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Background: Human serum is a major component of Plasmodium falciparum culture medium, and can be replaced with AlbuMAX™ II, a lipid-rich bovine serum albumin, for asexual cultures. However, gametocytes produced without serum are poorly infective to mosquitoes. Serum suffers from high cost, limited availability, and variability in quality. Methods: Several commercially-available media supplements were tested for their ability to support parasite growth and production of P. falciparum (3D7) gametocytes in standard RPMI1640 medium containing 0.5% AlbuMAX. The impact on asexual growth and gametocyte production with each supplement was assessed and compared to standard RPMI1640 medium containing 10% human serum, as well as to medium containing 0.5% AlbuMAX alone. The infectivity of gametocytes produced with one supplement to Anopheles gambiae sensu stricto was assessed by standard membrane feeding assay and measuring both prevalence of infection and oocyst intensity. Results: Supplementation of medium containing 0.5% AlbuMAX with five supplements did not affect asexual growth of P. falciparum, and four of the five supplements supported early gametocyte production. The supplement producing the highest number of gametocytes, ITS-X, was further investigated and was found to support the production of mature gametocytes. Infection prevalence and oocyst intensity did not differ significantly between mosquitoes given a membrane feed containing gametocytes grown in medium with 0.5% AlbuMAX + ITS-X and those grown in medium with 10% human serum. Infection prevalence and oocyst intensity was significantly higher in case of ITS–X supplementation when compared to AlbuMAX alone. Infectious gametocytes were also produced from two field clones using ITS–X supplementation. Conclusions: Serum-free medium supplemented with ITS-X was able to support the growth of gametocytes of P. falciparum that were as infectious to An. gambiae as those grown in medium with 10% serum. This is the first fully serum-free culture system able to produce highly infectious gametocytes, thereby removing the requirement for access to serum for transmission assays. [ABSTRACT FROM AUTHOR]

Background: The spread of antimalarial drug resistance parasites is a major obstacle in eliminating malaria in endemic areas. This increases the urgency for developing novel antimalarial drugs with improved profiles to eliminate both sensitive and resistant parasites in populations. The invention of the drug candidates needs a model for sensitive and resistant parasites on a laboratory scale. Methods: Repeated Incomplete Treatment (RIcT) method was followed in raising the rodent malaria parasite, Plasmodium berghei, resistant to sulfadoxine. Plasmodium berghei were exposed to an adequate therapeutic dose of sulfadoxine without finishing the treatment to let the parasite recover. Cycles of drug treatment and parasite recovery were repeated until phenotypic resistance appeared. Results: After undergoing 3–4 cycles, phenotypic resistance was not yet found in mice treated with sulfadoxine. Nevertheless, the molecular biology of dhps gene (the target of sulfadoxine) was analyzed at the end of the RIcT cycle. There was no mutations found in the gene target. Interestingly, the appearance of gametocytes at the end of every cycle of drug treatment and parasite recovery was observed. These gametocytes later on would no longer extend their life in the RBC stage, unless mosquitoes bite the infected host. This phenomenon is similar to the case in human malaria infections treated with sulfadoxine-pyrimethamine (SP). Conclusions: In this study, the antimalarial drug sulfadoxine induced gametocytogenesis in P. berghei, which could raise the risk factor for malaria transmission. [ABSTRACT FROM AUTHOR]

Abstract Background Human serum is a major component of Plasmodium falciparum culture medium, and can be replaced with AlbuMAX™ II, a lipid-rich bovine serum albumin, for asexual cultures. However, gametocytes produced without serum are poorly infective to mosquitoes. Serum suffers from high cost, limited availability, and variability in quality. Methods Several commercially-available media supplements were tested for their ability to support parasite growth and production of P. falciparum (3D7) gametocytes in standard RPMI1640 medium containing 0.5% AlbuMAX. The impact on asexual growth and gametocyte production with each supplement was assessed and compared to standard RPMI1640 medium containing 10% human serum, as well as to medium containing 0.5% AlbuMAX alone. The infectivity of gametocytes produced with one supplement to Anopheles gambiae sensu stricto was assessed by standard membrane feeding assay and measuring both prevalence of infection and oocyst intensity. Results Supplementation of medium containing 0.5% AlbuMAX with five supplements did not affect asexual growth of P. falciparum, and four of the five supplements supported early gametocyte production. The supplement producing the highest number of gametocytes, ITS-X, was further investigated and was found to support the production of mature gametocytes. Infection prevalence and oocyst intensity did not differ significantly between mosquitoes given a membrane feed containing gametocytes grown in medium with 0.5% AlbuMAX + ITS-X and those grown in medium with 10% human serum. Infection prevalence and oocyst intensity was significantly higher in case of ITS–X supplementation when compared to AlbuMAX alone. Infectious gametocytes were also produced from two field clones using ITS–X supplementation. Conclusions Serum-free medium supplemented with ITS-X was able to support the growth of gametocytes of P. falciparum that were as infectious to An. gambiae as those grown in medium with 10% serum. This is the first fully serum-free culture system able to produce highly infectious gametocytes, thereby removing the requirement for access to serum for transmission assays.

Abstract Background The spread of antimalarial drug resistance parasites is a major obstacle in eliminating malaria in endemic areas. This increases the urgency for developing novel antimalarial drugs with improved profiles to eliminate both sensitive and resistant parasites in populations. The invention of the drug candidates needs a model for sensitive and resistant parasites on a laboratory scale. Methods Repeated Incomplete Treatment (RIcT) method was followed in raising the rodent malaria parasite, Plasmodium berghei, resistant to sulfadoxine. Plasmodium berghei were exposed to an adequate therapeutic dose of sulfadoxine without finishing the treatment to let the parasite recover. Cycles of drug treatment and parasite recovery were repeated until phenotypic resistance appeared. Results After undergoing 3–4 cycles, phenotypic resistance was not yet found in mice treated with sulfadoxine. Nevertheless, the molecular biology of dhps gene (the target of sulfadoxine) was analyzed at the end of the RIcT cycle. There was no mutations found in the gene target. Interestingly, the appearance of gametocytes at the end of every cycle of drug treatment and parasite recovery was observed. These gametocytes later on would no longer extend their life in the RBC stage, unless mosquitoes bite the infected host. This phenomenon is similar to the case in human malaria infections treated with sulfadoxine-pyrimethamine (SP). Conclusions In this study, the antimalarial drug sulfadoxine induced gametocytogenesis in P. berghei, which could raise the risk factor for malaria transmission.

Abstract Background The incidence of malaria in Thailand has dramatically declined over the past two decades, and the goal is to eliminate malaria by 2025. Despite significant progress, one of the key challenges to malaria elimination are undetected gametocyte carriers. Human migration adds complexity to the malaria situation, as it not only sustains local transmission but also poses the risk of spreading drug-resistant parasites. Currently, no study has assessed the prevalence of gametocytes across multiple years in Plasmodium falciparum malaria patients in Thailand, and the risk factors for gametocyte carriage have not been fully explored. Methods Medical records of all P. falciparum malaria patients admitted from January 1, 2001 to December 31, 2020 at the Hospital for Tropical Diseases, Thailand, were retrospectively examined and a total of 1962 records were included for analysis. Both P. falciparum parasites and gametocytes were diagnosed by microscopy. A regression model was used to evaluate predictors of gametocyte carriage. Results The study demonstrated gametocyte prevalence in low malaria transmission areas. Nine risk factors for gametocyte carriage were identified: age between 15 and 24 years [adjusted odds ratio (aOR) = 1.96, 95% confidence interval (CI) 1.18−3.26], Karen ethnicity (aOR = 2.59, 95% CI 1.56−4.29), preadmission duration of fever > 7 days (aOR = 5.40, 95% CI 3.92−7.41), fever on admission (> 37.5 °C) (aOR = 0.61, 95% CI 0.48−0.77), haemoglobin ≤ 8 g/dL (aOR = 3.32, 95% CI 2.06−5.33), asexual parasite density > 5000−25,000/µL (aOR = 0.71, 95% CI 0.52−0.98), asexual parasite density > 25,000−100,000/µL (aOR = 0.74, 95% CI 0.53−1.03), asexual parasite density > 100,000/µL (aOR = 0.51, 95% CI 0.36−0.72), platelet count ≤ 100,000/µL (aOR = 0.65, 95% CI 0.50−0.85, clinical features of severe malaria (aOR = 2.33, 95% CI 1.76−3.10) and dry season (aOR = 1.41, 95% CI 1.10−1.80). An increasing incidence of imported transnational malaria cases was observed over the past two decades. Conclusions This is the first study to determine the prevalence of gametocytes among patients with symptomatic P. falciparum malaria, identify the risk factors for gametocyte carriage, and potential gametocyte carriers in Thailand. Blocking transmission is one of the key strategies for eliminating malaria in these areas. The results might provide important information for targeting gametocyte carriers and improving the allocation of resources for malaria control in Thailand. This study supports the already nationally recommended use of a single dose of primaquine in symptomatic P. falciparum malaria patients to clear gametocytes. Graphical Abstract

