Plasmodium vivax has been recently documented, the transmission of the two minor Plasmodium species, Plasmodium malariae and Plasmodium ovale spp., is poorly characterized in the region. This study aims to determine the prevalence of these minor species in the China–Myanmar border area and their genetic diversity.The Greater Mekong Subregion is aiming to achieve regional malaria elimination by 2030. Though a shift in malaria parasite species predominance by Plasmodium malariae and P. ovale infections were confirmed by sequencing the PCR products. The P. ovale subtypes were determined by sequencing the Pocytb, Pocox1 and Pog3p genes. Parasite populations were evaluated by PCR amplification and sequencing of the MSP-1 genes. Antifolate sensitivity was assessed by sequencing the dhfr-ts and dhps genes from the P. malariae and P. ovale isolates.Epidemiology study was conducted during passive case detection in hospitals and clinics in Myanmar and four counties in China along the China–Myanmar border. Cross-sectional surveys were conducted in villages and camps for internally displaced persons to determine the prevalence of malaria infections. Malaria infections were diagnosed initially by microscopy and later in the laboratory using nested PCR for the SSU rRNA genes. Plasmodium falciparum, 327 P. vivax, 66 P. falciparum/P. vivax mixed infections, 4 P. malariae and 3 P. ovale spp. P. vivax and P. falciparum accounted for >60 and ~30% of all malaria cases, respectively. In comparison, the prevalence of P. malariae and P. ovale spp. was very low and only made up ~1% of all PCR-positive cases. Nevertheless, these two species were often misidentified as P. vivax infections or completely missed by microscopy even among symptomatic patients. Phylogenetic analysis of the SSU rRNA, Pocytb, Pocox1 and Pog3p genes confirmed that the three P. ovale spp. isolates belonged to the subtype P. ovale curtisi. Low-level genetic diversity was detected in the MSP-1, dhfr and dhps genes of these minor parasite species, potentially stemming from the low prevalence of these parasites preventing their mixing. Whereas most of the dhfr and dhps positions equivalent to those conferring antifolate resistance in P. falciparum and P. vivax were wild type, a new mutation S113C corresponding to the S108 position in pfdhfr was identified in two P. ovale curtisi isolates.Analysis of 2701 blood samples collected from the China–Myanmar border by nested PCR targeting the parasite SSU rRNA genes identified 561 malaria cases, including 161 P. vivax has become the predominant species, the two minor parasite species also occurred at very low prevalence but were often misidentified or missed by conventional microscopy. These minor parasite species displayed low levels of polymorphisms in the msp-1, dhfr and dhps genes.The four human malaria parasite species all occurred sympatrically at the China–Myanmar border. While The online version of this article (doi:10.1186/s12936-016-1605-y) contains supplementary material, which is available to authorized users. Plasmodium species  and the zoonotic parasite Plasmodium knowlesi, which is found in many Southeast Asian countries [P. falciparum is also the most prevalent parasite in Africa, while P. vivax is the most widely distributed parasite outside of Africa [P. malariae and P. ovale spp. are much less prevalent and significantly under-studied. Plasmodium malariae is more or less sympatric with P. falciparum in distribution, mainly found in sub-Saharan Africa and southwest Pacific [P. ovale spp. were thought to have a much more limited distribution and mostly found in Africa and some islands of the west Pacific [P. vivax, and occurrence as mixed infections with the major parasite species [Plasmodium infections [P. ovale spp. into two distinct subspecies: P. ovale curtisi  and P. ovale wallikeri  [Poc and Pow are genetically distinct but morphologically indistinguishable [Malaria in humans is caused by four main ountries . The mosf Africa . Compare Pacific , 4. In c Pacific . Most pu species –10. The fections . Moleculnt type) . The nucuishable . Furtheruishable –17) as wuishable .Plasmodium parasites infecting humans co-exist [P. vivax and P. falciparum are the predominant parasite species. In contrast, other parasite species have been detected at much lower prevalence. Plasmodium malariae infections have been described in Cambodia [P. malariae may exhibit high genetic diversity [P. ovale spp. have been found in all nations of the GMS including Cambodia [P. ovale-like case was described in 1941 in Yunnan [P. ovale spp. reported in China in recent years were imported, mainly from Africa [P. ovale spp. were identified in autochthonous as well as imported cases [In the Greater Mekong Subregion (GMS) of Southeast Asia, which includes Cambodia, China, Laos, Myanmar, Thailand, and Vietnam, recent achievements in malaria control have motivated countries within this region to plan for regional malaria elimination by 2030. In this region, all five co-exist , and P. Cambodia , China [Cambodia , 23, LaoCambodia , MyanmarCambodia , 25, ThaCambodia , 26–28, Cambodia , 29, 30.iversity . SimilarCambodia , 22, LaoCambodia , 32, MyaCambodia , 33, 34,Cambodia , 26, 27,Cambodia . Althougn Yunnan , most, im Africa , 38. Boted cases .P. malariae and P. ovale infections often present with a low parasitaemia and occur as mixed infections with P. falciparum and P. vivax [In the final stage of malaria elimination, new strategies tailored for rapid identification of new cases, prevention of local spread, and efficient management of malaria introduction are critical. Especially, malaria in the GMS is highly heterogeneous with transmission foci located along international borders . Hence, P. vivax , 33, molThis molecular epidemiology study was carried out in two study sites on both sides of the China–Myanmar border. In Myanmar, passive case detection (PCD) was performed in a hospital located in the Laiza Township and several nearby malaria clinics, Kachin State, between May 2011 and December 2012. In 2012, to obtain malaria prevalence information, cross-sectional surveys (CSS) were conducted in ten surrounding villages. In addition, active case detection (ACD) was also conducted through weekly (April–September) or biweekly (October–March) home visits to assess clinical malaria. From 2010, both ACD and PCD activities were started in three nearby camps for internally displaced people (IDP), which were newly established as a result of internal military strife. The Myanmar study site is located in one of the designated Special Zones , where mIn Kachin, 1106 blood samples were obtained during PCD activities from febrile patients attending the hospital and malaria clinics, and 638 were from participants of two CSS . Nested PCR was performed to screen malaria infectious from these 2701 blood samples. Primers rPLU5 and rPLU6 were used for the primary PCR, whereas primers rFAL1/2, Pv18SF/R, Pm18SF/R, Po18SF/R, and Pk18SF/R, specific for each of the four human malaria parasite species as well as sted PCR . The PCRP. malariae and P. ovale isolates, several genes were amplified and sequenced using primers listed in Additional file cytochrome b (cytb), cytochrome oxidase subunit 1 (cox1) and glyceraldehyde-3-phosphate (g3p) genes of P. ovale spp., dihydropoteroate synthase (dhps) of P. malariae [dihydrofolate reductase (dhfr) and MSP-1 genes in both species [To further characterize the malariae , dihydro species –46. AmplP. malariae and P. ovale spp. were retrieved from the GenBank and PlasmoDB (http://www.plasmodb.org) for phylogenetic analysis. GenBank accession numbers of the genes included in sequence analysis are shown in Additional file Sequences were searched in the GenBank by using the BLAST program. Orthologous sequences of Plasmodium infections  determined by microscopy. For the CSS efforts, PCR detected 2.9% of participants carrying Plasmodium infections, an increase of 1% compared with that of microscopic diagnosis. In Yunnan, PCR also improved the RDT diagnosis sensitivity by 7.8%. In comparison, there were 41 discrepancies between the results of microscopy and PCR . Of 26 cases diagnosed as mixed-species infections by microscopy, 22 were P. falciparum and 4 were P. vivax single infections. In addition, nine slide-positive cases were identified as infections by a different species  in PCR-confirmed positive cases. In total, infections by the two minor Plasmodium species only made up 0.26% (7/2701) of all febrile cases at the hospitals and clinics. It is also noteworthy that all P. malariae and P. ovale cases were from PCD surveys. Most P. malariae and P. ovale patients presented with fever at the time of examination (>37.5 °C) and had 2–5 days of fever histories  and Pocox1 (861 bp), and one nuclear gene Pog3p (359 bp) from the three clinical isolates we amplified, sequenced and compared with the reference sequences  P. malariae isolates  and two (2/3) P. ovale isolates (C0100511 and M0500214), and sequenced. The assembled PmMSP-1 sequences were 5160 bp (M0N00648) and 5150 bp (M0N00290 and M0N00556) in length, whereas the PoMSP-1 sequences were both 5157 bp. Alignment of the PmMSP-1 sequences with the full-length sequence of a Cameroonian isolate MM1A (GenBank #FJ824669) [PmMSP-1 sequences from this study with the sequences of 11 Brazilian isolates by fragments showed that fragment 2 sequences corresponding to amino acids 637–858 in the MM1A sequence were the most polymorphic with 87.3–88.9 and 82.3–83.6% identities in the nucleotide and amino acid sequences, respectively   revealedPomsp-1 sequences encoding 1718 amino acids were 100% identical. They were >98% identical in nucleotide sequence with the Pocmsp-1 sequence from the Cameroonian isolates OM1A and OM1B (GenBank #FJ824670 and FJ824671), and ~91% identical with the Powmsp-1 from the Thai isolates . Comparison of the amino acid sequences showed a similar trend. Compared with the PoMSP-1 gene from the Thai isolate Po-7 (GenBank No. KC137346) [PoMSP-1 was observed among the M0500214, C0100511 and five Poc isolates  from Thailand.The two C137346) , there adhfr-ts gene from the four P. malariae isolates and three Poc isolates were amplified and sequenced. The four P. malariae dhfr-ts genes ranged in size from 1678 to 1779 bp, encoding proteins of 559–592 amino acids and corresponding to amino acids 13–604 of the PmDHFR-TS from the Thai isolate Pm3 (GenBank #EF188271) [The F188271)  (AdditioF188271) , 50. TwoF188271) , 51 werePodhfr-ts gene ranged from 1673 to 1757 bp and encode proteins of 557–585 amino acids, corresponding to amino acids 24–608 of the PocDHFR-TS from the Indian isolate Po2003 (GenBank #KP050405) [(Table The three sequences of the P050405)  (AdditioPmdhps gene was successfully amplified from the four P. malariae isolates. The partial Pmdhps gene was 962 bp in length and encoded 320 amino