The epidemiological characterization and geographic distribution of hepatitis D virus infection in Libya

Introduction North Africa is known to be endemic for hepatitis D virus. However, data one the prevalence of this virus in Libya are scanty. This study aimed to determine the prevalence of hepatitis D virus infection in Libya and analyze the demographic factors associated with the infection, and also to assess the variations across the regions and districts. Methods A total of 1873 samples collected from all over the country were tested for antibodies against hepatitis B surface antigen and the results were correlated with demographic and geographic variables. Results The overall prevalence of hepatitis D virus infection was 1.7%. The prevalence rate was significantly high among those aged over 40 years (P < 0.001) and it was associated with intravenous drug use and coinfection with human immunodeficiency virus and/or hepatitis C virus infection (P < 0.001). The prevalence rates varied with geographic location and differed markedly within the regions the country. The highest rate reported was in the central region of Libya, followed by the western and eastern regions. Conclusion Hepatitis D virus infection rate in Libya is considered to be low but is of some concern in some districts. This has been propagated by population displacement and African immigrants, indicating that a continuous epidemiological surveillance program should be implemented.


Introduction
Hepatitis D virus (HDV) is an RNA viroid that infects only people in the presence of hepatitis B virus (HBV), which expresses hepatitis B envelope protein. HDV is responsible for the most severe form of acute and chronic viral hepatitis [1][2][3][4]. Worldwide, it is estimated that 2-8% of chronic HBV carriers are co-infected with HDV, corresponding to 10-20 million patients. Furthermore, HDV is transmitted by the same routes as HBV, including blood transfusion, sexual contact, intravenous drug abuse and parenteral transmission [5][6][7]. In the early 1980s, surveys showed that HDV was endemic worldwide but its prevalence rates are geographically diverse [8,9]. High prevalence rates of HDV have been reported in North African countries [10]. A recent meta-analysis showed that HDV is found in 5% of the general population in 20.7% of liver disease patients. The highest rates were reported in Egypt, Sudan and Mauritania, followed by Tunisia, Morocco, Algeria and Libya. The findings of that study should be interpreted with caution due to the small number of individuals tested, their demographic heterogeneity, and the quality of the testing methods used [11,12]. Libya has been plagued by a civil war since 2011, which has caused high rates of mortality, injury, population displacement, and major damage to the health care system at the structural and organizational levels. In addition, the breakdown of the security situation led to a heavy influx of African immigrants [13,14].
Together, these circumstances have maintained high levels of viral hepatitis transmission through injuries, blood and sexual contacts, and even unsafe blood transfusion [15]. Data on the impact of HDV in the country is even scarcer particularly as screening for anti HDV is not routine in HBsAg-positive patients. Therefore, it becomes important to reappraise the level of HDV infection among Libyan patients in order to plan a more effective preventive strategy. Hence, the objectives of this study were to determine the prevalence of HDV infection among Libyan patients, analyze the potential risk and demographic factors associated with the infection, and investigate the geographic variation of HDV within the different regions and districts of the country.

Methods
Study population: the study involved 1873 HBsAg-positive serum samples that were collected from all Libyan regions and districts between 2015 and 2018. Appendix illustrates the geographic locations, regions, districts and population density. The national screening policy for HBV in Libya, which is uniform all over the country, includes screening of blood donors, preoperative patients, pregnant women and kidney and liver disease patients, as well as a mandatory pre-marriage screening. The study population included only those who were confirmed to be HBsAg-positive in the public health care system in Libya between 2015 and 2018, who were identified from the state wide data base with clear demographic and epidemiological information [11,12]. The data collected included region of origin, gender, age, level of education, marital status, and other related risk and demographic factors as previously described (12879_2013_2969_MOESM1_ESM.doc ) [11].
Laboratory diagnosis: the study included all the samples that were HBsAg-positive and had detectable HBV DNA according to preliminary screening using "Real Time HBV Viral Load Assay" (Abbott Laboratories, IL). Anti-HDV antibodies in EDTA plasma samples were detected by using an enzyme-linked immunosorbent assay (ETI-AB-DELTAK-2, Diasorin, Saluggia, Italy) [11].
Statistical analysis: data were coded and entered into a database, which was then cleaned and verified [12]. Demographic and risk factors were compared between patients using chi-square tests or Ethics approval and consent to participate: the study was approved by the Libyan National Ethical Committee (Approval No. LY NS, HDV-299-798-230). All participants signed an informed consent form witnessed by the local health office before collection of data and blood samples HIV [16,17].The questionnaire used to collect demographic and epidemiological data was anonymous and linked to the blood sample tube only by a code, as previously described [11].

Results
Prevalence of Hepatitis D Virus: a total of 1911 patients with a national number (i.e. Libyan citizens) were initially included in the study but 38 (1.98%) were excluded due to lack of personal information. HBV (55.9%) were males. No significant relationship between sex and HDV positivity was evident (P = 0.001). The mean age was 47.1 ± 13.4 years (median = 45.0). The highest HDV prevalence was among persons aged over 60 years (n = 13; 38.2%) followed by those aged 40-60 years (n = 11; 32.4%) and 21-40 years (n =7; 20.6%). It was lowest among those below 20 years of age (n = 3; 8.8%). The distribution of HDV seropositivity differed markedly from one region to another. The prevalence was higher in the central region (P < 0.001). Overall, HDV seropositivity was nearly twice more likely in the central region than in the other regions, and particularly compared to the eastern region. Study population characteristics: Table 1 shows the demographic and risk factor features in the study population. A variety of risk factors were found to be significantly associated with HDV infection. HDV seropositivity was more prevalent among those with no formal education (41.2%, P < 0.001) than among those with primary or secondary level education. Furthermore, marital status, family history of HBV infection and sex were found to be less associated with HDV seroprevalence (P = 0.001). A higher prevalence was found among those who had a history of intravenous drug use (35.3%; P < 0.001),

Discussion
In this large comprehensive nationwide study, we analyzed the seroprevalence of HDV among 1873 HBsAg-positive individuals collected from all the regions and districts of the country during four years. Anti-Delta antibodies was observed in all age groups, with an overall prevalence of 1.7%. The highest rate was reported among those aged over 40 years and the lowest among those aged below 20 years. This 1.7% prevalence rate is very low compared with other Arab countries, such as Saudi Arabia (13.6% ), Mauritania (9-67%) and the neighboring countries Egypt (23.53%), Tunisia, (7-44% ) and Sudan Furthermore, viral screening is mandatory in Libya for pregnant women, before surgical operations, and before a marriage contract is concluded. These measures have contributed immensely to reducing viral transmission within the Libyan population [11]. Notably, the use of highly effective anti-HBV vaccines has decreased both the prevalence and the spread of HBV and its associated pathogen, HDV.
This has led to the belief that HDV eradication is approaching.
By analyzing the demographic characteristics contributing to the emergence of HDV in Libya, evidence of familial transmission, influence of sex and prior hospitalization were not found to be significant. Moreover, no particular risk factor was found to be associated with HDV co-infection. Injection drug use was the most probable route of HDV transmission for most of the patients in our study (P < 0.001). Those coinfected with HIV or HCV accounted for 23.5% and 17.6%, respectively. In contrast, in Europe, prevalence rates of HDV antibodies approaching 15% have been reported among  Hepatitis D virus screening should be implanted in Libya.

Competing interests
The authors declare no competing interests.