Prevalence and Characteristics of Plasmodium vivax Gametocytes in Duffy-Positive and Duffy-Negative Populations across Ethiopia.

Plasmodium parasites replicate asexually in human hosts. The proportion of infections that carries gametocytes is a proxy for human-to-mosquito transmissibility. It is unclear which proportion of Plasmodium vivax infections in Duffy-negative populations carries gametocytes. We determined the prevalence and characteristics of P. vivax gametocytes in Duffy-positive and -negative populations across broad regions of Ethiopia. Finger-prick blood samples were collected for microscopic and molecular screening of Plasmodium parasites and Duffy status of individuals. Molecular screening of Plasmodium species and Duffy blood group genotyping was done using SYBR green and the Taqman quantitative polymerase chain reaction method. Of the 447 febrile patients who were shown to be P. vivax smear positive, 414 (92.6%) were confirmed by molecular screening as P. vivax and 16 (3.9%) of them were from Duffy-negative individuals. Of these, 5 of 16 (31.3%) Duffy-negative P. vivax-infected samples were detected with gametocytes. Of the 398 Duffy-positive P. vivax-infected samples, 150 (37.7%) were detected with gametocytes, slightly greater than that in Duffy-negative samples. This study highlights the presence of P. vivax gametocytes in Duffy-negative infections, suggestive of human-to-mosquito transmissibility. Although P. vivax infections in Duffy-negative individuals were commonly associated with low parasitemia, some of these infections were shown to have relatively high parasitemia and may represent a prominent erythrocyte invasion capability of P. vivax, and hidden reservoirs that can contribute to transmission. A better understanding of P. vivax transmission biology and gametocyte function particularly in Duffy-negative populations would aid future treatment and management of P. vivax malaria in Africa.

Epidemiology of Plasmodium vivax in Duffy negatives and Duffy positives from community and health centre collections in Ethiopia.

BACKGROUND: Malaria remains a significant cause of morbidity and mortality in Ethiopia with an estimated 3.8 million cases in 2021 and 61% of the population living in areas at risk of malaria transmission. Throughout the country Plasmodium vivax and Plasmodium falciparum are co-endemic, and Duffy expression is highly heterogeneous. The public health significance of Duffy negativity in relation to P. vivax malaria in Ethiopia, however, remains unclear. This study seeks to explore the prevalence and rates of P. vivax malaria infection across Duffy phenotypes in clinical and community settings. METHODS: A total of 9580 and 4667 subjects from community and health facilities from a malaria endemic site and an epidemic-prone site in western Ethiopia were enrolled and examined for P. vivax infection and Duffy expression from February 2018 to April 2021. Association between Duffy expression, P. vivax and P. falciparum infections were examined for samples collected from asymptomatic community volunteers and symptomatic subjects from health centres. RESULTS: Infection rate of P. vivax among Duffy positives was 2-22 fold higher than Duffy negatives in asymptomatic volunteers from the community. Parasite positivity rate was 10-50 fold higher in Duffy positives than Duffy negatives among samples collected from febrile patients attending health centres and mixed P. vivax and P. falciparum infections were significantly more common than P. vivax mono infections among Duffy negative individuals. Plasmodium vivax parasitaemia measured by 18sRNA parasite gene copy number was similar between Duffy positives and Duffy negatives. CONCLUSIONS: Duffy negativity does not offer complete protection against infection by P. vivax, and cases of P. vivax in Duffy negatives are widespread in Ethiopia, being found in asymptomatic volunteers from communities and in febrile patients from health centres. These findings offer evidence for consideration when developing control and intervention strategies in areas of endemic P. vivax and Duffy heterogeneity.

Genetic differentiation of Plasmodium vivax duffy binding protein in Ethiopia and comparison with other geographical isolates.

