Department of Pathology

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Hepatitis B virus mother-to-child transmission in Namibia: transmission dynamics and possibilities for elimination
(Stellenbosch : Stellenbosch University, 2019-12) Tamandjou Tchuem, Cynthia Raissa; Andersson, Monique I.; Preiser, Wolfgang; Wiysonge, Charles S.; Jacobs, Graeme Brendon; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: Introduction: Hepatitis B virus (HBV) remains endemic in sub-Saharan Africa (SSA). While the roll-out of pediatric HBV immunization from six weeks of age has had an impact on horizontal transmission of the virus, mother-to-child transmission (MTCT) has been identified as the driver of the current HBV epidemic in the region. Given the high likelihood of developing chronic HBV infection (CHB) if the infection is acquired during infancy, preventing HBV MTCT in SSA is vital. Where MTCT occurs, it is essential to identify the HBV-infected children for appropriate management, especially in the context of HIV. Current antiretroviral therapy (ART) for the management of HIV infection includes tenofovir for children ≥ 10 years old. Children below the age of 10 years are treated with lamivudine. However, many of the HIV/HBV co-infected children are left on lamivudine treatment for more than ten years and are at risk of developing HBV drug resistance and uncontrolled HBV infection. Uncontrolled HBV infection is a known factor for increased risk of severe liver damage. Aim: This research project aimed to (1) assess the molecular character of HBV and the liver health of HIV/HBV co-infected children who have been on long-term lamivudine treatment, (2) to determine the feasibility of a screen-treat-vaccinate intervention to prevent HBV MTCT, and (3) to measure the costs and health outcomes of combined prophylactic measures against HBV MTCT, in Namibia. Methodology: Three sub-studies were conducted as part of this research project, to answer each of its aims. The first sub-study involved HIV/HBV co-infected children and adolescents below the age of 18 years old, and who have been exposed to lamivudine. Venous blood samples were collected from these children for HBV serological testing (HBsAg, HBeAg, anti-HBe and anti-HBc total) using Murex ELISA assays. Dried blood spots (DBS) samples were used for HBV DNA levels measurement and genotyping. HBV DNA measurements were completed using the automated AmpliPrep/COBAS TaqMan HBV test V2.0. Genotyping and mutation analyses were performed using online tools. Liver health was assessed through AST platelet ratio index (APRI). An APRI score > 0.5 was considered a sign of liver fibrosis. Mothers attending with these children and adolescents were also enrolled in the study, to determine the role of HBV MTCT in these pediatric HBV infections. DBS were collected from these mothers for HBV molecular characterization as well. The second sub-study focused on pregnant women attending antenatal clinics (ANCs) in Windhoek. These women were recruited following informed consent and screened for HBV using the Alere DetermineTM HBsAg rapid test. HBsAg positive pregnant women were tested for further HBV serological markers (HBeAg, anti-HBe, anti-HBc IgM), and HBV viral loads were measured to determine the risk of MTCT. Positive mothers at high risk of MTCT were reviewed for antiviral prophylaxis and offered treatment where necessary. HBV-exposed babies were immunized as per Namibian guidelines, and followed-up to determine the rate of MTCT. The feasibility of offering routine antenatal HBV rapid testing was assessed quantitatively and qualitatively. The former involved determining the diagnostic accuracy of the rapid test used for HBsAg screening, and the latter focused on the perceptions of this antenatal care service by healthcare workers (HCWs). In the third sub-study, the costs and health outcomes of four interventions against HBV MTCT were assessed through a cost-effectiveness analysis. The interventions included: (1) universal birth dose (BD) vaccination, (2) BD vaccination and HBIG, (3) BD vaccination, HBIG, and maternal antiviral prophylaxis informed by sequential HBV viral load testing, and (4) BD vaccination, HBIG, and maternal antiviral prophylaxis informed by sequential HBeAg testing. All resources including consumables, HCW’s time, building space and facilities (and their quantity) were measured and valued to determine