Severity of anaemia is associated with bone marrow haemozoin in children exposed to Plasmodium falciparum

There are no large‐scale ex vivo studies addressing the contribution of Plasmodium falciparum in the bone marrow to anaemia. The presence of malaria parasites and haemozoin were studied in bone marrows from 290 anaemic children attending a rural hospital in Mozambique. Peripheral blood infections were determined by microscopy and polymerase chain reactions. Bone marrow parasitaemia, haemozoin and dyserythropoiesis were microscopically assessed. Forty‐two percent (123/290) of children had parasites in the bone marrow and 49% (111/226) had haemozoin, overlapping with parasitaemia in 83% (92/111) of cases. Sexual and mature asexual parasites were highly prevalent (62% gametocytes, 71% trophozoites, 23% schizonts) suggesting their sequestration in this tissue. Sixteen percent (19/120) of children without peripheral infection had haemozoin in the bone marrow. Haemozoin in the bone marrow was independently associated with decreased Hb concentration (P = 0·005) and was more common in dyserythropoietic bone marrows (P = 0·010). The results of this ex vivo study suggest that haemozoin in the bone marrow has a role in the pathogenesis of malarial‐anaemia through ineffective erythropoiesis. This finding may have clinical implications for the development of drugs targeted to prevent and treat malarial‐anaemia.     

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.     

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.     

Antibody responses to surface antigens of Plasmodium falciparum gametocyte‐infected erythrocytes and their relation to gametocytaemia

An essential element for continuing transmission of Plasmodium falciparum is the availability of mature gametocytes in human peripheral circulation for uptake by mosquitoes. Natural immune responses to circulating gametocytes may play a role in reducing transmission from humans to mosquitoes. Here, antibody recognition of the surface of mature intra‐erythrocytic gametocytes produced either by a laboratory‐adapted parasite, 3D7, or by a recent clinical isolate of Kenyan origin (HL1204), was evaluated longitudinally in a cohort of Ghanaian school children by flow cytometry. This showed that a proportion of children exhibited antibody responses that recognized gametocyte surface antigens on one or both parasite lines. A subset of the children maintained detectable anti‐gametocyte surface antigen (GSA) antibody levels during the 5 week study period. There was indicative evidence that children with anti‐GSA antibodies present at enrolment were less likely to have patent gametocytaemia at subsequent visits (odds ratio = 0·29, 95% CI 0·06–1·05; P = 0·034). Our data support the existence of antigens on the surface of gametocyte‐infected erythrocytes, but further studies are needed to confirm whether antibodies against them reduce gametocyte carriage. The identification of GSA would allow their evaluation as potential anti‐gametocyte vaccine candidates and/or biomarkers for gametocyte carriage.     

Increased pfmdr1 copy number in Plasmodium falciparum isolates from Suriname

Amplification of the pfmdr1 gene is associated with clinical failures and reduced in vivo and in vitro sensitivity to both mefloquine and artemether–lumefantrine in South‐East Asia. Several African countries have reported the absence or very low prevalence of increased copy number, whilst South American reports are limited to Peru without and Venezuela with increased pfmdr1 multiplication. The relative pfmdr1 copy numbers were assessed in 68 isolates from Suriname collected from different endemic villages (2005) and from mining areas (2009). 11% of the isolates harbour multiple copies of the pfmdr1 gene. Isolates originating from mining areas do not yet display a higher tendency for increased copy number and no significant differences could be registered within a time span of 4 years, but the mere presence of increased copy number warrants caution and should be considered as an early warning sign for emerging drug resistance in Suriname and South America.     

