Ivermectin is widely used in mass drug administrations for controlling neglected parasitic diseases, and can be lethal to malaria vectors that bite treated humans. Therefore, it could be a new tool to reduce plasmodium transmission. We tested the hypothesis that frequently repeated mass administrations of ivermectin to village residents would reduce clinical malaria episodes in children and would be well tolerated with minimal harms.ClinicalTrials.gov, number NCT02509481.We invited villages (clusters) in Burkina Faso to participate in a single-blind (outcomes assessor), parallel-assignment, two-arm, cluster-randomised trial over the 2015 rainy season. Villages were assigned (1:1) by random draw to either the intervention group or the control group. In both groups, all eligible participants who consented to the treatment and were at least 90 cm in height received single oral doses of ivermectin (150–200 μg/kg) and albendazole (400 mg), and those in the intervention group received five further doses of ivermectin alone at 3-week intervals thereafter over the 18-week treatment phase. The primary outcome was cumulative incidence of uncomplicated malaria episodes over 18 weeks  in an active case detection cohort of children aged 5 years or younger living in the study villages. This trial is registered with vs 24 events [2%] among 1265 in the control group; risk ratio 1·63 [1·01 to 2·67]; risk difference 1·21 [0·04 to 2·38], p=0·060), and no adverse reactions were reported.Eight villages agreed to participate, and four were randomly assigned to each group. 2712 participants , including 590 children aged 5 years or younger, provided consent and were enrolled between May 22 and July 20, 2015 , with 1447 enrolled into the intervention group and 1265 into the control group. 330 (23%) participants in the intervention group and 233 (18%) in the control group met the exclusion criteria for mass drug administration. Most children in the active case detection cohort were not treated because of height restrictions. 14 (4%) children in the intervention group and 10 (4%) in the control group were lost to follow-up. Cumulative malaria incidence was reduced in the intervention group  compared with the control group . The risk of adverse events among all participants did not differ between groups (45 events [3%] among 1447 participants in the intervention group Frequently repeated mass administrations of ivermectin during the malaria transmission season can reduce malaria episodes among children without significantly increasing harms in the populace.Bill & Melinda Gates Foundation. Current malaria control efforts primarily consist of symptomatic and preventive treatment with antimalarials, including artemisinin-based combination therapies, as well as vector control with long-lasting insecticidal mosquito nets and indoor residual spraying of insecticides. Although mortality caused by malaria has dropped an estimated 48% since 2000 and there are now fewer malaria-endemic regions,Evidence before this studyWe searched PubMed and Web of Science, with no language restrictions, for all studies on control of malaria with ivermectin up to April 1, 2015, with the terms “ivermectin”, “malaria”, and “anopheles”. In many laboratory studies , ivermectin, and similar endectocides, showed a significant ability to kill anopheline malaria vectors, including when added artificially to mosquito blood meals, when the mosquitoes were directly blood-fed on ivermectin-treated humans or animals, and when they indirectly ingested treated human or animal blood from artificial feeders. Several field experiments in Papua New Guinea and in west Africa showed a similar mosquitocidal effect on wild malaria vectors that were captured after they blood-fed on people who were treated with ivermectin in mass drug administration campaigns for control of lymphatic filariasis and onchocerciasis. Some of these studies showed temporary reductions in sporozoite infection in the surviving mosquitoes after the ivermectin treatment, and this effect was also predicted in published modelling studies. We found no studies or clinical trials examining the effect of ivermectin on clinical malaria incidence.Added value of this studyTo our knowledge, our trial is the first cluster-randomised clinical trial designed to test the safety and efficacy of repeated mass ivermectin administrations to control malaria among communities. Mass administrations of ivermectin repeated every 3 weeks during a rainy season significantly reduced the incidence of malaria episodes in children in the study villages by 20%, and caused no obvious drug-related harms to the village populace.Implications of all the available evidenceThese new data are an important addition to the growing number of reports showing that ivermectin could be an effective new malaria control tool. Because ivermectin has a unique mode of action relative to current vector control insecticides and antimalarial drugs, it could be a synergistic tool when paired with these other interventions to combat residual malaria transmission, helping to achieve malaria control or elimination in certain regions, and to combat the spread of both antimalarial drug-resistant parasites and insecticide-resistant vectors. Repeated mass administrations of ivermectin should also enhance elimination efforts of certain neglected tropical diseases. Similar double-blinded clinical trials are now needed, and from several malaria-endemic areas, to test the effects of ivermectin across different malaria ecologies. New doses or formulations should also be explored that could make the intervention more practical and effective. Finally, new clinical studies are now warranted for examining the suspected direct antimalarial effects of repeated ivermectin treatment in infected humans.Plasmodium falciparum parasites in surviving mosquitoes,Ivermectin is a well tolerated drug that is regularly distributed in mass drug administrations for the control of neglected tropical diseases, including lymphatic filariasis and onchocerciasis. It targets ligand-gated chloride channels in invertebrates, disrupting their neuromuscular transmission, and kills malaria vectors that blood feed on treated people and animals a week or more after drug treatment.Anopheles vectors blood feeding on them,Plasmodium and neglected tropical disease) indices were measured as secondary objectives.A scientific and policy working groupWe did a cluster-randomised, parallel-group trial in eight villages located around the town of Diebougou, in the Sud-Ouest administrative region of Burkina Faso . This rePlasmodium to all children, who are most at risk for disease in hyperendemic regions when they are infected with multiple Plasmodium clones over the rainy season.In government-conducted mass administrations of ivermectin for lymphatic filariasis control around the world, approximately 75% of a village's population are usually treated. People less than 90 cm in height, pregnant women, and women breastfeeding infants aged less than 1 week, are excluded per the drug indication. Importantly, most children aged 5 years or younger are not tall enough to be treated. However, high drug coverage is expected to lessen the ability of the local mosquito population to transmit Loa loa  and the Comité d'Ethique Institutionnel de L'Institut de Recherche en Sciences de la Santé (A03–2015/CEIRES).Villages (clusters) were randomly allocated (1:1) to two study groups—an intervention group and a control group. Randomisation was done at a public event at the Health District offices in Diebougou, attended by members of the study team, community health workers from all eight villages, and the local health clinic nurses. In front of the attendees, the words “treatment”  and “control” were written on four cards each, which were then sealed in identical opaque envelopes, mixed in a container, and randomly pulled from the container by each community health worker representing their village. Villages were assigned to the group written on the card selected by this representative.The study was blinded only to the outcomes assessor, who was allowed access only to coded group and participant data when doing the initial analyses.The treatment phase of the trial lasted 18 weeks . Both grMalaria incidence was actively monitored in the cohort by nurses who each travelled to their two assigned villages a minimum of three times every 2 weeks over the treatment phase, and screened the whole cohort each time. Although the original protocol specified that children should be visited twice a week, this frequency proved too logistically difficult and the protocol was amended. For suspected malaria episodes, nurses took a fingerprick blood sample, with which they did a rapid diagnostic test and made malaria slide smears. All positive malaria episodes were reported to the field physician, and uncomplicated episodes were immediately treated with artemether–lumefantrine and monitored for recovery in accordance with national malaria treatment guidelines. Complicated malaria or other complicated health problems were immediately reported to the study physicians, who visited and treated as able, and referred participants to the local community health centre or district hospital if necessary. Outside of regular nurse visits, parents or guardians of children in the cohort were told to call the clinical team via the community health worker if the child had any health problems. Additionally, all participants were encouraged to directly report any adverse events to the nurses or by mobile phone via the community health workers .Plasmodium. Rapid diagnostic tests were considered positive if any test line appeared , and incidence data were not modified in response to these results.An uncomplicated malaria episode was defined by a temperature of 38·0°C or higher (0·5°C was added to each thermometer recording to account for axillary readings) or history of fever in the last 24 h, and a positive rapid diagnostic test  for Demographic data, including age, height, and relevant medical history, were recorded from all participants on computer tablets. Entomological and parasitological data were also obtained from testing of blood or faecal samples from children and other select participants. Entomological sampling, which included mosquito capture by means of aspiration or light traps, was done for select households in each village at specified timepoints .P falciparum clones in each infection), molecular force of infection , presence of Wuchereria bancrofti in captured mosquitoes, and prevalence of soil-transmitted helminths in children aged 6–10 years.The primary outcome