acids, corresponding to amino acids 347–659 in PfDHPS. The four PmDHPS sequences from the China–Myanmar border area isolates were identical to the PmDHPS haplotype 1 reported earlier [The  earlier  (AdditioP. vivax has become the predominant parasite species followed by P. falciparum [P. malariae and P. ovale spp. were detected at very low frequencies in patients presenting in hospitals and clinics with acute febrile illness. It is important to note that three of the four P. malariae cases and one P. ovale case were misidentified as P. vivax infections by microscopy and treated with the chloroquine/primaquine combination, whereas two P. ovale cases were missed by microscopy and thus were not treated with anti-malarial drugs. Furthermore, PCR also identified more mixed-species infections, which may not have been treated properly depending on the results of the initial diagnosis. Thus, more sensitive diagnosis may well be needed for “border malaria” in order to completely eliminate malaria transmission.Accurate knowledge of the malaria epidemiology is essential for guiding control and elimination efforts. This study employed both ACD and PCD along the international borders between China and Myanmar, and used a molecular detection method in order to more accurately determine the infection prevalence among febrile patients and asymptomatic residents, and parasite species compositions. In this border region, malaria is seasonal with all four human malaria parasites. While lciparum , 53, thedhfr genes in Poc and Pow were highly conserved in codons potentially mediating pyrimethamine resistance, although isolates carrying double A15S and S58R mutations were observed in imported cases in Singapore [P. malariae cases and three P. ovale cases detected in this study did not show mutations at equivalent residues responsible for antifolate resistance in other Plasmodium, while two new mutations at positions L137P and K161R were observed in one P. malariae isolate and four new mutations were observed at positions T62A, S113C, Y231H and K370E in Podhfr. It is noteworthy that the S113C mutation in the Pocdhfr corresponds to amino acid S108 in Pfdhfr, the primary position associated with pyrimethamine resistance [Poc. Similarly, Pmdhps gene also showed limited genetic diversity and all four isolates from the China–Myanmar border were identical to the PmDHPS haplotype 1, which was most prevalent in Asian countries [Published data to date showed that the ingapore , 52. Thesistance , 54, butountries .Plasmodium species. P. falciparum and P. vivax MSP-1 genes display extensive genetic diversity, which is often used as a molecular marker for population studies. To date, limited studies on PmMSP-1 among Brazilian isolates and PocMSP-1 on Thai isolates detected low-level sequence diversity with most of variations located at interspecies variable domains of this gene [PmMSP-1 using the maximum likelihood method placed the China–Myanmar border isolates separated from the Brazilian isolates, indicating divergent parasite populations. Furthermore, the PocMSP-1 sequences from the China–Myanmar border isolates were highly conserved and also displayed a high level of similarity with the PocMSP-1 from the Thai isolates. Apparently, future studies with larger populations of the two minor parasite species are warranted.MSP-1 as a predominant merozoite surface antigen is present in all examined his gene , 48. PhyP. vivax predominance, while the two minor parasite species P. malariae and P. ovale spp. also occurred at very low prevalence. However, these minor species were most often misidentified or missed, highlighting a potential problem in malaria treatment. Molecular studies identified the P. ovale spp. as the subspecies Poc. Characterization of two antifolate genes dhfr and dhps revealed significant conservation at most positions possibly conferring antifolate resistance in P. falciparum and P. vivax, suggesting reduced impacts of antifolate selection on these two minor parasite species. Despite this, the S113C mutation, corresponding to S108 position in Pfdhfr, was first reported here in two Poc isolates from the China–Myanmar border area. Further analysis of the MSP-1 gene also revealed much lower levels of genetic diversity than their orthologs in P. falciparum and P. vivax populations, possibly due to the persistent low prevalence of these minor species in the GMS.Malaria epidemiology at the China–Myanmar border has shifted to


Ll in asymptomatic children living in two communities with varying malaria transmission patterns.Recent global reports on malaria suggest significant decrease in disease severity and an increase in control interventions in many malaria endemic countries, including Ghana. However, a major driving force sustaining malaria transmission in recent times is the asymptomatic carriage of malaria parasites, which can enhance immune responses against parasite antigens. This study determined the prevalence and relative avidities of naturally induced antibodies to EBA175RIII–VPlasmodium falciparum antigen, EBA175RIII–VLl was expressed in Lactococcus lactis, purified and used in indirect ELISA to measure total and cytophilic IgG concentrations and avidities in children aged between 6 and 12 years. The children were selected from Obom and Abura, communities with perennial and seasonal malaria transmission, respectively. Venous blood samples were collected in July and October 2015 and again in January 2016. The multiplicity of infection and the genetic diversity of EBA175RIII circulating in both sites were also assessed using polymerase chain reaction.An asexual stage Ll antibody concentrations than children living in Abura . Over the course of the study, the relative antibody avidities of EBA175RIII–VLl IgG antibodies were similar within and between the sites.Asymptomatic parasite carriage in the children from Obom decreased from July (peak season), through October and January, however parasite carriage in children from Abura was bimodal, with the lowest prevalence estimated in October. Antibody concentrations over the course of the study remained stable within each study site however, children living in Obom had significantly higher EBA175RIII–VNaturally acquired IgG concentrations but not relative antibody avidities to EBA175RIII–V were significantly higher in Obom where malaria transmission is perennial than in Abura, where malaria transmission is seasonal.The online version of this article (10.1186/s12936-017-2167-3) contains supplementary material, which is available to authorized users. Plasmodium falciparum, the major causative parasite for malaria in sub-Saharan Africa, results in the development of anti-disease immunity  × 100). Statistical significance was defined as Data from healthy (non parasitaemic) children were excluded from all analysis that compared antibody responses (concentration and avidity) between the three-time points.Plasmodium malariae mono infections in four children from Obom in July. Asymptomatic parasite carriage determined by PCR ranged between 59.4 and 68.8% in Obom and 18.9–75.3% in Abura  reduced in moving from July (the peak) through October (the end of the peak) to January (the off peak) season in both sites, with PD in July being significantly different from PD in both October and January in both Obom  and Abura .The study used samples from 137 school children between 6 and 12 years of age. The number of asymptomatic children detected by the microscopic evaluation of thick blood smears ranged between 10.9 and 71.9% in Obom and 17.8–58.9% in Abura (Table msp1 gene for asymptomatic parasite infection in Obom in July was 2.094 (1.873–2.341), which was significantly  higher than 1.397 (1.238–1.577) determined in the asymptomatic children living in Abura in the same month.The geometric mean multiplicity of infection  determined by genotyping the Agarose gel electrophoresis of the EBA175RIII PCR amplicons yielded fragments that were either ~ 740 bp  or ~ 820 bp . The band sizes are different from the 715 and the 795 bp fragments that are characterized by amplification of RIII using the primers EBA1, 2, 3 and 4  due to tL. lactis MG1363. Recombinant EBA175RIII–VLl (amino acid 760–1300 of EBA175) was purified from culture supernatants on a HisTrap FF Crude column  yielding a ~ 125 kDa product    in the median concentrations of antibodies against EBA175RIII–Vura Fig. a. Howeveura Fig. a. The trThe levels of IgG1 and IgG3 antibodies were significantly higher in asymptomatic children from Obom compared to the asymptomatic children from Abura  . Fewer children with high antibody avidities in July and October harboured parasites in the subsequent visit, October and January, respectively  higher in Obom than those measured in Abura  higher than those measured in Abura, even in July when the prevalence of asymptomatic parasite carriage estimated by both microscopy and PCR was higher than in Obom. This finding supports earlier studies that used naturally acquired antibody responses to merozoite antigens including the apical membrane protein 1 (AMA1) and the merozoite surface protein 1 (MSP1) to differentiate between communities with different malaria transmission intensities [Naturally-induced antibody responses to EBA175RIII–Vura Fig. a, b mostensities –57.No significant differences were observed in the median IgG concentrations of anti-EBA175RIII–V antibodies across the different malaria seasons in both Obom and Abura. This could be a result of boosting of antibody responses by the presence of infecting parasites in asymptomatic children, whose prevalence was maintained between October and January in both sites. This supports the suggestion that the presence of concurrent parasitaemia has been found to increase antibody responses to EBA175 antigens .The IgG subclass responses against EBA175 have been suggested to be predominantly cytophilic (IgG1 and IgG3) , 59. IgGLl was significantly inversely associated with parasite carriage during the subsequent quarterly visit   of IgG antibodies against EBA175RIII–Vit Table . The relst) Fig. c, d. ThePlasmodium falciparum parasites were the only species of malaria parasites characterized by PCR in the study. The children were also not followed actively or passively so could have had additional exposure to P. falciparum parasites that were not captured at the sampling time points.Naturally acquired IgG concentrations but not relative antibody avidities to EBA175RIII–V were significantly higher in Obom where malaria transmission is perennial than in Abura, where malaria transmission is seasonal.Additional file 1. Primers for cloning P. falciparum eba175RIII–V as well as for detecting and genotyping P. falciparum parasites.Additional file 2. IgG responses to EBA175 and the Glutamate Rich Protein (GLURP.RO) measured in plasma samples obtained from the school children in October 2015 and January 2016.


