A Cross-sectional Study to Detect Inactive Hepatitis B Carriers among Dialysis Patients in a Tertiary Care Hospital in Bangalore

INTRODUCTION

Hepatitis B Virus infection is a major blood-borne infection with high morbidity and mortality. It is currently affecting 2 billion people globally, with an estimated prevalence of 3.2% and an annual incidence rate of 1.5 million new cases per year, and it is responsible for 1 million deaths worldwide annually [1].

Hepatitis B Virus is known to cause chronic disease with an estimated burden of 300 million chronic hepatitis B carriers worldwide, and it is estimated to be the cause of 30% of cirrhosis and 53% of hepatocellular carcinoma (HCC) globally [2].

Around 15-40% of Chronic Hepatitis B carriers develop cirrhosis and end-stage liver failure or HCC in their lifetime, and the cause of death is mainly attributed to chronic HBV complications rather than acute HBV infection [3, 4].

Acute HBV infection becomes chronic in about 5%-10% of the general population whereas the rate is about 60%-80% in the case of dialysis patients due to an immunosuppressed state of kidney failure, long and frequent exposure of vasculature to the external environment, multiple blood transfusions, and increased rate of nosocomial HBV transmission in various dialysis settings [5].

Dialysis patients are at increased risk of latent HBV reactivation and development of fulminant hepatitis as they are unable to mount a proper immune response against HBV vaccination compared to the general population, and only 10%-50% achieve effective seroconversion following vaccination and are at higher risk of exposure to nosocomial HBV transmission [7, 8].

Chronic HBV infection is also known to induce kidney disease in various forms, such as membranous nephropathy, membranoproliferative glomerulonephritis, and polyarteritis nodosa. Cirrhosis is known to worsen kidney failure in around 10% of HBV-infected dialysis patients [9]. Treatment of chronic HBV infection in dialysis patients is also challenging, as most anti-viral drugs require a dose adjustment and have more toxic side effects compared to the general population [10].

Inactive hepatitis B carriers form a major part of chronic HBV carriers (36%), which are defined by the presence of Hepatitis B surface antigen (HBsAg), absence of hepatitis B envelope antigen (HBeAg), undetectable levels of HBV DNA and normal liver histology and other parameters [11, 12]. Though they have a low rate of seroconversion and excellent prognosis among the general population, they are known to undergo reactivation and develop into fulminant hepatitis and HCC [13] among dialysis patients due to various conditions such as spontaneous reactivation of latent HBV infection due to induced immunosuppression [14], advanced age, male gender, uremia and use of long-term steroids [15, 16].

Though the inactive HBV carriers remain latent for a longer period, the risk of HBV reactivation cannot be ruled out, as the HBV tends to remain silent or latent in hepatocytes for years owing to the unavailability of eradicative therapy for HBV infection at present [17].

An inactive hepatitis B carrier state is known to undergo spontaneous reactivation in patients who are immunocompromised due to various conditions such as acquired immunodeficiency syndrome (AIDS), malnutrition, chronic kidney disease, chronic autoimmune disorders such as Rheumatoid Arthritis, Systemic lupus erythematosus, receiving immunosuppressive therapies such as cancer chemotherapy, radiotherapy, high dose corticosteroids for a longer duration [18, 19 ].

Inactive HBV carriers have low HBsAg levels and undetectable HBV DNA levels because most Polymerase chain Reaction (PCR) Nucleic acid extraction and detection kits have a limit of detection between 250-1000 copies/ml, and HBV DNA levels may not be sufficient for detection by these kits. But low HBsAg positivity and HBV DNA negative reports get interpreted as false positive HBsAg reaction or past exposure, which may hold good in the general population, but results in hazardous complications in dialysis patients, as there are chances of reactivation of latent HBV infection among them [20]. This study aims to detect inactive Hepatitis B carriers among dialysis patients using serological and molecular methods and to follow them up at regular intervals for 1 year to rule out false positives, monitor HBsAg and HBV DNA levels, and prevent reactivation.

PATIENTS AND METHODS

Study design and source of data - This cross-sectional study was conducted in the department of microbiology of a tertiary care hospital in Bangalore, Karnataka, for a period of 1 year from January 2021 to December 2021.

120 patients on dialysis are selected for sample collection after applying the inclusion and exclusion criteria as follows-

Inclusion criteria are 1) Dialysis patients above 18 years of age who have given verbal consent.2) Dialysis patients not currently infected with Hepatitis A, C, D, and E viruses. 3) Dialysis patients who are not on antiviral drugs at present.

Exclusion criteria are 1) Dialysis patients who are co-infected with Human Immunodeficiency Virus (HIV) and 2) Dialysis patients with underlying primary diseases such as Autoimmune diseases, Coronary Artery Disease, Asthma, and Diabetes Mellitus.

Sample size estimation - the prevalence of Hepatitis B among renal dialysis patients was reported to be 22% [21]. Assuming similar proportions in our study with an absolute precision of 7.5% and the desired confidence interval of 95% (Alpha error -5%), the required sample size is 117 patients.

Sample collection- The required minimum number of participants was calculated to be 117. However, the present study encompasses 120 dialysis patients. About 4 mL of blood is collected from each of the 120 patients in yellow and purple top vacutainers and centrifuged at the rate of 4000 rotations per minute to separate serum and plasma, respectively. Serum samples were tested immediately in CLIA, and Plasma samples were transported to the PCR lab at 2-8℃ for immediate DNA extraction.

HBsAg detection - the VITROS HBsAg test is performed using the VITROS HBsAg reagent pack and VITROS Immunodiagnostic Products, HBsAg Calibrator on the VITROS ECIQ Immunodiagnostic systems, and the VITROS 3600 Immunodiagnostic system. An immunometric immunoassay technique is used, which involves a simultaneous reaction of HBsAg in the sample with mouse monoclonal anti-HBs antibody coated onto the wells and a horseradish peroxidase-labeled mouse monoclonal anti-HBs antibody in the conjugate. The bound HRP conjugate is estimated by a luminescent reaction. The amount of bound HRP conjugate is indicative of the level of HBsAg present in the sample. HBsAg level values were determined according to kit literature If the value is /=1 (>/= 1000 IU/ml), then serum is considered positive for HBV infection. The tested samples were stored at -20 ℃ for further reference.

HBV DNA detection is done by the PCR method using the THERMO-FISHER Quant Studio 3 Real-time PCR machine, where,

The extraction of plasma samples was carried out by the BIOBEE Viral DNA extraction kit and the extracted plasma samples were tested for the HBV DNA using the NEODX HBV DNA qualitative RT PCR detection kit and the master mix-template reaction mixture prepared using NEODX kit was loaded into the THERMO-FISHER Quant Studio 3 Real-time PCR machine where it undergoes denaturation, extension, and annealing in thermal cycles to amplify the HBV DNA if present in the sample and to analyze the final result and its validity.