BACKGROUND: Plasmodium vivax Duffy binding protein (PvDBP) is a merozoite surface protein located in the micronemes of P. vivax. The invasion of human reticulocytes by P. vivax merozoites depends on the parasite DBP binding domain engaging Duffy Antigen Receptor for Chemokine (DARC) on these red blood cells (RBCs). PvDBPII shows high genetic diversity which is a major challenge to its use in the development of a vaccine against vivax malaria. METHODS: A cross-sectional study was conducted from February 2021 to September 2022 in five study sites across Ethiopia. A total of 58 blood samples confirmed positive for P. vivax by polymerase chain reaction (PCR) were included in the study to determine PvDBPII genetic diversity. PvDBPII were amplified using primers designed from reference sequence of P. vivax Sal I strain. Assembling of sequences was done using Geneious Prime version 2023.2.1. Alignment and phylogenetic tree constructions using MEGA version 10.1.1. Nucleotide diversity and haplotype diversity were analysed using DnaSP version 6.12.03, and haplotype network was generated with PopART version 1.7. RESULTS: The mean age of the participants was 25 years, 5 (8.6%) participants were Duffy negatives. From the 58 PvDBPII sequences, seven haplotypes based on nucleotide differences at 8 positions were identified. Nucleotide diversity and haplotype diversity were 0.00267 ± 0.00023 and 0.731 ± 0.036, respectively. Among the five study sites, the highest numbers of haplotypes were identified in Arbaminch with six different haplotypes while only two haplotypes were identified in Gambella. The phylogenetic tree based on PvDBPII revealed that parasites of different study sites shared similar genetic clusters with few exceptions. Globally, a total of 39 haplotypes were identified from 223 PvDBPII sequences representing different geographical isolates obtained from NCBI archive. The nucleotide and haplotype diversity were 0.00373 and 0.845 ± 0.015, respectively. The haplotype prevalence ranged from 0.45% to 27.3%. Two haplotypes were shared among isolates from all geographical areas of the globe. CONCLUSIONS: PvDBPII of the Ethiopian P. vivax isolates showed low nucleotide but high haplotype diversity, this pattern of genetic variability suggests that the population may have undergone a recent expansion. Among the Ethiopian P. vivax isolates, almost half of the sequences were identical to the Sal-I reference sequence. However, there were unique haplotypes observed in the Ethiopian isolates, which does not share with isolates from other geographical areas. There were two haplotypes that were common among populations across the globe. Categorizing population haplotype frequency can help to determine common haplotypes for designing an effective blood-stage vaccine which will have a significant role for the control and elimination of P. vivax.

Comparative transcriptomics reveal differential gene expression among Plasmodium vivax geographical isolates and implications on erythrocyte invasion mechanisms.

The documentation of Plasmodium vivax malaria across Africa especially in regions where Duffy negatives are dominant suggests possibly alternative erythrocyte invasion mechanisms. While the transcriptomes of the Southeast Asian and South American P. vivax are well documented, the gene expression profile of P. vivax in Africa is unclear. In this study, we examined the expression of 4,404 gene transcripts belong to 12 functional groups and 43 erythrocyte binding gene candidates in Ethiopian isolates and compared them with the Cambodian and Brazilian P. vivax transcriptomes. Overall, there were 10-26% differences in the gene expression profile amongst geographical isolates, with the Ethiopian and Cambodian P. vivax being most similar. Majority of the gene transcripts involved in protein transportation, housekeeping, and host interaction were highly transcribed in the Ethiopian isolates. Members of the reticulocyte binding protein PvRBP2a and PvRBP3 expressed six-fold higher than Duffy binding protein PvDBP1 and 60-fold higher than PvEBP/DBP2 in the Ethiopian isolates. Other genes including PvMSP3.8, PvMSP3.9, PvTRAG2, PvTRAG14, and PvTRAG22 also showed relatively high expression. Differential expression patterns were observed among geographical isolates, e.g., PvDBP1 and PvEBP/DBP2 were highly expressed in the Cambodian but not the Brazilian and Ethiopian isolates, whereas PvRBP2a and PvRBP2b showed higher expression in the Ethiopian and Cambodian than the Brazilian isolates. Compared to Pvs25, gametocyte genes including PvAP2-G, PvGAP (female gametocytes), and Pvs47 (male gametocytes) were highly expressed across geographical samples.

Effectiveness of unsupervised primaquine regimen for preventing Plasmodium vivax malaria relapses in northeast Myanmar, a single-arm non-randomized observational study.

