Malaria Elimination in Africa: Rethinking Strategies for Plasmodium vivax and Lessons from Botswana.

The global malaria community has picked up the theme of malaria elimination in more than 90% of the world's population in the next decade. Recent reports of Plasmodium vivax (P. vivax) in sub-Saharan Africa, including in Duffy-negative individuals, threaten the efforts aimed at achieving elimination. This is not only in view of strategies that are tailored only to P. falciparum elimination but also due to currently revealed biological characteristics of P. vivax concerning the relapse patterns of hypnozoites and conservation of large biomasses in cryptic sites in the bone marrow and spleen. A typical scenario was observed in Botswana between 2008 and 2018, which palpably projects how P. vivax could endanger malaria elimination efforts where the two parasites co-exist. The need for the global malaria community, national malaria programs (NMPs), funding agencies and relevant stakeholders to engage in a forum to discuss and recommend clear pathways for elimination of malaria, including P. vivax, in sub-Saharan Africa is warranted.

Similar Ferroportin Q248H polymorphism prevalence in patients with Plasmodium falciparum malaria and control subjects in the low-endemic setting of Botswana.

INTRODUCTION: Recent evidence suggests that ferroportin (FPN) Q248H may confer a survival advantage against malaria by reducing erythrocytic intracellular iron in Africans. We investigated if FPN Q248H mutation, that is prevalent in Batswana, is a factor in limiting the susceptibility to Plasmodium falciparum malaria. METHODS: 264 archived dried blood spot samples (183 P. falciparum malaria cases and 81 controls, matched for geographical region and season for equal exposure) were genotyped. Human and P. falciparum DNA was extracted using Chelex-100 resin and P. falciparum molecular confirmation performed. Ferroportin Q248H mutation was identified by restriction fragment length polymorphism. The prevalence of the FPN Q248H mutation and allele frequency and the accompanying 95% confidence interval were calculated. A qPCR method was employed to estimate P. falciparum parasitaemia. Association between FPN and malaria susceptibility was tested using Pearson Chi-square test and Mood's median test was used to compare P. falciparum parasitaemias according to FPN Q248H mutation. RESULTS: All samples were successfully genotyped. The FPN Q248H allele frequency was 0.08 (95% CI: 0.05-0.11) in cases and 0.08 (95% CI: 0.02-0.14) in controls, consistent with Hardy-Weinberg equilibrium. The prevalence of FPN Q248H phenotype was comparable in patients with P. falciparum malaria and in un-infected individuals, 16.4% (95% CI: 11.0-21.8) vs 14.8% (95% CI: 7.1-22.5), P = 0.746. In addition, no association of presence of FPN Q248H with reduced parasitaemia was recorded, P = 0.837. CONCLUSION: In this small study, FPN Q248H polymorphism prevalence was comparable between patients with P. falciparum malaria and control subjects in the low-endemic setting of Botswana.

The Pan African Vivax and Ovale Network (PAVON): Refocusing on Plasmodium vivax, ovale and asymptomatic malaria in sub-Saharan Africa.

The recent World Malaria report shows that progress in malaria elimination has stalled. Current data acquisition by NMCPs depend on passive case detection and clinical reports focused mainly on Plasmodium falciparum (Pf). In recent times, several countries in sub-Saharan Africa have reported cases of Plasmodium vivax (Pv) with a considerable number being Duffy negative. The burden of Pv and Plasmodium ovale (Po) appear to be more than acknowledged. Similarly, the contribution of asymptomatic malaria in transmission is hardly considered by NMCPs in Africa. Inclusion of these as targets in malaria elimination agenda is necessary to achieve elimination goal, as these harbor hypnozoites. The Pan African Vivax and Ovale Network (PAVON) is a new consortium of African Scientists working in Africa on the transmission profile of Pv and Po. The group collaborates with African NMCPs to train in Plasmodium molecular diagnostics, microscopy, and interpretation of molecular data from active surveys to translate into policy. Details of the mission, rational and modus operandi of the group are outlined.

Recent Molecular Assessment of Plasmodium vivax and Plasmodium falciparum Asymptomatic Infections in Botswana.

