Identification and serological responses to a novel Plasmodium vivax merozoite surface protein 1 (PvMSP-1) derived synthetic peptide: a putative biomarker for malaria exposure.

Background: The integration of diagnostic methods holds promise for advancing the surveillance of malaria transmission in both endemic and non-endemic regions. Serological assays emerge as valuable tools to identify and delimit malaria transmission, serving as a complementary method to rapid diagnostic tests (RDT) and thick smear microscopy. Here, we evaluate the potential of antibodies directed against peptides encompassing the entire amino acid sequence of the PvMSP-1 Sal-I strain as viable serological biomarkers for P. vivax exposure. Methods: We screened peptides encompassing the complete amino acid sequence of the Plasmodium vivax Merozoite Surface Protein 1 (PvMSP-1) Sal-I strain as potential biomarkers for P. vivax exposure. Here, immunodominant peptides specifically recognized by antibodies from individuals infected with P. vivax were identified using the SPOT-synthesis technique followed by immunoblotting. Two 15-mer peptides were selected based on their higher and specific reactivity in immunoblotting assays. Subsequently, peptides p70 and p314 were synthesized in soluble form using SPPS (Solid Phase Peptide Synthesis) and tested by ELISA (IgG, and subclasses). Results: This study unveils the presence of IgG antibodies against the peptide p314 in most P. vivax-infected individuals from the Brazilian Amazon region. In silico B-cell epitope prediction further supports the utilization of p314 as a potential biomarker for evaluating malaria transmission, strengthened by its amino acid sequence being part of a conserved block of PvMSP-1. Indeed, compared to patients infected with P. falciparum and uninfected individuals never exposed to malaria, P. vivax-infected patients have a notably higher recognition of p314 by IgG1 and IgG3.

Molecular Epidemiology of Plasmodium species in Conflicted Federally Administered Tribal Area (FATA) Pakistan / Epidemiologia molecular de espécies de plasmódio em área tribal sob administração federal em conflito do Paquistão

Military conflicts have been significant obstacles in detecting and treating infectious disease diseases due to the diminished public health infrastructure, resulting in malaria endemicity. A variety of violent and destructive incidents were experienced by FATA (Federally Administered Tribal Areas). It was a struggle to pursue an epidemiological analysis due to continuing conflict and Talibanization. Clinical isolates were collected from Bajaur, Mohmand, Khyber, Orakzai agencies from May 2017 to May 2018. For Giemsa staining, full blood EDTA blood samples have been collected from symptomatic participants. Malaria-positive microscopy isolates were spotted on filter papers for future Plasmodial molecular detection by nested polymerase chain reaction (nPCR) of small subunit ribosomal ribonucleic acid (ssrRNA) genes specific primers. Since reconfirming the nPCR, a malariometric study of 762 patients found 679 positive malaria cases. Plasmodium vivax was 523 (77%), Plasmodium falciparum 121 (18%), 35 (5%) were with mixed-species infection (P. vivax plus P. falciparum), and 83 were declared negative by PCR. Among the five agencies of FATA, Khyber agency has the highest malaria incidence (19%) with followed by P. vivax (19%) and P. falciparum (4.1%). In contrast, Kurram has about (14%), including (10.8%) P. vivax and (2.7%) P. falciparum cases, the lowest malaria epidemiology. Surprisingly, no significant differences in the distribution of mixed-species infection among all five agencies. P. falciparum and P. vivax were two prevalent FATA malaria species in Pakistan's war-torn area. To overcome this rising incidence of malaria, this study recommends that initiating malaria awareness campaigns in school should be supported by public health agencies and malaria related education locally, targeting children and parents alike.