was cumulative incidence of uncomplicated malaria episodes over the 18-week intervention period in the cohort of children aged 5 years or younger, and the study was powered on this measure. Secondary outcomes included the number and type of adverse events among enrolled participants, obtained via passive case detection. Additional secondary outcomes included analyses of entomological data, as follows: human biting rate (number of mosquitoes that blood fed or attempted to blood feed per person per week), proportion of captured mosquitoes infected with sporozoites, entomological inoculation rate (product of the human biting rate and the proportion of mosquitoes infected with sporozoites), proportion of parous mosquitoes (a measure of the age of the mosquito population), and serological reactivity to an anopheles salivary gland protein. We also examined the following parasitological data: parasitaemia, multiplicity of infection  were analysed by χropriate . All anaClinicalTrials.gov (NCT02509481).This trial is registered with The funder of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.Between April 21 and May 6, 2015, we invited eight villages for participation. All villages that were asked to participate agreed to do so, and randomisation was done on May 21, 2015. The written informed consent or assent of participants was documented between May 22 and July 20, 2015 . Nearly all households and household residents of all study villages agreed to participate, and consent was obtained for all children aged 5 years or younger in participating households to be enrolled in the cohort . After rOverall, 648 uncomplicated malaria episodes were recorded among 327 children in the intervention group, and 647 episodes among 263 children in the control group . In the Malaria incidence was highest around week 11 of the trial, shortly after rainfall peaked . Malaria16Children in both groups had between zero and seven episodes of malaria . OverallW bancrofti microfilaria in the blood meals of captured mosquitoes  of 2712 participants , and fouAn exploratory cohort subgroup analysis was done because of the obvious but unexpected confounder that 69 (21%) of 327 children in the intervention group and 52 (20%) of 263 in the control group  were aged 4–5 years and were at least 90 cm in height, and were thus treated with ivermectin (four to six times in the intervention group and once in the control group). In these ivermectin-treated children, the risk difference between groups was of greater magnitude  than thaThis trial confirmed our hypothesis that 3-weekly repeated mass administrations of ivermectin to villages over the course of a rainy season in Burkina Faso can reduce the incidence of uncomplicated malaria episodes among children living in those villages. This treatment also did not obviously increase drug-related harms among those treated in the community, but more safety studies in large populations are needed. Many studies have shown that blood feeding on ivermectin-treated humans and animals significantly reduces the survival of malaria vectors,Anopheles biting in intervention group participants over the trial. Although other secondary entomological outcomes  did not differ between groups, these measures can be highly variable in time and space,The primary analysis of this study showed that the mean number of clinical malaria episodes per child was about 20% lower in the intervention group than in the control group over the 18-week treatment period. Because a similar reduction was observed in the subgroup analysis of children not treated with ivermectin, we infer that most of this reduction was due to a community-protective effect, as suggested from previous field dataConsidering the frequency distribution of malaria episodes, the greatest difference between the control and intervention groups was in the proportion of children who never had an episode during the trial, suggesting that the intervention most affects children with the lowest risk of disease. These children might have had low entomological inoculation rates in the first place, and particularly benefited as the number of infectious bites they received approached zero, meaning that they were unlikely to get infected over the trial period. Conversely, these data might also suggest that children who suffered multiple episodes had risk factors that countered the effect of mass administrations of ivermectin. Such risk factors might include living in a house that allows entry to many mosquitoes, or having behaviours that increase bite risk ; however, they might also include risk factors connected to failure of artemisinin-based combination therapies , or other unknown factors. Overall, these data suggest that repeated mass administrations of ivermectin as a tool for control of malaria transmission could be effective as an integrated intervention in malaria elimination campaigns, in which preventing all infectious bites is paramount.P falciparum clonal exposure or development. The scarce published data are conflicted as to whether standard single ivermectin treatments can affect the development of P falciparum in humans.In planning the trial, we did not expect that such a high proportion of children in the cohort (151 [22%] of 590) would be eligible to receive ivermectin treatment. When we accounted for this obvious confounder, we were surprised to see a considerably stronger effect among those from the intervention group (who were treated with ivermectin repeatedly) than among those from the control group (who were treated once), with 44% lower incidence in the intervention group. These novel data suggest that frequently repeated ivermectin treatments have an additional, direct effect on malaria incidence in children, and that this effect accounted for some of the overall intervention effect. The added effect was especially prevalent among children in the intervention group who had no malaria episodes during the study period, as this malaria episode frequency group comprised a higher proportion of children who received more than one ivermectin treatment (23 [36%] of 64) than did the other frequency groups  were classified as serious adverse events, suggesting that participants were more likely to report significant health problems to the study team than to report minor problems. This finding might also reflect a bias in the passive reporting system we used, in that serious adverse events resulting in hospitalisation or death were almost always reported to our study team by either the study populace or the local health authorities, in contrast to non-serious adverse events. However, the low overall number of adverse events was consistent with similar studies that examined the safety of frequently repeated mass administrations of ivermectin, even though our study was conducted with far more participants.Overall, the results from this study suggest that frequently repeated ivermectin mass drug administrations to village residents did not obviously increase drug-related harms in the treated population, and provided health benefits by significantly decreasing clinical malaria episodes in young children. These malaria-reducing effects seemed to be due to decreased malaria transmission from a community-protective effect, and possibly due to additional direct antimalarial effects in repeat-treated children. This study provides the first proof of principle of the antimalarial effects of ivermectin, but the intensive nature of our intervention is probably not optimal, and higher doseClinicalTrials.gov.Data collected from this study, including de-identified individual participant data, will be made available upon publication to members of the scientific and medical community for non-commercial use only, upon email request to the corresponding author. The study protocol, statistical analysis plan, and informed consent forms are available on


Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of clonally variant antigens on the surface of infected erythrocytes (IEs). The VAR2CSA subtype of PfEMP1 mediates IE binding in the placenta.Clinical immunity to malaria is associated with the acquisition of IgG specific for members of the  Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of clonally variant antigens on the surface of infected erythrocytes (IEs). The VAR2CSA subtype of PfEMP1 mediates IE binding in the placenta. VAR2CSA-specific IgG is normally acquired only after exposure to placental parasites. However, it was recently reported that men and children from Colombia often have high levels of functional VAR2CSA-specific IgG. This potentially undermines the current understanding of malaria immunity in pregnant women, and we thus conducted a study to assess further the levels of VAR2CSA-specific IgG in pregnant and nonpregnant Colombians. Plasma IgG against two full-length recombinant PfEMP1 proteins (one of the VAR2CSA type and one not) produced in baculovirus-transfected insect cells was detected frequently among Colombian men, children, and pregnant women with acute or previous malaria exposure. In contrast, IgG reactivity to a homologous full-length VAR2CSA-type protein expressed in Chinese hamster ovary (CHO) cells was low and infrequent among the Colombian plasma samples, as was reactivity to both corresponding native PfEMP1 proteins. Moreover, human and rabbit antibodies specific for Plasmodium vivax Duffy-binding protein (PvDBP), a protein with some homology to PfEMP1, did not react with VAR2CSA-type recombinant or native proteins, although the mouse monoclonal and PvDBP-specific antibody 3D10 was weakly reactive with recombinant proteins expressed in baculovirus-transfected insect cells. Our data indicate that the previously reported Colombian IgG reactivity to recombinant VAR2CSA is not malaria specific and that the acquisition of VAR2CSA-specific IgG is restricted to pregnancy, in Colombia and elsewhere.Clinical immunity to malaria is associated with the acquisition of IgG specific for members of the  Plasmodium falciparum in Africa  expressed on the infected erythrocyte (IE) surface are important targets  (The intensity of uncommon –15. It wncommon –. That sa (PvDBP) , which hThe above-described findings from Colombia either point to a completely new and unanticipated mode of acquisition of VAR2CSA-specific IgG or suggest that the current understanding of immunity to placental malaria is incomplete. We therefore set out to shed additional light on the prevalence and specificity of IgG recognizing VAR2CSA-type PfEMP1 in Colombian populations.P. falciparum-exposed women who are or have recently been pregnant, and the levels generally correlate with parity