Plasmodium vivax challenge infections in humans, which are essential for vaccine and drug testing. With the goal of accelerating clinical development of malaria vaccines, the outcome of infections experimentally induced in naïve and semi-immune volunteers by infected mosquito bites was compared.Significant progress has been recently achieved in the development of P. vivax sporozoite-infected Anopheles mosquitoes. Parasitemia levels, malaria clinical manifestations, and immune responses were assessed and compared.Seven malaria-naïve and nine semi-immune Colombian adults (n = 16) were subjected to the bites of 2–4 All volunteers developed infections as confirmed by microscopy and RT-qPCR. No significant difference in the pre-patent period  was observed but naïve volunteers developed classical malaria signs and symptoms, while semi-immune volunteers displayed minor or no symptoms at the day of diagnosis. A malaria-naïve volunteer developed a transient low submicroscopic parasitemia that cured spontaneously. Infection induced an increase in specific antibody levels in both groups.P. vivax vaccines in naïve and semi-immune volunteers under controlled conditions and would accelerate P. vivax vaccine development.Sporozoite infectious challenge was safe and reproducible in semi-immune and naïve volunteers. This model will provide information for simultaneous comparison of the protective efficacy of NCT01585077clinicaltrials.gov  Plasmodium vivax malaria research, significant efforts have been invested and progress has been achieved towards development of an effective P. vivax vaccine. Two P. vivax parasite antigens, the circumsporozoite (CS) protein P. vivax antigens that could be proposed for vaccine development Despite multiple technical and financial constraints for P. falciparum, the P. vivax CS protein is among the most promising vaccine candidates. P. vivax CS-derived subunit vaccine formulations based on Long Synthetic Peptides (LSP) formulated in Montanide adjuvant have been shown to be safe, well-tolerated and immunogenic in malaria-naïve volunteers As with The protective efficacy of malaria vaccine candidates that have proven to be safe and immunogenic in Phase I trials, can be tested in malaria-naïve volunteers in Phase IIa trials P. vivax in culture Because of the constraints to grow P. vivax challenge trials in naïve volunteers Taking advantage of experience provided by two previous P. vivax-infected donors. A separate IC was obtained from each volunteer for HIV screening. The clinical trial was registered on clinicaltrials.gov, registry number NCT01585077. The protocol for this trial and the supporting CONSORT checklist are available as supporting information  as described below. Duffy-positive phenotype (Fy+) was confirmed by DNA genotyping P. vivax-infected patients were recruited to serve as a potential parasite donor for mosquito infection.Sixteen healthy, Duffy-positive (Fy+) male and female volunteers, 18–45 years of age , were recruited for the study . PreviouMalaria-naïve volunteers were recruited in Cali (Colombia), a non-endemic city; those with previous malaria experience were recruited in Buenaventura, a malaria-endemic area on the Colombian Pacific Coast. Volunteers were extensively informed about the risks of participation and were provided sufficient opportunity to read the IC forms. Before signing the written consent, all volunteers had to pass an oral or written exam concerning the trial and its risks as described elsewhere P. vivax infection after signing a written IC at the out-patient malaria clinic of INSALPA (Buenaventura), and one of them was selected as a parasite donor based on laboratory results and the infection rate of the blood-feed mosquito batch.Thirty-five mL of whole blood was collected by venipuncture  from six patients diagnosed with P. vivax mono-infection was confirmed by real-time quantitative polymerase chain reaction (RT-qPCR) as previously reported Blood samples were collected and distributed as follows: 30 mL (sodium heparin tubes) for mosquito infection Anopheles albimanus mosquitoes were reared and infected at the MVDC insectary in Cali as previously described Sporozoite challenge of all volunteers was carried out on the same day by exposing volunteers to bites of 2–4 mosquitoes of the same infected batch P. vivax infection in challenged volunteers and parasite donor was diagnosed by thick blood smears (TBS), which were examined independently by two experienced microscopists P. vivax infection in the parasite donor was confirmed by Plasmodium RT-qPCR P. vivax and P. falciparum parasitemias for quantification A comprehensive clinical laboratory screening confirmed the health status of the volunteers one month prior to challenge and at the time of diagnosis, and again at three weeks and four months after treatment was completed. Screening assays consisted of the following: automated whole blood cell counts, urine analysis by dipstick , blood chemistry tests for creatinine, blood urea nitrogen (BUN), glycemia, total bilirubin, direct bilirubin, transaminases (ALT and AST), coagulation tests, and C-reactive protein.P. vivax blood-stage antigen preparations derived from infected patients or sporozoites obtained from experimentally infected mosquitoes P. vivax circumsporozoite protein (PvCS) and the merozoite surface protein-1 (PvMSP-1) described elsewhere PvCS corresponded to a chimeric synthetic polypeptide composed of the amino (N) flank, the VK210 and VK247 natural repeat variants, and the carboxyl (C) flanking sequences of the protein PvMSP-1 corresponded to a recombinant fragment (348 aa) from the N region of the protein, namely r200L Specific antimalarial antibodies were determined both by IFAT and enzyme-linked immuno-sorbent assay (ELISA) using sera collected from the bleedings described above for clinical laboratory tests. IFAT were performed using Study data were collected and managed using REDCap  electronic data capture tools P. vivax sporozoite challenge during June 2013. No age differences were observed among volunteers (ten men and six women), with the mean age of 28 years (range: 19–38) (All 16 volunteers were exposed to : 19–38) .P. vivax-infected mosquitoes used for the sporozoite challenge was fed on a blood sample that had a parasite density of 1,273 asexual parasites/µL and 199 gametocytes/µL. Approximately 80% of fed mosquitoes were determined to be infected, displaying an average of 409 sporozoites per salivary glands by day 14 post-blood feeding. Both groups of volunteers completed the biting process within a total period of about four hours. The majority of volunteers (15/16) completed the infective mosquito biting dose (3±1) in a single biting cycle, but one participant required two cycles   after sporozoite challenge. In naïve volunteers the mean pre-patent period was 12.5±0.8 days (range: 11-13 days), whereas in semi-immune volunteers the period was 12.9±0.3 days (range: 12–13 days). Retrospective RT-qPCR analysis indicated shorter pre-patent periods, 9.2±0.4 and 9.4±0.9 days for naïve and semi-immune volunteers, without significant difference between groups. A naïve volunteer developed transient low-level parasitemia detectable by RT-qPCR by day nine and cured spontaneously in four days. High Performance Liquid Chromatography (HPLC) used for antimalarial drug detection  was negative in this volunteer (data not shown). Median parasitemia determined by TBS was similar between naïve  and semi-immune volunteers . RT-qPCR showed that naïve volunteers presented a non-significant higher median parasite density than semi-immune volunteers on day nine, at the time of the first positive RT-qPCR . Median parasite density continued to be higher but not significant in naïve volunteers when parasitemia became patent by microscopy  .vs 67%; p = 0.143); nausea ; oropharyngeal dryness ; weakness ; and pruritus .Signs and symptoms most frequently observed were fever (body temperature ≥38°C), pallor, headache, nausea, chills, and malaise; all of the aforementioned were more frequent and severe in naïve volunteers. Fever was present in 100% of the naïve and in 33% of the semi-immune volunteers (p = 0.01) . AdditioP. vivax infection after being in a malaria-endemic area. The volunteer was treated with standard antimalarial therapy and successfully recovered, and has not presented a new infection as of today.Three months after treatment, a naïve volunteer presented with a 3, 110×103 and 96×103 platelets/µL, respectively) and four volunteers had mild (302N: 3.4×103 cell/µL) to moderate leukopenia . In contrast, none of the semi-immunes presented with thrombocytopenia and only one presented with mild leukopenia (327P: 3.3×103 cell/µL). Hematological and biochemical alterations resolved spontaneously after antimalarial treatment.All baseline clinical laboratory tests were normal before sporozoite challenge; however some biochemistry laboratory results showed abnormal values at the time of malaria diagnosis in a few volunteers with no significant difference . A naïveAs shown in PvCS and 45% to PvMSP-1. In the group of naïve volunteers, 5/7 developed specific antibodies to PvCS and PvMSP-1 antigens by the day of parasite patency; the entire group (7/7) became positive to PvMSP-1 by three weeks after challenge. A total of 6/9 (67%) and 8/9 (89%) of the semi-immune volunteers presented with or showed increases in anti-PvMSP-1 antibody levels at diagnosis and three weeks after challenge, respectively. In contrast, 5/7 naïve (71%) and 7/9 (78%) semi-immune volunteers presented with antibodies to PvCS three weeks after challenge. The percentage of positive naïve or semi-immune volunteers was similar after four months of follow-up. No differences in the frequency of responders were observed between the two groups after challenge. Antibody responses expressed as a reactivity index (RI) showed a significant increase between the pre-challenge day and four months after treatment in naïve volunteers with a mean value from 0 to 1.9, and 0 to 2.9 for PvCS and PvMSP-1, respectively. In semi-immune volunteers, the RI was similar for responses against the PvCS (from 1.5 to 1.8), whereas it increased in response to PvMSP-1 (0.8 to 2.5) at the same set points, although no significant differences were observed  and presented with low antimalarial antibody titers at the time of enrollment, suggesting that the frequency of previous malaria exposure and concomitant immunity were low. Therefore, it was not surprising that volunteers in both groups developed similar pre-patent periods, although it was interesting that both the clinical outcome of the infection and specific antibody responses in semi-immune and naïve volunteers were different. However, no association between onset of symptoms and antibody