Background: Malaria still presents as a major health problem in Indonesia and specifically in East Kalimantan. One common sign found in malaria patient is thrombocytopenia, the mechanism of which is still unclear. Several studies have suggested some mechanisms, one of which is splenomegaly. This study aimed to discover the association between thrombocytopenia and splenomegaly of malaria patients in East Kalimantan.Methods: This study was a descriptive retrospective study with clinical and laboratory data obtained from the medical records of malaria patients in four major public hospitals from January 2015 to July 2018. The association between thrombocytopenia with splenomegaly was analysed using Chi-Square test.Results: A total of 215 patients were included; 189 male (87.9%) and 26 female (12.1%). The etiologic agents found in these patients were Plasmodium vivax (43.2%), Plasmodium falciparum (42.8%), and mixed infection  (4.6%). The thrombocyte count was normal in 28 patients (13%) and decreased in 187 patients (87%). Among patients with thrombocytopenia, the percentage of mild, moderate and severe thrombocytopenia was 18.2%, 43.8% and 33%, respectively. Splenomegaly was found in only 11 patients (5.1%). We found no association between thrombocytopenia with splenomegaly (p=0.61).Conclusions: We conclude that splenomegaly, which was a rare clinical finding of these malaria patients, was not associated with thrombocytopenia. The 2007 and 2013 Basic Health Surveys of the Health Ministry show that the prevalence of malaria in Indonesia increased from 2.9% to 6.0%2.Data from the East Kalimantan Provincial Health Office indicated that there were 7,045 cases of malaria in 2010. This number fluctuated in the following years, with 3,021 cases in 2011, 9,966 cases in 2012 and 2,603 cases in 2013. In terms of Annual Parasitic Incidence (API), in 2014 East Kalimantan was still above the national average with an API of 2,04 per 1000 population, leading to it being categorized as a low cumulative incidence area5, and this is found in both infection withPlasmodium falciparum andPlasmodium vivax. The results from our previous study demonstrated that from 1041 malaria cases in East Kalimantan, 85% presented with thrombocytopenia of varying degrees4. Therefore, thrombocytopenia has been suggested as one important diagnostic parameter in malaria. The mechanism of thrombocytopenia in malaria is still unclear. Several theories, such as mechanical trapping of thrombocytes inside the spleen and immune response that attacks thrombocytes, has been proposed5. A study by Coelhoet al. found that platelet phagocytosis may contribute to thrombocytopenia in vivax malaria6.One common finding in malaria is decreased platelet count or so-called thrombocytopenia. This laboratory finding is often confused with other infectious diseases, especially dengue infection in which thrombocytopenia is a major diagnostic parameter. There are several studies that demonstrate thrombocytopenia in malaria patientsSplenomegaly is also one common clinical finding in malaria patients. The spleen is part of the reticuloendothelial system, which becomes active in order to get rid of plasmodium-infected erythrocytes. Splenomegaly can also contribute to increased destruction of thrombocytes due to mechanical trapping. Therefore, the aim of the present study was to determine the association of thrombocytopenia with splenomegaly in malaria patients in East Kalimantan.This study was a cross-sectional retrospective study. This study was approved by the Ethical Committee for Health Research at Abdul Wahab Syahranie Public Hospital Samarinda, East Kalimantan . Patient consent for the use of their data records was waived by the ethical committee due to the retrospective nature of the study.Data were collected between June and August 2018 from the medical records of patients with malaria during the period of January 2015 to July 2018. We collected clinical and laboratory data of both outpatients and inpatients diagnosed with malaria from four major hospitals in East Kalimantan: Abdul Wahab Sjahranie Hospital in Samarinda, Aji Putri Botung Hospital in Penajam Paser Utara, Abdul Rivai Hospital in Tanjung Redeb, and Panglima Sebaya Hospital in Tanah Grogot.All patients with malaria, both paediatric and adult patients, were included in the study. Patients were excluded from the study if the necessary data were incomplete or patients were discharged upon own’s request during treatment.In order to collect data, first, the hospital’s database was searched for patients diagnosed with malaria. Second, after identifying these patients, the relevant medical records were retrieved which contained age, gender, type of Plasmodium, thrombocyte count, and presence of splenomegaly. Type of Plasmodium was determined using microscopic method by obtaining thick blood smear and staining by Giemsa 3%. The thrombocyte count included in this study was from laboratory data at the time of hospital visit or admission before receiving any treatment. The thrombocyte count was determined using automated haematology analyser of different types and manufacturers available in each hospital.For descriptive data, we described patients’ characteristics that include age, sex, type of Plasmodium, and thrombocyte count on the first day admission. The association between thrombocytopenia and splenomegaly was analyzed by Chi-square test using SPSS 23.0 software. Results were considered statistically significant if p<0.05.Our study identified a total of 215 malaria patients from January 2015 to July 2018 from four hospitals in East Kalimantan. There were 87.9% (189/215) male and 12.1% (26/215) female patients .The association of thrombocytopenia with splenomegaly in malaria patients shown inData retrieved from the medical records of malaria patients in East Kalimantan, Indonesia, including age, gender, type of Plasmodium, thrombocyte count, and presence of splenomegalyClick here for additional data file.Copyright: © 2019 Siagian LRD et al.2019Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Plasmodium vivax (43.2%). This study found that thrombocytopenia affected the majority of malaria patients; thrombocytopenia occurred in 87% of patients, with moderate thrombocytopenia (43.8%) as the majority. This result is in concordance with another study by Arifet al., in India, that found that 79% of patients had moderate thrombocytopenia7, while Ansariet al. found 69.18%8. In the present study, the degree of thrombocytopenia classified to mild, moderate and severe was 18.2%, 43.8%, 33.0%, respectively. If we compare to study of Arifet al. that found 33.96%, 51.15% and 14.89%, respectively, we find that the moderate thrombocytopenia was the highest percentage in both studies7.We found that the incidence of malaria was higher in males than females in this group of patients, and the parasite infecting the majority of patients waset al. in Vietnam found that thrombocytopenia was a marker of disease severity in adults withPlasmodium falciparum infection, but has limited utility in prognostication, triage and management9. On the other hand, Raoet al. in India found that severe thrombocytopenia showed positive correlation with complicated malaria and become a good predictor for poor prognosis10. Thrombocyte has an important role in haemostasis. However, study by Abroet al. demonstrated that malaria patients with thrombocytopenia rarely presented with any bleeding manifestations even with platelet count as low as 9,000/μL11.A study by Hansonet al. found that only 20% malaria patients had splenomegaly, but there are no information about thrombocyte count in those malaria patients with splenomegaly12.Indeed, splenomegaly is a hallmark of malaria, but the result of this study found that only 11 patients (5.1%) showed splenomegaly. In India, Guptaet al. in 20136. Until now, definitive mechanism of thrombocytopenia in malaria is still unclear. However, some factors that contribute to thrombocytopenia have been reported, such as decreased thrombopoiesis, peripheral destruction induced byP. falciparum and disseminated intravascular coagulation11.In our study, we describe the thrombocyte count in malaria patients with splenomegaly. We found that there was no association between thrombocytopenia with splenomegaly (p=0.611). Therefore, we propose that thrombocytopenia is not caused by the mechanical trapping of thrombocytes in the spleen. This result suggests another mechanism of thrombocytopenia that involves immune process, as proposed by CoelhoeThis study has its limitations. Different types of automatic haematology analysers were used in the hospitals and this might contribute to variations in thrombocyte count. Therefore, we suggest the use of a single haematology analyser across hospitals to improve accuracy. In addition, we did not exclude other causes of thrombocytopenia that could coexist with malaria infection, such as viral infection and autoimmune diseases, which would be required to further validate our findings. Finally, the examination of splenomegaly was conducted by different physicians, which might contribute to subjective factors in determining splenomegaly, such as physician’s expertise and thoroughness, especially in cases with minor or subclinical splenomegaly.Overall, we conclude that splenomegaly, which was a rare clinical finding in this set of malaria patients, was not associated with thrombocytopenia.The data referenced by this article are under copyright with the following copyright statement: Copyright: © 2019 Siagian LRD et al.Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).https://doi.org/10.5256/f1000research.16606.d22512413F1000Research: Dataset 1. Data retrieved from the medical records of malaria patients in East Kalimantan, Indonesia, including age, gender, type of Plasmodium, thrombocyte count, and presence of splenomegaly, All my concerns have been addressed, and nothing further to add.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The report is a relatively straight-forward and short description of a prospective malaria patient cohort, with a specific focus on platelet numbers and possible relationships between thrombocytopenia and the presence of splenomegaly. This is an important question to ask in relation to thrombocytopenia in malaria, since the mechanisms involved are not well-defined – splenic trapping is certainly a valid mechanism to consider. Their platelet data support many previous studies of other patient cohorts showing thrombocytopenia is a common clinical feature in malaria. However, the relatively small number of patients exhibiting splenomegaly (11/204 or 5.1%) precludes making a definitive conclusion about the lack of association with thrombocytopenia. Therefore, I recommend this limitation be included in the abstract and final conclusions of the paper. Eg. Suggested wording: Splenomegaly, which was a rare clinical feature of this malaria patient cohort, was not associated with thrombocytopenia. The methods should include notes about how platelet counts were determined, and the fact that different hematological analyzers were used (this is currently mentioned in the Discussion).I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. How to define the confirmed cases of this study (microscopic ELISA or PCR).The methodologies to count the thrombocytopenia and splenomegaly.Plasmodium and the day of infection.It is important to separate the cases by species of Additional comments - there are some important things missing in this manuscript that need to be explained by the authors:The 1st paragraph; Please use the national Annual Parasite Incidence (API) below 1.00 per 1.000 population or the prevalence < 1%.The methodologies need to be addressed, because this is the most important; a) how did the authors use the definition of malaria cases in this study (giemsa 3%)? and b) for the methods of counting the parasite and thrombocytes , did the authors make QA for the test?P. vivax is correct – 42.0% (from page 3 in the discussion) or 43.2% (from Table 1)?Which prevalence ofIn the discussion I need more explanation about the aims of this study; "Discover the association between thrombocytopenia & splenomegaly”, please write about the following: a) the main function of thrombocytes and the kinds of situation risks decreased and for which patient conditions (acute or chronic diseases) and b) the authors should be addressing why their hypothesis was not considered significant.This study is important and has a great impact for clinicians in the subject, however I found many lacks in the assessment methodologies, especially:here for my full comments. Please find an annotated copy of the articleI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.


