Hepatitis E virus (HEV) infection is a global disease (1,2). HEV infection represents a major global public health issue especially in developing countries, where the global death rate is 1-15% and up to 30% in pregnant women (3). Every year there are an estimated 20 million hepatitis E infection (HEV) are registered over the world. it is known that chronic liver disease by hepatitis E develops in persons who are undergoing immunosuppression, including individuals infected with HIV(4). Recently, many studies have demonstrated cases of chronic HEV infection (characterized by detection of HEV RNA greater than or equal to 6 months in plasma) and cirrhosis in immunocompromised patients, including organ transplant recipients(5) patients with lymphoma and haematological malignancies (4,6 ) and in persons infected with the human immunodeficiency virus (HIV) (7). A number of studies have suggested that In persons infected with HIV may acquire HEV infection more often than individuals without HIV (8,9). The first verified case of chronic infection E in 2008 (10), which can to lead to liver damage and develops of hepatic fibrosis and even cirrhosis in immunosuppressed patients such as Patients with human immunodeficiency virus (HIV) infection and solid organ transplant recipients (11-14). Immunosuppression has been shown that facilitates chronicity of HEV infection, Therefore HIV infection is one of the possible causes for HEV persistence (15) and there are published reports of high HIV/HEV co-infection rates for particular regions (16,17). There are some data that suggest that HEV may promote the progression of liver disease due to other causes (18). There are only a few reports regarding HEV seroprevalence in Individuals who are immunocompromised. The seroprevalence of anti- HEV IgG in HIV-positive cohorts ranges varies from one geographic location to another from 1.5% to 11.2% (19,20). Incidence of infections caused by HEV, defined by detecting HEV RNA in the serum, is low, ranging from 0 to 1.3% (21-23).

However, studies regarding co-infection of HIV and HEV and HEV associated chronic liver disease in HIV-infected patients are limited in IRAN. The present study aimed to determine the prevalence of HEV RNA in HIV infected patients in Iran a country with moderately high prevalence of both infections. Therefore, in order to better understand the clinical impact of HEV infection in these populations we have conducted a study determining HEV RNA in HIV-infected patients.

In the present study, we determined whether HEV RNA were present in serum samples obtained from HIV-infected patients to investigate the prevalence of, and factors associated with, HEV infection in HIV-infected individuals. In addition, epidemiological, clinical and analytical factors were analyzed in order to identify potential risk factors associated with HEV seropositivity. The aim of this study was to define the degree of prevalence of HEV RNA in the group of HIV infected patients in Iran. For this study real-time RT-PCR assays targeting ..genes have been developed. We report 6 cases of hepatitis E infection in HIV-infected patients that  none of our patients received ribavirin treatment.

Methods and materials:    

Study population and samplecollection:

A total of 80 Iranian patients with HIV-1 infection attending the Tehran’s hospitals were enrolled in this study from February 2015 to April 2016. The exclusion criteria considered as patients who had been received anti-retroviral therapy. This study followed the principles of the Declaration of Helsinki and study has been approved by a local research ethics committee of the Iran University of Medical Sciences, Tehran, Iran. The participants were informed all aspects of the current study, and Informed consent was obtained from all of the participants prior to their enrollment for current study. About 5 ml of peripheral whole blood Sample from each participant was collected into a sterile EDTA-containing Vacutainer tube and plasma was separated from blood via centrifugation and frozen at -70 oC until analysis.

HEV RNA Extraction: Efficient HEV RNA extraction using the High Pure Viral Nucleic Acid Kit generates purified HEV RNA was extracted from 200 ml of plasma (Roche Diagnostics, Germany). RNA pellets eluted with the provided elution buffer and stored at -70°C until analysis.

cDNA Synthesis: For detection HEV RNA, Reverse transcription polymerase chain reaction (rReal Time PCR) was was performed by using the first strand cDNA synthesis kit by Revert AidcDNA synthesis kit from RNA templates (Thermoscientific, USA). In a nutshell, RNA samples were heated in 65°C for 10 minutes, then chilled on ice. The uniform suspension of bulk first-strand cDNA reaction mix was added according to the manufacturer’s protocol, then One μl of DTT solution, and 1 μl of random hexamer (24) primer (0.2 μg) were added to the RNA After heat denaturation and RNA and RT primers were mixed properly by pipetting up and down for several times, then incubated for an hour at 42°C. For Real time PCR, the QuantiTect Probe PCR Kit (Qiagen, Germany) was used, based on its instruction kit.

HIV-1 viral load quantification: Measurement of blood plasma HIV-1 RNA concentration performed by COBAS TaqMan 48 (Roche Diagnostics, Hacienda Drive Pleasanton, CA, USA) kit in the patient’s plasma samples (500 μl) and high pure extraction was used according to the manufacture’s recommendation. This method is a Real-Time PCR based on dual-labeled hybridization probe which targets the highly conserved region of HIV-1 gag gene. Limit of Detection of the COBAS TaqMan HIV-1 Test is 48 to 107 copies/mL.

