RESUMEN
Target-based approaches have traditionally been used in the search for new anti-infective molecules. Target selection process, a critical step in Drug Discovery, identifies targets that are essential to establish or maintain the infection, tractable to be susceptible for inhibition, selective towards their human ortholog and amenable for large scale purification and high throughput screening. The work presented herein validates the Plasmodium falciparum mRNA 5' triphosphatase (PfPRT1), the first enzymatic step to cap parasite nuclear mRNAs, as a candidate target for the development of new antimalarial compounds. mRNA capping is essential to maintain the integrity and stability of the messengers, allowing their translation. PfPRT1 has been identified as a member of the tunnel, metal dependent mRNA 5' triphosphatase family which differs structurally and mechanistically from human metal independent mRNA 5' triphosphatase. In the present study the essentiality of PfPRT1 was confirmed and molecular biology tools and methods for target purification, enzymatic assessment and target engagement were developed, with the goal of running a future high throughput screening to discover PfPRT1 inhibitors.
Asunto(s)
Antimaláricos , Descubrimiento de Drogas , Plasmodium falciparum , Antimaláricos/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/enzimología , Descubrimiento de Drogas/métodos , Humanos , Ensayos Analíticos de Alto Rendimiento/métodos , ARN Mensajero/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , Inhibidores Enzimáticos/farmacología , Ácido Anhídrido HidrolasasRESUMEN
BACKGROUND AND OBJECTIVES: Systemic lupus erythematosus (SLE) is an autoimmune condition that can highly impact patients' quality of life (QoL). However, there is a lack of knowledge about SLE, affecting the general population and health care professionals (HCPs) alike. This lack of knowledge has negative implications for patients and the healthcare system, worsening prognosis, negatively impacting QoL, and increasing healthcare utilization. The aim of this paper is to draw attention, according to the perspective of the participants of this study, to the lack of awareness of SLE and its consequences in Spain, and to suggest improvements. PATIENTS AND METHODS: This qualitative, descriptive, observational, multicenter, and cross-sectional study included 40 patients with moderate or severe SLE, recruited during their routine visits in six university hospitals in Spain. The study also included 11 caregivers and 9 HCPs. All participants were individually interviewed. Data from the interviews were coded and analyzed thematically by two anthropologists following a phenomenological perspective. RESULTS: Our study identified a lack of disease awareness among primary care physicians, emergency medicine doctors, and other specialists treating SLE symptomatology. This led to diagnostic delays, which had a clinical and emotional impact on patients. Furthermore, symptom awareness was found to be context dependent. Differences in symptom awareness between HCPs and patients led to a mismatch between the severity evaluation made by doctors and patients. Some HCPs did not consider the limitations of the current severity evaluation of SLE, and therefore attributed symptoms potentially caused by SLE to the unfavorable socioeconomic conditions patients lived in. Finally, a lack of social awareness among friends, family members, and romantic partners led to lower social support, increased isolation, and negative physical and emotional impact for patients. Gender differences in the provision of support were identified. CONCLUSION: This study highlights the need to increase SLE awareness among patients, HCPs, and the broader public in order to improve patient QoL. Being aware of the clinical and emotional impact of such lack of awareness, as well as the role played by context on the patient experience of SLE, is a crucial step towards achieving this goal.
