Plasmodium falciparum AMA1 and CSP antigen diversity in parasite isolates from southern Ghana.

Introduction: Diversity in malarial antigens is an immune evasion mechanism that gives malaria parasites an edge over the host. Immune responses against one variant of a polymorphic antigen are usually not fully effective against other variants due to altered epitopes. This study aimed to evaluate diversity in the Plasmodium falciparum antigens apical membrane antigen 1 (PfAMA1) and circumsporozoite protein (PfCSP) from circulating parasites in a malaria-endemic community in southern Ghana and to determine the effects of polymorphisms on antibody response specificity. Methods: The study involved 300 subjects, whose P. falciparum infection status was determined by microscopy and PCR. Diversity within the two antigens was evaluated by msp2 gene typing and molecular gene sequencing, while the host plasma levels of antibodies against PfAMA1, PfCSP, and two synthetic 24mer peptides from the conserved central repeat region of PfCSP, were measured by ELISA. Results: Of the 300 subjects, 171 (57%) had P. falciparum infection, with 165 of the 171 (96.5%) being positive for either or both of the msp2 allelic families. Gene sequencing of DNA from 55 clonally infected samples identified a total of 56 non-synonymous single nucleotide polymorphisms (SNPs) for the Pfama1 gene and these resulted in 44 polymorphic positions, including two novel positions (363 and 365). Sequencing of the Pfcsp gene from 69 clonal DNA samples identified 50 non-synonymous SNPs that resulted in 42 polymorphic positions, with half (21) of these polymorphic positions being novel. Of the measured antibodies, only anti-PfCSP antibodies varied considerably between PCR parasite-positive and parasite-negative persons. Discussion: These data confirm the presence of a considerable amount of unique, previously unreported amino acid changes, especially within PfCSP. Drivers for this diversity in the Pfcsp gene do not immediately seem apparent, as immune pressure will be expected to drive a similar level of diversity in the Pfama1 gene.

Non-Coding RNAs Potentially Involved in Pyrethroid Resistance of Anopheles funestus Population in Western Kenya.

Backgrounds: The resurgence of Anopheles funestus, a dominant vector of human malaria in western Kenya was partly attributed to insecticide resistance. However, evidence on the molecular basis of pyrethroid resistance in western Kenya is limited. Noncoding RNAs (ncRNAs) form a vast class of RNAs that do not code for proteins and are ubiquitous in the insect genome. Here, we demonstrated that multiple ncRNAs could play a potential role in An. funestusresistance to pyrethroid in western Kenya. Materials and Methods: Anopheles funestus mosquitoes were sampled by aspiration methods in Bungoma, Teso, Siaya, Port Victoria and Kombewa in western Kenya. The F1 progenies were exposed to deltamethrin (0.05%), permethrin (0.75%), DDT (4%) and pirimiphos-methyl (0.25%) following WHO test guidelines. A synergist assay using piperonyl butoxide (PBO) (4%) was conducted to determine cytochrome P450s' role in pyrethroid resistance. RNA-seq was conducted on a combined pool of specimens that were resistant and unexposed, and the results were compared with those of the FANG susceptible strain. This approach aimed to uncover the molecular mechanisms underlying pyrethroid resistance. Results: Pyrethroid resistance was observed in all the sites with an average mortality rate of 57.6%. Port Victoria had the highest level of resistance to permethrin (MR=53%) and deltamethrin (MR=11%) pyrethroids. Teso had the lowest level of resistance to permethrin (MR=70%) and deltamethrin (MR=87%). Resistance to DDT was observed only in Kombewa (MR=89%) and Port Victoria (MR=85%). A full susceptibility to P-methyl (0.25%) was observed in all the sites. PBO synergist assay revealed high susceptibility (>98%) to the pyrethroids in all the sites except for Port Victoria (MR=96%, n=100). Whole transcriptomic analysis showed that most of the gene families associated with pyrethroid resistance comprised non-coding RNAs (67%), followed by imipenemase (10%), cytochrome P450s (6%), cuticular proteins (5%), olfactory proteins (4%), glutathione S-transferases (3%), UDP-glycosyltransferases (2%), ATP-binding cassettes (2%) and carboxylesterases(1%). Conclusions: This study unveils the molecular basis of insecticide resistance in An. funestus in western Kenya, highlighting for the first time the potential role of non-coding RNAs in pyrethroid resistance. Targeting non-coding RNAs for intervention development could help in insecticide resistance management.

