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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.

Malaria is spread by the transmission of sexual stage parasites, called gametocytes. However, with Plasmodium falciparum, gametocytes can only be detected in peripheral blood when they are mature and transmissible to a mosquito, which complicates control efforts. Here, we identify the set of genes overexpressed in patient blood samples with high levels of gametocyte-committed ring stage parasites. Expression of all 18 genes is regulated by transcription factor AP2-G, which is required for gametocytogenesis. We select three genes, not expressed in mature gametocytes, to develop as biomarkers. All three biomarkers we validate in vitro using 6 different parasite lines and develop an algorithm that predicts gametocyte production in ex vivo samples and volunteer infection studies. The biomarkers are also sensitive enough to monitor gametocyte production in asymptomatic P. falciparum carriers allowing early detection and treatment of infectious reservoirs, as well as the in vivo analysis of factors that modulate sexual conversion., Malaria gametocytes are sexual-stage parasites transmitted from mammalian host’s blood back to their insect vector. Here, Prajapati et al. identify gametocyte-committed ring-stage biomarkers allowing to forecast malaria transmission potential.

Background Red blood cell (RBC) polymorphisms are suggested to influence the course of Plasmodium falciparum malaria. Whereas some variants have been found to be protective, others have been found to enhance parasite development. This study evaluated the effect of variant haemoglobin (Hb) and ABO blood groups on P. falciparum merozoite invasion, multiplication rates as well as gametocyte development. Methods Approximately 2.5 mL of venous blood was collected from each participant. Flow cytometry was used to determine the in vitro merozoite invasion rates of NF54 parasites into the blood of 66 non-parasitaemic individuals with variant Hb genotypes (HbSS, HbSC) and blood groups (A, B, O), which were then compared with invasion into HbAA blood. The ex vivo asexual parasite multiplication and gametocyte production rates of parasites from 79 uncomplicated malaria patients with varying Hb genotypes (HbAS, HbAC and HbAA) were also estimated using microscopy. Results Merozoite invasion rates were significantly reduced by about 50% in RBCs containing HbSS and HbSC relative to HbAA cells. The presence of blood group O and B reduced the invasion rates of HbSS by about 50% and 60%, respectively, relative to HbSC but the presence of blood group A removed the inhibitory effect of HbSS. The initial parasite densities in uncomplicated malaria patients with Hb genotypes HbAS and HbAC cells were similar but significantly lower than those with genotype HbAA. The ex vivo parasite multiplication rate, gametocytaemia and gametocyte conversion rates followed a similar trend but did not reach statistical significance (p > 0.05). Conclusions Parasite invasion rate into erythrocytes is dependent on both erythrocyte blood group antigen and haemoglobin genotype as blood group O and B provided protection via reduced merozoite invasion in RBCs containing HbSS relative to HbSC. Regardless of haemoglobin type, greater than 70% malaria patients had circulating ring stage parasites that differentiated into stage II gametocytes in 4 days.

Background Natural exposure to gametocytes can result in the development of immunity against the gametocyte by the host as well as genetic diversity in the gametocyte. This study evaluated the quantity and quality of natural immune responses against a gametocyte antigen, Pfs230 as well as the prevalence and diversity of gametocytes circulating in children living in two communities in southern Ghana. Methods Whole blood (2.5 ml) was collected from 137 non-febrile school children aged between 6 and 12 years old quarterly for a 6-month period. A drop of blood was used to prepare thick and thin blood films for parasite prevalence and density estimation. Subsequently, stored plasma samples were used in ELISAs assays to measure antibody responses and avidity against Pfs230. RNA was extraction from Trizol preserved packed cells and subsequently converted to complementary DNA (cDNA) which was used for reverse transcriptase PCR (RT-PCR) to determine gametocytes prevalence and diversity. Results Gametocyte carriage in the peak season (July) determined by Pfg377 RT-PCR was 49.2% in Obom and 22.2% in Abura, and was higher than that determined by microscopy. Gametocyte diversity was low and predominated by the same allele at both sites. The relative avidity index for antibodies measured in Abura was higher than that recorded in Obom at all time points although Pfs230 IgG concentrations were significantly high (P