Immunization of mice with Plasmodium TCTP delays establishment of Plasmodium infection

Translationally controlled tumour protein (TCTP) may play an important role in the establishment or maintenance of parasitemia in a malarial infection. In this study, the potential of TCTP as a malaria vaccine was investigated in two trials. In the initial vaccine trial, Plasmodium falciparum TCTP (PfTCTP) was expressed in Saccharomyces cerevisiae and used to immunize BALB/c mice. Following challenge with Plasmodium yoelii YM, parasitemia was significantly reduced during the early stages of infection. In the second vaccine trial, the TCTP from P. yoelii and P. berghei was expressed in Escherichia coli and used in several mouse malaria models. A significant reduction in parasitemia in the early stages of infection was observed in BALB/c mice challenged with P. yoelii YM. A significantly reduced parasitemia at each day leading up to a delayed and reduced peak parasitemia was also observed in BALB/c mice challenged with the nonlethal Plasmodium chabaudi (P.c.) chabaudi AS. These results suggest that TCTP has an important role for parasite establishment and may be important for pathogenesis.     

Melatonin activates FIS1, DYN1, and DYN2 Plasmodium falciparum related‐genes for mitochondria fission: Mitoemerald‐GFP as a tool to visualize mitochondria structure

Malaria causes millions of deaths worldwide and is considered a huge burden to underdeveloped countries. The number of cases with resistance to all antimalarials is continuously increasing, making the identification of novel drugs a very urgent necessity. A potentially very interesting target for novel therapeutic intervention is the parasite mitochondrion. In this work, we studied in Plasmodium falciparum 3 genes coding for proteins homologues of the mammalian FIS1 (Mitochondrial Fission Protein 1) and DRP1 (Dynamin Related Protein 1) involved in mitochondrial fission. We studied the expression of P. falciparum genes that show ample sequence and structural homologies with the mammalian counterparts, namely FIS1, DYN1, and DYN2. The encoded proteins are characterized by a distinct pattern of expression throughout the erythrocytic cycle of P. falciparum, and their mRNAs are modulated by treating the parasite with the host hormone melatonin. We have previously reported that the knockout of the Plasmodium gene that codes for protein kinase 7 is essential for melatonin sensing. We here show that PfPk7 knockout results in major alterations of mitochondrial fission genes expression when compared to wild‐type parasites, and no change in fission proteins expression upon treatment with the host hormone. Finally, we have compared the morphological characteristics (using MitoTracker Red CMX Ros) and oxygen consumption properties of P. falciparum mitochondria in wild‐type parasites and PfPk7 Knockout strains. A novel GFP construct targeted to the mitochondrial matrix to wild‐type parasites was also developed to visualize P. falciparum mitochondria. We here show that, the functional characteristics of P. falciparum are profoundly altered in cells lacking protein kinase 7, suggesting that this enzyme plays a major role in the control of mitochondrial morphogenesis and maturation during the intra‐erythrocyte cell cycle progression.     

Plasmodium falciparum infection transmitted by transfusion: A cause of hemophagocytic syndrome after bone marrow tranplantation in a non‐endemic country

A 27‐year‐old man with severe aplastic anemia underwent bone marrow transplantation from his HLA identical brother in July 2016. Conditioning included ATGAM 30 mg/kg for 3 days and Cyclophosphamide 50 mg/kg for 4 days. The patient received several platelet and red blood cell transfusions before and after the conditioning. The patient received broad spectrum antibiotics and caspofungin because persistant febrile neutropenia without bacteriological or mycological documentation. Hemophagocytic syndrome was diagnosed on day +12. Steroids at 1 mg/kg were started on day +12. Fever resolved the same day but resumed 3 days later associated to intravascular hemolysis with no schizocytes on blood smears and negative DAT. Thick blood film smears performed on day +26 revealed Plasmodium falciparum parasites (parasitemia = 20%). Except the level of parasitemia, there were no signs of gravity. Quinine was started on day 26 at a loading dose of 15 mg/kg followed by 8 mg/kg three times a day for 20 doses. Fever vanished after 2 days. Parasitemia cleared in 3 days and remained negative thereafter. Investigations revealed that the patient was transfused by a red cell unit harvested in a voluntary donor native of a malaria endemic country. PCR for P. falciparum performed in this donor in the frame of investigations was positive. The patient is alive with a normal blood count 1 year after BMT.