among such women . With the exception of exposed men  and pregnant women , the levels were not statistically significantly different from those observed in samples from nonpregnant Ghanaian women who had been pregnant one or more times previously (set 5). Plasma levels of FV2BIC-specific IgG were significantly lower among unexposed Colombians  than among exposed Colombians  but were still significantly higher than the levels among negative-control donors from Denmark   . As repo< 0.001) , levels < 0.001) . Althougmple set  from whiBIC), expressed as FV2BIC but representing a PfEMP1 antigen that is commonly recognized by plasma IgG from naturally exposed African donors. The expression of this PfEMP1 antigen is not restricted to parasites infecting pregnant women  that is not restricted to pregnancy  rather than in insect cells. Levels of IgG to this protein (FV2CHO) were generally low among all groups of Colombian donors  but not in the samples from children (set 1a and set 2a) or men (set 1b and set 2b). This suggests that some of the Colombian women had been exposed to VAR2CSA-positive IEs during their pregnancy. These findings suggest that most of the apparently VAR2CSA-specific IgG in the Colombian plasma samples actually recognized so-far-unidentified epitopes present only in the recombinant proteins produced in insect cells. In contrast, levels of IgG reacting with FV2BIC, FV2CHO, and the corresponding native protein on IEs were positively correlated among the samples from Ghana  ratherom Ghana , indicatBIC among the Colombian donors was indeed directed toward non-PfEMP1-specific epitopes that are present in recombinant proteins expressed in baculovirus-transfected insect cells but absent from proteins expressed in CHO cells and absent in native PfEMP1 proteins. To this end, we sequentially measured IgG reactivity against FV2BIC, FV2CHO, and FV6BIC in plasma pools transferred from one ELISA plate to the next . Those same authors proposed that the cross-reactivity is mediated by PvDBP  trial (set 3)  or nonirradiated (Duffy-negative [Fy−] donors [set 4c]) P. vivax-infected mosquitoes  and theby PvDBP . We ther (set 3) . In all  patency , meaningncy P. vivax BIC (endpoint titer of 0.08 μg/ml using mouse IgG as a reference) (CHO (endpoint titer of 10 μg/ml) (BIC (BIC (endpoint titer of 0.08 μg/ml), but 3D10 did not label the native counterpart of FV6, HB3VAR06 . It thus appears that 3D10, in addition to its specificity for PvDBP   but reac0 μg/ml)  and not ml)  does not necessarily react with a corresponding protein expressed in another system  or indeed with the native protein that it represents . A possible explanation for the discrepancy between FV2BIC IgG reactivity on the one hand and FV2CHO and IT4VAR04 reactivity on the other is the marked differences in protein glycosylation among malaria parasites (largely limited to the addition of glycosylphosphatidylinositol [GPI] anchors)  appeared to be a mix of both. We did not pursue the nature of the epitopes in the baculovirus proteins recognized by IgG in plasma from Colombia but not from Ghana, as this is likely to be a major undertaking that is well beyond the scope of the present work. For the same reasons, we did not investigate why these antibodies are common in Colombia while seemingly absent in Africa. It might be worth noting that the highest IgG responses to insect cell-expressed recombinant antigens among Colombian donors were found among children, both here and in the previous study. Exposure of Colombians to other pathogens or insects is another potential explanation that was not investigated here.A few years back, Gnidehou et al.  reporteden PvDBP . The stuen PvDBP , 17 thusen PvDBP . We theren PvDBP , 17, as en PvDBP , 43. We P. vivax. The rationale for the development of a VAR2CSA-based vaccine against placental malaria therefore stands  in Colombia as in Africa. Furthermore, acquired immunity to VAR2CSA-type PfEMP1 seems not to be influenced by exposure to e stands .The original studies from which the samples used here were obtained were approved by the Institutional Review Boards of the Malaria Vaccine and Drug Development Center (MVDC) and the Centro Médico Imbanaco, Cali, Colombia; by the Institutional Review Board of the Noguchi Memorial Institute for Medical Research, University of Ghana; and by the Regional Research Ethics Committees, Capital Region of Denmark. The Colombian clinical trials were registered at ClinicalTrials.gov (registration numbers NCT01585077 and NCT01082341). The present study was approved by the Institutional Review Board at MVDC.P. vivax. Tumaco  has a highly predominant Afro-descendant population, and P. falciparum dominates (∼85%) in this region. Quibdó  is also inhabited mainly by Afro-descendants, and most malaria episodes (∼70%) are caused by P. falciparum. Tierralta  is inhabited mostly by mestizo populations, with a small Amerindian Emberá Katío indigenous community; P. vivax dominates (∼85%) in this region.Stored plasma samples from four previous studies involving five locations of Colombia were used. Cali, Buenaventura, Tumaco, and Quibdó are located in the western and Pacific regions, while Tierralta is situated in the northwestern Caribbean region  48, 49), 4948, 4Several sets of samples were studied . The firP. vivax or P. falciparum malaria as well as a set of plasma samples from pregnant women with P. vivax or P. falciparum malaria (The second set of samples (set 2) was obtained from a passive case detection study conducted from 2011 to 2013 at malaria points of care  and from malaria , 52  trial carried out at MVDC in Cali. The unexposed  volunteers were from Cali, whereas the semi-immune volunteers were from Buenaventura. Their previous malaria exposure was confirmed by clinical history and by the presence of P. vivax-specific antibodies. Samples collected at enrollment, at patency (days 11 to 13), and 45 days after challenge (follow-up) were analyzed (The third set of samples (set 3) was collected from previously unexposed (set 3a) and semi-immune men and women (set 3b) participating in a controlled human analyzed .P. vivax sporozoites (PvRAS) (+) (set 4a and set 4b) and Duffy-negative (Fy−) individuals (set 4c) were collected at enrollment, after seven immunizations, and after PvCHMI with nonirradiated, P. vivax-infected mosquitoes.The fourth set of samples (set 4) was collected from previously unexposed adults during a clinical trial in Cali to assess the safety and protective efficacy of immunization with radiation-attenuated  (PvRAS) . SamplesPlasma samples from 32 nonpregnant women from Ghana with known reactivity to VAR2CSA-type PfEMP1  (set 5) BIC; GenBank accession number GU249598) and of the non-VAR2CSA-type PfEMP1 protein HB3VAR06  were produced in baculovirus-transfected Sf9 insect cells, as described previously (CHO), identical to FV2BIC but expressed in suspension-adapted CHO cells  according to the manufacturer's instructions, was also used. In brief, a full-length construct with a C-terminal histidine tag was cloned into the vector pcDNA3.4 and used to transfect ExpiCHO-S cells. Glycosylation sites were not removed. Secreted recombinant proteins were purified from the supernatants by using Ni2+ metal chelation agarose .The entire ectodomains of the VAR2CSA-type PfEMP1 protein IT4VAR04 (FV2eviously . A thirdP. vivax Duffy binding protein region II (PvDBP), produced in Escherichia coli, was a kind gift from John H. Adams (University of South Florida) (Recombinant Florida) .in vitro selection of particular PfEMP1 proteins expressed on the IE surface (see below), we used human monoclonal antibody PAM1.4, which is specific for a conformational epitope in several VAR2CSA-type PfEMP1 proteins, including IT4VAR04  was read at 450 nm, and the specific antibody levels were calculated in arbitrary units (AU), as described previously  and adults without previous malaria exposure , malaria-exposed nonpregnant women from Ghana , and unexposed Danish adults  were tested in three sequential ELISAs. In the first ELISA, levels of IgG specific for FV2BIC, FV2CHO, and FV6BIC were measured. After incubation (1 h), the depleted pools were transferred directly to new plates coated with the same protein used in the first ELISA. After incubation (1 h), the further depleted pools were transferred to FV2BIC-coated plates and once again incubated as described above. Further processing was performed as described above. For these experiments, the data were normalized by dividing the sample OD by the OD for the corresponding negative control (fold over control).Pooled plasma samples from Colombian pregnant women  were verified regularly.eviously . Erythroeviously . A simileviously . Transcrtime PCR  and flowtime PCR , respecttime PCR  and the sample − MFIblank)/(MFIpositive control − MFIblank)]. Negative cutoff values were calculated as the mean of rMFI values plus 2 SD obtained with the Danish control samples (set 6).Plasma IgG reactivity against IEs expressing IT4VAR04 or HB3VAR06 was analyzed by flow cytometry, as described previously , using asr) was used to assess the correlation between numeric variables. A chi-square test was used to compare differences in proportions. P values of <0.05 were considered statistically significant.Data were analyzed by using GraphPad Prism version 6.0 . All experiments reported here were repeated at least twice, with similar results. The Mann-Whitney U or Kruskal-Wallis test followed by Dunn's multiple-comparison test was used to compare two or more than two groups, respectively. Spearman's rank correlation (


