Risk Factors for Plasmodium falciparum Gametocyte Positivity in a Longitudinal Cohort

Malaria transmission intensity is highly heterogeneous even at a very small scale. Implementing targeted intervention in malaria transmission hotspots offers the potential to reduce the burden of disease both locally and in adjacent areas. Transmission of malaria parasites from man to mosquito requires the production of gametocyte stage parasites. Cluster analysis of a 19-year long cohort study for gametocyte carriage revealed spatially defined gametocyte hotspots that occurred during the time when chloroquine was the drug used for clinical case treatment. In addition to known risk factors for gametocyte carriage, notably young age (<15 years old) and associated with a clinical episode, blood groups B and O increased risk compared to groups A and AB. A hotspot of clinical P. falciparum clinical episodes that overlapped the gametocyte hotspots was also identified. Gametocyte positivity was found to be increased in individuals who had been treated with chloroquine, as opposed to other drug treatment regimens, for a clinical P. falciparum episode up to 30 days previously. It seems likely the hotspots were generated by a vicious circle of ineffective treatment of clinical cases and concomitant gametocyte production in a sub-population characterized by an increased prevalence of all the identified risk factors. While rapid access to treatment with an effective anti-malarial can reduce the duration of gametocyte carriage and onward parasite transmission, localised hotspots represent a challenge to malaria control and eventual eradication.     

Co-infection of Long-Term Carriers of Plasmodium falciparum with Schistosoma haematobium Enhances Protection from Febrile Malaria: A Prospective Cohort Study in Mali

Background

Malaria and schistosomiasis often overlap in tropical and subtropical countries and impose tremendous disease burdens; however, the extent to which schistosomiasis modifies the risk of febrile malaria remains unclear.

Methods

We evaluated the effect of baseline S. haematobium mono-infection, baseline P. falciparum mono-infection, and co-infection with both parasites on the risk of febrile malaria in a prospective cohort study of 616 children and adults living in Kalifabougou, Mali. Individuals with S. haematobium were treated with praziquantel within 6 weeks of enrollment. Malaria episodes were detected by weekly physical examination and self-referral for 7 months. The primary outcome was time to first or only malaria episode defined as fever (≥37.5°C) and parasitemia (≥2500 asexual parasites/µl). Secondary definitions of malaria using different parasite densities were also explored.

Results

After adjusting for age, anemia status, sickle cell trait, distance from home to river, residence within a cluster of high S. haematobium transmission, and housing type, baseline P. falciparum mono-infection (n = 254) and co-infection (n = 39) were significantly associated with protection from febrile malaria by Cox regression (hazard ratios 0.71 and 0.44; P = 0.01 and 0.02; reference group: uninfected at baseline). Baseline S. haematobium mono-infection (n = 23) did not associate with malaria protection in the adjusted analysis, but this may be due to lack of statistical power. Anemia significantly interacted with co-infection (P = 0.009), and the malaria-protective effect of co-infection was strongest in non-anemic individuals. Co-infection was an independent negative predictor of lower parasite density at the first febrile malaria episode.

Conclusions

Co-infection with S. haematobium and P. falciparum is significantly associated with reduced risk of febrile malaria in long-term asymptomatic carriers of P. falciparum. Future studies are needed to determine whether co-infection induces immunomodulatory mechanisms that protect against febrile malaria or whether genetic, behavioral, or environmental factors not accounted for here explain these findings.     

Adherence of infected erythrocytes to venular endothelium selects for antigenic variants of Plasmodium falciparum.