BACKGROUND: Plasmodium vivax presents a significant challenge for malaria elimination in the Greater Mekong Subregion (GMS). We evaluated the effectiveness of primaquine (PQ) for reducing relapses of vivax malaria. METHODS: Patients with uncomplicated P. vivax malaria from eastern Myanmar received chloroquine (CQ, 25 mg base/kg given in 3 days) plus unsupervised PQ (0.25 mg/kg/day for 14 days) without screening for glucose-6-phosphate dehydrogenase deficiency and were followed for a year. RESULTS: Totally 556 patients were enrolled to receive the CQ/PQ treatment from February 2012 to August 2013. During the follow-up, 38 recurrences were detected, presenting a cumulative rate of recurrence of 9.1% (95% confidence interval, 4.1-14.1%). Genotyping at the pvmsp1 and pvmsp3α loci by Amplicon deep sequencing and model prediction indicated that 13 of the 27 recurrences with genotyping data were likely due to relapses. Notably, all confirmed relapses occurred within the first six months. CONCLUSIONS: The unsupervised standard dose of PQ was highly effective as a radical cure for P. vivax malaria in eastern Myanmar. The high presumed effectiveness might have benefited from the health messages delivered during the enrollment and follow-up activities. Six-month follow-ups in the GMS are sufficient for detecting most relapses.

Detection of Duffy Blood Group Genotypes and Submicroscopic Plasmodium Infections Using Molecular Diagnostic Assays in Febrile Malaria Patients.

Background: Malaria remains a severe parasitic disease, posing a significant threat to public health and hindering economic development in sub-Saharan Africa. Ethiopia, a malaria endemic country, is facing a resurgence of the disease with a steadily rising incidence. Conventional diagnostic methods, such asmicroscopy, have become less effective due to low parasite density, particularly among Duffy-negative human populations in Africa. To develop comprehensive control strategies, it is crucial to generate data on the distribution and clinical occurrence of Plasmodium vivax and P. falciparum infections in regions where the disease is prevalent. This study assessed Plasmodium infections and Duffy antigen genotypes in febrile patients in Ethiopia. Methods: Three hundred febrile patients visiting four health facilities in Jimma town of southwestern Ethiopia were randomly selected during the malaria transmission season (Apr-Oct). Sociodemographic information was collected, and microscopic examination was performed for all study participants. Plasmodiumspecies and parasitemia as well as the Duffy genotype were assessed by quantitative polymerase chain reaction (qPCR) for all samples. Data were analyzed using Fisher's exact test and kappa statistics. Results: The Plasmodium infection rate by qPCR was 16% (48/300) among febrile patients, of which 19 (39.6%) were P. vivax, 25 (52.1%) were P. falciparum, and 4 (8.3%) were mixed (P. vivax and P. falciparum) infections. Among the 48 qPCR-positive samples, 39 (13%) were negative by microscopy. The results of bivariate logistic regression analysis showed that agriculture-related occupation, relapse and recurrence were significantly associated withPlasmodium infection (P<0.001). Of the 300 febrile patients, 85 (28.3%) were Duffy negative, of whom two had P. vivax, six had P. falciparum, and one had mixed infections.Except for one patient with P. falciparum infection, Plasmodium infections in Duffy-negative individuals were all submicroscopic with low parasitemia. Conclusions: The present study revealed a high prevalence of submicroscopic malaria infections. Plasmodium vivax infections in Duffy-negative individuals were not detected due to low parasitemia. Here, we recommend an improved molecular diagnostic tool to detect and characterize plasmodium infections, with the goal of quantifyingP. vivax infection in Duffy-negative individuals.

Distribution of Plasmodium falciparum K13 gene polymorphisms across transmission settings in Ghana.