Plasmodium malariae is the third most prevalent human malaria-causing species and has a patchy, but ample distribution in the world. Humans can host the parasite for years without presenting significant symptoms, turning its diagnosis and control into a difficult task. Here, we investigated the immunogenicity of recombinant proteins of P. malariae MSP1.Escherichia coli as GST-fusion proteins and immunized in BALB/c mice. The specificity, subtyping, and affinity of raised antibodies were evaluated by enzyme-linked immunosorbent assays. Cellular immune responses were analyzed by lymphoproliferation assays and cytokine levels produced by splenocytes were detected by cytometry.Five regions of PmMSP1 were expressed in 19 are strongly immunogenic in mice. After three doses, the induced immune responses remained high for 70 days. While antibodies induced after immunization with N-terminal and central regions showed similar affinities to the target antigens, affinities of IgG against PmMSP119 were higher. All proteins induced similar antibody subclass patterns , characterizing a mixed Th1/Th2 response. Further, autologous stimulation of splenocytes from immunized mice led to the secretion of IL2 and IL4, independently of the antigen used. Importantly, IgG from P. malariae-exposed individuals reacted against PmMSP1 recombinant proteins with a high specificity. On the other hand, sera from P. vivax or P. falciparum-infected individuals did not react at all against recombinant PmMSP1 proteins.We found that N-terminal, central regions, and PmMSP1P. malariae in epidemiological studies or in the differential diagnosis of malaria caused by this species. Immunization with recombinant PmMSP1 proteins resulted in a significant humoral immune response, which may turn them potential component candidates for a vaccine against P. malariae.Recombinant PmMSP1 proteins are very useful diagnostic markers of  Plasmodium malariae was the first malaria parasite observed in 1881 by Charles Laveran, Nobel Prize winner for discovering the cause of malaria [P. malariae is a cosmopolitan parasite that does not develop below 15°C and is described in tropical and subtropical regions of sub-Saharan Africa, South America, much of Southeast Asia, Indonesia, and in many islands of the western Pacific. In general, the distribution coincides with that of P. falciparum. In South America, P. malariae is associated with zoonotic infections, and is easily confounded with P. brasilianum, a parasite that infects twelve different genera of New World primates [P. malariae and P. brasilianum antigens in endemic areas [ic areas .P. malariae infections are usually low compared to those in patients infected with P. falciparum or P. vivax perhaps due to its longer developmental cycle , lower number of merozoites produced per erythrocyte cycle, and its preference for developing in older erythrocytes [P. malariae may persist asymptomatically in the infected individual for long periods [P. vivax and P. ovale infections, there is no evidence of latent liver stage forms (hypnozoites) [Parasitemias in  periods , but it P. malariae as one of the oldest parasites that cause malaria. The adaptive success is characteristic of the slow biological cycle that favors its survival for long periods in the host organism without being noticed [P. malariae  and cas is one of the major candidates for an antimalarial vaccine because it is expressed abundantly on the merozoite surface and capable of activating the host protective immunity . Studies monkeys ,16, P. mP. malariae MSP1 polypeptide, we evaluated the humoral and cellular immune responses of BALB/c mice immunized with five recombinant proteins, representing different portions of the PmMSP1 protein.In order to investigate the immunogenicity of the BamHI site). Recombinant plasmids were checked for their integrity by semiautomatic sequencing using the Big Dye Terminator v3.0 Cycle Sequencing Kit in an ABI Genetic Analyzer 3550 . The sequences were deposited in GenBank . The recombinant PvMSP119 and PfMSP119 proteins corresponding to the amino acid sequence of the 19-kDa fragment of MSP1 of the isolates Belém and S-20, respectively, were used as controls [The PmMSP1 fragments were obtained from sample I11, isolated in 2002 from an infected patient in Iporanga, a municipality located in Atlantic Forest Region of the State of São Paulo, Brazil . Five frcontrols . Then, tcontrols .19 and GST as control) of four female 6–8 weeks old BALB/c mice were used. The immunization was performed via the intraperitoneal (i.p.) route with 25 μg of recombinant protein in the presence of Freund’s incomplete adjuvant (Sigma) and 500 ng monophosphoryl lipid A adjuvant (MPLA) (Avanti)/dose. Three doses were administered at 15-day intervals. Blood samples were collected by a puncture of the submandibular artery of the immunized animals. Blood samples were collected before the first immunization and at the 14th, 28th, 45th and 70th day after the first immunization  values higher than 0.1. These values represent binding of IgG to the recombinant protein after subtraction of binding of the same serum to GST alone. The results are presented as the geometric mean ± standard deviation of four animals per group.Total IgG antibodies against F1, F2, F3, F4, PmMSP1eviously . RecombiDetection of IgG subclass responses was performed as described above, except that the secondary antibodies were specific for mouse IgG1, IgG2a, IgG2b and IgG3  diluted 1:5,000. The OD values of the sera against the fusion proteins were corrected by subtracting the OD values from the GST-coated wells.492 in the presence of ammonium thiocyanate x100 / OD492nm in the absence of ammonium thiocyanate.The affinities of the antibodies were assessed by a thiocyanate elution-based ELISA . The proCytokine levels secreted into the splenocyte culture supernatant stimulated for 72 h with the recombinant proteins (20 μg/mL) or ConA (2.5 μg /mL), were determined using a mouse Th1/Th2/Th17 cytometric bead array kit  on a LSR Fortessa BD flow cytometer in accordance with manufacturer’s instructions and data analysis was performed by FCAP Array v.3 software.Serum samples from 15 healthy individuals and 21 individuals with unrelated diseases  were obtained from the Biorepository of Samples, Laboratory of Protozoology, Tropical Medicine Institute, University of São Paulo. Serum samples from 16 individuals with malaria were obtained from the Biorepository of Samples of the Department of Parasitology, Biomedical Sciences Institute, University of São Paulo. Ethical clearance for the extension of the use of these sera was obtained from the Ethics Commission .P. malariae patients (1:500) diluted in PBS with 1% milk. Bound antibodies were detected with horseradish peroxidase-labeled anti-human IgG (Sigma Co.). The color of detected bands was developed after reaction using a chemiluminescence detection assay .For this analysis, the recombinant proteins were separated by SDS-PAGE (12%) diluted (vol/vol) in SDS-sample buffer , and boiled for 5 min at 100°C. Samples were loaded on a 12% polyacrylamide minigel . Antigens were separated by electrophoresis, transferred to nitrocellulose membranes , and blocked with PBS containing 5% non-fat milk for 1 h at room temperature. Membranes were incubated with a serum pool of a posteriori multiple comparisons between the means of the groups were performed with Tukey’s Test. To determine the differences between the sampling times ANOVA double-ranking was used, followed by a Bonferroni test. The cutoffs were established by using the mean plus three standard deviations of signal measured with negative sera.Statistical analysis was performed using the GraphPad Prism program version 5.0 for Windows . For the comparison of antibody titers of more than two groups at the same sampling time ANOVA analysis was used, and 2 asphyxiation as recommended [The mice used in this study were provided by the Department of Parasitology, Institute of Biomedical Sciences. All the procedures were submitted to the Ethics Committee of the Institute of Medicine Tropical and were approved under the registration number of the Committee on Ethics in the Use of Animals (CEUA) (CPE-IMT / 000304A). All animals immunized were females aged from 4 to 6 weeks and were maintained in a 12 h light/12 h dark cycle with free access to water and standard chow under appropriate environmental and hygienic conditions. Serum was harvested by submandibular vein bleeding. After trials, mice were euthanized using COommended . Ethical19 and with GST alone as a control, and