Plasmodium falciparum and P. vivax. Previous studies using PCR diagnostic assay had unraveled several interesting facts on distribution of malaria parasites in India. However, these studies had several limitations from small sample size to limited geographical areas of sampling. In order to mitigate these limitations, we have collected finger-prick blood samples from 2,333 malaria symptomatic individuals in nine states from 11 geographic locations, covering almost the entire malaria endemic regions of India and performed all the three diagnostic tests  and also have conducted comparative assessment on the performance of the three diagnostic tests. Since PCR assay turned out to be highly sensitive  among the three types of tests, we have utilized data from PCR diagnostic assay for analyses and inferences. The results indicate varied distributional prevalence of P. vivax and P. falciparum according to locations in India, and also the mixed species infection due to these two species. The proportion of P. falciparum to P. vivax was found to be 49:51, and percentage of mixed species infections due to these two parasites was found to be 13% of total infections. Considering India is set for malaria elimination by 2030, the present malaria epidemiological information is of high importance.Malaria is a vector-borne infectious disease, caused by five different species of the genus Plasmodium, and is endemic to many tropical and sub-tropical countries of the globe. At present, malaria diagnosis at the primary health care level in India is conducted by either microscopy or rapid diagnostic test (RDT). In recent years, molecular diagnosis (by PCR assay), has emerged as the most sensitive method for malaria diagnosis. India is highly endemic to malaria and shoulders the burden of two major malaria parasites,  P. vivax incidences  were used, which amplified a 1100-bp PCR product from the rRNA small subunit gene (18S rRNA). Following this, the second step uses two species specific primers; P. falciparum [rFAL1 (5’TTAAACTGGTTTGGGAAAACCAAATATATT3’) and rFAL2 (5’ACACAATGAACTCAATCATGACTACCCGTC3’) and P. vivax primer pairs [rVIV1 (5’CGCTTCTAGCTTAATCCACATAACTGATAC3’) and rVIV2 (5’ACTTCCAAGCCGAAGCAAAGAAAGTCCTTA3’)] for amplifying specific gene products for these species. Whereas a 120-bp PCR product of Plasmodium 18S rRNA indicates P. vivax infection, a 205-bp amplified product indicates P. falciparum infection  were purified using the ExoSap , and finally utilized for performing DNA sequencing using Big Dye Terminator Chemistry in a 96-capillary DNA analyzer (ABI 3730XL) under the facility of ICMR-NIMR, New Delhi.For each sample, PCR amplifications were carried out in a final volume of 25 μl, which included 10 pmol of each primer, 0.2 mM of dNTPs, 1 unit of Taq DNA polymerase (Banglore Genei), 1X Taq DNA polymerase buffer and 1μl of DNA. Cycling conditions for the first step included an initial denaturation at 95°C for 5 min followed by 30 cycles of 1 min denaturation at 94°C, 2 min annealing at 60°C and 2 min of extension at 72°C followed by 10 min final extension at 72°C. The cycling conditions for second step included an initial denaturation at 95°C for 5 min followed by 30 cycles of 1 min denaturation at 94°C, 2 min annealing at 55°C and 2 min of extension at 72°C followed by 10 min final extension at 72°C. After PCR amplification, 5 μl of PCR amplified product was electrophoresed on a 2% agarose gel and compared with a 100-bp ladder (Merck) to confirm the amplicon size. To identify false positive results and to check the primer specificity, two negative controls were used, one lacking DNA and the other one containing the genomic DNA of a healthy individual from Delhi. For further confirmation, PCR amplified products , of which 324 (42%) as P. falciparum mono infections, 398 (52%) as P. vivax mono infection and 42 (6%) as mixed infection  P. vivax mono infection and 58 (7%) as mixed infection  as P. vivax mono infection and 105 (13%) as mixed species infection . The newly generated DNA sequences have been submitted to the GenBank with accession number MG708202 –MG708221. The detail number of mono infections by the two parasites and also mixed infections by both these parasites in a single sample and the comparative assessment among all the 11 population samples have been provided in P. falciparum and P. vivax in India (see below). Considering both the mono and mixed infection detected with PCR diagnostic assay, 455 individuals were found to be infected with P. falciparum and 477 with P. vivax. Therefore, the proportion of P. falciparum to P. vivax infection from this study amounts to 49:51.In the present study, 2333 blood samples from malaria symptomatic individuals were collected at different time period (February 2012 to October 2015) adopting both active and passive surveillance methods. Malaria symptomatic individuals from as many as 11 different geographical locations (encompassing nine states) spanned vertically from Delhi in North and Tamil Nadu in the South and horizontally from Gujarat in the west and Asom in the east were collected, thus covering almost all the malaria endemic regions in India  in the pnfection . At the nfection . Howevernfection . As a coP. falciparum and P. vivax (mono or mixed) could be apprehended. Both the northern and southern Indian samples (DEL and CHE) were found to be under the sole dominance of P. vivax—not a single case of either P. falciparum mono or mixed infection could be detected , GUW (northeast) and SHA (north). However, like P. vivax, absolute mono infection of P. falciparum could not be detected from any population sample, but in three locations , dominancy of P. falciparum mono infection over the two other types of infections (P. vivax mono and mixed infections) could be noticed, confirming wide prevalence of P. falciparum mono infections in the central regions of India . For example, the highest proportion of mixed infection due to P. vivax and P. falciparum was found in a central Indian population, sampled from GAD (29%), followed by BET (17%). Incidences of mixed species infections were also found to be high in a south-west coastal city . Surprisingly, not a single case of mixed infection could be detected in the north eastern state of Asom, even though mono infections due to both P. falciparum and P. vivax are widely prevalent , regional biasness well exists on the prevalence of mixed malaria parasite infections due to these two species.Interestingly, wide prevalence of mixed infections was found in Indian locations, and with considerably high proportions (13%). With close observation on the distributional prevalence of mixed species infections, it was noted that mixed species infections are common in the central Indian locations  in unravelling differential prevalence of mono infections by of India  or have of India . For diaP. falciparum and mixed cases as mono P. vivax case. This can be ascribed to certain facts like (i) time of collection of blood sample, (ii) availability of all the life-cycle stages of P. vivax in peripheral blood samples in comparison to only ring and gametocyte stages of P. falciparum [P. falciparum and P. vivax [P. vivax gametocytes in comparison to P. falciparum [P. vivax can increase deformability of infected RBCs more effectively than P. falciparum [P. vivax mono infections and has also failed to detect few mono P. falciparum cases. Since the RDT kit  that we used here is based on histidine rich protein (hrp2) for diagnosis of P. falciparum and Lactate dehydrogenase (ldh) protein for diagnosis of P. vivax, and deletion and mutations are commonly reported in the Pfhrp-2 gene [P. falciparum and P. vivax [There might be several factors by which microscopy might have failed to detect some of the infections. For example, in the present study, microscopy has misdiagnosed several mono lciparum  (iii) siP. vivax  and (iv)lciparum . Furtherlciparum , making p-2 gene , misdiagp-2 gene  as well P. vivax , 20 thatP. falciparum and P. vivax in India is quite interesting, as it provides a unique epidemiological understanding on malaria in India. Out of the 827 malaria positive samples, 455 were found to be infected with P. falciparum and 477 with infection by P. vivax with a proportion of 49:51. Traditionally, with regard to malaria burden caused by the two-different species of malaria parasites , India is known to be majorly burdened with P. vivax malaria (3), but over time there has been a dramatic shift from P. vivax to P. falciparum malaria. The ratio of P. falciparum vs. P. vivax malaria was 0.41 in 1985 which had drastically shifted to 1.01 (in the ratio of 50:50) in 2007 [P. falciparum over P. vivax in India [P. vivax continues to be largely prevalent in specific Indian regions (e.g. northern and southern parts of India) where it is highly dominant [P. falciparum could be detected in either the northern or the southern Indian locations. Whether this observation will hold true with a much larger sample size is not known, as the present study is restricted to only Delhi (northern India) and Chennai (southern India) and that too with a limited sample size with non-uniformity of time of sample collection in all the locations. Furthermore, P. vivax is also appreciably prevalent in the north-eastern and western Indian locations  of mixed infection due to these two species. It was interesting to note that P. vivax is majorly distributed at the peripheral populations in each direction , whereas, P. falciparum is restricted to north-east, east, central and southwestern regions of India. Incidentally, major incidences of mixed infections take place in the central regions of India  is available yet. Since mixed infection cases put huge malaria burden, many more studies (with large sample size) are to be carried out in other parts of India that are known to be endemic to both P. falciparum and P. vivax. Furthermore, P. malariae is emerging as a potential threat and P. ovale cases are emerging in recent years across the malaria endemic regions of the globe [The present study involving large number of malaria samples covering nine different Indian states could unravel epidemiological information on the distributional prevalence of mono as well as mixed parasite infections in India. The results highlight wide distribution of mono infections of of India . Howeverhe globe , 58. In he globe .Historically, India has experienced the burden of malaria since time immemorial. Although malaria incidences were very high before coordinated interventions by the government, the launch of the National Malaria Control Program (NMCP) resulted in a drastic drop of malaria cases to less than 50,000 with no reported malaria mortality in 1961. However, malaria staged a dramatic comeback, with 6.45 million estimated malaria cases in 1976 . Neverth


