Malaria and HIV are major causes of morbidity and mortality in sub-Saharan Africa with both diseases highly endemic in Nigeria. This study was conducted to assess the effect of long lasting insecticide treated net (ITN) use and malaria preventive education on burden of malaria parasite among people living with AIDS (PLWHA) at Osogbo southwestern Nigeria.A descriptive cross-sectional study of newly recruited consenting PLWHA that were screened consecutively for malaria, those positive were treated with artemisinin combination therapy. All PLWHA were educated about malaria infection, given ITN and followed up monthly for three months when they were rescreened for malaria infection. Data collected was analyzed using descriptive and inferential statistics.A total of 392 (92%) PLWHA completed the study. Mean age of the respondents was 33 ± 11.6 years. They were 120 (31%) males and 272 (69%) females. Majority (80%) were married, over 33% completed secondary education while 21% had tertiary education. Most were traders (40%) and artisans (25%). About 60% had Plasmodium falciparum malaria parasitemia at baseline which drastically reduced to 5% at three months with ITN use and malaria prevention education.Malaria is a major preventable condition among PLWHA. Preventive education and ITN use reduced malaria parasite burden among this population. Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae [This is a descriptive cross-sectional study from June to December 2010. The study population included new PLWHA recruited consecutively by trained research assistants after they have consented to participate following through explanation of the study by the researchers to them and/or their family caregivers (where respondent was less than 18 years). The required sample size of 334 was calculated using an appropriate statistical formula for estimating the minimum sample size in descriptive health studies [n = Z2pq/d2]  and find2pq/d2]  where 32Ethical consideration included taking informed consent from respondents and their family caregiver, using serial numbers and not names to maintain confidentiality of information obtained. Permission to conduct the study was granted by the Osun State Hospital Ethics and Research Committee. Data collected were kept in a password computer.Data obtained was entered into SPSS version 16. Summary statistics using mean, median, standard deviation, range for continuous variables and frequency/percentages for categorical variables were generated. Chi square statistics was used to test association between categorical variables with p value less than 0.05 taken as significant.A total of 392 PLWHA out of 425 completed the study (92%). Thirty-three respondents opted out of the study for personal reasons such as unscheduled traveling. No respondent died or were lost to follow up during the study period. Mean age of the respondents was 33 years ± 11.6 years (Range = 13-60 years).Table Table Table Common reasons for inconsistent or non use of ITN included night duty (39%), reaction to the permethrin embedded in the net (27%) while some respondents never liked sleeping under ITN (23%).This study assessed the effect of ITN use and malaria preventive education on burden of malaria parasitemia among people living with AIDS. It reported that preventive education and ITN use reduced malaria parasitemia over a period of three months. The high malaria burden at baseline reduced remarkably by three months. Previous studies had shown this among pregnant women and children ,4.Majority of the study population had malaria at baseline. This finding is expected as the study area is an area of intense year round malaria transmission. Goselle et al. in 2009 in a study on malaria infection among PLWHA in Jos, Nigeria reported 32% while Saracino et al. in 2012 reported 26% in Beira, Mozambique . HoweverThis study reported that ITN use protects against malaria among the participants. This entails that malaria preventive education and ITN should be routinely given to PLWHA to prevent malaria among them, thereby improving their quality of life.Most respondents gave financial constraints and non availability as their reason for non use of ITN at baseline. Various studies on challenges to ITN use in this environment had reported this finding. For example, Olajide et al. in a study on challenges with ITN use among pregnant women reported that pregnant women did not sleep under ITN because of non availability (53%) and cost (24%) . This imThis study further showed that consenting PLWHA should be routinely visited at home to observe the conditions they lived. This will improve relationship between these PLWHA and the healthcare team. Studies had showed that the living conditions of patients affect their health and that improving these living conditions will improve their quality of life ,10.This study also reported that the respondents working in the formal sector with high parasitemia at baseline had no malaria parasitemia after three months of continuous ITN use at night and malaria preventive education. This was due to strict adherence to instructions given by their healthcare workers. This could be explained by higher formal education and better environmental conditions of respondents working in the formal sector. Studies had reported the positive influence of formal education on adherence to malaria preventive messages ,4.Also this study found that more than two-fifth of respondents who were not using ITN had malaria parasitemia at third month follow up. This implies that ITN use prevent exposure to mosquito bite hence thwarting the plasmodium organism transmission. Various studies had reported that mosquito bite is necessary for the transmission of the plasmodium organism -13.This study reported that the main reasons for inconsistent or non-use of ITN included night duty and reaction to permethrin embedded in the net. Permethrin impregnated ITN had been shown in various studies to prevent the plasmodium transmission however reaction to this chemical agent cannot be foreclosed ,14. SincLimitation to the study is that it is a preliminary study on effect of preventive education and ITN use on malaria parasitemia among PLWHA. Also, a convenience sample of PLWHA was selected.Preventive education and consistent ITN use reduced malaria infection among PLWHA. There should be ongoing preventive education on malaria with free provision of ITN with home visits by healthcare workers targeted at ensuring adherence to ITN use.The authors declare that they have no competing interests.SAO and NAA made substantial contributions to conception and design of the study while all the authors were involved in data collection, analysis and interpretation. All authors were involved in writing the manuscript and approved the final copy.SAO lectures at Department of Community Health, Faculty of Clinical Sciences, College of Health Sciences, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria. Areas of research interest include communicable and non communicable diseases, family issues; NAA lectures at Department of O&G, Osun State University, Osogbo, Nigeria. Areas of research interest include reproductive and infection epidemiology, gynaecological diseases; EAA is an associate professor at the Department of Community Health, Faculty of Clinical Sciences, College of Health Sciences, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria. Areas of research interest include communicable and non communicable diseases, family issues; ISM is a medical officer at the Antiretroviral clinic, State hospital, Osogbo, Nigeria.Study site: Antiretroviral clinic, State hospital, Osogbo, Osun state, Nigeria


Plasmodium falciparum infection, complement receptor-1 (CR1) on erythrocyte’s surface and ABO blood group play important roles in formation of rosettes which are presumed to be contributory in the pathogenesis of severe malaria. Although several studies have attempted to determine the association of CR1 polymorphisms with severe malaria, observations remain inconsistent. Therefore, a case control study and meta-analysis was performed to address this issue.In Common CR1 polymorphisms (intron 27 and exon 22) and blood group were typed in 353 cases of severe malaria (SM) , 141 un-complicated malaria and 100 healthy controls from an endemic region of Odisha, India. Relevant publications for meta-analysis were searched from the database.The homozygous polymorphisms of CR1 intron 27 and exon 22 (TT and GG) and alleles (T and G) that are associated with low expression of CR1 on red blood cells, conferred significant protection against CM, MOD and malaria deaths. Combined analysis showed significant association of blood group B/intron 27-AA/exon 22-AA with susceptibility to SM (CM and MOD). Meta-analysis revealed that the CR1 exon 22 low expression polymorphism is significantly associated with protection against severe malaria.The results of the present study demonstrate that common CR1 variants significantly protect against severe malaria in an endemic area. Plasmodium: Plasmodium falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. A recent report of World Health Organization (WHO) estimates about 3.3 billion individuals to be at risk worldwide and reports 216 million cases in 2010 P. falciparum parasite causes severe disease characterized by cerebral malaria (CM), multi-organ dysfunction (MOD), and non cerebral severe malaria (NCSM), which contributes to high mortality P. falciparum infected red blood cells (RBCs) with un-infected RBCs has been presumed to be one of the factors contributing to the development of severe disease P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of infected RBCs, binding to various molecules like complement receptor 1 (CR1/CD35) Malaria, a mosquito borne parasite infection, is caused by various species of genus CR1 is a membrane glycoprotein expressed on wide range of cells viz. red blood cells (RBCs), monocytes, granulocytes, glomerular podocytes, follicular dendritic cells, all B cells and some T cells P. falciparum malaria has been investigated earlier P. falciparum malaria. The association of intron 27 polymorphism with severe malaria has been contradictory. The CR1 low expression genotype (TT) has been shown to be associated with protection against cerebral malaria in an eastern Indian cohort P. falciparum infection The role of several common single nucleotide polymorphisms (SNPs) in severe P. falciparum malaria Blood group is another important factor determining rosetting capacity of infected RBCs and has been associated with pathophysiology of severe The observed relationship between CR1 polymorphisms and severe malaria remains inconsistent. Since previous studies were under powered, we conducted a tertiary care hospital based case-control study comprising of large number of patients to probe this relationship. A meta-analysis was further performed to summarize the earlier observed data . Since CThe study and its protocols was approved by the Institutional Human Ethics Committee of S.C.B. Medical College Cuttack. Blood samples were collected after written consent of the healthy controls and patients or accompanying person (in case of comatose patients).P. falciparumP. falciparum infection. P. falciparum infected individuals were categorized based on WHO guidelines. Uncomplicated malaria (UM) was defined as patients with fever and positive for P. falciparum infection. Severe malaria (SM) was categorized into three groups based on patients clinical features: 1) Cerebral malaria (CM), 2) Non cerebral severe malaria (NCSM) and 3) Multi-organ-dysfuction (MOD). CM was further defined as patients with altered sensorium, GCS  of ≤10. NCSM patients had one of the several manifestations of severe malaria without cerebral involvement, namely severe anaemia (haemoglobin <5 g/dl), acute renal failure (serum creatinine >3 mg/dl), jaundice (serum bilirubin >3 mg/dl), acute respiratory distress syndrome (PaO2/FIO2<200), haemoglobinuria (dark red or black colored urine positive for haemoglobin) and shock (systolic BP of <80 mm Hg). MOD was diagnosed based on presence of two or more organ involvement like CNS (GCS≤10), respiratory (PaO2/FIO2<200), renal failure (serum creatinine >3 mg/dl) and hepatic dysfunction  Plasmodium species, ii) chronic disease like tuberculosis, chronic renal failure, cirrhosis of liver and autoimmune diseases like systemic lupus erythematosus and rheumatoid arthritis. 