Background: The incidence of malaria in Thailand has dramatically declined over the past two decades, and the goal is to eliminate malaria by 2025. Despite significant progress, one of the key challenges to malaria elimination are undetected gametocyte carriers. Human migration adds complexity to the malaria situation, as it not only sustains local transmission but also poses the risk of spreading drug-resistant parasites. Currently, no study has assessed the prevalence of gametocytes across multiple years in Plasmodium falciparum malaria patients in Thailand, and the risk factors for gametocyte carriage have not been fully explored. Methods: Medical records of all P. falciparum malaria patients admitted from January 1, 2001 to December 31, 2020 at the Hospital for Tropical Diseases, Thailand, were retrospectively examined and a total of 1962 records were included for analysis. Both P. falciparum parasites and gametocytes were diagnosed by microscopy. A regression model was used to evaluate predictors of gametocyte carriage. Results: The study demonstrated gametocyte prevalence in low malaria transmission areas. Nine risk factors for gametocyte carriage were identified: age between 15 and 24 years [adjusted odds ratio (aOR) = 1.96, 95% confidence interval (CI) 1.18−3.26], Karen ethnicity (aOR = 2.59, 95% CI 1.56−4.29), preadmission duration of fever > 7 days (aOR = 5.40, 95% CI 3.92−7.41), fever on admission (> 37.5 °C) (aOR = 0.61, 95% CI 0.48−0.77), haemoglobin ≤ 8 g/dL (aOR = 3.32, 95% CI 2.06−5.33), asexual parasite density > 5000−25,000/µL (aOR = 0.71, 95% CI 0.52−0.98), asexual parasite density > 25,000−100,000/µL (aOR = 0.74, 95% CI 0.53−1.03), asexual parasite density > 100,000/µL (aOR = 0.51, 95% CI 0.36−0.72), platelet count ≤ 100,000/µL (aOR = 0.65, 95% CI 0.50−0.85, clinical features of severe malaria (aOR = 2.33, 95% CI 1.76−3.10) and dry season (aOR = 1.41, 95% CI 1.10−1.80). An increasing incidence of imported transnational malaria cases was observed over the past two decades. Conclusions: This is the first study to determine the prevalence of gametocytes among patients with symptomatic P. falciparum malaria, identify the risk factors for gametocyte carriage, and potential gametocyte carriers in Thailand. Blocking transmission is one of the key strategies for eliminating malaria in these areas. The results might provide important information for targeting gametocyte carriers and improving the allocation of resources for malaria control in Thailand. This study supports the already nationally recommended use of a single dose of primaquine in symptomatic P. falciparum malaria patients to clear gametocytes. [ABSTRACT FROM AUTHOR]

Background: The erythrocytic stage of the life cycle of the malaria parasite, Plasmodium falciparum, consists of trophozoite, schizont and gametocyte stages in humans. Various anti-malarial agents target different stages of the parasite to produce treatment outcomes. This study reports on the stage-specific anti-malarial activity of heptaphylline and imperatorin against human P. falciparum in addition to their cytotoxicity and selectivity indices (SI). Methods: The compounds were isolated from Clausena anisata using column chromatography and their structures elucidated using NMR spectroscopy. The anti-malarial activity was determined by measuring the trophozoitocidal, schizonticidal and gametocytocidal activities of the compounds using the SYBR green assay. Cytotoxicity was evaluated using the tetrazolium-based colorimetric assay. Results: Heptaphylline and imperatorin produced trophozoitocidal, schizonticidal and gametocytocidal activities with IC50s of 1.57 (0.2317)–26.92 (0.3144) µM with those of artesunate (the standard drug) being 0.00024 (0.0036)–0.0070 (0.0013) µM. In the cytotoxicity assay, the compounds produced CC50S greater than 350 µM and SI of 13.76–235.90. Also, the trophozoitocidal and schizonticidal activities of the compounds were more pronounced than their gametocytocidal activity. Imperatorin was 42.04% more trophozoitocidal than hepthaphyline. However, hepthaphyline has more schizonticidal and gametocytocidal properties than imperatorin. Conclusion: Heptaphylline and imperatorin are promising anti-malarial agents, since they possess potent anti-malarial activity with weak cytotoxicity on RBCs. However, imperatorin is a better anti-malarial prophylactic agent whereas heptaphylline is a better malaria treatment agent. [ABSTRACT FROM AUTHOR]

Abstract Background Plasmodium vivax is responsible for much of malaria outside Africa. Although most P. vivax infections in endemic areas are asymptomatic and have low parasite densities, they are considered a potentially important source of transmission. Several studies have demonstrated that asymptomatic P. vivax carriers can transmit the parasite to mosquitoes, but the efficiency has not been well quantified. The aim of this study is to determine the relationship between parasite density and mosquito infectivity, particularly at low parasitaemia. Methods Membrane feeding assays were performed using serial dilutions of P. vivax-infected blood to define the relationship between parasitaemia and mosquito infectivity. Results The infection rate (oocyst prevalence) and intensity (oocyst load) were positively correlated with the parasite density in the blood. There was a broad case-to-case variation in parasite infectivity. The geometric mean parasite density yielding a 10% mosquito infection rate was 33 (CI 95 9–120) parasites/µl or 4 (CI 95 1–17) gametocytes/µl. The geometric mean parasite density yielding a 50% mosquito infection rate was 146 (CI 95 36–586) parasites/µl or 13 (CI 95 3–49) gametocytes/µl. Conclusion This study quantified the ability of P. vivax to infect Anopheles dirus at over a broad range of parasite densities. It provides important information about parasite infectivity at low parasitaemia common among asymptomatic P. vivax carriers.

Background: Plasmodium vivax is responsible for much of malaria outside Africa. Although most P. vivax infections in endemic areas are asymptomatic and have low parasite densities, they are considered a potentially important source of transmission. Several studies have demonstrated that asymptomatic P. vivax carriers can transmit the parasite to mosquitoes, but the efficiency has not been well quantified. The aim of this study is to determine the relationship between parasite density and mosquito infectivity, particularly at low parasitaemia. Methods: Membrane feeding assays were performed using serial dilutions of P. vivax-infected blood to define the relationship between parasitaemia and mosquito infectivity. Results: The infection rate (oocyst prevalence) and intensity (oocyst load) were positively correlated with the parasite density in the blood. There was a broad case-to-case variation in parasite infectivity. The geometric mean parasite density yielding a 10% mosquito infection rate was 33 (CI 95 9–120) parasites/µl or 4 (CI 95 1–17) gametocytes/µl. The geometric mean parasite density yielding a 50% mosquito infection rate was 146 (CI 95 36–586) parasites/µl or 13 (CI 95 3–49) gametocytes/µl. Conclusion: This study quantified the ability of P. vivax to infect Anopheles dirus at over a broad range of parasite densities. It provides important information about parasite infectivity at low parasitaemia common among asymptomatic P. vivax carriers. [ABSTRACT FROM AUTHOR]

