The usage of LLITNs in malaria vector control has resulted in the reduction in malaria deaths among higher-risk groups (pregnant women and under-fives). However, there exists asymptomatic infection among older children, thereby making them a reservoir of malaria transmission. This study aimed at assessing the impact of LLITN usage on malaria prevalence among school-age children (SAC) in Ekiti, South Western Nigeria.  Cross-sectional, two-stage cluster sampling technique was used to collect data from SAC during May and June 2017. A total of 1313 (Oye LGA: 657 and Ikole LGA: 656) SAC in selected public primary schools participated in the study. Sociodemographic information as well as data on LLITN usage the previous night was obtained using pretested, semistructured questionnaires adapted from the standardized Malaria Indicator Survey (MIS) tools. Malaria infection was diagnosed by using the rapid diagnostic test (RDT) on blood samples that were collected by finger prick from each child. Data were analyzed using the Statistical Package for Social Sciences (SPSS) for Windows software version 26 . p < 0.001). Socioeconomic factors  showed significant associations with LLITN usage (p < 0.001) in both Oye and Ikole LGAs. Malaria prevalence was significantly low among SAC utilizing LLITNs in both Oye and Ikole LGAs (p < 0.001). There was a significant association between gender and malaria prevalence among SAC with males having higher prevalence than females (p < 0.001). Socioeconomic factors were significantly associated with malaria prevalence in both LGAs (p < 0.001).  Usage of LLITNs among SAC was significantly higher in Ikole LGA than in Oye ( The usage of LLITNs caused a significant reduction in malaria prevalence among the school-age children in the study areas; hence, sensitization on usage should be scaled up towards malaria elimination. Malaria is a leading cause of morbidity and mortality most especially among vulnerable groups in sub-Saharan Africa. An estimated 228 million cases of malaria occurred worldwide : 206–258 million), with 93% of the cases occurring in the World Health Organization (WHO) African Region. Nigeria constitutes one of the six countries that account for more than half of all malaria cases worldwide . While dThe effectiveness of LLITNs in malaria control and the role of schools have been demonstrated –11. WhilThe study was carried out among school-age children in Oye and Ikole local government areas (LGAs) in Ekiti State. The State is in one of the three major malaria epidemiological zones in Nigeria and is characterized by a tropical climate with an alternating rainy season (April–October) and dry season (November–March). Temperature ranges between 21°C and 28°C with high humidity, all favourable to malaria vector development.n = 1.962 pq/L2, where n = sample size, p = expected prevalence (0.5), q = 1−p (1–0.5), and L = limit.Sample size was calculated using the equation A community-based cross-sectional design was used in this study. For this study, only public, mixed primary schools were selected. The study flowchart is shown in Malaria infection in SAC was diagnosed by using the rapid diagnostic test (RDT) on blood samples that were collected by finger prick from each child. This was conducted and recorded by trained health personnel. The RDTs were the CareStart® Pf/Pv combo test , containing an alcohol swab, lancet, capillary tube, buffer, and test device.A total of 1313 (Oye LGA: 657 and Ikole LGA: 656) semistructured interviewer-guided questionnaires were administered to school-age children who met the inclusion and exclusion criteria in selected primary schools. Sociodemographic information, as well as data on LLITN usage the previous night, was obtained using pretested, semistructured questionnaires adapted from the standardized Malaria Indicator Survey (MIS) tools. Questionnaires were pretested in Ido/Osi local government area of Ekiti State and administered by trained interviewers comprising of fresh university graduates. Data collection occurred during the school term and lasted for two months during May and June 2017. The interviews were conducted in English and Yoruba languages. The interviewers were frequently supervised on the field by a designated team leader and researcher to monitor data collection and provide necessary feedback. Each respondent was interviewed for 20 minutes.p value of less than 0.001 was considered as statistically significant.Data were analyzed using the Statistical Package for Social Sciences (SPSS) for Windows software version 26 . Frequency tables were generated for relevant variables, and percentages were determined as appropriate. Malaria prevalence was calculated by dividing the number of SAC that have positive RDT result by the total number of pupils that were tested from each LGA. Pearson's chi-square test was used to assess the bivariate association between usage of LLITNs and malaria prevalence. Approval for the study was obtained from the Ministry of Health, Ekiti State, MOH/PRS/15/137, Ekiti State Primary Education Board (SPEB), EKSUBEB/ADM/79/123, and Local Government Education Authority Executive Secretaries. Education officers from the district education office in the LGAs assisted the team in locating the schools. The headteacher and teachers at each school that were enrolled in the study were shown approval letters and were informed about the purpose of the study. Awareness and sensitization for the program were carried out through the Parent-Teacher Association (PTA) forum before the study. Only pupils whose parents consented to the study participated in the study.In total, 1313 school children participated in the study: 657 and 656 in selected primary schools in Oye and Ikole LGAs, respectively. The sociodemographic characteristics of the participants are shown in p < 0.001)  in Oye LGA and (86.0%) in Ikole LGA, respectively. However, usage of LLITNs was significantly higher in Ikole than Oye LGA (< 0.001) .p < 0.001), while in Ikole LGA, mother's education, mother's occupation, ethnicity, household size, and access to electricity were significantly associated with LLITN usage (p < 0.001)  .Malaria prevalence in SAC was low in both Oye LGA (16.98%) and Ikole LGA (16.83%) . Sociodep < 0.001)  .Vector control through LLITNs constitutes one of the effective control measures in reducing deaths due to malaria . This stThe significant association between household size and LLITN usage observed in this study is consistent with reports from other studies from Ethiopia and Kenya –21 whereFurthermore, the significant association between access to electricity and LLITN usage is consistent with reports from Ethiopia and Tanzania , 25; howWith regard to mother's education, finding from this study showed that mothers of SAC with secondary education utilized LLITNs in Ikole LGA more than those with primary education. This is consistent with another study from elsewhere . Study hMoreover, LLITN usage was observed to be significantly high among school-age children whose mothers were self-employed. This may be attributed to self-employed mothers having more time for their children's care. This contradicts a report from another study where children of working mothers were observed to have an increased likelihood of LLITN usage .The finding from this study showed that malaria prevalence in the study locations is higher than 10.6% reported in Tanzania  and 14% Also, the significant association between malaria prevalence and socioeconomic factors  agrees with reports from other studies , 30, 31.Furthermore, the low malaria prevalence recorded in Oye and Ikole LGAs in this study may be attributed to the high usage of LLITNs observed among SAC in the study locations. This is consistent with other studies which have reported the effectiveness of net usage in the reduction of malaria cases among school-age children , 33–35, This study provides baseline information on the utilization of LLITNs among school-age children in Oye and Ikole LGAs and the impact of LLITN usage in reducing malaria infections among the study population.This is a cross-sectional study, which could only evaluate the association, instead of cause-and-effect relationships. LLITN usage the previous night was used as the basis to determine those who used LLITNs, and this might not be generalizable. The study was conducted in only two LGAs of Ekiti State, and therefore, the results may not be generalized to the country. The study was only based on interviews, and there were no observations conducted to validate the reported use of mosquito nets.This study showed a significant association between reduction in malaria infection and usage of LLITNs among school-age children in Oye and Ikole LGAs. This observation requires continuous educations on the usage of LLITNs in the study areas to sustain malaria infection control towards total elimination.


