Identification and Immune Assessment of T Cell Epitopes in Five Plasmodium falciparum Blood Stage Antigens to Facilitate Vaccine Candidate Selection and Optimization.

The hurdles to effective blood stage malaria vaccine design include immune evasion tactics used by the parasite such as redundant invasion pathways and antigen variation among circulating parasite strains. While blood stage malaria vaccine development primarily focuses on eliciting optimal humoral responses capable of blocking erythrocyte invasion, clinically-tested Plasmodium falciparum (Pf) vaccines have not elicited sterile protection, in part due to the dramatically high levels of antibody needed. Recent development efforts with non-redundant, conserved blood stage antigens suggest both high antibody titer and rapid antibody binding kinetics are important efficacy factors. Based on the central role of helper CD4 T cells in development of strong, protective immune responses, we systematically analyzed the class II epitope content in five leading Pf blood stage antigens (RH5, CyRPA, RIPR, AMA1 and EBA175) using in silico, in vitro, and ex vivo methodologies. We employed in silico T cell epitope analysis to enable identification of 67 HLA-restricted class II epitope clusters predicted to bind a panel of nine HLA-DRB1 alleles. We assessed a subset of these for HLA-DRB1 allele binding in vitro, to verify the in silico predictions. All clusters assessed (40 clusters represented by 46 peptides) bound at least two HLA-DR alleles in vitro. The overall epitope prediction to in vitro HLA-DRB1 allele binding accuracy was 71%. Utilizing the set of RH5 class II epitope clusters (10 clusters represented by 12 peptides), we assessed stimulation of T cells collected from HLA-matched RH5 vaccinees using an IFN-γ T cell recall assay. All clusters demonstrated positive recall responses, with the highest responses - by percentage of responders and response magnitude - associated with clusters located in the N-terminal region of RH5. Finally, a statistically significant correlation between in silico epitope predictions and ex vivo IFN-γ recall response was found when accounting for HLA-DR matches between the epitope predictions and donor HLA phenotypes. This is the first comprehensive analysis of class II epitope content in RH5, CyRPA, RIPR, AMA1 and EBA175 accompanied by in vitro HLA binding validation for all five proteins and ex vivo T cell response confirmation for RH5.

Assessment of molecular markers of anti-malarial drug resistance among children participating in a therapeutic efficacy study in western Kenya.

BACKGROUND: Anti-malarial drug resistance remains a major threat to global malaria control efforts. In Africa, Plasmodium falciparum remains susceptible to artemisinin-based combination therapy (ACT), but the emergence of resistant parasites in multiple countries in Southeast Asia and concerns over emergence and/or spread of resistant parasites in Africa warrants continuous monitoring. The World Health Organization recommends that surveillance for molecular markers of resistance be included within therapeutic efficacy studies (TES). The current study assessed molecular markers associated with resistance to Artemether-lumefantrine (AL) and Dihydroartemisinin-piperaquine (DP) from samples collected from children aged 6-59 months enrolled in a TES conducted in Siaya County, western Kenya from 2016 to 2017. METHODS: Three hundred and twenty-three samples collected pre-treatment (day-0) and 110 samples collected at the day of recurrent parasitaemia (up to day 42) were tested for the presence of drug resistance markers in the Pfk13 propeller domain, and the Pfmdr1 and Pfcrt genes by Sanger sequencing. Additionally, the Pfpm2 gene copy number was assessed by real-time polymerase chain reaction. RESULTS: No mutations previously associated with artemisinin resistance were detected in the Pfk13 propeller region. However, other non-synonymous mutations in the Pfk13 propeller region were detected. The most common mutation found on day-0 and at day of recurrence in the Pfmdr1 multidrug resistance marker was at codon 184F. Very few mutations were found in the Pfcrt marker (< 5%). Within the DP arm, all recrudescent cases (8 sample pairs) that were tested for Pfpm2 gene copy number had a single gene copy. None of the associations between observed mutations and treatment outcomes were statistically significant. CONCLUSION: The results indicate absence of Pfk13 mutations associated with parasite resistance to artemisinin in this area and a very high proportion of wild-type parasites for Pfcrt. Although the frequency of Pfmdr1 184F mutations was high in these samples, the association with treatment failure did not reach statistical significance. As the spread of artemisinin-resistant parasites remains a possibility, continued monitoring for molecular markers of ACT resistance is needed to complement clinical data to inform treatment policy in Kenya and other malaria-endemic regions.

