Detection of HBV DNA in ascitic uid of decompensated cirrhotic patients to assess infectivity: A novel pilot study

Background and aims: Body uids such as saliva, tears and urine from patients with hepatitis B virus (HBV) infection are well known to be infectious. However, the infectivity of internal body uids like ascitic uid from patients with HBV infection has not been established. So, we conducted this study to know the infectivity of ascitic uid for hepatitis B in decompensated cirrhosis by detecting HBV DNA in it. Methods: Patients with HBV related cirrhosis with ascites were enrolled. The levels of HBV DNA in the ascitic uid from these patients were quantied by real-time PCR, and compared with HBV DNA levels in serum. Clinical and laboratory parameters to predict HBV DNA positivity in ascitic uid were also assessed. Results : Twenty one patients (mean age 45.43±13 years) with HBV related cirrhosis with ascites were enrolled. HBV DNA in ascitic uid was detected in 4/21 (19 %) patients. The ascitic uid HBV DNA levels ranged from 4.8 to 6.4 log copies/mL (mean ± SD = 5.27 ± 0.55). High levels of serum HBV DNA was signicantly associated with HBV DNA detectability in ascitic uid (p=0.001). Patients with HBV DNA detected in ascitic uid had signicantly higher serum protein levels as compared to those having undetectable HBV DNA in ascitic uid (6.83 ± 0.33 versus 5.70 ± 0.77 g/dl, p=0.011). Conclusions : HBV DNA is detectable in ascitic uid in about one fth of HBV related cirrhosis patients with ascites so it may not be considered an important source for HBV transmission. High serum HBV DNA and high serum protein levels were positively associated with HBV DNA detectability in ascitic uid.


Introduction
Hepatitis B related cirrhosis is a major public health problem leading to signi cant morbidity & mortality.
About 4% of all cirrhosis is caused by hepatitis B (1). About 20% of patients with cirrhosis have ascites at their rst presentation (2). It is estimated that millions of therapeutic and diagnostic ascitic taps are performed each year in hepatitis B related cirrhosis patients worldwide, posing a healthcare hazard. The prevalence HBV in different category of healthcare workers ranges from 6-7% in various studies (3,4).
Occupational exposure is a major risk factor. HBV is transmitted e ciently by percutaneous and mucous membrane exposure to infectious body uids (5).
So, it is important to know the infectivity of ascitic uid to prevent horizontal transmission to health care workers via this route. As there is no good tissue culture for HBV to determine the infectivity of body uids, the presence of HBsAg or HBV DNA in them have been extrapolated as surrogate markers for their infectivity. As far as the presence of HBV DNA in ascitic uid is concerned, the current literature does not provide any useful information. However, presence of HBV DNA has been shown to be present in various other body uids like serum, saliva, nasopharyngeal uid, urine, semen, vaginal uids and tears in the HBV infected patients(6-8). A relationship between HBV DNA levels in different body uids and blood has also been reported along with a potential of horizontal as well as vertical transmission in different studies (7)(8)(9). Despite the availability of the evidence with respect to presence of HBV DNA in different body uids and its correlation with serum viral load, possibility of horizontal and vertical transmission and its predictive and prognostic value, there had been no study till date, assessing the HBV DNA in ascitic uid and its association with clinical pro le, predictive value and correlation with serum viral load.
With this background, the present study was carried out with the aim to know the infectivity of ascitic uid for hepatitis B in patients suffering from decompensated liver cirrhosis and the objectives were to determine the proportion of patients with HBV DNA detectable in ascitic uid to that with total number of HBV cirrhosis patients with ascites, to study the correlation of HBV DNA level in ascitic uid with serum viral load and to determine the predictors of HBV DNA detectability in ascitic uid.

Methods
Study design, setting, period and duration This was a pilot observational study in which patients admitted to the indoor facility of the department of Medicine, Medical and Surgical Gastroenterology of our institute from July 2020 to July 2021 were enrolled. Laboratory tests were done in collaboration with the department of Microbiology of our institute.

Patients
Patients more than 18 years with HBV related chronic liver disease with ascites, willing and able to provide consent were included in the study. Those patients who were not giving consent, critically ill, aged less than 18 years, without ascites or any local skin infection like cellulitis, abscess were excluded from the study.
Ascitic uid and serum collection and transportation 5 ml of ascitic uid and 5 ml of serum sample were taken into plain vial under aseptic conditions and samples were transferred to the Microbiology lab immediately and stored them at 4°C for 24 hours and then at -20°C for 5 days and processed for detection of HBV DNA viral load.
Detection of HBV DNA in serum and ascitic uid Hepatitis B DNA viral load assay in serum and ascitic uid were performed as per patent molecular protocol as described by Prakash S et al (10). The control were validated before the PCR run. RNAseP was checked for every clinical sample quality. The viral load HBV quanti cation was done by using standard protocol.

Data collection
Relevant demographic, clinical and laboratory data were collected for all patients on a pre-designed proforma. All patients underwent a detailed history, including presenting complaints, past and personal history, detailed history of recent exposure and risk factors. Findings of complete blood counts, liver and kidney function tests, ultrasound and/or CT abdomen, upper gastrointestinal endoscopy were recorded.