A parasitological survey of 3731 individuals in North Sumatera province used polymerase chain reaction to identify 1169 with parasitemia and highly specific amplification of  As Indonesia works toward the goal of malaria elimination, information is lacking on malaria epidemiology from some western provinces. As a basis for studies of antimalarial efficacy, we set out to survey parasite carriage in 3 communities in North Sumatera Province.Plasmodium species were identified using nested polymerase chain reaction (PCR) of ribosomal RNA genes and a novel assay that amplifies a conserved sequence specific for the sicavar gene family of Plasmodium knowlesi.A combination of active and passive detection of infection was carried out among communities in Batubara, Langkat, and South Nias regencies. Finger-prick blood samples from consenting individuals of all ages provided blood films for microscopic examination and blood spots on filter paper. P. knowlesi. Also present were Plasmodium vivax (14.3%), Plasmodium falciparum (10.5%) and Plasmodium malariae (3.4%).Of 3731 participants, 614 (16.5%) were positive for malaria parasites by microscopy. PCR detected parasite DNA in samples from 1169 individuals (31.3%). In total, 377 participants (11.8%) harbored sicavar is a specific and sensitive test for the presence of P. knowlesi DNA in humans. Subpatent and asymptomatic multispecies parasitemia is relatively common in North Sumatera, so PCR-based surveillance is required to support control and elimination activities.Amplification of  Plasmodium falciparum and Plasmodium vivax the 2 major reported causes [Plasmodium malariae malaria may require hospitalization in the eastern province of Papua [Plasmodium knowlesi, a parasite of long-tailed and pig-tailed macaques, is also known to infect humans. The morphological features in the blood stage are similar to those seen in P. falciparum and P. malariae, which in routine practice has led to frequent misdiagnosis [P. knowlesi parasitemia occurs in some individuals and has been reported to cause fatal disease [P. knowlesi infections are asymptomatic and submicroscopic across all age groups [P. knowlesi malaria have been documented in the province of Kalimantan, Indonesian Borneo [Malaria remains widespread across Southeast Asia. In Indonesia, 2 million cases of malaria are reported each year, with d causes . Among oiagnosis . High P. disease . Despitee groups . A smalln Borneo  and in An Borneo , but thiP. falciparum and P. vivax [The Ministry of Health of Indonesia has implemented malaria control, aiming for elimination by 2030. Malaria surveillance relies on passive case detection by microscopic examination and rapid diagnostic tests (RDTs) at primary health care centers . These tP. vivax . HoweverP. vivax . ModelinP. vivax , and in P. vivax . Hence, P. vivax .P. knowlesi infection, because the target region of the 18S ribosomal RNA (rRNA) can cross-react with P. vivax [P. knowlesi–specific nested PCR assay to ensure reliable determination of all Plasmodium species, including submicroscopic infection, in our study areas.In preparation for a study of antimalarial drug efficacy in vivo, we performed intensive malaria screening in 3 regencies of the Province of North Sumatera, western Indonesia. In addition to microscopy, we used established polymerase chain reaction (PCR) assays . HoweverP. vivax . Therefo2 with a population of 13 215 401. The province is subject to stable low malaria transmission and is currently planning for elimination by 2020. The 3 regencies were selected based on published malaria endemicity data [A parasitological survey was conducted between January and June 2015 among persons attending outpatient clinics temporarily established in >80 localities across 3 selected regencies in North Sumatera province, Indonesia: Batubara, Langkat, and South Nias regencies . North SThe study was approved by the ethics committees of the University of Sumatera Utara, Indonesia (identifier 401/KOMET/FK USU/2014) and the London School of Hygiene & Tropical Medicine, United Kingdom (identifier 8504‑01).Sampling strategies differed among the 3 sites, owing to contrasting geography and inconsistencies in access to health facilities. In Batubara, most communities had good access to a health clinic, which was open to patients from 8 am to noon, 6 days per week. We established a 24-hour, 7-day clinic for an 8-week period of screening, after intensive health promotion and education on malaria and sensitization concerning to our study objectives. This sensitization was facilitated by local leaders and carried out at the level of the whole community, but clinic attendance was entirely voluntary. Thus, sampling was not designed to reach the whole community. Local health clinics were also asked to refer patients with a malaria diagnosis to our team. Langkat is a forested area, with isolated villages that have poor access to health facilities; we therefore adopted a village-by-village approach, in which a sensitization meeting was followed by 2–3 days of screening, before the team moved to another village. The 31 communities we sampled in South Nias were spread across several islands, and sea journeys were required to move our team and the samples between each village and our study clinic/diagnostic laboratory, which was temporarily set up in a central location on Tello Island.Malaria testing was done in 3 groups of persons. First, patients with fever  or a history of fever in the preceding 48 hours who presented at the health clinics were tested for malaria. Tests were also offered to healthy individuals who, after our community sensitization activity, came for a voluntary malaria check. This second group includes children who volunteered during school sensitization visits (Batubara and South Nias only). Third, household members of any individuals who were slide positive for malaria parasites were subsequently visited and also offered malaria testing. Finger-prick blood samples were taken from all participants for thick and thin blood films for microscopy examination (single reading), and blood was also spotted onto filter papers (3MM Whatman) for molecular analysis. Participants confirmed as malaria positive by microscopy were clinically assessed and treated according to national guidelines, regardless of symptoms.Parasite DNA was extracted from filter papers using the Chelex method , 22.Plasmodium 18S rRNA was performed on all samples for species determination and detection of submicroscopic infections [P. falciparum, P. vivax, P. malariae, Plasmodium ovale spp. and P. knowlesi were included in all nested PCR assays.A conventional nested PCR assay targeting the genes encoding the fections , 18. PosP. knowlesi and P. vivax that occurs in the rRNA gene PCR assays, we developed a heminested PCR assay based on a conserved region of the amino-terminal exon of the 50–70 members of the gene family encoding P. knowlesi–specific schizont-infected cell agglutination variant antigens (SICAvar) [sicavar genes [To overcome the cross-reactivity between SICAvar) . The folP. falciparum, P. vivax, P. malariae, P. ovale curtisi, and P. ovale wallikeri), simian malaria parasites , clinical P. knowlesi isolates obtained from Kapit of Malaysian Borneo, and human DNA from malaria-free individuals to assess specificity. To determine the limit of detection of the assay, P. knowlesi culture of known parasitemia (kindly provided by F. Mohring) was serially diluted in whole human blood and spotted on filter paper, and DNA was extracted with a QIASymphony automated DNA extraction system. All field isolates were tested for P. knowlesi infection using this novel PCR assay. The sicavar amplicons from a subset of samples were verified by direct sequencing using BigDye Terminator v3.1 cycle sequencing kits and analysis on an ABI 3730 Sequencer (Applied Biosystems). Results were aligned and compared with the P. knowlesi strain H reference genome, using Geneious (version 8.0.5) and BLAST  software.This assay was tested against control DNA from all human malaria parasites . and both queries identified a number of distinct sequences in the reference genome (Supplementary Table 1).As further confirmation of our results, 7 ved exon . InteresP. falciparum and/or P. vivax, as expected. However in Batubara, P. knowlesi (39.7%) was more abundant than P. vivax (35.1%) among our tested participants , P. falciparum (n = 19), or P. malariae (n = 2) or various combinations of triple-species infection.After this successful validation of the SICAvar PCR assay, we were able to deduce robust estimates of the contribution of each species to malaria infections in each of our 3 sites. The most abundant in both Langkat and South Nias were icipants . Of patiPlasmodium infections by microscopy. Three species  were identified. A considerable number of participants with malaria-positive slides were negative by PCR subsequently performed on stored blood spots. Thus, the total number of patent infections confirmed by both microscopy and PCR decreased to 93 (8.1%), 74 (13.6%) and 169 (9.1%), respectively, indicating poor specificity of microscopy in South Nias in particular. Conversely, a substantial number of submicroscopic infections were identified by PCR alone, with a PCR-confirmed parasite carriage in 25.2%, 33.5%, and 34.8% of tested individuals, respectively, in the 3 sites , Langkat (n = 544), and South Nias (n = 1917) were included in the malaria screening (Supplementary Table 2). In these 3 regencies, 117 (9.2%), 98 (18.0%), and 397 (21.3%) individuals, respectively, were positive for P. vivax (n = 227) and P. knowlesi (n = 220). Submicroscopic infections of any species were more often found in older individuals, the mean age in this group being 23.0 years, . The mean age of individuals with patent infections was significantly lower, at 18.0 years  .PCR analysis revealed that the majority of the 1169 infected individuals (71.3%) harbored submicroscopic parasites . Among t t-test) . SubmicrPlasmodium species identified: P. falciparum, P. knowlesi, P. malariae, and P. vivax. Using a novel assay developed for this study, which detects a conserved motif in the multicopy sicavar gene family, we found that P. knowlesi was present in 377 individuals (10.1%), including both patent and subpatent infections. P. vivax and P. falciparum were both frequently detected, occurring in 11.3% and 8.2% of all individuals tested.In this study, a total of 3731 febrile and nonfebrile residents of 3 regencies in North Sumatera province were screened for malaria infection by both microscopy and PCR detection of parasite DNA. Microscopy identified 612 infected participants, whereas PCR identified 1169 individuals harboring ≥1 of the 4 P. knowlesi infection has been widely recorded in Southeast Asia, only a handful of confirmed cases have been from Indonesia, from Kalimantan, eastern Borneo [P. knowlesi, 15 P. vivax, and 8 P. falciparum infections, almost all of which were symptomatic. This contrasts with our findings of more frequent parasite carriage, and a significant proportion of subpatent and asymptomatic infections.Although n Borneo , 24, andn Borneo . This laPlasmodium species have decreased in prevalence over the past decade [P. falciparum and P. vivax remain common and it may be that acquired immunity to these human parasites protects individuals subsequently infected with P. knowlesi, although asymptomatic infections have also been reported in Sabah [Malaria transmission intensity is much lower in Aceh than in North Sumatera, and this neighboring province is closer to eliminating the disease. North Sumatera’s natural forests have been affected by deforestation in the last few years. Residents of rural districts may live in close proximity to semiforested, forested, or plantation areas, with a high likelihood of forest exposure, but may not have adequate access to health facilities and antimalarial medication. In the Aceh study, the more frequent occurrence of symptoms in persons with knowlesi malaria reflects observations in Malaysian Borneo where, as in Aceh, other t decade , 25. In in Sabah .P. knowlesi infections, and so we made some effort to validate its sensitivity and specificity . These findings suggest our assay is performing as hoped and is a useful tool for identifying P. knowlesi infections among complex mixtures of Plasmodium species.Our novel PCR assay identified an unexpectedly large number of cificity . SICAvaromosomes . SICAvaromosomes , 27 and d insert . ProbingPlasmodium spp. infections, but lacked accuracy in distinguishing among the 4 species present, as previously reported in Malaysia [P. malariae was only detected in 1 case and P. knowlesi was left unrecognized. In South Nias, microscopy results showed poor specificity (Supplementary Table 2), leading to a number of false-positives, whereas specificity was good at the other 2 sites. RDTs have limitations for detection of P. knowlesi because they include only P. falciparum and P. vivax parasite lactate dehydrogenase monoclonal antibodies [Plasmodium carriage is associated with subsequent transmission to mosquitoes [The national malaria control program focuses on case management through passive surveillance at primary health centers, deploying microscopy or RDTs to detect malaria cases to be treated . In our Malaysia . Althougtibodies . Most imsquitoes .P. vivax isolates with ribosomal gene PCR assays [P. knowlesi cytb DNA, which also provided satisfactory sensitivity. However, our sicavar target assay provides greater sensitivity for P. knowlesi identification in our hands and was effective in detecting submicroscopic parasites. This is a species-specific test, because the sicavar gene family is unique to P. knowlesi (P. knowlesi coinfections with P. vivax in the present study.On the other hand, molecular tests are highly sensitive and specific, provide the capacity to detect low-density infections missed by microscopy or RDTs, and are well established for detection of human malaria infections , 29. To R assays , 19, GhiR assays  have recknowlesi . BecauseP. falciparum contributed one-fifth of infections, and P. vivax was seen slightly more often. Interestingly, our findings also demonstrate that P. knowlesi carriage is not uncommon. Many P. knowlesi–infected individuals harbored additional Plasmodium species (P. vivax and P. falciparum are now very scarce [P. knowlesi infections in our study is consistent with recent findings in Malaysia [P. knowlesi.Despite the aim to achieve malaria elimination by 2020 in Sumatera, our study demonstrated that a substantial number of individuals in our study areas carried parasites.  species , in conty scarce , 7. Multy scarce . The obsAnopheles, thought to be the vector of P. knowlesi. These data may suggest that acquired immunity permits sustainable chronic infections with this simian parasite. Cross-protection among the 4 human Plasmodium species may maintain overall parasite density at low levels [P. knowlesi, particularly in settings where the closely related P. vivax is present. One weakness of our study is the lack of a systematic sampling procedure, which may have introduced bias; as we had not previously worked in these 3 regions, methods were adapted for each site to reflect the facilities available and particular local challenges. For South Nias, these included frequent sea journeys in small boats. Future studies could deploy a more systematic approach and collect sufficient data to better explain and characterize asymptomatic infections, now that community contacts have been established and baseline information is available.Macaques were present in all sites, and the communities shared established risk factors for malaria transmission by forest-dwelling w levels , which cPlasmodium species, including P. knowlesi, to malaria transmission in North Sumatera. There is an urgent need for the national malaria program to include in malaria guidelines the recommendation that microscopists are trained to identify P. knowlesi infection in Indonesian clinics. Molecular detection of infection is also needed, to strengthen control and elimination programs by accurately defining the true extent of the malaria reservoir, so as to achieve the current goal of elimination [Our study demonstrated the importance of submicroscopic infections of 4 mination .The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary TablesClick here for additional data file.


Reliable and comprehensive information on the burden of malaria is critical for guiding national and international efforts in malaria control. The purpose of this review is to provide an overview of published data and available information on malaria resulting from field studies/investigations conducted in the Republic of Congo (RoC) from 1992 to 2015, as baseline for assisting public health authorities and researchers to define future research priorities as well as interventions.This review considers data from peer-reviewed articles and information from the National Malaria Control Programme reports, based on field investigations or samples collected from 1992 to 2015. Peer-reviewed papers were searched throughout online bibliographic databases PubMed, HINARI and Google Scholar using the following terms: “malaria”, “Congo”, “Brazzaville”, “prevalence”, “antimalarial”, “efficacy”, “falciparum”, “genetic”, “diversity”. Original articles and reviews were included and selection of relevant papers was made.Twenty-eight published articles were included in this review and two additional records from the National Malaria Control Programme were also considered. The majority of studies were conducted in Brazzaville and Pointe-Noire.The present systematic review reveals that number of studies have been conducted in the RoC with regard to malaria. However, their results cannot formally be generalized at the country level. This suggests a need for implementing regular multisite investigations and surveys that may be representative of the country, calling for the support and lead of the Ministry of Health. Plasmodium that are transmitted to humans through the bites of infected Anopheles mosquitoes. Five different Plasmodium species have been demonstrated to infect humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi. Of these, P. falciparum is the most dangerous, with the highest rates of complications and mortality [Malaria is a life-threatening disease caused by protozoan parasites of the genus ortality .The scaling-up of interventions has reduced the number of malaria cases and deaths between 2000 and 2015 . In 20002 with a population estimated to be 4,800,000 inhabitants, with 61% of its total population living in the two biggest cities, namely Brazzaville and Pointe-Noire [P. falciparum is the predominant malaria parasite and Anopheles gambiae the predominant mosquito vector. The latest estimations from the National Malaria Control Programme (NMCP) indicate that clinical malaria account for 47.9% of all outpatient consultations in public hospitals, 64.8% of hospital admissions and 18.4% of deaths [The Republic of Congo (RoC) is one of the 54 countries where malaria transmission is still high . The coute-Noire . DespiteClearly, the country is still struggling with the control phase where expensiveness of the control program as well as its sustainability, resistance of the parasite to anti-malarial drugs and that of vectors to insecticides are some of the challenges. However, some research publications have reported a decrease in clinical malaria prevalence in Southern and Northern sentinel sites of the country , 10.The purpose of this review was to provide an overview of published data and available information from the NMCP of the MHP on malaria situation in RoC including prevalence/incidence trends, vectors, anti-malarial drug efficacy and parasite genetics based on field studies conducted from 1992 to 2015. This may assist in defining future research and intervention priorities.To collect research data on malaria in RoC, peer-reviewed articles have been retrieved from online bibliographic databases PubMed, HINARI and Google scholar using the following keywords: “malaria”, “Congo”, “Brazzaville”, “1992”, “prevalence”, “antimalarial”, “efficacy”, “falciparum”, “genetic”, “diversity”, “resistance”, “markers”. Reference lists of selected papers were used as leads for identification of additional studies. The Boolean operators “AND”, “OR” and “>” were used to combine two or three terms. In addition, reports from the NMCP were reviewed at the NMCP headquarters in Brazzaville. Predefined medical subject heading (MeSH) was not used to avoid restricting searches.Studies were included in the review if they explicitly reported on one of the considered aspect of malaria in RoC with: (1) samples collected in the country, 2) a clear description of the methods section  and (3) no review articles. An overall of 33 studies were retrieved from the search of peer-reviewed papers, of which five were excluded because of duplications  or they were review articles. Therefore, 28 studies including full text articles, and short reports written in English or in French were selected and reviewed. Two additional documents from the NMCP were considered  and P. ovale (0.7%) [P. ovale isolates  were further characterized by Oguiket et al. using conventional and real-time quantitative PCR methods to discriminate P. ovale curtisi and P. ovale wallikeri. The two isolates from Brazzaville were identified as P. ovale curtisi, while the remaining four isolates were identified as P. ovale wallikeri [P. vivax in RoC [P. vivax-specific antigens. Accordingly, they concluded that in conjunction with the frequent reports of travellers returning from western and central Africa with diagnosed P. vivax infections, their findings make an argument for the presence and continued transmission of P. vivax in RoC. How and where precisely is still unclear and deserves to be further investigated.Published articles included in this review were of samples collected from 1992 to 2015 (Table e (0.7%) . Six of x in RoC . The autP. falciparum compared to 59% of those residing in the peri-urban areas. With regard to PCR corrected results, the percentages rose to 42% P. falciparum positive for the urban residents and 75% positive for peri-urban residents, indicative a percentage of 11 and 20% of submicroscopic infections in these two populations, respectively. Another important study conducted before adoption of ACT was concomitantly done in different pediatric services of four hospitals in Brazzaville from January to August 2006 [The study with the largest sample size was conducted from 2003 to 2006 in southern part of Brazzaville just before introduction of ACT in the country . The autust 2006 . With thust 2006 .P. falciparum asymptomatic carriage using microscopy and PCR as diagnostic techniques. The reported prevalence of infection was 8.6 and 16% by microscopy and PCR, respectively [P. falciparum was the only species detected and the prevalence of infections among more than 3000 children and 700 pregnant women ranged from 8 to 29, and 8 to 24%, respectively [P. falciparum infection at 7 and 19% when using microscopy and PCR, respectively [Following the replacement of chloroquine with ASAQ or AL in 2006 for the treatment of uncomplicated malaria, the first study that reported the prevalence of malaria parasite infection was conducted in 2010 in a peri-urban area of southern Brazzaville , an areaectively . In a prectively . From Ocectively . The maiectively .Apart from the published articles, data available at the Ministry of Public Health and provided by the NMCP were also considered. In the national malaria report 2015 (released in 2016)  and the Anopheles gambiae sensu stricto (s.s.) was the only mosquito species identified, and it had a high P. falciparum infection rate (9.6%). In this study, insecticide susceptibility was also assessed and the authors mentioned that multiple insecticide resistance was detected in this vector population with full susceptibility observed to only one insecticide class, the organophosphate [Malaria entomological studies are very limited in the RoC. From 1992 to June 2016, only one published investigation was found . Conducthosphate .Anopheles gambiae s.l. and Anopheles funestus were reported as the predominant vectors, while other species including Anopheles coustani, Anopheles hancocki, Anopheles nili, Anopheles moucheti and Anopheles paludis were found to be potential secondary vectors [In 2014, the NMCP documented entomological surveys conducted in 2013 by Antonio-Nkondjio and Bitsindu in the Bouenza department located at 300 km southern Brazzaville.  