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Seroprevalence of HBV and HCV in primary hepatocellular carcinoma patients in Zimbabwe

Seroprevalence of HBV and HCV in primary hepatocellular carcinoma patients in Zimbabwe Background: Primary hepatocellular carcinoma (PHC) is one of the most common cancers in Zimbabwe. Hepatitis B virus (HBV) and hepatitis C virus (HCV) are suspected to play a major role in causing this cancer. The objective of this study was to determine the seroprevalence of HBV and HCV in PHC at Parirenyatwa Referral Hospital in Zimbabwe. We evaluated the serological markers of the two viruses in patients with PHC using commercially available enzyme-linked immunosorbent kits. Results: Out of the 60 patients with PHC, 48.3% were seropositive for HBV and 20.0% were seropositive for HCV. Co-infection by HCV and HBV was found in 8% of the patients. Only 13.3% of the health controls (blood donors) were positive for HBV. All the controls were negative for HCV. Conclusion: The high seropositivity of HBV and HCV in PHC in Zimbabwe suggested that the two viruses were a major cause of the cancer. Background sis and hepatocellular carcinoma [6,7]. Areas with high Liver cancer is one of the most common cancers in Zim- prevalence of HBV include Southeast Asia, China and babwe and Africa [1,2]. Hepatitis B virus (HBV) and hep- Africa, where approximately 10% of the population are atitis C virus (HCV) are suspected to play an important chronic carriers [5,8]. In persons infected with HBV, mor- aetiological role in liver cancer in this region and the con- bidity and mortality result when inflammatory liver dis- tinent. HBV is a partially double-stranded DNA virus and ease progresses to cirrhosis and primary hepatocellular is an important cause of morbidity and mortality world- carcinoma [8]. High-risk groups of HBV infection include wide [3,4]. Globally, it is estimated that about 2 billion intravenous drug users, attendees of sexually transmitted people have been infected with HBV and about 350 mil- disease clinics, homosexual men, patients undergoing lion people are chronically infected [5]. Most of the haemodialysis, children born to mothers who are hepati- chronically-infected people are found in developing tis B surface antigen positive and health care workers countries and are at a high-risk of developing liver cirrho- [6,7,9]. Page 1 of 6 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:15 http://www.infectagentscancer.com/content/4/1/15 HCV is a positive-stranded RNA virus which belongs to not significant (p > 0.05). The difference in sex between the Flaviviridae family [10]. Worldwide, the virus infects the two groups was also not significantly different (p > about 170 million people [11]. Infection by this virus can 0.05). be resolved spontaneously in only about 15-20% of cases and chronic infection is established in the remaining 80- HBsAg and HCV antibody status in PHC patients 85% [12]. Most of these chronic HCV infections usually The seroprevalence of HBV and HCV in patients with PHC lead to severe hepatitis, liver cirrhosis and hepatocellular and health blood donours is summarized in Table 2. Of carcinoma [13-17]. The risk of developing PHC ranges the 60 PHC cases, 29 patients (48%) were positive for from 1-5% after about two decades of chronic infection HBsAg of which 7 were females and 22 were males. The [18]. HCV infection usually occurs following direct age range of patients was 18-70 years and the mean was parenteral exposure to contaminated body fluids, espe- 43.8 years. Only 10 (34%) of the patients were over 45 cially blood [16,19]. The prevalence of both HBV and years of age. The mean age of HBsAg negative patients was HCV in PHC in Zimbabwe is poorly known. It was against 47.6 years. There was no significant discrepancy between this background that we set out to evaluate the presence of the mean ages of HBsAg positive and negative patients. antibodies to HBV surface antigen and HCV in patients Only 4 control subjects were positive for HBsAg. The rela- with PHC at Parirenyatwa Central Hospital in Zimbabwe. tive risk for developing PHC in patients with HBsAg was high when control subjects were considered as a reference Results group (odds ratio = 1.62, 95% CI 0.69-1.64). Patient data Data from hospital records showed that all the patients A total of 12 (20%) PHC patients were positive for anti- presented with abdominal distending discomfort, charac- HCV and 8 (67%) of these patients were over 45 years of terised by right upper quadrant pain and hepatic mass age (66.7%) and the other remaining 4 (33%) were below enlargement in some cases. Weight loss was observed in 45 years. All the control patients from blood donors were all the patients with almost a third showing that they were negative for anti-HCV antibodies. The difference in males jaundiced. Ascites and fever symptoms were not uncom- and females infected with HCV was not significant (p > mon in the patients on presentation or during hospitalisa- 0.05). The mean age of patients who were positive for tion. All the patients had impaired liver function in one or anti-HCV was 