HIV-HBV coinfection and liver fibrosis in long time monitored patients from the former Romanian pediatric HIV cohort – a small study single center experience

The aim of this study was to determine the degree of liver fibrosis with the help of a non-invasive method - transient elastography - in a long term monitored HIV-HBV coinfected group of patients (from the former Romanian pediatric cohort) and to assess the implication of various factors as potential risk factors for the development of liver fibrosis. We have reviewed a total of 636 patients (Px) that were in the evidence of the HIV Department of the “Victor Babes” Hospital of Infectious Diseases of Craiova, from which we randomly selected a group of 37 patients that are monthly attending the HIV department. Mean age of the patients was 34.03±2 years old and they were monitored for a long period of time, mean duration of monitorization was 26.11±4.31 years. Patients have been split into group A (13 Px) with various degrees of liver fibrosis and group B (24 Px) without liver fibrosis; the mean value of liver stiffness was higher in group A vs group B 11.27±5.72 vs.4.93±0.98 kPa, p <0.0001. Mean value of nadir CD4 was lower in group A. No significant differences were found between the 2 groups related to the number of associated risk factors, duration of treatment with Zidovudine, Didanosine


INTRODUCTION
Liver fibrosis (LF) is the consequence of chronic liver inflammation produced by various factors that lead to the accumulation of certain proteins (mainly collagen) in the liver.Long term development of LF concludes with the occurrence of cirrhosis and liver failure which, with the exception of liver transplantation cases, can lead to the death of the patients.Latest estimations of the burden of chronic liver diseases (including liver cancers, responsible for 600000 up to 900000 deaths) show that the condition is responsible for one up to two million deaths annually or 2-4% of all deaths worldwide [1][2][3].The most studied factors that impair liver function are chronic viral hepatitis, alcoholism and non-alcoholic hepatic steatosis.
HIV infection is also a global threat; currently there are about 39 million people infected and another 40 million lost their lives due to the infection [4].The current estimation of chronic HBV infection is 296 million people suffering (in 2019), with a 1.5 million newly infected annually [5].The exact number of HIV-HBV coinfections is not known, but a recent review of the worldwide data estimates a prevalence of HIV-HBV coinfections of 7.6% (IQR 5.6-12.1),or 2.7 million people coinfected, suggesting that 1 case in 100 people with HBV infection also have HIV infection as well [6].
A large number of children from Romania were infected with HIV in the past, most of them being born during 1987-1989 (the so called Romanian pediatric HIV cohort).It is not known the exact route of infection, but it's considered to be iatrogenic.Also, the time of infection is considered to be their first year of life.A significant proportion of those children were also coinfected with HBV.Today, most of the HIV infected people from our country under medical surveillance and monitoring are survivors from that cohort [7].

OBJECTIVE
To evaluate liver fibrosis in long-time monitored HIV-HBV coinfected patients (from the former Romanian HIV pediatric cohort) using liver transient elastography-Fibro Scan.

METHODS
This is a prospective study (January 2022-June 2023) performed at the "Victor Babes" Hospital of Infectious Diseases and Pulmonology from Craiova.We have reviewed a total of 636 patients parenterally infected with HIV during early childhood, from which 183 were alive at the beginning of 2022, 271 were deceased and 182 lost from follow up; from those alive, we have randomly selected a number of 37 HIV-HBV coinfected patients to perform liver transient elastography (Fibroscan).Level of fibrosis were quantified as follows: normal value (1.6-5.5 kPa), F1 grade (7-7.1 kPa), F2 grade (7.2-9.5 kPa), F3 grade (9.6-12.5 kPa) and F4 grade or cirrhosis (>12.5 kPa).Several factors (beside chronic HBV infection) were considered risk factors for liver fibrosis and / or inflammation (body mass index -BMI -, coinfections with hepatitis B, C and / or D virus, Cytomegalovirus infection, tuberculosis, antiretroviral treatments -drugs and duration -, antifungal and/or antineoplastic treatments, prophylaxis with Trimethoprim-Sulfamethoxazole, alcohol consumption, presence of dyslipidemia), introduced into a Microsoft Excel database and analyzed; every risk factor represents 1 point and a total score for risk factors were attributed to each participant; we have formed two main groups, A with patients with HIV-HBV coinfection and liver fibrosis and B with those coinfected, but without liver stiffness.Statistical comparisons were made using Chi2 with Yates' correction (two tailed) and unpaired t test the level of statistical significance being p<0.05.
Every participant signed up an informed consent for participation to the present study and publication of data.

RESULTS
Group A consists of 13 patients with liver fibrosis and group B consists of 24 individuals without liver stiffness.

Data regarding liver transient elastography
Mean value of liver stiffens is higher in group A vs group B (11.27 ± 5.72 vs 4.93 ± 0.98 kPa, p<0.0001).Data regarding liver fibrosis and grading are presented in Table 1.