Abstract Background A goal of malaria epidemiological interventions is the detection and treatment of parasite reservoirs in endemic areas—an activity that is expected to reduce local transmission. Since the gametocyte is the only transmissible stage from human host to mosquito vector, this study evaluated the pre and post presence of gametocytes during a mass screening and treatment (MST) intervention conducted during 2013 in East Nusa Tenggara, Indonesia. Methods RT-qPCR targeting pfs25 and pvs25 transcripts—gametocyte molecular markers for Plasmodium falciparum and Plasmodium vivax, respectively, was performed to detect and quantify gametocytes in blood samples of P. falciparum and P. vivax-infected subjects over the course of the MST study. The presence of both asexual and sexual parasites in microscopic and submicroscopic infections was compared from the start and end of the MST, using proportion tests as well as parametric and non-parametric tests. Results Parasite prevalence remained unchanged for P. falciparum (6% = 52/811 versus 7% = 50/740, p = 0.838), and decreased slightly for P. vivax (24% = 192/811 versus 19% = 142/740, p = 0.035) between the MST baseline and endpoint. No significant difference was observed in gametocyte prevalence for either P. falciparum (2% = 19/803 versus 3% = 23/729, p = 0.353, OR = 1.34, 95%CI = 0.69–2.63), or P. vivax (7% = 49/744 versus 5% = 39/704, p = 0.442, OR = 0.83, 95%CI = 0.52–1.31). Even though there was an insignificant difference between the two time points, the majority of parasite positive subjects at the endpoint had been negative at baseline (P. falciparum: 66% = 29/44, P. vivax: 60% = 80/134). This was similarly demonstrated for the transmissible stage—where the majority of gametocyte positive subjects at the endpoint were negative at baseline (P. falciparum: 95% = 20/21, P. vivax: 94% = 30/32). These results were independent of treatment provided during MST activities. No difference was demonstrated in parasite and gametocyte density between both time points either in P. falciparum or P. vivax. Conclusion In this study area, similar prevalence rates of P. falciparum and P. vivax parasites and gametocytes before and after MST, although in different individuals, points to a negligible impact on the parasite reservoir. Treatment administration based on parasite positivity as implemented in the MST should be reevaluated for the elimination strategy in the community. Trial registration Clinical trials registration NCT01878357. Registered 14 June 2013, https://www.clinicaltrials.gov/ct2/show/NCT01878357.

Abstract Background Mosquito feeding assays using venous blood are commonly used for evaluating the transmission potential of malaria infected individuals. To improve the accuracy of these assays, care must be taken to prevent premature activation or inactivation of gametocytes before they are fed to mosquitoes. This can be challenging in the field where infected individuals and insectary facilities are sometimes very far apart. In this study, a simple, reliable, field applicable method is presented for storage and transport of gametocyte infected blood using a thermos flask. Methods The optimal storage conditions for maintaining the transmissibility of gametocytes were determined initially using cultured Plasmodium falciparum gametocytes in standard membrane feeding assays (SMFAs). The impact of both the internal thermos water temperature (35.5 to 37.8 °C), and the external environmental temperature (room temperature to 42 °C) during long-term (4 h) storage, and the impact of short-term (15 min) temperature changes (room temp to 40 °C) during membrane feeding assays was assessed. The optimal conditions were then evaluated in direct membrane feeding assays (DMFAs) in Burkina Faso and The Gambia where blood from naturally-infected gametocyte carriers was offered to mosquitoes immediately and after storage in thermos flasks. Results Using cultured gametocytes in SMFAs it was determined that an internal thermos water temperature of 35.5 °C and storage of the thermos flask between RT (~ 21.3 °C) and 32 °C was optimal for maintaining transmissibility of gametocytes for 4 h. Short-term storage of the gametocyte infected blood for 15 min at temperatures up to 40 °C (range: RT, 30 °C, 38 °C and 40 °C) did not negatively affect gametocyte infectivity. Using samples from natural gametocyte carriers (47 from Burkina Faso and 16 from The Gambia), the prevalence of infected mosquitoes and the intensity of oocyst infection was maintained when gametocyte infected blood was stored in a thermos flask in water at 35.5 °C for up to 4 h. Conclusions This study determines the optimal long-term (4 h) storage temperature for gametocyte infected blood and the external environment temperature range within which gametocyte infectivity is unaffected. This will improve the accuracy, reproducibility, and utility of DMFAs in the field, and permit reliable comparative assessments of malaria transmission epidemiology in different settings.

Abstract Background Multi-genotype malaria infections are frequent in endemic area, and people commonly harbour several genetically distinct Plasmodium falciparum variants. The influence of genetic multiplicity and whether some specific genetic variants are more or less likely to invest into gametocyte production is not clearly understood. This study explored host and parasite-related risk factors for gametocyte carriage, and the extent to which some specific P. falciparum genetic variants are associated with gametocyte carriage. Methods Gametocytes and asexual forms were detected by light microscopy on thick smears collected between 2010 and 2012 in Nanoro, Burkina Faso. Merozoite surface protein 1 and 2 were genotyped by nested PCR on clinical samples. Associations between gametocyte carriage and factors, including multiplicity of infection, parasite density, patient age, gender, haemoglobin (Hb) level, and body temperature were assessed. The relationship between the presence of a particular msp1 and msp2 genetic variants and gametocyte carriage was also explored. Results Of the 724 samples positive to P. falciparum and successfully genotyped, gametocytes were found in 48 samples (6.63%). There was no effect of patient gender, age and body temperature on gametocyte carriage. However, the probability of gametocyte carriage significantly increased with increasing values of multiplicity of infection (MOI). Furthermore, there was a negative association between parasite density and gametocyte carriage. MOI decreased with parasite density in gametocyte-negative patients, but increased in gametocyte carriers. The probability of gametocyte carriage decreased with Hb level. Finally, the genetic composition of the infection influenced gametocyte carriage. In particular, the presence of RO33 increased the odds of developing gametocytes by 2 while the other allelic families K1, MAD20, FC27, and 3D7 had no significant impact on the occurrence of gametocytes in infected patients. Conclusion This study provides insight into potential factors influencing gametocyte production in symptomatic patients. The findings contribute to enhance understanding of risk factors associated with gametocyte carriage in humans. Trial registration NCT01232530.

BackgroundA vaccine that interrupts malaria transmission (VIMT) would be a valuable tool for malaria control and elimination. One VIMT approach is to identify sexual erythrocytic and mosquito stage antigens of the malaria parasite that induce immune responses targeted at disrupting parasite development in the mosquito. The standard Plasmodium falciparum membrane-feeding assay (SMFA) is used to assess transmission-blocking activity (TBA) of antibodies against candidate immunogens and of drugs targeting the mosquito stages. To develop its P. falciparum sporozoite (SPZ) products, Sanaria has industrialized the production of P. falciparum-infected Anopheles stephensi mosquitoes, incorporating quantitative analyses of oocyst and P. falciparum SPZ infections as part of the manufacturing process.MethodsThese capabilities were exploited to develop a robust, reliable, consistent SMFA that was used to assess 188 serum samples from animals immunized with the candidate vaccine immunogen, Pfs25, targeting P. falciparum mosquito stages. Seventy-four independent SMFAs were performed. Infection intensity (number of oocysts/mosquito) and infection prevalence (percentage of mosquitoes infected with oocysts) were compared between mosquitoes fed cultured gametocytes plus normal human O(+) serum (negative control), anti-Pfs25 polyclonal antisera (MRA39 or MRA38, at a final dilution in the blood meal of 1:54 as positive control), and test sera from animals immunized with Pfs25 (at a final dilution in the blood meal of 1:9).ResultsSMFA negative controls consistently yielded high infection intensity (mean = 46.1 oocysts/midgut, range of positives 3.7-135.6) and infection prevalence (mean = 94.2%, range 71.4-100.0) and in positive controls, infection intensity was reduced by 81.6% (anti-Pfs25 MRA39) and 97.0% (anti-Pfs25 MRA38), and infection prevalence was reduced by 12.9 and 63.5%, respectively. A range of TBAs was detected among the 188 test samples assayed in duplicate. Consistent administration of infectious gametocytes to mosquitoes within and between assays was achieved, and the TBA of anti-Pfs25 control antibodies was highly reproducible.ConclusionsThese results demonstrate a robust capacity to perform the SMFA in a medium-to-high throughput format, suitable for assessing large numbers of experimental samples of candidate antibodies or drugs.