Assessment of platelet indices and platelet activation markers in children with Plasmodium falciparum malaria.

BACKGROUND: Plasmodium falciparum malaria remains one of the world's major infectious diseases that cause most morbidity and mortality, particularly in children. In Ghana, most children below the ages of 5 years depending on the severity of the infection often lose their lives. However, it is still debatable why infection with falciparum malaria contributes to thrombocytopenia. METHODS: This study sought to investigate the expression of the various platelet indices and activation markers in children with falciparum malaria. Platelet indices (Platelet count [PLT], Plateletcrite [PCT], Mean Platelet Volume [MPV], Platelet Distribution Width [PDW] and Platelet-Large Cell Ratio [P-LCR]) and platelet surface membrane glycoproteins (GPIIb/IIIa [PAC-1], P-selectin [CD62p] and GPIV [CD36]) expressions were determined in children with falciparum malaria (cases) and healthy children (controls) using automated blood cell analysis and flow cytometry techniques, respectively. RESULTS: Except for P-LCR, all the other platelet indices (PLT, MPV, PDW, and PCT) were significantly lower in the cases than the controls (P < 0.05). Also, it was observed that the level of expression of the activation markers; PAC 1 and CD62p showed a significant (P < 0.05) decreased before and after activation in falciparum malaria cases than in the controls. On the contrary, CD36 expression in the controls did not differ significantly (p > 0.05) from the malaria cases. Platelet activation markers were known to be associated with increased risk of falciparum malaria with the mean fluorescence intensity of PAC1 (Odds Ratio [OR] 34.0, Relative Risk [RR] 4.47, 95% Confidence Interval [CI] 4.904-235.7; p < 0.0001) and CD36 (OR 4.2, RR 1.82, 95% CI 0.9824-17.96; p = 0.04). Moreover, the percentage expression of CD62p (OR 4.0, RR 1.80, 95% CI 0.59-27.24; p = 0.19) was also observed to be probably associated with increased risk of falciparum malaria although not statistically significant (p > 0.05). CONCLUSION: Plasmodium falciparum malaria has been known to be associated with platelet activation markers, which probably contributes to thrombocytopenia.

Malaria causes long-term effects on markers of iron status in children: a critical assessment of existing clinical and epidemiological tools.

BACKGROUND: Most epidemiological studies on the interplay between iron deficiency and malaria risk classify individuals as iron-deficient or iron-replete based on inflammation-dependent iron markers and adjustment for inflammation by using C-reactive protein (CRP) or α-1-acid glycoprotein (AGP). The validity of this approach and the usefulness of fibroblast growth factor 23 (FGF23) as a proposed inflammation-independent iron marker were tested. METHODS: Conventional iron markers and FGF23 were measured in children with acute falciparum malaria and after 1, 2, 4, and 6 weeks. Children, who were transfused or received iron supplementation in the follow-up period, were excluded, and iron stores were considered to be stable throughout. Ferritin levels 6 weeks after admission were used as a reference for admission iron status and compared with iron markers at different time points. RESULTS: There were long-term perturbations in iron markers during convalescence from acute malaria. None of the tested iron parameters, including FGF23, were independent of inflammation. CRP and AGP normalized faster than ferritin after malaria episodes. CONCLUSION: Malaria may bias epidemiological studies based on inflammation-dependent iron markers. Better markers of iron status during and after inflammation are needed in order to test strategies for iron supplementation in populations at risk of malaria.

Gametocyte Carriage, Antimalarial Use, and Drug Resistance in Cambodia, 2008-2014.