51.26 years and the age range was 16-70 more of serum aminotransferases, prolonged pro- years. The odds ratio or relative risk for the development thrombin time and albumin levels. Out of the 60 patients, of PHC in anti-HCV positive patients could not be ascer- 53 (88%) had serum gamma-glutamyl transferase (GGT) tained because none of the control individuals was posi- levels above the reference range, with 10 of them having tive and the calculation required an integer for the GGT values greater than 200 IU/L. Of all the patients, 30 reference sample. Of the 12 patients who were anti-HCV- (50%) had alanine transaminase (ALT) levels elevated positive, 4 had AFP levels greater than 400 ng/ml whilst above the upper limit of normal range. The age ranges of two had their AFP levels within the normal range (0-10 patients and controls are given in Table 1. ng/ml). The prevalence of HBsAg was significantly higher than that of anti- HCV both in PHC patients and controls The clinical management of the patients was also assessed (p < 0.05). The presence of both anti-HCV and HBsAg was from the records. Most of the patients were of poor social noted in 5 patients. It was also noted that more than half background, either in the lowest income group or not of the patients had infection with HBV and/or HCV. employed at all. Some of the males (n = 11) over 30 years of age had a history of alcohol drinking. Of all the The relative risk of developing PHC was strongly associ- patients, 7 (12%) had cirrhosis clearly indicated in their ated with HBV infection. The unadjusted odds ratio for records. Alpha-fetoprotein (AFP) levels were greater than PHC development was high (1.615; 95% CI 0.69 - 1.65) 400 ng/ml in 33 patients (55%) and 21 patients (35%) for overall HBV infection. For those patients with HBsAg with AFPs less than 400 ng/ml had confirmation of PHC alone, the risk for PHC development, using the cases by ultrasound and cytology. The difference in age distribu- which were negative for HBsAg as a reference group and tion in PHC patients and health individual controls was healthy individuals as controls, was still high (odds ratio Table 1: Sex and age parameters for PHC patients and health controls Parameter PHC patients (n = 60) Health Controls (n = 30) Age range 16 - 81 years 16 - 69 years Mean age 45.7 years 38.5 years Male to Female ratio 2:1 2.08:1 Page 2 of 6 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:15 http://www.infectagentscancer.com/content/4/1/15 Table 2: Seropositivity and negativity for Anti-HCV and HBsAg in PHC and health blood donors Parameter PHC (n = 60) Control s (n = 30) p value Anti-HCV positivity 12 (20%) 0 (0%) <0.05 Anti-HCV negativity 48 (80% 30 (100%) <0.05 HBsAg positivity 29 (48.3%) 4 (13.3%) <0.05 HBsAg negativity 31 (51.7%) 26 (86.7%) <0.05 Dual anti-HCV & HBsAg positivity 5 (8.3%) 0 (0%) - Anti-HCV and/or HBsAg positivity 36 (60%) 4 (13.3%) - Anti-HCV and/or HBsAg negativity 24 (40%) 26 (86.7%) - = 1.48: 95% CI 0.81-1.53). The relative risk for PHC devel- patients could not be established and hence an associa- oping in dual infection with HCV using non-HBsAg cases tion with HCV could not be studied. The biopsy rate is low as references and healthy individuals as controls, was low due to poor prognosis and late presentation. Only 6 (odds ratio = 0.818). The odds ratio in patients positive patients had histologically verified PHC status. The diag- for either anti-HCV and HBsAg or both was even higher nosis of PHC based on biochemical evaluation of AFP is (odds ratio = 1.875; 95% CI 0.54 - 1.88). controversial especially when levels less than 400 ng/ml are encountered. AFP levels were greater than 400 ng/ml Discussion in 33 patients, six had histological proof and the remain- Parirenyatwa Hospital is a supra-referral hospital in Zim- der were confirmed by cytology and ultrasound scanning babwe. Patients suspected to have primary hepatocellular when focal or multifocal lesions were suggested. The diag- carcinoma are referred from primary hospitals and clinics nosis techniques employed were convincing that the cases around the country to this referral hospital. Therefore, certainly had PHC. although our study had only 60 PHC patients, they were likely to be representative of the whole country. This study The risks of acquiring HBV and HCV infection could not strongly suggested that Hepatitis B and C viruses were be assessed for this study. The detection of viral markers important etiological factors of PHC in Zimbabwe. in two female teenagers with PHC suggested that they Although other factors such as exposure to aflatoxins could have acquired the viruses parenterally or vertically. (from mouldy grains and cereals) and alcohol consump- In previous studies of PHC, anti-HCV and HBsAg have tion were believed to be important, hospital records for been found in even younger patients of 16 years by biopsy most of the patients lacked the information. Alcohol con- [20,27]. The mean age of PHC patients of 45.7 years in sumption was documented in only 20% of the patients. our study agreed well with those found in studies in South Information on risks to