Os conflitos militares têm sido obstáculos significativos na detecção e tratamento de doenças infecciosas devido à diminuição da infraestrutura de saúde pública, resultando na endemicidade da malária. Uma variedade de incidentes violentos e destrutivos foi vivida pelas FATA (áreas tribais administradas pelo governo federal). Foi uma luta busca ruma análise epidemiológica devido ao conflito contínuo e à talibanização. Isolados clínicos foram coletados de agências Bajaur, Mohmand, Khyber e Orakzai, de maio de 2017 a maio de 2018. Para a coloração de Giemsa, amostras de sangue completo com EDTA foram coletadas de participantes sintomáticos. Isolados de microscopia positivos para malária foram colocados em papéis de filtro para futura detecção molecular plasmódica por reação em cadeia da polimerase aninhada (nPCR) de primers específicos de genes de subunidade ribossômica de ácido ribonucleico (ssrRNA). Desde a reconfirmação do nPCR, um estudo malariométrico de 762 pacientes encontrou 679 casos positivos de malária. Plasmodium vivax foi 523 (77%), Plasmodium falciparum 121 (18%), 35 (5%) eram com infecção de espécies mistas (P. vivax mais P. falciparum) e 83 foram declarados negativos por PCR. Entre as cinco agências da FATA, a agência Khyber tem a maior incidência de malária (19%), seguida por P. vivax (19%) e P. falciparum (4,1%). Em contraste, Kurram tem cerca de 14%, incluindo 10,8% casos de P. vivax e 2,7% P. falciparum, a epidemiologia de malária mais baixa. Surpreendentemente, não há diferenças significativas na distribuição da infecção de espécies mistas entre todas as cinco agências. P. falciparum e P. vivax foram duas espécies prevalentes de malária FATA na área devastada pela guerra no Paquistão. Para superar essa incidência crescente de malária, este estudo recomenda que o início de campanhas de conscientização sobre a malária na escola deve ser apoiado por agências de saúde pública e educação relacionada com a malária localmente, visando crianças e pais.

Cryptic erythrocytic infections in Plasmodium vivax, another challenge to its elimination.

Human malaria caused by Plasmodium vivax infection (vivax malaria) is a major global health issue. It is the most geographically widespread form of the disease, accounting for 7 million annual clinical cases, the majority of cases in America and Asia and an estimation of over 2.5 billion people living under risk of infection. The general perception towards vivax malaria has shifted recently, following a series of reports, from being viewed as a benign infection to the recognition of its potential for more severe manifestations including fatal cases. However, the underlying pathogenic mechanisms of vivax malaria remain largely unresolved. Asymptomatic carriers of malaria parasites are a major challenge for malaria elimination. In the case of P. vivax, it has been widely accepted that the only source of cryptic parasites is hypnozoite dormant stages. Here, we will review new evidence indicating that cryptic erythrocytic niches outside the liver, in particular in the spleen and bone marrow, can represent a major source of asymptomatic infections. The origin of such parasites is being controversial and many key gaps in the knowledge of such infections remain unanswered. Yet, as parasites in these niches seem to be sheltered from immune response and antimalarial drugs, research on this area should be reinforced if elimination of malaria is to be achieved. Last, we will glimpse into the role of reticulocyte-derived exosomes, extracellular vesicles of endocytic origin, as intercellular communicators likely involved in the formation of such cryptic erythrocytic infections.

HIF-1α regulated pathomechanism of low birth weight through angiogenesis factors in placental Plasmodium vivax infection.

Background: Malaria in pregnancy leads to placental malaria. The primary pathogenesis of the complex fetal implications in placental malaria is tissue hypoxia due to sequestrations of Plasmodium falciparum-infected erythrocytes in the placenta. However, the pathomechanism of placental Plasmodium vivax infection has not been thoroughly investigated. Hypoxia-inducible factor-1α (HIF-1α) is a key transcriptional mediator of the response to hypoxic conditions, which interacts with the change and imbalances of many chemical mediators, including angiogenic factors, leading to fetal growth abnormality. Methods: This study was conducted cross-sectionally in Maumere, Sikka Regency, East Nusa Tenggara Province, previously known as one of the malaria endemic areas with a high incidence of low birth weight (LBW) cases. This study collected peripheral and umbilical blood samples and placental tissues from mothers who delivered their babies with LBW at the TC Hiller Regional Hospital. All of the blood samples were examined for parasites by microscopic and PCR techniques, while the plasma levels of VEGF, PlGF, VEGFR-1, VEGFR-2, and HIF-1α were determined using ELISA. The sequestration of infected erythrocytes and hemozoin was determined from placental histological slides, and the expression of placenta angiogenic factors was observed using the immunofluorescent technique. Results: In this study, 33 cases had complete data to be analyzed. Of them, 19 samples were diagnosed as vivax malaria and none of falciparum malaria. There were significant differences in Δ 10th percentile growth curve of baby's body weights and also all angiogenic factors in placental tissues {VEGF, PlGF, and VEGFR-1, VEGFR-2, and HIF-1α} between those infected and not infected cases (p<0.05), but not for VEGF and VEGFR-2 in the plasma. Conclusion: This study indicated that Plasmodium vivax sequestration may promote LBW through alterations and imbalances in angiogenic factors led by HIF-1α.