High prevalence of impaired glucose metabolism among children and adolescents living with HIV in Ghana.

BACKGROUND: Antiretroviral therapy (ART)-associated metabolic abnormalities, including impairment of glucose metabolism, are prevalent in adults living with HIV. However, the prevalence and pathogenesis of impaired glucose metabolism in children and adolescents living with HIV, particularly in sub-Saharan Africa, are not well characterized. We investigated the prevalence of impaired glucose metabolism among children and adolescents living with perinatally infected HIV in Ghana. METHODS: In this multicentre, cross-sectional study, we recruited participants from 10 paediatric antiretroviral treatment clinics from January to June 2022 in 10 facilities in Greater Accra and Eastern regions of Ghana. We determined impaired glucose metabolism in the study sample by assessing fasting blood sugar (FBS), insulin resistance as defined by the homeostatic model assessment for insulin resistance (HOMA-IR) index and glycated haemoglobin (HbA1c) levels. The prevalence of impaired glucose metabolism using each criterion was stratified by age and sex. The phenotypic correlates of glucose metabolism markers were also assessed among age, sex, body mass index (BMI) and waist-to-hip ratio (WHR). RESULTS: We analysed data from 393 children and adolescents living with HIV aged 6-18 years. A little over half (205/393 or 52.25%) of the children were female. The mean age of the participants was 11.60 years (SD = 3.50), with 122/393 (31.00%) aged 6-9 years, 207/393 (52.67%) aged 10-15 years, and 62/393 (15.78%) aged 16-18 years. The prevalence rates of glucose impairment in the study population were 15.52% [95% confidence interval (CI): 12.26-19.45], 22.39% (95% CI: 18.54-26.78), and 26.21% (95% CI: 22.10-30.78) using HbA1c, HOMA-IR, and FBS criteria, respectively. Impaired glucose metabolism detected by FBS and HOMA-IR was higher in the older age group, whereas the prevalence of abnormal HbA1c levels was highest among the youngest age group. Age and BMI were positively associated with FBS and HOMA-IR (p < 0.001). However, there was negative correlation of WHR with HOMA-IR (p < 0.01) and HbA1c (p = 0.01). CONCLUSION: The high prevalence of impaired glucose metabolism observed among the children and adolescents living with HIV in sub-Saharan Africa is of concern as this could contribute to the development of metabolic syndrome in adulthood.

Genetic Diversity and Population Structure of Anopheles funestus in Western Kenya Based on Mitochondrial DNA Marker COII.

The mitochondrial marker, COII, was employed to assess the genetic structure and diversity of Anopheles funestus, a very important malaria vector in Africa that adapt and colonize different ecological niches in western Kenya. Mosquitoes were collected using mechanical aspirators in four areas (Bungoma, Port Victoria, Kombewa, and Migori) in western Kenya. Following morphological identification, PCR was used to confirm the species. The COII gene was amplified, sequenced, and analyzed to determine genetic diversity and population structure. A total of 126 (Port Victoria-38, Migori-38, Bungoma-22, and Kombewa-28) sequences of COII were used for population genetic analysis. Anopheles funestus had a high haplotype diversity (Hd = 0.97 to 0.98) but low nucleotide diversity (Π = 0.004 to 0.005). The neutrality test revealed negative Tajima's D and Fs values indicating an excess of low-frequency variation. This could be attributed to either population expansion or negative selection pressure across all the populations. No genetic or structural differentiation (Fst = -0.01) and a high level of gene flow (Gamma St, Nm = 17.99 to 35.22) were observed among the populations. Population expansion suggests the high adaptability of this species to various ecological requirements, hence sustaining its vectorial capacity and malaria transmission.