In 2016, we reported the presence of Plasmodium vivax in Botswana through active case detection. A real-time PCR was used during a similar study in 10 districts to assess changes in the P. vivax prevalence. We assessed 1,614 children (2-13 years of age) for hemoglobin (Hb; g/dL) and Plasmodium parasites. The median age of all participants was 5.0 years (25th percentile, 3 years; 75th percentile, 8 years). The median Hb (g/dL) level was 12.1, but 18.3% of the participants had anemia (Hb < 11.0 g/dL); these participants were clustered in the younger than 5 years age group in all districts (P < 0.001). The risk of anemia decreased with age 5 years or older (odds ratio [OR], 0.26; 95% confidence interval [CI], 0.197-0.34; P < 0.001). The prevalence rates of Plasmodium parasites were as follows: P. vivax, 12.7%; P. falciparum, 12.7%; P. malariae, 0.74%; and P. ovale (P. ovale curtisi), 0.68%. Mixed infection rates were as follows: P. falciparum and P. vivax, 2.35%; P. falciparum and P. ovale curtisi, 0.56%; P. vivax and P. malariae, 0.06%; and P. falciparum and P. malariae, 0.68%. The infections were largely asymptomatic (99.6%). Using logistic regression, the risk of infection with P. vivax was highest in Kweneng East (OR, 6.2; 95% CI, 2.9-13.1), followed by South East (OR, 5.6; 95% CI, 2.5-12.3) and Ngami (OR, 5.1; 95% CI, 2.2-12.0). Compared to the risk of infection for children younger than 5 years, the risk of infection decreased for children 5 years or older in regions with high rates of P. vivax and P. falciparum infections. P. vivax and P. falciparum have expanded within the asymptomatic population in Botswana; therefore, careful attention is required for their elimination.

Pregnancy-induced Cardiovascular Pathologies: Importance of Structural Components and Lipids.

Pregnancy leads to adaptations for maternal and fetal energy needs. The cardiovascular system bears the brunt of the adaptations as the heart and vessels enable nutrient supply to maternal organs facilitated by the placenta to the fetus. The components of the cardiovascular system are critical in the balance between maternal homeostatic and fetus driven homeorhetic regulation. Since lipids intersect maternal cardiovascular function and fetal needs with growth and in stress, factors affecting lipid deposition and mobilization impact risk outcomes. Here, the cardiovascular components and functional derangements associated with cardiovascular pathology in pregnancy, vis-à-vis lipid deposition, mobilization and maternal and/or cardiac and fetal energy needs are detailed. Most reports on the components and associated pathology in pregnancy, are on derangements affecting the extracellular matrix and epicardial fat, followed by the endothelium, vascular smooth muscle, pericytes and myocytes. Targeted studies on all cardiovascular components and pathological outcomes in pregnancy will enhance targeted interventions.

Glucose-6-phosphate dehydrogenase deficiency genotypes and allele frequencies in the Kavango and Zambezi regions of northern Namibia.

BACKGROUND: Namibia has made significant gains in the fight against malaria, with a target of elimination by 2023. We examined the genotype and allele frequencies of glucose-6-phosphate dehydrogenase (G6PD) deficiency to inform decisions on primaquine use, as we recently detected clusters of Plasmodium ovale curtisi in Kavango. METHODS: A multistaged cross-sectional sampling method was used to enrol 212 children 2-9 y of age from schools and clinics in the Okavango and Zambezi regions of northern Namibia. Genotypes for the 202 G→A and 376 A→G mutations were assigned by polymerase chain reaction restriction fragment length polymorphism. RESULTS: Of the 212 subjects enrolled, genotypes were available for 210, made up of 61 males and 149 females. G6PD-deficient males (hemizygotes) and females (homozygotes) constituted 3.27% (2/61) and 0.0% (0/149), respectively. Female heterozygotes (AA- and BA-) constituted 10.07% (15/149), while G6PD wild-type males (with A or B haplotype) and females (with AA, BB or AB haplotypes) consisted of 96.72% (59/61) and 89.93% (134/149), respectively. The A-, A and B allele frequencies were 0.0474, 0.3036 and 0.6490, respectively. Hardy-Weinberg equilibrium tests for female genotype frequencies did not show deviation (p=0.29). CONCLUSIONS: The frequency of G6PD deficiency alleles in males in the Kavango and Zambezi regions of northern Namibia constitute 3.27%, a first report to inform policy on primaquine role out.