levels or parasitemia was found.Both naïve and semi-immune volunteers presented with similar parasite densities (20–400 parasites/µL), but infections in naïve volunteers resulted in a significantly higher rate of severe headache and fever. In the scoring of AE, a bias may have occurred because symptoms were self-reported by volunteers; it is likely that both groups had different thresholds or responses to symptoms. It is intriguing that despite the low antibody levels and limited exposure to malaria, volunteers from Buenaventura had already developed a significant level of clinical immunity. Until a decade ago Buenaventura was among the regions with the highest malaria transmission in Colombia; however in the last few years, transmission has significantly decreased (∼80–90%) PvCS and PvMSP-1 proteins were low. The sporozoite inoculum during an Anopheles mosquito infectious-bite does not appear to depend on the mosquito sporozoite load. It is likely that 2–4 bites of An. albimanus, considered a “less efficient” vector than African or Asian Anopheles species, delivers only a limited number of sporozoites PvCS and anti-sporozoite antibodies in all naïve volunteers, although not enough to significantly boost the pre-existing antibody titers in the semi-immune group. Likewise, boosting of antibody responses to asexual blood-stage parasites may have also been compromised due to the careful monitoring and follow-up of volunteers with prompt curative treatment. Therefore, it is most likely that the milder clinical manifestations in semi-immune volunteers may have been due to the lower initial parasitemias detected by RT-qPCR but also possible to immune responses to parasite components other than PvCS and PvMSP1, including cytokines and innate immune responses. C-reactive protein measurements at the time of malaria diagnosis showed higher acute inflammatory responses in naïve compared to semi-immune volunteers , which appears to be associated with clinical findings in naïve volunteers. A comprehensive transcriptome analysis is being planned and would be the subject of further research. Antibody responses to asexual blood stage parasites and PvMSP-1, which are associated with a reduction in clinical manifestations Due to the short duration of infection and possibly the low-density parasite inoculum, specific antibody responses to the parasite blood stages as well as to P. vivax frequently induces thrombocytopenia which may be considered as predictive marker of malaria infection P. falciparum-infected mosquitoes It was also interesting to note that despite timely treatment, half of the naïve volunteers developed significant alterations in their hematological profiles, including thrombocytopenia and leukopenia, whereas only one semi-immune volunteer presented with mild leukopenia. It is known that According to FDA guidelines some laboratory test abnormalities could be classified as moderate or severe P. vivax infection three months after treatment, he reported to having been in Buenaventura, the same area where parasites used in the challenge were collected. Thus, whether this infection was a relapse or reinfection could not be confirmed.As in previous trials, parasitemias were cleared in most cases (13/15) within the first 24 hours of treatment in both groups P. vivax vaccines in volunteers previously exposed to malaria.Despite the great logistical challenges of this study, it's the potential usefulness of the mosquito challenge infection system could serve to accelerate the transition from Phase IIa to Phase IIb malaria vaccine trials. Furthermore, the model described here represents a cost- effective method for proof-of-principle malaria vaccine efficacy studies under conditions approximating Phase IIb. This phase is logistically and economically demanding as it requires well characterized endemic settings with sufficient malaria transmission to ensure that volunteers are exposed to natural mosquito challenge within a reasonable time. In addition, volunteers get exposed to undetermined genotypic/biological differences in circulating wild parasite isolates P. vivax irradiated-sporozoites. A controlled, size-limited Phase IIb vaccine study to evaluate the protective efficacy of a PvCS-based vaccine candidate is currently under development. Moreover, the model could contribute to efficacy studies on new antimalarial compounds, especially those with potential effects on P. vivax liver stages.In conclusion, the safety, reproducibility, and the narrow pre-patent window in this model would allow the use of relatively smaller-sized experimental groups to determine differences between control and immunized volunteers. The model should allow immediate testing of both pre-erythrocytic and asexual blood stages antigens in Phase II trials as well as human vaccination with Checklist S1CONSORT 2010 checklist of information to include when reporting a randomised trial.(DOC)Click here for additional data file.Table S1Screening of common infectious agents for parasite donors and recruited volunteers.(DOCX)Click here for additional data file.Table S2Sporozoite challenge summary.(DOCX)Click here for additional data file.Protocol S1Comparison of the susceptibility of naïve and pre-immune volunteers to the Infectious challenge with viable Plasmodium vivax sporozoites.(DOCX)Click here for additional data file.


Plasmodium falciparum infection in concurrent malaria-arbovirus infections in Kedougou region, southeastern Senegal.Concurrent malaria and arbovirus infections are common and represent an important public health concern in regions where both diseases are endemic. The present study investigates the genetic diversity and complexity of P. falciparum isolates of malaria and concurrent malaria-arbovirus infected patients, respectively, and followed by PCR-genotyping targeting the msp-1 (block2) and msp-2 (block3) allelic families.Parasite DNA was extracted from 60 to 27 sera samples collected from msp-1 allelic type both in malaria (94.91 %) and arbovirus-malaria (92.59 %) groups, whereas IC/3D7 was the most prevalent msp-2 allelic type in malaria (94.91 %) and arbovirus-malaria (96.29 %) groups. Frequencies of msp-1 and msp-2 allelic types were statistically comparable between the two groups  and were not associated with age. FC27 was strikingly the least prevalent in both groups and was absent in children under 5 years of age. The proportions of P. falciparum isolates from malaria-infected patients carrying the three msp-1 allelic types (67.44 %) or the two msp-2 allelic types (76.47 %) were significantly higher than those from arbovirus-malaria co-infected patients . The multiplicities of infection (MOI) were low and comparable for msp-1 (1.19 vs 1.22) and msp-2 (1.11 vs 1.10), respectively between malaria and arbovirus-malaria groups.The mean number of genotype per allelic family was comparable between the two groups. K1 was the predominant P. falciparum isolates from malaria and concurrent malaria-arbovirus infected patients in Kedougou. The MOI was low despite intense malaria transmission in Kedougou. The overall results suggest a limited or no influence of arbovirus infections on P. falciparum diversity and complexity of malaria infection.The study showed no difference in the genetic diversity between  Malaria and arboviruses infections are common arthropod-borne diseases in Africa and represent major public health problems, as both diseases have been independently associated with significant morbidity and mortality , 2. ConcPlasmodium falciparum malaria [While the interactions of malaria with parasitic infections, such as helminths, or viral infections, such as HIV, have been extensively documented –12, rela malaria , 15.Since malaria and arboviral infections often coincide geographically in the same tropical regions, the question arises whether arboviral infections modulate the immune responses towards the malaria parasite and affect the course and the complexity of malaria infection. A profound effect of arboviral infections on the immune system might be expected to influence the immune response against malaria parasites, thus affecting the course of infection and contributing to the complexity of the malaria disease.P. falciparum populations that are genetically distinct [P. falciparum merozoite surface proteins (msp-1 and msp-2) have been widely used as markers to investigate the genetic diversity, multiplicity of infection, level of malaria transmission, as well as relationship with immunity against malaria [In malaria endemic regions, studies have demonstrated that individuals can be simultaneously infected with multiple distinct –19. Suchdistinct –22. The  malaria , 24.P. falciparum isolates in concurrent malarial infection, this study investigated the genetic diversity of P. falciparum isolates from patients infected only with malaria parasites or co-infected with arboviruses and malaria parasites, in Kedougou, southeastern region of Senegal.As no study has been reported so far on the extent of the genetic diversity of A total of 101 sera from patients that have visited healthcare facilities in the Kedougou region between July 2009 and July 2013 were withdrawn from a collection established as part of a project investigating dengue infections in Kedougou region and investigated in this study. Due to the similar clinical presentation between malaria and arboviral infection, malaria diagnostic was systematically conducted to differentiate arboviral infection to malaria. During inclusion, all patients presented with acute febrile illness (AFI) which was defined as “any patient older than 1 year with a fever (temperature >38 °C) lasting for less than 2 weeks, exhibiting two or more of the following symptoms: headache, myalgia, eye pain, arthralgia, cough, nausea/vomiting, diarrhoea, jaundice, bleeding and neurological signs”. Clinical manifestations and socio-demographic data were recorded on a standardized interview form.Study objectives, benefits and risks were explained in French or local languages to all participants before inclusion. Written informed consent was obtained from adults and parents or legal guardians of children. The study was examined and approved by the Senegalese National Health Research Ethical Committee.P. falciparum malaria and arboviruses infections. Malaria diagnostic was initially performed by microscopic examination of Giemsa-stained thick blood smears and/or a rapid diagnostic test  while IgM antibodies capture ELISA, real-time RT-PCR and virus isolation methods [All 101 sera were systematically tested for  methods , 26 wereConsequently, “arbovirus confirmed case” was defined as “any AFI tested positive for any method used for detection of IgM and/or genome of ZIKV, DENV, CHIKV, RVFV, CCHFV, WNV or YFV” while malaria “confirmed case” was considered for “any AFI tested positive by RDT and/or microscopy”. Concurrent infection was defined as “any AFI confirmed for any arbovirus infection and malaria”.P. falciparum malaria patient was used as positive control.The detection of Plasmodium DNA in frozen serum samples has been reported earlier , 28. GenP. falciparum parasite DNA was based on nested PCR amplification with primers targeting the Plasmodium spp 18S small subunit ribosomal RNA gene as described by Snounou and others [P. falciparum using previously described primers pairs rFAL1 and rFAL2 [Qualitative detection of d others . The prid others , 31 and nd rFAL2 , 31 in and rFAL2 .Plasmodium falciparum isolates from malaria and concurrent arbovirus-malaria infected patients were analysed by amplification of the two highly polymorphic regions of msp-1 (block2) and msp-2 (block 3) using nested PCR as described previously [msp-1  and two families of msp-2 (FC27 and 3D7) are reported elsewhere [eviously , 33. Seqlsewhere . An initmsp family was calculated as the percentage of samples containing at least one allele from that family. The mean multiplicity of infection (MOI) or number of genotypes per infection [msp-1 or msp-2 by the number of samples positive for the same marker.The frequency of each nfection , 36 was Data were analysed using R statistical software version 3.1.1 (2014-07-10) . We perfP. falciparum and arboviruses infections. Among them, 32 were uniquely positive for P. falciparum based on RDT and/or microscopy examination  and 27 (B + C2), respectively  and sex .The sex ratios M/F were 1.40 (35/25) and 1.07 (14/13) for the malaria and arbovirus-malaria groups respectively  and two families of msp-2 (IC/3D7 and FC27) were detected in P. falciparum isolates from both malaria and arbovirus-malaria co-infected patients.Parasites DNA from all 60 msp-1 genotypes belonged to MAD20 allelic family while K1 and RO33 allelic families represented respectively 32.94 % (57/173) and 26.58 % (46/173) of the total msp-1 genotypes (Table msp-1 genotype and represented 40 % (34/85) of the overall detected msp-1 genotypes whereas K1 and RO33 allelic families represented 30.58 % (26/85) and 29.41 % (25/85), respectively  of the overall detected msp-2 genotype in both malaria and arbovirus-malaria groups with respectively 77.92 % (60/77) and 88.23 % (30/34) of the total detected msp-2 genotypes. FC27 allelic type represented only 22.07 % (17/77) and 11.76 % (4/34) of msp-2 genotypes in malaria and arbovirus-malaria groups, respectively . This suggests that the elevated number of genotypes observed in the malaria group could be due to the higher number of samples from malaria-infected patients rather than a lower genetic diversity of P. falciparum isolates from concurrent arbovirus-malaria infected patients.For a given msp-1 and IC/3D7 and FC27 allelic families of msp-2 were compared between malaria and arbovirus-malaria groups. In both groups, K1 and IC/3D7 families were respectively the predominant msp-1 and msp-2 allelic types , 78.33 vs 81.48 % for MAD20  and 73.33 vs 74.07 % for RO33   and 23.33 vs 14.81 % for FC27   was significantly higher than that from arbovirus-malaria co-infected patients (32.56 %) . A similar distribution was observed between P. falciparum isolates carrying the two msp-2 allelic types from simple malaria (76.47 %) and arbovirus-malaria (23.53 %) cases   and >15 years  showing the highest frequency  as well as the two msp-2 allelic types (IC/3D7 and FC27) were comparable within age groups.The frequency of specific ncy Fig. a. Frequemsp-2 allelic types of P. falciparum isolates from arbovirus-malaria co-infected patients according to age groups showed a slightly different pattern  was found to be 1.19 and 1.11 respectively for msp-1 and msp-2 in the malaria group .The mean multiplicity of oup Fig. a. In theely Fig. a. The diage Fig. b. The diP. falciparum highly underestimated malaria parasite carriage. Importantly, Plasmodium DNA was detected from frozen serum samples similar to previous reports [In resources-limited settings, given the limited number of laboratories diagnosing arboviral infections along with malaria infections, reports on malaria and arbovirus co-infections are scarce and their prevalence underestimated –5, 9, 38 reports , 28, proP. falciparum isolates from malaria-infected patients and concurrent arboviruses-malaria co-infected patients was investigated in Kedougou region with an ultimate goal of determining whether co-infection with an arbovirus contributed to the overall genetic diversity of P. falciparum isolates and complexity of malaria infections. Both malaria and arbovirus-malaria groups were comparable in term of age, sex and percentage of patients within defined age groups.In this study, the genetic diversity exhibited by P. falciparum isolates from malaria-infected patients. In this study, the frequency of all msp-1 allelic families  and IC/3D7 allelic family of msp-2 were high (>70 %) and comparable between the two study groups. The current parasite profile with the predominance of K1 and IC/3D7 allelic families was also reported in others studies [P. falciparum isolates carrying either the three msp-1 allelic types or the two msp-2 allelic types from malaria-infected patients compared to arbovirus-malaria co-infected patients, are probably uniquely due to elevated number of sample concerning simple infection compared to co-infection. The distribution of msp-1 and msp-2 families according to age groups was similar between malaria and arbovirus-malaria groups and no significant difference was found when comparing distribution of a given allelic family between the two groups despite the absence of FC27 family in younger children (<5 years).The comparable mean number of genotype for each locus between malaria and arbovirus-malaria groups suggested that the elevated number of genotypes observed in the malaria group could be related to the higher number of samples rather than an extended genetic diversity of  studies –43. The P. falciparum showed that samples with the highest MOI came from children with significantly lower antibody responses to specific antigens of the asexual parasite [The MOI reported in this study was not influenced by age similar to reports from others studies . This coparasite . Malariamsp-1 and msp-2 allelic families with respect to sampling period and health facilities/villages of origin are being investigated in a more extensive study using 160 P. falciparum isolates from Kedougou .Although the sera investigated in this study spanned a 5-year period and originated from different health facilities, possibly affecting the MOI as previously reported , the spaP. falciparum isolates and complexity of infection in concurrent arbovirus-malaria infections. The study revealed no influence of arboviral infection on the genetic diversity of P. falciparum isolates as well as complexity of P. falciparum malaria infections in Kedougou. The study did not find any msp-1 and/or msp-2 allelic type frequency, MOI or distribution of a given allelic type or MOI with age groups that would suggest a role of arboviruses infections on the genetic diversity of P. falciparum isolates and complexity of malaria infection in Kedougou. Other studies that examine P. falciparum population structure and, taking into account the nature of anti-malarial immune responses are needed to better document the influence of arboviruses infections on P. falciparum genetic diversity and complexity of malaria infection.This study provides the first information on the genetic diversity of


Abs) are critical for immunity to malaria. However, Plasmodium falciparum specific Abs decline rapidly in absence of reinfection, suggesting impaired immunological memory. This study determines whether residents of Sweden that were treated for malaria following international travel maintained long‐lasting malaria‐specific Abs and memory B cells (MBCs). We compared levels of malaria‐specific Abs and MBCs between 47 travelers who had been admitted with malaria at the Karolinska University Hospital between 1 and 16 years previously, eight malaria‐naïve adult Swedes without histories of travel, and 14 malaria‐immune adult Kenyans. Plasmodium falciparum‐lysate‐specific Ab levels were above naïve control levels in 30% of the travelers, whereas AMA‐1, merozoite surface protein‐142, and merozoite surface protein‐3‐specific Ab levels were similar. In contrast, 78% of travelers had IgG‐MBCs specific for at least one malaria antigen  suggesting that malaria‐specific MBCs are maintained for longer than the cognate serum Abs in the absence of re‐exposure to parasites. Five travelers maintained malaria antigen‐specific MBC responses for up to 16 years since the diagnosis of the index episode . Thus P. falciparum can induce long‐lasting MBCs, maintained for up to 16 years without reexposure.Antibodies ( Plasmodium falciparum transmission in parts of Africa, rendering formerly immune individuals or susceptible populations again There is long‐standing evidence that naturally acquired immunity to the erythrocytic stages of malaria, which limits morbidity and mortality in older children and adults, is strongly dependent on antibody  specific IgG Abs and MBCs between travelers that were treated for malaria at the Karolinska University Hospital, Stockholm, Sweden, between 1994 and 2010, malaria‐naïve Swedes living in Stockholm, and adults who lived in an endemic area of Kenya all their lives (“immune adults”).The aim of our study was to determine whether natural P. falciparum lysate were determined in 47 Swedish residents diagnosed with P. falciparum following travel in tropical countries (“travelers”). Antigen‐specific IgG MBCs and Abs were quantified using ELISpot and ELISA assays. The travelers were defined as residents of Sweden who were diagnosed and treated at Karolinska University Hospital, Stockholm, with acute malaria, as identified from the hospital records and were either born in Europe (n = 33) or in countries known to have malaria transmission (n = 14)  , MSP1‐42 kDa, merozoite surface protein‐3 (MSP3), and P. falciparum lysate‐specific Ab levels, where 30% of the travelers had levels above naive controls, all the other malaria‐specific Abs were at background levels among this group .Similarly, the proportions of Kenyan immune adults that were positive for P. falciparum specific MBC, these five that did not travel back provides the stronges tevidence for long‐term maintenance of MBCs in absence of reexposure to parasites. Each of them had maintained MBCs specific for at least one of three P. falciparum antigens tested for a median of 12 years (range 8–16) since the diagnosis and treatment of the index episode, confirming that humans can generate and