the resulting antibody responses were measured by ELISA using recombinant proteins. Results in To characterize the immune responses generated by immunization with PmMSP1, immunogenicity studies in mice were conducted. Groups of 4 BALB/c mice were immunized with F1, F2, F3, F4, PmMSP119 were boosted after the second immunization while specific antibody titers were similar  after the third immunization and were maintained until the end of the experiment (day 70)   (for the(day 70) .19 showed that all recombinant proteins elicited similar patterns of IgG subclasses. Antibodies of all isotypes were detected with the levels of IgG1, IgG2a and IgG2b higher when compared to IgG3 . Interestingly, the observed affinity was higher (2.096 M) for PmMSP119 .19, GST and concanavalin A stimulation are shown. In 19 showed a progressive decrease in fluorescence after 72 h, indicating proliferation. Almost no proliferation was observed for GST-, F1- and F4-peptide stimulation . In mulation .19 was higher than in the other groups , P. vivax (PvMSP119) and P. falciparum (PfMSP119) was evaluated by ELISA with five patient sera. As shown in 19, only the sera from patients I11, I16, I65  recognized the antigen (19). When the ELISA plate was sensitized with PvMSP119 (19) or PfMSP119 (19), only the sera from patients with P. vivax (C_Pv) or P. falciparum infection (C_Pf) reacted with their corresponding antigens. Some recognition by P. malariae patient sera was also recorded, possibly due to previous, not reported, contact with P. vivax and P. falciparum.The reactivity of MSP1P. malariae, P. vivax and P. falciparum by ELISA   . The cor n = 16) . Cut-offhttps://www.who.int/news-room/detail/23-04-2019-malaria-vaccine-pilot-launched-in-malawi). Of the 25 malaria vaccine projects under development in the world, four are in stage IIb or III [P. falciparum, which is probably insufficient in areas where a significant proportion of patients suffers from P. vivax or mixed infections. Furthermore, there are three other species that infect humans that have to be considered: P. malariae, P. ovale, and P. knowlesi.The development of effective methods for malaria diagnosis and control is still on the malaria research agenda. There is currently one vaccine  being applied in some African countries . After the second dose, the most immunogenic proteins were the F1  and PmMSP119  regions. These regions were also found to be the most immunogenic in other species of Plasmodium, such as P. vivax, where, using recombinant plasmids for the immunization of mice, the most antigenic plasmids were those that encoded the N-terminal and C-terminal regions [Regarding the response of antibodies against blood stage antigens, known for their importance in protecting against malaria , our res regions .P. falciparum blood stage is a bad predictor of protection [19 antigens [19 [P. yoelii [P. yoelii, protection occurs in the presence of elevated levels of IgG1, IgG2a and IgG2b, with a mixed Th1/Th2 immune response [Antibodies to MSP1 may persist for up to 30 years in some patients with malaria . Althougotection , it is botection . The devotection ), parameantigens ,36 or Pfgens [19 ,38,39 ingens [19 . It is i. yoelii ,42. In o. yoelii . Differeresponse ,45,46,4719 were those with the highest levels of IL-4. IL-10 cytokine is anti-inflammatory, secreted by activated Th2 cells, and is believed to limit harmful inflammatory responses during the blood stage of the parasite in mice [19. The cytokines produced by each subset promote the polarization, where the cytokines produced by Th1 cells negatively regulate the Th2 response, and vice versa [Cytokines play a critical role in determining the subclasses of IgG. IL-4 is secreted by Th2 cells and is associated with an IgG1 response, an indicator of a predominantly antibody-mediated response. As demonstrated by our results, the spleen cells of the mice immunized and stimulated with F1 and PmMSP1 in mice . IL-10 wce versa . TherefoP. malariae may be more widely evaluated as potential vaccine candidates against blood stage parasites. Similar results have been reported in studies involving mice immunized with MSP142 recombinant of P. falciparum and P. vivax [Many studies have shown that both cellular and humoral immunity are involved in protective immunity against malaria ,50,51. BP. vivax ,37.P. falciparum merozoite antigens were associated with protection from malaria [19) in the presence of the chaotropic agent ammonium thiocyanate. Our results showed that the antibodies generated with immunization with PmMSP119 showed the highest affinity, requiring a higher molarity of ammonium thiocyanate to dissociate 50% of the antibodies. In the sera of the animals immunized with the F1, F2, F3 and F4 proteins, the effect of ammonium thiocyanate in the antigen-antibody interaction was very similar. Antibodies with high affinity for the target antigen have also been reported for PvMSP119 [The affinity of an antibody for its corresponding antigen is an important determinant of the antibody’s biological efficacy ,53. For  malaria . Thus, wPvMSP119 ,58.P. malariae in western blot assays suggesting that the presence of the GST did not modify the epitopes of MSP1 recognized by human IgG. GST, originally from Schistosoma japonicum, was not recognized by these sera. Our experiments demonstrated that PmMSP119 was antigenic in natural infections, with species-specific recognition. Similar results were also found with human sera from Zimbabwe, Haiti, Cambodia and Mozambique, where species-specific reactivity and rare cross-reactivity of host serum to MSP119 antigens were demonstrated [19 and PfMSP119 also showed no reactivity with the respective heterologous antigen [19 for detection of antibodies in human malaria patients as well as its serological efficacy for detection of IgG and IgM among experimentally infected chimpanzees has also been demonstrated [PmMSP1 recombinant proteins were recognized by IgG antibodies from human sera from patients with malaria caused by nstrated ,60. Mice antigen . The usenstrated .P. malariae, P. vivax and P. falciparum, we observed that only sera from patients positive for P. malariae recognized the F1 and F2 proteins. This did not occur for F3 and F4, where the observed ODs were generally lower but sera positive for P. vivax and P. falciparum also recognized these proteins. Such cross-reactivity may be a consequence of the higher identity percentage that is observed between these regions and the heterologous proteins (from 50 to 60% at the amino acid level).Additionally, in our analysis of the reactivity of the F1, F2, F3 and F4 recombinant proteins with sera from positive patients for 19 by serum IgG from Brazilian individuals diagnosed with malaria due to P. malariae in parallel with sera from patients with other diseases, reaching 100% of sensitivity and specificity. In the particular case of malaria caused by P. vivax, studies on the naturally acquired humoral immune response against PvMSP1 showed that PvMSP119 is the most immunogenic portion of the molecule and is recognized by 90% of the patients in the region of the Brazilian Amazon with a specificity of 98.3% [19 is also strongly recognized by human sera from endemic regions of malaria [By ELISA, we also evaluated recognition of PmMSP1of 98.3% ,63. Studof 98.3% . PfMSP11 malaria .P. malariae or P. brasilianum in populations. Since the produced antigens are quite immunogenic, they may also prove useful for the rational design of vaccine formulations against P. malariae malaria. However, studies as a growth inhibition assay (GIA) and challenges by P. brasilianum of PmMSP1 protein immunized monkeys are still needed to know the true potential of these antigens as vaccine candidates.This study represents the first step in assessing humoral and cellular immune responses in animal models, as well as the naturally acquired response to the PmMSP1 antigen. The different recombinant proteins produced here may be useful in sero-epidemiological studies, both in humans as well as non-human primates from different geographic regions, highlighting possible targets in the development of more sensitive and specific diagnostic tests and enabling an evaluation of the exposure to S1 Fig19 and GST. Antibody titers were detected by ELISA at the 14th, 28th, 45th and 70th days after the first immunization. The results are expressed by geometric mean ± standard deviation pattern. * (p <0.05), ** (p <0.001) and *** (p <0.0001).F1, F2, F3, F4, PmMSP1(TIF)Click here for additional data file.S1 File(ZIP)Click here for additional data file.