Abstract Background PCR can be positive weeks after effective malaria treatment, potentially leading to over diagnose of recrudescence and re-infections. The DNA detected by PCR post-treatment might stem from residuals of destroyed asexual parasites, or from live gametocytes. The objective of this clinical observational study was to describe the presence of positive PCR for Plasmodium falciparum and Plasmodium vivax in follow-up samples post-treatment from returned travellers, and the proportion of positive PCR due to gametocytes. Methods Whole blood was collected during hospitalization and outpatient routine follow-up from 13 patients with imported malaria. DNA was extracted applying QIAamp DNA Blood Mini Kit, while mRNA was collected and extracted applying PAXgene Blood RNA Tubes and Kit. All DNA samples (N = 25) were analysed with a genus-specific cytb real-time SYBR PCR, and P. falciparum DNA samples (N = 22) were also analysed with a falciparum-specific varATS real-time TaqMan PCR. All the mRNA samples (N = 18) were analysed with both a genus-specific 18S rRNA RT-PCR and a gametocyte-specific Pfs25 (P. falciparum)/Pvs25 (P. vivax) RT-PCR. Results Latest samples were collected at day 1 (n = 2) and from day 11–54 (n = 11) after treatment. Genus DNA cytb PCR was positive up to 49 days after effective treatment, and 18S rRNA transcripts from active P. falciparum parasites were detectable for at least 11 days. Gametocyte-specific mRNA was detected at latest only two days after treatment. Among six patients with late positive PCR for P. falciparum, four had high parasitaemia at admittance (6–30%), while two had parasitaemia

Abstract 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.

Abstract Background The epidemiological control of malaria has been hampered by the appearance of parasite resistance to anti-malarial drugs and by the resistance of mosquito vectors to control measures. This has also been associated with weak transmission control, mostly due to poor control of asymptomatic patients associated with host-vector transmission. This highlights the importance of studying the parasite’s sexual forms (gametocytes) which are involved in this phase of the parasite’s life-cycle. Some African and Asian strains of Plasmodium falciparum have been fully characterized regarding sexual forms’ production; however, few Latin-American strains have been so characterized. This study was aimed at characterizing the Colombian FCB2 strain as a gametocyte producer able to infect mosquitoes. Methods Gametocyte production was induced in in vitro cultured P. falciparum FCB2 and 3D7 strains. Pfap2g and Pfs25 gene expression was detected in FCB2 strain gametocyte culture by RT-PCR. Comparative analysis of gametocytes obtained from both strains was made (counts and morphological changes). In vitro zygote formation from FCB2 gametocytes was induced by incubating a gametocyte culture sample at 27 °C for 20 min. A controlled Anopheles albimanus infection was made using an artificial feed system with cultured FCB2 gametocytes (14–15 days old). Mosquito midgut dissection was then carried out for analyzing oocysts. Results The FCB2 strain expressed Pfap2g, Pfs16, Pfg27/25 and Pfs25 sexual differentiation-related genes after in vitro sexual differentiation induction, producing gametocytes that conserved the expected morphological features. The amount of FCB2 gametocytes produced was similar to that from the 3D7 strain. FCB2 gametocytes were differentiated into zygotes and ookinetes after an in vitro low-temperature stimulus and infected An. albimanus mosquitoes, developing to oocyst stage. Conclusions Even with the history of long-term FCB2 strain in vitro culture maintenance, it has retained its sexual differentiation ability. The gametocytes produced here preserved these parasite forms’ usual characteristics and An. albimanus infection capability, thus enabling its use as a tool for studying sexual form biology, An. albimanus infection comparative analysis and anti-malarial drug and vaccine development.

Abstract Background Asymptomatic carriage of Plasmodium falciparum is widespread in adults and children living in malaria-endemic countries. This study identified the prevalence of malaria parasites and the corresponding levels of naturally acquired anti-parasite antibody levels in afebrile adults living in two communities in the Greater Accra Region of Ghana. Methods Two cross-sectional studies conducted in January and February 2016 and repeated in July and August 2016 recruited subjects aged between 6 and 75 years from high parasite prevalence (Obom) and low parasite prevalence (Asutsuare) communities. Whole blood (5 ml) was collected from each volunteer, plasma was aliquoted and frozen until needed. An aliquot (10 µl) of the blood was used to prepare thick and thin blood smears, 100 µl was preserved in Trizol and the rest was separated into plasma and blood cells and each stored at − 20 °C until needed. Anti-MSP3 and Pfs230 antibody levels were measured using ELISA. Results Asexual parasite and gametocyte prevalence were higher in Obom than Asutsuare. Antibody (IgG, IgG1, IgG3, IgM) responses against the asexual parasite antigen MSP3 and gametocyte antigen Pfs230 were higher in Obom during the course of the study except for IgM responses against Pfs230, which was higher in Asutsuare than in Obom during the rainy season. Antibody responses in Asutsuare were more significantly associated with age than the responses measured in Obom. Conclusion The pattern of antibody responses measured in people living in the high and low malaria transmission setting was similar. All antibody responses measured against the asexual antigen MSP3 increased, however, IgG and IgG1 responses against gametocyte antigen Pfs230 decreased in moving from the dry to the peak season in both sites. Whilst asexual and gametocyte prevalence was similar between the seasons in the low transmission setting, in the high transmission setting asexual parasite prevalence increased but gametocyte prevalence decreased in the rainy season relative to the dry season.

Abstract Background Primaquine is an important gametocytocidal drug that is combined with conventional malaria treatment for prevention of Plasmodium falciparum malaria transmission. Primaquine has been administered together on the first or the last day of conventional treatment but the impact of primaquine timing has never been examined. This study aimed to assess safety, efficacy and optimal timing of single full-dose (0.75 mg/kg) primaquine when added to a standard 6-dose regimen of artemether–lumefantrine (AL). Methods In an individual-level randomized controlled trial, enrolled participants who were G6PD normal and had uncomplicated P. falciparum malaria were randomly assigned to receive: AL only; AL and a single 0.75 mg/kg primaquine dose on the first day of AL (day 1); or AL and single 0.75 mg//kg primaquine on the last day of AL (day 3). On days 2, 3, 4, 8, 11 and 15, gametocytes were assessed and quantified by microscope and quantitative nuclear acid sequence based quantification (QT-NASBA). Results Overall, 111 participants aged between 3 and 17 years were randomly allocated to receive AL only (36) or combined with primaquine on day 1 (38), or primaquine on day 3 (37). Day 4 gametocyte prevalence in AL + day 1 primaquine was half the level seen in either AL + day 3 primaquine or AL only arm (11% [4/35] vs 26% [8/31] and 27% [8/30], respectively) albeit not statistically significant. A similar trend of lower gametocyte in the AL + day 1 primaquine verses AL + day 3 primaquine or AL only arm was observed in mean gametocyte density. Mean (sd) haemoglobin level in AL + day 3 primaquine arm recovered from -0.42(1.2) g/dl on day 2 to 0.35 (1.5) g/dl on day 15 of follow up. This was not the case in AL only and AL + day 1 primaquine arms during the same follow-up period, although the difference was not statistically significant (p = 318). No serious adverse events reported in the study. Across arms, 23% (26/111) of participants reported a total of 31 mild adverse events and the difference was not statistically significant (p = 0.477). Conclusion Primaquine administration on the first day of AL is well tolerated and as safe as later administration. Whilst the World Health Organization currently recommends a lower dose of primaquine (0.25 mg/kg), the findings are supportive of early primaquine administration when combined with artemisinin-combination therapy. ClinicalTrials.gov Registration NCT01906788

