Search

Abstract Background Host genetics influence the outcome of HCV disease. HCV is also highly mutable and escapes host immunity. HCV genotypes are geographically distributed and HCV subtypes have been shown to have distinct repertoires of HLA-restricted viral epitopes which explains the lack of cross protection across genotypes observed in some studies. Despite this, immune databases and putative epitope vaccines concentrate almost exclusively on HCV genotype 1 class I-epitopes restricted by the HLA-A*02 allele. While both genotype and allele predominate in developed countries, we hypothesise that HCV variation and population genetics will affect the efficacy of proposed epitope vaccines in South Africa. This in silico study investigates HCV viral variability within well-studied epitopes identified in genotype 1 and uses algorithms to predict the immunogenicity of their variants from other less studied genotypes and thus rate the most promising vaccine candidates for the South African population. Six class I- and seven class II- restricted epitope sequences within the core, NS3, NS4B and NS5B regions were compared across the six HCV genotypes using local genotype 5a sequence data together with global data. Common HLA alleles in the South African population are A30:01, A02:01, B58:02, B07:02; DRB1*13:01 and DRB1*03:01. Epitope binding to 13 class I- and 8 class –II alleles were described using web-based prediction servers, Immune Epitope Database, (IEDB) and Propred. Online population coverage tools were used to assess vaccine efficacy. Results Despite the homogeneity of genotype 1 and genotype 5 over the epitopes, there was limited promiscuity to local HLA-alleles.Host differences will make a putative vaccine less effective in South Africa. Of the 6 well-characterized class I- epitopes, only 2 class I- epitopes were promiscuous and 3 of the 7 class-II epitopes were better conserved and promiscuous. By fine tuning the putative vaccine using an optimal cocktail of genotype 1 and 5a epitopes and local HLA data, the coverage was raised from 65.85% to 91.87% in South African Blacks. Conclusion While in vivo and in vitro studies are needed to confirm immunogenic epitopes, in silico HCV epitope vaccine design which takes into account HCV variation and host allele frequency will maximize population coverage in different ethnic groups.

In 2023, passive laboratory-based surveillance showed an increase in hepatitis A virus (HAV) infection. We investigated hepatitis A incidence using the notifiable medical condition surveillance system (NMCSS) data and molecularly characterised positive blood samples from the Western Cape province for 2023. All HAV IgM seropositive cases from the NMCSS from 1 January to 31 October 2023 in South Africa were investigated. HAV RNA from blood samples that had tested positive for HAV IgM from Western Cape was amplified in the VP1/P2B junction and sequenced (3500Xl Genetic Analyser). Sequences were assembled, aligned (Sequencher) and analysed (Aliview 1.27 and MEGA11). Statistical analysis was performed using Excel and the CuSum2 Threshold to determine suspected outbreaks. In 2023, the incidence of HAV IgM was 6.28/100,000 in South Africa, with the highest incidence in Western Cape province (15.86/100,000). Children aged 5 to 14 years were affected the most in the Western Cape. The positive cases in the Western Cape were above the CuSum2 threshold from January to May 2023, with the highest incidence observed in the City of Cape Town Metropolitan (14.8/100,000). Genotyping was successfully performed on 92.7% (139/150) of serum samples, for which the IB sub-genotype was detected. Three primary mutations R63K, R71S and M104I were observed in more than 49% of the samples. Most of the samples sequenced belonged to patients residing in areas close to each other within the City of Cape Town Southern, Western, and Mitchells Plain sub-districts. The CuSum2 threshold method allowed the identification of suspected HAV outbreaks in the districts within the Western Cape in 2023 while genotyping identified clusters of sub-genotype IB. Genotyping could assist with determining the common source of infection during an outbreak, especially if coupled with epidemiological and geographical data. Further active surveillance can assist in investigating the HAV risk factors for targeted public health responses., (© 2024. The Author(s).)