Molecular detection of P. vivax and P. ovale foci of infection in asymptomatic and symptomatic children in Northern Namibia.

BACKGROUND: Knowledge of the foci of Plasmodium species infections is critical for a country with an elimination agenda. Namibia is targeting malaria elimination by 2020. To support decision making regarding targeted intervention, we examined for the first time, the foci of Plasmodium species infections and regional prevalence in northern Namibia, using nested and quantitative polymerase chain reaction (PCR) methods. METHODS: We used cross-sectional multi-staged sampling to select 952 children below 9 years old from schools and clinics in seven districts in northern Namibia, to assess the presence of Plasmodium species. RESULTS: The median participant age was 6 years (25-75%ile 4-8 y). Participants had a median hemoglobin of 12.0 g/dL (25-75%ile 11.1-12.7 g/dL), although 21% of the cohort was anemic, with anemia being severer in the younger population (p<0.002). Most of children with Plasmodium infection were asymptomatic (63.4%), presenting a challenge for elimination. The respective parasite prevalence for Plasmodium falciparum (Pf), Plasmodium vivax (Pv) and Plasmodium ovale curtisi (Po) were (4.41%, 0.84% and 0.31%); with Kavango East and West (10.4%, 6.19%) and Ohangwena (4.5%) having the most prevalence. Pv was localized in Ohangwena, Omusati and Oshana, while Po was found in Kavango. All children with Pv/Pf coinfections in Ohangwena, had previously visited Angola, affirming that perennial migrations are risks for importation of Plasmodium species. The mean hemoglobin was lower in those with Plasmodium infection compared to those without (0.96 g/dL less, 95%CI 0.40-1.52 g/dL less, p = 0.0009) indicating that quasi-endemicity exists in the low transmission setting. CONCLUSIONS: We conclude that Pv and Po species are present in northern Namibia. Additionally, the higher number of asymptomatic infections present challenges to the efforts at elimination for the country. Careful planning, coordination with neighboring Angola and execution of targeted active intervention, will be required for a successful elimination agenda.

Preliminary survey on Anopheles species distribution in Botswana shows the presence of Anopheles gambiae and Anopheles funestus complexes.

BACKGROUND: Botswana is one of the four front line malaria elimination countries in Southern Africa, with malaria control activities that include routine vector control. Past and recent studies have shown that Anopheles arabiensis is the only known vector of Plasmodium parasites in the country. This report presents a preliminary evaluation on Anopheles species composition in seven districts of Botswana with some inferences on their vectorial role. RESULTS: Overall, 404 Anopheles mosquito females were collected, of which 196 were larvae collected from several breeding sites, and 208 were adults obtained from indoor pyrethrum spray catches (PSC). Anopheles arabiensis (58.9%) accounted for the highest relative frequency in 5 out of 7 districts sampled. The other species collected, among those identified, were barely represented: Anopheles longipalpis type C (16.3%), Anopheles parensis (8.9%), Anopheles quadriannulatus (5.4%), and Anopheles leesoni (0.2%). PCR test for human ß-globin on mosquitoes collected by PSC showed that An. arabiensis and An. parensis had bitten human hosts. Moreover, An. arabiensis showed a non-negligible Plasmodium falciparum infection rate in two sites (3.0% and 2.5% in Chobe and Kweneng West districts, respectively). CONCLUSIONS: This work provides first time evidence of Anopheles diversity in several areas of Botswana. Anopheles arabiensis is confirmed to be widespread in all the sampled districts and to be vector of P. falciparum. Moreover, the presence of Anopheles funestus group in Botswana has been assessed. Further research, entomological surveillance activities and possibly vector control programmes need to be better developed and implemented as well as targeting outdoors resting vectors.