Nationwide Surveillance of Pfhrp2 Exon 2 Diversity in Plasmodium falciparum Circulating in Symptomatic Malaria Patients Living in Ghana.

Reports of increasing false-negative HRP2-based rapid diagnostic test results across Africa require constant monitoring of factors associated with these false-negative outcomes, as failure of this diagnostic tool will have severe consequences on malaria treatment and control programs. This study characterized the extent of genetic diversity in the Plasmodium falciparum histidine-rich protein 2 (Pfhrp2) gene in P. falciparum isolates from symptomatic malaria patients across the regions of Ghana. Exon 2 of Pfhrp2 was amplified from gDNA using polymerase chain reaction. All Pfhrp2-negative samples were subjected to Pf18S rRNA and Pfmsp2 gene amplifications. The amplified Pfhrp2 exon 2 fragments from clonal samples were sent for commercial Sanger sequencing. The type and number of PfHRP2 repeats, classified based on repeat types previously reported, were estimated from the sequence data and compared among geographical regions. About 81% (2,333/2,890) of the original microscopy positive DBS were available and used in this study. The Pfhrp2 exon 2 amplification was successful in 98.5% (2,297/2,333) of the tested samples, with band size ranging from 400 bp to 1,050 bp. A total of 13 out of the 24 previously reported repeat types were identified among the samples, with three samples lacking both type 2 and type 7 repeat motifs. This study suggested that the genetic diversity of Pfhrp2 exon 2 identified in P. falciparum circulating in symptomatic malaria patients in Ghana is unlikely to influence the sensitivity and specificity of HRP2 RDT-based diagnosis.

Association of TNF-Alpha, MBL2, NOS2, and G6PD with Malaria Outcomes in People in Southern Ghana.

Background: One major issue that has set back the gains of the numerous malaria control interventions that national malaria control programs have implemented is asymptomatic malaria. Certain host genetic factors are known to influence symptomatic malaria; however, not much is known about how host genetics influences the acquisition of asymptomatic malaria. Methods: Genomic DNA was extracted from whole blood collected from 60 symptomatic and 149 nonfebrile (asymptomatic, N = 109, and uninfected, N = 40) volunteers aged between 2 and 69 years from a high (Obom) and a low (Asutsuare) malaria transmission setting in Southern Ghana. Restriction fragment length polymorphism (RFLP) was used to determine polymorphisms at the MBL2 54, TNF-α 308, NOS2 954, and G6PD 202/376 gene loci. Results: Polymorphisms at the MBL2 54 and TNF-α 308 loci were significantly different amongst the three categories of volunteers in both Asutsuare (p = 0.006) and Obom (p=0.05). In Asutsuare, a low malaria transmission area, the allele G has significantly higher odds (3.15) of supporting asymptomatic malaria as against symptomatic malaria. There were significantly higher odds of TNF-α genotype GA being associated with symptomatic malaria as against asymptomatic malaria in both sites, Obom (p=0.027) and Asutsuare (p=0.027). The allele B of the G6PD gene was more prevalent in symptomatic rather than asymptomatic parasite-infected individuals in both Obom (p=0.001) and Asutsuare (p=0.003). Conclusion: Individuals in Southern Ghana carrying the TNF-α 308 GA genotype are more likely to exhibit symptoms of malaria when infected with the malaria parasite as opposed to harboring an asymptomatic infection. Also, the B allele of the G6PD gene is likely to prevent a P. falciparum-infected person from exhibiting symptoms and thereby promote asymptomatic parasite carriage.

Detecting asymptomatic carriage of Plasmodium falciparum in southern Ghana: utility of molecular and serological diagnostic tools.