the unit costs of HBsAg screening at the ANCs, providing antenatal treatment and administering pediatric immunoprophylaxis. Health outcomes were measured in terms of the number of pediatric HBV infections averted. The incremental cost-effectiveness ratios (ICERs) of these interventions were calculated and were used to compare each intervention to the previous less expensive one. Results: Fifteen HIV/HBV co-infected children/adolescents and six mothers attending with the children were enrolled in the first sub-study. Ten serum samples obtained from Windhoek were further tested for HBV serological markers; seven were HBeAg positive/anti-HBe negative (7/10; 70%), three were HBeAg negative (3/10; 30%), and all were reactive for anti-HBc (total) (10/10; 100%). Among HBeAg negatives, one was anti-HBe negative and two were anti-HBe positive. Eight of the fifteen children (8/15; 53.3%) were HBV DNA positive. The viral strains were grouped with genotype E (6/8; 75%) and genotype D3 (2/8; 25%) and harbored lamivudine drug-associated resistance variants and immune escape mutants. Liver health was assessed in nine children: five with detectable levels of HBV DNA and four with undetectable levels of HBV DNA. An abnormal APRI score of 0.713, was detected in one HBV DNA positive child (1/9; 11.1%). In the second sub-study an HBsAg seroprevalence of 5.4% was observed among 515 (28/515) pregnant women enrolled at ANCs in Windhoek. Three pregnant women (3/28; 10.7%) were positive for HBeAg; of whom one was HIV/HBV co-infected and the other two were HBV mono-infected. The two (2/28; 7.14%) HBV mono-infected/HBeAg-positive patients presented with viral load > 105 IU/ml, the study cut-off for antenatal treatment to prevent HBV MTCT; one received antiviral prophylaxis with tenofovir, the other was offered prophylaxis but did not receive it. Postpartum, 25 of the 28 HBV-exposed babies (25/28; 89.3%) were traced and followed-up to determine their HBV status and the rate of HBV MTCT. Fourteen (14/25; 56%) were males, and eleven were females (11/25; 44%). All babies had been vaccinated against HBV at birth, and 15 (15/25; 60%) had received hepatitis B immunoglobulin (HBIG). The 25 babies were tested for HBsAg at a median age of seven weeks (Range: 5.57 weeks – 20.29 weeks). All were non-reactive for HBsAg, including both babies born to the highly viremic women. With regards to the feasibility of HBV rapid testing as part of antenatal care services, the DetermineTM HBsAg rapid test had a 100% diagnostic sensitivity and specificity. HCWs found the test simple to use and showed a preference for rapid testing over laboratory testing for routine antenatal screening of HBV. They believed that this method would improve early diagnosis and treatment of HBV of pregnant women. In the cost-effectiveness analysis conducted in the third sub-study, a preventive strategy with universal BD vaccination alone was the cheapest option but was less effective. Adding HBIG to BD vaccination, and providing maternal antiviral prophylaxis was the most effective and the most costly strategy. The strategy that includes antiviral prophylaxis with sequential HBeAg testing added to BD vaccination and HBIG had an ICER of US$6 262.42 per infection averted, in comparison to the strategy including BD vaccination and HBIG only. The BD vaccination and HBIG strategy had an ICER of US$4 550.34/ pediatric HBV infection averted, in comparison to BD vaccination alone. These ICERs were highly sensitive to the prevalence of highly infectious pregnant women, the cost of the HBeAg test, and the effectiveness of each strategy for preventing MTCT in highly infectious pregnant women. Conclusion: Results from this research project reemphasized the issue of pediatric CHB, especially in HIV/HBV co-infected children. The data described in this study also showed that elimination of MTCT of HBV in Namibia is achievable, through routine antenatal HBsAg screening, treating pregnant women at high risk of MTCT, and providing HBV vaccination from birth. Screening using rapid testing was found cheap, and a feasible alternative for detecting HBV infection in pregnant women. The costs and health benefits of implementing antenatal antiviral prophylaxis and HBIG presented in the study provide background data for further assessment of the value for money of these interventions in SSA, and to explore alternatives excluding HBIG for HBV PMTCT.
