Hepatitis C genotype distribution in patient and blood donor samples in South Africa for the period 2008-2012.

There are limited molecular epidemiological studies of hepatitis C at a national level in South Africa. The introduction of newer treatment modalities for hepatitis C requires knowledge of the genotypes as these may have different prognostic and therapeutic implications. This retrospective study describes genotype distributions of patients attending specialist clinics and a blood donor group studied during the period 2008-2012 in South Africa. Residual samples from diagnostic viral load testing from specialist clinics in South Africa (n=941) and from the South African National Blood Service (n=294) were analysed quantitatively by real-time PCR and genotyped using the Versant line probe assay or sequencing. Genotype 1 was predominant in blood donors (34%), whilst genotype 5a was prevalent in patients (36%). In the blood donor group, genotype 4 was detected for the first time. Genotype 2 was rare in the patient group and not detected in blood donors. Genotype 1 was the predominant genotype in the younger age groups (less than 30 years), whereas genotype 5a was found at higher proportions in the older age groups for both the patient and blood donor groups, comprising more than 60% of genotypes in those older than 50 years. Genotypes 1 and 5 were at highest proportions across all provinces compared to other genotypes. In blood donors, genotype 1 was predominant among Caucasians (43%) and genotype 5a among Blacks (54%). Such information is required for planning the impact on the health sector with regard to newly emerging therapies for hepatitis C and burden of disease.

Positive views, attitudes, and acceptability toward mHealth applications in addressing queer sexual and reproductive health: Healthcare providers and the queer individuals.

Objective: In the age of digital health, mankind has resources to write over the historical narrative of queer individuals' healthcare exclusions. The main purpose of this study was to explore the perspectives of both healthcare providers (HCPs) and queer individuals regarding the use of web-based tools and mobile health applications (mHealth apps) in the context of addressing queer individuals' sexual and reproductive health services and needs (SRHSN). Methods: An overall study was conducted as an exploratory sequential mixed method. This article provides findings from the performed qualitative cycle. The selection method was led by purposeful sampling, which targeted 33 HCPs delivering SRHSN within the defined study settings. Additionally, respondent-driven sampling was employed to select 22 queer individuals. Throughout the study, semi-structured one-on-one face-to-face interviews were used to collect data. Results: Four major themes and related sub-themes emerged from HCPs and queer individuals: (a) aid queer individuals with consultations and treatment improvements, (b) drawing parallels with technology in other sectors, (c) enhancing knowledge and education, and (d) positive perception of technological advancements. Conclusions: In accordance with our findings, HCPs and queer individuals were all positive and sees mHealth apps as a tool to address SRHSN for homosexual people.

Expanding the epidemiological understanding of hepatitis C in South Africa: Perspectives from a patient cohort in a rural town.

BACKGROUND: The epidemiology of hepatitis C virus (HCV) in the general population of South Africa (SA) is incompletely understood. A high HCV prevalence in key populations is known, but data are limited in terms of a broader understanding of transmission risks in our general population. OBJECTIVES: To investigate a patient cohort with HCV infection clustering in a rural SA town, in order to identify possible HCV transmission risks, virological characteristics, phylogenetic data and treatment outcomes. METHODS: A cluster of patients with positive HCV serology, previously identified from laboratory records, were contacted by a local district hospital and offered confirmatory testing for HCV viraemia where needed. Those with confirmed HCV RNA were invited to a local hospital visit, where relevant demographic information was recorded, clinical assessment performed and a confidential questionnaire administered. HCV population-based sequencing was performed on HCV NS3/4A, NS5A and NS5B using polymerase chain reaction-specific or M13 universal primers, and sequences were aligned using BioEdit 7.2.5. Phylogenetic trees were constructed. Clinical assessments included liver fibrosis determination with FibroScan (cut-off ≥12.5 kPa = F4). Patients were offered treatment, and sustained virological response (SVR) was confirmed by undetectable HCV RNA at least 12 weeks after the end of treatment. RESULTS: Twenty-one patients, all from the same town, median (interquartile range (IQR)) age 64 (59 - 70) years, 57% female, were evaluated. Of these, 24% (n=5) were HIV co-infected, stable on antiretrovirals. The median (IQR) alanine aminotransferase level was 51 (31 - 89) U/L, with fibrosis distribution including 29% F1, 29% F2, 9% F3 and 33% F4 METAVIR fibrosis. Virologically, two genotypes were observed: 62% (n=13) genotype (GT) 1b and 38% (n=8) GT5a. No patient had ever used injecting drugs, 14% (n=3) had received blood products before 1992, and 9.5% (n=2) had undergone traditional healer-administered scarification. All (n=21) reported attendance at a single primary care clinic in the past, with most (n=20) recalling having received parenteral therapies at the clinic. Phylogenetic analysis of the HCV NS5A and NS5B regions confirmed GT1b and GT5a genotypes and formed two separate clusters within their respective genotypes, suggesting a common source for each genotype infection. Most patients received treatment with sofosbuvir/daclatasvir, 1 was treated with sofosbuvir/velpatasvir, and 1 was re-treated with sofosbuvir/velpatasvir/voxilaprevir. Per protocol SVR was 95%, with the non-SVR patient successfully re-treated. CONCLUSIONS: Data from a rural town cluster of patients suggest parenteral medical exposure as the probable common source of hepatitis C transmission risk. The cohort was of older age with a significant number having advanced fibrosis or cirrhosis, suggesting HCV acquisition in the distant past. Using a simplified care approach, treatment outcomes were very good.

