Prevalence and Associated Risk Factors of HTLV-1 and Co-infections of Blood-Borne Viruses in Birjand, Iran’s Eastern Border

1Birjand Infectious Disease Research Center, Birjand University of Medical Sciences, Birjand, Iran 2Blood Borne Infections Research Center, Academic Center for Education, Culture and Research (ACECR), Razavi Khorasan Branch, Mashhad, Iran 3Immunology Research Center, Inflammation and Inflammatory Diseases Division, Mashhad University of Medical Sciences, Mashhad, Iran 4Nursing and Midwifery Care Research Center, Mashhad University of Medical Sciences, Mashhad, Iran


Introduction
Hepatitis B virus (HBV), hepatitis D virus (HDV), hepatitis C virus (HCV) and human T-lymphotropic virus 1 (HTLV-1) are the most significant blood-borne viruses (BBVs) that can be associated with human cancers. These infections are major public health concerns since they are responsible for millions of deaths annually. 1,2 HBV and HCV infections represent a significant public health concern; HTLV-1, as an oncogenic virus, is moving progressively towards becoming a threatening health issue. Except HTLV-1, the prevalence of these infections is greater in some eastern provinces of Iran such as South Khorasan due to their closer ties to Afghanistan. [3][4][5] On the other hand, although co-infections of these viruses are not exactly common, they occur in some circumstances. 6 The sero-prevalence rate of HBV/HCV co-infection among individuals with seropositive hepatitis B surface antigen (HBsAg), chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in Asia and Western countries is estimated at 10%-15%. 7 This condition is the most common in injecting drug users, HIV-infected patients and people who need hemodialysis or blood transfusions. 8 Co-infection with hepatitis viruses (hepatotropic viruses) are known to influence progression and management as well as the outcome of retroviruses infections, such as HIV and HTLV-1. 9 HTLV-1 infection, as a neglected oncovirus, is becoming a major threat for public health because of stigmatic beliefs about it and its associated life-threatening diseases. Furthermore, it is a growing problem, which is Open Access associated with increased risk of spreading through organ transplantations, blood transfusion and human behavioral changes. 10 The North and Razavi Khorasan provinces, in close relationship with South Khorasan, constitute the main endemic area in Iran for HTLV-1 at 2%-7%. 11 Even a segregated population in the Razavi Khorasan, close to South Khorasan, is endemic (1.25%) for this virus. 12 Given the importance of global growth of HTLV-1 along with the danger of its co-infections with other common blood-borne oncogenic viruses, it could provide new insights into the world of microbial pathogenicity. Therefore, in this study, the incidence of HTLV-1 infection and co-infections of BBVs in Birjand, Iran's eastern border, were evaluated.

Study Design and Population
This cross-sectional study was conducted on 3441 subjects in urban areas of Birjand city, Iran with an age range of 14 to 70 years, who were chosen by simple random sampling using a random numbers table from a comprehensive study including 5235 subjects, which was performed in Birjand, South Khorasan, Iran, during 2013-2014. 5 The checklist of socio-demographic characteristics and behavioral risk factors were based on a review of the literature.

Sample Collection
After obtaining informed consent and completing the demographic data, 8 mL venous blood was collected by venipuncture from each of the participants in sterile tubes containing EDTA. The samples were stored at -30°C until further analysis.

Serological Assays
The serum samples were screened in this study for the presence of anti-HTLV-1 using Enzyme-linked Immunosorbent Assay (ELISA) according to the manufacturers' recommendations (Dia.pro, Italy). Furthermore, anti-HIV, anti-HCV, anti-HDV and anti-HBc antibodies as well as hepatitis B virus surface antigen (HBsAg) were evaluated by ELISA in our previous studies. 5,[13][14][15] It is noteworthy that the blood serum of patients with positive HBsAg was used in order to determine the prevalence of HDV infection. Finally, the positive anti-HCV serological results were confirmed using specific primers for HCV by polymerase chain reaction (PCR). 13 Statistical Analysis The data on demographic features, HTLV-1-related risk factors and other characteristics of the population were analyzed with the Pearson chi-square and Fisher's exact tests. The association of HTLV-1 with sociodemographic characteristics and behavioral risk factors was analyzed using a univariate logistic regression model, by estimating the odds ratio (OR) and 95% confidence interval (CI). Data were analyzed using SPSS version 21. A P value less than 0.05 was considered statistically significant.

Demographic Characteristics
This study was conducted on 3441 subjects in urban areas of Birjand, eastern Iran. The study population involved 1611 (46.8%) males and 1830 (53.2%) females. The mean age of participants was 38.67 ± 14.49 (range: 14 to 70) years. The data showed that 81.4% and 16.4% of the population were married and single, respectively; the others (2.2%) were widowed or divorced.
Risk Factors for HTLV-1 Infection As shown in Table 1, there was no evidence of a significant relationship between HTLV-1 infection and demographic features of patients including age, sex and education level (P>0.05). The associations of HTLV-1 infection with the behavioral risk factors are shown in Table 1  reported in 0.6% of cases with a history of hospitalization vs. 0.2% in those with no history. The results of univariate logistic regression analysis showed that the risk of HTLV-1 infection in patients with no history of hospitalization was 73% lower than those with a history of previous hospital admission (OR 0.27, 95% CI 0.07-0.97) ( Table 2). It is noteworthy that since the prevalence of HTLV-1 was low at some levels of the factors studied, ORs were reported only for those variables that could be fitted to the model correctly.