HBV and HCV infection such as Africa (45 years), Nigeria (46.7 years) and Southern Africa history of blood transfusion, use of intravenous drug (44.8 years) [20,23,27]. In Mozambique the mean age in abuse and traditional surgical procedures could also not Shangaan ethnic group was 33.4 years and 50% of the be obtained from the files. These parameters could have patients were below 30 years [21]. some influence on the prevalence of seropositivity of PHC patients to HBV and HCV. Aminotrasferase levels were The presence of antibodies to the HBsAg in some of the assessed in the records and most patients had abnormal PHC patients clearly indicated HBV infection. The pres- levels of one or more of ALT, AST and GGT being elevated. ence of HBsAg is normally used as a diagnostic marker to This is in agreement with studies that showed extensive indicate acute or chronic infection [28]. It could have hepatocellular damage in PHC patients [20-23]. All the been interesting if we had also looked at the presence of patients who were positive for anti-HCV and HBsAg had HBV DNA in the samples. However, in highly endemic elevated hepatic enzymes. Albumin was low and pro- areas, HBV DNA has also been detected in blood or liver thrombin time was prolonged in most of the cases. of patients without HBsAg [29]. The diagnosis of HBV by Hepatic aminotransferases were not measured in the con- detection of HBsAg is very reliable only in acute phase trols to evaluate hepatocyte damage in those who were infection and present infection [27,30]. It is not indicative HBsAg-positive. There is a strong link between HCV and of past infection. However most of the PHC studies done cirrhosis with 60-80% of HCV-positive PHC patients showed that HBsAg is expressed in 70-80% of HBV posi- being cirrhotic [24-26]. It could have been interesting to tive patients who were found to be positive for viral DNA note this association in cirrhotic HCV positive PHC cases. [30,31]. The reliability of HBsAg as a marker of HBV infec- Only seven of the patients whose cirrhotic condition was tion in PHC is based on the expression of this antigen well documented were in the study and anti-HCV anti- from the integrated viral genome in the hepatocyte DNA bodies were only found in two patients who also had during vigorous active hepatocyte proliferation and turn- HBsAg in their sera. The cirrhotic condition of other over. At least 80% of HBsAg carriers have evidence of Page 3 of 6 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:15 http://www.infectagentscancer.com/content/4/1/15 chromosomal integration of viral sequences, by Southern important factors in chronic liver disease and subse- hydridisation of tumour DNA [31,32]. Other markers that quently PHC development in Southern Africa. show past infection could have been assayed for to evalu- ate overall exposure. Our results confirmed previous Conclusion reports of high prevalence of HBsAg in patients with PHC HBV and HCV play a significant role in the aetiology of in sub-Sahara Africa, especially the southern parts hepatocellular carcinoma in Zimbabwe. [20,27,33]. Patients and Methods The presence of antibodies to HCV in some of the patients Study subjects also suggested that the virus was also an important aetio- The sixty patients were presented to Parirenyatwa Hospital logical agent in PHC in Zimbabwe. It was not clear between October 1999 and August 2000 and were diag- whether some of the patients with anti-HCV had already nosed as having PHC. Sera for the patients were collected resolved the virus. The resolution of HCV infection occurs from the Radioimmunoassay (RIA) Laboratory of the in 15-20% of patients [12,34]. It could have been an inter- Public Health Laboratory (PHL). The serum samples were esting study if we had also looked at the presence of HCV collected after determination of the levels of the tumour RNA in the samples. However, it takes between a decade marker, Alpha-fetoprotein (AFP). The sera were stored at - and three for the development of PHC from time of infec- 20°C until assayed for anti-HCV antibodies and HBsAg. tion with HCV and the viral RNA may disappear upon res- The medical records of the patients were also examined olution but the antibodies persist [35,36]. Detection of for diagnostic tests and evaluations suggesting or confirm- viraemia by polymerase chain reaction (PCR) could also ing primary hepatocellular carcinoma. The tests included have been done to detect cases where there was active viral ultrasound scanning, X-ray photography, needle aspirate replication. cytological proof and liver biopsy histology. Of the 60 patients recruited in this study, only 6 had liver biopsy In our study, 48.3% of 60 PHC patients were HBsAg pos- evaluation of PHC by histological examination. The rest itive and 20% were anti-HCV positive. In a previous study of the patients' diagnosis depended on ultrasound scan- in Zimbabwe, HBsAg was detected in 42.6% of the PHC ning, cytology (fine needle aspirate), clinical features and patients of the 62 patients studied [33]. Positivity for anti- biochemical evaluations of liver functions and AFP levels. HCV was