vectors .P. falciparum infections have been generated using samples from the RoC. A total of 8 published articles have been found, for which, different molecular markers have been used. The genes for the merozoite surface protein 1 and 2 have been used, msp2 alone has been used in four investigations [msp1 and msp2 in one study [msp1 in addition to the microsatellite markers [Number of information on the genetic diversity and the multiplicity of igations , 16, 34,ne study , and mic markers , 21.P. falciparum collected in 1998 at Pointe Noire, Durand et al. reported a high genetic diversity based on 28 microsatellites with a mean number of alleles per locus of 7.75 and the expected heterozygosity of 0.78 [P. falciparum isolates collected in urban and peri-urban areas in southern Brazzaville in 2005 [P. falciparum clinical isolates collected from children in south part of Brazzaville in 2005, the allelic specific msp1 and msp2 genotyping showed that malaria parasite population in Brazzaville is highly diverse. A total of 15 msp1 and 20 msp2 distinct alleles were identified. For msp1, K1 was found to be the predominant allelic type, whereas 3D7 family was the most prevalent for msp2. The overall mean MOI was 2.2 and 72% of the isolates carried more than one genotype [msp2 gene in P. falciparum isolates collected from Congolese children with asymptomatic infections [msp2 as molecular marker. A high genetic diversity of parasite population was observed with 21 different alleles detected (11 for 3D7 family and 10 for the FC27). Here, the MOI of 1.7 was determined and 54% the isolates harboured more than one msp2 genotype [In 2003, analysing 32 isolates of  of 0.78 . This tr in 2005 . In the  in 2005 , with 12genotype . In 2010fections  with reggenotype .msp2 gene revealed the presence of 11 (5 of 3D7 and 6 of FC27) and 22 alleles (13 of 3D7 and 9 of FC27) in P. falciparum isolates collected from 2011 to 2012 from children aged >15 years with uncomplicated malaria in Brazzaville and Pointe-Noire, respectively. The MOI was similar (about 1.7) in the two cities [P. falciparum infection using the same molecular marker, 11 alleles of the 3D7 family and 18 of the FC27 family were detected [msp2 genotype and the overall MOI was 1.6 [Recent studies among children and pregnant women from Brazzaville and Pointe-Noire conducted in 2011–2012 by Ntoumi et al.  provideddetected . This is was 1.6 .In the period 1992 to 2005, before adoption of ACTs, six efficacy in vivo studies had been conducted, of which one was based on the 1973 WHO protocol for asymptomatic children to assess chloroquine efficacy , and fouIn 2006, the government of the RoC adopted a new treatment policy for uncomplicated malaria with ASAQ and AL as the first and second-line drugs, respectively . HoweverP. falciparum parasites to standard anti-malarial drugs. The isotopic test was used in the two studies. The first study was conducted in Brazzaville in 1993 and 34 P. falciparum isolates were tested with chloroquine, quinine and mefloquine. In addition, halofantrine was also tested on 35 P. falciparum isolates. The resistance rates were 61.8, 17.7, 3 and 0% for chloroquine, quinine, mefloquine and halofantrine, respectively [P. falciparum isolates were collected from March 2005 to January 2006 and their sensitivity assessed against 11 drugs: chloroquine, quinine, mefloquine, atovaquone, dihydroartemisinin, doxycycline, cycloguanil, lumefantrine, monodesethylamodiaquine, halofantrine and pyrimethamine [Two studies have been conducted to assess the level of in vitro resistance of ectively . The secethamine . The in ethamine . These sP. falciparum resistance to anti-malarials with samples collected from 1999 to 2015 [P. falciparum chloroquine resistance transporter (pfcrt) gene, associated with chloroquine resistance, the dihydrofolate reductase (dhfr) and the dihydropteroate synthase (dhps) genes, which are linked with pyrimethamine resistance and sulfadoxine resistance, respectively, the P. falciparum Klech-13 (K13) propeller gene, which has recently been linked with artemisinin resistance, and MAL10-688956 and MAL13-1718319 single nucleotide polymorphisms (SNPs) which have also recently been proposed as molecular markers of artemisinin resistance which is defined as a delayed clearance of P. falciparum parasites following ACT. The most analysed molecular markers are pfcrt, dhfr and dhps for which the main findings are summarized in Table A total of eight articles have been found reporting data on molecular markers of  to 2015 , 32, 36.pfcrt gene has been studied in four articles and for all the K76T mutation was characterized. Nsimba et al. [P. falciparum isolates carrying the 76T mutation. This prevalence was similar to the rate of 98% reported by Mayengue et al. [The a et al.  with isoe et al. , 88–97% e et al. , and 92%e et al. .dhfr point mutations). In all of these studies, dhfr point mutations at codons 51, 59 and 108 were found at high frequencies while the mutation at codon 164 was absent. For dhps, high prevalence of the mutation at codon 437 was observed in the three studies, while higher frequency of the mutation at codon 436 and the presence of mutation at codon 540 were only observed in the recent study, with samples collected from pregnant women in 2012–2013 at Brazzaville [Molecular analysis of mutations associated with resistance to antifolates was performed by Nsimba et al. , NdoungaP. falciparum isolates collected in 2005–2006 in Brazzaville, Pointe-Noire and Gamboma, and none of the mutations associated with artemisinin resistance was found.The K13 propeller gene was characterized by Mita et al. , and MALThe purpose of this review is to provide an overview of published data and available information on malaria situation in RoC and to identify gaps in knowledge in order to contribute in research-based solutions adapted for the country. In the present review, as the first goal, we attempted to determine if there was any change in malaria epidemiology in RoC from 1992 to 2015, with regard to malaria parasites and vectors and in the presentation of the disease as well. The year 1992 was chosen because the first national policy for malaria control established by the NMCP was adopted that year. Besides, all the research institutions including those involved in malaria research were nationalized.As a summary, during almost 24 years, a total of 28 studies published in peer reviewed journals were conducted the RoC in relation to different aspects of malaria infection. Concerning malaria burden, lower rates of malaria parasite infection in children and adults were observed in studies conducted from 2009 to 2015 compared to those conducted from 1992 to 2006 regardless of the diagnostic method (microscopy or PCR). This is suggestive of a decline malaria prevalence and incidence during the past 7 years and might be attributable to the scaling-up of malaria interventions in the country with the support of Global fund for HIV/AIDS, malaria and tuberculosis, including the use ACT which are freely provided to children of ≤15 years in public health facilities since 2008 associated with mass distribution of LLINs from 2008 to 2012. Further evidence on sustainability of the trend to the years later is important to ascertain such attributes.Plasmodium species distribution, P. falciparum is by far the predominant malaria parasite occurring in the country accounting for almost 100% of malaria cases [P. falciparum in the RoC is in accordance with findings from studies conducted in other Central African countries [With regard to ia cases . This obountries , 40.P. falciparum was characterized trough different studies and the MOI was also determined. These two parameters are known to be good indicators of the level of premunition of populations living in endemic areas and correlate with the extent of the parasite population diversity as well as the transmission intensity [P. falciparum population has been observed in all the studies and this is representative of areas with holo- or hyper-endemic malaria transmission. The High genetic diversity remains similar before and after introduction of ACT in 2006 and scaling up of control measures in the RoC. This implies that most of the parasite clones are still persisting and the interventions did not have impact on specific P. falciparum clones. Lower values of the MOI (1.3–1.7) have been obtained in studies conducted after these interventions than those conducted before (2.2–2.3). This trend, in addition to low rates of parasite carriage observed in studies conducted after 2006 may suggest that the implemented control measures have resulted in a substantial decrease of transmission. However, this needs to be assessed by conducting entomological studies that allow determination of the EIR in each area, which correlate well with the level of malaria transmission.The genetic diversity of ntensity , 42. TheAn. gambiae complex specimens were collected and the specimen identification revealed that all were An. gambiae s.s., of which, 95.4% were further molecularly characterized as the S-form. Importantly, these vectors were found to be highly resistant to multiple insecticide classes. This review would classify entomological research as a key priority in the country. Moreove, as An. funestus, An. coustani and An.hancocki were found to be potential minor malaria vectors [Vector control is one of the main interventions for an effective malaria control programme. To be successful, this intervention needs to rely on availability of data on vector population, level of transmission and insecticide resistance. Unfortunately, only one publication was found  and thisP. falciparum K13 propeller gene in isolates from RoC [dhps mutation  [dhfr/dhps mutations might suggest that SP resistance become more pronounced.Since the utilization of ACT for the treatment of uncomplicated malaria, monitoring studies are required to detect any emergence of artemisinin resistant strains as it has been already reported from South East Asia , 44. Ovefrom RoC , and it 11–2012) , which w11–2012) , and higAs a second key priority highlighted by this review is the limited number of publications on malaria in pregnancy in RoC. For instance, there is no data on malaria parasites collected from pregnant women before the introduction of ACT and the first data were published in 2013 . The resA total of 28 peer reviewed articles and two official documents from the NMCP were included in this review and have shown that malaria is still endemic in the country. Unfortunately, the majority of studies were conducted in Brazzaville followed by Pointe-Noire (the two main cities). Therefore, results cannot formally be generalized. We note two major positive points: ASAQ and AL are highly efficacious in treatment of uncomplicated malaria and there is substantial reduction of malaria transmission since introduction of ACTs in the country. However, a strong resistance to SP is observed in parasites collected from Congolese pregnant women.


