Diagnostic performance of an ultra-sensitive RDT and a conventional RDT in malaria mass testing, treatment and tracking interventions in southern Ghana.

BACKGROUND: Application of numerous malaria control interventions has led to reduction in clinical malaria cases and deaths but also the realisation that asymptomatic parasite carriers play a key role in sustaining transmission. This study assessed the effectiveness of using the Ultra-sensitive NxTek eliminate RDT (uRDT) and conventional SD Bioline HRP2 RDT (cRDT) in diagnosing asymptomatic parasitaemia while measuring the impact of mass testing, treatment and tracking (MTTT) on the prevalence of asymptomatic malaria over a 1-year period in Ghana. METHODS: A total of 4000 targeted participants from two towns, Obom and Kofi Kwei, with their surrounding villages, were tested for asymptomatic malaria four times over the study period using uRDT (intervention) and the cRDT (control) respectively. Participants carrying malaria parasites were followed by home visit and phone calls for compliance to treatment, and filter paper blood blots collected from participants were used to determine true parasite carriage by PET-PCR. A mathematical model of the study site was developed and used to test the impact of test sensitivity and mass migration on the effect of MTTT. RESULTS: The start and end point sensitivities of the cRDT were 48.8% and 41.7% and those for the uRDT were 52.9% and 59.9% respectively. After a year of MTTTs, asymptomatic parasite prevalence, as determined by PCR, did not differ statistically in the control site (40.6% to 40.1%, P = 0.730) but decreased at the intervention site (55.9% to 46.4%, P < 0.0001). Parasite prevalence by RDT, however, indicated statistical reduction in the control site (25.3% to 22.3%, P = 0.017) and no change in the intervention site (35.1% to 36.0%, P = 0.614). The model predicted a mild effect of both diagnostic sensitivity and human movement in diminishing the impact of MTTT in the study sites. CONCLUSIONS: Asymptomatic parasite prevalence at the molecular level reduced significantly in the site where the uRDT was used but not where the cRDT was used. Overall, the uRDT exhibited higher sensitivity relative to the cRDT. Highly sensitive molecular techniques such as PET-PCR should be included in parasite prevalence estimation during MTTT exercises.

Biophysical Properties of Bifunctional Phage-Biosensor.

Biosensor research is a swiftly growing field for developing rapid and precise analytical devices for biomedical, pharmaceutical, and industrial use and beyond. Herein, we propose a phage-based biosensor method to develop a sensitive and specific system for biomedical detection. Our method is based on in vitro selected phages and their interaction with the targeted analytes as well as on optical properties that change according to the concentration of the model analyte. The green fluorescent protein (GFP) was chosen as our model analyte as it has its own well-known optical properties. Brilliant green was used as a reporter component for the sensor. Its presence enables a color intensity (absorbance) change when the analyte is present in the solution. Furthermore, the reporter dye functioned as a quencher for an additional lanthanide label in our assay. It mediated the specific phage-derived interference in the signal measured with the time-resolved luminescence. Most importantly, our results confirmed that the presented bifunctional phage with its liquid crystal properties enabled the measurement of GFP in a concentration-dependent, quantitative manner with a limit of detection of 0.24 µg/mL. In the future, our novel method to develop phage-based biosensors may provide highly sensitive and specific biosensors for biomedical or otherwise-relevant targets.

The Effectiveness of Varying Combination Ratios of A. cordifolia and M. indica against Field and Laboratory Strains of P. falciparum In Vitro.

BACKGROUND: Drug resistance in malaria is a global problem, with reports of Plasmodium parasites resistant to the current first-line antimalarial drug, artemisinin, expanding from Southeast Asia to Africa. There is therefore an urgent need to identify new drug candidates that will be effective against the existing malaria parasites. Drug combination therapy presents a myriad of advantages over monotherapy including delayed onset of resistance, potentiation, and synergism. This present study explored the effectiveness of combinations of aqueous extracts of Alchornea cordifolia (A. cordifolia) and Mangifera indica (M. indica) at clearing both laboratory and field isolates of P. falciparum. METHODS: Synchronized ring stage cultures of field (FA08) and laboratory strains (NF54 and CamWT_C580Y) of P. falciparum were subjected to combinations of different concentrations and ratios of aqueous extracts of A. cordifolia and M. indica. The growth inhibition of the individual plant extracts and their combinatory effects were studied in vitro using SYBR Green I drug assay. RESULTS: The A. cordifolia extract exhibited 50% inhibitory concentration (IC50) of 2.71, 7.80, and 3.56 µg/mL against the NF54, CamWT_C580Y, and FA08 parasite strains, respectively. Mangifera indica exhibited IC50 of 18.11, 20.08, and 10.23 µg/mL against the NF54, CamWT_C580Y, and FA08 parasite strains, respectively. Additive, synergistic and antagonistic interactions were observed at different combinations of A. cordifolia and M. indica extracts. CONCLUSION: A combination product containing A. cordifolia and M. indica has the potential to serve as an effective antimalarial as majority of the tested combinations of aqueous extracts of A. cordifolia and M. indica extracts exhibited synergistic effects in vitro against the NF54, CamWT_C580Y, and FA08 P. falciparum strains.

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.

Comparative analysis of asexual and sexual stage Plasmodium falciparum development in different red blood cell types.

BACKGROUND: Red blood cell (RBC) polymorphisms are suggested to influence the course of Plasmodium falciparum malaria. Whereas some variants have been found to be protective, others have been found to enhance parasite development. This study evaluated the effect of variant haemoglobin (Hb) and ABO blood groups on P. falciparum merozoite invasion, multiplication rates as well as gametocyte development. METHODS: Approximately 2.5 mL of venous blood was collected from each participant. Flow cytometry was used to determine the in vitro merozoite invasion rates of NF54 parasites into the blood of 66 non-parasitaemic individuals with variant Hb genotypes (HbSS, HbSC) and blood groups (A, B, O), which were then compared with invasion into HbAA blood. The ex vivo asexual parasite multiplication and gametocyte production rates of parasites from 79 uncomplicated malaria patients with varying Hb genotypes (HbAS, HbAC and HbAA) were also estimated using microscopy. RESULTS: Merozoite invasion rates were significantly reduced by about 50% in RBCs containing HbSS and HbSC relative to HbAA cells. The presence of blood group O and B reduced the invasion rates of HbSS by about 50% and 60%, respectively, relative to HbSC but the presence of blood group A removed the inhibitory effect of HbSS. The initial parasite densities in uncomplicated malaria patients with Hb genotypes HbAS and HbAC cells were similar but significantly lower than those with genotype HbAA. The ex vivo parasite multiplication rate, gametocytaemia and gametocyte conversion rates followed a similar trend but did not reach statistical significance (p > 0.05). CONCLUSIONS: Parasite invasion rate into erythrocytes is dependent on both erythrocyte blood group antigen and haemoglobin genotype as blood group O and B provided protection via reduced merozoite invasion in RBCs containing HbSS relative to HbSC. Regardless of haemoglobin type, greater than 70% malaria patients had circulating ring stage parasites that differentiated into stage II gametocytes in 4 days.

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.