Severe malaria has a poor prognosis with a morbidity rate of 80% in tropical areas. The early parasite detection is one of the effective means to prevent severe malaria of which specific treatment strategies are limited. Many clinical characteristics and laboratory testings have been used for the early diagnosis and prediction of severe disease. However, a few of these factors could be applied to clinical practice. MicroRNAs (miRNAs) were demonstrated as useful biomarkers in many diseases such as malignant diseases and cardiovascular diseases. Recently it was found that plasma miR-451 and miR-16 were downregulated in malaria infection at parasitic stages or with multi-organ failure involvement. MiR-125b, -27a, -23a, -150, 17–92 and -24 are deregulated in malaria patients with multiple organ failures. Here, the current findings of miRNAs were reviewed in relation to clinical severity of malaria infection and emphasized that miRNAs are potential biomarkers for severe malaria infection. Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax or Plasmodium knowlesi. A majority of malaria infections are caused by P. falciparum (over 90%), followed by others species [P. falciparum) and uncomplicated malaria [P. falciparum infection with one or more of the followings: coma , metabolic acidosis, organ failure, and severe anaemia [Malaria is a life-threatening arthropod-borne disease with high fatality rate in tropical countries. Almost one million patients with severe malaria are dying a year in the world . The dis species –5. The 2 malaria , 7. Diag anaemia , 9. High anaemia . Thus, e anaemia . Microsc anaemia . Nowaday anaemia –20.MicroRNA assay need only a small amount of blood with less invasive sampling . FurtherThe miRNAs testing started as the analysis of miRNAs obtained from cells or tissues. Recent studies have shown that many human body fluids contained miRNAs. Thus, body fluids are analysed as possible biomarkers to demonstrate the relationship between miRNAs and disease severity. There are still controversies about suitable sources to augment the usefulness of miRNAs as biomarkers , 23.Caenorhabditis elegans tissues [MiRNA was firstly extracted from  tissues . It is s tissues , 26. The tissues , 28. The tissues , 29, 30. tissues .Fig. 1BiCirculating miRNAs were studied in both plasma and serum. Mitchell et al.  found thPlasma miRNAs are circulating in four different molecular forms . The firKirschner et al.  describeThe evidence for interaction between host and pathogens via the miRNA pathway was documented in mammal infectious diseases . Nukui eFor an infection that can lead to cancer, there were two studies identified the relationship between viral infection and malignancy. Motawi et al. and Bandiera et al. showed a similar result that hepatitis C virus (HCV) had miR-34 and miR-122 related with a chronic condition and more tendency to turn to hepatocellular carcinoma , 45. TwoL. donovani-infected monocyte-derived dendritic cells. Two miRNAs act via the tumor growth factor-beta (TGF-β) signaling pathway [Cryptosporidium parvum infected epithelial cells with luciferase activity assay [Cryptosporidium spp. and Toxoplasma spp. are Apicomplexa parasites, related to Plasmodium [The majority of miRNAs studies on tropical diseases were done in schistosomiasis, leishmaniasis, cryptosporidium, and toxoplasma infection. Firstly, He et al. and Zhu et al. identified miR-223 and miR-454 as important molecules, respectively, for pathogenesis in schistosomes infection , 46, 47. pathway . For proty assay . Saçar ety assay  also repasmodium . HoweverP. falciparum Erythrocyte Membrane Protein-1 (PfEMP-1) [P. falciparum did not have miRNA-sequences in parasite genome [Plasmodium parasites changes alternative pathways other than miRNAs. The possibility is that Plasmodium is utilizing host miRNAs to regulate their gene expression [Malaria-infected RBCs can develop in malaria parasites compose with two asexual stages in human (blood steam and liver) and sexual stage in the mosquito. In human, these parasites invade into human red blood cells (RBCs) until the mature parasite sequestration using cytoadherence ligand of PfEMP-1) , 50. SplPfEMP-1) , 51–53. PfEMP-1) . Two groe genome . They mae genome , Epsteine genome , and hume genome . Howeverpression , 59, 60.P. falciparum malaria, Rathjen et al. found that the block of miR-451 synthesis pathway by knocking out Ago2 which produces mature miR-451 resulted in the development of severe anaemia in mice [P. falciparum parasites. Both studies did not found any Plasmodium specific miRNAs; those might be an effect from cells culture method. In erythroid cells, LaMonte et al. and Chapman et al. found that the levels of miR-233 and miR-451 were high in these parasite-infected cells when compared with normal [Plasmodium-infected RBCs. Similar to Chamnanchanunt et al. observed the lower levels of miR-451 and -16 in serums from 22 P. vivax patients than non-infected subjects [P. vivax patients [P. vivax malaria patients. The numbers of P. falciparum patients need to more large scale study.The correlation between reported miRNAs was reviewed according to malaria parasite species Table . For P.  in mice . Similar in mice . Xue et  in mice  also shoh normal , 62. Thesubjects . This grpatients . Reducinpatients . Interespatients  demonstrP. chabaudi malaria showed that 12 common miRNAs were downregulated in spleen and liver tissues [For an animal model of demonstration parasite induces organ failure, mice infected with  tissues . A study tissues . These f tissues . Thus, mThe knowledge of miRNAs as possible disease biomarkers in blood is a promising breakthrough especially the patients with malaria infection. For practical use, the disease criteria for severe falciparum malaria were applied to identify severe falciparum malaria patients from non-severe form  Table 22. The rePatients with severe malaria infection develop acidosis as a serious complication . It compAcute respiratory distress syndrome (ARDS) is the major complication of severe malaria , 74. ARDCerebral malaria is the condition of alternation of consciousness due to parasite sequestration and brain hypoxia . The hypKidney failure is a common complication in severe malaria patients . The mecThe optical microscopic examination is still the gold standard to detect malaria parasites. This method is straightforward and cheap. There are no currently available diagnostic techniques to predict the malaria severity that is very important for saving patient’s life. The new technology has more potential to help the physicians to manage severe malaria is recommended. New investigate applied into tropical diseases such as malaria infection. In vivo study of malaria infection was turn into clinical application. In addition, as a biomarker, it would have prognostic value, especially in early host responses. The objective of this review literature is to systematically review the available data on the relevance of candidate miRNAs among both in vitro and in vivo malaria study.A useful biomarker has to be an investigation, with high sensitivity, non-invasive and applicable to clinical management. There are more studies about miRNAs as a biomarker in many diseases, most of them related to malignant and cardiovascular diseases. For tropical infectious diseases, the knowledge of miRNAs raises to fill to explain the pathophysiology of diseases. The knowledge of miRNAs in malaria infection is still not enough if compared with antimalaria drug trial. The more studies were required to answer, (1) biomarker to predict malaria severity and further predictor, (2) development of the new treatment directed to malaria life cycle. Further studies will perform to fill the gap between bench and bed by accumulating knowledge in serious complications among malaria patients.