Malaria is a significant global health concern, with a majority of cases in Sub-Saharan African nations. Numerous antimalarial drugs have been developed to counter the rampant prevalence of Plasmodium falciparum malaria. Artemisinin-based Combination Therapy (ACT) has served as the primary treatment of uncomplicated malaria in Ghana since 2005. However, a growing concern has emerged due to the escalating reports of ACT resistance, particularly in Southeast Asia, and its encroachment into Africa. Specifically, mutations in the Kelch propeller domain on chromosome 13 (Pfk13) have been linked to ACT resistance. Yet, our understanding of mutation prevalence in Africa remains largely uncharted. In this study, we compared Pfk13 sequences obtained from 172 P. falciparum samples across three ecological and transmission zones in Ghana. We identified 27 non-synonymous mutations among these sequences, of which two of the mutations, C580Y (found in two samples from the central region) and Y493H (found in one sample from the north), had previously been validated for their association with artemisinin resistance, a phenomenon widespread in Southeast Asia. The Pfk13 gene diversity was most pronounced in the northern savannah than the central forest and south coastal regions, where transmission rates are lower. The observed mutations were not significantly associated with geographical regions, suggesting a frequent spread of mutations across the country. The ongoing global surveillance of artemisinin resistance remains pivotal, and our findings provides insights into the potential spread of resistant parasites in West Africa. Furthermore, the identification of novel codon mutations in this study raises their potential association to ACT resistance, warranting further investigation through in vitro assays to ascertain their functional significance.

Genomic analysis of Plasmodium vivax describes patterns of connectivity and putative drivers of adaptation in Ethiopia.

Ethiopia has the greatest burden of Plasmodium vivax in Africa, but little is known about the epidemiological landscape of parasites across the country. We analysed the genomic diversity of 137 P. vivax isolates collected nine Ethiopian districts from 2012 to 2016. Signatures of selection were detected by cross-country comparisons with isolates from Thailand (n = 104) and Indonesia (n = 111), representing regions with low and high chloroquine resistance respectively. 26% (35/137) of Ethiopian infections were polyclonal, and 48.5% (17/35) of these comprised highly related clones (within-host identity-by-descent > 25%), indicating frequent co-transmission and superinfection. Parasite gene flow between districts could not be explained entirely by geographic distance, with economic and cultural factors hypothesised to have an impact on connectivity. Amplification of the duffy binding protein gene (pvdbp1) was prevalent across all districts (16-75%). Cross-population haplotype homozygosity revealed positive selection in a region proximal to the putative chloroquine resistance transporter gene (pvcrt-o). An S25P variant in amino acid transporter 1 (pvaat1), whose homologue has recently been implicated in P. falciparum chloroquine resistance evolution, was prevalent in Ethiopia (96%) but not Thailand or Indonesia (35-53%). The genomic architecture in Ethiopia highlights circulating variants of potential public health concern in an endemic setting with evidence of stable transmission.

Comparison of Plasmodium Vivax Infections in Duffy Negatives From Community and Health Center Collections in Ethiopia.

Background: Malaria remains a significant cause of morbidity and mortality in Ethiopia with an estimated 4.2 million annual cases and 61% of the population living in areas at risk of malaria transmission. Throughout the country Plasmodium vivax and P. falciparum are co-endemic, and Duffy expression is highly heterogeneous. The public health significance of Duffy negativity in relation to P. vivax malaria in Ethiopia, however, remains unclear. Methods: A total of 9,580 and 4,667 subjects from community and health facilities from a malaria endemic site and an epidemic-prone site in western Ethiopia were enrolled and examined for P. vivax infection and Duffy expression. Association between Duffy expression, P. vivax and P. falciparum infections were examined for samples collected from asymptomatic community volunteers and symptomatic subjects from health centers. Results: Among the community-based cross-sectional samples, infection rate of P. vivax among the Duffy positives was 2-22 fold higher than among the Duffy negatives. Parasite positivity rate was 10-50 fold higher in Duffy positive than Duffy negatives among samples collected from the health center settings and mixed P. vivax and P. falciparum infections were significantly more common than P. vivax mono infections among Duffy negative individuals. P. vivax parasitemia measured by 18sRNA parasite gene copy number was similar between Duffy positives and Duffy negatives. Conclusions: Duffy negativity does not offer complete protection against infection by P. vivax, and cases of P. vivax in Duffy negatives are widespread in Ethiopia, being found in asymptomatic volunteers from communities and in febrile patients from health centers. These findings offer evidence for consideration when developing control and intervention strategies in areas of endemic P. vivax and Duffy heterogeneity.