Asunto(s)
Conocimientos, Actitudes y Práctica en Salud , Lupus Eritematoso Sistémico , Calidad de Vida , Índice de Severidad de la Enfermedad , Humanos , Lupus Eritematoso Sistémico/psicología , Lupus Eritematoso Sistémico/diagnóstico , España , Femenino , Estudios Transversales , Masculino , Adulto , Persona de Mediana Edad , Investigación Cualitativa , Personal de Salud/psicología , Anciano , Diagnóstico Tardío , Adulto Joven , ConcienciaciónRESUMEN
BACKGROUND: Belimumab was the first biological drug approved for Systemic Lupus Erythematosus (SLE). There is not a review focusing on all real-life experience with belimumab to date that could help to describe how this drug behaves in the Spanish clinical setting. OBJECTIVE: To describe the characteristics of SLE patients treated with belimumab added to standard of care in real-clinical setting in Spain. METHODS: We conducted a comprehensive scoping review of real-world data (RWD) according to PRISMA Scoping Reviews Checklist and the framework proposed by Arksey and O'Malley. PubMed and EMBASE were searched without language restriction and hand searches of relevant articles were examined. RESULTS: We included data from 222 patients treated with belimumab for SLE included in 19 RWD studies conducted in Spain. The mean age was 40.9 years, 84.2% were female, and baseline scores SELENA-SLEDAI ranged between 5.9 and 12. Lupus nephritis basal prevalence was of 2.7%. The main reason for belimumab initiation was previous treatments lack of efficacy (69.7%) and the most common laboratory abnormalities were hypocomplementemia (40.9%), ANA + (34.2%), and anti-DNA (33.3%). The addition of belimumab to standard therapy was associated with a reduction of daily glucocorticoids intake in 1.4-11.1 mg at 6 months. Belimumab discontinuation was observed in 18.6% of patients. CONCLUSION: Our study helps to further explore the profile of SLE patients most likely to be treated with belimumab.
Asunto(s)
Productos Biológicos , Lupus Eritematoso Sistémico , Adulto , Anticuerpos Monoclonales Humanizados , Productos Biológicos/uso terapéutico , Femenino , Humanos , Inmunosupresores/efectos adversos , Lupus Eritematoso Sistémico/inducido químicamente , Lupus Eritematoso Sistémico/tratamiento farmacológico , Masculino , España , Resultado del TratamientoRESUMEN
Phenotypic screening has produced most of the new chemical entities currently in clinical development for malaria, plus many lead compounds active against Plasmodium falciparum asexual stages. However, lack of knowledge about the mode of action of these compounds delays and may even hamper their future development. Identifying the mode of action of the inhibitors greatly helps to prioritise compounds for further development as novel antimalarials. Here we describe a whole-cell method to detect inhibitors of the mitochondrial electron transport chain, using oxygen consumption as high throughput readout in 384-well plate format. The usefulness of the method has been confirmed with the Tres Cantos Antimalarial Compound Set (TCAMS). The assay identified 124 respiratory inhibitors in TCAMS, seven of which were novel anti-plasmodial chemical structures never before described as mitochondrial inhibitors.
Asunto(s)
Antimaláricos/farmacología , Evaluación Preclínica de Medicamentos/métodos , Mitocondrias/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/instrumentación , Proteínas del Complejo de Cadena de Transporte de Electrón/antagonistas & inhibidores , Humanos , Concentración 50 Inhibidora , Malaria/tratamiento farmacológico , Malaria/parasitología , Malaria Falciparum , Oxígeno/metabolismo , Plasmodium falciparum/citologíaRESUMEN
BACKGROUND: Drugs that kill or inhibit Plasmodium gametocytes in the human host could potentially synergize the impact of other chemotherapeutic interventions by blocking transmission. To develop such agents, reliable methods are needed to study the in vitro activity of compounds against gametocytes. This study describes a novel assay for characterizing the activity of anti-malarial drugs against the later stages of Plasmodium falciparum gametocyte development using real-time PCR (qPCR). METHODS: Genes previously reported to be transcribed at the different sexual stages of the gametocytogenesis were selected for study and their mRNA expression was measured in a gametocytogenesis course by qPCR. Genes mainly expressed in the later stages of gametocyte development were used as a surrogate measurement of drug activity. To distinguish between cidal and static drug effects, two different experiments were performed in parallel, one with constant drug pressure throughout the experiment (144 h), and another in which the gametocyte cultures were exposed to the compound for only 48 h. RESULTS: Four P. falciparum genes coding for proteins Pf77, ROM3, Pfs25, and Pfg377 with transcription specific for late-stage gametocyte development were identified. The in vitro anti-malarial activity of compounds against such gametocytes was assessed by measuring mRNA levels of these genes using qPCR. The assay was validated against standard anti-malarial drugs (epoxomicin, dihydroartemisinin, chloroquine, thiostrepton, and methylene blue) and compounds from the GSK compound library with known anti-gametocyte activity. CONCLUSIONS: This study describes a novel assay for characterizing the activity of anti-malarial drugs against the later stages of P. falciparum gametocyte development using qPCR in genetically unmodified parasites. The method described is a reliable and user-friendly technique with a medium throughput that could be easily implemented in any laboratory.