in 23.8% using a lesser sensitive and specific sec- Data on age and sex were also extracted. Controls (health ond generation assay [33]. The two studies show that HBV blood donors) (n = 30) were included in the study. They remains the major infection of hepatitis viruses in the were mainly health blood donors from the National region. The prevalence of HBsAg in controls (13.3%) was Blood Transfusion Services. less than the prevalence of 24.2% observed in a study of 660 health individuals [37,38]. This probably was due to Laboratory tests of anti-HCV and anti-HBV serological the smaller proportion of the present study group (30 markers blood donors). The observation of no anti-HCV positive All samples (patients and controls) were tested for the cases in controls is agreement with data from the National anti-HCV using the commercially available MUREX anti- Blood Transfusion Service which show a prevalence of less HCV (Version 4.0) kit (Abbott Diagnostic Division, RSA) than 1% in healthy blood donors. However, another according to manufacturer's instructions. The MUREX study in Zimbabwe has observed an anti-HCV seropositiv- anti-HCV (Version 4.0) is a third generation solid phase ity of 8% in healthy rural individuals [22]. Co-infection enzyme immunoassay in which a mixture of highly puri- by HCV and HBV was found in this study. Although HBV fied viral antigens is coated into microwells of a microtitre and HCV may be independent risk factors of PHC, dual plate. The antigens contain sequences from the core infection is likely to increase the risk of primary hepatocel- (Structural), NS3 protease/helicase (non-structural), NS4 lular carcinogenesis. Some studies have also reported dual (non-structural) and the NS5 replicase (non-structural) infection by HBV and HCV in PHC [20,23,30]. However, regions of HCV. During incubation with samples any anti- the absence of serological markers for either HBV and/or HCV antibodies present in the sample will bind to the HCV in PHC in some PHC patients could not rule out the immobilized antigens. Following washing to remove role of the two viruses in carcinogenesis. Studies have unbound material, the captured anti-HCV antibodies are shown that hepatic tissues could have viral genomic incubated with peroxidase conjungated monoclonal anti- sequences in one-third or more of the serologically nega- human IgG. The conjugate will bind to the immobilized tive subjects [38]. It was also suspected that aflatoxin con- anti-HCV through the anti-human IgG moiety. After tamination could also have played a significant role in removal of excess conjugate, bound enzyme is detected by PHC development since Zimbabwe has been found to the addition of a solution containing 3,3;5,5- tetramethyl have this post-harvest problem [39]. This study and previ- benzidine (TMB) and hydrogen peroxide. The enzyme ous ones, therefore, demonstrate that HBV and HCV are peroxidase changes the substrate to a purple colour in the Page 4 of 6 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:15 http://www.infectagentscancer.com/content/4/1/15 wells, which contain anti-HCV positive samples. The Acknowledgements We thank the medical staff from the RIA Laboratory of the Public Health enzymatic reaction is terminated with sulphuric acid to Laboratory of Parirenyatwa Hospital for assisting us in collection of patient give an orange colour that is read spectrophotometrically. specimens which were used in the study. We also thank the staff from the The amount of conjugate bound and hence colour in the Zimbabwe National Blood Transfusion Services for providing specimens wells, is directly proportional to the concentration of anti- used as controls in the study. We also extend our heartfelt gratitude to bodies to HCV in the specimen. patients whose samples were used in the study. This work was supported financially by a grant from the University of Zimbabwe Research Board. All the samples were also tested for HBsAg using the Bioe- lisa Kit (Biokit, RSA) according to manufacturer's instruc- References tions. The Bioelisa Kit is an ELISA test kit for the detection 1. Chokunonga E, Levy LM, Bassett MT, Mauchaza BG, Thomas DB, Par- kin DM: Cancer incidence in the African population of of the hepatitis B surface antigen (HBsAg) in human sera Harare, Zimbabwe: second results from the cancer registry or plasma. The bioelisa HBsAg assay is a direct immu- 1993-1995. Int J Cancer 2000, 85:54-59. 2. Parkin DM, Sitas F, Chirenje M, Stein L, Abratt R, Wabinga H: Cancer noenzymatic method of the "sandwich" type in which in Indigenous Africans--burden, distribution, and trends. 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Cent Afr J Med 1989, 35:542-545. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 6 of 6 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Infectious Agents and Cancer Springer Journals

Seroprevalence of HBV and HCV in primary hepatocellular carcinoma patients in Zimbabwe

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Springer Journals
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Copyright © 2009 by Chin'ombe et al; licensee BioMed Central Ltd.
