Wuchereria bancrofti (Wb) and Mansonella perstans (Mp) are blood-borne filarial parasites that are endemic in many countries of Africa, including Mali. The geographic distribution of Wb and Mp overlaps considerably with that of malaria, and coinfection is common. Although chronic filarial infection has been shown to alter immune responses to malaria parasites, its effect on clinical and immunologic responses in acute malaria is unknown.To address this question, 31 filaria-positive (FIL+) and 31 filaria-negative (FIL−) children and young adults, matched for age, gender and hemoglobin type, were followed prospectively through a malaria transmission season. Filarial infection was defined by the presence of Wb or Mp microfilariae on calibrated thick smears performed between 10 pm and 2 am and/or by the presence of circulating filarial antigen in serum. Clinical malaria was defined as axillary temperature ≥37.5°C or another symptom or sign compatible with malaria infection plus the presence of asexual malaria parasites on a thick blood smear. Although the incidence of clinical malaria, time to first episode, clinical signs and symptoms, and malaria parasitemia were comparable between the two groups, geometric mean hemoglobin levels were significantly decreased in FIL− subjects at the height of the transmission season compared to FIL+ subjects . Plasma levels of IL-1ra, IP-10 and IL-8 were significantly decreased in FIL+ subjects at the time of presentation with clinical malaria .These data suggest that pre-existent filarial infection attenuates immune responses associated with severe malaria and protects against anemia, but has little effect on susceptibility to or severity of acute malaria infection. The apparent protective effect of filarial infection against anemia is intriguing and warrants further study in a larger cohort. In many regions of the world, including sub-Saharan Africa, concomitant infection with multiple parasites is common. In order to examine the effects of filariasis, a chronic helminth infection, on immune responses and clinical manifestations of acute malaria infection, the authors followed 31 filaria-infected (FIL+) and 31 filaria-uninfected (FIL–) individuals living in a malaria-endemic area of Mali through an entire malaria transmission season for the development of clinical malaria . Serum levels of inflammatory cytokines previously associated with severe malaria were decreased in FIL+ subjects at the time of acute clinical malaria. Although there were no differences between FIL+ and FIL– subjects with respect to the time of first episode of malaria or the number or severity of malaria episodes, filarial infection appeared to protect against the development of anemia during the malaria transmission season. These findings demonstrate that chronic filarial infection modulates the immune response to acute malaria. The apparent effect on anemia is intriguing and deserves further study. Filarial infections and malaria are coendemic in many areas of the world, including sub-Saharan Africa, where human coinfection with malaria and filarial parasites is common in vitro, including a recent study that showed an association of patent filarial infection with decreased malaria antigen-specific IL-12p70/IFNγ production A number of studies have demonstrated that chronic filarial infection can suppress human immune responses to malarial antigens The current study was designed to determine if pre-existent filarial infection alters the frequency and/or severity of clinical malaria in children and young adults in a coendemic area in Mali where malaria transmission follows a seasonal pattern. Cytokine profiles at the time of presentation with acute malaria were also compared between subjects with and without concomitant filarial infection.The study was designed as a matched prospective study of the effects of filarial infection on the incidence, severity and immune responses to malaria infection.Wuchereria bancrofti (Wb) and Mansonella perstans (Mp) microfilaremia in the population, but no evidence of onchocerciasis. A single round of mass drug administration (MDA) with ivermectin and albendazole was conducted by the National Program to Eliminate Lymphatic Filariasis prior to the study in May 2006, and study subjects participated in a second round of MDA during the study in August 2007. Malaria transmission in this region is seasonal (June–December) with a cumulative EIR of 19.2 infective bites/person in a neighboring village in 2000 The study was conducted in the villages of Tieneguebougou and Bougoudiana in the district of Kolokani, approximately 105 km northwest of Bamako, Mali, between June 2007 and December 2007. Prior studies in these villages had demonstrated a high prevalence of http://www.bio-rad.com), daytime thick smear for detection of malaria and/or Mp infection, and circulating antigen (CAg) ELISA for Wb . Based on these results, potential participants were divided into two groups: 1) FIL+: individuals with confirmed active filarial infection with Wb and/or Mp, as defined by the presence of CAg and/or microfilaremia (mf) and 2) FIL−: individuals without evidence of active filarial infection (negative for CAg and mf). Thirty-one FIL+ subjects were identified and matched to FIL− subjects on the basis of gender, age (within 4 years), and Hgb type. Glucose-6-phosphate dehydrogenase (G6PD) deficiency was assessed by restriction enzyme analysis The study flowchart and selection of study participants is shown in All participants were evaluated weekly throughout the malaria transmission season. At each visit, symptoms and signs of clinical malaria were assessed, and if present, blood was obtained from a finger stick for preparation of Giemsa-stained thick and thin blood smears for detection and speciation of malaria parasites and Hgb measurement. In addition, subjects were encouraged to present to the study physician for evaluation if they felt ill. Blood smears and Hgb determinations were also performed at the start of the study and monthly thereafter, regardless of the presence of clinical signs or symptoms. Reading of these monthly blood smears was deferred until the end of the study unless the subject was symptomatic at the time of the blood draw. To minimize the potential effects of concomitant helminth infection, all subjects were treated with single dose praziquantel (40 mg/kg) and albendazole (400 mg) at the start of the study. Albendazole treatment was repeated every two months during the study period.P. falciparum parasites on Giemsa stained thick blood films until 300 leukocytes were observed. All slides were read by two independent microscopists, and differences of >10% were resolved by an expert microscopist. Parasite densities were calculated by multiplying the mean value for the two readers by 25 to give parasites/microliter.Clinical malaria was defined as the presence of fever (axillary temperature ≥37.5°C) or another symptom or sign compatible with malaria infection plus the presence of asexual malaria parasites on a thick blood smear. Malaria parasitemia was determined by counting the number of asexual Cases of uncomplicated malaria were treated with standard recommended doses of artesunate-amodiaquine (4 mg/kg artesunate and 10 mg/kg amodiaquine per day for 3 days). Cases of therapeutic failure were treated with quinine (10 mg/kg/day for 3 days). On days 1, 2, 3, 7 and 14 following treatment, subjects were evaluated for clinical signs and symptoms, and malaria smears and Hgb measurements were performed. Plasma levels of interferon (IFN)γ, interleukin (IL)-1α, IL-1β, IL-1ra, IL-6, IL-8, IL-10, IL-12p70, IP-10, and tumor necrosis factor (TNF)α were measured in plasma samples obtained prior to treatment by suspension array in multiplex , according to the manufacturer's instructions. The limits of detection of the assay are 0.5 pg/ml for IFNγ, 0.63 pg/ml for IL-1α, 0.19 pg/ml for IL-1β, 10.97 pg/ml for IL-1ra, 0.79 pg/ml for IL-6, 0.32 pg/ml for IL-8, 0.41 pg/ml for IL-10, 0.23 pg/ml for IL-12p70, 1.14 pg/ml for IP-10, and 0.22 pg/ml for TNFα.The study (NCT00471666) was approved by the ethical review committees of the Faculty of Medicine, Pharmacy, and Dentistry at the University of Bamako  and of the National Institutes of Allergy and Infectious Diseases . Community permission for the study was obtained from village elders, and individual oral or written informed consent (for subjects ≥18 years of age) or parental informed consent and assent (for subjects <18 years of age) was obtained from all participants in French or Bambara, the local language. Oral consent was obtained for illiterate participants and parents of participants as approved by the ethical review committees and was documented by the signatures of a member of the study team and a witness.The ratio of the rates of clinical malaria events was calculated by Poisson regression adjusting for the time at risk Baseline characteristics for the two groups are shown in Filarial status was assessed at baseline and at 3 and 6 months. Among the 31 FIL+ subjects, 11 (35.5%) were infected with Wb and 20 (64.5%) with Mp. Two subjects had detectable Wb microfilariae in the peripheral blood. During the course of the study, 3 FIL+ subjects, all of whom were Mp-infected, became FIL− . As expeAlthough not all subjects were followed for the full 24 weeks season, 51.3 person-seasons of data were available for analysis. During the malaria transmission season, 38 (61%) subjects developed clinical malaria. There was no significant difference in the rates of clinical malaria between the FIL+ and FIL− groups  and FIL− (n = 14) subjects. Among the analytes measured, GM levels of IL-1ra, IP-10 and IL-8 were all significantly decreased in FIL+ subjects  as compared to FIL− subjects  and without (FIL−) chronic filarial infection were matched for age, gender and hemoglobin type and followed longitudinally through a malaria transmission season. No significant differences were detected between FIL+ and FIL− subjects with respect to the incidence of clinical malaria, time to first episode of clinical malaria, number of episodes of clinical malaria during the transmission season, parasitemia at first episode, or clinical signs and symptoms at first episode. A prospective study of similar design that examined the effects of coinfection with schistosomiasis on clinical malaria in 338 Malian children aged 4–14 years of age demonstrated a significant increase in the time to first episode of clinical malaria by 10–14 days in coinfected children (p = 0.04), but only in the 4–8 year old age group Despite the lack of an observable effect on the frequency or severity of clinical malaria, chronic filarial infection appeared to protect against the development of anemia during the height of the malaria transmission season . This isPrior studies from our group and others have demonstrated increased plasma levels of IL-10, as well as increases in regulatory T cell frequency and function, in children and young adults with helminth infection The association between high plasma IP-10 levels and more severe malarial anemia has been reported previously A major anticipated limitation of the present study was the relatively small sample size due to the difficulty in recruiting FIL+ subjects in the younger age groups. Consequently, in order to maximize the possibility of detecting significant differences between the FIL+ and FIL− groups, a matched design was selected. Potential confounding variables, such as baseline malaria parasitemia, intestinal helminth infection, G6PD deficiency, bednet use and location of residence, were also similar between the two groups. Although subjects were not tested for HIV infection, the prevalence of HIV is extremely low in rural Mali and no subjects with clinical evidence of immunodeficiency were identified in the screening. Additional factors that may have diminished differences between the FIL+ and FIL− groups include lasting effects of intestinal helminth infection following treatment, decreased severity of malaria infection due to earlier intervention as a result of the presence of health care personnel in the village (as has been seen in other studies) and the possibility that some FIL− subjects may have had occult (microfilaria-negative) Mp infection or have acquired filariasis during the course of the study. In fact, 8 FIL− subjects became CAg-positive during the study and 4 became microfilaria-positive for Mp. Although these subjects were likely infected at the start of the study, they were considered FIL− for the purposes of the study . In view of these considerations, it is likely that small  differences in responses between the FIL+ and FIL− groups were missed.In summary, although pre-existent filarial infection attenuates cytokine/chemokine responses known to be associated with severe malaria, it appears to have little effect on susceptibility to or severity of acute malaria infection in children and young adults living in a malaria-endemic area with seasonal transmission. The apparent protective effect of filarial infection on decreased hemoglobin levels at the height of malaria transmission is intriguing and warrants further study in a larger cohort.


