Presence Of Gametocytes Research Articles

Treatment of uncomplicated Plasmodium vivax with chloroquine plus radical cure with primaquine without G6PDd testing is safe in Arba Minch, Ethiopia: assessment of clinical and parasitological response

BackgroundEthiopia rolled out primaquine nationwide in 2018 for radical cure along with chloroquine for the treatment of uncomplicated Plasmodium vivax malaria in its bid for malaria elimination by 2030. The emergence of anti-malarial drug resistance would challenge the elimination goal. There is limited evidence on the emergence of chloroquine drug resistance. The clinical and parasitological outcomes of treatment of P. vivax with chloroquine plus radical cure using low dose 14 days primaquine were assessed in an endemic area of Ethiopia.MethodsA semi-directly observed 42-days follow up in-vivo therapeutic efficacy study was conducted from October 2019 to February 2020. Plasmodium vivax mono-species infected patients (n = 102) treated with a 14 days low dose (0.25 mg/kg body weight per day) primaquine plus chloroquine (a total dose of 25 mg base/kg for 3 days) were followed for 42 days to examine clinical and parasitological outcomes. Samples collected at recruitment and days of recurrence were examined by 18 S based nested polymerase chain reaction (nPCR) and Pvmsp3α nPCR-restriction fragment length polymorphism. Asexual parasitaemia and the presence of gametocytes were assessed on the scheduled days using microscopy. Clinical symptoms, haemoglobin levels, and Hillmen urine test were also assessed.ResultsOf the 102 patients followed in this study, no early clinical and parasitological failure was observed. All patients had adequate clinical and parasitological responses within the 28 days of follow up. Late clinical (n = 3) and parasitological (n = 6) failures were observed only after day 28. The cumulative incidence of failure was 10.9% (95% confidence interval, 5.8–19.9%) on day 42. Among the paired recurrent samples, identical clones were detected only in two samples on day 0 and day of recurrences (day 30 and 42) using Pvmsp3α genotyping. No adverse effect was detected related to the low dose 14 days primaquine administrations.ConclusionCo-administration of CQ with PQ in the study area is well tolerated and there was no recurrence of P. vivax before 28 days of follow up. Interpretation of CQ plus PQ efficacy should be done with caution especially when the recurrent parasitaemia occurs after day 28. Therapeutic efficacy studies with appropriate design might be informative to rule out chloroquine or primaquine drug resistance and/or metabolism in the study area.

Key cytokines and hematological parameters in patients with uncomplicated falciparum malaria in Hodeidah, Yemen

Immunity to malaria has a major role in controlling disease and pathogenesis with cytokine production being involved in almost every phase of the immune response. The present study aimed to assess hematological variables and to measure plasma levels of TNFα, IFNγ and IL10, their ratios, and their relation to parasitemia among patients with uncomplicated falciparum malaria in Hodeidah, Yemen. Forty patients with uncomplicated P. falciparum monoinfection and 40 healthy age and sex-matched controls were enrolled in the study. Parasitological diagnosis was confirmed, and parasite density was estimated. Plasma cytokine levels, hematologic parameters, and the presence of gametocytes were determined. Results revealed higher TNFα, IFNγ and IL10 in patients than in controls. A relatively higher IL10 production was demonstrated by the significantly lower TNFα/IL10 and IFNγ/IL10 ratios in patients than in controls. TNFα and IL10 correlated positively with parasite density. Lower Hb levels, RBC, lymphocyte and platelet counts, and higher neutrophil and reticulocyte counts were observed in patients compared to controls. Reticulocyte count was higher and IFNγ level was lower in the presence of gametocytes. Conclusively, uncomplicated falciparum malaria is associated with the ability to regulate the production of pro-inflammatory and anti-inflammatory cytokines. This mediates parasite clearance while simultaneously avoiding severe pathology.

Prevalence of Plasmodium falciparum gametocytaemia in asymptomatic school children before and after treatment with dihydroartemisinin-piperaquine (DP)

