Association of Previous Smoking Habit and Perceived Social Discrimination with the Risk of Multi-Drug Resistant Tuberculosis in Central Nepal.

BACKGROUND: Multidrug-resistant tuberculosis (MDR TB) caused by Mycobacterium tuberculosis resistant to both Isoniazid and Rifampicin with or without resistant to other drug, is among the most alarming pandemic problem. The objectives of this study was to assess the risk factors of MDR TB in Central Nepal. METHODS: A matched case control study was conducted among 186 cases of MDR TB and 372 non-MDR TB controls from central region of Nepal. Pretested questionnaires containing socio-economic, cultural & behavioral; environmental, biological and health service factors were used. Variables significant in bivariate analysis were entered in multiple regression models for further analysis. RESULTS: After adjusting for confounders, previous smoking habit (aOR= 4.5,(95%CI(1.24-16.2)) (p=0.04), and perceived social discrimination (aOR=5.83,95%CI (1.77-19.71)) (P=0.021) independently predicted greater MDR TB risk. CONCLUSIONS: Encouraging MDR TB cases for smoking cessation through awareness activities should be a priority. Stigma reduction programs should include the empowerment of patients and communities while promoting TBrelated research for further exploration into the risk factors of TB and associated stigma.

Spatio-temporal patterns of leptospirosis in Thailand: is flooding a risk factor?

We studied the temporal and spatial patterns of leptospirosis, its association with flooding and animal census data in Thailand. Flood data from 2010 to 2012 were extracted from spatial information taken from satellite images. The incidence rate ratio (IRR) was used to determine the relationship between spatio-temporal flooding patterns and the number of human leptospirosis cases. In addition, the area of flood coverage, duration of waterlogging, time lags between flood events, and a number of potential animal reservoirs were considered in a sub-analysis. There was no significant temporal trend of leptospirosis over the study period. Statistical analysis showed an inconsistent relationship between IRR and flooding across years and regions. Spatially, leptospirosis occurred repeatedly and predominantly in northeastern Thailand. Our findings suggest that flooding is less influential in leptospirosis transmission than previously assumed. High incidence of the disease in the northeastern region is explained by the fact that agriculture and animal farming are important economic activities in this area. The periodic rise and fall of reported leptospirosis cases over time might be explained by seasonal exposure from rice farming activities performed during the rainy season when flood events often occur. We conclude that leptospirosis remains an occupational disease in Thailand.

Screen of whole blood responses to flagellin identifies TLR5 variation associated with outcome in melioidosis.

Melioidosis is a severe infection caused by the flagellated bacterium Burkholderia pseudomallei. The nonsense polymorphism TLR51174C>T is associated with improved outcome in Thais with melioidosis. We hypothesized that other TLR5 variants may modulate the host response and determine outcome in melioidosis. We genotyped 12 TLR5 variants selected de novo from the HapMap database and examined the association of each with cytokines induced by flagellin stimulation of whole blood from healthy Thai subjects. We found a blunted cytokine response for three related markers that were in linkage disequilibrium (LD) with a non-synonymous variant, TLR51846T>C. Carriers of TLR51846T>C had broadly impaired cytokine responses induced by flagellin. TLR51846T>C was associated with protection against death in melioidosis patients (odds ratio: 0.62, 95% confidence interval: 0.42-0.93, P=0.021). We observed no impairment in TLR51846C-dependent nuclear factor κB activation, however, suggesting an alternative mechanism for the effect. We found that TLR51846T>C was in strong LD with TLR51174C>T. Many of the blunted cytokine responses observed and the association of TLR51846T>C with survival in melioidosis patients may be attributable to TLR51174C>T, but we could not exclude an independent effect of TLR51846T>C. These data identify novel associations for TLR51846T>C, enhance our understanding of TLR5 genetic architecture in Thais and highlight the role of TLR5 in melioidosis.

Adverse effects of falciparum and vivax malaria and the safety of antimalarial treatment in early pregnancy: a population-based study.

