A tropical disease of zoonotic origin, Leptospirosis is a public health concern, particularly in developing countries. Caused by the Leptospira species of pathogenic bacteria, Leptospirosis was primarily thought to be an infection related to jobs and rural environments, currently, this disease is growing in urban and rural areas [1, 2]. Concerning the impact of leptospirosis on people's health and well-being, and its correlation with low health levels, this infectious disease can cause the continuation of poverty . The epidemiology of leptospirosis is very complicated due to the multiplicity of factors that might affect the likelihood of infection on a regional basis . Risk factors for contracting Leptospirosis are generally associated with factors that contribute to the survival of the bacterium, i.e. exposure to water or dirt which has been contaminated by animal urine . Equally important determinants of disease transmission include skin contact with intermediate animals or their excrement, monsoon, and floods, and occupational exposure .
The clinical manifestation may range from a febrile illness of mild severity to high-grade fever with systemic complications and mortality. While Leptospirosis is rather an acute disease in humans, cases of prolonged illness have also been documented [7-9]. Manifestations of this disease vary from fever with an unknown source to potentially fatal complications including Villo disease and severe pulmonary hemorrhage syndrome, which may result in a mortality rate of 5-40%. As such, timely diagnosis coupled with the administration of effective antibiotics is of utmost importance . On a global scale, Leptospirosis is estimated to affect 1.03 million individuals, of whom 58,900 die annually. This marked prevalence is associated with roughly 2.90 million disability-adjusted life years (DALY) . The pathogenic Leptospira strain is able to survive in the external environment and kidney tubules of infected animals .
The host, humans in this case, usually becomes infected upon exposure to carrier animals or urine-contaminated environments .
Potential reservoirs of spirochetes  include wild animals such as rodents, along with canine, bovine, swine, and equine families of domesticated animals . Leptospirosis is often diagnosed based on serologic detection of antibodies, which are present at the detectable levels in the blood within 5 to 7 days following the onset of symptoms . Microscopic agglutination test (MAT) and ELISA are the most frequently preferred serologic tests for Leptospirosis diagnosis. Although leptospirosis has the highest prevalence in tropical regions, its prevalence has recently been reported to show an ascending trend in temperate regions as well, which is attributed to increased populations of rodents in urban areas, rainfalls, and floodings and global warming [15, 16].
As of today, the pathogenesis of leptospirosis is yet to be fully explained, as it appears to differ from the other bacterial infections to some extent. In spite of a marked rise in the count of white blood cells or leukocytosis in hospitalized cases, there is no evidence to suggest that neutrophils might be involved in the upregulation of cell markers associated with increased inflammatory response .
Generally regarded as an occupational disease associated with activities with certain levels of exposure to contaminated water and infected animals, Leptospirosis has also been reported in individuals performing recreational activities in humid environments without apparent exposure to presumed contaminated sites [18, 19]. Individuals who travel from other parts of the world to tropical regions are thought to be highly vulnerable to Leptospirosis .
As such, early diagnosis is a prerequisite for timely administration of antibiotics, which is highly important in cases with severe disease .
The disease could be prevented by avoiding contact with contaminants either directly or indirectly, which is not a feasible preventive measure for individuals whose job mandates continued exposure to such contaminants . Concerning the zoonotic nature of leptospirosis and its well-established prevalence in neighboring provinces, it is important to have a better understanding of the frequency of Leptospirosis in Zanjan province, which may prove useful in the proper management of patients presenting with fever and jaundice, and differentiation of the bacterial illness from hemorrhagic fevers of viral origin. Here, we sought to investigate the seroprevalence of Leptospira in healthy people referred to Shahid Motahari Laboratory, Zanjan, Iran.
Materials and Methods
The present work was a prospective cross-sectional investigation carried out from February to April 2020 to assess the seroprevalence of Leptospira among healthy people referred to Shahid Motahari Laboratory, Zanjan, Iran for checkup. This laboratory is located in a rural immigrant area. Inclusion criteria of participants in this study were (1) healthy people aged ≥ 18; and (2) willingness to participate in the study (all participants were asked to provide written informed consent). We did not limit our inclusion criteria to a particular sex or occupation. Participants were selected using simple random sampling method. The study design was approved by the Research Ethics Committee of Zanjan University of Medical Sciences (IR.ZUMS.REC.1398.460).
