Genotype/Subgenotype Distribution of Hepatitis B Virus in Hemodialysis Centers from Gaza Strip, Palestine

doi:10.21203/rs.3.rs-2239659/v1

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Genotype/Subgenotype Distribution of Hepatitis B Virus in Hemodialysis Centers from Gaza Strip, Palestine

https://doi.org/10.21203/rs.3.rs-2239659/v1

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Background: The human hepatitis B virus (HBV) is currently prevalent in 250 million people. Chronic hepatitis B (CHB) has been associated with cirrhosis, hepatocellular carcinoma (HCC), liver failure, and overall mortality. This study aimed to determine the prevalence of the hepatitis B virus, its genotype, mutations, and antiviral resistance in hemodialysis centers from the Gaza Strip, Palestine.

Results: From the 273 HD patients participating in the study from the four HD centers in Gaza Strip, HBcAb was detected in 19 patients (6.96%), where HBsAg was detected in 12 patients (4.4%). HBV DNA was detected in 17 of the 19 positive HBcAb patients (89.47%); sequencing of the positive DNA by Sanger methods for the partial polymerase gene of HBV shows that genotype D is the only detected genotype where subgenotype D1 is the common (82.35%) followed by D2 (17.65%); Detection of reverse transcriptase antiviral resistance was done for the nucleotide analog (NUCs) Lamivudine, Adefovir, Entecavir, Tenofovir, and Telbivudine and all cases were sensitive to the mentioned antiviral. Neighbor-joining tree analysis was used to construct a phylogenetic tree with 130 reference strains with genotype D retrieved from GenBank. HBsAg positive patients had ALT and AST mean values higher than negative patients with a highly statistically significant relationship (p<0.01).

Conclusion: Genotype D HBV is common in HD with emphasis on the significance of identifying genotype, mutation, and antiviral resistance among isolated positive samples because these elements are crucial to the progression of the illness, its severity, chronicity, and treatment response.

hepatitis B virus

Genotype

Hemodialysis

Palestine

Human hepatitis B virus (HBV) is a serious health problem that affects people all over the world [1]. Currently, HBV is widespread in 250 million individuals [2]. Cirrhosis, hepatocellular carcinoma (HCC), liver failure, and overall mortality have all been linked to chronic hepatitis B (CHB) [3]. HBV virions are double-shelled particles with a diameter of 40 to 42 nanometers (nm) has an outer lipoprotein envelope that comprises three related envelope glycoproteins (or surface antigens). The viral nucleocapsid, or core, is located inside the envelope and includes the viral genome along with 3.2 Kilobase (kb) of relaxed-circular, partially duplex DNA and a polymerase enzyme that produces viral DNA in infected cells [4-8].

Ten HBV genotypes (A–J) that differ in more than 8% of the genome have emerged as a result of genetic diversity [9,10], whereas forty sub-genotypes differ by at least 4% of the genome [11,12]. The intensity, risk of complications, response to treatment, and potential effectiveness of HBV immunization can all be influenced by structural and functional variations dependent on genotype [10]. Patients undergoing hemodialysis (HD) may be more susceptible to infections, particularly illnesses caused by blood-borne viruses, the most frequent condition that develops as a side effect of HD treatment is viral hepatitis, including HBV and HCV [13].

According to the Palestinian Ministry of Health (MOH) annual reports, four HD centers, which serve approximately 900 HD patients, are distributed throughout the Gaza Strip. In accordance with the most recent research published by ElOttol A. and his colleagues in 2008, HBV was detectable in 8.1% of HD patients [14,15]. Despite the high significance of this situation, due to our knowledge, no documented information or previous research about genotype distribution, antiviral resistance, and mutations in HBV among the Gazan population has been noted, and this is the first address on this issue.

Study population and sample size

According to MOH, there are approximately 900 HD patients, and the prevalence of HBV among them was estimated to be 8.1% according to the last study in 2008[15]; the total number of patients in our study was 273 patients (calculated by using Epi info programmed from the center for disease control and prevention (CDC).

Study period and ethical consideration

The study was started in October 2021 and was finished in September 2022. The Helsinki committee reviewed and approved the study's principles and protocols, and each patient verbally consented after being informed of the study's purpose and potential outcomes.