Background: The ability to culture Plasmodium falciparum continuously in vitro has enabled stable access to asexual and sexual parasites for malaria research. The portfolio of isolates has remained limited and research is still largely based on NF54 and its derived clone 3D7. Since 1978, isolates were collected and cryopreserved at Radboudumc from patients presenting at the hospital. Here, procedures are described for culture adaptation of asexual parasites, cloning and production of sexual stage parasites responsible for transmission (gametocytes) and production of oocysts in Anopheles mosquitoes. This study aimed to identify new culture-adapted transmissible P. falciparum isolates, originating from distinct geographical locations. Methods: Out of a collection of 121 P. falciparum isolates stored in liquid nitrogen, 21 from different geographical origin were selected for initial testing. Isolates were evaluated for their ability to be asexually cultured in vitro, their gametocyte production capacity, and consistent generation of oocysts. Results: Out of 21 isolates tested, twelve were excluded from further analysis due to lack of mature gametocyte production (n = 1) or generation of satisfactory numbers of oocysts in mosquitoes (n = 11). Nine isolates fulfilled selection criteria and were cloned by limiting dilution and retested. After cloning, one isolate was excluded for not showing transmission. The remaining eight isolates transmitted to Anopheles stephensi or Anopheles coluzzii mosquitoes and were categorized into two groups with a reproducible mean oocyst infection intensity above (n = 5) or below five (n = 3). Conclusions: These new P. falciparum culture-adapted isolates with reproducible transmission to Anopheles mosquitoes are a valuable addition to the malaria research tool box. They can aid in the development of malaria interventions and will be particularly useful for those studying malaria transmission. [ABSTRACT FROM AUTHOR]

Abstract Background The human infectious reservoir for malaria consists of individuals capable of infecting mosquitoes. Oocyst prevalence and density are typical indicators of human infectivity to mosquitoes. However, identification of oocysts is challenging, particularly in areas of low malaria transmission intensity where few individuals may infect mosquitoes, and infected mosquitoes tend to have few oocysts. Here, features that differentiate oocysts from other oocyst-like in mosquito midguts are explained and illustrated. In addition, the establishment and maintenance of infrastructure to perform malaria transmission experiments is described. This work may support other initiatives to set up membrane feeding infrastructure and guide oocyst detection in low transmission settings. Methods In 2014, an insectary was developed and equipped in Tororo district, Uganda. A colony of Anopheles gambiae s.s. mosquitoes (Kisumu strain) was initiated to support infectivity experiments from participants enrolled in a large cohort study. Venous blood drawn from participants who were naturally infected with malaria parasites was used for membrane feeding assays, using 60–80 mosquitoes per experiment. Approximately 9–10 days after feeding, mosquitoes were dissected, and midguts were stained in mercurochrome and examined by light microscopy for Plasmodium falciparum oocysts and similar structures. In supportive experiments, different staining procedures were compared using in vitro cultured parasites. Results A stable colony of the Kisumu strain of An. gambiae s.s. was achieved, producing 5000–10,000 adult mosquitoes on a weekly basis. Challenges due to temperature fluctuations, mosquito pathogens and pests were successfully overcome. Oocysts were characterized by: presence of malaria pigment, clearly defined edge, round shape within the mosquito midgut or on the peripheral tissue and always attached to the epithelium. The main distinguishing feature between artifacts and mature oocysts was the presence of defined pigment within the oocysts. Conclusions Oocysts may be mistaken for other structures in mosquito midguts. Distinguishing real oocysts from oocyst-like structures may be challenging for inexperienced microscopists due to overlapping features. The characteristics and guidelines outlined here support identification of oocysts and reliable detection at low oocyst densities. Practical advice on sustaining a healthy mosquito colony for feeding experiments is provided. Following the reported optimization, the established infrastructure in Tororo allows assessments of infectivity of naturally infected parasite carriers.

Abstract 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

Abstract While significant advances have been made in understanding Plasmodium falciparum gametocyte biology and its relationship with malaria parasite transmission, the gametocyte sex ratio contribution to this process still remains a relevant research question. The present review discusses the biology of sex determination in P. falciparum, the underlying host and parasite factors, the sex specific susceptibility to drugs, the effect of sex ratio dynamics on malaria parasite transmission and the development of gametocyte sex specific diagnosis tools. Despite the inherent differences across several studies and approaches, the emerging picture highlights a potentially relevant contribution of the P. falciparum gametocyte sex ratio in the modulation of malaria parasite transmission. The increasing availability of molecular methods to measure gametocyte sex ratio will enable evaluation of important parameters, such as the impact of drug treatment on gametocyte sex ratio in vitro and in vivo as well as the changes of gametocyte sex ratios in natural infections, key steps towards elucidating how these parameters affect parasite infectiousness to the mosquito vectors.

Abstract Background The development of malaria transmission-blocking strategies including the generation of malaria refractory mosquitoes to replace the wild populations through means of gene drives hold great promise. The standard membrane feeding assay (SMFA) that involves mosquito feeding on parasitized blood through an artificial membrane system is a vital tool for evaluating the efficacy of transmission-blocking interventions. However, despite the availability of several published protocols, the SMFA remains highly variable and broadly insensitive. Methods The SMFA protocol was optimized through coordinated culturing of Anopheles coluzzii mosquitoes and Plasmodium falciparum parasite coupled with placing mosquitoes under a strict dark regime before, during, and after the gametocyte feed. Results A detailed description of essential steps is provided toward synchronized generation of highly fit An. coluzzii mosquitoes and P. falciparum gametocytes in preparation for an SMFA. A dark-infection regime that emulates the natural vector-parasite interaction system is described, which results in a significant increase in the infection intensity and prevalence. Using this optimal SMFA pipeline, a series of putative transmission-blocking antimicrobial peptides (AMPs) were screened, confirming that melittin and magainin can interfere with P. falciparum development in the vector. Conclusion A robust SMFA protocol that enhances the evaluation of interventions targeting human malaria transmission in laboratory setting is reported. Melittin and magainin are identified as highly potent antiparasitic AMPs that can be used for the generation of refractory Anopheles gambiae mosquitoes.

Background: Mosquito feeding assays using venous blood are commonly used for evaluating the transmission potential of malaria infected individuals. To improve the accuracy of these assays, care must be taken to prevent premature activation or inactivation of gametocytes before they are fed to mosquitoes. This can be challenging in the field where infected individuals and insectary facilities are sometimes very far apart. In this study, a simple, reliable, field applicable method is presented for storage and transport of gametocyte infected blood using a thermos flask. Methods: The optimal storage conditions for maintaining the transmissibility of gametocytes were determined initially using cultured Plasmodium falciparum gametocytes in standard membrane feeding assays (SMFAs). The impact of both the internal thermos water temperature (35.5 to 37.8 °C), and the external environmental temperature (room temperature to 42 °C) during long-term (4 h) storage, and the impact of short-term (15 min) temperature changes (room temp to 40 °C) during membrane feeding assays was assessed. The optimal conditions were then evaluated in direct membrane feeding assays (DMFAs) in Burkina Faso and The Gambia where blood from naturally-infected gametocyte carriers was offered to mosquitoes immediately and after storage in thermos flasks. Results: Using cultured gametocytes in SMFAs it was determined that an internal thermos water temperature of 35.5 °C and storage of the thermos flask between RT (~ 21.3 °C) and 32 °C was optimal for maintaining transmissibility of gametocytes for 4 h. Short-term storage of the gametocyte infected blood for 15 min at temperatures up to 40 °C (range: RT, 30 °C, 38 °C and 40 °C) did not negatively affect gametocyte infectivity. Using samples from natural gametocyte carriers (47 from Burkina Faso and 16 from The Gambia), the prevalence of infected mosquitoes and the intensity of oocyst infection was maintained when gametocyte infected blood was stored in a thermos flask in water at 35.5 °C for up to 4 h. Conclusions: This study determines the optimal long-term (4 h) storage temperature for gametocyte infected blood and the external environment temperature range within which gametocyte infectivity is unaffected. This will improve the accuracy, reproducibility, and utility of DMFAs in the field, and permit reliable comparative assessments of malaria transmission epidemiology in different settings. [ABSTRACT FROM AUTHOR]