Hepatitis B virus-associated hepatocellular carcinoma in South Africa: epidemiology and impact of HIV-1 co-infection and immune dysregulation
(Stellenbosch : Stellenbosch University, 2016-12) Maponga, Tongai Gibson; Andersson, Monique I.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Virology
ENGLISH ABSTRACT : Co-infection with the human immunodeficiency virus (HIV) negatively impacts the natural progression of hepatitis B virus (HBV) infection, including causing rapid progression to liver fibrosis and hepatocellular carcinoma (HCC). In sub-Saharan Africa the overlap between high HIV and HBV prevalence may increase the incidence of HCC. The aim of this study was to investigate the effect of HIV co-infection on presentation of HCC among HBV-infected patients. Since HCC is thought to be driven by ongoing severe inflammation, the study also evaluated the association between the expression of markers of immune activation/exhaustion and liver inflammation in patients with chronic hepatitis B (CHB) to determine if the risk of hepatofibrosis is increased by exposure to gut microbial products and compared HIV-infected patients with HBV-infected and HIV-HBV co-infected patients. Ethical approval was obtained to conduct two sub-studies. The first sub-study (HCC Epidemiology Study) involved recruitment of patients diagnosed with HCC at oncology units of selected teaching hospitals in South Africa. A total of 107 HCC cases were recruited between December 2012 and October 2015. Demographic, laboratory and clinical data together with blood specimens were collected. Patients were tested for HBV, hepatitis C virus (HCV) and HIV. Molecular characterization of HBV and HCV was also performed. For the second sub-study (Liver Fibrosis and Immune Markers Study), 46 HBV/HIV co-infected; 47 HBV monoinfected; 39 HIV monoinfected and; 39 HIV-/HBV-uninfected controls were recruited following informed consent. All HIV-infected patients had been on highly active antiretroviral therapy (HAART) for ≥3 months. Liver stiffness measurements were taken using the Fibroscan 402. Cell-based immunomarkers of activation/exhaustion were measured using flow cytometry of fresh whole blood. Soluble serum/plasma immunomarkers were measured using ELISA and Luminex. HIV and HBV viral loads and genotyping of HBV were performed. Of 107 cases in the HCC Epidemiology study, 83 (78%) were male and 68/106 (64%, 95% CI: 59-77) were positive for HBsAg. HIV seropositivity was seen in 22/100 (22%, 95% CI: 14-30) of all HCC cases. Among HBsAg-positive HCC cases, 19/66 (29%, 95% CI: 18-40) were HIV-infected compared to only 3/34 (9%) among those that were HBsAg-negative, p=0.04. The proportion of females among the HBV/HIV co-infected HCC cases 6/18 (33%, 95% CI: 11-55) was significantly higher compared to those that were HBV-mono-infected 6/47 (13%, 95% CI: 3-23), p=0.005. HIV/HBV co-infected females presented younger, at mean age 36.8 years (95% CI: 32.2-41.5) compared to 50.5 years (95% CI: 30.2-70.8) in HBV-mono-infected women, p=0.09. Males continue to be disproportionally affected with HCC. There is a trend towards younger age at diagnosis of HCC among HIV-positive compared to HIV-negative women. The Liver Fibrosis and Immune Markers Study showed a high percentage of CD8+ T lymphocytes from co-infected subjects expressing HLA-DR/CD38 and PD-1 (p<0.05). Soluble CD14 and IP-10 were also significantly elevated in plasma of co-infected patients. Co-infected subjects exhibited delayed immune recovery with lower CD4/CD8 T cell ratio; CD4 cell counts and frequent HIV viremia compared to HIV mono-infected participants (p<0.05). The HBV mono-infected group had the highest proportion of participants with moderate/advanced liver fibrosis measured by Fibroscan, together with highest plasma concentrations of most of the cytokines measured. The results showed positive correlation between HIV and HBV viral replication and liver fibrosis. The results suggest that there is persistent T-lymphocyte dysregulation and delayed immune recovery in ART-experienced HBV/HIV co-infected patients. However this does not appear to be associated with severity of liver fibrosis in this cohort. HAART used in HIV is also effective against HBV and may therefore have led to control of viral replication leading to better fibrosis scores compared to the HBV mono-infected patients. Moderate/advanced liver fibrosis in HBV-mono-infection may well be an indicator of poor access to HBV screening and treatment.
