A Drug Repurposing Approach Reveals Targetable Epigenetic Pathways in Plasmodium vivax Hypnozoites.

Radical cure of Plasmodium vivax malaria must include elimination of quiescent 'hypnozoite' forms in the liver; however, the only FDA-approved treatments are contraindicated in many vulnerable populations. To identify new drugs and drug targets for hypnozoites, we screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library and a collection of epigenetic inhibitors against P. vivax liver stages. From both libraries, we identified inhibitors targeting epigenetics pathways as selectively active against P. vivax and P. cynomolgi hypnozoites. These include DNA methyltransferase (DNMT) inhibitors as well as several inhibitors targeting histone post-translational modifications. Immunofluorescence staining of Plasmodium liver forms showed strong nuclear 5-methylcystosine signal, indicating liver stage parasite DNA is methylated. Using bisulfite sequencing, we mapped genomic DNA methylation in sporozoites, revealing DNA methylation signals in most coding genes. We also demonstrated that methylation level in proximal promoter regions as well as in the first exon of the genes may affect, at least partially, gene expression in P. vivax. The importance of selective inhibitors targeting epigenetic features on hypnozoites was validated using MMV019721, an acetyl-CoA synthetase inhibitor that affects histone acetylation and was previously reported as active against P. falciparum blood stages. In summary, our data indicate that several epigenetic mechanisms are likely modulating hypnozoite formation or persistence and provide an avenue for the discovery and development of improved radical cure antimalarials.

PacBio assembly of a Plasmodium knowlesi genome sequence with Hi-C correction and manual annotation of the SICAvar gene family.

Plasmodium knowlesi has risen in importance as a zoonotic parasite that has been causing regular episodes of malaria throughout South East Asia. The P. knowlesi genome sequence generated in 2008 highlighted and confirmed many similarities and differences in Plasmodium species, including a global view of several multigene families, such as the large SICAvar multigene family encoding the variant antigens known as the schizont-infected cell agglutination proteins. However, repetitive DNA sequences are the bane of any genome project, and this and other Plasmodium genome projects have not been immune to the gaps, rearrangements and other pitfalls created by these genomic features. Today, long-read PacBio and chromatin conformation technologies are overcoming such obstacles. Here, based on the use of these technologies, we present a highly refined de novo P. knowlesi genome sequence of the Pk1(A+) clone. This sequence and annotation, referred to as the 'MaHPIC Pk genome sequence', includes manual annotation of the SICAvar gene family with 136 full-length members categorized as type I or II. This sequence provides a framework that will permit a better understanding of the SICAvar repertoire, selective pressures acting on this gene family and mechanisms of antigenic variation in this species and other pathogens.

Plasmodium knowlesi: a superb in vivo nonhuman primate model of antigenic variation in malaria.

Antigenic variation in malaria was discovered in Plasmodium knowlesi studies involving longitudinal infections of rhesus macaques (M. mulatta). The variant proteins, known as the P. knowlesi Schizont Infected Cell Agglutination (SICA) antigens and the P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) antigens, expressed by the SICAvar and var multigene families, respectively, have been studied for over 30 years. Expression of the SICA antigens in P. knowlesi requires a splenic component, and specific antibodies are necessary for variant antigen switch events in vivo. Outstanding questions revolve around the role of the spleen and the mechanisms by which the expression of these variant antigen families are regulated. Importantly, the longitudinal dynamics and molecular mechanisms that govern variant antigen expression can be studied with P. knowlesi infection of its mammalian and vector hosts. Synchronous infections can be initiated with established clones and studied at multi-omic levels, with the benefit of computational tools from systems biology that permit the integration of datasets and the design of explanatory, predictive mathematical models. Here we provide an historical account of this topic, while highlighting the potential for maximizing the use of P. knowlesi - macaque model systems and summarizing exciting new progress in this area of research.

B cell epitope mapping and characterization of naturally acquired antibodies to the Plasmodium vivax merozoite surface protein-3α (PvMSP-3α) in malaria exposed individuals from Brazilian Amazon.

