To investigate changes in malaria prevalence in Papua New Guinea after the distribution of long-lasting Insecticide-treated nets, starting in 2004, and the introduction of artemisinin-based combination therapy in 2011.Two malaria surveys were conducted in 2010–2011 and 2013–2014. They included 77 and 92 randomly selected villages, respectively. In each village, all members of 30 randomly selected households gave blood samples and were assessed for malaria infection by light microscopy. In addition, data were obtained from a malaria survey performed in 2008–2009.Plasmodium falciparum was more common than P. vivax overall, but not everywhere, and initially the prevalence of P. vivax infection decreased more slowly than P. falciparum infection. Malaria infections were clustered in households. In contrast to findings in 2008–2009, no significant association between net use and prevalence was found in the later two surveys. The prevalence of both fever and splenomegaly also decreased but their association with malaria infection became stronger.The prevalence of malaria below 1600 m in altitude decreased from 11.1%  in 2008–2009 to 5.1% (95% CI 3.6–7.4) in 2010–2011 and 0.9% (95% CI 0.6–1.5) in 2013–2014. Prevalence decreased with altitude. Large-scale insecticide-treated net distribution was associated with an unprecedented decline in malaria prevalence throughout Papua New Guinea, including epidemic-prone highland areas. The decline was accompanied by broader health benefits, such as decreased morbidity. Better clinical management of nonmalarial fever and research into residual malaria transmission are required. National campaigns were organized to distribute free, long-lasting insecticide-treated nets at the household level and, since late 2011, malaria rapid diagnostics tests, improved diagnostic microscopy and artemisinin-based combination therapy have increasingly been provided at public and church-run health-care facilities.P. falciparum was the most common species, followed by P. vivax, which dominated in several locations. A few P. malariae infections were found but P. ovale was not detected in any sample. Within 1 year of the initial insecticide-treated net campaign, a significant reduction in the prevalence, incidence and transmission of malaria was documented at selected sites, even though insect vectors tended to feed outdoors.In 2008–2009, towards the end of the first insecticide-treated net campaign, the Papua New Guinean Institute of Medical Research conducted a country-wide malaria indicator survey. It documented that 65% of households in areas covered by the campaign owned long-lasting insecticide-treated nets and that 33% of people were using them.,Subsequent national malaria surveys conducted by the Papua New Guinean Institute of Medical Research in 2010–2011 and 2013–2014 to evaluate the national malaria control programme provided evidence that coverage with long-lasting insecticide-treated nets had increased.National malaria surveys were conducted from November 2010 to August 2011 and from November 2013 to August 2014. In both surveys, five villages were randomly selected from each of the country’s 20 provinces – organized in four regions – using a list of villages identified in the 2000 national census – the most up-to-date.+ photometric analyser . Symptomatic household members were offered a malaria rapid diagnostic test and treatment or referral to the nearest health-care facility, where appropriate. Axillary temperature was measured using an electronic thermometer and children aged between 2 and 9 years had their spleen palpated. Each patient’s blood sample was accompanied by information on recent travel. The locations of the survey villages were determined using a hand-held Garmin eTrex Global Positioning System device .Data were collected using an adapted Malaria Indicator Survey questionnaire.,Malaria was diagnosed by light microscopy at the Papua New Guinean Institute of Medical Research following established procedures.2χ tests and logistic regression, and non-normally distributed variables were compared using the non-parametric Mann–Whitney U test. Data analyses were conducted using Stata/IC v. 14.0  and the survey design was taken into account by using Statas set of commands for survey data analysis (svy).Measures of the prevalence of malaria infection and morbidity were age-standardized using the standard population for Asia given by the International Network for the Demographic Evaluation of Populations and Their Health (INDEPTH).In the 2010–2011 survey, blood samples were collected from 10 060 individuals. Of the 77 villages included, 58 (75.3%) were below 1200 m in altitude, 5 (6.5%) were between 1200 and 1599 m and 14 (18.2%), with 1539 participants, were at 1600 m or higher . In the P < 0.001) and to 0.9% (95% CI: 0.6–1.5) in 2013–2014 (P < 0.001). The prevalence of P. falciparum infection was higher than that of P. vivax infection in all surveys  in 2008–2009 to 5.1% (95% CI: 3.6–7.4) in 2010–2011 . Between the 2008–2009 and 2010–2011 surveys, an increase in the prevalence of  regions  and in s regions , which lP = 0.004). In the 2010–2011 survey, the prevalence of P. falciparum infection was higher than that of P. vivax infection in highland villages: 0.6% (95% CI: 0.3–1.1) and 0.1% (95% CI: 0–0.7), respectively. In the 2013–2014 survey, the prevalence of P. falciparum infection was 0.1% (95% CI: 0–0.4), whereas no P. vivax infections were detected. Moreover, no P. malariae, P. ovale or mixed infections were found.In highland villages at 1600 m and above, the age-standardized prevalence of malaria decreased from 0.7% (95% CI: 0.3–1.2) in 2010–2011 to 0.1% (95% CI: 0–0.7) in 2013–2014  of P. vivax infections and 9.1% (95% CI: 3.4–22.0) of P. falciparum infections occurred in children aged under 5 years.Regression analysis findings are presented in nfection . In 2013Long-lasting insecticide-treated nets were used by 32.5% (95% CI: 27.0–38.4) of the population nationally in 2008–2009,P < 0.01 for all). Although the proportion of the population with a recent history of fever decreased over time , to be anaemic or, in those aged 2 to 9 years, to have splenomegaly (ver time , the assver time  and 3.7%ver time . The prever time  and agaiver time . Indepen–P. vivax than P. falciparum infections appeared to be transient and was followed by a clear reduction in both species, as observed elsewhere.,,,,,,P. falciparum was the dominant species in the highlands.Within 5 years, the prevalence of malaria in Papua New Guinea decreased from 11.1% to 0.9% and during 2013–2014 no parasites were detected by light microscopy in most provinces. This is a greater reduction than the 26% observed in Africa between 2000 and 2016.,The prevalence of fever and splenomegaly declined with that of parasite infection. However, the association between infection and symptoms became stronger over time, perhaps because the proportion of microscopically detectable infections that were symptomatic increased as transmission and immunity declined. The decrease in splenomegaly was most marked, which may reflect a reduction in chronic malaria infection.Between 2004 and 2012, the distribution of insecticide-treated nets to households was the only large-scale malaria intervention in Papua New Guinea.,,Anopheles populations, their biting preferences and their susceptibility to interventions.,,We found that individuals cohabiting with another infected person were more likely to carry parasites, possibly due to similar exposure patterns. There were fewer infections in high-quality houses occupied by better-off households, possibly because of economic factors or the building’s structure or location – most high-quality houses were in urban areas. Earlier studies in Papua New Guinea found conflicting evidence of the impact of housing, namely raised structures, on mosquito exposure.In our study, we used age-standardization to account for differences in the age-composition of participants between surveys and between participants who gave blood samples and the general population. However, as the 2008–2009 survey included only districts where nets were distributed, the national prevalence of parasite infection may have been underestimated. Data from sentinel sites showed that the prevalence after net distribution was 4.8% compared with 15.7% before.P. falciparum remained more common than P. vivax. Declining prevalence was accompanied by broader health benefits, such as decreased morbidity. However, nonmalarial fever now requires better clinical management. Research into the drivers of residual malaria transmission and the burden and role of submicroscopic parasite infection are crucial for better targeting of interventions and for eliminating the disease.In conclusion, increased use of long-lasting insecticide-treated nets in Papua New Guinea was associated with a rapid and significant decline in malaria prevalence – the lowest prevalence ever recorded was in 2013–2014. The decline also occurred in the epidemic-prone highlands. Light microscopy showed that


Plasmodium falciparum PfRH5 protein binds Ripr, CyRPA and Pf113 to form a complex that is essential for merozoite invasion of erythrocytes. The inter-genomic conservation of the PfRH5 complex proteins makes them attractive blood stage vaccine candidates. However, little is known about how antibodies to PfRH5, CyRPA and Pf113 are acquired and maintained in naturally exposed populations, and the role of PfRH5 complex proteins in naturally acquired immunity. To provide such data, we studied 206 Ghanaian children between the ages of 1–12 years, who were symptomatic, asymptomatic or aparasitemic and healthy. Plasma levels of antigen-specific IgG and IgG subclasses were measured by ELISA at several time points during acute disease and convalescence. On the day of admission with acute P. falciparum malaria, the prevalence of antibodies to PfRH5-complex proteins was low compared to other merozoite antigens . At convalescence, the levels of RH5-complex-specific IgG were reduced, with the decay of PfRH5-specific IgG being slower than the decay of IgG specific for CyRPA and Pf113. No correlation between IgG levels and protection against P. falciparum malaria was observed for any of the PfRH5 complex proteins. From this we conclude that specific IgG was induced against proteins from the PfRH5-complex during acute P. falciparum malaria, but the prevalence was low and the IgG levels decayed rapidly after treatment. These data indicate that the levels of IgG specific for PfRH5-complex proteins in natural infections in Ghanaian children were markers of recent exposure only. P. falciparum malaria is estimated to cost more than half a million lives every year, mainly in tropical Africa [l Africa . The disl Africa . ProtectThe asexual multiplication cycle initiates when a merozoite invades an erythrocyte. Despite the rapidity of invasion, it is a multi-step process that involves numerous parasite molecules, most of which are redundant and polymorphic . HoweverP. falciparum parasites [It is now known that the structured domain of PfRH5  binds to the erythrocyte receptor basigin, thereby forming the contact point that initiates parasite entry , 7. Two arasites –22. We tP. falciparum malaria  = 0.24), although the proportion was somewhat higher (34%) among asymptomatically infected children (AC) than in the other categories (<27%) (2) = 0.24). Nonetheless, the proportion of responders in the SM group was higher (28%) than the other groups (<13%). The median IgG levels were significantly higher in the parasitemic donors