Malaria, a febrile illness mostly confined to the tropical countries is transmitted by bite of infected female Anopheles mosquito. In 2015 alone, 88% of the malaria burden and 90% deaths due to malaria were confined to the African and Asian countries. Although number of tests are available for rapid diagnosis and screening for malaria, peripheral blood smear examination remains the gold standard. Leishman stain is recommended by WHO however herein we evaluate one of the alternative methods of staining which is simple and rapid. Fifty patients attending the various outpatient departments of the tertiary care hospital with fever and suspected to have malaria were selected. Two thin-air dried smears prepared from the peripheral venous blood from these subjects were stained by Leishman and Toluidine blue method. The findings of the slides by two independent qualified professionals were noted and the results were analyzed. A total of 14% (7/50) cases were diagnosed to have malaria. All the malaria cases which were positive in Leishman stain were also detected in Toluidine blue stain. Malarial parasites were clearly visible against the homogenously light green background in Toluidine blue. The detection of malarial parasite by Toluidine blue was quick, easy and confirmative. Toluidine blue stained peripheral blood smear allows for easy identification and speciation of malarial parasite at low magnification and in shorter period of time. Malaria is a febrile illness, often life threatening which is transmitted to people through the bite of infected female Anopheles mosquito. Worldwide in the year 2015 an estimated 21,2000,000 cases of malaria were seen while 429,000 deaths had occurred , 4.Similarly, acridine orange fluorescent microscopy can be used in the same setting but needs expertise and is expensive . Few autThe study was conducted over a period of one month at the All India Institute of Medical Sciences, India. Fifty patients attending the various outpatient departments of the tertiary care hospital with fever and suspected to have malaria were selected. Two peripheral blood thin smears were made from the blood collected in EDTA vacutainer within four hours of collection. After air drying one slide was subjected to staining by Leishman stain while the other by Toluidine blue stain. Leishman stain was prepared in a flask by mixing 1.5 grams of Leishman powder with 500 ml methanol followed by warming the mixture to 37 degree Celsius for overnight with occasional shaking. After cooling the stain was filtered and used. Leishman staining was done on one of the slides. The air dried smear was flooded with Leishman stain, after 2 minutes double quantity of distilled water was added and thoroughly mixed with the help of a pipette and kept for 8 minutes followed by washing in a stream of tap water. The back side of the slide was wiped clean with dry cotton and set upright for drying for 5 minutes . The Leind decade. The age wise distribution of cases is as shown in The patients belonged to all age groups from 2 months to 70 years with a mean of 27.4 years. Maximum number of cases (28%) was seen in the 2Malaria can be detected by different staining procedures some authors have reported the need for separate stains for thick and thin smears. Giemsa stain is the preferred method for thick smear where the focus is to easily detect the parasite of parasite and to increase the sensitivity, while Leishman stain is recommended for thin smears for identification of species. However, it is necessary to maintain the pH of 7.2 when using either of the two methods of staining. Toluidine blue stain which is used in this study showed comparable results as with Leishman stain in speciation and easy detection of parasites like Giemsa stain. Hence this single stain can be used on thin smears for both purposes. Moreover maintenance of optimal pH is not required making it useful for implementation in resource limited and field setting . Also, aToluidine blue stained peripheral blood smear thus allows for easy identification and species categorization of malarial parasite within shorter period of time and at a very low cost. Thus it can be superior in comparison to other stains for rapid detection of malarial parasite. The limitation of the study is that it is done in limited number of samples, so a large scale study is required for establishing the toluidine blue stain.Toluidine blue is a metachromatic dye used to stain structures like staining of connective tissue mucins, mast cell granules, amyloid microorganism like corynaebacterium diphtheria, helicobacter and mycobacterium and even malignant oral cells;Malaria screening and diagnosis is done by various methods like staining of thick blood films by Giemsa stain for screening and staining of thin blood films by Leishmna stain for species identification;The known methods are time consuming and also expensive.Screening and species identification can be done in the toluidine blue stained smear as only the malarial parasite is stained in the ghost of host cells;The toluidine blue method is cheap, expertise not required, less turnaround time and without compromise on the quality;The various species of malaria along with the various stages like gametocyte, ring forms are all visible in their fine morphological details.The authors declares no competing interests.


Plasmodium falciparum antigens in children with severe malaria would improve our understanding of vulnerability to severe malaria and how protective immunity develops. Using a protein microarray with 179 antigen variants from three VSA families as well as more than 300 variants of three other blood stage P. falciparum antigens, reactivity was measured in sera from Malian children with cerebral malaria or severe malarial anaemia and age-matched controls. Sera from children with severe malaria recognized fewer extracellular PfEMP1 fragments and were less reactive to specific fragments compared to controls. Following recovery from severe malaria, convalescent sera had increased reactivity to certain non-CD36 binding PfEMP1s, but not other malaria antigens. Sera from children with severe malarial anaemia reacted to fewer VSAs than did sera from children with cerebral malaria, and both of these groups had lacunae in their seroreactivity profiles in common with children who had both cerebral malaria and severe malarial anaemia. This microarray-based approach may identify a subset of VSAs that could inform the development of a vaccine to prevent severe disease or a diagnostic test to predict at-risk children.Variant surface antigens (VSAs) play a critical role in severe malaria pathogenesis. Defining gaps, or “lacunae”, in immunity to these  The burden of severe malaria in this region falls principally on young children, particularly in areas of high malaria transmission2. In Bandiagara, Mali, the average age of a child with any form of severe malaria is between 21 and 39 months3. Episodes of cerebral malaria and severe malarial anaemia are rare after age four, while the median age of children with uncomplicated P. falciparum malaria is 10 years4. Thus, immunity to severe malaria appears to develop before the more common and less pathogenic uncomplicated malaria. The basis of natural immunity to severe malaria appears to be, at least in part, the acquisition of antibodies to parasite antigens expressed on the infected erythrocyte surface. In epidemiological studies, having antibodies to these parasite-produced erythrocyte variant surface antigens (VSAs) was consistently associated with protection against clinical malaria8.Malaria kills hundreds of thousands of children in sub-Saharan Africa annually. Two manifestations of severe malaria - cerebral malaria and severe malarial anaemia - account for most of these deathsvar family of genes encodes one group of VSAs: P. falciparum erythrocyte membrane protein-1 (PfEMP1) antigens, large molecules expressed on the surface of the infected erythrocyte9 that bind to endothelial receptors16. Extracellular regions of PfEMP1s protruding from erythrocytes and exposed to the immune system are comprised of constituent domains that include cysteine interdomain rich region (CIDR) domains and Duffy-binding like (DBL) domains. Each parasite genome carries a large repertoire of about 60 var genes, but each infected erythrocyte expresses only one PfEMP1 at a time. PfEMP1s mediate sequestration, a phenomenon whereby an infected red blood cell adheres to the endothelial membrane of an end-organ’s capillary wall, preventing its passage through the reticuloendothelial system and thus avoiding splenic clearance. Sequestration allows P. falciparum to evade the immune system and, in the brain, is likely critical to cerebral malaria pathogenesis.The var genes could be responsible for this severe manifestation of malaria. In a case-control study of severe malaria conducted in Bandiagara, predominant expression of PfEMP1s that bind to the endothelial receptor CD36 was associated with hyperparasitemia, while expression of members of a distinct PfEMP1 group—which cannot bind to CD36 and have an as yet incompletely characterized set of targets—predominated in cerebral malaria episodes17. Non-CD36-binding PfEMP1s include subsets of PfEMP1s that have been associated with severe malaria23; VAR2CSA, which plays a primary role in pregnancy-associated malaria26; and conserved PfEMP1s encoded by var1 and var3, which do not have well-characterized host binding sites28. The host binding sites of non-CD36-binding PfEMP1s have not been completely characterized. Some of these PfEMP1s bind endothelial protein C receptor (EPCR), blocking normal activity of protein C to regulate inflammation and coagulation23. The PfEMP1 CIDRα1 domain has been implicated in this pathway, binding to EPCR. In addition, a subset of these PfEMP1s bind both EPCR and intercellular adhesion molecule 1 (ICAM-1) and is particularly expressed in cerebral malaria22. The potential protective role of antibodies directed against the constitutive CIDR or DBL domains of these PfEMP1s is unknown.The rapid acquisition of immunity to cerebral malaria in early childhood suggests that the lack of an immune response against a small subset of var genes may contribute to pathogenesis and that the development of immunity is rapid. Expression of subsets of var genes encoding non-CD36-binding PfEMP1s has been associated with severe malarial anaemia29. Mathematical modelling has suggested that immunity to non-cerebral severe malaria such as severe malarial anaemia should develop after just one or two infections30.Severe malarial anaemia studies also suggest that a smaller subset of var domain subtype motifs known as domain cassettes (DCs)31 and the much smaller STEVORs and RIFINs, which are encoded by the repetitive interspersed family (rif) and the sub-telomeric variable open reading frame (stevor) gene families, respectively. The array was probed with sera from a case-control study of severe malaria in Bandiagara to test the hypothesis that low seroreactivity to specific extracellular domains of PfEMP1 field variants is associated with vulnerability to cerebral malaria or to severe malarial anaemia. We predicted that severe malaria cases would have lower seroreactivity to a subset of PfEMP1 protein fragments than matched healthy controls and controls with uncomplicated malaria, as well as the convalescent sera of these cases. Children with severe malaria could thus be said to have serological “lacunae”, defined as gaps in the antibody repertoire reflecting a lack of exposure and/or a