Wuchereria bancrofti and Plasmodium falciparum malaria is high, particularly in Africa, and co-infection is common. However, the effects of filarial infection on the risk of severe malaria are unknown. We used the remaining serum samples from a large cohort study in Muheza, Tanzania to describe vector-borne filarial sero-reactivity among young children and to identify associations between exposure to filarial parasites and subsequent severe malaria infections. We identified positive filarial antibody responses  among infants as young as six months. In addition, we found a significant association between filarial seropositivity at six months of age and subsequent severe malaria. Specifically, infants who developed severe malaria by one year of age were 3.9 times more likely  to have been seropositive for filarial antigen at six months of age compared with infants who did not develop severe malaria.The disease burden of  S. stercoralis (a soil transmitted helminth), enhancing our understanding of co-infections in early childhood. We identified an increasing prevalence of filarial antibodies over time in a population of children as young as 6 months old. In addition, we found a significant association between filarial seropositivity at six months of age and subsequent severe malaria.In this paper, we used a multiplexed, serologic assessment to identify children with previous or current exposure to or infection with filarial parasites or  Plasmodium falciparum ; an; anOnchoexposure .The significant association between filarial serostatus at 6 months and subsequent severe malaria infection highlights the need for further investigation to assess whether the increased risk is due to a shared vector or if immune modulation is occurring. As such, a primary limitation to this analysis is the potential for confounders in the relationship between severe malaria and filarial serology. Both malaria and filarial parasites are transmitted by mosquitoes, and previous studies have suggested that the same mosquito species may transmit both infections , 41.S. haematobium co-infection in cohorts aged 3–15 [S. haematobium co-infections having little or no influence on severe malaria, while Ascaris, S. mansoni, filarial and Strongyloides co-infection may confer increased risk. The differences may also be attributable to the age of the children in this study, as the children in this cohort are younger and the effect of coinfection may be different, or the antibodies present may reflect maternal antibodies.The association between filarial seropositivity and severe malaria has been reported elsewhere in studies of older children and adults. Increased risk of severe malaria with helminth co-infection has been reported in children aged between 1–15 years in Senegal and Northern Senegal, and helminth coinfection has been associated with an increase in clinical malaria in children aged < 16 years in Zaire , 43, 44.ged 3–15 , 45, andged 3–15 , 47. TheWe assessed potential confounding for a variable indicating the presence of an insecticide treated bednet in the household and this variable was not significantly associated with filarial serology at 6 months or 1 year of age. A modest proportion of children were missing information for this variable. Even among children who had an insecticide treated net in their household, we are unaware of actual utilization rates or integrity of the bed nets, so actual assessment of treated bed net use may be imprecise.Brugia malayi antigens, based on the substantial antigenic cross-reactivity among all filarial species [W. bancrofti than with O. volvulus infection [W. bancrofti [O. volvulus [W. bancrofti or O. volvulus infection may also have a substantial impact on reducing severe malaria.This study has several additional limitations. First, we are unable to determine if the positive filarial serology indicates current or past infection. Because we do not have measures of current infection, we are unable to determine if our results correlate with infection or exposure and are limited to describing the associations with filarial sero-reactivity. However, one advantage of using serology is that we are able to observe cumulative exposure, rather than assessing exposure at a single point in time. Secondly, this well-established serologic assay specifically uses  species –10, 39, nfection –51 as doancrofti –54 and Ovolvulus , insuffiS1 TableSamples were the remaining sera from the Mother-Offspring Malaria Study (MOMS) Project conducted from 2002 to 2006 in Muheza, Tanzania.(TIF)Click here for additional data file.


Use of bednets reduces malaria morbidity and mortality. In Kilifi, Kenya, there was a mass distribution of free nets to children < 5 years in 2006. In 2009, a new policy was implemented to offer bednets to pregnant women and children < 5 years free of charge. Nets were again distributed to children and adults through national mass campaigns in 2012 and 2015. We aimed to evaluate trends in bednet ownership and usage, and the effect of bednets on the incidence of malaria hospitalization in children < 5 years within the Kilifi Health and Demographic Surveillance System (KHDSS).P. falciparum > 2500 parasitemia per μl) among children < 5 years were captured using a system of continuous vital registration that links admissions at Kilifi County Hospital to the KHDSS population register. Survival analysis was used to assess relative risk of hospitalization with malaria among children that reported using a bednet compared to those who did not.Bednet ownership and usage were assessed during eight routine enumeration rounds of the KHDSS between 2008 and 2015. Malaria admissions . An increase was also observed following the mass distribution campaigns in 2012  and 2015 . Among children <5 years who reported using a net the night prior to the survey, the incidence of malaria hospitalization per 1000 child-years was 2.91 compared to 4.37 among those who did not .We observed 63% and 62% mean bednet ownership and usage, respectively, over the eight-survey period. Among children < 5 years, reported bednet ownership in October–December 2008 was 69% and in March–August 2009 was 73%  contains supplementary material, which is available to authorized users. Malaria remains a global health problem with an annual incidence of 214 million cases and 438,000 deaths; more than 88% of the total morbidity is in the WHO Africa region –8. ModelIn Kilifi, Kenya, bednet coverage was estimated to be less than 6% in the early 1990s . A randoHousehold possession of bednets is an indicator of the extent to which distribution channels are enhancing coverage. However, use of bednets, rather than possession, is what affords protection and is therefore a more valid predictor of epidemiological impact , 17. The2 comprised of 15 administrative locations, containing 260,000 residents. The KHDSS area has been mapped using Magellan  and e-Trex  Geographic Positioning Systems technology, providing detailed information on topography, footpaths and roads, and human occupation of the area, including the coordinates of all homesteads. Fieldworkers visit every participating household approximately three times a year to monitor births, deaths, and migration events. Kilifi County Hospital (KCH) is located centrally within the county; it provides primary care and serves as the first-level referral centre for the KHDSS area. For each paediatric inpatient admission to KCH, clinical examination, demographic details, laboratory analysis and outcomes are recorded in a central database. Every child admitted to the hospital is investigated with a thick or thin blood smear for microscopy to ascertain Plasmodium infection, regardless of presentation.Kilifi County stretches along 65 km of Kenyan coastline, extends 90 km inland from the coast at the widest point, and covers diverse ecological zones, Fig. Data on bednet ownership and usage were collected during eight cross-sectional surveys integrated into the regular KHDSS enumeration in October 2008 – February 2009, March 2009 – August 2009, June 2010 – September 2010, May 2011 – September 2011, May 2012 – August 2012, May 2013 – October 2013, June 2014 – October 2014, and June 2015 – November 2015  parasites > 2500 parasites per μl [From the pediatric admissions database, we extracted patient records of KHDSS children < 5 years admitted at KCH with a positive malaria slide any time during one of the eight KHDSS enumeration rounds. We excluded patients who were not KHDSS residents at the time of admission. We defined clinical malaria as a slide positive for s per μl . We defiThe proportions of individuals who owned and slept under a bednet the night prior to the survey were computed. The Z-test was used to test for a statistically significant difference of any two proportions over the eight-survey period. Spearman’s rank correlation was used to assess the association between bednet ownership and usage over the eight-survey period. The validity of the data collected during the KHDSS routine enumerations was assessed by comparison with observations collected during the overlapping sero-surveys. We performed a paired analysis on resident response compared to fieldworker observation and used McNemar’s test to test the consistency of the responses. The null hypothesis of the McNemar’s test was that there was no difference between reported bed net ownership and the observations of bednet ownership. We used Gwet’s AC1 statistic to assess the reliability of resident response vs. fieldworker observation of a bednet. Gwet’s AC1 (a chance-corrected agreement statistic) was used to quantify the extent of agreement between two paired observation as the surrogate for accuracy , 21. BenP-value of 0.05 was considered significant.We used survival analysis to assess the effectiveness of bednet use in reducing malaria hospitalization over