For rapid initiation of anti-malarial treatment and prevention of complications, early diagnosis and risk stratification is important in patients with Plasmodium falciparum malaria. Routine laboratory values do not correlate well with disease severity. The aim of this study was to determine the diagnostic and prognostic value of several biomarkers related to inflammation; endothelial and cardiac dysfunction; coagulation, and haemolysis in imported P. falciparum malaria.In a prospective case-control study, 79 adult travellers with both uncomplicated and complicated P. falciparum malaria were included between 2007 and 2011. Forty-one healthy subjects were included as controls. Blood samples were obtained within 24 hours after first consultation to assess routine laboratory values as well as markers related to inflammation , endothelial activation , cardiac function , coagulation , and haemolysis (LDH). Prognostic performance was assessed using the receiver operating characteristic curve (area under the curve = AUROC).Twelve (15.2%) patients had severe P. falciparum malaria. In the patient group, significant thrombocytopaenia was found, all other markers but PAPP-A were significantly elevated. Diagnostic performance was best for CRP with an AUROC of 1.00, followed by MPO (0.99), D-dimers (0.98), elastase-2 (0.98), and sICAM-1 (0.98). Biomarker levels did not correlate well with disease severity.The combination of travel history, fever prior to blood sampling, and CRP serum levels above or below 10.8 mg/l upon hospital admission, best discriminated between malaria patients and control persons. None of the biomarkers studied predicted the presence or the development of malaria complications, neither at the time of admission, nor during hospitalization. Malaria remains the most important parasitic disease with an estimated number of deaths of 1.2 million in 2010, especially amongst children under five . PlasmodPrompt anti-parasitic treatment does not always prevent complicated courses of disease. Clinical symptoms can deteriorate rapidly, progressing to life-threatening conditions, such as coma, renal failure and pulmonary oedema ,6. The oBiomarkers are distinct biochemical substances that are indicators of a patient’s current state of health. They can be used to diagnose and predict the progress and outcome of many diseases. Ideally, a biomarker should fulfil the following criteria: i) ability to diagnose a disease; ii) ability to identify patients at risk; iii) ability to stratify patients depending on disease severity; iv) ability to provide prognosis; v) ability to provide guidance in treatment; and, vi) ability to identify the risk for long-term complications.The parasite’s virulence factors, as well as the host’s immune system, play an important role in the disease’s clinical manifestation and severity. Plasmodium falciparum malaria is a multisystem disorder, potentially affecting every organ of the body. Due to P. falciparum-specific pathophysiology, systemic inflammation, endothelial activation, coagulopathy, and circulatory dysfunction often occur.A panel of markers related to inflammation , endothelial activation , cardiac function , and coagulation  was selected. Lactate dehydrogenase (LDH) was determined as a marker for haemolysis. PAPP-A and myeloperoxidase had not been tested in malaria before.Regarding the role of these parameters, two aspects were examined in this case control study: i) the potential of diagnostic capacities, or whether there is an association with imported P. falciparum infection at all; and, ii) the association with disease severity. The hypothesis that particularly arises in this context is that if the biomarkers correlate with disease severity in P. falciparum malaria, according to World Health Organization (WHO) criteria or to national guidelines, patients with elevated markers are at risk even without consideration of the guidelines. However, to prove this hypothesis further prospective studies are needed.Between June 2007 and February 2011, patients with imported P. falciparum malaria were screened at the in- and outpatient departments of the Section for Tropical Medicine at the University Medical Centre Hamburg-Eppendorf, which is a tertiary hospital in Hamburg, Germany. Eligible patients had to be older than 18 years. Exclusion criteria were evidence of other significant infection, inability to give informed consent, and pregnancy. Complicated malaria was diagnosed according to the current guidelines of the German Society for Tropical Medicine and International Health (DTG) and modified WHO criteria  , ELH-sICAM1-001, , Human sVCAM-1 Platinum ELISA BMS232 , PMN Elastase kit BE59311 , endothelin kit BI-20052 , CardioMPO kit . The NT-proBNP ELISA used was Dimension Vista System by Siemens Healthcare Diagnostics Ltd, Camberley, UK. The commercial kits were used according to the protocols of the various manufacturers who ensured quality control and reproducibility. Reproducibility was verified by selected replicates, quantification was achieved by standard curves performed in duplicates. Blinded testing of the samples was performed. In order to assure the good quality of biomarker research, study design and procedures were conducted according to the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) checklist .® Chicago, IL, USA) was used for statistical analyses. In order to obtain information about the biomarkers’ potential for differentiation between malaria patients and controls, as well as between patients with uncomplicated and complicated malaria, results were graphically displayed in box plots. Next, the biomarkers were examined applying receiver operating characteristic (ROC) curves for illustration of sensitivity and specificity. The area under the ROC curve (AUROC) and their corresponding 95% confidence intervals were calculated. The highest AUROC possible was 1.0, defining the theoretically most perfect test with a sensitivity and specificity of 100%. An AUROC of 0.8-0.9 represented a good test, an area of 0.7-0.8 a fair test. A result <0.7 was considered a poor test, an area of 0.5 indicated a worthless test. Pearson’s correlation co-efficient was used to assess correlation amongst the different biomarkers. No significant association was found between test results and creatinine levels. Each marker’s best cut-off point was calculated by Youden’s index. A P-value of <0.05 was set as level for statistical significance. Due to the pronounced high collinearity, multivariate analysis was not done.SPSS statistics 17.0 software , HIV (n = 3), Herpes simplex viral pneumonia (n = 1), adnexitis (n = 1) and malignancy, i e, prostate cancer (n = 1). Two women were excluded because of pregnancy. Fifty-four (68.4%) and two (2.5%) of the patients were migrants from Africa and Asia, respectively, and twenty-three (29.1%) were Caucasian. The majority of patients (96.2%) acquired P. falciparum malaria in Africa, only one (1.3%) infection was acquired in Central America. A total of four (5.3%) patients reported to have taken adequate malaria prophylaxis: one patient with complicated and two patients with uncomplicated malaria had taken mefloquine, one patient with uncomplicated disease had taken atovaquone/proguanil. More than half of all malaria patients (64.8%) developed symptoms within the first seven days after returning from their journey, almost one quarter (23.0%) within seven to 14 days, and 5.4% and 6.8% within 14 to 21 days and >21 days, respectively. Nine (81.8%) of the patients with complicated disease developed symptoms within the first week after returning from their travels. Twelve (15.2%) patients were categorized as complicated malaria for the following reasons: parasitaemia >5% (n = 10), severe anaemia (n = 1), neurological symptoms (n = 4), and renal failure (n = 2). Four of the patients presented with more than one criterion defining complicated disease. None of the patients died. All 41 healthy controls were Caucasians from Germany. The mean age was 33.51 years (age range 18-72 years), twenty-four subjects (59%) were female.The heart rate and body temperature was significantly higher in the patient group compared with the control group: mean heart rate was 90 bpm versus 70 bpm P <0.001) and mean body temperature was 37.8°C versus 36.5°C (P <0.001), respectively. The mean systolic arterial blood pressure was similar in malaria patients and controls with a value of 119.6 mm Hg and 122.4 mm Hg, respectively (P = 0.283). The geometric mean parasite density (GMPD) was 17,349/μl in all malaria patients, and 255,946/μl and 10,635/μl (P <0.001) in complicated and uncomplicated cases, respectively. The mean/median levels of standard laboratory parameters characteristic for malaria like haemoglobin, leukocytes, platelets, as well as liver enzymes, total bilirubin and serum creatinine were significantly altered in the malaria patient group when compared with the healthy controls. Both the demographic and clinical characteristics, as well as basic laboratory results, are shown in Table  and meanThe following serum levels were significantly higher in the patient group when compared with the healthy controls: copeptin, CRP, MPO, elastase-2, endothelin-1, sICAM-1, sVCAM-1, NT-proBNP, MR-proANP, fibrinogen, D-dimers, and LDH. Median values for PAPP-A were similar in both groups. The diagnostic performance and accuracy was measured by AUROC and the corresponding 95% confidence intervals (95% CI). Five biomarkers showed the highest AUROC in all malaria patients compared to the control group: CRP (1.00), MPO (0.99), D-dimers (0.98), elastase-2 (0.98), and sICAM-1 (0.98) , complicated malaria (CM), and healthy controls.Click here for file


Plasmodium. Plasmodia are obligate intracellular parasites that are able to infect and replicate within the erythrocytes after a clinically silent replication phase in the liver. Four species  are traditionally recognized as responsible of natural infection in human beings but the recent upsurge of P.knowlesi malaria in South-East Asia has led clinicians to consider it as the fifth human malaria parasite. Recent studies in wild-living apes in Africa have revealed that P.falciparum, the most deadly form of human malaria, is not only human-host restricted as previously believed and its phylogenetic lineage is much more complex with new species identified in gorilla, bonobo and chimpanzee. Although less impressive, new data on biology of P.malariae, P.ovale and P.vivax are also emerging and will be briefly discussed in this review.Malaria is a vector-borne infection caused by unicellular parasite of the genus  Plasmodium are traditionally recognized as responsible of human malaria: Plasmodium falciparum, P.malariae, P.ovale and P.vivax.P. cynomolgi cynomolgi, P.cynomolgi bastianelli, P.simiovale, P.brasilianum, P.schwetzi, P.inui and P.knowlesi.2Four host-restricted or adapted species of Plasmodia have been regarded as belonging to the phylum Apicomplexa with an evolution from the Coccidian stem that encompass the progressive acquisition of more complicated phases in their life-cycles confined to a single host. Finally, the successful survival of these parasites requires completion of life cycle in two alternative hosts of evolutionarily distant species (i.e. human and mosquito). The genus Plasmodium is subdivided in the sub-genus Plasmodium and Laverania within the sub-order Haemosporidiidea(ridiidea.Plasmodium contains three genomes: 1) a nuclear genome that comprises 14 linear chromosomes;2) a linear mitochondrial genome that is one of the smallest known ; 3) a 35 kb circular plastid genome of red-algal origin, that is housed in the apicoplast.Anopheles mosquitos, and an endogenous asexual phase (named schizogony) which take place in the vertebrate host.Plasmodium is characterized by a tissue phase in the liver, followed by a blood phase, that is ultimately responsible of the appearance of clinical manifestations. It is generally believed that once an infectious female anopheline mosquito injects sporozoites in the blood, they are able to travel quickly to the liver where they invade the host’s hepatocytes. However, new findings seems to indicate the existence of a more complex invasion process characterized by the persistence of sporozoites in human dermis for hours with a slow release into capillaries as well as migration through lymphatic system.P.vivax/P.ovale and up to 30,000 in P.falciparum). This exoerythrocytic schizogony is characteristic of