Ecipients hospitalized in the departments of Medicine and Obstetrics and Gynaecology, at the Komfo Anokye Teaching Hospital (KATH) in Kumasi, Ghana as part of the Blood Organ Transmitted Infectious Agents (BOTIA) sample repository [18]. All 154 samples were selectedImpact of Hepatitis B on Plasmodium Infectionsat random from the repository using an online tool (http://www. randomizer.org/) to avoid selectional bias. Female recipients (N = 130; average age: 31.9 years) were mostly pregnant (N = 87), hospitalized for massive bleeding related to ectopic pregnancy (N = 16), Title Loaded From File post-partum hemorrhage (N = 10), abortion (N = 15), or other causes of anemia (N = 46). Non-pregnant women presented with hematological anemia (N = 5), gastro-Intestinal (GI) bleeding (N = 3) or other conditions including diabetes, polyps, fibroids and trauma (N = 35). Male recipients (N = 24; average age: 37.5 years) presented with hematological anemia (N = 3), GI bleeding (N = 7) or severe anemia (N = 10). Other conditions included renal failure and pneumonia (N = 4). EDTA-treated plasma and cellular fractions were separated and frozen at #240uC until tested as described previously [19]. After initial screening, 37 individuals were excluded from further analysis. Exclusion of these samples was based upon positivity with at least one of the following exclusion criteria: anti-HIV-1/2 (N = 11), anti-HCV (N = 5), receiving anti-malarial therapy (N = 13), diagnosed with sickle cell anemia (N = 13) 1313429 and Glucose6 Phosphate Dehydrogenase deficiency (G6PD) (N = 3).second nested PCR amplifying a 1,434 bp amplicon encompassing the entire pre-S/S gene [23]. In 6 HBsAg positive/HBV DNA unconfirmed samples a third nested-PCR was used to amplify a 276 bp fragment of the S gene [4]. The limit of detection (LOD) of the HBV qPCR assay was 10 IU/ml. The LODs for the heminested 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 1676428 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 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 Title Loaded From File realtime PCR targeting the Human Apoprotein B (HAPB) gene as described previously [24] to exclude the presence of potential amplification inhibitors.Ethics StatementApproval for the BOTIA repository and its use was obtained from the Kwame Nkrumah University of Science and Technology School of Medical Sciences committees for ethics and publication (Kumasi, Ghana). The BOTIA scientific committee approved the present study. Written informed consent was obtained from all participants prior to enrollment.Plasmodium DNA Detection with Species-specific Nested PCRsDNA was extracted from 200 ml red cell fractions using the QIAamp blood minikit (Qiagen) as per manufacturer’s instructions. All samples were tested twice, in duplicate using a genusspecific primer pair and four species-specific primer pairs (targeting the 18 s ribosomal DNA sequence of P.falciparum, P.vivax.Ecipients hospitalized in the departments of Medicine and Obstetrics and Gynaecology, at the Komfo Anokye Teaching Hospital (KATH) in Kumasi, Ghana as part of the Blood Organ Transmitted Infectious Agents (BOTIA) sample repository [18]. All 154 samples were selectedImpact of Hepatitis B on Plasmodium Infectionsat random from the repository using an online tool (http://www. randomizer.org/) to avoid selectional bias. Female recipients (N = 130; average age: 31.9 years) were mostly pregnant (N = 87), hospitalized for massive bleeding related to ectopic pregnancy (N = 16), post-partum hemorrhage (N = 10), abortion (N = 15), or other causes of anemia (N = 46). Non-pregnant women presented with hematological anemia (N = 5), gastro-Intestinal (GI) bleeding (N = 3) or other conditions including diabetes, polyps, fibroids and trauma (N = 35). Male recipients (N = 24; average age: 37.5 years) presented with hematological anemia (N = 3), GI bleeding (N = 7) or severe anemia (N = 10). Other conditions included renal failure and pneumonia (N = 4). EDTA-treated plasma and cellular fractions were separated and frozen at #240uC until tested as described previously [19]. After initial screening, 37 individuals were excluded from further analysis. Exclusion of these samples was based upon positivity with at least one of the following exclusion criteria: anti-HIV-1/2 (N = 11), anti-HCV (N = 5), receiving anti-malarial therapy (N = 13), diagnosed with sickle cell anemia (N = 13) 1313429 and Glucose6 Phosphate Dehydrogenase deficiency (G6PD) (N = 3).second nested PCR amplifying a 1,434 bp amplicon encompassing the entire pre-S/S gene [23]. In 6 HBsAg positive/HBV DNA unconfirmed samples a third nested-PCR was used to amplify a 276 bp fragment of the S gene [4]. The limit of detection (LOD) of the HBV qPCR assay was 10 IU/ml. The LODs for the heminested 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 1676428 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 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 realtime PCR targeting the Human Apoprotein B (HAPB) gene as described previously [24] to exclude the presence of potential amplification inhibitors.Ethics StatementApproval for the BOTIA repository and its use was obtained from the Kwame Nkrumah University of Science and Technology School of Medical Sciences committees for ethics and publication (Kumasi, Ghana). The BOTIA scientific committee approved the present study. Written informed consent was obtained from all participants prior to enrollment.Plasmodium DNA Detection with Species-specific Nested PCRsDNA was extracted from 200 ml red cell fractions using the QIAamp blood minikit (Qiagen) as per manufacturer’s instructions. All samples were tested twice, in duplicate using a genusspecific primer pair and four species-specific primer pairs (targeting the 18 s ribosomal DNA sequence of P.falciparum, P.vivax.