The study participants' and results' personal information was handled in strict confidence. To ensure accurate data collection and avoid any misunderstandings, the researchers completed a closed-ended, multiple-choice questionnaire during patient interviews.

Eligibility criteria

Patients attending HD centers for more than one month were accepted in the study; the four HD centers that participated in the study were Al-Shifa hospital center in the middle of Gaza City, Shuhada’a Al- Aqsa Hospital center in Dair El-Balah, Nasser hospital center in Khan Yunis city, and Abu- Yousef Al-Najar Hospital center in Rafah city.

Samples collection

Prior to starting dialysis, two blood samples from each HD patient were taken in plain tubes to avoid heparin interfering with subsequent procedures. Within two hours, serum from one tube was separated and tested for HBcAb, HBsAg, and serum alanine and aspartate aminotransferases (ALT, AST). Serum isolated from the second tube was frozen at -70°C to be used for PCR analysis and a subsequent confirmatory serology test in a sterile, DNAse, and RNAse-free, firmly capped tube.

Virology

Hepatitis B core antibodies (HBcAb) determination was done by using Eco test one step HBcAb antibody test device, which is a rapid chromatography immunoassay to the qualitative determination of HBcAb in plasma (assure tech co. ltd, China), Non-reactive samples were considered negative for HBcAb and not retested further, while a reactive sample was retested to confirm the result by using MAGLUMI HBcAb IgG (CLIA) kit (Snibe diagnostic, China) a reactive sample were considered positive.

Hepatitis B surface Antigen (HBsAg) determination was done by using Eco test one-step HBsAg test device, which is a rapid chromatography immunoassay to the qualitative determination of HBsAg in plasma (assure tech co. ltd, China), A non-reactive sample was ruled to be negative for HBsAg and did not require further testing, whereas a reactive sample was retested to confirm the outcome using MAGLUMI HBsAg (CLIA) (Snibe diagnostic, China) a reactive sample was considered positive.

Positive HBcAb was tested for Hepatitis B surface antibodies (HBsAb) by using MAGLUMI HBsAb (CLIA) (Snibe diagnostic, China); a reactive sample was considered positive. In all tests, a positive and negative control was included to ensure quality.

Chemistry

All samples were tested for serum alanine and aspartate aminotransferase levels using kinetic methods (Diasys, Germany); For both ALT and AST, the upper limits of normal were determined at 40 and 37 IU/ml, respectively. [16].

Molecular Detection of HBV DNA

Following the manufacturer's instructions, viral DNA was isolated from serum samples using the exgene TM DNA/RNA extraction kit (GeneAll biotechnology, South Korea).

The primers were designed to specifically bind the conserved region among the various HBV genotypes as follows: External (F) primer (5`-CTGCT-GGTGGCTCCAGTT-3′); External (R) primer (5′-GCTAGGAGTTCC-GCAGTATGG-3′) [17]; Internal (F) (5`-ATCCTGCTGCTATGCCTCAT-3′); and Internal (R) primer (5`-TTGCGTCAGCAAACACTTGG-3′) (home designed).

The initial reactions were conducted in quantities of 20μl volumes, using 8μl extracted DNA in the presence of 1μl of each HBV external primer and 10μl of master mix (2X) (Promega, USA). The reaction cycling conditions were: 94°C, 3 minutes;( 94°C 30 seconds; 56°C for 50 seconds; and 72°C for 50 seconds) for 35 cycles; Finally, the reactions were completed at 72°C for 10 minutes and reserved at 4°C.

To increase the PCR test's sensitivity and specificity, a second nested PCR was run in each case. The reactions were conducted in 24μl volumes with 10μl of the first PCR DNA template and, 1μl of each internal HBV primer, 12μl of master mix (2X). The reaction cycling conditions were 94°C for 3 minutes; then (94°C for 30 seconds, 60°C for 50 seconds, and 72°C for 50 seconds) for 35 cycles; The reactions were allowed to finish at 72°C for 10 minutes before being held at 4°C. The results were examined using a 2% agarose gel, a 100 bp ladder, and ethidium bromide dye. Positive results for the first and second PCRs were assessed to have appeared when bands measuring 1230 bp and 786 bp, respectively.