Direct-acting antiviral therapy for hepatitis C: The initial experience of the University of Cape Town/Groote Schuur Hospital Liver Clinic, South Africa.

BACKGROUND: An estimated 600 000 South Africans are chronically infected with hepatitis C virus (HCV). To date, accurate prevalence data are lacking, but emerging data suggest a significant burden in key populations. Historically, pegylated interferon and ribavirin treatment was challenging, with access limited. The advent of all-oral, short-course direct-acting antiviral (DAA) therapy has revolutionised the management of HCV, being well tolerated and highly effective, although initial cost was a prohibitive factor. OBJECTIVES: To report our initial 2-year experience with DAA therapy at the University of Cape Town/Groote Schuur Hospital Liver Clinic, South Africa (SA). METHODS: Patients who were viraemic for HCV were offered access to DAA therapy. All relevant demographic, virological, serological and clinical laboratory data were captured in a registry. Liver fibrosis was assessed non-invasively with the FibroScan. DAA regimens were prescribed according to current guidance based on HCV genotype (GT), prior treatment history and degree of fibrosis. On treatment, virological response was recorded and a sustained virological response (SVR) was defined as an undetectable HCV RNA at least 12 weeks after the end of treatment. RESULTS: We report on the first 210 patients treated. Their median (interquartile range (IQR)) age was 52 (42 - 61) years and 65% were male, with men significantly younger than women at 50 (42 - 59) years v. 58 (47 - 67) years, respectively (p=0.001). All GTs were observed, with 1 and 5 most prevalent at 45% and 20%, respectively, and GTs 2, 3 and 4 frequencies of 7%, 11% and 17%, respectively. Extensive subtype diversity for GTs 2 and 4 was present. The median (IQR) HCV viral load was log10 5.9 IU/mL (5.4 - 6.5). A significant proportion of patients (39%) had advanced fibrosis or cirrhosis, with 11% F3 fibrosis and 28% F4. Of those with cirrhosis, 12% were decompensated with Childs-Pugh B or C disease. Of the patients, 19% were HIV co-infected and 2% HBV co-infected. In total, 13% were treatment experienced. The majority of patients were treated with sofosbuvir and ledipasvir (38%), daclatasvir (36%) or velpatasvir (± voxilaprevir, 9%). Less frequent combinations included partitaprevir, ritonavir, ombitasvir ± dasbuvir (11%) and sofosbuvir/ribavirin (5%). The per-protocol SVR was 96% (98% if sofosbuvir/ribavirin is excluded). The majority of treatment failures occurred with GT-4, notably subtype 4r. Mild side-effects were reported in 10% of patients, with none discontinuing therapy. CONCLUSIONS: DAA therapy for HCV in a pan-genotypic group of patients, many with advanced liver disease, was highly effective. Our outcomes correspond with existing trial and real-world data for similar treatment. DAA therapy and access need rapid upscaling in SA, especially targeting key populations at point of care.

Fulminant hepatitis B virus (HBV) infection in an infant following mother-to-child transmission of an e-minus HBV mutant: Time to relook at HBV prophylaxis in South African infants.