100 healthy controls (HC) from a similar geographical background were enrolled. None of the healthy reported history of clinical malaria in the last 5 years. They were essentially healthy and negative for P. falciparum infection. The risk of exposure to malaria was similar for both HC and patients. P. falciparum infected patients and HC were not related to each other. About 5 ml of venous blood was collected in EDTA vials from all enrolled patients.The study was conducted at SCB Medical College and Hospital, Cuttack, Odisha. The state is considered endemic for malaria with more than 85% cases attributed to DNA was isolated from whole blood by QIAamp DNA Blood Mini Kit (QIAGEN). Samples were genotyped for intron27 HindIIIA>T, exon22 3650A>G, gene by PCR-RFLP as described earlier P. falciparum infected patients (n = 388) and healthy controls (n = 100) were typed by commercial haemagglutination kit .Blood group of An extensive search was performed by two authors AKP and MP on Pubmed and Science direct with a combination of following key words: “CR1”, “polymorphism”, “falciparum” and “malaria”. To find out additional relevant reports, references of original studies were screened thoroughly. Eligible studies included in this meta-analysis supported the following criteria: (a) association of intron 27 and/or exon 22 polymorphism and severe malaria (b) use of a case-control protocol (c) genotypes frequencies or Odds ratio (OR) and 95% confidence interval (CI) values and (d) published in English language.http://biostat.mc.vanderbilt.edu/twiki/bin/view/Main/PowerSampleSize) A power calculation was performed a priori using PS: Power and Sample Size Calculation Software  indicates heterogeneity among studies and combined OR was calculated by random effect model (the Mantel-Haenszel method) 2 statistics was used to quantify inter study variability. It ranges between 0% and 100%, where a value of 0% indicates no observed heterogeneity, and larger values indicate an increasing degree of heterogeneity P value less than 0.10 was considered to be significant.All statistical analysis for meta-analysis was performed by comprehensive meta-analysis (CMA) V2 software. The association of intron 27 and exon 22 with severe malaria was assessed by calculation of OR and 95% CI in various models: (i) the allele contrast , (ii) homozygous comparison , (iii) heterozygous comparison , (iv) the dominant model  and (v) recessive model . Heterogeneity between the studies was tested by chi-square based Cochran’s Q statistic. A significant P. falciparum-infected patients were enrolled in the present study, including 353 severe (SM) and 141 uncomplicated patients (UM). Severe P. falciparum malaria patients were further subdivided into cerebral malaria (CM) (n = 97), multi-organ dysfunction (MOD) (n = 129) and non-cerebral severe malaria (NCSM) (n = 127). 100 healthy controls (HC) were included. The difference of mean age between all clinical categories and HC was comparable. Significantly higher levels of haemoglobin were observed in HC than UM and other clinical categories of severe malaria (P<0.0001)  .To assess the prevalence of CR1 polymorphism (exon 22 and intron 27) in studied population, 100 healthy controls were genotyped by PCR-RFLP and the results are shown in P. falciparum malaria. Two functional CR1 polymorphisms (exon 22 and intron 27) in P. falciparum infected subjects were typed and results are shown in We used a case control study to examine whether common polymorphism at CR1 gene is associated with severe For the CR1 exon 22 polymorphism, the frequency of the GG genotype and variant allele G were significantly lower in SM compared to UM . The SM patients were further divided into sub-clinical categories and compared to UM. Prevalence of homozygous mutant (GG) and allele G were significantly lower in CM  and MOD  compared to UM. HC displayed significantly higher frequency of allele ‘G’ and GG genotype than CM and MOD (data not shown). Genotype and allele distribution among NCSM, UM and HC were comparable .For CR1 intron 27 polymorphism, frequency of low CR1 expressing genotype ‘TT’ and allele ‘T’ were significantly higher in UM than SM , CM  and MOD . Furthermore, prevalence of heterozygous (AT) also significantly higher in UM compared to SM , CM  and MOD . The genotype and allele distribution of CR1 intron 27 polymorphism were comparable in NCSM, UM and HC .Since both polymorphisms of CR1 were associated with protection against severe malaria, we analysed the distribution of CR1 haplotypes in different clinical categories to study their probable association. The prevalence of haplotype ‘A-A’ was significantly higher in SM , CM  and MOD  compared UM. Distribution of haplotypes in UM and NCSM was comparable .P. falciparum infected patients were typed for blood groups comprising of CM (n = 75), MOD (n = 93), NCSM (n = 101) and UM (n = 119). Blood group ‘B’ was significantly higher in SM , CM , MOD  and NCSM  compared to UM in current study  with protection against severe malaria, possible role of these variants to the disease outcome was explored. For CR1 exon 22 polymorphism, prevalence of both heterozygous (AG) and homozygous (GG) genotypes were significantly higher in survivors than non-survivors . For CR1 intron 27 polymorphism, the frequency of AT and TT genotype were also higher in survivors than non-survivors  suggesting protection against mortality. Interestingly, the mutant alleles of both polymorphisms also showed higher prevalence in survivors  . We furtWe also analysed the prevalence of different blood groups in survivors . AlthougP. falciparum malaria and prognosis. A combined analysis of these two important factors was done based on the hypothesis that they contribute to rosette formation which has a significant role in the pathogensis of severe malaria.We had earlier demonstrated an independent association of CR1 polymorphisms, haplotypes and ABO blood groups with predisposition to severe As shown in Characteristics of eligible studies are shown in Funnel plot and Egger’s test were conducted to assess the publication bias in the reports included for meta-analysis. The shape of funnel plots did not reveal any evidence of asymmetry . Egger’s test further strengthens statistical evidence of funnel plot symmetry and did not show any evidence of publication bias .2 statistics. Results are shown in Heterogeneity among studies was tested by Q test and IMeta-analysis result of exon 22 polymorphism is shown in Meta-analysis of CR1 intron 27 polymorphism is shown in P. falciparum malaria in an endemic region of Odisha, India. Our data suggest that mutants of CR1 polymorphims (exon 22 and intron 27) and blood group ‘O’ protect P. falciparum infected subjects from severe disease and mortality. Furthermore, a meta-analysis revealed a significant association of exon 22 polymorphism with severe malaria in most endemic regions of the world.In this case-control study we investigated the role of common CR1 variants and blood groups in P. falciparum infection P. falciparum infection. Data of the present study showed significant association of CR1 (intron 27 and exon 22) mutants with protection against CM and MOD. In an earlier report association of CR1 variants with CM but not with MOD had been demonstrated Malaria has a strong selective pressure on human genome. In endemic areas, it positively affects blood cell polymorphisms which protects against P. falciparum malarin in different endemic geographical regions CR1 intron 27 and exon 22 variants are associated with severe P.falciparum infection in malaria endemic areas.Meta-analysis is a powerful tool which combines finding of independent similar studies and derive a definitive conclusion Supplement S1PRISMA 2009 checklist.(DOC)Click here for additional data file.


Malaria rapid diagnostic tests (RDTs) are a useful tool in endemic malaria countries, where light microscopy is not feasible. In non-endemic countries they can be used as complementary tests to provide timely results in case of microscopy inexperience. This study aims to compare the new VIKIA Malaria Ag Pf/Pan™ RDT with PCR-corrected microscopy results and the commonly used CareStart™ RDT to diagnose falciparum and non-falciparum malaria in the endemic setting of Bamako, Mali and the non-endemic setting of Lyon, France.Blood samples were collected during a 12-months and six-months period in 2011 from patients suspected to have malaria in Lyon and Bamako respectively. The samples were examined by light microscopy, the VIKIA Malaria Ag Pf/Pan™ test and in Bamako additionally with the CareStart™ RDT. Discordant results were corrected by real-time PCR. Sensitivity, specificity, positive predictive value and negative predictive value were used to evaluate test performance.Plasmodium falciparum in Lyon and Bamako, respectively, performing similar to PCR-corrected microscopy.Samples of 877 patients from both sites were included. The VIKIA Malaria Ag Pf/Pan™ had a sensitivity of 98% and 96% for P. falciparum, making it a valuable tool in malaria endemic and non-endemic regions.The VIKIA Malaria Ag Pf/Pan™ performs similar to PCR-corrected microscopy for the detection of  Malaria diagnostic plays a key role in malaria control and elimination programmes in order to avoid unnecessary anti-malarial therapy, to prevent drug resistance and to enhance case finding. The World Health Organization (WHO) estimated that in the African region, approximately one half of suspected malaria cases received parasitological confirmation . The numThe commonly accepted gold standard diagnostic method for detecting malaria was, for decades, microscopic reading of Giemsa-stained thick and thin blood films. Microscopy constrains in malaria endemic regions are the need for skilled laboratory technicians, good quality reagents and well-maintained microscopes as well as its time consumption . Even inMalaria rapid diagnostic tests (RDTs) are an alternative diagnostic method for endemic regions, where microscopy has not been implemented, as well as for non-endemic countries, where they are able complement microscopy in screening febrile travellers. The first commercial RDT was marketed in 1994, and more than 200 devices from more than 60 manufacturers are now available in the world market. Some of them were successfully evaluated by international organizations -12. SeveThe first part of the study was performed during a 12-months period from January to December 2011 in the non-endemic setting of Lyon, France. Study site was the Parasitology Department of the Lyon University Hospital, were symptomatic travellers, returning from malaria endemic countries, are routinely checked for malaria. Blood samples were collected from febrile patients suspected of malaria due to recent history of travel abroad. Malaria diagnosis was carried out with Giemsa-stained thick and thin blood smears and real time PCR was performed as previously described . No specPlasmodium falciparum accounts for 95% of all malaria infections at the country level. Inoculation rates vary between 30 and 50 infective bites per human per transmission season in the periurban area.The second part of the study was conducted between July and December 2011, in a level two general health centre (Centre de Santé de Référence de la commune VI), where minimal laboratory equipment is available, in the city of Bamako, Mali. This structure is located in a district area of approximately 800,000 inhabitants, with limited access to a higher level hospital facility. Malaria is hyperendemic in the peripheral villages, mesoendemic in the periurban area and hypoendemic in the city of Bamako . MalariaAll patients presenting with a temperature >37.5°C and with suspected malaria were included into the study after informed consent was obtained. Exclusion criteria were symptoms associated with signs of severe malaria according to the definitions of the WHO , and absThick and thin films were prepared on a single slide. After thin films were fixed in methanol and air-dried, slides were stained in 10% Giemsa solution for 15 min. The parasitaemia was recorded as number of asexual parasites counted per 200 white blood cells present in the thick smear. The thick-film was considered to be negative, if no parasite had been found in 100 fields at 1,000 × magnification. All the thick and thin blood smears were read immediately by two local investigators in both sites, and secondly read by an expert at the Parasitology Department of the Lyon University Hospital, as a quality control. All microscopists were blinded to the results of the RDTs.The VIKIA Malaria Ag Pf/Pan™ test was performed by medical or pharmaceutical students at the Lyon study site, and by local community health workers at the Bamako study site, immediately after the samples were collected. The Lyon study site performed only the VIKIA Malaria Ag Pf/Pan™  test, while in Bamako the CareStart Malaria™ , a commonly used RDT in Mali, was used as an additional control. Both tests were used according to manufacturer’s recommendation , G21MR ). The VIKIA Malaria Ag Pf/Pan™ test was read at different time points , while the CareStart Malaria™ test was read after 20 minutes as recommended. Community health