Plasmodium diagnosis by microscopy and PCR, and an extensive clinical questionnaire completed. Malaria prevalence ranged from 3–8% by PCR in community surveys , with Plasmodium vivax dominating in Chennai (70.8%) and Nadiad (67.9%), and Plasmodium falciparum in Rourkela (77.3%). A proportional high burden of asymptomatic and submicroscopic infections was detected in community surveys in Chennai  and Rourkela . In clinic studies, a proportional high burden of infections was identified as submicroscopic in Rourkela  and Chennai . In the community surveys, anemia and fever were significantly more common among microscopic than submicroscopic infections. Exploratory spatial analysis identified a number of potential malaria hotspots at all three sites. There is a considerable burden of submicroscopic and asymptomatic malaria in malarious regions in India, which may act as a reservoir with implications for malaria elimination strategies.Malaria in India, while decreasing, remains a serious public health problem, and the contribution of submicroscopic and asymptomatic infections to its persistence is poorly understood. We conducted community surveys and clinic studies at three sites in India differing in their eco-epidemiologies: Chennai (Tamil Nadu), Nadiad (Gujarat), and Rourkela (Odisha), during 2012–2015. A total of 6,645 subject blood samples were collected for  As the seventh largest and second most populous country in the world, the subcontinent contributes nearly half of the global population at risk of Plasmodium vivax1. Malaria surveillance is primarily conducted by active case detection by accredited social health activists (ASHAs) and passive case detection at Primary Health Centres (PHCs), Community Health Centres (CHCs), or malaria clinics. These surveillance programs included over 120 million blood smear examinations in 2014, equivalent to sampling ~10% of the population2. Although blood smear microscopy remains the most common diagnostic method, the number of malaria rapid diagnostic tests (RDTs) supplied by local and regional manufacturers has more than doubled since 20102. Molecular methods such as polymerase chain reaction (PCR) are expensive and require sophisticated laboratory equipment, making them unfeasible for routine diagnosis.Malaria remains a major public health burden in the south-east Asia region, and approximately 80% of malaria cases of the nine countries in this region occur in IndiaNational Strategic Plan for Malaria Elimination in India 2017–2022, whose goal is to eliminate malaria in states with low level malaria  or bring states with higher levels of malaria to pre-elimination levels by 20223. The epidemiology of malaria in India is complex. Both P. falciparum and P. vivax can be present, with falciparum malaria tending to dominate in the eastern and northeastern provinces, and P. vivax in other parts of the country. Malaria elimination strategies in India will need to be fine-tuned according to the endemicity at the state and district levels. For this purpose, it is important to know the real burden of malaria, i.e., not only what is routinely detected, but also what is asymptomatic (infections in people who do not feel sick and so who do not visit a clinic for diagnosis or treatment), or below the level of detection of the current common diagnostic tools of microscopy (submicroscopic) or RDTs. Recent reviews have shown the importance of submicroscopic infections for both P. falciparum and P. vivax5, although studies from India are lacking. To understand the local epidemiology of malaria in India and the submicroscopic and asymptomatic burden further, we conducted a series of cross-sectional and clinic-based epidemiology studies at three sites in India with different malaria endemicities as part of an International Center for Excellence for Malaria Research6. A better understanding of the epidemiology of malaria in India is critical for allocating resources and assessing the impact of malaria control efforts. Here we describe the prevalence of malaria, species of Plasmodium parasite, presence of submicroscopic and asymptomatic infections, and identify hotspots, as well as assess the factors associated with malaria for each setting.India has joined other countries on the path to malaria elimination and has recently developed a five-year Plasmodium diagnosis by microscopy, rapid diagnostic test (RDT), and PCR, and for testing of hemoglobin level. The study populations enrolled at the three sites differed in their characteristics .Our three study sites were chosen because of their different eco-epidemiologies: Chennai, in the southern state of Tamil Nadu; Nadiad in the western state of Gujarat,; and Rourkela in the eastern state of Odisha Fig. . We condcs Table , e.g., mP. falciparum the dominant species in Rourkela and P. vivax more common at the other sites. Risk factors identified for malaria  included (1) rainy season (as measured by PCR and microscopy) at all sites  in Chennai and Rourkela  in our community surveys to be 2.7% in Chennai, 7.5% in Nadiad, and 8.3% in Rourkela . Additionally, asymptomatic P. falciparum infections were associated with the rainy season, being male, and ITN (insecticide treated net) use, and asymptomatic P. vivax infections with repellent (protective) and recent antimalarial use  microscopic and 15 or 88.2% submicroscopic), 64.4% of all malaria infections in Rourkela , and 21.4% of all malaria infections in Nadiad . Submicroscopic P. falciparum infections were more common among males at both sites, and submicroscopic P. vivax infections were associated with the rainy season and age <15 years compared to age ≥15 years in Rourkela , and 30.7% of the cases in Rourkela (31 infections), although they were not species-specific  and locally (within the immediate surroundings of each data point), of Plasmodium infections identified by PCR in the community studies. Global and Local Moran’s I analyses identified areas where greater concentrations of malaria occurred than would be expected from a random distribution of cases , 0.125 (p = 0.0014), and 0.049 (p = 0.0002) respectively , indicating that Nadiad and Rourkela exhibit overall clustering (significant at the 5% level), whereas Chennai exhibited overall dispersal (not significant at the 5% level). This is notable because a vector-borne disease such as malaria typically would be expected to exhibit a strong clustered autocorrelation due to the spatial relationships between cases and the transmission of the parasite by Anopheles mosquito8.Using Exploratory Spatial Data Analysis (ESDA)ses Fig. . The Cheses Fig. , Nadiad ses Fig.  and Rourses Fig.  sites ex7, a single illustrative site was characterized further from each of the locations. In Chennai  for further investigation, comprising an area known for housing fishermen in close proximity to the Besant Nagar beach. In Nadiad , malaria prevalence as detected by PCR was 21.1% (222 infections) in Chennai, 9.6% (66 infections) in Nadiad, and 5.0% (93 infections) in Rourkela; submicroscopic infections were 18.9% (42 infections) in Chennai, 1.5% (1 infection) in Nadiad, and 45.2% (42 infections) in Rourkela of the total malaria detected Fig. . Althougi.e., active) surveys, the point prevalence was low in all sites, with P. vivax dominating in Chennai and Nadiad, and P. falciparum in Rourkela, confirming the species distribution historically reported. In Chennai the majority of infections were submicroscopic (17 or 70.8%) and asymptomatic (17 or 70.8%), and similarly in Rourkela most infections were asymptomatic (65 or 64.4%) and 31 (35.6%) were not detected by microscopy. In contrast, Nadiad subjects that were asymptomatic (12 or 21.4%) or submicroscopic (2 or 3.6%) constituted only a minority of infections detected by community surveys. In our clinic studies, the majority of malaria infections were detectable by microscopy. However, approximately one in five infections in Chennai and two in five infections in Rourkela were submicroscopic, with troubling implications for malaria treatment and reservoirs of transmission. Moreover, just like symptomatic infections, asymptomatic infections detected in the community survey in Nadiad were associated with a significantly increased risk of anemia compared to persons without Plasmodium infections.To gain an understanding of the local epidemiology and burden of malaria in India, we conducted active and passive case detection studies in community and clinic settings at three sites. In the community , but no association with anemia was detected11. In three villages in Chhattisgarh, a survey in 2016 during the low transmission season detected 10% subpatent and 18% asymptomatic malaria12. A high submicroscopic or asymptomatic burden has been reported in studies elsewhere in Asia14; and a recent study in three southeast Asian countries found four times higher prevalence of submicroscopic Plasmodium infections with a sensitive PCR method compared with microscopy, indicating that the burden may still be underestimated15. We could retrieve few studies reporting on the effects of submicroscopic malaria from areas with both malaria parasite species: in Brazil, submicroscopic P. vivax was associated with anemia16, but this association was not seen in our studies, or in a study in Indonesia13.Very few surveys in India have used molecular methods such as PCR to assess the submicroscopic burden of malaria, and in those cases (as in our study) the results were variable and dependent upon the setting. For example, a survey in June 2012 in Purulia district, West Bengal, identified 6.9% P. falciparum; compared to adults, young children (<5 years) were significantly more likely to have microscopic P. falciparum and microscopic and submicroscopic P. vivax, whereas older children (5–14 years) were more at risk for P. falciparum. In areas with multiple malaria parasite species, it has been suggested that the acquisition of immunity to P. vivax develops faster, with a peak of P. vivax at younger ages compared to P. falciparum, and an earlier ability to keep parasite densities of P. vivax infections under control18. Age patterns similar to our observations have been described in studies from Bangladesh, Papua New Guinea, Thailand, and the Amazon in Brazil21. The reasons for the faster acquisition of clinical immunity to P. vivax are not well understood, but repeated exposures from hypnozoites might be a contributing factor23.In our community surveys, age was a risk factor for malaria mainly in Rourkela, the site with the highest prevalence of 24 and Ethiopia25, and has been attributed to more outdoor activity by men in areas, such as forests, with infected mosquitoes. However, the increased risk was also observed in our site in the urban city of Chennai that has few forested areas. This higher risk might be due to different use of malaria prevention methods, distinctive attractiveness to mosquitoes, different travel patterns by gender, or hormonal or host-genetic factors24. A better understanding of the factors contributing to the gender gap may assist in identifying more efficient ways to prevent malaria among males, by determining, e.g., if the increased risk among males is