Hepatitis E Virus real time PCR assay: A Real Time PCR assay was developed for detection of HEV RNA. The primers amplify a bp region containing the . variable regions of the .. Primer’s sequences and their position with melting temperature are shown in . So, after alignment of complete genomes, HEV consist of Nucleotide sequences based on pubmed database. Reactions contained 5µl of cDNA, 2.5 mmol/L MgCl2, 800 mmol/L of dNTPs, 100 ng of each primer,30 ng of probe and 1.5 units of QuantiTect Probe PCR Kit (Qiagen, Germany) to a total volume of 15 µL. Thermal cycling conditions were as follows: 95°C for 10 min; 40 cycles of 95°C for 15 sec, 60°C for 40 sec, Quantitative determination of the amplified products have done by the BioRad CFX-96 instrument (BioRad, USA). In order to synthesis our ideal genes, tests should be done by two pairs of forward and reveres primers. Our specific probes were designed by different fluorescent labels to track our targets separately.

Statistical analysis:The statistical analysis was performed using the Statistical Package for Social Sciences software version 21 (SPSS Inc, Chicago, IL, USA). Categorical variables were compared by Fisher’s exact test or the chi-square test as appropriate. Continuous variables was analyzed using Student’s t test. Data are presented as absolute counts, proportions [95% binomial exact confidence intervals (CI)], medians [interquartile range (IQR)], and means [standard deviation (SD)]. For all comparisons, p-value less than 0.05 was considered statistically significant.

Results:

The study population consisted of 80 participants with HIV-1 infection that were enrolled in the current study . The mean age of the patients was 36.51 ± 12.75 (range 4-64) years. Among 150 participants, 95 (63.3 %) were male and 53 (35.3 %) were female. . Real-Time PCR assay for HEV nucleic acid detection results in 6 (.%) positive samples out of 80 subjects, including . males (69/2%) and .. females (30/8%). Based on the analysis by Fisher exact test, no significant association was observed between HEV and gender of the patients (p value= 0.79) (Table 2). In our study, half of the HIV-1 infected patients were over 30 years of age, while the other half were under 30 years. The mean age for 6 HEV positive patients and 74 HEV negative ones was 40.9 and 35.8 years respectively. There was no significant correlation between age and HEV ‘s RNA positivity (p value= 0.18) (Table 3). By measuring the viral load, we could examine the relationship between HIV viral load and HEV infection. The average HIV viral load in positive and negative HEV patients was 14471.92 and 17016.66 respectively but t-test analysis showed no association between HEV -positive RNA and HIV Viral Load (p value= 0.61) (Table 4).

 Discussion:

The aim of this study was to evaluate the presence of HEV RNA in blood samples which have been collected from HIV-1-infected patients in Tehran, Iran. In current study we used Real-Time PCR method mainly because it has been shown to be more sensitive and reliable than other methods for identify the infection. Since HEV could be potentially inhibited by anti-retroviral therapy (ART), especially ribavirin (25), none of our HIV-1-infected patients received ribavirin treatment. Thus, our data is not influenced by the viral suppression of antiretroviral therapy.

immunosuppressive agents have been shown to facilitate severity or risk of chronicity of hepatitis E infection, HIV infection being one of the possible causes that may lead to HEV persistence (15), and there are Studies of high prevalence of HEV/ HIV co-infection for particular regions (16,17). Moreover, an relationship between exposure to HEV and cirrhosis has been reported in patients with cirrhosis (26), and chronic HEV infection is a leading cause of chronic liver disease in an HIV co-infection patient has been recently described (15).

The presence of HEV RNA indicates current infection. This study is the first report to show HEV RNA in HIV-1-infected patients in Tehran, Iran. In this cross-sectional analysis, the overall HEV molecular infection was 7.5% (6/80). The seroprevalence of anti- HEV IgG in HIV-positive cohorts ranges varies from one geographic location to another from 1.5% to 11.2% (19, 20). Incidence of infections caused by HEV, defined by detecting HEV RNA in the serum, is low, ranging from 0 to 1.3% (21-23).

In our study, we find HEV-RNA in 6 HIV patients, Unlike studies by Amitis Ramezani et al did not identify any case of HEV-RNA. An explanation for this finding could be the viral suppression observed in HIV-1-infected patients with antiretroviral therapy in their study. our results adapted with results of studies by Madejon et al., (27) Renou et al. (28) and Pischke et al. (29)Therefore, our data also support HEV infection as a viral hepatitis among HIV patients with With relatively moderate prevalence. Hepatitis E virus infection recently has been described as an emerging infection among patients with immunosuppressing conditions of such human immunodeficiency infection (30-32). In current study we found a moderate prevalence (7.5%) of HEV RNA among HIV positive individuals attending the Tehran’s hospitals in Iran. Although this rate is higher than the previous studies among Similar patient population in the industrialized countries (33-35), it is lower than the rates of HEV infection reported among some population groups HEV endemic areas of Africa (36) and Asia (15). Carry et al. (39) and Keane et al. (40) have also suggested that the chronic HEV infection may be averted by use of highly active antiretroviral. A sufficiently large sample size is also necessary to establish this finding.

The main conclusion of our study is that HEV infection important to consider in the differential diagnosis of otherwise unexplained chronic hepatitis in Iranian HIV-1-infected patients. Furthermore, our study revealed that HEV infection has moderate prevalence in the HIV-infected population of Tehran. Due to the HEV infection that may cause rapidly progressing chronic hepatitis in immunosuppressed HIV co-infection patients, with development of cirrhosis in the short term, Screening for HEV in HIV-infected individuals presenting Symptoms of Hepatitis or with hepatic fibrosis of unknown origin is warranted.