Background: A goal of malaria epidemiological interventions is the detection and treatment of parasite reservoirs in endemic areas—an activity that is expected to reduce local transmission. Since the gametocyte is the only transmissible stage from human host to mosquito vector, this study evaluated the pre and post presence of gametocytes during a mass screening and treatment (MST) intervention conducted during 2013 in East Nusa Tenggara, Indonesia. Methods: RT-qPCR targeting pfs25 and pvs25 transcripts—gametocyte molecular markers for Plasmodium falciparum and Plasmodium vivax, respectively, was performed to detect and quantify gametocytes in blood samples of P. falciparum and P. vivax-infected subjects over the course of the MST study. The presence of both asexual and sexual parasites in microscopic and submicroscopic infections was compared from the start and end of the MST, using proportion tests as well as parametric and non-parametric tests. Results: Parasite prevalence remained unchanged for P. falciparum (6% = 52/811 versus 7% = 50/740, p = 0.838), and decreased slightly for P. vivax (24% = 192/811 versus 19% = 142/740, p = 0.035) between the MST baseline and endpoint. No significant difference was observed in gametocyte prevalence for either P. falciparum (2% = 19/803 versus 3% = 23/729, p = 0.353, OR = 1.34, 95%CI = 0.69–2.63), or P. vivax (7% = 49/744 versus 5% = 39/704, p = 0.442, OR = 0.83, 95%CI = 0.52–1.31). Even though there was an insignificant difference between the two time points, the majority of parasite positive subjects at the endpoint had been negative at baseline (P. falciparum: 66% = 29/44, P. vivax: 60% = 80/134). This was similarly demonstrated for the transmissible stage—where the majority of gametocyte positive subjects at the endpoint were negative at baseline (P. falciparum: 95% = 20/21, P. vivax: 94% = 30/32). These results were independent of treatment provided during MST activities. No difference was demonstrated in parasite and gametocyte density between both time points either in P. falciparum or P. vivax. Conclusion: In this study area, similar prevalence rates of P. falciparum and P. vivax parasites and gametocytes before and after MST, although in different individuals, points to a negligible impact on the parasite reservoir. Treatment administration based on parasite positivity as implemented in the MST should be reevaluated for the elimination strategy in the community. Trial registration Clinical trials registration NCT01878357. Registered 14 June 2013, https://www.clinicaltrials.gov/ct2/show/NCT01878357. [ABSTRACT FROM AUTHOR]

Background: Daily periodicity in the diverse activities of parasites occurs across a broad taxonomic range. The rhythms exhibited by parasites are thought to be adaptations that allow parasites to cope with, or exploit, the consequences of host activities that follow daily rhythms. Malaria parasites (Plasmodium) are well-known for their synchronized cycles of replication within host red blood cells. Whilst most species of Plasmodium appear sensitive to the timing of the daily rhythms of hosts, and even vectors, some species present no detectable rhythms in blood-stage replication. Why the intraerythrocytic development cycle (IDC) of, for example Plasmodium chabaudi, is governed by host rhythms, yet seems completely independent of host rhythms in Plasmodium berghei, another rodent malaria species, is mysterious. Methods: This study reports a series of five experiments probing the relationships between the asynchronous IDC schedule of P. berghei and the rhythms of hosts and vectors by manipulating host time-of-day, photoperiod and feeding rhythms. Results: The results reveal that: (i) a lack coordination between host and parasite rhythms does not impose appreciable fitness costs on P. berghei; (ii) the IDC schedule of P. berghei is impervious to host rhythms, including altered photoperiod and host-feeding-related rhythms; (iii) there is weak evidence for daily rhythms in the density and activities of transmission stages; but (iv), these rhythms have little consequence for successful transmission to mosquitoes. Conclusions: Overall, host rhythms do not affect the performance of P. berghei and its asynchronous IDC is resistant to the scheduling forces that underpin synchronous replication in closely related parasites. This suggests that natural variation in the IDC schedule across species represents different parasite strategies that maximize fitness. Thus, subtle differences in the ecological interactions between parasites and their hosts/vectors may select for the evolution of very different IDC schedules. [ABSTRACT FROM AUTHOR]

Background: Multi-genotype malaria infections are frequent in endemic area, and people commonly harbour several genetically distinct Plasmodium falciparum variants. The influence of genetic multiplicity and whether some specific genetic variants are more or less likely to invest into gametocyte production is not clearly understood. This study explored host and parasite-related risk factors for gametocyte carriage, and the extent to which some specific P. falciparum genetic variants are associated with gametocyte carriage. Methods: Gametocytes and asexual forms were detected by light microscopy on thick smears collected between 2010 and 2012 in Nanoro, Burkina Faso. Merozoite surface protein 1 and 2 were genotyped by nested PCR on clinical samples. Associations between gametocyte carriage and factors, including multiplicity of infection, parasite density, patient age, gender, haemoglobin (Hb) level, and body temperature were assessed. The relationship between the presence of a particular msp1 and msp2 genetic variants and gametocyte carriage was also explored. Results: Of the 724 samples positive to P. falciparum and successfully genotyped, gametocytes were found in 48 samples (6.63%). There was no effect of patient gender, age and body temperature on gametocyte carriage. However, the probability of gametocyte carriage significantly increased with increasing values of multiplicity of infection (MOI). Furthermore, there was a negative association between parasite density and gametocyte carriage. MOI decreased with parasite density in gametocyte-negative patients, but increased in gametocyte carriers. The probability of gametocyte carriage decreased with Hb level. Finally, the genetic composition of the infection influenced gametocyte carriage. In particular, the presence of RO33 increased the odds of developing gametocytes by 2 while the other allelic families K1, MAD20, FC27, and 3D7 had no significant impact on the occurrence of gametocytes in infected patients. Conclusion: This study provides insight into potential factors influencing gametocyte production in symptomatic patients. The findings contribute to enhance understanding of risk factors associated with gametocyte carriage in humans. Trial registration NCT01232530. [ABSTRACT FROM AUTHOR]

Abstract Background The study of malaria transmission requires the experimental infection of mosquitoes with Plasmodium gametocytes. In the laboratory, this is achieved using artificial membrane feeding apparatus that simulate body temperature and skin of the host, and so permit mosquito feeding on reconstituted gametocyte-containing blood. Membrane feeders either use electric heating elements or complex glass chambers to warm the infected blood; both of which are expensive to purchase and can only be sourced from a handful of specialized companies. Presented and tested here is a membrane feeder that can be inexpensively printed using 3D-printing technology. Results Using the Plasmodium falciparum laboratory strain NF54, three independent standard membrane feeding assays (SMFAs) were performed comparing the 3D-printed feeder against a commercial glass feeder. Exflagellation rates did not differ between the two feeders. Furthermore, no statistically significant difference was found in the oocyst load nor oocyst intensity of Anopheles stephensi mosquitoes (mean oocyst range 1.3–6.2 per mosquito; infection prevalence range 41–79%). Conclusions Open source provision of the design files of the 3D-printed feeder will facilitate a wider range of laboratories to perform SMFAs in laboratory and field settings, and enable them to freely customize the design to their own requirements.