BACKGROUND: Asymptomatic malaria infections can serve as potential reservoirs for malaria transmission. The density of parasites contained in these infections range from microscopic to submicroscopic densities, making the accurate detection of asymptomatic parasite carriage highly dependent on the sensitivity of the tools used for the diagnosis. This study sought to evaluate the sensitivities of a variety of molecular and serological diagnostic tools at determining the prevalence of asymptomatic Plasmodium falciparum parasite infections in two communities with varying malaria parasite prevalence. METHODS: Whole blood was collected from 194 afebrile participants aged between 6 and 70 years old living in a high (Obom) and a low (Asutsuare) malaria transmission setting of Ghana. Thick and thin blood smears, HRP2 based malaria rapid diagnostic test (RDT) and filter paper dried blood spots (DBS) were prepared from each blood sample. Genomic DNA was extracted from the remaining blood and used in Plasmodium specific photo-induced electron transfer polymerase chain reaction (PET-PCR) and Nested PCR, whilst the HRP2 antigen content of the DBS was estimated using a bead immunoassay. A comparison of malaria parasite prevalence as determined by each method was performed. RESULTS: Parasite prevalence in the high transmission site of Obom was estimated at 71.4%, 61.9%, 60%, 37.8% and 19.1% by Nested PCR, the HRP2 bead assay, PET-PCR, HRP2-RDT and microscopy respectively. Parasite prevalence in the low transmission site of Asutsuare was estimated at 50.1%, 11.2%, 5.6%, 0% and 2.2% by Nested PCR, the HRP2 bead assay, PET-PCR, RDT and microscopy, respectively. The diagnostic performance of Nested PCR, PET-PCR and the HRP2 bead assay was similar in Obom but in Asutsuare, Nested PCR had a significantly higher sensitivity than PET-PCR and the HRP2 bead assay, which had similar sensitivity. CONCLUSIONS: Nested PCR exhibited the highest sensitivity by identifying the highest prevalence of asymptomatic P. falciparum in both the high and low parasite prevalence settings. However, parasite prevalence estimated by the HRP2 bead assay and PET-PCR had the highest level of inter-rater agreement relative to all the other tools tested and have the advantage of requiring fewer processing steps relative to Nested PCR and producing quantitative results.

Nationwide molecular surveillance of three Plasmodium species harboured by symptomatic malaria patients living in Ghana.

BACKGROUND: Clinical presentations of malaria in Ghana are primarily caused by infections containing microscopic densities of Plasmodium falciparum, with a minor contribution from Plasmodium malariae and Plasmodium ovale. However, infections containing submicroscopic parasite densities can result in clinical disease. In this study, we used PCR to determine the prevalence of three human malaria parasite species harboured by suspected malaria patients attending healthcare facilities across the country. METHODS: Archived dried blood spots on filter paper that had been prepared from whole blood collected from 5260 patients with suspected malaria attending healthcare facilities across the country in 2018 were used as experimental material. Plasmodium species-specific PCR was performed on DNA extracted from the dried blood spots. Demographic data and microscopy data for the subset of samples tested were available from the original study on these specimens. RESULTS: The overall frequency of P. falciparum, P. malariae and P. ovale detected by PCR was 74.9, 1.4 and 0.9%, respectively. Of the suspected symptomatic P. falciparum malaria cases, 33.5% contained submicroscopic densities of parasites. For all regions, molecular diagnosis of P. falciparum, P. malariae and P. ovale was significantly higher than diagnosis using microscopy: up to 98.7% (75/76) of P. malariae and 97.8% (45/46) of P. ovale infections detected by PCR were missed by microscopy. CONCLUSION: Plasmodium malariae and P. ovale contributed to clinical malaria infections, with children aged between 5 and 15 years harbouring a higher frequency of P. falciparum and P. ovale, whilst P. malariae was more predominant in individuals aged between 10 and 20 years. More sensitive point-of-care tools are needed to detect the presence of low-density (submicroscopic) Plasmodium infections, which may be responsible for symptomatic infections.

Molecular Epidemiology of HIV-1 in Ghana: Subtype Distribution, Drug Resistance and Coreceptor Usage.