Steps include- 1) Nucleic acid extraction- HBV DNA extraction from plasma samples was done immediately using a BIOBEE viral DNA extraction kit. 250 microliters of plasma are mixed with 250 microliters of lysis buffer, 5 microliters of carrier RNA, and 10 microliters of Proteinase in a sterile microcentrifuge tube and incubated at room temperature for 5 minutes. The tube is then centrifuged at 8000 rotations per minute (rpm) for 5 minutes to get the lysate. 400 microliters of this lysate are mixed with 400 microliters of binding buffer in a new 1.5 ml microcentrifuge tube and then centrifuged at 8000 rpm for 30 seconds. After discarding the flow-through, 450 microliters of wash buffer 1 are added and centrifuged at 8000 rpm for 30 seconds. The process is repeated with wash buffer 2 also. After discarding the flow through, the spin column is air-dried at 8000 rpm for 3 minutes. Then the spin column is shifted to a new 1.5 ml microcentrifuge tube, and 50 microliters of elution buffer is added and centrifuged at 8000 rpm for 1 minute. The lysate collected in the microcentrifuge tube will have viral DNA if present. The extracted samples (templates) were stored at -80 ℃. HBV DNA detection from those templates is done using the NEODX HBV DNA qualitative RT PCR detection kit. 2) Master Mix preparation (120 reactions) (Tables 1 and 2) - Thaw contents of the kit at room temperature till equilibrated and the reaction mix is prepared for 100 and 20 reactions as below. 3) Template addition- 15 microliters of reaction mix are prepared for each of 120 PCR reaction tubes with 1 tube each for negative and positive controls to which 10 microliters each of sample, negative and positive control is added to respective tubes and centrifuged and transferred to PCR room where tubes are placed in the sample holder and programmed as below (Table 3) and the result is generated and data is interpreted for each run (Table 4 and 5). Follow-up – All 120 participants of the study were followed up regularly for a year at regular intervals of 2 months to monitor HBsAg and HBV DNA levels by CLIA and PCR, respectively. This follow-up was necessary to assess latent HBV reactivation among the dialysis participants. But the follow-up was solely based on HBsAg and HBV DNA parameters and other serological markers of Hepatitis B infection such as HBeAg (Hepatitis B pre-core antigen), anti-HBS (Hepatitis B antibody) and biochemical tests such as LFT (Liver function test), serum albumin to globulin ratio among others were not performed as patients did not have any clinical features of Hepatitis B infection and also due to financial constraints of the study.

Statistical method- Descriptive statistics of the HBV positives were analyzed and summarized in terms of percentage in Microsoft Excel. Statistical significance between continuous variables was tested using the Chi-squared test, and categorical variables were tested using the t-test. Statistical analysis was done using Statistical Package for the Social Sciences for Windows (version 21.0; Armonk, NY: IBM Corp., USA). A P value of

RESULTS

Out of 120 dialysis patients, 7 (5.8%) patients are found to be positive for HBsAg by CLIA, and no patient is found to be positive for HBV DNA by PCR (P value = 0.03). Out of 7 (5.8%) HBsAg-positive patients, 5(4.2%) belonged to the age group of 59-68 years, and 2(1.7%) belonged to the age group of 49-58 years (P value = 0.05).Out of 7 (5.8%) positives, 4(3.3%) are males and 3(2.6%) are females (P value = 0.0001).Out of 7 (5.8%) positives, 5 (4.2%) underwent dialysis for >50 months, and 2 (1.7%) underwent dialysis for 40-49 months (P value = 0.04). Out of 7 (5.8%) positives, 4 (3.3%) underwent dialysis 3 times per week, and 3 (2.6%) 2 times a week (P value=0.02). [Table 6]. These patients are followed up for 1 year at regular intervals of 2 months to monitor HBsAg and HBV DNA levels and to rule out any possibility of latent viral reactivation. Over the past year, the HBsAg levels remained low reactive (

Prevalence of Inactive Hepatitis B carriers = Number of cases (patients with positive HBsAg and negative HBV DNA) during a specified time/total study population*100 7/120*100=5.8%.

Table 1: Master Mix preparation for 100 reactions (1st batch)

Table 2: Master Mix preparation for 20 reactions

Table 3: Steps of Programming the PCR Instrument

Min – minutes

Sec - seconds

 Table 4: Result interpretation of HBV

Ct – Cycle threshold

Invalid – Repeat extraction and detection

Table 5: Data interpretation for HBV

FAM, Texas Red/Rox – genes tested

IC – Internal control

 Table 6: Baseline demographic characteristics of Participants

Age (years), Duration of dialysis (months), Frequency of dialysis (number of dialysis sessions/week), HBsAg – Hepatitis B surface antigen, CLIA – Chemiluminescence immunosorbent assay, HBV DNA – Hepatitis B virus Deoxyribonucleic acid, PCR – Polymerase Chain Reaction.

DISCUSSION