According to Araghian et al. and Naddaf et al. studies [23, 24] that the seroprevalence of leptospirosis in Iran was reported at about 48-50%, and assuming a confidence level of 98%, and a margin of error (precision) of 5%, the estimated sample size was determined to be 181.
Blood samples and data collection
Two milliliters of blood sample were collected from a peripheral vein of each participant under aseptic precautions, coagulated at room temperature and then centrifuged at 4500 rpm for 5 min. The obtained serum samples were stored at -20 °C for serological analysis. All participants were interviewed using a questionnaire including age, gender, educational level, occupation, history of fever or underlying diseases, and contact history of exposure to domestic animals.
Anti-Leptospira IgG antibodies were measured in sera of participants using "NovaLisa Leptospira IgG ELISA" kit (NovaTec Immundiagnostica GmbH, Dietzenbach, Germany). Briefly, Leptospira antigen-coated microwells were incubated with 100 µl of serum diluted with a ratio of 1:100 using IgG sample diluent for 1 h at 37 °C. Once the wells were washed using phosphate buffer (0.2 M), bound antibodies were detected with Leptospira anti-IgG Conjugate (100 µl per well) and 3,3',5,5'-tetramethylbenzidine (TMB) substrate (100 µl per well). Following 30 minutes from the addition of the Stop solution, the light absorbance of the specimens was measured at wavelengths 450 and 620 nm. Positive and negative controls, along with cut-off controls and substrate blank samples were analyzed in each run. Results were expressed by Cut-off calculation. The cut-off was set to mean absorbance value of the cut-off control determinations. Results were reported to Unit [NovaTec Unit = NTU] as follows: [NovaTec Unit = NTU] = patient absorbance value × 10/ Cut-off. The results were interpreted as follows: NTU> 11 was interpreted as positive; NTU between 9-11 was interpreted as equivocal and NTU< 9 was interpreted as negative. Based on the information provided by the manufacturer, the "NovaLisa Leptospira IgG" kits were > 98% sensitive and 97.4% specific in terms of diagnostic accuracy.
The data were analyzed using IBM SPSS version 17.0 (SPSS, Inc., Chicago, IL). Statistical significance was determined based on the chi-square test. A P-value < 0.05 was assumed to be the threshold of significance.
Results and Discussion
A total of 181 participants, comprising 91 males (50.3%) and 90 females (49.7%), with a mean age of 40.6, were included in the study. The Anti-Leptospira IgG results were categorized as positive, equivocal, or negative. Among the 181 serum samples, 43 (23.7%) tested positive for anti-Leptospira IgG, 36 (19.9%) were equivocal, and 102 (56.4%) were negative.
Table 1 lists the sociodemographic characteristics of the participants and their correlation with anti-Leptospira IgG. In terms of age distribution, 60 participants (33.1%) were aged 18-30 years old, 69 participants (38.1%) were aged 31-60 years old, and 52 participants (28.7%) were over 60. The analysis revealed no significant difference between age and gender concerning anti-Leptospira IgG, based on the chi-square test (P> 0.05). Regarding occupation, approximately 44.2% of participants were housewives, and 36.5% were employed. However, our investigation did not identify any statistically significant correlation between occupation and anti-Leptospira IgG, based on the chi-square test (P> 0.05). Regarding medical history, 99.4% of participants had no prior history of fever, and 71.8% had no underlying diseases. Nevertheless, our analysis found no significant association between these risk factors and anti-Leptospira IgG, based on the chi-square test (P> 0.05). Out of 181 participants, 59 (32.6%) had a village living history and 27.6% of individuals had a contact history of exposure to domestic animals.
Table 1: Demographic information of the participants
However, none of these factors were found to be significantly correlated with anti-Leptospira IgG, based on the chi-square test (P> 0.05).
