A simple and rapid external standard calibration HPLC method for determination of lumefantrine in dried blood spot samples from malaria patients in Botswana.

A simple external calibration liquid chromatography-diode array detector method was developed, validated, and applied for the determination of lumefantrine (Lum) in dried blood spot (DBS) samples collected from malaria patients in Botswana. The samples were validated in accordance with the United States Food and Drug Administration guidelines for bioanalytical methods after sample preparation using solid-liquid extraction. Separation was achieved using an XTerra C18 column (50 × 4.6 mm, 5 µm), and a binary solvent system of acetonitrile and water adjusted to pH 2.3 was used as the mobile phase. The validated method was applied for the determination of Lum in DBS samples collected from malaria patients infected with Plasmodium falciparum in Botswana. The calibration curve was linear between 0.5 and 12 µg/mL with a coefficient of determination (R2 ) of 0.9996. The limit of detection and the lower limit of quantification were 0.5 and 1.4 µg/mL, respectively. The efficiency of extraction measured as percentage recovery ranged between 84.2% and 107.8% at the three quality control (QC) levels, that is, low QC, mid QC, and high QC. In conclusion, data suggest that the method is suitable for the determination of trace Lum in biofluids and can also be used for therapeutic drug monitoring and pharmacokinetic profiling.

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.

Improving the reading skills of children with neurodevelopmental disabilities: Preliminary study from Botswana.

In Botswana, Special Needs Education has been implemented for 25 years with some success but there is still a need for evidence-based methods like Frequency Building, behavioural fluency, and Precision Teaching to be used to measure and improve school performance and learning. We explored the impact of these behavioural technologies on reading performances of four children with learning disorders (ADHD, speech impairment and acquired brain disorder) in a special school in Gaborone. At the assessment, two children were unable to read letter sounds and two could not read sight words. Reading performances were measured with frequency and displayed on a standard celeration chart. During the intervention, the length of the tasks was reduced and then augmented. Findings revealed that after 3 months of intervention children significantly increased their score stimulating self-confidence and enthusiasm during activities. This work demonstrates that behavioural technologies can be applied in Africa without using expensive or time-consuming resources.

Molecular and morphological identification of suspected Plasmodium vivax vectors in Central and Eastern Sudan.

BACKGROUND: In spite of the global effort to eliminate malaria, it remains the most significant vector-borne disease of humans. Plasmodium falciparum is the dominant malaria parasite in sub-Saharan Africa. However, Plasmodium vivax is becoming widely spread throughout Africa. The overuse of vector control methods has resulted in a remarkable change in the behaviour of mosquito that feeds on human as well as on vector composition. The aim of this study was to identify Anopheles mosquito species in vivax malaria endemic regions and to investigate their role in P. vivax circumsporozoite protein (Pvcsp) allele diversity. METHODS: Mosquito samples were collected from Central Sudan (Rural Khartoum and Sennar) and Eastern Sudan (New Halfa, Kassala state) using pyrethrum spray catch (PSC) and CDC light traps. Mosquitoes were identified using appropriate morphological identification keys and Anopheles gambiae complex were confirmed to species level using molecular analysis. A subset of blood-fed anopheline mosquitoes were dissected to determine the presence of natural infection of malaria parasites. In addition, the rest of the samples were investigated for the presence of Pvcsp gene using nested-PCR. RESULTS: A total of 1037 adult anopheline mosquitoes were collected from New Halfa (N = 467), Rural Khartoum (N = 132), and Sennar (N = 438). Morphological and molecular identification of the collected mosquitoes revealed the presence of Anopheles arabiensis (94.2%), Anopheles funestus (0.5%), and Anopheles pharoensis (5.4%). None of the dissected mosquitoes (N = 108) showed to be infected with malaria parasite. Overall P. vivax infectivity rate was 6.1% (63/1037) by Pvcsp nested PCR. Co-dominance of An. arabiensis and An. pharoensis is reported in Sennar state both being infected with P. vivax. CONCLUSION: This study reported P. vivax infection among wild-caught anopheline mosquitoes in Central and Eastern Sudan. While An. arabiensis is the most abundant vector observed in all study areas, An. funestus was recorded for the first time in New Halfa, Eastern Sudan. The documented Anopheles species are implicated in Pvcsp allele diversity. Large-scale surveys are needed to identify the incriminated vectors of P. vivax malaria and determine their contribution in disease transmission dynamics.

A fast-screening dispersive liquid-liquid microextraction-gas chromatography-mass spectrometry method applied to the determination of efavirenz in human plasma samples.