Gametocytes are the malaria parasite stages responsible for transmission from humans to mosquitoes. Gametocytemia often follows drug treatment, especially as therapies start to fail. We examined Plasmodium falciparum gametocyte carriage and drug resistance profiles among 824 persons with uncomplicated malaria in Cambodia to determine whether prevalent drug resistance and antimalarial use has led to a concentration of drug-resistant parasites among gametocyte carriers. Although report of prior antimalarial use increased from 2008 to 2014, the prevalence of study participants presenting with microscopic gametocyte carriage declined. Gametocytemia was more common in those reporting antimalarial use within the past year, and prior antimalarial use was correlated with higher IC50s to piperaquine and mefloquine, as well as to increased pfmdr1 copy number. However, there was no association between microscopic gametocyte carriage and parasite drug resistance. Thus, we found no evidence that the infectious reservoir, marked by those carrying gametocytes, is enriched with drug-resistant parasites.

Rapid Diagnostic for Point-of-Care Malaria Screening.

Despite significant success in therapeutic development, malaria remains a widespread and deadly infectious disease in the developing world. Given the nearly 100% efficacy of current malaria therapeutics, the primary barrier to eradication is lack of early diagnosis of the infected population. However, there are multiple strains of malaria. Although significant efforts and resources have been invested in developing antibody-based diagnostic methods for Plasmodium falciparum, a rapid and easy to use screening method capable of detecting all malaria strains has not been realized. Yet, until the entire malaria-infected population receives treatment, the disease will continue to impact society. Here, we report the development of a portable, magneto-optic technology for early stage malaria diagnosis based on the detection of the malaria pigment, hemozoin. Using ß-hematin, a hemozoin mimic, we demonstrate detection limits of <0.0081 µg/mL in 500 µL of whole rabbit blood with no additional reagents required. This level corresponds to <26 parasites/µL, a full order of magnitude below clinical relevance and comparable to or less than existing technologies.

Assessment of Myocardial Function and Injury by Echocardiography and Cardiac Biomarkers in African Children With Severe Plasmodium falciparum Malaria.

OBJECTIVES: Perturbed hemodynamic function complicates severe malaria. The Fluid Expansion as Supportive Therapy trial demonstrated that fluid resuscitation, involving children with severe malaria, was associated with increased mortality, primarily due to cardiovascular collapse, suggesting that myocardial dysfunction may have a role. The aim of this study was to characterize cardiac function in children with severe malaria. DESIGN: A prospective observational study with clinical, laboratory, and echocardiographic data collected at presentation (T0) and 24 hours (T1) in children with severe malaria. Cardiac index and ejection fraction were calculated at T0 and T1. Cardiac troponin I and brain natriuretic peptide were measured at T0. We compared clinical and echocardiographic variables in children with and without severe malarial anemia (hemoglobin < 5 mg/dL) at T0 and T1. SETTING: Mbale Regional Referral Hospital. PATIENTS: Children 3 months to 12 years old with severe falciparum malaria. INTERVENTIONS: Usual care. MEASUREMENTS AND MAIN RESULTS: We enrolled 104 children, median age 23.3 months, including 61 children with severe malarial anemia. Cardiac troponin I levels were elevated (> 0.1 ng/mL) in n equals to 50, (48%), and median brain natriuretic peptide was within normal range (69.1 pg/mL; interquartile range, 48.4-90.8). At T0, median Cardiac index was significantly higher in the severe malarial anemia versus nonsevere malarial anemia group (6.89 vs 5.28 L/min/m) (p = 0.001), which normalized in both groups at T1 (5.60 vs 5.13 L/min/m) (p = 0.452). Cardiac index negatively correlated with hemoglobin, r equals to -0.380 (p < 0.001). Four patients (3.8%) had evidence of depressed cardiac systolic function (ejection fraction < 45%). Overall, six children died, none developed pulmonary edema, biventricular failure, or required diuretic treatment. CONCLUSIONS: Elevation of cardiac index, due to increased stroke volume, in severe malaria is a physiologic response to circulatory compromise and correlates with anemia. Following whole blood transfusion and antimalarial therapy, cardiac index in severe malarial anemia returns to normal. The majority (> 96%) of children with severe malaria have preserved myocardial systolic function. Although there is evidence for myocardial injury (elevated cardiac troponin I), this does not correlate with cardiac dysfunction.