In the context of polio eradication efforts, accurate assessment of vaccination programme effectiveness is essential to public health planning and decision making. Such assessments are often based on zero-dose children, estimated using the number of children who did not receive the first dose of the Diphtheria-Tetanus-Pertussis containing vaccine as a proxy. Our study introduces a novel approach to directly estimate the number of children susceptible to poliovirus type 2 (PV2) and uses this approach to provide district-level estimates for South Africa of susceptible children born between 2017 and 2022. We used district-level data on annual doses of inactivated poliovirus vaccine (IPV) administered, live births, and population sizes, from 2017 through 2022. We imputed missing vaccination data, implemented flexible assumptions regarding dose distribution in the eligible population, and used estimated efficacy values for one, two, three, and four doses of IPV, to compute the number of susceptible and immune children by birth year. We validated our approach by comparing an intermediary output with zero-dose children (ZDC) estimated using data reported by WHO/UNICEF Estimates of National Immunization Coverage (WUENIC). Our results indicate high heterogeneity in susceptibility to PV2 across South Africa's 52 districts as of the end of 2022. In children under 5 years, PV2 susceptibility ranged from approximately 30 % in districts including Xhariep (31.9 %), Ekurhuleni (30.1 %), and Central Karoo (29.8 %), to less than 4 % in Sarah Baartman (1.9 %), Buffalo City (2.1 %), and eThekwini (3.2 %). Our susceptibility estimates were consistently higher than ZDC over the timeframe. We estimated that ZDC decreased nationally from 155,168 (152,737-158,523) in 2017 to 108,593 in 2021, and increased to 127,102 in 2022, a trend consistent with ZDC derived from data reported by WUENIC. While our approach provides a more comprehensive profile of PV2 susceptibility, our susceptibility and ZDC estimates generally agree in the ranking of districts according to risk., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hepatitis A (HAV) presents a significant global health concern with diverse clinical manifestations primarily transmitted through fecal-oral routes, emphasizing the critical role of sanitation and water cleanliness in transmission dynamics. Age-related variations, notably asymptomatic presentation in children, add complexity. The World Health Organization's (WHO) endemicity classification aids in understanding prevalence and control strategies. This study examines 2023 South African epidemiological data on HAV cases, evaluating age distribution, incidence rates, and provincial disparities. Data from the national surveillance system and weather services were analyzed. Findings reveal distinct age-related trends, with the highest seroprevalence observed in the 5-9 age group with the most burdened areas located in the Western Cape, KwaZulu-Natal, and Gauteng provinces. Furthermore, seasonal rainfall variations correlate with increased incidence in Western Cape and KZN. The amalgamation of results suggest a potential epidemiological shift, emphasizing the need for updated immunization strategies. Noteworthy patterns, like the rise in 5-9-year-olds, may be influenced by factors such as school clustering and migration. Provincial disparities and the impact of climatic events underscore the necessity for dynamic vaccination strategies and surveillance network enhancements. This study highlights the urgency for improved understanding and response to HAV in South Africa.

Hepatitis B, a potentially life-threatening viral infection of the liver, remains a global public health concern despite the availability of effective vaccines for over three decades. The aim of our study was to provide national data on active hepatitis B infections in the public health sector of South Africa. We conducted retrospective analyses on national laboratory data over the period 2015 to 2019. We identified 176,530 cases who tested positive for HBsAg (active infection) with a test positivity rate of 9.02%. Of these active infections, 11,355 (6.43%) were found to be chronically infected. We linked 24,839 (14.07%) and 2,461 (21.67%) HBeAg positive results to all active HBV infections and identified chronic infections respectively. Clearance of HBsAg was observed in 5,569 cases, inclusive of clearance in 135 chronic cases. Active HBV infections were significantly higher in men than women over the five years (p < 0.0001). Among individuals who were vaccine-eligible as infants (0 to 19 years old), we observed 4,981 active HBV infections, including 1,131 infections under five years old, majority of which (65.78%) were under one year old. In the under five-year age group, the HBsAg population positivity rate was 0.02% and test positivity rate was 4.83%. Among all women with active HBV infections (78,935), 85.17% were of reproductive age and of these, 13.73% were HBeAg positive. Without a birth dose of the HBV vaccine, lack of routine HBsAg screening at antenatal care, and HBsAg and HBeAg prevalence among women of reproductive age, it is likely that the majority of cases under five years old were vertically infected. Optimal HBV vaccine coverage, inclusive of a birth dose, is key to eliminating horizontal and vertical transmission of HBV. Early identification of HBV chronicity through real time data analysis is fundamental in reducing the risk of liver cirrhosis and hepatocellular