Plasmodium vivax Duffy binding protein: baseline antibody responses and parasite polymorphisms in a well‐consolidated settlement of the Amazon Region

Objective  To investigate risk factors associated with the acquisition of antibodies against Plasmodium vivax Duffy binding protein (PvDBP) – a leading malaria vaccine candidate – in a well‐consolidated agricultural settlement of the Brazilian Amazon Region and to determine the sequence diversity of the PvDBP ligand domain (DBP_II) within the local malaria parasite population. Methods  Demographic, epidemiological and clinical data were collected from 541 volunteers using a structured questionnaire. Malaria parasites were detected by conventional microscopy and PCR, and blood collection was used for antibody assays and molecular characterisation of DBP_II. Results  The frequency of malaria infection was 7% (6% for P. vivax and 1% for P. falciparum), with malaria cases clustered near mosquito breeding sites. Nearly 50% of settlers had anti‐PvDBP IgG antibodies, as detected by enzyme‐linked immunosorbent assay (ELISA) with subject’s age being the only strong predictor of seropositivity to PvDBP. Unexpectedly, low levels of DBP_II diversity were found within the local malaria parasites, suggesting the existence of low gene flow between P. vivax populations, probably due to the relative isolation of the studied settlement. Conclusion  The recognition of PvDBP by a significant proportion of the community, associated with low levels of DBP_II diversity among local P. vivax, reinforces the variety of malaria transmission patterns in communities from frontier settlements. Such studies should provide baseline information for antimalarial vaccines now in development.     

Stronger host response per parasitized erythrocyte in Plasmodium vivax or ovale than in Plasmodium falciparum malaria

Objective and methods Fever tends to start at a lower level of parasitemia in Plasmodium vivax or ovale than in P. falciparum malaria, but hyperparasitemia and complications are more likely to occur in P. falciparum malaria. Therefore, we compared the relationship between parasitemia and host response parameters before therapy in 97 patients with P. faciparum malaria (18 with complications), and 28 with P. vivax or ovale malaria. Results In both types of malaria, parasitemia correlated with blood levels of tumour necrosis factor alpha (TNF‐alpha), lactate dehydrogenase (LDH), Thrombin–antithrombin III (TAT) and elastase, and these parameters were higher in P. falciparum malaria than in P. vivax or ovale malaria. In contrast, the ratios of TNF‐alpha, TAT, elastase, and LDH per parasitized erythrocyte were higher in P. vivax or ovale malaria than in uncomplicated P. falciparum malaria. They were lowest in complicated disease. Multivariate regression analysis confirmed that parasitemia did not affect these differences. Conclusion The host response may reach full strength at lower parasitemia in Plasmodium vivax or ovale, than in P. falciparum malaria. With hyperparasitemia in P. falciparum malaria, the host response seems to be unable to control parasite multiplication.     

Neutrophil alterations in pregnancy‐associated malaria and induction of neutrophil chemotaxis by Plasmodium falciparum

Pregnancy‐associated malaria (PAM) is a severe form of the disease caused by sequestration of Plasmodium falciparum‐infected red blood cells (iRBCs) in the developing placenta. Pathogenesis of PAM is partially based on immunopathology, with frequent monocyte infiltration into the placenta. Neutrophils are abundant blood cells that are essential for immune defence but may also cause inflammatory pathology. Their role in PAM remains unclear. We analysed neutrophil alterations in the context of PAM to better understand their contribution to disease development. Pregnant women exposed to Plasmodium falciparum had decreased numbers of circulating neutrophils. Placental‐like BeWo cells stimulated with malaria parasites produced the neutrophil chemoattractant IL‐8 and recruited neutrophils in a trans‐well assay. Finally, immunostaining of a PAM placenta confirmed neutrophil accumulation in the intervillous space. Our data indicate neutrophils may play a role in placental malaria and should be more closely examined as an etiological agent in the pathophysiology of disease.     

High proportion of mixed‐species Plasmodium infections in India revealed by PCR diagnostic assay

Accurate diagnosis is the key to effective treatment and control of malaria. We screened 180 microscopically diagnosed Indian malaria‐positive blood samples for pure and mixed infections by Plasmodium falciparum and Plasmodium vivax. An unusually high proportion of mixed infections was detected, signifying the sensitivity of PCR assay over traditional microscopic diagnosis.     