Sequencing and phylogeny

The positive 786bp product was purified by using Wizard^® SV Gel and PCR Clean-Up System (Promega, USA) according to the manufacturer's recommendation. Sequencing of the purified PCR products was conducted by Pronto Diagnostics (Tel Aviv-Yafo), using the same forward primer as was used in the second PCR.

HBV genotyping and sequence analysis

Genotype determination of the purified PCR products and drug resistance were conducted by using Genafer/Geno2pheno [hbv] 2.0 software (https://hbv.geno2pheno.org/index.php), the nucleotide sequence was compared to those from GenBank by using Basic Local Alignment Search Tool (BLASTN) program (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) Polygenetic analyses were performed by using OMEGA11 program on the translated HBV pol gene (702 bp, position 481–1182). Muscle algorithm alignment was used to determine the genetic distance, the divergence of nucleotide substitutions per 100 nucleotide sites, and neighbor-joining tree analysis (https://www.megasoftware.net/).

During the study, a total of 273 patients from four centers participated; 143 (52.4%) patients came from the Al-Shifa hospital in Gaza City, followed by 30 (11.0%) from the Shuhada Al-Aqsa Hospital in Dair El-Balah, 49 (17.9%) from the Nasser hospital in Khan Yunis, and 51 (18.7%) from the Abu-Yousef Al-Najar Hospital in Rafah. Among the 237 individuals tested, 149 (54.6%) were men, and 124 (45.4%) were women. The patient ages ranged from 14 to 84 years old, with a mean age (x̄) of 51.25 years with a Standard division (SD) of 17.5 years. The socio-economical and health status of all participants are described in (Table 1) and (Table 2), respectively.

Prevalence of HBV by serology test

From the 273 patients participate in the study, HBcAb was detected in 19 (7%) patients, 14 (9.7%) out of 143 patients from Al-Shifa hospital, 5 (16.6%) out of 30 patients from Shuhada Al-Aqsa Hospital, and 0 patients out of 49 and 51 patients from both Nasser and Abu- Yousef Al-Najar hospitals, on the other hands HBsAg was detected in 12 (4.4%) patients, 8 (5.6%) patients from Al-Shifa hospital ,4 (13.3%) patients from Shuhada Al-Aqsa Hospital, and 0 patients from both Nasser and Abu- Yousef Al-Najar hospitals (table 3). From the19 positive HBcAb patients, two (14.2%) out of the 14 patients from Al-Shifa hospital, and zero (0 %) of the 5 patients of Shuhada Al-Aqsa Hospital were positives for HBsAb respectively.

Table 1: The socio-economic characteristics of the study participant (n=273)

Characteristic		number	Percent (%)
Place of Residence	North of Gaza	50	18.3
	Gaza city	93	34.1
	Middle of Gaza	30	11.0
	khan Yunis	49	17.9
	Rafah	51	18.7
Age Groups	less than 20	10	3.7
	20-40	69	25.3
	41-60	95	34.8
	61-80	94	34.4
	more than 80	5	1.8
Education Level	illiterate	19	7.0
	primary	38	13.9
	preparatory	60	22.0
	secondary	91	33.3
	university	65	23.8
Sport	yes	20	7.3
Sport	no	253	92.7
Smoking	yes	32	11.7
Smoking	no	241	88.3
work	no	267	97.8
work	yes	6	2.2

Table 2: health status characteristics of participant patients.

Characteristic		number	percent
Period of HD	1-24 months	89	32.6
	>24-48 months	68	24.9
	>48-72 months	50	18.3
	>72 months	66	24.2
No. of HD/week	one	3	1.1
	two	51	18.7
	three	216	79.1
	four	3	1.1
Stable HD center	yes	265	97.1
Stable HD center	no	8	2.9
HBV vaccine	less than one years	6	2.2
	from one to less than five years	9	3.3
	from 5 years to less than ten years	6	2.2
	more than ten years	36	13.2
	no	191	70.0
	don't know	25	9.2
Previous surgery	no	148	54.2
	yes, since less than one year	42	15.4
	yes, since from 1 year to less than five years	49	17.9
	yes, for more than five years	34	12.5
Blood unit receiving/month	one	61	22.3
	two	16	5.9
	three	2	0.7
	less than 1	27	9.9
	no	167	61.2
Treatment abroad	Not treated	214	78.4
	EGYPT	43	15.8
	KSA	5	1.8
	UAE	1	.4
	JORDAN	7	2.6
	IRAQ	1	0.4
	OMAN	1	0.4
	Germany	1	0.4

