Incidence of Occult Hepatitis B virus infection among Sudanese patient with Hepatocellular Carcinoma and Liver Cirrhosis in the absence of Hepatitis C virus

doi:10.21203/rs.3.rs-2320343/v1

This study was undertaken to determine the incidence of Occult HBV Infection (OBI) in Sudanese patients living with LC and HCC in absence of hepatitis C virus (HCV). The plasma of 200 recruited patients were tested for HBsAg, anti HBcAg and HBeAg using ELISA kits. The DNA and RNA were extracted from the samples which tested negative for HBsAg and positive for HBcAg by using viral DNA and RNA extraction kit. Real time PCR was performed for detection of HBV and HCV followed by Multiplex Nested PCR with specific HBV genotype primers. Only 2% HCC patients and 3% LC patients were seronegative for both HBsAg. However, high seroprevalence of HBsAg and HBeAg were recorded in HCC patients group compared to LC group with no statistical significant differences. In regards to HBV genotypes, D, E and mixed D/E genotypes were detected in all positive samples with absence of HCV. Statistical analysis was performed using IBM SPSS version 20.0 OBI was detected in hepatocellular carcinoma and liver cirrhosis patients. These results highlighted the importance of integrating effective OBI screening techniques such as real time PCR and multiplex nested PCR for proper diagnosis and clinical management of liver diseases.

Hepatitis B virus

occult HBV

Hepatocellular Carcinoma and Liver Cirrhosis

multiplex nested PCR

Sudan

Chronic Hepatitis B virus (HBV) infection is one of the global leading causes of liver cirrhosis (LC) and hepatocellular carcinoma (HCC) representing serious public health concerns. In 2017 the World Health Organization (WHO) estimated approximately 350 million of the world population were infected with HBV ^1,2,3. As a special phase, Occult HBV infection (OBI) is recognized to be a risk factor for progression of the clinical manifestations of HBV infection ⁴. Moreover the OBI is characterized by high detectable level of HBV-DNA in hepatic tissues accompanied by negative results of hepatitis B surface antigen (HBsAg) and low or undetectable serum HBV-DNA ⁴. Both seropositive (the presence of antibody against viral core – Anti-HBc- without detectable HBsAg) and seronegative results can be found in OBI individuals, making the HBV-DNA the only marker of HBV infection ⁵. It’s now well-known from a variety of studies that such seronegative results were due to the evolved mutation on the surface gene (escape mutant) that modified the HBsAg and making difficulties in estimating HBV infection incidence by commercially available detection assays ^{6, 7}.

Despite that OBI patients demonstrated almost the minimal clinical implication; the resulting persistent infection receives a big concerns as it represent a potential risk of transmitting the infection to healthy people ⁵. However, the prevalence of OBI varies among the populations in different geographical regions and with the sensitivity and specificity of serological tests and nucleic acid-based assays used for detection. Generally, based on the endemicity of HBV infection, the prevalence of OBI is higher in HBV endemic regions compared to other regions with low/ intermediate HBV prevalence rate ⁸. Considering Africa at intermediate endemicity (2–7%), the OBI is fairly prevalent across the continent ranging from approximately 7–50% in low-risk populations to approximately 6–30% in high risk populations ^{9, 10, 11}. In Sudan, several studies have investigated the prevalence of OBI among various risk groups. A recent study by Ibrahim and colleagues ¹² reported high prevalence of OBI among renal transplant’s Sudanese patients. Furthermore systematic review and meta-analysis studies were conducted and were published in literature between 2009 and 2019. These studies provided an update of the OBI epidemiology in Sudan and estimated high pooled prevalence of OBI, with 15.51%. Individuals within high-risk populations comprised those of blood donors, hemodialysis patients, febrile patients, HIV-positive patients, HCV-infected patients and renal transplant recipients from seven states in Sudan¹³.