Potential hidden Plasmodium vivax malaria reservoirs from low parasitemia Duffy-negative Ethiopians: Molecular evidence.

BACKGROUND: The interaction between the Plasmodium vivax Duffy-binding protein and the corresponding Duffy Antigen Receptor for Chemokines (DARC) is primarily responsible for the invasion of reticulocytes by P. vivax. The Duffy-negative host phenotype, highly prevalent in sub-Saharan Africa, is caused by a single point mutation in the GATA-1 transcription factor binding site of the DARC gene promoter. The aim of this study was to assess the Duffy status of patients with P. vivax infection from different study sites in Ethiopia. METHODS: A cross-sectional study was conducted from February 2021 to September 2022 at five varying eco-epidemiological malaria endemic sites in Ethiopia. Outpatients who were diagnosed with P. vivax infection (pure and mixed P. vivax/P. falciparum) by microscopy and Rapid Diagnostic Test (RDT) were subjected to PCR genotyping at the DARC promoter. The associations between P. vivax infection, host genotypes and other factors were evaluated. RESULT: In total, 361 patients with P. vivax infection were included in the study. Patients with pure P. vivax infections accounted for 89.8% (324/361), while the remaining 10.2% (37/361) had mixed P. vivax/P. falciparum infections. About 95.6% (345/361) of the participants were Duffy-positives (21.2% homozygous and 78.8%, heterozygous) and 4.4% (16/361) were Duffy-negatives. The mean asexual parasite density in homozygous and heterozygous Duffy-positives was 12,165 p/µl (IQR25-75: 1,640-24,234 p/µl) and11,655 p/µl (IQR25-75: 1,676-14,065 p/µl), respectively, significantly higher than that in Duffy-negatives (1,227p/µl; IQR25-75: 539-1,732p/µl). CONCLUSION: This study confirms that Duffy-negativity does not provide complete protection against P. vivax infection. The development of P. vivax-specific elimination strategies, including alternative antimalarial vaccines should be facilitated by a better understanding of the epidemiological landscape of vivax malaria in Africa. More importantly, low parasitemia associated with P. vivax infections in Duffy-negative patients may represent hidden reservoirs of transmission in Ethiopia.

Significant number of Plasmodium vivax mono-infections by PCR misidentified as mixed infections (P. vivax/P. falciparum) by microscopy and rapid diagnostic tests: malaria diagnostic challenges in Ethiopia.

BACKGROUND: Plasmodium vivax malaria is now recognized as a cause of severe morbidity and mortality, resulting in a substantial negative effect on health especially in endemic countries. Accurate and prompt diagnosis and treatment of P. vivax malaria is vital for the control and elimination of the disease. METHODS: A cross-sectional study was conducted from February 2021 to September 2022 at five malaria endemic sites in Ethiopia including Aribaminch, Shewarobit, Metehara, Gambella, and Dubti. A total of 365 samples that were diagnosed positive for P. vivax (mono and mixed infection) using RDT, site level microscopists and expert microscopists were selected for PCR. Statistical analyses were performed to calculate the proportions, agreement (k), frequencies, and ranges among different diagnostic methods. Fisher's exact tests and correlation test were used to detect associations and relationship between different variables. RESULTS: Of the 365 samples, 324 (88.8%), 37(10.1%), 2 (0.5%), and 2 (0.5%) were P. vivax (mono), P. vivax/Plasmodium falciparum (mixed), P. falciparum (mono) and negative by PCR, respectively. The overall agreement of rapid diagnostic test (RDT), site level microscopy and expert microscopists result with PCR was 90.41% (k: 0.49), 90.96% (k: 0.53), and 80.27% (k: 0.24). The overall prevalence of sexual (gametocyte) stage P. vivax in the study population was 215/361 (59.6%). The majority of these 215 samples (180; 83.7%) had below 1000 parasites/µl, with only four samples (1.9%) had ≥ 5000 parasites/µl. The gametocyte density was found to be weakly positive but statically significant with asexual parasitaemia (r = 0.31; p < 0.001). CONCLUSION: Both microscopy and RDT showed moderate agreement with PCR in the detection and identification of P. vivax (mono) and P. vivax/P. falciparum (mixed) infections. Therefore, to achieve malaria elimination goals, strengthening routine malaria diagnostic methods by implementing diagnostic tools with a good performance in detecting and accurately identifying malaria species in clinical settings is recommended.