than in the HC and FC donors (2) = 0.22) among the different donor categories. However, the median IgG levels was markedly higher in the severe malaria (SM) donors in comparison to the febrile (FC) and afebrile (HC) children without detectable parasitemia (P<0.05)  . The medC donors . For Pf1(P<0.05) . For the(P<0.05) . With GL(P<0.05) .While it is difficult to draw firm conclusions from these results, the data suggest that responses to each of the tested antigens indicate parasite exposure more than anything else.P. falciparum and that the immune responses induced by infection are relatively transient. To obtain more direct information about the duration of the responses, we also measured IgG levels to the three PfRH5-complex antigens  in samples obtained two (Day 14) and six weeks (Day 42) after the initial diagnosis of P. falciparum malaria  than in children without detectable parasitemia (donor categories FC and HC) . This su malaria . In the  malaria  where th malaria , althoug malaria .P. falciparum malaria [in vitro growth inhibition assay (GIA) and converting the AU into concentration of PfRH5 specific IgG [Levels of IgG specific for several merozoite-specific antigens have been associated with clinical protection from  malaria . Further malaria . In the ific IgG . Resultsific IgG  and therIn this study, we analyze antibodies specific for three potential blood stage malaria vaccine candidates. It is the first longitudinal study comparing the acquisition and kinetics of naturally acquired antibodies towards PfRH5, Pf113 and CyRPA in the same cohort of children. Furthermore, it is the first study reporting on detectable levels of naturally acquired CyRPA antibodies. Several reports do exist on naturally acquired PfRH5-reactive antibodies –21, 32, The prevalence of anti-Pf113 antibodies found in this study was slightly lower but comparable to levels observed in studies from Kenya and Gabon (50 and 51% respectively). This slight difference probably reflects differences in endemicity in the different areas or the age range of the children. In Imboumy-Limoukou et al, 2016 the most prevalent subclass for the Pf113 specific antibodies were IgG4, whereas we found IgG1 and IgG3 to be most abundant similarly to all other antigens tested here. That fact that IgG1 and IgG3 were the most abundant subclasses in our cohort points to a possibility of an antibody-mediated protection not only via blocking invasion, but also by compliment deposition and opsonization for phagocytosis.P. falciparum malaria. In other studies comparing the prevalence and levels of PfRH5 specific antibodies to other merozoite antigens such as Apical Membrane Antigen 1(AMA1) and Merozoite Surface Protein 1 (MSP1), the same pattern was seen [Aotus monkeys, vaccinations with both PfRH5 protein as well as viral vectored PfRH5 induced protective antibodies, supporting PfRH5 as a good immunogen [The invasion complex antigens PfRH5 and CyRPA appeared to be less immunogenic than all the other studied merozoite surface antigens in this cohort of naturally exposed children with was seen , 19, butmmunogen . A respemmunogen . The disP. falciparum antigens.In general, we observed a rapid reduction in antibody levels to the three studied antigens after malaria exposure. The malaria induced antibodies to PfRH5-complex proteins appears to be secreted by short lived plasma cells. But it is clear from our data, as well as the results of others , that thP. falciparum, although cases of P. ovale and P. malariae infection are occasionally seen [The study was conducted in Hohoe, a town located about 220 km northeast of Accra, in an area of tropical semi-deciduous forest vegetation. Malaria transmission intensity in the area is high and has two seasonal peaks: a major one in April-July and a minor one in September-November. The dominant malaria species is lly seen .P. falciparum malaria were recruited to the study after informed consent had been obtained from a parent or legal guardian. Malaria patients (N = 108) were those where presence of P. falciparum infection was confirmed by a positive rapid diagnostic test (RDT) and by light microscopy with a parasite density > 2,500 parasites/μL. These malaria patients were subdivided into those with severe malaria  or uncomplicated malaria , respectively. SM was defined by the presence of one or more of the following without any other obvious unrelated cause: (i) impaired consciousness (Blantyre coma score ≤3) lasting longer than 30 minutes, (ii) acidosis, defined as respiratory distress , (iii) hypoglycemia (blood glucose <2.2 mM), (iv) hemoglobin concentration <5 g/dL, (v) >2 convulsions within the last 24 h, (vi) shock, and/or (vii) hyperparasitemia . Malaria patients without any of these SM criteria were classified as UM. Febrile patients without detectable parasitemia were grouped as febrile controls . All children were treated according to country guidelines. SM and UM children were seen again 14 (Day 14) and 42 days (Day 42) after the day of diagnosis (Day 0). In addition, 88 clinically healthy and age-matched children from within the Hohoe community were recruited in a similar manner, and grouped as either asymptomatic  or uninfected  controls. AC children were RDT-positive, and some had low parasitemia , whereas UC children were negative by microscopy and RDT. The main clinical characteristics of all the study participants are summarized in A total of 118 febrile  children aged 1–12 years reporting to Hohoe Municipal Hospital on clinical suspicion of One  or one to three venous blood samples  were collected in EDTA Vacutainers from each participant, in addition to the clinical parameters summarized in Drosophila melanogaster Schneider 2 stable cell line system as described in detail previously [Escherichia coli as described previously [Recombinant PfRH5 was expressed using eviously  and usedeviously . CyRPA aeviously  were expeviously . Recombieviously . The receviously .Plasma levels of IgG specific for PfRH5, GLURP-R0, GLURP-R2, and EBA175 were determined by ELISA as described for other malaria antigens in . In summsample−ODblank)/(ODpositive control−ODblank). The cutoff for seropositivity was defined as normalized ELISA OD + (3 x mean standard deviations) of the non-exposed sera samples.In all cases, antibody levels are presented as arbitrary units calculated as (ODOne-way ANOVA and Dunn’s post-hoc test were used to evaluate differences in antibody levels among donor groups, while Mann-Whitney’s test was used to assess malaria severity-related differences in antibody levels. Pairwise associations between D0 antibody levels to different antigens, and between D0 antibody levels and log-transformed parasitemias were assessed by Spearman’s rank correlation test. Temporal changes in antibody levels were evaluated using the running means method described elsewhere .


P. vivax in Qatar, and its ability to produce gametocytes, compared to parasites in main sites of imported cases, the Indian subcontinent (india) and East Africa (Sudan and Ethiopia). High diversity was seen among imported P. vivax in Qatar, comparable to parasites in the Indian subcontinent and East Africa. Limited genetic differentiation was seen among imported P. vivax, which overlapped with parasites in India, but differentiated from that in Sudan and Ethiopia. Parasite density among imported cases, ranged widely between 26.25–7985934.1 Pv18S rRNA copies/µl blood, with a high prevalence of infections carried gametocytes detectable by qRT-PCR. Parasitaemia was a stronger predictor for P. vivax gametocytes density . The extensive diversity of imported P. vivax and its ability to produce gametocytes represent a major threat for re-introduction of malaria in Qatar. The genetic relatedness between P. vivax reported in Qatar and those in India suggest that elimination strategy should target flow and dispersal of imported malaria into the region.Malaria control program in the Arabian Peninsula, backed by adequate logistical support, has interrupted transmission with exception of limited sites in Saudi Arabia and sporadic outbreaks in Oman. However, sustained influx of imported malaria represents a direct threat to the above success. Here we examined the extent of genetic diversity among imported  The escalating volume of international travel among people in endemic areas, driven by desire to improve quality of life and political unrest, has increased the risk of malaria importation into non-endemic areas, including Europe2.Global concerted and collaborative efforts, backed by WHO, have resulted in remarkable reduction in the burden of malaria, and disease elimination in many areas. Nonetheless, imported malaria represents a significant threat to the above achievements3, and active case detection to define existing malaria foci. Consequently, this has brought local transmission to a halt in many countries in the region, including Bahrain, Kuwait and the United Arab Emirates (UAE), and more recently Oman3. However, some sites in Yemen and southern Saudi Arabia remain malarious, with a high prevalence of drug-resistant Plasmodium falciparum4. The success of control programs in the region has prompted the health authorities in GCC countries to shift strategies towards malaria elimination and prevention of re-introduction in receptive areas5.Local malaria transmission in the Gulf Cooperation Council (GCC) countries has been interrupted as a result of sustained vector control7. The risk of malaria resurgence depends on the combined effect of receptivity and vulnerability. Receptivity is a function of the presence of local vectors and environmental conditions that favour malaria transmission, while vulnerability reflects the probability of importation of malaria parasites into a country8. The region is highly vulnerable to re-introduction of malaria, given the high volume of immigration from malaria endemic regions. This has been implicated in the persistence of high rates of imported malaria in many GCC countries9. For example, while a significant reduction in locally acquired malaria cases was observed between 2000 and 2014 in Saudi Arabia, imported cases remained consistent averaging between 250 and 830 cases per year10. Imported malaria is also common in Qatar, Bahrain, Kuwait and the UAE13, and has been implicated in repeated malaria outbreaks in Oman14. In Qatar, malaria transmission has been interrupted since 197015, however, a constant influx of imported malaria by the large number of immigrant workers from the Indian Subcontinent, and Sub-Saharan Africa, poses a risk for re-introduction16. Receptivity of the country to a re-introduction of malaria is evident by the presence of two potential vectors, Anopheles stephensi and A. multicolour17. The proportion of all imported malaria reported amongst travellers in Qatar has increased over the past decade, with P. vivax being the main prevalent parasite11.However, the above success is vulnerable to a possible resurgence of local transmission caused by imported parasites via asymptomatic travellers from malaria-endemic areasP. falciparum and P. vivax infections to produce gametocytes is well documented19. Currently there is no clear information on the actual magnitude of asymptomatic parasite reservoir in areas where malaria has been eliminated in the GCC countries and its population structure. Thus, the present study examined the source of imported P. vivax into the transmission free setting of Qatar, assessed its genetic complexity and its ability to produce gametocytes and transmit malaria. In addition, we compared the extent of