BACKGROUND: The effects of malaria and its treatment in the first trimester of pregnancy remain an area of concern. We aimed to assess the outcome of malaria-exposed and malaria-unexposed first-trimester pregnancies of women from the Thai-Burmese border and compare outcomes after chloroquine-based, quinine-based, or artemisinin-based treatments. METHODS: We analysed all antenatal records of women in the first trimester of pregnancy attending Shoklo Malaria Research Unit antenatal clinics from May 12, 1986, to Oct 31, 2010. Women without malaria in pregnancy were compared with those who had a single episode of malaria in the first trimester. The association between malaria and miscarriage was estimated using multivariable logistic regression. FINDINGS: Of 48,426 pregnant women, 17,613 (36%) met the inclusion criteria: 16,668 (95%) had no malaria during the pregnancy and 945 (5%) had a single episode in the first trimester. The odds of miscarriage increased in women with asymptomatic malaria (adjusted odds ratio 2·70, 95% CI 2·04-3·59) and symptomatic malaria (3·99, 3·10-5·13), and were similar for Plasmodium falciparum and Plasmodium vivax. Other risk factors for miscarriage included smoking, maternal age, previous miscarriage, and non-malaria febrile illness. In women with malaria, additional risk factors for miscarriage included severe or hyperparasitaemic malaria (adjusted odds ratio 3·63, 95% CI 1·15-11·46) and parasitaemia (1·49, 1·25-1·78 for each ten-fold increase in parasitaemia). Higher gestational age at the time of infection was protective (adjusted odds ratio 0·86, 95% CI 0·81-0·91). The risk of miscarriage was similar for women treated with chloroquine (92 [26%] of 354), quinine (95 [27%) of 355), or artesunate (20 [31%] of 64; p=0·71). Adverse effects related to antimalarial treatment were not observed. INTERPRETATION: A single episode of falciparum or vivax malaria in the first trimester of pregnancy can cause miscarriage. No additional toxic effects associated with artesunate treatment occurred in early pregnancy. Prospective studies should now be done to assess the safety and efficacy of artemisinin combination treatments in early pregnancy.

katG (SER 315 THR) gene mutation in isoniazid resistant Mycobacterium tuberculosis.

UNLABELLED: BACKGROUND Isoniazid (INH) together with Rifampicin (RFP) forms the cornerstone of a short chemotherapy course for tuberculosis (TB) treatment. Mutation at codon 315 of katG gene is most prevalent in isoniazid resistant Mycobacterium tuberculosis (MTB) and is high in area with high TB incidence. Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) has been found to be a reliable and effective tool for the identification of the specific gene alteration. OBJECTIVE: The objective of this study was to screen Ser315Thr mutation of KatG gene of INH resistant MTB strain by PCR-RFLP technique. METHODS: Altogether 37 INHr MTB isolates obtained from German Nepal Tuberculosis Project (GENETUP) Kathmandu Nepal was included in the study. Deoxyribonucleic Acid (DNA) extraction was performed according to protocol of SORPOCLEAN from the culture isolates. Amplification of the fragment with katG codon 315 was performed in a Biometra Thermocycler using primers. The amplified fragment was cleaved with MspI. The restriction fragments obtained were electrophoresed in a 2% agarose gel and were visualized using transilluminator. RESULTS: The katG Ser315Thr mutation was observed in 23 (62.2%) out of 37 INH resistant isolates. The drug susceptibility profile of INHr MTB isolates showed all isolates to be resistant to INH and RFP whereas 26 and 27 MTB isolates were resistant to Ethambutol (EMB) and Streptomycin (S) respectively. Seventeen (17) patients were harbouring katG gene mutated strain among Ethambutol and Streptomycin resistant cases. CONCLUSION: The study identified high prevalence of Ser315Thr mutation in katG. The isolates harbouring this mutation were also simultaneously resistant to RFP. Ser315Th could be a potential genetic marker for predicting MDR-TB.

Hepatitis B among pregnant women attending health care facilities in rural Bangladesh.