Malaria infection is a serious health problem killing millions in tropical developing countries including Ethiopia. The present study focused on assessing malaria prevalence and identification of determinants in Shewa Robit, northcentral Ethiopia. p < 0.05 was used to indicate the level of significance.  A cross-sectional study was conducted among 422 participants who visited Shewa Robit Health Center between 01/10/2017 and 30/04/2018, using a simple random sampling. Sociodemographic characteristics were recorded using a pre-tested semi-structured questionnaire and infection was confirmed by microscopic examination. Data were analyzed using the Statistical Program for Social Sciences (SPSS) version 20 and P.vivax was the most frequently detected species . Interestingly, 73.2% (n = 309) of the participant did not utilize LLINs due to the fear of toxicity , misconception , and shortage . The data showed age, gender, marital status, family size, usage of LLINs and application of IRS, proximity to mosquito breeding sites and less robust and porous walls were the determinants of the infection in the study area.  Eighty-one (19.0%) microscopically confirmed malaria cases were recorded, P. vivax being the most common causative agent. Environmental and behavioral factors related to LLIN are the potential determinants of malaria. Continued public health interventions, targeting proper utilization of bed nets, drainage of stagnant water, and improved public awareness about reducing the risk of insect bites have the potential to minimize the infection. The prevalence of malaria in the study population was high and  Malaria is recognized around the world as a debilitating and terrible infectious disease that kills millions and causes serious complications such as severe anemia, cerebral involvement, acute renal failure, and hypoglycemia , 2. It iMalaria is widespread in almost 45 African nations, including Ethiopia, with nearly 3 million cases each year, and morbidity and mortality rates are growing substantially , 7. ResePrevious research confirmed that age, sex, marital status of the respondent , proximiAs part of the global Roll Back Malaria project, Ethiopia has a long-term goal to eliminate malaria , and in Malaria remains one of the most serious public health concerns in Ethiopia, despite significant efforts to combat it . MeanwhiThe terrain of Shewa Robit is favorable for the transmission of malaria, which is one of the top ten causes of morbidity . Despite the high rate of malaria infection, literature analyzes showed a paucity of recently updated data on the prevalence and risk factors in the study area . In factA cross-sectional study was conducted among all malaria suspected outpatients who visited Shewa Robit Health Center located in Shewa Robit town, from 01/10/2017 to 30/04/2018. The town is located 225 km northeast of Addis Ababa, in the Amhara Regional State at an elevation of about 1,280 meters above sea level. The town lies at a longitude and latitude of 10°060 N39°590 E and 10.1°N39.983°E, respectively. The climate is tropical with an annual temperature ranging from 28 to 37°C with an annual rainfall of 1000 mm. It has nine administrative units (Kebele) with a total population of 50,528, of which 25,890 (51.2%) are women. Malaria is one of the top ten diseases in the town and is reported throughout the year , and theAll suspected cases with a febrile illness (>37.5 C) who have been living in the administrative units of Shewa Robit for at least six months were included in the study. However, those who underwent chemotherapy with antimalarial drugs three months prior to the start of the study were excluded.The sample size was calculated using a single population formula. A prevalence of 50% was chosen, as there were no specific reports in the study area . In order to calculate sample size, the value of Z chosen was 1.96 at 95% CI and a 5% margin of error. Therefore, the final sample size was consolidated to 422, after adding 10% non-response rate.To start with, all suspected outpatients i.e, these suspected cases with febrile illness attended to the health center were stratified according to sex and age, and a random sampling technique was used to select each participant from the kebeles chosen.Plasmodium infection. Thin films were fixed with 100% methanol, and both thin and thick films were stained with 3% Giemsa according to the protocol . Participants were informed of the minor risks involved in this study, which was also conducted in accordance with the Declaration of Helsinki .n = 184) of them were under 5 years. Furthermore, 57.1% (n = 241) were unmarried with a family size of more than five members. Majority  of the respondents were an attendants of secondary education and above whereas, 33.9% (n = 143) of the participant were illiterate. Most of the study participants were farmers and merchants ie, 23.2% (n = 98) and 16.1% (n = 68) respectively by occupation with monthly family income of less than 18.30 USD  and 35.1% (n = 20), respectively. However, a lower incidence of infection was observed in January with an infection rate of only 4% (n = 2). In particular, the findings revealed that P. falciparum infection peaked in October and November with an infection rate of 43.5 (n = 10) and 20% (n = 4), respectively. The lowest infection with P. falciparum was recorded in January, February, and April with nil incidence. However, the infection rate caused by the malaria infection by P. vivax went up to the maximum in January, February and April , while the lower infection caused by P. vivax was recorded in March with a transmission rate of 60% (n = 3). Similarly, mixed infection was recorded in October and November with a prevalence of 21.7% (n = 5) and 5% (n = 1) respectively (Although the ectively .It was found that 289 (75.3%) of the participants had a history of malaria infection in their households. Although (83.6%) of the participant have access to long-lasting insecticide nets (LLINs), they sleep under the net daily (26.8%) and during the high transmission season (43.1%). However, most of the study participants (73.1%) did not use LLINs for two reasons, fear of toxicity (37.45%) and misconception (21.6%) due to the belief that the net did not prevent infection .n = 81) of the participants had malaria parasites in their blood that could be seen microscopically. The most prevalent Plasmodium species found in individuals with positive laboratory test results were, P. vivax 71.6% (n = 58), P. falciparum 21% (n = 17) and mixed infection (recurrence of both species) accounts 7.4% (n = 6), as shown in The findings show that 19%  and 2.20 (CI 95%: 1.2–4.06) more likely to be infected with malaria respectively. The result confirmed that usage of LLTN reduces malaria infection. Study participants who did not use LLTN (CI 95%: 0.69–2.83) were 1.4 more likely to be infected with malaria as compared to their counterparts. Furthermore, study participants who refused IRS  were 2.6 times more likely than their peers to develop malaria infection. The presence of a mosquito-nesting site close to the house and holes between the house wall and the roof had a strong relationship with the occurrence of malaria infection. According to the findings, study participants who had proximity to mosquito location were 3.91 times more likely to contract malaria than their peers CI 95%: 1.87, 5.18). However, the chance of malaria infection was 2.1 higher in the participant who lived in a house with holes between the wall and the roof (CI 95%: 1.13–3.67) as shown in n = 422), with P. vivax being the most prevalent species, is lower than the previous findings from Wolaita Zone (33.27%) [This study evaluated the prevalence of malaria infection in Shewarobait, Ethiopia from October 2017 to April 2018. The result showed that malaria is still one of the most serious public burdens in the study area. In addition, it was evident that age of the participants, sex, marital status, family size, utilization of LLINs and IRS, proximity to mosquito breeding site, and presence of holes on the wall were determinants of malaria transmission. In the current study, the overall percentage of malaria cases detected was 81 (19%) (33.27%) , and Hal(33.27%)  Kenya (1(33.27%) , Kenya ((33.27%) . Interes(33.27%)  respecti(33.27%) , Kombolc% , and H(33.27%) . However(33.27%)  and Wola(33.27%) .Plasmodium species detected among the participants in the current study participants was P. vivax. This is in agreement with previous report from Jimma Town [P. vivax over P. falciparum in recent years [P. vivax against chloroquine [The predominant mma Town , Aresi Nmma Town , Hallabamma Town , there ent years . This cooroquine .Plasmodium species compared to their counterparts (p < 0.001). These findings were supported by the Hamusite report, northwest Ethiopia [Plasmodium parasites.According to the present study, 72.3% of participants had a history of malaria infection however, only 63 (20.7%) were infected with malaria. In particular, individuals who had a family history of malaria were 1.53 times more likely to be infected by Ethiopia . This miDifferent sociodemographic and other factors had been analyzed by taking into consideration of prevalence of malaria infection. Of these factors, the age was one of the significantly associated factors. Here, the odds of having malaria infection were 2.31 and 4.05 more likely among participants in the age group 5–14 years and above 15 compared to others. This aspect of the study is comparable to a previous work conducted in Woreta , KombolcThe odds of being infected with malaria were 1.4 and 2.6 times higher among participants who did not use LLIN and apply IRS, respectively, and this is consistent with the results of other studies conducted in Jimma  and ShewPlasmodium species.This is a cross-sectional study that addresses percentage prevalence and cannot account for seasonal transmission trends. In addition, the study is based on a single institution and has a shorter duration, including a smaller sample size. All surveys are self-reported without confirmation of bednet ownership and usage, and frequently application of IRS and RDTs. In addition, no PCR tests were performed to identify the infection and the P. vivax being the most common causative agent. The main infection factors linked to the infection in the study area were age, sex, marital status, family size, use of LLIN and IRS, presence of mosquito breeding sites, and openings on their wall/roof. In addition, the main reason for rejecting LLIN is misconceptions about the toxicity of the treated net. The burden of malaria could be reduced by focusing on changing the attitudes towards malaria prevention and control through continued health education.The study population who attended the Shewa Robit Health Center had a high incidence of malaria, with