diversity and genetic relatedness of imported parasites into Qatar with that in three countries of origin of immigrant malaria into Qatar, in Africa (Sudan and Ethiopia) and the Indian Subcontinent (India). Such knowledge would allow regional and national control programs to develop targeted policies to reduce the size of infectious reservoir, define the source of outbreaks and limit the risk of re-introduction of malaria.The impact of imported malaria, often as asymptomatic infection, on local epidemiology and local transmission is not well defined in Qatar and surrounding regions. However the ability of asymptomatic P. vivax cases (n = 318 [70.9%]), while P. falciparum (n = 118 [26.3%]) and mixed infection, with P. falciparum and P. vivax, was less frequent (n = 12 [2.7%])  were found to be positive. Species-specific PCR revealed a high prevalence of P. vivax cases were young males (93.7%), mean age 32 years, from the Indian Subcontinent (n = 264 [83%]); including, India (n = 148 [46.5%]), Pakistan  and Nepal . However, a small proportion of imported P. vivax cases were from Africa (n = 53 [16.7%]), including Sudan , and other African countries . On the contrary, the vast majority of imported P. falciparum cases were from Africa , with a fewer number of patients from the Indian Subcontinent  and others (n = 7 [6%])  were successfully examined by qPCR and qRT-PCR to quantify total parasitaemia and gametocyte density, respectively. Total parasite density estimated as transcript of Pv18s rRNA ranged between 26.2 and 7985934.1 copies/µl of blood (median = 1029.0).Ninety-five imported P. vivax isolates examined by RT-qPCR, 94 (98.9%) had detectable transcripts of Pvs25, indicative of the presence of gametocytes. The copy number of Pvs25 transcript, among the examined isolates ranged from 0.33 to −78441.24 copies of Pvs25/µl of blood (median = 300.3).Out of 95 Pv18srRNA and Pvs25 transcripts , suggesting an association between parasitaemia and gametocyte density , Ethiopia (n = 87) and India (n = 40). There was a high degree of polymorphism in all microsatellites, with number of alleles per locus varied from 8 for MS1 to 38 for MS8 among imported parasites in Qatar, 6 for MS7 to 22 for MS20 among parasites in Sudan, 4 for MS7 to 22 for MS8 among parasites in Ethiopia and 6 for MS5 to 19 for MS8 among Indian isolates  similar to that seen in endemic sites in Sudan (HE = 0.726), Ethiopia (HE = 0.684) and India (HE = 0.797) , Ethiopia (n = 34), Sudan (n = 33) and India (n = 30)  in Sudan each shared a distinct genotype.The predominant allele at each locus in each isolate was used to construct multi-locus genotypes (haplotypes). Each SA). This determines whether there is an association between alleles on different loci, and if the above high levels of diversity observed in P. vivax populations could be explained by frequent genetic exchange.The multi-locus genotype (haplotypes) data was used to estimate the standard index of association  compared to parasites from Ethiopia (0.0367) and India (0.0265) (Table Limited linkage disequilibrium (LD) was seen among the imported cases in Qatar Iiopia 0.067 and InP. vivax in Qatar, the isolates were divided into three populations  based on the nationality of patients. Pairwise analysis showed a close relationship between parasites from Pakistan and India (FST = 0.0008), compared to that seen between parasites from Sudan and India (FST = 0.0037) or Sudan and Pakistan (FST = 0.0011). Nonetheless, imported P. vivax from the three countries can be considered as one population. Similar close genetic relatedness was seen between imported P. vivax in Qatar, treated as one population, and isolates collected in India (FST = 0.0594). However, imported P. vivax in Qatar was slightly diverged from local parasite in Sudan (FST = 0.1435) and Ethiopia (FST = 0.1896).To assess the genetic relatedness among imported P. vivax population was distanced from the imported parasites in Qatar, while the Sudanese and Ethiopian parasites were slightly overlapped  Fig. . The Ethped Fig. . AnalysiP. vivax in Qatar and the East African isolates (Sudan and Ethiopia) when K = 2. However, when K = 3 there was some overlap between Sudanese isolates and imported cases in Qatar in agreement with the FST analysis. However, when K = 4, a clear overlap between the imported parasites to Qatar and parasite in Sudan and Ethiopia , Sudan (60.6%), and Pakistan (50.5%). Similar, high MOI was seen among local P. vivax in Sudan (44.70%), Ethiopia (67%) and India (62.5%).MOI defined as presence of more than one allele in any locus, was high among imported P. vivax from India (1.6), Sudan (0.7), and Pakistan (1.2) compared to that seen among endemic parasites in Sudan (2.4), Ethiopia (2.00) and India (2.00).Moreover, the minimum number of clones (MNC), estimated as the minimum number of alleles per locus, across all loci, was relatively low among imported 10 and sporadic outbreaks in Oman14 represent the final stage in the battle to eliminate endemic malaria in the region. However, the above success is threatened by the high influx of imported malaria by young immigrants from endemic countries in the Indian subcontinent  and East Africa (Sudan).The GCC countries have achieved great success in the fight against malaria. Four out of the six states accomplished complete interruption of malaria transmission and a malaria-free status, while limited sites in southwest Saudi ArabiaP. vivax malaria into GCC countries while Africa is the main origin of P. falciparum malaria. For some decades, the oil-rich region has been a popular destination for temporary labour workers, seeking employment opportunities. The high migratory turnover of immigrants from malarious areas is accompanied by increased reports of imported malaria in GCC countries9, reflecting a high vulnerability for re-introduction of malaria in areas where the disease has been eliminated. Though, receptivity, defined as the presence of the Anopheles vector, is limited as the result of current efficient vector control programs20. Nonetheless, autochthonous malaria cases were seen near livestock farms and building sites, where a large number of immigrants work, and environmental conditions favour vector habitats14. Analysis of a recent malaria outbreak in Oman identified Anopheles culicifacies as the vector involved14. Therefore, to minimize vulnerability, to re-introduction of malaria, a stringent surveillance system, based on highly sensitive molecular tools, should be structured to ensure timely detection and management of outbreaks seeded by imported malaria.The findings of the present study are in line with previous reports, demonstrating that the Indian Subcontinent is the most likely source for imported P. vivax in Qatar. Out of 95 P. vivax isolates successfully examined by RT-PCR, 83 (87.37%) harboured gametocytes evident by detectable transcripts of Pvs25 gene. This accords well with the findings of the high prevalence of low-density gametocyte carriage among P. vivax infection in many endemic sites21. P. vivax isolates examined in the present study were obtained from clinical cases. However, imported malaria often exists as asymptomatic low-level parasitaemia detectable only by PCR22 that may easily be missed by traditional microscopy and serological surveillance methods. An additional feature of asymptomatic infection, of particular relevance to prevention of re-introduction in areas where malaria has been eliminated, is its extended longevity. Asymptomatic P. falciparum infection can persist for several years, and discovered only when an individual develops clinical malaria23. Clinical presentations of P. falciparum malaria among immigrants have been found to delay by as long as 8 years after patients have left malaria-endemic areas24. Thus, asymptomatic parasite carriers can sustain long-term threat to malaria elimination programs.The vulnerability to re-introduction of local transmission is emphasised by the high prevalence of gametocytes carriage among imported 25. Amplification-based techniques are sensitive enough to detect and quantify gametocytes as low as 0.02–10 gametocytes per microliter26. Such low gametocytes densities are unlikely to be detected by microscopy and can make significant contributions to transmission27. In addition, parasites can increase gametocyte conversion rate in response to clues for transmission opportunity28. For example, the reappearance of Anopheles mosquitoes following the long dry period in areas of seasonal transmission can promote surge in gametocyte densities among asymptomatic parasite carriers26. This may explain the success of imported asymptomatic malaria to raise regular outbreaks in receptive sites, with low mosquito density in some GCC countries such as Oman14 and southern Europe29. Thus, molecular surveillance of migrants from endemic areas, in GCC countries and other receptive areas, can estimate the risk of re-introduction and allow targeted control measures to interrupt potential transmission.The presence of gametocyte among low-density asymptomatic parasite carriers is well documentedP. vivax parasites into Qatar [average HE = 0.782], similar to that in endemic sites where the majority of imported cases originate, in India [average HE = 0.797] and East Africa . This is consistent with previous reports on diversity of P. vivax in the Indian Subcontinent30 and East Africa31. The high level of diversity seen among imported P. vivax in Qatar reflects a constant flow of importation of diverse strains from multiple origins. This hypothesis is supported by the close genetic relatedness of imported P. vivax in Qatar and local parasite in India as shown by FST and PCoA analysis  and enhance the parasite diversity, as there is a direct relationship between the expected level of diversity and Ne32. In addition, the appearance of drug resistance in countries of origin such as India33, Pakistan34 and Ethiopia35 can augment the ability of the parasite to establish and evolve in the face of current control measures, if local transmission arises.High level of diversity was seen among imported P. vivax is higher than that seen amongst P. falciparum reported by immigrants in Qatar at the same time of the present study36. This is in line with data from different endemic areas that examined sympatric P. vivax and P. falciparum populations37. This has been attributed to the fact that the global P. vivax population is older, efficient in transmission, very diverse and less structured than P. falciparum38. This is conistsnt with the findings of low level of genetic differentiation between imported P. vivax in Qatar and local parasites in India (FST = 0.0594), and moderate differentiation between imported parasites in Qatar and that in East Africa  and Alkhor Hospital; Hamad General Hospital in Al-Doha city and Alkhor Hospital in the north area are the two main hospitals within HMC that assigned to admit patients with malaria, were examined microscopically for malaria between January 2013 and October 2016. All cases were diagnosed using conventional microscopic examination of Giemsa stained thick (100 fields) and thin blood (1000 RBCs). A total of 448 (76.8%) were found to be positive for the malaria parasites.All subjects were interviewed using a structured questionnaire to collect demographic information, including age, gender, nationality, history of travel and treatment. Patients were given treatment as per the Ministry of Public Health, Qatar.P. vivax infection, in Qatar. In addition, we also used 137 confirmed infected samples from Sudan, collected in