The greatest HIV-1 genetic diversity is found in West/Central Africa due to the pandemic's origins in this region, but this diversity remains understudied. We characterized HIV-1 subtype diversity (from both sub-genomic and full-genome viral sequences), drug resistance and coreceptor usage in 103 predominantly (90%) antiretroviral-naive individuals living with HIV-1 in Ghana. Full-genome HIV-1 subtyping confirmed the circulating recombinant form CRF02_AG as the dominant (53.9%) subtype in the region, with the complex recombinant 06_cpx (4%) present as well. Unique recombinants, most of which were mosaics containing CRF02_AG and/or 06_cpx, made up 37% of sequences, while "pure" subtypes were rare (<6%). Pretreatment resistance to at least one drug class was observed in 17% of the cohort, with NNRTI resistance being the most common (12%) and INSTI resistance being relatively rare (2%). CXCR4-using HIV-1 sequences were identified in 23% of participants. Overall, our findings advance our understanding of HIV-1 molecular epidemiology in Ghana. Extensive HIV-1 genetic diversity in the region appears to be fueling the ongoing creation of novel recombinants, the majority CRF02_AG-containing, in the region. The relatively high prevalence of pretreatment NNRTI resistance but low prevalence of INSTI resistance supports the use of INSTI-based first-line regimens in Ghana.

Profiling the Quality and Quantity of Naturally Induced Antibody Responses Against Pfs230 and Pfs48/45 Among Non-Febrile Children Living in Southern Ghana: A Longitudinal Study.

A clear understanding of the properties of naturally induced antibody responses against transmission-blocking vaccine candidates can accelerate the understanding of the development of transmission-blocking immunity. This study characterized the naturally induced IgG responses against two leading transmission-blocking vaccine antigens, Pfs230 and Pfs48/45, in non-febrile children living in Simiw, Ghana. Consecutive sampling was used to recruit 84 non-febrile children aged from 6 to 12 years old into the 6-month (November 2017 until May 2018) longitudinal study. Venous blood (1 ml) was collected once every 2 months and used to determine hemoglobin levels, P. falciparum prevalence using microscopy and polymerase chain reaction, and the levels and relative avidity of IgG responses against Pfs230 and Pfs48/45 using indirect ELISA. IgG levels against Pfs230 and Pfs48/45 decreased from the start (November) to the middle (January) and end (March) of the dry season respectively, then they began to increase. Participants, especially older children (10-12 years old) with active infections generally had lower antibody levels against both antigens. The relative avidities of IgG against both antigens followed the trend of IgG levels until the middle of the dry season, after which the relative avidities of both antigens correlated inversely with the antibody levels. In conclusion, although IgG antibody levels against both Pfs48/45 and Pfs230 began to increase by the early rainy season, they were inversely correlated to their respective relative avidities.

Persistent Plasmodium falciparum infections enhance transmission-reducing immunity development.

Subclinical infections that serve as reservoir populations to drive transmission remain a hurdle to malaria control. Data on infection dynamics in a geographical area is required to strategically design and implement malaria interventions. In a longitudinal cohort, we monitored Plasmodium falciparum infection prevalence and persistence, and anti-parasite immunity to gametocyte and asexual antigens for 10 weeks. Of the 100 participants, only 11 were never infected, whilst 16 had persistent infections detected by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), and one participant had microscopic parasites at all visits. Over 70% of the participants were infected three or more times, and submicroscopic gametocyte prevalence was high, ≥ 48% of the parasite carriers. Naturally induced responses against recombinant Pfs48/45.6C, Pfs230proC, and EBA175RIII-V antigens were not associated with either infection status or gametocyte carriage, but the antigen-specific IgG titers inversely correlated with parasite and gametocyte densities consistent with partial immunity. Longitudinal analysis of gametocyte diversity indicated at least four distinct clones circulated throughout the study period. The high prevalence of children infected with distinct gametocyte clones coupled with marked variation in infection status at the individual level suggests ongoing transmission and should be targeted in malaria control programs.

Larval ecology and bionomics of Anopheles funestus in highland and lowland sites in western Kenya.