The Plasmodium vivax Merozoite Surface Protein-3α (PvMSP-3α) is considered as a potential vaccine candidate. However, the detailed investigations of the type of immune responses induced in naturally exposed populations are necessary. Therefore, we aim to characterize the naturally induced antibody to PvMSP-3α in 282 individuals with different levels of exposure to malaria infections residents in Brazilian Amazon. PvMSP3 specific antibodies (IgA, IgG and IgG subclass) to five recombinant proteins and the epitope mapping by Spot-synthesis technique to full-protein sequence of amino acids (15aa sequence with overlapping sequence of 9aa) were performed. Our results indicates that PvMSP3 is highly immunogenic in naturally exposed populations, where 78% of studied individuals present IgG immune response against the full-length recombinant protein (PVMSP3-FL) and IgG subclass profile was similar to all five recombinant proteins studied with a high predominance of IgG1 and IgG3. We also observe that IgG and subclass levels against PvMSP3 are associated with malaria exposure. The PvMSP3 epitope mapping by Spot-synthesis shows a natural recognition of at least 15 antigenic determinants, located mainly in the two blocks of repeats, confirming the high immunogenicity of this region. In conclusion, PvMSP-3α is immunogenic in naturally exposed individuals to malaria infections and that antibodies to PvMSP3 are induced to several B cell epitopes. The presence of PvMSP3 cytophilic antibodies (IgG1 and IgG3), suggests that this mechanism could also occur in P. vivax.

Promiscuous T-cell epitopes of Plasmodium merozoite surface protein 9 (PvMSP9) induces IFN-gamma and IL-4 responses in individuals naturally exposed to malaria in the Brazilian Amazon.

Plasmodium vivax merozoite surface protein (PvMSP9) stimulates both cellular and humoral immune responses in individuals who are naturally infected by this parasite species. To identify immunodominant human T-cell epitopes in PvMSP9, we used the MHC class II binding peptide prediction algorithm ProPred. Eleven synthetic peptides representing predicted putative promiscuous T-cell epitopes were tested in IFN-gamma and IL-4 ELISPOT assays using peripheral blood mononuclear cells (PBMC) derived from 142 individuals from Rondonia State, Brazil who had been naturally exposed to P. vivax infections. To determine whether the predicted epitopes are preferentially recognized in the context of multiple alleles, MHC Class II typing of the cohort was also performed. Five synthetic peptides elicited robust cellular responses, and the overall frequencies of IFN-gamma and IL-4 responders to at least one of the promiscuous peptides were 62% and 46%, respectively. The frequencies of IFN-gamma and IL-4 responders to each peptide were not associated with a particular HLA-DRB1 allelic group since most of the peptides induced a response in individuals of 12 out of 13 studied allelic groups. The prediction of promiscuous epitopes using ProPred led to the identification of immunodominant epitopes recognized by PBMC from a significant proportion of a genetically heterogeneous population exposed to malaria infections. The combination of several such T-cell epitopes in a vaccine construct may increase the frequency of responders and the overall efficacy of subunit vaccines in genetically distinct populations.

The genome of the simian and human malaria parasite Plasmodium knowlesi.

Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the 'kra' monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated, and it has a close phylogenetic relationship to Plasmodium vivax, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or 'hypnozoite' in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome and other sequenced Plasmodium genomes. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.

Naturally acquired humoral and cellular immune responses to Plasmodium vivax merozoite surface protein 9 in Northwestern Amazon individuals.

Antibody and T-cell reactivities to Plasmodium vivax merozoite surface protein 9 (PvMSP9) were evaluated in a cross-sectional study of individuals naturally exposed to malaria infections living in Ribeirinha, a native riverine community and in Colina, a transmigrant community, Rondonia, Brazil. The antibody responses to PvMSP9-RIRIIand PvMSP9-Nt domains in Ribeirinha were higher compared with Colina and correlated with age and time of malaria exposure. IgG2 was most prevalent for PvMSP9-RII in both communities, and IgG1 was the predominant isotype for PvMSP9-Nt and PvMSP9-RIRII in Ribeirinha. IFN-gamma and IL-4 predominated in Ribeirinha, while IFN-gamma predominated in Colina. Variation in exposure to P. vivax likely accounts for the differences observed in cytokine and antibody levels between the two populations studied.

Preclinical assessment of the receptor-binding domain of Plasmodium vivax Duffy-binding protein as a vaccine candidate in rhesus macaques.

The receptor-binding domain of Plasmodium vivax Duffy-binding protein, region II (PvRII), is an attractive candidate for a vaccine against P. vivax malaria. Here, we have studied the safety and immunogenicity of recombinant PvRII in Macaca mulatta (rhesus monkeys). Recombinant PvRII with a C-terminal 6-histidine tag was expressed in E. coli, recovered from inclusion bodies, refolded into its functional conformation, purified to homogeneity and formulated with three adjuvants, namely, Alhydrogel, Montanide ISA 720 and the GSK proprietary Adjuvant System AS02A for use in immunogenicity studies. All the PvRII vaccine formulations tested were safe and highly immunogenic. The overall magnitude of the antibody response was significantly higher for both Montanide ISA 720 and AS02A formulations in comparison with Alhydrogel. Furthermore, there was a significant correlation between antibody recognition titers by ELISA and binding inhibition titers in in vitro binding assays. The PvRII vaccine formulations also induced IFN-gamma recall responses that were identified using ex vivo ELISPOT assays. These results provide support for further clinical development of a vaccine for P. vivax malaria based on recombinant PvRII.