An investigation of hepatitis B virus in antenatal women tested for human immunodeficiency virus, in the Western Cape Province of South Africa
(Stellenbosch : Stellenbosch University, 2012-03) Maponga, Tongai Gibson; Andersson, Monique I.; Preiser, Wolfgang; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology.
ENGLISH ABSTRACT: Hepatitis B virus (HBV) immunisation protocols in much of Africa are based on data from the pre-human immunodeficiency virus (pre-HIV) era that indicated that HBV transmission occurs predominantly horizontally between siblings and play-mates rather than vertically from mother to child. The immunosuppression associated with HIV infection however may release HBV from immune control resulting in higher HBV viral loads, which may increase the risk of perinatal mother to child transmission of HBV. The aim of this study was to determine the prevalence and characteristics of chronic HBV infection in HIV-infected pregnant women compared to HIV-uninfected pregnant women in the Western Cape province of South Africa. Ethical approval was obtained to conduct a retrospective, matched case-control, unlinked anonymous study using residual plasma samples from the 9355 pregnant women included in the Western Cape's 2008 National HIV and Syphilis Antenatal Survey. Samples were tested for HBsAg on the AxSYM (Abbott, Chicago, IL) and confirmed by neutralization. Confirmed HBsAg-positive samples were tested for HBeAg, anti-HBe and anti-HD (Diasorin, Saluggia, Italy) and had HBV viral load and genotyping done. In addition, HBsAg-negative samples were tested for anti-HBc. Samples from 1549 HIV-infected pregnant women were included and matched to the same number of samples from age- and race-matched HIV-uninfected women. Median age of 26 years, parity and education were similar in the two groups. The prevalence of HBsAg was 3.4% for the HIV-infected group and 2.9% for the HIV-uninfected group. HBV DNA loads of greater than 104 IU/ml were detected in 32.1% of HBsAg-positive, HIV/HBV co-infected women, and in 14.3% HBsAg positive, HBV mono-infected women. Among the HIV-infected group 18.9% of HBsAg-positive were HBeAg positive, with a median viral load of 7.93 log10 IU/ml; whilst 15.5% HIV-uninfected women were positive for HBeAg with a median viral load of 6.07 log10 IU/ml. Genotype A was seen in 92.6% of the isolates while 7.4% of the isolates were genotype D. Serum total anti-HBc antibodies that are a marker of past infection were detected in 42.2% of HIV-infected and in 24.1% of HIV-uninfected women that were negative for HBsAg. No positive sample for anti-HD was seen among all HBsAg-positive samples. This data indicates that there is increased exposure to HBV in HIV-infected pregnant women than in HIV-uninfected women and that a greater proportion of HIV-infected pregnant women compared to HBV mono-infected pregnant women may be at increased risk of transmitting HBV to their infants. Further studies are needed to determine the rate of vertical transmission of HBV in the HIV era.