exposure-related on account of different habits before bed time compared to women.Males were at higher risk of malaria at all of our sites. The difference in malaria risk by gender among adults has been reported before in AsiaP. vivax in the community surveys in Chennai and Nadiad, perhaps indicating that not all malaria detected in these locations originated at those sites. Although ITNs were only used in Rourkela, they were more a marker of malaria risk than associated with protection, as has been previously reported26, whereas the use of repellents was associated with reduced malaria risk in Nadiad and Rourkela.Other risk factors for malaria included travel for P. falciparum infections suggested that submicroscopic carriers actually predominate in areas of lower transmission, which would accord with our observations at our other sites27.At our site in Nadiad, almost all infections in the clinic study and community survey were microscopic. Nadiad has a history of low malaria prevalence, so residents can be expected to have low immunity, which may explain why most infections will develop parasitaemias detectable by microscopy. However, even in this area with sustained low transmission, one out of five malaria infections identified in the community surveys was asymptomatic. A review of submicroscopic Plasmodium infections. Recent use of an antimalarial drug can reduce Plasmodium infections to submicroscopic levels as part of the normal process of clearing parasites. However, persistence of submicroscopic levels can occur after treatment when drug resistance is present or when inadequate doses of antimalarials are used. In areas with both P. vivax and P. falciparum, treatment of P. falciparum or mixed species infections has been associated with an increased risk of the occurrence of P. vivax28. Recent antimalarial use was associated with symptomatic and asymptomatic P. vivax infections in community studies in Nadiad and symptomatic P. vivax in community studies in Rourkela, and with submicroscopic malaria in clinic studies in Chennai. P. falciparum resistance to chloroquine is well known, but P. vivax resistance to chloroquine has been infrequently reported in India30; currently the combination artesunate-sulfadoxine-pyrimethamine is used with good reported efficacy in our study sites31.Several factors have been associated with submicroscopic and asymptomatic 32, we did not find a large discrepancy in number detected by the two methods  in the literature in recent years, leading to substantial improvements in risk mapping for malaria and other diseases such as dengue36, this work has almost exclusively been undertaken using historical data that have been aggregated to the district or village level (e.g.34). In contrast to this, our analysis of individually georeferenced PCR results allowed analysis to take place without the generalisation and aggregation that are typically exhibited in datasets relating to disease prevalence. Accordingly, specific AOIs have been identified for further analysis, rather than simply the district within which hotspots are contained. Once identified, AOIs can undergo further scrutiny either statistically, using techniques such as Geographically Weighted Regression in order to examine the relationship between hotspots and social, entomological, or environmental factors; or in the field, by visiting the locality in order to identify behaviours or factors that may be contributing to these hotspots, and propose interventions in order to reduce risk. Additional collection of temporal data too will enable changes in risk factors, malaria incidence, and the resulting autocorrelation results over time to be discerned, as reported by other studies36.Our spatial data analysis demonstrated how local spatial autocorrelation techniques on high resolution  datasets can be used to identify specific areas of interest (AOIs) in which there are unexpectedly high concentrations of 4. The microscopists at our sites were trained and experienced, and the PCR tests were were performed by experienced molecular biology technicians at each site and repeated at least twice with cross-checking of a subset in a second lab. Parasite genetic diversity may affect submicroscopic infections and this is still under study at our sites, and associations between other host genetic factors such as hemoglobinopathies were not examined. We did not determine if persons identified with asymptomatic infections subsequently developed symptoms, or if they were able to clear the parasites. The surveys in Chennai and Rourkela were underpowered and it is possible that some associations with malaria were not significant because of low numbers. Second, because of the timing of the interviews during the daytime, our surveys did not capture all household members; in Nadiad and Chennai, men and children were underrepresented compared to the census  and of asymptomatic infections (range 18–74%) should not be underestimated in light of malaria elimination efforts. This is especially true for 40. Malaria transmission (predominantly P. vivax) in Chennai is perennial and peaks between July-October. The Besant Nagar Malaria Clinic is in a predominantly residential neighbourhood composed of middle- and upper-class dwellings, along with a few slums and a large coastal fishing community. Subjects were enrolled from the Besant Nagar Malaria Clinic and the surrounding Besant Nagar catchment area. Nadiad city, population ~225,000, is located in the district of Kheda in the central part of Gujarat State. Nadiad receives the majority of its rain during the southwest monsoon season (June-September)40. Malaria is considered to be unstable (hypo-endemic), with P. vivax and P. falciparum prevalence rates oscillating throughout the year based on the rainfall. The National Institute of Malaria Research (NIMR) Malaria Clinic is located in the Civil Hospital of Nadiad in a predominantly residential neighbourhood. Subjects were enrolled at the Civil Hospital outpatient clinic, and community surveys were conducted in areas adjacent to Nadiad town. Rourkela city, population ~483,000, is located in the Sundargarh district of the eastern state of Odisha. Rourkela receives rains during the southwest monsoon season (June-September) and some rainfall during the northeast monsoon (December-January)40. Historically, malaria transmission has been meso- to hyper-endemic in this region, with P. falciparum as the major infecting species, accounting for >40% of the total cases in the country. Subjects were enrolled from a malaria clinic at Sector 1 in Rourkela, whereas community surveys were conducted in areas close to forested regions around Rourkela.Chennai city, the capital of the southern state of Tamil Nadu and the sixth most populous city with ~7 million people, is located on the coast of the Bay of Bengal. The main rainfall period is from October-December as part of the northeast monsoon, but rains also fall during the southwest monsoon between July-AugustP. vivax: chloroquine 25 mg/kg over three days and primaquine 0.25 mg/kg for 14 days; P. falciparum artesunate 4 mg/kg for 3 days in combination with sulfadoxine 25 mg/kg and pyrimethamine 1.25 mg/kg on the first day and primaquine 0.75 mg/kg)41. A census was conducted before the start of the community surveys. The community surveys were conducted in random household samples of the census and were conducted over the course of two years spread around the seasons; If a household was not available, the next household on the list was used. All members available in the household were sampled during day time hours of 7AM to 3 PM. We did not return to a household to find missing household members. Compared to the census, children were under-represented in Chennai and Nadiad, whereas women were overpresented in all sites . All samples were amplified by PCR at least twice, and a third time if any discrepancy, and 10% sent for cross-checking at a central lab facility. Positive controls consisting of known P. falciparum, P. vivax and P. malariae DNA and negative controls (no DNA) were used for every amplification experiment.Hemoglobin level was assessed using HemoCue . Thin and thick smears were Giemsa stained and at least 300 fields in the thick smear were examined by microscopy using a 100x oil immersion objective. Parasites were counted on the thick smear against 200–500 leucocytes. The results were expressed as parasites per microliter of blood, using the white blood cell (WBC) count if known, or assuming 8,000 WBCs per microliter of blood. Slides were routinely read by two microscopists, and a third microscopist was used if there was disagreement. All samples, whether positive or negative by microscopy, underwent DNA extraction by QIAamp DNA blood Mini Kits . A modified nested, multiplex-PCR method targeting the 18 S small subunit ribosomal DNA gene (SSU rDNA) was used for species-specific detection of 44, and data were exported into Stata  for analysis. We classified parasite infections detected by PCR but not by microscopy as ‘submicroscopic’. We defined malaria as symptomatic if the subject was Plasmodium-positive by PCR and had documented fever (a body temperature of ≥37.5 °C) or a history of fever in the past 48 hours. For anemia, an age- and gender-appropriate definition was used45: <11 g/dl if <5 years, <11.5 g/dl if 5–11 years, <12 g/dl if 12–14 years or >15 years and female,<13 g/dl if male and >15 years. In all three study areas, risk factors for submicroscopic and asymptomatic malaria were examined among community survey participants, and for submicroscopic malaria among clinic participants. Risk factors included those known from literature, such as age, gender, a history of travel in the past two weeks, a history of malaria in the past year, antimalarial use in the past two weeks, reported use of repellents , and season at the time of visit. Generalized linear regression with a log link and binomial distribution was used for multivariate analyses, and Poisson regression with a robust variance estimator was used for models which did not converge46. Factors with a p-value < 0.15 in the univariate model were included in the multivariate model, and factors with a p-value > 0.08 were removed from the multivariate model. All community survey analyses were weighted by age and gender, using the household information obtained from the census (svy procedure in Stata 14.2). To assess a potential effect of malaria infections on fever and anemia, we combined the data in forest plots using fever and anemia among persons without malaria by PCR as the reference group (random effects model); the I2 was used as an indicator of heterogeneity between studies.A secure, web-based REDCap (Research Electronic Data Capture) database was used to capture and store all subject data and test results7 to visualize spatial distributions of a subset of 3,001 subjects enrolled in the community studies at Chennai (n = 907), Rourkela (n = 1306) and Nadiad (n = 788). Local Indicators of Spatial Association (LISA) were calculated using Local Moran’s I analysis47, operationalized using the PySAL library for Python . As with similar spatial analysis techniques (e.g. Gi and Gi*48), Moran’s I may be used to test the null hypothesis of no local spatial association. Pseudo-significance levels were calculated using 9999 random permutations of the analysis. We constructed a ‘spatial weights’ matrix, containing a representation of the neighbourhood structure of the study area and containing all of the points that fall within a certain distance threshold of another point, referred to as the ‘neighbours’ for that point, with each value representing a standardised distance between the points. This approach minimises sensitivity to variations in the distance threshold because closer points are more important than those further away. For the purposes of this analysis, the threshold at each site is calculated as the smallest distance at which each point is ensured at least one neighbour, and the matrix is row standardised  to facilitate interpretation of the statistics47.We used Exploratory Spatial Data Analysis (ESDA)Institutional Review Board approval was obtained from New York University, and Institutional Ethical Clearance from NIMR. Informed consent was obtained from all subjects or, if subjects were under 18, from a parent and/or legal guardian. All experiments were performed in accordance with the relevant guidelines and regulations.https://clinepidb.org), part of the EuPathDB project49.Supplementary information accompanies this paper. Data are available through the Clinical Epidemiology Database, ClinEpiD (SUPPLEMENTARY MATERIAL