Statistical analysis
Continuous data were summarized as mean ± SE (standard error of the mean) whereas discrete (categorical) in number (n) and percentage (% cases, it was not detected ( Table 1). The calculated 95% con dence intervals for the sample size used in the study was in the range 5.45% to 41.91%. In all the cases, serum HBV DNA level was higher than the ascitic uid level except one case, in which serum HBV DNA was undetectable (Case 2) ( Figure 1).

Discussion
HBV is one of the most common underlying causes of decompensated liver cirrhosis, especially in tertiary care hospitals where people often report to a healthcare facility only when the disease is in its advanced stage. Ascites is one of the most commonly encountered complications of cirrhosis. It might be proposed that after viral translocation, virus may enter the systemic bloodstream and access ascitic uid.
In the present study, only a total of 21 patients ful lling the eligibility criteria could be enrolled owing to COVID-19 pandemic, thus the present study re ects a pilot study to assess the issue in question. We enrolled patients with decompensated cirrhosis only. The mean age of patients was 45.43 years and mean BMI was 20.2 kg/m 2 . The study sample was predominantly male (85.7%) and was dominated by a lower socioeconomic class (85.7%) marked by high prevalence of uneducated patients (61.9%). HBV DNA was found to be positive in 4 (19.0%) ascitic uid specimens only. Detection of HBV DNA in different body uids in serum positive patients has been reported to vary substantially(6). Detection rates in different body uids might be dependent on the interaction of these body uids with the bloodstream. It would also be pertinent to mention here that in the present study, HBV DNA in serum was found in only 17 (81%) cases. Moreover, there were three cases having low serum HBV DNA expression (<2000 IU/L). It was also observed that one of the patients with undetectable serum HBV DNA had detectable HBV DNA in ascitic uid. This nding indicates thst it is di cult to state that the detectability of HBV DNA is solely dependent on serum positivity. However further studies are needed to reach any de nite conclusion.
Although, some case reports like the one by Fagan and Coworkers et. al (11) who detected HBV DNA in the urine, semen and saliva of a HBV patient who failed to have detectable HBV DNA levels in serum. It is important to understand the reason for this discrepancy which might be dependent upon heterogeneity in the assay procedures used for detection of HBV DNA in different body uids.
As far as replicability is concerned, there seems to be high inconsistencies among different studies(6-8,12-16). In the present study, we could detect HBV DNA in only 19% of ascitic uid specimens in decompensated cirrhosis patients. The calculated 95% con dence intervals for the sample size used in the present study was in the range 5.45% to 41.91%, thus offering a vast range for replication of results. It must be seen that the previous studies have reported detection rates in different uids to be highly diversi ed. A number of previous studies have tried to explore the predictive or prognostic role of detection of HBV DNA in body uids(6). In the present study, we also assessed the relationship of HBV DNA detection in ascitic uid with various clinico-demographic and outcome factors but failed to nd a signi cant association except a signi cant difference in serum protein and HBV DNA levels. Serum protein levels of those with HBV DNA positivity in ascitic uid were signi cantly higher as compared to those having undetectable HBV DNA in ascitic uid.
Given the fewer number of cases with detectable HBV DNA in ascitic uid (n=4/21), the statistical signi cance of any association needs to be viewed with caution. Most of the previous studies did not report an association of HBV DNA detection in different body uids with clinico demographic pro le or outcome.
Other internal body uids like pleural uid, crebrospinal uid and pericardial uids may be considered equivalent to ascitic uid. One case report described presence of HBsAg in pleural uid(18). However, no attempts have been made to look for presence of HBV DNA in them and their potential for infectivity may be explored in further studies.
Only raised serum protein and high serum HBV DNA level were positively associated with detection of HBV DNA in ascitic uid. All other clinical and laboratory parameters were found not signi cantly associated. Further studies are needed to nd the pathophysiological signi cance of this nding.
Detection of HBV DNA in ascitic uid is comparable to detection of complete virion in it, as no good virus culuture is available. However, studies aimed to detect complete virion in body uids including ascitic uid may be planned to further substantiate the concept of body uids as potential sources of horizontal transmission.
There were several limitations of the study. Smaller sample size, mostly descriptive data, absence of HBeAg and ascitic uid parameters were few of them. However, the ndings of the present study, within limitations, provide the rst evidence of detectability of HBV DNA in ascitic uid in patients with decompensated cirrhosis. Further studies with larger sample size are required to explore the replicability of these results and their clinical implications to establish the usefulness of such investigations in clinical or epidemiological context.

Conclusions
Our study concluded that, HBV DNA is detectable in ascitic uid in about one fth of patients with HBV related decompensated cirrhosis with ascites. So ascitic uid may not be considered an important source for HBV transmission for epidemiological purposes. However, healthcare workers and laboratory staff may be warned while handling ascitic uid of HBV infected persons. Serum HBV DNA and protein levels are the determining factors for HBV DNA positivity in the ascitic uid.  Figure 1 Comparison of HBV DNA levels between serum and ascitic uid in detected cases