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Biomedicine; Cancer Research; Infectious Diseases; Oncology
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1750-9378
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10.1186/1750-9378-4-15
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19814789
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Abstract

Background: Primary hepatocellular carcinoma (PHC) is one of the most common cancers in Zimbabwe. Hepatitis B virus (HBV) and hepatitis C virus (HCV) are suspected to play a major role in causing this cancer. The objective of this study was to determine the seroprevalence of HBV and HCV in PHC at Parirenyatwa Referral Hospital in Zimbabwe. We evaluated the serological markers of the two viruses in patients with PHC using commercially available enzyme-linked immunosorbent kits. Results: Out of the 60 patients with PHC, 48.3% were seropositive for HBV and 20.0% were seropositive for HCV. Co-infection by HCV and HBV was found in 8% of the patients. Only 13.3% of the health controls (blood donors) were positive for HBV. All the controls were negative for HCV. Conclusion: The high seropositivity of HBV and HCV in PHC in Zimbabwe suggested that the two viruses were a major cause of the cancer. Background sis and hepatocellular carcinoma [6,7]. Areas with high Liver cancer is one of the most common cancers in Zim- prevalence of HBV include Southeast Asia, China and babwe and Africa [1,2]. Hepatitis B virus (HBV) and hep- Africa, where approximately 10% of the population are atitis C virus (HCV) are suspected to play an important chronic carriers [5,8]. In persons infected with HBV, mor- aetiological role in liver cancer in this region and the con- bidity and mortality result when inflammatory liver dis- tinent. HBV is a partially double-stranded DNA virus and ease progresses to cirrhosis and primary hepatocellular is an important cause of morbidity and mortality world- carcinoma [8]. High-risk groups of HBV infection include wide [3,4]. Globally, it is estimated that about 2 billion intravenous drug users, attendees of sexually transmitted people have been infected with HBV and about 350 mil- disease clinics, homosexual men, patients undergoing lion people are chronically infected [5]. Most of the haemodialysis, children born to mothers who are hepati- chronically-infected people are found in developing tis B surface antigen positive and health care workers countries and are at a high-risk of developing liver cirrho- [6,7,9]. Page 1 of 6 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:15 http://www.infectagentscancer.com/content/4/1/15 HCV is a positive-stranded RNA virus which belongs to not significant (p > 0.05). The difference in sex between the Flaviviridae family [10]. Worldwide, the virus infects the two groups was also not significantly different (p > about 170 million people [11]. Infection by this virus can 0.05). be resolved spontaneously in only about 15-20% of cases and chronic infection is established in the remaining 80- HBsAg and HCV antibody status in PHC patients 85% [12]. Most of these chronic HCV infections usually The seroprevalence of HBV and HCV in patients with PHC lead to severe hepatitis, liver cirrhosis and hepatocellular and health blood donours is summarized in Table 2. Of carcinoma [13-17]. The risk of developing PHC ranges the 60 PHC cases, 29 patients (48%) were positive for from 1-5% after about two decades of chronic infection HBsAg of which 7 were females and 22 were males. The [18]. HCV infection usually occurs following direct age range of patients was 18-70 years and the mean was parenteral exposure to contaminated body fluids, espe- 43.8 years. Only 10 (34%) of the patients were over 45 cially blood [16,19]. The prevalence of both HBV and years of age. The mean age of HBsAg negative patients was HCV in PHC in Zimbabwe is poorly known. It was against 47.6 years. There was no significant discrepancy between this background that we set out to evaluate the presence of the mean ages of HBsAg positive and negative patients. antibodies to HBV surface antigen and HCV in patients Only 4 control subjects were positive for HBsAg. The rela- with PHC at Parirenyatwa Central Hospital in Zimbabwe. tive risk for developing PHC in patients with HBsAg was high when control subjects were considered as a reference Results group (odds ratio = 1.62, 95% CI 0.69-1.64). Patient data Data from hospital records showed that all the patients A total of 12 (20%) PHC patients were positive for anti- presented with abdominal distending discomfort, charac- HCV and 8 (67%) of these patients were over 45 years of terised by right upper quadrant pain and hepatic mass age (66.7%) and the other remaining 4 (33%) were below enlargement in some cases. Weight loss was observed in 45 years. All the control patients from blood donors were all the patients with almost a third showing that they were negative for anti-HCV antibodies. The difference in males jaundiced. Ascites and fever symptoms were not uncom- and females infected with HCV was not significant (p > mon in the