Erythrocytes (E) infected with asexual forms of malaria parasites exhibit surface antigenic variation. In Plasmodium falciparum infections, the variant Ag is the P. falciparum E membrane protein 1 (PfEMP1). This molecule may also mediate the adherence of infected E to host venular endothelium. We show here that parasite lines selected for increased adherence to endothelial cells have undergone antigenic variation. Three adherent lines selected from the same P. falciparum clone reacted with the same agglutinating antiserum that failed to agglutinate the parental clone. Immunoprecipitation experiments with the agglutinating anti-serum demonstrated that the selected lines expressed cross-reactive forms of PfEMP1 that were of higher m.w. and antigenically distinct from PfEMP1 of the parental clone. When one of the adherent lines was cloned in the absence of selection, a range of variant antigenic types emerged with differing cytoadherence phenotypes. These findings show that selection for cytoadherence in vitro favors the emergence of antigenic variants of P. falciparum and suggest that the requirement for cytoadherence in vivo may restrict the range of antigenic variants of P. falciparum in natural infections.     

Commitment to gametocytogenesis in Plasmodium falciparum

To achieve transmission, a subpopulation of asexually dividing bloodstream forms of the human malaria parasite Plasmodium falciparum withdraws from the cell cycle to develop into gametocytes - cells specialized for sexual reproduction and invasion of the mosquito vector. For natural selection to maximize transmission to new hosts, a balance must have evolved between asexual replication and sexual differentiation. Here, Mike Dyer and Karen Day consider observations on the process of commitment to gametocytogenesis and use this information as the framework for a model that begins to explain the control of the dynamics between asexual and sexual development.     

Resistance to a protein farnesyltransferase inhibitor in Plasmodium falciparum

The post-translational farnesylation of proteins serves to anchor a subset of intracellular proteins to membranes in eukaryotic organisms and also promotes protein-protein interactions. Inhibition of protein farnesyltransferase (PFT) is lethal to the pathogenic protozoa Plasmodium falciparum. Parasites were isolated that were resistant to BMS-388891, a tetrahydroquinoline (THQ) PFT inhibitor. Resistance was associated with a 12-fold decrease in drug susceptibility. Genotypic analysis revealed a single point mutation in the beta subunit in resistant parasites. The resultant tyrosine 837 to cysteine alteration in the beta subunit corresponded to the binding site for the THQ and peptide substrate. Biochemical analysis of Y837C-PFT demonstrated a 13-fold increase in BMS-388891 concentration necessary for inhibiting 50% of the enzyme activity. These data are consistent with PFT as the target of BMS-388891 in P. falciparum and suggest that PFT inhibitors should be combined with other antimalarial agents for effective therapy.     

Limitations of microscopy to differentiate Plasmodium species in a region co-endemic for Plasmodium falciparum, Plasmodium vivax and Plasmodium knowlesi

Background

The question whether Plasmodium falciparum infection affects the fitness of mosquito vectors remains open. A hurdle for resolving this question is the lack of appropriate control, non-infected mosquitoes that can be compared to the infected ones. It was shown recently that heating P. falciparum gametocyte-infected blood before feeding by malaria vectors inhibits the infection. Therefore, the same source of gametocyte-infected blood could be divided in two parts, one heated, serving as the control, the other unheated, allowing the comparison of infected and uninfected mosquitoes which fed on exactly the same blood otherwise. However, before using this method for characterizing the cost of infection to mosquitoes, it is necessary to establish whether feeding on previously heated blood affects the survival and fecundity of mosquito females.

Methods

Anopheles gambiae M molecular form females were exposed to heated versus non-heated, parasite-free human blood to mimic blood meal on non-infectious versus infectious gametocyte-containing blood. Life history traits of mosquito females fed on blood that was heat-treated or not were then compared.

Results

The results reveal that heat treatment of the blood did not affect the survival and fecundity of mosquito females. Consistently, blood heat treatment did not affect the quantity of blood ingested.

Conclusions

The study indicates that heat inactivation of gametocyte-infected blood will only inhibit mosquito infection and that this method is suitable for quantifying the fitness cost incurred by mosquitoes upon infection by P. falciparum.     

Malaria in the Post-Partum Period; a Prospective Cohort Study

Background

Several studies have shown a prolonged or increased susceptibility to malaria in the post-partum period. A matched cohort study was conducted to evaluate prospectively the susceptibility to malaria of post-partum women in an area where P.falciparum and P.vivax are prevalent.