Background. We performed 2 cross-sectional studies in Ménaka in the Northeastern Mali across 9 sites in different ecological settings: 4 sites have permanent ponds, 4 without ponds, and one (City of Ménaka) has a semipermanent pond. We enrolled 1328 subjects in May 2004 (hot dry season) and 1422 in February 2005 (cold dry season) after the rainy season. Objective. To examine the seasonality of malaria parasite prevalence in this dry northern part of Mali at the edge of the Sahara desert. Results. Slide prevalence was lower in hot dry than cold dry season . Gametocyte rate increased to 0.91% in February. Four species were identified. Plasmodium falciparum was most prevalent (74.13 and 63.72%). P. malariae increased from 9.38% to 22.54% in February. In contrast, prevalence of P. vivax was higher (10.31%) without seasonal variation. Smear positivity was associated with splenomegaly (P = 0.007). Malaria remained stable in the villages with ponds (P = 0.221); in contrast, prevalence varied between the 2 seasons in the villages without ponds (P = 0.004). Conclusion. Malaria was mesoendemic; 4 species circulates with a seasonal fluctuation for Plasmodium falciparum. Malaria is still an overwhelming public health problem in the sub-Saharan Africa, more than 90% of the disease burden occurs in Africa . ChildreP. malariae represents only 2 to 3% of infected patients and no infection with P. vivax was observed in dark skin subjects during all parasitological investigations carried out so far in Mali. The northern Mali is land of nomadic populations (Tuaregs and Maures) who move around ponds, oueds, and water forages; no epidemiological studies were performed in a prospective manner to identify dynamic of circulating parasites in those areas and to evaluate parasitological parameters of malaria in the North of Mali. Thus, we carried out a cross-sectional study to examine the seasonality of malaria parasite prevalence during dry hot (May 2004) and cold (February 2005) seasons in the District of Ménaka located in the North-Eastern part of Mali.Thus, distribution of malaria relies on ecological factors such as rainfall in the Sahel; anopheline mosquito, the vector of malaria, responsible for transmitting the parasite from one host to another needs enough water to breed. Based on previous studies , 7, it h2. Since 1998, 18% of the populations settled permanently in cities of Ménaka, Andéramboukane, and Inékar. The rest of the population (82%) settled in the nomadic camps around the ponds. The main ethnic group is Kel Tamacheq (nomadic inhabitants), they are about 95%, and the minority groups are seminomadic Sonrhaïs, Haoussas, and Maures. The Daoussahaqs are persons of mixed race of Touaregs and Sonrhaï and speak a dialect close to these two languages. They represent the majority of the active population in sectors of agriculture and cattle breeding [The District of Ménaka is located in the northern-East between longitude 1°20 and 4°15 East and latitude 15°20 and 18° north. It is limited by Tinessako  in the North, Niger in the East, and by Ansongo in the West . The heibreeding .We have carried out a random sampling by relying on subjects who were present at the time of our passage in each site until we reached the estimated number of subjects planned for the study. Based on our stratification, 108 subjects may constitute one unit. A total of 1328 subjects of 12 clusters (108 units per cluster) have been screened for malaria infection in May 2004 in 9 rural and district sites of Ménaka prefecture. We performed 8 months after a second passage in February by including 1422 subjects with 108 units per cluster.Each subject was examined by a physician at teach study site (village). Body axillary's temperature was recorded using an electronic thermometer  and spleen enlargement was assessed based on Hackett's criteria .P. falciparum parasites in fields containing 300 white blood cells, and multiplied that number by 25 to estimate the number of asexual P. falciparum parasites per μL  in phosphate buffer (pH 7.0) and examined using oil immersion magnification (1000x). Each slide was examined by two microscopists, who estimated the parasite density by counting the number of asexual 0 per μL—). SlidesFreshly blotted filter papers obtained from each subject were dried in a closed cardboard box to protect them from flies and dust. Each filter paper was then labeled with the subject's study number, placed in an envelope labeled with the same number, and stored at ambient temperature in a file cabinet.A capillary tube  covered of heparin (anticoagulant) was filled of blood to the feature of calibration. Then, one of the ends was stopped with wax. The blood contained in each capillary tube of each subject was then separated after centrifugation with the Thermo MicroMB centrifuge . Reading was performed according to instructions' of manufacturer using circular microcapillary tube reader . In this study, anemia was defined when the hematocrit rate is less than 33 .Plasmodium vivax parasite DNA was extracted from filter paper blots with Chelex-100 method as previously described [P. vivax that flank the repeat region, Nested PCR amplification was performed as described by Alger [escribed . Using pby Alger .t-test was performed to compare if there was a difference in terms of the size population between the dry hot season (2004) and dry cold season (2005). The odds ratio test has been estimated to seek the relationship between malaria species and symptoms. A P value less than 0.05 was considered as significant.Excel software (Microsoft office 2003) has been used for data entry, the statistical analysis with the SPSS software (Version 11.0 for Window). Pearson chi-square test or Fisher test has been used for prevalence comparison between data obtained in 2004 and 2005. Unpaired Protocol was first submitted and approved by the Ministry of Health  and then explained to village's council. After its approval, each participant was then informed about the protocol; care was given to all study participants. Oral informed consent or assent (for children less than 18 years of age) was then obtained from each participant.P value = 0.868 (unpaired t-test). Similarly, subjects with age greater than 9 years were also comparable between the 2 passages (May 2004 and February 2005) with P value = 0.866  were in the school summer break which occurs during the malaria transmission season (June-September) and were not accessible. However, data collected during the dry and cold periods allowed us to examine the fluctuation of parasitological parameters among populations in the 9 sites of Ménaka. There was not a significant difference between our study populations during the 2 passages . Thus, 1 = 0.866 .P < 0.0001). The same trends were observed with the type of skin (derma); there were more subjects with dark skin in the areas with ponds than areas without ponds during the years 2004 and 2005 (P < 0.0001). There were more females in the areas with ponds than areas without ponds in 2004 (P = 0.002); in contrast there was no significant difference between gender and presence of (absence) ponds in 2005 (P = 0.625).Comparing the data obtained from the studied populations in 2004 and 2005 , there wP = 0.0259). However, we could not find any statistically difference between gametocyte prevalence between the 2 periods although the prevalence was higher in cold season with 0.91% (13 out of 1415) versus 0.46% (6 out of 1293) in dry season (P = 0.2364).Parasitological data showed that the 4 species were circulating in Ménaka. Overall prevalence varied from 4.95% in May 2004 (with 64 positive slides in 1293 screened subjects) to 7.06% in February 2005 (with 97 positive smears among 1422 subjects). This seasonal variation in prevalence of malaria infection was statistically significant  as well as in cold period (2005), but with a higher proportion in dry season 74.13% versus 63.72% in cold season. In 64 infections (2004), 50 were due to P. falciparum .lciparum . This prlciparum . This vaP. malariae infection increased considerably in cold season. Six infections were due to this species among 64 infected subjects with the 4 species (9.38%) in dry season while among 97 infected persons in cold period, 21 were caused by P. malariae (21.64%). There was a significant difference in prevalence of P. malariae infection between May 2004 and February 2005 (P = 0.0419).Prevalence of Plasmodium ovale and P. vivax infections represented 3.13% (2/64) and 7.81% (5/64), respectively, in dry season. In cold season, even though the frequency of P. vivax was higher 10.31% (10/97), there was not a statistically variation between May 2004 and February 2005 (P > 0.05).P. falciparum and P. malariae was identified in dry season (2004) among the 64 positive slides (1.56%), and the number increased to 7 out of 97 positive slides in cold season (2005) with 7.21% (P = 0.147). One case of mixed of 3 species  was identified only in the cold season in 2005 .Prevalence of enlarged spleen was higher during the dry season with 5% (64/1264) than during the cold period with 2.9% (41/1397), and this difference was statistically significant  and 10.5% and 22.2%, respectively, for anemia and fever  versus 1.94 (14/718), respectively, and this difference was significant (P = 0.000001). Interestingly between the cold (2005) and dry (2004) seasons, children from 0 to 9 years of age and subjects with age greater than 9 years were equally infected; no significant difference was found (P = 0.0753). In addition, a different picture was observed in May during the dry season; children (0–9 years of age) were frequently infected with 8.69% (50/575) of malaria parasites than older subjects (age > 9 years) with 1.94% (14/718). Difference was statistically significant with P = 0.0001 (P = 0.2890).Relationship between