BackgroundAsymptomatic Plasmodium carriers form the majority of malaria-infected individuals in most endemic areas. A proportion of these asymptomatically infected individuals carry gametocytes, the transmissible stages of malaria parasites, that sustain human to mosquito transmission. Few studies examine gametocytaemia in asymptomatic school children who may form an important reservoir for transmission. We assessed the prevalence of gametocytaemia before antimalarial treatment and monitored clearance of gametocytes after treatment in asymptomatic malaria children. MethodsA total of 274 primary school children were screened for P. falciparum parasitaemia by microscopy. One hundred and fifty-five (155) parasite positive children were treated under direct observation with dihydroartemisinin-piperaquine (DP). Gametocyte carriage was determined by microscopy seven days prior to treatment, day 0 before treatment, and on days 7, 14 and 21 post initiation of treatment. ResultsThe prevalence of microscopically-detectable gametocytes at screening (day −7) and enrolment (day 0) were 9% (25/274) and 13.6% (21/155) respectively. Following DP treatment, gametocyte carriage dropped to 4% (6/135), 3% (5/135) and 6% (10/151) on days 7, 14 and 21 respectively. Asexual parasites persisted in a minority of treated children, resulting in microscopically detectable parasites on days 7 (9%, 12/135), 14 (4%, 5/135) and 21 (7%, 10/151). Gametocyte carriage was inversely correlated with the age of the participants (p = 0.05) and asexual parasite density (p = 0.08). In a variate analysis, persistent gametocytaemia 7 or more days after treatment was significantly associated with post-treatment asexual parasitaemia at day 7 (P = 0.027) and presence of gametocytes on the day of treatment (P < 0.001). ConclusionsThough DP provides both excellent cure rates for clinical malaria and a long prophylactic half-life, our findings suggest that after treatment of asymptomatic infections, both asexual parasites and gametocytes may persist in a minority of individuals during the first 3 weeks after treatment. This indicates DP may be unsuitable for use in mass drug administration strategies towards malaria elimination in Africa.

Prevalence of Haemoproteus columbae in domestic pigeons (Columba livia domestica) reared in a coastal enclave of Southern India

Haemoproteus columbae is a species of malarian parasite affecting pigeons coming under the family Plasmodiidae. A study was conducted to know the prevalence of Haemoproteus columbae in domestic pigeons of the coastal enclave of Puducherry, India, using thin blood smears stained with Giemsa and Leishman stain, which was confirmed by the presence of gametocytes in the RBCs. A total of 50 blood samples were collected and examined during the study, out of which 46 (92%) samples were found to be positive for Haemoproteus columbae in the adolescent and adult age group of birds, and the vector Pseudolynchia canariensis were also collected from the birds during the study period.

Prevalence of asymptomatic P. falciparum gametocyte carriage among school children in Mbita, Western Kenya and assessment of the association between gametocyte density, multiplicity of infection and mosquito infection prevalence.

Background: Asymptomatic Plasmodium falciparum gametocyte carriers are reservoirs for sustaining transmission in malaria endemic regions. Gametocyte presence in the host peripheral blood is a predictor of capacity to transmit malaria. However, it does not always directly translate to mosquito infectivity. Factors that affect mosquito infectivity include, gametocyte sex-ratio and density, multiplicity of infection (MOI), and host and vector anti-parasite immunity. We assess the prevalence of gametocyte carriage and some of its associated risk factors among asymptomatic schoolchildren in Western Kenya and to further analyse the association between gametocyte density, multiplicity of infection (MOI) and mosquito infection prevalence. Methods: P. falciparum parasite infections were detected by RDT (Rapid Diagnostic Test) and microscopy among schoolchildren (5-15 years old). Blood from 37 microscopy positive gametocyte carriers offered to laboratory reared An. gambiae s.l. mosquitoes. A total of 3395 fully fed mosquitoes were screened for Plasmodium sporozoites by ELISA. P. falciparum was genotyped using 10 polymorphic microsatellite markers. The association between MOI and gametocyte density and mosquito infection prevalence was investigated. Results: A significantly higher prevalence of P. falciparum infection was found in males 31.54% (764/2422) ( p-value < 0.001) compared to females 26.72% (657/2459). The microscopic gametocyte prevalence among the study population was 2% (84/4881). Children aged 5-9 years have a higher prevalence of gametocyte carriage (odds ratios = 2.1 [95% CI = 1.3-3.4], P = 0.002) as compared to children aged 10-15 years. After offering gametocyte positive blood to An. gambiae s.l. by membrane feeding assay, our results indicated that 68.1% of the variation in mosquito infection prevalence was accounted for by gametocyte density and MOI (R-SQR. = 0.681, p<0.001). Conclusions: We observed a higher risk of gametocyte carriage among the younger children (5-9 years). Gametocyte density and MOI significantly predicted mosquito infection prevalence.

Gametocyte carriage of Plasmodium falciparum (pfs25) and Plasmodium vivax (pvs25) during mass screening and treatment in West Timor, Indonesia: a longitudinal prospective study

BackgroundA 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.MethodsRT-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.ResultsParasite 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.ConclusionIn 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.