carcinoma., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Moonsamy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Hepatitis B virus (HBV), a global public health threat, is targeted for elimination by 2030. As national HBV prevalence and incidence is lacking for South Africa, our study aimed to provide such data in the public health sector., Methods: We analysed laboratory-confirmed HBV data from 2015 to 2019 to determine annual prevalence and incidence rates of HBV infection per 100,000 population, HBsAg and anti-HBc IgM test positivity rates, and HBsAg and anti-HBc IgM testing rates per 100,000 population. Time trend and statistical analyses were performed on HBsAg and anti-HBc IgM test positivity rates., Results: The national prevalence rate of HBV infection per 100,000 population increased from 56.14 in 2015 to 67.76 in 2019. Over the five years, the prevalence rate was higher in males than females, highest amongst individuals 25 to 49 years old and highest in Gauteng province. The HBsAg test positivity rate dropped from 9.77% in 2015 to 8.09% in 2019. Over the five years, the HBsAg test positivity rate was higher in males than females, amongst individuals 25 to 49 years old and amongst individuals of Limpopo province. Amongst HBsAg positive children under 5 years old, the majority (65.7%) were less than a year old. HBsAg testing rates per 100,000 population were higher in females under 45 years of age and in males 45 years and above. The national incidence rate of acute HBV infection per 100,000 population dropped from 3.17 in 2015 to 1.69 in 2019. Over the five-year period, incidence rates were similar between males and females, highest amongst individuals 20 to 39 years old and highest in Mpumalanga province. Amongst individuals 20 to 24 years old, there was a substantial decline in the incidence and anti-HBc IgM test positivity rates over time. Anti-HBc IgM testing rates per 100,000 population were higher in females under 40 years of age and in males 40 years and above., Conclusion: Critical to hepatitis B elimination is strengthened infant vaccination coverage and interruption of vertical transmission. Transmission of HBV infection in adults may be reduced through heightened awareness of transmission routes and prevention measures., (© 2021. The Author(s).)

As South Africa transitions from endemic to intermediate endemicity, hepatitis A surveillance needs strengthening to monitor trends in disease incidence and to identify outbreaks. We used passive laboratory-based surveillance data from the National Health Laboratory Services to calculate national hepatitis A incidence and to establish thresholds for outbreaks. Incidence was calculated by age and geographic location. The static threshold used two or three standard deviations (SDs) above the mean hepatitis A incidence in 2017-2019, and a cumulative summation (CuSum2) threshold used three SDs above the mean of the preceding seven months. These thresholds were applied to hepatitis A data for 2020. From 2017 to 2020, the mean incidence of hepatitis A IgM was 4.06/100,000 and ranged from 4.23 to 4.85/100,000 per year. Hepatitis A incidence was highest in the Western Cape province (WCP) (7.00-10.92/100,000 per year). The highest incidence was in the 1-9-year-olds. The incidence of hepatitis A in 2020 exceeded the static threshold in two districts of the WCP: Cape Winelands in January and Overberg district in August. The provincial incidence did not exceed the static and CuSum2 thresholds. District-level analysis using either threshold was sensitive enough to monitor trends and to alert district health authorities, allowing early outbreak responses.

Background and Aims: To enhance screening and diagnosis in those at-risk of hepatitis C virus (HCV), efficient and improved sampling and testing is required. We investigated the performance of point-of-care (POC) tests and dried blood spots (DBS) for HCV antibody and HCV RNA quantification in individuals at higher risk for HCV (people who use and inject drugs, sex workers and men who have sex with men) in seven South African cities., Methods: Samples were screened on the OraQuick HCV POC test (471 whole blood and 218 oral fluid); 218 whole blood and DBS paired samples were evaluated on the ARCHITECT HCV antibody (Abbott) and HCV viral load (COBAS Ampliprep/COBAS TaqMan version 2) assays. For HCV RNA quantification, 107 dB were analyzed with and without normalization coefficients., Results: POC on either whole blood or oral fluid showed an overall sensitivity of 98.5% (95% CI 97.4-99.5), specificity of 98.2% (95% CI 98.8-100) and accuracy of 98.4% (95% CI 96.5-99.3). On the antibody immunoassay, DBS showed a sensitivity of 96.0% (95% CI 93.4-98.6), specificity of 97% (95% CI 94.8-99.3) and accuracy of 96.3% (95% CI 93.8-98.8). A strong correlation ( R 2 = 0.90) between viral load measurements for DBS and plasma samples was observed. After normalization, DBS viral load results showed an improved bias from 0.5 to 0.16 log10 IU/mL., Conclusion: The POC test performed sufficiently well to be used for HCV screening in at-risk populations. DBS for diagnosis and quantification was accurate and should be considered as an alternative sample to test. POC and DBS can help scale up hepatitis services in the country, in light of our elimination goals., Competing Interests: Andrew Scheibe received an honourarium from Gilead for presenting work in this field., (© 2021 The Authors. Health Science Reports published by Wiley Periodicals LLC.)

