An overview of artemisinin-resistant malaria and associated Pfk13 gene mutations in Central Africa.

Malaria caused by Plasmodium falciparum is one of the deadliest and most common tropical infectious diseases. However, the emergence of artemisinin drug resistance associated with the parasite's Pfk13 gene, threatens the public health of individual countries as well as current efforts to reduce malaria burdens globally. It is of concern that artemisinin-resistant parasites may be selected or have already emerged in Africa. This narrative review aims to evaluate the published evidence concerning validated, candidate, and novel Pfk13 polymorphisms in ten Central African countries. Results show that four validated non-synonymous polymorphisms (M476I, R539T, P553L, and P574L), directly associated with a delayed therapy response, have been reported in the region. Also, two Pfk13 polymorphisms associated to artemisinin resistance but not validated (C469F and P527H) have been reported. Furthermore, several non-validated mutations have been observed in Central Africa, and one allele A578S, is commonly found in different countries, although additional molecular and biochemical studies are needed to investigate whether those mutations alter artemisinin effects. This information is discussed in the context of biochemical and genetic aspects of Pfk13, and related to the regional malaria epidemiology of Central African countries.

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.

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.

Prevalence of G6PD deficiency and associated haematological parameters in children from Botswana.

Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is commonly seen in malaria endemic areas as it is known to confer a selective advantage against malaria. Recently, we reported a high proportion of asymptomatic reservoir of Plasmodium vivax in Botswana, that calls for intervention with primaquine to achieve radical cure of vivax malaria. Considering that individuals with this enzyme deficiency are at risk of haemolysis following primaquine treatment, assessment of the population for the relative frequency of G6PD deficiency is imperative. Samples from 3019 children from all the districts of Botswana were successfully genotyped for polymorphisms at positions 202 and 376 of the G6PD gene. Haematological parameters were also measured. The overall population allele frequency (based on the hemizygous male frequency) was 2.30% (95% CI, 1.77-2.83), while the overall frequency of G6PD-deficient genotypes A- (hemizygote and homozygote genotypes only) was 1.26% (95% CI, 0.86-1.66). G6PD deficiency is spread in Botswana according to the historical prevalence of malaria with a North-West to South-East decreasing gradient trend. There was no association between G6PD status and P. vivax infection. G6PD A- form was found to be associated with decreased RBC count and haemoglobin levels without a known cause or illness. In conclusion, we report for the first time the prevalence of G6PD deficiency in Botswana which is relevant for strategies in the malaria elimination campaign. Further work to examine the activities of the enzyme in the Botswana population at risk for malaria is warranted.

Human cytochrome P450 2B6 genetic variability in Botswana: a case of haplotype diversity and convergent phenotypes.

Identification of inter-individual variability for drug metabolism through cytochrome P450 2B6 (CYP2B6) enzyme is important for understanding the differences in clinical responses to malaria and HIV. This study evaluates the distribution of CYP2B6 alleles, haplotypes and inferred metabolic phenotypes among subjects with different ethnicity in Botswana. A total of 570 subjects were analyzed for CYP2B6 polymorphisms at position 516 G > T (rs3745274), 785 A > G (rs2279343) and 983 T > C (rs28399499). Samples were collected in three districts of Botswana where the population belongs to Bantu (Serowe/Palapye and Chobe) and San-related (Ghanzi) ethnicity. The three districts showed different haplotype composition according to the ethnic background but similar metabolic inferred phenotypes, with 59.12%, 34.56%, 2.10% and 4.21% of the subjects having, respectively, an extensive, intermediate, slow and rapid metabolic profile. The results hint at the possibility of a convergent adaptation of detoxifying metabolic phenotypes despite a different haplotype structure due to the different genetic background. The main implication is that, while there is substantial homogeneity of metabolic inferred phenotypes among the country, the response to drugs metabolized via CYP2B6 could be individually associated to an increased risk of treatment failure and toxicity. These are important facts since Botswana is facing malaria elimination and a very high HIV prevalence.

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.