Methods

In an area of low seasonal malaria transmission on the Thai-Myanmar border pregnant women attending antenatal clinics were matched to a non-pregnant, non-post-partum control and followed up prospectively until 12 weeks after delivery.

Results

Post-partum women (n = 744) experienced significantly less P.falciparum episodes than controls (hazard ratio (HR) 0.39 (95%CI 0.21–0.72) p = 0.003) but significantly more P.vivax (HR 1.34 (1.05–1.72) p = 0.018). The reduced risk of falciparum malaria was accounted for by reduced exposure, whereas a history of P.vivax infection during pregnancy was a strong risk factor for P.vivax in post-partum women (HR 13.98 (9.13–21.41), p<0.001). After controlling for effect modification by history of P.vivax, post-partum women were not more susceptible to P.vivax than controls (HR: 0.33 (0.21–0.51), p<0.001). Genotyping of pre-and post-partum infections (n⊕ = ⊕10) showed that each post-partum P.falciparum was a newly acquired infection.

Conclusions

In this area of low seasonal malaria transmission post-partum women were less likely to develop falciparum malaria but more likely to develop vivax malaria than controls. This was explained by reduced risk of exposure and increased risk of relapse, respectively. There was no evidence for altered susceptibility to malaria in the post-partum period. The treatment of vivax malaria during and immediately after pregnancy needs to be improved.     

Anemia and Dental Caries in Pregnant Women: a Prospective Cohort Study

The objective was to evaluate the effect of anemia during pregnancy on the risk of dental caries development in pregnant women. A prospective cohort including a sample of pregnant women in a prenatal care unit of São Luís, Brazil, was done. The incidence of dental caries during pregnancy, according to Nyvad’s criteria, was the outcome. The main independent variables were serum iron, ferritin, hemoglobin, erythrocyte, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red cell distribution width (RDW). Pregnant women (n = 121) were evaluated at two moments: up to 16th week of gestational age (T1) and in the last trimester of pregnancy (T2). Crude and adjusted associations were estimated by the incidence ratio risk (IRR) and respective 95% confidence intervals (95%CI). After adjustment, higher serum concentrations of ferritin (IRR = 0.97, 95%CI 0.95–0.99) in T1, and Fe (IRR = 0.99, 95%CI 0.98–0.99), ferritin (IRR = 0.99, 95%CI 0.98–0.99), erythrocyte (IRR = 0.71, 95%CI 0.50–0.99), hemoglobin (IRR = 0.84, 95%CI 0.73–0.96), hematocrit (IRR = 0.93, 95%CI 0.88–0.98), MCV (IRR = 0.91, 95%CI 0.86–0.96), and MCH (IRR = 0.83, 95%CI 0.74–0.93) in T2, were associated with fewer incidence of dental caries in pregnant women. Iron deficiency anemia during pregnancy is a risk factor for the incidence of dental caries in these women.     

Exposure to Secondhand Smoke and Risk of Tuberculosis: Prospective Cohort Study

Background

Prospective evidence on the association between secondhand-smoke exposure and tuberculosis is limited.

Methods

We included 23,827 never smokers from two rounds (2001 and 2005) of Taiwan National Health Interview Survey. Information on exposure to secondhand smoke at home as well as other sociodemographic and behavioral factors was collected through in-person interview. The participants were prospectively followed for incidence of tuberculosis through cross-matching the survey database to the national tuberculosis registry of Taiwan.

Results

A total of 85 cases of active tuberculosis were identified after a median follow-up of 7.0 years. The prevalence of exposure to secondhand smoke at home was 41.8% in the study population. In the multivariable Cox proportional hazards analysis, secondhand smoke was not associated with active tuberculosis (adjusted hazard ratio [HR], 1.03; 95% CI, 0.64 to 1.64). In the subgroup analysis, the association between secondhand smoke and tuberculosis decreased with increasing age; the adjusted HR for those <18, > = 18 and <40, > = 40 and <60, and > = 60 years old was 8.48 (0.77 to 93.56), 2.29 (0.75 to 7.01), 1.33 (0.58 to 3.01), and 0.66 (0.35 to 1.23) respectively. Results from extensive sensitivity analyses suggested that potential misclassification of secondhand-smoke exposure would not substantially affect the observed associations.

