Preerythrocytic, live-attenuated Plasmodium falciparum vaccine candidates by design

Falciparum malaria is initiated when Anopheles mosquitoes transmit the Plasmodium sporozoite stage during a blood meal. Irradiated sporozoites confer sterile protection against subsequent malaria infection in animal models and humans. This level of protection is unmatched by current recombinant malaria vaccines. However, the live-attenuated vaccine approach faces formidable obstacles, including development of accurate, reproducible attenuation techniques. We tested whether Plasmodium falciparum could be attenuated at the early liver stage by genetic engineering. The P. falciparum genetically attenuated parasites (GAPs) harbor individual deletions or simultaneous deletions of the sporozoite-expressed genes P52 and P36. Gene deletions were done by double-cross-over recombination to avoid genetic reversion of the knockout parasites. The gene deletions did not affect parasite replication throughout the erythrocytic cycle, gametocyte production, mosquito infections, and sporozoite production rates. However, the deletions caused parasite developmental arrest during hepatocyte infection. The double-gene deletion line exhibited a more severe intrahepatocytic growth defect compared with the single-gene deletion lines, and it did not persist. This defect was assessed in an in vitro liver-stage growth assay and in a chimeric mouse model harboring human hepatocytes. The strong phenotype of the double knockout GAP justifies its human testing as a whole-organism vaccine candidate using the established sporozoite challenge model. GAPs might provide a safe and reproducible platform to develop an efficacious whole-cell malaria vaccine that prevents infection at the preerythrocytic stage.     

Retrospective analysis of vivax malaria patients presenting to tertiary referral centre of Uttarakhand

Introduction

Despite the high prevalence of Plasmodium vivax (P vivax) malaria, research into its complications has lagged disproportionately as compared to Plasmodium falciparum (P falciparum) malaria.

Material and methods

The present retrospective observational study was conducted on cases with P vivax mono-infection presenting with severe malaria on the basis of one or more criteria as per the World Health Organization guidelines being used for severe falciparum malaria in children, as well as other manifestations been classified as complicated malaria, during an outbreak of malaria in a single tertiary referral hospital of north India.

Results

Seventy-four patients of acute malaria presented during the outbreak, of which 50 cases with P vivax mono-infection were included for the study. Complicated malaria was diagnosed in 41/50 cases, with thrombocytopenia being the commonest manifestation. Other presentations of severe malaria in our patients were liver dysfunction (with or without jaundice) 31/50 cases, respiratory involvement 14/50 cases, renal impairment 11/50 cases, circulatory collapse (Shock) 8/50 cases, severe anaemia 3/50 cases and central nervous system (CNS) involvement 2/50 cases.

Conclusion

The term “benign tertian malaria” no longer holds true for P vivax mono-infection. The authors wish to open a new front for researches on the possible genotypic abnormalities that the parasite or its carrier might have acquired over decades and has transformed into a species with the malignant potential of P falciparum.     

Plasmodium vivax cerebral malaria complicated with venous sinus thrombosis in Colombia

Complicated malaria is usually due to Plasmodium falciparum. Nevertheless, Plasmodium vivax is infrequently related with life-threatening complications. Few cases have been reported of severe Plasmodium vivax infection, and most of them from Southeast Asia and India. We report the first case of cerebral malaria due to Plasmodium vivax in Latin America, complicated with sagittal sinus thrombosis and confirmed by a molecular method.     