Malaria transmission in African highland areas can be prone to epidemics, with minor fluctuations in temperature or altitude resulting in highly heterogeneous transmission. In the Kenyan Highlands, where malaria prevalence has been increasing, characterising malaria incidence and identifying risk factors for infection is complicated by asymptomatic infection.This all-age cohort study, one element of the Malaria Transmission Consortium, involved monthly follow-up of 3155 residents of the Kisii and Rachuonyo South districts during June 2009–June 2010. Participants were tested for malaria using rapid diagnostic testing at every visit, regardless of symptoms.Plasmodium falciparum infection was 0.2 cases per person, although infections were clustered within individuals and over time, with the majority of infections detected in the last month of the cohort study. Overall, incidence was higher in the Rachuonyo district and infections were detected most frequently in 5–10-year-olds. The majority of infections were asymptomatic (58%). Travel away from the study area was a notable risk factor for infection.The incidence of Identifying risk factors for malaria infection can help to guide targeting of interventions to populations most likely to be exposed to malaria. Establishing risk factors for infection may help to target interventions to areas or populations where residual transmission remains.Malaria in African highland areas is typically characterised as unstable or epidemic. Small changes in altitude and temperature, land use and population movement, can result in highly heterogeneous transmission.9 despite traditional dogma dictating that the relatively high altitude should have been a barrier to malaria transmission. In more recent years, it appears that malaria in this region has become more stable with a reservoir of asymptomatic malaria infection.11 The increase in prevalence in the region over the past few decades has generally been attributed to drug failure12 and a change in mosquito behaviours.14 In addition, intensification of agricultural production over the past 50 years has changed the entomological landscape of the area1 resulting in renewed transmission in areas that were previously malaria-free.The population residing in the western highlands in Kenya has been subject to malaria epidemics since the 1930s,15 two school surveys in 2009 and 2010,16 health facility surveys in 2011 and 2012,17 and a cluster randomised trial in 2012.19 In addition, a cohort study was set up to estimate the incidence of infection in the population. This paper reports on results from the cohort study which ran during 2009 and 2010. The aim of the cohort was to help describe transmission in the area using extended follow-up and to identify risk factors associated with new malaria infections.In order to characterise transmission in the region, the Malaria Transmission Consortium (MTC) initiated a programme which consisted of five repeated cross-sectional surveys between 2009 and 2012,15 which summarises results from the baseline cross-sectional study, which took place in June 2009. Briefly, the cohort study took place during 2009 and 2010 in two contiguous districts  close to Lake Victoria in a highland fringe area of Kenya  have been promoted by the Ministry of Health across both districts, with routine distribution occurring to targeted vulnerable populations through antenatal and child health clinics. Indoor residual spraying (IRS) was implemented in both districts in 2008 using lambda-cyhalothrin capsule suspension , although coverage varied between districts. P. falciparum-specific Rapid Diagnostic Test (RDT)  and given treatment  regardless of result in order to ensure all infections were cleared. Baseline data were collected regarding household characteristics such as household assets and construction, travel behaviour, bed net ownership, and use and time since IRS. GPS coordinates were taken for each household.The study population was randomly selected from 40 enumeration areas (EAs) which were identified using probability proportional to size; approximately 12 compounds were randomly picked per EA and all inhabitants were asked to take part in the cohort, representing approximately 10% of the population of EAs overall. The cohort study took place for 12 months from May 2009–June 2010. Inclusion criteria for the cohort included living in a selected compound and being aged >6 months. Women who were pregnant at enrolment were excluded from the cohort. At recruitment, all participants were tested using a malaria Follow-up visits occurred every month and involved testing for malaria by RDT and for anaemia using HemoCue . Participants found positive by RDT were treated according to national guidelines and those found anaemic were treated with iron. Participants were asked to seek treatment for illness between monthly visits and these data were captured by field workers and incorporated into the final dataset.Monthly rainfall in mm was collected from one central point in the study area.Each participant was assigned a unique ID to enable linkage between visits. Data were collected on written forms and double-entered using Microsoft Excel. The dataset was then cleaned and analysed using STATA v.14 .P. falciparum infection (as determined by PfHRP-2 RDT result) or confirmed test result at the health facility for those individuals who fell ill in between monthly visits, and incidence of moderate/severe anaemia measured using HemoCue, classified by a haemoglobin level <11 g/dl. Altitude was divided into 50 m categories. A socioeconomic status (SES) variable was calculated using Principal Component Analysis (PCA) including household construction and asset ownership variables. The resulting score was divided into quintiles. Chi2 tests were used to compare proportions.Primary outcome measures were incidence of Records within two weeks of a RDT-positive visit (and treatment) were removed from the analysis due to the prophylactic effect of AL. Any visits with >6 weeks follow-up time between them were also censored. Total person-time was calculated from date of enrolment to the last visit (minus any long follow-ups or prophylaxis time). Survival analysis was performed using Poisson regression with an exponentiated distribution accounting for clustering at the EA level and multiple failures. Individual risk factors including age, infection status at recruitment and intervention use were investigated in uni- and multi-variable analysis in a forward stepwise manner.The study was approved by the ethical committees of the London School of Hygiene and Tropical Medicine (protocol #5423) and the Kenya Medical Research Institute (protocol #1517). Meetings were held with district administrative representatives, chiefs and health teams to inform them about the study prior to onset. Community meetings took place to inform potential participants. Individual written consent was obtained from participants >18 years and from those considered mature minors according to national guidelines. Consent for children <13 years of age was provided by a parent/guardian and children aged 13–17 years signed assent forms accompanied by parental/guardian consent.Recruitment for the cohort took place during May–June 2009. The characteristics of the study population at recruitment are summarised in Table Household wealth varied between the two districts with a higher proportion of households in Rachuonyo classified at the lowest socioeconomic status . Approximately 42% (n=1320) of participants reported using a net the previous night and 48% of households reported spraying within the previous 12 months. Due to the programmatic design of the IRS campaign, which aimed for blanket coverage of certain higher prevalence districts and targeted spraying in others, more households had been sprayed in Rachuonyo within the last 12 months (73% compared with 23% in Kisii).At baseline, 12% (n=372) of participants reported having a fever within the previous month. Reported fevers were most common in people aged >20 years (16% reported fever compared with 7% of children aged 10–15 years). Current temperature was not recorded at the baseline visit. Also, 489 (15.5%) of participants were anaemic, with prevalence highest in children aged <5 years (37%). Forty-eight (1.5%) of participants were RDT-positive at baseline. Seventeen (35%) of RDT-positive individuals were anaemic. Forty (83%) of the malaria infections were in children aged ≤15 years.P. falciparum infections were detected in 500 participants (16%) (either during follow-up visits or through presentation to clinic), the equivalent of 0.2 cases per person. The average number of days to first infection was 283 (32–383). A larger number of infections was detected in Rachuonyo (n=539) compared with Kisii (n=82). The highest number of infections was detected in the final month of the cohort study (June 2010) (n=177), with the increase appearing closely correlated to an increase in rainfall across the study area . Infections were generally more common in younger participants; 18% of children aged ≤15 years had an infection compared with 13% of participants aged >15 years (p=0.001).Three hundred and sixty-two infections (58%) were classified as asymptomatic (no fever at time of visit or within 48 h before). The highest proportion of asymptomatic infections were in those aged 15–20 years where 68% of infections were asymptomatic, compared with 52% in those aged <5 years. Anaemia was much more prevalent in those aged <5 years, where it was detected at 29% of visits (n=1888), compared with just 5% (n=223) in those aged 10–15 years.The incidence rate (per 1000 people) and hazard ratio (HR) was calculated for each possible risk factor for infection