This study assessed hepatitis B prevalence among pregnant women attending health care facilities in rural Bangladesh. Blood samples were collected from 480 participants. HBsAg was positive in 0.4% of subjects, anti-HBc was positive in 21.5% and anti-HBs was positive in 8.5% of subjects. HBsAg was more prevalent among the older age group. Hepatitis B has a low prevalence among pregnant women in rural Bangladesh. Existing hepatitis B vaccination schedule in the Expanded Program on Immunization (EPI) to vaccinate the children in rural Bangladesh is appropriate.

Community awareness and perceptions of health sector preparedness and response to Cyclone Nargis.

Community awareness, preparedness and response to public health emergencies are essential for a successful response to public health emergencies. This study was carried out to determine community awareness and perceptions regarding health sector preparedness and response to Cyclone Nargis in Myanmar. Six focus group discussions were carried out in 3 villages severely affected by Cyclone Nargis. Thematic content analysis was carried out to determine community perceptions. Focus group participants, consisting of community members, community leaders and government personnel, were aware of the cyclone, but were unaware of its intensity and where it would make landfall. There was inadequate knowledge on how to prepare for a cyclone. There was some training on cyclone preparation but coverage was not wide enough. Participants received service and relief from health sector; they had a positive attitude toward health services provided to them. However, 5 out of 6 focus groups stated most villagers were not interested in health education. Only a few participants had some knowledge on how to prepare for a cyclone. Based on these results, there are evident weaknesses on how to prepare for cyclones. Community preparedness is essential to prevent disasters with cyclones, such as with Cyclone Nargis.

Risk factors of multidrug resistant tuberculosis in central Nepal: a pilot study.

INTRODUCTION: Tuberculosis is the most widespread infectious disease in Nepal and poses a serious threat to the health and development of the country. Incidences of drug resistant tuberculosis in Nepal are increasing and this tuberculosis a major threat to successfully controlling tuberculosis. OBJECTIVE: The general objective of the study was to assess the risk factors of multi-drug resistant tuberculosis among the patients attending the National Tuberculosis Centre, Bhaktpur Nepal. METHODS: An observational study/ case-control study with a total number of 55 multi-drug resistant tuberculosis cases and 55 controls. The study was conducted among the patient attending in the National Tuberculosis Centre, Bhaktpur Nepal for six months, between May-October 2010. sImulti-drug resistant tuberculosis wasThe collected data was analysed in SPSS 11.5 version. The association between categorical variables were analysed by chi-square tests, OR and their 95% CI were measured. RESULTS: The total number of patients used for the study was 110, of which among them 55 were cases and 55 were controls . Our study revealed that there were significant associations between history of prior TB MDR-TB OR = 2.799 (95 % CI 1.159 to 6.667) (p = 0.020); smoking habit OR = 2.350 and (95%CI 1.071 to 5.159) (p = 0.032); social stigma social stigma OR 2.655 (95%CI r 1.071 to 5.159) (p = 0.013); knowledge on MDR-TB OR = 9.643 (95% CI 3.339 to 27.846) (p less than 0.001)and knowledge on DOTS Plus OR = 16.714 (95% CI is ranging from 4.656 to 60.008) (p less than 0.001). However, there was no association found between alcohol drinking habits and ventilation in the room. CONCLUSION: Our study revealed that there were significant associations between history of prior tuberculosis, smoking habit social stigma social stigma, knowledge on multi-drug resistant tuberculosis and knowledge on DOTS Plus with multi-drug resistant tuberculosis However there was no association between alcohol drinking habit and ventilation in room with multi-drug resistant tuberculosis.

Obstetric ultrasound scanning by local health workers in a refugee camp on the Thai-Burmese border.