Prevalence and risks of Hepatitis E virus infection in blood donors from the Western Cape, South Africa
(Stellenbosch : Stellenbosch University, 2016-03) Lopes, Tatum; Andersson, Monique I.; Preiser, Wolfgang; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: Hepatitis E virus (HEV) is an important cause of enterically transmitted acute hepatitis worldwide and is a locally acquired disease in both developing and developed nations. Different genotypes in these two regions display the different characteristics of this interesting infection. HEV is classified into four major genotypes and it can present as two contrasting clinical entities. HEV genotypes 1 (HEV1) and 2 (HEV 2) are related to waterborne transmission and poor sanitation. Whereas genotypes 3 (HEV3) and 4 (HEV4) are associated with zoonotic transmission mainly through pigs, wild boar and deer. HEV infections in Africa are thought to be caused by HEV1 and HEV2. The seroprevalence of HEV has been described in Southern Africa, but all more than 10 years ago when assays were not well developed. South Africa has three HEV reports, describing a hospital outbreak, and the seroprevalence in specific communities of South Africa. The seroprevalence from these studies ranged from 2% to 10.7% however no genotyping was done. Researchers have reported evidence of direct and indirect transfusion-transmitted HEV infection being a potential risk to recipients of blood transfusions. Asymptomatic HEV infections in blood donors increase the likelihood that blood or blood products are contaminated with HEV viral particles. Hence, there is a greater chance of infecting high-risk recipient groups with compromised immune systems. Therefore, the main aim of this study was to determine the prevalence of past and active HEV infection in blood donors from the Western Cape. We also investigated which risk factors are associated with infection. Our study population consisted of 10,250 blood donors that were tested as two sub-studies. For study group 1 we recruited 250 donors to complete an HEV risk questionnaire. Thereafter these donors were tested using an indirect Wantai ELISA (Fortress Diagnostics) for anti-HEV IgG detection. Statistical analysis was done to determine which demographics and risk factors were associated with past HEV infection. In addition, to this, their plasma donations were pooled, prior to extraction and amplified with an in-house real-time reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) to detect HEV RNA. The 10,000 blood donors of study group 2 were tested as individual donations using a commercial Procleix HEV nucleic acid testing (NAT) assay to qualitatively detect HEV RNA by transcription-mediated amplification (TMA). Thereafter repeat-reactive donations were quantified using our in-house real-time RT-qPCR. The total anti-HEV IgG seroprevalence of our study was found to be 42.4% in blood donors (study group 1). Risk analysis revealed that eating turkey (p=0.001) and organ meat (p=0.026) and canoeing (p=0.017) were significantly associated with past HEV infection. Whereas direct contact with rabbits (p=0.045) or chickens (p=0.020) were statistically significantly different means of HEV exposure associated with HEV3 and HEV4. Furthermore, we found that the total HEV RNA prevalence was 0.009% (1/10,250). Studies are needed to further assess the risk of HEV blood-borne transmission and to understand the epidemiology of HEV in our setting.
The prevalence of Hepatitis B virus infection in an HIV-exposed paediatric cohort from the Western Cape, South Africa
(Stellenbosch : Stellenbosch University, 2012-12) Chotun, Bibi Nafiisah; Andersson, Monique I.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology Medical Virology..
ENGLISH ABSTRACT: Despite the availability of Hepatitis B virus (HBV) vaccination for over three decades, this infection remains a major public health problem. Whilst the WHO recommends giving a birth dose of the vaccine, in South Africa, routine infant HBV vaccination commences at six weeks of age. This schedule is based on data from the pre-HIV era which showed transmission occurred via the horizontal, rather than the vertical route. In the era of HIV however, maternal HIV co-infection may release HBV from immune control, resulting in higher HBV loads and increasing the risk of vertical transmission. The aim of this study was to determine the prevalence and character of HBV infection in HIV-exposed infected and uninfected infants. Residual plasma samples from routine HIV nucleic acid testing of 1000 HIV-exposed infants aged between 0 and 18 months from the Western Cape were tested. Samples were tested for HBsAg by ELISA (Murex HBsAg Version 3) and confirmed by neutralisation. HBV DNA was quantified using an in-house real-time PCR assay. Infants with HBsAg positive samples were followed up and a blood sample was collected from mother and child. Those HBsAg positive samples were tested for HBeAg/antiHBe (Diasorin) and HBsAg negative samples were tested for antiHBs. HBV DNA was quantified. The surface gene was sequenced and the HBV genotype determined by phylogenetic analysis using HepSEQ (www.hepseq.org.uk). Whole genome sequencing was also performed. Of 1000 samples tested, four samples were positive for HBsAg and/or HBV DNA, indicating a prevalence of HBV transmission of 0.4%. At follow-up, two of three infected infants were positive for HBsAg, with HBV viral loads of greater than 108 IU/ml. The third infant was found to have cleared his infection and the fourth child was lost to follow up. These infected infants had all received HBV vaccination. All four mothers were HBeAg positive. Sequencing analysis showed the HBV strains from the two infants and four mothers belonged to subgenotype A1. The two mother-child paired sequences were identical. The data from this study shows that vertical transmission of HBV infection in HIV-exposed infants from the Western Cape is occurring, despite vaccination. Data from the Western Cape, showing an HBV prevalence of 3.4% in HIV-infected pregnant women, and those presented here suggest a vertical transmission rate of HBV of 12%. This is despite the widespread use of tenofovir and lamivudine in HIV-infected women with low CD4 counts. This study provides data supporting calls to bring HBV vaccination closer to the time of birth. Further work is urgently needed to confirm these findings and to determine the rates of transmission in HIV-unexposed infants.