Molecular evidence of high rates of asymptomatic P. vivax infection and very low P. falciparum malaria in Botswana.

BACKGROUND: Botswana is one of eight SADC countries targeting malaria elimination by 2018. Through spirited upscaling of control activities and passive surveillance, significant reductions in case incidence of Plasmodium falciparum (0.96 - 0.01) was achieved between 2008 and 2012. As part of the elimination campaign, active detection of asymptomatic Plasmodium species by a highly sensitive method was deemed necessary. This study was carried out to determine asymptomatic Plasmodium species carriage by nested PCR in the country, in 2012. METHOD: A cross-sectional study involving 3924 apparently healthy participants were screened for Plasmodium species in 14 districts (5 endemic: Okavango, Ngami, Tutume, Boteti and Bobirwa; and 9 epidemic: North East, Francistown, Serowe-Palapye, Ghanzi, Kweneng West, Kweneng East, Kgatleng, South East, and Good Hope). Venous blood was taken from each participant for a nested PCR detection of Plasmodium species. RESULTS: The parasite rates of asymptomatic Plasmodium species detected were as follows: Plasmodium falciparum, 0.16 %; Plasmodium vivax, 4.66 %; Plasmodium malariae, (Pm) 0.16 %; Plasmodium ovale, 0 %, mixed infections (P. falciparum and P. vivax), 0.055 %; and (P. vivax and P. malariae), 0.027 %, (total: 5.062 %). The high proportion of asymptomatic reservoir of P. vivax was clustered in the East, South Eastern and Central districts of the country. There appeared to be a correlation between the occurrence of P. malariae infection with P. vivax infection, with the former only occurring in districts that had substantial P. vivax circulation. The median age among 2-12 year olds for P. vivax infection was 5 years (Mean 5.13 years, interquartile range 3-7 years). The odds of being infected with P. vivax decreased by 7 % for each year increase in age (OR 0.93, 95 % CI 0.87-1.00, p = 0.056). CONCLUSION: We have confirmed low parasite rate of asymptomatic Plasmodium species in Botswana, with the exception of P.vivax which was unexpectedly high. This has implication for the elimination campaign so a follow up study is warranted to inform decisions on new strategies that take this evidence into account in the elimination campaign.

Extracellular hemoglobin: the case of a friend turned foe.

Hemoglobin (Hb) is a highly conserved molecule present in all life forms and functionally tied to the complexity of aerobic organisms on earth in utilizing oxygen from the atmosphere and delivering to cells and tissues. This primary function sustains the energy requirements of cells and maintains cellular homeostasis. Decades of intensive research has presented a paradigm shift that shows how the molecule also functions to facilitate smooth oxygen delivery through the cardiovascular system for cellular bioenergetic homeostasis and signaling for cell function and defense. These roles are particularly highlighted in the binding of Hb to gaseous molecules carbon dioxide (CO2), nitric oxide (NO) and carbon monoxide (CO), while also serving indirectly or directly as sources of these signaling molecules. The functional activities impacted by Hb outside of bioenergetics homeostasis, include fertilization, signaling functions, modulation of inflammatory responses for defense and cell viability. These activities are efficiently executed while Hb is sequestered safely within the confines of the red blood cell (rbc). Outside of rbc confines, Hb disaggregates and becomes a danger molecule to cell survival. In these perpectives, Hb function is broadly dichotomous, either a friend in its natural environment providing and facilitating the means for cell function or foe when dislocated from its habitat under stress or pathological condition disrupting cell function. The review presents insights into how this dichotomy in function manifests.

Haptoglobin, inflammation and disease.

Haptoglobin is an acute phase protein that scavenges haemoglobin in the event of intravascular or extravascular haemolysis. The protein exists in humans as three main phenotypes, Hp1-1, Hp2-2 and Hp2-1. Accumulated data on the protein's function has established its strong association with diseases that have inflammatory causes. These include parasitic (malaria), infectious (HIV, tuberculosis) and non-infectious diseases (diabetes, cardiovascular disease and obesity) among others. Phenotype-dependent poor disease outcomes have been linked with the Hp2-2 phenotype. The present review brings this association into perspective by looking at the functions of the protein and how defects in these functions associated with the Hp2 allele affect disease outcome. A model is provided to explain the mechanism, which appears to be largely immunomodulatory.