Quantitative cytokine level of TNF-α, IFN-γ, IL-10, TGF-ß and circulating Epstein-Barr virus DNA load in individuals with acute Malaria due to P. falciparum or P. vivax or double infection in a Malaria endemic region in Indonesia.

Plasmodium falciparum Malaria and Epstein-Barr Virus (EBV) infection are risk factors in the development of Burkitt's lymphoma. In Indonesia, 100% of the population is persistently infected with EBV early in life and at risk of developing EBV-linked cancers. Currently, 10.7 million people in Indonesia are living in Malaria-endemic areas. This cross-sectional study was initiated to investigate how acute Malaria dysregulates immune control over latent EBV infection. Using blood and plasma samples of 68 patients with acute Malaria and 27 healthy controls, we measured the level of parasitemia for each plasmodium type (P. falciparum, P. vivax, and mixed) by microscopy and rapid test. The level of 4 regulatory cytokines was determined by quantitative ELISA and the level of circulating EBV genome by real-time PCR targeting the single copy EBNA-1 sequence. All Plasmodium-infected cases had high-level parasitemia (>1000 parasites/ul blood) except for one case. EBV-DNA levels were significantly more elevated in P. falciparum and P. vivax infections (P<0.05) compared to controls. EBV-DNA levels were not related to age, gender, Malaria symptoms, or plasmodium type. TNF-α and IL-10 levels were increased in Malaria cases versus controls, but IFN-γ and TGF- ß levels were comparable between the groups. Only TNF-α levels in P. falciparum cases showed a clear correlation with elevated EBV DNA levels (R2 = 0.8915). This is the first study addressing the relation between EBV (re)activation and cytokine responses during acute Malaria, revealing a clear correlation between pro-inflammatory cytokine TNF-α and EBV-DNA levels, specifically in P. falciparum cases, suggesting this cytokine to be key in dysregulating EBV homeostasis during acute P. falciparum Malaria.

Rosettes integrity protects Plasmodium vivax of being phagocytized.

Plasmodium vivax is the most prevalent cause of malaria outside of Africa. P. vivax biology and pathogenesis are still poorly understood. The role of one highly occurring phenotype in particular where infected reticulocytes cytoadhere to noninfected normocytes, forming rosettes, remains unknown. Here, using a range of ex vivo approaches, we showed that P. vivax rosetting rates were enhanced by plasma of infected patients and that total immunoglobulin M levels correlated with rosetting frequency. Moreover, rosetting rates were also correlated with parasitemia, IL-6 and IL-10 levels in infected patients. Transcriptomic analysis of peripheral leukocytes from P. vivax-infected patients with low or moderated rosetting rates identified differentially expressed genes related to human host phagocytosis pathway. In addition, phagocytosis assay showed that rosetting parasites were less phagocyted. Collectively, these results showed that rosette formation plays a role in host immune response by hampering leukocyte phagocytosis. Thus, these findings suggest that rosetting could be an effective P. vivax immune evasion strategy.

Platelet disturbances correlate with endothelial cell activation in uncomplicated Plasmodium vivax malaria.

Platelets drive endothelial cell activation in many diseases. However, if this occurs in Plasmodium vivax malaria is unclear. As platelets have been reported to be activated and to play a role in inflammatory response during malaria, we hypothesized that this would correlate with endothelial alterations during acute illness. We performed platelet flow cytometry of PAC-1 and P-selectin. We measured platelet markers (CXCL4, CD40L, P-selectin, Thrombopoietin, IL-11) and endothelial activation markers (ICAM-1, von Willebrand Factor and E-selectin) in plasma with a multiplex-based assay. The values of each mediator were used to generate heatmaps, K-means clustering and Principal Component analysis. In addition, we determined pair-wise Pearson's correlation coefficients to generate correlation networks. Platelet counts were reduced, and mean platelet volume increased in malaria patients. The activation of circulating platelets in flow cytometry did not differ between patients and controls. CD40L levels (Median [IQ]: 517 [406-651] vs. 1029 [732-1267] pg/mL, P = 0.0001) were significantly higher in patients, while P-selectin and CXCL4 showed a nonsignificant trend towards higher levels in patients. The network correlation approach demonstrated the correlation between markers of platelet and endothelial activation, and the heatmaps revealed a distinct pattern of activation in two subsets of P. vivax patients when compared to controls. Although absolute platelet activation was not strong in uncomplicated vivax malaria, markers of platelet activity and production were correlated with higher endothelial cell activation, especially in a specific subset of patients.