the eight KHDSS survey periods. The child years of observation under each period were computed using survival analysis methods. Age in years was used as the underlying time scale, with entry time defined as the start of each survey period or date of birth or date of in-migration, and exit time as 60 months of age, age at malaria diagnosis or death, or at end of the survey period or outmigration, whichever occurred first. The malaria hospitalization incidence rate was calculated as the number of admissions per 1000 child-years of observation. Two KHDSS surveys overlapped mass bednet distribution campaigns in the study community. To minimize misclassification of the exposure status  arising from the July 2012 and October 2015 mass bednet distributions, we restricted the analysis to the survey period prior to the distribution campaigns. Given that the hospital malaria episodes were clustered within patients, we allowed for clustering by using a survival analysis with robust standard errors. The robust standard errors were used to account for the clustering effect in the estimation of the standard errors . The rathttps://africaopendata.org/dataset/kenya-counties-shapefile).Data was stored in MySQL, cleaned and analyzed using STATA 13.1 . The map was drawn using STATA and the Shapefiles downloaded from an open source website (p < 0.001). The overall proportion of bednet ownership in 2008 was 58% and in 2015 was 57% prior to the distribution campaign and 91% thereafter, with similar results observed for reported bednet usage . An increase was also observed following the mass distribution campaigns in 2012  and 2015   for each of the four periods during which the KHDSS survey and the serosurvey overlapped Table . The agrAmong children aged < 5 years, the overall malaria hospitalization rate was computed per 1000 child years of observation during each of the eight enumeration surveys Table . The incMalaria remains a major cause of morbidity and mortality, particularly in sub-Saharan Africa. Many countries, including Kenya, have attempted to reduce the burden of malaria through policies and practices that promote bednet ownership and use. We report that bednet ownership and use remained relatively stable over the 8-year period and temporarily increased corresponding to mass distribution campaigns. We found that bednet use was associated with a 33% reduction in the incidence of malaria hospitalization among children < 5 years.Pooled data from controlled trials of insecticide treated nets have consistently demonstrated reductions in mortality, morbidity and malaria parasitemia of 17, 50 and 13%, respectively , 25. AltThe protective association of bednet use has been shown to vary by setting, making a multi-faceted approach to malaria control essential . In our Observing a mounted bednet is considered a more reliable indicator of bednet use than relying solely on self-reported use; however, the time required to do a thorough assessment of the household, along with local customs that typically prohibit strangers from entering the household, can preclude conducting such surveys at scale. In a contemporaneous survey of a randomly selected subset of children under 16 years in the KHDSS, we observed a bednet in 86% of children for whom net ownership was reported as “yes”. This suggests that self-reported net-use the night prior to the survey may overestimate actual net use to a small degree in our setting but is a reasonable proxy. The median time lapse between the routine KHDSS enumeration rounds and sero-study interviews was 1 month . We considered the time lapse to be reasonable to make a comparison between observed and reported bednet ownership and usage; however, the gap in time may have contributed to the lack of perfect agreement in the data.Our study has several other limitations. Because we did not require each resident to be present to respond to the questions, data on bednet ownership and usage may have been incorrectly reported. We attempted to minimize this potential misclassification by instructing fieldworkers to interview only residents of the same homestead regarding bednet ownership and usage. Although non-significant, there was a trend toward a reduced protective effect as the period of observation was lengthened 1, 2, and 3 months before and after the KHDSS enumeration period. This suggests that limiting the analysis to the KHDSS enumeration periods was an appropriate choice to minimize misclassification of exposure status that could arise due to variation in bednet use or ownership over time. In this study, we asked about individual bednet ownership – defined as availability of a bednet for an individual’s usual sleeping space – and bednet use the night prior to enumeration. This limits comparability to studies that report ownership at a household level. While we found good agreement between bednet ownership reported during the KHDSS routine enumerations and bednet observation during the sero-survey, this does not directly validate reported bednet usage estimates. The results on the incidence of malaria hospitalization may be biased and confounded by other unmeasured factors . Therefore, the estimates obtained could be an overestimation or underestimation of the true incidence. Because we included all bednets, regardless of quality or insecticidal efficacy, we likely underestimated the protection afforded by usage of a high-quality, long-lasting insecticide treated net.The strengths of the study include use of demographic surveillance data of a sizeable population, the longitudinal nature of the study, which allowed us to evaluate trends in bednet ownership and use over time in association with changes in government policy on provision of bednets and mass distribution campaigns, and the validation of the demographic surveillance data using the sero-survey data. In addition, the diagnostic and laboratory evaluation of children admitted to KCH was the same throughout the study period.Bednet use was associated with a 33% reduction in the incidence of malaria hospitalization in children < 5 years. On longitudinal surveillance, we observed increasing bednet ownership and usage following mass distribution campaigns. A high proportion of people who reported owning a bednet also reported using a bednet, suggesting that efforts to increase net ownership would result in increased net usage. Ownership and usage declined in the years following mass distribution campaigns; to achieve and sustain universal coverage, expansion of continuous distribution channels or other distribution methods should be considered.Additional file 1:Timeline for the KHDSS bednet survey, sero-survey and the mass distribution. (DOCX 260 kb)Additional file 2:Baseline characteristics, bednet ownership and usage, and hospital admission among residents of KHDSS during 8 rounds of enumeration. (XLS 76 kb)Additional file 3:Data validation: reported and observed bednet ownership status among KHDSS residents by survey period and age categories. (XLS 37 kb)Additional file 4:Sensitivity analysis: Incidence of malaria hospitalization among children < 5 years in the KHDSS by pattern of reported bednet ownership and usage, defining malaria three different ways. (XLS 37 kb)Additional file 5:Sensitivity analysis: Incidence of malaria hospitalization (parasitemia > 2500) among children < 5 years in the KHDSS by pattern of reported bednet ownership and usage, using incrementally longer periods of observation. (XLS 39 kb)


Malaria is highly prevalent in many parts of India and the Indian subcontinent. Mangaluru, a city in the southwest coastal region of Karnataka state in India, and surrounding areas are malaria endemic with 10–12 annual parasite index. Despite high endemicity, to-date, very little has been reported on the epidemiology and burden of malaria in this area.A cross-sectional surveillance of malaria cases was performed among 900 febrile symptomatic native people (long-time residents) and immigrant labourers (temporary residents) living in Mangaluru city area. During each of dry, rainy, and end of rainy season, blood samples from a group of 300 randomly selected symptomatic people were screened for malaria infection. Data on socio-demographic, literacy, knowledge of malaria, and treatment-seeking behaviour were collected to understand the socio-demographic contributions to malaria menace in this region.Plasmodium vivax is predominant species compared to Plasmodium falciparum. The infection frequency was found to be high during rainy season. Infections were markedly higher in males than females, and in adults aged 16–45 years than both younger and older age groups. Also, malaria incidence was high among immigrants compared to native population. In both groups, infection rate was directly correlated with their literacy level, knowledge on malaria, dwelling environment, and protective measures used. There was also a significant difference in treatment-seeking behaviour between these two groups.Malaria is prevalent in Mangaluru region throughout the year and Malaria incidences in Mangaluru region are predominantly localized to certain hotspot areas within the city, where socioeconomically underprivileged and immigrant labourers are densely populated. These areas have inadequate sanitation and constant water stagnation, harbouring high vector density and contributing to high infection incidences. Additionally, people in these areas seldom practice preventive measures such as using bed nets. The high incidences of malaria in adults are due to minimal cloth wearing, and long working hours stretching to late evenings in places with high vector density. Instituting heightened preventive public measures by governments and creating awareness on using preventive protective and environmental hygienic measures through educational programmes may substantially reduce the