Serosurvey of hepatitis A virus and E virus infection among municipal sweepers working in the largest city in the south of Iran

ABSTRACT This study was conducted to determine the exposure rate of Hepatitis A and Hepatitis E viruses in urban solid waste collectors/sweepers in the south of Iran. The 385 samples (serums) were collected from Shiraz Municipality waste sweepers.. A questionnaire was used to gather data on their demographic and occupational characteristics, as well as their awareness of viral hepatitis disease. The viral seroprevalence was determined by commercial IgG ELISA kit. All participants were male, mean age of 41 ± 8 years. ELISA assay showed that all of them were positive for anti-HAV IgG. Also, 62 out of 385 individuals were positive for anti-HEV IgG. The statistical analysis showed that the frequency of HEV IgG antibody among age groups 20–30, 31–40, 41–50 and >50 years old had an increasing trend, 4.5%, 10.1%, 17.4%, and 36.7%, respectively, indicating age factor significance (p = .001). Based on some investigated factors including the duration of work experience, current and previous jobs, habitation, personal hygiene status, and knowledge on viral hepatitis diseasees/their transmission, there was no statistically significant difference between anti-HEV IgG positive versus negative sweepers. The results indicated a slighty higher frequency of anti-HAV and anti-HEV IgG among sweepers compared to other pre-investigated population. It doesn’t seem that garbage collecting/sweeping could be a significant risk factor for HAV and HEV infection.


Introduction
Hepatitis A (HAV) and Hepatitis E (HEV) infections are still worldwide concerns of acute hepatitis. Both of viruses transmit oral-fecally and cause a self-limiting 1hepatic disease without any longitudinal sequels. [1] The global disparity in HAV and HEV prevalence depends on the degree of hygiene and health, in addition of some factors, such as socioeconomic status. [2] HAV is mainly transmitted through contaminated food,water and through person to person transmission. [3] Given to the presence of inactivated vaccine and increased sanitation status, the prevalence of HAV virus is decreasing in the developed societies, however the rate is still high. [4,5] However, there are evidence that in developing countries the number of people infected with HAV remain significantly high. [2] In one study, the anti-HAV antibody was found in 99.2% of the people >30 years old in Fars, Iran. [6] Moreover, another study in Shiraz indicated that the lower rate of about <70% seroprevalence, among 15-yearold subjects. [7] The prevalence of HAV infection is mainly affected by general hygiene, especially with regard to toilet facilities, water supplies, and food preparation methods, indicating living standards and socioeconomic status. [8] Many studies have indicated that some jobs are at higher risk of HAV and HEV infections due to the path of transmission. Some workers such as military personnel, social workers and health workers are more at risk of being infected with these viruses due to frequent contaminants, water, and sewage. In particular, sweepers could be at higher risk of infection by HAV since they are in daily contact with contaminats. [9] Sweeping and collecting garbage are among the careers in which the spread of oral-fecal infections might be more common. A related study on sweepers has indicated that they are at high risk of infection with Toxocara infection. Therefore, oral-fecal contamination by several microorganisms circulating among humans in this way is common in the mentioned group. [10] HEV is spread through 2 different epidemiological patterns through contaminated water or under cooked meat from zoonosis resources. [11,12] In the developing area, the main route of HEV infection is the consumption of contaminated water. HEV has a different serological worldwide pattern which reaches 15-20% in the developing countries, but it is still about 5% in the developed countries. [13,14] Iran, as one of the Middle-East countries, is an endemic area for HEV infections, despite the frequent reports of various seroprevalence rates in different provinces. The previous studies have reported that the prevalence of HEV is roughly estimated near 10% with a maximum and minimum of 46% and 1%, respectively. [15,16] A recent study also emphasized that HEV infection is still endemic herein. [17] Raw sewage and water supplies contain HEV isolates; therefore, street sweepers could be at risk of HEV exposure. [18] So far, any study which is addressing the rate of HEV antibody or genome in sweepers is rare unless in Pakistan that high prevalence of HEV RNA genome (45%) in water sewerage were detected. [19] Therefore, the present study aimed to determine the seroprevalence of HAV and HEV in waste collectors of Shiraz, southern Iran.