Asunto(s)
Antimaláricos/aislamiento & purificación , Antimaláricos/farmacología , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Plasmodium falciparum/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Supervivencia Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Plasmodium falciparum/fisiologíaRESUMEN
BACKGROUND: Quantitative real-time PCR (qPCR) is now commonly used as a method to confirm diagnosis of malaria and to differentiate recrudescence from re-infection, especially in clinical trials and in reference laboratories where precise quantification is critical. Although anti-malarial drug discovery is based on in vivo murine efficacy models, use of molecular analysis has been limited. The aim of this study was to develop qPCR as a valid methodology to support pre-clinical anti-malarial models by using filter papers to maintain material for qPCR and to compare this with traditional methods. METHODS: FTA technology (Whatman) is a rapid and safe method for extracting nucleic acids from blood. Peripheral blood samples from mice infected with Plasmodium berghei, P. yoelii, or P. falciparum were kept as frozen samples or as spots on FTA cards. The extracted genetic material from both types of samples was assessed for quantification by qPCR using sets of specific primers specifically designed for Plasmodium 18S rRNA, LDH, and CytB genes. RESULTS: The optimal conditions for nucleic acid extraction from FTA cards and qPCR amplification were set up, and were confirmed to be suitable for parasite quantification using DNA as template after storage at room temperature for as long as 26 months in the case of P. berghei samples and 52 months for P. falciparum and P. yoelii. The quality of DNA extracted from the FTA cards for gene sequencing and microsatellite amplification was also assessed. CONCLUSIONS: This is the first study to report the suitability of FTA cards and qPCR assay to quantify parasite load in samples from in vivo efficacy models to support the drug discovery process.
Asunto(s)
Malaria/diagnóstico , Malaria/parasitología , Plasmodium/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Animales , ADN Protozoario/sangre , ADN Protozoario/aislamiento & purificación , Límite de Detección , Ratones , ParasitologíaRESUMEN
This study analyzed the relationship between intermittent preventive treatment with sulfadoxine-pyrimethamine (SP) (IPTp-SP), the rate of multiple resistant parasites and of submicroscopic gametocyte carriage among pregnant women at the beginning of IPTp implementation in Gabon (2005) and six years after (2011). The detection of pfdhfr and pfdhps gene mutations was performed by PCR-RFLP in Plasmodium (P.) falciparum positive samples collected from pregnant women in 2005 and 2011. Gametocytes carriage was detected by Pfs25mRNA amplification using QT-NASBA. Data were analyzed according to the time of collection (study period) and IPTp-SP doses. The proportion of isolates with at least a triple Pfdhfr mutation (n = 39/42, 92.9% versus 100%, n = 78/78)) and of those isolates with the S108N/C59R/N51I/S436A/A437G multiple mutation (17.9% versus 75.6%) significantly increased between 2005 and 2011 (p<0.01). Mutations I164L and A581G were not found, while higher proportions of 436 and 437 mutations were detected in 2011.A trend toward a higher frequency of isolates with five mutations was observed in women who received two SP doses (p<0.01). Pfs25mRNA was found in 6.8 % (n = 3/44) and 34.6% (n = 27/78) of the samples collected in 2005 and 2011 respectively (p<0.01). In 2011, 74.0% (n = 20/27) of women with detected submicroscopic gametocytes carried parasites with the S108N/C59R/N51/S436A/A437G multiple mutation. All the ten delivering women who received three IPTp-SP doses had a submicroscopic Plasmodium falciparum infection, but none had detected gametocytes. Following IPTp-SP implementation, an increase in the frequency of multiple mutant parasites and of submicroscopic gametocyte carriage was observed among pregnant women living in Gabon.