Prevalence of asymptomatic P. falciparum gametocyte carriage among school children in Mbita, Western Kenya and assessment of the association between gametocyte density, multiplicity of infection and mosquito infection prevalence.

Background: Asymptomatic Plasmodium falciparum gametocyte carriers are reservoirs for sustaining transmission in malaria endemic regions. Gametocyte presence in the host peripheral blood is a predictor of capacity to transmit malaria. However, it does not always directly translate to mosquito infectivity. Factors that affect mosquito infectivity include, gametocyte sex-ratio and density, multiplicity of infection (MOI), and host and vector anti-parasite immunity. We assess the prevalence of gametocyte carriage and some of its associated risk factors among asymptomatic schoolchildren in Western Kenya and to further analyse the association between gametocyte density, multiplicity of infection (MOI) and mosquito infection prevalence. Methods: P. falciparum parasite infections were detected by RDT (Rapid Diagnostic Test) and microscopy among schoolchildren (5-15 years old). Blood from 37 microscopy positive gametocyte carriers offered to laboratory reared An. gambiae s.l. mosquitoes. A total of 3395 fully fed mosquitoes were screened for Plasmodium sporozoites by ELISA. P. falciparum was genotyped using 10 polymorphic microsatellite markers. The association between MOI and gametocyte density and mosquito infection prevalence was investigated. Results: A significantly higher prevalence of P. falciparum infection was found in males 31.54% (764/2422) ( p-value < 0.001) compared to females 26.72% (657/2459). The microscopic gametocyte prevalence among the study population was 2% (84/4881). Children aged 5-9 years have a higher prevalence of gametocyte carriage (odds ratios = 2.1 [95% CI = 1.3–3.4], P = 0.002) as compared to children aged 10-15 years. After offering gametocyte positive blood to An. gambiae s.l. by membrane feeding assay, our results indicated that 68.1% of the variation in mosquito infection prevalence was accounted for by gametocyte density and MOI (R-SQR. = 0.681, p < 0.001). Conclusions: We observed a higher risk of gametocyte carriage among the younger children (5-9 years). Gametocyte density and MOI significantly predicted mosquito infection prevalence.

Plasmodium falciparum Genetic Diversity in Panamá Based on glurp, msp-1 and msp-2 Genes: Implications for Malaria Elimination in Mesoamerica.

Panamá, together with all the nations in Mesoamerica, has committed to eliminate malaria from the region by 2020. As these countries approach malaria elimination and local transmission decreases, an active molecular surveillance to identify genotypes circulating along the border areas is particularly needed to accurately infer infection origin, drug resistance and disease propagation patterns in the region. This study evaluated the genetic diversity and allele frequencies of msp-1, msp-2 and glurp genes using different molecular analyses (nested PCR, PCR-restriction fragment length polymorphism (RFLP) and sequencing) from 106 autochthonous and imported P. falciparum isolates collected from different endemic areas in Panamá between 2003 and 2019. We also explored if P. falciparum genotypes assessed with these molecular markers were associated with relevant malaria epidemiological parameters using a multiple correspondence analysis. A strong association of certain local haplotypes with their geographic distribution in endemic areas, but also with parasite load and presence of gametocytes, was evidenced. Few multiclonal infections and low genetic diversity among locally transmitted P. falciparum samples were detected, consequent with the low transmission intensity of this parasite in Panamá, a pattern likely to be extended across Mesoamerica. In addition, several imported cases were genetically dissimilar to local infections and representative of more diverse extra-continental lineages.

Prevalence, characteristics and risk factors of imported and local malaria cases in North-Western Province, Zambia: a cross-sectional study