Plasmodium falciparum infection prevalence and persistence, and anti-parasite immunity to gametocyte and asexual antigens for 10 weeks. Of the 100 participants, only 11 were never infected, whilst 16 had persistent infections detected by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), and one participant had microscopic parasites at all visits. Over 70% of the participants were infected three or more times, and submicroscopic gametocyte prevalence was high, ≥ 48% of the parasite carriers. Naturally induced responses against recombinant Pfs48/45.6C, Pfs230proC, and EBA175RIII–V antigens were not associated with either infection status or gametocyte carriage, but the antigen-specific IgG titers inversely correlated with parasite and gametocyte densities consistent with partial immunity. Longitudinal analysis of gametocyte diversity indicated at least four distinct clones circulated throughout the study period. The high prevalence of children infected with distinct gametocyte clones coupled with marked variation in infection status at the individual level suggests ongoing transmission and should be targeted in malaria control programs.Subclinical infections that serve as reservoir populations to drive transmission remain a hurdle to malaria control. Data on infection dynamics in a geographical area is required to strategically design and implement malaria interventions. In a longitudinal cohort, we monitored  This plateau in malaria prevalence is attributed to factors such as high subclinical infections that act as potential reservoirs for transmission in endemic populations3. Renewed and intensified approaches are needed to successfully eliminate the disease. Reports from health facilities shows that Ghana has a high microscopic P. falciparum infection prevalence in children under 5 years, which ranges from 4 to 33% depending on the location4. Areas of high parasite prevalence can serve as infection hotspots that maintain transmission in neighboring communities with lower prevalence. In high parasite prevalence settings (endemic populations) where only a fraction of the infected individuals are thought to be responsible for most of the Plasmodium transmission, monitoring the dynamics of P. falciparum infections can aid in the identification of the reservoir source5.The scale-up of malaria interventions over the past decade has led to decreased rates of malaria morbidity and mortality, but these declines have slowed in the past 3 years7. Mature, stage V gametocytes first appear in the peripheral blood 10 days after committed merozoites emerge from the liver8, and immature gametocytes continue to be produced during each erythrocytic asexual cycle. Continual gametocytogenesis provides a consistent supply of mature gametocytes capable of undergoing sexual recombination once taken up in a blood meal by a mosquito10. Therefore, multiclonal parasites in subclinical infections can increase population diversity. Asexual clones of P. falciparum can persist for weeks at the subpatent or subclinical levels to provide a continuous source of gametocytes12. Monitoring the clonal dynamics of gametocytes provides insight into the clones available for transmission between individuals via mosquitoes and their spread through the community.As subclinical infections are not treated in most hyper-endemic areas, these individuals will continue to produce gametocytes, the transmissible form of the parasiteP. falciparum parasite contributes to the acquisition of stage-related immunity. Antibodies elicited against the various stage-specific antigens serve as serological markers that can be used to measure exposure and test for anti-parasite or transmission-reducing activity14. The levels of host IgG and IgM antibodies against parasite antigens at the individual or population level provide an important metric to monitor infection progression and transmission dynamics. Antibody levels are also influenced by the age of the host and transmission intensity16. It has been shown that for an individual to be protected against clinical malaria, periodic infections are needed, even if they are sporadic or subclinical17. Protection against clinical malaria involves various mechanisms that contribute to parasite clearance. Several parasite antigens have been implicated in protection, including the conserved region III–V of PfEBA175 (EBA175RIII–V), which is expressed on the surface of the merozoite. Anti-EBA175RIII–V responses have been shown to prevent red blood cell (RBC) invasion19. While anti-gametocyte immunity can prevent the completion of the parasite development in the mosquito, thereby reducing or preventing malaria transmission20. Specific epitopes of Pfs48/45 and Pfs230 antigens, which are expressed during gametocyte development and exposed on the extracellular surface of the gamete during the transition to zygote formation have been shown to be the targets of transmission-blocking monoclonal antibodies21. Recently, tools have been developed to evaluate gametocyte production, maturation, and immunity within the human host and the contribution of this immunity in reducing transmission needs to be tested in the field25.Exposure to different stages of the P. falciparum infections on malaria transmission is difficult to evaluate as neither these infections nor mosquito bite exposure are routinely quantified in endemic populations. Closely monitoring parasite infection dynamics including identifying and targeting carriers with persistent infections could break the transmission cycle in the defined population. This study assessed P. falciparum infection dynamics at the individual level and monitored infection heterogeneity and sexual-stage clones during the off-peak season. Additionally, the association of infection frequencies and antibody responses to recombinant EBA175RIII–V asexual stage and gametocyte-specific Pfs48/45.6C and Pfs230proC were examined.The effect of subclinical P. falciparum infection dynamics in 100 children aged 6–12 years just after the peak malaria season in Ghana. Samples were collected every 14 days for 10 weeks; children present on five or more sampling days were included in the analysis. Feverish children with high body temperature were referred for clinical management. Monthly hemoglobin levels were assessed for each participant to monitor anemia prevalence in the study population. Plasmodium falciparum infections were detected at both patent and subpatent levels. Parasite positivity was also assessed using RDT kits  participants in the study was slightly more than the males. The monthly mean hemoglobin (Hb) level was estimated at 11.4 g/dl, and one-third of the participants (31/99) had low Hb levels (< 10 g/dl). Nonetheless, the Hb levels did not differ between time points (0.7933) or infection groups p = 0.2296)  were infected  in all the patent (n = 178) and subpatent (n = 172) infections  on the sampling days. Gametocytes were detected by microscopy in three infections and two children had gametocytes that persisted over the duration of the study Table . Whereasts Table . We furt50 on thePfg37727. Eleven distinct clones  were observed during the study   or anti-IgG to the asexual antigen (EBA175RIII–V), the participants were divided into four groups based on the parasite and gametocyte persistence. (1) Participants that remained uninfected throughout the study (n = 11). (2) Those with sporadic infections . (3) Those with persistent infections  or (4) a subpopulation of the persistently infected group that also had persistent gametocyte carriage were also analyzed separately   and the same gametocyte clone was rarely detected in consecutive samples. These findings suggest that following infection with a particular clone, parasites emerge from the liver, invade RBCs and continue asexual replication while producing a subpopulation of gametocyte committed parasites each 48-h cycle. The erythrocytic cycle continues until the infection is controlled and that clone is cleared by the immune response. When the person is infected with another clone the cycle of increasing parasitemia and gametocyte production followed by clearance is repeated. The results obtained are not consistent with a subpopulation of individuals infected with multiple clones acting as focal infectious reservoirs for the rest of the population.Waxing and waning parasitemia and gametocytemia in a majority of the study participants is consistent with partial immunity coupled with ongoing transmission32. However, despite the concurrent increase in microscopic parasite and gametocyte detection on week 8, overall the geometric mean gametocyte densities in both patent and subpatent infections were similar throughout the study and both populations would be expected to contribute to the infectious reservoir34. The factors regulating gametocyte production have been examined in vitro, in rodent malaria, and symptomatic infections37, but not yet carefully examined in asymptomatic infections. Even though low Hb levels have been linked to gametocyte prevalence in symptomatic infections38, the lack of association with Hb levels observed in this study suggest identifying differences in gametocyte production in asymptomatic infections will be an important focus for future studies.Further support for ongoing transmission through the population was the appearance of a set of new gametocyte clones on week 6 and peaking at week 8. The peak in clonal diversity coincided with a peak in gametocyte prevalence as well as parasite detection by microscopy. This could suggest the introduction of new parasite strains into the population that replicated to higher densities before being controlled, which resulted in higher numbers of infections detected by microscopy and the production of more gametocytes. Such a correlation between gametocyte prevalence and microscopic parasite prevalence has been reported previously44. Antibodies against asexual stage antigens including EBA175 RIII–V that inhibit merozoite invasion have been shown to correlate with protection45. Here IgG titers against EBA175RIII–V as well as two sexual-stage antigens, Pfs48/45.6C and Pfs230proC, were inversely correlated with parasite and gametocyte density consistent with the children having partial immunity. The specific contribution of anti-EBA175RIII–V antibodies to protection is difficult to predict from this study, especially given the significant inverse correlation between parasitemia and anti- Pfs48.45.6C and Pfs230proC IgG titers even though Pfs48/45 and Pfs230 are not expressed by asexual parasites. In fact, unless the RBCs are lysed in the human host, Pfs48.45 and Pfs230 antigens are not accessible to antibodies until the parasite emerges from the RBC as a gamete in the mosquito midgut48. Consequently, it is likely that the increased IgG titers observed are markers of a more robust general anti-parasite immune response that effectively reduces parasitemia and consequently gametocyte levels50. Further inquiry into the specific immune response associated with the clearance of different clones is needed to better understand the development of protection. At the population level, the median antibody titer against EBA175RIII–V antigen was maintained through the 10 weeks in individuals that were either persistently or sporadically infected, consistent with the longevity of these antibodies in earlier studies47. However, the 11 subjects that remained uninfected, the median EBA175RIII–V antibody levels were consistently lower at all time points.In areas of ongoing transmission, protection against clinical disease has been associated with the development of antibodies specific for parasite specific-antigens54. Prior reports have suggested that persistent antigen exposure for longer than 10 weeks is needed to induce a stable long-term antibody response56, which could have happened during a prior malaria season. Although the immunogenicity of these antigens is well documented, immune responses have been reported to be higher in asymptomatic vs symptomatic infections, adults vs children, high vs low parasite prevalence population, and high vs low transmission intensities or zones58. However, the variation in antibody responses between studies makes it difficult to draw strong conclusions60, and suggest additional factors that have not yet been identified are likely to contribute to the responses. Given the persistent spread of the same set of four clones from person to person during the 10-week study period, it is unlikely that the population has strong transmission-blocking immunity. One possibility is that Pfs48.45 and Pfs230 are polymorphic in these distinct gametocyte clones64. Another reason comes from recent work screening monoclonal antibodies against Pfs48/4565 and Pfs23067, that suggests there are a limited number of transmission-blocking epitopes and different polyclonal serum samples contain differing levels of blocking and non-blocking antibodies. Future investigation of the infectiousness of the individuals with gametocytes and high antibody titers would continue to advance our understanding of the factors underlying transmission and guide the development of effective transmission-blocking interventions.Although this difference in anti-EBA175RIII–V antibody concentration was not significant, possibly due to the small number of persistently uninfected individuals, lower antibody levels in the uninfected volunteers could suggest that periodic exposure to antigen is needed to maintain high antibody titers. Continued follow-up through the rest of the dry season would be needed to evaluate whether levels continue to decline in the absence of reinfection. In contrast, anti- Pfs48/45.6C or Pfs230proC titers remained constant in persistently uninfected individuals as well as those that did and did not have parasites or gametocytes through the study period. This pattern is consistent with the presence of long-lived antibody-secreting plasma cells and have been observed previouslyTogether, the high prevalence of asexual and sexual parasites in school children enrolled in the study with variable carriage of distinct parasite strains at the individual level is consistent with ongoing malaria transmission in a partially immune population. The relatively low individual MOI (< 1.5) and lack of persistent gametocyte carriage by any one individual argues against a focal infectious