The molecular mechanism and pathogenesis of OBI varies depending on the viral-host interactions that mediated by the immune response ¹⁴. The diverse molecular interactions were found to suppress the replication of HBV-DNA and the secretion of HBsAg, making difficulties in diagnosis and complex dynamics of OBI transmission ^{13, 15}. However, among the HBV genotypes, the A, C, G, E and D are the most genotypes that have been detected in patients with OBI worldwide¹⁶. The most efficient molecular biology technique in identifying these genotypes is the Multiplex Nested PCR ^12,17. In Sudan, several studies have used this technique to investigated the prevalence and the role of OBI in various targeted groups, for instance; renal transplant receipts, ^12,17 blood donors ^18,19 and hemodialysis patients ¹⁸. Across these studies there were no reports on the prevalence of occult HBV infections in patients living with liver-related complications of cirrhosis and hepatocellular carcinoma. Thus, this study described the association of hepatocellular carcinoma and liver cirrhosis and OBI in the absence of HCV infection in Sudanese patients. The study was set out to understand the incidence/prevalence and the role of OBI in Sudanese patients specially those at risk and with such complications which could lead to the development of better detection methods and to provide effective clinical recommendations.

Descriptive Statistics:

A total of (200) of patients (100 with HCC and 100 with LC were enrolled in this study. In HCC patient group, about 36% (36 out of 100) of the participants were female and 64% (64out of 100) were male, whereas in LC patient groups the females and males were representing 44% and 56% of the 100 participants, respectively. The age of participants from both groups was between 33 years to 73 years (Table 2).
Table 1: Primers used for the first PCR program

First PCR round for Universal primer
	primer name	primer type	Sense/ antisense	primer sequence 5'-3'	primer spanning position	amplicon size
P1	universal outer primer		sense	5'-TCA CCA TAT TCT TGG GAA CAA GA-3'	(2823–2845 nt)	1063bp
S1	universal outer primer		antisense	5'-CGA ACC ACT GAA CAA ATG GC-3	(685-704 nt)
Second PCR round
MIX A	BA1R	specific type A	sense	5'-CTC GCG GAG ATT GAC GAG ATG T-3'	(113-134 nt,)	68 bp
	BB1R	specific type B	sense	5'-CAG GTT GGT GAG TGA CTG GAG A-3'	(324-345 nt,)	281 bp
	BC1R	specific type C	sense	5'-GGT CCT AGG AAT CCT GAT GTT G-3	(165-186 nt)	122bp
	B2	specific genotype A to C	antisense	5'-GGC TCM AGT TCM GGA ACA GT-3'	(67–86, nt,)
MIX B	BD1	specific type D	sense	5'-GCC AAC AAG GTA GGA GCT-3'	(2979–2996 nt,)	119bp
	BE1	Specific type E	sense	5'-CAC CAG AAA TCC AGA TTG GGA CCA-3'	(2955–2978 nt)	167bp
	BF1	specific type F	sense	5'-GYT ACG GTC CAG GGT TAC CA-3'	3032–3051 nt)	97 bp
	B2R	Specific types D to F	antisense	5'-GGA GGC GGA TYT GCT GGC AA-3'	(3097-3078 nt)

Table 2

The Frequency of age groups and Gender in the HCC and LC groups
Groups		HCC	LC
Total Number of Subjects		100	100
Gender (%)	Females	36.0%	44.0%
Gender (%)	Males	64.0%	56.0%
Age Groups (%)	Age < 40	14.0%	17.0%
	Age: 40–60	56.0%	64.0%
	Age > 60	30.0%	19.0%

The Frequency of HBsAg and anti HBeAg:

All the samples were seropositive for HBV DNA and antiHBcAg. Only one individual was HBsAg seronegative among HCC patients and three among LC patients as illustrated in Table 3 and 4. Similarly, the ELISA test revealed that 44% of the Hepatocellular Carcinoma group (Table 3) and 44% of the Liver Cirrhosis group were negative for HBeAg (Table 4). In HCC patients (Fischer exact test, P-value = 1.000) or LC patients (Fischer exact test, P-value = 0.253), there was no significant difference between HBsAg and HBeAg results. However, one HCC patient was reported to be negative for both HBsAg and HBeAg (Table 3).

Table 3: Cross Tabulation between the frequencies of HBsAg and HBeAg in Hepatocellular carcinoma patients:

Table 4: Cross Tabulation between the frequencies of HBsAg and HBeAg in Liver Cirrhosis patients:

The frequency of HBV genotypes in HCC and LC groups:

In the HCC group, one sample was genotype E and the other was mixed genotype D/E, while in the LC group, one sample was genotype E, one was genotype D, and the other was mixed genotype D/E. (Fig. 1).