Table 3: Prevalence of HBcAb, HBsAg among HD patients

hospitals	HBsAg	HBcAb
hospitals	HBsAg	positive	negative
Al-Shifa	Positive	8	0
Al-Shifa	negative	6	129
Shuhada Al-Aqsa	Positive	4	0
Shuhada Al-Aqsa	negative	1	25
Nasser	Positive	0	0
Nasser	negative	0	49
Abu- Yousef Al-Najar	Positive	0	0
Abu- Yousef Al-Najar	negative	0	51

Prevalence of HBV by nested PCR test and genotype detection

HBV DNA was detected in 17 (89.47%). Of the19 positive HBcAb patients, five patients from Shuhada Al-Aqsa hospitals (100%),12 patients from Al-Shifa hospital (85.71%), and two patients from Al-Shifa hospital were negative for PCR, distribution of positive HBV DNA are shown in table 4, hepatitis B virus genotypes determined among the positive HBV DNA show that genotype D is the only detected genotype in Gazan HD centers were subgenotype D1 is the most common prevalent 14 out of the 17 positive HBV DNA (82.35%) followed by D2 in three samples (17.65%), the two negative PCR samples were positive for HBsAb indicate the previous infection with HBV (Table 4).

Positive HBV DNA sequences were submitted to GenBank and given the accession numbers OP184982, OP184983, OP184990, OP184992, OP184996, OP184998, OP185001, OP185002, OP185011, OP185012, OP185014, OP185019, OP185022, OP185026, OP185028, OP185031, OP219675.

Table 4: Prevalence of HBcAb, HBV DNA among HD patients

hospitals	HBV DNA			HBcAb positive
Al-Shifa	Positive	D1	10		12
		D2	2
	negative		2		2
Shuhada Al-Aqsa	Positive	D1	4		5
		D2	1
	negative		0		0
Nasser	Positive	D1	0		0
		D2	0
	negative		0		0
Abu- Yousef Al-Najar	Positive	D1	0
		D2	0		0
	negative		0

Antiviral susceptibility and mutational detection

Detection of reverse transcriptase antiviral resistance was done by using Genafer/Geno2pheno (HBV) 2.0 software for the nucleotide analog (NUCs) Lamivudine, Adefovir, Entecavir, Tenofovir, and Telbivudine by detection of protein mutations, all cases were sensitive for the mentioned antiviral, mutation detected are summarized in (Table 5).

Phylogenetic analysis

A phylogenetic tree was created using neighbor-joining tree analysis, the divergence of nucleotide substitutions per 100 nucleotide sites, and muscle algorithm alignment to calculate the genetic distance. as illustrated in figure 1.

ALT and AST Results

ALT mean values for HBsAg positive patients were 42.08 ± 22.6 IU/ml, and for HBsAg negative patients, they were 17.74 ± 6.91 IU/ml. There was highly statistically significant (p< 0.01). Additionally, AST mean values for HBsAg-positive patients were 34.67 ±15.89 IU/ml and 18.67± 6.91 IU/ml for HBV-negative patients. There was highly statistically significant (p< 0.01).