age groups and parasite prevalence was assessed ; we obse= 0.0001 . CompariP = 0.221) was observed. In contrast, there was a statically difference in the prevalence of malaria between cold and dry season in the villages without ponds (P = 0.004). By comparing the prevalence of malaria infection in the villages with ponds and villages without ponds, we did find a difference; the prevalence was higher in sites with ponds than without ponds (P = 0.0289).We have investigated whether there was a difference in prevalence of parasite infection based on presence of ponds in study's sites. When we compared malaria prevalence in sites with ponds, no statistical difference in May and February  and Salvador-1 (429 basepairs) genotypes have been identified in a single individual, confirming that P. vivax was circulating in Mali  than in those with other malaria species (9.7%).We assessed the relationship between anemia and malaria species (P = 0.003). This fluctuation could be explained by ecological factors such as ponds that contribute to the transmission of malaria parasites beside the rainy season [We performed 2 cross-sectional studies in Ménaka, which is situated in the Northern part of Mali at the borders with Niger and Algeria. In terms of sample size, the population screened during the passages of May 2004 and February 2005 was comparable . Howevery season –19. ThisP = 0.221). In contrast, there was a significant difference in prevalence of malaria infection in the sites without ponds (P = 0.004), the prevalence being higher in cold season than during the hot period. Those data taken together will allow one to hypothesize that ponds serve as breeding sites for mosquitoes, which are able to maintain transmission of malaria parasites in absence of rains in the dry part of the country. Guthmann et al. [Higher prevalence of malaria infection obtained in February 2005 three months after the conclusion of the rainy period may be associated with the presence of ponds in sites with higher prevalence. In addition, prevalence was stable between hot dry period and cold dry season, and no statistically significant variation in parasite prevalence was observed between sites with ponds , followed by P. malariae, which increased from 9.38 during the dry hot season (May) to 22.54% after the rainy season in February. The high prevalence of P. malariae observed in February was unusual; all studies performed in Mali show that the prevalence was always less than 10% [P. malariae than those found in South associated mainly with the rainfall. However, entomological studies are needed to identify anopheline species, which breed around the ponds and assess their vectorial capacity. The only data on anopheline mosquitoes were from the north of Mali and obtained between Gao and the border of Algeria; they indicated that Anopheles gambiae complex and Anopheles pharaonsis were present [P. vivax and P. ovale remained stable over the 2 periods. Interestingly, the prevalence of P. vivax was higher with ~10% of infection compared with to the data collected along the Transsaharan road from Bourem to Tessalit at the border with Algeria. In that region, Koita [P. vivax. The stability in the prevalence of P. vivax and P. ovale could be explained in part by recurrent infection with the release of hypnozoites [P. vivax at the edge of Sahara desert. Two genotypes Belem (366 basepairs) and Salvador-1 (429 basepairs) have been identified in a single infection. Presence of P. vivax parasite in that region was expected, because in that part of Mali live Duffy positive population, who are susceptible to P. vivax infection [P. vivax infections in 9 countries in West and Central Africa. This higher prevalence could be explained by the existence of a proportion of Duffy positive subjects in our study population which turns around 40% ; however the proportion of infected people with the four species varied seasonally. than 10% . Thus, wnfection . Our resnfection  found onP. malariae. We observed that children less than 9 years of age were frequently exposed than older subjects during the dry season; however we did not find a difference among our age-groups after the rainy season. This suggests that age is not a factor in preventing Plasmodium infection; each subject was at risk for infection at this period of year.Although prevalence of anemia was lower compared to that found in South of the country , subjectP. falciparum. The prevalence of P. vivax (9.2%) and P. ovale remained stable and was not affected by the season. Two genotypes of Plasmodium vivax, Belem and Salvator-1, were identified. The presence of P. vivax species is related to the presence of Duffy positive populations in that area of Mali. The results suggest that the presence (or absence) of ponds is an overwhelmingly important factor. In contrast to the situation in the more southern (savannah) part of Mali, they also suggest that the significance of seasonal changes is apparent only in communities without ponds.Four species of malaria parasite are in circulation in the northeastern of Mali with a seasonal fluctuation of


P. vivax malaria in three medical college hospitals, one in the union territory of Chandigarh and the other two in Gujarat, that is, Ahmedabad and Surat. The clinical presentation, treatment, and outcome were recorded. As per WHO criteria for severity, 19 of 110 patients had severe disease—six patients had clinical jaundice with hepatic dysfunction, three patients had severe anemia, three had spontaneous bleeding, two had acute respiratory distress syndrome, and one had cerebral malaria, hyperparasitemia, renal failure, circulatory collapse, and metabolic acidosis. All patients with severe P. vivax malaria survived, but one child with cerebral malaria had neurological sequelae. There was wide variation in the antimalarial treatment received at the three centres. Plasmodium vivax malaria can no longer be considered a benign condition. WHO guidelines for treatment of P. vivax malaria need to be reinforced.This was a retrospective study done on 110 patients hospitalized with  Plasmodium vivax being responsible for more than 50% of the cases. Although earlier regarded as causing a benign infection, there is increasing evidence that the overall burden, economic impact, and severity of P. vivax have been underestimated [P. vivax malaria [India accounts for 77% of the total malaria in south-east Asia with stimated . There a malaria –4.P. vivax malaria with 14 days of primaquine for radical cure. The WHO guidelines for the treatment of malaria recommend that intravenous artesunate should be used for the treatment of severe malaria  for a minimum period of 24 hours. Following initial parenteral treatment, once the patient can tolerate oral therapy, it is essential to continue and complete treatment with an effective oral antimalarial using a full course of an effective ACT (artesunate plus amodiaquine or artemether plus lumefantrine or dihydroartemisinin plus piperaquine) or artesunate (plus clindamycin or doxycycline) or quinine (plus clindamycin or doxycycline) [Also, malaria case management remains a vital component of malaria control strategies. The WHO guidelines for the treatment of malaria recommend chloroquine (except in areas of chloroquine resistance) as first-line treatment for uncomplicated cycline) .P. vivax malaria and whether WHO guidelines for treatment of P. vivax malaria were being adhered to.The present study was done to evaluate the clinical manifestations and outcome in patients hospitalized with P. vivax malaria, were studied. The period from May to December was chosen since it coincides with the monsoons in India, a period when transmission of malaria is at its highest [This was a retrospective study done using data collected from the medical records of three tertiary care centres. One of the hospitals was in Chandigarh, a union territory in the northern region of India, whereas two of the hospitals were in Gujarat , a state in the western region of India. Records of patients admitted to the ward or intensive care unit of the respective hospitals, from May to December 2012, with a diagnosis of  highest , 7.P. vivax malaria were included in the study. Patients of P. falciparum malaria, those with mixed infections and those with other coexisting infections like typhoid fever, dengue, and leptospirosis, were excluded.Adults and children, with smear-positive A case record form was developed and used to retrieve data from the inpatient records. The first three authors were responsible for extracting data from the medical records of their respective hospitals.Ethics committee approval was obtained from all three hospitals for retrieval of data from the medical records. The confidentiality of the patients' record and identity of the treating physician was adequately protected using codes.Information extracted from the records included patient's age, sex, the presenting complaints, findings on clinical examination, hematological and biochemical investigations, treatment given, course in the hospital, and outcome.P. vivax malaria, the WHO guidelines for classification of severe P. falciparum malaria were used to identify patients with severe disease [In the absence of guidelines to classify severe  disease .The data was pooled at the coordinating centre at the National Institute for Research in Reproductive Health, Mumbai. Data entry and analysis were done using Microsoft Excel 2007. Descriptive statistics in terms of proportions for qualitative variables were applied for analysis of data.P. vivax malaria in the three centres were studied. The ages of the patients are summarized in Medical records of a total of 110 patients hospitalized for The presenting symptoms are summarised in P. vivax malaria had severe disease (Tables Nineteen of 110 patients (17.2%) hospitalized