Pyrimethamine-resistant dihydrofolate reductase enzymes of Plasmodium falciparum are not enzymatically compromised in vitro.

Plasmodium falciparum, the protozoan that causes the most lethal form of human malaria, has been controlled principally by two safe, affordable drugs, chloroquine and sulfadoxine-pyrimethamine (SP). Studies in the laboratory and in the field have demonstrated that resistance to SP depends on non-synonymous point mutations in the dihydrofolate reductase (DHFR), and dihydropteroate synthase (DHPS) coding regions. Parasites that carry dhfr genes with 3 or 4 point mutations (51I/59R/108N triple mutation or 51I/59R/108N/164L quadruple mutation) are resistant to pyrimethamine in vitro and patients infected with these parasites respond poorly to SP treatment. The wide spread of these pyrimethamine-resistant alleles demonstrates the increased fitness over drug-sensitive alleles in the presence of the drug. However, it is not clear whether these alleles might reduce the fitness of parasites in the absence of drug pressure. As a first step, we compared the kinetic properties of the wild type, and three mutant alleles to determine whether the native DHFR-thymidylate synthase form of the mutant proteins showed compromised activity in vitro. The mutant enzymes had K(m) values for their substrate, dihydrofolate that were significantly lower than the wild type, k(cat) values in the same range as the wild type enzyme, and k(cat)/K(m) values higher than wild type. In contrast, the K(m) values for the NADPH cofactor were higher than wild type for the mutant enzymes. These observations suggest that the fitness of these parasites may not be compromised relative to those that carry the wild type allele, even without sustained SP drug pressure.

Correlates of hepatitis C virus infection among incarcerated Ghanaians: a national multicentre study.

A national multicentre cross-sectional study was undertaken on the correlates of hepatitis C virus (HCV) infection in a sample of inmates from eight Ghanaian prisons. A total of 1366 inmates from eight of the ten regional central prisons in Ghana were enrolled between May 2004 and December 2005. Subjects voluntarily completed a risk-factor questionnaire and provided blood specimens for unlinked anonymous testing for the presence of antibodies to HCV. These data were analysed using both univariate and multivariate techniques. The median age of participants was 36.5 years (range 16-84 years). Of the 1366 inmates tested, HCV seroprevalence was 18.7%. On multivariate analysis, the independent determinants of HCV infection were being incarcerated for longer than the median time served of 36 months [odds ratio (OR) 5.8; 95% confidence interval (95% CI) 5.0-6.9], history of intravenous drug use (OR 4.5; 95% CI 3.8-5.4) and homosexuality (OR 3.1; 95% CI 2.5-3.9). Consistent with similar studies worldwide, the prevalence of HCV in prison inmates was higher than the general population in Ghana, suggesting probable transmission in prisons in Ghana through intravenous drug use and unsafe sexual behaviour.

Haptoglobin gene promoter polymorphism and haplotypes are unique in different populations.

We investigated the distribution of haptoglobin (HP) alleles and haplotypes among Africans (Ghanaians), Europeans (from South Africa), and Chinese. HP*1F was present only in Africans and Europeans, whereas HP*del was unique to Chinese. Six base substitutions at the promoter region were population specific. Only 3 out of 18 haplotypes were shared among the populations. A probable application of HP in human population genetics appears legitimate.

Haptoglobin 2-2 phenotype is a risk factor for type 2 diabetes in Ghana.

