Evidence of Hantavirus circulation among municipal street sweepers, southwest of Iran

Abstract

Hantaviruses are rodent-borne zoonosis pathogens that cause hemorrhagic fever with renal syndrome (HFRS) and Hantavirus cardiopulmonary syndrome (HCPS) in humans. Rodents spread the virus via their excretions. The outbreak of Hantaviruses pose a significant public health problem. The epidemiology and history of Hantaviruses in Iran is not clear and regardless of the data from the few available studies, little is known about its epidemiology in this country. Herein, we discuss the prevalence of IgG antibody against Hantavirus serotypes in 385 street sweepers from southwest of Iran. Serum samples were investigated, using Hantavirus Pool 1 "Eurasia" IgG kit and Pool 2 "America" ELISA IgG kit (Euroimmun, Germany) to detect IgG antibodies against Old and New World Hantaviruses. The results showed a specific IgG antibody in two samples (0.5%). Both of seropositive cases had specific IgG antibody against Old World Hantaviruses. The data of the current study along with the previous data, indicate the circulation of Hantaviruses in Iran. Hence, the risk of Hantavirus infection in high-risk groups should be considered as a serious health issue.

Introduction

Hantaviruses are one of the most important rodent-transmitted zoonotic viruses [1]. They are enveloped, single-stranded, segmented RNA viruses, classified in Hantaviridae family within the order Bunyavirales and genus Orthohantavirus [2]. Hitherto, more than 28 pathogenic Hantaviruses have been discovered. Human infection with pathogenic Hantaviruses can result in two lethal disease i.e.hemorrhagic fever with renal syndrome (HFRS) with a case fatality rate of 12% and Hantavirus cardiopulmonary syndrome (HCPS) with a case fatality rate of 50% [3–5]. HFRS is reported from many countries in Eurasia, and is associated with oldword Hantaviruses such as Hantaan, Puumala and Dobrava. HCPS is endemic in the Americas and is associated with new-world hantaviruses such as Sin Nombre, Black Creek Canal and Andes [6].

Rodents are the primary reservoirs of Hantaviruses in nature and shed the virus into their defecations such as urine, droppings and saliva [7]. Human infection usually develops through inhalation of aerosolized rodent excreta. Consequently, the more exposure to rodent excreta, the higher risk of infection. Animal trappers, farmers, campers, forestry workers, military staff and street sweepers are the high risk group for Hantavirus infection [5, 8].

Owing to the fact that both incidence and geographical distribution of Hantavirus infection have increased in recent years, these rodent borne viruses have received much attention as a threat to word's public health [9].

Although it is believed that Hantaviruses are endemic in most parts of the world, the majority of data on the prevalence of Hantaviruses have been published from the Americas, Europe and eastern Asia [10]. Apart from few studies in Iran, little is known about the epidemiology of Hantaviruses in this country.

With respect to the prevalence of Hantavirus in Iran, Dr. Amin-ol Ashrafi reported an epidemic suspicious to unknown HFRS related with Asian HRS, in a group of patients from Azarbayjan Iran, 1969 [8, 11]. Although this report was not confirmed by laboratory diagnosis, it is the first study stating the importance of HFRS in the country. In a study by Chinikar et al., 2014, Hantavirus antibodies and genome was evaluated in sweepers from Isfahan who had developed acute nephropathy by molecular and serological assays [8]. In a recent study published in 2019, Hantavirus seropositivity (antibodies against Hantaan and Puumala viruses) was confirmed amongst Iranian patients with viral hemorrhagic fever symptoms [12].

Also, the data on Hantavirus prevalence is insufficient in the neighboring countries. However, Turkey is considered as one of the sporadic areas for the infection, since they have detected less than 10 cases related to the disease [13]. Nonetheless, China [14] and Russia are considered as areas with high prevalence, with 100 and 100.000 detected cases, respectively [15]. In these countries, Hantavirus infections were mostly associated with HFRS.

Therefore, the aim of the current study was to primarily evaluate the seroprevalence of Hantavirus amongst sweepers as a high-risk group in Shiraz, the biggest southern city in Iran.

Materials and methods

Samples

This Cross-sectional study was conducted on municipality of Shiraz sweepers, the biggest city in southern Iran, 2018. A total of 385 sweepers from the 10 districts of Shiraz were enrolled randomly. After explaining the study objectives, an informed consent was signed by each participant. Their blood samples were collected and sera were isolated.

The demography, sanitation status at work considering face mask wearing and using gloves as well as hand/face washing data were considered in the questionnaire. In order to categorize the rate of dust exposure, sweepers with > 2 h working with heavy aerosolized soil were selected as the high exposure individuals while those with less than one hour classified in the low exposure group.

The study protocols and ethics were confirmed by the local Ethics Committee of Shiraz University of Medical Sciences (code: IR.sums.med.rec.1396.s10).

ELISA assay

Hantavirus Pool 1 "Eurasia" IgG kit and Pool 2 "America" ELISA IgG kit (Euroimmun, Germany) were used to detect IgG antibodies against OldWorld Hantaviruses and New-World Hantaviruses, respectively. ELISA tests were performed according to the manufacturer’s instructions. Samples with a ratio of  ≥ 1.1 were considered as seropositive. Samples with a ratio of ≥ 0.8 to < 1.1 were regarded as borderline, and samples with a ratio of < 0.8 were considered as negative.