The findings revealed that 23.7% of the 181 serum samples were positive for anti-Leptospira IgG antibodies, 19.9% were equivocal, and 56.4% were negative. The average age of the participants was 40.6 years old, and no significant correlation was observed between age, gender, rural residence, or other demographic characteristics and anti-Leptospira IgG levels. According to our findings, 43 (23.7%), 36 (19.9%), and 102 (56.4%) subjects had positive, suspected, and negative leptospirosis serology, respectively, which, to some extent, is in agreement with the results of Regmi et al.'s study 21% and Delmas et al. (19%) [25, 26]. Meanwhile, the overall prevalence of leptospirosis in an investigation by Gonwong et al. in Thailand was reported to be 28%, which is higher than the overall prevalence of leptospirosis in the present study.
Factors such as high rodent population, high humidity, close contact, and constant contact with animals and working without protection have been reported as the most pressing predisposing factors for leptospirosis in Thailand . In some studies, including the investigation performed by Vargas et al. in Colombia, the overall prevalence of leptospirosis was lower than in the present study (12.2%) . In the current study, we did not observe any relationships between gender and the results of leptospirosis serological tests, which fell in line with the findings of Regmi et al.'s study and Alikhani et al.'s study . While other investigations have reported differences in terms of the prevalence of leptospirosis and mentioned that leptospirosis is more common in men [30, 31].
Nevertheless, we were not able to find any significant relationships between gender and positive serological results, which could be due to the presence of women in agriculture and animal husbandry almost equal to men in the studied geographical area. No relationship was found between age and the results of serological tests, which is in agreement with the findings reported by Alikhani et al. . While other studies including the study of Gonwong et al. in Thailand  and Delmas et al. in France  reported a significant correlation between age and the results of serological tests. The highest number of positive cases were reported among individuals aged 31-60, while the study of Etienne et al. reported the highest number of positive cases among participants aged 51-61 . Our findings did not indicate any relationship of potential significance between the level of education and the results of serological tests, while the study conducted by Gonwong et al. reported a significant inverse correlation between education and the results of leptospirosis serological tests . No relationship was found between job status and the results of leptospirosis serological tests, while most of the investigations conducted demonstrated a significant relationship between job and leptospirosis. As Regmi et al.'s study  reported the most positive cases of leptospirosis serology in farmers; Etienne et al.'s study  also reported the most positive cases of leptospirosis serology among livestock farmers.
No significant relationship was found between the results of leptospirosis serological tests in terms of residence, which is in agreement with the findings of other studies [31, 33].
While Gonwong et al.'s study in Thailand demonstrated positive cases of leptospirosis in rural areas . In other studies in Tanzania and Nicaragua, a statistically significant relationship has been reported between the place of residence and the results of leptospirosis serological tests [33, 34]. Similarly, we did not observe any correlations between the history of contact with domestic animals and the results of leptospirosis serological tests. While other studies such as Regmi et al.'s study have found a significant relationship between the history of contact with domestic animals and the results of leptospirosis serological tests [35, 36].
According to the high seroprevalence of Leptospira in healthy people in our study, disease control and prevention policies are necessary for people with high risk of infection. Furthermore, comprehensive studies using Microscopic Agglutination Technique (MAT) are suggested for the leptospirosis diagnosis due to its diagnostic accuracy for seroepidemiologic identification of the disease. MAT uses panels of live Leptospira, ideally recent isolates, representing circulating serovars from the patient's area of residence. ELISA-based serological tests with complementary molecular detection (PCR) are a practical and robust alternative method for diagnosing leptospirosis with new infections in countries with a higher burden of the disease. It will be possible to prevent the disease by increasing the awareness of the people at risk and the personnel of the regional health and treatment systems, and by preventing human contact with contaminated materials, as well as by vaccinating livestock and animals.
No potential conflict of interest was reported by the authors.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
All authors contributed to data analysis, drafting, and revising of the paper and agreed to be responsible for all the aspects of this work.
HOW TO CITE THIS ARTICLE
Manizhe Jozpanahi, Rojin Ahmadi, Afsaneh Ayaz, Fakhri Haghi*. Eroepidemiology of Leptospirosis among Healthy People in Zanjan, Northwest of Iran. J. Med. Chem. Sci., 2024, 7(1) 116-123.