maintain long‐lived P. falciparum specific MBCs in the complete absence of reexposure to parasites.We identified 17 travelers that were unlikely to have been reexopsed to malaria during the intervening period between treatment of the index episode in Sweden and this study. Twelve of these traveled to countries with extremely low transmission in Asia, while five  did not travel at all  with MBCs to at least two P. falciparum antigens, and five of the nine (55.6%) with MBCs to all three P. falciparum antigens had Abs to P. falciparum lysate, respectively.Interestingly, only one of the 11 travelers with no MBCs to any of the te Table . In contP. falciparum specific MBC responses were associated with the time since malaria diagnosis, being born in an endemic country, parasitemia at diagnosis, and previous episodes .In multivariable linear regression analysis, none of the es Table . Age at The levels of anti‐TT Abs were similar across the three groups Fig. . HoweverP. falciparum infection followed by subsequent prolonged periods of nonexposure to malaria antigen resulted in the expansions and maintenance of these atypical MBCs as well as other B‐cell subsets by comparing their relative proportions in the peripheral bloods of naive controls, travelers, and immune adults. B‐cell subsets were defined as described elsewhere +CD27−CD21+CD10−; plasma cells, CD19+CD27+CD21−CD20−; immature B cells, CD19+CD10+; classical MBC, CD19+CD27+CD21+CD10‐; atypical MBC, CD19+CD27−CD21−CD10−; and activated MBC, CD19+CD27+CD21−CD20+CD10−. Figure We next determined whether exposure to The immune adults had an expanded atypical MBC population relative to the naive controls and the travelers. There were, however, no differences in the proportions of atypical MBCs between the travelers and the naive controls Fig. A. SimilaThere were no significant differences between the travelers and either of the naive controls or immune adults for activated B cells, classical MBCs, plasma cells, immature and naïve B cells . In addition, we found similar frequencies of atypical MBC between exposed travelers and naïve controls, confirming that persistent and/or chronic P. falciparum exposure is required for their maintenance We found that travelers returning to Sweden with acute malaria generated and maintained P. falciparum antigens, especially in young children  in the complete absence of persisting exposure are reassuring in the face of reports of declining P. falciparum transmission in Africa P. falciparum antigens is not as short‐lived as previously presumed. Nonetheless, future studies should investigate why not all the travelers in the current study, or the individuals in previous studies P. falciparum specific MBC, in spite of clear evidence for prior exposure.Although 17 of the travelers had only traveled back to countries with extremely low transmission in Asia and had maintained long‐lived P. falciparum specific MBCs in the absence of reexposure is consistent with a previous report suggesting that naturally acquired immunity to malaria could persist for several years of nonexposure P. falciparumP. falciparum specific MBCs is yet to be demonstrated in prospective studies. Although we reported a lack of prospective association with protection for anti‐MSP1 and AMA1 IgG MBCs in a recent study The finding of long‐lived P. falciparum specific Abs in endemic areas, P. falciparum specific MBCs can be maintained independently of sustained exposure to the parasite. This finding has major implications on future immunoepidemiological studies. Such studies would gain more information on immunological memory, or previous antigenic exposures by combining serological analysis of antigen‐specific Abs with quantification of their cognate MBCs. Otherwise, studies investigating humoral responses to P. falciparum antigens in areas of declining P. falciparum transmission by serology alone would greatly underrepresent past P. falciparum specific Ab responses. Having shown that P. falciparum specific MBCs can be long‐lived in the absence of reexposure (in agreement with recent studies), future studies should now investigate the possible protective role for P. falciparum specific MBCs in prospective studies of individuals exposed to endemic malaria. Such studies will help identify protective long‐lived IgG MBC specificities that can then be targeted for induction by vaccination.In summary, we conclude that although persistent exposure to malaria antigen is required for the maintenance of circulating Ethical approval was obtained from the Central Ethical Review Board, Stockholm, and the Kenyan Medical Research Institute (KEMRI) National Ethics Committee. Written informed consent was subsequently obtained from the respective participants.The study was done at the Karolinska University Hospital in Stockholm. The hospital serves ∼ 2 million people living in Stockholm. The Department of Infectious Diseases treats up to 50 cases of malaria in returning travelers per year. In addition, samples from malaria immune adults were obtained from the KEMRI, Centre for Geographic Medicine Research Coast situated at Kilifi District Hospital, Kenya. The hospital serves ∼ 240 000 people living in Kilifi District. The immune adults were life‐long residents of Junju location, which remains stably endemic with two high‑transmission seasons .P. falciparum malaria episode to Karolinska University Hospital between 1994 and 2010 were invited to participate in a cross‐sectional study of malaria‐specific B‐cell memory in May 2011. An invitation letter was sent out to 270 patients along with a description and aim of the study. Out of 270 invitees, 47 individuals consented to participate and came to a visit at the hospital for blood sampling and to fill out a questionnaire. Additionally, for comparison we recruited eight malaria‐naïve Swedish born adults and 14 malaria immune adults from the Kenyan coast , MSP1 42 kDa, and merozoite surface protein‐3 (MSP3) to which circulating IgG Abs have been associated with clinical protection in previous studies P. falciparum antigens were provided by Dr. Louis Miller , while P. falciparum lysate, was made from the ITO parent strain sonicated on ice P. falciparum and TT antigens using a standard ELISA protocol, as described elsewhere P. falciparum lysate and the accompanying red blood cell control lysate at 1 in 500 dilution in bicarbonate buffer (100 μL/well). A 100 μL/well of 1 in 1000 dilution of test plasma in 0.3% phosphate‐buffered saline (PBST) +EDTA was added after plates had been washed three times with 0.05% Tween in PBST, and thereafter blocked with 10% fetal calf serum (FCS)/PBS (200 μL/well). Plates with test plasma were then incubated for 1.5 h at room temperature in a humidified chamber and then washed five times before the addition of alkaline phosphatase (AP) labeled goat anti‐human IgG Ab (Sigma) conjugate at 1:2000 dilution 0.05% PBST at 100 μL/well. After 1‐h incubation with the conjugate, the plates were washed five times and the human IgG complexed with the AP labeled conjugate revealed with p‐nitrophenyl phosphate (Sigma). The substrate reaction was stopped with 50 μL per well of 3M NaOH, after which the plates were left for 5 min in the dark before being read at 405/570 nm. Purified hyperimmune IgG was used as a standard for the P. falciparum specific ELISAs. Anti‐TT IgG Abs were quantified against hyperimmune plasma from an adult, who received booster immunizations with the TT vaccine prior to this study. Ab concentrations were expressed in arbitrary units determined against the respective standard curves on each plate.Plasma samples were tested in duplicates for human IgG Abs specific for Plasmodium falciparum specific MBCs were quantified by a recently optimized ELISpot assay 6cells/mL of RPMI complete media with 2.5 μg/mL CpG oligodeoxynucleotide‐2006 (Eurofins MWG/Operon); 1/10 000 dilution SAC, 1/100 000 dilution pokeweed mitogen (Sigma‐Aldrich); and 50 ng/mL of IL10 (R&D Systems) in 24‐well plates. The 96‐well ELISpot plates (Millipore Multiscreen‐HA) were precoated by incubating plates overnight at 4°C with either: 10 μg/mL polyclonal goat Ab specific for human IgG  to detect all IgG‐secreting cells; 1% BSA as a nonspecific protein control or 5 μg/mL of tetanus toxoid (TT), MSP1 or AMA1 in PBS. Plates were blocked by incubation with a solution of 10% FCS in RPMI for 2 h at 37°C. PBMCs from 5‐day cultures were serially diluted by a factor of a ½ in duplicates or triplicates (depending on the numbers of cultured PBMC available) to final concentrations of between 0.125 × 104 and 1 × 104 PBMC/well to detect total IgG+ ASCs, and between 0.062 × 105 and 2 × 105 PBMC per well to detect antigen‐specific ASCs. ELISpot plates were kept at 37°C in a 5% CO2 incubator for 5 h and then washed four times with PBS and four times with PBS‐0.05% Tween‐20. AP‐conjugated goat anti‐human IgG Fc Ab (Jackson ImmunoResearch Laboratories) diluted 1:1000 in PBS‐0.05% Tween‐20 with 1% FCS was added to wells and incubated overnight at 4°C. Plates were washed four times with PBS‐0.05% Tween‐20, four times with PBS, and three times with distilled water before adding BCIP/NBT 100 μL per well (Bio‐Rad). The plates were dried in the dark and spots scanned with the ImmunoSpot series 4 analyzer (Cellular Technologies LTD (CTL), Germany) and results analyzed using Immunospot version 5 software . We determined the threshold criteria for positivity at eight spots per 1 × 106 PBMCs, based on the upper range of the frequency obtained for 1% BSA coating.Briefly, PBMCs were stained with a panel of monoclonal Abs as described elsewhere p‐values were considered significant if p < 0.05.Log transformed Ab and MBC data were analyzed using Stata version 11  and GraphPad Prism for Macintosh version 5.01 . Multivariable linear regression analysis was used to test the predictive value for origin of the travelers , time since malaria diagnosis, age at admission with malaria, parasitemia at diagnosis, previous episodes, and duration of symptoms before curation. Kruskal–Wallis test (with Dunn's correction for multiple comparisons) was used to compare continuous variables between groups. For all tests, two‐tailed The authors declare no financial or commercial conflict of interest. This paper is published with permission from the Director of KEMRI.MBCmemory B cellAMA1apical merozoite antigen‐1MSP1merozoite surface protein‐1MSP3merozoite surface protein‐3PBSTphospahte buffered saline/tweenAs a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Table S1. Characteristics of study participantsFigure S1. Total IgG MBC frequencies in the travelers are similar to those in the naïve and immune adult controls.Click here for additional data file.Peer review correspondenceClick here for additional data file.