with e Tables  and 4. JThe treatment and outcome of patients with severe disease is given in The five patients with severe anemia received packed cell transfusions. Three patients with thrombocytopenia  had bleeding manifestations and received platelet transfusions. Two patients with ARDS required ventilation. The one patient with acute renal failure required dialysis, with indications being anuria and hyperkalemia.P. vivax malaria including those with severe disease survived. One child with cerebral malaria had sequelae in the form of blindness which was still present at one-month followup.All patients with P. vivax malaria had evidence of severe disease. Only three large studies describing the whole spectrum of severe manifestations with P. vivax malaria could be identified from India; two were from Bikaner by Kochar et al. [P. vivax malaria in Bikaner. The criteria for hospitalizing patients with P. vivax malaria were not well defined in our study or in the published studies and were at the discretion of the admitting physician.Nineteen of 110 17.2%) hospitalized patients with % hospitar et al. , while tr et al. . Severe P. vivax indicates that severe anemia is not due to destruction of infected RBCs alone. Malariotherapy studies have shown that, for every infected RBC destroyed during vivax infection, 32 noninfected RBCs are removed from the circulation, compared to the loss of 8 RBCs for every infected erythrocyte in falciparum malaria. Cytokine-related dyserythropoiesis also probably contributes to anemia [Severe anemia (hemoglobin < 5 G/dL) necessitating packed red cell transfusion was seen in five of nineteen patients with severe malaria in this study. The low parasite biomass of vivax malaria. Bleeding manifestations necessitating platelet transfusions were seen in only three patients. The frequency of thrombocytopenia, that is, platelet count <1,50,000/cu·mm, in malaria ranges from 24% to 94% in the literature and this frequency is not different in the two species. However, occurrence of bleeding even in severe illness is low and a conservative approach is adopted in most cases. Platelet counts usually revert to normal after effective antimalarial treatment [Thrombocytopenia was seen in 34.55% of patients hospitalized with vivax malaria in our study. Cytoadherence phenomena are believed to be central to the etiology of cerebral malaria in falciparum infection, but their role in P. vivax malaria remains unclear [Cerebral malaria was seen in three of nineteen patients with severe  unclear .P. falciparum malaria. Case reports of ARDS with P. vivax malaria are also being increasingly reported [One patient each from Chandigarh and Surat had acute respiratory distress syndrome (ARDS) and required ventilatory support. ARDS is considered to be the most severe form of acute lung injury in malaria and has been reported most commonly with reported .vivax malaria. Hepatic dysfunction has been also described in 20–58% of patients with vivax malaria in Bikaner and Mumbai [Clinical jaundice with hepatic dysfunction was seen in eight of nineteen patients with severe d Mumbai .P. vivax malaria be treated along the lines of severe falciparum malaria with artemisinin-based combination therapies (ACT) [The WHO guidelines recommend that severe es (ACT) .P. vivax malaria, whereas in Chandigarh artesunate only was used in four patients. ACT was used in four patients (three in Chandigarh and one in Surat). The rationale for using antimalarial combination therapy is the simultaneous use of two or more blood schizontocidal medicines with independent modes of action and, thus, different biochemical targets in the parasite. This is postulated to prevent or delay the appearance of resistance [There was wide variation in the therapy for severe disease in the three centres. Chloroquine was used as the antimalarial of choice in Sola even in patients with severe disease. In Surat, both artesunate and chloroquine were used in patients with severe sistance .vivax [Drug resistance to the first-line drug chloroquine has been reported from Papua New Guinea, Indonesia, parts of Asia including India, and South America –14. ChloP. vivax malaria [P. vivax malaria in Sola and Surat received primaquine in the correct dosage.WHO guidelines recommend primaquine for 14 days for radical cure in  malaria . None ofP. vivax malaria at any of the three centres. One child with cerebral malaria in Chandigarh had neurological sequelae at discharge. A mortality of 6% was noted in children in Bikaner, whereas it was 5% and 0.9% in adults in Bikaner and Mumbai, respectively [There were no deaths in any of the patients hospitalized with ectively –4.The limitations of this study are as follows: it is a retrospective study with a small sample size and uses data retrieved from the medical records. The problem of mixed infection has not been addressed adequately since this was a retrospective study and PCR is not done routinely at the three centres. A prospective multicentric study with an additional centre in Mumbai is under way and will no doubt clarify the picture better.Plasmodium vivax can result in severe disease and can no longer be considered to be a benign condition in India. There is considerable variation in the treatment of P. vivax malaria. Adherence to WHO guidelines for treatment is recommended.


Plasmodium falciparum is a complex process that involves two families; Erythrocyte Binding-Like (EBL) and the Reticulocyte Binding-Like (PfRh) proteins. Antibodies that inhibit merozoite attachment and invasion are believed to be important in mediating naturally acquired immunity and immunity generated by parasite blood stage vaccine candidates. The hypotheses tested in this study were 1) that antibody responses against specific P. falciparum invasion ligands (EBL and PfRh) differ between symptomatic and asymptomatic individuals living in the low-transmission region of the Peruvian Amazon and 2), such antibody responses might have an association, either direct or indirect, with clinical immunity observed in asymptomatically parasitaemic individuals.Erythrocyte invasion by P. falciparum invasion ligands of the EBL  and PfRh families  in 45 individuals infected with P. falciparum from Peruvian Amazon. Individuals were classified as having symptomatic malaria (N=37) or asymptomatic infection (N=8).ELISA was used to assess antibody responses  against recombinant 19 (as a control). IgG1 responses against EBA-181, PfRh2a and PfRh2b were significantly higher in the asymptomatic individuals. Total IgG antibody responses against PfRh1, PfRh2a, PfRh2b, PfRh5, EBA-175, EBA-181 and MSP119 proteins were negatively correlated with level of parasitaemia. IgG1 responses against EBA-181, PfRh2a and PfRh2b and IgG3 response for PfRh2a were also negatively correlated with parasitaemia.Antibody responses against both EBL and PfRh family proteins were significantly higher in asymptomatic compared to symptomatic individuals, demonstrating an association with clinical immunity. Significant differences in the total IgG responses were observed with EBA-175, EBA-181, PfRh2b, and MSP1P. falciparum invasion ligand proteins higher than those found in symptomatic (non-immune) patients. While these findings will have to be confirmed by larger studies, these results are consistent with a potential role for one or more of these invasion ligands as a component of an anti-P. falciparum vaccine in low-transmission malaria-endemic regions.These data suggest that falciparum malaria patients who develop clinical immunity (asymptomatic parasitaemia) in a low transmission setting such as the Peruvian Amazon have antibody responses to defined  Plasmodium falciparum is a complex process including attachment, reorientation, penetration, and formation of a parasitophorous vacuole. Several merozoite proteins that have a role during the initial steps of attachment and invasion have been extensively studied, including members of the Merozoite Surface Protein family (MSP), AMA-1, Erythrocyte Binding-Like proteins , and the Reticulocyte Binding-Like or Reticulocyte Homologue proteins  [Erythrocyte invasion by d PfRh5) . Moreoved PfRh5) , by enabPlasmodium vivax, but P. falciparum is still responsible for the major cases of severe malaria, and these infections continue to persist even though control measures are in place [P. falciparum transmission clinical immunity to symptomatic malaria is thought to be acquired only after repeated exposure [Malaria in the Amazonian region is hypoendemic and characterized by a low transmission . The malin place . Previouin place ,10. Impoexposure . In contexposure -15. Thisexposure .P. falciparum, this study aimed to test the hypothesis that antibody responses against P. falciparum invasion ligands belonging to both EBL and PfRh protein families might differ between symptomatic (Sym) and asymptomatic (Asy) individuals living in the low-transmission region of the Peruvian Amazon, and hence potentially contributing to explaining mechanisms of clinical immunity observed in the Asy individuals. Recombinant P. falciparum proteins corresponding to the known EBL and PfRh invasion ligands were used to determine the total IgG and IgG isotype-specific antibody responses in both study groups.Given the epidemiological observations indicating clinical immunity against This study was approved by the Universidad Peruana Cayetano Heredia Institutional Review Board (Comite de Etica) in Lima, Peru, and by the New York Blood Center’s IRB (protocol #415). Informed consent was obtained from each adult individual or from the parents or guardians of children less than 18 years of age. The cross-sectional study took place in the Peruvian Amazon region of Loreto in 2008–2010.P. falciparum were collected over the period of 2008–2010 from 14 different communities in Loreto. The