risk of contracting infections in these areas and spreading to other areas. Malaria is one of the most prevalent parasitic diseases worldwide, currently accounting for an estimated 200–300 million clinical cases every year and about half a million deaths worldwide . ApproxiMalaria is highly endemic and persistent throughout the year in several parts of southwestern regions of India, including a substantial portion of Karnataka state , 8. MangMalaria persists in Mangaluru area throughout the year with peak infections in the rainy season. Several localities in the city and suburban areas harbor slum dwellers and socio-economically disadvantaged people. Because of this and wide-spread stagnant water, these localities are hotspots for malaria infection throughout the year with high incidence rates in the rainy season . DespiteMangaluru city is situated 12.91°N, 74.85°E on the basin of rivers Netravathi and Gurupura in the Arabian peninsula of Dakshina Kannada district , August–September (rainy reason), and November–December (the end of rainy season). Multi stage random sampling technique was used in recruiting individuals. A one-sample study design within a population of 5,00,000 city residents was used to determine the sample size, assuming that the malaria risk prevalence is between 20 and 30% of the city population. The study was focused on selected malaria hotspots, where socioeconomically disadvantaged and immigrant workers live. In each season, 300 individuals were randomly recruited based on household sampling frame. Altogether, 900 individuals were screened during the calendar year of 2014. Individuals diagnosed to have malaria infection and did not receive treatment were referred to nearby government hospitals or health care facilities for treatment.Plasmodium falciparum and lactate dehydrogenase of Plasmodium vivax in human blood. Individuals who were positive for infection and those who have already been diagnosed in hospitals and clinics, and those were undergoing treatment at the time of study were recruited.Symptomatic individuals were screened for malaria infection using Giemsa-stained thin and thick blood film examination, and by using a malaria Ag Pf/Pv rapid diagnostic test kit , which detects histidine-rich protein II antigen of A semi-structured interview questionnaire was used to collect information of the study population that included socio-demographic factors, resident status (whether they are long-time residents or migrated recently to this region for the livelihood), recent travel history, and education level. Information about knowledge on the disease, how it spreads and transmits, preventive measures, and where to get diagnosed and obtain proper treatment was collected. Other information collected was treatment-seeking behaviours, such as measures taken after being sick, symptoms experienced during sickness, and previous history of malaria, if any.Statistical analysis of data was performed using Statistical Package for the Social Sciences (SPSS) for Windows, Version 23.0. Chicago, SPSS Inc. Statistical significance was derived using Chi square test and by logistic regression.The study was conducted during February to December 2014 at six different localities, where low-income group dwellers in Mangaluru city corporation limits reside. These localities are considered as malaria hotspots in the city , 67 (14.9%) and 15 (3.3%), respectively, had P. vivax, P. falciparum, and P. vivax and P. falciparum mixed infections  than females 120 (26.7%). Infection in immigrant labourers 272 (60.5%) was higher compared to native population 177 (39.4%) and in both groups, P. vivax was the predominant infection (P = 0.001). Ratio of male to female among infected immigrants was ~ 4:1 as compared to 2:1 in native group. With regard to occupational status, highest prevalence of infection 250 (55.7%) was seen among construction workers compared to 17 (3.8%) in hotel workers, 40 (8.9%) in porters, 24 (5.3%) in students, and 118 (26.3%) in all other occupations  and at the end of rainy season (November–December), respectively, 179 (59.6%) and 146 (48.7%) as compared to 124 41.3%) during the dry season (February–March)  were construction workers as compared to 10 (3.7%) hotel workers, 2 (0.7%) students, 22 (8.1%) porters, and 27 (9.9%) other occupations Table . On the P. vivax infection and artemisinin-based combination therapy comprising artesunate, sulfadoxine and pyrimethamine is prescribed for uncomplicated P. falciparum infection. Primaquine is prescribed as a radical treatment for both the types of infections [Treatment-seeking behaviour of all the 900 study participants was recorded. In both native and immigrant groups, the preferred treatment was anti-malarial drugs. In this region, chloroquine is the prescribed drug of treatment for uncomplicated fections . Among iRegarding treatment-seeking, of 177 total infected native individuals, 94 (53.1%) waited 2–4 days for taking treatment after feeling sick, about 47 26.5%) went on the same day for medication, and 37 (20.9%) did not take any treatment  office of Dakshina Kannada District, which shows that ~ 80% P. vivax and ~ 20% P. falciparum infections. The data recorded by DVBDCP indicate that Mangaluru city area has been having a similar ratios of P. falciparum and mixed infections since 1990 [P. falciparum and P. vivax infection throughout India is in the ratio of 1.5:1. However the proportion of P. vivax and P. falciparum infection varies in different parts of India. The prevalence of P. vivax and P. falciparum infections in Indo-Gangetic plains and Northern hilly states, Northwestern and Southwestern regions is 80–90% P. vivax and 10–20% P. falciparum respectively; however, in the forest areas of South Eastern regions inhabited by ethnic tribes, the situation is markedly different with P. vivax to P. falciparum prevalence ratio of 1:3 [The aim of this study was to understand the burden of malaria in Mangaluru city area. For the past several decades, malaria is endemic in this city and its surrounding areas with peak infections occurring during rainy season. Although o of 1:3 , 11, 12.In Mangaluru, the incidence of malaria infection is highest among immigrant workers compared to native individuals. This is due to a rapid urbanization and construction activity, which provides many employment opportunities for immigrant workers. So, labourers mainly from Northern and Northeastern states of India, where malaria is endemic, come to Mangaluru. They reside in temporary accommodations, often in the construction sites with poor living conditions, disposing to malaria infection. Water stored at the construction sites and stagnant water in building under constructions provides ideal breeding conditions for the mosquito and spreading of the disease. Additionally, limited or lack of health knowledge on malaria contributes to accelerated transmission of infection. Individuals who travel to malaria endemic places in other parts of Karnataka and India also are the source of spreading malaria in Mangaluru. Especially, the migrant workers from Northern or Northeastern parts of India where malaria is highly endemic are high carriers of infection, contributing markedly to spread of malaria locally –16. ThusUnlike in Africa and several others parts of endemic regions in the world, where infection is predominantly in the age group of < 12 years , 18, theAs in the case of other malaria endemic countries or other parts of India , 22, theAlthough the majority of study group preferred the prescribed drug treatment, a significant number of both native and migrant groups preferred self-medication, such as steam inhalation for nasal congestions, and herbal preparation for fever during initial period of experiencing symptoms. In the migrant population, who sought drug treatment, the majority went to the informal medicine providers, such as drug stores and ended up in buying antipyretics, analgesics, antiemetic and antihistamines. Thus, educating vulnerable people on malaria knowledge and on implementing preventative measures, and the necessity of seeking early diagnosis and prompt treatment may prove to be effective in controlling malaria in Mangaluru area.P. vivax and P. falciparum infections occur throughout the year at ~ 80 and ~ 20% proportions, respectively. Overall, the infection rate is significantly higher among immigrant and socioeconomically disadvantaged temporary resident laborers, who work for house and building construction industries, hotels, and hostels, where stagnant water harbours high vector density. In both native and immigrant classes of people, males were highly disposed to the infection due to their long working hours that include late evening when feeding activity of vector is high. Also, due to hot and humid weather condition, the workers wear minimal clothing. Unsatisfactory levels of malaria knowledge and lack of preventive protective measures are substantial risk factors in immigrant population. Efforts in seeking treatment and knowledge about malaria infection are low among immigrant population compared to native people. Immigrant individuals are also largely unaware of the government facilities that provide free diagnosis and treatment for malaria. Their lack of knowledge appears to be mainly attributed to limited levels of health education activities in malaria hotspot localities in the city. Therefore, conducting educational programmes on malaria awareness and implementation of public control measures are likely to significantly reduce infection rates in Mangaluru.Malaria is highly prevalent in Mangaluru and persists throughout the year with maximum level of infection cases seen during the rainy season. Both