Association between Gender and HBsAg in HCC and LC patients:

Among the 100 individuals in HCC patients, all the females were seropositive HBsAg whereas 3.1% were tested negative. In contrast, the high HBsAg seropositive results were recorded in males (98.2%) compared to females (95.5%) (Figs. 2 and 3).The difference between gender types were found to be not significant in the two patients groups (HCC: P-value = 0.535, LC: P-value = 0.581)

Association between Age Groups and HBsAg in HCC and LC patients:

In the HCC group, the large number of seropositive results were recorded in patients with age greater than 60 years (100%) whereas the HBsAg seronegative results were highly recorded in patients’ age lower than 40 (7.1%) compared to other age groups From the Fischer exact test there was no association between age groups and HBsAg as illustrated in Fig. 4. Similarly, as shown in Fig. 5, the differences between age groups and HBsAg detection in LC patients groups were statistically not significant. It is found that OBI occurred exclusively in the 40–60 age group.

Occult HBV infection accelerates the progression of liver fibrosis, cirrhosis, and finally leading to hepatocellular carcinoma (HCC) (20). In Sudan the incidence of OBI is low frequency; according to result were conducted 2020 by Ibrahim and colleagues which is exhibit 18 were OBI in renal transplant patients. (12) .This study analyzed the occult HBV-genotypes and HCV in liver cirrhosis HCC patients. Indicated that only5 (2were HCC and 3 were Liver Cirrhosis) out 200 samples were OBI (positive HBV DNA, negative for HBsAg, and that all positive for anti HBcAg), and 4 out of 5 were positive for HBeAg indicating that they active occult HBV carrier.

In addition, the prevalence of OBI was moderately low (2.5%) as it has been diagnosed by molecular biology technique. This agrees with a study had carried out by Alejandra Duque and collogues in Colombia (21) where they found OBI occurred in 13% of HCC and Liver Cirrhosis patients. However, it was much lower than studies have been conducted in japan(22) and Italy ( 23) and disagree with other study was conducted in Egypt by (20) which they found OBI were22% in HCC and LC Egyptian patient .

This study showed us males were more susceptible to OBI in HCC (64.0%) and LC (56.0) group while the female were less susceptible to OBI in HCC (36.0%) and LC (44.0%) among the 100 individuals in HCC patients, all the females were seropositive HBsAg whereas 3.1% were tested negative. In contrast, the high HBsAg seropositive results were recorded in males (98.2%) compared to females (95.5%) (Figs. 2 and 3).The difference between gender types were found to be not significant in the two patients groups (HCC: P-value = 0.535, LC: P-value = 0.581) this result agree with other study were conducted in by (24) in Japan.

On the other hands In the HCC group, the large number of seropositive results were recorded in patients with age greater than 60 years (100%) whereas the HBsAg seronegative results were highly recorded in patients’ age smaller than 40 (7.1%) compared to other age groups From the Fischer exact test there was no association between age groups and HBsAg as illustrated in Fig. 4. Similarly, as shown in Fig. 5, the differences between age groups and HBsAg detection in LC patients groups were statistically not significant. It is found that OBI occurred exclusively in the 40–60 age group this result agree with other result was conducted in japan by Nakano and collogues (24).

This study exhibit that all the samples were positive for HBV DNA and antiHBcAg. Only two individual were HBsAg seronegative among HCC patients and three among LC patients as illustrated in Table 3 and 4 this the result was similar to other study was conducted in Italy (23), the ELISA test revealed that 44% of the Hepatocellular Carcinoma group (Table 3) and 44% of the Liver Cirrhosis group were negative for HBeAg (Table 4). In HCC patients (Fischer exact test, P-value = 1.000) or LC patients (Fischer exact test, P-value = 0.253), there was no significant difference between HBsAg and HBeAg results. However, one HCC patient was reported to be negative for both HBsAg and HBeAg (Table 3).

