassette. GUS assay performed using various plant parts of event-10 showed constitutive expression of the transgene. In view of cotton breeding, among all the events, the event-10 was found to be phenotypically significant with earliness of 12 days in flowering and 15 days in maturity and yield enhancement of 27%. In addition, the event-10 showed no square dropping and allowed the plants to bear more number of bolls. Based on these results, event-10 was chosen to carry out the inheritance study of expressed characters in the progeny.

Comparative study of cerebrospinal fluid adenosine deaminase activity in patients with meningitis.

Cerebrospinal fluid (CSF) adenosine deaminase (ADA) activity in tubercular meningitis (TBM) patients (n=20), non-tubercular meningitis (NTBM) patients (n=10) and non-tubercular non-meningitis (NTBNM) cases (n=15) were measured by the method based on Berthlot's reaction. The mean CSF ADA activity in TBM (13.62 +/- 8.45 IU/L) was found to be significantly higher as compared to NTBM (6.51 +/- 2.41 IU/ L, p<0.001) and NTBNM (2.35 +/- 1.16 IU/L, p<0.0001) respectively. The sensitivity and specificity of CSF ADA activity was 85.0% and 88.0% respectively at cut-off value of 6.97 IU/L to diagnose tubercular meningitis. The specificity and sensitivity of CSF ADA for TBM was found to be 85.0% and 70.0% as compared to NTBM and 85.0% and 100.0% as compared to NTBNM. We propose that estimation of that ADA activity in CSF of TBM patients, using a cut off value 6.97 IU/L can diagnose differentially tubercular meningitis. Since, most developing countries have the dubious distinction of having higher prevalence and incidence of tubeculosis and lack of well equipped laboratory services for proper diagnosis of tubercular meningitis, measurement of CSF ADA activity can be a better and reliable approach for the rapid diagnosis and management of tubercular meningitis vis a vis other types of meningitis.

Antimicrobial susceptibility patterns of Salmonella enterica serotype typhi in eastern Nepal.

The aim of the present study was to evaluate antimicrobial susceptibility patterns with special reference to multidrug resistance, susceptibility to ciprofloxacin, and bacteriophage typing of Salmonella enterica serotype Typhi isolated from blood sent for culture in a tertiary-care teaching hospital in eastern Nepal during January 2000-December 2004. In total, 132 strains of S. enterica Typhi, isolated from 2,568 blood culture samples collected from cases of suspected enteric fever, were tested for susceptibility to commonly-used antimicrobials by the disc-diffusion method. There were 35 multidrug-resistant strains. None of the isolates were resistant to ciprofloxacin. Of 52 isolates tested for minimum inhibitory concentration (MIC) of ciprofloxacin, 36 (69.23%) showed reduced susceptibility (MIC >0.25 mg/L). Of 112 strains tested for nalidixic acid susceptibility, 86 (76%) were resistant. Strains with reduced susceptibility to ciprofloxacin and resistance to nalidixic acid could be correlated. The commonest phage type was El. Nalidixic acid susceptibility could be a useful screening test for the detection of decreased susceptibility of S. Typhi to ciprofloxacin, a drug which is commonly used even for minor ailments in this area.

Diagnostic utility of adenosine deaminase (ADA) activity in pleural fluid and serum of tuberculous and non-tuberculous respiratory disease patients.

