Characterization of hepatitis E virus in southern Africa

M.Sc. (Med.), Faculty of Health Sciences, University of the Witwatersrand, 2004Endemic circulation of hepatitis E virus (HEV) in Namibia was suspected from serological data during an outbreak of non-A non-B hepatitis in Rundu, in 1995. The source of the outbreak was surmised to be the water supply, which had been compromised approximately six months earlier. This study examines nucleotide and amino acid sequence data obtained from the open reading frame 2 (ORF2) region towards the carboxy terminal end (3’-end) of the HEV genome extracted from the stool of Namibian patients infected during this outbreak. The overall aim is to establish a polymerase chain reaction (PCR) for molecular diagnosis of HEV, to isolate HEV from specimens collected from acute viral hepatitis outbreaks such as this one in Namibia, to characterize, at the genomic level, the strain of the virus involved, to compare the strain to those involved in other outbreaks of HEV in southern Africa and to relate this information to available data from other parts of the world. A nested reverse transcriptase-polymerase chain reaction (RT-PCR) was developed for molecular diagnosis of HEV and four representative HEV isolates from this outbreak have been successfully amplified, sequenced and analysed over a 451 base pair (bp) region of a subgenomic fragment from the 3’-end of the genome in ORF2. Phylogenetic analysis showed the four Namibian HEV isolates clustered with a Mexican isolate in genotype II and shared a 85.8-86.3 percent (%) nucleotide identity with the 1987 Mexican isolate but were only 77.6-79.6 % similar to other African isolates. HEV isolated from the same region of Namibia in 1983 was reported to cluster into genotype I and this analysis clearly indicates the difference between the strains involved in the two outbreaks. Virus from sporadic cases of HEV isolated in 1997/8 in Nigeria were also found to be from genotype II. This is the first study performed in South Africa to isolate, amplify by PCR and sequence the HEV. It is also the first to characterize HEV as the causative agent of the hepatitis outbreak that occurred in Namibia in 1995. As it reports the presence of a second unique HEV strain in southern Africa, we conclude that HEV genotypes may be more widely distributed than previously thought

Standardization of cytokine flow cytometry assays

BACKGROUND: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). RESULTS: Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results (CD4(+)cytokine(+ )cells and CD8(+)cytokine(+ )cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17–44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5–20%, depending upon the experiment. The inter-lab C.V. was lowest (18–24%) for samples with a mean of >0.5% IFNγ + T cells, and highest (57–82%) for samples with a mean of <0.1% IFNγ + cells. CONCLUSION: ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays