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.

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 as microscopy, 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 Plasmodium 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. Plasmodium species and parasitaemia as well as the Duffy genotype were assessed by quantitative polymerase chain reaction (qPCR) for all samples. Data were analysed 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 with Plasmodium 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 parasitaemia. 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 parasitaemia. In this study, an improved molecular diagnostic tool was used to detect and characterize Plasmodium infections, with the goal of quantifying P. vivax infection in Duffy-negative individuals. Advanced molecular diagnostic techniques, such as multiplex real-time PCR, loop-mediated isothermal amplification (LAMP), and CRISPR-based diagnostic methods. These techniques offer increased sensitivity, specificity, and the ability to detect low-parasite-density infections compared to the employed methodologies.

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.

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.

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.

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.

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.

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.

Plasmodium vivax From Duffy-Negative and Duffy-Positive Individuals Share Similar Gene Pools in East Africa.

Plasmodium vivax malaria was thought to be rare in Africa, but an increasing number of P. vivax cases reported across Africa and in Duffy-negative individuals challenges this dogma. The genetic characteristics of P. vivax in Duffy-negative infections, the transmission of P. vivax in East Africa, and the impact of environments on transmission remain largely unknown. This study examined genetic and transmission features of P. vivax from 107 Duffy-negative and 305 Duffy-positive individuals in Ethiopia and Sudan. No clear genetic differentiation was found in P. vivax between the 2 Duffy groups, indicating between-host transmission. P. vivax from Ethiopia and Sudan showed similar genetic clusters, except samples from Khartoum, possibly due to distance and road density that inhibited parasite gene flow. This study is the first to show that P. vivax can transmit to and from Duffy-negative individuals and provides critical insights into the spread of P. vivax in sub-Saharan Africa.

Genetic variation of Plasmodium falciparum histidine-rich protein 2 and 3 in Assosa zone, Ethiopia: its impact on the performance of malaria rapid diagnostic tests.

BACKGROUND: Rapid diagnostic tests (RDT) are commonly used for the diagnosis of malaria caused by Plasmodium falciparum. However, false negative results of RDT caused by genetic variation of P. falciparum histidine-rich protein 2 and 3 genes (pfhrp2/3) threaten existing malaria case management and control efforts. The main objective of this study was to investigate the genetic variations of the pfhrp2/3 genes. METHODS: A cross-sectional study was conducted from malaria symptomatic individuals in 2018 in Assosa zone, Ethiopia. Finger-prick blood samples were collected for RDT and microscopic examination of thick and thin blood films. Dried blood spots (DBS) were used for genomic parasite DNA extraction and molecular detection. Amplification of parasite DNA was made by quantitative PCR. DNA amplicons of pfhrp2/3 were purified and sequenced. RESULTS: The PfHRP2 amino acid repeat type isolates were less conserved compared to the PfHRP3 repeat type. Eleven and eight previously characterized PfHRP2 and PfHRP3 amino acid repeat types were identified, respectively. Type 1, 4 and 7 repeats were shared by PfHRP2 and PfHRP3 proteins. Type 2 repeats were found only in PfHRP2, while types 16 and 17 were found only in PfHRP3 with a high frequency in all isolates. 18 novel repeat types were found in PfHRP2 and 13 novel repeat types were found in PfHRP3 in single or multiple copies per isolate. The positivity rate for PfHRP2 RDT was high, 82.9% in PfHRP2 and 84.3% in PfHRP3 sequence isolates at parasitaemia levels > 250 parasites/µl. Using the Baker model, 100% of the isolates in group A (If product of types 2 × type 7 repeats ≥ 100) and 73.7% of the isolates in group B (If product of types 2 × type 7 repeats 50-99) were predicted to be detected by PfHRP2 RDT at parasitaemia level > 250 parasite/µl. CONCLUSION: The findings of this study indicate the presence of different PfHRP2 and PfHRP3 amino acid repeat including novel repeats in P. falciparum from Ethiopia. These results indicate that there is a need to closely monitor the performance of PfHRP2 RDT associated with the genetic variation of the pfhrp2 and pfhrp3 gene in P. falciparum isolates at the country-wide level.

Detection of high prevalence of Plasmodium falciparum histidine-rich protein 2/3 gene deletions in Assosa zone, Ethiopia: implication for malaria diagnosis.

BACKGROUND: Rapid diagnostic tests (RDTs) targeting histidine rich protein 2(HRP2) are widely used for diagnosis of Plasmodium falciparum infections. Besides PfHRP2, the PfHRP3 antigen contributes to the detection of P. falciparum infections in PfHRP2 RDTs. However, the performance HRP2-based RDT is affected by pfhrp2/3 gene deletions resulting in false-negative test results. The objective of this study was to determine the presence and prevalence of pfhrp2/3 gene deletions including the respective flanking regions among symptomatic patients in Assosa zone, Northwest Ethiopia. METHODS: A health-facility based cross-sectional study was conducted in febrile patients seeking a malaria diagnosis in 2018. Blood samples were collected by finger-prick for microscopic examination of blood smears, malaria RDT, and molecular analysis using dried blood spots (DBS) prepared on Whatman filter paper. A total of 218 P. falciparum positive samples confirmed by quantitative PCR were included for molecular assay of pfhrp2/3 target gene. RESULTS: Of 218 P. falciparum positive samples, exon 2 deletions were observed in 17.9% of pfhrp2 gene and in 9.2% of pfhrp3 gene. A high proportion of deletions in short segments of pfhrp2 exon1-2 (50%) was also detected while the deletions of the pfhrp3 exon1-2 gene were 4.1%. The deletions were extended to the downstream and upstream of the flanking regions in pfhrp2/3 gene (above 30%). Of eighty-six PfHRP2 RDT negative samples, thirty-six lacked pfhrp2 exon 2. Five PfHRP2 RDT negative samples had double deletions in pfhrp2 exon 2 and pfhrp3 exon2. Of these double deletions, only two of the samples with a parasite density above 2000 parasite/µl were positive by the microscopy. Three samples with intact pfhrp3 exon2 in the pfhrp2 exon2 deleted parasite isolates were found to be positive by PfHRP2 RDT and microscopy with a parasite density above 10,000/µl. CONCLUSION: This study confirms the presence of deletions of pfhrp2/3 gene including the flanking regions. Pfhrp2/3 gene deletions results in false-negative results undoubtedly affect the current malaria control and elimination effort in the country. However, further countrywide investigations are required to determine the magnitude of pfhrp2/3 gene deletions and its consequences on routine malaria diagnosis.

Population structure and diversity of Plasmodium falciparum in children with asymptomatic malaria living in different ecological zones of Ghana.

BACKGROUND: Genetic diversity in Plasmodium falciparum populations can be used to describe the resilience and spatial distribution of the parasite in the midst of intensified intervention efforts. This study used microsatellite analysis to evaluate the genetic diversity and population dynamics of P. falciparum parasites circulating in three ecological zones of Ghana. METHODS: A total of 1168 afebrile children aged between 3 to 13 years were recruited from five (5) Primary schools in 3 different ecological zones (Sahel (Tamale and Kumbungu), Forest (Konongo) and Coastal (Ada and Dodowa)) of Ghana. Asymptomatic malaria parasite carriage was determined using microscopy and PCR, whilst fragment analysis of 6 microsatellite loci was used to determine the diversity and population structure of P. falciparum parasites. RESULTS: Out of the 1168 samples examined, 16.1 and 39.5% tested positive for P. falciparum by microscopy and nested PCR respectively. The genetic diversity of parasites in the 3 ecological zones was generally high, with an average heterozygosity (He) of 0.804, 0.787 and 0.608 the rainy (peak) season for the Sahel, Forest and Coastal zones respectively. The mean He for the dry (off-peak) season were 0.562, 0.693 and 0.610 for the Sahel, Forest and Coastal zones respectively. Parasites from the Forest zone were more closely related to those from the Sahel than from the Coastal zone, despite the Coastal zone being closer in physical distance to the Forest zone. The fixation indexes among study sites ranged from 0.049 to 0.112 during the rainy season and 0.112 to 0.348 during the dry season. CONCLUSION: A large asymptomatic parasite reservoir was found in the school children during both rainy and dry seasons, especially those in the Forest and Sahel savannah zones where parasites were also found to be related compared to those from the Coastal zone. Further studies are recommended to understand why despite the roll out of several malaria interventions in Ghana, high transmission still persist.

The biology of unconventional invasion of Duffy-negative reticulocytes by Plasmodium vivax and its implication in malaria epidemiology and public health.

Plasmodium vivax has been largely neglected over the past century, despite a widespread recognition of its burden across region where it is endemic. The parasite invades reticulocytes, employing the interaction between Plasmodium vivax Duffy binding protein (PvDBP) and human Duffy antigen receptor for chemokines (DARC). However, P. vivax has now been observed in Duffy-negative individuals, presenting a potentially serious public health problem as the majority of African populations are Duffy-negative. Invasion of Duffy-negative reticulocytes is suggested to be through duplication of the PvDBP and a novel protein encoded by P. vivax erythrocyte binding protein (EBP) genes. The emergence and spread of specific P. vivax strains with ability to invade Duffy-negative reticulocytes has, therefore, drawn substantial attention and further complicated the epidemiology and public health implication of vivax malaria. Given the right environment and vectorial capacity for transmission coupled with the parasite's ability to invade Duffy-negative individuals, P. vivax could increase its epidemiological significance in Africa. In this review, authors present accruing knowledge on the paradigm shift in P. vivax invasion of Duffy-negative reticulocytes against the established mechanism of invading only Duffy-positive individuals and offer a perspective on the epidemiological diagnostic and public health implication in Africa.

Prevalence and distribution of G6PD deficiency: implication for the use of primaquine in malaria treatment in Ethiopia.

BACKGROUND: G6PD enzyme deficiency is a common enzymatic X-linked disorder. Deficiency of the G6PD enzyme can cause free radical-mediated oxidative damage to red blood cells, leading to premature haemolysis. Treatment of Plasmodium vivax malaria with primaquine poses a potential risk of mild to severe acute haemolytic anaemia in G6PD deficient people. In this study, the prevalence and distribution of G6PD mutations were investigated across broad areas of Ethiopia, and tested the association between G6PD genotype and phenotype with the goal to provide additional information relevant to the use of primaquine in malaria treatment. METHODS: This study examined G6PD mutations in exons 3-11 for 344 febrile patient samples collected from seven sites across Ethiopia. In addition, the G6PD enzyme level of 400 febrile patient samples from Southwestern Ethiopia was determined by the CareStart™ biosensor. The association between G6PD phenotype and genotype was examined by Fisher exact test on a subset of 184 samples. RESULTS: Mutations were observed at three positions of the G6PD gene. The most common G6PD mutation across all sites was A376G, which was detected in 21 of 344 (6.1%) febrile patients. Thirteen of them were homozygous and eight were heterozygous for this mutation. The G267+119C/T mutation was found in 4 (1.2%) individuals in South Ethiopia, but absent in other sites. The G1116A mutation was also found in 4 (1.2%) individuals from East and South Ethiopia. For the 400 samples in the south, 17 (4.25%) were shown to be G6PD-deficient. G6PD enzyme level was not significantly different by age or gender. Among a subset of 202 febrile patients who were diagnosed with malaria, 11 (5.45%) were G6PD-deficient. These 11 infected samples were diagnosed with Plasmodium vivax by microscopy. Parasitaemia was not significantly different between the G6PD-deficient and G6PD-normal infections. CONCLUSIONS: The prevalence of G6PD deficiency is modest among febrile patients in Ethiopia. G6PD deficiency testing is thus recommended before administrating primaquine for radical cure of P. vivax infected patients. The present study did not indicate a significant association between G6PD gene mutations and enzyme levels.

Epidemiological risk factors for clinical malaria infection in the highlands of Western Kenya.

BACKGROUND: Understanding the complex heterogeneity of risk factors that can contribute to an increased risk of malaria at the individual and household level will enable more effective use of control measures. The objective of this study was to understand individual and household factors that influence clinical malaria infection among individuals in the highlands of Western Kenya. METHODS: This was a matched case-control study undertaken in the Western Kenya highlands. Clinical malaria cases were recruited from health facilities and matched to asymptomatic individuals from the community who served as controls. Each participant was screened for malaria using microscopy. Follow-up surveys were conducted with individual households to collect socio-economic data. The houses were also checked using pyrethrum spray catches to collect mosquitoes. RESULTS: A total of 302 malaria cases were matched to 604 controls during the surveillance period. Mosquito densities were similar in the houses of both groups. A greater percentage of people in the control group (64.6%) used insecticide-treated bed nets (ITNs) compared to the families of malaria cases (48.3%). Use of ITNs was associated with lower level of clinical malaria episodes (odds ratio 0.51; 95% CI 0.39-0.68; P < 0.0001). Low income was the most important factor associated with higher malaria infections (adj. OR 4.70). Use of malaria prophylaxis was the most important factor associated with less malaria infections (adj OR 0.36). Mother's (not fathers) employment status (adj OR 0.48) and education level (adj OR 0.54) was important malaria risk factor. Houses with open eaves was an important malaria risk factor (adj OR 1.72). CONCLUSION: The identification of risk factors for clinical malaria infection provides information on the local malaria epidemiology and has the potential to lead to a more effective and targeted use of malaria control measures. These risk factors could be used to assess why some individuals acquire clinical malaria whilst others do not and to inform how intervention could be scaled at the local level.

Role of Plasmodium vivax Duffy-binding protein 1 in invasion of Duffy-null Africans.

The ability of the malaria parasite Plasmodium vivax to invade erythrocytes is dependent on the expression of the Duffy blood group antigen on erythrocytes. Consequently, Africans who are null for the Duffy antigen are not susceptible to P. vivax infections. Recently, P. vivax infections in Duffy-null Africans have been documented, raising the possibility that P. vivax, a virulent pathogen in other parts of the world, may expand malarial disease in Africa. P. vivax binds the Duffy blood group antigen through its Duffy-binding protein 1 (DBP1). To determine if mutations in DBP1 resulted in the ability of P. vivax to bind Duffy-null erythrocytes, we analyzed P. vivax parasites obtained from two Duffy-null individuals living in Ethiopia where Duffy-null and -positive Africans live side-by-side. We determined that, although the DBP1s from these parasites contained unique sequences, they failed to bind Duffy-null erythrocytes, indicating that mutations in DBP1 did not account for the ability of P. vivax to infect Duffy-null Africans. However, an unusual DNA expansion of DBP1 (three and eight copies) in the two Duffy-null P. vivax infections suggests that an expansion of DBP1 may have been selected to allow low-affinity binding to another receptor on Duffy-null erythrocytes. Indeed, we show that Salvador (Sal) I P. vivax infects Squirrel monkeys independently of DBP1 binding to Squirrel monkey erythrocytes. We conclude that P. vivax Sal I and perhaps P. vivax in Duffy-null patients may have adapted to use new ligand-receptor pairs for invasion.

Multiplicity and molecular epidemiology of Plasmodium vivax and Plasmodium falciparum infections in East Africa.

BACKGROUND: Parasite genetic diversity and multiplicity of infection (MOI) affect clinical outcomes, response to drug treatment and naturally-acquired or vaccine-induced immunity. Traditional methods often underestimate the frequency and diversity of multiclonal infections due to technical sensitivity and specificity. Next-generation sequencing techniques provide a novel opportunity to study complexity of parasite populations and molecular epidemiology. METHODS: Symptomatic and asymptomatic Plasmodium vivax samples were collected from health centres/hospitals and schools, respectively, from 2011 to 2015 in Ethiopia. Similarly, both symptomatic and asymptomatic Plasmodium falciparum samples were collected, respectively, from hospitals and schools in 2005 and 2015 in Kenya. Finger-pricked blood samples were collected and dried on filter paper. Long amplicon (> 400 bp) deep sequencing of merozoite surface protein 1 (msp1) gene was conducted to determine multiplicity and molecular epidemiology of P. vivax and P. falciparum infections. The results were compared with those based on short amplicon (117 bp) deep sequencing. RESULTS: A total of 139 P. vivax and 222 P. falciparum samples were pyro-sequenced for pvmsp1 and pfmsp1, yielding a total of 21 P. vivax and 99 P. falciparum predominant haplotypes. The average MOI for P. vivax and P. falciparum were 2.16 and 2.68, respectively, which were significantly higher than that of microsatellite markers and short amplicon (117 bp) deep sequencing. Multiclonal infections were detected in 62.2% of the samples for P. vivax and 74.8% of the samples for P. falciparum. Four out of the five subjects with recurrent P. vivax malaria were found to be a relapse 44-65 days after clearance of parasites. No difference was observed in MOI among P. vivax patients of different symptoms, ages and genders. Similar patterns were also observed in P. falciparum except for one study site in Kenyan lowland areas with significantly higher MOI. CONCLUSIONS: The study used a novel method to evaluate Plasmodium MOI and molecular epidemiological patterns by long amplicon ultra-deep sequencing. The complexity of infections were similar among age groups, symptoms, genders, transmission settings (spatial heterogeneity), as well as over years (pre- vs. post-scale-up interventions). This study demonstrated that long amplicon deep sequencing is a useful tool to investigate multiplicity and molecular epidemiology of Plasmodium parasite infections.

Frequent Spread of Plasmodium vivax Malaria Maintains High Genetic Diversity at the Myanmar-China Border, Without Distance and Landscape Barriers.

Background: In Myanmar, civil unrest and the establishment of internally displaced person (IDP) settlements along the Myanmar-China border have impacted malaria transmission. Methods: Microsatellite markers were used to examine source-sink dynamics for Plasmodium vivax between IDP settlements and surrounding villages in the border region. Genotypic structure and diversity were compared across the 3 years following the establishment of IDP settlements, to infer demographic history. We investigated whether human migration and landscape heterogeneity contributed to P. vivax transmission. Results: P. vivax from IDP settlements and local communities consistently exhibited high genetic diversity within populations but low polyclonality within individuals. No apparent genetic structure was observed among populations and years. P. vivax genotypes in China were similar to those in Myanmar, and parasite introduction was unidirectional. Landscape factors, including distance, elevation, and land cover, do not appear to impede parasite gene flow. Conclusions: The admixture of P. vivax genotypes suggested that parasite gene flow via human movement contributes to the spread of malaria both locally in Myanmar and across the international border. Our genetic findings highlight the presence of large P. vivax gene reservoirs that can sustain transmission. Thus, it is important to reinforce and improve existing control efforts along border areas.

Plasmodium malariae Prevalence and csp Gene Diversity, Kenya, 2014 and 2015.

In Africa, control programs that target primarily Plasmodium falciparum are inadequate for eliminating malaria. To learn more about prevalence and genetic variability of P. malariae in Africa, we examined blood samples from 663 asymptomatic and 245 symptomatic persons from western Kenya during June-August of 2014 and 2015. P. malariae accounted for 5.3% (35/663) of asymptomatic infections and 3.3% (8/245) of clinical cases. Among asymptomatic persons, 71% (32/45) of P. malariae infections detected by PCR were undetected by microscopy. The low sensitivity of microscopy probably results from the significantly lower parasitemia of P. malariae. Analyses of P. malariae circumsporozoite protein gene sequences revealed high genetic diversity among P. malariae in Africa, but no clear differentiation among geographic populations was observed. Our findings suggest that P. malariae should be included in the malaria elimination strategy in Africa and highlight the need for sensitive and field-applicable methods to identify P. malariae in malaria-endemic areas.

Analysis of asymptomatic and clinical malaria in urban and suburban settings of southwestern Ethiopia in the context of sustaining malaria control and approaching elimination.

BACKGROUND: Malaria intervention in Ethiopia has been strengthened significantly in the past decade. The Ethiopian government recently stratified the country based upon annual parasite incidence into malaria free, low, moderate and high transmission strata. Districts with low transmission were targeted for indigenous transmission elimination. Surveillance on malaria disease incidence is needed for planning control and elimination efforts. METHODS: Clinical malaria was monitored prospectively in health facilities in Jimma town, Oromia Region, southwestern Ethiopia from July 2014 to June 2015. Seasonal cross-sectional parasite prevalence surveys in local communities were conducted in 2014 and 2015 in eight kebeles. Case report forms were administered to obtain sociodemographic and epidemiological information from patients. RESULTS: A total of 1434 suspected malaria cases were examined from the health facilities and 428 confirmed malaria cases were found. Among them, 327 (76.4 %) cases were Plasmodium vivax, 97 (22.7 %) were Plasmodium falciparum, and 4 (0.9 %) were mixed infection of P. vivax and P. falciparum. The annual malaria incidence rate was 1.7 cases per 1000 people at risk. Parasite prevalence in the community was less than 3 %. Household ownership of insecticide-treated nets (ITNs) was 47.3 % (1173/2479) and ITN usage was 37.9 %. All ITNs were long-lasting insecticidal nets, and repellent use was not found in the study area. Being male and traveling were the significant risk factors for P. falciparum malaria. For P. vivax malaria, risk factors included occupation and history of malaria illness during the preceding 30 days. CONCLUSION: Epidemiological evidence suggested low clinical malaria incidence and prevalence in Jimma town. More aggressive measures may be needed to further suppress vivax transmission. Strategies should be planned targeting sustained control and elimination.