lack of an effective antibody response to a particular PfEMP1 variant. Given the extraordinary diversity of PfEMP1s, allele-specific immune recognition is likely, whereas cross-reactivity may be present in serorecognition of less diverse antigens. Such serological lacunae would also be present with fragments from the RIFIN and STEVOR VSA families, but not consistently present in serological responses to protein fragments of malaria vaccine candidate blood stage antigens apical membrane 1 (AMA1), merozoite surface protein 1 (MSP1), or reticulocyte binding-like homologue protein (RH5). Our results demonstrated that variant surface antigen lacunae predominated across case-control and acute-convalescent comparisons, with gaps in PfEMP1 seroreactivity particularly suggestive of vulnerability to severe malaria.We designed a protein microarray populated with fragments of variant surface antigens from the 3D7 reference genome, including PfEMP1 variants that have many of the commonly occurring consecutive From 2000 to 2003, 78 children with severe malaria were enrolled that met criteria for cerebral malaria (CM) and/or severe malarial anaemia (SMA), including 43 CM cases, 24 SMA cases, and 11 cases with both CM and SMA (referred to as CM + SMA cases) , the entire 3D7 version of the MSP protein divided into two fragments, and 15 full-length RH5 proteins, all cloned from parasites isolated from field samples , the sera of uncomplicated malaria controls reacted more intensely to 107 protein fragments than did sera of cases , and eight DBL-CIDR domain couplets, including six DBLδ1-CIDRβ1 domain couplets, none of which were DBLα-CIDRα1 domain couplets.In contrast, children with severe malarial anaemia had comparatively fewer gaps in PfEMP1 immunity compared to their uncomplicated malaria controls. The 13 differentially reactive variant surface antigen fragments in the SMA analysis included 12 extracellular PfEMP1s and 1 STEVOR, but no RIFINs. PfEMP1 lacunae included six fragments from non-CD36-binding PfEMP1s overall, four DBL-DBL domain couplets ]. For CM case comparisons (n = 39), 26 lacunae were identified , only two lacunae were identified: one DBL-CIDR domain couplet and one intracellular PfEMP1 fragment, both from CD36-binding PfEMP1s.For CM + SMA comparisons (n = 11), all 11 lacunae identified were from extracellular PfEMP1 fragments, four of which were non-CD36-binding PfEMP1s and were also found in the healthy control-CM comparison (listed below). Eight DBL-CIDR domain couplets were identified, none of which included DBLα-CIDRα1 domain couplets.var3-encoded PfEMP1 PFA0015c; the proximal portion of PF13_0003, a DC16 PfEMP1; and the DC19 portion of the Group B PfEMP1 PFB1055c. Neither CM nor CM + SMA acute illness comparisons shared lacunae with SMA acute illness comparisons.Only five lacunae were shared between healthy control comparisons with CM and CM + SMA cases, all of which were extracellular PfEMP1 fragments, including four non-CD36-binding PfEMP1 fragments Table : a DBLβ-23. It is bound more readily by parasites from children with severe malaria than parasites from uncomplicated malaria23. CIDRα1-containing PfEMP1 fragments have been implicated in binding human brain endothelial cells in vitro20. Significant CIDRα1 expression has been observed in Tanzanian and Beninese children with cerebral malaria32 as has elevated expression of the non-CD36-binding PfEMP1s (a group that includes CIDRα1-containing PfEMP1 fragments) in cerebral malaria cases in Mali17 and Kenya29.We sought to identify PfEMP1 fragments bound more intensely by sera following an episode of severe malaria than when a child was acutely ill. We were particularly interested in increased seroreactivity to CIDRα1-containing PfEMP1s. EPCR is a target of the CIDRα1 domain31 , and the fourth was a proximal DBLα-CIDRα1 domain couplet that is part of the DC4-containing PFD1235w, which binds both EPCR and ICAM-122.In comparisons between matched acute and convalescent sera from CM children, only four protein fragments, all from non-CD36-binding PfEMP1s containing proximal DBLα-CIDRα1 domain couplets, elicited more intense responses by convalescent sera than acute sera Fig. ; n = 27.var1-encoded PFE1640w, the DC4-containing PFD1235w, and the DC8-associated MAL6P1.316) and one proximal DBLα-CIDRδ domain couplet, the DC16 portion of PF13_0003.Eight protein fragments elicited more intense responses by convalescent sera than acutely ill sera from SMA children (n = 12). This included two AMA1 fragments, two MSP1-19 fragments, and four PfEMP1 fragments, all from non-CD36-binding PfEMP1s, including three with proximal DBLα-CIDRα1 domain couplets. These four PfEMP1 fragments included three DBL-DBL domain couplets ; an additional non-CD36-binding PfEMP1, , had different fragment lacunae identified for the CM comparison and for the SMA comparison Table .The limited number of convalescent samples prohibited meaningful comparison of acute versus convalescent sera of CM + SMA children (n = 2).For each severe malaria type, we compared seroreactivity from the group with acute disease with seroreactivities of the healthy control and uncomplicated malaria groups as well as with convalescent seroreactivity in order to identify lacunae that were present across multiple serology group comparisons.Seventeen lacunae were identified across more than one CM comparison Fig. . All of var1-encoded PFE1640w), an MSP1 fragment, and two AMA1s that were different concentrations of a 3D7-based vaccine preparation , one non-CD36-binding extracellular PfEMP1 fragment  in seroreactivity were consistently identified for several malaria blood stage vaccine candidate antigens, supporting the central role of VSAs in immunity against severe malaria. Children with severe malarial anaemia had fewer VSA fragment lacunae than did children with cerebral malaria. Children who had both cerebral malaria and severe malarial anaemia shared some lacunae with children who had only cerebral malaria and children who had only severe malarial anaemia, suggesting that there are both syndrome-specific and non-specific targets of protective immunity.Identifying specific gaps in immunity to parasite variant surface antigens (VSA) in children with severe malaria has the potential to pinpoint antigens critical to cerebral malaria and severe malarial anaemia pathogenesis. Antigens targeted by protective natural immunity may be promising targets for a vaccine to prevent severe malaria in children. Using a custom protein microarray to measure seroreactivity to fragments of the major Convalescent sera following recovery from cerebral malaria or severe malarial anaemia reacted more intensely than acute sera to certain non-CD36-binding PfEMP1s; again, this was not the case for other malaria antigens, potentially implicating this subset of VSAs in the pathogenesis of severe disease. All such fragments for cerebral malaria cases were from CIDRα1-containing PfEMP1s; three out of four PfEMP1s in severe malarial anaemia cases were from CIDRα1-containing PfEMP1s. However, only one differentially seroreactive fragment contained the actual CIDRα1 domain; the rest contained consecutive pairs of DBL domains distal to the CIDRα1 domain.30, recent studies support the idea that acquisition of immunity to severe malaria may be more gradual33. Indeed, studies have identified subsets of children who experienced multiple episodes of severe malaria35. An alternative explanation is that the specific PfEMP1 domains that are exposed to the immune system and generate antibody responses that are temporally associated with the acquisition of natural immunity are not the PfEMP1 domains that are critical to receptor binding and severe malaria pathogenesis. This may be the case with VAR2CSA domains associated with immunity to pregnancy-associated malaria, which appear to differ from those VAR2CSA domains critical to binding placental receptors36. In such a scenario, CIDRα1 domains associated with EPCR binding would not be associated with immune system exposure and a subsequent increase in seroreactivity, while other constitutive domains on the same PfEMP1 would be exposed to and elicit a response by the immune system. This would explain why fragments identified in the acute-convalescent comparison were primarily DBL-DBL domain couplets distal to CIDRα1 domains. Alternatively, the CIDRα1 domain may be exposed to the immune system, but somehow fails to elicit an appropriate response, in contrast to neighboring constitutive domains.One potential explanation for the failure to react more intensely to a domain that may be critical to severe malaria pathogenesis is that a single episode of severe malaria may be insufficient to develop protective immunity to severe malaria in all individuals. While mathematical modelling has indicated that immunity to severe malaria may be acquired after one or two infections33, we found that serological profiles of children who had recovered from severe malaria still did not resemble those of their matched peers who experienced episodes of uncomplicated malaria. Other potential explanations for why a child does not develop severe malaria, such as earlier treatment, may also explain differences in severity of disease.One potential explanation for why children who served as controls had uncomplicated malaria is that they had immunity against severe malaria, so that infection produced only relatively mild symptoms. If this were the case, comparisons of sera from cases and controls may have identified gaps in immunologic protection in children with severe malaria. Lacunae that were “filled in” after an episode of either cerebral malaria or severe malarial anaemia consisted of a few extracellular PfEMP1 fragments, while many additional immunity gaps remained in comparison to uncomplicated malaria controls. Whether this post-episode immunity is sufficient to protect these children against subsequent severe malaria episodes is unclear. Consistent with the models predicting a more gradual acquisition of protective immunity to severe malariaComparisons with healthy controls may be harder to interpret than comparisons with uncomplicated malaria controls, given that the immune status of healthy controls is unknown. Healthy controls could have protective immunity against severe malaria, or they may simply be unexposed to infection. In comparison to healthy controls, children with cerebral malaria had multiple PfEMP1 lacunae, as did, to a lesser extent, children with both cerebral malaria and severe malarial anaemia. In contrast, children with severe malarial anaemia had only two lacunae compared to healthy controls, making it appear that these children differed from healthy controls to a lesser extent than children with cerebral malaria. Alternatively, decreased exposure and thus decreased acquisition of immunity in the healthy control group could explain this observation, with “healthy” unexposed children and those with anaemia being more similar because both have less immunity. Indeed, healthy controls recognized fewer merozoite proteins than severe malarial anaemia cases, suggesting a low level of malaria exposure in this group, in contrast to the healthy controls for cerebral malaria subjects.37. This difference in reactivity to intracellular PfEMP1s suggests a greater degree of malaria exposure in controls with uncomplicated malaria versus matched cerebral malaria cases.Lacunae that recurred across different analyses were predominantly seen with respect to extracellular PfEMP1s, suggesting that these antibody responses play a critical role in