reservoir that could be targeted for treatment. Rather, the data suggest that most of the population could contribute to transmission and is susceptible to the introduction of new parasites strains. This pattern of transmission highlights the importance of an effective vaccine to provide long-lasting immunity. However, the high prevalence of asymptomatic parasites after the peak malaria season in individuals with consistent antibody titers against asexual and sexual antigens demonstrates the challenge to vaccine development. More extensive analysis of the immune response before and after parasite clearance is needed to understand strain-specific clearance and develop strategies to induce antibodies that effectively block infection and transmission to successfully control malaria.69. The samples used in this study were collected from November 2017 to January 2018 during the off-peak malaria transmission season. The participants were school children from two communities (50 each from Obom and Simiw) as described in our previous studies71. The communities share similar malaria transmission patterns where infections peak between June and August. The main occupation of the inhabitants from both communities is predominantly farming4. The sites and the population were chosen based on observed near similar infection prevalence4.A subset of 100 children aged between 6 to 12 years was selected from a larger cohort of an ongoing malaria transmission study. This age group was selected based on the reported high susceptibility to clinical malaria and parasitemiaP. falciparum infection was done by spotting 5 μl of whole blood on the HRP2 RDT kit on days 0 and 70 as well as selectively on any other sample collection day  for participants with body temperatures above 37.5 °C. The remaining whole blood was centrifuged and the plasma saved at − 20 °C for the immunological assays. The axillary body temperature of the participants was measured using a digital thermometer at each time point.Venous blood samples were collected fortnightly at six-time points translating into days 0, 14, 28, 42, 56, and 70. A milliliter of blood was obtained from each participant at all-time points. The samples were processed as follows; 100 μl of whole blood was preserved (Trizol) for gene transcripts analysis, 100 μl of whole blood spotted on filter paper (Whatman 3 mm) for parasite identification, about 10 μl of whole blood for Giemsa-stained smears for microscopic parasite identification and quantification. Hemoglobin levels were measured monthly  by spotting 10 μl of whole blood on a Urit 12 HB meter . On-site diagnosis of Plasmodium falciparum identification and density were determined using light microscopy. The parasites were counted against 200 white blood cells (WBC) for the Giemsa stained thick smears following the standard protocols by WHO72, by three malaria microscopists. Due to the difficulty in detecting microscopic parasites at low parasite densities in the asymptomatic infections and to increase the sensitivity and limit of detection of the parasite in the blood films to 90% and under field conditions74, the cut-off for parasite positivity by microscopy was set at ≥ 80/µl (parasite per microliter of blood).Pf18S rRNA and Pfs25 mRNA transcripts respectively as previously described32. Briefly, RNA was extracted from the Trizol preserved whole blood samples using the Quick RNA miniprep kit . DNase 1 on-column treatment was performed before RNA elution to minimize gDNA contamination. RNA was converted to cDNA using the Protoscript II first-strand cDNA synthesis kit  with both oligo dT and random hexamer primers. ABI Fast SYBR Green 2X RT-PCR kit was used for the RT-PCR reactions. The amplifications were performed on ABI Quanti-Studio 5 systems . cDNA from synchronized and hemocytometer quantified NF54 (high gametocyte producer), RCM47 (gametocyte deficient) and 3D7 (low gametocytes producer) strains (matured gametocytes or early rings) served as parasite controls, while no template control (NTC) and no reverse transcriptase control (No RTC) from the cDNA prep and water were used as negative controls. Any cycle threshold (CT) value below those of the negative controls was considered positive for the specific gene. All samples were run in triplicates, and mean CT values used for the analysis were from at least duplicate readings with a standard deviation below 0.05. A standard curve using cDNA generated from RNA isolated from a known number of purified ring stage parasites or stage V gametocytes was used to calculate parasite density.Total parasites and gametocytes were detected by measuring Pfs25 transcripts (gametocyte positive infections). Diversity was assessed by amplifying region 3 of the gametocyte-specific gene, Pfg377, from cDNA using the nested RT-PCR protocol of Menegon et al.27, previously described57. The cDNA templates were amplified using AmpliTaq Gold Fast PCR Master Mix . cDNA and gDNA from laboratory cultured gametocytes from the 3D7 P. falciparum parasite isolate were used as positive controls for the reactions whereas NTC, no RTC and nuclease free water were used as negative controls. Allele sizes were determined against the 50 bp  ladder used on the 2% ethidium bromide-stained agarose gel.Gametocyte diversity was assessed only in randomly selected samples that tested positive for P. falciparum recombinant Pfs48/45.6C and Pfs230proC antigens and IgG antibodies against EBA175RIII–V were measured by an indirect enzyme-linked immunosorbent assay75. Briefly, the antigens were diluted to 1 µg/ml and coated at 100 µl/well on a plate . Recombinant polyclonal human IgG  at a starting concentration of 1000 ng/ml was used as the standard calibrator to measure the total IgG. Hyper-reactive sera from individuals with high asexual [Merozoite surface protein 3 (MSP3)] or sexual (Pfs48/45) stage antibody titers were used as standard calibrators for IgM at a dilution of 1:200 (100 μl/well) and positive controls. Sera from malaria naïve individuals with low antibody titers to the test antigens were negative controls for the assays. Plasma samples and controls were diluted at 1:200 for the assays. Color development was stopped with 100 µl/well of 0.2 M H2SO4 and optical densities (OD) read at 450 nm using ELISA plate reader . The measured OD values were converted into concentration using the ADAMSEL software (Edmond J. Remarque). Antibody titers  were reported as concentrations in actual units (AU).Plasma IgG and IgM of naturally induced antibody titers against P. falciparum infections, parasite density was calculated based on the assumption of the standard 8000 leukocytes per microliter of blood from three microscopists’ readings. The cycle threshold (CT) values of the no template control were used to determine the cut-off for the presence or absence of parasite and gametocyte at submicroscopic level. The detection limit of Pf18S rRNA (CT ≤ 34) and Pfs25 (CT ≤ 37) mRNA transcripts estimated 0.08 and 0.02 parasites/gametocytes per µL reference to the standard curve of parasite ring  and gametocyte stages, below the CT of the negative controls76. Nonlinear regression was used to assess the influence of patent infections on gametocyte positivity. The gametocyte multiplicity of infection (MOI) was estimated from the total number of gametocyte alleles in the parasite-infected population at each sampling point. Numeric values including sex, age, temperature, hemoglobin levels, P. falciparum and gametocyte prevalence were represented in counts, geometric mean (GM) or percentage (%). The difference in parasite infections, densities, gametocyte positivity and antibody  titers  at the time points were determined by non-parametric statistical methods by the Fisher’s exact Chi-squared and Kruskal Wallis tests. A p-value < 0.05 was considered statistically significant. Dynamics in the infections between the time points were analyzed using STATA (version 15). Data analyses were performed with Prism version 8.01 (GraphPad Software).For microscopic The study received ethical approval from the Institutional Review Board of the Noguchi Memorial Institute for Medical Research, University of Ghana (protocol # 024/14-15), permission from the Ghana Education Service, and the Head of the schools were also sought. Written parental informed consent was obtained from a parent or guardian before a child was enrolled. Prior to consenting and enrollment, the study’s objectives, methods, anticipated benefits, and potential hazards were explained to the parents/guardians. The parents/guardians were encouraged to ask questions and clarification about any aspect of the study that was unclear. The parents/guardians were also informed about their liberty to withdraw their children at any time point without penalty. The participant information was treated as confidential. All methods were performed in accordance with the relevant guidelines and regulations governing the study.Supplementary Information.


Long-lasting insecticidal nets (LLINs) have successfully reduced malaria in sub-Saharan Africa, but their effectiveness is now partly compromised by widespread resistance to insecticides among vectors. We evaluated new classes of LLINs with two active ingredients with differing modes of action against resistant malaria vectors.Plasmodium falciparum malaria infection by rapid diagnostic tests. The primary outcome was malaria infection prevalence at 24 months after LLIN distribution, comparing each of the dual-active-ingredient LLINs to the standard pyrethroid-only LLINs in the intention-to-treat population. The primary economic outcome was cost-effectiveness of dual-active-ingredient LLINs, based on incremental cost per disability-adjusted life-year (DALY) averted compared with pyrethroid-only LLINs, modelled over a 2-year period; we included costs of net procurement and malaria diagnosis and treatment, and estimated DALYs in all age groups. This study is registered with ClinicalTrials.gov (NCT03554616), and is ongoing but no longer recruiting.We did a four-arm, cluster-randomised trial in Misungwi, Tanzania. Clusters were villages, or groups of hamlets, with at least 119 households containing children aged 6 months to 14 years living in the cluster's core area. Constrained randomisation was used to allocate clusters (1:1:1:1) to receive one of four types of LLIN treated with the following: α-cypermethrin only (pyrethroid-only [reference] group); pyriproxyfen and α-cypermethrin (pyriproxyfen group); chlorfenapyr and α-cypermethrin (chlorfenapyr group); or the synergist piperonyl butoxide and permethrin (piperonyl butoxide group). At least one LLIN was distributed for every two people. Community members and the field team were masked to group allocation. Malaria prevalence data were collected through cross-sectional surveys of randomly selected households from each cluster, in which children aged 6 months to 14 years were assessed for 84 clusters comprising 39 307 households were included in the study between May 11 and July 2, 2018. 