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  • Study design and study participants: A cross-sectional survey was carried out recruiting a purposive sample of ST consumers in Dhaka, Bangladesh. We recruited 200 current ST users aged 18 years or older, who were non-smokers (either never smokers or former smokers who have not smoked for at least past one year). Current ST users were defined as those using ST products for at least one year, with a minimum of one can or pouch of ST used per week. Those with a history of other substance abuse or psychiatric illnesses were excluded. Participants were recruited purposively from two urban cities in Dhaka, namely Mohakhali and Mirpur, through convenience sampling techniques, including snowball sampling and community referrals. Trained field researchers recruited participants by visiting houses in these communities, at community gatherings, ST shops, and through personal referrals. Researchers first approached local leaders in the study areas and people buying ST products from shops and explained the study objectives. Local leaders and ST users gave references of their relatives, friends, and neighbours who use ST. We recruited male and female ST users in equal proportion. Taka 200 (GBP 1.5) was paid to respondents as opportunity cost of their time. Interviews and saliva sample collections were conducted at ST users’ home or other mutually convenient place, such as their workplace. Informed written consent was obtained from all study participants. Data were collected between September and November 2014. This study was approved by the Bangladesh Medical Research Council (BMRC) and the Research Governance Committee at the University of York, UK. A survey containing a set of questionnaires was administered by trained interviewers to collect information on the; (a) socio-demographic characteristics, including age, sex, education, and household asset ownership; and b) tobacco use behaviours, i.e. quantity (in grams) of ST used per week, number of chews/dips per day (DPD) in the past week, duration of ST use in months, type of ST products used, swallowing of tobacco juices, urge and strength of urge to use ST, Fagerström Test for Nicotine Dependence (FTND- ST), Tobacco Dependence Screener (TDS), attempt to cut down on ST use in the past, and past smoking status. ST products are generally sold in cans and packets that vary in weights. We standardized our quantity estimates by showing participants cans and packets of 10, 20 and 30 grams. We asked the respondent to point out to the size of cans and packets they generally buy and how long does it take for them to consume these. From this information, we estimated the amount of ST in grams consumed during an average week. Measures of dependence, FTND-ST and TDS were adapted through the process of translation and back-translation, and cross-cultural validation for use in Bangladeshi culture and language. Translation and back-translation was done by bilingual and bicultural translators outside the research team [16]. To measure cotinine concentration, saliva samples were collected from all participants at the interviews using Salivette, oral swab and swab storage tube. Saliva samples were taken at least 30 minutes after eating, drinking or taking medication. A sterile cotton swab was tipped under the tongue without touching, and was left there until it became soggy. This took up to five minutes. The swab was then directly spat into the kit and closed firmly with the stopper. The saliva samples were kept at the study centre at room temperature for a maximum of four days before being shipped to the UK and subsequently tested for cotinine at ABS Laboratories, UK. The samples were frozen at -20 degrees immediately on receipt in the UK and then thawed for analysis. Cotinine was determined using a validated Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay in human saliva using cotinine-d3 to internally standardize the procedure. The method with a calibration range of 1 to 750 ng/mL was applied to this study as the samples were from subjects that chewed tobacco. Cotinine was extracted from human saliva using 50 μL of sample using liquid/liquid extraction with ethyl acetate. The solvent was removed under nitrogen and the extract re-suspended in methanol for quantitative determination using Hydrophilic interaction liquid chromatography (HILIC) on a Thermo perfluorinated phenyl (PFP) column using LC-MS/MS with multiple reaction monitoring (MRM) on an Agilent 1100 LC system interfaced to an ABSciex API 4000 tandem mass spectrometer. Analytical acceptance criteria were considered as specified by the Food and Drug Administration [14]. Exploratory analysis of all variables was performed. Geometric mean was calculated for salivary cotinine concentration across socio-demographic and tobacco use behaviours. We also explored whether differences in proportion of tobacco use behaviours exist across sex of the participants. Simple and multiple linear regression analyses were conducted to compute crude and adjusted estimates for association of variables with saliva cotinine levels. Assumption of normality was not met for the bivariate analysis and saliva cotinine levels were positively skewed; therefore we applied logarithmic transformation to salivary cotinine levels for statistical analyses. Regression diagnostics were performed to evaluate multicollinearity and influential observations. We set the cut off value of Variance Inflation Factor >5 to exclude the factors from the final model [17]. None of the variables met this criterion. However only those variables that were significantly associated with cotinine levels in simple linear regression were included in the final model due to the limited sample size. All analyses were performed using SAS v.9.4 (SAS Institute Inc. Cary, NC, USA) and a level of 0.05 was used for statistical significance.
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