The prevalence of hepatitis B virus (HBV) infection in pregnant women is high in South Africa (SA), yet prophylaxis to prevent mother-to-child transmission (MTCT) falls short of international recommendations. We describe a 10-week-old infant who developed fulminant hepatic failure following MTCT. The mother was hepatitis e-antibody positive and had a viral load of only 760 IU/mL. Genetic analysis of virus from mother and infant showed that both had the G1896A mutation in the preC/C gene, which truncates hepatitis e antigen (HBeAg) during translation, causing an HBeAg-negative phenotype. HBeAg attenuates antiviral immune responses, and its absence was probably responsible for the infant's fulminant hepatitis, due to an uncontrolled immune attack on infected liver cells. Pregnant women are not tested for HBV infection in SA and MTCT rates are unknown. Addition of a birth dose of vaccine, HBV screening of pregnant women and antiviral prophylaxis to positive mothers should be prioritised.

Isotachophoresis of cerebrospinal fluid.

Unconcentrated cerebrospinal fluid (CSF) was mixed in a constant ratio with carrier ampholytes and spacer amino acids and analysed by isotachophoresis. Examination of the cerebrospinal fluids from 113 patients showed characteristic patterns for normal, acute viral and acute bacterial meningitis. CSF from cases of subacute sclerosing panencephalitis (SSPE) and multiple sclerosis (MS) differed from these and from each other in the gamma-globulin region. This technique provides useful information on the integrity of the blood-brain barrier and is able to determine whether immunoglobulins present arise within the central nervous system or enter through a damaged barrier.

Review of the epidemiology, molecular characterization and tropism of the hepatitis G virus/GBV-C.

The hepatitis G virus and GBV-C are recently discovered variants of the same virus belonging to the family Flaviviridae (HGV/GBV-C). Although initially thought to be a hepatitis virus, it has been shown to have no association with liver disease. This paper reviews the data relating to the discovery, global prevalence, natural history, disease association, molecular features, replication and tissue tropism of HGV/GBV-C.

Investigation into a school enterovirus outbreak using PCR detection and serotype identification based on the 5' non-coding region.

A summer camp was followed by an outbreak of illness involving around 90 children. Investigations included individual questionnaires, inspection of the camp facilities, and laboratory analysis of water and clinical samples. Contamination of drinking and swimming water was demonstrated. An enterovirus was detected by polymerase chain reaction (PCR) and/or culture in 4/4 cerebrospinal fluid samples, 9/15 (60%) stool samples from symptomatic children and 2/9 (22%) stool samples from asymptomatic children. The virus was identified as an echovirus 3 by sequencing and phylogenetic analysis of a short 5' non-coding region (NCR) PCR product. Viruses from the outbreak clustered closely and an echovirus 3 from a temporally associated non-outbreak case could be readily distinguished. Despite the lack of a standardized approach, direct molecular detection and identification of enteroviruses is an efficient epidemiological tool. Here the 5'-NCR was successfully used for both detection and 'serotyping', and the close genetic relatedness of isolates was proven.

Genotyping of hepatitis C virus in South Africa.

The six major hepatitis C virus genotypes were investigated by using samples from 79 seropositive and PCR-positive blood donors from three different regions of South Africa as well as 9 patients with chronic renal failure, 19 with liver disease, and 23 with hemophilia. PCR products of the genome were typed by restriction fragment length polymorphic analysis by RsaI-HaeIII and MvaI-HinfI double digestion. Type 5 occurred in 40% of this population group; type 1 occurred in 33%; and types 2, 3, and 4 were found in 13.8, 7.7, and 2.3%, respectively.

GBV-C/HGV genotypes: proposed nomenclature for genotypes 1-5.

The GB virus-C and hepatitis G virus (GBV-C/HGV) are variants of the same flavivirus. This proposal attempts to clarify the conflicting nomenclature for GBV-C/HGV genotypes. The first three genotypes described were genotype 1 (West Africa); genotype 2 (US/Europe) and genotype 3 (Asia). Subsequently, two groups published data from South Africa and Southeast Asia both stating the presence of a novel "4th genotype." These isolates are distinct phylogenetically. It is proposed that the nomenclature for genotypes 1-3 remains as per previous publications, and that the Southeast Asian isolates be known as genotype 4, and the South African isolates as genotype 5.

Molecular epidemiology of hepatitis C virus infection at Ga-Rankuwa Hospital.