Whatman qualitative filter paper, grade 3 between 2013–2014, 87 confirmed infected samples from Ethiopia collected in Whatman qualitative filter paper, grade 3 between 2010–2013 and 40 confirmed P. vivax samples from India collected between 2014–2016.The present study focused on analysis of 265 isolates obtained from patients with 35. Samples were collected after obtaining a written informed consent from the patients or guardians. All experiments in this study were performed in accordance with relevant guidelines and regulations of the above institutes.Ethical approvals were obtained from the Institutional Review Board of WCM-Q and HMC (Protocol no. 14-00097), ethical committee of the Postgraduate Institute of Medical Education and Research, Chandigarh, India (PGI/IEC/2014/88), Research Review Committee of Institute of Endemic Diseases, University of Khartoum (certificate research number 9/2016), and the Ethiopian isolates were reviewed and approved by the respective Ethical Boards of the Addis Ababa University College of Natural Sciences, Ethiopia (RERC/002/05/2013) and National Research Ethics Review Committee of Ethiopia (Ref.no. 3.10/580/06)P. vivax isolates in Qatar and parasites collected in India, using QIAamp DNA Blood Mini Kit as described by the manufacturer . DNA was isolated from blood collected on filter paper  from Sudan and Ethiopia using Chelex extraction method40.DNA was extracted from 200 µl of venous blood from the imported P. vivax isolate obtained from imported cases in Qatar using SV Total RNA Isolation kit . Conventional PCR was used to confirm the absence of co-extracted genomic DNA, and then RNA samples were converted to cDNA using the High Capacity cDNA Reverse Transcription Kit .RNA was extracted from 100 µl blood of P. vivax was confirmed using species-specific PCR as described by41. Total parasitaemia was quantified as copy number of the 18S rRNA transcripts in reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays based on TaqMan probe chemistry, primers and probes sequences are provided in Table 419. Pv18s rRNA copy numbers were estimated using in-plate standard curve generated from a 10-fold serial dilution  of a purified PCR product. Gametocytes were detected and quantified using RT-qPCR, targeting transcripts of Pvs25 gene, as described elsewhere21, with the use of modified probes and primers sequences (Table Species identification of 2 (1.5 mM), dNTPs (50 µM of each dNTPs) and 10 pmole of each primer. PCR conditions were, 94 °C/3 min, 35 cycles at 94 °C/30 s, 59.8 °C/30 s and 72 °C/45 s, and a final extension step at 72 °C/5 min42.We examined 9 polymorphic single copy tri-nucleotide repeats microsatellites , distributed across the whole parasite genome. In 25 μl premix containing Taq polymerase (2U), MgCl43.PCR products were analysed using ABI 3130XL Genetic analyser  with relative to an internal reference Genescan 500 LIZ . GeneMapper v4.0  for allele calling and quantification of peak height44. The minimum number of alleles across the 9 loci in each sample was calculated and this index value represented the minimum number of clones (MNC) (genotypes) per isolate. Then the average MNC for all isolates in each region was calculated44.The multiplicity of infection (MOI) refers to presence of more than one allele per isolate at least in one locus. To avoid overestimation of MOI, multiple alleles per locus were scored if electrophoretic peaks corresponding to minor alleles were ≥33% the height of the predominant allele45. This included determining the number of alleles per locus, and expected heterozygosity (HE). These two parameters were used to assess the level of polymorphism at each locus and determine diversity. HE was calculated using the formula for ‘unbiased heterozygosity’ also termed haploid genetic diversity, HE = [n/(n − 1)][1 − ∑p2] where n is the number of isolates and p the frequency of each different allele at a locus46.Genetic diversity parameters were calculated for the entire dataset using GenAlex v6.5P. vivax populations in different regions comprised a single panmictic population with a high degree of genetic exchange. Multilocus linkage disequilibrium  of the alleles at pairs of loci was estimated as the standard index of association (ISA) using the program LIAN version 3.547. The software tests for independent assortment of alleles by determining the number of loci at which each pair of MLGs differs, and from the distribution of mismatch values a variance VD  is calculated, which is then compared with the variance expected for LE, termed Ve. The null hypothesis that VD = Ve is tested by a Monte Carlo simulation and a parametric method and the results provide 95% confidence limits, which are denoted LMC and LPARA, respectively. If there is limited or no association between alleles at different loci, indicating panmixia, a value close to zero is obtained, whereas if association is detected, a value significantly greater than 0 is obtained, indicating non-panmixia47. The variance of pairwise difference (VD) between the data and that predicated for panmixia14 and L (LMC & LPARA), were calculated in order to test the hypothesis of panmixia. Therefore, when the VD value exceeds the L value, LD is indicted and the null hypothesis of panmixia is discarded. When the VD is less than L, LE is indicated and the null hypothesis of panmixia is accepted47.The predominant allele in each of the examined 9 microsatellites was used to construct multi-locus haplotypes to determine whether the FST) using Gene pop web interface48. Molecular variance (AMOVA) was calculated to estimate the variation within and between population and Principal component analysis (PCoA) was used to visualise the relationship between MLGs using GenAlEx 6.545.The genetic association between population pairs was evaluated by Wright’s fixation index , consisting of a burn-in period of 10,000 iterations followed by 100,000 Markov Chain Monte Carlo (MCMC) iterations, assuming a mixture model and correlated allele frequencies. K was defined according to49 by calculating the rate of change of K, ΔK using STRUCTURE HARVESTER v0.6.9450. Additional analysis of structure results was conducted using CLUMMP51 and DISTRUCT52. CLUMMP aligns the cluster assignment across replicate analyses while DISTRUCT performs a graphical display of the aligned cluster assignments.Structure software was used to elucidate the genetic structure and to detect the most likely number of clusters (K)


Plasmodium falciparum infection. While the majority recover, long-term cognitive impairment has been highlighted as a significant and neglected problem. Persistent or serious deficits in processes such as attention or behavioural inhibition should be manifest in changes to performance on oculomotor tasks. Therefore we investigated the impact of cerebral malaria on the development of reflexive pro-saccades and antisaccades. In a longitudinal study, 47 children previously admitted with retinopathy-confirmed cerebral malaria (mean age at admission 54 months), were compared with 37 local healthy controls (mean ages at first study visit 117 and 110 months respectively). In each of three or four test sessions, over a period of up to 32 months, participants completed 100 prosaccade tasks and 100 antisaccade tasks. Eye movements were recorded using infrared reflectance oculography; prosaccade, correct antisaccade and error prosaccade latency, and antisaccade directional error rate were calculated. Hierarchical linear modelling was used to investigate the effect of age and the influence of cerebral malaria on these parameters. Data were also collected from an independent, older group (mean age 183 months) of 37 local healthy participants in a separate cross-sectional study. Longitudinal data exhibited the expected decrease in latency with age for all saccade types, and a decrease in the antisaccade directional error rate. Hierarchical linear modelling confirmed that age had a statistically significant effect on all parameters (p< = 0.001). However, there were no statistically significant differences between the cerebral malaria and control groups. Combining groups, comparison with the literature demonstrated that antisaccade directional error rate for the Malawi sample was significantly higher than expected, while latencies for all saccade types were indistinguishable from published. The high directional error rate was also confirmed in the older, healthy Malawian participants from the cross sectional study. Our observation of similar oculomotor performance in cerebral malaria and control groups at long follow-up periods suggests that cerebral malaria survivors are not at a generally increased risk of persistent cognitive deficits. Our data raise questions about the prevailing hypothesis that cerebral malaria has gross impacts on the development of processes such as attention and behavioural inhibition. More importantly, our novel finding of a clear difference in antisaccade performance between all of the Malawi participants and published data suggests that the Malawian paediatric population as a whole faces serious challenges to cognitive development beyond cerebral malaria.Paediatric cerebral malaria is the most serious complication of  Malaria presents a substantial global health challenge, with an estimated annual incidence of approximately two million cases and over 600 000 deaths per year. Most deStudying cognition in cerebral malaria survivors is challenging. Neuropsychological tests developed in one cultural context cannot simply be transferred to another, even once linguistic barriers have been overcome ,12. CognAn alternative is the measurement of the parameters of saccadic eye movements, an approach widely used to investigate cognition and specific neural circuits  19] and and19] aIf cerebral malaria survivors are at risk of long-term impairments in cognition, particularly related to executive function, then we would expect to see performance deficits in saccade tasks compared to a non-cerebral malaria local control group. We therefore conducted a longitudinal case control study of retinopathy-confirmed cerebral malaria using both prosaccade and antisaccade tasks. As many of our participants were relatively young even at the end of the longitudinal study, a smaller cross-sectional study was conducted in a group of normally developing, older participants to confirm the developmental course of the parameters of interest in the population we were examining.All experiments were performed in accordance with the ethical standards laid down in the Declaration of Helsinki and were approved by the University of Malawi College of Medicine Research Ethics Committee. Study information was provided to parents, guardians and participants in the local language and written, informed consent obtained.Participants were recruited from two pre-existing cohorts of children from previous studies in Blantyre, Malawi. The Blantyre Malaria Project Epilepsy Study (BMPES ) recruitWe also examined a second group of participants who were originally recruited for the Brain Imaging in Normal Kids (BRINK) study . The BRI2 target spots subtending approximately 0.1°, in a horizontal line, centrally and at 10° to left and right of centre. As the stimuli moved with the head, participants were not head-fixed; they sat in a comfortable position approximately 1.5m in front of a near-white surface. For both pro- and antisaccades the stimulus was identical. After a randomised fixation period (1s-2s), the central fixation target was extinguished, and a single eccentric target appeared at 10° to