Vivax infection alters peripheral B‐cell profile and induces persistent serum IgM

B cell–mediated humoral responses are essential for controlling malarial infection. Studies have addressed the effects of Plasmodium falciparum infection on peripheral B‐cell subsets but not much is known for P. vivax infection. Furthermore, majority of the studies investigate changes during acute infection, but not after parasite clearance. In this prospective study, we analysed peripheral B‐cell profiles and antibody responses during acute P. vivax infection and upon recovery (30 days post‐treatment) in a low‐transmission area in India. Dengue patients were included as febrile‐condition controls. Both dengue and malaria patients showed a transient increase in atypical memory B cells during acute infection. However, transient B cell‐activating factor (BAFF)–independent increase in the percentage of total and activated immature B cells was observed in malaria patients. Naïve B cells from malaria patients also showed increased TLR4 expression. Total IgM levels remained unchanged during acute infection but increased significantly at recovery. Serum antibody profiling showed a parasite‐specific IgM response that persisted at recovery. A persistent IgM autoantibody response was also observed in malaria but not dengue patients. Our data suggest that in hypoendemic regions acute P. vivax infection skews peripheral B‐cell subsets and results in a persistent parasite‐specific and autoreactive IgM response.     

Automated detection of malaria‐associated intraleucocytic haemozoin by Cell‐Dyn CD4000 depolarization analysis

Summary Laboratory tests for malaria are only performed if there is clinical suspicion of the disease, and a missed diagnosis contributes substantially to morbidity and mortality. Malaria parasites produce haemozoin, which is able to depolarize light and this allows the automated detection of malaria during routine complete blood count analysis (CBC) with some Abbott Cell‐Dyn instruments. In this study, we evaluated the Cell‐Dyn CD4000 with 831 blood samples submitted for malaria investigations. Samples were categorized as malaria negative (n = 417), convalescent malaria (n = 64) or malaria positive (n = 350) by reference to thin/thick film microscopy, ‘rapid test’ procedures, polymerase chain reaction analysis and clinical history. With regard to CD4000 depolarization analysis, a malaria positive CD4000 pattern was ascribed to samples that showed one or more abnormal depolarizing purple events, which corresponded to monocytes containing ingested malaria pigment (haemozoin). Positive CD4000 patterns were observed in 11 of 417, 50 of 64 and 281 of 350 of malaria negative, convalescent malaria and malaria positive samples respectively. The specificity and positive predictive values for malaria (active and convalescent) were very high (97.4 and 96.8%, respectively), while sensitivity and negative predictive values were 80.0 and 83.0% respectively. Depolarization analysis was particularly effective for Plasmodium falciparum malaria but there was lower detection sensitivity for White compared with Black African patients. CD4000 90° depolarization vs 0° analysis revealed a proportion of samples with small nonleucocyte‐associated depolarizing particles. Appearance of such events in the form of a discrete cluster was associated with P. vivax rather than P. falciparum infection.     

Emergence of a new focus of Plasmodium malariae in forest villages of district Balaghat,Central India: implications for the diagnosis of malaria and its control

Objective During an epidemiological study (January–July 2012) on malaria in forest villages of Central India, Plasmodium malariae‐like malaria parasites were observed in blood smears of fever cases. We aimed to confirm the presence of P. malariae using molecular tools i.e. species‐specific nested polymerase chain reaction (PCR) and DNA sequencing. Methods All fever cases or cases with history of fever in 25 villages of Balaghat district were screened for malaria parasite using bivalent rapid diagnostic test and microscopy after obtaining written informed consent. Nested PCR was employed on microscopically suspected P. malariae cases. DNA sequences in the target region for PCR diagnosis were analysed for all the suspected cases of P. malariae. Results Among the 22 microscopy suspected P. malariae cases, nested PCR confirmed the identity of P. malariae in 19 cases. Among these 14 were mono P. malariae infections, three were mixed infection of P. malariae with Plasmodium falciparum and two were mixed infection of P. malariae with Plasmodium vivax. Clinically P. malariae subjects generally presented with fever and headache. However, the typical 3‐day pattern of quantum malaria was not observed. The parasite density of P. malariae was significantly lower than that of P. vivax and P. falciparum. Discussions Plasmodium malariae may have been in existence in forest villages of central India but escaped identification due to its close resemblance to P. vivax. The results re‐affirm the importance of molecular methods of testing on routine basis for efficacious control strategies against malaria.     