Patients and blood sampling
This is a cross-sectional study on sweepers/waste collectors in Shiraz, the biggest city in the south of Iran, early 2019. A total of 385 sweepers from all 10 districts of city were enrolled based on general announcement. All the cases were recruited by a general announcement and during the bimonthly routine health check in related centers of different districts. In each districts, near 40-50 individuals were enrolled if their blood samples were available. It took 3-4 months that all samples were collected. They consented to participate in study before filing in the questionnaire file. Their blood samples were collected and sera were isolated. All sampling stages were performed on participants who signed the written consent forms. The study was approved based on the ethical standards of the Shiraz University of Medical Sciences, with the code number of IR.SUMS.MED.REC.1389.259. 5 ml blood sample was collected from each patient. The sera were separated from the whole blood by centrifugation at 3000 RPM, and the separated sera were stored at −20°C until use. The questionnaire contained questions on demographic characteristics, sanitation status at work, wearing mask and/or gloves, hand washing, and other factors.

ELISA test to determine past virus exposure
In order to investigate the exposure rate of the participants to HAV and HEV viruses, the IgG antibody against them was determined by commercial Enzyme linked Immunosorbent assay kits (Dia.Pro. Milano, Italy), according to the manufacturer's instructions. Index values for seropositivity in each test were calculated based on the instructions of the kits.

Statistical analysis
Statistical analysis was performed by using SPSS (version 16.0; SPSS Inc., Chicago, IL, USA). In addition to the descriptive test, Chi-square test was used to compare different variables and find out the association of two-way tables. A p-value less than 0.05 was considered significant.

Results
All 385 street sweepers were men with a mean age of 40.98 ± 8.437 years old (Table 1). Surprisingly, all individuals who were positive for IgG Ab against HAV indicated evidence of past infection with HAV. Therefore, no more statistical analysis regarding the HAV and regarding demography and sanitation status at work was performed.
Moreover, the results indicated that 16.1% (62/385) of street sweepers were positive for IgG antibody against HEV. The mean age of street sweepers that were positive and negative for IgG Ab against HEV were 46.31 ± 8.338 and 39.95 ± 8.073 years, respectively, that was statistically significant (p˂0.01).
Also, statistical analysis showed that the frequency of IgG antibody against HEV among the age group of 20-30, 31-40, 41-50, and over 50 years old were 4.5%, 10.1%,17.4%, and 36.7% that was statistically significant (P < .001), with an increasing trend. Based on the sweeper's work experience, the results among anti-HEV seropositive individuals indicated that 16.9% (52/307) of street sweepers had five or more than 5 years, and 13% (10/77) had less than 5 years of work experience in all professions, and the difference was not significant (p > 0.05). According to the relationship between the sweepers' current and previous jobs, 16.7% (52/307) of anti-HEV seropositive street sweepers had been working for more than 5 years as a sweeper; 11.8% (6/51) of them had previous work experience the same as the current job, and 19% (4/21) had unrelated previous work experience. As to the sweeper's living place, 16.4% (55/336) of the anti-HEV seropositive street sweepers lived in urban areas and 14.3% (5/35) in rural areas.
One of the parameters considered in this study was anti-HEV seropositive individuals' personal hygiene. The results revealed that 16.7% (48/ 288) of them washed their face and hands twice or more, and 18.2% (4/22) did so fewer than twice, and the difference was not statistically significant The highest prevalence of anti-HEV IgG was detected in municipal street sweepers in districts 1 (27.8%) and 2 (24.1%), and the lowest prevalence was discovered in districts 4 (8.9%) and 8 (9.8%).
In addition, another point which is addressed in this study was the status of overall knowledge of participants about some general aspects of viral hepatitis. The analysis of the questionnaire showed that out of all sweepers in terms of transmission routes of hepatitis viruses 15% (9/60) from HEV seropositive and 13.3% (42/316) from HEV seronegative group were aware of the correlation between needle stick, drug abuse, and tattooing with hepatitis transmission. Also, 13.3% (8/60) seropositive and 11.7% (37/316) seronegative group knew about the importance of blood transfusion in hepatitis viruses infection, moreover just 6.7% (4/60) from seropositive, and 8.2% (26/316) from seronegative group knew about the available hepatitis vaccination. In addition, none of the seropositive street sweepers had information about available treatment modalities for viral hepatitis, whereas it was 0.6% (2/316) for seronegative ones.