Regarding to the occult HBV genotypes this study showed us In the HCC group, one sample was genotype E and the other was mixed genotype D/E, while in the LC group, one sample was genotype E, one was genotype D, and the other was mixed genotype D/E. (Fig. 1). This result is similar to results were conducted in Sudan (12) (17) showed us the common genotype of HBV in Sudan were D, E and mixed infection D and E genotypes and in Egypt (20).

In This study all occult HBV patient were negative for HCV indicating that Occult Hepatitis B virus cusses of Hepatocellular Carcinoma and Liver Cirrhosis in absence of hepatitis C virus in Sudanese Patients this finding supporting the uses of molecular techniques such as real time PCR and multiplex PCR in Sudan for routine diagnosis spicily in blood donors and other groups under risk of HBV infection such as renal transplant patients, hemodialysis patients, liver diseases and immune suppressive drugs users .

In this study, the incidence of OBI was found high HCC and Liver Cirrhosis patients in the Khartoum-Sudan by using real time PCR and multiplec nested PCR. However, there are no studies about OBI frequency in different Sudan subpopulations with or without risk factors for HBV infection HCC and Liver Cirrhosis patients. Generally, these findings are useful for future studies since there is minimum information available OBI infection in Sudan. We recommended to use the real time PCR, multiplex nested PCR than ELISA in OBI diagnosis. Further studies should be performed in Sudanese patients to determine OBI incidence and prevalence in populations with different risk factors. Furthermore, evaluation and follow-up of patients with OBI should be performed in order to determine the clinical implications of OBI in patient with liver disease.

Specimen collection and processing:

This descriptive cross-sectional study was conducted on patients admitted to National Center for Gastroenterology Ibnsena Hospital-Khartoum State during the period of January-March 2019. A total of 200 blood samples (100) from patient with hepatocellular carcinoma and 100 with liver cirrhosis) were collected. Prior to sampling, sociodemographic data (i. e. age, gender, original residence and clinical manifestation) were collected through structured questionnaire.

Samples Processing:

Whole blood was drawn into a tube containing EDTA and was then centrifuged at 3000 rpm for five minutes for separation plasma.

Enzyme Linked Immunosorbent Assay

The plasma samples were tested for HBsAg, anti HBcAg and HBeAg using commercial ELISA kit (fortress U.K) following the manufacturer instructions.

Viral Dna And Rna Extraction

The viral DNA and RNA were extracted from samples that were shown seronegative for HBsAg and seropositive for HBcAg using viral DNA and RNA extraction kit (intron biotechnology, Korea).

Real time PCR for detection HBV and HCV.

For HBV and HCV detection, a commercially available quantitative Real time PCR kit (Sansure biotechnology, china) was used according to manufacturer instruction (Fig. 7and 8)

Multiplex Nested Pcr

Multiplex nested PCR with specific HBV genotype primers to amplify the HBV surface and polymerase regions. The nested PCR was performed in thermocycler machine (Techne, England) to amplify the HBV DNA using previously designed primers specific for HBV surface (S) and polymerase (P) regions (Naito et al., 2001). The PCR reactions were carried out in a 25µl reaction mixture containing 5µl of DNA, 2µl from 10pmol of each primer (forward and reverse) (Table 1), 5µl of 2mM dNTP mix, 25m M MgCl2, 2.5U Taq, 1x buffer (intron biotechnology, Korea) and 13µl ddH₂O. Optimized amplification conditions were as follows: initial denaturation at 94°C for 5 minutes, followed by 40 cycles consisted of denaturation at 94°C for 1 minute, annealing at 55°C for 1 minutes and elongation at 72°C for 2 minutes. Genotype specific primers were used in Nested PCRs to determine the six HBV genotypes (A to F) according to differences in the sizes of amplified DNA (Table 1) (25). For this purpose two reactions were performed in different mixtures for each sample: mix A: applied for identification of genotypes A, B, C and mix B: for genotypes D, E, F. Thus a total of 3µL aliquot of the first-round PCR product was added to each of mix 1 and mix 2. For each nested PCR mixture, 3µl of DNA were added to a mixture includes 2µl 10 pmol of each primers forward and reverse primer, 5 µl of 2mM dNTP mix, 25 mM MgCl₂, 2.5 U Taq, 1x buffer (intron biotechnology, Korea) and 15µl ddH₂O. The nested PCRs were amplified with the following conditions; preheating at 94°C for 5 minutes, 25 cycles of amplification at 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, with the final elongation at 72°C for 5 minutes. Then, PCR products were electrophoresed in a 2% agarose gel and the bands were visualized using a UV gel documentation system (Uni EQUIP, Munich, Germany). The sizes of PCR products were estimated according to the migration pattern of a 100bp DNA ladder and genotypes were then determined according to the amplified size of PCR product (Fig. 6).