BACKGROUND: An. funestus is a major Afrotropical vector of human malaria. This study sought to investigate the larval ecology, sporozoite infection rates and blood meal sources of An. funestus in western Kenya. METHODS: Larval surveys were carried out in Bungoma (Highland) and Kombewa (lowland) of western Kenya. Aquatic habitats were identified, characterized, georeferenced and carefully examined for mosquito larvae and predators. Indoor resting mosquitoes were sampled using pyrethrum spray catches. Adults and larvae were morphologically and molecularly identified to species. Sporozoite infections and blood meal sources were detected using real-time PCR and ELISA respectively. RESULTS: Of the 151 aquatic habitats assessed, 62/80 (78%) in Bungoma and 58/71(82%) in Kombewa were positive for mosquito larvae. Of the 3,193 larvae sampled, An. funestus larvae constitute 38% (1224/3193). Bungoma recorded a higher number of An. funestus larvae (85%, 95%, CI, 8.722-17.15) than Kombewa (15%, 95%, CI, 1.33-3.91). Molecular identification of larvae showed that 89% (n = 80) were An. funestus. Approximately 59%, 35% and 5% of An. funestus larvae co-existed with An. gambiae s.l, Culex spp and An. coustani in the same habitats respectively. Of 1,221 An. funestus s.l adults sampled, molecular identifications revealed that An. funestus constituted 87% (n = 201) and 88% (n = 179) in Bungoma and Kombewa, respectively. The Plasmodium falciparum sporozoite rate of An. funestus in Bungoma and Kombewa was 2% (3/174) and 1% (2/157), respectively, and the human blood index of An. funestus was 84% (48/57) and 89% (39/44) and for Bungoma and Kombewa, respectively. CONCLUSION: Man-made ponds had the highest abundance of An. funestus larvae. Multiple regression and principal component analyses identified the distance to the nearest house as the key environmental factor associated with the abundance of An. funestus larvae in aquatic habitats. This study serves as a guide for the control of An. funestus and other mosquito species to complement existing vector control strategies.

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 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.

Large Variations in Malaria Parasite Carriage by Afebrile School Children Living in Nearby Communities in the Central Region of Ghana.

BACKGROUND: Indicators of successful malaria control interventions include a reduction in the prevalence and densities of malaria parasites contained in both symptomatic and asymptomatic infections as well as a reduction in malaria transmission. Individuals harboring malaria parasites in asymptomatic infections serve as reservoirs for malaria transmission. This study determined the prevalence of asymptomatic malaria parasite carriage in afebrile children attending six different schools in two districts, the Cape Coast Metropolitan Assembly (CCMA) and the Komenda Edina Eguafo Abirem (KEEA) of the Central Region of Ghana. METHODS: This cross sectional study recruited afebrile children aged between 3 and 15 years old from six randomly selected schools in the Central Region of Ghana. Finger-pricked blood was collected and used to prepare thick and thin blood smears as well as spot a strip of filter paper (Whatman #3). Nested PCR was used to identify Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax in DNA extracted from the filter paper spots. The multiplicity of P. falciparum infection was determined using merozoite surface protein 2 genotyping. RESULTS: Out of the 528 children sampled, PCR identified 27.1% to harbor Plasmodium parasites in asymptomatic infections, whilst microscopy identified malaria parasites in 10.6% of the children. The overall PCR estimated prevalence of P. falciparum and P. malariae was 26.6% and 1.3%, respectively, with no P. ovale or P. vivax identified by PCR or microscopy. The RDT positivity rate ranged from 55.8% in Simiw to 4.5% in Kuful. Children from the Simiw Basic School accounted for 87.5% of all the asymptomatic infections. The multiplicity of P. falciparum infection was predominantly monoclonal and biclonal. CONCLUSIONS: The low prevalence of asymptomatic malaria parasite carriage by the children living in the Cape Coast Metropolis suggests that the malaria control interventions in place in CCMA are highly effective and that additional malaria control interventions are required for the KEEA district to reduce the prevalence of asymptomatic malaria parasite carriers. No molecular evidence of P. ovale and P. vivax was identified in the afebrile children sampled from the selected schools.

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.