In this cross sectional study, subjects were recruited by simple random sampling from PERSIAN Guilan Cohort Study (PGCS) population. The full details of the PGCS have been described elsewhere .In short, The PGCS was a multistage probability sample of the northern Iranian population in which 10520 individuals from 35 to 70 years of age were enrolled since 2014,, as part of the Prospective Epidemiological Research Studies in Iran (PERSIAN)[15, 16].
The sample size was estimated by G*Power version 126.96.36.199 software. The sample size was estimated to be 571 participants, based on effect size (f 2) of 0.05 and the number of related factors (25 independent variable), with a confidence level of 95% and test power of 80%.
The inclusion criteria were participation in PGCS and willingness to complete the questionnaires. The exclusion criteria were 20% missing data in the questionnaire.
Data were collected using a phone interview format by trained interviewers during two periods. First period conducted between 03/23/20 and 03/30/20, at the first peak of COVID-19 pandemic in Iran. Second period conducted between 05/3/20 and 05/10/20, at the first subsidence of COVID-19 pandemic in Iran.
In the first period of study, general characteristics of participants were collected, including age, education, gender, occupation, residency, marital status, underling disease (Includes cardiovascular disease, uncontrolled high blood pressure, uncontrolled diabetes, respiratory diseases and BMI > 40), immune deficiency condition ( corticosteroid use, chemotherapy, malignancies, organ transplants and HIV), pregnancy and past history of COVID-19 infection in participants or their families.
Information about hand-washing behavior and believes were collected twice, in the first period of study (peak of COVID-19 pandemic) and second period of study (subsidence of COVID-19 pandemic).
A data gathering questionnaire was designed to assess the hand-washing behavior and believes, based on recommendations on appropriate hand-washing by WHO and CDC  and Obsessive-Compulsive Disorder criteria in the DSM 5 . Content validity of the questionnaire was confirmed by 20 experts with CVI = 0.8 and CVR = 0.71. A pilot study was conducted with 40 participants prior to the study in order to confirm the face validity and reliability of the questionnaire. The results of this pilot study confirmed the face validity and reliability of the questionnaire with Cronbach’s alpha of 0.86.
Information about hand-washing behavior and believes was collected in following areas:
Section 1 collected data on frequency of hand-washing per day
Section 2 collected data on type of detergent used for hand-washing (categories as only soap, soap + alcohol or bleach)
Section 3 collected data on procedure of hand-washing by 5 yes or no questions to define if they include each step, as follows: 1) Wet hands with running, clean water, 2) rubbing hands together with the soap to lather them, including under the nails, between the fingers, and backs of the hands, 3) Scrub the hands for at least 20 seconds, 4) Rinse the hands well under running, clean water, 5) Dry hands by a clean towel. Each yes answer got one point and the total score was 5.
Section 4 collected data on circumstances of hand-washing by 14 yes or no questions to define if they include each condition, as follows: 1)Before, 2)during, 3)after preparing food, 4)before eating, 5)before, 6)after caring for someone who is sick at home, 7)before, 8)after treating a wound or cut, 9)after toilet using, 10)after or cleaning up a child who has used the toilet or changing diapers, 11)after sneezing, coughing or blowing nose, 12)after animal waste, animal feed, or touching an animal, 13)after pet treats or handling pet food, 14)after touching garbage. Each yes answer got one point and total score was 14.
Section 5 collected data on worries about hand-washing or obsessive-like behaviors by 8 yes or no questions to define if they experience each item, as follows: 1) worry about remaining a contamination on a properly washed hand, 2) afraid or anxious about hands are not clean enough, even after repeated rinsing, 3)wash in a special order, 4) starts washing again for fear of mistake, 5) avoid places or situations where may be exposed to germs or soil, 6)avoid touching surfaces in public places, 7) red, cracked and scaly hands due hand-washing, 8)effect of pollution concerns and efforts to reduce them in daily relationships and activities. Each yes answer got one point and total score was 8.
Change in type of detergents, procedure and circumstances of hand-washing and obsessive-like behaviors, over time (at the subsidence versus the peak of COVID-19 pandemic) were examined by McNamar tests. Change in hand washing frequency (the frequency was categorized to, 0–4 times, 5–8 times, 9–12 times, more than 13 times) over time was examined by Wilcoxon test. Change in score of obsessive-like behaviors and score of procedure and circumstances of hand-washing over time was examined by paired T test.
Independent T test and chi-square test were used to examine the differences of procedure and circumstances of hand-washing score, obsessive-like behaviors score and reduction in hand hygiene behaviors between male and female.
Additionally, to identify factors related to decrease in hand-washing frequency at least one time, simple and multiple logistic regression models stratified by gender were used. Factors with a p value < 0.05 on a univariate analysis were entered into a multivariate analysis. Unadjusted and adjusted odds ratios (ORs) with 95% confidence intervals (CIs) were calculated.
The SPSS version 17.0 software (SPSS Inc., Chicago, IL, USA) was used to for all data analysis.