A screening for malaria parasites was conducted with asymptomatic residents in Colombia. A descriptive study was carried out in December 2012 in four municipalities of Urabá region in Colombia. A convenience sample of 400 subjects was selected. Participants responded to a survey regarding epidemiological data and blood samples were taken from capillary blood obtained by finger prick for thick smear, rapid diagnostic test (RDT), and polymerase chain reaction (PCR). P. falciparum, 3 with P. vivax), and 3 were confirmed by PCR. In 399 thick blood smears examined 5 (1.3%) were positive , and 3 were confirmed by PCR. In 227 samples, PCR showed 6 (2.6%) positive samples. The parasitaemia was below 1,440 parasites/μL. The best agreement between diagnoses was found between the RDT and thick blood smears (Kappa = 0.75). 399 subjects aged 0.2-98 years were studied ). Episodes of malaria in the last year confirmed by thick film were reported by 47 participants (12%). In 399 samples tested by RDT 4 (1%) were positive (1 with Plasmodial afebrile infection was found in 2% of the studied population, by three diagnostic methods, in residents from a low endemic malaria region in Colombia. Malaria is an infectious disease caused by various species of Plasmodium, characterized in humans by an acute febrile syndrome with symptoms of general illness such as chills, headache, musculoskeletal pain, and weakness; with low frequency, it presents without fever.Although the malarial transmission has been declining in prevalence around the world, this protozoan disease remains as an important cause of fatal cases; 429 000 deaths were reported in 2015, most of them (92%) in Africa [ Plasmodium spp. have not been rigorously defined and generally are referred to as asymptomatic malaria [Asymptomatic infections by malaria –6, which malaria  or contr malaria . Plasmodium falciparum infections in endemic areas [μL of blood [μL [Asymptomatic infections or asymptomatic carriers can be either microscopic or submicroscopic ; the incic areas , 11; thiof blood  in contrlood [μL . P. falciparum and 2.9% of P. vivax infected subjects were febrile [ P. falciparum infection [Some studies indicate that asymptomatic infections are more frequent than symptomatic ones in most malarial endemic settings , 14–16.  febrile  and in Snfection . In contnfection –19. In lnfection . These cnfection .The presence of asymptomatic plasmodial infections in Colombia has been explored in few studies, most of them conducted in Tierralta, Buenaventura, and Tumaco , 22–25.  Plasmodium spp. has been poorly studied in general population. The aim of this study was to explore the presence of asymptomatic carriers of Plasmodium spp. and describe their epidemiological profile in four municipalities in an endemic region for malaria in Colombia.In these malarial zones, the frequency of asymptomatic infections by P. vivax and P. falciparum endemicity is low. Patients were enrolled between 2012 and 2013 in the municipalities of Turbo , Necoclí , San Pedro de Urabá , and Mutatá .This descriptive study was conducted in Urabá region, Colombia, whereA convenience sample of 400 subjects was established. The researchers visited the residents in their home in the rural area. People of any age and sex were invited to participate in the study, who did not show symptoms of the disease in the week before the sample was taken. Demographic aspects as age, sex, place of residence, occupation, residence time in endemic regions for malaria and history of illness, previous malaria episodes, and antimalarial drugs used in the last month were obtained.μL, and the presence of gametocytes was checked. The thick film was considered to be negative, if no parasite had been found in 200 high-power fields, with 1000X magnification.From each patient capillary blood was obtained by finger prick for microscopic diagnosis, rapid diagnosis test (RDT), and polymerase chain reaction (PCR). Thick blood smear was prepared according to World Health Organization recommendations  and staiThe RDTs were prepared and read according to manufacturer's instructions  by trained medical students who were blinded to blood smear results. P. falciparum.For molecular diagnosis, a sample of blood was taken in Whatman filter paper No. 3, individually stored in a plastic bag and sent to the Laboratory of Malaria Group of the University of Antioquia. The PCR diagnosis is described briefly. The filter paper was added with 0.5% saponin and washed 3 times with 1X PBS and Chelex 100 was added; it was denatured at different temperatures (56°C for 15 minutes and 100°C) and finally the supernatant was recovered after a 15-minute centrifugation at 13000 rpm. Genotyping was done using a PCR protocol developed by Singh et al.  by a firA descriptive statistical analysis was done with IBM® SPSS® statistics program, 23rd version (licensed to Universidad de Antioquia). The Kolmogorov-Smirnov test was used to assess normality for quantitative variables; the median values were compared with Mann–Whitney U test when variables were not normal. Associations between qualitative variables were analyzed with the Chi-square test. The adopted level of statistical significance was 5% .From a proposed sample of 400 participants, 399 asymptomatic participants were enrolled in the study: 99 (24.8%) in Turbo, 111 (27.8%) in Necoclí, 35 (8.8%) in Mutatá, and 154 (38.6%) in San Pedro de Urabá. Some demographic aspects are described in  P. falciparum, 3 with P. vivax), the rapid diagnostic tests were positive in 4 subjects , and the PCR results were positive in 6 subjects . The frequency of asymptomatic infections detected with any test was 2.0% (8/399) and the frequency of asymptomatic infections by PCR was 2.6% (6/227). The age range of the asymptomatic patients was between 2 and 33 years; 75% were under 15 years.Blood smears and rapid diagnostic tests were performed to all subjects; the PCR was made in 227 subjects. A positive result in any test was considered as an asymptomatic infection and was classified as submicroscopic infection when blood smear was negative and PCR was positive. The blood smear was positive in 5 participants  were submicroscopic infections, which were presented in subjects of the mestizo ethnic group, with ages of 8, 12, and 26 years.The median value of the age in noninfected participants was 22 years ; in the infected subjects the median was 10 , without statistical difference . Most of the infected subjects were males, students, and of mestizo ethnic group .The median of the time of residency in malarial endemic region was 7. 4 years (3.1–11.6) for the infected subjects against 17.4 years (15.7–17.1) in the noninfected group. P. vivax was the most common agent identified in asymptomatic subjects; no gametocytes were observed.Identification of asymptomatic carriers of Plasmodium parasites is an important issue for public health due to the epidemiological impact of asymptomatic infections in the perpetuation of the disease in the population and the adverse effects during pregnancy as anemia and low birth weight , 28–30 aDifferent studies defined asymptomatic malaria as the absence of fever, but clinical manifestations of Plasmodium infection include other findings that usually are not explored and are explained by an increased level of inflammatory markers  that cou Plasmodium spp. varies according to the endemicity. In high transmission settings it has been reported that frequencies rise up to 73.7% and 82,0% [ P. vivax in Cauca and 6,7% were found to be by P. falciparum in Nariño [The frequency of asymptomatic infections bynd 82,0% , 35, diand 82,0% , 17. Asynd 82,0% , 16. In nd 82,0% . In Colond 82,0%  and 3,9%n Nariño . In thisIt has been described from previous studies that asymptomatic infections are related to low parasitaemia ; a similThe importance of asymptomatic patient as a reservoir of the disease rises in two important questions: the duration of infection and the presence of gametocytes . The lon P. falciparum the infection had a median duration of 194 days (95% CI: 191-196) [In the natural history of the infection, some of the asymptomatic carriers can become symptomatic. In Brazil it was found that 10.7% of asymptomatic individuals became symptomatic in a period of two months ; other o191-196) .A study conducted in Uganda showed that 39% of children between 6 months and 5 years with subpatent infection became symptomatic in 20 weeks in comparison to 82% of children with patent infection  concludi P. falciparum and 4 days for P. vivax) this is likely an infrequent occurrence [In this study, asymptomatic infection was found in 8 subjects; however, because there was no follow-up, it raises the question of whether this parasitaemia is detected in the last moment of the incubation period; given the short time between the completion of the prepatent period and the end of the incubation period (1 day forcurrence .μL [μl of blood in approximately 5 μl of whole blood [μL for most sensitive methods) [An important conclusion of these findings is that the active search of cases diagnosed by light microscopy is insufficient to find all the asymptomatic carriers; the lower threshold of parasite detection in the field by microscopy has been estimated in 50-100 parasites/μL  similar le blood , in contmethods)  that themethods) . Observamethods) ; these fIt is important to keep in mind that, in the areas where parasite prevalence established by light microscopy is less than 10%, the proportion of submicroscopic infections is estimated up to 70-80% and these infections would be responsible for more than 50% of human to mosquito infection . Based o P. vivax was the most common agent implicated in asymptomatic malaria; this in contrast with other results of asymptomatic malaria in Latin America. In Brazil, P. falciparum was diagnosed more frequently (37.5%) than P. vivax (18.5%) [ P. vivax infection was slightly higher [ P. vivax is the most common Plasmodium parasite in Brazil. Observations in Colombia show that asymptomatic infections are more prevalent in P. vivax than in P. falciparum infections; in Tierralta 64.5% of asymptomatic infections were caused by P. vivax, 29% by P. falciparum, and 6,5% by mixed infection of these species; asymptomatic cases were more common in men than in women [ P. falciparum was present in 56% of asymptomatic plasmodial infections in women diagnosed by qPCR, P. vivax in 41,5%, and mixed infection in 2.5% [ P. vivax was 93%, 66%, and 50%, respectively [ P. vivax was more prevalent in Tierralta but P. falciparum was more prevalent in Tumaco [ P. vivax was more prevalent in Tierralta and P. falciparum was more common in Buenaventura, and despite malarial decreasing trend in the region, the proportion of P. vivax did not change significantly between 2011 and 2013 [An interesting observation is that we found that (18.5%)  and othey higher ; maybe tin women . In cont in 2.5% . Anotherectively . Anothern Tumaco . A crossand 2013 . This diIt has been described that adults are more likely to develop submicroscopic infections than children , 52. ParIn areas of low malaria endemicity, several studies show that subclinical infections with very low parasite loads are quite common , and notWe found the presence of asymptomatic plasmodial infections in a malarial region of low endemicity in Colombia, with an important component of submicroscopic malaria, which showed that light microscopy is an insufficient technique for active patient screening and to eradicate malaria. Asymptomatic infections were found especially in children, which may indicate a high exposure to the parasite and demand surveillance through more sensitive diagnostic methods in this risk group and in the general population.