Discussion
Given the importance of hepatitis in international communities, understanding the exposure rate in some careers not only can contribute to global prevention but also would suggest more possible risk factors. In this current study, for the first time, the seroprevalence of hepatitis E and A viruses was investigated among sweepers from all districts of Shiraz municipality, the largest city in the south of Iran, 2019.
Previous studies reported that the seroprevalence of HAV among adults (around 30 years old) in Iran and Fars province was over 82.6%, [20] and 99%, [6] respectively, which was much similar to our study that revealed a 100% rate among street sweepers.
As the second step, this study showed that 16.1% of the investigated sweepers were positive for anti-HEV IgG antibody. At first glance, sweeping as a career seems to be at high risk of oral-fecal viral transmission however limited study take this issue into account. [21] A study carried out by Asaei et al. in Shiraz reported the sero-prevalence of 13.4% for total anti-HEV among the healthy population. [13] Recently, it has been reported that 10.4% (26/151) of HIV infected patients in Fars Province were positive for anti-HEV antibody. [22] A recent study in India indicated a higher concentration of fecal pollution, as marked by E.coli detection, at the end of the daily work activities. [23] Also, another study on our investigated sweepers has indicated a high risk of contamination by Toxocara parasite. [10] In Egypt, parasitic infestations including Entameba Histolytica and Hymenolepis Nana were detected more significantly in stool analysis of street sweepers than university employees. [24] The findings of Kretchy et al. revealed that solid waste collectors should be provided with proper protective measures and sanitation facilities, and they should be trained on hygienic behaviors to prevent oral-fecal contamination by food/water-borne contaminants. [25] In the case of HEV, few studies have been evaluated to determine its seroprevalence among street sweepers or waste collectors. In 2003, Vadiya et al. showed that 56.5% of sewage workers were anti-HEV IgG positive; this is significantly higher exposure rate than the relevant normal population. Therefore, a career with direct contact with sewage or sewage-contaminated materials can be considered as a risk factor. [26] In addition, a study by Farshadpour et al. has shown a significant difference between the prevalence of HEV antibodies and the habitation place. [27] Meanwhile, the study of Saffar et al. in 2009 revealed a significant relationship between seroprevalence of this virus and habitation in rural areas. [28] In our study, totally 16.4% of street sweepers were habitants of rural areas, and of them 14.3% were HEV seropositive, which has no significant association.
As a new finding, the study revealed that only a small portion of both IgG positive and negative groups were aware of hepatitis vaccination. In addition, they have rarely heard about the available treatment of hepatitis. The awareness of employee of at risk career should be increased by simple modalities.
Age is a common factor contributes to exposure rate of pathogen. The statistical analysis showed that the frequency of HEV IgG antibody among age had an increasing trend (from 4.5% to 36.7%), which indicate its significance. Asaei's study revealed that the seroprevalence of anti-HEV IgG is directly related to age in sewage collectors, which is in accordance with our results. [13] Also, in Vaidya's study, a significant increase of anti-HEV IgG was reported in those in contact with urban garbage for more than 5 years (67.6%) compared to those who had worked for less than 5 years (38.9%). [26] In the present study, the anti-HEV IgG seroprevalence was a bit higher in people who had over 5 years of experience comparing to people with less experience; however, no statistically significant relationship was observed with the HEV seroprevalence.
Although, sanitation such as hand washing has been recommended in most articles as an effective way for public health. [29,30] In contrast, we did not find a relationship between the seroprevalence of HEV and hand washing. The present study also confirmed that 17.1% of street sweepers who washed their hands with soap and water and 15.8% of those who used only water to wash their hands and faces were positive for anti-HEV IgG. However, it is clearly indicated that sanitation status makes this population more vulnerable to infection by food/waterborne microorganisms, as demonstrated previously. [25] Also, 16.7% of street sweepers who washed their hands and face more than twice during and after finishing their work and 18.3% of those who did so fewer than twice were positive, respectively. However, no significant relationship was found between the sero-prevalence of anti-HEV and the number and method of washing hands and faces. On the other hand, Dahiya et al. pointed out the importance of using mask to prevent HEV infection which is transmitted through the mouth. [31] In the present study, 14.9% of street sweepers who always used mask during sweeping and 19.4% of those who rarely did so were HEV seropositive. However, no significant difference was found between the seroprevalence of anti-HEV and wearing face mask.
The absence of molecular assays to find the status of virus genome among the individuals and non-sweeper men in the control group was a limitation of the present study. In conclusion, although our results showed that 100% and 16.1% of street sweepers were positive for anti-HAV and HEV IgG Ab respectively, which was slightly higher than the normal population. However, it does not seem that garbage collecting/sweeping career is significant risk factors of HAV and HEV infection.
In conclusion, based on unknown parameters, the frequency of HEV IgG antibody among sweepers is not significantly different from normal population.