BackgroundImported malaria is a major challenge for countries that are in malaria elimination stage such as Zambia. Legitimate cross-border activities add to the risk of transmission, necessitating determination of prevalence, characteristics and risk factors of imported and local malaria.MethodsThis cross-sectional study was conducted in 103 consented child and adult patients with clinical malaria symptoms, from selected health facilities in north-western Zambia. Patient demographic data and blood samples for malaria microscopy and full blood count were obtained. Chi-square and penalized logistic regression were performed to describe the characteristics and assess the risk factors of imported and local malaria in North-Western Province.ResultsOverall, malaria prevalence was 78.6% with 93.8% Plasmodium falciparum and 6.2% other species. The local cases were 72 (88.9%) while the imported were 9 (11.1%) out of the 81 positive participants. About 98.6% of the local cases were P. falciparum compared to 55.6% (χ2 = 52.4; p < 0.01) P. falciparum among the imported cases. Among the imported cases, 44% were species other than P. falciparum (χ2 = 48; p < 0.01) while among the local cases only 1.4% were. Gametocytes were present in 44% of the imported malaria cases and only in 2.8% of the local cases (χ2 = 48; p < 0.01). About 48.6% of local participants had severe anaemia compared to 33.3% of participants from the two neighbouring countries who had (χ2 = 4.9; p = 0.03). In the final model, only country of residence related positively to presence of species other than P. falciparum (OR = 39.0, CI [5.9, 445.9]; p < 0.01) and presence of gametocytes (OR = 23.1, CI [4.2, 161.6]; p < 0.01).ConclusionMalaria prevalence in North-Western Province is high, with P. falciparum as the predominant species although importation of Plasmodium ovale and Plasmodium malariae is happening as well. Country of residence of patients is a major risk factor for malaria species and gametocyte presence. The need for enhanced malaria control with specific focus on border controls to detect and treat, for specific diagnosis and treatment according to species obtaining, for further research in the role of species and gametocytaemia in imported malaria, cannot be overemphasized.

UNCOMMON PRESENTATION OF A COMMON DISEASE AS PUO

We report a case of pyrexia of unknown origin in a 35 yr old male . He presented to us with fever since 2 months which was associated with chills and rigor . He is non alcoholic , non smoker . Mild Hepatosplenomegaly was found on initial physical examination. Before he was referred to our hospital, he was investigated in outside hospital . The serial Rapid Diagnostic Test and microscopic peripheral blood smears for plasmodium were negative . He was treated symptomatically . In view of non subsiding fever patient was referred to higher centre . He presented to our institute for further evaluation . His laboratory findings found pancytopenia. Bone marrow aspiration(BMA) was done. BMA showed Gametocytes of P. falciparum. The presence of gametocytes of P.Falciparum in the bone marrow ,not in peripheral smear and presenting as PUO is rare .

Assays for quantification of male and female gametocytes in human blood by qRT-PCR in the absence of pure sex-specific gametocyte standards

BackgroundMalaria transmission from humans to Anopheles mosquitoes requires the presence of gametocytes in human peripheral circulation, and the dynamics of transmission are determined largely by the density and sex ratio of the gametocytes. Molecular methods are thus employed to measure gametocyte densities, particularly when assessing transmission epidemiology and the efficacy of transmission-blocking interventions. However, accurate quantification of male and female gametocytes with molecular methods requires pure male and female gametocytes as reference standards, which are not widely available.MethodsqRT-PCR assays were used to quantify levels of sex-specific mRNA transcripts in Plasmodium falciparum female and male gametocytes (pfs25 and pfMGET, respectively) using synthetic complimentary RNA standards and in vitro cultured gametocytes. Assays were validated and assay performance was investigated in blood samples of clinical trial participants using these standards and compared to absolute quantification by droplet digital PCR (ddPCR).ResultsThe number of transcript copies per gametocyte were determined to be 279.3 (95% CI 253.5–307.6) for the female-specific transcript pfs25, and 12.5 (95% CI 10.6–14.9) for the male-specific transcript pfMGET. These numbers can be used to convert from transcript copies/mL to gametocyte/mL. The reportable range was determined to be 5.71 × 106 to 5.71 female gametocytes/mL for pfs25, and 1.73 × 107 to 1.73 × 101 male gametocytes/mL for pfMGET. The limit of detection was 3.9 (95% CI 2.5–8.2) female gametocytes/mL for pfs25, and 26.9 (95% CI 19.3–51.7) male gametocytes/mL for PfMGET. Both assays showed minimal intra-assay and inter-assay variability with coefficient of variation < 3%. No cross-reactivity was observed in both assays in uninfected human blood samples. Comparison of results from ddPCR to qRT-PCR assays on clinical blood samples indicated a high-level agreement (ICC = 0.998 for pfs25 and 0.995 for pfMGET).ConclusionsThis study reports the validation of qRT-PCR assays that are able to accurately quantify female and male P. falciparum gametocytes at sub-microscopic densities. The assays showed excellent reproducibility, sensitivity, precision, specificity, and accuracy. The methodology will enable the estimation of gametocyte density in the absence of pure female and male gametocyte standards, and will facilitate clinical trials and epidemiological studies.