each species and takes a minimum maturation time of 6 days in P.falciparum and a maximum of 16 days in P.malariae. The mature schizont ruptures (together with the infected hepatocytes) releasing merozoites that once in the blood actively invade erythrocytes using an acto-myosin motor system. The tissue phase extinguishes itself at this point with the notable exception of P.vivax and P.ovale (and certain other primate species such as P.cynomolgi) who are able to persist in the liver as “hypnozoites” that are eventually responsible of the phenomenon of relapse. The blood cycle begins when the merozoites invade the erythrocytes and the period of time from the infection by mosquito bite and the first appearance of the trophozoites in the erythrocytes is called “prepatent period” ; this period is characteristic of each species and is constant. The prepatent period lasts 9 days in P.falciparum, 11–13 days in P.vivax, 10–14 days in P.ovale, 15 days in P.malariae and 9–12 days in P.knowlesi  contrasted this view.Plasmodium species.The life cycle of all species of human malaria parasites is characterized by an exogenous sexual phase (named sporogony), in which multiplication occurs in several species of knowlesi . Within Anopheles mosquito during a blood meal. A mosquito blood meal is, on average, 2 to 3 μL, and should contain at least one male and one female gametocyte to be infective.The sexual development of malaria parasite (sporogonic cycle) will be completed only when mature female and male gametocytes are ingested by a biologically suitable species of female Anopheles gambiae  one of the major malaria vector in Africa. They showed that individuals with higher skin microbial diversity and higher abundance of Pseudomonas and Variovorax spp. were less attractive to the mosquitoes and hypothesized an in-built defence system controlled by the Major Histocompatibility Complex genes.8Host location by the mosquito is mediated by physical  and chemical cues that play a role during orientation and landing. i.e. female) is released by the erythrocytes to became a macrogamete (within 5 minutes) whereas the male counterpart (microgametocyte) divides its nucleus more slowly (about 20 minutes) into eight flagellated microgametes responsible of the fertilization of macrogamete a process that takes one hour.Plasmodium life cycle. The sporozoites actively escape from the oocyst and only 25 % of those liberated by oocyst migrate through the hemocoelomic fluid to the acinal cells of salivary glands, where after about a day of residence, they became highly infective.Once in the midgut of the insect the macrogametocyte (Plasmodium (Laverania) falciparum  is the highly pathogenic and most deadly parasite causing malaria in humans. It was discovered in 1880 by Charles Alphonse Laveran, a French Army Surgeon, deployed in Constantine (Algeria) and originally named by himself Oscillaria malariae.P.falciparum a phenomenon that was subsequently explained by Maccallum.14P.falciparum malaria with 2.57 billion people living in area at risk for transmission of this infection.16Eighty-five countries are actually classified as endemic for P.falciparum clinical malaria is a difficult task complicated by several factors such as inadequate and incomplete national reporting systems and inaccurate diagnoses with possible overestimate disease rates. By using improved cartographic and epidemiological data sources combined with geostatistical space-time joint simulation framework, Hay and coworkers, estimated that in 2007 there were 451  million clinical cases of P.falciparum malaria.18The estimate of the burden of P.falciparum has been sequenced and published in 2002 being a major advance in the struggle against this lethal parasite.P.falciparum, 81.6 % have been shown to be conserved with those of P.vivax and only 334 genes (18.5%) were unique to the former species.P.vivax and P.knowlesi genomes, P.falciparum shows an higher content of A+T and despite having the smallest genome size it displays the highest content of simple sequence repeats (SSRs) that are supposed to be responsible of genome complexity responsible for rapid evolutionary adaptation  that is known to be encoded by about 60 members of the specific var gene family, is considered the principal virulence factor of P.falciparum because PfEMP1 proteins, expressed on the surface of infected erythrocytes, are able to mediate adhesion to both uninfected and host endothelial cells.19The whole genome of aptation .21 The pP.falciparum is a rapid process that is usually completed in five and half days with the production of a schizont, characterized by bizarre forms, containing a very high number of merozoites. Following the rupture of liver schizonts, each released merozoite invade an erythrocyte a complex process that requires a recognition between receptors (on erythrocyte) and ligands (on merozoite). In the case of P.falciparum, early studies identified as possible receptors for merozoite invasion sialic acid, glycophorin A, B and C.P.falciparum adhesin PfRh4, the complement receptor 1 (CR1) has been identified in 2010.basigin, an antigen of the Ok blood group, as the receptor essential for the erythrocyte invasion by P.falciparum.P.falciparum strain.27The exoerythrocytic schizogony of P.falciparum develop in the internal organs and do not appear until about eight to ten days after the start of parasitaemia. P.falciparum gametocytogenesis has been characterized through five morphologically distinct substages.P.falciparum gametocytes (stage I–IV) are sequestered away from circulation and only the mature crescent-shaped gametocytes (stage V) are released in peripheral blood where they finally become infectious to mosquitoes.29Erythrocytic schizogony is characterized by the appearance in the blood of young rings whereas the maturation stages are rarely seen in the peripheral blood. There is no enlargement of the infected erythrocytes during the development and the mature schizont usually contains 8–32 merozoites. The gametocytes of P.falciparum was considered to be a chimpanzee parasite, P.reichenowi, which was believed to have diverged from its human counterpart 5–7 Myears.Plasmodium DNA, using a single-genome amplification strategy, challenged this view.P.falciparum sequences constitute a single lineage nested within the G1 clade of gorilla parasites thus suggesting that human P.falciparum is of gorilla origin and not of chimpanzee.P.falciparum, once considered strictly human specific, can infect bonobos, chimpanzees and gorillas and thus these African apes might serve also as possible reservoir for the malignant form of human malaria.Laverania subgenus have been identified in just one year providing a new representation of the phylogeny with two groups. Group A that includes three species : P.GorA (that infects Gorillas), P.gaboni and P.billbrayi (both infecting chimpanzees) and group B that includes P.falciparum and P.reichenowi and P.GorB (that infects gorillas) and P.billcollinsi (that infects chimpanzees).P.falciparum was transferred by gorilla to human and on the basis of their analysis of 45 orthologous gene sequences, indicated that ape infections with P.falciparum might be a very recent phenomena.40Until the last year, the closest ancestor of Plasmodium vivax  is responsible of the so called “benign tertian fever” although the lack of life-threatening complications associated with this infection have been recently questioned by several reports.P.malariae in 1886 by Camillo Golgi who described the typical “tertian” and “quartan” fever paroxisms and confuted the Laveran’s postulate of the existence of a single malaria species.Haemamoeba vivax and H.malariae.P.vivax Salvador I strain was sequenced and published in 2008: with its 26.8-megabase (Mb) nuclear genome is higher than that of P.falciparum and displays chromosomes that are unique among human Plasmodium species with an isochore structure.Plasmodium genome sequenced to date  probably implicated in virulence/antigenicity of P.vivax.46 to date . A recenPlasmodium vivax is present throughout the tropics with low rate of infection in western and central sub-Saharan Africa. According to recent estimates 40% of the world’s population (2.6 billion people) is at risk of P.vivax transmission with between 130–435 million clinical episodes of vivax malaria each year.P.vivax has been, as in the case of P.falciparum, a debatable question with different hypotheses; the abundance of simian malaria species observed in Southeast Asia together with morphological and biological characteristics shared by P.vivax with macaque parasites where the arguments used to place the origin of P.vivax in Southeast Asia.i.e. the lack of the Duffy blood group antigen) among human population throughout sub-Saharan Africa has been indicated to argue an African origin for P.vivaxP.vivax was introduced into Homo sapiens in Asia by a species of Plasmodium parasitic to macaques.53P.vivax, once in the liver, differentiate either into early, primary tissue schizonts or into hypnozoites which are responsible for late relapse of the infection. The term “hypnozoite” was probably coined and adopted for malaria by Markus in 1978 whereas the biological proof of the existence of hypnozoites is the direct consequence of the work by Krotoski and coworkers.56Sporozoites of P.vivax malaria was described at the end of nineteen century by Thayer, Bignami and Manson as outlined in the recent elegant review by White.57The biological determinant that supports the active or dormant development pathway is still unknown although the relapsing nature of P.vivax all forms can be found in the peripheral blood and in most stages the appearance is larger that in the other species of human Plasmodia. It is also responsible of the enlargement of host cells thus increasing its deformability.i.e reticulocytes) an issue that seems to limit its reproductive capacity with level of parasitaemia that rarely exceed 2 % of circulating erythrocytes. The invasion of erythrocytes by P.vivax merozoites requires interaction with the Duffy antigen receptor for chemokines (DARC) with Duffy-negative individuals considered to be naturally resistant to this human malaria parasite.P.vivax malaria in those geographical areas. However, in recent years several investigators either in Africa and South America were able to demonstrate that P.vivax has evolved and adapted in a way that circumvent this pathway and it is able to invade erythrocytes also in individuals who lack the Duffy antigen on their red cells.49During the erythrocytic development of P.vivax and can be detected within 3 days after the first asexual parasites are observed.29Gametocytes production probably start with the first generation of merozoites of P.vivax is whether or not it exists as a single species or on the contrary different species or subspecies are represented.P.vivax comprises two distinct lineages with phenotypic difference with respect to their preferred mosquito vector and with a distinct chromosomal translocation.P.vivax Chesson, P.vivax Madagascar, St.Elizabeth, P.vivax North Korean, P.vivax Hibernans) are responsible of different presentation.P.vivax) usually cause a primary illness two weeks after mosquito inoculation with subsequent relapse after an interval of 7–10 months but with shortened subsequent inter-relapse intervals. On the contrary the P.vivax hibernans (distributed in northern Europe and Russia) presented a primary infection 8–10 months after inoculation. Finally, the P.vivax Chesson strain shows very frequent relapses and high relapse rate. The exact trigger for the activation of hypnozoites is not understood and various theories such external stresses, seasonal stimuli or mosquito bites, have been proposed.64One of the most intriguing and debatable issue regarding Anopheles mosquito, after fertilization the sexual cycle takes 8–10 days at 28 °C and 16 days at 20 °C whereas below 15 °C the completion of the sporogonic cycle is unlikely.1In the Plasmodium ovale  was discovered in 1922 by Stephens who observed it in the blood of an East african patient with malaria erythrocytes with oval shape and fimbriated edges and named the parasite P.ovale.P.ovale belong to 2 genetic haplotypes named classic and variant.P.ovale curtisi (classic type) and P.ovale wallikeri (variant type) in honour of 2 malariologists : Christofer Curtis (1939–2008) and David Walliker (1940–2007).P.ovale is distributed in sub-Saharan Africa, South-east Asia , Middle