OBJECTIVES: Ultrasound examination of the fetus is a powerful tool for assessing gestational age and detecting obstetric problems but is rarely available in developing countries. The aim of this study was to assess the intraobserver and interobserver agreement of fetal biometry by locally trained health workers in a refugee camp on the Thai-Burmese border. METHODS: One expatriate doctor and four local health workers participated in the study, which included examinations performed on every fifth pregnant woman with a singleton pregnancy between 16 and 40 weeks' gestation, and who had undergone an early dating ultrasound scan, attending the antenatal clinic in Maela refugee camp. At each examination, two examiners independently measured biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC) and femur length (FL), with one of the examiners obtaining duplicate measurements of each parameter. Intraobserver measurement error was assessed using the intraclass correlation coefficient (ICC) and interobserver error was assessed by the Bland and Altman 95% limits of agreement method. RESULTS: A total of 4188 ultrasound measurements (12 per woman) were obtained in 349 pregnancies at a median gestational age of 27 (range, 16-40) weeks in 2008. The ICC for BPD, HC, AC and FL was greater than 0.99 for all four trainees and the doctor (range, 0.996-0.998). For gestational ages between 18 and 24 weeks, interobserver 95% limits of agreement corresponding to differences in estimated gestational age of less than +/- 1 week were calculated for BPD, HC, AC and FL. Measurements by local health workers showed high levels of agreement with those of the expatriate doctor. CONCLUSIONS: Locally trained health workers working in a well organized unit with ongoing quality control can obtain accurate fetal biometry measurements for gestational age estimation. This experience suggests that training of local health workers in developing countries is possible and could allow effective use of obstetric ultrasound imaging.

Quantification of artemisinin in human plasma using liquid chromatography coupled to tandem mass spectrometry.

A liquid chromatographic tandem mass spectroscopy method for the quantification of artemisinin in human heparinised plasma has been developed and validated. The method uses Oasis HLB mu-elution solid phase extraction 96-well plates to facilitate a high throughput of 192 samples a day. Artesunate (internal standard) in a plasma-water solution was added to plasma (50 microL) before solid phase extraction. Artemisinin and its internal standard artesunate were analysed by liquid chromatography and MS/MS detection on a Hypersil Gold C18 (100 mm x 2.1 mm, 5 microm) column using a mobile phase containing acetonitrile-ammonium acetate 10mM pH 3.5 (50:50, v/v) at a flow rate of 0.5 mL/min. The method has been validated according to published FDA guidelines and showed excellent performance. The within-day, between-day and total precisions expressed as R.S.D., were lower than 8% at all tested quality control levels including the upper and lower limit of quantification. The limit of detection was 0.257 ng/mL for artemisinin and the calibration range was 1.03-762 ng/mL using 50 microL plasma. The method was free from matrix effects as demonstrated both graphically and quantitatively.

Pharmacokinetics of high-dose oseltamivir in healthy volunteers.

The effects of loading doses and probenecid coadministration on oseltamivir pharmacokinetics at four increasing dose levels in groups of eight healthy adult Thai volunteers (125 individual series) were evaluated. Doses of up to 675 mg were well-tolerated. The pharmacokinetics were dose linear. Oseltamivir phosphate (OS) was rapidly and completely absorbed and converted (median conversion level, 93%) to the active carboxylate metabolite. Median elimination half-lives (and 95% confidence intervals [CI]) were 1.0 h (0.9 to 1.1 h) for OS and 5.1 h (4.7 to 5.7 h) for oseltamivir carboxylate (OC). One subject repeatedly showed markedly reduced OS-to-OC conversion, indicating constitutionally impaired carboxylesterase activity. The coadministration of probenecid resulted in a mean contraction in the apparent volume of distribution of OC of 40% (95% CI, 37 to 44%) and a reduction in the renal elimination of OC of 61% (95% CI, 58 to 62%), thereby increasing the median area under the concentration-time curve (AUC) for OC by 154% (range, 71 to 278%). The AUC increase for OC in saliva was approximately three times less than the AUC increase for OC in plasma. A loading dose 1.25 times the maintenance dose should be given for severe influenza pneumonia. Probenecid coadministration may allow considerable dose saving for oseltamivir, but more information on OC penetration into respiratory secretions is needed to devise appropriate dose regimens.

Major pitfalls in the measurement of artemisinin derivatives in plasma in clinical studies.