147 230 LLINs were distributed among households between Jan 26 and Jan 28, 2019. Use of study LLINs was reported in 3155 (72·1%) of 4378 participants surveyed at 3 months post-distribution and decreased to 8694 (40·9%) of 21 246 at 24 months, with varying rates of decline between groups. Malaria infection prevalence at 24 months was 549 (45·8%) of 1199 children in the pyrethroid-only reference group, 472 (37·5%) of 1258 in the pyriproxyfen group , 512 (40·7%) of 1259 in the piperonyl butoxide group , and 326 [25·6%] of 1272 in the chlorfenapyr group . Skin irritation or paraesthesia was the most commonly reported side-effect in all groups. Chlorfenapyr LLINs were the most cost-effective LLINs, costing only US$19  more to public providers or $28 (11–120) more to donors per DALY averted over a 2-year period compared with pyrethroid-only LLINs, and saving costs from societal and household perspectives.After 2 years, chlorfenapyr LLINs provided significantly better protection than pyrethroid-only LLINs against malaria in an area with pyrethroid-resistant mosquitoes, and the additional cost of these nets would be considerably below plausible cost-effectiveness thresholds ($292–393 per DALY averted). Before scale-up of chlorfenapyr LLINs, resistance management strategies are needed to preserve their effectiveness. Poor textile and active ingredient durability in the piperonyl butoxide and pyriproxyfen LLINs might have contributed to their relative lack of effectiveness compared with standard LLINs.Joint Global Health Trials scheme , US Agency for International Development, President's Malaria Initiative. Evidence before this studyWe searched PubMed on May 12, 2021, with no language or date restrictions. We used the search term “randomised controlled trial” and “malaria” and “net” or “long lasting insecticidal net”, combined with the terms “piperonyl butoxide” or “pyriproxyfen” or “chlorfenapyr”. We identified two cluster-randomised controlled trials that evaluated the efficacy with regard to malaria outcomes of long-lasting insecticidal nets (LLINs) combining the synergist piperonyl butoxide and a pyrethroid insecticide (one conducted in Tanzania and one in Uganda), and a meta-analysis of those results in a Cochrane review. One randomised controlled trial was found assessing the effectiveness of LLINs treated with pyriproxyfen and pyrethroid, in Burkina Faso, but we found no trials of LLINs combining a pyrethroid and chlorfenapyr. The Tanzanian study was a four-arm superiority trial comparing piperonyl butoxide LLINs (Olyset Plus) with a standard pyrethroid-only LLIN (Olyset net), with or without indoor residual spraying, using a two-by-two factorial design. The Ugandan trial compared piperonyl butoxide LLINs of two types, Olyset Plus (permethrin and piperonyl butoxide) and Permanet 3.0  with standard LLINs of the same brands but without the piperonyl butoxide. Both trials showed that piperonyl butoxide LLINs reduced malaria prevalence in children compared with the standard LLIN, up to 18 months post-intervention in Uganda and 21 months in Tanzania. The Cochrane review concluded that, for these two trials, the odds of malaria infection was 31% lower in the piperonyl butoxide LLIN groups than in the standard LLIN groups at 21–25 months. Intensity of insecticide resistance in malaria vectors was high in both study areas. The trial in Burkina Faso used a stepped-wedge design and showed a small (12%) but significant reduction in clinical malaria incidence in children in an area with pyriproxyfen LLINs compared with those residing in the standard pyrethroid-only LLIN group, whereas there was no effect of the pyriproxyfen LLINs on malaria prevalence.Added value of this studyTo our knowledge, this study is the first to assess the effectiveness and cost-effectiveness of the three classes of dual-active-ingredient LLINs currently available for malaria control. The use of a mixture of active ingredients on an LLIN has the potential to be more effective in controlling malaria transmitted by insecticide-resistant vectors. This strategy can, in turn, delay the evolution of resistance, and therefore extend the effective lifespan of both active ingredients. We showed for the first time, since the adoption of pyrethroid insecticides for malaria control 40 years ago, that another insecticide, chlorfenapyr, when used for net treatment, is a safe, effective, and cost-effective alternative to standard pyrethroid-only LLINs. Pyrethroid LLINs treated with pyriproxyfen, an insect growth regulator designed to sterilise female mosquitoes, did not provide additional protection against malaria compared with standard pyrethroid-only LLINs. Moderate use of this LLIN in the trial, low residual bioefficacy of pyriproxyfen on the surface of the net fabric, or pyriproxyfen resistance in malaria vectors, or a combination of these factors, might explain this result. Finally, our trial confirmed the superior effectiveness of piperonyl butoxide LLINs compared with standard pyrethroid-only LLINs, but over a limited period of 12 months, possibly due to low textile durability leading to rapid reduction in the use of this net in the community.Implications of all the available evidenceThis study has several major implications. First, LLINs treated with both chlorfenapyr and a pyrethroid should be deployed in areas of insecticide resistance, with careful consideration of strategies for resistance management. Second, further investigation of the role of cross-resistance in pyriproxyfen LLINs is warranted to understand the utility of these nets for malaria control in different ecological and epidemiological settings. Finally, funders, national malaria control programmes, and stakeholders should urgently advocate and set up incentive structures to promote better textile and active ingredient durability on LLINs to maximise the potential of these tools for sustained malaria control in the face of rising insecticide resistance.Long-lasting insecticidal nets (LLINs) have successfully controlled malaria in sub-Saharan Africa for two decades, but their effectiveness might now be compromised by widespread resistance to pyrethroid insecticides in Anopheles vectors.LLINs combining a pyrethroid and a second insecticide with a different mode of action have also been developed. This class of LLINs might be more effective than standard pyrethroid-only LLINs in controlling pyrethroid-resistant malaria vectors, and have the potential to delay the evolution of resistance, thereby extending the effective lifespan of this class of product.Our trial compared the effectiveness of these three classes of dual-active-ingredient LLINs to pyrethroid-only LLINs in an area of Tanzania with pyrethroid-resistant mosquitoes. We assessed malaria infection prevalence through repeated cross-sectional surveys. We also evaluated clinical malaria incidence using a nested cohort study, malaria transmission through entomological monitoring, and cost-effectiveness by modelling from incidence estimates.We did a four parallel-arm, superiority, cluster-randomised, controlled trial in the Misungwi district of Mwanza, northwest Tanzania. Previous vector control interventions in the region included indoor residual spraying and a universal LLIN coverage campaign in 2015, as well as distribution of LLINs to primary school students in 2018 and to pregnant women during antenatal care visits.Clusters were allocated to one of four study groups: the pyrethroid-only (reference) group, which received LLINs containing the pyrethroid α-cypermethrin ; the pyriproxyfen group, with LLINs combining pyriproxyfen (5·5 g/kg) and α-cypermethrin ; the chlorfenapyr group, with LLINs combining chlorfenapyr (4·8 g/kg) and α-cypermethrin ; and the piperonyl butoxide group, with LLINs combining piperonyl butoxide (10 g/kg) and the pyrethroid permethrin .A cluster was defined in the protocol as a village or group of hamlets with a minimum of 150 households containing children aged 6 months to 14 years living in the core area; this number was later reduced to 119 households during mapping and census. Clusters were designed with core and buffer areas, with all houses in core areas located at least 600 m from any houses in adjacent clusters to reduce contamination of intervention effects between study groups. Houses allocated to cluster buffer areas received the same LLINs as those distributed in the core area, but outcome monitoring was done only in households in the core areas. Each household in the study area was enumerated and mapped using GPS handheld units and a short Open Data Kit programmed questionnaire.We did cross-sectional surveys to collect data on household factors and malaria infection prevalence at baseline (October 2018) and at 12 months , 18 months , and 24 months  after LLIN distribution . At eachTo assess malaria case incidence, 35 households per cluster were enrolled after LLIN distribution in year 1. 40 households were selected at random, and these households were visited in order of the randomisation until we obtained 35 consenting households, at which point enrolment stopped. Five additional households were selected if needed to reach 35 consenting households. From each household, one child aged 6 months to 10 years was selected at random and actively followed up for 1 year. A second independent cohort of 40 children per cluster (number increased because of the low incidence in the first year) was recruited 1 year after distribution and also followed up for 1 year. A similar recruitment process was followed, with 40 households initially selected, and an additional 20 selected at random until 40 consented.Routine mosquito collections were conducted in eight randomly selected households in 28 clusters per month (seven clusters per intervention group) for one night. Each collection round in the 84 clusters lasted for 3 months, with successive rounds over a 2-year period; five additional houses were also randomly selected but only visited if any of the eight selected households were ineligible or unavailable for inclusion. From the five additional households, the one the closest  to the ineligible household from the original eight selected was visited.Inclusion criteria for prevalence and incidence data collection were households with at least one child of appropriate age who permanently resided in the selected household and an adult caregiver who could provide written consent. All households in the core area were eligible for mosquito collection. Exclusion criteria were dwellings not found or vacant, no adult caregiver capable of giving informed consent, or eligible children who were severely ill.Institutional review boards of the Tanzanian National Institute for Medical Research (reference NIMR/HQ/R.8a/Vol.IX/2743), Kilimanjaro Christian Medical University College (2267), London School of Hygiene & Tropical Medicine (14952), and University of Ottawa (H-05-19-4411) granted ethical approval. During the household census, leaders of each hamlet were informed about the distribution and asked to consent on behalf of the community. Household members could decide whether to receive a study LLIN. Written informed consent was obtained from an adult guardian for selected children and from any adult living in households selected for mosquito collection.The protocol for this study has been published previously.Anopheles gambiae sensu stricto (ss) and Anopheles funestus (± 15 percentage points). Using Stata (version 11), 200 000 random allocations were generated and tested against the restriction criteria; of the 10 077 acceptable allocations, one was selected at random. The validity of the randomisations was verified by checking the frequency of allocations to the same study group of all pairs of clusters.An independent statistician conducted constrained randomisation to allocate the 84 clusters to the four study groups at a ratio of 1:1:1:1, ensuring that absolute differences in cluster means between study groups were within the specified ranges for each of the following variables: population size (range ± 9000 people); malaria infection prevalence at baseline (± 5 percentage points); socioeconomic status, measured as the percentage of households in the poorest tercile based on the wealth index of the entire study area (± 10 percentage points); LLIN use, measured as the percentage of residents who reported using a net last night (± 10 percentage points); and cluster suitability The inhabitants of each cluster and the field staff who were responsible for enrolment and collected data were masked to the type of LLIN allocated. LLINs of each type were similar in appearance apart from a colour-coded loop and a unique identifying code.All households enumerated in the core and buffer areas of clusters were allocated at least one LLIN for every two people.Plasmodium falciparum malaria infection using rapid diagnostic tests  and for haemoglobin concentration . Children who tested positive for malaria were treated with artemether–lumefantrine. Any child presenting with minor illness, such as helminth infection, upper respiratory tract infection, mild anaemia, or diarrhoea, was treated. Children with severe symptoms were referred to the nearest health facility. Cross-sectional prevalence surveys were not affected by the SARS-CoV-2 pandemic.During cross-sectional household surveys, information on household characteristics, LLIN use, and recent malaria cases were collected using electronic forms in tablets with Open Data Kit. At 12 and 24 months, net textile integrity was assessed by counting holes in a subsample of households (13 of 45 households per cluster). All selected children who came for the clinical appointment were assessed for All cohort children were treated with artemether–lumefantrine following recruitment. After 2 weeks, clearance of malaria infection was verified by rapid diagnostic tests and blood slides. Cohort children were asked to visit, with their guardian, a central point in each cluster every 2 weeks, or monthly during the dry season (July–August). Children with fever (tympanic temperature ≥37·5°C) or a history of fever in the past 48 h were tested for malaria parasites by rapid diagnostic test . Positive tests