Different hepatitis C virus (HCV) genotypes may be associated with viral load, severity of liver disease, hepatocellular carcinoma, geographical location and sensitivity to interferon. In this study the prevalence of HCV-RNA and distribution of HCV genotypes was investigated in anti-HCV-positive patients admitted to Ga-Rankuwa Hospital during 1994 and 1995. One hundred and forty-nine sera from three groups of anti-HCV-positive patients (N = 78) were analysed. These included: (i) patients with various liver diseases; (ii) patients admitted to the renal unit; and (iii) a miscellaneous group of patients for whom HCV antibody screening was requested. Twenty-six patients (33%) tested positive for viral RNA. Restriction fragment length polymorphism (RFLP) analysis of these patients showed that HCV genotypes 1, 2 and 5 were present. Type 2 (35%) was the dominant genotype in the region served by Ga-Rankuwa Hospital. In a large proportion of viral RNA-positive patients (27%), the polymerase chain reaction product could not be digested with one of the sets of enzymes, and therefore could not be classified into genotypes 1 to 6. Further studies are now in progress to enhance our current knowledge of the epidemiology of HCV infection.

An electron microscopic demonstration of immune complexes of hepatitis B e-antigen using colloid gold as a marker.

Immune complexes of the hepatitis B e-antigen (HBeAg) could be labeled and thus visually identified in the electron microscope by using antibody to HBeAg (anti-HBe) tagged with colloidal gold particles. Circulating immune complexes of HBeAg were detected in sera from patients with acute hepatitis B infections as well as from asymptomatic carriers of hepatitis B surface antigens (HBsAg). Sera positive for rheumatoid factor frequently contained mixed aggregates in which immune complexes of HBsAg were closely bound to immune complexes of HBeAg.

Hepatitis B virus associated membranous glomerulonephritis.

The incidence of persistent hepatitis B surface (HBs) antigenaemia was studied in 114 nephrotic children with glomerulonephritis. Twenty five (24 boys) of 28 cases of membranous glomerulonephritis were HBs antigen (HBsAg) carriers. Only 9 of the remaining 86 patients with nephropathies other than membranous glomerulonephritis were HBsAg positive. HBsAg immune complexes were seen in the sera by electron microscopy. On radioimmunoassay both HBsAg and antibody (anti-HBs), and HBeAg and antibody (anti-HBe) were often detected concurrently, HBsAg was not shown in the glomerular capillary wall. HBs antigenaemia persisted in 80% of patients after recovery from glomerulonephritis but remission of the proteinuria correlated well, although not fully, with seroconversion to anti-HBe. The natural history of hepatitis B virus (HBV) associated glomerulonephritis in childhood is one of slow recovery. A few patients are left with mild asymptomatic proteinuria but progressive renal failure is rare. The 14% incidence of membranous glomerulonephritis in nephrotic children in this area is much higher than that found by the international study of kidney disease in children in well developed countries and is probably related to a high HBV carrier rate. A search for HBV markers should be included in the investigation of persistent glomerulonephritis, particularly in countries with a high prevalence of HBV carriers.

Prevalence of human parvovirus B19 and TT virus in a group of young haemophiliacs in South Africa.

A well recognized hazard of transfusion with blood or blood products is the acquisition of a viral infection. Parvovirus B19 and transfusion transmitted virus (TTV) are two of several non-enveloped viruses that may on rare occasions be present in coagulation factor concentrates. The prevalence of these viruses in the South African Haemophilia population has not previously been studied. Thirty-nine Haemophiliac children were investigated for evidence of parvovirus and TTV infection. 26 boys with Haemophilia A had been treated with cryoprecipitate or intermediate purity factor VIII, and 13 boys with Haemophilia B had received prothrombin complex concentrates. All the plasma products were prepared from South African donors and were virally inactivated by heat or solvent/detergent since 1992. A control group of 32 children who had not been transfused were also studied. IgG antibodies to B19 were present in 29 of the 39 patients (74%), 18/26 (69%) with Haemophilia A and 12 of the 13 (85%) with Haemophilia B. None of the patients was IgM antibody positive but two children were PCR positive for B19 DNA. Of the control children, 47% had IgG antibodies to B19, but none were IgM antibody or B19 DNA positive. TTV viral DNA was found in 10.2% of patients and in 9% of the control group. The results indicate that our locally produced plasma products are not a significant source of TTV transmitted infection but may contribute to infection by B19 parvovirus.

Molecular characterization of the 5' non-coding region of South African GBV-C/HGV isolates: major deletion and evidence for a fourth genotype.