East, the Indian subcontinent, Papua New Guinea and Irian Jaya and East Timor in Indonesia ; it has never been reported from South America.P.ovale in Africa might exceeds 15 million cases annually.i.e. the interval between sporozoites inoculation and the first detection of parasites in peripheral blood) of P.ovale is between 12 and 20 days, with a median of 14,5 days.P.ovale infection probably as the consequence of restricted development in younger erythrocytes; in a study regarding 90 patients the maximum parasite levels ranged between 380 and 27,660/μL with a mean maximum parasite level of 6,944/μL.P.ovale on the infected erythrocytes are similar to those seen in P.vivax whereas the schizonts and gametocytes may resemble those of P.malariae.Stevens, 922 was dP.ovale is generally regarded as responsible of a relapsing infection originating from dormant (“hypnozoites”) exoerythrocytic stages in the liver.Anopheles mosquitoes infected with a Liberian strain of P.ovale.P.vivax misidentified as P.ovale.P.ovale infection, hypnozoites have never been demonstrated by biological experiments but in their paper failed to cite the work by Garnham.P.ovale has been described to be in the range of 17 to 255 days;P.ovale, a primary attack was observed after 53 months.76P.ovale a condition that raise the possibility that cross-species exchange might be more important than previously thought with the potential role of African great apes as reservoir for human malaria parasites.77Recently in a study conducted in Cameroon, Duval and coworkers found for the first time two chimpanzees infected by human Plasmodium malariae , responsible of the “quartan malaria”, is present worldwide in all major malaria-endemic regions but with a scattered distribution.P.malariae are most common in sub-Saharan Africa and southwest Pacific and less frequently encountered in Asia, Middle East, Central and South America. As for P.vivax also for P.malariae the relationship between the life cycle of development (respectively 48- and 72-hours) and the periodicity of the fever paroxysm were elegantly explained by Camillo Golgi in 1886 although the two parasites were identified as separate species by Grassi and Feletti.44Anopheles mosquito (15 days) and in human . P.malariae is considered to be the precursor of P.brasilianum a parasite that infects New World monkeys and has naturally adapted to it;Plasmodia are able to infect either humans and monkeys. P.malariae is responsible of low grade parasitaemia, rarely exceeding 30,000 parasites per microliter, probably as a consequence of the low number of merozoites produced per erythrocytic cycle together with the 72-hour developmental cycle and the preference to infect older erythrocytes. The pre-patent period for P.malariae is extremely variable with a range of 16 to 59 days. No quiescent liver stage forms have been identified for P.malariae but this parasite is able to persist in the blood with low level parasitaemia for extremely long periods and perhaps for the entire life of the human host causing recrudescence even after more than 30–40 years or longer.P.malariae infection was linked to nephrotic syndrome in Nigerian children about 50 years ago, and it is believed to be caused by immune complex deposition on the basement membrane.P.vivax ; a characteristic feature of P.malariae is the appearance of band form with the trophozoite that stretch across the entire width of the cell. The mature schizont has an average of 8 merozoites that are sometimes arranged symmetrically around the centre with a daisy appearance.Anopheles mosquito takes 30–35 days at 20°C but may be as short as 14 days at 28°C.The parasite is characterized by a slow development either in the mosquito 5 days anmosquito 5 days anmosquito 5 days anPlasmodium knowlesi (Sinton and Mulligan 1932) is a simian plasmodium that was probably first described by the Italian malariologist Giuseppe Franchini in the blood of Macaca fascicularis. P.knowlesi in honour and acknowledging the original work of Dr Knowles.P.knowlesi was employed in the treatment of general paresis of insane (i.e. neurosyphilis) until 1955 when it was finally abandoned due to the increased virulence of the organisms after multiple passages in humans.P.knowlesi from P.malariae and to identify it as an important cause of human malaria not only in the Peninsular Malaysian Borneo but also in other parts of South-east Asia.P.knowlesi is more closely related with P.vivax than other humans Plasmodia and in a similar way the process of merozoites invasion of erythrocytes requires the interaction of Duffy-bindings proteins (DBP) with the Duffy antigen receptor for chemokines (DARC).P.vivax exists such as absence of a dormant liver stage, host blood cell preference and length of asexual cycle. The genome of P.knowlesi has been sequenced and described showing important differences with those of P.falciparum and P.vivax  and pig-tailed macaques (M.nemestrina) are the main natural hosts of P.knowlesi.P.falciparum whereas during subsequent stage of maturation the intra-erythrocyte parasites resemble the band forms observed in P.malariae infection.P.falciparum malaria have been described for P.knowlesi with a possible lethal outcome.95Long-tailed (Plasmodia: P.falciparum and P.vivax. Moreover, the introduction of molecular techniques has greatly improved the identification of malaria parasites at the species level. In this regard, the availability of species-specific polymerase chain reaction allowed Balbir Singh and his coworkers to recognize the significant role of P.knowlesi, a natural plasmodia of macaques, as a cause of human malaria in the Southeast Asia. This discovery, together with the possibility to use non-invasive methods to supply animal DNA, renewed the interest to study malaria parasite of apes increasing the membership clade of P.falciparum and P.ovale and finally showing that P.falciparum is diverse and not human-host restricted.Our knowledge of the biology of human malaria parasites has dramatically improved in the last few years thanks to the whole sequencing of DNA of the two most important human


We examined the relationship between insecticide-treated mosquito nets (ITNs), malaria parasite infection, and severe anemia prevalence in children in Luangwa District, Zambia, an area with near-universal ITN coverage, at the end of the 2008 and 2010 malaria transmission seasons. Malaria parasite infection prevalence among children < 5 years old was 9.7%  over both survey years. Prevalence of severe anemia among children 6–59 months old was 6.9% (95% CI = 5.4–8.5%) over both survey years. Within this context of near-universal ITN coverage, we were unable to detect a significant association between malaria parasite or severe anemia prevalence and ITNs (possession and use). In addition to maintaining universal ITN coverage, it will be essential for the malaria control program to achieve high ITN use and laboratory diagnosis and treatment of all fevers among all age groups to further reduce the malaria burden in this area. Plasmodium falciparum endemic settings.1Community randomized controlled trials have shown insecticide-treated mosquito nets (ITNs) to be an effective strategy for preventing malaria morbidity and all causes of mortality among children in There has recently been a call for universal coverage of ITNs across malaria endemic countries in Africa, defined in the Global Malaria Action Plan as 100% coverage of ITNs among at-risk populations.This paper examines the relationship between ITN possession and use and the outcomes of malaria parasite infection and severe anemia in children in Luangwa District, Zambia, in 2008 and 2010, an area with near-universal coverage of ITNs. The objectives of this paper are to (1) report malaria parasite infection prevalence and severe anemia prevalence in children by ITN possession and use exposure and (2) determine whether ITN possession and use exposure are associated with reduced risk of malaria parasite infection or severe anemia among children within this context while controlling for potential confounding factors.Luangwa district is located in Eastern Lusaka Province and is situated along the Luangwa River at the confluence with the Zambezi River. The entire district, with a total population of approximately 34,000 people, is classified as rural by the Zambian Central Statistics Office. The only town is Luangwa Boma, located at the southernmost end of the district. Villages are primarily situated along the Luangwa River, with fishing and small-scale agriculture being the primary livelihoods of the population. The district is served by nine rural health centers, two of which have both inpatient and outpatient services.The district has a history of high malaria transmission, with a marked seasonal pattern after the rains from December to April. Luangwa district was one of the first districts in Zambia to be targeted for the scale-up of ITNs, serving as the pilot for mass distribution in 2005. Approximately 16,000 ITNs, of which 14,000 were long-lasting ITNs (LLINs), were distributed through mass free distribution and resale at heavily subsidized prices from 2005 to 2006 (LLINs and ITNs are henceforth referred to here as ITNs). As a result, there were enough ITNs to achieve a ratio of three nets per household in the district by the end of 2006. No indoor residual spraying had been done in Luangwa District. Rapid diagnostic tests (RDTs) have been used since 2006 throughout the public sector for malaria diagnosis, with artemisinin-combination therapy (ACT)  used as the first-line treatment of malaria since 2004.As described elsewhere,The Luangwa baseline survey followed the standardized malaria indicator survey (MIS) protocol that was used for the 2008 and 2010 Zambia National MIS. Blood slides were sought for all children < 5 years old within selected households. Malaria blood slides were read at the National Malaria Control Center, Ministry of Health, by two trained laboratory technicians using standard methods.http://www.rollbackmalaria.org/toolbox/tool_MISToolkit.html). ITN possession and use among all household residents were ascertained with a net roster and a registry of all household residents. Exposure of a child to household protection by an ITN (ITN possession) is defined in the following two ways: (1) the proportion of sampled children living in a household with more than or equal to one ITN, and (2) the proportion of sampled children living in households with at least one ITN per two residents , which provides ideal coverage for a household. Exposure of a child to personal protection by an ITN  is defined in the following two ways: (1) the proportion of sampled children living in a household reported to have slept under an ITN the night before the survey, and (2) the proportion of children living in a household where any ITN was used by any household member the night before the survey. ITN possession analyses are among all children providing a blood sample . ITN use analyses are among all children providing a blood sample as well as among children providing a blood sample living in households with greater than or equal to one ITN to assess the effect of personal protection independent of having access to an ITN in the household (N = 969).Standardized household and women's questionnaires were used to collect information in accordance with the MIS protocol  of Tulane University, the University of Zambia, and the Partnership for Appropriate Technology in Health (PATH).2 statistics were used to test for differences in severe anemia and parasite prevalence by ITN possession and use variables and between anemia and parasite prevalence. Logistic regressions were used to test whether ITN possession and use predicted parasite prevalence in children < 5 years old or severe anemia among children 6–59 months while controlling for age and sex of the child, location in the district, and household wealth; a total of four regressions models were performed. Wald statistics were used to identify variable significance in the models, with the probability of committing a type-1 error (α) set at 0.05. Standard errors for all point estimates and regression parameters were empirically estimated to account for correlated data at the household level.Stata 10.1  was used to perform all statistical analyses. Data from the 2008 and 2010 surveys were pooled after determining that the association between ITN exposure (household possession and use) and the primary