A bioanalytical method for the analysis of artesunate (ARS) and its metabolite dihydroartemisinin (DHA) in human plasma using protein precipitation and liquid chromatography coupled to positive tandem mass spectroscopy was developed. The method was validated according to published US FDA-guidelines and showed excellent performance. However, when it was applied to clinical pharmacokinetic studies in malaria, variable degradation of the artemisinins introduced an unacceptable large source of error, rendering the assay useless. Haemolytic products related to sample collection and malaria infection degraded the compounds. Addition of organic solvents during sample processing and even low volume addition of the internal standard in an organic solvent caused degradation. A solid phase extraction method avoiding organic solvents eliminated problems arising from haemolysis induced degradation. Plasma esterases mediated only approximately 20% of ex vivo hydrolysis of ARS into DHA. There are multiple sources of major preventable error in measuring ARS and DHA in plasma samples from clinical trials. These various pitfalls have undoubtedly contributed to the large inter-subject variation in plasma concentration profiles and derived pharmacokinetic parameters for these important antimalarial drugs.

Development and validation of a high-throughput zwitterionic hydrophilic interaction liquid chromatography solid-phase extraction-liquid chromatography-tandem mass spectrometry method for determination of the anti-influenza drug peramivir in plasma.

An assay for the analysis for the quantification of the anti-influenza drug peramivir in human plasma using high-throughput zwitterionic (ZIC) hydrophilic interaction liquid chromatography (HILIC) solid-phase extraction (SPE) in a 96-wellplate format and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. The ZIC-HILIC SPE efficiently removed sources of interference present in the supernatant after protein precipitation of plasma proteins. The main advantage of the ZIC-HILIC SPE sample preparation step was that it allowed load and elution conditions to be optimised to extract only peramivir and minimize co-extraction of lipophilic phospholipids. The method was validated according to published US Food and Drugs Administration guidelines and showed excellent performance. The assay was validated over two calibration ranges (0.952-500 and 50-50,000 ng/mL) to support analysis of peramivir after intra-venous administration. The lower limit of quantification for peramivir in plasma was 1 ng/mL and the upper limit of quantification was 50,000 ng/mL. The within-day and between-day precisions expressed as RSD, were lower than 8% at all tested quality control concentrations and below 11% at the lower limit of quantification. Validation of over-curve samples ensured that it would be possible with dilution if samples went outside the calibration range.

A liquid chromatographic-tandem mass spectrometric method for determination of artesunate and its metabolite dihydroartemisinin in human plasma.

A bioanalytical method for the analysis of artesunate and its metabolite dihydroartemisinin in human plasma using high throughput solid-phase extraction in the 96-wellplate format and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. The method was validated according to published FDA guidelines and showed excellent performance. The within-day and between-day precisions expressed as RSD, were lower than 7% at all tested concentrations including the lower limit of quantification. Using 50 microl plasma the calibration range was 1.19-728 ng/ml with a limit of detection at 0.5 ng/ml for artesunate and 1.96-2500 ng/ml with a limit of detection at 0.6 ng/ml for dihydroartemisinin. Using 250 microl of plasma sample the lower limit of quantification was decreased to 0.119 ng/ml for artesunate and 0.196 ng/ml dihydroartemisinin. Validation of over-curve samples in plasma ensured that accurate estimation would be possible with dilution if samples went outside the calibration range. The method was free from matrix effects as demonstrated both graphically and quantitatively.

Development and validation of a liquid chromatographic-tandem mass spectrometric method for determination of oseltamivir and its metabolite oseltamivir carboxylate in plasma, saliva and urine.

A bioanalytical method for the analysis of oseltamivir (OP) and its metabolite oseltamivir carboxylate (OC) in human plasma, saliva and urine using off-line solid-phase extraction and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. OP and OC were analysed on a ZIC-HILIC column (50 mm x 2.1 mm) using a mobile phase gradient containing acetonitrile-ammonium acetate buffer (pH 3.5; 10mM) at a flow rate of 500 microL/min. The method was validated according to published FDA guidelines and showed excellent performance. The lower limit of quantification for OP was determined to be 1, 1 and 5 ng/mL for plasma, saliva and urine, respectively and for OC was 10, 10 and 30 ng/mL for plasma, saliva and urine, respectively. The upper limit of quantification for OP was determined to be 600, 300 and 1500 ng/mL for plasma, saliva and urine, respectively and for OC was 10,000, 10,000 and 30,000 ng/mL for plasma, saliva and urine, respectively. The within-day and between-day precisions expressed as R.S.D., were lower than 5% at all tested concentrations for all matrices and below 12% at the lower limit of quantification. Validation of over-curve samples ensured that it would be possible with dilution if samples went outside the calibration range. Matrix effects were thoroughly evaluated both graphically and quantitatively. No matrix effects were detected for OP or OC in plasma or saliva. Residues from the urine matrix (most likely salts) caused some ion suppression for both OP and its deuterated internal standard but had no effect on OC or its deuterated internal standard. The suppression did not affect the quantification of OP.