individuals were classified into two groups: 37 individuals with symptomatic malaria  and eight individuals with asymptomatic infection . Asymptomatic individuals were identified by active case detection after carrying out a malaria diagnostic survey in communities that reported a malaria outbreak. Passive case detection was used to enroll symptomatic individuals who attended the local Health Care Center for diagnosis and treatment and were diagnosed with P. falciparum infection by light microscopy. The microscopic diagnosis in all individuals enrolled was confirmed as P. falciparum single infection by real time PCR [P. falciparum. Individuals were excluded if they used anti-malarial medication over the two weeks before enrollment and sample collection. No follow up was done. Blood samples were collected before all individuals were treated for malaria with mefloquine/artesunate following the National Drug Policy Guidelines of the Ministry of Health of Peru.A total of 45 plasma samples from individuals infected with P. falciparum 3D7 laboratory strain were used for ELISA. The C-terminal region of EBA-181 (aa 1235–1345), PfRh1 (aa 1318–1574), PfRh2a (aa 2813–2933), PfRh2b (aa 2735–3133) and PfRh5 (aa 31–174) were expressed in Escherichia coli BL21. PfRh2b and PfRh5 were expressed as fusion proteins with glutathione S-transferase (GST) using the pGEX 4T-3 expression vector . The E. coli expressed PfRh4 (aa 1160–1370) protein was a kind gift from Deepak Gaur  and Louis Miller  [Pichia pastoris and were a kind gift from David Narum and Louis Miller  [19; MRA-55, MR4, ATCC Manassas Virginia) was also used as a control [The following recombinant EBL and PfRh proteins corresponding to the h5 aa 31–4 were exE. coli extract prior to dilution and incubation with the bound antigen to remove antibodies to potential E. coli contaminants in the recombinant protein preparations. Briefly, 96-well ELISA plates  were coated with 1 μg/mL of the recombinant protein (0.5 μg/mL for PfRh4) in 0.05 M carbonate buffer, pH 9.6 overnight at 4°C. The next day, plates were washed 6 times with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST) and blocked for 1.5-2 h with blocking buffer (3% BSA in PBST) at 37°C. Plates were washed with PBST and test and control plasma samples (1:200) diluted in blocking buffer were added into each well in duplicate for 2 h at 37°C. The plates were then washed 6 times with PBST and the bound IgG antibodies were detected after 1 h of incubation at 37°C using HRP-conjugated goat anti-human  IgG . IgG1 and IgG3 antibodies were detected after incubation for 1 hr at 37°C with mouse monoclonal antibodies against human IgG1 or IgG3  diluted 1:1,000 in blocking buffer, followed by HRP-conjugated anti-mouse (1:1250). After 6X washing with PBST, bound antibodies were detected by adding the SureBlue tetramethyl benzidine (TMB) substrate . The reaction was stopped by the addition of 2N H2SO4, and the Optical Density (OD) at 450 nm was measured using a SpectraMax 190 ELISA Reader . For the GST-fusion PfRh2b and PfRh5 proteins, each plasma sample was also tested against recombinant GST as a negative control and the corresponding absorbance was deducted from all test values for the fusion proteins. The negative control human serum was composed of a pool of sera from five healthy adult volunteers living in USA who had never been exposed to malaria. Positive responders were defined as those that gave an OD value greater than the mean plus three standard deviations (SD) of the reactivity observed with negative control serum .Peruvian plasma samples were analyzed for total IgG, IgG1 and IgG3 reactivity to the recombinant EBL and PfRh ligands by ELISA as previously described . Plasma p value < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism software .Fisher’s exact test (2x2 contingency tables) was used to compare differences in the seroprevalence between groups. The Mann–Whitney test was used to compare medians between different groups. Correlations between antibody response (as indicated by OD) and parasitaemia or age were examined using Spearman’s test. A p < 0.0001).Two groups of individuals were studied; individuals with symptomatic malaria  and individuals with asymptomatic infection . There were no significant differences in the sex distribution or median of age between the groups. Parasitaemia was lower in the Asy  than in the Sym individuals  . Notably, there were fewer responders in both groups against EBA-140 (25% and 5.4%) and PfRh5 (25% and 8.1%) (Table p = 0.0006), EBA-181 (p = 0.0009) and PfRh2b (p = 0.007) as well as against the control merozoite protein, MSP119 (p = 0.0171). Significantly, a higher total IgG response, as indicated by the OD value, was observed in Asy than Sym individuals for EBA-175 , EBA-181 , PfRh1  and PfRh2b  as well as for MSP119  , PfRh2a , PfRh2b , PfRh5 , EBA-175 , EBA-181  as well as for MSP119 . The total IgG responses against EBA-140 and PfRh4 ligands were not associated with levels of parasitaemia . A significant negative correlation was observed between the total IgG levels and parasitaemia for the following invasion ligands: PfRh1 , PfRh2a  and PfRh2b . The prevalence of IgG3 responders was higher in Asy than Sym for PfRh2a  and PfRh5 .IgG1 and IgG3 are the predominant isotypes produced in response to merozoite invasion ligands -6, and tvs. 0.09; p = 0.0006) and PfRh2b  were significantly higher in the Asy than the Sym group. Notably, both IgG1 and IgG3 were dominant in the Asy group for PfRh2a .Differences in IgG1 and IgG3 responses between the Asy \and Sym individuals were also observed , PfRh2a  and PfRh2b , and the IgG3 levels for PfRh2a .Notably, IgG1 and IgG3 levels were also significantly associated with lower parasitaemia Figure . A negatP. falciparum merozoite ligands in asymptomatic vs symptomatic populations living in a malaria hypoendemic area. Sera from P. falciparum-infected individuals from the Peruvian Amazon were tested against all PfRh and EBL family members known to be involved in P. falciparum invasion. This study’s main finding is that antibody responses against the EBL and PfRh proteins were significantly higher in asymptomatic than symptomatic parasitaemic individuals, suggesting a potential association with the development of clinical immunity. Whether these anti-invasion ligand antibodies directly mediate protective immunity or are simply statistically associated with clinical immunity is beyond the scope of this pilot study. Significant differences in the total IgG responses were observed against EBA-175, EBA-181, PfRh2b and MSP119. IgG1 responses against EBA-181, PfRh2a and PfRh2b were also significantly higher in the asymptomatic individuals. Furthermore, elevated total IgG antibody responses against PfRh1, PfRh2a, PfRh2b, PfRh5, EBA-175, EBA-181 and MSP119 proteins were positively associated with decreased parasitaemia. IgG1 response against EBA-181, PfRh2a and PfRh2b and IgG3 response for PfRh2a were also negatively correlated with parasitaemia. Thus, these results suggest that total and IgG subclass-specific responses to distinct merozoite antigens are significantly associated with protection from high-density parasitaemia and symptomatic malaria.This study is the first to comprehensively analyse the profile of naturally acquired antibodies against the two major families of P. falciparum in some malaria endemic areas  in individuals living in a hypoendemic malaria region of Brazil were much lower thanin individuals living in a hyperendemic area of Cameroon [IgG responses to some but not all EBL antigens has been associated with protection against symptomatic iewed in ). Naturaiewed in . Nonetheiewed in -23 althoc origin ,25. Studc origin ,19. A co19, MSP-2 and AMA-1 [in vitro more effectively than antibodies induced by other vaccine candidates, such as AMA-1 and MSP-1, increased the appeal of using PfRh5 as an anti-blood-stage malaria vaccine [Immune responses against PfRh invasion ligands have been less studied ,26,27. And AMA-1 , but, itnd AMA-1 . Antibodnd AMA-1 ,27. The nd AMA-1 , and EBAnd AMA-1 . The abivs. 38% and IgG3 75% vs. 30%). In contrast, the response against PfRh2b in the Peruvians (31%) was higher than that reported in individuals with malaria in Senegal (8.9%) and Tanzania (5.6%) and lower than those observed in Papua New Guinea (85%) [vs. 70%) [c binding and are potentially associated with functional antibody-dependent cellular cytotoxicity [Three studies have previously examined the presence of antibodies against PfRh ligands ,26,27, bea (85%) ,26. Howeea (85%) . Seropretoxicity ,30. Noneeruvians % was higtoxicity  and the P. falciparum invasion ligands. These results are consistent with a potential role for one or more of these invasion ligands as a component of an anti-P. falciparum vaccine in the low-transmission areas of the Amazon region, but may argue against a one-size fits all strain-transcending blood stage malaria vaccine.Although the contribution of the anti-invasion EBL and/or PfRh ligand antibody responses to protective immunity needs to be confirmed in future studies by functional assays such as growth inhibition assays, the present study strongly suggests that the Peruvian population present in a low transmission setting has a capacity to develop clinical immunity that is likely associated with antibody responses to defined EBL: Erythrocyte binding-like; PfRh: Reticulocyte binding-like; OD: Optical density; Asy: Asymptomatic; Sym: Symptomatic; ADCI: Antibody-dependent cell-mediated inhibition.The authors declare that they have no competing interests.KT, DG, JMV and SL participated in the design of the study. EV, KT, VN, JB collected the samples. MLP, NT and CL designed or carried out the ELISA studies. EV, MLP, JMV and SL drafted the manuscript. All authors read and approved the final manuscript.