workers in Bamako and medical and pharmaceutical students in Lyon were trained by the same investigator to use both tests according to standard operating procedures. Each staff member was qualified for the global study protocol and the specific SOPs before the start of the study. An external quality monitoring in Bamako was conducted twice during the study. Study quality improvements were added to SOPs when deficiencies were identified.All discordant results between microscopy and the two RDTs were resolved by PCR and test characteristics were recalculated according to the PCR-corrected results. DNA of blood samples from the Lyon study site was extracted using a spin-column procedure . DNA from blood spots on filter papers from the Bamako study site was extracted using Instagene Matrix resin™  . SpeciesThe data collected were computerized using Excel program . Statistical analysis was performed with Stata 12 . Sensitivity was calculated as the proportion of positive test results obtained among samples containing malaria parasites as identified by PCR-corrected microscopy. Specificity was calculated as the proportion of negative test results among samples negative in PCR-corrected microscopy. Positive and negative predictive values were obtained by the proportion of true-positive results among all positive samples and the proportion of true negative results among all negative samples, respectively. Sensitivity, specificity, positive and negative predictive values were determined for both RDTs at all reading time points. A 95% confidence interval (95% CI) was given for each parameter. Samples with mixed infections were not considered in the test performance calculations. Proportions were compared using the McNemar test with p-values <0.05 considered as being significant.Permission to perform the study was obtained from the Ministry of Health in Bamako, Mali. Informed written consent was taken from all study participants and caregivers of all children under 18 at the Bamako study site.Plasmodium falciparum was the most frequently detected species  followed by Plasmodium ovale , Plasmodium vivax  and Plasmodium malariae . Mixed infections were detected in five cases (7.0%). A parasitaemia lower than 1,000 parasites/μl was observed in 16 patients (23%), a high parasitaemia above 100,000 parasites/μl was detected in 5 patients (7%).155 patients were included at the Parasitology Department of the Lyon University Hospital. PCR-corrected microscopy revealed 69 (44.5%) malaria positive blood samples. The majority of patients were of African ethnicity (68%), 19 patients were Caucasian (28%) and the ethnicity was unknown for three patients (4%). According to the information given by the patients, anti-malarial chemoprophylaxis was regularly taken by 32% (n=22). Most of malaria infected travellers returned from West Africa , followed by Central Africa , East Africa , India , South-East Asia  and Central Asia . Plasmodium falciparum diagnosis was confirmed in 48/49 patients and for 5/5 patients with mixed infections . Four negative samples and two P. ovale samples were false positive for P. falciparum after 15 minutes, increasing to three P. ovale samples positive for P. falciparum after 20 minutes. False negative results for all Plasmodium species decreased from seven to four when readings were performed at 15 and 30 minutes respectively. The test results did not differ regarding the level of parasitaemia. Differences between the VIKIA Malaria Ag Pf/Pan™ and corrected microscopy were not significant for the detection of P. falciparum (p=0.07), but significant for the detection of non-falciparum species (p=0.03).ns Table , when rens Table . MalariaAt the “Centre de santé de reference de la commune VI” of Bamako, 727 patients were included during the study period. The mean age was 23.5 ± 14.9  and the mean body temperature was 37.8°C  with P. ovale and two patients (1.5%) with P. malariae. Differences in the mean age between malaria and none-malaria patients was not significant (p<0.05). The mean body temperature at admission was higher in malaria patients than in non-malaria patients .135/727 (18.6%) patients were positive for malaria parasites according to PCR-corrected microscopy of thick and thin blood smears Table . None ofPlasmodium falciparum diagnosis was confirmed in 125/130  and 123/130  patients using the CARE-START Malaria™ and VIKIA Malaria Ag Pf/Pan™ test respectively (Table 2). For non-falciparum species the CARE-START Malaria™ identified 3/5 patients  correctly, while the VIKIA Malaria Ag Pf/Pan™ identified 4/5 patients after 15 minutes and 5/5 patients  after 30 minutes reading time. Considering the low number of non-falciparum species detected among these patients, no definitive conclusion can be drawn from these results.Malaria diagnosis  was confirmed in 128/135 patients in both the CARE-START Malaria™ and VIKIA Malaria Ag Pf/Pan™ test after a 30 minutes reading time (Table 4). The CARE-START Malaria™ test produced 18 false positive and 7 false negative results for all Plasmodium species. Similarly, 18 false positive results and 8 false negative results after 15 minutes, decreasing to seven false negative results between 30 and 60 minutes, were obtained from the VIKIA Malaria Ag Pf/Pan™ test. For both tests the number of false negative decreased to three for a parasitaemia >200 parasites/μL and to zero when parasitaemia was >1000 parasites/μL.P. falciparum (p=0.4), but for non-falciparum species (p=0.03) compared to PCR-corrected microscopy method.No significant difference in the detection of falciparum (p=0.13) and non-falciparum (p=0.5) Malaria between the two RDTs was observed. The performance of the VIKIA Malaria Ag Pf/Pan™ test was not significantly different for Local staffs, with limited laboratory skills, were easily trained to the use of the VIKIA Malaria Ag Pf/Pan™. No difficulties in tests readings were noted. Tests were stored at room temperature during the study period, without detected alteration; however no specific study was conducted on the test stability. Technical instructions, provided with the tests, were considered to be clear and explicit for performing the test by local users.P. falciparum malaria in endemic as well as in non-endemic settings and fulfils the requirements for a useful RDT, according to WHO standards. The test performance is comparable to the commonly used CARE-START Malaria™ RDT. At the Lyon and the Bamako sites, the test showed similar results as the PCR-corrected microscopy for P. falciparum detection. The best reading time is between 20 and 30 minutes, and should not be reduced to 15 minutes in order to limit false negative results. Similar results with the VIKIA Malaria Ag Pf/Pan™ were obtained from a study in Cambodia, showing a sensitivity of 93.4% and a specificity of 98.6% for falciparum malaria [The new VIKIA Malaria Ag Pf/Pan™ RDT showed sensitivities above 94% in Mali and 98% in France with specificities above 93% for the detection of  malaria .A meta-analysis calculated a mean sensitivity and specificity of 95.0% and 95.2% respectively for HRP-2 based assays . HoweverIn Bamako, the study was performed under field conditions, carried out by non-expert community health workers. The conditions were representative for a typical medical reference centre in low-income countries; therefore, the data obtained with both RDTs for malaria diagnosis were entirely satisfactory.P. falciparum parasites show deletions or mutations of the hrp-2 gene, leading to false negative results with RDTs [hrp-2 expression in false negative samples was not tested, thus the possibility that these parasites are responsible for some false negative results cannot be excluded. It has as well been suggested that anti-hrp-2 antibodies in humans may explain negative test results despite significant parasitaemia [It is known that a small number of ith RDTs ,20. The sitaemia .P. ovale, P. malariae or even Plasmodium knowlesi. Considering the low number of samples with non-falciparum malaria at both study sites, statistically valid knowledge has not been obtained.A reduced sensitivity for non-falciparum species in combined hrp-2/aldolase RDTs has been noticed in many studies -24, withP. falciparum in U.S. laboratories [This study confirms previous abundant literature showing that RDTs are not able to replace microscopy as the gold standard for malaria diagnosis in most cases. However, RDTs have a justified place in laboratories of endemic as well as non-endemic countries. Main challenges faced in non-endemic regions are a low parasite density, altered parasite morphology due to the use of chemoprophylaxis or empiric therapy and inexperience in identifying malaria parasites due to the small number of cases seen . Those pratories , and fairatories ,9. ThereIn malaria endemic countries, RDTs may contribute to rationalization of treatment of febrile illness and reduce anti-malarial drug consumption in areas where microscopy diagnosis is not available, not reliable or not performed immediately. Successful use of RDTs can be observed in Zambia, where the consumption of artemisinin-based combination therapy could be reduced as a result .VIKIA Malaria Ag Pf/Pan™ showed a similar level of performance than comparative RDTs in endemic and in non-endemic settings. Community health workers were as efficient as medical students in managing malaria cases using both RDTs tested. This new RDT, recently commercially available, could be used to implement efficient malaria diagnosis in low-income endemic countries or complement PCR/microscopy diagnosis in non-endemic regions.This study was supported by IMACCESS. The fund providers had no role in data collection and analysis, decision to publish, or preparation of the manuscript.DE contributed to interpretation of results and drafted the manuscript. SP and ALB designed and coordinated the study and prepared the manuscript. GB was involved in the laboratory work. BT, BC, NC and FS supervised the field study. MB assisted with data entry and laboratory work. All authors read and approved the final manuscript.


Plasmodium falciparum. Drugs currently in use are associated with poor outcome in an increasing number of cases and new drugs are urgently needed. The potential of the medicinal plant Azadirachta indica (Neem) for the treatment of experimental cerebral malaria was evaluated in mice.Cerebral malaria is a rapidly developing encephalopathy caused by the apicomplexan parasite Plasmodium berghei ANKA. Infected mice were administered with Azadirachta indica ethanolic extract at doses of 300, 500, or 1000 mg/kg intraperitoneally (i.p.) in experimental groups, or with the anti-malarial drugs chloroquine  or artemether , in the positive control groups. Treatment was initiated at the onset of signs of brain involvement and pursued for five days on a daily basis. Mice brains were dissected out and processed for the study of the effects of the extract on pyramidal cells’ fate and on markers of neuroinflammation and apoptosis, in the medial temporal lobe.Experimental cerebral malaria was induced in mice by infection with Azadirachta indica ethanolic extract mitigated neuroinflammation, decreased the severity of brain oedema, and protected pyramidal neurons from apoptosis, particularly at the highest dose used, comparable to chloroquine and artemether.Azadirachta indica ethanolic extract has protective effects on neuronal populations in the inflamed central nervous system, and justify at least in part its use in African and Asian folk medicine and practices.The present findings suggest that  Plasmodium falciparum. Cerebral malaria mainly occurs in resource-poor tropical countries, and mostly affects children, t, tA. indAzadirachta indica extract did not protected infected mice from death from cerebral malaria, although the highest doses slightly delayed death time. However, A. indica extract displayed neuroprotective effects mediated probably by mitigating oedema build up, and modulating both neuroinflammatory triggers of apoptosis and apoptosis signaling pathways. The use of extracts of A. indica in African and Asian folk medicine and practices for the treatment of malaria and cerebral malaria is justified. Characterization of the active principle(s) accounting for these effects will probably provide new drugs, which are particularly needed in the current context of increased chemoresistance to chloroquine.The authors declare that they have no competing interests.MF, EAK, SAK, MAA, AM, PFSE, and YHA participated in the design of the experiment. SB and MF performed the experiments. MF, EAK, SAK, MAA, AM, SB, YHA and PFSE participated in the preparation of the manuscript. MF and PFSE analysed the data, prepared the figures, and were in charge of the manuscript submission. All authors read and approved the final manuscript.


Plasmodium falciparum makes polyparasitism a common phenomenon in Sub Saharan Africa. The devastating effects of helminths-Plasmodium co-infections on primary school health have raised global interest for integrated control. However little is known on the feasibility, timing and efficacy of integrated helminths-Plasmodium control strategies. A study was conducted in Zimbabwe to evaluate the efficacy of repeated combined school based antihelminthic and prompt malaria treatment.The geographical congruency in distribution of helminths and Plasmodium and helminths-Plasmodium co-infections was determined by parasitological examinations at baseline and at each treatment point. The prevalence of S. haematobium, S. mansoni, STH, malaria, helminths-Plasmodium co-infections and helminths infection intensities before and after treatment were analysed.A cohort of primary schoolchildren (5-17 years) received combined Praziquantel, albendazole treatment at baseline, and again during 6, 12 and 33 months follow up surveys and sustained prompt malaria treatment. Sustained prompt malaria treatment was carried out throughout the study period. Children's infection status with helminths, S. haematobium, S. mansoni, hookworms and P. falciparum infection in primary schoolchildren by 73.5%, 70.8%, 67.3% and 58.8% respectively . More importantly, the prevalence of STH + schistosomes, P. f + schistosomes, and P. f + STHs + schistosomes co-infections were reduced by 68.0%, 84.2%, and 90.7%, respectively. The absence of anti-helminthic treatment between the 12 mth and 33 mth follow-up surveys resulted in the sharp increase in STHs + schistosomes co-infection from 3.3% at 12 months follow up survey to 10.7%, slightly more than the baseline level (10.3%) while other co-infection combinations remained significantly low. The overall prevalence of heavy S. haematobium, S. mansoni and hookworms infection intensities were significantly reduced from: 17.9-22.4% to 2.6-5.1%, 1.6-3.3% to 0.0% and 0.0-0.7% to 0.0% respectively.Longitudinal data showed that two rounds of combined Praziquantel and albendazole treatment for schistosomiasis and STHs at 6 monthly intervals and sustained prompt malaria treatment significantly reduced the overall prevalence of plasmodium co-infections in primary school children. In areas of stable malaria transmission, active case finding is recommended to track and treat asymptomatic malaria cases as these may sustain transmission in the community.Biannual Integrated school based antihelminthic and sustained prompt malaria treatment has a potential to reduce the burden of helminths- Ascaris lumbricoides (173 million) and Trichuris trichiura (182 million) among others .Plasmodium falciparum and helminths -Plasmodium falciparum co-infections are known to cause anaemia  and red anaemia . Schistoover 415 000 annual deaths and 43.5 million DALYS . Hence, Plasmodium falciparum co-infections in primary schoolchildren and the proven geographical congruency in the distribution of helminths and Plasmodium falciparum in the developing world [Plasmodium falciparum co-infections, rigorous community based efficacy and feasibility studies have been recommended for any integrated control strategy in order to inform on the best practices that will improve the quality of life in primary schools [Owing to the burden of morbidity imposed by helminth-ng world ,37, the ng world  suggesteng world . On the ng world ,40. Howe schools ,37-41.Plasmodium falciparum co-infections in rural and commercial farming areas in Zimbabwe. The effect of treatment intervals on prevalence of polyparasitism and on helminths infection intensities was also assessed.A study was therefore conducted to evaluate the parasitological efficacy of integrated school based antihelminthic praziquantel and albendazole treatment as well as prompt malaria treatment on the prevalence of schistosomiasis, STHs, malaria and helminths -A school based longitudinal intervention study that involved examination and treatment of the study population for malaria, schistosomiasis and STHs at baseline, and again during 6, 12 and 33 months follow up surveys was conducted among primary school age children (5-17 years). Sustained prompt malaria treatment was carried out throughout the study period." The Baseline survey was conducted in June to July 2004. Follow up studies were conducted in November to December 2004, June 2005 and March 2007 respectively. The study population comprised of children living in Nyamaropa rural area located in Shamva district, Mashonaland Central province and Burma Valley commercial farming area located in Mutare district, Manicaland province. The study areas are described in detail elsewhere . Inhabit2 p(1-p), where n is the sample size required, z = 1.96 is the z-score associated with a 95% confidence interval (CI), Δ is the margin of error = 0.05 and p is the prevalence of disease. Based on previous studies in Mutare District, the prevalence of hookworm, 61.7% [S. haematobium (53.1%) observed in Chiweshe and Bushu areas adjacent to Nyamaropa in Shamva district, Mashonaland Central [The sample size was obtained using n = (z/Δ)m, 61.7% , S. haemm, 61.7%  and malam, 61.7%  were use Central  was usedMultistage sampling technique was used to select districts, wards and schools in which the study was conducted. Using rotary method, Mutare district was randomly selected out of the 7 districts in Manicaland province. Ward 7 was randomly selected from the Mutare district out of which 3 primary schools were also selected. Shamva district was randomly selected out of 7 districts of Mashonaland Central province and ward 10 was selected from 29 wards in the district. Nyamaropa Primary School, with an enrolment of over 700 pupils and the only school in Ward 10 was included in the study. Every child from Grade 1 to 6 attending each of the selected schools was eligible for the study. Excluded from the study were Grade 7 children since they could not be followed up for two years as they would have transferred to secondary schools. Also excluded were severely sick children, those who could not provide stool, urine or blood samples and those not willing to participate in the study.All demographic data were obtained by interviewing children. Age of also obtained from the class registers provided by teachers when available.S. haematobium and intestinal helminths  was based on the detection of worm eggs in urine and faeces, respectively. The urine filtration technique as described by Mott et al [Urine and faecal samples were collected between 10:00 am and 2:00 pm in separate wide mouth plastic specimen bottles correspondingly labelled with the laboratory identification numbers assigned to each individual. The samples were processed within two hours of collection. Diagnosis of tt et al  was usedtt et al .S. mansoni, hookworm, T. trichiura, A. lumbricoides), two methods, Kato Katz and the formal ether concentration technique, were used. Results from the two techniques were combined to determine intestinal helminths infection status. In addition results from the two Kato Katz slides prepared on two successive days (one slide/day) were used to determine egg intensities for intestinal helminths. On day one of urine collection, a stool sample was collected from each participant as well. The stool was processed using the formal ether concentration technique as described by Cheesbrough [T. trichiura, A. lumbricoides and S. mansoni ova. Stools samples collected from each individual on day 2 and 3 were processed using the Koto Katz technique as described by Katz et al [S. mansoni infection. In determining the infection status of intestinal helminths, a person who had ova in stool as observed using either the formal ether concentration technique, Kato Katz technique or had ova detected in stool using both techniques was considered positive for the parasite species demonstrated by the ova observed. The individual was considered negative if no ova were detected in stool using both techniques. The number of ova observed for each intestinal helminths using the Kato Katz technique was multiplied by 24 in order to get the number of eggs per gram stool (epg). Since two slides were examined on two successive days the mean egg intensity was calculated by determining the mean of the sum of ova detected on day 2 and day 3 (two days).In order to improve sensitivity in diagnosis of intestinal helminths  as anticoagulant. Thick smears of malaria slides were prepared from fresh blood and the remaining blood was used for plasma separation being used for immunological studies elsewhere. sa stain . The preThe research team provided basic health education about malaria; schistosomiasis and STHs in schools. Children were taught about malaria risks and were asked to continuously seek prompt malaria treatment based on recognition of signs and symptoms of the disease that include fever, headache, nausea, general malaise and joint pains. Leaflets with basic information about malaria, STHs and schistosomiasis where distributed to children in schools involved in the study for children to learn basic life skills to avoid diseases under investigation. At each school, teachers were taught about the life cycles of malaria, schistosomiasis, STHs and signs and symptoms of these diseases so that they would teach their children regularly during their free or study periods. A flip chart with basic information about bilharzia was given to the school headmaster who was responsible for circulating it in turns to each class from grade 1 to 7. Class teachers were asked to continue school health education on schistosomiasis, STHs and malaria and to encourage children to seek medical care promptly when they felt signs and symptom of malaria.Children infected with any of the schistosome species and STHs were treated with Praziquantel at 40 mg/kg body and a single albendazole tablet 400 mg as a single dose by the state registered nurse. Bread and orange juice (500 ml/child) were given as supplementary food following swallowing of tablets in order to reduce the nauseating effect of Praziquantel and to aid absorption. Combined deworming refers to treatment of schistosomiasis and STHs at the same treatment time point: baseline, 6, 12 and 33 months follow up surveys. Treatment for malaria was based on parasitological results from the microscopic examination of Giemsa stained blood thick smears at baseline, and also during 6, 12 and 33 moths follow up surveys when the research team was in the field. All children who had either gametocytes or ring forms in their blood thick smears were taken to the nearest clinic for treatment. During the time when the research team was not in the field participants also sought prompt malaria treatment based on their ability to recognise signs and symptoms of the disease as taught in school. Malaria was treated with a combination of chloroquine, sulphadoxine and pyrimethamine (SP) according to local malaria case management guidelines .A register was left with the nurse in charge of Burma Valley clinic who also served as our research nurse during the field visits. All children reporting for malaria treatment in the absence of the research team were recorded in the register. The research team collected this information on subsequent follow up surveys. Also children were asked if they had received malaria treatment during the period in between the surveys.The Medical Research Council of Zimbabwe gave ethical approval for the study. In addition, Provincial and District Medical and Education Directors, chiefs, councillors and village head-men granted permission. General information regarding the nature of study and objectives was explained to the community and study participants. Feedback and consent was sought at schools, farms and village meetings. Inclusion of children into the study took place after free individual, parental and school authority informed consent. Children joined the study voluntarily and were allowed to drop out at any time they wished without any prejudice.2- test was used where appropriate. McNemar's test was used to test for significant difference in prevalence of parasites between baseline and 33 months follow up surveys Helminths infection intensities were classified according to the World Health Organization guidelines [S. haematobium infection was classified into light infection (1-49e/10 ml urine) and heavy infection (≥ 50e/10 ml urine). S. mansoni infection determined using the Kato Katz technique was stratified into light (1-99epg stool), moderate (100-399epg stool), heavy infection (≥ 400epg stool). Hookworm infection intensity determined using the Kato Katz technique was stratified into light infection (1-1999epg stool), moderate infection (2000 - 3999epg stool) and heavy infection (≥ 4000epg stool). Single infection referred to infection with schistosomes (S. haematobium and or S. mansoni), any one of or all of the STHs or P. falciparum. Co-infection referred to infection with schistosome + STH, P. falciparum + schistosomes, P. falciparum + STHs or P. falciparum + STHs + schistosomes.Data was captured using SPSS version 8. Frequency tables together with 95%CI were obtained. The percentage reduction of the prevalence of parasites from baseline to 33 months follow up survey and geometric mean egg counts was calculated. χidelines . S. haemPlasmodium co-infections, parasites infection combinations were stratified at each treatment point into the following groups: (0) non infected individuals, (1) schistosomiasis infection only, (2) STHs infection only, (3) P. falciparum infection only, (4) infection with schistosomes + STHs, (5) infection with P. falciparum + schistosomes, (6) infection with P. falciparum + STHs, (7) infection with P. falciparum + STHs + schistosome.In order to assess the prevalence and effect of integrated school based deworming and prompt malaria treatment on helminths - Table P. falciparum was determined from those children who were successfully followed up at all surveys and had all parasitological results (n = 682).The effect of integrated school based deworming and prompt malaria treatment on the prevalence of schistosomes, STHs and P. falciparum, schistosomes and STHs in Burma Valley farming area and on schistosomiasis only in Nyamaropa rural area at each follow up time point is shown in table The effect of intervention on the prevalence of P. falciparum from baseline to 33 months follow up survey (28.3% to 8.1%) with the percentage prevalence reduction of 54.4%, 56.2% and 71.9% from baseline to 6, 12 and 33 months follow up surveys respectively. McNemar's test demonstrated a significant reduction in prevalence of malaria from baseline to 33 months follow up survey, p < 0.001. There was no significant difference in the prevalence of malaria between males and females at each treatment point in time. Throughout the study period, malaria was not diagnosed parasitologically in the rural area.There was a significant decrease in prevalence of S. haematobium in both rural and commercial farming areas. The observed overall percentage reduction (PR) of S. haematobium was 75.9% in the commercial farming area and 72.0% in the rural area during the first 12 months of intervention. The absence of anti-helminthic treatment between the 12 mth and 33 month follow-up surveys resulted in rebounding of S. haematobium prevalence in both areas although it did not reach the baseline level  to 12 months (12.4%), PR = 66.2%, after which it increased to 25.7% at 33 months follow up survey  at base line to 15.6% (95%CI = 11.5-20.6) at 12 months follow up survey. It rebounded to 30.5% (95%CI = 25.0-36.5) as a result of the absence of treatment between 12 months and 33 months follow up surveys. Heavy infection declined progressively from baseline 21.8% (95%CI = 16.9-27.2) to 2.7% (95%CI = 1.1-5.4) at 12 months follow up survey after which it slightly increased to 3.4% (95%CI = 1.6-6.4) at 33 months follow up survey.Figure S. mansoni and its infection intensities in both Burma Valley farming area and Nyamaropa rural area is illustrated in tables The effect of intervention on the prevalence of 2 = 3.4, p = 0.063).The overall prevalence of hookworm in Burma Valley farming area was 23.8% (95%CI = 19.8-28.2) at baseline . However the PR declined to 82.5% with delayed treatment at 33 months follow up survey.The prevalence percentage reduction of co-infection with schistosomes + STHs was 70.5% after two rounds of treatment in the first year. However, the proceeding 21 months of delayed treatment resulted in an exceptional increase in prevalence of schistosome + STHs co-infection (10.7%). Following two rounds of treatment in the first year the percentage prevalence reduction of Overall the prevalence of children infected with at least two parasites at baseline, 6, 12 and 33 months follow up surveys in the commercial farming area were 33.1% (1139/420), 5.5% (23/420), 6.4% (27/420) and 15.0% (63/420) respectively.Following baseline treatment and subsequent school health education, the following participants reported to the nearest health facility  between baseline and six months follow up survey (n = 8), between 6 months and 12 months follow up survey (n = 24) and between 12 months and 33 months follow up survey (n = 56).Plasmodium co-infections. Results from our study demonstrate that two rounds of deworming and sustained prompt malaria treatment significantly reduce the proportions of children with helminths -Plasmodium falciparum co-infections. The observed low level of helminths-Plasmodium falciparum co-infection during the proceeding 21 months  could have been due to the initial combined treatment intervention and school based health education leading to change of children's behaviour towards the preventive practices. Thus the intervention could be promising as it managed to reduce the proportion of helminths-Plasmodium falciparum co-infection, hence the multiplicative effect of co-infection on malaria clinical outcomes and morbidity [Our study investigated efficacy of combined school based antihelminthic treatment with Praziquantel and albendazole; prompt malaria treatment and health education in control of schistosomiasis, STHs, malaria and on suppression of helminths - orbidity ,28,49,50Plasmodium falciparum co-endemic areas would result in reduction of morbidity associated with helminths Plasmodium falciparum co-infections or helminths co-infections [Plasmodium falciparum co-infections for control [If similar but large scale intervention could demonstrate similar results to our study then application of such a strategy in helminths -fections ,30,32-34fections ,36,40. Tfections  could al control ,37,41,52S. mansoni and hookworm light infections (figures The observed higher prevalence of co-infection with schistosomes + STHs in Burma Valley farming area following 21 months of delayed treatment than that observed at pre-treatment survey as well as the unprecedented increase in prevalence of  figures  and 5 in figures , 4 and 5Currently, conflicting suggestions on the timing of regular deworming do exist, with the World Health Organization recommending regular annual de-worming in areas where the prevalence of schistosomiasis is ≥ 50% and the prevalence of STHs ≥ 20%, whilst the best practice paper on deworming considers it cost effective if annual deworming is done when the prevalence of STHs is ≥ 40% ,41. EmpiThe prevalence of STHs > 50% was observed in school children attending Kinyasini and Chaani approximately 6 months after a decade of sustained regular school based de-worming . This waS. mansoni in Nyamaropa and Burma Valley commercial farming areas at 33 months follow up survey demonstrate the effect of heterogeneities in water contact patterns in different communities and the spatial heterogeneity in the distribution of infected snails on the epidemiology of schistosomiasis [The observed significant difference in prevalence of somiasis -57. In aPlasmodium falciparum parasites at each treatment point parasitologically. These could be a few asymptomatic malaria cases whose immunity could be able to control parasite load and tolerate parasitaemia without clinical manifestation of the disease [Although the research team and school teachers educated children about malaria disease, its signs and symptoms, a strategy implemented continuously through out the study to promote children to recognise malaria and seek prompt treatment, Table  disease . Thus in disease ,60 couldP. falciparum parasites but did not seek treatment. The three primary schools in Burma Valley are served with Burma Valley clinic that is located at the centre and is approximately 200 m away from Msapa primary school, and about 2 to 3 km away from Kaswa and Valhalla primary schools. The schools are therefore in close proximity to the clinic, thus distance can not be attributed to low compliance observed. More studies in diverse endemic communities are recommended in order to explore the role of school based health education on prompt malaria treatment. Although our study show that integrated school based parasite control can reach a wide population at risk of infection and resulted in reduction of prevalence of polyparasitism, teachers were not able to provide treatment since the local policy stipulates that only medically qualified personnel prescribe and administer drugs regardless of safety guaranteed. In order to realise maximum benefit from school based integrated treatment approach, school teachers need to be allowed to administer treatment in situations where drugs are declared safe like praziquantel and albendazole as this is not only cost effective but also a quick strategy for reaching children living in hard to reach areas who are most commonly overburdened with curable neglected tropical disease.The health seeking behaviour of children for malaria treatment as demonstrated by records made at the clinic is difficult to attribute it to school health education intervention since the figures of children who reported for prompt malaria treatment were rather low at each follow up survey. There was also no control arm. We observed that at each follow up survey, some children harboured Plasmodium co-infections. Whilst the results show some potential, it should be noted that the study did not have an untreated control arm due to ethical reasons, a weakness that begs for care in interpreting our results. Further, rigorous studies are therefore recommended in diverse areas in order to inform public health managers on appropriate and cost effective integrated control measures in areas co-endemic for NTDs and malaria.To our knowledge, this is the first study to investigate the efficacy of combined school based control strategy for helminths - plasmodium co-infections in primary school children. In areas of stable malaria transmission, active case finding is recommended to track and treat asymptomatic malaria cases as these may sustain transmission in the community.Biannual Integrated school based anthelminthic and sustained prompt malaria treatment has a potential to reduce the burden of helminths-The authors declare that they have no competing interests.TM, NM, NK, KCB and FM contributed to the concept and design of the study protocol; NM, DS, SZ, TM, NHP and VC carried out the clinical assessment and parasitology; TM, NM, MG, and MPM carried out the analysis and interpretation of the data; NM and TM drafted the manuscript. All authors read and approved the final manuscript. NM and TM are guarantors of the paper.The UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases Grant A60125, ENHR-Ministry of Health and child Welfare, Harare, Zimbabwe, Fogarty Grant for field work to NK and DS. International Foundation for Science Grantee: W/4321-1 to TM.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-698X/11/9/prepub