We have investigated the role of haptoglobin gene polymorphisms in 129 type 2 diabetic patients and 87 non-diabetic subjects, classified by the ADA criteria, in Ghana. The diabetic subjects were recruited consecutively from the National Diabetic Management and Research Center of the University of Ghana Medical School, Korle-Bu, Accra, Ghana and were categorized by their haptoglobin phenotypes. The haptoglobin 2 allele was determined to be a risk factor for type 2 diabetes in Ghana (OR = 6.1, 95% CI = 1.8-21.2; P = .0.001) while the Hp1 allele appeared protective (OR = 0.56, 95% CI = 0.31-1.0; P = .06). The deleterious role of the Hp2 allele was further evidenced by the reduced risk associated with Hp2-1M mutant heterozygotes, who produce less Hp2 protein than the normal Hp2-1 heterozygote. (OR = 0.52, 95% CI = 0.27-1.0; P = 0.06). The subjects with the homozygous Hp2 allele were also hypertensive and overweight. There was no difference (p > 0.05) in the levels of triglycerides, total cholesterol, LDL and HDL between diabetic subjects with different haptoglobin phenotypes. We conclude that hypertensive and overweight individuals with the Hp2-2 phenotype in Ghana are at a high risk of developing type 2 diabetes and may require a more aggressive management.

Prevalence of human immunodeficiency virus, hepatitis B virus, hepatitis C virus and syphilis among prison inmates and officers at Nsawam and Accra, Ghana.

Although the high prevalence of blood-borne viral infections and syphilis in correctional facilities has been well documented globally, such data are sparse from Africa, and there has been no such data from Ghana. This study sought to estimate the prevalence of human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and syphilis among prison inmates and officers at prisons in Nsawan and Accra, Ghana. Prisoners and officers in 3 of the 46 prisons in Ghana were surveyed from May 2004 to May 2005. Subjects voluntarily completed a risk-factor questionnaire and provided blood specimens for unlinked anonymous testing for the presence of antibodies to HIV, HCV and Treponema pallidum, the causative agent of syphilis, and the surface antigen of hepatitis B virus (HBsAg). Almost 16% (3770) of the total of 23,980 prison inmates in Ghana were eligible, and 281 (7.5%) of those eligible took part, whilst almost 23% (1120) of the total of 4910 prison officers were eligible, and 82 (7.3%) of those eligible took part. For the 281 inmates tested, HIV seroprevalence was 19.2%, 17.4% had HBsAg, HCV seroprevalence was 19.2% and reactive syphilis serology was noted in 11%. For the 82 officers tested, HIV seroprevalence was 8.5%, 3.7% had HBsAg, HCV seroprevalence was 23.2% and reactive syphilis serology was noted in 4.9%. The data indicate a higher prevalence of HIV and HCV in correctional facilities (both prison inmates and officers) than in the general population in Ghana, suggesting their probable transmission in prisons in Ghana through intravenous drug use, unsafe sexual behaviour and tattooing as pertains to prisons worldwide.

A novel I247T missense mutation in the haptoglobin 2 beta-chain decreases the expression of the protein and is associated with ahaptoglobinemia.

We have identified a novel base substitution at codon 247 in the beta-chain of the haptoglobin 2 ( Hp(2)) allele in a Ghanaian with the Hp0 (ahaptoglobinemic) phenotype. The heterozygous T-->C substitution caused reduced expression of the protein when the mutant was transfected into COS7 cells. The base substitution resulted in a missense change of the non-polar amino acid isoleucine to the polar amino acid threonine at a position in the beta-chain that is highly conserved among several species. We had previously identified a mutation in the Hp gene promoter region for the same individual, which gives her genotype as -61C Hp(2)/-61C Hp(2)(I247T). Since the -61C mutation also leads to low Hp expression, the genotype represents the first and most definitive ahaptoglobinemic case reported in Africa.

Isolation and characterization of chicken GA-binding protein.

We have purified and characterized chicken liver nuclear proteins that bind to Ets binding sites (EBSs) of ribosomal protein (r-protein) gene promoters. We employed supershift assays and antibodies to mouse GA binding protein (GABP), to show that the proteins were similar to alpha and beta subunits of GABP. Western blot analysis identified 54- and 38-kDa proteins as the alpha type, and a 46-kDa protein as the beta type. When compared with nuclear extracts (NEs) of other species, we observed that the 38-kDa protein was unique to chicken, and appears to be derived from the 54-kDa protein. The 54- and 46-kDa proteins were highly expressed in chicken tissues and were major components through higher animals, indicating that both proteins have a conserved role.