Validation and application of a liquid chromatographic-mass spectrometric method for determination of artesunate in pharmaceutical samples.

A simple and rapid liquid chromatographic-mass spectrometric assay for the evaluation of artesunate in vials for injection has been developed and validated. The content of each vial was dissolved in 3.0 mL of methanol using a SGE analytical syringe (1.0 mL). Each sample was diluted to a theoretical concentration of 1000 ng/mL and analysed in triplicate. Three replicates of calibration standards at concentrations 500, 1000 and 1500 ng/mL were used to construct a calibration curve. Artesunate was analysed by liquid chromatography with atmospheric pressure chemical ionisation (APCI) mass spectrometric (MS) detection on a Hypersil Gold column (100 mm x 4.6 mm) using a mobile phase containing methanol-ammonium acetate 10 mM pH 5.3 (70:30, v/v) at a flow rate of 1 mL/min. The assay was implemented for the analysis of artesunate for injection purchased from Guilin Pharmaceutical Company in China.

Safety of benzyl benzoate lotion and permethrin in pregnancy: a retrospective matched cohort study.

OBJECTIVE: To assess the safety of benzyl benzoate lotion (BBL) and permethrin, topical treatments for scabies, during pregnancy. DESIGN: A retrospective controlled cohort study. POPULATION: Refugee and migrant women attending antenatal clinics (ANC) on the Thai-Burmese border between August 1993 and April 2006. METHODS: Women treated with either BBL (25%) or permethrin (4%) were identified from a manual search of antenatal records. Each case of scabies was matched with four scabies-free controls for gravidity, age, smoking status, malaria, period of treatment and gestational age at treatment. Conditional Poisson regression was used to estimate risk ratios for outcomes of pregnancy (proportion of abortions, congenital abnormalities, neonatal deaths, stillbirths and premature babies), mean birthweight and estimated median gestational age, for scabies and scabies-free women, independently for BBL and permethrin. RESULTS: There were no statistically significant differences in pregnancy outcomes between women who were treated with either BBL (n = 444) compared with their matched controls (n = 1,776) or permethrin (n = 196) treated women and their matched controls (n = 784). Overall, only 10.9% (n = 66) of treatments were in the first trimester. Retreatment rates were higher with BBL 16.4%, than permethrin 9.7%, P = 0.038. Scabies was more common during cooler periods. CONCLUSION: We found no evidence of adverse effects on pregnancy outcome due to topical 25% BBL or 4% permethrin.

C-reactive protein, interleukin-6, and tumor necrosis factor-alpha levels in overweight and healthy adults.

This study aimed to 1) compare levels of high sensitivity c-reactive protein (hs-CRP), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) between overweight Thais and apparently healthy controls, and 2) investigate the association between serum hs-CRP, IL-6, and TNF-alpha levels and other biochemical parameters. A total of 180 health-conscious adults aged 25-60 years, who resided in Bangkok, participated in this study. No significant difference was found in age and sex between the overweight subjects and controls. Serum levels of hs-CRP, IL-6, TNF-alpha, glucose, lipid profile, body mass index (BMI), waist circumference (WC), hip circumference (HC) and waist hip ratio (WHR) were determined in these volunteers. The mean levels of white blood cells (WBC), uric acid, total cholesterol (TC), triglyceride (TG), and hs-CRP were significantly higher in the overweight subjects than those in the controls, whereas high density lipoprotein-cholesterol (HDL-C) values were significantly higher in the controls than the overweight subjects (p < 0.05). Hs-CRP levels were significantly positively correlated with levels of TG, BMI, WC, HC and WHR. HDL-C levels were significantly negative correlated with hs-CRP levels. In conclusion, the prevalence of elevated serum hs-CRP levels was higher in overweight subjects than controls. However, more data in larger and other population groups are needed to confirm this study.