Conclusions

The results from this prospective cohort study did not support an overall association between secondhand smoke and tuberculosis. However, the finding that adolescents might be particularly susceptible to secondhand smoke''s effect warrants further investigation.     

Plasmodium falciparum resistant to chloroquine and to pyrimethamine in Comoros

We report the outcome of chloroquine treatment and the prevalence of mutations at codon 86 of the pfmdr1 gene, at codon 76 of the pfcrt gene, and at codon 108 of the pfdhfr gene in clinical isolates of Plasmodium falciparum collected from 30 children under 10 years of age living in the Comoros Union. This in vivo study was carried out in February and March 2001 in Moroni. Chloroquine treatment failed in 23 children (76.6%; 95% confidence interval: 57.7 to 90.1%). Subsequent genotyping showed that all P. falciparum isolates (100%) harboured a tyrosine residue at position 86 in pfMDR1. 83.3% (25/30) of these isolates harboured a mutation at position 76 in pfCRT and half (15/30) of these isolates also harboured a mutation at position 108 in pfDHFR. Chloroquine resistance is a real concern in the Comoros Union. The prevalence of pfDHFR mutant parasites is alarming. The alternative drugs proposed as a replacement for chloroquine as first-line treatment in Comoros, and the strategy to monitor the drug susceptibility of Plasmodium sp in this part of the Indian Ocean sub-region are discussed.     

Longitudinal study of Plasmodium falciparum and Plasmodium vivax in a Karen population in Thailand

Background

Pregnancy malaria is caused by Plasmodium falciparum -infected erythrocytes binding the placental receptor chondroitin sulfate A (CSA). This results in accumulation of parasites in the placenta with severe clinical consequences for the mother and her unborn child. Women become resistant to placental malaria as antibodies are acquired which specifically target the surface of infected erythrocytes binding in the placenta. VAR2CSA is most likely the parasite-encoded protein which mediates binding to the placental receptor CSA. Several domains have been shown to bind CSA in vitro; and it is apparent that a VAR2CSA-based vaccine cannot accommodate all the CSA binding domains and serovariants. It is thus of high priority to define minimal ligand binding regions throughout the VAR2CSA molecule.

Methods

To define minimal CSA-binding regions/peptides of VAR2CSA, a phage display library based on the entire var2csa coding region was constructed. This library was screened on immobilized CSA and cells expressing CSA resulting in a limited number of CSA-binding phages. Antibodies against these peptides were affinity purified and tested for reactivity against CSA-binding infected erythrocytes.

Results

The most frequently identified phages expressed peptides residing in the parts of VAR2CSA previously defined as CSA binding. In addition, most of the binding regions mapped to surface-exposed parts of VAR2CSA. The binding of a DBL2X peptide to CSA was confirmed with a synthetic peptide. Antibodies against a CSA-binding DBL2X peptide reacted with the surface of infected erythrocytes indicating that this epitope is accessible for antibodies on native VAR2CSA on infected erythrocytes.

Conclusion

Short continuous regions of VAR2CSA with affinity for multiple types of CSA were defined. A number of these regions localize to CSA-binding domains and to surface-exposed regions within these domains and a synthetic peptide corresponding to a peptide sequence in DBL2 was shown to bind to CSA and not to CSC. It is likely that some of these epitopes are involved in native parasite CSA adhesion. However, antibodies directed against single epitopes did not inhibit parasite adhesion. This study supports phage display as a technique to identify CSA-binding regions of large proteins such as VAR2CSA.     

Stage-dependent toxicity of N-acetyl-glucosamine to Plasmodium falciparum

The effects of N-acetyl-glucosamine on growth of synchronized cultures of Plasmodium falciparum were assessed by morphological observations and by measurement of parasite incorporation of 3H-hypoxanthine. Inhibition of 3H-hypoxanthine incorporation was more marked during the later stages of the erythrocytic cycle. At concentrations of the sugar below 20 mM, however, the deleterious effects were mainly a result of failure of released merozoites to invade erythrocytes, rather than a failure of schizonts to mature or release merozoites. These results are compatible with the hypothesis that a lectin-like substance on the merozoite interacts with a surface glycoprotein on the red cell and that sugar residues on this glycoprotein may be involved in this recognition.     

RNA helicase-related genes of Plasmodium falciparum and Plasmodium cynomolgi

RNA helicases play many essential roles including cell development and growth. Using degenerate oligonucleotide primers designed to amplify DNA fragments flanked by the highly conserved helicase motifs VLDEAD and YIHRIG and genomic DNAs from the malarial parasites as a template, we have cloned two putative RNA helicase genes (546 and 540 bp) from P. falciparum and one gene (546 bp) from P. cynomologi. Southern blot analysis revealed that these could be multiple and single-copy genes in P. falciparum and P. cynomolgi, respectively. Several members of the RNA helicase gene family share sequence identity with malarial parasite's helicases ranging from 30 to 76%, suggesting that they are functionally related. The discovery of such a multitude of putative RNA helicase genes in malarial parasites suggested that RNA helicase activities may be involved in many essential biological processes. Further characterization of these helicases may also help in designing parasite-specific inhibitors/drugs which specifically inhibit the parasite's growth without affecting the host.     

Appropriateness,Reasons and Independent Predictors of Consultations in the Emergency Department (ED) of a Dutch Tertiary Care Center: A Prospective Cohort Study

Objective

Consultations occur frequently in the emergency department (ED) of tertiary care centres and pose a threat for patient safety as they contribute to ED lengths of stay (LOS) and overcrowding. The aim of this study was to investigate reasons and appropriateness of consultations, and the relative impact of specialty and patient characteristics on the probability of a consultation, because this could help to improve efficiency of ED patient care.

Methods

This prospective cohort study included ED patients presenting to a Dutch tertiary care centre in a setting where ED physicians mostly treat self-referred and undifferentiated patients and other specialists treat referred patients. Consultations were defined as appropriate if the reason of consultation corresponded with the final advice, conclusion or policy of the consulted specialty. Multivariable logistic regression analysis was used to assess the relative contribution of specialty and patient characteristics on consultation.

Results

In the 344 (24% (95% CI 22 to 26%)) of the 1434 inclusions another specialty was consulted, resulting in a 55% increase of ED LOS. ED physicians more often consulted another specialty with a corrected odds ratio (OR) of 5.6 (4.0 to 7.8), mostly because consultations were mandatory in case of hospitalization or outpatient follow-up. Limited expertise of ED physicians was the reason for consultation in 7% (5 to 9%). The appropriateness of consultations was 84% (81 to 88%), similar between ED physicians and other specialists (P = 0.949). The patient characteristics age, comorbidity, and triage category and complaint predicted consultation.

Conclusion

In a Dutch tertiary care centre another specialty was consulted in 24% of the patients, mostly for an appropriate reason, and rarely because of lack of expertise. The impact of consultations on ED LOS could be reduced if mandatory consultations are abolished and predictors of a consultation are used to facilitate timely consultation.     