Asunto(s)
Portador Sano/parasitología , Dihidropteroato Sintasa/genética , Malaria Falciparum/parasitología , Proteínas Mutantes/genética , Plasmodium falciparum/enzimología , Complicaciones Infecciosas del Embarazo/parasitología , Tetrahidrofolato Deshidrogenasa/genética , Antimaláricos/uso terapéutico , Quimioprevención/métodos , ADN Protozoario/genética , Combinación de Medicamentos , Femenino , Gabón , Frecuencia de los Genes , Humanos , Malaria Falciparum/prevención & control , Mutación , Plasmodium falciparum/genética , Plasmodium falciparum/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Embarazo , Pirimetamina/uso terapéutico , Sulfadoxina/uso terapéuticoRESUMEN
Screening of the GSK corporate collection, some 1.9 million compounds, against Plasmodium falciparum (Pf), revealed almost 14000 active hits that are now known as the Tres Cantos Antimalarial Set (TCAMS). Followup work by Calderon et al. clustered and computationally filtered the TCAMS through a variety of criteria and reported 47 series containing a total of 522 compounds. From this enhanced set, we identified the carbamoyl triazole TCMDC-134379 (1), a known serine protease inhibitor, as an excellent starting point for SAR profiling. Lead optimization of 1 led to several molecules with improved antimalarial potency, metabolic stabilities in mouse and human liver microsomes, along with acceptable cytotoxicity profiles. Analogue 44 displayed potent in vitro activity (IC50 = 10 nM) and oral activity in a SCID mouse model of Pf infection with an ED50 of 100 and ED90 of between 100 and 150 mg kg(-1), respectively. The results presented encourage further investigations to identify the target of these highly active compounds.
Asunto(s)
Antimaláricos/síntesis química , Antimaláricos/farmacología , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/farmacología , Triazoles/síntesis química , Triazoles/farmacología , Animales , Antimaláricos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Técnicas In Vitro , Malaria/tratamiento farmacológico , Malaria/psicología , Malaria Falciparum/tratamiento farmacológico , Ratones , Ratones SCID , Microsomas Hepáticos/metabolismo , Plasmodium berghei/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Inhibidores de Serina Proteinasa/metabolismo , Relación Estructura-Actividad , Triazoles/metabolismoRESUMEN
BACKGROUND: Considering malaria prevalence declines in parts of sub-Saharan Africa, such as Gabon, identification of the human infectious reservoir is important for successful malaria control. Microscopic and sub-microscopic parasites contribute to malaria transmission. The aim of the present study was to evaluate the proportion of microscopic and sub-microscopic gametocyte carriers among febrile patients in two different areas of Gabon. METHODS: Samples from febrile children aged less than 11 years old were collected from February 2008 to January 2009 at two health centres of Gabon. Patients were screened for the presence of asexual Plasmodium falciparum parasites. Gametocyte carriage was determined by microscopy and QT-NASBA. RESULTS: Gametocytes were detected in 5.3% (n = 16/304) of children by microscopy compared to 45.7% (n = 139/304) by QT-Nasba. Sub-microscopic gametocyte carriage (ie microscopy negative and QT-Nasba positive) was found in 89.2% (n = 124/139) of patients. Among patients with microscopically detected trophozoites, the proportion of sub-microscopic gametocyte (SMG) carriers was 58.4% (n = 118/202) and 6% in samples from children with negative slides (p < 0.01). In Oyem, where malaria prevalence is three-fold higher than in Owendo, SMG carriage was more frequent (49.0% vs 32.6% in Owendo; p < 0.01). CONCLUSION: Sub-microscopic gametocytaemia is common among Gabonese febrile children. They might strongly contribute to maintain malaria transmission. However, further analysis of sub-microscopic parasite carriage among asymptomatic individuals will be helpful to better characterize malaria transmission.