Co-infection of Blood Borne Viruses
The occurrence of HTLV-1, HDV and HCV co-infections with HBV among the population are shown in Table 3. The results indicated that simultaneous presence of HBV and HCV was the most common co-infection among 85 HBsAg positive patients (2.35% with 95% CI, 0.002-0.072). It is noteworthy that no co-infection was detected except for the simultaneous presence of HBV and other viruses (HTLV-1, HDV and HCV).

Discussion
BBVs (BBVs) are still one of the most important global public health concerns. In addition to HBV and HCV, the Khorasan provinces are an endemic region for HTLV-1, which is the most significant blood-borne oncogenic virus. 2,10 In the North and Razavi Khorasan provinces, the prevalence of HTLV-1 has been reported at 2-3% 11 ; however, the prevalence of this virus in South Khorasan is yet to be evaluated.
In the present study, the sero-prevalence of HTLV-1 was evaluated among a population in South Khorasan, eastern Iran with a long border with the Afghanistan. According to the serology tests, anti-HTLV-1 was 0.3%. Furthermore, the sero-prevalence of anti-HIV, anti-HCV, anti-HDV, anti-HBc and HBsAg in our previous studies was reported at 0%, 0.2%, 1.2%, 15% and 1.6%, respectively. 5,[13][14][15] The results of our studies show that the prevalence of bloodborne viral infection in South Khorasan is close to that observed in the overall Iranian population. [16][17][18] However, the prevalence of HTLV-1 was 10 times lower than North and Razavi Khorasan provinces (0.3% vs. 2-3%), 11 and even 4 times lower than a segregated population in this region close to South Khorasan. 12 On the other hand, in other studies on subjects at risk of these viruses in northeastern Iran, 1,19-21 the sero-prevalence of blood-borne oncogenic viruses (HCV, HBV, HTLV-1 and KSHV infections) was reported to be considerably higher than our study. With respect to the present study, it is more likely that the source of HTLV-1 infection in Iran, as an archetypic virus, is in Razavi Khorasan, Neyshabour or Mashhad.
Previous studies have also shown that different risk factors can be involved in the incidence of BBV infections. Therefore, knowledge on these factors can help in prevention and control of infections caused by BBVs. Our study indicated that the occurrence of HTLV-1 infection was associated only with history of hospitalization, such that the risk of HTLV-1 infection in patients with no history of hospitalization was 73% lower than those with a history of previous hospital admission. Accordingly, the authorities should pay more attention to HTLV-1 screening related to blood banking and surgical activities, as this region has a close relationship with the Razavi Khorasan, an endemic area for this virus. In a study in Khorasan Razavi province, the main risk factors for HTLV-1 infection were hospitalization, traditional cupping, age, marital status, history of blood transfusion and surgery. However, occupational infection has not been reported so far for HTLV-1 in a cohort study of 10 years. 24 In a previous study conducted among Peruvian women, history of blood transfusion, chronic scabies, having a relative with leukemia, age ≥38 years, young age at first intercourse, having more than four lifetime partners, educational status and having more than four pregnancies were significantly associated with HTLV-1 infection. 25 It seems that the risk factors for BBV infections such as HTLV-1 can be different depending on the study population and the cultural, educational, behavioral, socio-economic, and geographical variables. Finally, in the present study, HBV-HTLV-1, HBV-HDV and HBV-HCV co-infections were found at 1.17%, 1.17% and 2.35%, respectively. In a previous study, 26 a co-infection rate of 0.5% was found for HBV/HCV, while a rate of 1.1% was reported for HBV/HTLV-1 co-infection, which is nearly the same as our study. On the other hand, in a survey conducted in Australia in 2017, 27 the sero-prevalence of HTLV-1 among HBVinfected patients was 14.4%, which is much higher than the 1.17% found in our study. These discrepancies may be the result of heterogeneity in patient populations studied in different geographical regions. Taken together, many studies suggest that physiological, iatrogenic and acquired immune-compromise along with environmental changes are the main factors for emerging and re-emerging microbial co-infections. However, host ecology and virusvirus interactions are important drivers of the occurrence and spread of viral co-infection. 28 A limitation of the present study was the impossibility of Generalized Estimating Equation (GEE) analysis due to lack of identification of clusters at the time of data collection. In addition, given the rarity of the infection and lack of a sufficient number of positive cases in the age subgroups, it was not possible for us to use a direct standardization method for adjusting the incidence rate.
In conclusion, apart from HTLV-1, which had a lower prevalence compared to the neighboring provinces, the prevalence of other blood-borne oncogenic viruses in South Khorasan is close to that observed in the overall Iranian population. Our results suggest that the most important risk factor for HTLV-1 infection was history of hospitalization. Consequently, our findings reinforce the importance of improving our knowledge on BBV risk of hospitalization and behavioral intention, particularly in general populations to prevent the emergence of new cases.

Authors' Contribution
GRS, AE and ZA collected the data and provided the first reports. MZ designed and supervised the research. MY and SAGH interpreted the analysis and wrote the final manuscript. MHN, GHA, RR, NVZ and AM contributed to the discussion and critical revision of the manuscript.

Conflict of Interest Disclosures
None declared.

Ethical Statement
The study was approved by the Bio-medical Ethics Committee of Birjand University of Medical Sciences (IR.BUMS.REC.1394.381).