Adherence of Plasmodium falciparum infected erythrocytes to CHO-745 cells and inhibition of binding by protein A in the presence of human serum

Adhesion of erythrocytes infected with the malaria parasite Plasmodium falciparum to human host receptors is a process associated with severe malarial pathology. A number of in vitro cell lines are available as models for these adhesive processes, including Chinese hamster ovary (CHO) cells which express the placental adhesion receptor chondroitin-4-sulphate (CSA) on their surface. CHO-745 cells, a glycosaminoglycan-negative mutant CHO cell line lacking CSA and other reported P. falciparum adhesion receptors, are often used for recombinant expression of host receptors and for receptor binding studies. In this study we show that P. falciparum-infected erythrocytes can be easily selected for adhesion to an endogenous receptor on the surface of CHO-745 cells, bringing into question the validity of using these cells as a tool for P. falciparum adhesin expression studies. The adhesive interaction between CHO-745 cells and parasitized erythrocytes described here is not mediated by the known P. falciparum adhesion receptors CSA, CD36, or ICAM-1. However, we found that CHO-745-selected parasitized erythrocytes bind normal human IgM and that adhesion to CHO-745 cells is inhibited by protein A in the presence of serum, but not in its absence, indicating a non-specific inhibitory effect. Thus, protein A, which has been used as an inhibitor for a recently described interaction between infected erythrocytes and the placenta, may not be an appropriate in vitro inhibitor for understanding in vivo adhesive interactions.     

Stage-Dependent Toxicity of N-Acetyl-Glucosamine to Plasmodium falciparum1

The effects of N-acetyl-glucosamine on growth of synchronized cultures of Plasmodium falciparum were assessed by morphological observations and by measurement of parasite incorporation of ³H-hypoxanthine. Inhibition of ³H-hypoxanthine incorporation was more marked during the later stages of the erythrocytic cycle. At concentrations of the sugar below 20 mM, however, the deleterious effects were mainly a result of failure of released merozoites to invade erythrocytes, rather than a failure of schizonts to mature or release merozoites. These results are compatible with the hypothesis that a lectin-like substance on the merozoite interacts with a surface glycoprotein on the red cell and that sugar residues on this glycoprotein may be involved in this recognition.     

Assessment of therapeutic response of Plasmodium vivax and Plasmodium falciparum to chloroquine in a Malaria transmission free area in Colombia

In order to determine the frequency of therapeutic failures to chloroquine (CQ) in patients with malaria due to either Plasmodium falciparum or P. vivax, and to explore the usefulness of a malaria-free city as a sentinel site to monitor the emergence of drug resistance, 53 patients (44 infected with P. vivax and 9 with P. falciparum) were evaluated at the Laboratory of Parasitology, Universidad del Valle in Cali, Colombia. Patients received 25 mg/kg of CQ divided in three doses over 48 h; they were followed during 28 days according to WHO/PAHO protocols. While therapeutic failures to CQ in the P. vivax group were not detected, the proportion of therapeutic failures in the P. falciparum group was high (78%) and consistent with the reports from endemic areas in Colombia. The diverse origin of cases presenting therapeutic failure confirmed that P. falciparum resistant to CQ is widespread in Colombia, and further supports the change in the national antimalarial drug scheme. Monitoring of drug resistance in malaria free areas would be useful to identify sites requiring efficacy evaluation, and in some situations could be the most appropriate alternative to collect information from endemic areas where therapeutic efficacy studies are not feasible.     

Adherence of Plasmodium falciparum-Infected Erythrocytes to Chondroitin 4-Sulfate

Adherence of Plasmodium falciparum-infected erythrocytes (PRBCs) to the microvascular endothelium of specific organs and consequent sequestration is believed to be responsible for the development of malaria pathology. A number of studies have shown that cell adhesion molecules expressed on the surface of endothelial cells mediate the adherence. Recent studies indicate that a subpopulation of PRBCs adhere to chondroitin 4-sulfate (C4S). This adhesion can be effectively inhibited by C4S oligosaccharides. In pregnant women, the placenta specifically selects C4S-adherent PRBCs, and thus these phenotypes multiply and sequester in the intervillous spaces. Over successive pregnancies, women develop a protective humoral response to the C4S-adhesion phenotype. Disruption of C4S-mediated PRBCs adhesion using either a C4S oligosaccharide mimetic or an antiC4S-adhesion vaccine can be an efficient strategy for the treatment of malaria caused by C4S-adherent P. falciparum.