either the left or right . Note that in both prosaccade and antisaccade tasks the target appeared immediately after fixation target extinction; synchronous rather than gap tasks were used.Horizontal eye movements were recorded binocularly with a miniaturised head-mounted infrared saccadometer . This sampled infrared reflectance signals at 1KHz, and low-pass filtered them at 250 Hz with 12-bit resolution. The device incorporated three low-power red lasers projecting red 13 cd/mIn the longitudinal study, participants completed 100 prosaccade and 100 antisaccade trials in two separate blocks in each testing session. While initially it had been hoped to counterbalance block order, participants found this confusing. So for the bulk of testing the prosaccade block was completed first. Prior to each block, participants completed 10 calibration trials, and were given detailed task instructions and stepped through the task. They were asked to describe, by pointing with their finger, what they had been instructed to do. For prosaccade trials, they were instructed to look at targets appearing to the left and right of centre as soon as they saw them. Other than this they were given no instructions about speed or accuracy. For antisaccade trials, care was taken to explain that they were to look to the mirror image position of the target, that is in the opposite direction to the target, but the same distance from fixation as the target. This was done while demonstrating the relative positions of the central fixation target, the target position, and where they were required to look. Participants were carefully observed throughout testing. If there was any doubt that they had understood the tasks instructions, the run was stopped, and the task explained again. Exactly the same procedures were followed during the single test session in the cross-sectional study on the BRINK cohort.Oculomotor data were stored on the Saccadometer handset, and downloaded using Latency Meter 4 software. Parameters were collated using MS Excel. For each participant and each testing session we computed the median prosaccade (Pro), correct antisaccade (CorAS) and error prosaccade (ErrPS) latency. We also calculated mean saccade amplitude for each type of saccade. For each participant and session, the antisaccade directional error rate (DER) was calculated.In an initial analysis of the longitudinal data, and to aid visualization, least squares linear regressions of each participant’s session data on age were calculated, and the slope and Pearson correlation coefficient used to summarize each regression. To explicitly investigate the influence of cerebral malaria, the latency of each type of saccade  and the DER were analysed using growth curve modelling ,43. Thisπi1 reflects i-th child slope of DER profile curve, π2 reflects the mean increase in DER due Malaria, β00 reflects the grand mean DER at the centred age of the sample, and β01 reflects the grand mean slope of the child’s DER profile. The variability of the random terms (ri0 and ri1) show the amount of variability within participants (of their intercepts and slopes). The models were fit using the restricted maximisation likelihood in the R statistical package (function “lme”).Level 1 describes the values of DER on the level of all participants (i.e. the between participant differences), while Level 2 describes the values of DER at the level of individual participants (i.e. the within-participant differences). In this model Malaria is a dichotomous predictor  and Age is centered at 132 months. The parameter In the longitudinal study, data were available from 47 retinopathy-confirmed cerebral malaria survivors and 37 control participants , all witWe observed the expected development in performance in prosaccade tasks ; as age As it takes additional time to programme and execute an antisaccade compared to a prosaccade, CorAS latency is expected to be greater than ErrPS latency. This pattern was observed in our dataset, and did not differ between groups . We compAs prosaccade errors in the antisaccade task are assumed to be ineffectively inhibited reflexive responses toward target onsets, there should be a clear correlation across participants between ErrPS latency and the latency of saccades in the prosaccade task itself (Pro). For both groups we observed statistically significant correlations between the latencies of these two types of saccade across participants  aged 192 months (16y) or over at the time of testing. The intersubject mean of individual median Pro latencies was 237±45ms, while CorAS and ErrPS latencies were 351±117ms and 253±51ms respectively. The mean DER was 58±22%. We compared these data with older participants from the longitudinal study. Given that there was no difference in the performance of cerebral malaria and control groups, we calculated mean latencies and DER for all participants whose average age over testing sessions was greater than 144 months N = 15; . The groFor all of the saccade parameters, performance in the BRINK group tended to be worse . For Pro and CorAS latency the group differences were not statistically significant . For ErrPS latency and DER the differences were statistically significant .Given the absence of a difference between the cerebral malaria and control groups for DER, we fitted a single function to all of the raw data to provide a single description of the influence of age on DER. For this we used the mean from the linear hierarchical model (±95% CI) because it was our best fitting model of DER . This siWe also compared Pro and CorAS latency from our data with data from the literature. This analysis was limited because, particularly for the antisaccade literature, latency is reported less consistently than DER. As listed in The developmental trajectory of reflexive prosaccade (Pro) latency, correct antisaccade (CorAS) and error prosaccade (ErrPS) latency, and of antisaccade directional error rate (DER) have been widely reported. Latencies steadily decrease with age, reaching a minimum in mid to late adolescence; the antisaccade directional error rate begins to decline around the age of 8 years, and shows a particularly marked decrease up to the age of approximately 14 years ,20,22,44Longitudinal analysis of saccade data from retinopathy-confirmed cerebral malaria survivors and age-matched local controls, demonstrated the expected decrease in latency with age for all saccade types examined and no difference in saccade behaviour or in its longitudinal development between the groups. We confirmed detailed relationships between saccade latency parameters and again found that they did not differ between the groups. Antisaccade DER also declined with age with no apparent difference between the groups. Hierarchical linear modelling confirmed that age had a statistically significant effect on all parameters, while having had cerebral malaria did not.While there is a clear qualitative Figs  and 3 anFor our samples, over the age range of 138 to 150 months, the mean antisaccade DER was 58.9% and 56.9% for cases and controls respectively, with a standard deviation of 19.4%. If antisaccade DER between groups is compared using a 1-tailed alternative hypothesis t-test, assuming a level of significance of 0.05, then group differences of 10%, 15% or 20%, would be detectable with powers of 75.2%, 96.7% and 99.9% respectively. This provides some assurance that we have sufficient statistical power to detect a magnitude of group difference (15%) that would generally be considered functionally important. Note that this reduces the power calculation to point estimates at a specific age. The growth curve modelling extends this by taking the longitudinal nature of the data into account.This lack of effect of cerebral malaria is surprising, and seemingly at odds with a body of literature suggesting a range of sequelae following cerebral malaria. However, there are important differences between this and previous studies. Many cerebral malaria studies have used standard clinical criteria ,49 for pWe also excluded participants with epilepsy in contrast to a number of studies reporting persistent neurocognitive impairments post-infection ,54. The Studies also differ with respect to both the age of infection and coma in cases, and the timing of assessments post-coma. In malaria endemic areas in sub-Saharan Africa with high rates of transmission, cerebral malaria mainly occurs in children aged less than 4 years. The average age at admission in our cerebral malaria group was approximately 4.5y. This was partly determined by the timing of our study relative to the original recruitment of participants into the BMPES study, and our need to test participants in the age range where saccade behaviour is developing. In children <3 years of age, cerebral malaria  has been reported to adversely affect general cognition and attention , as wellThe timing of post-coma or post-discharge assessments and follow-up periods vary across studies; this may be important as there is a temporal pattern to the emergence of sequelae. Across a number of studies ,51,60 a Two studies examined outcomes in retinopathy positive cerebral malaria survivors from Blantyre, Malawi ,61. BirbTiming of assessments is important because, particularly for cognitive functions such as attention, development continues into late adolescence and early adulthood ,63. It iThe other surprising result the emerges from our analysis is that when we combined the saccade data from cerebral malaria and control participants in the longitudinal study into a single “Malawi” dataset, we found that antisaccade DER was much higher than expected. This was confirmed using a weighted mixed-effects linear model to compare our data with data extracted from the literature. Not only was DER in the Malawi data higher across the range of ages we studied, but for that part of the age range for which we have most data, the lack of overlap of the 95% confidence intervals suggests that the difference is robust. This was not replicated for prosaccade and antisaccade latency where comparisons demonstrated that prosaccade and antisaccade latency at 11 years of age was statistically indistinguishable between the Malawi dataset and data drawn from the literature. The main weakness in this analysis is the relatively small number of studies we were able to identify which had used a synchronous antisaccade task as opposed to a gap task. The type of task used is an important consideration as DER is higher in gap tasks. However, agreement between the studies we were able to identify was reasonable despite a degree of methodological heterogeneity.Antisaccade DER reaches a plateau in late adolescence, as does saccade latency ,64. In tOur motivation in testing saccade development, and in particular in testing antisaccades, was to investigate cognition. It might be argued that saccade tasks are not associated closely enough with the aspects of cognitive function reported to be affected by cerebral malaria, compared to other types of neuropsychological testing. However, deficits in attention ,59,65, wFurther complementary testing of cognition at older ages in well-characterised cerebral malaria survivors will be required to establish precisely the long-term risks of cerebral malaria in early childhood, particularly in those apparently making good recoveries. It would also be useful to have saccade data from other populations for comparison both with our Malawi data and that published in the literature which is dominated by participants drawn from Western populations.While malaria in general and cerebral malaria in particular continue to pose serious health challenges, there is a need to study factors beyond cerebral malaria that might explain the poor antisaccade performance we have uncovered. While there are some population differences in saccade processing that do not appear to have an environmental cause –72, thes