were confirmed by microscopy. Children with a positive rapid diagnostic test or minor illness were treated by trained study nurses. A questionnaire was administered to enquire about LLIN use the night before the visit, adverse events, travel history, and visit to health facilities within the past 2 weeks. Twice a year, community health workers visited the children in their houses to verify reported LLIN use. Because of the SARS-CoV-2 pandemic, from March to September, 2020, community health workers visited cohort children at home to assess fever (axillary temperature) the day before central point study visits. Study nurses only met with children with fever or history of fever to test them for malaria. COVID-19-safe protocols were followed during these visits.P falciparum circumsporozoite protein by ELISA.A gambiaeA funestusIndoor mosquito density was monitored from January, 2019, to December, 2020  using CeMosquito collections were suspended from March to May, 2020, because of the SARS-CoV-2 pandemic; to meet sample size requirements and compensate for the lost round during the high mosquito density season, the number of collection rounds was increased from four to five in 2020.The primary outcome was the prevalence of malaria infection (positive rapid diagnostic test) in children aged 6 months to 14 years at 24 months post-intervention (distribution of LLINs); secondary comparisons of this outcome were made at 12 and 18 months. The primary malaria transmission outcome was the entomological inoculation rate defined as the number of mosquito vectors tested positive for malaria over 24 months, and the primary economic outcome was cost-effectiveness based on incremental cost per disability-adjusted life-years (DALYs) averted. Secondary outcomes were the incidence of malaria cases (temperature ≥37·5°C or fever within 48 h and positive rapid diagnostic test) in children aged 6 months to 10 years, measured over 24 months of follow-up; the prevalence of moderate and severe anaemia (defined as haemoglobin concentration <8 g/dL) in children 6 months to 4 years measured at 12, 18, and 24 months; and vector density , sporozoite rate , and resistance to insecticides (proportion of vectors dead or sterile after exposure to insecticide). Adverse events and severe adverse events were recorded in the study cohort and in the population during cross-sectional surveys. Cause of death in the cohort was investigated by interviewing the child's guardian after receiving consent. Other secondary outcomes, including malaria prevalence, anaemia, and entomological inoculation rate at 30 and 36 months post-intervention will be published separately; data collection is ongoing.The required sample size was calculated using the method of Hayes and Moulton,For the entomological survey, a mean entomological inoculation rate of 1·76 infective mosquitoes per month per household in the reference group was assumed, with a between-cluster coefficient of variation of 40%.Statistical analyses were done with Stata (version 15). Malaria infection prevalence, malaria case incidence, anaemia, and entomological inoculation rate were compared between study group in the intention-to-treat population, defined as children allocated to LLIN type as randomised regardless of type of LLIN used. A per protocol analysis was done for malaria infection prevalence and included only children who actually used the allocated study nets. For the intention-to-treat and per protocol analyses, malaria infection prevalence at 12, 18, and 24 months was analysed by mixed-effects logistic regression. Cumulative incidence of malaria infection was analysed by mixed-effects Poisson regression with individual follow-up time specified as offset; following antimalarial treatment, 14 days follow-up time was censored to account for the prophylactic effect of the treatment. Blood slide confirmation of positive rapid diagnostic test results ensured that only those positive for parasites were counted as cases. The entomological inoculation rate was estimated as the number of sporozoite-infected mosquitoes per household per night and weighted to account for the proportion of mosquitoes processed for sporozoite detection. Differences in Anopheles density and entomological inoculation rate between study groups were estimated by mixed-effects negative binomial regression, and differences in sporozoite rate by mixed-effects logistic regression. Each model included fixed effects for time (survey timepoint or study year), study group, and time-by-study group interaction, and adjusted for the cluster-level variables used in restricted randomisation according to the prespecified analysis plan. A block-exchangeable correlation structure was used to account for the within-period and between-period intracluster correlations for all outcomes .ClinicalTrials.gov, number NCT03554616.This trial is registered with We modelled cost-effectiveness over the 2-year trial period. Malaria incidence estimates for each trial year were combined with probabilities of progression to severe disease and death on the basis of secondary sources . Using aWe took a disaggregated societal perspective, meaning that we considered costs to donors, to public providers (donors and Tanzanian government combined), and to households, both separately and together. Costs of net distribution were excluded because they were identical across strategies. Costs of net procurement were obtained from The Global Fund to Fight AIDS, Tuberculosis, and Malaria. The cost per case of malaria was assumed to be the same across trial groups, with donors bearing the costs of publicly funded rapid diagnostic tests and antimalarial medicines, the Tanzanian government bearing the costs of publicly funded consultations and hospitalisations, and households bearing the costs of time spent ill and out-of-pocket payments. Details of parameter values and sources are provided in We used Monte Carlo simulation to conduct probabilistic analyses, which reflect combined uncertainty in stochastic parameters. Analyses were re-run, varying one key parameter at a time, to examine the robustness of results to plausible variations in individual parameters. A threshold analysis identified the price of each net at which cost-effectiveness conclusions would change. Analyses were done in Microsoft Excel with Visual Basic for Applications. Results are presented on the cost-effectiveness plane and compared against plausible cost-effectiveness thresholds.The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.Selection of villages and households was done during the census between May 11 and July 2, 2018. Enrolment of children for the cohort was done in March, 2019. The study area included 39 307 households across 72 villages. These households formed 84 clusters, which were evenly distributed between the four groups , characteristics of the surveyed households (n=2656) and children (n=5019) were similar between the four groups, with malaria infection detected in 1948 (44·2%) of 4403 children tested and LLIN use reported by 11 314 (60·7%) of 18 654 participants across all age groups . Across For the cohort, 2982 children aged 6 months to 10 years were recruited in March, 2019, and 3371 in February, 2020, giving a total follow-up time of 5565·5 child-years. Baseline characteristics are reported in A funestus. Across nine further collection rounds, entomological data were collected in 6043 households  to 5·9 (20·9) across study groups (n=1340 households), and 4700 (94·5%) of 4973 Anopheles collected were identified as useholds .3 months after LLIN distribution, 3155 (72·1%) of 4378 of study participants surveyed reported using the assigned study LLINs the previous night; this proportion was similar between study groups . Most of2)The proportion of study LLINs that were torn  was 0·46 per child-year in the pyrethroid-only reference group, 0·42 per child-year in the pyriproxyfen group , 0·23 per child-year in the chlorfenapyr group , and 0·33 per child-year in the piperonyl butoxide group  and in the piperonyl butoxide group in year 1 only  compared with that in the pyrethroid-only group. No significant difference was observed in the pyriproxyfen group compared with the pyrethroid-only group at any timepoint .Table 4EA funestus, with mortality below 85% after exposure to 10 times diagnostic concentrations of α-cypermethrin or permethrin. Exposure to pyriproxyfen sterilised only 127 (24%) of 536 of A funestus and 23 (21%) of 112 A gambiae sensu lato. No resistance was observed against chlorfenapyr insecticide , followed by piperonyl butoxide LLINs (37 DALYs averted [72] per 10 000 population), while pyriproxyfen LLINs incurred 9 more DALYs [71] per 10 000 population than did pyrethroid-only LLINs . PyrethrAnopheles mosquitoes, we found that only the chlorfenapyr LLINs provided significantly better protection over 2 years than did pyrethroid LLINs; children aged 6 months to 14 years had 55% lower odds of having malaria 2 years after LLIN distribution, and children aged 6 months to 10 years had 44% lower malaria incidence over the 2 years. The entomological inoculation rate, an indicator of malaria transmission, was also 85% lower in the chlorfenapyr group than in the pyrethroid-only group, arising from reduced vector population density and longevity. Added protection provided by piperonyl butoxide LLINs lasted for only 1 year, with clinical malaria incidence in children nearly halved and a 73% reduction in entomological inoculation rate, but with no evident effect on malaria prevalence. Although the point estimates for malaria prevalence and entomological inoculation rate were lower in the pyriproxyfen LLIN group compared with the pyrethroid-only reference group during the first year, these differences were not statistically significant. Chlorfenapyr LLINs were the most effective and cost-effective of the dual-active-ingredient LLINs; in this context, they would be less costly than even standard pyrethroid LLINs from societal and household perspectives, and well within plausible cost-effectiveness thresholds for public providers. Piperonyl butoxide LLINs were more cost-effective than pyrethroid-only nets, but unlikely to be cost-saving from a societal perspective.In this trial assessing three types of dual-active-ingredient LLINs in an area of Tanzania with high pyrethroid resistance among A gambiaeA funestusThis trial provides the first evidence of the additional effectiveness and cost-effectiveness of chlorfenapyr LLINs relative to pyrethroid LLINs for malaria control. Several small-scale studies had already shown the efficacy of these nets against entomological outcomes, reporting higher mortality in resistant Kawada and colleaguesThe piperonyl butoxide LLIN (Olyset Plus) showed additional effectiveness compared with the pyrethroid-only LLIN over a shorter period (12 months) than in previous RCTs (21–25 months).Our trial's combined assessment of epidemiological, entomological, economic, and LLIN durability outcomes significantly advances the evidence base on all three classes of dual-active-ingredient LLIN currently available for malaria control. Nonetheless, limitations remain. First, the rapid decrease in use of study LLINs, within a context of high overall net use, might partly explain the relative lack of effectiveness of piperonyl butoxide LLINs and pyriproxyfen LLINs over the 2 years compared with pyrethroid-only LLINs. Second, the size of the cluster buffer areas, despite being maximised given the study area, might not have totally prevented contamination of effects between intervention groups. Any residual overspill would have biased results towards the null. Third, if chlorfenapyr LLINs maintain their relative effectiveness in a third year of use, our findings will have underestimated the cases averted and associated reductions in malaria treatment costs.This study represents the first time, to our knowledge, since the initial deployment of pyrethroid-treated nets 40 years ago that a new class of insecticide (the pyrrole chlorfenapyr) has proven safe and suitable for use on LLINs, with demonstrable effectiveness in controlling malaria. Unlike all other insecticides used in malaria control, chlorfenapyr has a non-neural mode of action and no known resistance in mosquitoes. However, caution is needed; massive scale-up of standard pyrethroid-only LLINs 10–20 years ago led to the spread of pyrethroid resistance.Deidentified data and data dictionary will be made available at the end of the third year of trial follow-up upon reasonable request to the corresponding author.We declare no competing interests.