Plasmodium infections in community surveys is essential to successful malaria control. Microscopy and rapid diagnostic tests (RDTs) are the main techniques used to diagnose malaria in field-based surveys. While microscopy is still considered the gold standard, RDTs are growing in popularity as they allow for rapid and inexpensive diagnosis. Using data from a prevalence survey conducted in north-western Angola in 2010, the authors aimed to compare the performance of microscopy and RDTs in identifying Plasmodium falciparum infections, using polymerase chain reaction (PCR) as the gold standard.Accurate identification of P. falciparum infections, were utilized. The sensitivity, specificity, positive, and negative predictive values (PPV and NPV) of microscopy and Paracheck-Pf® were compared using the McNemar’s test and the weighted generalized score Chi-squared test for paired data.Results from 3,307 subjects , 1,134 school-aged children (six to 15 year olds) and 948 mothers/caregivers (>15 years of age)), tested for P. falciparum infections determined by PCR and microscopy was 15.9% and by Paracheck- Pf® was 16.3%. Compared to microscopy, Paracheck-Pf® had significantly higher sensitivity (72.8% versus 60%), specificity (94.3% versus 92.5%), PPV (70.7% versus 60%) and NPV (94.8% versus 92.5%). Both tests had significantly lower sensitivity in mothers (36.8% for microscopy and 43.7% for Paracheck-Pf®) than in their children (68.4% in zero to five years-old and 60.6% in six to 15 years-old for microscopy and 80.4% in zero to five year-olds and 76.5% in six to 15 year-olds for Paracheck-Pf®).The prevalence of Both microscopy and RDTs performed suboptimally when compared to PCR. False negativity could be associated with the low parasite density profile of the samples. False positivity may be related to the well-described limitations of those techniques such as level of expertise of microscopists or persistent antigenicity from previous infections in the case of RDTs. Nevertheless, RDTs had enhanced performance comparatively to microscopy in detecting malaria infections, favouring their use in community cross-sectional malaria surveys, where expert performance of microscopy is hard to accomplish. Plasmodium infections is crucial for providing realistic estimates of the burden of malaria and preventing misinformed interventions [Active surveillance through field-based surveys is considered a powerful tool for estimating the burden of malaria, as it identifies sustained foci of transmission perpetuated by asymptomatic carriers and low parasitaemia infections -3. Accurventions ,5.Plasmodium species at low cost [in situ[Plasmodium infections in malaria surveillance programmes in Tanzania, Gambia, Bangladesh and in the 2006–2007 malaria indicator survey in Angola [Microscopy has been the method of choice in determining the prevalence of malaria in epidemiologic surveys, allowing quantification and differentiation of low cost -8. More  [in situ,9,10. RDn Angola -14.pfhrp-2 gene and the prozone effect may lead to false negatives [The choice between microscopy and RDTs is not always clear-cut as the performance of both diagnostic techniques in operational conditions varies depending on transmission intensity, prevalence of infections, and parasite density ,15-17. Megatives -25. Addiegatives . Consideegatives ,18,27-29egatives -32. Alteegatives -37. Neveegatives ,38-40.® in the detection of Plasmodium falciparum infections, using PCR as the gold standard.Using data from a prevalence survey conducted in north-west Angola, this study compared the performance ), of microscopy and Paracheck-PfHealth Research Centre in Angola, translated) [P. falciparum but all other human species are present either alone or in mixed infections. Almost 90% of malaria infections are due to P. falciparum alone, 6.5% to P. falciparum and P. malariae together, 3.7% due to P. ovale curtisi or P. ovale wallikeri alone or in combination with other species and 1.1% due to P. vivax alone [The study was conducted in Bengo province, north-western Angola, in the Health and Demographic Surveillance System (HDSS) study area covered by the CISA project (nslated) . This isnslated) . The mainslated) . A recenax alone .® and PCR were collected from children and their mothers/caregivers, during a baseline field-based prevalence survey implemented between May and August 2010, as described by Sousa-Figueiredo et al.[Finger-prick blood samples for microscopy, Paracheck-Pfdo et al.. Of the ®. All laboratory technicians were supplied with an operational procedures manual regarding laboratory and field diagnosis.The laboratory diagnosis team was composed of five technicians with pre-university training and previous work experience in public and private health units. A five-day retraining course was provided by the CISA project resident laboratory experts and included theoretical and practical sessions on: finger prick blood collection, thick and thin blood smear preparation, Giemsa staining and slide reading, following standard operational procedures, according to the Basic Laboratory Methods in Medical Parasitology manual from WHO . Field wP. falciparum parasites by two independent technicians (double-blind). Discordance in the diagnostic (positivity/negativity) was solved by a third reader and discordance in parasite counts was solved by calculating the mean of the two readings. The agreement between the two laboratory technicians performing the 2 independent readings was 99.9%, Kappa = 0.997, P < 0.001. Assexual parasitaemia was quantified against 200 to 500 leucocytes, assuming a white blood cell count of 8,000/μl as recommended by WHO [Thick and thin blood smears were made on the same slide, air dried and transported to the CISA laboratory where they were stored. The slides were stained with 10% Giemsa for 15 minutes and screened for d by WHO . A slide® test was performed accordingly to the manufacturer .The Paracheck-Pf® 3MMChr filter paper, air dried and stored at 4°C in the CISA laboratory until DNA extraction. Total DNA was extracted using the QIAmp DNA Mini Kit , following the manufacturer’s instructions. Nested PCR was performed using primers complementary to the Plasmodium small subunit ribosomal RNA (SSU-rRNA) gene, as described previously [Blood samples were spotted onto Whatmaneviously ,46 and ieviously .® software version 9.3. Sensitivity, specificity, positive, and negative predictive values (PPV and NPV) of microscopy and Paracheck-Pf®, with PCR as the gold standard, were determined using 2×2 contingency tables and compared using the McNemar’s test (sensitivity and specificity) or the weighted generalized score Chi-squared test (PPV and NPV) for paired data [95) were calculated for each measure listed above. For each of the two diagnostic techniques, the Pearson Chi-squared test was used to assess difference in sensitivity, specificity, and predictive values across the three age groups. Statistical significance was set at p < 0.05.Data were analysed using SASred data . Exact 9Ethical approval was obtained from the Angolan Ministry of Health Ethics Committee. Written informed consent was obtained before inclusion in the study and anti-malarial treatment with ACTs was provided by a nurse or physician when participants had a positive rapid test result. Participants who mentioned feeling unwell were advised to go to the nearest health centre; those deemed with a serious illness were observed by the physician on site if present or transported by the research team to the reference hospital.® performed in relation to PCR as the gold standard. In 3,307 samples screened, 525 (15.9%) were identified as positive P. falciparum infections by PCR and microscopy and 540 (16.3%) by Paracheck-Pf®.Table ® had significantly higher sensitivity, specificity and predictive values  and 2,572 out of 2,782 PCR-negative samples , with a PPV of 60.0% and NPV of 92.5%. Paracheck-Pf® correctly identified 382 out of 525 PCR-positive P. falciparum infections  and 2,624 out of 2,782 PCR-negative samples , with a PPV of 70.7% and NPV of 94.8%  than in their children  (p < 0.0001). No significant change in the specificity of microscopy was observed across the three age groups. Similarly, the sensitivity of Paracheck-Pf® was lower in mothers  than in their children  (p < 0.0001)  combo tests would allow minimizing HRP-2 associated limitations such as false negativity, related to gene deletions or prozone effect; and false positivity associated to persistent antigenicity, extending the feasibility of their use in this context [Plasmodium infections can be developed.In conclusion, given the observed higher sensitivity, specificity and predictive values of RDTs, the data presented here suggest that, for community-based surveys with similar levels of endemicity and transmission rates and where adequate expert performance of microscopy is hard to accomplish, the use of RDTs to determine the prevalence of  context . However context . A systeThe authors declare that they have no competing interests.SVN was responsible for the study design and coordination and contributed to drafts of the manuscript. CFV and DG carried out the PCR analysis and supervised the microscopy work and field diagnosis. CFV drafted the manuscript. YVS performed the statistical analysis and contributed to drafts of the manuscript. JEP contributed to the statistical analysis and drafts of the manuscript. All authors read and approved the final draft of the manuscript.