As part of efforts to eliminate malaria, Vanuatu has piloted the implementation of enhanced malaria surveillance and response strategies since 2011. This involves passive case detection (PCD) in health facilities, proactive case detection (Pro-ACD) and reactive case detection (Re-ACD) in communities using malaria rapid diagnostic tests (RDTs). While RDTs improve case management, their utility for detection of malaria infections in ACDs in this setting is unclear.The utility of malaria RDTs as diagnostic tools was evaluated in PCD, in five rounds of Pro-ACDs and five rounds of Re-ACDs conducted in Tafea and Torba Provinces between 2011 and 2014. The number of malaria infections detected by RDTs was compared to that detected by PCR from collected used-RDTs.P. falciparum infections. In five rounds of Re-ACD, RDTs did not identify any additional infections while PCR detected only one among 173 subjects screened.PCD in Tafea Province (2013) showed a RDT-positive rate of 0.21% (2/939) and a PCR-positive rate of 0.44% (2/453), indicating less than 1% of suspected malaria cases in Tafea Province were due to malaria. In Pro-ACDs conducted in Tafea and Torba Provinces, RDT-positive rates in 2013 and 2014 were 0.14% (3/2145) and 0% (0/2823), respectively, while the corresponding PCR-positive rates were 0.72% (9/1242) and 0.79% (9/1141). PCR identified villages in both provinces appearing to be transmission foci with a small number of low-density infections, mainly PCD and Pro-ACDs demonstrate that both Tafea and Torba Provinces in Vanuatu has achieved very low malaria prevalence. In these low-transmission areas, conducting Pro-ACD and Re-ACDs using RDTs appears not cost-effective and may have limited impact on interrupting malaria transmission due to the small number of infections identified by RDTs and considerable operational resources invested. More sensitive, field deployable and affordable diagnostic tools will improve malaria surveillance in malaria elimination settings. P. falciparum and P. vivax, with higher malaria prevalence in northern provinces. Over the past decade the malaria incidence rate in Vanuatu has declined due to intensified control measures and the country met the set target of >75% decrease in incidence between 2000 and 2015 . Th. ThP. faptomatic ,16. Suchptomatic . Pro-ACDRe-ACD is case investigation for each notified case of confirmed malaria in the field (index case), which includes obtaining the details of the confirmed case, reviewing local transmission potential and conducting ACD in the vicinity of the case, which involves the testing of household members and neighbours of the index case . During Although ACD is recommended by the WHO , severalP. falciparum and P. vivax coexist in Vanuatu, combo RDTs (pfHRP2/PAN pLDH) detecting both species were selected and deployed over the whole country. The first brand of RDT introduced in 2009 was the ICTTM Combo test, but this brand was replaced by CareStartTM in 2012 on the basis of higher sensitivity of the later brand, particularly in detecting P. vivax, as documented by the WHO-FIND malaria RDT product testing programme [Malaria RDTs were introduced in Vanuatu to improve case management and surveillance as part of the intensified malaria control and elimination program launched in 2009. As rogramme .While quality RDTs are recommended and have been used as the standard diagnostic tool for routine malaria case management and PCD, it is unclear whether RDTs are useful tools in surveillance at low transmission settings where a substantial proportion of low density and sub-microscopic infections were reported ,16. It iMolecular tests can be used to estimate the total number of, and the prevalence of malaria infections in the community. These tests usually rely on extra blood collected, i.e. in addition to blood required for smears and RDTs, during the surveillance. In recent years, used-RDTs have been shown to be a useful source for DNA extraction for PCR ,27. TherIn this paper, we evaluate the utility of RDTs as surveillance tools in PCD and ACDs conducted in two provinces in Vanuatu. This was achieved by comparing the number and prevalence of malaria infection detected by RDTs to those detected by PCR from used-RDTs that were collected during the same community surveys. The findings will inform future malaria elimination strategies.The surveillance activities described in this paper were conducted in Tafea and Torba provinces, which are located towards the southern and the northern part of Vanuatu, respectively. For all surveys conducted, a written informed consent was obtained from each participant. Information on name, residence, age and sex of the tested participants was recorded prior to testing. Following registration, participants had a finger prick performed with a blood lancet and a few drops of blood were collected for the malaria tests. RDT positive cases were investigated, clinically assessed and given appropriate treatment as per national guidelines.Tafea province: The national malaria control program in Vanuatu has targeted Tafea Province for malaria elimination since 2008. Enhanced malaria case surveillance and response operations were implemented since 2011 as part of the transition from control to elimination. RDTs  Combo G0131, AccessBio) were used in PCD, two rounds of Pro-ACDs and five rounds of Re-ACDs conducted in Tafea Province. Post purchasing, lot testing of RDTs were performed by the WHO-FIND malaria RDT lot testing laboratories.2013 PCD. In 2013, all patients presenting with fever to a number of health facilities on Tanna Island, including one hospital, 11 dispensaries, four health centres and 28 aid posts, were tested for malaria with RDTs.2013 Pro-ACD. In Nov 2013, a house-hold survey was conducted in 10 villages on Tanna Island. These villages were selected because of recent malaria incidences.2014 Pro-ACD. A house-hold survey covering one village on Erromango Island and eight villages on Tanna Island was conducted between 6 May and 18 Aug 2014. Four of the eight villages surveyed on Tanna Island were included in the 2013 Pro-ACD.2013 and 2014 Re-ACDs. Re-ACDs were conducted in different locations on Tanna Island, Tafea province in Vanuatu. Every RDT-positive case is classified as an index case which triggers a case investigation in the village of residence during which recent travel history is collected. Cases are classified as local if individuals did not report any overnight stay outside the village of residence 14 days prior to the diagnosis. All households within a 500 m radius of each index case that was notified to the provincial malaria team was visited by a survey team within 5 days after case notification. GPS coordinates were used to map the tested household. All individuals were finger-pricked for RDT testing and preparation of 2 blood spots dried on filter papers (≥20μL each). Four rounds of Re-ACDs were conducted in Nov 2013, during which 7 to 52 individuals were screened with a RDT at each Re-ACD. The fifth Re-ACD was conducted in Oct 2014 in Lounanen where a total of 47 individuals from 11 house-holds were screened.Torba province: The national malaria program conducted three rounds of Pro-ACD surveys to detect malaria cases and point prevalence in selected islands in Torba Province.The 2011 Pro-ACD. In Dec 2011, Pro-ACDs were conducted on two islands in the Torres Island group: Toga Island and Loh Island. ICT Malaria Combo Cassette test ML02 (ICT Diagnostics) was used in this survey. A total of 485 individuals were included in the survey; 292 individuals were registered on Toga Island and 193 individuals on Loh Island.TM Malaria HRP2/pLDH (Pf/PAN) Combo G0131 (AccessBio).The 2013 Pro-ACD. In Aug 2013 a follow-up Pro-ACD was conducted on Toga Island and Loh Island, and also on Hiw Island and Tegua Island of the Torres Island group, Torba Province. A total of 891 individuals were screened with CareStartTM Malaria HRP2/pLDH (Pf/PAN) Combo G0131 (AccessBio).The 2014 Pro-ACD. Another house-hold survey was conducted between 18 Nov and 8 Dec 2014 on the Banks Island group of Torba Province including islands of Vanua Lava, Gaua, Motalava and Mota. A total of 1705 among 1827 registered residents (93.32% coverage) were screened with CareStartUsed-RDTs were packed and stored with desiccant at room temperature until shipment to AMI in Brisbane, Australia.Used-RDTs were sorted and numbered. Approximately 50% of used-RDTs from each Pro-ACD were selected for PCR. However, when the total number of RDTs collected from a survey exceeded 1500, approximately 30% of RDTs were selected for PCR. Depending on the proportion of RDTs to be processed, between 1/3 and 1/2 of used-RDTs were randomly selected from each household or each village. 100% of used-RDTs from Re-ACDs were used for PCR because the total number of RDTs collected from Re-ACDs is relatively small.The selected RDTs were disassembled, nitrocellulose strips removed, cut to small pieces (2–5 mm each) and placed in Eppendorf tubes. Genomic DNA was extracted from these pieces using QIAamp DNA Mini Kits and a QIAcube robot  following manufacturer’s instructions. DNA was eluted in 100 μL volume.Plasmodium spp. as previously described [A multiplex PCR amplifying the parasite 18s RNA gene was performed to determine the presence of escribed . 