patients on presentation or during hospitalisa- 0.05). The mean age of patients who were positive for tion. All the patients had impaired liver function in one or anti-HCV was 51.26 years and the age range was 16-70 more of serum aminotransferases, prolonged pro- years. The odds ratio or relative risk for the development thrombin time and albumin levels. Out of the 60 patients, of PHC in anti-HCV positive patients could not be ascer- 53 (88%) had serum gamma-glutamyl transferase (GGT) tained because none of the control individuals was posi- levels above the reference range, with 10 of them having tive and the calculation required an integer for the GGT values greater than 200 IU/L. Of all the patients, 30 reference sample. Of the 12 patients who were anti-HCV- (50%) had alanine transaminase (ALT) levels elevated positive, 4 had AFP levels greater than 400 ng/ml whilst above the upper limit of normal range. The age ranges of two had their AFP levels within the normal range (0-10 patients and controls are given in Table 1. ng/ml). The prevalence of HBsAg was significantly higher than that of anti- HCV both in PHC patients and controls The clinical management of the patients was also assessed (p < 0.05). The presence of both anti-HCV and HBsAg was from the records. Most of the patients were of poor social noted in 5 patients. It was also noted that more than half background, either in the lowest income group or not of the patients had infection with HBV and/or HCV. employed at all. Some of the males (n = 11) over 30 years of age had a history of alcohol drinking. Of all the The relative risk of developing PHC was strongly associ- patients, 7 (12%) had cirrhosis clearly indicated in their ated with HBV infection. The unadjusted odds ratio for records. Alpha-fetoprotein (AFP) levels were greater than PHC development was high (1.615; 95% CI 0.69 - 1.65) 400 ng/ml in 33 patients (55%) and 21 patients (35%) for overall HBV infection. For those patients with HBsAg with AFPs less than 400 ng/ml had confirmation of PHC alone, the risk for PHC development, using the cases by ultrasound and cytology. The difference in age distribu- which were negative for HBsAg as a reference group and tion in PHC patients and health individual controls was healthy individuals as controls, was still high (odds ratio Table 1: Sex and age parameters for PHC patients and health controls Parameter PHC patients (n = 60) Health Controls (n = 30) Age range 16 - 81 years 16 - 69 years Mean age 45.7 years 38.5 years Male to Female ratio 2:1 2.08:1 Page 2 of 6 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:15 http://www.infectagentscancer.com/content/4/1/15 Table 2: Seropositivity and negativity for Anti-HCV and HBsAg in PHC and health blood donors Parameter PHC (n = 60) Control s (n = 30) p value Anti-HCV positivity 12 (20%) 0 (0%) <0.05 Anti-HCV negativity 48 (80% 30 (100%) <0.05 HBsAg positivity 29 (48.3%) 4 (13.3%) <0.05 HBsAg negativity 31 (51.7%) 26 (86.7%) <0.05 Dual anti-HCV & HBsAg positivity 5 (8.3%) 0 (0%) - Anti-HCV and/or HBsAg positivity 36 (60%) 4 (13.3%) - Anti-HCV and/or HBsAg negativity 24 (40%) 26 (86.7%) - = 1.48: 95% CI 0.81-1.53). The relative risk for PHC devel- patients could not be established and hence an associa- oping in dual infection with HCV using non-HBsAg cases tion with HCV could not be studied. The biopsy rate is low as references and healthy individuals as controls, was low due to poor prognosis and late presentation. Only 6 (odds ratio = 0.818). The odds ratio in patients positive patients had histologically verified PHC status. The diag- for either anti-HCV and HBsAg or both was even higher nosis of PHC based on biochemical evaluation of AFP is (odds ratio = 1.875; 95% CI 0.54 - 1.88). controversial especially when levels less than 400 ng/ml are encountered. AFP levels were greater than 400 ng/ml Discussion in 33 patients, six had histological proof and the remain- Parirenyatwa Hospital is a supra-referral hospital in Zim- der were confirmed by cytology and ultrasound scanning babwe. Patients suspected to have primary hepatocellular when focal or multifocal lesions were suggested. The diag- carcinoma are referred from primary hospitals and clinics nosis techniques employed were convincing that the cases around the country to this referral hospital. Therefore, certainly had PHC. although our study had only 60 PHC patients, they were likely to be representative of the whole country. This study The risks of acquiring HBV and HCV infection could not strongly suggested that Hepatitis B and C viruses were be assessed for this study. The detection of viral markers important etiological factors of PHC in Zimbabwe. in two female teenagers with PHC suggested that they Although other factors such as exposure to aflatoxins could have acquired the viruses parenterally or vertically. (from mouldy grains and cereals) and alcohol consump- In previous studies of PHC, anti-HCV and HBsAg have tion were believed to be important, hospital records for been found in even younger patients of 16 years by biopsy most of the patients lacked the information. Alcohol