Plasmodium sexual differentiation is required for malaria transmission, yet much remains unknown about its regulation. Here, we quantify early gametocyte-committed ring (gc-ring) stage, P. falciparum parasites in 260 uncomplicated malaria patient blood samples 10 days before maturation to transmissible stage V gametocytes using a gametocyte conversion assay (GCA). Seventy six percent of the samples have gc-rings, but the ratio of gametocyte to asexual-committed rings (GCR) varies widely (0–78%). GCR correlates positively with parasitemia and is negatively influenced by fever, not hematocrit, age or leukocyte counts. Higher expression levels of GDV1-dependent genes, ap2-g, msrp1 and gexp5, as well as a gdv1 allele encoding H217 are associated with high GCR, while high plasma lysophosphatidylcholine levels are associated with low GCR in the second study year. The results provide a view of sexual differentiation in the field and suggest key regulatory roles for clinical factors and gdv1 in gametocytogenesis in vivo., Here, the authors quantify early gametocyte-committed ring (gc-ring) stage Plasmodium falciparum parasites in 260 malaria patients 10 days before maturation to transmissible stage V gametocytes, and show that the ratio of circulating gc-rings is positively correlated with parasitemia and negatively correlated with body temperature.

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.

Plasmodium falciparum infections presenting either as symptomatic or asymptomatic may contain sexual stage parasites (gametocytes) that are crucial to malaria transmission. In this study, the prevalence of microscopic and submicroscopic asexual and gametocyte parasite stages were assessed in asymptomatic children from two communities in southern Ghana. Eighty children aged twelve years and below, none of whom exhibited signs of clinical malaria living in Obom and Cape Coast were sampled twice, one during the rainy (July 2015) and subsequently during the dry (January 2016) season. Venous blood was used to prepare thick and thin blood smears, spot a rapid malaria diagnostic test (PfHRP2 RDT) as well as prepare filter paper blood spots. Blood cell pellets were preserved in Trizol for RNA extraction. Polymerase chain reaction (PCR) and semi-quantitative real time reverse transcriptase PCR (qRT-PCR) were used to determine submicroscopic parasite prevalence. In both sites 87% (95% CI: 78-96) of the asymptomatic individuals surveyed were parasites positive during the 6 month study period. The prevalence of asexual and gametocyte stage parasites in the rainy season were both significantly higher in Obom than in Cape Coast (P < 0.001). Submicroscopic gametocyte prevalence was highest in the rainy season in Obom but in the dry season in Cape Coast. Parasite prevalence determined by PCR was similar to that determined by qRT-PCR in Obom but significantly lower than that determined by qRT-PCR in Cape Coast. Communities with varying parasite prevalence exhibit seasonal variations in the prevalence of gametocyte carriers. Submicroscopic asymptomatic parasite and gametocyte carriage is very high in southern Ghana, even during the dry season in communities with low microscopic parasite prevalence and likely to be missed during national surveillance exercises.

Background During a Plasmodium infection, exposure of human host immune cells to both the asexual and the sexual stages of the parasite elicit immune responses. These responses may be protective and prevent the development of high parasitaemia and its associated clinical symptoms, or block the transmission of malaria to an uninfected person. This study aimed at examining the dynamics of naturally acquired immune responses against the asexual and sexual forms of Plasmodium falciparum as well as assessing differences in the multiplicity of infection (MOI) in asymptomatic Ghanaian children living in two communities with varying malaria transmission intensities. Methods School children aged between 6 and 12 years were recruited from Obom, a high malaria prevalence setting and Abura, a low malaria prevalence setting and enrolled in monthly multiple cross sectional surveys between February and May 2015. Filter paper blood blots (DBS) as well as thick and thin blood smears were made from finger-pricked blood at each visit. Plasmodium falciparum parasite prevalence was determined by microscopy and PCR. Serum eluted from the DBS were used to assess anti-Pfs230 (sexual stage) and anti-MSP3 (asexual stage) antibody levels using indirect ELISA and DNA extracted from the DBS used to assess MOI. Results Malaria parasite point prevalence and MOI throughout the study was higher in Obom than Abura. The trend of parasite prevalence estimated by microscopy was similar to that determined by PCR in Obom but not in Abura. The trend of MSP3 antibody seroprevalence followed that of PCR-estimated parasite prevalence in Obom, while in Abura the trend of Pfs230 antibody seroprevalence followed that of PCR-estimated parasite prevalence. Conclusions Microscopy can more accurately predict changes in parasite prevalence in high transmission settings than low transmission settings. In high transmission settings, P. falciparum parasite prevalence can predict antibody seroprevalence to MSP3, whilst in low transmission settings, seroprevalence against Pfs230 may be a useful predictor of parasite prevalence. Electronic supplementary material The online version of this article (10.1186/s13071-017-2607-5) contains supplementary material, which is available to authorized users.