Thrombocytopenia in children with malaria–A study from coastal Karnataka,India

ObjectiveTo study the occurrence and severity of thrombocytopenia in children with malaria.MethodsIt was a retrospective study, done at Fr Muller Medical College Hospital Mangalore, in Karnataka, India. Data regarding all positive cases of malaria < 15 years admitted in the hospital between January 2010 to June 2011 were obtained. Patients were further assessed for thrombocytopenia and its severity. Data were analysed by Chi square test using SPSS version 13.0.ResultsA total of 159 cases were included in the study with a mean age of presentation of 9 years. Plasmodium vivax was identified in 106 (66%) patients while Plasmodium falciparum in 26 (16%) and mixed infection in 27 (18%) patients. Thrombocytopenia was observed in 113 (71%) cases, of which 35 (31%) cases had mild, 49 (43%) cases moderate and 29 (26%) cases had severe thrombocytopenia. Thrombocytopenia was equally found in vivax and falciparum infection with no significant difference in severity between vivax and falciparum species.ConclusionsThrombocytopenia is frequently seen in malaria and it is not dependent on type of malaria. In any acute febrile illness, thrombocytopenia should alert one to the possibility of malaria.     

Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes

Research into the aetiological agent of the most widespread form of severe malaria, Plasmodium falciparum, has benefitted enormously from the ability to culture and genetically manipulate blood-stage forms of the parasite in vitro. However, most malaria outside Africa is caused by a distinct Plasmodium species, Plasmodium vivax, and it has become increasingly apparent that zoonotic infection by the closely related simian parasite Plasmodium knowlesi is a frequent cause of life-threatening malaria in regions of southeast Asia. Neither of these important malarial species can be cultured in human cells in vitro, requiring access to primates with the associated ethical and practical constraints. We report the successful adaptation of P. knowlesi to continuous culture in human erythrocytes. Human-adapted P. knowlesi clones maintain their capacity to replicate in monkey erythrocytes and can be genetically modified with unprecedented efficiency, providing an important and unique model for studying conserved aspects of malarial biology as well as species-specific features of an emerging pathogen.     

SERO-EPIDEMIOLOGICAL STUDIES ON POPULATION GROUPS PREVIOUSLY EXPOSED TO MALARIA

Application of the indirect-fluorescent- antibody (I.F.A.) test to the study of malaria antibodies in a group of Indian and Pakistani immigrants in Bradford revealed that about 50% showed a positive response at a low titre against Plasmodium falciparum and P. vivax. There is little evidence of persistence of malaria infection in this group, but the test indicates the long duration of antibody acquired ten to fifteen years ago. In a group of potential blood-donors whose whole blood was not used for transfusion because of their possible previous exposure to malaria, only 24% were found to have a positive I.F.A. test using P. falciparum and P. malariœantigens. A negative I.F.A. test is a good criterion of the absence of previous malaria infection.     

Phytochemical licochalcone A enhances antimalarial activity of artemisinin in vitro

Resistance to synthetic first-line antimalarial drugs is considered to be a major cause of increased malaria morbidity and mortality. Use of artemisinin-based combination therapies (ACTs) is being encouraged to reduce the malaria mortality in areas of falciparum resistance. Artemisinin is a natural product at times in short supply. With projected rise in demand of artemisinin there is an unmet need for alternate ACTs. Novel compounds that reduce dependance on artemisinin are required. In vitro cultures of Plasmodium falciparum provide a screen system for identifying and evaluating new drug combinations. Interactions of two phytochemicals, artemisinin and licochalcone A, has been studied against synchronized erythrocytic stages of chloroquine-sensitive 3D7 and chloroquine-resistant RKL 303 strains of P. falciparum. These two compounds in combination show synergistic antiplasmodial activity in vitro on these strains. Artemisinin but not licochalcone A interferes with hemozoin formation. Neither of the phytochemicals alone or in combination obstructs sorbitol-induced hemolysis.     