Rice‐produced MSP142 of Plasmodium falciparum elicits antibodies that inhibit parasite growth in vitro

Many malaria antigens contain multiple disulphide bonds involved in the formation of inhibitory B‐cell epitopes. Producing properly folded malaria antigens in sufficient quantities for vaccination is often a challenge. The 42‐kDa fragment of Plasmodium falciparum merozoite surface protein 1 (MSP1₄₂) is such a kind of malaria antigen. In this study, we investigated the expression of MSP1₄₂ in a rice system (9522, a cultivar of Oryza sativa ssp. japonica), which was used as a bioreactor for protein production. The MSP1₄₂ gene was synthesized according to rice‐preferred codons and transformed into rice plants via an Agrobacterium‐mediated method. The recombinant antigen was efficiently expressed in rice seeds with a level up to 1.56% of total soluble protein and was recognized by both the conformational monoclonal antibody 5.2 (mAb5.2) and the pooled sera of P. falciparum malaria patients. Rabbits were immunized intramuscularly with the purified MSP1₄₂ formulated with Freund's adjuvant. High antibody titres against MSP1₄₂ were elicited. The rabbit immune sera reacted well with the native protein of P. falciparum parasite and strongly inhibited the in vitro growth of blood‐stage P. falciparum parasites, demonstrating that transgenic rice can become an efficient bioreactor for the production of malaria vaccine antigens.     

Evaluation of Plasmodium vivax ELISA for the blood screen

Plasmodium vivax malaria is the indigenous strain in the Republic of Korea (ROK). Plasmodium vivax can be transmitted through the transfusions of various blood components, which became a severe problem with the safety of blood transfusions and blood‐related products in ROK. We evaluated a P. vivax‐specific enzyme‐linked immunosorbent assay (Genedia Malaria Ab ELISA 2.0, Green Cross, ROK) with blood samples from four groups: 251 samples from P. vivax‐infected patients, 39 samples from post‐treatment patients upon follow‐up, 200 samples from healthy volunteers and 421 samples from domestic travellers to and from high endemic areas of ROK. The positive cases from the ELISA test were confirmed by both Giemsa microscopic and polymerase chain reaction methods. The clinical sensitivity and specificity of detecting P. vivax with ELISA test were 94.4% and 99.0%, respectively. Thirteen of 421 domestic travellers (3.0%) to endemic areas tested positive. The results indicate the effectiveness of detecting antibodies against P. vivax in blood with Genedia Malaria Ab ELISA 2.0 test in a large blood screen setting.     

Development of a Polymerase Chain Reaction (PCR) method based on amplification of mitochondrial DNA to detect Plasmodium falciparum and Plasmodium vivax

In this study we standardized a new technical approach in which the target mitochondrial DNA sequence (mtDNA) is amplified using a simple but sensitive PCR method as a tool to detect Plasmodium falciparum and Plasmodium vivax. Specific primers were designed to hybridize with cytochrome c oxidase genes of P. falciparum (cox III) and P. vivax (cox I). Amplification products were obtained for all positive samples, presenting homology only for species-specific mtDNA. Sensitivity and specificity were 100%. The applicability of the method was tested in a cross-sectional study, in which 88 blood samples from individuals naturally exposed to malaria in the Brazilian Amazon region were analyzed. Based on the results, the sensitivity and specificity were 100% and 88.3%, respectively. This simple and sensitive PCR method can be useful in specific situations and in different settings of malaria management, in endemic as well as non-endemic areas (travelers), and in clinical or epidemiological studies, with applications in malaria control programs.     

Preclinical efficacy and immunogenicity assessment to show that a chimeric Plasmodium falciparum UB05‐09 antigen could be a malaria vaccine candidate

Although it is generally agreed that an effective vaccine would greatly accelerate the control of malaria, the lone registered malaria vaccine Mosquirix? has an efficacy of 30%‐60% that wanes rapidly, indicating a need for improved second‐generation malaria vaccines. Previous studies suggested that immune responses to a chimeric Plasmodium falciparum antigen UB05‐09 are associated with immune protection against malaria. Herein, the preclinical efficacy and immunogenicity of UB05‐09 are tested. Growth inhibition assay was employed to measure the effect of anti‐UB05‐09 antibodies on P. falciparum growth in vitro. BALB/c mice were immunized with UB05‐09 and challenged with the lethal Plasmodium yoelii 17XL infection. ELISA was used to measure antigen‐specific antibody production. ELISPOT assays were employed to measure interferon‐gamma production ex vivo after stimulation with chimeric UB05‐09 and its constituent antigens. Purified immunoglobulins raised in rabbits against UB05‐09 significantly inhibited P. falciparum growth in vitro compared to that of its respective constituent antigens. A combination of antibodies to UB05‐09 and the apical membrane antigen (AMA1) completely inhibited P. falciparum growth in culture. Immunization of BALB/c mice with recombinant UB05‐09 blocked parasitaemia and protected them against lethal P. yoelii 17XL challenge infection. These data suggest that UB05‐09 is a malaria vaccine candidate that could be developed further and used in conjunction with AMA1 to create a potent malaria vaccine.     