Table 5: Mutation detection among HBV isolated samples.

accession number	reverse transcriptase (RT) domain	Small Hepatitis B (SHB) protein	Escape mutation
OP184982	F122I, H124Y, N248H, S317A	----	-
OP184983	H124Y, Y135S, F221Y, N248H	T127P, L213I	-
OP184990	F122I, H124Y, N248H, S317A	----	-
OP184992	F122I, H124Y, N248H, S317A	----	-
OP184996	F122I, H124Y, D134H, Y135P, C136S, S137T, S219A, F221Y, N248H, K275Q, S317A	T127P, A128V, S210R, L213I	128V
OP184998	H124Y, Y135S, I163S, V214G, S219A, N248H, I266R, Q267H	T127P, S155A, Y206V, S210R	-
OP185001	F122I, H124Y, T128I, Y135S, N248H, S317A	P120S, T127P	120S
OP185002	F122I, H124Y, T128I, Y135S, N248H, S317A	P120S, T127P	120S
OP185011	F122I, H124Y, Y135S, N248H, V253I, C256S, S317A	T127P, V184A	-
OP185012	F122I, H124Y, Y135S, N248H, V253I, C256S, S317A	T127P, V184A	-
OP185014	F122I, H124Y, Y135S, Q149K, A211T, L220I, L229M, S246H, N248H, I266R, M309K, S317A, L336M	T127P, F220L	-
OP185019	F122I, H124Y, H126Q, Y135S, N248H, S317A	T118K, T127P	118K
OP185022	F122I, H124Y, Y135S, N248H, S317A	T127P	-
OP185026	Y135S, N139S, N248H, V278C, C303R, M309K, L336M	T127P, T131A	-
OP185028	Y135S, S189P, H197P, N248H, V278C	T127P, F170S, T189P	-
OP185031	Y135S, N248H, V278C	T127P	-
OP219675	S219A, N248H	S210R

Hepatitis B virus infection is a significant health problem among HD patients all over the world, as they may affect the course of HBV-associated chronic liver disease and its outcome, as well as a patient's responsiveness to antiviral medications; genotypes have drawn increased attention [18].

According to our knowledge, this is the first study in the Gaza Strip to determine the genotype of HBV among Gazan patients and to detect mutation and antiviral resistance by using molecular biology techniques. In this study, HBcAb was detected in 19 (6.96%) of the 273 HD patients who participated in the study; the most prevalent center was from Shuhada Al-Aqsa Hospital 5 out of 30 patients (16.67%), followed by Al-Shifa hospital 14 out of 143 patients (9.79%), were HBcAb was not detected in patients from both Nasser and Abu- Yousef Al-Najar hospitals.

Of the 19 HBcAb positive HBsAg was only detected in 12 patients (63.16%), 4 out of 5 patients (80%) from Shuhada Al-Aqsa Hospital, 12 out of 14 patients (63.16%) from Al-Shifa hospital, HBsAg was not detected in HBcAb negative patients in the four centers, so the prevalence of HBsAg among the 273 patents was (4.39%) and this prevalence for HBsAg lower than previous studies (5.9%), (8.1%), (10%), (11.8%) in Jordan, Palestine, Saudi Arabia, and Bahrain, respectively [19,15,20,21].

By using nested PCR technique among the 19 positive HBcAb patients, 17 (89.47%) Of them had positive DNA results, while only two patients from Al-Shifa hospital were negative for PCR and positive for HBsAb, indicating previous HBV infection.

The prevalence of HBV among HD patients was 6.23% by using nested PCR compared to 6.96% by using the HBcAb test and 4.4% by using the HBsAg test; these results demonstrate the importance of using nucleic acid techniques in combination with serology tests for the detection of HBV.

HBV genotype was determined by using Sanger Sequencing, where hepatitis B genotype D was the only detected genotype among the 17 positive PCR samples; this result was convenient with another study in the west bank, Palestine [22], also our study agreed with many studies in the Mediterranean region as Saudi Arabia, Emirates, Egypt, Jordan, Syria, and Lebanon were the genotype D is the dominant [23-30]. Subgenotype D1 was the most common, detected in 14 (82.35%) samples, followed by D2 in three samples (17.5%), and this result agrees with the study in the west bank, Turkey, Greece, Iran, Pakistan, Egypt and Lebanon [22,31-36].

Antiviral susceptibility was determined by detecting protein mutation in the polymerase gene by using Genafer/Geno2pheno (HBV) 2.0 software, where all the positive samples were found to be sensitive to the antiviral Lamivudine, Adefovir, Entecavir, Tenofovir, and Telbivudine.