A multiplex assay for the sensitive detection and quantification of male and female Plasmodium falciparum gametocytes

BackgroundThe transmission of malaria to mosquitoes depends on the presence of gametocytes that circulate in the peripheral blood of infected human hosts. Sensitive estimates of the densities of female gametocytes (FG) and male gametocytes (MG) may allow the prediction of infectivity to mosquitoes and thus a molecular estimate of the human infectious reservoir for transmission.MethodsA novel multiplex qRT-PCR assay with intron-spanning primers was developed for the parallel quantification of FG and MG. CCp4 (PF3D7_0903800) transcripts specific for FG and PfMGET (PF3D7_1469900) transcripts specific for MG were quantified in total nucleic acids. The assay was validated on sex-sorted gametocytes from culture material and on samples from clinical trials with gametocytocidal drugs. Synthetic RNA standards were generated for the two targets genes and calibrated against known gametocyte quantities.ResultsThe limit of detection was determined at 0.1 male and 0.1 female gametocyte/µL, which was equal to the limit of quantification (LOQ) for MG, while the LOQ for FG was 1 FG/µL. Results from previously reported clinical trials that used separate gametocyte qRT-PCR assays for FG (targeting Pfs25) and MG (targeting PfMGET) were reproduced with the multiplex assay. High levels of agreement between separate assays and the multiplex approach were observed (R2 = 0.9473, 95% CI 0.9314–0.9632, for FG measured by transcript levels of Pfs25 in qRT-PCR or CCp4 in multiplex; R2 = 0.8869, 95% CI 0.8541–0.9197, for MG measured by PfMGET in either single or multiplex qRT-PCR). FG and MG transcripts were detected in pure ring stage parasites at 10,000- and 100,000-fold reduced frequency for CCp4 and PfMGET, respectively. The CCp4 and PfMGET transcripts were equally stable under suboptimal storage conditions.ConclusionsGametocyte densities and their sex ratios can be determined in the presented one-step multiplex assay with higher throughput than single assays. The interpretation of low gametocyte densities at asexual parasite densities above 1000 parasites/µL requires caution to avoid false positive gametocyte signals from spurious transcript levels in ring stage parasites.

Commit, hide and escape: the story of Plasmodium gametocytes.

Malaria is the major cause of mortality and morbidity in tropical countries. The causative agent, Plasmodium sp., has a complex life cycle and is armed with various mechanisms which ensure its continuous transmission. Gametocytes represent the sexual stage of the parasite and are indispensable for the transmission of the parasite from the human host to the mosquito. Despite its vital role in the parasite's success, it is the least understood stage in the parasite's life cycle. The presence of gametocytes in asymptomatic populations and induction of gametocytogenesis by most antimalarial drugs warrants further investigation into its biology. With a renewed focus on malaria elimination and advent of modern technology available to biologists today, the field of gametocyte biology has developed swiftly, providing crucial insights into the molecular mechanisms driving sexual commitment. This review will summarise key current findings in the field of gametocyte biology and address the associated challenges faced in malaria detection, control and elimination.

Detection of malaria parasites after treatment in travelers: A 12-months longitudinal study and statistical modelling analysis

The rapid clearance of malaria parasite DNA from circulation has widely been accepted as a fact without being systemically investigated. In this longitudinal study, we examined the duration of PCR positivity as well as the presence of gametocytes in adult travelers treated for Plasmodium falciparum malaria in a malaria-free setting, using microscopy, species-specific qPCR, merozoite surface protein 2 (msp2)-genotyping PCR, and gametocyte-specific qPCR. Venous blood was collected at the time of admission and prospectively up to one year. Patients were treated with a full regimen of six doses of artemether-lumefantrine (AL). In 31 successfully treated individuals, asexual parasites were seen by microscopy until two days after treatment, whereas parasite DNA was detected by msp2- and species-specific PCR up to days 31 and 42, respectively. Statistical modelling predicted 26% (± 0•05 SE) species-specific PCR positivity until day 40 and estimated 48 days for all samples to become PCR negative. Gametocytes were detected by microscopy and PCR latest two days after treatment. CT values correlated well with microscopy-defined parasite densities before but not after treatment started. Duration of PCR positivity was correlated neither with the initial (asexual) parasite densities nor with the initial presence of gametocytes.These results reveal that PCR positivity can persist several weeks after treatment without evidence of viable sexual or asexual parasites, and that the removal of dead parasites and their debris is not as rapid as it is believed, indicating that PCR may overestimate post-treatment parasite prevalence in epidemiological studies, and underestimate drug efficiency in clinical management and trials. This report underlines an important diagnostic matter essentially in infectious diseases and particularly in malaria, and points out the need for detection tool as sensitive as PCR and as accurate as microscopy

Predominance of asymptomatic and sub-microscopic infections characterizes the Plasmodium gametocyte reservoir in the Peruvian Amazon