Table . There wIncidence was higher in Rachuonyo compared with Kisii Figure . People 21 malaria surveillance systems need to switch from facility-based aggregated data to focus on individual infections and risk factors. Cohort studies are one way to identify potential risk factors for ongoing transmission in a community. This cohort study in a highland area of western Kenya highlights temporal and spatial differences in P. falciparum incidence risk over relatively short times and distances, with repeat infections among individuals and substantial clustering within households.As transmission reduces and becomes more heterogeneous,23 due to lower temperatures increasing the time required for sporogeny within the mosquito, which consequently reduces the likelihood of transmission cycle completion as mosquitoes may not live long enough to permit parasite development. Previous studies in the Kenyan Highlands have detected differences in malaria prevalence over relatively small changes in altitude,24 with Githeko et al. describing a 16% reduction in prevalence for every 50 m increase in altitude, and Baidjoe et al. reporting prevalence variations between areas with as little as a 10 m difference in altitude.26 In this cohort study, as with the baseline cross-sectional survey which took place in the same clusters,15 no difference in parasite measures was detected within the relatively small range of altitude of the study site. Serological measures in the same clusters, however, indicated that altitude may have had an impact on longer-term malaria exposure, with lower seroprevalence detected in the highest altitude bands.26The study took place at a median altitude of 1500 m above sea level. High altitude has a notable impact on malaria transmissionP. falciparum incidence. Although highland areas have potentially lower receptivity to malaria due to cooler temperatures, vulnerability to imported infections is a risk due to regular population movement to areas of lower altitude  for commercial purposes. Travel has been noted as a risk factor in other highland areas of Kenya28 and elsewhere in East Africa.29 In the baseline cross-sectional survey, travel was not found to be associated with higher infection prevalence,15 which the authors hypothesised was due to the majority of travel potentially being to non-endemic Nairobi. The identification of risk factors is potentially more precise using incident measures, which detect new infections, as compared with prevalence, which identifies ongoing infections, which could have been present for a longer time period and as a result of other risk behaviours. In addition, the longitudinal nature of a cohort study means further nuance may be detected using risk factor analysis. The importance of travel as a risk factor within our cohort may have been exacerbated by the so-called ‘cohort effect’. Our cohort population represented approximately 10% of the population within the study area and the increased surveillance (and subsequent treatment) within this group may have resulted in an overall transmission reduction in the area.Overnight travel away from home was a notable risk factor for higher 31). We suspect that the lack of protection in this cohort is due to the use of bed nets being based on a question asked at enrolment, rather than during each follow-up visit. While use of a net the night before enrolment may be a useful proxy, perhaps in this study it was not representative of actual net use, which is known to be seasonal in some settings. Another reason for the lack of apparent association with protection could be due to people not using bed nets when they travel, which was a clear risk factor in this cohort. Educational campaigns encouraging net use have taken place across the study area to increase usage. One area of focus could be in encouraging the use of interventions during travel. A subsequent study looking at the combination of IRS and ITNs in the Rachuonyo district found that using a net and having IRS provided significantly greater protection against infection than just using ITNs alone.32Intervention use did not appear to reduce infection in this cohort. IRS targeted to high prevalence areas took place during the study period which meant that living in a sprayed household was associated with infection. Also, use of bed nets did not appear to be protective, which is in contrast to many studies showing the use of bed nets to be protective against infection , which can assist with planning timings for efficient vector control. Cohort studies work best in stable populations and are not always ideal in highland areas where population movement can be high; however, loss to follow-up in this study was minimal.35 Typically, programmes measure malaria incidence through passive detection at health facilities, and as such only include symptomatic infections that occur in the healthcare-seeking population. In areas of moderate to high transmission, exposure-based immunity is likely to mean that passive detection will predominantly only include cases in children. Actively measured incidence (i.e. testing people regardless of symptoms), such as occurred in this cohort study, is likely to generate results that more accurately reflect the transmission of malaria in a population. However, infections in previously exposed individuals may potentially be of lower parasite density and could remain undetected by the diagnostic used in this study, RDT. In the baseline cross-sectional survey, 65% of all infections were only detectable by molecular methods,15 which suggests there is a substantial reservoir of low-density infection in the population. Potentially, further risk factors may have been identified within the cohort if low-density infections had also been detected, and the age distribution of infections may also shift.The relationship between prevalence and incidence is complex.25 In another highland site in Kenya, the highly heterogeneous distribution of vectors led to large differences in infection prevalence between sites only a few km apart,8 and drainage has also been indicated as a cause of heterogeneous transmission in other areas in Kenya.10 Despite only being separated by a road, the difference in transmission between the two districts was stark and may be related to different topologies.Malaria infections were highly clustered temporally and spatially. Little difference in incidence was evident between the two districts until rainfall increased from March onwards when rains began, at which point the population in Rachuonyo experienced much higher incidence than those living in Kisii. Rainfall increases the availability of mosquito-breeding sites, which can be very focal depending on the topology of the area, with breeding sites often being confined to lower altitudes where drainage is less efficient.P. falciparum infection, which although the main Plasmodium species in the area, means that we did not assess risk factors associated with the transmission of the other potential species present, P. malariae and P. ovale. In addition, the RDT is also likely to have missed a proportion of the low-density infections present in the population, as well as potentially missing parasites with HRP-2/3 deletions.The diagnostic used in this study presents some limitations. It only detects Numerous surveys have taken place within this region of the Kenyan Highlands in order to characterise transmission and identify risk factors in an area where transmission is known to be heterogeneous and unpredictable. Transmission in this cohort was remarkably clustered, both temporally and spatially, with the majority of the population not experiencing any exposure. Identifying those that remain at risk of infection, such as those who experienced repeated infections, can help to guide more targeted, cost-efficient intervention approaches. Net use was relatively low with the highest risk group (school-age children) being least likely to report using a net. At this low usage, the added mass effect when net use is high, will not be achieved. Increasing net use within populations living in high-risk geographical areas, such as the Rachuonyo district, and within populations at higher risk, such as people travelling overnight to higher endemic areas, could help further reduce transmission in the region.Supplementary DataClick here for additional data file.


In Republic of Congo, malaria diagnosis still widely relies on microscopy. We aimed to evaluate the performance of routine microscopy for malaria diagnosis at three different health centers in Brazzaville. A total of 259, 416, and 131 patients with clinical signs of uncomplicated malaria were enrolled at the Hôpital de Mfilou, Centre de Santé Intégré “Maman Mboualé,” and Laboratoire National de Santé Publique, respectively. Two thick blood smears were prepared for each patient, the first being examined by routine microscopists and the second by expert. At the Hôpital de Mfilou, sensitivity was 62.1% and specificity was 67.3%. Positive and negative predictive values were 55.6% and 72.9%, respectively. At the Centre de Santé Intégré “Maman Mboualé,” sensitivity was 94.2% and specificity was 33.6%. Positive and negative predictive values were 50% and 89.1%, respectively. At the Laboratoire National de Santé Publique, sensitivity and specificity were high with 91.7% and 94.9%, respectively. Positive and negative predictive values were 64.7% and 99.1%, respectively. The performance of routine malaria microscopy in Brazzaville remains inaccurate with large variations among different health centers. Therefore, repeated training including supervision and evaluation would improve routine malaria diagnosis for better management of malaria in Brazzaville, the Republic of Congo. Malaria remains a public health problem in the majority of countries in sub-Saharan Africa. Global morbidity and mortality are estimated at 212 million malaria cases and 429,000 deaths attributable to malaria, respectively, in 2015, with 70% of all malaria deaths occurring among children under the age of five. Approximately 90% of malaria cases and 92% of deaths occurred in the WHO African Region. Thirteen countries, mainly in sub-Saharan Africa, accounted for 76% of malaria cases and 75% deaths globally [ Plasmodium falciparum to chloroquine and the inefficacy of sulphadoxine-pyrimethamine and amodiaquine either singly or in combination for the treatment of uncomplicated malaria have been well documented [In the Republic of Congo, where malaria is still the leading cause of attendance in health facilities, the high level of resistance ofcumented –5. Thus,cumented . To avoicumented . Laboratcumented –11.In the Republic of Congo, diagnosis of malaria is still widely based on microscopy in the majority of health facilities, and confirmatory testing rate with RDTs remains low. A study conducted in Brazzaville has shown that utility of RDTs as a diagnostic tool was poor, due to lack of experience of staff and their lacking availability . This obOver the last five years the Ministry of Health has initiated the “PARAMED” project which aims at capacity building of laboratory technicians was involved in routine malaria microscopy. First, individual