outcomes were homogeneous across survey rounds. ArcGIS was used to divide Luangwa District into north (≥ 50 km from the southern border) versus south (< 50 km from the southern border); in this analysis, the distance division is based on the parasite prevalence described elsewhere and is intended to capture variation in ecological conditions,N = 483 in 2008; N = 431 in 2010). The total number of eligible children in these households was 1,440, of which consent from a parent or guardian for their child to provide a blood sample was received for 1,190 children, representing a non-response rate of 15.2%.Of a total of 1,595 households sampled in 2008 and 2010, 914 households contained children < 5 years old . No other significant differences in ITN household possession were detected.Among all children < 5 years old in the samples, 81.1%  slept in a house possessing ≥ 1 ITN. Among children < 5 years old that provided a blood sample for microscopy diagnosis, 81.4% (95% CI = 78.5–84.3%) slept in a house with ≥ 1 ITN . ITN hou2 = 44.9; P < 0.01); no other significant differences in intrahousehold access to ITNs were detected.Among children providing a blood sample for microscopy diagnosis, 19.1% (95% CI = 16.8–21.8%) slept in a house with ≥ 1 ITN per two occupants . The proITN use the previous night among children in ITN-owning households increased from just over one-half  in 2008 to 85.5% (95% CI = 81.2–87.8%) in 2010. The combined proportion of children sleeping under an ITN the previous night was 68.0% (95% CI = 64.6–71.4) . Among cAt the end of the malaria transmission season, the malaria parasite infection prevalence was 7.0% (95% CI = 5.0–9.0%) among children < 5 years old in 2008 and 12.6% (95% CI = 9.9–15.3%) in 2010. The overall parasite infection prevalence was 9.7% (95% CI = 8.0–11.4%) across both years. Bivariate analyses showed no difference in parasite prevalence between children in households with or without an ITN or between children who used an ITN or did not use an ITN the previous night .At the end of the malaria transmission season, the prevalence of severe anemia among children 6–59 months was 5.8% (95% CI = 3.8–7.9%) in 2008, 8.0% (95% CI = 5.7–10.3%) in 2010, and 6.9% (95% CI = 5.4–8.5%) over both years. Bivariate analyses showed no difference in severe anemia between children in households with or without an ITN or between those that used or did not use an ITN the previous night .2 = 73.9; P < 0.001) across both years. Overall, 38.5% of children with severe anemia also had a malaria parasite infection, whereas 26.3% of children with a malaria parasite infection also had severe anemia.There was a strong positive correlation between the prevalence of malaria parasite infection and severe anemia , controlling for child age and sex, location of household in the district, survey year, and household wealth, suggest that neither ITN possession nor ITN use was significantly associated with malaria parasite infection prevalence or severe anemia in children in this context  and 3. CAdditional regressions were performed using ITN to occupant ratio as a second test variable and proxy for ITN ownership and whether anyone in the house slept under an ITN as a test variable and proxy for ITN use (results not shown); results of these additional regressions were similar to the results that we present here and also showed that ITN possession and use were not significantly associated with malaria parasite infection prevalence or severe anemia in this context.Results from the 2008 and 2010 household surveys show that Luangwa District, Zambia, has achieved near-universal coverage of ITNs, with > 80% of households possessing ≥ 1 ITN. The majority of children in the sample (58.4%) lived in households with adequate intra-household access to an ITN, defined as ≥ 1 ITN for every two household occupants. ITN use among those in ITN-owning households increased from just over one-half (52.8%) in 2008 to over three-quarters (85.5%) in 2010.At the end of the malaria transmission seasons in 2008 and 2010, the prevalence of children with a malaria parasite infection in Luangwa District was only 9.7%, whereas the prevalence of severe anemia among these children aged 6–59 months was 6.9%.Results of the 2010 Zambia National MIS are not yet available. However, the prevalence of malaria parasite infection in Luangwa in 2008 (7.0%), with near-universal coverage of ITNs, was almost one-half that of the rest of rural Zambia during the malaria transmission season in 2008, which had a parasite infection prevalence of 12.4% and ITN household possession of 63.9%.102 = 73.9; P < 0.001). This suggests that, even with high ITN coverage in areas of once stable P. falciparum transmission, chronic malaria parasite infections remain a significant risk factor for severe anemia in children 6–59 months.Although one might expect that the association of severe anemia in children with a malaria parasite infection to decrease in such an area of near-universal coverage of ITNs, there remains a strong association among these conditions within this area of intervention-suppressed transmission  reduction in malaria parasite prevalence among children in ITN-possessing households compared with those in households without an ITN. This is less than one-third of the 17% reduction detected by the trials, which compared children in villages with and without ITNs.12Our analyses were also unable to detect a significant protective effect of sleeping under an ITN the previous night against malaria parasite infection or severe anemia prevalence among all children  and among those within ITN-owning households . We identified four studies that published data on the association between ITN use and malaria parasite infection prevalence under program conditions. Reductions in malaria parasite infection prevalence among children who slept versus did not sleep under ITNs ranged from 39% to 63%; all were significant.Within this area of near-universal ITN coverage, we hypothesize that our null results are explained in part by the community effect of ITNs, where children unprotected by ITNs benefited from an overall suppression of transmission.This study has several important limitations. First, this study was slightly underpowered to detect such small differences in the prevalence of malaria parasite infection and severe anemia by ITN possession and use. Second, although there is anecdotal evidence that malaria transmission was once high within this area of Zambia, we could not identify published data that quantified the level of malaria transmission before the ITN scale-up in 2005; thus, an assessment of the relative decline in malaria transmission associated with the ITN scale-up was precluded. Lastly, we did not collect parasite or hemoglobin data from the population older than 4 years and therefore, were unable to assess the relationship between ITNs and malaria parasite infections among this population.Our results suggest that, with near-universal coverage of ITNs, malaria parasite infection prevalence remains at 10% in this area of Zambia. Such prevalence represents a substantial pool of parasites that will sustain transmission. In addition to maintaining universal ITN coverage, it will be essential for the malaria control program to achieve high ITN use and laboratory diagnosis and treatment of all fevers among all age groups to further reduce the malaria burden in this area.


Plasmodium spp. antibodies, while these individuals were negative by microscopy. The antibody test described here is useful to elucidate malaria exposure in microscopy-negative travellers from endemic countries.In this study, we show that 40.29% of travellers with a possible history of malaria exposure were positive for anti- Plasmodium, which is transmitted between humans by the bite of infected female Anopheles mosquitoes. These parasites have a complex life cycle, both in the invertebrate vector and vertebrate hosts. In the human body, parasites multiply in hepatocytes, and then invade red blood cells (RBCs), initiating blood stage infection, which corresponds to the symptomatic period of the disease [Malaria is an infectious disease caused by a protozoan parasite of the genus  disease .Malaria remains one of the most serious public health problems not only in endemic countries, where 2 billion people (approximately 40% of the world's population) are at risk of contracting the disease, but also in nonendemic areas, where the increasing number of imported malaria cases is worrying , 4. In PPlasmodium that are capable of infecting humans, P. falciparum, P. ovale, P. vivax, P. malariae, and P. knowlesi, specific antibodies are produced one or two weeks after the initial infection and persist for three to six months after parasite clearance [Following infection with any of the five species of learance . These alearance , 7.Plasmodium spp. antibodies in serum samples from travellers with possible clinical signals and symptoms of malaria. Using an ELISA-based commercial immunoassay kit to measure antimalarial antibodies, we determined the raw serological profile of these individuals. Additionally, we compare the latter serological profile with the gold-standard laboratory diagnosis, based on direct microscopy.Thus, in the present study, we aim to evaluate the prevalence and the level of anti-Plasmodium spp. travelled back to Portugal from malaria-endemic regions of Africa, Brazil, Ecuador, India, Indonesia, Thailand, and Haiti, either as residents or tourists, and most of them are adults. Subjects for this study were actively recruited after being seen for symptoms of malaria at the Clinical Unit for Tropical Diseases .The population for this study consisted of 335 individuals with possible clinical history of malaria and 23 healthy individuals . All of the 435 subjects who have had potential exposure to n = 45); subjects potentially exposed to the parasite but displayed negative microscopy were categorized into group 2 (n = 290); and finally, healthy naïve subjects were categorized into group 3 (n = 23).Following microscopic examination of Giemsa-stained blood films, subjects who were potentially exposed to the parasite and had concomitant positive microscopy were categorized into group 1 , and two blood smears were prepared (thick and thin blood films). The haematological data was obtained from an automatic Coulter Sysmex K-1000 analyzer (Emílio de Azevedo Campos). Both blood films were stained by Giemsa's staining method and were observed on an optical microscope. The thick blood film was used to attain a qualitative diagnosis for malarial infection, and the thin blood film was used to identify the of blood . SamplesPlasmodium spp.  antibodies were analysed from serum samples collected from all individuals (n = 358). The Newmarket Laboratories Malaria EIA kit  was used in this study for evaluating the prevalence of total antimalarial antibodies in the depicted groups of subjects. This system is based on the binding of anti-Plasmodium spp. antibodies  by use of four recombinant antigens that detect antigens from P. falciparum, P. ovale, P. vivax, and P. malariae. The test was performed as recommended by the manufacturer, as follows. 50 μL of individual undiluted sera samples were added in each single well. For each assay plate, 50 μL of positive and negative controls were also dispensed. The negative control was tested in triplicate and the positive control in duplicates. After mixing on a plate shaker for 30 seconds, the plate was covered and incubated for 30 minutes at 37°C before being washed 5 times with wash buffer. 