Clinical features of children hospitalized with malaria--a study from Bikaner, northwest India

Severe Plasmodium vivax malaria in adults has been reported from Bikaner (northwestern India) but the reports on children are scanty. This prospective study was done on 303 admitted children of malaria. The diagnosis was done by peripheral blood smear and rapid diagnostic test. Further confirmation of severe P. vivax monoinfection was done by polymerase chain reaction (PCR). The proportion of P. falciparum, P. vivax, and mixed (P. falciparum and P. vivax) infection was 61.01%, 33.99%, and 4.95%, respectively. Severe disease was present in 49.5% (150/303) children with malaria, with the risk greatest among P. vivax monoinfection (63.1% [65/103]) compared with P. falciparum, either alone (42.7% [79/185]; odds ratio [OR] = 2.3 [95% confidence interval (CI) = 1.40-3.76], P = 0.001) or mixed infections (40% [6/15]; OR = 2.57 [95% CI = 0.88-7.48]). In children < 5 years of age, the proportion of severe malaria attributable to P. vivax rose to 67.4% (31/46) compared with 30.4% (14/46) of P. falciparum (OR = 4.7 [95% CI = 2.6-8.6], P < 0.0001) and 2.2% (1/46) of mixed infection (OR = 92 [95% CI = 24.6-339.9], P < 0.0001). The proportion of patients having severe manifestations, which included severe anemia, thrombocytopenia, cerebral malaria, acute respiratory distress syndrome, hepatic dysfunction, renal dysfunction, abnormal bleeding was significantly high in association with P. vivax monoinfection in 0-5 year age group, while the same was significantly high in association with P. falciparum monoinfection in 5-10 year age group. Similarly P. vivax monoinfection had greatest propensity to cause multiorgan dysfunction in 0-5 year age group (34.1% [17/41], P < 0.0001) in comparison to P. falciparum monoinfection, which had similar propensity in 5-10 year age group (36.8% [35/95], P = 0.039). Plasmodium vivax monoinfection was almost equally serious to cause significant mortality in comparison to P. falciparum (case fatality rate of severe P. vivax was 3.9% versus 3.2% of severe P. falciparum malaria; P = 1.0). This study reaffirms the evidence of severe P. vivax malaria in children in Bikaner.     

Malaria severity status in patients with soil-transmitted helminth infections

Objective

To investigate the possible impact of soil-transmitted helminth (STH) infection on malaria severity, level of parasitaemia and clearance/reduction of Plasmodium parasites following treatment with anti-malarial drugs.

Methods

458 voluntary malaria patients who visited the Alaba Kulito Health Center, southern Ethiopia, for medical treatment in November and December 2007 were included in this study. Giemsa-stained thick and thin blood films were used for the determination of parasitaemia and identification of Plasmodium species, respectively. Stool sample was collected from these patients and diagnosed for intestinal helminths using Kato-Katz technique. Haemoglobin concentration was measured using a portable spectrophotometer (HemoCue HB 201). Malaria parasite clearance was checked on day 3 post-treatment.

Findings

The prevalence of co-infection of malaria with the major soil-transmitted helminths (STHs), i.e., with hookworm species, Ascaris lumbricoides and Trichuris trichiura was 9.6%, 6.3% and 2.1%, respectively. About 8.1% of the study subjects had severe malaria. Intensity of hookworm infection showed positive association with malaria parasite densities (F = 3.510, P = 0.033). STHs infection in general was negatively correlated with the symptoms of severe malaria (OR = 0.317, 95% CI = 0.315-0.86, P = 0.01), but a small proportion (4.5%) of malaria patients who were concurrently harboring one or more intestinal helminths had severe malaria. Only few malaria patients (2.3%) co-infected with STHs were found positive for Plasmodium parasites on day 3 post-treatment.

Conclusion

The present findings indicate that soil-transmitted helminths have very little contribution to malaria severity in co-infected individuals. The findings also indicate that STHs do not have significant impact on clearance rate of Plasmodium falciparum and Plasmodium vivax when treated with anti-malarial drugs.     

Plasmodium vivax malaria complicated by hemophagocytic syndrome in an immunocompetent serviceman

We describe a 23-year-old retired military officer who was immunocompetent but diagnosed with hemophagocytic syndrome (HPS) by Plasmodium vivax infection. Initially, the patient was suspected to have toxic hepatitis related to heavy drinking. But abnormal hematologic findings required a further bone marrow examination and the diagnosis of HPS was made. Antimalarial chemotherapy then brought complete remission. Plasmodium falciparum, a species causing more severe malarial infection, was listed as one of the major causes of HPS. However, P. vivax was not mentioned, and only one case was reported in the literature. In this study, we suggest that P. vivax malaria should be included in the differential diagnosis of HPS, even in an immunocompetent person.     