Adenosine deaminase activity (ADA) was assayed in pleural fluid and serum of 42 subjects with pleural effusion. Twenty-nine of them had TB pleural effusion and the remaining 13 had pleural effusion due to non-TB respiratory diseases. Serum adenosine deaminase activity were also measured in 32 pulmonary tuberculosis patients without pleural effusion and equal numbers of healthy controls without systemic diseases for comparative analysis. The patients attending the medicine out-patient department (MOPD) of the B. P. Koirala Institute of Health Sciences, Dharan, Nepal were taken as study subjects. Serum and pleural fluid ADA activities were assayed spectrophotometrically by the method of Guisti and Gallanti. The mean serum ADA activity was significantly increased in patients with tubercular pleural effusion (34.53 +/- 10.27 IU/l) compared to pulmonary tuberculosis patients without pleural effusion (26.54 +/- 4.76 IU/l), (p = 0.004), those with non-TB respiratory disease (16.71 +/- 5.16 IU/l), (p = 0.0001) and healthy controls (15.53 +/- 4.4 IU/l) (p = 0.0001). The mean ADA in the pleural fluid of tubercular pleural effusion patients (90.29 +/- 54.80 IU/l) was significantly higher compared to those with non-TB respiratory disease (24.43 +/- 9.28 IU/l) (p = 0.0001). Using the lowest cutoff value for enzyme activity in the serum of patients with TB pleural effusion (25 IU/l), a test sensitivity of 72.41% and specificity of 81.53% were obtained. Using the lowest cutoff value for enzyme activity in pleural fluid of patients with TB pleural effusion (45 IU/l) the sensitivity and specificity for diagnosis were 76.10% and 100%, respectively. Therefore, the measurement of ADA in tubercular pleural effusion has a utility in the diagnosis of tuberculosis when other clinical and laboratory tests are negative.

Prevalence of Shigella species and their antimicrobial resistance patterns in Eastern Nepal.

The study was conducted to determine the prevalence of Shigella species and their antimicrobial resistance patterns in eastern Nepal. Stool samples submitted to the diagnostic laboratory of B.P. Koirala Institute of Health Sciences, Nepal, during August 2000-July 2004, were cultured for Shigella species and were confirmed by biochemical and serological tests. Of 53 Shigella species isolated, Shigella dysenteriae type 1 was the most predominant isolate (73.7%), followed by S. flexneri (23%) and S. boydii (4%). The majority (79%) of Shigella species were isolated from children aged less than five years. An overall high resistance was observed for trimethoprim-sulphamethoxazole, ampicillin, nalidixic acid, mecillinam, and ciprofloxacin. There was a statistically significant (p < 0.001) increasing trend in the prevalence of ciprofloxacin resistance in S. dysenteriae type 1. The results suggest reconsideration of the empiric use of these antimicrobial agents for shigellosis. A further study is required to evaluate additional antimicrobial agents.

Comparison of needle biopsy with pleural fluid analysis for the evaluation of pleural lesions.

A total of 60 pleural biopsies were received at Nidaan Clinic, Kathmandu during the period of two years from January 2000 to December 2002. The age of the patient ranged from 8 years to 82 years. Male: female ratio seen was 3:1. The most common lesion was found to be chronic nonspecific; pleuritis seen in 50.0% cases followed by tuberculous pleuritis in 36.7% cases, metastatic adenocarcinoma in 8.3% cases, metastatic small cell carcinoma in 3.3% cases and anaplastic large cell carcinoma in 1.7% cases. 63.3% cases of chronic nonspecific pleuritis showed transudative pleural fluid effusion. None of the cases of tuberculous pleuritis had transudative pleural effusion. 2 cases (40.0%) of metastatic adenocarcinoma had transudative pleural fluid effusion which suggests the need to perform pleural biopsy in all the cases of transudative pleural effusion whether encountered for malignancy or not.

Surgical drainage of primary iliopsoas abscess--safe and cost-effective treatment.

The report evaluates surgical drainage (SD) as a primary treatment of primary iliopsoas abscess (PIA). Seventy-two patients, who underwent SD for PIA at B P Koirala Institute of Health Sciences, Dharan, Nepal were studied. SD was performed through a lower abdominal, extra peritoneel, muscle splitting incision. Ultrasonography was used to diagnose the abscess in 53/54 patients (98%). Staphylococcus aureus was the most frequent organism grown in 45/65 patients (69%). The mean duration of drainage was 3.2 +/- 1.4 days (range, 1-7 days). The treatment was successful in resolving the abscesses in all patients. The mean hospital stay was 9.0 +/- 5.4 days (range, 3-40 days). Two patients (2.8%) developed a recurrence, 10 months and 1 year after the operation, respectively. Another patient developed an incisional hernia. There were no deaths. The average cost of treatment to the patient was approximately Nepali rupees 2800 (US$ 40). Surgical drainage appears to be a cost-effective and safe treatment for PIA.