New laboratory perspectives for evaluation of vivax malaria infected patients: a useful tool for infection monitoring

ABSTRACT In recent years, the number of cases with severe Plasmodium vivax malaria has shown an increasing trend. It is, therefore, important to identify routine laboratory markers that best characterize the acute disease phase and can serve as a tool for clinical follow-up of patients. In a cohort study, we followed 87 patients with acute P. vivax monoinfection acquired in an endemic region of the Brazilian Amazon. Forty-two different biochemical and hematological parameters frequently tested in clinical routine were evaluated at the acute phase and the convalescent phase. A total of 42 laboratory tests were performed: biochemical parameters measured were serum lipids levels, aminotransferases, bilirubin, amylase, glucose, urea, creatinine, albumin, globulin, uric acid, C-reactive protein, and alpha-1-acid glycoprotein. Hematological parameters included total and differential white blood cell and platelet counts, hemoglobin concentration, mean platelet volume, platelet width distribution, and plateletcrit. Our results show that several biochemical and hematological parameters were associated with acute phase P. vivax malaria and these parameters reverted to normal values in the convalescent phase. The use of these parameters during diagnosis and follow-up of the infection is a useful clinical tool to evaluate the clinical course and therapeutic response of patients with uncomplicated vivax malaria.

Increased platelet distribution width and reduced IL-2 and IL-12 are associated with thrombocytopenia in Plasmodium vivax malaria

BACKGROUND Thrombocytopenia in malaria involves platelet destruction and consumption; however, the cellular response underlying this phenomenon has still not been elucidated. OBJECTIVE To find associations between platelet indices and unbalanced Th1/Th2/Th17 cytokines as a response to thrombocytopenia in Plasmodium vivax infected (Pv-MAL) patients. METHODS Platelet counts and quantification of Th1/Th2/Th17 cytokine levels were compared in 77 patients with uncomplicated P. vivax malaria and 37 healthy donors from the same area (endemic control group - ENCG). FINDINGS Thrombocytopenia was the main manifestation in 55 patients, but was not associated with parasitaemia. The Pv-MAL patients showed increases in the mean platelet volume (MPV), which may be consistent with larger or megaplatelets. Contrary to the findings regarding the endemic control group, MPV and platelet distribution width (PDW) did not show an inverse correlation, due the increase in the heterogeneity of platelet width. In addition, the Pv-MAL patients presented increased IL-1β and reduced IL-12p70 and IL-2 serum concentrations. Furthermore, the reduction of these cytokines was associated with PDW values. MAIN CONCLUSIONS Our data demonstrate that an increase in MPV and the association between reductions of IL-2 and IL-12 and PDW values may be an immune response to thrombocytopenia in uncomplicated P. vivax malaria.

Evaluación de la calidad del diagnóstico de malaria en la red local de laboratorios y en los laboratorios intermedios en el contexto de la eliminación de la enfermedad en Ecuador / Quality evaluation of malaria diagnosis in the local laboratories network and in intermediate laboratories in a setting towards the disease elimination in Ecuador