con- [20,27]. The mean age of PHC patients of 45.7 years in sumption was documented in only 20% of the patients. our study agreed well with those found in studies in South Information on risks to HBV and HCV infection such as Africa (45 years), Nigeria (46.7 years) and Southern Africa history of blood transfusion, use of intravenous drug (44.8 years) [20,23,27]. In Mozambique the mean age in abuse and traditional surgical procedures could also not Shangaan ethnic group was 33.4 years and 50% of the be obtained from the files. These parameters could have patients were below 30 years [21]. some influence on the prevalence of seropositivity of PHC patients to HBV and HCV. Aminotrasferase levels were The presence of antibodies to the HBsAg in some of the assessed in the records and most patients had abnormal PHC patients clearly indicated HBV infection. The pres- levels of one or more of ALT, AST and GGT being elevated. ence of HBsAg is normally used as a diagnostic marker to This is in agreement with studies that showed extensive indicate acute or chronic infection [28]. It could have hepatocellular damage in PHC patients [20-23]. All the been interesting if we had also looked at the presence of patients who were positive for anti-HCV and HBsAg had HBV DNA in the samples. However, in highly endemic elevated hepatic enzymes. Albumin was low and pro- areas, HBV DNA has also been detected in blood or liver thrombin time was prolonged in most of the cases. of patients without HBsAg [29]. The diagnosis of HBV by Hepatic aminotransferases were not measured in the con- detection of HBsAg is very reliable only in acute phase trols to evaluate hepatocyte damage in those who were infection and present infection [27,30]. It is not indicative HBsAg-positive. There is a strong link between HCV and of past infection. However most of the PHC studies done cirrhosis with 60-80% of HCV-positive PHC patients showed that HBsAg is expressed in 70-80% of HBV posi- being cirrhotic [24-26]. It could have been interesting to tive patients who were found to be positive for viral DNA note this association in cirrhotic HCV positive PHC cases. [30,31]. The reliability of HBsAg as a marker of HBV infec- Only seven of the patients whose cirrhotic condition was tion in PHC is based on the expression of this antigen well documented were in the study and anti-HCV anti- from the integrated viral genome in the hepatocyte DNA bodies were only found in two patients who also had during vigorous active hepatocyte proliferation and turn- HBsAg in their sera. The cirrhotic condition of other over. At least 80% of HBsAg carriers have evidence of Page 3 of 6 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:15 http://www.infectagentscancer.com/content/4/1/15 chromosomal integration of viral sequences, by Southern important factors in chronic liver disease and subse- hydridisation of tumour DNA [31,32]. Other markers that quently PHC development in Southern Africa. show past infection could have been assayed for to evalu- ate overall exposure. Our results confirmed previous Conclusion reports of high prevalence of HBsAg in patients with PHC HBV and HCV play a significant role in the aetiology of in sub-Sahara Africa, especially the southern parts hepatocellular carcinoma in Zimbabwe. [20,27,33]. Patients and Methods The presence of antibodies to HCV in some of the patients Study subjects also suggested that the virus was also an important aetio- The sixty patients were presented to Parirenyatwa Hospital logical agent in PHC in Zimbabwe. It was not clear between October 1999 and August 2000 and were diag- whether some of the patients with anti-HCV had already nosed as having PHC. Sera for the patients were collected resolved the virus. The resolution of HCV infection occurs from the Radioimmunoassay (RIA) Laboratory of the in 15-20% of patients [12,34]. It could have been an inter- Public Health Laboratory (PHL). The serum samples were esting study if we had also looked at the presence of HCV collected after determination of the levels of the tumour RNA in the samples. However, it takes between a decade marker, Alpha-fetoprotein (AFP). The sera were stored at - and three for the development of PHC from time of infec- 20°C until assayed for anti-HCV antibodies and HBsAg. tion with HCV and the viral RNA may disappear upon res- The medical records of the patients were also examined olution but the antibodies persist [35,36]. Detection of for diagnostic tests and evaluations suggesting or confirm- viraemia by polymerase chain reaction (PCR) could also ing primary hepatocellular carcinoma. The tests included have been done to detect cases where there was active viral ultrasound scanning, X-ray photography, needle aspirate replication. cytological proof and liver biopsy histology. Of the 60 patients recruited in this study, only 6 had liver biopsy In our study, 48.3% of 60 PHC patients were HBsAg pos- evaluation of PHC by histological examination. The rest itive and 20% were anti-HCV positive. In a previous study of the patients' diagnosis depended on ultrasound scan- in