GB virus C/hepatitis G virus (GBV-C/HGV) has been characterised as a novel flavivirus, and to date three known genotypes have been cloned. Greater genetic variation of GBV-C/HGV has been demonstrated in West African isolates, but no major deletions have been shown in the 5' non-coding region (NCR). The 5'NCR regulates protein translation via an internal ribosomal entry site (IRES). We cloned, sequenced, and analysed a 344-bp polymerase chain reaction (PCR) product, representing >60% of the 5'NCR, from 32 GBV-C/HGV PCR-positive volunteers. Wild-type virus amplicons were detected in all samples. However, 5/32 (15.6%) also amplified another fragment of between 205 and 231 bp. Sequence analysis showed all cloned PCR fragments to be GBV-C/HGV-specific. A typical deletion of 113-131 bp with minor variation was detected in isolates generating the smaller bands. RNA secondary structure analysis showed the deletions to be over domains II and III. This finding suggests that nucleotides 303-444 may be non-essential for 5'NCR functioning. Phylogenetic analysis demonstrated a novel fourth South African genotype, distinct from genotypes 1-3 with DNA distances of >0.1000. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) values for the wild-type and mutant samples were normal. This study documents the first major deletion in the 5'NCR of GBV-C/HGV, and suggests that bases 303-444 may not be essential for viral replication and ribosomal entry. A fourth GBV-C/HGV genotype appears to predominate in South Africa.

Hepatitis C virus infection rate in volunteer blood donors from the Western Cape--comparison of screening tests and PCR.

INTRODUCTION: Hepatitis C virus (HCV) antibody seroprevalence studies overestimate the true infection rate. No data exist on the incidence of HCV or its clinical features in blood donors of sub-Saharan Africa. AIMS: To establish the true incidence of HCV infection in volunteer blood donors in the Western Cape, and compare risk factors and clinical and biochemical features of viraemic and non-viraemic subjects. METHODS: All donors attending the Western Province Blood Transfusion Service between December 1992 and August 1994 were screened prospectively for anti-HCV using the Abbott second-generation assay. Positive donors were evaluated clinically and biochemically. Their sera were examined for HCV-RNA by the polymerase chain reaction (PCR). RESULTS: Of 66314 donors screened, 275 (0.41%) were anti-HCV-positive. Of these 13.6% were PCR-positive (0.056% of all donors). PCR-positive patients had more risk factors for HCV acquisition (P < 0.01), symptoms of hepatitis (P = 0.02) and clinical signs of liver disease (P = 0.05) and higher alanine (P < 0.0001) and aspartate aminotransferase levels (P < 0.0001) than PCR-negative donors. However, clinical and biochemical features did not discriminate adequately between PCR-positive and negative donors. Liver biopsies performed in 9 of 13 PCR-positive cases showed mild inflammation, but no cirrhosis.

Evidence that the GBV-C/hepatitis G virus is primarily a lymphotropic virus.

GB virus-C and the hepatitis G virus (GBV-C/HGV) are variants of the same positive sense RNA flavivirus, initially thought to be associated with hepatitis. The tissue tropism of GBV-C/HGV in normal subjects has not been evaluated to date using an extended tissue spectrum. Therefore, the sites of GBV-C/HGV replication were investigated in serum and twenty-three tissues collected during post-mortem examination of four apparently healthy individuals who died accidental deaths, who were infected with GBV-C/HGV. All were anti-HIV and anti-HCV negative and three out of four were HBsAg negative. Tissues were collected carefully to prevent cross contamination. A highly strand-specific RT-PCR assay was employed for the detection of either GBV-C/HGV positive strand RNA (virion) or negative strand RNA (replicative intermediary). Strand specificity of the RT-PCR assay was assessed with synthetic positive-and negative strand GBV-C/HGV RNA generated from a plasmid, using T7 and T3 RNA polymerases. The spleen and bone marrow biopsies were found to be uniformly positive for both negative-and positive strand GBV-C/HGV RNA. In addition, one cadaver was positive for both RNA strands in the kidney, and another positive for both in the liver. No negative strand RNA was detected in the following: brain, muscle, heart, thyroid, salivary gland, tonsil, lung, lymph nodes, gall bladder, pancreas, oesophagus, stomach, small bowel, large bowel, adrenal gland, gonad, aorta, skin and cartilage. This preliminary study concludes that GBV-C/HGV is a lymphotropic virus that replicates primarily in the spleen and bone marrow.