Diagnostic performance of an ultrasensitive HRP2-based malaria rapid diagnostic test kit used in surveys of afebrile people living in Southern Ghana.

BACKGROUND: The Alere™ Malaria Ag P.f Ultra-sensitive RDT (UsmRDT) kit is an HRP2-based malaria rapid diagnostic test (RDT) with enhanced sensitivity relative to the SD Bioline Malaria Ag P.f RDT (mRDT) kit. However, the diagnostic performance of the UsmRDT kit has not been evaluated in Ghana. METHODS: A total of 740 afebrile participants aged between 3 and 88 years old were recruited from the Central and Greater Accra Regions of Ghana during the off-peak malaria season. Axillary body temperature was measured, and a volume of 1 ml venous blood was drawn from each participant. Prior to separating the blood into plasma and packed cell pellets via centrifugation, the blood was spotted onto one UsmRDT and one mRDT kit and also used to prepare thick and thin blood smears as well as filter paper blood spots. Plasmodium falciparum specific polymerase chain reaction (PCR) was performed on gDNA extracted from 100 µl of the whole blood. RESULTS: The overall positivity rate for microscopy, PCR, UsmRDT and mRDT kit were 20.4%, 40.8%, 31.3% and 30.8%, respectively. Overall, the UsmRDT identified 9.3% (28/302) more PCR positive samples than the mRDT kits. All samples that were negative by the UsmRDT kit were also negative by the mRDT kit. Overall, the sensitivity and specificity of the UsmRDT was 73% (221/302) and 89% (388/436), respectively, while that for the mRDT kit was 58% and 90%, respectively. CONCLUSION: Although the UsmRDT kit was not as sensitive as PCR at detecting asymptomatic P. falciparum carriage, it correctly identified P. falciparum in 9.3% of the study participants that were not captured by the mRDT kit. In malaria endemic settings, the UsmRDT would provide an added advantage by identifying more asymptomatic P. falciparum carriers than the mRDT kit for targeted treatment interventions.

The transcriptome of circulating sexually committed Plasmodium falciparum ring stage parasites forecasts malaria transmission potential.

Malaria is spread by the transmission of sexual stage parasites, called gametocytes. However, with Plasmodium falciparum, gametocytes can only be detected in peripheral blood when they are mature and transmissible to a mosquito, which complicates control efforts. Here, we identify the set of genes overexpressed in patient blood samples with high levels of gametocyte-committed ring stage parasites. Expression of all 18 genes is regulated by transcription factor AP2-G, which is required for gametocytogenesis. We select three genes, not expressed in mature gametocytes, to develop as biomarkers. All three biomarkers we validate in vitro using 6 different parasite lines and develop an algorithm that predicts gametocyte production in ex vivo samples and volunteer infection studies. The biomarkers are also sensitive enough to monitor gametocyte production in asymptomatic P. falciparum carriers allowing early detection and treatment of infectious reservoirs, as well as the in vivo analysis of factors that modulate sexual conversion.

The In Vitro Antiplasmodial Activities of Aqueous Extracts of Selected Ghanaian Herbal Plants.

BACKGROUND: The asexual and sexual stages (gametocytes) of Plasmodium falciparum parasites are known to respond differently to antimalarial drugs. Herbal products with extended treatment regimens and inadequate dosing information are widely used to treat malaria in Ghana. This study set out to determine the in vitro activity of selected herbal extracts on the development of asexual and sexual stage malaria parasites. METHODS: The 72-hour SYBR Green 1-based in vitro drug assay was used to determine the asexual parasite growth inhibitory effects exhibited by aqueous extracts of Alchornea cordifolia, Polyalthia longifolia, Moringa oleifera, and Mangifera indica on the NF54, CamWT_C580Y, and IPC 4912 strains of Plasmodium falciparum. The effects of exposure of asexual and early-stage NF54 gametocytes to varying concentrations of the aqueous herbal extracts were assessed by microscopy after 7 days of continuous culturing in the presence of the herbal extract. Qualitative and quantitative phytochemical screening were also performed on the herbal extracts. RESULTS: In the SYBR Green 1 assay, aqueous extracts of Alchornea cordifolia exhibited moderate (IC50 of 5.8, 17.4, and 15.8 µg/ml) and Mangifera indica exhibited low (IC50 of 65.4, 96.7, and 81.7 µg/ml) activities against the NF54, Cam WT_C580Y, and IPC 4912 parasites, respectively, whilst Polyalthia longifolia and Moringa oleifera were inactive. Long-term treatment of NF54 parasites with 1 mg/ml of Polyalthia longifolia produced the highest densities of gametocytes and the least (56%) inhibition of asexual parasites on Day 7. Long-term treatment of NF54 parasites with 10 µg/ml Alchornea cordifolia resulted in complete parasite (asexual and gametocyte) clearance on Day 7. Conclusions. Alchornea cordifolia exhibited a 'moderate' activity against the three parasites tested in the 72-hour SYBR Green 1 assay and also effectively cleared both asexual parasites and gametocytes. Long-term treatment of malaria parasites with herbal extracts mimics a treatment regimen and should be used to determine the antimalarial properties of herbal extracts.