Zimbabwe, HBsAg was detected in 42.6% of the PHC ning, cytology (fine needle aspirate), clinical features and patients of the 62 patients studied [33]. Positivity for anti- biochemical evaluations of liver functions and AFP levels. HCV was in 23.8% using a lesser sensitive and specific sec- Data on age and sex were also extracted. Controls (health ond generation assay [33]. The two studies show that HBV blood donors) (n = 30) were included in the study. They remains the major infection of hepatitis viruses in the were mainly health blood donors from the National region. The prevalence of HBsAg in controls (13.3%) was Blood Transfusion Services. less than the prevalence of 24.2% observed in a study of 660 health individuals [37,38]. This probably was due to Laboratory tests of anti-HCV and anti-HBV serological the smaller proportion of the present study group (30 markers blood donors). The observation of no anti-HCV positive All samples (patients and controls) were tested for the cases in controls is agreement with data from the National anti-HCV using the commercially available MUREX anti- Blood Transfusion Service which show a prevalence of less HCV (Version 4.0) kit (Abbott Diagnostic Division, RSA) than 1% in healthy blood donors. However, another according to manufacturer's instructions. The MUREX study in Zimbabwe has observed an anti-HCV seropositiv- anti-HCV (Version 4.0) is a third generation solid phase ity of 8% in healthy rural individuals [22]. Co-infection enzyme immunoassay in which a mixture of highly puri- by HCV and HBV was found in this study. Although HBV fied viral antigens is coated into microwells of a microtitre and HCV may be independent risk factors of PHC, dual plate. The antigens contain sequences from the core infection is likely to increase the risk of primary hepatocel- (Structural), NS3 protease/helicase (non-structural), NS4 lular carcinogenesis. Some studies have also reported dual (non-structural) and the NS5 replicase (non-structural) infection by HBV and HCV in PHC [20,23,30]. However, regions of HCV. During incubation with samples any anti- the absence of serological markers for either HBV and/or HCV antibodies present in the sample will bind to the HCV in PHC in some PHC patients could not rule out the immobilized antigens. Following washing to remove role of the two viruses in carcinogenesis. Studies have unbound material, the captured anti-HCV antibodies are shown that hepatic tissues could have viral genomic incubated with peroxidase conjungated monoclonal anti- sequences in one-third or more of the serologically nega- human IgG. The conjugate will bind to the immobilized tive subjects [38]. It was also suspected that aflatoxin con- anti-HCV through the anti-human IgG moiety. After tamination could also have played a significant role in removal of excess conjugate, bound enzyme is detected by PHC development since Zimbabwe has been found to the addition of a solution containing 3,3;5,5- tetramethyl have this post-harvest problem [39]. This study and previ- benzidine (TMB) and hydrogen peroxide. The enzyme ous ones, therefore, demonstrate that HBV and HCV are peroxidase changes the substrate to a purple colour in the Page 4 of 6 (page number not for citation purposes) Infectious Agents and Cancer 2009, 4:15 http://www.infectagentscancer.com/content/4/1/15 wells, which contain anti-HCV positive samples. The Acknowledgements We thank the medical staff from the RIA Laboratory of the Public Health enzymatic reaction is terminated with sulphuric acid to Laboratory of Parirenyatwa Hospital for assisting us in collection of patient give an orange colour that is read spectrophotometrically. specimens which were used in the study. We also thank the staff from the The amount of conjugate bound and hence colour in the Zimbabwe National Blood Transfusion Services for providing specimens wells, is directly proportional to the concentration of anti- used as controls in the study. We also extend our heartfelt gratitude to bodies to HCV in the specimen. patients whose samples were used in the study. This work was supported financially by a grant from the University of Zimbabwe Research Board. All the samples were also tested for HBsAg using the Bioe- lisa Kit (Biokit, RSA) according to manufacturer's instruc- References tions. The Bioelisa Kit is an ELISA test kit for the detection 1. Chokunonga E, Levy LM, Bassett MT, Mauchaza BG, Thomas DB, Par- kin DM: Cancer incidence in the African population of of the hepatitis B surface antigen (HBsAg) in human sera Harare, Zimbabwe: second results from the cancer registry or plasma. The bioelisa HBsAg assay is a direct immu- 1993-1995. Int J Cancer 2000, 85:54-59. 2. Parkin DM, Sitas F, Chirenje M, Stein L, Abratt R, Wabinga H: Cancer noenzymatic method of the "sandwich" type in which in Indigenous Africans--burden, distribution, and trends. 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Cent Afr J Med 1989, 35:542-545. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 6 of 6 (page number not for citation purposes)

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