microscopists from various healthcare sites were selected and trained in malaria light microsocpy. Subsequently, after their returning to their original health-facility laboratories they would know how to perform microscopy and spread relevant knowledge to their peers. However, the impacts of these trainings are not well documented. Consequently, little is known about the actual performance of malaria routine microscopy in different healthcare level facilities in Brazzaville. Thus, the present study aimed at evaluating the performance of routine microscopy for malaria diagnosis at three different health centers in Brazzaville, the Republic of Congo.Brazzaville, the political capital, hosts 38% (1 642 105 inhabitants) of the total population of the Republic of Congo, estimated at 4 312 715 inhabitants. With the population expansion due to urbanization, Brazzaville is now divided into nine districts: Bacongo, Makelekele, Poto-Poto, Moungali, Ouenze, Talangaï, Mfilou, Madibou, and Djiri. The present study was conducted in three centers: Centre de Santé Intégré (CSI) “Maman Mboualé” located in the district of Talangaï, in the north part of city, Hôpital de Mfilou located in the district of Mfilou, in the south part of the city, and the Laboratoire National de Santé Publique (LNSP), the national reference laboratory located, in the center part of city, in the district of Poto-Poto. P. falciparum. Two rainy seasons are observed each year with the main one during the months of February to May and a short one from October to December [Malaria transmission in the study areas varies from low and moderate to intense with meso-hyper-to perennial endemicity. Malaria infection is primarily due toDecember . InsteadThe quality control of microscopy reading was performed at the laboratory of the Centre Hospitalier Universitaire de Brazzaville (CHU), which is the big referral hospital with a reference laboratory in Brazzaville. Expert reading was performed by the head of the laboratory, who was also trainer in PARAMED, with the urge experience on parasitology and blinded from the results obtained in all three study health centers.The study was approved by the institutional “Comité d'Ethique de la Recherche en Sciences de la Santé” (N° 032/CERSSA-2015). In the early project stage, first, a meeting with the site actors including the head of each laboratory and microscopists was organized for the purpose of presenting the project objectives, methodology, and expected results, as well as to obtain microscopists consent. Second, a meeting was held in the various study sites, assuring the availability of material for thick blood smears preparation during the study execution. Also, training was provided ensuring the standardization of thick blood preparation and parasite density assessments in all sites.From May 2015 to May 2016, patients with clinical signs of uncomplicated malaria, presenting at the laboratory of one of the three study sites, were invited to participate in this study. Exclusion criteria comprised pregnancy, severe malaria, or other severe illness as judged by the attending physician.A number of representative patients to be included each month, per week, and per day have been estimated by the statistician taking into account the proportion of malaria reported in each health center, one year before starting the study. In sample size calculations, using a power of 80% and a significance level of 5%, the SCHWARZ method yielded a minimum number of 310, 200, and 100 patients to be recruited at the CSI “Maman Mboualé,” Hôpital de Mfilou, and the LNSP, respectively. Recruited patients were randomly selected from Monday to Friday. At the CSI “Maman Mboualé,” a minimum of 7 patients were recruited per week, with at least one patient per day, whereas at the Hôpital de Mfilou the minimum number was 5 patients per week, with one per day. For the LNSP, 3 patients were enough, with one included on Monday, on Wednesday, and on Friday. After informed consent was obtained, records were made on patient demographics, fever or history of fever in the last 48 hours, other signs of malaria, provenance, and previous antimalarial drugs intake used of bed net treated. The axillary temperature was taken for fever confirmation.μl of blood using the proposed method of the WHO [At each study site, two thick blood smears were prepared for each patient, with one being read immediately to inform the patient of the respective result. Before reading, thick blood smears were dried and stained with 10% Giemsa solution  in pH 7.2, for approximately 10 min. The stain was gently washed away by adding drops of clean water and the slide was completely dried before examination. Thick blood smears were assessed by micrsocopists until 200 leucocytes had been counted. Parasite density was calculated for each patient assuming an average of 8000 leucocytes per  the WHO . IndividData were entered and verified in Microsoft Excel  and validated in EpiInfo for Windows version 3.5.1. Data were analyzed using the SPSS 16.0 for Windows . Age was expressed as median (range), while categorical variables were expressed as percentages. Correlation coefficients were used to compare parasites densities from health centers and quality control expert. The Bayesian theory was used to estimate the sensitivity, specificity, positive predictive value, and negative predictive value as described by Joseph et al. . 95% conA total of 259, 416, and 131 patients with suspected malaria were enrolled at the Hôpital de Mfilou, CSI “Maman Mboualé,” and the LNSP, respectively.Out of 259 patients enrolled at the Hôpital de Mfilou, gender and age were recorded for 257 of them, with 131 being female and 126 male. Thirty-eight patients had an age below 5 years. With regards to CSI “Maman Mboualé,” out of the 416 recruited patients, 410 had records on gender with 207 and 203 being female and male, respectively, and 99 patients were children under 5 years of age .Among 131 patients recruited at the LNSP, 68 and 63 were female and male, respectively, and all of them had an age above 5 years.p = 0.240). Thus, the performance at the Hôpital de Mfilou in terms of sensitivity and specificity was 62.1% (CI: 52.5% -70.9%) and 67.3% (CI: 59.6% -74.2%), respectively. Positive and negative predictive values were 55.6% (CI 46.5% - 64.4%) and 72.9% (CI 65.1% - 79.5%), respectively  were reported positive by routine microscopists, whereas 103 (39.8%) slides were positive by the quality control expert. Of the 144 slides that were negative by the routine microscopy, 39 were identified positive by the quality control expert. False positive proportions  and false negative proportions  were not statistically significant (ectively . p< 0.001. Routine microscopists had a performance of high sensitivity at 94.2% (CI: 89. 6%- 96.8%), but of low specificity at 33.6% (CI: 29.0%- 38.7%). Positive and negative predictive values were 50% (CI: 44. 6%- 55.4%) and 89.1 (81.1%- 93.9%), respectively.With regard to CSI “Maman Mboualé,” 324 (77.9%) slides were reported positive by routine microscopists, while only 172 (41.3%) slides were positive by the quality control expert. Out of the 92 slides that were rated negative by the routine microscopists, 10 were revealed positive by the quality control expert . At this p< 0.001.At the LNSP, 17 (12.98%) out of 131 slides were reported positive by the routine microscopists, whereas 12 (9.2%) were rated positive by the quality control expert. Out of the 114 slides that were rated negative by routine microscopists, only one was revealed positive by the quality control expert . HoweverBoth sensitivity and specificity were high at this study site with 91.7% (64.6% -98.5%) and 94.9% (89.4%-97.7%), respectively. Positive and negative predictive values were 64.7% (CI: 41.3%- 82.7%) and 99.1 (95.2%- 99.8%), respectively .P. falciparum was the only species identified in all positive slides confirmed by the quality control expert. However, P. malariae was detected by routine microscopists in one and two slides of 64 and 162 P. falciparum confirmed positives slides from Hôpital de Mfilou and CSI “Maman Mboualé,” respectively. At the LNSP, out of 11 confirmed positive slides, one slide was positive for Plasmodium ovale by the routine microscopists.p= 0.001) and Hôpital de Mfilou (p= 0.009) compared to that with the LNSP (p= 0.048).All three study sites assessed parasite density different comparatively to the quality control expert . HoweverThis study demonstrates the inaccurate performance of routine microscopy in the three health centers compared to expert microscopy, indicating poor performance, where large quantities of blood smear examination and permanent involvement of several microscopists were necessary.Both sensitivity and specificity were significantly higher at the LNSP, which is the reference laboratory at the national level. This performance may be explained by the fact that the LNSP is the national site for microscopist training and as such having qualified trainers and technicians who are also working in parasitological diagnostic routine. Other factors that contribute to this performance were favorable quality of equipment and the relative low number of slides prepared ranging from one to ten, related to low malaria transmission as it was observed in Kenya . HoweverIn contrast to LNSP, different performances have been found at the Hôpital de Mfilou and at the CSI “Maman Mboualé.” The lower sensitivity and specificity of routine microscopy were observed at the Hôpital de Mfilou and the CSI “Maman Mboualé.” Both over- and underdiagnoses of malaria are common in malaria-endemic African countries such as Tanzania, Uganda, Burkina Faso, Kenya, and Côte d'Ivoire , 16–19. The PARAMED training lasted only 1 month with many modules. The short duration of training of microscopists may also impact acquired knowledge. Therefore, permanent training including laboratory supervision as well as improvement of infrastructure quality and implementation of internal and/or external quality control are needed for achieving improvement of malaria diagnosis by microscopy as observed in Angola . The othThe responsibility of the National Malaria Control Program in the Republic of Congo may be in increasing training on the use of RDTs and implementing their systematic use in the algorithm of malaria diagnosis in the country.By considering the proportion of positive slides determined by the expert reader, the prevalence of malaria was estimated at 39.77%, 41.34%, and 9.16% at the Hôpital de Mfilou, the CSI “Maman Mboualé,” and the LNSP, respectively. Interestingly, malaria prevalence did not differ between the northern and the southern health centers; however these prevalence cases were high compared to those found previously in the north and south part of Brazzaville , 23. ThiThis study showed that the performance of routine malaria microscopy in Brazzaville remains substandard between different health centers. Therefore, repeated training including supervision and evaluation at the national level as well as implementation and use of RDTs would improve routine malaria diagnosis for better management and control of malaria in Brazzaville.