First record of the Asian malaria vector Anopheles stephensi and its possible role in the resurgence of malaria in Djibouti,Horn of Africa

Anopheles stephensi is an important vector of urban malaria in India and the Persian Gulf area. Its previously known geographical range includes southern Asia and the Arab Peninsula. For the first time, we report A. stephensi from the African continent, based on collections made in Djibouti, on the Horn of Africa, where this species’ occurrence was linked to an unusual urban outbreak of Plasmodium falciparum malaria, with 1228 cases reported from February to May 2013, and a second, more severe epidemic that emerged in November 2013 and resulted in 2017 reported malaria cases between January and February 2014. Anopheles stephensi was initially identified using morphological identification keys, followed by sequencing of the Barcode cytochrome c-oxidase I (COI) gene and the rDNA second internal transcribed spacer (ITS2). Positive tests for P. falciparum circumsporozoite antigen in two of six female A. stephensi trapped in homes of malaria patients in March 2013 are evidence that autochthonous urban malaria transmission by A. stephensi has occurred. Concurrent with the second malaria outbreak, P. falciparum-positive A. stephensi females were detected in Djibouti City starting in November 2013. In sub-Saharan Africa, newly present A. stephensi may pose a significant future health threat because of this species’ high susceptibility to P. falciparum infection and its tolerance of urban habitats. This may lead to increased malaria outbreaks in African cities. Rapid interruption of the urban malaria transmission cycle, based on integrated vector surveillance and control programs aimed at the complete eradication of A. stephensi from the African continent, is strongly recommended.     

Comparing the Impact of Artemisinin-Based Combination Therapies on Malaria Transmission in Sub-Saharan Africa

Artemisinin-based combination therapies (ACTs) are currently considered the first-line treatments for uncomplicated Plasmodium falciparum malaria. Among these, artemether-lumefantrine (AL) has been the most widely prescribed ACT in sub-Saharan Africa. Recent clinical trials conducted in sub-Saharan Africa have shown that dihydroartemisinin-piperaquine (DP), a most recent ACT, may have a longer post-treatment prophylactic period and post-treatment infection period (duration of gametocyte carriage) than AL. Using epidemiological and clinical data on the efficacy of AL and DP, we developed and parameterized a mathematical transmission model that we used to compare the population-level impact of AL and DP for reducing P. falciparum malaria transmission in sub-Saharan Africa. Our results showed that DP is likely to more effectively reduce malaria incidence of clinical episodes than AL. However in low P. falciparum transmission areas, DP and AL are likely to be equally effective in reducing malaria prevalence. The predictions of our model were shown to be robust to the empirical uncertainty summarizing the epidemiological parameters. DP should be considered as a replacement for AL as first-line treatment of uncomplicated malaria in highly endemic P. falciparum communities. To optimize the effectiveness of ACTs, it is necessary to tailor treatment policies to the transmission intensity in different settings.     

African apes as reservoirs of Plasmodium falciparum and the origin and diversification of the Laverania subgenus