50 μL of diluted horseradish peroxidase-conjugated antibody were added to each well, and the plates were incubated for 30 minutes at 37°C. The wells were washed again 5 times, and 50 μL of substrate solution were added to each well. The plate was then covered and incubated in the dark for 30 minutes. Finally, 50 μL of 0.5 M sulphuric acid were added to each well to stop the reaction, and the absorbance was read at 450 nm, with a reference wavelength of 620 nm. The antibody index was obtained by dividing the OD value of each sample (at 490 nm) by the cut-off value, which was calculated as the mean of the negative control value plus 0.100, according to the manufacturer.Total anti-Plasmodium spp. antibodies. The Malaria EIA kit is based on presence of antibodies  reactive to four recombinant antigens to detect P. falciparum, P. ovale, P. vivax, and P. malariae. The cut-off value was calculated as the mean of the negative control value plus 0.100. To validate the assay, the optical density (OD) of each negative control should be lower or equal to 0.080, and the OD of each positive control should be greater than or equal to 1.000. The antibody index was obtained by dividing the OD value of each sample (at 490 nm) by the cut-off value. The samples with indexes lesser than or equal to 1 were considered negative. Moreover, the samples with an antibody index greater than 1 were considered positive for the presence of antimalarial antibodies. This immunoassay does not distinguish between IgG, IgM, and IgA antibodies, or between antibodies to P. falciparum, P. vivax, P. ovale, and P. malariae.After establishing the study groups, a commercial immunoassay Malaria EIA kit  was used to determine total anti-Plasmodium spp.) were determined in patients with possible clinical history of malaria. These patients were actively recruited in the Clinical Unit for Tropical Diseases . The malaria antibody EIA  is based on binding of anti-Plasmodium antibodies present in a serum sample to antigens immobilized on a solid phase. The antigens are four recombinant types specific for P. falciparum with cross-reactivity for P. ovale and P. malariae and one specific antigen for P. vivax. The test detects total immunoglobulin (Ig) antibodies against P. falciparum and P. vivax and shows 80% cross-reactivity with P. ovale and 67% with P. malariae. The malaria antibody EIA was performed as recommended by the manufacturer.In this study, the prevalence and level of total antimalarial antibodies , compared with a third group consisting of nonexposed individuals.Plasmodium spp. and microscopically positive for malaria), group no. 2 , and group no. 3 . From all individuals potentially exposed to Plasmodium spp. (n = 335), 13.43% (n = 45) had positive blood films. On the other hand, 40.29% (n = 135) had positive ELISA serological reactions to Plasmodium spp. (ium spp. . As expen = 45), 80% (n = 36) showed some level of antimalarial antibodies (n = 290), 34.1% (n = 127) had antimalarial antibodies and 65.9% (n = 191) had no serological reaction to Plasmodium spp.  and group no. 2 (negative blood film) are detailed in Figures tibodies . Furtherium spp. .The use of immunofluorescence to determine the prevalence of antimalarial antibodies has been used to estimate malaria endemicity , 10, butHowever, antibody detection is not a substitute for blood film examination in the diagnosis of an acute attack of malaria, and it is mainly used in the screening of prospective blood donors to avoid transfusion-transmitted malaria , 18. A bPlasmodium spp. antibodies when titrated by a commercial immunoassay Malaria EIA kit, while the remaining 9 (20%) subjects had no significant levels of anti-Plasmodium spp. antibodies, when compared to the control group (group no. 3). These 9 individuals might be at the initial phase of infection or at an acute phase of the disease, which would explain why no antibodies have been detected in their serum, while they had microscopically positive slides or a false positive microscopically. In any of these cases, antibody levels are too low to be detected by this method or have not been yet produced. On the other hand, 200 out of 335 (66%) subjects did not show significant levels of anti-Plasmodium spp. antibodies with the commercial kit used in this study. Moreover, 135 (40.29%) subjects had significant levels of anti-Plasmodium spp. antibodies, when compared to the control group (group no. 3). In our study, the presence of antibodies in 34.1% of the subjects with negative microscopy confirms the microscopy limitations, and that an additional antibody test is an asset to know about contact with malaria parasite in travellers from endemic countries. Furthermore, it is often difficult to distinguish between species, especially if the patient has already been treated or has done chemoprophylaxis.Thirty-six out of 45 (80%) subjects from group 1 displayed levels of anti-This work has been complemented with new studies that evaluated the serological reactivity in these patients and were identified major antigenic proteins that are responsible for the production of the antibodies detected in this study .Plasmodium spp. antibodies, while 40.29% of these individuals were negative by microscopy. The antibody test described here is useful to elucidate malaria exposure in microscopy-negative travellers from endemic countries.This study showed that 40.29% of travellers with a possible history of malaria exposure were positive for anti-


Plasmodium falciparum in Nigeria. Using clinical criteria, 217 children were recruited into the study out of which 106 (48.8%) were positive by TFM, 84 (38.7%) by RDT, and 125 (57.6%) by PCR. Using a composite reference method generated from the three diagnostic methods, 71 (32.7%) patients were found to be truly infected and 90 (41.5%) truly uninfected, while 56 (25.8%) were misidentified as infected or noninfected. When each of the 3 diagnostic methods was compared with the composite reference, PCR had sensitivity of 97.3%, specificity of 62.5%, positive predictive value (PPV) of 56.8%, and negative predictive value (NPV) of 97.8%; microscopy had sensitivity of 77.2%, specificity of 72%, PPV of 66.9%, and NPV of 81.1%, while RDT had sensitivity of 62.3%, specificity of 87.4%, PPV of 67.7%, and NPV of 84.5%. PCR test performed best among the three methods followed by TFM and RDT in that order. The result of this study shows that clinical diagnosis cannot be relied upon for accurate diagnosis of P. falciparum in endemic areas.This study compares the performance of clinical diagnosis and three laboratory diagnostic methods (thick film microscopy (TFM), rapid diagnostic test (RDT), and polymerase chain reaction (PCR)) for the diagnosis of  Malaria remains an important public health concern in countries where transmission occurs regularly as well as in areas where transmission has been largely controlled or eliminated. It was estimated that there are 39 million children under 5 years of age who experience 33.7 million malaria episodes and 152,000 childhood deaths from malaria each year in areas suitable for seasonal malaria chemoprevention . μL (depending on the microscopist expertise) [Plasmodium falciparum and Parasite-Specific Lactate Dehydrogenase (pLDH) or Plasmodium aldolase from the parasite glycolytic pathway found in all species [μL of blood [In most endemic countries malaria diagnosis depends mainly on clinical evidence and in some cases thick film microscopy (TFM) and rapid diagnostic technique (RDT) may be used for laboratory confirmation. Microscopy remains the gold standard for malaria diagnosis and it is less costly with a threshold sensitivity of 5 to 50 parasite/pertise) . The majpertise) . RDT, anpertise) . Preferr species . However species . Polymerof blood . PCR is of blood . It is aof blood . HoweverClinical diagnosis is imprecise but remains the basis for therapeutic care for the majority of febrile patients in malaria endemic areas, where laboratory support is often out of reach. Clinical diagnosis also referred to as presumptive diagnosis is the least expensive and most commonly used method and is the basis for self-treatment in endemic countries. Overlap of malaria symptoms with other tropical diseases like typhoid fever, respiratory tract infections and viral infections impairs the specificity of presumptive diagnosis thereby encouraging indiscriminate use of antimalarials in endemic areas. Accuracy of clinical diagnosis varies with the level of endemicity, malaria season, and age group. Therefore no single clinical algorithm can be regarded as a universal predictor .P. falciparum malaria in Nigeria. This paper reports the comparative performance of clinical diagnosis, TFM, RDT, and PCR in the diagnosis of The study was carried out in Osogbo located in the Western part of Nigeria. Osogbo is the state capital of Osun State, Nigeria, and it represents a typical urban setting in Nigeria. Malaria is present throughout the year with a marked increase during the raining season. Patients (ages 4 months to 20 years) who were clinically diagnosed for malaria at the outpatient departments of General Hospital Asubiaro and LAUTECH Health Centre in Osogbo were recruited into the study. Exclusion criteria used were complete absence of malaria symptoms and unwillingness to participate. All the patients that were clinically diagnosed were subsequently confirmed using TFM, RDT, and PCR before treatment. Ethical approval was obtained from the ethical committee of Osun State Hospital Management Board, Osogbo.Clinical diagnosis based on fever (temperature ≥ 37.5°C) and/or history of fever was carried out by physicians at the outpatient departments of the hospitals. Other symptoms considered for clinical diagnosis include headache, joint pains, body weakness, cough, diarrhea, loss of appetite/refusal of feeds, abdominal pain, and generalized body weakness.μL of blood using Paracheck  according to manufacturer's instruction. A drop of blood was used for microscopic examination of malaria parasites using thick films method stained with 5% Giemsa for 30 minutes. Parasites were counted against 200 white blood cells (WBCs) from the thick film. The parasite density was obtained by assuming a total WBC count of 8000/mL and 4.5 million RBC/mL and at least 200 fields were examined before being taken as a negative result [5 mL of blood was collected aseptically from antecubital vein of consenting febrile patients, into EDTA bottle. RDT was performed on about 5 e result . μL of blood was dotted on Whatman 3 mm filter paper and air-dried at room temperature for PCR. Parasite genomic DNA was extracted from blood samples collected on filter paper using methanol extraction method as previously described [P. falciparum stevor gene. The PCR reaction involves a primary and nested reaction to enhance specificity. Primary amplification was performed with reaction mixture of 25 μL containing 2.5 μL 10x reaction buffer, 5 μL of Magnesium chloride, 0.75 μL of each primers , 0.2 μL of DNTPs, 9.05 μL of water, 0.25 μL of Taq polymerase, and 5 μL of DNA extract. The PCR programme was as follows: 93°C for 3 minutes, 22 cycles of 30 seconds at 93°C, 50 sec at 50°C, and 30 sec at 72° and final extension period of 3 minutes at 72°C. 2.0 μL of the first PCR product was used in the second round amplification which was performed with a reaction mixture of 25 μL containing 2.5 μL 10x reaction buffer, 2.5 μL of magnesium chloride, 0.4 μL of each DNTPs, 0.25 μL of Taq polymerase, 1.0 μL of each primers , and 15.35 μL of water. DNA extracted from FCR P. falciparum laboratory adapted strain was used as positive control and water as negative control. PCR products were subjected to electrophoresis on 1.5% agarose gels and visualized using Syngene gel documentation system  after staining with ethidium bromide. The primer sequences for the stevor PCR are as previously described [10 escribed . PCR wasData obtained was analyzed using SPSS package version 16.0. The sensitivity, specificity, and predictive values of each of the three test methods were calculated by comparing to a composite reference gold standard generated from the three methods. The composite reference method was defined as a method that is positive for malaria parasites by all the three methods  and also negative for malaria parasites by all the three methods. This gives the method 100% hypothetical sensitivity, specificity, and positive and negative predictive values. The sensitivity, specificity, and predictive values of each of the 3 methods were then calculated using the formulasP = 0.0005) when the prevalence of 3 methods  was compared  for the detection of malaria parasites in Nigeria. A total of 217 individuals clinically diagnosed for malaria were recruited into the study. Of these, 103 were males and 114 were with a male to female ratio of 0.9. The mean age of the patients was 8 years ± 3.04 and the mean axillary temperature was 38.2°C ± 0.96. The general characteristics of the patients are shown in compared . P. falciparum 90 (41.5%) truly uninfected while 56 (25.8%) were misidentified as infected or noninfected by the three methods. When each of the 3 diagnostic methods was compared with the composite reference method, PCR had sensitivity of 97.3%, specificity of 62.5%, positive predictive value (PPV) of 56.8%, and negative predictive value (NPV) of 97.8%; microscopy had