Pulmonary Edema Due to <Emphasis Type="Italic">Plasmodium vivax</Emphasis> Malaria in an American Missionary

Abstract Pulmonary edema is a recognized complication of Plasmodium falciparum malaria but is uncommon with Plasmodium vivax infection. We report the case of a non-immune adult with imported P. vivax malaria who developed pulmonary edema during treatment. The case was further complicated by a recurrent malaria episode after failure of acute quinine and doxycycline treatment followed by terminal primaquine therapy. Prompt recognition and appropriate management of pulmonary edema is needed for optimal outcomes of P. vivax infection, as well as awareness of the potential failure of terminal therapy for liver hypnozoites.     

Cytokine profile of Plasmodium falciparum-specific T cells in non-immune malaria patients

CD3⁺ T cells are important sources of both pro‐ and anti‐inflammatory cytokines during Plasmodium falciparum malaria. We studied the frequency of interleukin‐2 (IL‐2), gamma interferon (IFN‐γ), tumour necrosis factor‐alpha (TNF‐α) and IL‐10 expressing CD3⁺ cells in 10 non‐immune malaria patients with uncomplicated malaria and in one patient with cerebral malaria after P. falciparum‐specific and non‐specific mitogenic stimulation. Analysis by fluorescence‐activated cell sorting was performed after drug‐induced clearance of parasites to allow previously sequestered T cells to be detected in peripheral blood. CD3⁺ cells of patients responded to P. falciparum infected erythrocytes with significant increases in the percentage of IL‐2, IFN‐γ, and TNF‐α, but also IL‐10, positive cells. CD3⁺ cells from malaria‐naïve donors were also responsive to specific stimulation albeit to a much lesser extent. Mitogenic stimulation of PBMC revealed no significant differences between cells of patients and controls. CD3⁺ cells of the patient with cerebral malaria were hyporesponsive both to the infecting parasite isolate as well as to our laboratory‐adapted P. falciparum isolate, whereas two patients with uncomplicated disease were more responsive to their infecting parasites than to the laboratory‐adapted isolate. The results indicate that the increased responsiveness of in vivo primed compared to malaria‐naïve CD3⁺ cells is Plasmodium‐specific and biased towards production of IFN‐γ and TNF‐α.     

Fibrinolysis,inhibitors of blood coagulation,and monocyte derived coagulant activity in acute malaria

Different parameters of fibrinolytic systems like t-PA, PAI, D-dimer, and inhibitors of blood coagulation, i.e., protein C (PC), protein S(PS), and antithrombin III (AT-III), have been studied in cases of acute malaria due to Plasmodium falciparum and plasmodium vivax infection, and these patients were followed up. It was observed that the plasma PAI-1 was very high in cases of P. falciparum malaria infection as compared to normal controls and P. vivax infection. The changes in complicated cases of P. falciparum were remarkable as compared to uncomplicated ones. The PC, PS, and AT-III levels were also low in P. falciparum, particularly so in complicated cases, and were normal in P. vivax infection. The factor VIII R:Ag levels were invariably high in acute malaria. On follow-up of some of these cases the values came back to normal after the antiparasite treatment. The monocyte procoagulant activity was found to be significantly higher in P. falciparum infection as compared to that of P. vivax infection. All these findings therefore contribute towards the production of a hypercoagulable state in P. falciparum infection and partly explain the complications of P. falciparum infection like cerebral malaria. Am. J. Hematol. 54:23–29, 1997 © 1997 Wiley-Liss, Inc.     

Patterns of pseudo‐reticulocytosis in malaria: fluorescent analysis with the Cell‐Dyn® CD4000

This study of Plasmodium falciparum malaria evaluated patterns of fluorescent reticulocyte measurements as determined with the Abbott Cell‐Dyn^® CD4000. The parasitaemia of positive samples (n=180) ranged from 0.04% to 25.5%, with those (19/180) showing gametocytes having lower parasitaemia levels (mean 0.31%, median 0.2%) compared to those that did not (mean 2.59%, median 0.8%). There was a reasonable association (R²=0.60) between parasitaemia level and CD4000 reticulocyte percentages, although there was overall a small statistical bias towards higher parasitaemia estimates determined microscopically. Consistently high immature reticulocyte fraction (IRF) values of >0.5 were observed in cases with a parasitaemia exceeding 5%, while samples with lower parasitaemia levels showed more variable IRF values. Visual examination of CD4000 reticulocyte histograms revealed that 81/100 malaria‐positive samples with an IRF above 0.5 showed the presence of a fluorescent population `spike' consistent with the staining of intracellular malaria parasites. Only three of the 80 malaria‐positive samples with an IRF below 0.5, and none of the 237 malaria‐negative samples, showed this histogram pattern. These observations indicate that samples with malaria parasites give erroneously high CD4000 reticulocyte estimates that essentially comprise the sum total of true reticulocytes and parasite‐infected red cells (pseudo‐reticulocytes). This limitation is common to all automated reticulocyte procedures but recognizing the differences between homogenous staining parasitized red cells and heterogeneous staining reticulocytes has potential applications in monitoring parasitaemia levels both at patient presentation and during subsequent treatment.     