Data regarding risk factors for liver fibrosis and/or inflammation
The average number of considered risk factors for liver fibrosis and / or inflammation is similar in both groups (8.84 ± 1.4 vs 8.33 ± 2.18, p is not statistically significant).Details of every considered item are presented in Table 2. Certain antiretrovirals (Lamivudine -3TC, Emtricitabine -FTC, Tenofovir -TNF) have also active against HBV.Data regarding anti HBV therapy are shown in Table 3.There are no statistically significant differences between group A and B as regarding the duration of treatment with AZT, ddI, ddC, d4T, NNRTI or RTV, but the average length of treatment is higher for group A.

DISCUSSION
From the 636 HIV-infected patients identified with parenteral transmission during the early childhood, a number of 153 were chronically infected with HBV (only 439 were tested), that is 24.06% from the total, or 53,5% from those tested.Thus makes HBV infection a prime factor to be considered in relation with liver fibrosis.
There are many causes and additional risk factors for liver fibrosis in people living with HIV: demographic (older age, male gender), individual (heavy alcohol use, tobacco use, herbal medicine use, non-adherence to prescribed antiretroviral regimen), HIV-related factors (increased viral load, low CD4 cell count, use of AZT, ddI, ddC or d4T), coinfections (hepatitis B ± D, hepatitis C, schistosomiasis), metabolic (type 2 diabetes mellitus, obesity, dyslipidemia, metabolic syndrome), as well as certain ge-  netic factors [8][9][10][11][12].The number of risk factors is translated into the number of liver related events and it seems that the higher the number, the higher the probability of liver fibrosis development [12].In patients without HIV infection, metabolic factors (if steatosis is present) are more important than viral factors; also, in spite of effective antiviral therapy, progression of HBV infection might remain present at a low level and leads to liver fibrosis [13,14].Highly effective antiretroviral therapy seems to lower the risk of liver stiffness development [15].Our study has included some of those factors (hepatitis C or D, use of certain antiretrovirals, obesity, dyslipidemia or heavy alcohol use), but we have not found statistical differences.We have also considered other factors that might lead to liver fibrosis and/or inflammation (CMV exposure, antibacilar treatment, antifungal treatment, chemotherapy, prophylaxis with Trimethoprim/sulfamethoxazole), but we failed to notice any statistically significant differences.The number of considered risk factors are similar for both groups.While those risk factors were considered individually, it was impossible to study their overlapping and effects on liver function.
It was established that the use of Tenofovir/ Emtricitabine, entry inhibitors or integrase strand transfer inhibitors are associated with better liver function; also, "older" antiretrovirals have a higher negative impact on liver function than the "newer" ones [16][17][18][19].
Our data show that for the group A (those with liver fibrosis) there is a statistically significant difference as compared with group B for the treatment with 3TC (longer in group A) and Tenofovir (longer in group B).We suspect that longer use of 3TC produced the specific YMDD mutation inside HBV genotype and it would have been interesting to detect that alteration, however we were not able to work this out for the present study.

Limitations of the study
The authors acknowledge that they have included only a small number of cases; also, the information about HBV viremic levels and resistance to certain antivirals are not available.

CONCLUSION
In this study, we conducted an evaluation of liver fibrosis using transient elastography on patients living with HIV plus HBV coinfection from the former Romanian pediatric HIV cohort due to its unique-ness characterized by a long period of evolution of the disease but also a long period of time under antiretroviral therapy.Because we are talking about patients born in the early 90's, they went under antiretroviral regimens with substances that nowadays are associated with liver fibrosis development.We assesed de implication of various antiretrovirals as risk factors for liver fibrosis development and we found a strong association between liver fibrosis and longer use of Lamivudine, while longer use of Tenofovir-based therapy was characteristic of patients without liver fibrosis.We also considered other risk factors like past or present various individual medical history, other coinfections, metabolic factors, however no statistically significant differences were found between patients who developed liver fibrosis and those who did not.Liver fibrosis was identified in a significant percentage of patients living with HIV/HBV coinfection.In conclusion, this study underscores the importance of monitoring liver fibrosis in HIV/HBV coinfection for a better management of these complex patients.
Institutional Review Board Statement: This study was approved by the Committee of Ethics and Academic and Scientific Deontology of Craiova, Romania (approval no.78/07.09.2020).Access to the database for this study was approved by the Ethics Committee of "Victor Babes" Clinical County of Infectious Diseases and Pulmonology.

TABLE 3 .
Duration of anti HBV therapy in considered groups and statistical significance

TABLE 1 .
Data regarding liver fibrosis and grading for the considered group

TABLE 2 .
Risk factors for liver fibrosis and / or inflammation for the two studied groups