ASSESSMENT OF THREE BLOOD GENOMIC-DNA PREPARATION METHODS FOR MALARIA MOLECULAR DIAGNOSIS.

Species-specific PCR techniques are highly sensitive and reliable alternatives to classical methods for malaria diagnosis and speciation, especially in endemic regions under advanced control or elimination programs where asymptomatic and low-density infections are increasingly reported. Nevertheless, the performance of these techniques is directly affected by the quality of isolated DNA templates. A Plasmodium falciparum/vivax-specific diagnostic Nested-PCR (Pƒ/Pv N-PCR) was used to assess three DNA preparation methods, Qiagen® Mini-Chromatographic kit (QIAmp®) and Jena-Biosciences® DNA isolation kit (JB®) for genomic DNA extraction from EDTA-preserved whole blood samples, and Whatman-FTA® purification reagent (FTA®) for DNA preparation from dry blood spots (DBS) collected onto FTA®- cards. A total of 84 out of 137 blood specimens collected from malaria suspicious febrile patients who visited five health care centres in south-western endemic localities of Saudi Arabia were found P. falciparum positive by at least one method. Among these, only 76 (90%) were reported P. falciparum malaria positive by two expert microscopists. No other species of Plasmodium were detected. Pƒ/Pv N-PCR revealed 84/84 (100%), 75/84 (89%), and 81 (96%) P. falciparum positive samples using DNA templates prepared by QIAmp®, JB®, and FTA® purification methods, respectively. Therefore, Pƒ/Pv N-PCR, when applied to QIAmp® DNA templates showed to be a highly sensitive diagnostic method, particularly useful for submicroscopic specimens from clinically malaria suspicious patients in endemic areas. On the other hand, Pƒ/Pv N-PCR of FTA®-DBS DNA templates revealed 5 positive cases missed by microscopy, encouraging its use as an affordable field semi-adapted protocol for malaria active screening, especially in remqte rural regions with limited laboratory infrastructure.

Performance of Microscopy for the Diagnosis of Malaria and Human African Trypanosomiasis by Diagnostic Laboratories in the Democratic Republic of the Congo: Results of a Nation-Wide External Quality Assessment.

The present External Quality Assessment (EQA) assessed microscopy of blood parasites among diagnostic laboratories in the Democratic Republic of the Congo. The EQA addressed 445 participants in 10/11 provinces (October 2013-April 2014). Participants were sent a panel of five slides and asked to return a routinely stained slide which was assessed for quality of preparation and staining. Response rate was 89.9% (400/445). For slide 1 (no parasites), 30.6% participants reported malaria, mostly Plasmodium falciparum. Only 11.0% participants reported slide 2 (Plasmodium malariae) correctly, 71.0% reported "malaria" or "Plasmodium falciparum" (considered acceptable). Slide 3 contained Plasmodium falciparum (109/µl) and Trypanosoma brucei brucei trypomastigotes: they were each reported by 32.5% and 16.5% participants respectively, 6.0% reported both. Slide 4 (Trypanosoma) was recognised by 44.9% participants. Slide 5 (Plasmodium ovale) was correctly reported by 6.2% participants, another 68.8% replied "malaria" or "Plasmodium falciparum" (considered acceptable). Only 13.6% of routine slides returned were correctly prepared and stained. The proportion of correct/acceptable scores for at least 4/5 slides was higher among EQA-experienced participants compared to first time participants (40.9% versus 22.4%, p = 0.001) and higher among those being trained < 2 years ago compared to those who were not (42.9% versus 26.3%, p = 0.01). Among diagnostic laboratories in Democratic Republic of the Congo, performance of blood parasite microscopy including non-falciparum species and Trypanosoma was poor. Recent training and previous EQA participation were associated with a better performance.

Immunomagnetic capture and colorimetric detection of malarial biomarker Plasmodium falciparum lactate dehydrogenase.