In Uganda, parasite-based diagnosis is recommended for every patient suspected to have malaria before prescribing anti-malarials. However, the majority of patients are still treated presumptively especially in low-level health units. The feasibility of implementing parasite-based diagnosis for uncomplicated malaria in rural health centres (HCs) was investigated with a view to recommending measures for scaling up the policy.Thirty HCs were randomized to implement parasite-based diagnosis based on rapid diagnostic tests [RDTs] (n = 10), blood microscopy (n = 10) and presumptive diagnosis (control arm) (n = 10). Feasibility was assessed by comparing the proportion of patients who received parasite-based diagnosis; with a positive malaria parasite-based diagnosis who received artemether-lumefantrine (AL); with a negative malaria parasite-based diagnosis who received AL; and patient waiting time. Clinicaltrials.gov: NCT00565071.102, 087 outpatients were enrolled. Patients were more likely to be tested in the RDT 44, 565 (96.6%) than in microscopy arm 19, 545 (60.9%) [RR: 1.59]. RDTs reduced patient waiting time compared to microscopy and were more convenient to health workers and patients. Majority 23, 804 (99.7%) in presumptive arm were prescribed AL. All (100%) of patients who tested positive for malaria in RDT and microscopy arms were prescribed anti-malarials. Parasitological-based diagnosis significantly reduced AL prescription in RDT arm [RR: 0.62] and microscopy arm [RR: 0.72] compared to presumptive treatment. Among patients not tested in the two intervention arms, 12, 044 (96.1%) in microscopy and 965 (61.6%) in RDT arm were treated with AL [RR: 1.56]. Overall 10, 558 (29.4%) with negative results  were prescribed AL.It was more feasible to implement parasite-based diagnosis for malaria using RDT than with microscopy. A high proportion of patients with negative malaria results are still prescribed anti-malarials. There is need to increase access to parasite-based diagnosis where microscopy is used. In order to fully harness the benefits of parasitological confirmation of malaria, it is necessary to reduce the prescription of anti-malarials in negative patients. Malaria presents a diagnostic challenge in most endemic countries in sub-Saharan Africa, yet early diagnosis and appropriate treatment is a basic tenet of current malaria policy . In sub-The role of laboratory diagnosis of malaria is primarily to support clinical care , and theBecause parasite detection is performed by someone other than the prescriber, there is a tendency to ignore microscopy results provided by the laboratory -4. Yet hAlthough the sensitivity and specificity of malaria microscopy and RDTs have been assessed in different studies ,19 and tThis stratified cluster randomized trial  was implemented in 15 out of 20 sub-county government HCs randomly selected in a district of low and 15 in a district of high malaria transmission intensity from March 2010 to July 2011. Stratification was based on transmission intensity. HCs were the primary sampling units. In each district, HCs were randomized to two intervention arms (microscopy and RDT) and control arm (presumptive diagnosis), resulting into five HCs per arm. This trial took a cluster design to avoid contamination between control and intervention group subjects and to allow service providers to operate as they would normally on a day-to-day basis.The trial was carried out in Bushenyi and Iganga districts in Uganda, and commenced before the two districts were partitioned. However, partitioning did not affect the delivery of health services by the time the study was closed. Bushenyi district has a population of 731, 392. The district experiences low and unstable malaria transmission, with people of all ages being at risk. It is epidemic-prone, with occasional malaria outbreaks occurring shortly after the rains. Iganga district has a total population of 540, 939 with 15 HCs at sub-county level. The district experiences very high malaria transmission intensity. Malaria is the leading cause of morbidity and outpatient attendance for all age groups. Additional description of the study setting has been published elsewhere .Health centres were allocated to the three diagnostic arms following simple randomization. In Bushenyi district, three of the five HCs randomized to the microscopy arm were already offering the service. The study strengthened the laboratory of Kabira HC with a microscope and supplies. In Iganga district, two HCs were offering microscopy services but the study strengthened the laboratory for one . In the other three HCs, the posts for laboratory personnel were already filled although the laboratories were not functional. Therefore, the district provided microscopes and laboratory supplies. In the RDT arm, three selected HCs in Bushenyi district and two in Iganga had microscopy services. However, RDTs were introduced and both clinicians and laboratory personnel were trained. They were informed during training that their HCs had been randomized to use RDTs. However, they were free to test patients with microscopy if needed. In the presumptive arm although one HC in Bushenyi and two in Iganga had the laboratory assistants post filled, the laboratories were not functional. They either had no space or laboratory equipment and supplies.Before the study commenced RDTs, AL, laboratory reagents and supplies from the National Medical Stores were delivered to the districts medical stores. This was meant to integrate the study into the district health services delivery system. It was also a strategy for continuity of services from the time of closing the study. RDTs were packaged in cartons of 1, 000 tests. In each carton, there were 40 boxes of 25 tests. Also in the box were 25 alcohol swabs, lancets and a 10 ml bottle of clearing buffer. Each sachet of RDT contained the test device, a loop for collecting blood and a descant. The study HCs were to make requisitions for AL, RDTs, and laboratory supplies together with antibiotics, analgesics and sundries following the usual guidelines. HCs opened up stock cards for RDTs that were updated regularly.®Paracheck. In the microscopy arm, members were in addition trained in: 1) finger prick for collection of blood, 2) thick/thin blood smear preparation, 3) staining smears, and 4) blood smear reading. In the microscopy and RDT arms, data collection commenced after inter-reader reliability reached a very good agreement (kappa coefficient = 0.97). The staff in HCs with microscopy or RDT were instructed to treat patients who have positive results with anti-malarials. Those with negative results were to receive alternative medications after further assessment. Staff in the control arm were only re-oriented to the current malaria treatment guidelines. HC outpatient registers were modified to record additional variables such as the presenting complaints, drugs dispensed and to indicate those prescribed but out-of-stock. The trained staff were charged with training those that were off-duty on the day of training. Further clarifications were provided during supervision. Supervision by the study team and the district laboratory focal persons was carried out weekly during the first two months and monthly thereafter. The supervisory visits were aimed at troubleshooting problems related to the skills of performing an RDT and interpretation of results, quality of the test, quality of blood smears, reagents, status of the microscopes and recording of results. In addition to monthly support supervision, the district laboratory focal person provided further visits during the routine quarterly schedules.Clinicians  and laboratory assistants received a one-day refresher training on-site. The training and subsequent study procedures were a scale-up of the activities performed during the assessment of the accuracy of these diagnostic techniques . All staPatients presenting with fever (by statement or measured) were enrolled to receive service without parasitological confirmation of malaria. Patients were treated on the basis of signs and symptoms only.Patients were enrolled on the basis of fever (by statement or measured). Microscopy was performed by laboratory assistants. These laboratory personnel have two to three years of pre-service training. Thick and thin blood smears were prepared by finger-prick using sterile blood lancets on separate frosted slides. Standard staining was performed using the Field's stain method. Laboratory assistants were only familiar with this staining technique. Blood films were read at magnification x1, 000. Each film was graded as positive  or negative  based on inspection of 200 fields. Microscopy test results were recorded in the laboratory registers. Patients received the results and treatment on the same day of visit.®Paracheck" device . ® Paracheck Pfis based on the detection of histidine rich protein-2 (Pf HRP-2) produced by Plasmodium falciparum trophozoites and young gametocytes. The specimens were drawn by trained clinicians or laboratory assistants using a simple finger-prick. The test preparation and interpretation were done following manufacturer's instructions and standard operating procedures prepared for this study. The test was considered positive when the antigen line was visible in the test window, negative when only the control band was visible. RDT results were recorded in the outpatient registers. Also patients received the results and treatment on that day of visit.Patients presenting with fever (by statement or measured) underwent rapid testing with the "The duration of outpatient visit was assessed in six HCs using a random sample of 1627 consenting patients. When a patient arrived at the HC, time was recorded by the research assistant on a "time sheet." The "time sheet" was then given to the patient. Thereafter, the time was recorded by clinicians and laboratory personnel at every point of service delivery. The "time sheet" was finally retained at the dispensing window. For the laboratory personnel, the effective contact time was comprised of: drawing a sample from the patient, slide preparation, scanning the 200 film fields until declaring a slide negative and reporting of results. With regard to RDT, the effective contact time was comprised of: drawing blood samples from patients, applying samples onto the test, test reading and reporting of results.Data collection was carried out weekly by the research assistants by extracting information from the laboratory and outpatient registers from March 2010 to July 2011.p-values) were set at 0.05 and confidence intervals (CI) were calculated at the 95% level. Socio-demographic and symptom data was presented using descriptive statistics: distribution by age, number and percent of patients with positive and negative results by transmission setting and diagnostic method and prescribed drugs.The collected data were manually checked and cleaned. Data were double entered by two trained database assistants in a customized entry template with in-built consistency checks in EpiData 3.1 software . The two data sets were validated to check for entry errors. Before analysis in Stata version 10 , the data was declared a cluster design using the "svyset command" with HCs as primary sampling units. Further, the Poisson regression model was fitted while accounting for clustering. Probability values . The study was registered with the Clinicaltrials.gov (NCT00565071).The study was carried out in 30 sub-county level government HCs. Each diagnostic arm had five HCs located in an area of low malaria transmission and another five in a setting of high transmission intensity. Overall, 102, 087 outpatients presenting with fever were enrolled. This included 52, 116 (51%) in low and 49, 971 (49%) in high transmission settings. Those enrolled in the presumptive arm were 23, 884, RDT 46, 131 and microscopy arm 32, 072  counselling and testing. Therefore, they were experienced in testing HIV using whole blood on rapid test strips, devices or cassettes that work on the same principle as malaria RDTs. At this level of the health care services delivery system the staff in the RDT arm mentioned that one day of training is adequate, mainly focusing on practical (preparation of test and interpretation of results). On the first day, few staff especially nursing assistants had a challenge in collecting blood for the RDT using a loop in children under five years. However, they had gained adequate skills by the end of the first week. In the microscopy arm, all staff attended the practical session. Although they were able to prepare a thick blood smear, reading and interpretation of results was the responsibility of the laboratory personnel. Eight of the HCs (four in each district) in the microscopy arm did not have electricity. They used natural light when scanning for malaria parasites. In the remaining two HCs, black-out was frequent and electricity as source of light for malaria microscopy was unreliable.The effective average contact time of clinicians with febrile outpatients (excluding time for investigation) was 11.4 min [95%CI: 11.1-11.7], and was similar in the three arms Table . The meaOverall, 64, 110 in the two intervention arms were tested Table . The proOverall, 28, 289 (44.2%) [95%CI: 30.8-57.5] had a positive test result. In the low transmission setting, the proportion of those who tested positive in the RDT arm was 3, 313 (20.5%) [95%CI: 17.4-23.6] and microscopy arm 1, 548 (14.0%) [95%CI: 11.2-16.7]. The proportion of those testing positive in the RDT arm 19, 414 (68.6%) [95%CI: 62.2-74.9] in the high transmission setting was also significantly higher than in the microscopy arm 4, 014 (47.4%) [95%CI: 35.7-59.1]. Generally, the risk of having a positive test was higher among children under-five years of age 10, 900 (61.1%) compared to patients five years old and above 17, 124 (37.4%) . This relationship was maintained in the low transmission area , and increased in the high transmission setting .All patients in the control arm were treated with anti-malarials Figure . PrescriAll patients in both microscopy and RDT arms with positive results received anti-malarial treatment Figure . OverallOverall, 10, 558 (29.5%) were treated with AL. Patients with RDT negative results were more often prescribed AL in the high transmission setting 3, 301 (37.1%) compared to those in the low transmission area 1, 744 (13.6%) . In the microscopy arm, there was a two to five-fold likelihood of prescribing AL to patients with negative results in the high transmission area 3, 436 (77.2%) than in the low transmission setting 2, 002 (21.0%) . This implies that the likelihood of not accepting negative microscopy results was higher than that for RDT in a setting of high malaria transmission.Within the low transmission setting, children under-five years of age with negative microscopy results were more often prescribed AL compared to those in the RDT arm . A similar pattern in AL prescription was observed among patients five years and above. In the high transmission setting there was no statistically significant difference in AL prescription among children under-five years when those treated under microscopy were compared to those under RDT. Among patients five years and above in the high transmission setting 2, 055 (30.2%) in the RDT arm and 2, 298 (74.9%) in microscopy arm were prescribed AL .This study provides data on the comparative feasibility of microscopy and RDT among outpatients attending government rural primary health care centres located within areas of varying transmission intensities. The study findings indicate that RDT was more feasible than microscopy and prescribers were unlikely to adhere to negative results especially in the microscopy arm.It is reported here that patients attending HCs with RDT as the method for malaria investigation had a higher probability of getting a parasitological test done regardless of age and transmission setting. Out of 133 clinical and laboratory staffs trained in 30 HCs, 56 were in the RDT arm and they performed the testing of patients during the whole period of study implementation. In the microscopy arm, the staff either had clinical or laboratory roles, although clinicians were able to prepare usable thick smears. Microscopy was the sole responsibility of the laboratory assistants. This meant that the RDT diagnostic services were not interrupted even if the laboratory assistants were away on leave. Performing RDT takes a much shorter time than microscopy. Therefore given a similar time, more patients could be tested in RDT compared to microscopy. RDTs reduced the patient waiting time compared to microscopy and were thus more convenient for health workers and patients. An earlier investigation  also repThe risk of a febrile patient not getting a malaria diagnostic service was higher in HCs with microscopy and even far higher among patients under-five years of age. The current malaria treatment policy in Uganda and WHO  recommenWith routine use of parasitological confirmation of malaria, prescription of AL was reduced by 28.1% between presumptive and microscopy; and by 38% between presumptive and RDT. This benefit was also reported in other studies ,24, but In the preparation for the study, RDTs, AL and laboratory supplies were delivered by the study team to the district medical stores. However, AL stock-out occurred in the high transmission setting that resulted into a reduction in the number of outpatient attendance. Subsequently this impacted on the number of patients enrolled in the high transmission setting. RDTs were in-stock throughout the study implementation period. The training of staff for this study took one day. However, other cadres of staff without such skills as HIV testing might require slightly longer period (three to five days). To acquire adequate skills for example in testing malaria with RDT, it is important to make frequent supervisory visits and tapering the number of visits with time. In this study, it was planned to have three diagnostic arms . Further research should consider incorporating the arm of RDT plus microscopy.RDT was more feasible than microscopy and patients with negative results received significantly more AL in the microscopy arm compared to those in the RDT arm. To realise the benefits of parasitological confirmation of malaria, service providers need to adhere to test results and they need guidance regarding management of patients with negative results.The authors declare that they have no competing interests.All authors conceived and designed the study; VB and FN collected, analysed, interpreted the data and drafted the manuscript; PM critically revised the manuscript. All authors read and approved the final manuscript


Plasmodium falciparum malaria is responsible for over 250 million clinical cases every year worldwide. Severe malaria cases might present with a range of disease syndromes including acute respiratory distress, metabolic acidosis, hypoglycaemia, renal failure, anaemia, pulmonary oedema, cerebral malaria (CM) and placental malaria (PM) in pregnant women. Two main determinants of severe malaria have been identified: sequestration of parasitized red blood cells and strong pro-inflammatory responses. Increasing evidence from human studies and malaria infection animal models revealed the presence of host leucocytes at the site of parasite sequestration in brain blood vessels as well as placental tissue in complicated malaria cases. These observations suggested that apart from secreting cytokines, leucocytes might also contribute to disease by migrating to the site of parasite sequestration thereby exacerbating organ-specific inflammation. This evidence attracted substantial interest in identifying trafficking pathways by which inflammatory leucocytes are recruited to target organs during severe malaria syndromes. Chemo-attractant cytokines or chemokines are the key regulators of leucocyte trafficking and their potential contribution to disease has recently received considerable attention. This review summarizes the main findings to date, investigating the role of chemokines in severe malaria and the implication of these responses for the induction of pathogenesis and immunity to infection. Anopheles mosquitoes that are infected with parasites of the genus Plasmodium. Most cases of severe disease are caused by Plasmodium falciparum, which is endemic in sub-Saharan Africa and throughout the tropics. The blood stage of the parasite is largely responsible for malaria-associated pathology , which allows them to bind to endothelial cells, sequester in vascular beds and avoid clearance in the spleen. Although the precise mechanisms leading to severe malaria syndromes are not completely understood, it is accepted that sequestration of parasitized red blood cells (pRBC) is a major determinant of disease development. Parasite sequestration is thought to induce obstructions in blood flow resulting in hypoxia and haemorrhages  . To date, nearly 50 chemokines have been identified  as well  Luster, .Table 1et al.Chemokines and chemokine receptors are essential in two distinct processes of leucocyte migration. The first is the directional migration and positioning of leucocytes within lymphoid organs as well as in peripheral tissues. In this context, cells encounter chemoattractant signals in a complex spatial and temporal pattern, in which cells follow a gradient of increasing concentration towards the source of the chemokine  may in part contribute to CCL5 suppression during SMA, as children with high levels of intramonocytic pfHz have been shown to have lower circulating CCL5 levels than children with only low levels of intramonocytic pfHz , CXCL10 , CXCL4 and CXCL8 have been found to be significantly elevated in CM cases compared with mild malaria cases or healthy control individuals , CCL4, CXCL8, CXCL9, CXCL13 and CXCL16 have been observed in the placentae of women with PM  infection  in the recruitment of inflammatory leucocytes to the brain of malaria-infected mice has been extensively investigated . As CXCL−/− mice or mice receiving anti-CXCL10 neutralizing antibodies over the course of P. berghei-ANKA infection were found to have reduced cerebral intravascular inflammation and were protected against fatality , which binds CXCR3B, a spliced variant of the chemokine receptor CXCR3, was found to be induced in the brain during P. berghei-ANKA  that were identified as biomarkers of CM in humans (Armah et al.et al.Current views in the field propose that the same inflammatory responses that contribute to severe malaria might be also involved in the control of parasitaemia. This concept has discouraged the use of anti-cytokine treatments (van Hensbroek Supported by the Australian Government National Health and Medical Research Council IRIISS and Project Grant 1031212.