Plasmodium falciparum in vitro culture samples.Current available malaria diagnostic methods each have some limitations to meet the need for real-time and large-scale screening of asymptomatic and low density malaria infection at community level. It was proposed that malaria parasite-specific low molecular-weight metabolites could be used as biomarkers for the development of a malaria diagnostic tool aimed to address this diagnostic challenge. In this study, high resolution metabolomics (HRM) was employed to identify malaria parasite-specific metabolites in in vitro 48-hour time-course asynchronized culture system of P. falciparum. Liquid chromatography coupled with high resolution mass spectrometry was applied to discover potential parasite-specific metabolites in the cell culture supernatant. A metabolome-wide association study was performed to extract metabolites using Manhattan plot with false discovery rate (FDR) and hierarchical cluster analysis. The significant metabolites based on FDR cutoff were annotated using Metlin database. Standard curves were created using corresponding chemical compounds to accurately quantify potential Plasmodium-specific metabolites in culture supernatants.Supernatants were collected at 12 hours interval from 3% haematocrit Plasmodium infected culture based on Manhattan plot with FDR q=0.05. A two way hierarchical cluster analysis showed a clear segregation of the metabolic profile of parasite infected supernatant from non-infected supernatant at four time points during the 48 hour culture. Among the 1025 annotated metabolites, the intensities of four molecules were significantly increased with culture time suggesting a positive association between the quantity of these molecules and level of parasitaemia: i) 3-methylindole, a mosquito attractant, ii) succinylacetone, a haem biosynthesis inhibitor, iii) S-methyl-L-thiocitrulline, a nitric oxide synthase inhibitor, and iv) O-arachidonoyl glycidol, a fatty acid amide hydrolase inhibitor, The highest concentrations of 3-methylindole and succinylacetone were 178 ± 18.7 pmoles at 36 hours and 157±30.5 pmoles at 48 hours respectively in parasite infected supernatant.The number of significant metabolite features was 1025 in the supernatant of the in vitro culture supernatants. Further study in malaria infected human is needed to determine presence of the molecules and its relationship with parasite densities.HRM with bioinformatics identified four potential parasite-specific metabolite biomarkers using The online version of this article (doi:10.1186/s12936-015-0651-1) contains supplementary material, which is available to authorized users. Plasmodium falciparum is responsible for most malaria-attributed morbidity and mortality. Over the past decade, successful scale-up of malaria control has resulted in substantial reductions in malaria cases and deaths [Among four species of human malaria parasites, d deaths ,2. As mad deaths ,4. Currein vitro 48-hour time-course culture system of P. falciparum using high-resolution metabolomics (HRM) [et al. identified the potential mechanism of cerebral malaria pathogenesis which was associated with the depletion of arginine [et al. identified two urinary metabolites in Plasmodium berghei infected mice, while a study by Sengupta et al. suggested urinary ornithine seems to have the potential as biomarkers of Plasmodium vivax malaria [P. vivax malaria respectively, their analytical approaches could be used in mining for biomarkers of P. falciparum malaria infection. A study by Teng et al. showed strain-specific differences in a range of metabolites in erythrocytes infected with P. falciparum from in vitro culture and further highlighted the variation in levels of choline and phosphocholine among the strains [et al. investigated global mass spectrometry-based metabolomic profiling between in vitro P. falciparum infected and uninfected erythrocytes [As the first step for proof of concept, this study was designed to identify parasite specific low molecular-weight metabolites in an cs (HRM) -7. Earli malaria ,10. Alth strains . In addihrocytes . They deP. falciparum specific waste products, low molecular-weight metabolites, in the supernatant from the erythrocyte culture system. The rationale for profiling low molecular-weight metabolites in culture supernatants, first, was based on the hypothesis that parasite-specific small molecular wastes could be secreted into urine, saliva or sweat at high concentrations in malaria infected human and the ultimate goal is to use the small molecules as potential biomarkers for development of non-invasive and sensitive malaria diagnostic tools. This study demonstrated HRM could achieve a relatively comprehensive and quantitative analysis of Plasmodium-specific metabolites in supernatant from parasite infected culture system, and explores low molecular-weight biomarkers [Plasmodium.In contrast to the above reports, this study was designed to explore omarkers ,13 assocP. falciparum was used. The asynchronized blood stage parasites were cultured as described [In this study, asynchronized culture was employed. The purpose of using asynchronized culture was to capture small metabolite molecules that might be commonly released by all stages of parasites. The laboratory-adapted 3D7 strain of escribed  in RPMI Plasmodium metabolic pathways. Examination of m/z of metabolites in the KEGG [Plasmodium metabolomics pathways shows that less than 10% of metabolites are redundant with others in terms of elemental composition [All the samples were run in duplicate. One hundred μl aliquots of supernatant samples were treated with acetonitrile , spiked with 2.5 μl internal standard mix, and centrifuged at 14,000 × g for 5 minutes at 4°C to remove protein as described previously . Then LTthe KEGG ,19 humanposition ,19. Idenposition ,21. DireAnalyses were performed based on the results from both biological and technical replicates. Total features of culture supernatant were collected after processing mass spectral data with apLCMS. The features from duplicate LCMS analyses were averaged, log2 transformed and quantile normalized for subsequent statistical and bioinformatics analyses including univariate analysis, Manhattan plot, and false discovery rate (FDR)  to deterPlasmodium intermediary metabolites are shown in black dots in pathway maps; most human and known Plasmodium metabolic pathways are represented.The database of Kyoto Encyclopedia of Genes and Genomes (KEGG) was utilized to map the features distribution on both human and plasmodium metabolic pathways. Detected m/z features matching known human and 2>0.99. 3-methylindole and succinylacetone in supernatants from parasite culture were treated using 100 μl in microcentrifuge tubes. These tubes were centrifuged at 14,000× g at 4 degrees Celsius for 5 minutes. 10 μl of the supernatant was injected in mass spectrometry and resulting areas were noted. Concentrations of the compounds were calculated using the standard curve.3-methylindole (M51458) and succinylacetone (D1415) were purchased from Sigma-Aldrich . A standard curve was made by known concentrations (0.1-0.5nmole) of the reagent grade compounds in cell culture media. Areas from the samples were plotted against concentrations and a standard curve with an rPlasmodium-infected cultures at all points . A Manhattan plot  determined by FDR, the ion intensities of several metabolites were increased with culture time in infected culture supernatants but not in non-infected culture supernatants, suggesting a positive association between the quantity of these molecules released and level of parasitaemia. These metabolites were: i) 3-methylindole  were found to be potential biomarkers from the parasite during the erythrocytic stage culture system. This was based on the fact that the ion intensities of the four molecules were increased with culture time, suggesting a positive association between relative quantity of these molecules and level of parasitaemia. In addition, 3-methylindole and succinylacetone molecules in the supernatant samples from 3% haematocrit 48 hour time-course cultures were further quantified. The concentration of succinylacetone peaked at 48 hours compared to 36 hours for 3-methylindole. As 3-methylindole is volatile molecule, it is possible that 3-methylindole was evaporated due to extended culture to 48 hours. Importantly, 3-methylindole has been shown to stimulate an odorant receptor to attract malaria mosquito vector [Use of HRM is a uniquely good approach to identify putative biomarkers for a complex disease like malaria since fe cycle ,34. This systems . Furthero vector , potentio vector . A studyP. falciparum infected people from Africa is underway. The ongoing study will provide information on concentration and stability of the molecules in malaria infected people and their association with parasite densities.Another potential biomarker identified was succinylacetone which is known to inhibit haem biosynthesis (delta-aminolevulinate dehydrolase inhibitor) . SuccinyPlasmodium sp. to develop blood stage parasites [An apparent disappearance of two amino acids, arginine and isoleucine, was observed in the 48 hr culture system. Several important host-parasite interaction studies elucidated a mechanism that isoleucine was taken by arasites ,39. Malaarasites . IsoleucIn summary, HRM coupled with network and pathway analysis using the significant metabolites from culture supernatants of infected erythrocytes and incorporating the broader human and malaria parasite metabolomic knowledge identified four potential parasite-specific biomarkers. The findings from the current study may provide improved opportunities for innovative prevention and management programmes such as development of new malaria diagnostic tools.