We report a sensitive, magnetic bead-based colorimetric assay for Plasmodium falciparum lactate dehydrogenase (PfLDH) in which the biomarker is extracted from parasitized whole blood and purified based on antigen binding to antibody-functionalized magnetic particles. Antigen-bound particles are washed, and PfLDH activity is measured on-bead using an optimized colorimetric enzyme reaction (limit of detection [LOD] = 21.1 ± 0.4 parasites/µl). Enhanced analytical sensitivity is achieved by removal of PfLDH from the sample matrix before detection and elimination of nonspecific reductases and species that interfere with the optimal detection wavelength for measuring assay development. The optimized assay represents a simple and effective diagnostic strategy for P. falciparum malaria with time-to-result of 45 min and detection limits similar to those of commercial enzyme-linked immunosorbent assay (ELISA) kits, which can take 4-6 h. This method could be expanded to detect all species of malaria by switching the capture antibody on the magnetic particles to a pan-specific Plasmodium LDH antibody.

Hotspots of Malaria Transmission in the Peruvian Amazon: Rapid Assessment through a Parasitological and Serological Survey.

BACKGROUND: With low and markedly seasonal malaria transmission, increasingly sensitive tools for better stratifying the risk of infection and targeting control interventions are needed. A cross-sectional survey to characterize the current malaria transmission patterns, identify hotspots, and detect recent changes using parasitological and serological measures was conducted in three sites of the Peruvian Amazon. MATERIAL AND METHODS: After full census of the study population, 651 participants were interviewed, clinically examined and had a blood sample taken for the detection of malaria parasites (microscopy and PCR) and antibodies against P. vivax (PvMSP119, PvAMA1) and P. falciparum (PfGLURP, PfAMA1) antigens by ELISA. Risk factors for malaria infection (positive PCR) and malaria exposure (seropositivity) were assessed by multivariate survey logistic regression models. Age-specific seroprevalence was analyzed using a reversible catalytic conversion model based on maximum likelihood for generating seroconversion rates (SCR, λ). SaTScan was used to detect spatial clusters of serology-positive individuals within each site. RESULTS: The overall parasite prevalence by PCR was low, i.e. 3.9% for P. vivax and 6.7% for P. falciparum, while the seroprevalence was substantially higher, 33.6% for P. vivax and 22.0% for P. falciparum, with major differences between study sites. Age and location (site) were significantly associated with P. vivax exposure; while location, age and outdoor occupation were associated with P. falciparum exposure. P. falciparum seroprevalence curves showed a stable transmission throughout time, while for P. vivax transmission was better described by a model with two SCRs. The spatial analysis identified well-defined clusters of P. falciparum seropositive individuals in two sites, while it detected only a very small cluster of P. vivax exposure. CONCLUSION: The use of a single parasitological and serological malaria survey has proven to be an efficient and accurate method to characterize the species specific heterogeneity in malaria transmission at micro-geographical level as well as to identify recent changes in transmission.

Assessment of humoral immune responses to blood-stage malaria antigens following ChAd63-MVA immunization, controlled human malaria infection and natural exposure.

The development of protective vaccines against many difficult infectious pathogens will necessitate the induction of effective antibody responses. Here we assess humoral immune responses against two antigens from the blood-stage merozoite of the Plasmodium falciparum human malaria parasite--MSP1 and AMA1. These antigens were delivered to healthy malaria-naïve adult volunteers in Phase Ia clinical trials using recombinant replication-deficient viral vectors--ChAd63 to prime the immune response and MVA to boost. In subsequent Phase IIa clinical trials, immunized volunteers underwent controlled human malaria infection (CHMI) with P. falciparum to assess vaccine efficacy, whereby all but one volunteer developed low-density blood-stage parasitemia. Here we assess serum antibody responses against both the MSP1 and AMA1 antigens following i) ChAd63-MVA immunization, ii) immunization and CHMI, and iii) primary malaria exposure in the context of CHMI in unimmunized control volunteers. Responses were also assessed in a cohort of naturally-immune Kenyan adults to provide comparison with those induced by a lifetime of natural malaria exposure. Serum antibody responses against MSP1 and AMA1 were characterized in terms of i) total IgG responses before and after CHMI, ii) responses to allelic variants of MSP1 and AMA1, iii) functional growth inhibitory activity (GIA), iv) IgG avidity, and v) isotype responses (IgG1-4, IgA and IgM). These data provide the first in-depth assessment of the quality of adenovirus-MVA vaccine-induced antibody responses in humans, along with assessment of how these responses are modulated by subsequent low-density parasite exposure. Notable differences were observed in qualitative aspects of the human antibody responses against these malaria antigens depending on the means of their induction and/or exposure of the host to the malaria parasite. Given the continued clinical development of viral vectored vaccines for malaria and a range of other diseases targets, these data should help to guide further immuno-monitoring studies of vaccine-induced human antibody responses.