Statistical Analysis:

Descriptive indices such as frequencies, percentages, mean and median were calculated. Data was analyzed using software package. Categorical results were examined using a Pearson’s Chi square test and Fisher's Exact Test. The strength of significant was done by calculating the contribution to chi square of each cell. Then the post hoc test was done by multiple regression approach to analyze contingency tables as described by (Beasley and Schumacker 1995) and adjusted (P value = 0.008) was used to compare different groups to each other. All of these statistical analyses were performed by statistical package of social science (IBM SPSS version 20.0) for windows considering P values ≤ 0.05 as statistically significant

Acknowledgment

All authors would like to thanks ibn sena hospital administration and national center for gasteroentology for permission for sample collection and special thanks to National University Biomedical Research Institute (NUBRI), National University, Sudan for fund and support.

Conflict of interest statement

All authors have no competing interest declared.

Funding

This work has been supported by National University Biomedical Research Institute (NUBRI), National University, Sudan.

Ethical consideration

The study was approved by the Ethical Review Committee (ERC) of National University biomedical research institute, Khartoum State, Sudan. Informed consents were obtained from adult patients or from parents or legal guardians of children. All methods were performed in accordance with the relevant guidelines and regulations.

Data availability

The datasets used and analysed during the current study available from the corresponding author on reasonable request.