Prevalence and distribution of Plasmodium vivax Duffy Binding Protein gene duplications in Sudan.

Plasmodium vivax Duffy Binding Protein (PvDBP) is essential for interacting with Duffy antigen receptor for chemokines (DARC) on the surface of red blood cells to allow invasion. Earlier whole genome sequence analyses provided evidence for the duplications of PvDBP. It is unclear whether PvDBP duplications play a role in recent increase of P. vivax in Sudan and in Duffy-negative individuals. In this study, the prevalence and type of PvDBP duplications, and its relationship to demographic and clinical features were investigated. A total of 200 malaria-suspected blood samples were collected from health facilities in Khartoum, River Nile, and Al-Obied. Among them, 145 were confirmed to be P. vivax, and 43 (29.7%) had more than one PvDBP copies with up to four copies being detected. Both the Malagasy and Cambodian types of PvDBP duplication were detected. No significant difference was observed between the two types of duplications between Duffy groups. Parasitemia was significantly higher in samples with the Malagasy-type than those without duplications. No significant difference was observed in PvDBP duplication prevalence and copy number among study sites. The functional significance of PvDBP duplications, especially those Malagasy-type that associated with higher parasitemia, merit further investigations.

Comparative transcriptomics reveal differential gene expression in Plasmodium vivax geographical isolates and implications on erythrocyte invasion mechanisms.

Plasmodium vivax uses Duffy binding protein (PvDBP1) to bind to the Duffy Antigen-Chemokine Receptor (DARC) to invade human erythrocytes. Individuals who lack DARC expression (Duffy-negative) are thought to be resistance to P. vivax. In recent years, P. vivax malaria is becoming more prevalent in Africa with a portion of these cases detected in Duffy-negatives. Apart from DBP1, members of the reticulocyte binding protein (RBP) and tryptophan-rich antigen (TRAg) families may also play a role in erythrocyte invasion. While the transcriptomes of the Southeast Asian and South American P. vivax are well documented, the gene expression profile of P. vivax in Africa and more specifically the expression level of several erythrocyte binding gene candidates as compared to DBP1 are largely unknown. This paper characterized the first P. vivax transcriptome in Africa and compared with those from the Southeast Asian and South American isolates. The expression of 4,404 gene transcripts belong to 12 functional groups including 43 specific erythrocyte binding gene candidates were examined. Overall, there were 10-26% differences in the gene expression profile amongst the geographical isolates, with the Ethiopian and Cambodian P. vivax being most similar. Majority of the gene transcripts involved in protein transportation, housekeeping, and host interaction were highly transcribed in the Ethiopian P. vivax. Erythrocyte binding genes including PvRBP2a and PvRBP3 expressed six-fold higher than PvDBP1and 60-fold higher than PvEBP/DBP2. Other genes including PvRBP1a, PvMSP3.8, PvMSP3.9, PvTRAG2, PvTRAG14, and PvTRAG22 also showed relatively high expression. Differential expression was observed among geographical isolates, e.g., PvDBP1 and PvEBP/DBP2 were highly expressed in the Cambodian but not the Brazilian and Ethiopian isolates, whereas PvRBP2a and PvRBP2b showed higher expression in the Ethiopian and Cambodian than the Brazilian isolates. Compared to Pvs25, the standard biomarker for detecting female gametocytes, PvAP2-G (PVP01_1440800), GAP (PVP01_1403000), and Pvs47 (PVP01_1208000) were highly expressed across geographical samples. These findings provide an important baseline for future comparisons of P. vivax transcriptomes from Duffy-negative infections and highlight potential biomarkers for improved gametocyte detection.

Genetic variations of Plasmodium falciparum circumsporozoite protein and the impact on interactions with human immunoproteins and malaria vaccine efficacy.