Resumen Introducción. El cumplimiento de la meta de eliminación de la malaria en Ecuador en el 2020 exige contar con la capacidad requerida para el diagnóstico microscópico ajustado a los estándares de calidad de la Organización Mundial de la Salud (OMS) y de la Organización Panamericana de la Salud (OPS) y proveer el tratamiento adecuado a los pacientes. Objetivo. Conocer la idoneidad o competencia de los microscopistas de la red pública local para el diagnóstico parasitológico de la malaria y el desempeño de los laboratorios intermedios de referencia. Materiales y métodos. Se hizo un estudio descriptivo de corte transversal a partir de la información obtenida en los talleres de evaluación de idoneidad en el diagnóstico microscópico de la red de laboratorios en las coordinaciones zonales de salud utilizando un panel de láminas para evaluar la concordancia del diagnóstico. Además, se calificó el desempeño de los laboratorios intermedios en el diagnóstico en el marco del programa de evaluación externa del desempeño. Los resultados se compararon con los obtenidos por el laboratorio supranacional de Perú. Resultados. En los 11 talleres realizados, se evaluó la idoneidad de 191 microscopistas, de los cuales 153 (80,1 %) aprobaron las pruebas. Las medianas de los indicadores fueron las siguientes: concordancia entre la detección y el resultado, 100 % (Q1- Q3: 96-100); concordancia en la especie, 100 % (Q1- Q3: 93-100); concordancia en el estadio, 93,0 % (Q1- Q3: 86-95) y concordancia en el recuento, 77 % (Q1- Q3: 71-82). En el programa de evaluación externa de desempeño, los tres laboratorios intermedios obtuvieron una concordancia del 100 % en el resultado y una del 96 % en la especie. Conclusiones. Los indicadores de competencia de la red local y de desempeño de los laboratorios intermedios alcanzaron altos estándares de calidad acordes con el proceso de entrenamiento implementado en el país.

Abstract Introduction: To reach the goal of malaria elimination in Ecuador for the year 2020, it is necessary to have a laboratory network with the capacity to perform microscopic diagnosis according to the WHO/PAHO quality standards and to provide the adequate treatment of cases. Objective: To determine the level of competence for parasitological diagnosis of the microscopists from the local public network and the performance of intermediate reference laboratories. Materials and methods: We conducted a cross-sectional study based on the information collected in workshops carried out to appraise the competence for microscopic diagnosis of the local laboratory network (zonal health coordinating offices 1 to 8) using a slide panel to evaluate diagnosis agreement, as well as the diagnostic performance of the intermediate laboratories using an external quality assessment program. The results were compared against the reference standards of the supranational laboratory in Perú. Results: We evaluated the competencies of 191 microscopists in 11 workshops and 153 (80.1%) of them were approved. The medians of the indicators were the following: concordance for parasite detection, 100% (Q1- Q3: 96-100), concordance for species identification, 100% (Q1- Q3: 93-100), and concordances for stage identification, 93.0% (Q1- Q3: 86-95) and parasite counting, 77.0% (Q1- Q3: 71-82). In the external quality assessment, the three intermediate laboratories obtained 100% in parasite detection concordance and 96% for species detection concordance. Conclusions: The results for the primary network and the performance indicators for the intermediate laboratories showed the high-quality standards of the training program implemented in the country.

Preliminary assessment of anti-α-Gal IgG and IgM levels in patients with patent Plasmodium vivax infection

Anti-α-Gal responses may exert a protective effect in falciparum malaria. However, the biological role of such antibodies is still unknown during Plasmodium vivax infections. We investigated IgG and IgM responses to α-Gal in individuals with vivax malaria. Anti-α-Gal IgG and IgM levels were higher in these patients than in controls, but no significant correlation was found between parasitaemia and anti-α-Gal response, nor between this response and ABO blood group status. This is the first study to investigate anti-α-Gal antibodies in P. vivax-infected patients; a larger survey is necessary to achieve a better understanding of host immune response during vivax malaria.

Decreased uric acid levels in the acute phase of Plasmodium vivax malaria

Abstract INTRODUCTION Uric acid is one of the compounds associated with the inflammatory process in malaria. It acts as an indicator of cellular damage by activating the immune response and inflammatory process. METHODS: We measured serum concentrations of uric acid in 60 symptomatic patients before and after treatment for malarial infections caused by Plasmodium vivax. RESULTS: Lower serum concentrations of uric acid were found during the acute phase of P. vivax malaria compared to those in its convalescent phase (p < 0.0001). CONCLUSIONS: Patients in the acute phase of malaria had lower uric acid levels than those in its convalescent phase.

Screening and identification of potential novel biomarker for diagnosis of complicated Plasmodium vivax malaria.