We demonstrate the suitability of a fast, green, easy-to-perform, and modified sample extraction procedure, i.e., dispersive liquid-liquid microextraction (DLLME) for the determination of efavirenz (EFV) in human plasma. Data acquisition was done by gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring (SIM) mode. The simplicity of the method lies in, among others, the avoidance of the use of large organic solvent volumes as mobile phases and non-volatile buffers that tend to block the plumbing in high-performance liquid chromatography (HPLC). Chromatographic and mass spectral parameters were optimized using bovine whole blood for matrix matching due to insufficient human plasma. Method validation was accomplished using the United States Food and Drug Administration (USFDA) 2018 guidelines. The calibration curve was linear with a dynamic range of 0.10-2.0 µg/mL and an R2 value of 0.9998. The within-run accuracy and precision were both less than 20% at the lower limit of quantification (LLOQ) spike level. The LLOQ was 0.027 µg/mL which compared well with some values but was also orders of magnitude better than others reported in the literature. The percent recovery was 91.5% at the LLOQ spike level. The DLLME technique was applied in human plasma samples from patients who were on treatment with EFV. The human plasma samples gave concentrations of EFV ranging between 0.14-1.00 µg/mL with three samples out of seven showing concentrations that fell within or close to the recommended therapeutic range.

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 Plasmodium vivax infection in Duffy negative symptomatic individuals from Dschang, West Cameroon.

BACKGROUND: Plasmodium vivax infection is known to be rare in West/Central Africa, the most accepted explanation being the lack of expression of erythroid Duffy antigen in the local human populations. Duffy negativity prevents the parasite to exploit the entry mechanism on the red blood cell surface. However, there are a growing number of reported vivax infections in Duffy-negative individuals. Data on P. vivax circulation in Cameroon are limited. The aim of the study was to evaluate the P. vivax presence, and its association with the Duffy genotype in West Cameroon. RESULTS: Overall, 484 blood samples were collected consecutively from febrile outpatients attending the Dschang's Hospital (West Cameroon) during a 3-months period. Plasmodium vivax infection was detected by PCR in 5.6% (n = 27/484) of the cases, representing 38.6% (n = 27/70) of all Plasmodium infections detected. All P. vivax infected individuals showed a Duffy-negative genotype, and the frequency of Duffy-positive individuals in the whole tested population was 1.7%. CONCLUSIONS: The results of this study confirm the circulation of P. vivax in Cameroon, as well as that the lack of expression of Duffy-antigen does not confer full protection against vivax malaria acquisition.

Biocide resistance in Klebsiella pneumoniae: a narrative review.

Klebsiella pneumoniae is among the World Health Organization's list of priority pathogens, notorious for its role in causing healthcare-associated infections and neonatal sepsis globally. Containment of K. pneumoniae transmission depends on the continued effectiveness of antimicrobials and of biocides used for topical antisepsis and surface disinfection. Klebsiella pneumoniae is known to disseminate antimicrobial resistance (AMR) through a large auxiliary genome made up of plasmids, transposons and integrons, enabling it to evade antimicrobial killing through the use of efflux systems and biofilm development. Because AMR mechanisms are also known to impart tolerance to biocides, AMR is frequently linked with biocide resistance (BR). However, despite extensive research on AMR, there is a gap in knowledge about BR and the extent to which AMR and BR mechanisms overlap remains debatable. The aim of this paper is to review and summarise the current knowledge on the determinants of BR in K. pneumoniae and highlight content areas that require further inquiry.

Pharmacogenetics of Breast Cancer Treatments: A Sub-Saharan Africa Perspective.

Breast cancer is the most frequent cause of cancer death in low- and middle-income countries, in particular among sub-Saharan African women, where response to available anticancer treatment therapy is often limited by the recurrent breast tumours and metastasis, ultimately resulting in decreased overall survival rate. This can also be attributed to African genomes that contain more variation than those from other parts of the world. The purpose of this review is to summarize published evidence on pharmacogenetic and pharmacokinetic aspects related to specific available treatments and the known genetic variabilities associated with metabolism and/or transport of breast cancer drugs, and treatment outcomes when possible. The emphasis is on the African genetic variation and focuses on the genes with the highest strength of evidence, with a close look on CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4/5, CYP19A1, UGT1A4, UGT2B7, UGT2B15, SLC22A16, SLC38A7, FcγR, DPYD, ABCB1, and SULT1A1, which are the genes known to play major roles in the metabolism and/or elimination of the respective anti-breast cancer drugs given to the patients. The genetic variability of their metabolism could be associated with different metabolic phenotypes that may cause reduced patients' adherence because of toxicity or sub-therapeutic doses. Finally, this knowledge enhances possible personalized treatment approaches, with the possibility of improving survival outcomes in patients with breast cancer.

Mosquito vector diversity and abundance in southern Botswana, in a global context of emerging pathogen transmission.