Malaria transmission requires that Anopheles mosquitoes ingest Plasmodium gametocyte stages circulating in the human bloodstream. In the context of malaria elimination, understanding the epidemiology of gametocytes relative to all Plasmodium infections and the contribution of asymptomatic and sub-microscopic parasite carriers to the gametocyte reservoir is necessary, especially in low endemic settings with predominance of P.vivax. A 13-month longitudinal study was conducted in two communities (n = 1935 individuals) of Loreto Department, Peru, with five active screenings for Plasmodium infections and gametocyte stages by quantitative real-time PCR (qPCR) and reverse transcription (RT)-qPCR, respectively. Parasite prevalence by qPCR was 7.2% for P.vivax (n = 520/7235; range by survey 6.0%-8.1%) and 3.2% for P.falciparum (n = 235/7235; range by survey 0.4%-7.7%). Sub-microscopic infections accounted for 73.5% of P.vivax (range by survey 60%-89%) and almost the totality of P.falciparum cases. Gametocytes were found in 28.4% P.vivax infections (range by survey 18.7%-34.1%), with a peak of 61.5% in one community at the start of the transmission season. About 59.8% of all P.vivax gametocyte carriers were asymptomatic and 31.9% were sub-microscopic. Age patterns for gametocyte prevalence paralleled asexual stage infections and peaked among >15–25 year old individuals. Asexual parasite density was found to be the strongest predictor for P.vivax gametocyte presence in longitudinal multivariate analysis (odds ratio 2.33 [95% confidence interval 1.96, 2.78]; P<0.001). Despite significant differences in seasonality patterns and P.vivax prevalence found at the local scale, sub-microscopic and asymptomatic infections predominate and contribute significantly to the gametocyte reservoir in different communities of the Peruvian Amazon. Control and elimination campaigns need sensitive tools to detect all infections that escape routine malaria surveillance, which may contribute to maintain transmission in the region.

Recognition of Plasmodium falciparum mature gametocyte-infected erythrocytes by antibodies of semi-immune adults and malaria-exposed children from Gabon

BackgroundTransmission of malaria from man to mosquito depends on the presence of gametocytes, the sexual stage of Plasmodium parasites in the infected host. Naturally acquired antibodies against gametocytes exist and may play a role in controlling transmission by limiting the gametocyte development in the circulation or by interrupting gamete development and fertilization in the mosquito following ingestion. So far, most studies on antibody responses to sexual stage antigens have focused on a subset of gametocyte-surface antigens, even though inhibitory Ab responses to other gametocyte antigens might also play a role in controlling gametocyte density and fertility. Limited information is available on natural antibody response to the surfaces of gametocyte-infected erythrocytes.MethodsAb responses to surface antigens of erythrocytes infected by in vitro differentiated Plasmodium falciparum mature gametocytes were investigated in sera of semi-immune adults and malaria-exposed children. In addition, the effect of immunization with GMZ2, a blood stage malaria vaccine candidate, and the effect of intestinal helminth infection on the development of immunity to gametocytes of P. falciparum was evaluated in malaria-exposed children and adults from Gabon. Serum samples from two Phase I clinical trials conducted in Gabon were analysed by microscopic and flow-cytometric immunofluorescence assay.ResultsAdults had a higher Ab response compared to children. Ab reactivity was significantly higher after fixation and permeabilization of parasitized erythrocytes. Following vaccination with the malaria vaccine candidate GMZ2, anti-gametocyte Ab concentration decreased in adults compared to baseline. Ab response to whole asexual stage antigens had a significant but weak positive correlation to anti-gametocyte Ab responses in adults, but not in children. Children infected with Ascaris lumbricoides had a significantly higher anti-gametocyte Ab response compared to non-infected children.ConclusionThe current data suggest that antigens exposed on the gametocyte-infected red blood cells are recognized by serum antibodies from malaria-exposed children and semi-immune adults. This anti-gametocyte immune response may be influenced by natural exposure and vaccination. Modulation of the natural immune response to gametocytes by co-infecting parasites should be investigated further and may have an important impact on malaria control strategies.

High prevalence of Plasmodium falciparum gametocyte infections in school-age children using molecular detection: patterns and predictors of risk from a cross-sectional study in southern Malawi.