BACKGROUND: In the recent years Plasmodium vivax has been reported to cause severe infections associated with mortality. Clinical evaluation has limited accuracy for the early identification of the patients progressing towards the fatal condition. Researchers have tried to identify the serum and the plasma-based indicators of the severe malaria. Discovery of MicroRNA (miRNA) has opened up an era of identification of early biomarkers for various infectious and non-infectious diseases. MicroRNAs (miRNA) are the small non-coding RNA molecules of length 19-24 nts and are responsible for the regulation of the majority of human gene expressions at post transcriptional level. METHODS: We identified the differentially expressed miRNAs by microarray and validated the selected miRNAs by qRT-PCR. We assessed the diagnostic potential of these up-regulated miRNAs for complicated P. vivax malaria. Futher, the bioinformtic analysis was performed to construct protein-protein and mRNA-miRNA networks to identify highly regulated miRNA. RESULTS: In the present study, utility of miRNA as potential biomarker of complicated P. vivax malaria was explored. A total of 276 miRNAs were found to be differentially expressed by miRNA microarray and out of which 5 miRNAs (hsa-miR-7977, hsa-miR-28-3p, hsa-miR-378-5p, hsa-miR-194-5p and hsa-miR-3667-5p) were found to be significantly up-regulated in complicated P. vivax malaria patients using qRT-PCR. The diagnostic potential of these 5 miRNAs were found to be significant with sensitivity and specificity of 60-71% and 69-81% respectively and area under curve (AUC) of 0.7 (p < 0.05). Moreover, in silico analysis of the common targets of up-regulated miRNAs revealed UBA52 and hsa-miR-7977 as majorly regulated hubs in the PPI and mRNA-miRNA networks, suggesting their putative role in complicated P. vivax malaria. CONCLUSION: miR-7977 might act as a potential biomarker for differentiating complicated P. vivax malaria from uncomplicated type. The elevated levels of miR-7977 may have a role to play in the disease pathology through UBA52 or TGF-beta signalling pathway.

Role of IL-1ß, IL-6 and TNF-α cytokines and TNF-α promoter variability in Plasmodium vivax infection during pregnancy in endemic population of Jharkhand, India.

BACKGROUND: The combinatorial effects of Plasmodium infection, perturbation of inflammatory responses and the dichotomic role of TNF promoter polymorphism has potential clinical and physiological relevance during pregnancy. OBJECTIVE AND METHODS: This coordinated orchestration instigated us to investigate the circulating level of inflammatory cytokines (IL-1ß, TNF-α and IL-6) employing ELISA in a stratified group of samples and the plausible genetic association of TNF-α -308 G/A using PCR-RFLP/sequencing during Plasmodium vivax infection in pregnancy. RESULTS: We observed significantly elevated concentrations of IL-1ß were observed, followed by IL-6 and TNF-α in women with malaria (WWM) and in malaria in pregnancy (MIP). Further, elevated IL-1ß, followed by TNF-α and IL-6 were detected in the non-infected pregnancy group. The differential dynamics of inflammatory cytokine concentration during each trimester of pregnancy with and without P. vivax infection were detected. For the first time, a high level of IL-6 was observed in the first trimester of MIP and high IL-1ß in healthy pregnancies. In the second trimester, however, we observed a high level of IL-1ß in the MIP group compared to a sustained high level of IL-1ß in the healthy pregnancy group. In the third trimester, high IL-1ß was sustained in the MIP group and healthy pregnancies acquired a high TNF-α level. The genotypic distribution for the TNF-α promoter -308 G/A position was observed to be nonsignificant and mildly associated during MIP (OR = 1.4) and in WWM (OR = 1.2). Moreover, based on genotypic distribution, we observed a well-correlated and significantly elevated TNF-α concentration in the mutant homozygote genotype (AA; p = 0.001) followed by heterozygotes (GA; p = 0.0001) and ancestral genotypes (GG; p = 0.0001) in both MIP and WWM subjects. CONCLUSION: The observation of elevated IL-1ß and IL-6 in MIP and TNF-α in WWM may be regarded as a prognostic inflammatory marker of infection and pregnancy. Most particularly, the TNF-α concentration and its polymorphic variability in the promoter region may indicate genetic susceptibility and mildly influence the risk for P. vivax infection during pregnancy and in women with malaria.

Correlation of APRIL with production of inflammatory cytokines during acute malaria in the Brazilian Amazon.