Franco E, Bagnato B, Marino MG, Meleleo C, Serino L, etal Hepatitis B: epidemiology and prevention in developing countries. World J Hepatol.; 74–80 (2012).
Maucort-Boulch D, de Martel C, Franceschi S, Plummer M. Fraction and incidence of liver cancer attributable to hepatitis B and c viruses worldwide. Int J Cancer; 7 142:2471- (2018)
WHO – World Health Organization. Global hepatitis report,. Available online: https://www.who.int/hepatitis/publications/global-hepatitis-report2017/en /.(2017)
Raimondo G, Locarnini S, Pollicino T, Levrero M, Zoulim F, et al. Update of the statements on biology and clinical impact of occult hepatitis B virus infection. J Hepatol.;71:397–408. (2019).
Zobeiri M. Occult hepatitis B: clinical viewpoint and management. Hepat Res Treat; 259148. (2013).
Gerlich WH, Bremer C, Saniewski M. Schüttler CG, Wend UC, Willems WR, et al. Occult hepatitis B virus infection: detection and significance. Dig Dis;; 28(1):116-25. (2010).
Torbenson M, Thomas DL. Occult hepatitis B. Lancet Infect Dis.;2:479–86.( 2002)
Gizachew Beykaso,Andargachew Mulu,Mirutse Giday,Nega Berhe,Markos Selamu, et al. "Occult Hepatitis B Virus Infection and Its Risks of Cryptic Transmission in Southern Ethiopia." Infection and Drug Resistance 15: 619. (2022).
Abd El Kader Mahmoud O, Abd El Rahim Ghazal A, El Sayed Metwally D, Elnour AM, Yousif GE. Detection of occult hepatitis B virus infection among blood donors in Sudan. J Egypt Public Health Assoc.;88: 14–18. (2013) pmid:23528527
Sondlane TH, Mawela L, Razwiedani LL, Selabe SG, Lebelo RL, Rakgole JN, et al. High prevalence of active and occult hepatitis B virus infections in healthcare workers from two provinces of South Africa. Vaccine.;34: 3835–3839. (2016) pmid:27265453
Spearman CW, Afihene M, Ally R, Apica B, Awuku Y, Cunha L, et al. Hepatitis B in sub-Saharan Africa: strategies to achieve the 2030 elimination targets. Lancet Gastroenterol Hepatol.;2: 900–909. (2017) pmid:29132759
Ibrahim, S. A., Mohamed, S. B., Kambal, S., Diya-Aldeen, A., Ahmed, S., Faisal, B., ... & Mohamed, O. Molecular Detection of Occult Hepatitis B virus in plasma and urine of renal transplant patients in Khartoum state Sudan. International Journal of Infectious Diseases, 97, 126-130. (2020).
Eltom, K., Albeely, A., El Hussein, A. R. M., Elkhidir, I. M., etal.. Occult hepatitis B virus infection in Sudan: A systematic review and meta‐analysis. JGH Open, 4(5), 800-807 (2020).
Said ZN. An overview of occult hepatitis B virus infection. World J Gastroenterol.;17:1927–38. (2011)
Samal J, Kandpal M, Vivekanandan P. Molecular mechanisms underlying occult hepatitis B virus infection. Clin. Microbiol. Rev.; 25: 142–63. (2012)
Makvandi M. Update on occult hepatitis B virus infection. World J Gastroenterol.; 22(39):8720. (2016)
Mustafa M, Enan K, Elkhidir I, Hussein AR. Occult hepatitis B virus and hepatitis B genotypes among renal transplant patients in Khartoum State, Sudan. J. Emerg. Dis. Virol. ; 5: 21–5. (2019).
Abakar AE. Sero-molecular epidemiology of occult hepatitis B virus infection among haemodialysis patients and blood donors, Gezira Hospital for renal diseases and surgery, Gezira State, Sudan (2013–2017) (Doctoral dissertation, University of Gezira).
Hassan AG, Yassin ME, Mohammed AB, Molecular Detection BNM. Sero-frequency rate of occult hepatitis B virus among blood donors in Southern Darfur State (Sudan). Afr. J. Med. Sci 2 (9). (2017).
Hassan, Z.K., Hafez, M.M., Mansor, T.M. , Zekri,A.R. Occult HBV infection among Egyptian hepatocellular carcinoma patients. Virol J 8, 90 (2011). https://doi.org/10.1186/1743-422X-8-90
Alejandra Duque-Jaramillo, Biol.,1 Julio C. Rendón, etal , MSc, PhD. Occult Hepatitis B Virus Infection in Liver Transplant Patients, Rev Col Gastroenterol ;31 (4) ; 345- 351.( 2016).
Jun Muto, Masaya Sugiyama, Ken Shirabe, Motokazu Mukaide, Ikue Kirikae-etal .Frequency and Characteristics of Occult Hepatitis B Infection Among Hepatocellular Carcinoma Patients in Japan . Annals of hepatology , 17 (4): 596-603 (2018)
Nicola Coppola1, Lorenzo Onorato1, Valentina Iodice1,2 Mario Starace1, Carmine Minichini1, etal . Occult HBV infection in HCC and cirrhotic tissue of HBsAg negative patients: a virological and clinical study Oncotarget,; 7 ( 38) 62706- 62714.( 2016).
Nakano, M., Kawaguchi, T., Nakamoto, S., Kawaguchi, A., Kanda, etal ."Effect of occult hepatitis B virus infection on the early-onset of hepatocellular carcinoma in patients with hepatitis C virus infection". Oncology Reports 30, no. 5 .2049-2055. (2013): https://doi.org/10.3892/or.2013.2700
Naito, H., Hayashi, S., & Abe, K.. Rapid and specific genotyping system for hepatitis B virus corresponding to six major genotypes by PCR using type-specific primers. Journal of clinical microbiology, 39(1), 362-364. (2001).

No competing interests reported.

Incidence of Occult Hepatitis B virus infection among Sudanese patient with Hepatocellular Carcinoma and Liver Cirrhosis in the absence of Hepatitis C virus

Status:

Version 1

Abstract

Figures

Introduction

Results

Descriptive Statistics:

The Frequency of HBsAg and anti HBeAg:

The frequency of HBV genotypes in HCC and LC groups:

Association between Gender and HBsAg in HCC and LC patients:

Association between Age Groups and HBsAg in HCC and LC patients:

Discussion

Conclusion

Materials And Methods