BackgroundIn endemic areas, many people experience asymptomatic Plasmodium infections, particularly older children and adults, but their transmission contribution is unknown. Though not the exclusive determinant of infectiousness, transmission from humans to mosquitoes requires blood meals containing gametocytes. Gametocytes often occur at submicroscopic densities, challenging measurement in human populations. More sensitive molecular techniques allow better characterization of gametocyte epidemiologic patterns.MethodsApproximately 30 households were selected from each of eight sites in southern Malawi during two cross-sectional surveys. Blood was sampled from 623 people during the dry season and 896 the following rainy season. Among people PCR-positive for Plasmodium falciparum, mature gametocytes were detected by qRT-PCR. Regression models evaluated predictors of gametocyte carriage and density in the total population and among those with PCR-positive infections.ResultsThe prevalence of gametocyte carriage by molecular testing was 3.5% during the dry season and 8.6% during the rainy season, and by microscopy 0.8 and 3.3%, respectively. Nearly half of PCR-positive infections carried gametocytes, regardless of recent symptom status. Among P. falciparum-infected people, only living in unfinished houses and age were significantly associated with gametocyte presence. Infected people in unfinished houses had higher odds of carrying gametocytes (OR 2.24, 95% CI 1.16–4.31), and 31% (95% CI 3–65%) higher gametocyte density than those in finished houses. School-age children (5–15 years), had higher odds than adults (≥16 years) of having gametocytes when infected (OR 2.77, 95% CI 1.47–5.19), but 31% (95% CI 11–47%) lower gametocyte density. Children <5 years did not have significantly higher odds of gametocyte carriage or density when infected than adults.ConclusionsSchool-age children frequently carry gametocytes in communities of southern Malawi and represent an under-recognized reservoir of infection. Malaria elimination strategies should address these frequently asymptomatic reservoirs, especially in highly endemic areas. Improved household construction may also reduce the infectious reservoir.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1587-9) contains supplementary material, which is available to authorized users.

Assessment of submicroscopic infections and gametocyte carriage of Plasmodium falciparum during peak malaria transmission season in a community-based cross-sectional survey in western Kenya, 2012.

BackgroundAlthough malaria control intervention has greatly decreased malaria morbidity and mortality in many African countries, further decline in parasite prevalence has stagnated in western Kenya. In order to assess if malaria transmission reservoir is associated with this stagnation, submicroscopic infection and gametocyte carriage was estimated. Risk factors and associations between malaria control interventions and gametocyte carriage were further investigated in this study.MethodsA total of 996 dried blood spot samples were used from two strata, all smear-positives (516 samples) and randomly selected smear-negatives (480 samples), from a community cross-sectional survey conducted at peak transmission season in 2012 in Siaya County, western Kenya. Plasmodium falciparum parasite presence and density were determined by stained blood smear and by 18S mRNA transcripts using nucleic acid sequence-based amplification assay (NASBA), gametocyte presence and density were determined by blood smear and by Pfs25 mRNA-NASBA, and gametocyte diversity by Pfg377 mRNA RT-PCR and RT-qPCR.ResultsOf the randomly selected smear-negative samples, 69.6 % (334/480) were positive by 18S-NASBA while 18S-NASBA detected 99.6 % (514/516) smear positive samples. Overall, 80.2 % of the weighted population was parasite positive by 18S-NASBA vs 30.6 % by smear diagnosis and 44.0 % of the weighted population was gametocyte positive by Pfs25-NASBA vs 2.6 % by smear diagnosis. Children 5–15 years old were more likely to be parasitaemic and gametocytaemic by NASBA than individuals >15 years old or children <5 years old while gametocyte density decreased with age. Anaemia and self-reported fever within the past 24 h were associated with increased odds of gametocytaemia. Fever was also positively associated with parasite density, but not with gametocyte density. Anti-malarial use within the past 2 weeks decreased the odds of gametocytaemia, but not the odds of parasitaemia. In contrast, recent anti-malarial use was associated with lowered parasite density, but not the gametocyte density. Use of ITNs was associated with lower odds for parasitaemia in part of the study area with a longer history of ITN interventions. In the same part of study area, the odds of having multiple gametocyte alleles were also lower in individuals using ITNs than in those not using ITNs and parasite density was positively associated with gametocyte diversity.ConclusionA large proportion of submicroscopic parasites and gametocytes in western Kenya might contribute to the stagnation in malaria prevalence, suggesting that additional interventions targeting the infectious reservoir are needed. As school aged children and persons with anaemia and fever were major sources for gametocyte reservoir, these groups should be targeted for intervention and prevention to reduce malaria transmission. Anti-malarial use was associated with lower parasite density and odds of gametocytaemia, but not the gametocyte density, indicating a limitation of anti-malarial impact on the transmission reservoir. ITN use had a protective role against parasitaemia and gametocyte diversity in western Kenya.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1482-4) contains supplementary material, which is available to authorized users.