In October 2021, the world's first malaria vaccine RTS,S was endorsed by WHO for broad use in children, despite its low efficacy. This study examined polyclonal infections and the associations of parasite genetic variations with binding affinity to human leukocyte antigen (HLA). Multiplicity of infection was determined by amplicon deep sequencing of PfMSP1. Genetic variations in PfCSP were examined across 88 samples from Ghana and analyzed together with 1655 PfCSP sequences from other African and non-African isolates. Binding interactions of PfCSP peptide variants and HLA were predicted using NetChop and HADDOCK. High polyclonality was detected among infections, with each infection harboring multiple non-3D7 PfCSP variants. Twenty-seven PfCSP haplotypes were detected in the Ghanaian samples, and they broadly represented PfCSP diversity across Africa. The number of genetic differences between 3D7 and non-3D7 PfCSP variants does not influence binding to HLA. However, CSP peptide length after proteolytic degradation significantly affects its molecular weight and binding affinity to HLA. Despite the high diversity of HLA, the majority of the HLAI and II alleles interacted/bound with all Ghana CSP peptides. Multiple non-3D7 strains among P. falciparum infections could impact the effectiveness of RTS,S. Longer peptides of the Th2R/Th3R CSP regions should be considered in future versions of RTS,S.

Implementing landscape genetics in molecular epidemiology to determine drivers of vector-borne disease: A malaria case study.

This study employs landscape genetics to investigate the environmental drivers of a deadly vector-borne disease, malaria caused by Plasmodium falciparum, in a more spatially comprehensive manner than any previous work. With 1804 samples from 44 sites collected in western Kenya in 2012 and 2013, we performed resistance surface analysis to show that Lake Victoria acts as a barrier to transmission between areas north and south of the Winam Gulf. In addition, Mantel correlograms clearly showed significant correlations between genetic and geographic distance over short distances (less than 70 km). In both cases, we used an identity-by-state measure of relatedness tailored to find highly related individual parasites in order to focus on recent gene flow that is more relevant to disease transmission. To supplement these results, we performed conventional population genetics analyses, including Bayesian clustering methods and spatial ordination techniques. These analyses revealed some differentiation on the basis of geography and elevation and a cluster of genetic similarity in the lowlands north of the Winam Gulf of Lake Victoria. Taken as a whole, these results indicate low overall genetic differentiation in the Lake Victoria region, but with some separation of parasite populations north and south of the Winam Gulf that is explained by the presence of the lake as a geographic barrier to gene flow. We recommend similar landscape genetics analyses in future molecular epidemiology studies of vector-borne diseases to extend and contextualize the results of traditional population genetics.

Prevalence and characteristics of Plasmodium vivax Gametocytes in Duffy-positive and Duffy-negative populations across Ethiopia.

Plasmodium parasites replicate asexually in the human host. The proportion of infections that carries gametocytes is a proxy for human-to-mosquito transmissibility. It is unclear what proportion of P. vivax infections in Duffy-negatives carries gametocytes. This study aims to determine the prevalence of P. vivax in Duffy-negatives across broad regions of Ethiopia and characterize parasite stages. Finger-prick blood samples were collected for microscopic and molecular screening of Plasmodium parasites and Duffy status of individuals. Molecular screening of plasmodium species and Duffy blood group genotyping was done using SYBR green and Taqman qPCR method. Among the total 447 samples, 414 (92.6%) were P. vivax confirmed and, 16 (3.9%) of them were from Duffy-negatives. Of these, 5/16 (31.3%) Duffy-negative P. vivax-infected samples were detected with gametocytes. Of the 398 Duffy-positive P. vivax-infected samples, 150 (37.7%) were detected with gametocytes, slightly higher than that in Duffy-negatives. This study highlights the presence of P. vivax gametocytes in Duffy-negative infections, suggestive of human-to-mosquito transmissibility. Although P. vivax infections in Duffy-negatives are commonly associated with low parasitemia, some of these infections were shown with relatively high parasitemia and may represent better erythrocyte invasion capability of P. vivax and hidden reservoirs that can contribute to transmission. A better understanding of P. vivax transmission biology and gametocyte function particularly in Duffy-negative populations would aid future treatment and management of vivax malaria in Africa.