Background: People who use drugs including people who inject drugs (PWUD/ID), sex workers (SWs) and men who have sex with men (MSM) are at increased risk of HIV and viral hepatitis infection. Limited epidemiological data on the infections exists in key populations (KPs) in South Africa. We investigated the prevalence of hepatitis B (HBV), hepatitis C (HCV) and HIV and selected risk factors among these KPs to inform effective responses., Methods: We used convenience sampling to recruit a targeted 3500 KPs accessing HIV-related health services across Cape Town (SWs, MSM, PWUD/ID), Durban (SWs, PWUD/ID), Pietermaritzburg (SWs), Mthatha (SWs), Port Elizabeth (SWs), Johannesburg (MSM) and Pretoria (MSM and PWUD/ID) into a cross-sectional survey. An interviewer questionnaire to assess socio-demographic characteristics, drug use and sexual risk practices, was administered. HBV surface antigen (HBsAg); HCV antibody, viral load and genotype, and HIV antibody, was tested., Results: Among the 3439 people included in the study (1528 SWs, 746 MSM, 1165 PWUD/ID) the median age was 29 years, most participants were black African (60%), and 24% reported homelessness. 82% reported substance use in the last month, including alcohol (46%) and heroin (33%). 75% were sexually active in the previous month, with condom use at last sex at 74%. HIV prevalence was 37% (highest among SWs at 47%), HBsAg prevalence 4% (similar across KPs) and HCV prevalence was 16% (highest among PWUD/ID at 46%)., Conclusions: HBV, HCV and HIV pose a health burden for KPs in South Africa. While HIV is key for all included KPs, HCV is of particular importance to PWUD/ID. For KPs, HBV vaccination and behavioural change interventions that support consistent condom and lubricant access and use are needed. Coverage of opioid substitution therapy and needle and syringe services, and access to HCV treatment for PWUD/ID need to be expanded.

Introduction and Methods: Hepatitis B is a vaccine preventable disease and is notifiable in South Africa. Hepatitis B vaccination was incorporated into the Expanded Programme on Immunisation in South Africa in 1995. We used a convenience sample from community-based febrile rash surveillance in 2013 to estimate hepatitis B sero-prevalence. Of samples serologically negative for acute measles infection, 450 samples spanning nine provinces of South Africa were tested for hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (anti-HBs) and hepatitis B core antibody (anti-HBc)., Results: Two children (2/450; 0.4%) tested positive for HBsAg. Three hundred and three children (67.3%) had evidence of vaccine induced immunity. Vaccine induced immunity was present in 80.2% of 1-5 year olds, but only 60.3% of 10-14 year olds. Natural immunity, indicating exposure to circulating hepatitis B, was present in 13/450 (2.9%) children., Conclusion: Chronic hepatitis B in South African has decreased in prevalence from highly endemic levels prior to vaccine introduction to approximately 0.4% in this sample, demonstrating impact of a successful vaccination programme 18 years after introduction. Decreased vaccine-induced immunity with increasing age may reflect waning antibody titres over time., Competing Interests: The authors perceive no conflicts of interest. The employer of all authors, the National Institute for Communicable Diseases, is funded by the South African government (www.nicd.ac.za). Dr Suchard administered during the conduct of the study; grants from Sanofi Pasteur, speaker honoraria from Aspen- GSK and Paediatric management group, consultancy fees from Prepex Circ Med Tech ltd and personal fees from World Health Organization, outside the submitted work; In addition, Dr. Suchard has an expired provisional South African patent 2010 “Method for Diagnosing a Disease” PA151392/P unrelated to the current work. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Dr. Cohen reports grants from Sanofi Pastuer, non-financial support from Parexel, grants from US CDC, outside the submitted work. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: People who inject drugs (PWID) are at high risk for hepatitis C (HCV), hepatitis B (HBV) and HIV without accessible harm reduction programmes. Coverage of needle and syringe and opioid substitution therapy (OST) services in South Africa is below global recommendations and no hepatitis services exist for PWID. We assessed HCV, HBV and HIV prevalence and risk factors among PWID accessing harm reduction services in Cape Town, Durban and Pretoria to inform policy and programming., Methods: We conducted a cross-sectional survey among PWID in these cities between August 2016 and October 2017. Participants were opportunistically sampled while accessing services. Study team members administered a questionnaire that assessed sociodemographic characteristics, drug use and sexual risk practices. We tested for HCV (antibody, viral load and genotype), HBV surface antigen (HBsAg) and HIV. Bivariate and multivariate analyses assessed associations with HCV serostatus., Results: Nine hundred and forty-three PWID were included in the per protocol analysis. The majority (87%, 819/943) were male, the overall median age was 29 and most lived on the street (66%, 626/943). At last injection, 77% (722/943) reported using a new needle and syringe and 17% (163/943) shared equipment. HIV prevalence was 21% (196/926), HBsAg positivity 5% (47/936), HCV seroprevalence 55% (513/937), HCV viraemic prevalence (proportion tested with detectable HCV) 43% (404/937) and HCV viraemic rate (proportion HCV antibody positive with detectable HCV) 79% (404/513). HCV genotype 1a (73%, 270/368) was the most prevalent. In multivariate analysis, HCV infection was positively associated with residing in Pretoria (adjusted odds ratio (aOR) 1.27, 95% CI 1.21-1.34), living on the street (aOR 1.90, 95% CI 1.38-2.60), frequent injecting (aOR 1.58, 95% CI 1.15-2.16) and HIV infection (aOR 1.69, 95% CI 1.15-2.47), and negatively associated with black race (aOR 0.52, 95% CI 0.36-0.74) and sexual activity in the previous month (aOR 0.61, 95% CI 0.42-0.88)., Conclusions: HCV and HIV are major health threats affecting PWID in these cities. Access to OST and needle and syringe services needs to be increased and integrated with HCV services. Social and structural factors affecting PWID who live on the street need to be addressed.

Background: Data on viral hepatitis in South Africa is scarce. Although viral hepatitis A, B and C are notifiable conditions in South Africa, discrepancies have been noted in the number of viral hepatitis cases notified by the National Department of Health (NDOH) compared with laboratory confirmed cases from the National Institute for Communicable Diseases (NICD). The aim of the study was to assess the knowledge, attitudes and practices of health care professionals on the notification of viral hepatitis A, B and C., Methods: A descriptive, cross-sectional study on 385 health care professionals was conducted at Charlotte Maxeke Johannesburg Academic and Tshwane District hospitals in Gauteng province, South Africa, between March and May 2015. A pre-tested, structured questionnaire with 21 (6 demographic and 15 knowledge, attitudes, and practice (KAP)) questions was used to collect information from invited participants. A score was assigned to each KAP question and a mean (SD) score was calculated for each section. Data were analyzed using descriptive statistics in STATA version 13., Results: Of the total 385 respondents, 65% ( n  = 250) were nurses and 35% ( n  = 135) were doctors. The overall mean knowledge score for health care professionals was 2.0 ± 1.6 (mean ± SD) out of a score of 6 regarding viral hepatitis notification. Overall mean scores of practice and attitude towards notification were higher at 2.9 ± 0.4 and 3.3 ± 0.7, out of a score of 4 and 5, respectively. Lack of training, poor knowledge, a complex process and excessive workload were some of the reasons for poor notification of viral hepatitis., Conclusions: Overall, knowledge on notification of viral hepatitis was poor among health care professionals. Adequate training on viral hepatitis, notification process, roles and responsibilities of health care professionals to notify and the implication of viral hepatitis notifications is recommended to improve reporting rate of notifiable diseases and referrals to increase linkage to care., Competing Interests: The authors declare that they have no competing interests.The study was approved by the Faculty of Health Science Research Ethics Committee, University of Pretoria (reference number 401/2014). Permission to conduct the research study at both the hospitals was granted by the hospital clinical managers. Consent forms were obtained from the study participants.Permission to publish the study results was obtained from all stakeholders involved in the project. However, there are no participants details reported in the study.