Anopheles at different altitudinal transects, and the risk of malaria if any in the highland of Dirashe Woreda, South Ethiopia.The transmission of malaria is heterogeneous, and varies due to altitude. The information on whether the transmission of malaria is indigenous or imported to highland areas is scarce. Therefore, this study aimed to assess the species composition and infection rates of Anopheles mosquitoes were sampled using Centers for Disease Control and Prevention (CDC) light traps from thirty houses (ten houses from each village). The circum-sporozoite proteins (CSPs) rate and entomological inoculation rate (EIR) of Anopheles mosquitoes were estimated. For the epidemiological survey, malaria cases were collected from laboratory registration books of selected health facilities from (August 2015-April 2016). A cross-sectional survey was done to collect data on malaria vector control activities in each village (August-September 2015).This study was conducted in Gato , Onota  and Layignaw-Arguba  from August 2015 to April 2016. Anopheles mosquitoes comprising Anopheles arabiensis, An. demeilloni, An. cinereus, An. pharoensis, An. funestus-group, An. pretoriensis, An. christyi, An. ardensis and An. tenebrosus were identified in the study area. Anopheles arabiensis was the dominant species in Gato, whereas An. demeilloni was the dominant species in Layignaw-Arguba. Five mosquitoes, three An. arabiensis from Gato and two An. demeilloni from Layignaw-Arguba, were positive for Plasmodium falciparum CSPs. Plasmodium falciparum CSP rate was 0.4% (95% CI: 0.08–1.15) for An. arabiensis in Gato, and it was 0.64% (95% CI: 0.08–2.3) for An. demeilloni from Layignaw-Arguba. The P. falciparum EIR of An. arabiensis was 8.6 (95% CI: 2.4–33.4) infectious bites/person/nine-months in Gato. Plasmodium falciparum parasite was dominant in Gato (88%) and Onota (57.5%), whereas in Layignaw-Arguba P. vivax (59.4%) occurred most frequently. Increased malaria cases were observed in children age 5–14 years in Gato (P < 0.05), whereas in Onota and Layignaw-Arguba there was no statistically significant difference in malaria cases among the age groups. Households owning at least one long lasting insecticidal net were 92.7% in the study area, and 77.6% slept under the net during the preceding night of the survey. About 64.4% of the households in Gato were protected by the indoor residual spray. However, the spraying was done when the density of local malaria vectors was low.One thousand two hundred sixty-eight Plasmodium CSP positive Anopheles species and the presence of malaria in children under five years in high altitude Layignaw-Arguba may justify the existence of indigenous malaria transmission and the need for effective malaria control. Further investigation and confirmation using more sensitive molecular techniques are however needed to consider An. demeilloni as a proven vector of malaria in Ethiopia.Incrimination of  Malaria is a major public health problem of the world with the highest disease burden in sub-Saharan Africa . In EthiAnopheles mosquitoes [The areas with altitude 2000 m above sea level (masl) have been considered as malaria-free, but currently areas above 2400 masl experience local malaria transmission , 4. The squitoes , 7. Moresquitoes  includinsquitoes . Travellsquitoes . TherefoAnopheles species such as An. arabiensis , An. christyi, An. demeilloni and An. coustani have been documented in highland areas [Anopheles species from lowland and highland of Dirashe. Anopheles arabiensis, An. funestus, An. demeilloni and An. pharoensis were documented in low altitude, An. demeilloni and An. cinereus at mid altitude, and An. demeilloni from the high altitude of Dirashe. However, no adequate entomological and parasitological studies were conducted in the area to determine whether the transmission of malaria is indigenous or imported to the highlands. In the previous study conducted by Gone et al. [Plasmodium parasites, and therefore, the existing evidence may not be sufficient to conclude the presence of local malaria transmission in the highlands of the study area. Hence, the present study aimed to investigate variation in species composition, sporozoite infection rates of Anopheles mosquitoes, and estimate the risk of malaria at different altitudinal transects of Dirashe Woreda in south Ethiopia using circum-sporozoite proteins (CSPs), entomological inoculation rates (EIR), and malaria parasite prevalence.In Ethiopia, highlands ranging from an altitude of 1500–2500 m experienced a huge malaria epidemic in 1958 . Also, And areas , 12, 13.kebeles (villages) at different altitudinal transects in Dirashe Woreda (district) south Ethiopia. These were Gato , Onota  and Layignaw-Arguba  . Gato is one of the productive villages of the area using irrigation water from the River Yanda which crosses the village from West to East. Onota has varying topography with steep slopes and some plateau in the middle. Surface water is scarce in Onota. On the other hand, Layignaw-Arguba has several swampy areas, many springs, and ponds with a variable topography, vegetation cover and land use.This study was conducted in three de) Fig. . The altThe annual rainfall ranges between 600 and 1600 mm, and the annual temperature ranges between 15.1 and to 27.5 °C. There is a bimodal rainy season with the long rainy months from March to June and the short rainy months from September to October . Health services are provided by one primary hospital, four health centres, fourteen health posts and thirty-two private health facilities. The total population of the district is estimated to be 130,276 . The population of Gato is 12,327. Onota has a population of 6381, whereas the population of Layignaw-Arguba is 6569. The total number of households in the three villages is 5159; Gato has 2516, Onota 1302 and Layignaw-Arguba 1341 .Anopheles mosquitoes were collected twice a month using Centers for Disease Control and Prevention (CDC) light traps. Ten houses were selected randomly from each of the three villages. After obtaining consent from the head of the households, CDC light traps were installed in the sleeping places of the selected houses (from 18:00 to 06:00 h) at about 1.5 m above the floor near the foot end of the person sleeping under the insecticide untreated bednet [Anopheles mosquitoes were then identified morphologically to species under a stereomicroscope using a morphological identification key [Anopheles mosquitoes was determined under the microscope into unfed, freshly-fed, half-gravid and gravid. Finally, each female Anopheles mosquito was preserved individually in Eppendorf tube containing silica gel for detection of CSPs using enzyme-linked immunosorbent assay (ELISA) [A longitudinal entomological study was conducted from August 2015 to April 2016. Adult d bednet . A totalAn. gambiae (presumably An. arabiensis) and An. demeilloni were analysed for CSP detection using ELISA [P. falciparum and P. vivax_210 CSPs in Arba Minch University Medical Entomology Laboratory.The two dominant species, ng ELISA . The heaP. falciparum, P. vivax and mixed infections.The total number of outpatient attendant cases suspected of having malaria was confirmed by microscopic or rapid diagnostic test (RDT) in the selected health centres and health posts. The selection of the health facilities was not random but was based on where most people go for health care. Two health centres and four health posts were used for the survey. Malaria positive cases were categorised using standard age groups for malaria as < 1 year, 1–4 years, 5–14 years and 15+. All microscopic or RDT positive malaria cases were identified and classified into http://www.cdc.gov/epiinfo). The total number of households in the three villages was 5139. To determine the sample size of the survey, 50% malaria control intervention coverage, the margin of error of 4% and 95% confidence level (α = 5%) was considered. Therefore, the total sample size calculated was 538 households. Thus, the sample size of each village was calculated proportionally based on their total number of households. Hence, the number of households needed for the survey in Gato was 262, and it was 136 in Onota and 140 in Layignaw-Arguba.The sample size required for a cross-sectional survey to assess the vector control intervention coverage and use rate was determined using Epi Info™ . Analysis of variance was used to compare the density of was used . Thus, twas used . The altAnopheles mosquitoes comprising nine species were collected. Of these, seven species were found in Gato, three species in Onota and four species in Layignaw-Arguba. Anopheles gambiae (s.l.) (An. arabiensis) was the predominant species , followed by An. demeilloni . The other species were An. cinereus, An. pharoensis, An. funestus group, An. pretoriensis, An. christyi, An. ardensis and An. tenebrosus . Anopheles arabiensis was the most common species  in Gato while An. demeilloni was the most prevalent species in Onota , and in Layignaw-Arguba . No An. arabiensis were collected in Layignaw-Arguba and this species was among the rare species in Onota. Anopheles christyi was collected in Layignaw-Arguba only.The density of Anopheles mosquitoes varied significantly . Anopheles mosquitoes were collected mainly from November to January, and in April in Gato, from February to April in Onota, and from January to April in Layignaw-Arguba.The abundance of mosquitoes showed seasonal variations Fig. . The monAnopheles mosquitoes collected by CDC light traps, the majority  were unfed, and 467 (36.8%) were freshly-fed. Only a small proportion of Anopheles mosquitoes were half-gravid and gravid. In Gato, of 828 collected adult Anopheles females, 499 (60.3%) were unfed and 265 (32.0%) were freshly-fed. In Onota 71.9% (23 of the 32) were unfed. The trend was similar in Layignaw-Arguba where many female Anopheles were unfed , and freshly-fed   . The overall CSP rate of Anopheles mosquitoes for P. falciparum was 0.45% (95% CI: 0.06–1.1%) (5 of 1102). Plasmodium falciparum CSP rate of An. arabiensis from Gato was 0.4% (95% CI: 0.08–1.15%) (3 of the 754) but, no An. arabiensis was positive for P. vivax CSP. In Onota, one An. arabiensis and 19 An. demeilloni were tested for CSP, and all were negative. In Layignaw-Arguba, two An. demeilloni were positive for P. falciparum CSP. Hence, the P. falciparum CSP rate of An. demeilloni was 0.64% (95% CI: 0.08–2.3%) (2 of the 311), but no P. vivax positive An. demeilloni were detected.A total of 1102 CDC light traps collected SA Table . Five AnP. falciparum EIR of An. arabiensis was 8.6 (95% CI: 1.7–24.8) infectious bites per person (ib/p)/nine months , when the density of An. arabiensis was also higher compared to the other months. The P. falciparum EIR in October was 2.9 (95% CI: 0.07–14.7) for An. arabiensis. The EIR was not estimated for An. demeilloni because the species is not so far incriminated as a malaria vector, and no conversion factor was established to estimate the human biting rate from CDC light traps.The overall estimated P. falciparum, P. vivax or mixed infection. Plasmodium falciparum accounted for 34.3% (139 of the 405), whereas P. vivax accounted 18.3% (74 of the 405). Mixed infection of P. falciparum and