Background Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked genetic disorder that results in impaired enzyme activity. Although G6PD deficiency is globally distributed it is more prevalent in malaria-endemic countries. Several mutations have been identified in the G6PD gene, which alter enzyme activity. The G6PD genotype predominantly found in sub-Saharan Africa is the G6PDB (G6PD376A) with (G6PD376G) and G6PDA- (G6PD376G/202A, G6PD376G/542T, G6PD376G/680T and G6PD376G/968C) occurring at lower frequencies. Aim The aim of this study was to identify the prevalence of G6PD deficiency and asymptomatic Plasmodium falciparum carriage in children living in southern Ghana and determine whether G6PD deficiency influences asymptomatic carriage of P. falciparum parasites. Methods Blood samples were obtained once a month from 170 healthy Ghanaian school children aged between 5 and 12 years from Basic schools in two communities Obom and Abura with similar rainfall patterns and malaria peak seasons. G6PD enzyme activity was assessed using the qualitative G6PD RDT kit (AccessBIO). G6PD genotyping and asymptomatic parasite carriage was determined by PCR followed by restriction fragment length polymorphism (RFLP) of DNA extracted from dried blood spots. Results The only sub-Saharan G6PD A- allele detected was the A376G/G202A found in 12.4 % (21/170), of the children and distributed as 4.1 % (7/170) A-, 1.8 % (3/170) A-/A- homozygous deficient males and females and 6.5 % (11/170) A/A- and B/A- heterozygous deficient females. Phenotypically, 10.6 % (15/142) of the children were G6PD deficient. The asymptomatic carriage of P. falciparum by PCR was 50, 29.4, 38.2 and 38.8 % over the months of February through May 2015, respectively, and 28.8, 22.4, 25.9 and 5.9 % by microscopy during the same periods. Conclusions G6PD deficiency was significantly associated with a lowered risk of PCR-estimated asymptomatic P. falciparum carriage in children during the off peak malaria season in Southern Ghana. Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1440-1) contains supplementary material, which is available to authorized users.

Background The recently introduced SYBR Green1 (SG) assay for testing parasites susceptibility to anti-malarial drugs needs further improvement. This has been necessitated by various setbacks, the major one being the low fluorescence intensity associated with it use. This shortcoming diminishes the anticipated hope that this novel method was going to replace the more traditional ones, such as the isotopic and microscopy. In order to restore confidence in its use, series of experiments to determine conditions that give the best fluorescence intensity were conducted. Methods Conditions that yield the maximum fluorescent signal were ascertained by measuring the fluorescence after incubation of Plasmodium falciparum culture at different parasites concentration with lysis buffer containing SYBR Green (LBS) at different time period. In order to ascertain the effect of freeze–thaw on fluorescence intensity, P. falciparum culture was frozen for 1 h, thawed, incubated with LBS and the fluorescence measured. The optimized conditions determined in this study were then used to assess the susceptibility of clinical isolates of P. falciparum to artesunate, chloroquine and mefloquine. The concentration of anti-malarial drug inhibiting parasite growth by 50 % (IC50) for each drug was estimated using the online ICEstimator. The IC50 generated using the optimized SG method determined in this study was compared with that obtained using microscopic method and the previously reported standard SG method. Results Over all, the SG method was found to be easy to perform and sensitive. Freeze–thaw of parasite culture followed by incubation with lysis buffer containing the dye for 3 h was consistently observed to give the highest fluorescence signal. The IC50 values for chloroquine, mefloquine and artesunate determined were consistent and comparable with that determined with the previously reported standard SG method and the microscopic method. Conclusion The authors conclude that freezing and thawing of parasite culture, followed by incubation with LBS in the dark for 3 h provided a significant improvement in fluorescence signal. The IC50 generated using the improved SG method is comparable with that from microscopy and the standard method.