Statistical analysis

The descriptive analysis was employed when comparing the data. Based on data normality test, the Shapiro–Wilk, the Kruskal–Wallis tests were used to compare groups. P value < 0.05 was considered to be statistically significant. All statistical analyses were carried out by GraphPad Prism™ version 5.

Results

Demographic results

All the participants were male with the mean age 41.72 ± 16.52 (range: 24–64) years. Totally, 77 out of the 385 (20%) sweepers had job experience less than 5 years. Their work schedule was from early morning until 2 P.M, and their main task was sweeping the streets. They had not been admitted to clinical center or were not suspected to hemorrhagic fever related infections, during the last year.

ELISA

The ELISA results showed that only 2 out of 385 cases (0.5%) were Hantavirus IgG antibody positive (optical density of samples were 0.2 and 0.3 and OD of positive control was 0.7; 450–620 nm). Both seropositive samples were reactive against Eurasia Hantaviruses, but not to American ones. Therefore, further statistical analysis was merely considered by descriptive rather than analytical.

Sanitation status and rodent/dust disposal

Overall, 49 out of the 385 sweepers (12.7%) were living in rural and 336 (87.3) in the urban areas. Two cases with positive ELISA result were living in the urban areas.

Of all samples, 54 (14%) had low exposure to dust, whereas 331 (86%) had high exposure to dust during work. Both seropositive sweepers belonged to the high exposure group.

Totally, 41.8% of the sweepers had seen rodents at their workplace. One of seropositive sweepers had seen rodents at work and had high exposures to dust during work.

The majority of sweepers (92.8%) including the seropositive individuals did not have a history of acute pulmonary as well as kidney distress for at least within the past two years.

In total, 71.7% of the sweepers always wore face masks during work, but 28.3% used face masks occasionally. Of the two seropositive sweepers, one who was exposed to dust and rodents declared that he always wears mask at work, but the other one did not consider wearing a mask at work.

The assessment of hand/face washing showed that 84.7% of sweepers washed their hands and face more than twice a day at work, and 94.8% used soap. The two hanta seropositive sweepers also used soap to wash their hands and faces.

Discussion

Hantaviruses are transmitted by rodents' feces and secretions. Epidemiological studies have linked Hantavirus infection with outdoor activities, especially where people are exposed to infected materials. In the present study, we investigated Hantavirus seropositivity amongst street sweepers as one of the high-risk groups in Fars province, Iran. Our results showed a seropositivity rate of 0.5% (2 of 385) which is much lower than the rate reported by Chinikar et al., 2014 [8], where they found IgG and IgM antibodies against hantaviruses in 4.5% (9 of 200) street sweepers in Isfahan, central Iran. This difference can be explained by two reasons. First and foremost, the participants in the study by Chinikar et al. were selected amongst the individuals with recent renal failure whereas all cases in our study had not declared any medical conditions compatible with HFRS. Second, these two studies were conducted in different geographical areas. Our results however are in line with the seroprevalence of less than 1% in a very small sample from Shiraz, indicated by another study investigating antibodies against Hantaviruses in Iranian patient suspected of viral hemorrhagic fevers [12]. However, as the first study on sweepers population from a big city the very low rate of Hantavirus exposure was not supposed to be achieved. Due to the short-term viremia in humans, diagnosis is usually based on serology which the data are affected by antigen cross-reactivity. These cross-reactivity may disturb the interpretation of serological results, especially in areas where multiple Hantaviruses co-circulate in the region.

Rodents are considered reservoirs for Hantavirus. Therefore, the more exposure to infected rodents excreta means the higher risk of Hantaviruses transmission [16]. In the present study, one of the two seropositive cases reported the presence of rodents in their workplaces. In the study by Chinikar et al., the majority of seropositive cases (44.4%) reported high prevalence of rodents in their workplace [8]. Although the prevalence of rodents is directly associated with the risk of hantavirus infection, adopting mitigating measures such as using face masks could have been helpful in reducing the probability of inhaling infected aerosolized rodent's excreta [17]. In this study, while 86% of cases were exposed to dust, most of them (> 70%) always used face masks while working. In addition, sanitation factors, such as wearing mask and handwashing would inhibit the distribution of pathogens including Hantavirus [17].

Species diversity of Hantavirus reservoirs in Iran is considerable. Approximately, 79 rodent species from 31 genera and eight families have been found in Iran. In spite of availability of little data on the epidemiology of Hantavirus in Iran, the presence of the reservoirs of OldWorld Hantaviruses has been documented in the country. For instance, Rattus norvegicus and Rattus rattus (reservoirs of Seoul hantavirus) are reported in a wide geographical range in Iran [18, 19]. Rodents from genus Apodemus and Microtus have also been reported in the western and northern parts of Iran [20–22]. Therefore, regardless of low infection rates reported by the present study as well as the previous investigations in Iran, the risk of Hantavirus infection should be considered as a serious health concern in high-risk groups. Performing studies both on human and rodent populations can shed a light on the epidemiology and the burden of Hantaviruses in Iran.