P. vivax was predominant . More mixed infections were documented by RDT test  in the lowland Gato . Plasmodium falciparum (including the mixed infection) was the predominant species  in Gato. In Onota P. falciparum was also dominant species , whereas more P. vivax  cases were reported from Layignaw-Arguba. More malaria cases were observed in Gato than in Onota and Layugnaw-Arguba  but, the difference was not statistically significant between Onota and Layignaw-Arguba . Although malaria cases occurred in all age groups, more malaria cases were observed in children age 5–14 years in Gato . However, there was no statistically significant difference in number of malaria cases between Onota  and Layignaw-Arguba  among the age groups  were positive for P. falciparum and P. vivax were reported in Gato where microscope and RDT were routinely used for malaria parasite detection. August–September 2015 and January-April 2016 were the main malaria transmission seasons in Gato  had at least one LLIN. Of these, 160 (36.2%) had only one LLIN, and 282 (64.8%) had more than one LLIN, and the mean number was 2.1 LLINs (95% CI: 2.0–2.2). About half of the LLINs  were 2–3 years old with a mean age of 2.3 years (95% CI: 2.2–2.4)  households owned more than one LLINs, and the rest 16 (7.0%) had one LLIN, with the mean number of 2.8 LLINs (95% CI: 2.7–2.9). In Onota 66 (65.3%) of the households owned more than one LLIN and the remaining 35 (34.7%) had only one LLIN, with the mean number of 1.8 LLIN (95% CI: 1.6–1.9). In Layignaw-Arguba, most households  were provided with only one LLIN .There was a difference in LLINs ownership and usage at different altitudes. The overall LLIN usage was significantly higher in Gato (86.4%) compared to 71.3% in Onota and 64.6% in Layignaw-Arguba  households were re-plastered after the spray. IRS was conducted when the density of An. arabiensis was very low, and malaria cases have already been built-up in the population  were sprayed with propoxur 50% water-dispersible powder with the residual efficacy of 3–6 months at 2 g/m.4% were Anopheles mosquitoes at different altitudinal transects of Dirashe Woreda. Anopheles arabiensis, An. demeilloni, An. cinereus, An. pharoensis, An. funestus-group, An. pretoriensis, An. christyi, An. ardensis and An. tenebrosus were documented in the villages. Anopheles arabiensis was the dominant and the principal malaria vector in lowland Gato as reported in Chano Mille, south Ethiopia [An. demeilloni was dominant and positive for P. falciparum CSPs. The estimated P. falciparum EIR of An. arabiensis was 8.6 infective bites per person/nine months from Gato. Malaria cases were identified in all age groups in the three villages; however, more malaria cases were documented in children age 5–14 years in Gato. Plasmodium falciparum was the dominant parasite species in Gato, whereas in Layignaw-Arguba P. vivax was dominant species.There was variation in species composition and infection rate of Ethiopia , whereasAnopheles mosquito was based on the morphological characteristics which may result in misclassification as we did not use polymerase chain reaction (PCR) because of the limitation of resources and accesses.This study has several strengths and limitations. The entomological and epidemiological data were used to assess malaria transmission patterns at different altitudinal transects. The limitation of the study was the use of secondary data from health facilities. The identification of Anopheles mosquito was higher in Gato than Onota and Layignaw-Arguba. This finding agreed with the previous study done in the district [Anopheles demeilloni was the dominant species next to An. christyi in Western Kenya highlands [Anopheles cinereus was the second most common species in Layignaw-Arguba and Onota as it was previously documented in the highlands [P. falciparum CSP protein-positive An. cinereus was reported in Eritrea [Anopheles christyi was reported from highland, and it was the second common species next to An. arabiensis elsewhere in Ethiopia [An. christyi was reported from Akaki, in Ethiopia [An. christyi, An. demeilloni and An. cinereus [Anopheles mosquitoes were collected in the dry season; it may be due to drying of streams and formation of small water bodies which provide suitable breading habitats for Anopheles mosquitoes. Similar results have been reported from highland of South-central Ethiopia [The density of district . Anophelighlands . Anophelighlands , 13. So  Eritrea . AnophelEthiopia . The humcinereus . Most AnEthiopia .Anopheles arabiensis from Gato and An. demeilloni from Layignaw-Arguba were positive for P. falciparum CSPs. The sporozoite rate of An. arabiensis for P. falciparum in Gato was 0.4%, which is similar to 0.32% from Chano-Mille in southern Ethiopia [P. falciparum CSP rate of An. demeilloni in Layignaw-Arguba was 0.64%. In Western Kenya, a species morphologically identified as the member of An. demeilloni group was positive for P. falciparum CSPs by ELISA and PCR [Anopheles demeilloni has received attention because it was relatively abundant and its infectivity rate was similar to An. funestus group in western Kenya [An. demeilloni for comparison. However, confirmation using a more sensitive method such as PCR may be advisable.Ethiopia  and 0.3%Ethiopia . The P.  and PCR . Anophelrn Kenya , 25. Thern Kenya . This isP. falciparum EIR of An. arabiensis was 8.6 infectious bites per person/nine months that were higher than expected for malaria interruption (< 1 infective bites per person per year) [An. arabiensis was higher than the annual EIR of 3.66 infective bites per person per year from central highlands of southern Ethiopia [The estimated er year) . The ninEthiopia . HoweverEthiopia  since thAn. arbiensis and the warmer climate [P. falciparum was the dominant malaria parasites in Gato. On the other hand, P. vivax was the predominant parasite in the highland Layignaw-Arguba, as reported by other studies in Ethiopia [P. vivax which can develop in the vectors at a low temperature and can survive at high altitude and cool climate [In the current study, more malaria cases were documented in the lowland Gato. It could be due to the existence of an efficient malaria vector  climate . MoreoveEthiopia , 12. It  climate , 30. We  climate –33. The  climate .An. demeilloni may imply the indigenous malaria transmission. Similarly, highlands of Ethiopia ranging from an altitude of 1500–2500 masl have experienced malaria epidemics [Malaria in Layignaw-Arguba could be either indigenous, imported or both. The detection of malaria in age group <1 year who are less likely to travel to lowland and incrimination of pidemics . There ipidemics . The caupidemics . The incpidemics  and shorpidemics . HoweverAn. arabiensis to early evening before people go to bed or the vector feeding outdoors [The current study revealed that 93% of the households owned at least one LLIN and 64% of the households owned more than one LLIN which is much higher than the report of Ethiopian malaria indicator survey of 2011 . Despiteoutdoors . Moreoveoutdoors  and 62% outdoors .Anopheles mosquitoes during the house spray was low. On the other hand, one month after the spray from October through January the number of indoor Anopheles mosquitoes was increased. This suggests that the residual efficacy of propoxur may be less than the expected duration or the sprayer may use doses less than the recommended. It may not be due to the resistance of An. arabiensis because they are susceptible to propoxur in most parts of the country [The percentage of households protected by IRS in Gato (IRS was applicable only in this village) was 64.4% which is lower than 80%; the recommended coverage for effective use of IRS at the community level . This fi country . MoreoveAnopheles mosquitoes at different altitudinal transects. Anopheles arabiensis was the most dominant and the principal vector of malaria in Gato with an estimated P. falciparum EIR of 8.6 infective bites/ person/nine months. On the other hand, An. demeilloni was the predominant and positive for P. falciparum CSPs in Layignaw-Arguba. The incrimination of Plasmodium-positive Anopheles and presence of malaria in children under five years in Layignaw-Arguba may justify the risk of local malaria transmission in the highland of Dirashe Woreda, south Ethiopia. Hence, malaria control intervention strategies could include the highland village of the district. However, there is a need for further investigation and confirmation using more sensitive molecular techniques to consider An. demeilloni as a proven vector of malaria in Ethiopia.There was variation in species composition and infection rate of


Plasmodium carriage were investigated to estimate the parasites reservoir among asymptomatic individuals living in low transmission areas in Dielmo and Ndiop, Senegal during the dry season.In the progress towards malaria elimination, the accurate diagnosis of low-density asymptomatic infections is critical. Low-density asymptomatic submicroscopic malaria infections may act as silent reservoirs that maintain low-level residual malaria transmission in the community. Light microscopy, the gold standard in malaria diagnosis lacks the sensitivity to detect low-level parasitaemia. In this study, the presence and prevalence of submicroscopic Plasmodium carriage by real time PCR (qPCR) in comparison to microscopy considered as gold standard.A total of 2,037 blood samples were collected during cross-sectional surveys prior the malaria transmission season in July 2013 (N = 612), June 2014 (N = 723) and June 2015 (N = 702) from asymptomatic individuals living in Dielmo and Ndiop, Senegal. Samples were used to determine the prevalence of submicroscopic Plasmodium carriage was 3.75% (23/612), 12.44% (90/723) and 6.41% (45/702) in 2013, 2014 and 2015, respectively. No Plasmodium carriage was detected by microscopy in 2013 while microscopy-based prevalence of Plasmodium carriage accounted for only 0.27% (2/723) and 0.14% (1/702) in 2014 and 2015, respectively. Plasmodium falciparum accounted for the majority of submicroscopic infections and represented 86.95% (20/23), 81.11% (73/90) and 95.55 (43/45) of infections in 2013, 2014 and 2015 respectively.The prevalence of submicroscopic Plasmodium carriage is common in the study areas during the dry season indicating that traditional measures are insufficient to assess the scale of parasite reservoir when transmission reaches very low level. Control and elimination strategies may wish to consider using molecular methods to identify parasites carriers to guide Mass screening and Treatment strategies.Low-density submicroscopic asymptomatic  Plasmodium falciparum remains a significant public health problem despite largely successful control efforts that have led to a sharp decrease of malaria transmission, morbidity and mortality in recent years [Malaria due to nt years . Accordint years . This suMost malaria surveillance systems, including the system in Senegal, are predicated on detection of symptomatic malaria cases at health facilities using either clinical diagnosis alone or with parasitological confirmation by light microscopy (LM) or rapid diagnostic tests (RDT). In most settings, where the goal is to significantly reduce malaria morbidity and mortality, quality assured LM and RDT have been shown to effectively detect parasites in the majority of symptomatic patients and thus guide treatments. In contrast, as malaria transmission declines and countries progress towards elimination, LM and RDT are insufficiently sensitive to detect low-level