5–15 μLThe survey protocols were approved by the Vanuatu Ministry of Health Ethics Committee and by the WHO Western Pacific Regional Office Ethical Review Committee (2014.6.VAN.1.MVP). The storage and processing of samples for PCR at AMI was approved by Australian Defence Human Research Ethics Committee (ADHREC 505–07). All participants of the active surveys provided a written consent as per approved survey protocols.Graphpad Prism version 6.05 was used for data analyses and graph production. The overall malaria point prevalence for Tafea and Torba province were calculated as the mean and 95% confidence intervals based on the village and island prevalence data. 95% confidence intervals for sensitivity and specificity of RDTs were calculated using modified Wald method.P. falciparum (non-Pf) infection and one positive for both Pf/PAN indicating either Pf infection or a mixed species infection. Two additional cases were detected by microscopy (one Pv and one Pf). PCR of 453/939 (48%) used-RDTs which included two positive RDTs detected P. falciparum DNA from three RDTs: one of the two positive RDTs that was positive for Pf and two of the 451 negative RDTs. Assuming the same PCR positive rate applies to the remaining negative RDTs, the total number of malaria infections in 939 patients would be estimated to be 5 (4+1). This indicates that malaria was the cause in only 0.53% (5/939) of fever patients on Tanna Island, Tafea Province.Between July-Dec 2013, 939 symptomatic patients on Tanna Island were tested for malaria using CareStart RDTs. Only two positive RDTs were observed: one positive for PAN band indicating non-Pf, but negative on 2nd RDT), giving a point prevalence of 0.16% (2/1250). PCR of 713 used RDTs (57% of used RDTs) identified a total of 6 P. falciparum infections across 5 of the 10 villages, giving a point prevalence of 0.84% (6/713), which is 5.25 fold higher than that of RDTs  used-RDTs identified plasmodial DNA in 7 individuals . Five ofP. falciparum DNA from 11 negative RDTs in 274 (56%) of used-RDTs screened, giving a point prevalence of 4.38% (House-hold surveys were conducted on Loh and Toga Islands using ICT combo RDT. 14/485 (2.89%) residents screened gave a positive RDT result: eight positive for Pf, four positive for PAN and two positive for Pf/PAN. However, only one of the 14 positive RDTs had detectable plasmodial DNA by PCR indicating 13 false positive RDT results. PCR detected of 4.38% .P. falciparum infections from 529 negative RDTs (59% RDTs screened), giving a point prevalence of 0.57%  individuals screened. This RDT was negative by PCR. PCR detected 3 of 0.57% . PCR detP. vivax infections, giving a point prevalence of 0.39%  were tested by PCR, of which 30 RDTs were positive by PCR. The malaria prevalence in Tafea and Torba Provinces determined by RDTs and PCR from used-RDTs of the five Pro-ACDs is illustrated in In the context of the Pro-ACDs, the sensitivity and specificity of the CareStart RDT compared to PCR was 0% (95% CI: 0–19.79%) and 99.88% (95% CI: 99.64–99.98%), respectively. The overall false positive rate is 0.12% (95% CI: 0.02–0.36%). The ICT RDT had a sensitivity and specificity of 8.33% (95% CI:<0.01–37.53%) and 95.04% (95% CI:91.62–97.15%), with a false positive rate of 4.74% (95% CI: 2.72–8.02%). The results suggest that RDTs are not sensitive at detecting the small number of asymptomatic infections in active surveillance.P. falciparum DNA in one individual who lived in Eniou village  screened, nor did PCR from 47 used-RDTs  of fever in patients in Tafea Province was due to malaria and greater than 99% of fever cases should be investigated for other causes. Those patients missed by RDTs were likely to have parasite densities below the detection threshold of RDTs at the time, and may be detected by RDTs during follow up visits. The PCD results demonstrated a very low level of malaria incidence rate on Tanna Island.RDTs and PCR from used-RDTs were also applied for detecting malaria cases in 5 rounds of Pro-ACD conducted in Tafea and Torba Province between 2011 and 2014. These Pro-ACDs all confirmed the low malaria transmission status in both Provinces. The overall malaria point prevalence detected during Pro-ACDs by RDTs for Tafea and Torba provinces was 0.16% and 0.11%, respectively in 2013, and 0% for both provinces in 2014. The corresponding point prevalence determined by PCR from used-RDTs was 0.75% and 0.48%, respectively, in 2013, and 0.67% and 0.22% in 2014. The higher detection rates by PCR indicated the presence of low density plasmodium infections in these communities, which is consistent with findings in similar settings ,16. ImpoP. falciparum on RDTs could have resulted from circulating PfHRP2 antigen of recent infections. It should also be noted that false positive Pf and PAN results could also relate to the false positive rate of the RDT product. For instance, the CareStart RDTs used since 2013 have a low false positive rate of 0.4%, which means that for every 1000 RDTs tested, 4 false positive results may be expected. The false positive rates observed in this study for this RDT were lower than 0.4%. Therefore, it would be a good practice that RDT positive results in Pro-ACDs are verified using quality microscopy. Confirmation of infection may trigger focal mass screening and treatment (MSAT) using a more sensitive diagnostic tool such as PCR or LAMP followed by treatment with ACT, or trigger deployment of alternative measures such as mass drug administration.Compared to PCR results, RDTs gave some false positive results: ICT produced 10 false positives in 485 tests performed in 2011, while CareStart gave three false positives in 2145 tests in 2013, 0 in 2823 tests in 2014. False positive Plasmodium infections detected than other villages during 2013 and 2014, which may indicate active local transmission occurring around these villages. In Torba Province, it is likely that active transmission occurs in Loh and Toga islands as well as in Vuries village on Vanua Lava Island. These foci should be targeted for intensified surveillance and case investigations to confirm the presence of locally transmitted cases. Favouring factors that may contribute to the sustained transmission such as entomological, geographical, population, occupation and intervention coverage, should be investigated.PCR from used-RDTs has helped to retrospectively detect possible transmission foci in both provinces. In Tafea Province, Imaio and Isaka villages on Tanna Island have more P. falciparum infections in both provinces. In Tafea Province, PCR of 713 used-RDTs from the 2013 Pro-ACD produced 6 P. falciparum positive results, and PCR of 633 used-RDTs from the 2014 Pro-ACD produced 5 P. falciparum, 1 P. vivax and 1 P. malariae. Combined, eleven of the 13 PCR positive infections were P. falciparum. This trend is consistent with the 2008 baseline prevalence survey in Tafea Province, where 68% of microscopy negative/PCR positive infections were P. falciparum [P. falciparum was identified by PCR in 12/12, 3/3 and 0/2 infections, respectively. These findings are consistent with the recently reported distributions of parasite densities in asymptomatic malaria in Southeast Asia where a higher proportion of very low density P. falciparum infections were identified by an ultra-sensitive PCR compared to P. vivax [One interesting observation of the Pro-ACDs is the evidence of persistence of low density P. vivax .Plasmodium species and the proportion of asymptomatic and sub-patent infections. In relatively high transmission settings of Africa where P. falciparum dominates, RDTs have been reported to consistently provide higher parasite prevalence than microscopy [The value of RDT as a surveillance tool in Pro-ACD could be judged by its impact on reducing malaria burden, interrupting local malaria transmission and cost-effectiveness, all of which can be influenced by the transmission intensity, the dominant croscopy , likely croscopy . On the croscopy . These dcroscopy . In thatcroscopy .In Tafea and Torba Provinces in Vanuatu, the overall RDT positive rates in 2013 and 2014 were 0.14% (3/2145) and 0% (0/2823), respectively. Despite using a good performing RDT, the RDTs detected less than 20% of plasmodium infections identified by PCR. Because very considerable efforts and resources were invested in organising and conducting these Pro-ACDs, and because very few infections were detected by RDTs in the target communities which accounted for only a small proportion of true infections as judged by PCR, it appears that RDTs used alone as surveillance tools for Pro-ACD in locations with very low point prevalence of malaria, such as Tafea and Torba Provinces, may add limited value to interrupting malaria transmission. Although positive RDTs in Pro-ACDs could potentially be used as a trigger for more intensified malaria surveillance and treatment activities, RDTs as surveillance tools to detect asymptomatic infections appear not cost-effective.Our study showed that PCR from used-RDTs did increase the detection rate by 2–5 fold compared to RDT alone. It demonstrates that used-RDTs provide a valuable source of DNA for molecular epidemiological studies and that the results help to assess malaria transmission status and to identify transmission foci. In addition, used-RDTs can serve as a source for serological studies for estimating sero-prevalence and for monitoring changes in transmission intensity . TherefoThis study has some limitations. Although the sensitivity of PCR from used-RDTs was reported to be identical to that from filter papers , it shouRDTs have been used in several countries for Re-ACDs and demonstrated ability to detect additional infections located in households within 140m, 500m or 1 km radius of index cases ,36,37. VThe use of RDTs in Pro-ACDs conducted in Tafea and Torba Province, Vanuatu revealed that malaria point prevalence rates have been brought down to at a level below 1% in both provinces since 2013. This was confirmed by retrospective PCR from used-RDTs. RDT and PCR nevertheless identified several possible malaria transmission foci in both provinces. However, in these very low transmission settings, implementation of Pro-ACD and Re-ACD using RDT as a detection tool to find asymptomatic malaria infections appears not to be cost effective and may have very limited impact on the overall effort towards interrupting malaria transmission. A simple, sensitive and easy-to-perform molecular diagnostic tool is required for effective malaria surveillance in malaria elimination settings.