defining vulnerability to severe malaria; intracellular PfEMP1s were identified as lacunae solely in comparisons of cerebral malaria and uncomplicated malaria in sera collected at the acute visit. We have previously shown that intracellular PfEMP1 seroreactivity increases with age and over the course of a malaria season29. In this study, following an episode of cerebral malaria or severe malarial anaemia, children “filled in” several PfEMP1 lacunae, but the majority of lacunae that separated them from uncomplicated malaria controls remained unfilled. An ordered pattern of acquisition of immunity to particular PfEMP1 domains was seen in young Tanzanian children as they aged39, with early acquisition of immunity to non-CD36-binding PfEMP1 domains. We found that differences between sera from acute cerebral malaria and severe malarial anaemia cases and their convalescent sera were primarily due to non-CD36-binding PfEMP1s, particularly non-CD36-binding PfEMP1s with CIDRα1 domains. However, acute sera from severe malaria cases also differed with controls with respect to CD36-binding PfEMP1 fragments. The extent to which seroreactivity to CD36-binding PfEMP1 fragments is important in protection against severe malaria is unclear and requires further investigation.Previous work has suggested expression of distinct subsets of non-CD36-binding PfEMP1s in cerebral malaria and severe malarial anaemia casesNext steps to identify lacunae critical to protection against severe malaria include identifying the PfEMP1 variants expressed in children with severe malaria, determining their complete sequences, and characterizing which PfEMP1 domains are critical to protection against such infections. An approach combining next generation sequencing to identify these PfEMP1s and then populating a protein microarray with protein fragments of their constitutive domains will provide a powerful tool to assay the immunity of children with severe malaria during an acute attack and determine which lacunae are subsequently filled. With this approach, we may better identify particular VSA fragments with differential seroreactivity that may allow us to identify children vulnerable to cerebral malaria or severe malarial anaemia.31. In particular, DC8, DC13, and DC17 have been associated with severe malaria, but are either not found or are incomplete in the 3D7 var gene repertoire. Future protein microarray studies of severe malaria should include representative fragments from these and other DCs underrepresented in 3D7.This study had some limitations. A protein microarray was populated with PfEMP1 fragments from the reference strain 3D7, representing only a subset of the known DBL and CIDR domain types, as well as domain cassettesE. coli-based cell-free system that allows formation of disulphide bonds. Using this system, we have observed epidemiological patterns matching what would be predicted in the acquisition of natural immunity, including patterns of recognition of VAR2CSA, the leading PfEMP1 candidate for a malaria vaccine, in malaria-exposed Malian women of differing gravidities36. Nevertheless, protein conformations may not completely match native PfEMP1 conformations.Protein fragments on our microarray had an upper size limit of about 1000 amino acids. Domain-domain interactions that do not fit within these constraints would not be identified with this approach. In addition, the fidelity of protein conformation could be an issue when using an Development of an effective severe malaria vaccine relies on identification of malaria proteins critical to the acquisition of immunity to specific severe malaria syndromes. Identification of these proteins requires a broad approach examining multiple antigens at once, a strength of microarray-based methods. This approach may allow further winnowing of the thousands of known variant surface antigens to determine a much smaller subset eliciting increased seroreactivity by children protected against severe malaria that may form the basis of a vaccine or therapy. This could also allow for development of an antibody test to identify those children most susceptible to severe malaria in a population.Anopheles gambiae is the principal malaria vector. Malaria transmission is highly seasonal, with minimal transmission at the height of the dry season in March; less than one infected mosquito bite per person per month as the transmission season starts and ends in June and December, respectively; and a peak of up to 60 infected bites per person per month40. P. falciparum represents 97% of malaria infections with 3% due to P. malariae and rare infections with P. ovale. Despite a seasonal transmission pattern, the malaria burden is heavy: children aged less than 10 years had an average of two clinical malaria episodes in the 1999 transmission season40, and severe malaria afflicts 1 in 50 children aged less than 6 years each year3. Older children and adults are relatively protected against malaria disease, but remain susceptible to infection.The study was conducted in Bandiagara, a town of 13,634 inhabitants (2002 census) in the Dogon Country in east-central Mali. Bandiagara is relatively dry, with a mean annual rainfall of 600 mm. The protocol was approved by institutional review boards of the Faculty of Medicine, Pharmacy and Dentistry, Bamako, Mali and the University of Maryland, Baltimore. Written informed consent was obtained from parents or guardians of all study participants. All methods were performed in accordance with the relevant guidelines and regulations.41. An episode of cerebral malaria was defined using the standard WHO definition of cerebral malaria: a Blantyre Coma Score of <342, with no other obvious cause of coma43. Severe malarial anaemia was defined as a haemoglobin ≤ 5 g/dL. An additional subset of severe malaria cases was defined as cases with characteristics of both cerebral malaria and severe malarial anaemia. No subjects received blood transfusions as part of their care. As previously described, each index case was age-, residence-, and ethnicity-matched to a case of uncomplicated malaria and a healthy control44. Age categories were defined as 3–5 months, 6–11 months, 1 year, 2 years, 3–4 years, 5–6 years, 7–8 years, 9–10 years, 11–12 years, and 13–14 years. Residence was defined as one of eight distinct sectors of Bandiagara town or, in the case of children from outer villages, the specific village of origin. Uncomplicated malaria was defined as P. falciparum parasitemia and an axillary temperature of 37.5 °C detected by active surveillance, or parasitemia and symptoms leading to treatment-seeking behavior in the absence of other clear cause of fever on passive surveillance.Cases of severe malaria from Bandiagara and surrounding areas were admitted to the Bandiagara Malaria Project ward from October 1999 to December 2002. Cases were classified as severe malaria based on modified criteria put forth by the World Health OrganizationMatched uncomplicated malaria controls were enrolled from the population of children presenting to the daily Bandiagara Malaria Project clinic. Healthy controls were enrolled by traveling to the home of a child with severe malaria. We then followed a standardized routine of exiting the front entrance of the child’s housing compound and making consecutive left turns until another compound with an eligible control was identified. Children were enrolled as healthy controls if they were asymptomatic for acute illness, had no evidence or history of chronic illness, and if they were found to be aparasitemic upon examination.Sera was obtained at enrolment (June to December) and, when possible, in the following dry season, a period extending from January to June , typically several months after the acute illness.A protein microarray was made with 170 fragments of PfEMP1s, six full-length STEVOR antigens, and three full-length RIFIN antigens, all based on the 3D7 reference genome  in vivo recombination cloning, (3) in vitro transcription/translation, and (4) microarray chip printing45.Protein microarray construction followed a four-step process that includes: (1) PCR amplification of each complete or partial P. falciparum protein microarrays7. “Recognition” of a protein fragment was defined as fluorescence intensity significantly greater than that of the malaria-naïve control group, based on a two-sample Kolmogorov-Smirnov test, as per previous array analyses36. Comparisons of recognized proteins between severe malaria cases and controls in Fig. Fluorescence intensity was defined as the raw signal intensity reduced by the mean for the no-DNA negative controls. We used data analysis techniques that have previously been used for evaluating humoral immune responses in P. falciparum antigen variant was compared for severe malaria cases versus controls to identify protein fragments eliciting more reactivity by sera from control groups versus severe malaria cases, thereby defining “lacunae” in antibody responses of children with severe malaria. The significance of these comparisons was determined by a Wilcoxon-signed rank test. Similarly, fluorescence intensity for each P. falciparum antigen variant was compared for severe malaria at the time of illness versus convalescence to identify “lacunae” in antibody responses when presenting with severe malaria.Fluorescence intensity for each 46. Statistical analyses were primarily conducted using SAS 9.2 and the Real Statistics Resource Pack software (Release 3.8).All P-values presented were two-sided. In identifying lacunae, consistent with other similar published analyses, we did not adjust P-values for multiple comparisons, instead reporting all results with a P < 0.05 and noting comparisons with a P < 0.01 or P < 0.001, as in other microarray analyses47. This analysis was performed for individual protein fragments  approach, generating receiver operating characteristic curve plots for each fragment and comparing results by the area-under-the-curvets Table  and for ts Table .The microarray dataset of fluorescence intensities to parasite protein fragments generated during during the current study are attached as a supplementary data file Database .Supplementary informationData file S1Database S1