The mutation was detected by using the same program in the polymerase gene in all samples, and the following mutations were determined A211T, C136S, C256S, C303R, D134H, F122I, F221Y, H124Y, H126Q, H197P, I163S, I266R, K275Q, L220I, L229M, L336M, M309K, N139S, N248H, Q149K, Q267H, S137T, S189P, S219A, S246H, S317A, T128I, V214G, V253I, V278C, Y135P, Y135S) eight of these mutations have been detected in the previous study in the west bank of Palestine(F221Y, H124Y, Y135S, S137T, F221Y, N248H, C256S, M309K) [22]; Small Hepatitis B (SHB) protein mutation has detected in 14 cases out of 17 positive cases T127P, L213I, A128V, S210R, S155A, Y206V, P120S, V184A, F220L, T118K, T131A, F170S, T189P), two of these mutations were detected in the previous study in the west bank. Palestine (S210R and L213I) [22].

Escape mutation (A128V, P120S, T118K) was detected in four cases of the positive PCR samples; this escape mutation was negative for HBsAg and positive for HBcAb, which may indicate occult HBV infection; many studies in the world illustrate that HBV-carrying mutant escaping from antibodies has decrease affinity for HBsAb binding which may lead to occult HBV infection and ineffective vaccination [37-42], the previous study demonstrated that the presence of aa substitutions in the range of 120 to 123 is crucial for HBsAg antigenicity and substantially linked with decreased immunoassay detection [43], another study elucidate that antigenicity and immunogenicity of HBsAg are significantly influenced by the biochemical characteristics of the aa residues at positions 122 and 145. Additionally, the neutralization and elimination of HBsAg during HBV infection depends on the existence of appropriate anti-HBs antibodies [44].

Determination of liver enzymes (ALT, AST) shows a statically significant difference between positive and negative HBV patients. The previous study agreed with this finding, where positive HBV patients have higher liver enzymes level [15].

Our study demonstrates the prevalence of HBV among HD patients and also the importance of using PCR for detecting occult HBV among the population, especially high-risk groups such as HD patients. The study focuses on the importance of detecting genotype, mutation, and antiviral resistance among isolated positive samples where these factors play a vital role in the progress of the disease, the severity, the chronicity, and treatment response, so the treatment plan should base on genotyping with mutation and resistant detection. Our study recommended that a national plan should be implanted for the diagnosis and treatment of HBV depending on serological and molecular technology to increase sensitivity and specificity for detecting positive cases that decrease unsafe blood transfusion, Ineffective treatment, and great suffering of the patients with the high cost and prolong hospital stay.

Ethics approval and consent to participate

A formal letter of approval to conduct the study were obtained from the Helsinki Committee, and Ministry of Health in Gaza strip, Palestine.

Every patient in the study was given a consent form about the study. This form included the purpose of the research, confidentiality of information, and so on; The interviewer was read the consent form for each patient.

Consent for publications

Written informed consent provided by the patients were obtained for the publication.

Availability of data and materials

Due to the privacy of patients, the related data cannot be available for public access but can be obtained from Abed Elkader Y. ElOttol upon reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This research was funded by the researcher Abed Elkader Y. ElOttol.

Authors’ contributions

AYO contributed to the design of the study, carried out and directly funded the research, computed the statistical analysis, and created the manuscript. MAA contributed to the design of the study; provided guidance for the theoretical and practical work. FAS contributed to the design of the study; and supervised the molecular genetic studies. MJA contributed to the molecular genetic studies; SFA contributed to the molecular genetic studies; AMT contributed to the serological and biochemical testing of the studies; DOK participated in the serological and biochemical testing of the studies; NMAZ contributed to the design of the study; supervised on the theoretical and practical work and the statistical analysis. All authors have read and approved the final manuscript.

Acknowledgments

Our sincere gratitude goes to the University College of Science and Technology- Khan Younis, the Palestinian Ministry of Health, especially all workers and patients at the HD centers, to AlDiea Medical laboratory team, and to the Molecular Biology department team at the Islamic University-Gaza for all their helpful and friendly support to complete this work.

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Genotype/Subgenotype Distribution of Hepatitis B Virus in Hemodialysis Centers from Gaza Strip, Palestine

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