An ambiguous mutation index of 2.04×10-3 nts/site was detected in HIV-1 recent infections which is equivalent to the HIV-1 substitution rate (2×10-3 nts/site/year) reported before. However, significantly higher index (14.41×10-3 nts/site) was revealed with established infections. Using this substitution rate, 75.2% subjects in HIVDR-TS with the exception of the Vietnam dataset and 3.3% those in HIVDR-baseline were classified as recent infection within one year. We also calculated mutation scores at amino acid level at HIVDR sites based on ambiguous or fitted mutations. The overall mutation scores caused by ambiguous mutations increased (0.54×10-23.48×10-2/DR-site) whereas those caused by fitted mutations remained stable (7.50-7.89×10-2/DR-site) in both recent and established infections, indicating that t-HIVDR exists in drug-naïve populations regardless of infection status in which new HIVDR continues to emerge. Our findings suggest that characterization of ambiguous mutations in HIV may serve as an additional tool to differentiate recent from established infections and to monitor HIVDR emergence.Detection of recent HIV infections is a prerequisite for reliable estimations of transmitted HIV drug resistance (t-HIVDR) and incidence. However, accurately identifying recent HIV infection is challenging due partially to the limitations of current serological tests. Ambiguous nucleotides are newly emerged mutations in quasispecies, and accumulate by time of viral infection. We utilized ambiguous mutations to establish a measurement for detecting recent HIV infection and monitoring early HIVDR development. Ambiguous nucleotides were extracted from HIV-1  With over 8.2 million HIV-infected patients on antiretroviral therapy (ART) in low- and middle-income countries at the end of 2011, emergence and transmission of HIV drug resistance (HIVDR) are ongoing public health challenges in the battle against HIV/AIDS . BecauseEstimates of transmitted HIVDR (t-HIVDR) and HIV incidence have been two important surrogates in measuring ART efficacy and prevention program effectiveness . To assepol-gene /NSEQ)×100% . At the AA level, we scored 1 for a pure mutated AA and 0.5 for an ambiguous mutated AA because of its ambiguity, and calculated the total DR mutation score at each of the HIVDR sites  with the-3 nts per site per year , 0.590 [baseline], and 0.110 [VN-TS])  , indicatTo obtain the DR-score attributed to the ambiguous or non-ambiguous mutated nucleotides, we stratified the non-synonymous DR-associated mutations at amino acid (AA) level based on the 2013 HIVDR list  Figure . Results-2 per DR site) (p=0.681); in contrast, the index of ambiguous mutated AAs increased significantly from 0.54×10-2 per DR site for threshold to 4.30-3.48×10-2 per DR site for Vietnam and baseline datasets (p<0.001), indicating that background of t-HIVDR existed in ART-naïve populations of these cohorts. Under such background, new HIVDR continued to develop in the studied populations.By calculating the overall DR-associated mutations derived from pure mutated AAs or ambiguous mutated AAs , we founSubtype of HIV sequences in each dataset was primarily determined using the online REGA HIV subtyping tool . A singlp=0.16) or subtype C (p=0.107); however, significant differences were noticed between subtypes B and A (p=0.001) or between subtype B and CRF01_AE  and established infections (HIVDR baseline), and demonstrated the lower ambiguous mutation index was associated significantly with recent infections. We defined 2.04×10-3 nts/site), which clearly differentiated them from the recent infections in the threshold subset, and served as a great calibrator for determining the ambiguous mutation index for recent infections. Based on our analyses, HIV infection status could range from 1 to 10 years for the majority of subjects in the Nigeria dataset, which reflects the reality because the subjects enrolled for ART included established and chronic infections according to the Nigeria HIV treatment guidelines [The dataset from Nigerian HIVDR monitoring baseline survey representing established infections exhibited a significantly higher mutation index  similar to the one found in the Nigeria baseline dataset was somewhat not surprising because in Vietnam the two highest HIV risk groups were IDUs and commercial sex workers [Although the Vietnam dataset was also from HIVDR-TS, only 10.3% of the subjects could be identified for being recently infected. The overall high ambiguous mutation index  but a constant level of fitted DR mutations (7.50-7.89×10-2) from recent to established infections, indicating that t-HIVDR background exists in ART-naïve populations in which new HIVDR mutations continue to emerge over time. We also identified multiple DR sites that had mutational scores increased, remained stable or deceased over the time of infections. This dynamic distribution profile may be valuable for predicting early development of possible DRs in HIV-infected populations which provide data to decision maker for regimen considerations and/or selections.Emergence and transmission of HIVDR is an on-going concern in the scale-up ART programs in resource-limited settings. To increase the elements of HIVDR surveillance and monitoring, we utilized the ambiguous mutation approach to monitor early emerged DR mutations and distinguish them from fitted DR mutations. Our data revealed an increase trend in DR mutations caused by ambiguous mutations , which is consistent with a recent study [p=0.001) and CRF01-AE (p=0.011) did show higher ambiguity when they were compared to subtype B. We believe that the discrepant results may be attributed to the smaller sample size that we have for these subtypes (Our observation on subtype preference in viral ambiguity showed no significant difference between subtype B and overall non-B subtypes (nt study . Howeversubtypes . In this However, there are limitations in our study. We characterized ambiguous mutations with datasets based on recent and established infections. However, due to the limited epidemiological data, we couldn’t conduct analysis and interpret the data based on transmission modes. We could only confirm the heterosexual transmission route for the women recruited at ANC clinics because they were all at their first pregnancy and diagnosed the first time with HIV infections. Therefore, the founder effect would likely be the viral replication mechanism, and the ambiguous mutation detected in the HIVDR-TS datasets by Sanger sequencing would reflect the genetic diversity in the viral population. However, for the dataset with suspected IDUs, Sanger sequencing might not be able to resolve the genetic diversity due to the multi-virion infection nature. With the progress on new generation sequencing technologies, e.g. deep or single genome sequencing, the multi-virion infection could be resolved at individual variant level to reflect the true genetic diversity -46. We i In summary, we characterized ambiguous mutations in HIV-1 protease and reverse transcriptase gene regions with likely recent and established infections. We defined an ambiguous mutation index for detecting HIV recent infections and characterized the distribution of ambiguous mutations for monitoring the early development of HIVDR. Our data suggest that molecular characterization of ambiguous mutations in HIV-1 may serve as an additional tool along with serologic assays to differentiate recent from established infections, evaluate infection status, and monitor the early development of HIVDR.


Malaria parasites and their products can activate a specific immune response by stimulating cytokine production in the host’s immune cells. Transcription nuclear factor kappa B (NF-κB) is an important regulator for the control of many pro-inflammatory genes, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF). The activation and expression of NF-κB p65 in peripheral blood mononuclear cells (PBMCs) of malaria patients were investigated and correlated with the levels of IL-10 and TNF to study the nature of NF-κB p65 and its linkage to inflammatory cytokines.Plasmodium vivax (n = 11), uncomplicated Plasmodium falciparum (n = 11), and complicated Plasmodium falciparum (n = 11). Peripheral blood was collected at admission and on day 7 for PBMC isolation. Healthy subjects were used as a control group. The expressions of NF-κB p65 in the PBMCs from malaria patients and the plasma levels of IL-10 and TNF were measured by using enzyme-linked immunosorbent assay (ELISA). The immunofluorescence technique was used to determine NF-κB nuclear translocation.The sample group comprised 33 patients admitted with malaria caused by P. vivax and uncomplicated P. falciparum had significantly elevated phospho-NF-κB p65 levels in the PBMCs compared with those of healthy controls. However, patients with complicated P. falciparum malaria had decreased levels of phospho-NF-κB p65. On day 7 post-treatment, significantly increased phospho-NF-κB p65 was found in the PBMCs of patients with complicated P. falciparum, compared with healthy controls. The plasma level of IL-10 was elevated in day 0 in patients with complicated P. falciparum malaria and was found to be negatively correlated with phospho-NF-κB p65 level . However, there was no correlation between phospho-NF-κB p65 expression and TNF level in patients with complicated P. falciparum malaria.At admission, patients with P. falciparum at admission could be due to a suppressive effect of high IL-10 associated with complicated P. falciparum malaria.This is the first report demonstrating alterations in NF-κB p65 activity in the PBMCs of malaria patients. The altered lower features of NF-κB p65 in the PBMCs of patients with complicated  Nuclear factor kappa B (NF-κB) plays a crucial role in immune and inflammatory responses, through the regulation of various genes involved in pro-inflammatory cytokines, adhesion molecules, chemokines, inducible enzymes, and apoptosis . MammalsP. vivax and P. falciparum malaria infections [P. falciparum infection in malaria-naïve individuals has shown a coordinated increase in the level of pro-inflammatory cytokines, including IFN-γ, IL-12 and IL-8 in the serum [in vitro studies have demonstrated that haemozoin (HZ) [The process of malaria pathogenesis is very complex and still poorly understood. Several studies have indicated that the expression of adhesion molecules on vascular endothelial cells and the production of pro-inflammatory cytokines are closely linked to the activation of NF-κB protein transcription. It is already known that circulating mononuclear cells are involved in the immune responses and production of pro-inflammatory cytokines. Accumulated evidence has demonstrated that excessive production of pro-inflammatory mediators leads to systemic and organ-related pathological conditions. Previous studies have reported that levels of endogenous pyrogens such as interleukin-6 (IL-6), IL-1β and IL-8 were elevated in fections -7. The ifections . A recenhe serum . Previouoin (HZ)  and glycoin (HZ)  can stimoin (HZ) . Using aoin (HZ) . Aside fPlasmodium vivax malaria (n = 11), classified based on a positive blood smear with P. vivax by microscopic examination, (2) uncomplicated Plasmodium falciparum malaria (n = 11), classified based on a positive blood smear with P. falciparum, and no evidence of severe or complicated malaria, and (3) complicated Plasmodium falciparum malaria (n = 11), defined by the WHO criteria [P. falciparum in blood smears, with other causes of coma excluded. Eleven healthy volunteers living in Bangkok, a non-endemic malaria area, were recruited as the control group. This group had no history of malaria infection. Written informed consent was obtained from all patients or their legal representatives before enrollment in the study. The study protocol was approved by the Ethics Committee, Faculty of Tropical Medicine, Mahidol University (MUTM 2010-053-01).Thirty-three malaria patients admitted to the Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Thailand, were included in this study. The patients were divided into three groups: (1) criteria . ComplicFive milliliters (ml) of peripheral blood was collected in heparinized tubes from the malaria patients on day 0 (pre-treatment) and day 7 (post-treatment) for PBMC isolation. In addition, 20 ml of whole blood was obtained from the healthy controls for PBMC isolation, which were employed as unstimulated cells, to investigate the ability of malaria-patient sera to induce NF-κB activation. To prepare the sera from malaria patients, clotted blood was centrifuged at 1,700 g for 10 min. The supernatant representing the serum was harvested and stored in an aliquoted state at −80°C. The serum was heat-inactivated at 56°C for 30 min before use.PBMCs were isolated from freshly heparinized blood by gradient centrifugation, using Isoprep® separation medium , according to the manufacturer’s instructions. The heparinized blood samples were centrifuged at 1,700 g for 5 min and plasma was removed and stored at −80°C until use for cytokine measurements. The remaining blood samples were diluted with an equal volume of phosphate-buffered saline (PBS) (pH 7.4), layered carefully into a conical tube containing Isoprep® solution, then centrifuged at 1,200 g for 25 min. After centrifugation, the PBMCs were removed and washed three times with PBS. The cells were stained with trypan blue and counted with a haemocytometer to determine the number of viable PBMCs.2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin) and a protease inhibitor cocktail  for 5 min. The lysates were briefly sonicated on ice, centrifuged at 14,000 g for 10 min at 4°C, and the supernatants were harvested. Protein concentrations in each sample were determined by Bradford assay , using bovine serum albumin (BSA) as the standard.PBMCs were suspended in ice-cold lysis buffer   according to the