Malaria rapid diagnostic tests (RDTs) are nowadays widely used in malaria endemic countries as an alternative to microscopy for the diagnosis of malaria. However, quality control of test performance and execution in the field are important in order to ensure proper use and adequate diagnosis of malaria. The current study compared the performance of a histidine-rich protein 2-based RDT used at peripheral health facilities level in real life conditions with that performed at central reference laboratory level with strict adherence to manufacturer instructions.Febrile children attending rural health clinics were tested for malaria with a RDT provided by the Ministry of Health of Burkina Faso as recommended by the National Malaria Control Programme. In addition, a blood sample was collected in an EDTA tube from all study cases for retesting with the same brand of RDT following the manufacturer’s instructions with expert malaria microscopy as gold standard at the central reference laboratory. Fisher exact test was used to compare the proportions by estimating the p-value (p ≤ 0.05) as statistically significant.In total, 407 febrile children were included in the study and malaria was diagnosed in 59.9% (244/407) of the cases with expert malaria microscopy. The sensitivity of malaria RDT testing performed at health facilities was 97.5% and comparable to that achieved at the laboratory (98.8%). The number of malaria false negatives was not statistically significant between the two groups (p = 0.5209). However, the malaria RDT testing performed at health facilities had a specificity issue (52.8%) and was much lower compared to RDT testing performed at laboratory (74.2%). The number of malaria false positives was statistically significantly different between the two groups (p = 0.0005).Malaria RDT testing performed at the participating rural health facilities resulted in more malaria false positives compared to those performed at central laboratory. Several factors, including storage and transportation conditions but also training of health workers, are most likely to influence test performance. Therefore, it is very important to have appropriate quality control and training programmes in place to ensure correct performance of RDT testing. Plasmodium parasites in Giemsa-stained thick or thin blood slides still remains the gold standard for malaria diagnosis  and the mean axillary temperature was 37.7 °C (standard deviation: 0.78 °C). Males represented 56.8% (231/407) of the study population  was 77.4% (315/407). No test failures were reported at health facilities. Retesting the collected blood samples in the laboratory of CRUN (Lab-PfHRP2) revealed that 69.3% (282/407) was RDT positive , with a geometric mean and median parasite density of 22,839.4 parasites/µl  and 39,847 , respectively. Co-infections were found in 5 cases: there were 4 co-infections with Plasmodium malariae and 1 case with Plasmodium ovale. There was no need for a third reader as the two expert technicians agreed on the initial microscopy result.The number of PfHRP2 RDT supplied by the NMCP and performed by the nurses at health facilities level (HF-PfHRP2) and 59.0% (240/407) with the PfHRP2 RDT purchased by CRUN and performed by trained laboratory technicians at laboratory level (Lab-PfHRP2) (Table PfHRP2 and Lab-PfHRP2 , as well as the number of false positives   was 58.5% 238/407) with the 38/407 wiPfHRP2-RDT supplied by the NMCP and performed by the nurses at health facilities level (HF-PfHRP2) was 97.5% and 52.8%, respectively when using expert microscopy as reference (Table PfHRP2 RDT purchased by CRUN and performed by trained laboratory technicians at laboratory level (Lab- PfHRP2) was 98.4% and 74.2%, respectively (using expert microscopy as gold standard). The positive predictive value of the HF-PfHRP2 was 75.6% and 85.1% for the Lab-PfHRP2 , that between expert microscopy and Lab-PfHRP2 was “good”  and that between HF-PfHRP2 and Lab-PfHRP2 also “good”  (Table 4). Only 49.5% (38/77) of malaria false positives found with HF-PfHRP2 were also tested positive with Lab-PfHRP2  were also tested positive with Lab-PfHRP2  of the RDT was worrying low (52.8%) when the test was performed by the nurses at the health facilities level. This is also reflected in the observed agreement between the tests. Overall, the agreement between expert microscopy and Lab-HRP2 was good, but moderate between HF-RDT and expert microscopy. This could be explained by the fact that the HF-RDT was more often false positive. In general, the specificity of the HRP2-based RDTs is being questioned particularly under low transmission conditions . This isHRP2 persistence after a successful treatment is often used as an explanation for the lower specificity of RDTs that are based on the detection of this specific antigen –19. ThisP. falciparum specific HRP2-based RDT can stand up to 40 °C for 24 months [PfHRP2 RDT.According to the WHO testing report and the RDT manufacturer information note, the 4 months . However4 months . The par4 months , 39, 40.Finally, the blood specimen used to perform the malaria RDTs in the field was a capillary sample and the one used to perform malaria RDTs in the central laboratory was from venous blood. It has been reported that the sensitivity of malaria tests (i.e. microscopy) depend on the site of blood collection, in particular in asymptomatic malaria cases. Capillary blood tends to be more sensitive than venous blood . HoweverRapid diagnostic tests are a valuable tool for the diagnosis of malaria in settings where expert microscopy is not available. However, some external factors could negatively influence the performance of these RDTs in the field. As long as these factors remain, causes of fever might not be correctly diagnosed and results in inappropriate prescription of anti-malarials and antibiotics in fear of overlooking a treatable infection.


Although mobile and migrant populations are considered an important group in malaria elimination settings, there is currently a lack of understanding about foreign migrant workers in the Lao People’s Democratic Republic (Lao PDR). The present study aimed to document the migration characteristics, positive rate of malaria infection, and preventive and treatment-seeking behavior for malaria among foreign migrant workers in the malaria-endemic districts of Savannakhet province, Lao PDR.A community-based survey was undertaken in four districts of Savannakhet province between February and June, 2015. Questionnaire-based interviews and blood examinations, including rapid diagnostic tests and PCR assays, were conducted with 391 migrant workers who were registered at local police departments.Most of the study participants were men (75.7%) and Vietnamese (92.6%). The median age (interquartile range) was 31 (25 to 41) years old. Most common occupation was factory worker (47.6%), followed by trader/shopkeeper (21.5%) and plantation worker/farmer (16.4%). The median length of stay (interquartile range) in the districts was 405 (183 to 1207) days. The majority of the participants (85.9%) had not worked in a province other than the study province, nor had the majority (92.6%) worked in a foreign country other than the Lao PDR. Although most of the participants (62.7%) reportedly used a bed net daily, these nets were mostly conventional untreated ones. No one tested positive for malaria. However, 10.0% of the participants reported a malaria-like illness episode that had occurred in the Lao PDR. The most common measure taken for the episode was to visit a hospital/health center in the Lao PDR, followed by conducting self-medication alone. Forty-one participants reported an experience of working in the forest while living in the Lao PDR.Foreign migrant workers who are registered at local police departments are unlikely to play a major role in maintaining local transmissions and spreading drug-resistant malaria in the study province. However, some of them were involved in forest-related activities, suggesting that these workers are potentially at risk of malaria. The Lao National Malaria Control Program should educate foreign migrant workers about the risk of malaria when living in Lao PDR. Plasmodium falciparum resistance to antimalarial medicines had reached alarming levels in certain areas of the GMS, and the only solution is to eliminate P. falciparum from the GMS. The GMS countries have been working to eliminate all species of human malaria by 2030 [The burdens of malaria have decreased in the Lao People’s Democratic Republic (Lao PDR) in recent years: estimated malaria cases dropped from 51,000 in 2010 to 27,390 in 2016 [The Asian Highway and GMS economic corridors that cut through endemic areas in GMS countries including Lao PDR are contributing to the increased population movement between and within GMS countries . In respThe National Malaria Control Program of the Lao PDR has paid special attention to migrant workers for a number of reasons. First, migrant workers are likely to be involved in spreading drug-resistant malaria from one area to another. Second, migrant workers are considered to be at high risk of contracting malaria. In a large outbreak that occurred in Attapeau province, migrants, both from other provinces in the Lao PDR and from neighboring countries, accounted for the majority of the reported cases , 5. Fina2) in the Lao PDR, it is bordered by Vietnam to the east and Thailand to the west. Approximately 90% of all malaria cases in the Lao PDR occur in the five southernmost provinces, of which Savannakhet is one [Among the 18 provinces of the Lao PDR, Savannakhet province is a major province to which Vietnamese emigrate [There is currently a lack of understanding about foreign migrant workers in the Lao PDR in terms of designing and implementing malaria control strategies to better target them. Therefore, the present study aimed to document the migration characteristics, positive rate of malaria infection, and preventive and treatment-seeking behavior for malaria among foreign migrant workers in the four malaria-endemic districts of Savannakhet province, Lao PDR.The present study was conducted in the four districts of Thapangthong, Nong, Xepon, and Phin in Savannakhet province, whose populations in 2015 were 40,584, 29,622, 56,213, and 65,085, respectively . Prior tIn each district, the research team visited the local police office, where local residents are registered, to collect information on the residences and workplaces of foreign migrant workers. Then, the team visited the residences and/or workplaces of these workers with the help of the local authorities, including village heads. When an eligible foreign migrant worker was absent, multiple attempts were made to contact him/her.The research team included in the present study all of the foreign migrant workers on whom information was provided by the local police office. Foreign migrant workers were excluded if they were aged less than 15 years old or refused to participate in the study. Out of a total of 520 registered foreign migrant workers , 392 participated in the study (participation rate 75.4%). The majority of eligible foreign