We investigated two mitochondrial genes (cytb and cox1), one plastid gene (tufA), and one nuclear gene (ldh) in blood samples from 12 chimpanzees and two gorillas from Cameroon and one lemur from Madagascar. One gorilla sample is related to Plasmodium falciparum, thus confirming the recently reported presence in gorillas of this parasite. The second gorilla sample is more similar to the recently defined Plasmodium gaboni than to the P. falciparum–Plasmodium reichenowi clade, but distinct from both. Two chimpanzee samples are P. falciparum. A third sample is P. reichenowi and two others are P. gaboni. The other chimpanzee samples are different from those in the ape clade: two are Plasmodium ovale, and one is Plasmodium malariae. That is, we have found three human Plasmodium parasites in chimpanzees. Four chimpanzee samples were mixed: one species was P. reichenowi; the other species was P. gaboni in three samples and P. ovale in the fourth sample. The lemur sample, provisionally named Plasmodium malagasi, is a sister lineage to the large cluster of primate parasites that does not include P. falciparum or ape parasites, suggesting that the falciparum + ape parasite cluster (Laverania clade) may have evolved from a parasite present in hosts not ancestral to the primates. If malignant malaria were eradicated from human populations, chimpanzees, in addition to gorillas, might serve as a reservoir for P. falciparum.     

Prospective risk of morbidity in relation to malaria infection in an area of high endemicity of multiple species of Plasmodium.

In an area of Papua New Guinea with high prevalence of Plasmodium falciparum (39.6%), Plasmodium vivax (18.3%), and Plasmodium malariae (13.8%), cross-sectional analysis found P. falciparum infection to be independent of the other species despite heterogeneities in transmission. Plasmodium vivax and P. malariae infections were negatively correlated. Plasmodium malariae infection was positively associated with homologous infection four months previously and with prior P. falciparum, but not P. vivax infection. There were no other indications that any Plasmodium species protected against heterologous infection. Prospective analysis of health-center morbidity supported the idea that P. malariae infection protects against disease, but indicated greater protection against non-malaria than P. falciparum-associated fevers. Plasmodium vivax appeared to protect against P. falciparum disease but not against other forms of morbidity. Covariate adjustment had considerable effects on estimated relationships between species, and confounding variables may account for many differences among reports of inter-species interactions in human malaria.     

Demographic risk factors for severe and fatal vivax and falciparum malaria among hospital admissions in northeastern Indonesian Papua

Between January 1998 and December 2000, the Jayapura Provincial Public Hospital in northeastern Indonesian New Guinea (Papua) admitted 5,936 patients with a diagnosis of malaria. The microscopic diagnosis at admission was Plasmodium falciparum (3,976, 67%), Plasmodium vivax (1,135, 19%), Plasmodium malariae (8, < 1%), and mixed species infections (817, 14%). Approximately 9% (367) of patients were classified as having severe malaria (277 P. falciparum, 36 P. vivax, 53 mixed infections, and 1 P. malariae) and 88 died (79 P. falciparum/mixed infections and 9 P. vivax). Risk of fatal outcomes among severe malaria patients was indistinguishable between those with falciparum versus vivax malaria (OR = 0.89; P = 0.771). Compared with non-pregnant women, pregnant women showed no higher risk of severe malaria (P = 0.643) or death caused by severe malaria (P = 0.748). This study compares admissions per population (based on census data), parasitemia, morbidity, and mortality among children versus adults, pregnant versus non-pregnant women, and urban/suburban versus rural residents.     

Falciparum malaria parasitemia index for predicting severe malaria

Introduction: While hyperparasitemia is considered an important indicator for the development of severe malaria, there is currently no consensus on the quantitative definition of hyperparasitemia. This study was conducted to establish a cutoff point for peripheral parasitemia among patients with Plasmodium falciparum malaria, to define severe malaria. Methods: The clinical presentations of 200 uncomplicated P. falciparum malaria, and 189 severe P. falciparum malaria, patients, admitted to the Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, were analyzed. Results: A peripheral parasitemia of 0.5% was found to be the optimal cutoff point for defining severe malaria, demonstrating highest sensitivity (85.1%), specificity (62.0%), and accuracy (73.2%). Conclusion: Symptoms of severe falciparum malaria depend on many factors. For the definition of hyperparasitemia in areas of low or seasonal transmission, peripheral parasitemia of 0.5% might be considered a cutoff point for discrimination between severity levels. This value might be useful for the clinical management of malaria, particularly in hypo‐endemic areas, unstable transmission areas, and other areas with similar transmission patterns.     