INTRODUCTION: A proliferation-inducing ligand (APRIL) and B cell activation factor (BAFF) are known to play a significant role in the pathogenesis of several diseases, including BAFF in malaria. The aim of this study was to investigate whether APRIL and BAFF plasma concentrations could be part of inflammatory responses associated with P. vivax and P. falciparum malaria in patients from the Brazilian Amazon. METHODS: Blood samples were obtained from P. vivax and P. falciparum malaria patients (n = 52) resident in Porto Velho before and 15 days after the beginning of treatment and from uninfected individuals (n = 12). We investigated APRIL and BAFF circulating levels and their association with parasitaemia, WBC counts, and cytokine/chemokine plasma levels. The expression levels of transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) on PBMC from a subset of 5 P. vivax-infected patients were analyzed by flow cytometry. RESULTS: APRIL plasma levels were transiently increased during acute P. vivax and P. falciparum infections whereas BAFF levels were only increased during acute P. falciparum malaria. Although P. vivax and P. falciparum malaria patients have similar cytokine profiles during infection, in P. vivax acute phase malaria, APRIL but not BAFF levels correlated positively with IL-1, IL-2, IL-4, IL-6, and IL-13 levels. We did not find any association between P. vivax parasitaemia and APRIL levels, while an inverse correlation was found between P. falciparum parasitaemia and APRIL levels. The percentage of TACI positive CD4+ and CD8+ T cells were increased in the acute phase P. vivax malaria. CONCLUSION: These findings suggest that the APRIL and BAFF inductions reflect different host strategies for controlling infection with each malaria species.

Further evaluation of the NWF filter for the purification of Plasmodium vivax-infected erythrocytes.

BACKGROUND: Isolation of Plasmodium-infected red blood cells (iRBCs) from clinical blood samples is often required for experiments, such as ex vivo drug assays, in vitro invasion assays and genome sequencing. Current methods for removing white blood cells (WBCs) from malaria-infected blood are time-consuming or costly. A prototype non-woven fabric (NWF) filter was developed for the purification of iRBCs, which showed great efficiency for removing WBCs in a pilot study. Previous work was performed with prototype filters optimized for processing 5-10 mL of blood. With the commercialization of the filters, this study aims to evaluate the efficiency and suitability of the commercial NWF filter for the purification of Plasmodium vivax-infected RBCs in smaller volumes of blood and to compare its performance with that of Plasmodipur® filters. METHODS: Forty-three clinical P. vivax blood samples taken from symptomatic patients attending malaria clinics at the China-Myanmar border were processed using the NWF filters in a nearby field laboratory. The numbers of WBCs and iRBCs and morphology of P. vivax parasites in the blood samples before and after NWF filtration were compared. The viability of P. vivax parasites after filtration from 27 blood samples was examined by in vitro short-term culture. In addition, the effectiveness of the NWF filter for removing WBCs was compared with that of the Plasmodipur® filter in six P. vivax blood samples. RESULTS: Filtration of 1-2 mL of P. vivax-infected blood with the NWF filter removed 99.68% WBCs. The densities of total iRBCs, ring and trophozoite stages before and after filtration were not significantly different (P > 0.05). However, the recovery rates of schizont- and gametocyte-infected RBCs, which were minor parasite stages in the clinical samples, were relatively low. After filtration, the P. vivax parasites did not show apparent morphological changes. Culture of 27 P. vivax-infected blood samples after filtration showed that parasites successfully matured into the schizont stage. The WBC removal rates and iRBC recovery rates were not significantly different between the NWF and Plasmodipur® filters (P > 0.05). CONCLUSIONS: When tested with 1-2 mL of P. vivax-infected blood, the NWF filter could effectively remove WBCs and the recovery rates for ring- and trophozoite-iRBCs were high. P. vivax parasites after filtration could be successfully cultured in vitro to reach maturity. The performance of the NWF and Plasmodipur® filters for removing WBCs and recovering iRBCs was comparable.

Field performance of malaria rapid diagnostic test for the detection of Plasmodium falciparum infection in Odisha State, India.