manufacturer's protocol. Total cell proteins (10 μg/well) were added into a 96-well microplate coated with total-/phospho-NF-κB p65 mouse monoclonal antibody and incubated for 2 h at 37°C. The plate was then washed with PBS containing 0.05% (w/v) Tween-20 (PBS-T) and 100 μl of total-/phospho-NF-κB p65 rabbit monoclonal antibody was added to the wells and incubated for 1 h at 37°C to detect the captured total-/phospho-NF-κB p65 protein. After washing with PBS-T, 100 μl of anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase (HRP) was added and incubated for 30 min at 37°C. To develop the reaction, 100 μl of 3,3’,5,5’-tetramethylbenzidine (TMB) substrate  was added and incubated for 10 min at 37°C. Finally, 100 μl of 0.18 M sulfuric acid was added to stop the reaction. The optical density (OD) of the yellow-colored product was determined with a microplate reader at 450 nm. All assays were carried out in duplicate.TM reagent  and observed under a fluorescence microscope  connected to a digital camera  and a standard UV filter set. Normal PBMCs stimulated with 50 ng/ml of TNF for 30 min were used as a positive control and the omission of primary antibody was used as a negative control. For quantitative analysis, PBMCs (approximately 200–300 cells) were randomly examined by fluorescence microscopy under high power (x400) and cells stained positive for nuclear NF-κB p65 were counted. To calculate the percentage of cells with NF-κB nuclear translocation, the number of positive nuclear stained cells was divided by the total number of cell and the result was multiplied by 100.The translocation of NF-κB p65 from the cytoplasm to the nucleus was examined by immunofluorescence. PBMCs were smeared on adhesive slides coated with 3-aminopropyltriethoxysilane  and fixed with 3.7% formaldehyde in PBS (pH 7.4) for 20 min at room temperature. After fixation, the cells were permeabilized with 0.5% Triton X-100 for 10 min. After washing with PBS, the slides were blocked with 5% BSA for 30 min at room temperature and incubated with mouse anti-human NF-κB p65 (F-6) monoclonal antibody (1:200)  for 1 h at 37 °C. The slides were then incubated with goat anti-mouse antibodies conjugated with Alexa-488 (1:1000)  for 45 min at 37°C. Finally, the slides were mounted with FluorSaveThe levels of IL-10 and TNF in plasma was determined by the Human IL-10 and TNF ELISA Development Kit , a quantitative sandwich enzyme immunoassay using a purified rabbit antibody against IL-10 or by TNF pre-coated onto an ELISA plate , and then incubated overnight at room temperature. Human recombinant IL-10 and TNF protein at serial concentrations and 2-fold diluted plasma samples were incubated into the wells. Assay standards and samples were added to duplicate wells in the plate, which was incubated for 2 h at room temperature. After washing, either a biotinylated purified rabbit anti-human IL-10 or TNF antibody as the detection antibody was added to each well at a concentration of 50 ng/ml and incubated for 2 h at room temperature. Then, an avidin-horseradish peroxidase (HRP) conjugate was added to the wells and incubated for 30 min at room temperature. For color development, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), or ABTS liquid substrate , was added and incubated at room temperature. Absorbance was read at 405 nm with wavelength correction set at 650 nm. The sensitivity of the kit was within the range of 32–2000 pg/ml for IL-10 and 16–2000 pg/ml for TNF.6 cells) were separately stimulated by 10% sera from healthy controls and patients with P. vivax, uncomplicated P. falciparum, and complicated P. falciparum malaria for 0, 30, and 60 min, at 37°C, 5% CO2. TNF (50 ng/ml) was used as positive control. After incubation, PBMCs were washed with PBS and total proteins were isolated to detect the phospho-NF-κB p65 using sandwich ELISA.To investigate whether the sera from malaria patients could induce the expression of NF-κB p65 in PBMCs of healthy volunteers, PBMCs . Data was analysed by statistical analysis performed using SPSS version 17.0 software . The p value < 0.05 was considered significantly different.Data were expressed as mean ± standard error of the mean (SEM). The normality of distribution was determined by the Kolmogorov-Smirnov test. Differences in NF-κB p65 levels between groups were compared by unpaired Student’s P. falciparum malaria compared to those with uncomplicated P. falciparum infection (p = 0.001). Patients diagnosed with P. vivax malaria were administered chloroquine and primaquine, whereas patients with P. falciparum malaria were treated with artesunate and mefloquine. On day 7 post-treatment, no asexual forms of malaria parasites were found in the peripheral blood of all malaria patient groups. Complications in patients with complicated P. falciparum malaria included pulmonary oedema , cerebral malaria , acute renal failure , shock , anaemia , and acidosis .The clinical and laboratory parameters of malaria patients and healthy controls are shown in Table p > 0.05) on both day 0 and day 7  and uncomplicated P. falciparum malaria , compared to the healthy controls (0.104 ± 0.04). However, the phospho-NF-κB p65 levels were significantly lower in the PBMCs of patients with complicated P. falciparum malaria on admission compared to day 7 post-treatment . Among the patient groups, phospho-NF-κB p65 activity was significantly higher in the PBMCs of patients with uncomplicated P. falciparum malaria, compared to patients with P. vivax (p = 0.01) or complicated P. falciparum malaria (p = 0.001)  , whereas phospho-NF-κB p65 levels in PBMCs from patients with uncomplicated P. falciparum malaria showed an increasing trend, but this was not statistically significant (p = 0.210) when compared to healthy controls (0.103 ± 0.01). In addition, no difference in the level of phospho-NF-κB p65 was found between uncomplicated and complicated P. falciparum malaria on day 7 (p > 0.05). No correlation was observed in any group of patients between the level of phospho-NF-κB p65 and other clinical parameters such as age, days of fever, malaria parasite density, RBC, WBC, haemoglobin, haematocrit, and platelet levels.The total-NF-κB p65 levels detected in the PBMCs was similar in all experimental groups   and uncomplicated P. falciparum (p = 0.002) patients during the acute illness (day 0). The mean plasma IL-10 on day 0 for P. vivax was 437.44 ± 104.60 pg/ml as compared with uncomplicated P. falciparum (630.13 ± 183.78 pg/ml) and complicated P. falciparum malaria patients  . On day 7, the mean plasma IL-10 in the three malaria groups declined significantly compared to their levels on day 0 , 464.30 ± 169.00 pg/ml for uncomplicated P. falciparum (p = 0.041), and 331.26 ± 61.41 pg/ml for complicated P. falciparum malaria patients (p = 0.010)). The mean plasma levels of IL-10 in P. vivax patients were not significantly different from those observed in uncomplicated P. falciparum patients on both day 0 (p > 0.05) and day 7 (p > 0.05), and on day 7 of patients with complicated P. falciparum malaria (p > 0.05) , uncomplicated P. falciparum (331.70 ± 77.55 pg/ml), and complicated P. falciparum (479.64 ± 133.53 pg/ml), compared with the healthy controls . The mean TNF concentration of patients with complicated P. falciparum was significantly higher than patients with P. vivax malaria (p < 0.05)   was established during acute illness. No correlation was observed between phospho-NF-κB p65 in PBMCs and plasma TNF in any of the malaria groups.In complicated P. vivax, uncomplicated P. falciparum, and complicated P. falciparum malaria) at 30 min after stimulation . However, this effect seems to be transient. In the present study, the level of phospho-NF-κB p65 induced by sera from patients showed a tendency to return to the pre-stimulation state after 60 min -induced enhancement of inflammatory cytokines [P. falciparum glycosylphosphatidylinositol (GPI) stimulating monocytes and macrophages, leading to the activation of NF-κB downstream signaling pathways induced expression of pro-inflammatory mediators, such as TNF, IL-6, IL-12, and nitric oxide (NO) [This is the first report showing NF-κB expression in the PBMCs of malaria patients and its correlation with IL-10 and TNF by using sandwich ELISA. The use of ELISA has become a powerful method for measuring protein phosphorylation. ELISA is more quantitative than Western blotting and possesses high specificity and sensitivity due to the use of two antibodies specific for the target protein employed together in the sandwich. NF-κB p65 activation was increased in the PBMCs of ytokines ,16-18, aytokines , and P. ide (NO) . Recent ide (NO) ,20 and nide (NO) . These fide (NO)  and HZ [ide (NO)  transmitP. falciparum at admission, consistent with the reduced mean percentage of NF-κB p65 nuclear translocation evidenced by the immunofluorescence study. These findings agree with previous reports which demonstrated that PBMCs from patients with sepsis and major trauma reduced the active form of NF-κB p65 on the day of admission [P. falciparum wherein NF-κB p65 could be repressed by cytokines [P. falciparum malaria [P. falciparum malaria prior to treatment and the levels were found to return to normal after malaria treatment [P. falciparum malaria patients is linked to plasma IL-10 levels, IL-10 levels were determined in the malaria groups. In this study, the plasma level of IL-10 was significantly elevated in complicated P. falciparum malaria infection and had a negative correlation with phospho-NF-κB p65 expression at admission. When the IL-10 levels were high, the phospho-NF-κB p65 levels were low. This correlation was not found in P. vivax or uncomplicated P. falciparum infections. At admission, the plasma levels of IL-10 in patients with complicated P. falciparum malaria were 4.3 times and 3 times higher than in patients with P. vivax and uncomplicated P. falciparum, respectively. This observation suggests that decreased levels of NF-κB p65 in the PBMCs of complicated P. falciparum patients during acute infection could be due to a negative feedback loop mechanism, or the consequence of high levels of IL-10, an important anti-inflammatory cytokine associated with severe disease. It has been shown that IL-10 inhibits NF-κB activation rapidly and in a dose-dependent manner [P. falciparum malaria declined 5.7 times from the level on day 0, to the same levels as the P. vivax and uncomplicated P. falciparum malaria groups. This trend is similar to results in a previous report [P. falciparum malaria at day 7 post-treatment.NF-κB p65 activity was decreased in PBMCs from patients with complicated dmission ,24. The ytokines . Studiesytokines ,27 and Iytokines  reportedytokines -28. Normytokines . It has  malaria . Elevatereatment . To inves report . The lowP. falciparum malaria. The PBMCs from healthy controls are naïve and activation is short-lived. The effect might not be long enough to initiate a complex response to malaria serum. In contrary, the PBMCs collected from complicated P. falciparum malaria patients was sensitized by prolonged infection and response mechanisms induced by other cell signaling processes. The present study elucidates that sera from malaria patients can induce NF-κB p65 activation in naïve PBMCs. It would be helpful to further analyse the expression of NF-κB in PBMCs from malaria patients after stimulation with malaria sera to determine the event of “desensitization” in malaria. Further work to investigate whether pre-incubation of the malaria sera with an anti-IL-10 neutralizing monoclonal antibody would suppress NF-κB activation in PBMCs from complicated P. falciparum patients will be of interest.Furthermore, the study investigated whether malaria patient serum could induce NF-κB p65 activation in unstimulated PBMCs. Significantly, transiently increased levels of phospho-NF-κB p65 were found in the healthy PBMCs 30 min after stimulation with malaria serum, consistent with previous studies on endothelial cells , monocytThe authors declare that they have no competing interests.CP carried out the ELISA and immunofluorescence work, preliminary data analysis, and wrote the first draft of the manuscript. SK, NT and KN collected clinical data, supervised patient recruitment, critically analysed, and interpreted the data. YM, UC, EP and RU participated in the study design and manuscript preparation and revision. PV formulated the research idea, designed the experiments, gave laboratory and technical support, supervised, and revised the manuscript. All authors have approved the final version of the manuscript.