A study of the prevalence of Hepatitis B virus infection in the infants of HIV-positive mothers participating in P1041 in South Africa
(Stellenbosch : Stellenbosch University, 2014-12) Tamandjou Tchuem, Cynthia Raissa; Andersson, Monique I.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: Despite the decreased rate of HBV horizontal transmission in South Africa (SA) due to the HB vaccine, the risk of perinatal transmission remains of concern, especially in HIV/HBV co-infected women. Loss of HBV immune control, resulting in higher HBV replication and thus increasing the risk of transmission is described in HIV/HBV co-infected women. Chronic hepatitis is a well-recognized risk factor for hepatocellular carcinoma (HCC). The presence of specific HBV mutations has been reported in chronic and HCC patients and is used in algorithms for the prediction of HCC in CHB patients in Asia. While these mutations are extensively described in male patients, little is known regarding the antenatal and paediatric populations. This study aimed to determine the prevalence of HBV infection in HIV-exposed infants and to investigate the presence of HCC-related mutations in pregnant women and HIV-exposed children in SA. Residual samples of infants born to HIV-infected mothers were collected from the P1041 study previously conducted in SA. HBV markers (HBsAg, anti-HBs and anti-HBc) were tested on the Architect (Abbott). HBsAg positive samples were tested for HBV DNA to determine HBV viral loads. HBV strains were characterised by sequencing of the HBsAg gene and genotypes were determined by phylogenetic analysis using HepSEQ (www.hepseq.org.uk). For the HCC-related mutations investigation, samples and data were collected from three HBV-related studies: the NHLS Paediatric Study, an Antenatal Study and the current study. Pre-S, basal core promoter (BCP) and pre-core data was collected from all samples. Multiple alignments were formed and the nucleotide sequences of these extracts were translated into protein sequences. These protein sequences were compared manually to the HBV reference genes to identify HCC-related mutations. Of 850 HIV-exposed infants tested, three infants were positive for both HBsAg and HBV DNA. Two samples show evidence of past, but cleared HBV infection. Sequence analysis showed that the infants were infected with a subgenotype A1. At follow up, only one infant and mother were able to be traced and contacted. The infant was HIV-infected and had been on an ART regimen, including lamivudine for two years. HBV testing showed that the infant was HBsAg positive and had an undetectable viral load. Core sequence analysis showed clustering between mother and infant sequences. Transmission of mutant HBV previously associated with HCC prompted the question of what the prevalence of mutations in the antenatal and paediatric population is. In this investigation of HCC-related mutations study, a higher prevalence of combined pre-S, BCP and pre-core mutations was found in HIV-infected as compared to HIV-uninfected women. This study shows that vertical transmission is occurring in HIV-exposed infants in SA despite HB vaccination. Data described in this study suggests the importance of HB vaccination closer to the time of birth in SA. Moreover, data on the higher prevalence of HCC-related mutations in HIV-infected pregnant women provide a background for further longitudinal studies to confirm these findings and their implications in SA.

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