Stress dependent infection cost of the human malaria agent Plasmodium falciparum on its natural vector Anopheles coluzzii.

Unraveling selective forces that shape vector-parasite interactions has critical implications for malaria control. However, it remains unclear whether Plasmodium infection induces a fitness cost to their natural mosquito vectors. Moreover, environmental conditions are known to affect infection outcome and may impact the effect of infection on mosquito fitness. We investigated in the laboratory the effects of exposition to and infection by field isolates of Plasmodium falciparum on fecundity and survival of a major vector in the field, Anopheles coluzzii under different conditions of access to sugar resources after blood feeding. The results evidenced fitness costs induced by exposition and infection. When sugar was available after blood meal, infected and exposed mosquitoes had either reduced or equal to survival to unexposed mosquitoes while fecundity was either increased or decreased depending on the blood donor. Under strong nutritional stress, survival was reduced for exposed and infected mosquitoes in all assays. We therefore provide here evidence of an environmental-dependant reduced survival in mosquitoes exposed to infection in a natural and one of the most important parasite-mosquito species associations for human malaria transmission.

Population pharmacokinetic assessment of the effect of food on piperaquine bioavailability in patients with uncomplicated malaria.

Previously published literature reports various impacts of food on the oral bioavailability of piperaquine. The aim of this study was to use a population modeling approach to investigate the impact of concomitant intake of a small amount of food on piperaquine pharmacokinetics. This was an open, randomized comparison of piperaquine pharmacokinetics when administered as a fixed oral formulation once daily for 3 days with (n=15) and without (n=15) concomitant food to patients with uncomplicated Plasmodium falciparum malaria in Thailand. Nonlinear mixed-effects modeling was used to characterize the pharmacokinetics of piperaquine and the influence of concomitant food intake. A modified Monte Carlo mapped power approach was applied to evaluate the relationship between statistical power and various degrees of covariate effect sizes of the given study design. Piperaquine population pharmacokinetics were described well in fasting and fed patients by a three-compartment distribution model with flexible absorption. The final model showed a 25% increase in relative bioavailability per dose occasion during recovery from malaria but demonstrated no clinical impact of concomitant intake of a low-fat meal. Body weight and age were both significant covariates in the final model. The novel power approach concluded that the study was adequately powered to detect a food effect of at least 35%. This modified Monte Carlo mapped power approach may be a useful tool for evaluating the power to detect true covariate effects in mixed-effects modeling and a given study design. A small amount of food does not affect piperaquine absorption significantly in acute malaria.

Laboratory tests for malaria: a diagnostic conundrum?

OBJECTIVES: To detect malarial parasites using the peripheral blood smear (PBS) and to compare the PBS with the immunochromatographic antigen test (i.e. OptiMAL and polymerase chain reaction (PCR)). METHODS: Six ml of blood was collected from each of 170 patients clinically suspected of having malaria. These samples were used to perform PBS examination, the OptiMAL test and PCR by standard protocol. RESULTS: PBS examination found malarial parasites in 86 (50.6%) samples. In comparison, 71 (41.8%) samples were positive by OptiMAL test whereas PCR could detect malarial parasites in only 44 (25.9%) samples. All 84 (49.4%) samples which were negative by PBS were negative by both OptiMAL and PCR. The sensitivity and specificity were respectively 85.54% and 100% for OptiMAL and 51.12% and 100% for PCR. CONCLUSION; Depending on the tests' operational feasibility, and the availability of adequate trained personnel, equipment and laboratory management systems, and considering its sensitivity and cost-effectiveness, peripheral blood smear remains the test of choice for malaria, especially in endemic areas.