sensitivity of 77.2%, specificity of 72%, PPV of 66.9%, and NPV of 81.1%, while RDT had sensitivity of 62.3%, specificity of 87.4%, PPV of 67.7%, and NPV of 84.5% (Using a composite reference (gold standard) method generated from the three diagnostic methods, only 71 (32.7%) patients were found to be truly infected, with of 84.5% .Correlation of RDT and PCR to parasite density observed by microscopy is shown in  P. falciparum in a malaria endemic area. Each of these methods has particular attributes that stands them out in different settings. Clinical diagnosis, for instance, is commonly used because it is cheap and allows for prompt treatment of the patient [μL (0.002% parasitemia) and of giving rapid results (15 to 20 min) [μL of blood [This study provides a dataset for judging the performance of clinical diagnosis against TFM, RDT, and PCR for the detection of patient . Nonspec patient . RDTs ar 20 min) . They ar 20 min) . The maiof blood ; howeverOur results show that the continuous practice of using clinical diagnosis as the basis for antimalarial treatment in endemic area is by far not an effective diagnostic method in our study area. Out of the 217 (100%) patients that were clinically diagnosed for malaria, 104 (49.8%), 83 (38.2%), and 123 56.7%) were positive by TFM, RDT, and PCR, respectively. Invariably irrespective of the laboratory method, about half of the patients who were diagnosed as having malaria through clinical diagnosis (syndrome approach) and who should have received antimalarial turned out to be parasite-negative. There is therefore an urgent need to review the clinical diagnosis procedure. Although it may be argued that in some cases especially in children, promptness of malaria treatments reduces the progression of simple malaria to severe malaria, which still encourages syndromic approach to malaria diagnosis. Nevertheless malaria over diagnosis is still a major public health problem in Africa with studies suggesting between 50% and 99% of those prescribed antimalarial to be test negatives depending on endemicity of the clinical setting 6.7% were, 19. TheμL and have insufficient accuracy [In this study routine microscopic examination of Giemsa-stained blood smears which is considered as the gold standard for malaria diagnosis had a sensitivity of 77.2% and was able to detect more parasites than the RDT (sensitivity 62.3%). Though the specificity of microscopy (72%) was not as high as that of RDT 87.4%); nevertheless, it has high sensitivity, possibility for quantification of parasitemia, and easy handling which is a good advantage. Detection of parasites depends on many factors including the amount of blood processed and the competence of the microscopist, among others . Also th7.4%; nevaccuracy . RDTs haμL and identification to the species level make this an excellent technique against which to compare the sensitivity and specificity of other nonmolecular methods [Different PCR based methods have been constantly shown to be powerful tools for malaria diagnosis with better sensitivity than conventional microscopy and antigen-based diagnostic tests , 22. Mos methods .Greater percentage of children presented at general outpatient department of the hospital in our study with fever were diagnosed for malaria . Available records also show that at least 50% of the population of Nigeria suffer from at least one episode of malaria each year accounting for over 45% of all out-patient visits . The impIn conclusion our study revealed the need for complete shift from symptom-based diagnosis to parasite-based diagnosis. This can bring significant improvement to tropical fever management and reduce drug wastage and also help to curtail development of malaria drug resistance.


Pregnant women remain are at an increased risk of malaria with primigravidae being at the highest risk. Genetic polymorphism of the Fc receptor IIa for immunologlobulin (Ig) G (FcγRIIa) determines IgG subclass binding. Protection against pregnancy-associated malaria (PAM) is associated with the production of IgG specific for apical membrane antigen-1 (AMA-1). The present study was undertaken to examine the relationship between specific IgG/IgG subclasses and malaria infection. The second aim of the study is to examine the association between FcγRIIa R/H131 polymorphism in correlation with specific anti-malarial IgG antibodies of AMA-1 distribution and asymptomatic malaria infection among Saudi women living in the southern part of Saudi Arabia.Plasmodium falciparum malaria infection were consecutively enrolled onto the study. These pregnant women were asymptomatic and attending routine antenatal clinics. The levels of plasma antibodies (IgG and subclasses AMA-1) were measured using indirect enzyme-linked immunosorbent assays (ELISA). Genotyping of FcγRIIa-R/H131 dimorphism was performed using gene-specific polymerase chain reaction (PCR) amplification with allele-specific restriction enzyme digestion (BstU1) of the PCR product.One hundred and twenty pregnant women living in an area of meso-endemic P value <0.001, respectively). The FcγRIIa-R/R131 genotype and R131 were found to be statistically significantly more prevalent in the ASM group when compared to the MFC group . However, the H/H131 genotype showed statistically significant association with MFC .A total of sixty-two (52%) pregnant women was diagnosed with asymptomatic malarial infection (ASM) compared with 58 (48%) malaria free controls (MFC). In the ASM group, there were high levels of anti-malarial IgG1 and IgG3, when compared to MFC (The study revealed that the ASM patients had higher anti-malarial IgG and IgG subclasses antibody levels when compared to the MFC. The FcγRIIa-R/R131 genotype and R131 allele were found to be statistically prevalent in the ASM when compared to the MFC group. The individuals carrying H/H131 were consistently associated with higher levels of anti-malarial IgG subclasses. Plasmodium falciparum malaria is largely a childhood disease due to the gradual achievement of protective immunity. Each year, more than 125 million pregnant women are at risk of malaria infection with devastating consequences for the newborn and first and second time mothers     Table . For they) Table . The mosP. falciparum parasite density. Interestingly, there was statistically significant association between higher levels of IgG, cytophilic IgG1 and IgG3 subclass antibodies and a reduction in parasite density . The OR for IgG = 0.13; 95% CI: (0.02- 0.71) and P value = 0.005. The OR for IgG1 = 0.13; 95% CI: (0.03- 0.59) and P value = 0.008]. The OR for IgG3 = 0.5; 95% CI: (0.14- 1.74) and P value = 0.034]  , P value= 0.001 and adjusted OR= 5.11; 95% CI (1.74- 15.00), P value= 0.003) , P value = 0.03] , OR 3.31, 95% CI (1.59–6.56), P <0.001 (Table All subjects within the two study groups were genotyped for the G494A single nucleotide polymorphism (R131H) in the FcγRIIa gene (rs1801274). Both allele and genotype frequencies were found to be in Hardy-Weinberg equilibrium  test showed that IgG3 antibodies specific to AMA-1 malarial antigen were associated with individuals carrying the R/R131 genotype than H/H131 genotype in the ASM group    Table . High leP. falciparum in pregnant Saudi Women. This report investigated the relationship between specific IgG, IgG subclasses (AMA-1) antibodies, and FcγRIIa R/H131 polymorphism in association with asymptomatic malaria infection among Saudi women living in southern KSA. The risk of maternal infection was highest during the first and second trimester. One possible explanation may be that CD4+ and CD25+ regulatory T cells, which increase during the third trimester, modulate the immune response [+ and CD25+ regulatory T cells gradually returns to normal level after pregnancy, thus reflecting the pattern of increased incidence of malaria during the first and second trimesters [Despite the importance of malaria in pregnancy, this may be the first report on the immunology of response . The levimesters . Specifiimesters .et al. reported the first trimester as the main risk period for malaria infection [When assessing the relationship between specific IgG, IgG subclasses (AMA-1) antibodies and pregnancy duration, it appears that there is a significant difference in IgG and IgG2 between the study groups and the duration of pregnancy. Furthermore, the levels of anti-malarial IgG and IgG subclasses were higher in the third trimester than the second and first trimesters. This finding may offer an explanation to the lower prevalence of malaria infection in the third trimester when compared to the first and second. In support of this finding, a previous study in Mali by Dicko nfection . Furthernfection .The current study showed accumulative association of high levels of IgG and IgG subclass antibodies with ASM patients. In support of this finding, a previous cohort study in Gabon showed that pregnant women who had malaria infection had higher levels of specific IgG of variant surface antigens (VSA) antibody compared to women who had no malaria infection .P. falciparum blood-stage AMA-1 antigen. The cytophilic antibodies (IgG1 and IgG3) are currently thought to be instrumental in protective immunity, while non-cytophilic antibodies against the same epitopes may block the protective activity of cytophilic ones [The correlation of anti-malarial IgG, IgG subclass antibodies and malaria infection showed that the serum levels of IgG1 and IgG3 subclass antibodies were found to be higher than IgG2 and IgG4 antibodies, for lic ones ,23,45. Slic ones ,45,46. Flic ones ,20,47.When assessing the relationship between anti-malarial IgG, IgG subclass antibodies and the risk of parasite density, there were higher levels of anti-malaria AMA-1 antibody IgG, IgG1 and IgG3. This may have played a crucial role in parasite reduction. The suggested mechanism by which these subclasses are protective involves their binding to the FcRs on monocytes, leading to antibody dependent cell mediated inhibition of parasite replication ,45. ThisThe importance of FcγRIIa with regards to malaria comes from its role as a link between the humoral and cellular branches of the immune system . In the The present study was undertaken to examine the relationship between polymorphism in the gene for primary cellular IgG receptor, FcγRIIa and PAM. This study has provided evidence that the high levels of IgG subclasses are associated with FcγRIIa-R/R131 genotype among ASM women in southern Saudi. The results are in contrast with a study in western Kenya which described evidence that the IgG2-binding FcγRIIa-H/H131 genotype is associated with increased susceptibility to placental malaria in HIV-positive women .Although, the FcγRIIa are important molecules in malaria outcome; several studies have attempted to associate mutations in these genes with increased susceptibility to developing different forms of malaria. However, the results are contradictory and no conclusion has yet been determined. This confliction may be due to differences in the degrees of malarial endemicity, and genetic background of the individual as well as the differences in the study designs.Taken together, this study revealed that the ASM patients showed higher anti-malarial IgG and IgG subclass antibody levels when compared to the MFC. Furthermore, FcγRIIa-R/R131 genotype and R131 allele were associated with ASM patients. However, the FcγRIIa-H/H131 genotype is associated with higher levels of anti-malarial IgG subclass. Thus, the higher IgG subclass antibody levels seen in the ASM may be due to the relatively high prevalence of the individuals carrying the H/H131 genotype in this study group. Expectedly, the result of the present study suggests that the magnitude and the quality of anti-malarial humoral response is not the only key element for relative reduction in malaria infection in the ASM group. Further studies are needed to elucidate if the FcγRIIa-R/H131 polymorphism is a contributing factor to the differential susceptibility to malaria seen in different groups.The authors declare that they have no competing interests.OH and GA designed the study and carried out the sampling; AN and GA performed the ELISA; AN performed the genotyping of FcγRIIa polymorphism and participated in the statistical analysis. Both AN and GA drafted the manuscript. AN, AAG and GA set up the framework, financed and revised the manuscript. All authors participated in the manuscript preparation, read and approved the final version of the manuscript.