Asunto(s)
Portador Sano/epidemiología , Malaria Falciparum/epidemiología , Plasmodium falciparum/aislamiento & purificación , Portador Sano/parasitología , Niño , Preescolar , Femenino , Gabón/epidemiología , Humanos , Lactante , Malaria Falciparum/parasitología , Masculino , Microscopía , Técnicas de Amplificación de Ácido Nucleico , PrevalenciaRESUMEN
In order to maximise compliance, the future antimalarial treatment should ideally require just a single-dose administration. This, in turn, demands new fast-acting effective drugs. Currently, methods to measure the in vitro killing rate of antimalarials are based on parasite growth. We have developed and validated a method to determine and classify antimalarial agents based on their cidal or static activity following quantitative Real Time PCR (RT-PCR) analysis. The method described here is a fast, reliable and user-friendly technique with a medium throughput. Metabolic activity of the parasite is followed by measuring mRNA expression levels of several genes during 5 parasite life cycles. mRNA from the parasite culture is then retrotranscribed to cDNA and quantified by RT-PCR. This new method provides a rapid and reproducible way to accurately measure the antimalarial activity of new compounds in vitro against Plasmodium falciparum.
Asunto(s)
Antimaláricos/farmacología , Evaluación Preclínica de Medicamentos/métodos , Plasmodium falciparum/efectos de los fármacos , ARN Mensajero/análisis , Antimaláricos/clasificación , Gametogénesis/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Genes Protozoarios/genética , Plasmodium falciparum/genética , ARN Mensajero/metabolismoRESUMEN
The resistance of malaria parasites to current anti-malarial drugs is an issue of major concern globally. Recently we identified a Plasmodium falciparum cell membrane aspartyl protease, which binds to erythrocyte band 3, and is involved in merozoite invasion. Here we report the complete primary structure of P. falciparum signal peptide peptidase (PfSPP), and demonstrate that it is essential for parasite invasion and growth in human erythrocytes. Gene silencing suggests that PfSPP may be essential for parasite survival in human erythrocytes. Remarkably, mammalian signal peptide peptidase inhibitors (Z-LL)(2)-ketone and L-685,458 effectively inhibited malaria parasite invasion as well as growth in human erythrocytes. In contrast, DAPT, an inhibitor of a related gamma-secretase/presenilin-1, was ineffective. Thus, SPP inhibitors specific for PfSPP may function as potent anti-malarial drugs against the blood stage malaria.
Asunto(s)
Antimaláricos/farmacología , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Diseño de Fármacos , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/enzimología , Inhibidores de Proteasas/farmacología , Secuencia de Aminoácidos , Animales , Antimaláricos/química , Antimaláricos/uso terapéutico , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/metabolismo , Carbamatos/química , Carbamatos/farmacología , Carbamatos/uso terapéutico , Dipéptidos/química , Dipéptidos/farmacología , Dipéptidos/uso terapéutico , Eritrocitos/parasitología , Humanos , Malaria Falciparum/sangre , Datos de Secuencia Molecular , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Inhibidores de Proteasas/química , Inhibidores de Proteasas/uso terapéutico , Conformación Proteica , ARN Interferente Pequeño/genéticaRESUMEN
BACKGROUND: The human GIL blood group is encoded by the aquaporin 3 (AQP3), a water-glycerol channel present in human red cell (RBC) membranes. So far no molecular investigation of this gene has been performed in an African population. STUDY DESIGN AND METHODS: To analyze the genetic variability of the AQP3 gene in African and European persons, all exons including the boundaries to introns and the promoter region were sequenced. AQP3 mRNA levels in children affected by uncomplicated and by severe malarial anemia were determined by real-time polymerase chain reaction (RT-PCR). RESULTS: Two mutations in the promoter region and three synonymous mutations in the exons 1, 2, and 4 were found. The promoter polymorphisms, however, were not associated with the expression level of AQP3 measured in whole blood. Significant differences in AQP3 expression were found between malaria diseases severity groups (p = 0.017). CONCLUSION: AQP3 is a highly conserved gene. Although reticulocytes are highly abundant in malarial anemia, low AQP3 expression was observed. This may be a result of an impaired erythropoiesis.