migrant workers, who were not included, were absent mainly either because they were staying in Vietnam at the time of the visit or because they appeared to be no longer living in the study districts. Of the participants, only one participant who withdrew from an interview survey was excluded. Thus, the present study analyzed the data of 391 participants.n = 77) in the first study district, i.e., Thapangthong district. The reason for adding the questions was that the research team realized the need for collecting information more comprehensively, as no participant was found to be positive by rapid diagnostic test and a few participants reported a malaria-like illness episode in the first district. The blood examination included a rapid diagnostic test  and PCR assay. The PCR assays were performed as described elsewhere [A community-based survey was performed between February and June, 2015. The survey included a questionnaire-based interview with a blood examination of each study participant. The questionnaire was first developed in English and then was translated into Lao, Vietnamese, and Chinese languages. Whenever a participant was able to communicate in Lao language, Lao version questionnaire was used. Healthcare professionals including two fluent Vietnamese speakers, who belong to a Lao governmental institution, worked as surveyors. Prior to data collection, the surveyors were trained for a day on data collection procedures. The questionnaire included questions on migration characteristics, socio-demographic and economic characteristics, behavior for malaria prevention and treatment, general healthcare-seeking behavior, and forest activity. Most of the questions were presented with response options: For example, the response options for most of the questions on preventive behavior were “Everyday,” “Often,” “Sometimes,” and “Never.” Questions pertaining to general healthcare-seeking behavior and forest activity were added to the questionnaire during the data collection stage, meaning that these questions were not asked of participants , gender , occupation , use of bed net , and experience of forest activity (yes or no). The odds ratio (OR) and 95% confidence interval (CI) of the outcome  for each factor were estimated using a mixed-effect logistic regression analysis, with the district-level clustering being treated as a random effect. For the multivariate analysis, all of the factors were entered into the model. These analyses were performed using the STATA MP 12 software program .Bivariate and multivariate analyses were carried out to examine factors associated with each participant’s self-reported malaria-like illness episode that occurred in the Lao PDR. Factors examined in the analyses were age .Most of the participants (75.7%) were men, with a median age of 31 years old  or by a family member 33.2%) (Table % (Table Although most of the participants (62.7%) used a bed net daily, non-users (10.2%) and infrequent users (7.2%) also existed Table . The usen = 253, 73.3%) were conventional non-treated nets (data not shown in tables). Only 49 (14.2%) of the participants used an insecticide-treated net. The insecticide-treated status was unclear for the 43 (12.5%) remaining bed nets. Over half of the bed nets used were obtained in Vietnam , whereas fewer bed nets were obtained in the Lao PDR  and Thailand . The place of origin of 102 (29.6%) bed nets was unclear.Of the 345 bed nets that the participants used, most , followed by conducting self-medication alone  and taking a rest alone . Among the 24 participants who visited a hospital/health center, nearly half  conducted self-medication prior to the visit.Thirty-nine participants (10.0%) reported a malaria-like illness episode that occurred while living in the Lao PDR Table . The mosn = 28, 71.8%) who reported a malaria-like illness episode had not worked in a province other than the study province (data not shown in tables).The majority of the participants  reported that they had not experienced seeking healthcare service from Lao medical staff . In response to the hypothetical question “If you get sick, then would you like to utilize a Lao hospital/health center?”, 61.8% of the participants answered “Yes,” whereas 18.2% answered “No” and 18.5% answered “Not sure.”Of the 314 participants from the Xepon, Nong, and Phin districts, the majority , plantation building , swidden , seeking forest products , and others .Of the 314 participants from Xepon, Nong, and Phin districts, 41 (13.1%) reported an experience of working in the forest while living in the Lao PDR (data not shown in tables). The objectives of the forest work were wood cutting  and forest activity , whereas the association between malaria-like illness episodes and occupation (construction workers/wood cutters) became insignificant in the multivariate analysis .Plasmodium infections. One study that was conducted with 891 villagers in 10 villages in Xepon district reported a positive rate of 6% [In the present study involving 391 foreign migrant workers from Thapangthong, Xepon, Nong, and Phin districts in 2015, no participants tested positive for malaria, even though a number of studies that were conducted with the general Laotian population in these districts between 2013 and 2015 reported substantial positive rates of te of 6% . TherefoNone of the participants in the present study were infected with malaria at the time of blood examination. A possible explanation for the absence of positive participants is that the present study did not include unregistered/illegal foreign migrant workers, of whom there are estimated to be 24,000 among the 54,000 foreign migrants in the Lao PDR . These wA further study is necessary that targets unregistered/illegal foreign migrant workers in the Lao PDR. Sampling will be a challenge, however, because these workers are hard to reach. A study conducted in Thailand to explore knowledge, perception, and practices pertaining to malaria  includedAmong the study participants, 62.7% used a bed net daily. This percentage is lower than those reported from studies conducted with the general Laotian population in the same districts: 82.7% in villages with high incidence and 82.3% in villages with low incidence of malaria in Xepon  and 76.8In the present study, 10% of the participants reported a malaria-like illness episode that had occurred in the Lao PDR. Because 86% of the participants had no experience of working in a province other than the study province, they may have contracted malaria-like illness in the study province. Some of these malaria-like illness episodes could be due to malaria because such illness episodes were significantly associated with the involvement in forest-related activities. In the study province, involvement in forest-related activities is known to be a risk factor of malaria , 16.Approximately 95% of the study participants had reportedly never experienced malaria in their mother countries. Thus, they may not have been aware of the risk of malaria while living in the Lao PDR. Foreign migrant workers who live in malaria-endemic districts in the Lao PDR should be educated about the risk of contracting malaria when working in the forest in the Lao PDR.Approximately 60% of the participants used a Lao healthcare facility such as a health center or hospital for treatment of a self-reported malaria episode. This percentage could be reasonable because more than half of the participants had been living in the Lao PDR for over a year and thus might not have had a critical language barrier, which is known to be a major barrier for migrants for the use of the healthcare services of a host country –19. AnotThe present study has some limitations. First, because the survey period did not coincide with the high transmission season (often between June and August) , the preThe foreign migrant workers who are registered at local police departments are unlikely to play a major role in maintaining local transmissions and spreading drug-resistant malaria in the study province. However, some of them were involved in forest-related activities that were significantly associated with malaria-like illness episodes, suggesting that they are potentially at risk of malaria. For treatment of these episodes, the majority of the study participants used a local Lao healthcare facility. The use of a bed net was common, although their nets were mostly conventional untreated ones. Because 94.6% of the participants had not experienced malaria in their mother countries, they may not be very aware of the risk of malaria while living in the Lao PDR. Therefore, the Lao National Malaria Control Program should educate foreign migrant workers about the risk of acquiring malaria in the Lao PDR. Because the present study included only registered foreign migrant workers, a further study that targets unregistered foreign migrant workers is also warranted.


Plasmodium vivax than P. falciparum. Early radical cure and prophylaxis are warranted in malaria endemic areas.In Papua, splenectomized individuals have greater risk of malaria in the 12 months following splenectomy but not of mortality. Malaria risk was higher for  Plasmodium vivax malaria is unknown. We quantified the Plasmodium species-specific risks of malaria and other outcomes following splenectomy in patients attending a hospital in Papua, Indonesia.Splenectomy increases the risk of severe and fatal infections; however, the risk of Records of all patients attending Mitra-Masyarakat Hospital 2004–2013 were reviewed, identifying those who underwent splenectomy. Subsequent risks of specific clinical outcomes within 12 months for splenectomized patients were compared to nonsplenectomized patients from their first recorded hospital admission. In addition, patients splenectomized for trauma 2015–2016 were followed prospectively for 14 months.P < .001). The AHR was 7.8 (95% CI: 5.0–12.3) for P. vivax and 3.0 (95% CI: 1.7–5.4) for P. falciparum (both P < .001). Splenectomized patients had greater risk of being hospitalized for any cause  and diarrheal . In the 14-month prospective cohort, 12 episodes of P. vivax and 6 episodes of P. falciparum were observed in 11 splenectomised patients.Of the 10774 patients hospitalized during 2004–2013, 67 underwent splenectomy. Compared to nonsplenectomized inpatients, patients undergoing splenectomy had a 5-fold higher rate of malaria presentation within 12 months : 3.4–7.3], P. vivax than P. falciparum. Eradication of P. vivax hypnozoites using primaquine  and subsequent malaria prophylaxis is warranted following splenectomy in malaria-endemic areas.Splenectomy is associated with a high risk of malaria, greater for  Plasmodium and Babesia ) between July 2015 and November 2016 were followed from their day of splenectomy for 14 months. Malaria encounters were recorded by monitoring visits to healthcare facilities and conducting monthly interviews. Patients were checked for peripheral parasitemia on the day of surgery and were treated according to local treatment guidelines. Data from these patients are presented using descriptive statistics only.This study was approved by the Ethics Committees of the