Background: The continued spread of infectious diseases by mosquitoes remains a formidable obstacle to the well-being of the people all over the world. Arboviruses are spread from one vertebrate host to another by vectors through intricate transmission cycles that involve the virus, the vertebrate host, and the vector. It is essential to acquire a better understanding of the current abundance and distribution of major vectors in order to adequately prepare for the possibility of arbovirus outbreaks. This is because the abundance and distribution of these major vectors determines the human populations that are at risk for the diseases that they transmit. The effects of climate change on the amount of mosquitoes and their ability to survive the seasons have had a substantial impact on the spread of diseases that are transmitted by vectors in many different parts of Botswana. Methods: The purpose was to collect mosquito samples in Gaborone and the neighboring areas in southern Botswana, including border stations. We collected different stages of the mosquito from each place, raised them to maturity, and then identified them. Both morphological and genetic studies were utilized in order to successfully identify the organism. The species of Culex mosquitoes accounted for 88.3% of the 5177 mosquitoes that were collected and identified, whereas the species of Aedes aegypti and Anopheles mosquitoes accounted for 11.5% and 0.2% respectively. Conclusions: These findings give entomological baseline data that will aid in the study of vectorial patterns and the estimation of future arboviral hazards provided by mosquitoes. Additionally, these findings document the diversity and abundance of mosquito species.

Dengue and Chikungunya virus circulation in Cameroon and Gabon: molecular evidence among symptomatic individuals.

We report the molecular evidence of dengue virus (DENV) and chikungunya virus (CHIKV) infection in symptomatic individuals in Cameroon and Gabon, respectively. Arthropod-borne viruses (arboviruses) are distributed in the tropical or subtropical regions, with DENV having the highest burden. The morbidity and mortality related to arboviral diseases raise the concern of timely and efficient surveillance and care. Our aim was to assess the circulation of arboviruses [DENV, CHIKV, Zika virus (ZIKV)] among febrile patients in Dschang (West Cameroon) and Kyé-ossi (South Cameroon, border with Gabon and Equatorial Guinea). Dried blood spots were collected from 601 consenting febrile patients, and 194 Plasmodium spp.-negative samples were tested for the molecular detection of cases of DENV, CHIKV and ZIKV infection. Overall, no case of ZIKV infection was found, whereas one case of DENV infection and one case of CHIKV infection were detected in Dschang and Kyé-ossi, respectively, with the CHIKV-infected patient being resident in Gabon. Our findings suggest the need to establish an active surveillance of arbovirus transmission in Cameroon and bordering countries.

Plasmodium vivax Infections Detected in a Large Number of Febrile Duffy-Negative Africans in Dschang, Cameroon.

The Duffy blood group is a critical receptor for Plasmodium vivax (P. vivax) invasion of red blood cells, and consequently, P. vivax infections were considered rare in sub-Saharan Africa where the prevalence of Duffy-negativity is high. However, recently, P. vivax infections have been found in Duffy-negative Africans throughout the malaria transmission area of sub-Saharan Africa, raising important questions concerning the molecular composition of these P. vivax clones and the red blood cell receptors that facilitate their invasion. Here, we describe an unusually high number of P. vivax infections in febrile Duffy-negative Africans in Dschang, Cameroon (177 of 500 outpatients), as compared with Santchou (two of 400 outpatients) and Kyé-ossi (two of 101 outpatients), in other areas in Cameroon. In the discussion, we speculate on the possible reasons why Dschang might account for the unusually large numbers of P. vivax infections in Duffy-negative individuals living there.

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.

Contrasting epidemiology and genetic variation of Plasmodium vivax infecting Duffy-negative individuals across Africa.

OBJECTIVES: Plasmodium vivax malaria was thought to be rare in Africans who lack the Duffy blood group antigen expression. However, recent studies indicate that P. vivax can infect Duffy-negative individuals and has spread into areas of high Duffy negativity across Africa. Our study compared epidemiological and genetic features of P. vivax between African regions. METHODS: A standardized approach was used to identify and quantify P. vivax from Botswana, Ethiopia, and Sudan, where Duffy-positive and Duffy-negative individuals coexist. The study involved sequencing the Duffy binding protein (DBP) gene and inferring genetic relationships among P. vivax populations across Africa. RESULTS: Among 1215 febrile patients, the proportions of Duffy negativity ranged from 20-36% in East Africa to 84% in southern Africa. Average P. vivax prevalence among Duffy-negative populations ranged from 9.2% in Sudan to 86% in Botswana. Parasite density in Duffy-negative infections was significantly lower than in Duffy-positive infections. P. vivax in Duffy-negative populations were not monophyletic, with P. vivax in Duffy-negative and Duffy-positive populations sharing similar DBP haplotypes and occurring in multiple, well-supported clades. CONCLUSIONS: Duffy-negative Africans are not resistant to P. vivax, and the public health significance of this should not be neglected. Our study highlights the need for a standardized approach and more resources/training directed towards the diagnosis of vivax malaria in Africa.

