ABSTRACT
BACKGROUND: Malaria and HIV, 2 of the world's deadliest diseases, share a lot of territory in sub-Saharan Africa. OBJECTIVE: This study seeks to investigate the effect of HIV on the immune response to malaria infection among pregnant women in Kumba in the southwest region (SWR) of Cameroon. The study aims to determine the prevalence of malaria infection, assess the occurrence of Plasmodium falciparum genetic diversity, and evaluate the antibody (immunoglobulin [Ig]G and IgM: apical membrane antigen-1 [AMA1], merozoite surface protein [MSP]1, MSP2, MSP3, and erythrocyte-binding antigen [EBA]175) and cytokine (interleukin [IL]-10, tumor necrosis factor alpha [TNF-α], and interferon gamma [IFNγ]) response to malaria infection among pregnant women with and without HIV in Kumba. METHODS: The study will be a hospital-based cross-sectional design that will run from March 2022 to February 2023. It will recruit pregnant women with and without HIV who are in their third trimester of pregnancy. The study will be carried out in 5 health institutions in Kumba: General Hospital Kumba, Presbyterian Hospital Kumba, District Hospital Kumba-town, Kossala Integrated Health Center Kumba, and Catholic Hospital Kumba. About 3 mL of the mother's venous blood, placental blood, and baby cord blood will be collected from each pregnant women at the point of delivery. Microscopy, rapid diagnostic tests (RDTs), and nested polymerase chain reaction (PCR) will be performed to identify the malaria parasite in all the samples, and nested PCR targeting the different genetic diversity markers for P. falciparum will also be performed. Furthermore, sequencing will be performed to study the nucleotide sequence of different alleles, and the genetic diversity of the alleles responsible for malaria infection among pregnant women will be assessed. A multiplex assay will be conducted to analyze the peripheral blood plasma and cord blood plasma for the cytokine and total antibody response to malaria infection among pregnant women with and without HIV. The questionnaire for data collection will be pretested at the Kumba District Hospital, and ethical clearance will be obtained from the University of Buea and the Regional Delegation of Public Health for the SWR. Data will be analyzed using SPSS Statistics and STATA. All P values <.05 will be considered statistically significant. BioEdit 7.0.0 software will be used to align the nucleotide sequences of different genes after sequencing. Phylogenetic tree searching will be conducted using the maximum-likelihood (ML) method in MEGA V6.0. RESULTS: The project started in March 2022 and will end in February 2023. Presently, three-fourth of the project funding has been disbursed to date. A total of 218 participants have been enrolled: 193 (88.5%) women without HIV and 25 (11.5%) women with HIV. Between February 2023 and March 2024, the following results will be ready for publication: maternal-neonatal malaria prevalence among pregnant women and babies in Kumba, the effect of HIV on (1) P. falciparum genetic diversity among pregnant women in Kumba, (2) the maternal and neonatal immune response to MSP1, MSP2, and EBA175 IgG antibody response to P. falciparum-caused malaria infection among pregnant women, and (3) the maternal and neonatal pro-inflammatory and anti-inflammatory cytokine response to malaria infection. CONCLUSIONS: HIV infection increases the prevalence of malaria infection among pregnant women and also influences the genetic diversity of P. falciparum, with MSP1 alleles being the most prevalent. HIV infection also reduces the antibody response to malaria infection, as well as altering the level of pro-inflammatory and anti-inflammatory responses to malaria infection. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/38213.
ABSTRACT
BACKGROUND: Sampling of saliva for diagnosing Plasmodium falciparum infections is a safe, non-invasive alternative to sampling of blood. However, the use of saliva presents a challenge because lower concentrations of parasite DNA are present in saliva compared to peripheral blood. Therefore, a sensitive method is needed for detection of parasite DNA in saliva. This study utilized two recently reported "ultra-sensitive" PCR assays based on detection of the P. falciparum mitochondrial cox3 gene and the multi-copy nuclear varATS gene. The ultra-sensitive assays have an advantage over standard 18S rRNA gene-based PCR assay as they target genes with higher copy numbers per parasite genome. Stored saliva DNA samples from 60 Cameroonian individuals with infections previously confirmed by 18S rRNA gene PCR in peripheral blood were tested with assays targeting the cox3 and varATS genes. RESULTS: Overall, the standard 18S rRNA gene-based PCR assay detected P. falciparum DNA in 62% of the stored saliva DNA samples, whereas 77 and 68% of the samples were positive with assays that target the cox3 and varATS genes, respectively. Interestingly, the ultra-sensitive assays detected more P. falciparum infections in stored saliva samples than were originally detected by thick-film microscopy (41/60 = 68%). When stratified by number of parasites in the blood, the cox3 assay successfully detected more than 90% of infections using saliva when individuals had > 1000 parasites/µl of peripheral blood, but sensitivity was reduced at submicroscopic parasitemia levels. Bands on electrophoresis gels were distinct for the cox3 assay, whereas faint or non-specific bands were sometimes observed for varATS and 18S rRNA that made interpretation of results difficult. Assays could be completed in 3.5 and 3 h for the cox3 and varATS assays, respectively, whereas the 18S rRNA gene assays required at least 7 h. CONCLUSIONS: This study demonstrates that a PCR assay targeting the cox3 gene detected P. falciparum DNA in more saliva samples than primers for the 18S rRNA gene. Non-invasive collection of saliva in combination with the proposed cox3 primer-based PCR assay could potentially enhance routine testing of P. falciparum during disease surveillance, monitoring, and evaluation of interventions for malaria elimination.