Plasmodium falciparum chimeric protein which contains a fragment of the sexual-stage Pfs48/45-6C protein genetically fused to GMZ2, a fusion protein of GLURP and MSP-3, that has been shown to be well tolerated, safe and immunogenic in clinical trials performed in a malaria-endemic area of Africa. However, there is no data available on the antigenicity or immunogenicity of GMZ2.6c in humans. Considering that circulating parasites can be genetically distinct in different malaria-endemic areas and that host genetic factors can influence the immune response to vaccine antigens, it is important to verify the antigenicity, immunogenicity and the possibility of associated protection in individuals living in malaria-endemic areas with different epidemiological scenarios. Herein, the profile of antibody response against GMZ2.6c and its components  in residents of the Brazilian Amazon naturally exposed to malaria, in areas with different levels of transmission, was evaluated.The GMZ2.6c malaria vaccine candidate is a multi-stage This study was performed using serum samples from 352 individuals from Cruzeiro do Sul and Mâncio Lima, in the state of Acre, and Guajará, in the state of Amazonas. Specific IgG, IgM, IgA and IgE antibodies and IgG subclasses were detected by Enzyme-Linked Immunosorbent Assay.The results showed that GMZ2.6c protein was widely recognized by naturally acquired antibodies from individuals of the Brazilian endemic areas with different levels of transmission. The higher prevalence of individuals with antibodies against GMZ2.6c when compared to its individual components may suggest an additive effect of GLURP, MSP-3, and Pfs48/45 when inserted in a same construct. Furthermore, naturally malaria-exposed individuals predominantly had IgG1 and IgG3 cytophilic anti-GMZ2.6c antibodies, an important fact considering that the acquisition of anti-malaria protective immunity results from a delicate balance between cytophilic/non-cytophilic antibodies. Interestingly, anti-GMZ2.6c antibodies seem to increase with exposure to malaria infection and may contribute to parasite immunity.The data showed that GMZ2.6c protein is widely recognized by naturally acquired antibodies from individuals living in malaria-endemic areas in Brazil and that these may contribute to parasite immunity. These data highlight the importance of GMZ2.6c as a candidate for an anti-malarial vaccine.The online version contains supplementary material available at 10.1186/s12936-021-04020-6. Plasmodium, remains a tremendous burden to global public health, causing 405,000 deaths annually and generating morbidity to more than two hundred million of individuals [Plasmodium species able to infect humans, Plasmodium falciparum is responsible for most of the severe cases and deaths [P. falciparum malaria infection in most parts of the world; however, resistance to artemisinin and to its partner combination drugs has developed in the Greater Mekong Subregion (GMS) and more recently in South America [P. falciparum are targets of protective immunity.Malaria, an infectious parasitic disease caused by blood-borne apicomplexan parasites of the genus ividuals . Among td deaths . Control America –7, highl America . MalariaP. falciparum chimeric protein that contains a fragment of the sexual-stage Pfs48/45-6C protein genetically fused to recombinant protein GMZ2, an asexual-stage vaccine construct consisting of the N-terminal region of the Glutamate-Rich Protein (GLURP) and the C-terminal region of Merozoite Surface Protein-3 (MSP-3) [The GMZ2.6c malaria vaccine candidate is a multi-stage  (MSP-3) , 10  or synthetic lipid adjuvant (SLA) induce high parasite-specific antibody titers, IFN-γ responses in CD4 + TH1 cells, and a high percentage of multifunctional CD4 + T cells expressing IFN-γ and TNF in response to GMZ2.6c [GLURP is expressed in both pre-erythrocytic and erythrocytic stages in the vertebrate host and may participate in the binding to receptors on erythrocytes surface during merozoite invasion and formation of the parasitophorous vacuole; MSP-3 is expressed in the erythrocytic stage and has a crucial role not only in binding to the host red blood cell, but also in protecting the parasite against haem that is released during parasite egression; Pfs48/45 is expressed on the surface of gametocytes and gametes playing an important role in fertilization, thus considered a candidate for the transmission-blocking vaccine –15. GLURo growth –26. A re GMZ2.6c .Until now, there is no data available on the antigenicity or immunogenicity of GMZ2.6c in humans. However, clinical trials performed in a malaria-endemic area of Africa showed that GMZ2, the vaccine construct of GLURP and MSP-3, was well tolerated, safe, and immunogenic –31. The Considering that circulating parasites can be genetically distinct in different malaria-endemic areas and host genetic factors can influence the immune response to vaccine antigens, it is important to verify the antigenicity, immunogenicity, and the possibility of associated protection in malaria-endemic areas with different epidemiological scenarios. The goal of this work is, therefore, to evaluate the profile of antibody response against the chimeric protein GMZ2.6c and its components  in residents of the Brazilian Amazon, naturally exposed to malaria, in areas with different levels of malaria transmission.rd and 6th main hotspots, among the 17 municipalities that concentrate 80% of falciparum malaria transmission in the whole Amazon. The Guajará municipality (02º58′18’’S/57º40′38’W), a medium-risk area in the state of Amazonas  and Mâncio Lima (07º36′49’’S/72º53′47’’W), are high-risk areas situated at the Juruá Valley, state of Acre, and correspond, respectively, to the 3Serum samples were obtained from 299 malaria exposed individuals from Cruzeiro do Sul , Mâncio Lima  and Guajará . In addition, serum samples from individuals who have never had malaria were included in the study as area control . All individuals from this group were negative for the presence of malaria parasites as assessed by thick blood films. Non-endemic control blood samples from 40 individuals from the laboratory staff  who had neither history of malaria nor contact with malaria transmission areas, were included as Rio de Janeiro Controls.The study protocol was reviewed and approved by the Oswaldo Cruz Foundation Ethical Committee , which included obtaining the following patients’ written consents for research use of their blood samples. Written informed consent was obtained from all adult donors or from donor’s parents in the cases of children. All the procedures adopted in this study fully complied with specific federal permits issued by the Brazilian Ministry of Health.Donors who provided informed consent also completed an epidemiological survey. In order to evaluate the degree of malaria exposure, subjects responded to questions related to personal data such as: age, time of residence in the endemic area, number of previous malaria episodes, the time elapsed from the last infection, use of malaria prophylaxis, and presence of symptoms.Plasmodium sp) and species , as previously described [P. vivax and/or P. falciparum at the time of blood collection were subsequently treated by the chemotherapeutic regimen recommended by the Brazilian Ministry of Health [Venous peripheral blood (20 ml) was collected into heparin or EDTA tubes for antibodies analyses and molecular diagnosis, respectively. The plasma was stored at –20 °C, and the pellets, containing peripheral blood cells collected into EDTA tubes, were mixed with equal volumes of a cryopreservation solution (0.9% NaCl/4.2% sorbitol/20% glycerol) and were stored at –70 °C for later DNA extraction for molecular diagnosis. Thin and thick blood smears were examined for identification of malaria parasites by a technician experienced in malaria diagnosis from the Laboratory of Malaria Research (Fiocruz), which is the headquarters of the CEMART , a reference center for malaria diagnosis in the Extra-Amazonian Region for the Brazilian Ministry of Health. Malaria diagnosis was performed in Giemsa-stained thin and thick blood smears, and parasitological evaluation was done by examination of 200 fields at 1,000 × magnification under oil-immersion. Thin blood smears of the positive samples were examined for species identification. The parasitaemia was expressed as the number of parasites/μl of blood in the thick blood smear. The number of parasites/μl of blood was calculated by multiplying the number of parasites counted against 500 leucocytes, and the number of leukocytes of the subject and dividing the product by 500. To increase the sensitivity of the parasite detection, molecular analyses were performed in all samples. Briefly, DNA was extracted from the blood samples using the QIAamp DNA blood midi kit  according to manufacturer instructions, and Polymerase Chain Reaction (PCR) was performed using specific primers for genus . GMZ2.6c and its fragments were expressed in Lactococcus lactis strain MG1363 and purified as previously described [L. lactis containing pSS4 was cultured in LAB medium supplemented 5 mM cysteamine/0.5 mM cystamine. The recombinant protein in the supernatant of the culture was purified by affinity chromatography with a 5 ml HisTrap™ HP column  followed by a 5 ml HiTrap NHS-activated HP column containing monoclonal antibody mAb45.1 (epitope I), according to the manufacturer . To assess purity in purified proteins, reversed-phase HPLC was performed, showing a relative purify ˃ 95%. Production of GLURP-R0, MSP3 and Pfs48/45-6C was done as previously described [The multi-stage GMZ2.6c construct was created from GLURPescribed , 38.Microtiter 96-well plates  were coated with the recombinant proteins at an optimal dilution using phosphate-buffered saline at pH 7.2 (PBS) or a carbonate-bicarbonate buffer at pH 9.6 at 100 µl/well overnight at 4 °C  data bank software. Epi-Info and GraphPad Instat  statistical software programs were used for data analyses. Kruskal–Wallis followed by Mann–Whitney pairwise tests were used to analyse the differences in distributions of continuous numerical variables, and the chi-square analysis was applied to test independence of prevalence among groups. The Spearman rank coefficient test was used to analyse the correlation between variables.Data were stored in the Epi-Info 6 .The main characteristics of the studied groups are shown in Table All individuals of the CO group were residents of the studied areas, CZS, ML or GJ but reported no previous malaria episode, and none had symptoms or circulating parasites at the time of collection. CO group showed a higher frequency of females and a lower mean of age and time of residence in malaria endemic area than CZS, ML and GJ groups .Considering the prevalence of individuals with detectable antibodies that