Malaria haplotype frequency estimation.

We present a Bayesian approach for estimating the relative frequencies of multi-single nucleotide polymorphism (SNP) haplotypes in populations of the malaria parasite Plasmodium falciparum by using microarray SNP data from human blood samples. Each sample comes from a malaria patient and contains one or several parasite clones that may genetically differ. Samples containing multiple parasite clones with different genetic markers pose a special challenge. The situation is comparable with a polyploid organism. The data from each blood sample indicates whether the parasites in the blood carry a mutant or a wildtype allele at various selected genomic positions. If both mutant and wildtype alleles are detected at a given position in a multiply infected sample, the data indicates the presence of both alleles, but the ratio is unknown. Thus, the data only partially reveals which specific combinations of genetic markers (i.e. haplotypes across the examined SNPs) occur in distinct parasite clones. In addition, SNP data may contain errors at non-negligible rates. We use a multinomial mixture model with partially missing observations to represent this data and a Markov chain Monte Carlo method to estimate the haplotype frequencies in a population. Our approach addresses both challenges, multiple infections and data errors.

Assessment of microbial larvicide spraying with Bacillus thuringiensis israelensis, for the prevention of malaria.

OBJECTIVES: The aim of this study was to assess the contribution of microbial larvicide spraying, Bacillus thuringiensis israelensis, as prevention strategy against malaria. METHODS: An experimental study consisted in spraying B. thuringiensis israelensis in a district during 1 year has been conducted. Another district (control) was not sprayed. Eight hundred and two children were evaluated, thick drop and swab examination was performed for those presenting with fever. The larval density was calculated in their habitats as well as larvicide remanence. Capture of mosquitoes with human bait allowed determining human exposure to bites at night, and identifying anopheles after dissection. RESULTS: The incidence of pediatric malaria was 13.8% in the sprayed district and 31.4% in the control district. The parasitic load ranged from 2000 to 42,000 parasites/µL in the sprayed district and 2000 to 576,000 parasites/µL in the control district. Plasmodium falciparum was the most frequent (97.8%) plasmodial species. In the control district, at least 20 larvae by liter of water were counted; anopheles larvae were found in 11 larval habitats out of 15 (73.33%). The human exposure to anopheles bites at night was 14.25 in the sprayed district and 33.13 in the control district. The remanence of B. thuringiensis israelensis was estimated at 9 days in the sprayed district. CONCLUSION: The larvicide B. thuringiensis israelensis may be used in vector control strategy for the prevention of malaria.

Assessment of two malaria rapid diagnostic tests in children under five years of age, with follow-up of false-positive pLDH test results, in a hyperendemic falciparum malaria area, Sierra Leone.