BACKGROUND & OBJECTIVES: Rapid diagnostic tests (RDTs) have become an essential surveillance tool in the malaria control programme in India. The current study aimed to assess the performance of ParaHIT-f, a rapid test in diagnosis of Plasmodium falciparum infection through detecting its specific antigen, histidine rich protein 2 (PfHRP-2), in Odisha State, India. METHODS: The study was undertaken in eight falciparum malaria endemic southern districts of Odisha State. Febrile patients included through active case detection, were diagnosed by Accredited Social Health Activists (ASHAs) for P. falciparum infection using the RDT, ParaHIT-f. The performance of ParaHIT-f was evaluated using microscopy as the gold standard. RESULTS: A total of 1030 febrile patients were screened by both microscopy and the RDT for P. falciparum infection. The sensitivity of ParaHIT-f was 63.6% (95% CI: 56.0-70.6) and specificity was 98.9% (95% CI: 97.9-99.5), with positive and negative predictive values (PPV and NPV) of 92.6% (95% CI: 86.0-96.3) and 93.0% (95% CI: 91.0-94.5), respectively. When related to parasitaemia, the RDT sensitivity was 47.8% at the low parasitaemia of 4 to 40 parasites/µl of blood. INTERPRETATION & CONCLUSIONS: The results showed that the performance of the RDT, ParaHIT-f, was not as sensitive as microscopy in detecting true falciparum infections; a high specificity presented a low frequency of false-positive RDT results. t0 he sensitivity of ParaHIT-f was around 60 per cent. It is, therefore, essential to improve the efficiency (sensitivity) of the kit so that the true falciparum infections will not be missed especially in areas where P. falciparum has been the predominant species causing cerebral malaria.

Genetic diversity of chloroquine-resistant Plasmodium vivax parasites from the western Brazilian Amazon

The molecular basis of Plasmodium vivax chloroquine (CQ) resistance is still unknown. Elucidating the molecular background of parasites that are sensitive or resistant to CQ will help to identify and monitor the spread of resistance. By genotyping a panel of molecular markers, we demonstrate a similar genetic variability between in vitro CQ-resistant and sensitive phenotypes of P. vivax parasites. However, our studies identified two loci (MS8 and MSP1-B10) that could be used to discriminate between both CQ-susceptible phenotypes among P. vivax isolates in vitro. These preliminary data suggest that microsatellites may be used to identify and to monitor the spread of P. vivax-resistance around the world.

[Plasmodium vivax specific peptides prediction and screening based on repetitive protein sequences and linear B cell epitope].

OBJECTIVE: To establish a method based on repetitive protein sequences and linear B cell epitope to predict and screen specific peptides of Plasmodium vivax. METHODS: A P. vivax protein sequence database was reconstructed based on PlasmoDB data, and a customized software for searching of repetitive sequences was used to count the repetition times of each 16 aa peptide in the whole database, and the highly repetitive peptides were chosen to predict the potential linear B cell epitopes. The repetitive peptides with P. vivax specificity were selected as candidate antigen peptides to synthesize and to couple with KLH carrier protein for immunizing BALB/c mice. After the immunization, the antibody titers of the immunized mice were detected. RESULTS: The repetitive information of 16 aa peptides was analyzed by screening of the total 5 432 peptide sequences in the P. vivax database. A total of 22 peptides were identified as candidate peptides from the top 1 000 repetitive peptides by linear B cell epitope prediction on the BcePred website. Through clustering analysis and similarity comparison, five potential P. vivax specific peptides were selected, synthesized and then coupled with KLH to immunize the mice. The antibody titers of the immunized mice induced by the 5 peptides were all above 1 : 9 000. CONCLUSION: The method for predicting and screening of specific peptides of P. vivax based on repetitive protein sequences and linear B cell epitope has been established successfully, and all the 5 peptides obtained by the method can induce the high titer antibody in mice.

Influence of age on the haemoglobin concentration of malaria-infected patients in a reference centre in the Brazilian Amazon

Anaemia is amongst the major complications of malaria, a major public health problem in the Amazon Region in Latin America. We examined the haemoglobin (Hb) concentrations of malaria-infected patients and compared it to that of malaria-negative febrile patients and afebrile controls. The haematological parameters of febrile patients who had a thick-blood-smear performed at an infectious diseases reference centre of the Brazilian Amazon between December 2009-January 2012 were retrieved together with clinical data. An afebrile community control group was composed from a survey performed in a malaria-endemic area. Hb concentrations and anaemia prevalence were analysed according to clinical-epidemiological status and demographic characteristics. In total, 7,831 observations were included. Patients with Plasmodium falciparum infection had lower mean Hb concentrations (10.5 g/dL) followed by P. vivax-infected individuals (12.4 g/dL), community controls (12.8 g/dL) and malaria-negative febrile patients (13.1 g/dL) (p < 0.001). Age, gender and clinical-epidemiological status were strong independent predictors for both outcomes. Amongst malaria-infected individuals, women in the reproductive age had considerably lower Hb concentrations. In this moderate transmission intensity setting, both vivax and falciparum malaria are associated with reduced Hb concentrations and risk of anaemia throughout a wide age range.