recognize GMZ2.6c, regardless antibody class , GMZ2.6c was shown to be largely recognized by naturally acquired antibodies in the CZS, ML and GJ groups, and these three groups showed comparable prevalence of individuals presenting GMZ2.6c specific antibodies: 103/124 (83%), 65/88 (74%), and 63/87 (72%), respectively. The analyses of the prevalence of individuals with detectable antibodies that recognize GMZ2.6c components, regardless antibody class , is shown in Fig. No volunteers from the CO group, who reported no previous malaria episodes, had antibodies against GMZ2.6c, GLURP, MSP-3, or Pfs48/45. Likewise, none of the 40 Rio-controls, with no history of malaria or previous travel to malaria transmission areas, had detectable antibodies to GMZ2.6c, GLURP, MSP-3, or Pfs48/45.The prevalence of individuals with IgG, IgM, IgA, and IgE anti-GMZ2.6c antibodies in CZS, ML, and GJ groups is shown in Fig. As showed in Fig. P < 0.0001, CZS and GJ, IgG1 versus IgG2 and IgG4; P = 0.01, ML IgG1 versus IgG2; P < 0.001, ML IgG1 versus IgG4; P < 0.0001, CZS and GJ IgG3 versus IgG2 and IgG4; P = 0.01, ML IgG3 versus IgG2; P < 0.0001ML IgG3 versus IgG4) , while the frequency of individuals with IgG3 antibodies was not different between the groups. CZS and ML groups also showed higher levels of IgG1 and IgG3 than of IgG2 and IgG4 antibodies . The GJ group showed higher levels of IgG3 than of IgG2 and IgG4 antibodies   Fig. . The CZSes) Fig. .Fig. 7PrAs showed in Table P. falciparum-infected and 181 non-infected individuals, regardless of whether individuals belong to the CZS, ML or GJ group. Levels of anti-GMZ2.6c and anti-GLURP IgG and IgM antibodies were higher in infected than in non-infected individuals . Levels of cytophilic (IgG1 and IgG3) antibodies against these antigens were also higher in the P. falciparum-infected than in the non-infected  group. No difference was observed in antibody levels against MSP-3 and Pfs48/45 between infected and non-infected individuals  with the number of previous malaria infections. Levels of anti-GMZ2.6c IgG3 antibodies were also negatively associated  to parasitaemia. Individuals with higher levels (> 75th percentile) of anti-GLURP IgG3 antibodies were also older and presented a higher time of residence in the malaria-endemic areas   groups were all considered in a single group. Individuals with higher levels (> 75th percentile) of IgG and IgG3 antibodies against GMZ2.6c were older and presented a higher time of residence in malaria-endemic areas than individuals with lower IgG and IgG3 antibody levels (< 75th percentile)  Fig. . IgG ant02) Fig. , but didPlasmodium. In this sense, the recombinant GMZ2.6c protein has shown to be a promising multi-stage anti-malarial vaccine candidate. In this work, the antibody response profiles to the recombinant Plasmodium falciparum GMZ2.6c vaccine candidate in individuals living in Brazilian malaria-endemic areas, with different levels of transmission, was evaluated. The humoral response against the GMZ2.6c protein and its fragments, GLURP, MSP-3, and Pfs48/45, was assessed in individuals living in three cities of the Brazilian Amazon: Cruzeiro do Sul and Mâncio Lima, in the state of Acre, and Guajará, in the state of Amazonas.Given the complexity of the life cycle and of the parasite-host interactions, as well as the extensive parasite genetic variability, and the mechanisms it uses to evade the host immune response, an ideal malaria vaccine should be directed against antigens expressed in different development stages of the P. falciparum and P. vivax, but P. vivax was the most prevalent infecting plasmodial species, reflecting the current malaria scenario in Brazil, where P. vivax is responsible for 89% of infections [Individuals were not differently distributed by sex, age, origin and previous exposure to malaria in Cruzeiro do Sul (CZS), Mâncio Lima (ML), and Guajará (GJ), although individuals from ML have reported a higher number of previous infections, even though ML is a highly endemic city like CZS. Most of the individuals reported previous infections by both fections .Most individuals from CZS, ML, and GJ had anti-GMZ2.6c antibodies, regardless of the immunoglobulin class, indicating that GMZ2.6c is widely recognized for naturally acquired antibodies from resident individuals. The antigenicity of GMZ2.6c seems to be independent of the endemicity levels, since the prevalence of individuals with antibodies that recognized GMZ2.6c was similar in the three study sites, with different endemicity levels.P. vivax. The other individuals reported at least one P. falciparum infection. The non-detection of these antibodies could also be related to functional exhaustion among T and B cell subsets, well-described in viral infections [P. falciparum genetic polymorphisms, intensity of infection, coinfections, malnutrition, or human host genetic factors.Sixty-eight individuals  had no antibodies that recognized GMZ2.6c . The non-detection of anti-GMZ2.6c antibodies in these individuals could be associated to the absence of a previous contact with the parasite. However, of these, only 4 reported no previous malaria episodes and 10 reported only previous infection by fections –43 and efections –49. BesiClinical trials in naive subjects and in individuals naturally exposed to malaria in Africa to evaluate the safety and immunogenicity of GMZ2 showed a more potent response against GMZ2 when compared to its individual components , 28. In Independent studies have evaluated the potential of humoral immune response against GLURP and MSP-3 in conferring clinical immunity to malaria in African endemic areas with different transmission intensities. A wide variety of results regarding the natural immunogenicity of GLURP and MSP-3 was observed. Some studies have reported the higher immunogenicity of MSP-3 when compared to GLURP-R0 , 51, whiIn the present study, the prevalence of individuals with anti-Pfs48/45 antibodies ranged from 24 to 40%. These prevalences are lower than those (74.7%) observed in the Central Region of Ghana for the same Pfs48/45 construct (Pfs48/45.6c) (37), possibly reflecting differences in exposure to gametocytes as previously reported in a longitudinal study in a hypo-endemic area in Tanzania .P. falciparum isolates circulating at variable levels in the Brazilian Amazonian Region may explain this difference in immune response in the CZS group, since genetic polymorphisms in B epitopes may influence the immunological properties of the antigen [In this work, many individuals from the CZS, ML and GJ groups had IgG and IgM antibodies recognizing GMZ2.6c, contrasting with an uncommon IgA and IgE specific response. The data presented here also showed that anti-GMZ2.6c IgG antibodies were more frequent and present higher levels in the CZS group when compared to the ML and GJ groups. It is widely accepted that IgG antibodies are the main mediators of protection against clinical malaria , 55, and antigen . This reP. falciparum-specific IgE are proposed to be associated with protection as well as to participate in the pathogenesis of malaria [Anopheles salivary gland IgE level is positively correlated with the antiplasmodial IgE antibody levels, and a hypothesis proposes that, during Anopheles bite, the injection of saliva containing pharmacologically active proteins and peptides predisposes individuals to the development of an antiplasmodial IgE response [The role of IgE antibodies in the outcome of malaria infection remains controversial and poorly understood. The levels of  malaria –63. Thusresponse , 65.P. falciparum merozoites [It has been proposed that cytophilic IgG1 and IgG3 isotypes are more correlated with protective responses than the IgG2 and IgG4 (non-cytophilic) antibodies , 55. Herrozoites , 69, 70.Studies have shown that the inherent characteristics of plasmodial antigens can influence the molecular events that lead to the preferential induction of different IgG subclasses. The presence of both repetitive and polymorphic amino acid sequences predominantly induces an IgG3 response, whereas the absence of such polymorphic repeats predominantly induces IgG1 responses. Polymorphic antigens with no repetitive sequence induce an IgG1 + IgG3 response, with the predominance of IgG3, whereas conserved repetitive antigens tend to induce IgG1 or IgG1 + IgG3 responses , 72. TheIgG and IgM antibodies levels against GMZ2.6c and GLURP were higher in parasitized individuals when compared to non-parasitized subjects. This result may reflect the stimulation of the immune system leading to the production of antiplasmodial antibodies, including anti-GLURP antibodies, due to infection. This booster, in response to infection, seems to induce a predominantly IgG1 and IgG3 antibodies that recognize GMZ2.6c and GLURP.P. falciparum and of the anti-Pfs48/45 antibodies in inhibiting fertilization has been currently conducted in the laboratory and could also help to clarify this issue.Classically, levels of antibodies against several plasmodial antigens seem to be associated with age and time of exposure to malaria in endemic regions , 82–88. The analysis of the antibody response against the individual  proteins constituting the GMZ2.6c vaccine candidate did not provide evidence of a possible protective role of these antibodies, since no relationship was verified between prevalence or levels of specific antibodies and the presence or absence of parasites in the blood, the presence or absence of symptoms at the time of collection, parasitaemia, number of previous malaria episodes, time elapsed since the last malaria episode, current or last infecting plasmodial species. Associations between high anti-GLURP and anti-MSP3 antibody levels and low parasite densities in malaria patients living in the Central Region of Ghana have however been reported .Taken together, the data presented here showed that GMZ2.6c protein is widely recognized by naturally acquired antibodies from individuals living in endemic areas of Brazil with different levels of transmission. The higher prevalence of individuals with antibodies that recognize GMZ2.6c and the higher levels of these antibodies when compared to their individual components may suggest an additive effect of GLURP, MSP-3, and Pfs48/45 when inserted in a same construct. Also, anti-GMZ2.6c antibodies seem to increase with exposure to malaria infection and may contribute to parasite immunity. These data highlight the importance of GMZ2.6c as a candidate for an anti-malarial vaccine. Additional studies in other endemic areas, with populations with different genetic backgrounds, besides the evaluation of the functional role of anti-GMZ2.6c antibodies, are important to confirm the potential of GMZ2.6c as an anti-malarial vaccine.Additional file 1: Table S1. Antigens and Enzyme-Linked Immunosorbent Assay antibody assays.