Malaria in Mauritania: the first cases of malaria endemic to Nouakchott

The current situation of endemic malaria in Mauritania is not clear since, in most health centres, suspected malaria cases are not confirmed by parasitological analysis and diagnosis is based on clinical symptoms alone. To obtain reliable data about malaria in this country, thin and thick blood smears were taken from patients with symptoms compatible with the illness, who attended two hospitals: Polyclinic of Nouakchott, which serves one-third of the country's population, where a malaria infection rate of 18.5% (77 of 446) was recorded; Plasmodium falciparum caused 61.85% of these, P. vivax 35.5% (28/77). In Kaedi Regional Hospital, provincial capital of the endemic Gorgol region, a prevalence of 25.49% (106 of 416) was recorded, with P. falciparum as the sole pathogenic species. Of the 77 cases of malaria diagnosed in Nouakchott, nine (seven of P. falciparum and two of P. vivax) were considered as endemic to the city. These cases were all children under 8 years of age except for one adult who had never left the capital, and this is the first time that cases endemic to this city have been detected.     

Maternal Clinical Findings in Malaria in Pregnancy in a Region of Northwestern Colombia

In malaria-endemic regions of Latin America, little is known about malaria in pregnancy. To characterize the clinical and laboratory findings of maternal infection, we evaluated 166 cases of pregnant women infected with Plasmodium spp. in a prospective study conducted in northwestern Colombia during 2005–2006. A total of 89.8% (149 of 166) had fever or a history of fever in the past 48 hours, 9.0% (15 of 166) had severe malaria, of which 66.7% was caused by Plasmodium vivax and 33.3% by P. falciparum. Hepatic dysfunction was the main complication (9 of 15) observed. The proportion of severe cases was similar for both species (P = 0.41). In malaria-endemic areas of Colombia, malaria in pregnancy has a broad clinical spectrum. In pregnant women, P. vivax infection frequently leads to organ-specific complications.     

Modulation of cerebral malaria by curcumin as an adjunctive therapy

Cerebral malaria is the most severe and rapidly fatal neurological complication of Plasmodium falciparum infection and responsible for more than two million deaths annually. The current therapy is inadequate in terms of reducing mortality or post-treatment symptoms such as neurological and cognitive deficits. The pathophysiology of cerebral malaria is quite complex and offers a variety of targets which remain to be exploited for better therapeutic outcome. The present review discusses on the pathophysiology of cerebral malaria with particular emphasis on scope and promises of curcumin as an adjunctive therapy to improve survival and overcome neurological deficits.     

African great apes are natural hosts of multiple related malaria species,including Plasmodium falciparum

Plasmodium reichenowi, a chimpanzee parasite, was until very recently the only known close relative of Plasmodium falciparum, the most virulent agent of human malaria. Recently, Plasmodium gaboni, another closely related chimpanzee parasite, was discovered, suggesting that the diversity of Plasmodium circulating in great apes in Africa might have been underestimated. It was also recently shown that P. reichenowi is a geographically widespread and genetically diverse chimpanzee parasite and that the world diversity of P. falciparum is fully included within the much broader genetic diversity of P. reichenowi. The evidence indicates that all extant populations of P. falciparum originated from P. reichenowi, likely by a single transfer from chimpanzees. In this work, we have studied the diversity of Plasmodium species infecting chimpanzees and gorillas in Central Africa (Cameroon and Gabon) from both wild-living and captive animals. The studies in wild apes used noninvasive sampling methods. We confirm the presence of P. reichenowi and P. gaboni in wild chimpanzees. Moreover, our results reveal the existence of an unexpected genetic diversity of Plasmodium lineages circulating in gorillas. We show that gorillas are naturally infected by two related lineages of parasites that have not been described previously, herein referred to as Plasmodium GorA and P. GorB, but also by P. falciparum, a species previously considered as strictly human specific. The continuously increasing contacts between humans and primate populations raise concerns about further reciprocal host transfers of these pathogens.