Background. We describe an irregular migrant who returned to Sri Lanka after a failed people smuggling operation from West Africa. Results. On-arrival screening by Anti-Malaria Campaign (AMC) officers using a rapid diagnostic test (RDT)  indicated a negative result. On day 3 after arrival, he presented with fever and chills but was managed as dengue (which is hyperendemic in Sri Lanka). Only on day 7, diagnosis of Plasmodium falciparum malaria was made by microcopy and CareStart RDT. The initially negative RDT was ascribed to a low parasite density. Irregular migration may be an unrecognized source of malaria reintroduction. Despite some limitations in detection, RDTs form an important point-of-entry assessment. As a consequence of this case, the AMC is now focused on repeat testing and close monitoring of all irregular migrants from malaria-endemic zones. Conclusion. The present case study highlights the effective collaboration and coordination between inter-governmental agencies such as IOM and the Ministry of Health towards the goals of malaria elimination in Sri Lanka. Malaria is an important disease along international and internal borders that continues to contribute to a large burden of disease in the South East Asian Region (SEAR). Due to extensive efforts, progress is being made. During the 2000–2011 period, the number of confirmed cases of malaria declined by 24% and deaths by 68%. Of the 10 malaria-endemic countries in the region, Sri Lanka has reached elimination phase of malaria, whereas Bhutan is in pre-elimination phase.Sri Lanka has been heralded as a success story in malaria control in Asia . In 2008Infection is mostly encountered among travelers who return from endemic countries or among military personnel serving in the northeast of the country , 4. The These routes may act as potential source of malaria reintroduction, retarding elimination goals of the nation's Anti-Malaria Campaign (AMC). Rapid diagnostic tests (RDTs) for malaria if performed correctly offer excellent diagnostic capability for screening of “at-risk” groups at ports of entry . HoweverBackground and Travel History. A 42-year-old male farmer of Tamil ethnicity from the Eastern Province of Sri Lanka joined a group of other irregular migrants that left on a flight to Benin in December 2011. All had paid large sums of money to a human smuggler, who had assured them safe passage to Canada and legal work permits on arrival. They arrived in Mali on 25th of December 2011 where another group of people smugglers arranged them to stay in a small shelter for one month. After a month, they travelled via flight to Benin, where they joined other cohorts of irregular migrants from Sri Lanka. The shelters they lived in had only basic facilities with no protection from mosquitoes. The smugglers intended to reach a quota of at least 900 people from Sri Lanka before charting a fishing vessel to enter Canadian ports. The scheme had already proven successful on previous occasions. During his time in Benin, the farmer recalled a heavy presence of mosquitoes and regular episodes of fever among the smuggled cohort. Smugglers had refused medical assistance in fear of alerting authorities. One death of a person due to “fever-like illness” was also reported although no medical details were available. The captives managed to escape and had alerted domestic and international law enforcement authorities who eventually informed the International Organization for Migration (IOM) to support the safe return of all irregular migrants from West Africa to Sri Lanka. Plasmodium falciparum-specific histidine-rich protein 2 (HRP2) and pan-Plasmodium-specific parasite lactate dehydrogenase (pLDH) performed on venous blood. For those with a positive RDT, the procedure prescribed further microscopic examination of blood smears at the National Malaria Reference Laboratory; patients with P. falciparum are treated with artemether-lumefantrine (Coartem) for 3 days and primaquine stat dose. For Plasmodium vivax infections, treatment consists of chloroquine for 3 days and primaquine for 14 days. All infected patients are treated at a specialist Infectious Disease Hospital in Colombo, and in case of children less than 12 years, at the Lady Ridgway Children's Hospital. Since the current patient showed a negative result for the CareStart RDT, he was allowed to proceed home.Upon arrival at Bandaranaike International Airport in Colombo (day 1), all irregular migrants escorted by IOM were screened by Anti-Malaria Campaign officers (AMC) of the Ministry of Health. According to AMC standard procedure, screening for Malaria was done by a rapid diagnostic test (RDT) and CareStart Malaria HRP2/PLDH  for the rapid qualitative determination of On day 3 after arrival, the patient presented to a private clinic with symptoms of high fever, headache, chills, rigors, and preceding cold sweats. He was referred on day 4 to a Government Peripheral Hospital with suspicion for dengue fever. In view of clinical suspicion for rapid dengue progression, the attending doctor transferred the patient to Batticaloa Teaching Hospital in line with current policy of dengue management in Sri Lanka . A timelHospital Course. For 48 hours upon admission, the patient received supportive treatment for the presumed diagnosis of dengue infection. As part of treatment follow-up, platelet counts were daily measured. Due to low platelet counts during the first two to three days, the initial suspicion was maintained for dengue. However, thrombocytopenia due to Plasmodium infection was suggested after the dengue antibody test showed a negative result and when the platelet count began to rise again after two days of hospital admission. By the end of day 7, venous blood was submitted to the hospital laboratory for RDT testing (CareStart) and microscopic examination. The slides were later sent to reference laboratory at AMC in Colombo for confirmation. Giemsa-stained thick and thin blood films were examined for the detection of malarial parasites. The RDT was positive for P. falciparum and was confirmed by the microscopy (double chromatin ring stage and “appliqué” form of trophozoites), and the parasite density was calculated as 36,180 asexual parasites/µL, which equals 0.7% of red blood cell infected. CareStart RDT revealed the presence of a visible HRP2 and pan-pLDH test line.P. falciparum was made, intravenous quinine (800 mg q8h) was administered. However, this protocol was changed to treatment according to AMC guidelines, that is, artemether-lumefantrine (Coartem) for 3 days and primaquine stat dose. Daily followup was carried out for the early detection of complications and the therapeutic response to drugs. Parasites disappeared within 5 days of treatment, and the patient recovered well and was discharged at day 14. There was no recrudescence at the followup 10 days after the start of treatment. Once the diagnosis of The present report highlights the delay of malaria diagnosis in an immigrant returning from a malaria-endemic region to a country in the near-elimination phase of malaria.RDTs Do Not Completely Rule Out Malaria. Despite microscopy remaining the “gold standard” for malaria diagnosis, studies in nonendemic and endemic countries have proven the effectiveness of RDTs [P. falciparum and P. vivax. at parasite densities of 200 asexual parasites/µL [P. falciparum at parasite densities above 100 and 1,000/µL to be 94.3% and 99.3%, respectively; for the detection of P. vivax, overall sensitivity was 77.6%, increasing to 90.2% at parasite densities above 500/µL [ of RDTs . A recen of RDTs . As some of RDTs . For thisites/µL . Anothere 500/µL .P. falciparum dips to 69.9% at parasite densities below 100/µL [µL.Malaria RDTs are a cost-effective and convenient screening technology, particularly at use in “point-of-entry” settings such as international airports. However, RDTs do have several limitations as described in this present case. We hypothesize the most likely cause for the initially negative CareStart RDT in the patient upon arrival at Colombo airport to be low parasite density. Indeed, the diagnostic sensitivity of CareStart RDT for the detection of w 100/µL . At the As a consequence of this case, the AMC revised its follow-up procedure by undertaking repeat RDT testing of all irregular migrants channeled via IOM within 2 weeks of their arrival at home. This practice has since been extended to Sri Lanka's United Nations Peace Keepers returning from endemic areas. A network of AMC program officers and IOM staff at district level also conduct followup of the returnee cases.Malaria Should Not Be Overlooked in Patient Management. Sri Lanka is also a hyperendemic country for dengue with repeated outbreaks occurring throughout the year [the year . Due to the year . HoweverImplications for Malaria Elimination. An increasing trend of irregular migrant flows has been reported in Sri Lanka since the end of civil conflict in 2009. IOM estimates a total of 900 Sri Lankans to be stranded in West Africa in 2012 alone. From January to July 2012, 14 cases of Plasmodium falciparum were detected in 437 returnees from people smuggling operations from West Africa [t Africa . Beyond As a consequence of this case, AMC revised its follow-up procedure by undertaking repeat RDT testing of all irregular migrants channeled via IOM within 2 weeks of their arrival at home. This practice has since been extended to Sri Lanka's United Nations Peace Keepers returning from endemic areas. The case study also highlights the importance of effective collaboration and coordination between intergovernmental agencies such as IOM and Ministry of Health towards the goals of malaria elimination by the end of 2012 [


Koraput, a predominantly tribe-inhabited and one of the highly endemic districts of Odisha State that contributes a substantial number of malaria cases to the India’s total. Control of malaria in such districts would contribute to change the national scenario on malaria situation. Hence, a study was carried out to measure the magnitude of malaria prevalence in the district to strengthen the malaria control activities.Prevalence of malaria was assessed through a sample blood survey (SBS) in seven randomly selected community health centres (CHCs). Individuals of all age groups in the villages selected (one in each subcentre) were screened for malaria infection. Both thick and thin smears were prepared from blood samples collected by finger prick, stained and examined for malaria parasites searching 100 fields in each smear. The results of a blood survey  carried out, as a part of another study, during 1986–87 covering a population of 17,722 spread in 37 villages of Koraput district were compared with the current survey results. Software SPSS version 16.0 was used for data analysis.Plasmodium falciparum was the major malaria parasite species accounted for 89.1%  of the total positives; Plasmodium vivax and Plasmodium malariae accounted for 9.3% (184) and 0.2% (5), respectively. Gametocytes were found in 7.7% (n = 152) of the positive cases. The majority of parasite carriers (78.9%) were afebrile. The 1986–87 blood survey showed that of 10,733 people screened, 833 (7.8%) were positive for malaria parasites, 714 (85.7%) with P. falciparum, 86 (10.3%) with P. vivax, 12 (1.4%) with P. malariae and 21 (2.5%) with mixed infections.During the current study, blood survey was done in 135 villages screening 12,045 individuals  and among them, 1,983 (16.5%) were found positive for malaria parasites. The results of the current study indicated a rising trend in transmission of malaria in Koraput district compared to the situation during 1986–87 and indicated the necessity for a focused and reinforced approach for the control of the disease by improving people’s access to