Prevalence of G6PD deficiency and distribution of its genetic variants among malaria-suspected patients visiting Metehara health centre, Eastern Ethiopia.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) is cytosolic enzyme, which has a vital role for the integrity and functioning of red blood cells. Lower activity of this enzyme leads to the occurrence of acute haemolytic anaemia after exposure to oxidative stressors like primaquine. Primaquine is an important drug for the radical cure of Plasmodium vivax and blocking transmission of Plasmodium falciparum, and thereby enhancing malaria elimination. However, there is a need to identify G6PD deficient individuals and administer the drug with caution due to its haemolytic side effects. The main objective of this study is to determine the prevalence of G6PD deficiency among malaria-suspected individuals. METHODS: A facility-based cross-sectional study was conducted from September 2020 to September 2021 in Metehara Health Centre, Eastern Ethiopia. A structured questionnaire was used to collect the socio-demographic and clinical information of the study participants. Capillary and venous blood samples were collected based on standard procedures for onsite screening, dried blood spot preparation, and malaria microscopy. The G6PD enzyme activity was measured by careSTART™ G6PD biosensor analyzer. Data was entered and analysed by SPSS. RESULTS: A total of 498 study participants were included in the study, of which 62% (309) were males. The overall prevalence of G6PD deficiency based on the biosensor screening was 3.6% (18/498), of which 2.9% and 4.8% were males and females, respectively. Eleven of the G6PD deficient samples had mutations confirmed by G6PD gene sequencing analysis. Mutations were detected in G267 + 119C/T, A376T, and ChrX:154535443. A significant association was found in sex and history of previous malaria infection with G6PD deficiency. CONCLUSIONS: The study showed that the G6PD deficient phenotype exists in Metehara even if the prevalence is not very high. G267 + 119C/T mutation is the predominant G6PD variant in this area. Therefore, malaria patient treatment using primaquine should be monitored closely for any adverse effects.

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.

Low Density Plasmodium Infections and G6PD Deficiency Among Malaria Suspected Febrile Individuals in Ethiopia.

The identification and management of low parasitemia infections have become increasingly challenging for malaria control and elimination. Submicroscopic Plasmodium infections and G6PD deficiency among febrile patients require more sensitive diagnostic methods to improve detection and careful treatment regime of these infections. In Ethiopia, information on the low density submicroscopic malarial infections and frequency of G6PD deficiency (G6PDd) is scarce. In this study, 297 malaria suspected febrile patient samples were collected from health facilities of Bonga town in southwestern Ethiopia. The positivity rates of Plasmodium infection were determined by microscopy and quantitative PCR. G6PD activity level was determined by careSTART™ G6PD biosensor and the frequency of three common variants: G6PD*A (A376G), G6PD*A- (G202A) and Mediterranean (C563T) were investigated. G6PD gene sequencing was performed to detect mutations in exons 2-11 for both G6PD normal and deficient samples based on the phenotypic assay. More than twice Plasmodium infected samples was detected by qPCR (52/297; 17.4%) than microscopy (21/297; 7.0%). About 31 (10%) of the infections were submicroscopic. Bednet usage and age had a significant association with Plasmodium infection. Of the 271 participants who were tested for G6PD phenotype, 19 (7.0%) had low G6PD level. No mutations were observed in A376G, G202A, and C563T in the G6PDd samples, but three novel non-synonymous mutations in exon 2 including a C to T transition at position ChrX:6504 (Arg to Thr), G to T at ChrX:6369 (Ser to IIe), and G to C at ChrX:6664 (Gln to His) were detected. A high number of submicroscopic Plasmodium infections observed in this study pose a challenge for accurate and timely diagnosis, which could hinder malaria control efforts. G6PD deficiency in malaria patients pose danger when treating patients with primaquine. The three novel mutations detected in exon 2 of the G6PD gene merit further investigation on the hemolytic risk when exposed to oxidative antimalarials, their prevalence, and clinical significance.