University of Gadjah Mada , Indonesia, and Menzies School of Health Research, Australia . In the retrospective cohort, hospital records were anonymized with informed consent not requested because data were collected as part of routine hospital surveillance. Informed consent was obtained in the prospective cohort.Between April 2004 and December 2013, 162966 patients attended RSMM hospital of whom 56458 (34.6%) were admitted at least once and 67 patients underwent splenectomy . A totalOf the 67 splenectomized patients, 43 (64.2%) were male, 44 (65.7%) were highland-Papuans, and 4 (6.0%) were lowland-Papuans; The nonsplenectomized patients had similar demographics to that of the cases, with a median age at first admission of 26.8 , children compared to adults , and highlanders compared to non-Papuans . The risk of representing with malaria was also elevated in those presenting initially with malaria and this was apparent for all species; In total, 56.1% (6041/10774) of patients represented to hospital within 4 years following initial presentation, of whom 86.2% (5208/6041) did so within 12 months. The overall risk of representing with any malaria within 12 months was 13.5%  and was greater in females compared to males  with P. vivax within 12 months of splenectomy compared to 8.4% (901/10707) and 4.9% (523/10707) in the control group, respectively (P < .001). The risk of malaria was similar in the subgroup of 50 patients splenectomized due to trauma . Having detectable parasitemia at initial presentation was a risk factor for representing with any malaria in the control group  but not in the splenectomy group . In total, 70.0% (20/29) of the first malaria episodes post-splenectomy occurred within the first 3 months, compared to 48.8% .Overall 43.3% (29/67) of splenectomized patients represented with malaria within 12 months of surgery compared to 13.3% (1426/10707) of those in the control group , and this was more apparent with P. vivax  compared to P. falciparum  of those with P. vivax and 0% (0/2) of those with mixed species infections.After controlling for confounding factors, splenectomy was a significant risk factor for hospital representation with any malaria  compared to that for P. falciparum  per thousand patient-years in splenectomy patients compared to 222 (95% CI: 213–232) per thousand patient-years in the nonsplenectomy controls  in splenectomy patients compared to 48.1% (5153/10707) in controls  patients were admitted due to trauma and were considered nonsplenectomy trauma controls . BaselinP. vivax and 3 P. falciparum) and were given antimalarial treatment. In the 14 months following splenectomy, 8 of 11 (72.7%) patients returned with 18 episodes of symptomatic microscopically confirmed malaria: 8 (72.7%) patients with 12 episodes of P. vivax and 6 (54.5%) with 6 episodes of P. falciparum infection, none severe. Four of these 8 patients (50%) returned within 3 months of surgery, on day 8 (P. vivax), day 18 , day 32  and month 3 (P. vivax), respectively. At 14 months of follow-up no patient had died.Eleven patients underwent splenectomy at RSUD Hospital, all due to trauma, of which 9 (81.8%) were male, 6 (54.5%) were highland-Papuans, and 2 (18.2%) were lowland-Papuans . The medP. vivax compared to P. falciparum. These retrospective findings were supported by data from a prospective cohort of 11 patients followed after splenectomy. Splenectomy patients were at increased risk of representation due to any cause, admission with diarrheal illness or pneumonia, but not mortality or other causes of infection.Our large observational study quantifies the risk of morbidity and mortality in splenectomized individuals living in a malaria-endemic region. Splenectomized patients were at 5-fold higher risk of malaria within 12 months of surgery, with the risk being far greater for Several case reports and small series highlight an increased risk of malaria after splenectomy; however, these reports are limited to no more than 33 splenectomized individuals , 22–26. P. vivax has a greater propensity for splenic pooling [P. vivax recurrence compared to P. falciparum. Interestingly, after splenectomy a higher proportion of patients in the retrospective study had more than one episode of P. falciparum compared to P. vivax, suggesting that P. vivax attacks may have originated from preexisting infections or relapses that were then cleared, whereas P. falciparum recurrences were attributable to an increased susceptibility to new infections.The greatest risk of malaria occurred in the first 3 months following splenectomy. In 2005 the incidence of malaria in Papua was approximately 876 per 1000 patient-years . This co pooling , this coP. falciparum attacks or P. vivax relapses after surgery do not differ from the overall population or from pre-surgery [P. falciparum in trauma patients undergoing primary surgery [A third explanation for the increased risk of malaria is that attributable to trauma itself. The process of splenectomy is known to be followed by malaria in previously asymptomatic individuals . Further-surgery . Only on surgery . Only 11 surgery ; thus, sP = .085 and .350, respectively). Our study was also underpowered to determine an increased risk of mortality following splenectomy, with only 4 (6.0%) patients known to have died within 12 months of splenectomy. The only outcome that reached statistical significance was a 3.5-fold increased risk of diarrheal admission. Salmonellosis is caused by encapsulated bacteria, and splenectomy patients are known to be at a high risk of disease [Previous studies have identified higher risk of sepsis including pneumonia following splenectomy , 37, 38. disease , 40; howOur study has several limitations. Although our analysis was observational and free from coercive biases associated with many clinical study designs, our data are subject to the effects of residual confounding. Retrospective analyses were confined to RSMM presentations and did not consider presentations at other health facilities or emigration from the catchment area during the follow-up period. However, it is likely that this attrition bias would apply equally to the cases and controls. Furthermore, our findings were confirmed in the 11 patients followed prospectively. In the retrospective analysis, malaria occurring within 14 days of splenectomy was not captured and diagnosis by the attending physician was determined without microbiological testing.P. vivax than for P. falciparum. Current guidelines recommend that patients undergoing splenectomy should be offered vaccinations and lifelong prophylactic and standby antibiotics. Our study suggests that in malaria-endemic regions these individuals should also be offered early radical cure of malaria with an artemisinin combination therapy plus, in vivax-endemic regions, 14 days primaquine. In addition, malaria prophylaxis should be offered thereafter to prevent reinfection.In conclusion, splenectomized individuals in a malaria- endemic area are at 5-fold greater risk of malaria within 12 months of surgery, with 70% of events occurring in the first 3 months. The risk is greater for Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary MaterialClick here for additional data file.


Plasmodium knowlesi has added to the challenges of prompt and accurate malaria diagnosis and surveillance. In this study from Aceh Province, Indonesia, a malaria elimination setting where P. knowlesi endemicity was not previously known, we report the laboratory investigation and difficulties encountered when using molecular detection methods for quality assurance of microscopically identified clinical cases. From 2014 to 2015, 20 (49%) P. falciparum, 16 (39%) P. vivax, 3 (7%) P. malariae, and 2 (5%) indeterminate species were identified by microscopy from four sentinel health facilities. At a provincial-level reference laboratory, loop-mediated isothermal amplification (LAMP), a field-friendly molecular method, was performed and confirmed Plasmodium in all samples though further species-identification was limited by the unavailability of non-falciparum species-specific testing with the platform used. At a national reference laboratory, several molecular methods including nested PCR (nPCR) targeting the 18 small sub-unit (18S) ribosomal RNA, nPCR targeting the cytochrome-b (cytb) gene, a P. knowlesi-specific nPCR, and finally sequencing, were necessary to ultimately classify the samples as: 19 (46%) P. knowlesi, 8 (20%) P. falciparum, 14 (34%) P. vivax. Microscopy was unable to identify or mis-classified up to 56% of confirmed cases, including all cases of P. knowlesi. With the nPCR methods targeting the four human-only species, P. knowlesi was missed (18S rRNA method) or showed cross-reactivity for P. vivax (cytb method). To facilitate diagnosis and management of potentially fatal P. knowlesi infection and surveillance for elimination of human-only malaria in Indonesia and other affected settings, new detection methods are needed for testing at the point-of-care and in local reference laboratories.The discovery of the life-threatening zoonotic infection  Plasmodium knowlesi, a malaria parasite of macaques, was recently discovered to infect humans. This emerging disease is important because it has potential for causing severe disease and death, and it is a threat to malaria elimination efforts in the region. In this report from Aceh Province, Indonesia, where P. knowlesi was only recently discovered, the authors report on the laboratory challenges of distinguishing this species from other human species. Using several different molecular methods, they investigated 41 malaria cases which by microscopy, were initially reported as: P. falciparum (49%), P. vivax (39%), P. malariae (7%), and indeterminate (5%). Only after using a P. knowlesi-specific nPCR method and sequencing, did they find that nearly half were P. knowlesi. Consistent with a sparse literature, a field-friendly molecular method (genus-specific LAMP) reliably detected P. knowlesi, while use of a more standard reference laboratory molecular method (18S rRNA nPCR targeting the four human-only species) missed the infections. Also another reference laboratory molecular method (cytb nPCR) mis-classified P. knowlesi infections as P. vivax due to cross-reactivity. To address the emerging threat of P. knowlesi, new detection methods are needed for point-of-care and reference testing.In Southeast Asia,  Plasmodium knowlesi is a newly emergent zoonotic human malaria species previously thought to only infect macaques. Since the first report of a human case from Peninsular Malaysia in 1965 [ in 1965 , endemic in 1965 –5, and m in 1965 .P. knowlesi infection is important for clinical and public health reasons. Infection in humans is most often uncomplicated, but 6–9% of symptomatic patients develop severe malaria and 0.3–1.8% of cases die [P. knowlesi infection is associated with a number of different risk factors than infections caused by other Plasmodium species [The identification of ases die . Fatal oases die , 12. Fro species   as P. falciparum, and the remaining 34 unspecified or mixed P. falciparum/P. vivax [The study was