Background Based on report of declining efficacy of chloroquine, Ghana shifted to the use of artemisinin-based combination therapy (ACT) in 2005 as the first-line anti-malarial drug. Since then, there has not been any major evaluation of the efficacy of anti-malarial drugs in Ghana in vitro. The sensitivity of Ghanaian Plasmodium falciparum isolates to anti-malarial drugs was, therefore, assessed and the data compared with that obtained prior to the change in the malaria treatment policy. Methods A SYBR Green 1 fluorescent-based in vitro drug sensitivity assay was used to assess the susceptibility of clinical isolates of P. falciparum to a panel of 12 anti-malarial drugs in three distinct eco-epidemiological zones in Ghana. The isolates were obtained from children visiting health facilities in sentinel sites located in Hohoe, Navrongo and Cape Coast municipalities. The concentration of anti-malarial drug inhibiting parasite growth by 50% (IC50) for each drug was estimated using the online program, ICEstimator. Results Pooled results from all the sentinel sites indicated geometric mean IC50 values of 1.60, 3.80, 4.00, 4.56, 5.20, 6.11, 10.12, 28.32, 31.56, 93.60, 107.20, and 8952.50 nM for atovaquone, artesunate, dihydroartemisin, artemether, lumefantrine, amodiaquine, mefloquine, piperaquine, chloroquine, tafenoquine, quinine, and doxycycline, respectively. With reference to the literature threshold value indicative of resistance, the parasites showed resistance to all the test drugs except the artemisinin derivatives, atovaquone and to a lesser extent, lumefantrine. There was nearly a two-fold decrease in the IC50 value determined for chloroquine in this study compared to that determined in 2004 (57.56 nM). This observation is important, since it suggests a significant improvement in the efficacy of chloroquine, probably as a direct consequence of reduced drug pressure after cessation of its use. Compared to that measured prior to the change in treatment policy, significant elevation of artesunate IC50 value was observed. The results also suggest the existence of possible cross-resistance among some of the test drugs. Conclusion Ghanaian P. falciparum isolates, to some extent, have become susceptible to chloroquine in vitro, however the increasing trend in artesunate IC50 value observed should be of concern. Continuous monitoring of ACT in Ghana is recommended.

Background Plasmodium falciparum gametocytes are vital to sustaining malaria transmission. Parasite densities, multiplicity of infection as well as asexual genotype are features that have been found to influence gametocyte production. Measurements of the prevalence of Plasmodium sp. gametocytes may serve as a tool to monitor the success of malaria eradication efforts. Methods Whole blood was collected from 112 children aged between 6 months and 13 years with uncomplicated P. falciparum malaria attending three health facilities in southern Ghana from June to August, 2014 before (day 0) and 4 days after completion of anti-malaria drug treatment (day 7). Malaria parasites were observed by microscopy and polymerase chain reaction (PCR); submicroscopic gametocyte carriage was measured by a Pfs25 (PF3D7_1031000) mRNA real time reverse transcriptase polymerase chain reaction (RT-PCR). Parasite genotyping was performed on gDNA extracted from dried filter paper blood blots by amplification of the polymorphic regions of msp1 (PF3D7_0930300) and msp2 (PF3D7_0206800) using PCR. Results Microscopy estimated 3.1% (3/96) of the total population to carry gametocytes on day 0, which decreased to 2.1% (2/96) on day 7. In contrast, reverse transcriptase-real time PCR (RT-PCR) analysis of a subset of 35 samples estimated submicroscopic gametocyte carriage to be as high as 77% (27/35) using primers specific for Pfs25 (CT