Plasmodium falciparum schizonts and merozoites: MSPDBL1  and MSPDBL2 (MSP3.8), which possess Duffy binding-like (DBL) domains, and SURFIN4.2, encoded by a member of the surface-associated interspersed (surf) multigene family. After testing the antigenicities of these reagents by murine immunization and parasite immunofluorescence, we analyzed naturally acquired antibody responses to the antigens in two cohorts in coastal Kenya in which the parasite was endemic (Chonyi [n = 497] and Ngerenya [n = 461]). As expected, the prevalence and levels of serum antibodies increased with age. We then investigated correlations with subsequent risk of clinical malaria among children <11 years of age during 6 months follow-up surveillance. Antibodies to the polymorphic central region of MSPDBL2 were associated with reduced risk of malaria in both cohorts, with statistical significance remaining for the 3D7 allelic type after adjustment for individuals' ages in years and antibody reactivity to whole-schizont extract . For the MSPDBL1 Palo Alto allelic-type antigen, there was a protective association in one cohort , whereas the other antigens showed no protective associations after adjustment. These findings support the prediction that antibodies to the polymorphic region of MSPDBL2 contribute to protective immunity.Prospective studies continue to identify malaria parasite genes with particular patterns of polymorphism which indicate they may be under immune selection, and the encoded proteins require investigation. Sixteen new recombinant protein reagents were designed to characterize three such polymorphic proteins expressed in  Plasmodium falciparum, which causes most disease and mortality. Trials of the lead preerythrocytic stage candidate vaccine—RTS, S/ASO2—have shown partial protection of short duration, suggesting that addition of antigens of the blood stage may be needed to achieve higher levels of efficacy   , 22. RecFD1160w) , 24, witFD1160w) , and genin vitro , 26.P. falciparum schizonts and was then assayed for reactivity with naturally acquired antibodies in cohorts of individuals living in two villages in coastal Kenya where the parasite is endemic. Antibodies against one allelic form of MSPDBL2 were significantly associated with protection from malaria in both cohorts, even after adjusting for potential confounding variables, such as age and exposure, while only one other recombinant antigen showed a protective association in one cohort and the remaining 14 in neither cohort.In this study, 16 new recombinant proteins based on polymorphic and conserved parts of these antigens were designed and expressed. Each of the antigens elicited murine antibodies reactive with http://www.homeoffice.gov.uk/science-research/animal-research/.Ethical approval for the study on samples from human subjects was obtained from the Kenya National Research Ethics Committee, the University of Oxford, and the London School of Hygiene and Tropical Medicine. Written informed consent was obtained from a parent or guardian of each child contributing a blood sample and also from participating adults. Murine antibodies were obtained commercially by immunization of mice under commercial subcontract, and all animal work protocols were approved and licensed by the United Kingdom Home Office as governed by law under the Animals (Scientific Procedures) Act of 1986, in strict accordance with the Code of Practice Part 1 for the housing and care of animals (21 March 2005), available at Escherichia coli, and 7 larger fragments with predicted intramolecular disulfide bonds were expressed in baculovirus.Sixteen new constructs were designed ; 9 smallP. falciparum genes mspdbl1  and mspdbl2  and a C-terminal intracellular sequence encoded by exon 2 of 4.2surf  were PCR amplified from regions of each gene showing minimal polymorphism . Four allelic constructs  were PCR amplified from a polymorphic extracellular sequence encoded within exon 1 of 4.2surf  . DNA for each construct was PCR amplified from 3D7 genomic DNA (and K1 for 4.2surf), cloned into the pGEM-T Easy TA vector (Promega), and sequence verified. Correct sequence inserts were subcloned into the pGEX-2T expression vector , sequenced again to ensure fidelity, and transformed into E. coli BL21(DE3) cells for expression. Expression and affinity purification were performed as described previously for other glutathione S-transferase (GST) fusion proteins  and two polymorphic antigens based on the mspdbl2 gene , covering the central DBL region, were expressed using a baculovirus expression system   using an Akta cross-flow .Five polymorphic antigens based on the n system . PCR priAN35552) . BrieflyGroups of 5 CD1 outbred mice were immunized with 25 μg of each of the 16 recombinant antigens emulsified in Freund's complete adjuvant following a 60-day protocol , and boosting immunizations were performed twice more at 28-day intervals in Freund's incomplete adjuvant. Sera were collected before immunization and on days 14 and 42, and final serum collection was 7 days after the last immunization.P. falciparum 3D7 parasites using IFA. Parasite cultures with a large proportion of schizonts were washed in phosphate-buffered saline (PBS)-1% bovine serum albumin (BSA) and resuspended to 2.5% hematocrit, and 15-μl aliquots were spotted onto multiwell slides , which were then air dried and stored at −40°C with desiccant until required. Following a recommended fixation protocol  were performed with each of the 16 antigens using protocols similar to those previously described for other merozoite antigens , with ea malaria , 32. Ant malaria , 30.All analysis was performed with Stata/IC 11.2 . Generalized linear models were used to determine the associated risk ratio (RR) between the presence or absence of detectable serum antibodies  and occurrence of subsequent clinical malaria episodes. Individuals' ages in years and antibody reactivity to cultured parasite schizont extract were used in multivariate analyses to adjust for the confounding effects of variation among individuals in previous exposure to malaria.P. falciparum antigens, 16 recombinant proteins based on polymorphic and conserved sequences were designed  and two of mspdbl2 (3D7 and T9/96) were baculovirus expressed, whereas all other antigens were expressed as GST-tagged fusion proteins in E. coli. All 16 recombinant antigens were assessed for size and purity by SDS-PAGE . We previously noted that only a small proportion of mature schizont stage parasites were positive by IFA for the MSPDBL2 antigen (immature stage parasites were negative), using sera raised to the conserved N- and C-terminal antigens  and Ngerenya (n = 461). Antibody prevalence and OD levels were higher in Chonyi (high transmission) than in Ngerenya (low transmission) for most antigens tested. There was high antibody prevalence against polymorphic parts of MSPDBL1 and MSPDBL2, increasing in young children and rapidly approaching 100% in young adults . In addition, high correlation coefficients were observed in comparison of reactivity profiles between MSPDBL1 and MSPDBL2, whereas cross correlations between either of them and the SURFIN4.2 antigens were much lower . Competition ELISAs using sera from two adult donors with high levels of antibodies were performed to test for cross-reactivity between antigens. Although most antibodies in these sera were specific for each separate antigen, with reactivity against allele-specific as well as conserved epitopes, some antibodies were cross-reactive between MSPDBL1 and MSPDBL2, and heat denaturation of competing antigens confirmed that some reactivity was against conformational epitopes .P. falciparum by slide examination at the time of serum collection in October 2000 , following an approach taken in a previous study  and the Ngerenya cohort , and the adjusted relative risk estimates are summarized in P < 0.05), but this association was not significant in Chonyi. More significantly, antibodies to the 3D7 allelic type of DBLMSP2 were associated with a reduced subsequent risk of malaria in each cohort  (Tests for associations between serum IgG antibody reactivities and occurrence of clinical malaria during follow-up over the following 28 weeks focused on individuals who were <11 years old and asymptomatically positive for us study . This grus study , 30, 33.P. falciparum genes expressed at the schizont and merozoite stages that show evidence of being under balancing selection in populations where the parasite is endemic. A similar approach has been used to identify regions of more intensively studied candidate antigens (This study involved the design of antigenic reagents based on recently described proteins encoded by three antigens , 27, 33 antigens –39 or seantigens , 40.P. falciparum (Here, human serum IgG prevalence was highest to the polymorphic regions of MSPDBL1 and MSPDBL2 in both of the Kenyan cohorts studied. Consistent with the fact that MSPDBL2 showed the strongest indication of balancing selection in a genome-wide analysis of lciparum , we obselciparum . These rlciparum  suggest lciparum , 25 alsoP. falciparum lines (Comparisons of serological reactivity profiles demonstrated high correlations between the DBL regions of the MSPDBL1 and MSPDBL2 antigens, reported to be important for erythrocyte binding , 24, andum lines , showingum lines , 42, altum lines  and inveum lines . HoweverStudies such as these of antibody reactivity to recombinant proteins, even when associated with epidemiological outcomes, do not directly demonstrate a mechanism of immunity. It was previously shown  that eacClearly, the appropriate selection of antigens is an essential step in the design and development of vaccines. Here, we have described the generation of a new panel of antigen reagents as part of a process to identify potential candidates that are targets of naturally acquired immunity. For one of these antigens, based on the 3D7 allelic type of the DBL region of MSPDBL2, antibodies were associated with reduced prospective risk of malaria in two different populations where the parasite is endemic. This is already a higher level of reproducibility than normally shown in studies of other candidate antigens (