Characterization and application of multiple genetic markers for Plasmodium malariae.

Plasmodium malariae, a protozoan parasite that causes malaria in humans, has a global distribution in tropical and subtropical regions and is commonly found in sympatry with other Plasmodium species of humans. Little is known about the genetics or population structure of P. malariae. In the present study, we describe polymorphic genetic markers for P. malariae and present the first molecular epidemiological data for this parasite. Six microsatellite or minisatellite markers were validated using 76 P. malariae samples from a diverse geographical range. The repeat unit length varied from 2 to 17 bp, and up to 10 different alleles per locus were detected. Multiple genotypes of P. malariae were detected in 33 of 70 samples from humans with naturally acquired infection. Heterozygosity was calculated to be between 0.236 and 0.811. Allelic diversity was reduced for samples from South America and, at some loci, in samples from Thailand compared with those from Malawi. The number of unique multilocus genotypes defined using the 6 markers was significantly greater in Malawi than in Thailand, even when data from single genotype infections were used. There was a significant reduction in the multiplicity of infection in symptomatic infections compared with asymptomatic ones, suggesting that clinical episodes are usually caused by the expansion of a single genotype.

[Emergency ambulance management of acute pain in children: a national survey]. / Douleur aiguë de l'enfant dans l'aide médicale d'urgence. Evaluation des pratiques.

OBJECTIVES: To assess the knowledge and skills of physicians staffing mobile intensive care units (emergency ambulances) in the management of severe acute pain in children. METHODS: Questionnaire-based telephone interviews with emergency physicians of all urban emergency ambulance services (n=360). This questionnaire covered knowledge of procedures for assessment of pain, definition of severe acute pain and its, treatment, availability of morphine and similar drugs, local guidelines and the physicians' opinion of the national guidelines. RESULTS: Physicians from all but one ambulance service responded. Forty-nine percent were unaware of the French Society of Anesthesiology and Intensive Care guidelines, and 63% had no local guidelines. Eight percent defined severe acute pain correctly and 10% defined the therapeutic objective correctly. Forty-seven percent used morphine (which was available for 93%), and 7% and 13% respectively followed guidelines about doses and waiting periods between administrations. CONCLUSION: This survey showed inadequate knowledge about the management (assessment and treatment) of severe acute pain in children in prehospital emergency settings. Training in this area is essential.

[National survey of emergency management of acute pain in prehospital setting]. / Prise en charge de la douleur aiguë sévère chez l'adulte en médecine extrahospitalière: enquête nationale auprès des médecins de Smur.

OBJECTIVES: Assessment of skill of physicians staffed Mobile Intensive Care Units (MICU) in severe acute pain (SAP) management. METHODS: This study was conducted with a phone questionnaire addressed to emergency physicians of all metropolitan Smur (N =360). This questionnaire included medical teaching, professional position, means of severe acute pain assessment and definition of SAP, treatment, analgesic drug availability, locals guidelines and personal assessment of national guidelines were studied. RESULTS: Exhaustivity was more than 99% (359/360). Eighty percent of physicians were specialized in emergency care, 8% were anaesthesiologists (or intensivists), 78% worked in emergency department and 76% were full time. Forty nine percent of physicians did not know French Society of Anesthesiology and Intensive Care guidelines and 63% did not have locals analgesics guidelines. To define SAP, Visual Analogic Scale (VAS) or Numeric Scale (NS) >6/10 were mentioned only by 17%. Therapeutic efficiency was assessed and defined by VAS or NS <3/10 by 14%. Morphine was available in 90% of MICU, fentanyl in 79% and nalbuphine in 64%. Morphine was used in first intention by 71% of physicians. Guidelines about doses and waiting periods between 2 administrations were followed respectively by 6% and 28%. Ninety percent of physicians combined at least two treatments, 58 % combined at least three and 39%, at least four. CONCLUSION: This survey showed a low knowledge about severe acute pain management in out-of-hospital setting, both for pain assessment and treatment. There were few locals guidelines to overcome this deficiency. A training work is essential to improve care of acute pain in out-of-hospital setting.

[Victims of fall from height. Study of 287 patients and determination of clinical prognostic factors]. / Patients victimes de chutes de grande hauteur. Etude d'une cohorte de 287 patients et détermination des facteurs pronostiques cliniques.