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Background: In August 2014, the National Institute for Communicable Diseases (NICD) in South Africa established a modular high-biosafety field Ebola diagnostic laboratory (SA FEDL) near Freetown, Sierra Leone in response to the rapidly increasing number of Ebola virus disease (EVD) cases., Methods and Findings: The SA FEDL operated in the Western Area of Sierra Leone, which remained a "hotspot" of the EVD epidemic for months. The FEDL was the only diagnostic capacity available to respond to the overwhelming demand for rapid EVD laboratory diagnosis for several weeks in the initial stages of the EVD crisis in the capital of Sierra Leone. Furthermore, the NICD set out to establish local capacity amongst Sierra Leonean nationals in all aspects of the FEDL functions from the outset. This led to the successful hand-over of the FEDL to the Sierra Leone Ministry of Health and Sanitation in March 2015. Between 25 August 2014 and 22 June 2016, the laboratory tested 11,250 specimens mostly from the Western Urban and Western Rural regions of Sierra Leone, of which 2,379 (21.14%) tested positive for Ebola virus RNA., Conclusions: The bio-safety standards and the portability of the SA FEDL, offered a cost-effective and practical alternative for the rapid deployment of a field-operated high biocontainment facility. The SA FEDL teams demonstrated that it is highly beneficial to train the national staff in the course of formidable disease outbreak and accomplished their full integration into all operational and diagnostic aspects of the laboratory. This initiative contributed to the international efforts in bringing the EVD outbreak under control in Sierra Leone, as well as capacitating local African scientists and technologists to respond to diagnostic needs that might be required in future outbreaks of highly contagious pathogens.

Hepatitis C virus (HCV) exists as six major genotypes that differ in geographical distribution, pathogenesis, and response to antiviral therapy. In vitro replication systems for all HCV genotypes except genotype 5 have been reported. In this study, we recovered genotype 5a full-length genomes from four infected voluntary blood donors in South Africa and established a G418-selectable subgenomic replicon system using one of these strains. The replicon derived from the wild-type sequence failed to replicate in Huh-7.5 cells. However, the inclusion of the S2205I amino acid substitution, a cell culture-adaptive change originally described for a genotype 1b replicon, resulted in a small number of G418-resistant cell colonies. HCV RNA replication in these cells was confirmed by quantification of viral RNA and detection of the nonstructural protein NS5A. Sequence analysis of the viral RNAs isolated from multiple independent cell clones revealed the presence of several nonsynonymous mutations, which were localized mainly in the NS3 protein. These mutations, when introduced back into the parental backbone, significantly increased colony formation. To facilitate convenient monitoring of HCV RNA replication levels, the mutant with the highest replication level was further modified to express a fusion protein of firefly luciferase and neomycin phosphotransferase. Using such replicons from genotypes 1a, 1b, 2a, 3a, 4a, and 5a, we compared the effects of various HCV inhibitors on their replication. In conclusion, we have established an in vitro replication system for HCV genotype 5a, which will be useful for the development of pan-genotype anti-HCV compounds., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Previous studies in South African patients have shown that hepatitis C virus (HCV) prevalence is low (2.6%) and genotype 5 is the dominant genotype. This is the first sequence-based genotype study from this country on two different patient groups (haemophiliacs (H) and liver disease (LD) patients) over two time periods (2000-2002 and 2007). Genotype 5 was found to be the dominant genotype in the LD groups, but genotype 1 dominated in the H group. Genotype 2, and a higher prevalence of genotype 3a, was present in the H group when compared to previous South African studies. Genotypes 3 and 4 have been described here in the LD groups for the first time in South Africa, with subtypes 4c and 4g being identified. A separate cluster of genotype 5 sequences were found to have only one adenine at nucleotide position 107 of the 5'UTR, as previously reported. Subtyping in the less conserved NS5B region was used to further characterize the 2000-2002 isolates and validate 5'UTR typing. The study shows that by using reliable methods of genotyping, the prevalence and frequencies of HCV genotypes can be monitored in South Africa for better diagnosis and treatment of the disease.