parasitaemia, thus causing gross underestimation of parasite prevalence in areas where most infections are subpatent –6.Plasmodium parasites, the rapid advances in molecular biology and nucleic acid testing methods and their routine application in clinical studies and epidemiological surveys have enabled the detection of low-density submicroscopic infections as reported in several studies [Although LM remains the gold standard for the diagnosis of malaria and quantification of  studies –10. More studies , 12.To date, the importance of sub-microscopic infections for malaria transmission is still unclear . HoweverPlasmodium parasites carriage in the wider catchment population; therefore mitigating against some of the biases associated with passive case detection [Cross-sectional surveys that sample from asymptomatic individuals provide an operationally attractive method to estimate the prevalence of etection , 17.P. falciparum prevalence in asymptomatic individuals estimated by LM has been shown to decline from 85% to 1% in 0–3 years old children and 34% to 2% in adults > 50 years between 1990 and 2010 [In Dielmo and Ndiop, two Senegalese villages where longitudinal studies on determinants of malaria infections have been conducted since early 1990s , the disand 2010 .Plasmodium spp parasites carriage in community-based cross sectional surveys conducted prior the malaria transmission season in two Senegalese villages, Dielmo and Ndiop, where all malaria indicators have declined substantially over the last decade.The present study sought to determine the prevalence of submicroscopic The study was conducted in Dielmo and Ndiop, two extensively described malaria endemic villages situated in Fatick region, Senegal , 22. In Plasmodium carriage by LM and quantitative Real Time PCR (qPCR)  .The study falls within the ongoing Dielmo/Ndiop project, a longitudinal prospective study of malaria infection and the determinants of the disease in a community that has been conducted since early 1990s. The study objectives, benefits and risks were explained in Serere and wolof (the most commonly spoken languages in the areas) or French  to adults participants and legal guardians of minors before inclusion. Written informed consent was obtained from adults and legal guardians of minors participants. The study was examined and approved by the Senegalese National Health Research Ethics Committee.For microscopy determination of malaria parasites, thin and thick blood smears were prepared, air-dried, fixed in methanol, stained for 20–30 minutes in Giemsa and microscopically examined for malaria parasites by experienced microscopists at the Institute for Research and Development. At least two hundred oil-immersion fields were examined at 100X magnification before a slide was considered negative.Plasmodium spp was carried out by qPCR following genomic DNA isolation of Plasmodium parasites using QIAamp DNA Blood Mini Kit  according to manufacturer’s instructions. DNA from blood samples of known microscopically-confirmed P. falciparum, P. malariae, P. ovale and P. vivax infected patients served for positive controls in all amplifications; sterile water and uninfected samples served for negative control to ensure lack of contamination [The detection of mination .Plasmodium spp was based on two steps real time PCR as previously described [Plasmodium parasites were detected by a “screening real-time PCR” with genus-specific primers targeting the Plasmodium Cytochrome B gene using the Eva Green dye (Solis Biodyne) followed by melt curve analysis of resulting amplicons. Amplification was conducted in a 20 μl reaction mixture containing 5 μl of genomic DNA, 4 μl of 5X Evagreen qPCR Master Mix (Solis Biodyne) and 10 μM of each primer. Amplification was run in a Bio-Rad CFX96 real time thermal cycler using previously described conditions [P. falciparum, P. vivax, P. malariae, and P. ovale). Primers sequences and real-time PCR conditions used to characterize Plasmodium species were as described by Canier et al. [P. knowlesi, the fifth human malaria parasite [The qPCR-based detection and characterization of escribed , 25. In nditions . In the r et al. . The preparasite  was not Plasmodium carriage was defined as an infection with Plasmodium detected by qPCR but not by LM.Submicroscopic Plasmodium carriage was analyzed according to three defined age groups , sampling period (2013 to 2015), and village of origin (Dielmo and Ndiop) taking into account entomological inoculation rate (EIR) determined as detailed elsewhere [Plasmodium carriage between villages and across sampling periods. Statistical significance was considered when P values were less than or equal to 0.05.Data were analyzed using R statistical software. Distribution of submicroscopic lsewhere , 27. FisPlasmodium parasites , 2014 (N = 723) and 2015 (N = 702) were investigated in this study for the presence of arasites . Female arasites . Particiarasites .Plasmodium species, DNA obtained from known microscopy and nested PCR-confirmed P. falciparum, P. vivax, P. malariae and P. ovale infected samples were used as positive controls [Plasmodium DNA results in a melt curve appearing with a melt peak temperature between 76.20°C and 78.80°C  in 4.65% (14/301) and 2.89% (9/311) of samples from Dielmo and Ndiop, respectively (Plasmodium positive samples (1 in Dielmo and 1 in Ndiop) among the 723 samples screened representing a prevalence of 0.28% (1/355) and 0.27% (1/368) in Dielmo and Ndiop, respectively. The 2-microsocopy positive samples originated from a 9.7 years old male from Dielmo and a 0.6 years old female from Ndiop. The qPCR-based molecular diagnostic successfully amplified Plasmodium DNA in 12.72% (92/723) of samples collected in 2014 of which 6.22% (45/723) originated from Dielmo and 6.50% (47/723) from Ndiop (Plasmodium carriage in Dielmo and Ndiop were 12.67% (45/355) and 12.77% (47/368) respectively  and Ndiop  among the corresponding 702 samples with available LM data. Sub-microscopic Plasmodium carriage represented 6.41% (45/702) of the overall tested samples with available microscopic data in 2015. All microscopy-positive samples from 2014 and 2015 sampling were also positive by qPCR.In 2013, no nd Ndiop  while qPectively . In 2014om Ndiop . The speectively . In 2015 species  and micrP falciparum infections accounted for the majority of submicroscopic Plasmodium carriage representing 86.95% (20/23), 87.77% (79/90) and 95.55% (43/45) of detected Plasmodium in 2013, 2014 and 2015 respectively  and 2.22% (2/90) of sub-microscopic Plasmodium spp in 2013 and 2014 respectively while P. malariae was not detected in 2015  and 10% (9/90) of sub-microscopic infections in 2013 and 2014, respectively  of sub-microscopic infected samples , villages (Dielmo and Ndiop) and sampling period (2013 to 2014). Submicroscopic Plasmodium carriage was similarly distributed across the three-years sampling periods  and in both villages (Dielmo and Ndiop) and was characterized by an age-dependent carriage of Plasmodium parasites with frequencies being highest in older individuals (> 15 years) and lowest in younger ones (≤ 5 years) (The carriage of submicroscopic 5 years) . The uni5 years) . StatistPlasmodium carriage was not significantly different between Dielmo and Ndiop in 2013 , 2014  and 2015  despite variation of transmission as determined by the annual entomological inoculate rate (EIR)  and Ndiop , similar to the EIR  . By cont the EIR .Plasmodium infection  to reduce and subsequently suppress the reservoir. The function “reservoir” is multifactorial and involves humans , vectors and Plasmodia. From the parasite perspective, the rapid and accurate diagnostic of low-density submicroscopic infections that are usually missed by conventional RDT and LM diagnostic methods remains a challenge. In fact, despite their many advantages, the limitations and shortcomings of both RDT and LM, to detect low-density parasitaemia are well documented elsewhere [Given the renewed focus on continuous reduction of malaria transmission, morbidity and mortality in many endemic countries ultimately leading to elimination of the disease, malaria control efforts deserve to focus on lsewhere , 9. The lsewhere , 9, thusPlasmodium cytochrome b gene [Plasmodium carriage in Dielmo and Ndiop, two malaria endemic villages where all malaria indicators have declined substantially over the last decade. The study revealed substantial submicroscopic Plasmodium carriage among asymptomatic individuals at various proportions in both villages prior the malaria transmission season. These findings are important as they provide a basis for identifying and defining priority areas for control interventions when elimination strategies are being considered.In the present study, a reliable and cost effective molecular-based malaria diagnostic targeting the e b gene  was usedPlasmodium carriage in 2014 and 2015, respectively compared to 12.72% and 6.01% prevalence revealed by qPCR.The report of similar findings from others studies conducted in different malaria transmission contexts , 28, 29 Plasmodium carriage was age-dependent, similar to reports from others studies that have shown that individuals older than 15 years were several times more likely to carry submicroscopic parasites than younger individuals, thus arguing for a link between increasing age and lower parasite densities [Plasmodium carriage in a community.In this study, submicroscopic ensities , 30. ThePlasmodium carriage observed between the two studied villages despite the difference in malaria transmission intensity, could suggest that submicroscopic Plasmodium carriage is not related to malaria transmission intensity. This is supported by the fact that the significant difference of prevalence observed across sampling period (2013 to 2015) both in Dielmo and Ndiop was unrelated to the malaria transmission intensity.The absence of significant difference in the prevalence of submicroscopic P. falciparum carriage in the study areas may have important implications for malaria control measures in Senegal since such infections may be important contributors to the infectious reservoir as documented elsewhere [The higher prevalence of submicroscopic lsewhere , 31, 32.lsewhere , 34. Thelsewhere , 36.From a clinical point of view, low-density infection may have significant public health importance, as studies conducted by Bousema and Drakeley have shown that mosquitoes feeding on LM parasite-negative individuals can become infected with malaria . From thFurther studies to unveil the magnitude of low-density submicroscopic malaria parasites carriage at the country level both within asymptomatic and symptomatic individuals coupled with entomological investigations addressing their role in sustaining malaria transmission are needed to guide accelerated efforts towards malaria elimination in Senegal.Plasmodium carriage among asymptomatic individuals, thus indicating that estimates based on routinely used diagnostic techniques such as RDT and LM, grossly underestimate true malaria parasite prevalence. The findings reported in this study warn against a relaxation of control efforts once prevalence of infection appears low according to LM. The results have also important practical implication for potential malaria elimination strategies in the areas as they could guide a mass screening and treatment approach to accompany elimination efforts in the areas that can be subsequently extended countrywide.The community-based surveys conducted prior the malaria transmission season in Ndiop and Dielmo revealed substantial submicroscopic