diagnosis and treatment and ensuring implementation of the intervention measures with adequate coverage and compliance. Plasmodium falciparum malaria and 25.5% of malaria deaths (n = 239) reported in the country during 2008[P. falciparum infection[P. falciparum malaria and 6.3% of deaths due to malaria, during 2008[The goals set by the World Health Assembly and the Roll Back Malaria (RBM) Partnership to reduce the number of malaria cases and deaths recorded in 2000 by 50% or more by the end of 2010, and by 75% or more by 2015 have not yet been achieved[ths n = 29 reporteing 2008. The disths n = 29 reporte013’ and 19010’ north latitude and 8205’ and 83013’ east longitude). Some 21%  of the total area is covered with forest[Koraput district has a population of 13,05,492 (2008) spread in an area of 8,807 sq km, and is situated in the southern part of Odisha State (18The climate of the district is characterized by hot summer (March–June), rainy (July–October) and cold (November–February) seasons. The monsoon generally breaks during the later part of June each year. Average annual rainfall recorded during 2007 was 2,278.0 mm, of which >80% occurred during July to September under the influence of the south-west monsoon. Maximum rainfall was in August. The mean minimum temperature varied between 14.0°C (January–February) and 27.5°C (April–May) and mean maximum temperature between 27.0°C (December) and 41.5°C (May)[Of the 61 tribes living in Odisha State, 51 inhabit Koraput district, constituting more than 55% of the district’s total population, distributed in 14 CHCs. The important aboriginal tribes in the district include Porojas, Kondhs, Bondas, Gadabas, Koyas, Bhumiyas, Amanathyas and Gonds. The major occupation of the people is agriculture and collection of forest products. The people of the district, in general, are economically poor, their literacy rate is 35.7% against the State’s 63.1%[Based on topography, the villages in the district could be broadly classified into three ecotypes: hilltop, foothill and plain. Villages that are situated on hilltops or on slopes are grouped into hilltop ecotype. Perennial streams are the only source of water in these villages. Foothill ecotype includes villages that are located within 0.5 km from foot of hills. Streams, rivulets, terraced paddy fields, wells and ponds are the major breeding habitats in this ecotype. Villages located on flat but undulating land and at least 2 km away from foot of hills are the plain ecotype. Wells, ponds and paddy fields are the major source of Anopheline breeding in this ecotype.To assess malaria prevalence, among the 14 CHCs of Koraput district, seven CHCs were randomly selected and a sample blood survey was carried out in the CHCs. The total population of the seven CHCs was 5,04,207 comprising 140 subcentres (subdivision of a CHC) and 2,107 villages. All subcentres in each of the seven CHCs were covered for the blood survey by selecting randomly at least one village in each subcentre. Due to logistic problems, five selected villages could not be surveyed. The required sample size for the blood survey with 95% confidence was worked out to be 13,000 assuming an average API of 20 and allowing an error of 12%. The sample size for each CHC was estimated based on proportion to the population.Informed consent was obtained from the village committee first and then from the persons from who finger-prick blood samples were collected. In the case of children, informed consent was obtained from their parents/caregivers. Ethical clearance was obtained from the ethical committee of Vector Control Research Centre, Puducherry for conducting the blood survey. During the time of blood smear collection, body temperature of fever and non-fever cases was recorded using a clinical thermometer. Afebrile parasitaemia is defined as any parasite load (by microscopy) greater than zero without fever or negative history of fever during the previous 48 hours from the time of taking blood smear. Presumptive radical treatment at appropriate doses of chloroquine and primaquine was administered to all fever cases as per the guidelines of the national programme. Thick and thin smears were prepared on clean slides using the blood samples obtained by finger prick. All blood smears were stained with Giemsa and examined for malaria parasites at 5x100 magnifications searching 100 fields in each thick smear. As a quality control procedure, all positive and 10% of the negative slides were cross-examined following the national guidelines[Using the sample blood survey data, overall parasite prevalence was estimated. The number of parasites in thick smears was graded as 1+, 2+, 3+ and 4+. The bloχ2 test was used. To assess risk factors for malaria positivity, logistic regression analysis was carried out. Probability level of P < 0.05 was used for statistical significance.Data were entered into Microsoft Excel spreadsheet and statistical analyses were carried out using SPSS version 16.0. To compare the malaria parasite prevalence between different categories, During September 1986 to July 1987, as a part of another study, a blood survey was conducted covering a population of 17,722 in 37 villages of Koraput district and screening a total of 10,733 persons with a coverage of 60.6%, and the results have been discussed elsewhere[χ2 = 1,243; P < 0.001). Overall, SPR among males (18.1%) was significantly  higher than that of females (14.9%)  variation in SPRs between the three ecotypes. However, there was no significant  variation in malaria parasite species distribution between the ecotypes.Malaria SPRs, with 95% confidence interval, across the three ecotypes, hilltop, foothill and plain villages were 27.63% (30.4-24.85%), 17.88% (18.79-16.97%) and 11.57% (12.53-10.61%), respectively. There was a significant . Individual co-efficient of independent variables was also found to be statistically significant (P < 0.05). The estimated odds ratio (OR) with 95% confidence interval for females was 0.84 (0.76-0.93) indicating that the risk of getting malaria among females is significantly (P < 0.001) less than that of males (OR = 1.0). Similarly the OR for foothill  and plain areas  were significantly (P < 0.001) less than that of hilltop villages (OR = 1).Logistic regression analysis was carried out by considering SPR  as dependent variable and the other study variables, such as age, gender and ecotype, as independent variables. The overall fit of the logistic model was found to be good fit . Three human malaria parasite species were detected during the study and P. falciparum was the predominant one accounting for 89.1%  of the total infections, Plasmodium vivax and Plasmodium malariae accounted for 9.3% (184) and 0.2% (5), respectively. Mixed infection of more than one species was found in 27  infected individuals. Mixed P. falciparum and P. vivax infection was the most common (92.6%), followed by P. falciparum and P. malariae (7.4%) , 14.7% (27) and 40% (two) had gametocytes, respectively. Among the total P. falciparum cases , the highest gametocyte rate of 8.6% (25 out of 292) was found among one to four years age group followed by 8.2% (27 out of 330) among five to nine years. The gametocyte rate among 10–14 and >14 years was 3.7% and 6.8%, respectively. Among the infants, the gametocyte rate was 5.1% (two out of 39). However, there was no evidence of age dependence  on gametocyte rate. Similarly, among the total P. vivax infections (184), 14.1% was with gametocytes. The highest gametocyte rate of 28.6% (two out of seven) was recorded among infants followed by 23.7% (nine out of 38) among five to nine years. There was no significant difference (P > 0.05) in gametocyte rates between different age groups.In total, 152 (7.7%) malaria positive cases had gametocytes in their blood. Among the P. falciparum and P. vivax cases, 1+ density was the highest followed by 2+, 3+ and 4+  in the parasite density pattern between P. falciparum and P. vivax. Age-wise distribution of infected individuals by parasite density is given in Table4+ Table. There wP. falciparum, 77.7% P. vivax and 80% P. malariae infections). However, in the case of mixed infections, the trend was in reverse order, 40.7% was afebrile and 59.3% was febrile. Age-wise analysis of malaria positives showed that afebrile cases ranged from 73.2% to 81.1% among the five age groups  and there was no significant  difference between them (TableAfebrile parasitaemia was confirmed in 78.9% of the cases and the remainder (21.1%) were febrile. There was no significant difference (P > 0.05) in proportion of afebrile cases with the three parasite species  higher (16.5%) than that (7.8%) reported 22 years ago, i.e., during 1986–87, increased from 7.8% to 16.5%. The proportion of P. falciparum and P. vivax remained almost same without variation. However, the prevalence of P. malariae reduced from 1.4% to 0.2%. Infant parasite rate increased from 15.5% to 18.1%. The prevalence of afebrile carriers in the community remained almost at the same level (78.0% in 1986–87 and 78.9% in 2009). Parasite rate among one to nine years was higher (20.0%) in the 2009 survey compared to the survey in 1986–87 (9.4%). However, the gametocyte rate in the community was found higher in the earlier survey (15.3%) compared to the present survey (7.7%)  had higher prevalence of malaria compared to adults. Similar observation was made in Assam,19,20, AP. falciparum infection was the prevalence of low number of gametocyte carriers (7.0%) though a large proportion of population had the infection. This could be due to the consequence of launching Enhanced Malaria Control Project (EMCP) in India in April 1997 covering 100 districts (including Koraput district) and 19 urban areas in eight peninsular states[P. falciparum) was administered to all suspected/ clinical malaria cases and this could be the reason for the low prevalence of P. falciparum infection with gametocytes. The proportion of overall gametocyte carriers  was also higher (15.3%) in the earlier survey[In the present study, one of the interesting aspects of r states. Under tr states. As mostThere is no doubt that malaria continues to be a major public health problem causing morbidity and mortality among the tribes in the district and several factors are responsible for the persistence of the disease. As most of the villages in the district recorded API well above two during the last two decades, indoor residual spraying (IRS) of insecticide had been the mainstay of vector control by the malaria control programme in the country. The villages of the district have been receiving yearly two rounds of IRS with DDT at a dosage of 1 g/sq m till date. Active P. falciparum strain was detected with increasing frequency in many districts of Odisha State as well as in the country[P. falciparum cases in the entire country will be artemisinin-based combination therapy (ACT)[However, since the villages in the district are scattered and most of them are located on hilltops or slopes of hillocks, communication facilities and health care services were poor and inadequate and often led to breakdown of regular malaria control operations. Insecticide spraying operations could not be carried out in time due to difficult terrain and inaccessibility of many villages. Even if sprayed, the coverage and quality of spraying were poor due to lack of supervision. More than 55% of the houses could not be sprayed due to refusal. Moreove country. Hence, py (ACT). The intThe authors declare that they have no competing interests.SSS and KG conceived and designed and performed the study. PV compiled and analysed the data. SSS and KG drafted the manuscript. PJ critically reviewed the manuscript. All authors contributed to the writing of the manuscript and approved the final version.