Computational protocol: Hepatitis B Virus Infection Does Not Significantly Influence Plasmodium Parasite Density in Asymptomatic Infections in Ghanaian Transfusion Recipients

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Protocol publication

[…] Viral DNA was extracted from 200 µl of plasma, taken from each of the 117 blood recipient’s using the QIAamp DNA minikit (Qiagen, Crawley, UK) according to manufacturers instructions. HBV DNA was tested by using a real-time qPCR assay targeting HBV S-gene and confirmed with a hemi-nested PCR within the basic core promoter/pre-core region (BCP/PC) and/or a second nested PCR amplifying a 1,434 bp amplicon encompassing the entire pre-S/S gene . In 6 HBsAg positive/HBV DNA unconfirmed samples a third nested-PCR was used to amplify a 276 bp fragment of the S gene . The limit of detection (LOD) of the HBV qPCR assay was 10 IU/ml. The LODs for the hemi-nested assays were, 50 IU/ml for the BCP and S-specific assays and 100 IU/ml for the pre-S/S PCR assay. Sequences of BCP, Pre-S/S and S PCR amplicons were obtained by direct sequencing of PCR products. Amplified products were purified from agarose gel excised bands using Wizard gel and PCR purification kits (Promega, Wallisellen, Switzerland). Ghanaian sequences were aligned with reference HBV genotypes A–H sequences using the CLUSTAL W software implemented within Mac Vector version 10.0.2 software (MacVector). Phylogenetic analysis was performed using the PAUP 4.01b10 software. To confirm the reliability of phylogenetic trees, bootstrap re-sampling was performed for each analysis (1000 replicates). Samples negative by nucleic acid testing were further tested with a real-time PCR targeting the Human Apoprotein B (HAPB) gene as described previously to exclude the presence of potential amplification inhibitors. […]

Pipeline specifications

Software tools Clustal W, MacVector, PAUP*
Application Phylogenetics
Organisms Hepatitis B virus, Homo sapiens
Diseases Hepatitis B, Infection, Malaria, Coinfection