The objectives of this study were to determine the proportion of malaria, bacteraemia, scrub typhus, leptospirosis, chikungunya and dengue among hospitalized patients with acute undifferentiated fever in India, and to describe the performance of standard diagnostic methods.During April 2011–November 2012, 1564 patients aged ≥5 years with febrile illness for 2–14 days were consecutively included in an observational study at seven community hospitals in six states in India.Leptospira IgM ELISA, Scrub typhus IgM ELISA and Chikungunya IgM ELISA were routinely performed at the hospitals.Malaria microscopy, blood culture, Dengue rapid NS1 antigen and IgM Combo test, Leptospira Microscopic Agglutination Test (MAT), malaria PCR and malaria immunochromatographic rapid diagnostic test (RDT) Parahit Total™ were performed at the coordinating centre. Convalescence samples were not available.Second line testing, Dengue IgM capture ELISA (MAC-ELISA), Scrub typhus immunofluorescence (IFA), Case definitions were as follows: Leptospirosis: Positive ELISA and positive MAT. Scrub typhus: Positive ELISA and positive IFA. Dengue: Positive RDT and/or positive MAC-ELISA. Chikungunya: Positive ELISA. Bacteraemia: Growth in blood culture excluding those defined as contaminants. Malaria: Positive genus-specific PCR.P. falciparum. Dengue was diagnosed in 16% (244/1564). Bacteraemia was found in 8% (124/1564), and among these Salmonella typhi or S. paratyphi constituted 35%. Scrub typhus was diagnosed in 10%, leptospirosis in 7% and chikungunya in 6%. Fulfilling more than one case definition was common, most frequent in chikungunya where 26% (25/98) also had positive dengue test.Malaria was diagnosed in 17% (268/1564) and among these 54% had Malaria and dengue were the most common causes of fever in this study. A high overlap between case definitions probably reflects high prevalence of prior infections, cross reactivity and subclinical infections, rather than high prevalence of coinfections. Low accuracy of routine diagnostic tests should be taken into consideration when approaching the patient with acute undifferentiated fever in India. Infectious diseases are the leading causes of morbidity and death in India . Field sThe main objective of this study was to determine the proportion of AUF caused by malaria, bacteraemia, scrub typhus, leptospirosis, chikungunya and dengue among patients admitted to community hospitals in India. A secondary objective was to describe the performance of routine diagnostic methods.During April 2011–November 2012, patients aged ≥5 years admitted with AUF were consecutively included from the following secondary, community (100 to 500) bed hospitals: Baptist Christian Hospital in Tezpur , Duncan Hospital in Raxaul , Christian Hospital in Mungeli , B.K. Walawalkar Hospital in Ratnagiri , Rural Development Trust Hospital in Anantapur , Christian Fellowship Hospital in Oddanchatram  and Bethesda Hospital in Ambur  , Chikungunya IgM ELISA , Dengue rapid NS1 antigen and IgM/IgG Combo test  and blood cultures. Convalescence serology testing was not performed due to logistic challenges. In order to improve detection of IgM antibodies serological testing was delayed until five days of fever, if possible.The following laboratory tests were performed at the study hospitals as part of routine investigation: Malaria blood smears, Scrub typhus IgM ELISA , Blood was cultured with conventional methods, or automated , and if growth was detected, the isolate was identified at each site and frozen, then in Transport swab  sent to the reference laboratory for re-identification and confirmation.Leptospira IgM ELISA, Chikungunya IgM ELISA and Dengue NS1/IgM Combo test, only if not performed at local site. Dengue IgM capture ELISA (MAC-ELISA) was performed at reference laboratory on all samples. Scrub typhus immunofluorescence (IFA) was performed on all IgM ELISA positives and some ELISA negatives. Leptospira Microscopic Agglutination Test (MAT) was performed on all IgM ELISA positives and some ELISA negatives.The following investigations were performed at the reference laboratory at CMC: Scrub typhus IgM ELISA , Plasmodium genus-specific PCR targeting mitochondrial genome [The immunochromatographic malaria rapid diagnostic test (RDT) Parahit Total™  was performed on all samples. A l genome  was perfl genome .Case definitions were as follows:Leptospirosis: Positive ELISA and positive MAT.Scrub typhus: Positive ELISA and positive IFA.Dengue: Positive RDT and/or positive MAC-ELISA.Chikungunya: Positive ELISA.Bacteraemia: Growth of bacteria not considered to be contaminants in blood culture.Malaria: Positive malaria genus-specific PCR.All tests were performed as per the standard protocol provided by the manufacturers. Some serology tests were performed as a quality control with the same method both at the local centre and at the reference laboratory. In these cases a positive result was defined as two positive results or one positive and one equivocal, a negative result defined as two negatives or one negative and one equivocal, while one positive and one negative result was defined as discrepant.Chi-square test was used to assess differences between proportions.A total of 1564 patients were included, with mean  age 34  years. Among these 632 (40%) were women and 895 (57%) were men, and 1219 (78%) lived in rural areas. Table Plasmodium falciparum. Details of malaria results in this study have been reported previously [Salmonella typhi or S. paratyphi constituted 35% (44/124), Staphylococcus aureus 19% (24/124), E. coli 9% (11/124) and Streptococcus pneumoniae 6% (7/124).As per case definition, malaria positivity was found in 17% (268/1564), dengue in 16% (244/1564), scrub typhus in 10% (159/1564), bacteraemia in 8% (124/1564), leptospirosis in 7% (116/1564) and chikungunya in 6% (98/1564). Among malaria cases, 54% (145/268) were Table The highest prevalence of malaria was found in West-, North- and Central India . The highest prevalence of dengue was found in South- and West India , and a high prevalence of chikungunya (18%) was also found in Anantapur. The highest prevalence of scrub typhus was found in North- and North East India (Tezpur 22% and Raxaul 15%). Tezpur also had the highest prevalence of leptospirosis (15%). Raxaul in North India had as high prevalence as 20% of bacteraemia.P. falciparum. Among patients with P. falciparum and bacteraemia, Staphylococcus aureus or Enterobacteriacae including Salmonella typhi and S. paratyphi were identified. Among patients with positive malaria microscopy confirmed by PCR, 5% (3/66) had bacteraemia.More than one case definition was found in a high number of patients. The overlap between diagnoses is shown in detail in Table The association between positive serology  and malaria and septicaemia is shown in Table Table Using IFA as gold standard, positive predictive value for Scrub typhus IgM ELISA was 61% (159/260), and negative predictive value was 89% (95/107).Leptospira IgM ELISA was 65% (116/179) while negative predictive value was as low as 38% (20/52). The study was not designed to evaluate sensitivity and specificity, as gold standard tests were not performed on all ELISA negatives.Compared to MAT, positive predictive value for Using malaria PCR as gold standard, the sensitivity of routine microscopy was 29% (66/228) and RDT 24% (65/268), as reported previously .Sensitivity and specificity for dengue tests were not calculated because gold standard was positive RDT and/or MAC ELISA, and the two tests are expected to be positive during different intervals of the illness. Indeed, only 46% (57/124) of RDT positives were positive by MAC ELISA, probably reflecting early infections detected only by NS1Ag.This study of aetiology of undifferentiated fever in rural India using standard diagnostic tests, revealed a high prevalence of malaria and dengue. However, there was a strikingly high prevalence of overlap of case definitions. An overlap with one or more other case-definition was found for all diagnosed diseases, ranging from 33% (bacteraemia) to 57% (chikungunya)  among patients included, and among these 54% (145/268) were eviously . As repoSalmonella typhi or S. paratyphi were found in 35% (44/124), reflecting the high prevalence of enteric fever in India. Enteric fever is closely associated with poor sanitation, lack of safe water supply and treatment failures due to antimicrobial resistance and is still reported as the most common blood stream infection in India and in South Asia [S. aureus (19% 24/124), followed by E.-coli  and S. pneumoniae .Blood stream infection with pathogenic bacteria was diagnosed in 8% (124/1564), and among these uth Asia –13. The Dengue and severe dengue because of immune enhancement due to a previous infection with another serotype is an increasing problem in India , 15. IndRapid tests combining detection of non-structural protein 1 (NS1) antigen and IgM/IgG are used in routine diagnostics, as they have high sensitivity both during the viremic early phase of infection when NS1 is produced and after more than five days when IgM can be detected . IgM capChikungunya was reported in Ahmedabad in India in 2006 [A large outbreak of  in 2006 . Sharing in 2006 . Sporadi in 2006 , with hiLeptospirosis is transmitted by urine from infected animals  and is endemic particularly in the Andaman and Nicobar group of islands (“Andaman haemorrhagic fever”) . In AUF Leptospira is unreliable, and the gold standard is therefore serology confirmed by MAT. MAT detects IgM and IgG antibodies against a pool of live antigens from different Leptospira serovars. A MAT titer >100 is considered positive, but a fourfold rise in convalescence titer or a high single acute phase titer (>200–1600 depending on endemicity) supports the diagnosis [Leptospira IgM ELISA has been reported positive in 40% and 5% one and six years after infection respectively [Culturing iagnosis . Followiiagnosis . In one iagnosis . In the iagnosis . Leptospectively . DiscrimScrub typhus is transmitted by mites who live on rats. The disease is, similar to leptospirosis, associated with agricultural work and rural dwelling . Two stuSerology confirmed by IFA, ideally confirmed by rise of titer in convalescent samples and/or by cut-off values based on endemicity, remains the mainstay of diagnostics since isolation of the bacteria is not possible and PCR from blood has low sensitivity . SensitiAlthough background positivity or cross reactivity in serology, and potential subclinical infections in malaria, may have given positive test results in some cases, some of the overlapping aetiologies have probably been due to true clinically relevant coinfections.Coinfections could occur principally by two different mechanisms; by contracting multiple infections at the same time, or increased pathogenicity of a simultaneous subclinical infection due to immune reactions.Salmonella spp. or other Gram-negatives, are found both in P. falciparum and P. vivax malaria [The risk of bacterial sepsis is increased in severe malaria, through immune mediated barrier dysfunction in the gut and bacterial translocation, as well as IL-10 mediated decreased control of bacteraemia , 38. In  malaria , 40, whi malaria –43, and In a study among Thai rice farmers with leptospirosis diagnosed with 4-fold rise in titer or a single high titer, as many as nine among 22 patients had coinfection with scrub typhus confirmed by serology and eschar or clinical characteristics . AlthougTrue coinfections with malaria and scrub typhus, diagnosed by clinical characteristics and eschar or PCR, have also been reported in India –47. HoweIn the mosquito borne infections dengue, malaria and chikungunya, outbreaks occur during rainy seasons and although the specific vector is different for malaria, coinfections are not unlikely. This was shown in a study from India during a dengue outbreak, where 7% 27/367) of dengue cases had coinfection with malaria . As much67 of denAedes aegypti and A. albopictus, and are present in similar geographical regions. In the present study, dengue and chikungunya both had high prevalence in Anantapur, supporting the notion that coinfections as well as cross reactivity could explain some overlap between dengue and chikungunya.A high level of coinfections with dengue and chikungunya was shown during a dengue outbreak in Delhi in 2006 using PCR as the method for detection. Among 17 chikungunya positive patients, six were co-infected with dengue virus . Ten perA high prevalence of malaria and dengue, and a high overlap between case definitions were found in this study. The overlap probably reflects an undefined level of previous infections, cross reactivity and subclinical infections in the population, rather than high prevalence of coinfections. These limitations of routine diagnostic tests should be taken into consideration when approaching the patient with acute undifferentiated fever in India.