Plasmodium falciparum. Among 5796 individuals aged greater than six months, PCR prevalence of malaria infection was 5%, 10%, and 20% in dry, and 9%, 15%, and 32% in rainy seasons in Blantyre, Thyolo, and Chikhwawa, respectively. Over 88% of those infected were asymptomatic. Participants aged 6–15 years were at higher risk of infection  and asymptomatic infection  than younger children in all settings. School-age children used bednets less frequently than other age groups. Compared to young children, school-age children were brought less often for treatment and more often to unreliable treatment sources. Conclusion: School-age children represent an underappreciated reservoir of malaria infection and have less exposure to antimalarial interventions. Malaria control and elimination strategies may need to expand to include this age group.Malaria surveillance and interventions in endemic countries often target young children at highest risk of malaria morbidity and mortality. We aimed to determine whether school-age children and adults not captured in surveillance serve as a reservoir for malaria infection and may contribute to malaria transmission. Cross-sectional surveys were conducted in one rainy and one dry season in southern Malawi. Demographic and health information was collected for all household members. Blood samples were obtained for microscopic and PCR identification of  Plasmodium infections, who are less often symptomatic, may play an important role in continued transmission. These groups have not traditionally been the focus of intensive detection and control strategies. To achieve malaria control and ultimately elimination, it may be important to identify and characterize these potential reservoirs of infection and transmission.Despite widespread expansion of malaria control measures in the past decade, malaria remains a leading cause of morbidity and mortality, with children under five years of age and pregnant women most severely affected in Africa . While sOlder children and adults may serve as transmission reservoirs because acquired antimalarial immunity leads to persistent asymptomatic or minimally symptomatic infections that are less likely to be treated with antimalarial drugs than acute febrile illnesses that usually occur in young children . AsymptoThe epidemiological importance of older age groups may increase as elimination efforts intensify and transmission patterns change . For exaTo better characterize infections, utilization of interventions, and health care seeking behaviors in all age groups, the Malawi International Center of Excellence for Malaria Research (ICEMR) has undertaken biannual community-based household surveillance in low and high transmission settings and seasons, using both microscopic and molecular malaria diagnostics. We hypothesized that older children not routinely included in malaria surveillance represent a reservoir of asymptomatic infections, and that this age-group has limited access to prevention and treatment interventions.Cross-sectional household surveys were conducted at the end of the dry season 2012 (September-October) and end of the rainy season 2013 (April-May), when malaria transmission is expected to be at its lowest and highest, respectively. Three districts in southern Malawi were chosen to represent the range of malaria transmission settings. Blantyre city is an urban highland setting with expected low transmission; Thyolo is a rural highland area with expected low to moderate transmission; and Chikhwawa is low-lying and rural with high transmission.One compact segment containing 30 households in each of 10 enumeration areas (EAs) per district was selected using two-stage cluster sampling [Prior permission to survey each village was provided by village leaders. Written informed consent and assent were obtained for all adults and children, as appropriate. Questionnaires were administered in the local dialect. The study received ethical approval from University of Maryland Baltimore and Michigan State University Institutional Review Boards and University of Malawi College of Medicine Research and Ethics Committee.Household members were defined as individuals who slept in the house for at least two weeks of the past month. Interview data about any household member (present or absent) was collected from a consenting adult household member; specimens were collected from those present at the time of survey.http://opendatakit.org) and managed using electronic data capture [Questionnaires were adapted from standardized MIS tools. Data were collected on android-based tablets using OpenDataKit  or report of fever or rigors within the past 48 hours. Submicroscopic malaria infection was defined as malaria infection identified by PCR from individuals with malaria slides negative by microscopy. Review of at least 100 microscopic fields was required before a blood smear was determined negative for parasites.Malaria infection and intervention prevalence estimates by district, season and age group were compared using Pearson chi-square tests or Fisher’s exact test. Heterogeneity of prevalence across EA level within each district was assessed using logistic regression with likelihood-ratio tests. District comparisons for continuous variables were made using t-tests or Wilcoxon rank-sum tests, as appropriate. Individual counts per household and net were estimated using overdispersed Poisson regression.th percentile EAs); medium (25-75th percentile EAs); or high (>75th percentile EAs). District and season were forced into all models, and best subset regression was used to select final models based on p-value <0.05 or substantial change in the coefficients of variables included in models. Potential interactions were evaluated for each model. Since community IRS campaigns occurred in Chikhwawa district only after the 2012 rainy season, a Chikhwawa-specific model was fit to assess the effect of IRS on infection.Predictors of parasitemia and access to malaria interventions were identified using multivariate mixed models, with random intercept to account for hierarchical clustering by households and EA. Parasitemia outcomes modeled were: PCR-detected infection, asymptomatic infection among all infections, and submicroscopic infection among all infections. Intervention outcomes modeled were: net use among all individuals, treatment seeking among individuals with history of fever within two weeks, and source of treatment  among those with a history of seeking treatment. The primary predictor variable of interest was age group. To adjust for community-level malaria prevalence, we used data from the prior rainy season 2012 to create EA prevalence categories: low .During the two surveys, 1,833 households  and 7,653 individuals  were surveyed. Age distributions were similar across districts . HousehoMolecular results were obtained from 5,796 individuals. The prevalence of malaria infection in Blantyre, Thyolo, and Chikhwawa was 5%, 10%, and 20% in dry, and 9%, 15%, and 32% in rainy seasons. Rainy season prevalence was significantly higher than dry season prevalence in all districts . ParasitOverall, rainy season parasite prevalence was 14% among young children, 31% among school-age children, and 14% among adults (P<0.001). While multiple factors were associated with infection in bivariate analysis, multivariable analysis in all three districts, controlling for season and EA-level prevalence, revealed that school-age children had 4.8  fold increased odds of infection compared to both younger children and adults . Factorsage-district-interaction = 0.001), and were not observed in Blantyre. Males had twice the odds of asymptomatic infection as females . The association of age and likelihood of asymptomatic infection among those infected was not affected by other socioeconomic variables or net use.Almost all infected individuals were asymptomatic; the proportion of asymptomatic infections among infected individuals was similar across seasons (P = 0.88) and districts (P = 0.59) . Among mIn both crude and adjusted analyses, submicroscopic infections represented the majority of infections in the dry (55%), but not in the rainy season . In the rainy season, the proportions of submicroscopic infections were comparable between school-aged and younger children, but in the dry season, school-age children were more likely to have submicroscopic infection than younger children . In adults, most infections were submicroscopic in both dry and rainy seasons . SubmicrThe reported prevalence of sleeping under a net the previous night was 67%, 64%, and 76%, in the rainy season and 61%, 49%, and 63% in the dry season in Blantyre, Thyolo, and Chikhwawa, respectively. Prevalence of household net ownership and individual net use during the rainy season are shown by district in Overall, 22% of individuals had a history of fever within the previous two weeks; of these 18% reported seeking treatment . Across There were significant differences in malaria treatment seeking by age: school-age children were brought for any treatment less frequently (P<0.001) and were less likely to be brought for treatment within 48 hours of onset of fever than younger children (P = 0.008, In this large cross-sectional study, school-age children had the highest prevalence of malaria infection and were not likely to report associated symptoms. School-age children were also more likely to be submicroscopic carriers in the dry season, and to have lower use of both nets and treatment. These findings were consistent across settings and seasons despite significantly different infection prevalence by setting and season.This is the first study to link community prevalence of malaria among school-age children to decreased access to malaria interventions. The results are consistent with results from more limited prior studies ,15–17, bIn Malawi, as in other endemic regions, school-age children sleep under nets less often than any other age group. A study examining national survey data from 18 African malaria-endemic countries identified net use patterns similar to those seen in our study: mothers slept under nets with their young children, while other household members slept under nets less frequently [The semi-immune status of older children and adults spares them from the clinical symptoms that commonly prompt care-seeking. The fact that older children often appear less sick may also lead caregivers to seek care from a nearby shop rather than traveling to a formal government or private health center. Despite free access to government health services in Malawi, we found that local shops were used by nearly half of the population with fever, and shops were more likely used when treatment was sought early. Local shops are a common source of care for febrile individuals in endemic countries ,20, and Beyond lack of access to prevention and treatment, entomologic factors such as predominance of outdoor biting vectors may be related to the observed elevated malaria prevalence in school-age children and adults . Older cLogistical constraints may have introduced non-response and recall biases to our results, but should not have impacted the validity of our main conclusions. Because our cross-sectional survey teams worked during the day, school-age children and adults at work were more likely to be absent than younger children and mothers. The survey team made particular efforts to capture household members by alerting communities of the planned survey date in advance and waiting at the site for school-age children and working adults who returned at the end of the day. The proportion of school-age children who did and did not provide a blood specimen was similar. Adult men appear to have been consistently under-represented; if they were at higher risk of malaria infection, we may have underestimated the prevalence of malaria infection in this age group. Recall bias may have resulted in an underestimate of the proportion of asymptomatic malaria in younger children. A household head was asked about use of bednets, recent symptoms and treatment seeking for all household members; therefore, symptoms may have been identified more readily in young children, who are more likely to be at home with a parent all of the time. The definition of asymptomatic malaria based on lack of fever and rigors is specific. Exclusion of additional malaria-related symptoms would have made the definition of asymptomatic more sensitive; however, detailed symptom history was not available. Many investigators have noted the complexities of defining asymptomatic malaria infection, especially in high transmission settings ,23. In tThis study highlights the importance of including school-age children and adults in malaria surveillance activities, given their contribution to the pool of asymptomatic reservoirs of infection and potentially of transmission. Affordable and sensitive diagnostic methods are needed to detect low levels of infection. In addition, control measures directed toward high-transmission areas defined by political boundaries rather than environmental or socioeconomic risk factors may not capture hot spots for malaria infection. Appropriate surveillance and intervention strategies should be implemented to identify all infected individuals and capture the heterogeneity of malaria transmission within close geographic proximity.There is urgent need to target school-age children for malaria prevention efforts. Educational efforts may be highly effective in promoting bednet use and improving health-seeking behavior in this high prevalence population to maximize utilization of existing resources. With high prevalence of submicroscopic infection identified in this study and lack of a point-of-care test to reliably detect low density infections, mass drug administration programs may prove more useful than screening and treatment approaches for a school- or community-based intervention targeting all infected individuals.School-age children are a key reservoir for malaria infection and this population should be included in surveillance and control activities in all transmission settings and seasons in malaria-endemic countries. Prevention and treatment programs are utilized less commonly in school-age compared to younger children; novel interventions and strategies to target this group are needed.