Asymptomatic carriage of Plasmodium falciparum by individuals with variant blood groups and haemoglobin genotypes in southern Ghana.

BACKGROUND: The ABO and the Rhesus blood group systems, as well as various abnormal haemoglobin (Hb) variants (haemoglobinopathies) are known to influence malaria parasite carriage and disease severity in individuals living in malaria endemic areas. This study identified the blood group and Hb variant distribution and Plasmodium falciparum infection status of afebrile individuals living in southern Ghana. METHODS: Afebrile participants were recruited from Obom (358) in the Greater Accra Region and Ewim (100) and Simiw (329) in the Central Region of Ghana. Venous blood (1 ml) was collected into EDTA vacutainer tubes. Three 20 µl drops of blood were used for blood group analysis using the tile method. Another 500 µl aliquot was used for the qualitative sickling test using sodium metabisulphite and haemoglobin electrophoresis. Genomic DNA was extracted from 100 µl of whole blood and used in P. falciparum species-specific PCR. RESULTS: The most abundant blood group and abnormal haemoglobin variant in both sites was blood group O + (47.4%) and HbAS (15.8%). A total of 13 (1.7%) of the participants had full haemoglobinopathies (SS, SC and CC), whilst 196 (25.4%) were carriers (AS and AC). Although there was a significantly higher prevalence of sickling positive participants from the Central Region, genotyping identified a similar prevalence of each of the abnormal haemoglobin genes in both sites. Asymptomatic parasite carriage estimated by PCR was 40.9% in the Central Region and 41.8% in the Greater Accra Region. CONCLUSIONS: Asymptomatic carriage of P. falciparum parasite in the study population was not associated with any particular blood group variant or haemoglobin genotype.

Publisher Correction: Plasmodium falciparum sexual differentiation in malaria patients is associated with host factors and GDV1-dependent genes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Plasmodium falciparum sexual differentiation in malaria patients is associated with host factors and GDV1-dependent genes.

Plasmodium sexual differentiation is required for malaria transmission, yet much remains unknown about its regulation. Here, we quantify early gametocyte-committed ring (gc-ring) stage, P. falciparum parasites in 260 uncomplicated malaria patient blood samples 10 days before maturation to transmissible stage V gametocytes using a gametocyte conversion assay (GCA). Seventy six percent of the samples have gc-rings, but the ratio of gametocyte to asexual-committed rings (GCR) varies widely (0-78%). GCR correlates positively with parasitemia and is negatively influenced by fever, not hematocrit, age or leukocyte counts. Higher expression levels of GDV1-dependent genes, ap2-g, msrp1 and gexp5, as well as a gdv1 allele encoding H217 are associated with high GCR, while high plasma lysophosphatidylcholine levels are associated with low GCR in the second study year. The results provide a view of sexual differentiation in the field and suggest key regulatory roles for clinical factors and gdv1 in gametocytogenesis in vivo.

Variations in the quality of malaria-specific antibodies with transmission intensity in a seasonal malaria transmission area of Northern Ghana.

INTRODUCTION: Plasmodium falciparum induced antibodies are key components of anti-malarial immunity in malaria endemic areas, but their antigen targets can be polymorphic. Induction of a high proportion of strain-specific antibodies will limit the recognition of a broad diversity of parasite strains by these responses. There are indications that circulating parasite diversity varies with malaria transmission intensity, and this may affect the specificity of elicited anti-malarial antibodies. This study therefore assessed the effect of varying malaria transmission patterns on the specificity of elicited antibody responses and to identify possible antibody correlates of naturally acquired immunity to malaria in children in an area of Ghana with seasonal malaria transmission. METHODS: This retrospective study utilized plasma samples collected longitudinally at six time points from children aged one to five years. Multiplex assays were used to measure antibody levels against four P. falciparum AMA 1 variants (from the 3D7, FVO, HB3 and CAMP parasite strains) and the 3D7 variant of the EBA 175 region II antigen and the levels compared between symptomatic and asymptomatic children. The relative proportions of cross-reactive and strain-specific antibodies against the four AMA 1 variants per sampling time point were assessed by Bland-Altman plots. The levels of antibodies against allelic AMA1 variants, measured by singleplex and multiplex luminex assays, were also compared. RESULTS: The data show that increased transmission intensity is associated with higher levels of cross-reactive antibody responses, most likely a result of a greater proportion of multiple parasite clone infections during the high transmission period. Anti-AMA1 antibodies were however associated with a history of infection rather than protection in this age group. CONCLUSION: The data contribute to understanding the underlying mechanism of the acquisition of strain-transcending antibody immunity following repeated exposure to diverse parasite strains.