A simple and rapid liquid chromatographic assay for evaluation of potentially counterfeit Tamiflu.

A simple and rapid liquid chromatographic assay for the evaluation of potentially counterfeit oseltamivir (Tamiflu has been developed and assessed. The assay uses approximately 1mg Tamiflu powder when used for authentication and content estimate. The procedure was validated using 50 replicates analysed during five independent series with a total R.S.D. of 11.2%. The assay can also be used to monitor the exact content of oseltamivir in Tamiflu capsules. One Tamiflu capsule was transferred to a 250mL volumetric flask and 150mL water was added. The flask was placed in an ultrasonic bath at 40 degrees C for 20min to dissolve the capsule. The solution was allowed to cool to room temperature before the flask was filled up to the mark (250mL). A small aliquot was centrifuged and then directly injected into the LC-system for quantification. Oseltamivir was analysed by liquid chromatography with UV detection on a Hypersil Gold column (150mmx4.6mm) using a mobile phase containing methanol-phosphate buffer (pH 2.5; 0.1M) (50:50, v/v) at a flow rate of 1.0mL/min. The assay was implemented for the analysis of Tamiflu purchased over the Internet and at local pharmacies in Thailand and Vietnam.

Elimination of visceral leishmaniasis in Nepal: pipe-dreams and possibilities.

INTRODUCTION: Visceral Leishmaniasis (VL) re-emerged in the Indian subcontinent in the mid-1970s after an almost complete absence in the previous fifteen or so years. The disease was first noted in Nepal in 1978 and, since 1980, it has been reported regularly in increasing numbers. Elimination of visceral leishmaniasis by 2015 has been identified as regional priority program in the level of high political commitment. OBJECTIVE: The objectives of this study are the comprehensive assessment of information related to VL on the basis of past research studies conducted in Nepal, and an assessment of the prospects of control measures. MATERIALS AND METHODS: This was time line comprehensive VL epidemiological assessment study based on the research conducted by main author during the past ten years. During the period the studies were conducted using cross sectional, case control and exploratory study design. The statistical analysis was done using qualitative and quantitative methods. RESULTS: In our study in the visceral leishmaniasis endemic district, Siraha, in the population of 112,029, a total of 996 clinically suspected cases were reported (with fever of long duration and splenomegaly, with no malaria) during 1998-2002. In all, 283 subjects were found positive for visceral leishmaniasis by rK39 and 284 had positive bone marrow. There was no detectable difference in the density of Phlebotomus argentipes between high, and moderate incidence village development committees (VDC: the smallest administrative unit), but collections in the low incidence areas (in winter) were negative. P. argentipes was never numerous (maximum 4.4 females collected per man-hour), and was much less common than P. papatasi. Peaks of abundance were recorded in the March and September collections. We have found that the numbers of reported cases of visceral leishmaniasis in Nepalese villages was unaffected by indoor residual spray (IRS) indicated by parallel trends in case numbers by time series analysis in treated and untreated villages. A series of maps through ten years clearly showed that the infection can move rapidly between villages, and it is impossible to predict where transmission will occur from year to year. CONCLUSION: If maximum benefit in relation to cost is the goal, it may be preferable to put all possible efforts into active case detection (ACD) with free treatment. ACD should involve the network of Village Health Workers or Female Community Health Volunteers and the rK39 dipstick test at health centre level. Surveillance of disease and vector, communication for behavioural impacts and insecticide spraying should be important component of elimination program. If IRS is to be a part of the intervention, it is essential that it is carried out effectively, both in areas where the disease has been reported and in neighbouring areas. Integrated vector management need to be monitored for its application and effectiveness for VL elimination.