With low and markedly seasonal malaria transmission, increasingly sensitive tools for better stratifying the risk of infection and targeting control interventions are needed. A cross-sectional survey to characterize the current malaria transmission patterns, identify hotspots, and detect recent changes using parasitological and serological measures was conducted in three sites of the Peruvian Amazon.P. vivax  and P. falciparum  antigens by ELISA. Risk factors for malaria infection (positive PCR) and malaria exposure (seropositivity) were assessed by multivariate survey logistic regression models. Age-specific seroprevalence was analyzed using a reversible catalytic conversion model based on maximum likelihood for generating seroconversion rates . SaTScan was used to detect spatial clusters of serology-positive individuals within each site.After full census of the study population, 651 participants were interviewed, clinically examined and had a blood sample taken for the detection of malaria parasites (microscopy and PCR) and antibodies against P. vivax and 6.7% for P. falciparum, while the seroprevalence was substantially higher, 33.6% for P. vivax and 22.0% for P. falciparum, with major differences between study sites. Age and location (site) were significantly associated with P. vivax exposure; while location, age and outdoor occupation were associated with P. falciparum exposure. P. falciparum seroprevalence curves showed a stable transmission throughout time, while for P. vivax transmission was better described by a model with two SCRs. The spatial analysis identified well-defined clusters of P. falciparum seropositive individuals in two sites, while it detected only a very small cluster of P. vivax exposure.The overall parasite prevalence by PCR was low, i.e. 3.9% for The use of a single parasitological and serological malaria survey has proven to be an efficient and accurate method to characterize the species specific heterogeneity in malaria transmission at micro-geographical level as well as to identify recent changes in transmission. P. falciparum) in 2005 to 11,604 cases  in 2010, [Despite several decades of intense control efforts, malaria remains an important public health problem in Peru , mainly Although Loreto is considered as a low endemic area , previouCross-sectional surveys using serological markers are increasingly proposed as useful alternatives for estimating MTI in low transmission settings, especially for the identification of malaria hotspots and related risk factors –19. Age-Following the recent changes in malaria control activities in the Peruvian Amazon region, we conducted a cross-sectional survey combining molecular and serological markers with the aim of characterizing the current malaria transmission patterns, detecting hotspots, and identifying recent changes in species-specific MTI in three sites around Iquitos city.nd and 48th km. Several fish ponds are found along this section of the road. Unlike site A, communities in sites B and C have an important proportion of semi-closed wooden houses and no electricity. The study population was predominantly mestizos—referring to all Peruvians that cannot be clearly identified as belonging to any ethnic minority population- living mainly from small-scale agriculture, fishing, and small commercial activities.The study was conducted in three different sites in San Juan (south of Iquitos city) , one of Anopheles darlingi is the main malaria vector and is highly anthropophilic [P. vivax and P. falciparum infections at district level occur at a ratio 5:1 and all age groups are at risk of infection, though adults more than children. Malaria surveillance relies on passive case detection (PCD) with microscopy. Patients presenting with fever or any other symptoms compatible with malaria are systematically tested by microscopy at health facilities and treated with chloroquine (CQ) for 3 days , plus primaquine (PQ) for 7 days (0.5 mg/kg/day) if P. vivax malaria is confirmed, or with mefloquine (MQ) (12.5 mg/kg/day for 2 days) plus artesunate (AS) (4 mg/kg/day for 3 days) if P. falciparum malaria is confirmed. These treatment guidelines are in place since 2001, and all health facilities should perform directly observed therapy (DOT).The climate is tropical, warm and humid with a rainy season from November to May and a dry season from June to October. The annual average temperature is around 27°C, the relative humidity above 80%, and an average annual rainfall of 4 meters . Malariapophilic . P. vivaA complete census of the study population with collection of information on socio-demographic variables  and malaria preventive measures (bed net use), was conducted two weeks prior to the survey. Each house was identified with a unique number and geo-referenced using a handheld Global Positioning System (GPS) device .The survey was done in November 2012. Each household was visited and all available children between six months and seven years old, plus one randomly selected individual above 7, were enrolled after providing written informed consent/assent. Whenever selected individuals were absent, the household was revisited within the next two days to maximize subject participation. Each participant had the axillary temperature taken, malaria symptoms were recorded, and a finger-prick blood sample collected for immediate microscopy (thick and thin blood smears) and on filter paper  for later serological and molecular tests. Filter paper dried blood samples were individually stored at 4°C with desiccant until processed at Institute of Tropical Medicine Alexander von Humboldt, Lima (ITM-AvH). All individuals with a malaria infection detected by LM were treated according to the national guidelines.Thick and thin smears were stained for 10 minutes with a 10% Giemsa solution, and parasite density was computed after counting the number of asexual parasites for 200 white blood cells (WBC) in the thick smear and assuming a concentration of 8,000 WBCs/μl. Slides were read during the survey and then a day later by an expert at microscopy at the reference laboratory in Iquitos. A slide was declared negative if no malaria parasite was found after examining 100 fields. Quality control was done blindly on all positive slides and 10% of randomly chosen negative slides by a senior technician at ITM-AvH. Any discordant results were reread by a second senior technician until agreement.P. falciparum and P. vivax specific primers.Parasite DNA was extracted using the Saponin lysis Chelex 100 method  and storP. vivax merozoite surface protein-1 [P. falciparum glutamate-rich protein-fragment R2 (PfGLURP-R2) [P. vivax and five P. falciparum infected patients, and from five control individuals without recent (negative PCR) and past history of malaria infection were diluted at 1:400 in PBS-Blotto, as positive and negative control respectively. Goat anti-human IgG (H+L) specific peroxidase  diluted to 1:20,000 in PBS-Tween was used as conjugate and incubated for 1 h before development of the ELISA using 200μl ABTS substrate-chromogen solution. Optical Density (OD) values at 405 nm , were corrected by subtracting the mean OD of the antigen negative control wells from the mean OD of the corresponding antigen containing wells. Analyses with duplicate OD values that differed by more than 1.5-fold were rejected and rerun. To ensure a standardization of the sample results across ELISA plates, the percent positivity (PP) of each specimen was calculated using the OD of the positive control serum as 100%. Quality control was done blindly at ITM-AvH on 5% of randomly chosen samples. The criterion for positivity was determined for each antigen of each species applying a mixture model to the PP data which assumed two inherent Gaussian distributions: a narrow distribution or seronegatives, and a broader distribution of seropositives. Cut-off value was calculated as the mean plus 3 standard deviations of the narrow distribution [Antibodies for specific IgG responses to MSP-119)  and apicMSP-119) , and to LURP-R2)  and apicLURP-R2)  were detLURP-R2) . Accordiribution .Survey data were double entered and cross-checked in Excel , and data analysis was performed with R v.2.15 software . Baseline characteristics between sites were compared using the Chi-squared test. Species-specific parasite prevalence (by LM and by PCR) as well as seroprevalence were estimated overall and by site.Uni- and multivariate adjusted analyses were performed using survey logistic regression to determine risk factors for species-specific malaria outcomes , adjusting for the following potential confounders: site, age, gender, out-door occupation, predominant material in walls and roof, availability of electricity, and past history of malaria. Malaria infection was defined as an individual with a positive PCR result, regardless of symptoms; while malaria exposure to any of the two species was defined as an individual with a positive serology to any of the two antigens for each species. Factors with p-values <0.1 for the likelihood ratio test in the univariate analysis were considered for inclusion in the multivariate adjusted model. Using the manual backward methods, final models retained all factors that were significantly associated with the malaria outcome (p <0.05). Interactions were systematically checked for up to order two. All analyses took into account the following survey design characteristics: community as strata, household as primary sampling unit, and corresponding sampling weights (w) for children <7 years (w = 1) and for individuals ≥7 years .The QGIS software QGIS v.2.6  was used to map all surveyed individuals  and the a) a seroconversion rate  or force of infection (FOI) defined as the annual rate at which individuals change from seronegative to seropositive); and b) a seroreversion rate  defined as the annual rate at which seropositive individuals revert to a seronegative state. The main assumptions of the model are the following [Seroprevalence was stratified into yearly age-decile groups and then analyzed using a reverse catalytic conversion model  which alollowing : all indSince study communities were established in the early 1980s, the first analysis excluded individuals older than 30 years old  in order to comply with the first assumption mentioned above. The dataset of all sites was first analyzed using a reversible catalytic model that assumed a stable transmission throughout time estimating SCR and SRR for the study population up to age 30 years. Then, the specific data set from each site was analyzed using two reversible catalytic models, one that assumed a constant-SCR over time and another that assumed a change in SCR at a given time point. All these models included the population SRR as common parameter. Profile likelihood plots were applied to determine the most likely time point for change in the transmission prior to survey , which wP. vivax (APIv) and P. falciparum (APIf) per site in the year of the survey.Additionally, routine surveillance data, namely reported clinical malaria episodes through PCD provided by the Regional Direction of Health Loreto, were used to explore the association between malaria incidence trends in the department (between 1990 and 2013), [Ethical clearance was obtained from the Ethics Review Board of the Universidad Peruana Cayetano Heredia, Lima, Peru (SIDISI code: 059745). Permissions were received from health and local authorities after explaining the purpose and procedures of the study. Signed informed consent was obtained prior to participation and blood sampling by all adults and the parents of all participating children <18 years. Besides their parent's consent, children older than 7 years provided a signed informed assent.A total of 348 houses and 1,649 individuals were identified during the census, with the following site specific distribution: Site A = 129 houses (H) and 655 individuals (Ind); Site B = 80 H and 377 Ind; Site C = 139 H and 617 Ind. The overall ratio of female to male was 1.04; half of the population was less than 15 years old. The survey enrolled 651 individuals: 304 (72.4%) of the total 420 censored children between 6 months and 7 years, and 347 (31.2%) individuals of the total individuals ≥ 7 years. P. vivax and 10 P. falciparum infections), and by PCR 10.9%  (vs 1.1%), while in site B P. falciparum was predominant (11.2%) over P. vivax (2.1%). The species specific seroprevalence showed similar heterogeneity between sites. P. vivax seroprevalence was equally high in sites B (42.3%) and C (46.7%), and relatively low in site A (16.4%) (p<0.001). P. falciparum seroprevalence was 36.7% in site B, 16.7% in site A and 18.7% in site C (p<0.001). Interestingly for both species, sites with the highest seroprevalence were respectively the ones with the highest prevalence by PCR, and also the ones with the highest API estimated from PCD records in 2012. Gametocytes were