High Rates of Asymptomatic, Sub-microscopic Plasmodium vivax Infection and Disappearing Plasmodium falciparum Malaria in an Area of Low Transmission in Solomon Islands.

IntroductionSolomon Islands is intensifying national efforts to achieve malaria elimination. A long history of indoor spraying with residual insecticides, combined recently with distribution of long lasting insecticidal nets and artemether-lumefantrine therapy, has been implemented in Solomon Islands. The impact of these interventions on local endemicity of Plasmodium spp. is unknown.MethodsIn 2012, a cross-sectional survey of 3501 residents of all ages was conducted in Ngella, Central Islands Province, Solomon Islands. Prevalence of Plasmodium falciparum, P. vivax, P. ovale and P. malariae was assessed by quantitative PCR (qPCR) and light microscopy (LM). Presence of gametocytes was determined by reverse transcription quantitative PCR (RT-qPCR).ResultsBy qPCR, 468 Plasmodium spp. infections were detected (prevalence = 13.4%; 463 P. vivax, five mixed P. falciparum/P. vivax, no P. ovale or P. malariae) versus 130 by LM (prevalence = 3.7%; 126 P. vivax, three P. falciparum and one P. falciparum/P. vivax). The prevalence of P. vivax infection varied significantly among villages (range 3.0–38.5%, p<0.001) and across age groups (5.3–25.9%, p<0.001). Of 468 P. vivax infections, 72.9% were sub-microscopic, 84.5% afebrile and 60.0% were both sub-microscopic and afebrile. Local residency, low education level of the household head and living in a household with at least one other P. vivax infected individual increased the risk of P. vivax infection. Overall, 23.5% of P. vivax infections had concurrent gametocytaemia. Of all P. vivax positive samples, 29.2% were polyclonal by MS16 and msp1F3 genotyping. All five P. falciparum infections were detected in residents of the same village, carried the same msp2 allele and four were positive for P. falciparum gametocytes.Conclusion P. vivax infection remains endemic in Ngella, with the majority of cases afebrile and below the detection limit of LM. P. falciparum has nearly disappeared, but the risk of re-introductions and outbreaks due to travel to nearby islands with higher malaria endemicity remains.

The effect of dose on the antimalarial efficacy of artemether–lumefantrine: a systematic review and pooled analysis of individual patient data

Artemether-lumefantrine is the most widely used artemisinin-based combination therapy for malaria, although treatment failures occur in some regions. We investigated the effect of dosing strategy on efficacy in a pooled analysis from trials done in a wide range of malaria-endemic settings. We searched PubMed for clinical trials that enrolled and treated patients with artemether-lumefantrine and were published from 1960 to December, 2012. We merged individual patient data from these trials by use of standardised methods. The primary endpoint was the PCR-adjusted risk of Plasmodium falciparum recrudescence by day 28. Secondary endpoints consisted of the PCR-adjusted risk of P falciparum recurrence by day 42, PCR-unadjusted risk of P falciparum recurrence by day 42, early parasite clearance, and gametocyte carriage. Risk factors for PCR-adjusted recrudescence were identified using Cox's regression model with frailty shared across the study sites. We included 61 studies done between January, 1998, and December, 2012, and included 14,327 patients in our analyses. The PCR-adjusted therapeutic efficacy was 97·6% (95% CI 97·4-97·9) at day 28 and 96·0% (95·6-96·5) at day 42. After controlling for age and parasitaemia, patients prescribed a higher dose of artemether had a lower risk of having parasitaemia on day 1 (adjusted odds ratio [OR] 0·92, 95% CI 0·86-0·99 for every 1 mg/kg increase in daily artemether dose; p=0·024), but not on day 2 (p=0·69) or day 3 (0·087). In Asia, children weighing 10-15 kg who received a total lumefantrine dose less than 60 mg/kg had the lowest PCR-adjusted efficacy (91·7%, 95% CI 86·5-96·9). In Africa, the risk of treatment failure was greatest in malnourished children aged 1-3 years (PCR-adjusted efficacy 94·3%, 95% CI 92·3-96·3). A higher artemether dose was associated with a lower gametocyte presence within 14 days of treatment (adjusted OR 0·92, 95% CI 0·85-0·99; p=0·037 for every 1 mg/kg increase in total artemether dose). The recommended dose of artemether-lumefantrine provides reliable efficacy in most patients with uncomplicated malaria. However, therapeutic efficacy was lowest in young children from Asia and young underweight children from Africa; a higher dose regimen should be assessed in these groups. Bill & Melinda Gates Foundation.