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. [ABSTRACT FROM AUTHOR]

Background: Asymptomatic carriage of Plasmodium falciparum is widespread in adults and children living in malaria-endemic countries. This study identified the prevalence of malaria parasites and the corresponding levels of naturally acquired anti-parasite antibody levels in afebrile adults living in two communities in the Greater Accra Region of Ghana. Methods: Two cross-sectional studies conducted in January and February 2016 and repeated in July and August 2016 recruited subjects aged between 6 and 75 years from high parasite prevalence (Obom) and low parasite prevalence (Asutsuare) communities. Whole blood (5 ml) was collected from each volunteer, plasma was aliquoted and frozen until needed. An aliquot (10 µl) of the blood was used to prepare thick and thin blood smears, 100 µl was preserved in Trizol and the rest was separated into plasma and blood cells and each stored at − 20 °C until needed. Anti-MSP3 and Pfs230 antibody levels were measured using ELISA. Results: Asexual parasite and gametocyte prevalence were higher in Obom than Asutsuare. Antibody (IgG, IgG1, IgG3, IgM) responses against the asexual parasite antigen MSP3 and gametocyte antigen Pfs230 were higher in Obom during the course of the study except for IgM responses against Pfs230, which was higher in Asutsuare than in Obom during the rainy season. Antibody responses in Asutsuare were more significantly associated with age than the responses measured in Obom. Conclusion: The pattern of antibody responses measured in people living in the high and low malaria transmission setting was similar. All antibody responses measured against the asexual antigen MSP3 increased, however, IgG and IgG1 responses against gametocyte antigen Pfs230 decreased in moving from the dry to the peak season in both sites. Whilst asexual and gametocyte prevalence was similar between the seasons in the low transmission setting, in the high transmission setting asexual parasite prevalence increased but gametocyte prevalence decreased in the rainy season relative to the dry season. [ABSTRACT FROM AUTHOR]

Background: The epidemiological control of malaria has been hampered by the appearance of parasite resistance to anti-malarial drugs and by the resistance of mosquito vectors to control measures. This has also been associated with weak transmission control, mostly due to poor control of asymptomatic patients associated with host-vector transmission. This highlights the importance of studying the parasite's sexual forms (gametocytes) which are involved in this phase of the parasite's life-cycle. Some African and Asian strains of Plasmodium falciparum have been fully characterized regarding sexual forms' production; however, few Latin-American strains have been so characterized. This study was aimed at characterizing the Colombian FCB2 strain as a gametocyte producer able to infect mosquitoes. Methods: Gametocyte production was induced in in vitro cultured P. falciparum FCB2 and 3D7 strains. Pfap2g and Pfs25 gene expression was detected in FCB2 strain gametocyte culture by RT-PCR. Comparative analysis of gametocytes obtained from both strains was made (counts and morphological changes). In vitro zygote formation from FCB2 gametocytes was induced by incubating a gametocyte culture sample at 27 °C for 20 min. A controlled Anopheles albimanus infection was made using an artificial feed system with cultured FCB2 gametocytes (14–15 days old). Mosquito midgut dissection was then carried out for analyzing oocysts. Results: The FCB2 strain expressed Pfap2g, Pfs16, Pfg27/25 and Pfs25 sexual differentiation-related genes after in vitro sexual differentiation induction, producing gametocytes that conserved the expected morphological features. The amount of FCB2 gametocytes produced was similar to that from the 3D7 strain. FCB2 gametocytes were differentiated into zygotes and ookinetes after an in vitro low-temperature stimulus and infected An. albimanus mosquitoes, developing to oocyst stage. Conclusions: Even with the history of long-term FCB2 strain in vitro culture maintenance, it has retained its sexual differentiation ability. The gametocytes produced here preserved these parasite forms' usual characteristics and An. albimanus infection capability, thus enabling its use as a tool for studying sexual form biology, An. albimanus infection comparative analysis and anti-malarial drug and vaccine development. [ABSTRACT FROM AUTHOR]

Background: Primaquine is an important gametocytocidal drug that is combined with conventional malaria treatment for prevention of Plasmodium falciparum malaria transmission. Primaquine has been administered together on the first or the last day of conventional treatment but the impact of primaquine timing has never been examined. This study aimed to assess safety, efficacy and optimal timing of single full-dose (0.75 mg/kg) primaquine when added to a standard 6-dose regimen of artemether–lumefantrine (AL). Methods: In an individual-level randomized controlled trial, enrolled participants who were G6PD normal and had uncomplicated P. falciparum malaria were randomly assigned to receive: AL only; AL and a single 0.75 mg/kg primaquine dose on the first day of AL (day 1); or AL and single 0.75 mg//kg primaquine on the last day of AL (day 3). On days 2, 3, 4, 8, 11 and 15, gametocytes were assessed and quantified by microscope and quantitative nuclear acid sequence based quantification (QT-NASBA). Results: Overall, 111 participants aged between 3 and 17 years were randomly allocated to receive AL only (36) or combined with primaquine on day 1 (38), or primaquine on day 3 (37). Day 4 gametocyte prevalence in AL + day 1 primaquine was half the level seen in either AL + day 3 primaquine or AL only arm (11% [4/35] vs 26% [8/31] and 27% [8/30], respectively) albeit not statistically significant. A similar trend of lower gametocyte in the AL + day 1 primaquine verses AL + day 3 primaquine or AL only arm was observed in mean gametocyte density. Mean (sd) haemoglobin level in AL + day 3 primaquine arm recovered from -0.42(1.2) g/dl on day 2 to 0.35 (1.5) g/dl on day 15 of follow up. This was not the case in AL only and AL + day 1 primaquine arms during the same follow-up period, although the difference was not statistically significant (p = 318). No serious adverse events reported in the study. Across arms, 23% (26/111) of participants reported a total of 31 mild adverse events and the difference was not statistically significant (p = 0.477). Conclusion: Primaquine administration on the first day of AL is well tolerated and as safe as later administration. Whilst the World Health Organization currently recommends a lower dose of primaquine (0.25 mg/kg), the findings are supportive of early primaquine administration when combined with artemisinin-combination therapy. ClinicalTrials.gov Registration NCT01906788 [ABSTRACT FROM AUTHOR]

Abstract Background The malaria infection status of mosquitoes is commonly determined by microscopic detection of oocysts on the dissected mosquito midgut. This method is labour-intensive, does not allow processing of large numbers of mosquitoes and can be challenging in terms of objective classification of oocysts. Here, a semi-high-throughput bead-beating ELISA method is proposed for detection of the circumsporozoite protein (CSP) followed by confirmation by quantitative PCR (qPCR). Methods Cultured Plasmodium falciparum gametocytes were offered to Anopheles stephensi mosquitoes and examined by microscopy. After bead-beating, mosquito homogenate was examined by CSP-ELISA and 18S qPCR. As negative controls, mosquitoes that were offered a heat-inactivated gametocyte blood meal were used. The CSP-ELISA/qPCR methodology was applied to high and low-intensity infections of cultured P. falciparum gametocytes. A similar methodology optimized for P. vivax was used on mosquitoes that were offered blood from Ethiopian donors who were naturally infected with P. vivax. Results There was considerable variation in CSP-ELISA signal and qPCR values in mosquitoes with low oocyst intensities. There was a strong agreement mosquito positivity by CSP-ELISA and by qPCR in mosquitoes that fed on cultured P. falciparum material (agreement 96.9%; kappa = 0.97) and naturally infected P. vivax parasite carriers [agreement 92.4% (kappa = 0.83)]. Conclusions The proposed bead-beating CSP-ELISA/qPCR methodology considerably increases throughput for the detection of mosquito infection. qPCR remains necessary to confirm infections in mosquitoes with low CSP-ELISA signal. This methodology may prove particularly useful for studies where very low mosquito infection prevalence is expected and study sites where experience with oocyst detection is limited.