found in 4 (44.4%) of the total 9 microscopically confirmed P. falciparum infections in site B, as well as in 4 (57.1%) of the total 7 microscopically confirmed P. vivax infections in site C. The unique microscopically confirmed P. falciparum infection in site C had also gametocytes. Most of PCR-positive infections were sub-microscopic (>70%) and asymptomatic (>75%) at the time of the survey, regardless of the Plasmodium species  , with th species . SpeciesP. vivax exposure, the multivariate adjusted risk factor analysis showed that site and age remained independently associated with the P. vivax seropositivity: individuals living in Site B  and in Site C  had higher odds of P. vivax seropositivity than those in Site A, and those older than seven years had increasingly higher odds (test for trend p < 0.001) of P. vivax seropositivity compared to younger children  were more likely to be exposed to P. falciparum than those in Site A, adults >30 years  were more exposed than children < 7 years, and people working outdoor were three times more exposed to P. falciparum . Despite being significantly associated with both P. vivax  and P. falciparum exposure  in the univariate analysis, the history of malaria in the past year was not included in the multivariate analysis, since it is not a risk factor for malaria infections per se but rather a consequence of previous exposure to given risk factors.Given the small number of PCR-positive infections by species, multivariate adjusted risk factor analysis was carried out only for any parasite infection. Only site and age remained independently associated with malaria infection: while individuals living in Site B  and in Site C  were more likely to be infected than those living in Site A, individuals >30 years had higher odds of malaria infection than children < 7 years . Considering children . For P. P. falciparum was not randomly distributed. In site A, the most likely spatial cluster of P. falciparum seropositive individuals , located on the western part of the village close to Nanay river  had a radius of 1.3 km including 50 individuals  in 30 households   of the 14 P. falciparum mono-infections detected by PCR. Site C had no significant spatial cluster of P. falciparum exposure but was the only site where a most likely cluster of P. vivax-exposure  could be identified (P. vivax seropositive but none of them was infected (PCR negative). Adding age group and/or outdoor occupation  as covariates in SaTScan analysis to locate species-specific exposure clusters had no effect on results.Figs ay river , had a 1e total) . This clentified . This waP. falciparum age seroprevalence was better described by a model with only one SCR (λ = 0.08) and one SRR (ρ = 0.384), while the overall P. vivax age seroprevalence was best modeled with two SCR  and one SRR (ρ = 0.096) indicating an increase in MTI in 2010 for this species . The reversible catalytic models for P. falciparum showed a stable transmission in each study site, with higher transmission intensity in Site B (λ = 0.129) than in sites A (λ = 0.061) and C (λ = 0.075). Conversely, best models for P. vivax differed by site. While site C had a change in P. vivax MTI in 2010 with a significant threefold increase , sites A and B experienced significant drops in SCR but at different times: 13–14 years before the survey in site A , and 7–8 years prior survey in site B . Moreover for both species, sites with the highest SCRs (SCR after the time cut-off in models with two SCRs) were respectively the ones with the highest API.Age seroprevalence curves of individuals aged ≤ 30 years confirmed a significant increase of malaria seroprevalence with age, but with different species specific patterns . The oveP. falciparum per site , it allowed identifying a decrease in SCR for P. falciparum 32 years before the survey when the analysis included all the sites  , P. vivax seroprevalences curves in site A was best described by a model assuming a decrease in malaria transmission 37 years prior survey . Conversely, model results for site B indicated a constant P. vivax SCR over time (λ = 0.04).Although the inclusion of seroprevalence data of individuals aged >30 years did not change the assumption of a stable transmission over time for  = 0.03) . For P. P. vivax and 50,000 P. falciparum cases were recorded. After the epidemic, incidence dropped drastically and stabilized between 2000 and 2001 at around 30,000 total annual cases, and at 45,000 and 55,000 cases between 2002 and 2005. During this post-epidemic period, a new antimalarial treatment policy (2001–2004) was progressively implemented as previously described. During the Global Fund-PAMAFRO Project (2005–2010), intense control efforts resulted in a marked decline of malaria incidence from 54,291 reported clinical cases in 2005 to 11,604 cases in 2010. However, since 2011 , malaria incidence is doubling each year. Noteworthy, P. falciparum infections usually followed similar trends than P. vivax, accounting for 20–25% of the total malaria cases, with exception of 1996–1998 when they represented almost 45% of the cases in Loreto. Between 2002 and 2013, though San Juan and Iquitos districts followed similar trends of malaria incidence than Loreto department  in these two districts.The retrospective analysis (1990–2013) of annual malaria incidence in Loreto department  showed apartment , P. falcP. falciparum and P. vivax. However, species-specific parasitological and serological metrics  were in good agreement with IPA estimates by species.The combined analysis of parasitological and serological measures collected during a single cross-sectional survey allowed for an in-depth characterization of the malaria transmission patterns in three different sites in an endemic district of the Peruvian Amazon. Most malaria infections detected by PCR were sub-microscopic and asymptomatic. The site specific analysis of both prevalence and seroprevalence data showed a high degree of heterogeneity at micro-geographic level and in time for both P. falciparum, and most of them were undetectable by microscopy and free of malaria-related symptoms. As reported by other countries in the region, asymptomatic malaria infections (including submicroscopic infections) are common in low endemic settings, representing a “hidden” human reservoir likely to contribute to maintain transmission [P. vivax was always the predominant species.Although the survey was conducted in November, before the usual peak of clinical malaria cases, an important number of malaria-infected individuals were identified, mainly by smission –42. Prevsmission ,8,43; hoP. vivax over P. falciparum reported cases, parasitological and serological measures indicated that species-specific malaria transmission may vary widely across sites. Indeed, site C had similar prevalence of both species by PCR, but the higher seroprevalence and SCR estimates for P. vivax compared to those of P. falciparum suggested a very recent increase in P. falciparum infections. Moreover, in site B the higher P. falciparum seroprevalence and SCR compared to those of P. vivax suggested that the predominance of P. falciparum infections detected by PCR was not new. Additionally, the identification of P. falciparum gametocyte carriers by microscopy suggested ongoing transmission of this species in the site. These hypothesis were further supported by the newly generated site incidence data on P. falciparum (IPAf) and P. vivax (IPAv) in 2012. These results highlight the added value of combining parasitological and serological metrics in accurately understanding the local epidemiology since data from the health information system are often missing or not very reliable.Although routine surveillance data at district and department level showed a predominance of P. falciparum were significantly higher in rural communities along the Iquitos-Nauta road than in periurban areas, with large monthly variations ranging from 0 to 12 infective bites per person per month [An. darlingi, and were significantly associated with malaria infection [The differences in species-specific seroprevalence across sites were in line with findings from previous entomological studies, all reporting a high heterogeneity in malaria transmission in the peri-Iquitos region ,44,45. Der month . Deforesnfection ; while snfection ,48.P. falciparum and P. vivax [P. vivax relapse patterns may influence serological profiles, the quantification of their effect on the production of malaria antibodies remains unclear due to the difficulty to classify a recurrent parasitaemia as relapse, new infection or recrudescence [Genetic factors may play a role in human susceptibility to malaria infection and human infectiousness to mosquitoes ,50. HoweP. vivax ,52. Evendescence ,52.Species specific heterogeneity was not only observed at small-scale geographical level but also over time as illustrated by the respective SCR estimates calculated by models including and excluding individuals older than 30 years old. A strong advantage of SCRs is the ability to reconstruct the history of malaria exposure in a population , but oneP. vivax seroprevalence curves showed that the significant decline in malaria transmission in sites A and B occurred at different points in time. While in site A, this time point coincided with the drastic reduction in P. vivax incidence after the late 90s epidemic; in site B, the change was concordant with the start in 2005 of the Global Fund-PAMAFRO project which prioritized comprehensive malaria control interventions in rural communities [P. vivax in site C, i.e. increase in MTI two years before the survey. The fact that the catalytic model did not detect similar changes in SCRs for P. falciparum transmission as for P. vivax, could be due to the lack of power due to the lower levels seropositivity for P. falciparum and therefore insufficient sample size. Moreover, the independent association of outdoor economic activities with P. falciparum exposure would influence the model fit to age-seroprevalence data, since the assumption of the catalytic model that P. falciparum transmission is homogeneous in the community would not be fulfilled [On the other hand, SCR estimates generated using seroprevalence data of individuals aged ≤ 30 years would be more accurate to identify more recent changes in malaria transmission. Indeed, reversible catalytic models used to generate munities ,53. Sinculfilled .Effective methods for quantifying micro-geographical variations in malaria exposure should be able to identify stable hotspots of transmission over time . The spaP. falciparum exposure: in site A, this cluster covered the area closest to the river which floods every year during the rainy season; in site B, the cluster included mainly households from Villa Buen Pastor, the community closest to natural water bodies and surrounding secondary vegetation, and with most individuals having outdoor occupations (i.e. farmers and guards). The quasi absence of clustering in site C may be explained by the local mosquito and human mobility patterns. Indeed the presence of several permanent breeding sites (i.e. fish ponds) along the Iquitos-Nauta road could result into a wider dispersal of An. darlingi and numerous movements of infected individuals through the unique and highly accessible road could further increase the dispersal of parasites. Although a similar explanation could be advanced for the absence of P. vivax clusters in site A and B, it is difficult to know how the relapse patterns can affect the spatial analysis using serological data.Our spatial analysis using serological data allowed the identification of clusters with the highest Plasmodium species [Our study has some limitations. First, despite the use of two antigens of proven sensitivity for each  species ,57, regi species ,22. Thou species  in Peruv species ,58,59. O species . Third, The use of a single parasitological and serological malaria survey has proven to be an efficient and accurate method to characterize the species specific heterogeneity in malaria transmission at micro-geographical level as well as to identify recent changes in transmission. This approach, once optimized by a validated set of best antigens by geographical area, represents a reliable and effective tool to accurately identify and characterize hotspots of transmission and consequently help decision-makers to better design and deliver targeted interventions. This is particularly important in low transmission settings moving towards malaria elimination such as the Peruvian Amazon.S1 FigBlack dots represent observed prevalence, whilst the blue lines represent the maximum likelihood model. Seroconversion rates ) are plotted on the graphs. Two seroconversion rates (λ1 and λ2) are plotted in graphs if likelihood ratio tests indicate change at a certain point in calendar time.(PNG)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.