BACKGROUND: Most malaria rapid diagnostic tests (RDTs) use HRP2 detection, including Paracheck-Pf(R), but their utility is limited by persistent false positivity after treatment. PLDH-based tests become negative more quickly, but sensitivity has been reported below the recommended standard of 90%. A new pLDH test, CareStart three-line P.f/PAN-pLDH, claims better sensitivity with continued rapid conversion to negative. The study aims were to 1) compare sensitivity and specificity of CareStart to Paracheck-Pf(R) to diagnose falciparum malaria in children under five years of age, 2) assess how quickly false-positive CareStart tests become negative and 3) evaluate ease of use and inter-reader agreement of both tests. METHODS: Participants were included if they were aged between two and 59 months, presenting to a Médecins Sans Frontières community health centre in eastern Sierra Leone with suspected malaria defined as fever (axillary temperature > 37.5 degrees C) and/or history of fever in the previous 72 hours and no signs of severe disease. The same capillary blood was used for the RDTs and the blood slide, the latter used as the gold standard reference. All positive participants were treated with supervised artesunate and amodiaquine treatment for three days. Participants with a persistent false-positive CareStart, but a negative blood slide on Day 2, were followed with repeated CareStart and blood slide tests every seven days until CareStart became negative or a maximum of 28 days. RESULTS: Sensitivity of CareStart was 99.4% (CI 96.8-100.0, 168/169) and of Paracheck-Pf(R), 98.8% (95% CI 95.8-99.8, 167/169). Specificity of CareStart was 96.0% (CI 91.9-98.4, 167/174) and of Paracheck-Pf(R), 74.7% (CI 67.6-81.0, 130/174) (p < 0.001). Neither test showed any change in sensitivity with decreasing parasitaemia. Of the 155 eligible follow-up CareStart participants, 63.9% (99/155) had a false-positive test on day 2, 21.3% (33/155) on day 7, 5.8% (9/155) on day 14, 1.9% (3/155) on day 21 and 0.6% (1/155) on day 28. The median time for test negativity was seven days. CareStart was as easy to use and interpret as Paracheck-Pf(R) with excellent inter-reader agreement. CONCLUSIONS: Both RDTs were highly sensitive, met WHO standards for the detection of falciparum malaria monoinfections where parasitaemia was >100 parasites/mul and were easy to use. CareStart persistent false positivity decreased quickly after successful anti-malarial treatment, making it a good choice for a RDT for a hyperendemic falciparum malaria area.

The blood donor in sub-Saharan Africa: a review.

The high prevalence of numerous endemic and epidemic diseases such as malaria, HIV infection and viral hepatitis in some areas of sub-Saharan Africa (SSA) affects the health status of blood donors. Considering the difficulties in ensuring sufficient and safe blood supply, analysing epidemiological factors that impact blood donors in this community may further bring light on issues of supply and safety, and help in planning for its rational use. This review does not aim to propose new strategies but describes the main characteristics of blood donors in SSA as collected from different reports. Data were mainly obtained from the reports of the World Health Organization and national blood transfusion programmes and also from relevant literature and conference reports. Several characteristics are common in blood donors, such as the predominance of young adult males, the high frequency of Transmission-transmitted Infections (TTIs) and some erythrocytic phenotypes. The data indicate variations in the level of improvement of blood collection and blood safety from one area to another, particularly in the field of donor motivation or screening strategies for TTIs. These data could be useful to supplement previous reports and to provide updates for governments and international organizations' programs involved in the improvement of blood safety in Africa.

Assessment of the prozone effect in malaria rapid diagnostic tests.

BACKGROUND: The prozone effect (or high doses-hook phenomenon) consists of false-negative or false-low results in immunological tests, due to an excess of either antigens or antibodies. Although frequently cited as a cause of false-negative results in malaria rapid diagnostic tests (RDTs), especially at high parasite densities of Plasmodium falciparum, it has been poorly documented. In this study, a panel of malaria RDTs was challenged with clinical samples with P. falciparum hyperparasitaemia (> 5% infected red blood cells). METHODS: Twenty-two RDT brands were tested with seven samples, both undiluted and upon 10 x, 50 x and 100 x dilutions in NaCl 0.9%. The P. falciparum targets included histidine-rich protein-2 (HRP-2, n = 17) and P. falciparum-specific parasite lactate dehydrogenase (Pf-pLDH, n = 5). Test lines intensities were recorded in the following categories: negative, faint, weak, medium or strong. The prozone effect was defined as an increase in test line intensity of at least one category after dilution, if observed upon duplicate testing and by two readers. RESULTS: Sixteen of the 17 HRP-2 based RDTs were affected by prozone: the prozone effect was observed in at least one RDT sample/brand combination for 16/17 HRP-2 based RDTs in 6/7 samples, but not for any of the Pf-pLDH tests. The HRP-2 line intensities of the undiluted sample/brand combinations with prozone effect (n = 51) included a single negative (1.9%) and 29 faint and weak readings (56.9%). The other target lens (P. vivax-pLDH, pan-specific pLDH and aldolase) did not show a prozone effect. CONCLUSION: This study confirms the prozone effect as a cause of false-negative HRP-2 RDTs in samples with hyperparasitaemia.