Abstract Background Plasmodium gametocytes are sexual stages transmitted to female Anopheles mosquitoes. While Plasmodium parasites can be differentiated microscopically on Giemsa-stained blood smears, molecular methods are increasingly used because of their increased sensitivity. Molecular detection of gametocytes requires methods that discriminate between asexual and sexual stage parasites. Commonly tested gametocyte-specific mRNAs are pfs25 and pfs230 detected by reverse transcription polymerase chain reaction (RT-PCR). However, detection of these unspliced mRNA targets requires preceding DNase treatment of nucleic acids to eliminate co-purified genomic DNA. If gametocyte-specific, spliced mRNAs could be identified, DNase treatment could be eliminated and one-step multiplexed molecular methods utilized. Results Expression data was used to identify highly-expressed mRNAs in mature gametocytes that were also low in antisense RNA expression in non-gametocyte stages. After testing numerous candidate mRNAs, the spliced female Pf3D7_0630000 mRNA was selected as a Plasmodium falciparum gametocyte-specific biomarker compatible with Plasmodium 18S rRNA RT-PCR. This mRNA was only detected in samples containing mature gametocytes and was absent in those containing only asexual stage parasites or uninfected human blood. PF3D7_0630000 RT-PCR detected gametocytes across a wide range of parasite densities in both spiked and clinical samples and agreed with pfs25 RT-PCR, the gold standard for RT-PCR-based gametocyte detection. PF3D7_0630000 multiplexed with Plasmodium 18S rRNA RT-PCR was more sensitive than other spliced mRNA targets for one-step RT-PCR gametocyte detection. Conclusions Because the spliced target does not require DNase treatment, the PF3D7_0630000 assay can be multiplexed with Plasmodium 18S rRNA for direct one-step detection of gametocytes from whole human blood.

Abstract Background Blocking malaria transmission is an important step in eradicating malaria. In the field, transmission requires the production of sexual stage Plasmodium parasites, called gametocytes, which are not effectively killed by the commonly used anti-malarials allowing individuals to remain infectious after clearance of asexual parasites. Methods To identify new gametocytocidal compounds, a library of 45,056 compounds with diverse structures was screened using a high throughput gametocyte viability assay. The characteristics of active hits were further evaluated against asexual stage parasites in a growth inhibition assay. Their cytotoxicity were tested against mammalian cells in a cytotoxicity assay. The chemical scaffold similarity of active hits were studied using scaffold cluster analysis. Results A set of 23 compounds were identified and further confirmed for their activity against gametocytes. All the 23 confirmed compounds possess dual-activities against both gametocytes responsible for human to mosquito transmission and asexual parasites that cause the clinical symptoms. Three of these compounds were fourfold more active against gametocytes than asexual parasites. Further cheminformatic analysis revealed three sets of novel scaffolds, including highly selective 4-1H-pyrazol-5-yl piperidine analogs. Conclusions This study revealed important new structural scaffolds that can be used as starting points for dual activity anti-malarial drug development.

Background: The malaria infection status of mosquitoes is commonly determined by microscopic detection of oocysts on the dissected mosquito midgut. This method is labour-intensive, does not allow processing of large numbers of mosquitoes and can be challenging in terms of objective classification of oocysts. Here, a semi-high-throughput bead-beating ELISA method is proposed for detection of the circumsporozoite protein (CSP) followed by confirmation by quantitative PCR (qPCR). Methods: Cultured Plasmodium falciparum gametocytes were offered to Anopheles stephensi mosquitoes and examined by microscopy. After bead-beating, mosquito homogenate was examined by CSP-ELISA and 18S qPCR. As negative controls, mosquitoes that were offered a heat-inactivated gametocyte blood meal were used. The CSP-ELISA/qPCR methodology was applied to high and low-intensity infections of cultured P. falciparum gametocytes. A similar methodology optimized for P. vivax was used on mosquitoes that were offered blood from Ethiopian donors who were naturally infected with P. vivax. Results: There was considerable variation in CSP-ELISA signal and qPCR values in mosquitoes with low oocyst intensities. There was a strong agreement mosquito positivity by CSP-ELISA and by qPCR in mosquitoes that fed on cultured P. falciparum material (agreement 96.9%; kappa = 0.97) and naturally infected P. vivax parasite carriers [agreement 92.4% (kappa = 0.83)]. Conclusions: The proposed bead-beating CSP-ELISA/qPCR methodology considerably increases throughput for the detection of mosquito infection. qPCR remains necessary to confirm infections in mosquitoes with low CSP-ELISA signal. This methodology may prove particularly useful for studies where very low mosquito infection prevalence is expected and study sites where experience with oocyst detection is limited. [ABSTRACT FROM AUTHOR]

Background: Plasmodium gametocytes are sexual stages transmitted to female Anopheles mosquitoes. While Plasmodium parasites can be differentiated microscopically on Giemsa-stained blood smears, molecular methods are increasingly used because of their increased sensitivity. Molecular detection of gametocytes requires methods that discriminate between asexual and sexual stage parasites. Commonly tested gametocyte-specific mRNAs are pfs25 and pfs230 detected by reverse transcription polymerase chain reaction (RT-PCR). However, detection of these unspliced mRNA targets requires preceding DNase treatment of nucleic acids to eliminate co-purified genomic DNA. If gametocyte-specific, spliced mRNAs could be identified, DNase treatment could be eliminated and one-step multiplexed molecular methods utilized. Results: Expression data was used to identify highly-expressed mRNAs in mature gametocytes that were also low in antisense RNA expression in non-gametocyte stages. After testing numerous candidate mRNAs, the spliced female Pf3D7_0630000 mRNA was selected as a Plasmodium falciparum gametocyte-specific biomarker compatible with Plasmodium 18S rRNA RT-PCR. This mRNA was only detected in samples containing mature gametocytes and was absent in those containing only asexual stage parasites or uninfected human blood. PF3D7_0630000 RT-PCR detected gametocytes across a wide range of parasite densities in both spiked and clinical samples and agreed with pfs25 RTPCR, the gold standard for RT-PCR-based gametocyte detection. PF3D7_0630000 multiplexed with Plasmodium 18S rRNA RT-PCR was more sensitive than other spliced mRNA targets for one-step RT-PCR gametocyte detection. Conclusions: Because the spliced target does not require DNase treatment, the PF3D7_0630000 assay can be multiplexed with Plasmodium 18S rRNA for direct one-step detection of gametocytes from whole human blood. [ABSTRACT FROM AUTHOR]

Background: An effective malaria transmission-blocking vaccine may play an important role in malaria elimination efforts, and a robust biological assay is essential for its development. The standard membrane-feeding assay (SMFA) for Plasmodium falciparum infection of mosquitoes is considered a "gold standard" assay to measure transmissionblocking activity of test antibodies, and has been utilized widely in both non-clinical and clinical studies. While several studies have discussed the inherent variability of SMFA within a study group, there has been no assessment of interlaboratory variation. Therefore, there is currently no assurance that SMFA results are comparable between different studies. Methods: Mouse anti-Pfs25 monoclonal antibody (mAb, 4B7 mAb), rat anti-Pfs48/45 mAb (85RF45.1 mAb) and a human polyclonal antibody (pAb) collected from a malaria-exposed adult were tested at the same concentrations (6-94 μg/mL for 4B7, 1.2-31.3 μg/mL for 85RF45.1 and 23-630 μg/mL for human pAb) in two laboratories following their own standardized SMFA protocols. The mAbs and pAb, previously shown to have strong inhibition activities in the SMFA, were tested at three or four concentrations in two or three independent assays in each laboratory, and percent inhibition in mean oocyst intensity relative to a control in the same feed was determined in each feeding experiment. Results: Both monoclonal and polyclonal antibodies dose-dependently reduced oocyst intensity in all experiments performed at the two test sites. In both laboratories, the inter-assay variability in percent inhibition in oocyst intensity decreased at higher levels of inhibition, regardless of which antibody was tested. At antibody concentrations that led to a >80 % reduction in oocyst numbers, the inter-laboratory variations were in the same range compared with the inter-assay variation observed within a single laboratory, and the differences in best estimates from multiple feeds between the two laboratories were <5 percentage points. Conclusions: This study confirms previous reports that the precision of the SMFA increases with increasing percent inhibition. Moreover, the variation between the two laboratories is not greater than the variation observed within a laboratory. The findings of this study provide guidance for comparison of SMFA data from different laboratories. [ABSTRACT FROM AUTHOR]