conducted in Aceh Besar District, Aceh Province, Sumatra island, Indonesia, a low-transmission setting that aims to eliminate malaria by 2020. The 2013 incidence of malaria was 0.4/1000, and 68 (39%) of cases were reported as After written consent was obtained and prior to treatment, venous blood was collected and partly used to prepare dried blood spots (DBS) using Whatman 3MM paper. DBS along with remaining whole blood were initially stored at 4°C, transferred to -20°C within a week of collection, and then stored at -80°C. Antimalarial treatment was based on microscopy results and according to Indonesian government’s national policy.Ethical approval for the study was obtained from the National Institute of Health, Research and Development of the Indonesian Ministry of Health (number LB.02.01/5.2/KE.111/2014 and LB.02.01/5.2/KE.211/2015) and IRB Committee of the University of California, San Francisco. Written informed consent was obtained from all adults or a parent or guardian for participants less than 18 years of age.P. falciparum and P. vivax malaria before the study was launched [P. vivax.For quality assurance of microscopy performed at health centers, blood smears were re-read by certified microscopists at the provincial laboratory according to national guidelines. For further quality assurance at the provincial-level, LAMP was selected due to its field-friendly platform. Initial extraction of DNA and LAMP testing were performed at the provincial laboratory. DNA was extracted from DBS using the Saponin/Chelex method . Pan-LAMlaunched . As suchcytb gene followed by AluI enzyme digestion for species identification of the four main human species was used initially, as previously described [P. knowlesi by a field microscopist, as well as limited data on the performance of the cytb nPCR method for detection of P. knowlesi, additional methods were employed including nPCR testing targeting the 18S rRNA gene for the four human-only species [P. knowlesi-specific nPCR [P. knowlesi specific nPCR, DNA was extracted from whole blood using the QIAamp DNA Mini kit  and Sanger targeted genome sequencing [Further molecular testing was performed at the Malaria Pathogenesis laboratory at the Eijkman Institute in Jakarta, using chelex-extracted DNA from a second DBS. Genus-specific PCR targeting the mitochondrial escribed . After a species , and P. fic nPCR  for all quencing  was perfP. falciparum and P. vivax classification were based on species-specific positivity by both cytb and 18S rRNA nPCR, and P. knowlesi classification was based on genus-specific PCR positivity by both cytb and 18S rRNA nPCR and P. knowlesi-specific nPCR positivity. With regards to diagnostic performance for species identification, we were not able to calculate sensitivity, specificity, or negative predictive value (NPV) due to having not included a representative sample of microscopy-negative infections. However, positive predictive values (PPV) were calculated.Results from microscopy and each molecular method were compared to a gold standard established through serial molecular testing: P. vivax by microscopy) was excluded as the DBS had insufficient blood for subsequent molecular testing. The 41 cases included: 20 P. falciparum (49%), 16 P. vivax (39%), 3 P. malariae (7%), and 2 with indeterminate morphology (5%) , 32,157  and 3,842 . The parasite densities of the indeterminate samples were 803 and 1,473, respectively. Microphotography of the indeterminate samples showed resemblance to other species  . Parasit species .cytb PCR genus-specific testing and using the AluI restriction digest reaction for species identification, 8 (19.5%) were classified as P. falciparum, 33 (80.5%) as P. vivax. By 18S rRNA nPCR, there were 8 P. falciparum (19.5%), 14 P. vivax (34.1%), and 19 (46.3%) did not amplify. P. knowlesi-specific nPCR was positive in 19/41 (46.3%), of which 11 underwent sequencing and showed 100% identity to a published P. knowlesi 18S rRNA gene sequence .Genus-specific Pan-LAMP testing at the provincial laboratory was positive in all 41 isolates , P. vivax (n = 6), or P. malariae (n = 3), and 2 were unable to be classified. There were also 4 P. vivax cases that were mis-classified as P. falciparum by microscopy. Sixty percent (12/20) of cases identified by microscopy as P. falciparum were either P. vivax or P. knowlesi; 37.5% (6/16) of cases identified by microscopy as P. vivax were P. knowlesi. All P. malariae and indeterminate species by microscopy were P. knowlesi.Microscopy was unable to classify or mis-classified 23 of 41 (56%) malaria cases confirmed by the gold standard of serial molecular testing . These iP. knowlesi mono-infection as P. falciparum but otherwise correctly identified all the P. falciparum cases.Genus-specific testing by LAMP identified all infections, though species identification was limited by the unavailability of non-falciparum species-specific testing with the platform used. Pf-LAMP testing mis-classified one P. vivax by cytb PCR, 58% (19/33) were later confirmed as P. knowlesi and showed a similar banding pattern to P. vivax  for species identification by different diagnostic methods using the gold standard of serial molecular testing are shown in P. knowlesi in Indonesia outside Borneo, and an epidemiological investigation revealed that forest exposures are a key risk factor for this zoonotic infection [P. knowlesi cases as P. malariae or P. falciparum, as commonly reported elsewhere, but also as P. vivax, which has been less commonly reported [P. knowlesi infection was not straightforward. All P. knowlesi cases did not amplify with a standard nPCR method (18S rRNA) targeting the four human-only species. With the cytb method, there was cross-reactivity with P. vivax for all P. knowlesi cases. We highlight the difficulties of P. knowlesi diagnosis at the point-of-care and reference laboratory levels in a setting where endemicity was not previously known and bring attention to an emerging challenge for malaria elimination.To support malaria diagnosis and surveillance in Aceh Province, a low transmission setting in Indonesia that is aiming for malaria elimination, we utilized molecular testing for quality assurance of microscopy-confirmed cases from health facilities. As previously published, this work resulted in the first report of nfection . In thisreported . The PPVP. knowlesi, a fifth human species previously thought to only infect macaques, has created an additional challenge for species identification. Microscopy is difficult because the morphology at different stages resembles other malaria species [P. knowlesi detection is poor, leaving no other useful point-of-care diagnostic test [P. knowlesi [P. knowlesi. In our study, the investigation into P. knowlesi was prompted by the observation by an astute microscopist of unusual morphology in two malaria cases, as well as the known local presence of pig-tailed and long-tailed macaques and Anopheles leucosphyrus, a known vector on Sumatra island [The recent discovery and emergence of  species . The diatic test –29. Despknowlesi , this ina island .P. knowlesi-specific commercial kit was not available for use in our study, and evaluations of other P. knowlesi-specific LAMP assays have reported cross-reactivity with P. vivax [P. knowlesi-specific PCR method utilized in this study did not cross-react with P. vivax infections, with excellent specificity as observed previously [P. knowlesi either does not amplify (as occurred in this study) or is mis-classified as P. vivax due to high sequence homology at the target sequences [cytb nPCR method that we used, our finding of cross-reactivity between P. knowlesi and P. vivax has not been previously reported, but can also be explained by high sequence homology at the target mitochondrial sequences. Others have reported P. knowlesi amplification using P. vivax-specific PCR targeting the mitochondrial gene cox1 [P. knowlesi detection in mixed species settings have recently been developed [For quality control in reference laboratories, none of the nucleic acid-based methods for both genus and species-specific identification were found to be suitable. With LAMP, a molecular detection method that has been promoted for use in resource-limited settings due to the rapid turnaround time and simple methods, genus-specific testing was reliable, as has been reported from Malaysia . HoweverP. vivax . The P. eviously . The nPCequences , 32. Witene cox1 . Other meveloped , 34, 35 P. knowlesi detection is of both clinical and public health significance. In Malaysia, where the clinical disease has been well studied, P. knowlesi is associated with at least as high a risk of severe disease compared with P. falciparum [P. malariae” as “P. knowlesi”, and enhanced implementation of standardized referral and clinical protocols, including first-line use of artemisinin-based combination therapy and early intravenous artesunate [P. knowlesi as P. vivax, as occurred at the point of care in our study, also results in unnecessary treatment with primaquine, an antimalarial not indicated for P. knowlesi, but necessary for radical cure of the latent liver stages with P. vivax. In our study, we did not experience any severe adverse events from the unnecessary use of primaquine, but use in subjects with underlying severe glucose-6-phosphate dehydrogenase deficiency is known to be associated with life-threatening hemolysis.The challenge of accurate lciparum  and in elciparum , 37. Follciparum . These htesunate , 36. ProP. knowlesi distribution and incidence elsewhere in Indonesia, particularly across Kalimantan, Sulawesi and other regions of Sumatra, where modelling predicts a high risk of human infection [P. knowlesi is critical to the design and implementation of effective malaria interventions. In a related study in Aceh Province and also in Malaysia, adult males with forest-related and agricultural occupational exposure are at significantly higher risk of being infected with P. knowlesi [P. knowlesi-transmitting mosquitos, the interface between humans and macaques, and to individual risk factors for infection identified in different settings. Further investigation into the epidemiology and transmission of P. knowlesi in Aceh Besar is needed.While only recently recognized in areas of Aceh and North Sumatra, there has been little molecular surveillance of nfection . From a knowlesi , 13. IntP. knowlesi are well established. Our challenges using LAMP and PCR for species identification in a setting with previously unknown P. knowlesi endemicity add to a growing literature on the limitations of molecular methods as well. For settings approaching malaria elimination and/or where epidemiological conditions are predicted to support P. knowlesi transmission to humans, quality assurance of malaria diagnosis and species identification is essential, but at present, practical and accurate methods are not available for local and peripheral reference laboratories. Development, evaluation and implementation of improved P. knowlesi detection methods for use at both the point-of-care and in local reference laboratories are needed.Limitations of microscopy to identify