A whole‐killed,blood‐stage lysate vaccine protects against the malaria liver stage

Although the attenuated sporozoite is the most efficient vaccine to prevent infection with the malaria parasite, the limitation of a source of sterile sporozoites greatly hampers its application. In this study, we found that the whole‐killed, blood‐stage lysate vaccine could confer protection against the blood stage as well as the liver stage. Although the protective immunity induced by the whole‐organism vaccine against the blood stage is dependent on parasite‐specific CD4⁺ T‐cell responses and antibodies, in mice immunized with the whole‐killed, blood‐stage lysate vaccine, CD8⁺, but not CD4⁺ effector T‐cell responses greatly contributed to protection against the liver stage. Thus, our data suggested that the whole‐killed, blood‐stage lysate vaccine could be an alternative promising strategy to prevent malaria infection and to reduce the morbidity and mortality of patients with malaria.     

Sensitive detection and accurate monitoring of Plasmodium vivax parasites on routine complete blood count using automatic blood cell analyzer (DxH800(TM))

Introduction: Plasmodium vivax malaria is one of the most important infectious diseases plaguing humanity and causes significant mortality and morbidity worldwide. The gold standard of P. vivax malaria diagnosis is the microscopy of blood smears. Although microscopy is a rapid, cost‐effective, and readily applicable method, it has many disadvantages, including low sensitivity, specificity, and precision. Therefore, there is a clear need for an effective screening test for P. vivax malaria detection both in high‐prevalence areas and developed countries. Methods: A total of 1761 complete blood count (CBC) samples generated by the automated hematology analyzer (DxH 800?; Beckman Coulter Inc., Miami, FL, USA) were retrospectively analyzed. The sample pool contained 123 samples from 52 P. vivax malaria patients and 1504 nonmalarial samples including 509 patients with leukopenia (white blood cell <2000/μL) and 134 normal subjects. Results: The P. vivax malaria samples exhibited easily recognizable typical malaria signals on the nucleated red blood cell (nRBC) plots (sensitivity 100%) in DxH 800?. All 1504 samples without P. vivax infection were negative for malaria signal (specificity 100%). The size of P. vivax malaria signals correlated roughly with the parasite burden. Conclusion: DxH800? provides very sensitive and specific, easily recognizable P. vivax malaria signals on routine CBC without need for the additional reagents or special procedures.     

Artefactually‐normal automated platelet counts due to malaria‐infected RBC

Protein aggregates, red cell or white cell fragments are known to interfere with platelet counts in automated blood analysers, both by aperture impedance and optical technologies. When a falsely high value is suspected, interference by pseudo‐platelet particles can be confirmed by systematic examination of stained blood films. The method that best avoids these sources of interference is the reference, immunological platelet count. We describe a case of treated malaria with a false normal platelet count. The blood smear revealed small red cells, infected by trophozoites of Plasmodium falciparum, that interfered with the platelet count. The Cell Dyn^® 4000 shows different patterns of interference by infected red cells in its impedance and optical counts, and thrombocytopenia was suspected immediately. This was confirmed by a phase‐contrast microscopic platelet count.     

Development of PCR-RFLP assay for the discrimination of Plasmodium species and variants of P. vivax (VK210, VK247 and P. vivax-like) in Anopheles mosquitoes

The identification of Plasmodium species in Anopheles mosquitoes is an integral component of malaria control programs. We developed a new assay to identify Plasmodium falciparum, Plasmodium malariae, and Plasmodium vivax variants. Specific primers were designed to hybridize to CS gene-specific regions. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used to distinguish the P. vivax variants VK210, VK247, and P. vivax-like. The new PCR–RFLP assay revealed good agreement when compared with a nested PCR using artificially infected Anopheles mosquitoes. This sensitive PCR–RFLP method can be useful when detection of Plasmodium species and P. vivax variants is required and may be employed to improve the understanding of malaria transmission dynamics by Anopheles species.