Prevalence of Cryptosporidium parvum, Giardia intestinalis and molecular characterization of group A rotavirus associated with diarrhea in children below five years old in Gaborone, Botswana.

INTRODUCTION: Cryptosporidium, Giardia and rotaviruses are amongst the leading causes of acute gastroenteritis in children ≤5 years worldwide. The purpose of this study was to determine the occurrence of Cryptosporidium parvum, Giardia intestinalis and molecular characteristics of rotaviruses after Rotarix® introduction in Botswana. METHODS: in this case study, 200 diarrheic stool specimens and 100 control samples from children under five years old were collected between March and November, 2017. Samples were analyzed by modified Ziehl Neelsen staining technique for cryptosporidium, wet mount procedure for Giardia and negative samples were confirmed by immunochromatographic assay. Specimens were analyzed for rotavirus by ELISA, PAGE, RT-PCR, sequencing of VP7 and VP4 antigen followed by phylogenetic analysis. RESULTS: prevalence rates of 20.5%, 16.5% and 11.0% in diarrhea cases were observed for Cryptosporidium parvum, Giardia intestinalis and rotavirus, respectively. Four percent of diarrheic specimens had multiple infections. The predominant rotavirus genotype was GIP[8] (7/15) followed by G2P[4] (2/15) and G3P[8] (1/15). Twenty percent of specimens were non-typeable. One mixed strain, G1+G2P[4,8] (2/15), was detected. Phylogenetic analysis of VP4 and VP7 sequences clustered Botswana rotavirus strains within G1 lineages 1 and 2, G3 lineage 1, P[8] lineage 3 and P[4] lineage 5 together with Southern African strains. CONCLUSION: this study provides important information on occurrence and demographic risk groups for Cryptosporidium parvum, Giardia intestinalis and rotavirus in young children as well as genetic diversity of rotaviruses after vaccine introduction in Botswana. Constant monitoring of circulating rotavirus strains is essential in assessing effectiveness of current vaccines in Botswana.

Past and current biological factors affecting malaria in the low transmission setting of Botswana: A review.

Malaria continues to be one of the top infectious agents contributing to morbidity and mortality in sub-Saharan Africa. Annually, Botswana accounts only for a small proportion of cases (<<1%). Despite significantly reduced incidence rate, the country still experiences sporadic outbreaks that hamper the goal of malaria elimination. This review evaluated previous and current biological factors that impact malaria in Botswana, specifically focussing on the vectors, the parasite and the host. This was accomplished via a literature review evaluating these variables in Botswana. Current literature suggests that Anopheles arabiensis is the main malaria vector in the country. Several other potential vectors have been found widely distributed throughout Botswana in high numbers, yet remain largely unstudied with regards to their contribution to the country's malaria burden. We also report the most up to date list of all Anopheles species that have been found in Botswana. Plasmodium falciparum is responsible for the vast majority of symptomatic malaria in the country and some drug resistance markers have been documented for this species. Plasmodium vivax has been reported in asymptomatic subjects, even though a large proportion of the Botswana population appears to be Duffy antigen negative. Very little is known about the true distribution of P. vivax and no point of care testing infrastructure for this species exists in Botswana, making it difficult to tailor treatment to address possible recrudescence or relapse. Due to a genetically diverse population with a substantial Khoisan contribution into the Bantu genetic background, several phenotypes that potentially impact prevalence and severity of malaria exist within the country. These include sickle cell trait, Glucose-6-Phosphate Dehydrogenase deficiency, and Duffy negativity. This review highlights the information that currently exists on malaria in Botswana. It also postulates that a comprehensive understanding of these aforementioned biological factors may help to explain malaria persistence in Botswana.

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.

The peopling of the last Green Sahara revealed by high-coverage resequencing of trans-Saharan patrilineages.

BACKGROUND: Little is known about the peopling of the Sahara during the Holocene climatic optimum, when the desert was replaced by a fertile environment. RESULTS: In order to investigate the role of the last Green Sahara in the peopling of Africa, we deep-sequence the whole non-repetitive portion of the Y chromosome in 104 males selected as representative of haplogroups which are currently found to the north and to the south of the Sahara. We identify 5,966 mutations, from which we extract 142 informative markers then genotyped in about 8,000 subjects from 145 African, Eurasian and African American populations. We find that the coalescence age of the trans-Saharan haplogroups dates back to the last Green Sahara, while most northern African or sub-Saharan clades expanded locally in the subsequent arid phase. CONCLUSIONS: Our findings suggest that the Green Sahara promoted human movements and demographic expansions, possibly linked to the adoption of pastoralism. Comparing our results with previously reported genome-wide data, we also find evidence for a sex-biased sub-Saharan contribution to northern Africans, suggesting that historical events such as the trans-Saharan slave trade mainly contributed to the mtDNA and autosomal gene pool, whereas the northern African paternal gene pool was mainly shaped by more ancient events.

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.