INTRODUCTION: Fall from height is a common cause of severe blunt urban trauma but this mechanism of trauma is poorly documented. OBJECTIVE: To describe initial clinical parameters, injuries and outcome in patients victims of falls from height and to determinate clinical prognostic factors. STUDY DESIGN: Retrospective study from January 1998 to May 1999 and prospective study from June 1999 to September 2000. Inclusions: patients older than 12 year-old victims of falls with height of more than 3 m. PATIENTS AND METHODS: Studied parameters: demographic data, fall circumstances, prehospital clinical evaluation (Glasgow coma scale (GCS), Arterial Blood Pressure (ABP), Heart Rhythm (ER) and revised trauma score (RTS) initial and patients outcome. RESULTS: Two hundred and eighty seven patients were included during 32.5 months; 33% were women and 67% men. Mean age: 37 +/- 16 years. Median height of fall: three stairs (3-4). Final mortality: 34% including 22% death on site with or without resuscitation, 5% before hospitalization, 8% during the first day and 2% later. Independent prognostic factors were GCS (OR = 0.40; IC: 0.25 - 0.65; P = 0.0002), initial ABP (OR = 0.94; IC: 0.90 - 0.98; P = 0.008) and RTS (OR = 2.93; 1.19-7.24; P = 0.02). CONCLUSION: Immediate mortality after fall from heights is high and remains low after day 3. GCS, ABP and RTS are independent prognostic factors.

Disposable laryngoscope blades do not interfere with ease of intubation in scheduled general anaesthesia patients.

BACKGROUND AND OBJECTIVE: Intubation of the trachea has been a risky cross-contamination procedure over the past decade because no perfect decontamination procedures exist. Infectious agents found on laryngoscopic devices have the potential for devastating spread of the human immunodeficiency virus, hepatitis viruses B and C and transmissible non-conventional agents. The purpose of this prospective observational study was to assess the quality of endotracheal intubation with disposable laryngoscope blades, under normal intubating conditions. METHODS: Anaesthetists were asked to complete daily questionnaires regarding the difficulty of intubation experienced using the Vital View disposable laryngoscope blade (Vital Signs Inc, Totowa, NC, USA). The choice of the type of blade (conventional or disposable blade) for the first attempt at intubation depended only on the operating room assignment. Glottic visualization during laryngoscopy was assessed by the modified Cormack and Lehane classification. Difficult tracheal intubation was evaluated by the intubation difficulty scale (> 5, procedure involving moderate to major difficulty). RESULTS: The anaesthetic staff recorded 219 intubations. One hundred-and-nineteen of first attempts at laryngoscopy were with disposable blades (DB group) and another 100 with conventional blades (CB group). There were no significant differences between the two groups for Cormack and Lehane score 3, for intubation difficulty scale scores > 5 and for intubation difficulty scale score 0. There were 12 blade changes before successful intubation. CONCLUSIONS: In routine use, the Vital View disposable laryngoscope blade appears to be an efficient device because it does not modify the ease of endotracheal intubation in most cases. Nonetheless, it may be advisable to maintain conventional laryngoscopes in reserve for difficult intubations.

[In-hospital cardiac arrest resuscitation: medical and paramedical theory skill assessment in an university hospital]. / Prise en charge des arrêts cardiaques intrahospitaliers: évaluation des connaissances théoriques du personnel médical et paramédical.

OBJECTIVES: Theory knowledge and attitude assessment about in-hospital cardiac arrest (CA) basic life support by hospital staff. METHODS: We tested medical and paramedical working staff in a 450 beds university hospital with anonymous questionnaire based on training for basic life support. Questions were about presence of a formation before, clinical signs reached in front of unconscious patient, attitude in front of cardiac-arrest, practical experience with basic life support and ward emergency trolley. RESULTS: Five hundred and seventy one on 996 people answered to handed out questionnaires (57%): 158 from medical staff (Med group) and 413 from nurses "Pmed". Seventy one percent people from "Med" group and 64% from "Pmed" received one time at least training about cardiac-arrest. Front of unconscious patient, no spontaneous breath was reached explicitly by 55% people from Med group and 19% from "Pmed" group and central pulse was reached explicitly by 70% people from "Med" group and 18% from "Pmed" group. Front of CA, 50% people from "Med" group released airway, 75% began ventilation and 86%, External Heart Compression (EHC) and 42% called for rescue. There were respectively 29, 47, 64 and 60% people from "Pmed" group. Eighty-one percent people from "Med" group thought they knew to do ventilation and 82% did it one time at least. Eighty-eight thought they knew to do EHC and 85% did it one time at least. They were respectively 67, 76, 73 and 78% people from Pmed group. Sixty-four per cent of Med group people know that there is emergency trolley in there department versus 89% for "Pmed" group. CONCLUSION: This study showed that theoretical knowledge of hospital staff about cardiac arrest diagnostic and management are insufficient from the point of view of national and international guidelines. Analysis is difficult because of weak response number and knowledge people overestimation.