Asunto(s)
Acuaporina 3/genética , Población Negra/genética , Antígenos de Grupos Sanguíneos/genética , Población Blanca/genética , Secuencia de Aminoácidos , Acuaporina 3/química , Acuaporina 3/inmunología , Secuencia de Bases , Antígenos de Grupos Sanguíneos/química , Antígenos de Grupos Sanguíneos/inmunología , ADN Complementario/genética , Regulación de la Expresión Génica , Salud , Humanos , Malaria/genética , Datos de Secuencia Molecular , Mutación/genéticaRESUMEN
In Plasmodium falciparum small solutes like water, ammonium, glycerol and others are transported by a parasite-encoded channel into the parasite. The gene encoding this channel is termed P. falciparum aquaglyceroporin (PfAQP) and is a single-copy gene and highly homologous to other aquaporins from other protozoa. Aquaporins are considered to be attractive targets for drug treatment and more so since the human and parasite aquaporins show considerable sequence differences. To investigate whether PfAQP may be suitable as a conserved target for potential aquaporin blocking agents we determined the DNA sequences of PfAQP from 65 parasite strains, either from in vitro cultured laboratory strains or from parasites obtained in an malaria-endemic region of Gabon. Only two non-synonymous mutations were found and functionally tested by a methylamine efflux assay. The efflux activity of all variants tested was similar. The lack of functionally variability suggests an invariable protein core, which may restrict parasite populations from evading therapeutic pressure if PfAQP inhibitors will be found.
Asunto(s)
Variación Genética , Plasmodium falciparum/genética , Porinas/genética , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , ADN Protozoario/química , ADN Protozoario/genética , Gabón , Humanos , Malaria Falciparum/parasitología , Metilaminas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Missense , Plasmodium falciparum/aislamiento & purificación , Análisis de Secuencia de ADNRESUMEN
A series of 1-aminopropan-2-ols were synthesized and evaluated against two strains of malaria, Plasmodium falciparum FCR3 (chloroquine-resistant) and 3D7 (chloroquine-sensitive). Microwave-assisted ring opening of epoxides (aryl and alkyl glycidyl ethers, glycidol, epichlorohydrin) with various amines without catalysts generated the desired library of beta-amino alcohols rapidly and efficiently. Most of the compounds showed micromolar potency against malaria, with seven of them having IC50 values between 1 and 10 microM against both Plasmodium falciparum strains.
Asunto(s)
Antimaláricos/síntesis química , Compuestos Epoxi/efectos de la radiación , Microondas , Plasmodium falciparum/efectos de los fármacos , Propanolaminas/síntesis química , Animales , Antimaláricos/química , Antimaláricos/farmacología , Línea Celular Tumoral , Células Cultivadas , Cloroquina/farmacología , Resistencia a Medicamentos , Compuestos Epoxi/química , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Humanos , Concentración 50 Inhibidora , Propanolaminas/química , Propanolaminas/farmacología , Relación Estructura-ActividadRESUMEN
Multigene families are a common feature in Plasmodia spp. and constitute a substantial content of the parasite genome. Here, we analyse the structural organisation and sequence diversity of two further members of the Trp-rich multigene family of P. falciparum. The complete DNA sequence of both genes was determined from a series of laboratory adapted and field isolates. Based on the amino acid sequences, we have termed them tryptophan-rich antigen-3 (TrpA-3) and lysine-tryptophan-rich antigen (LysTrpA). Analysis of the genes using reverse transcriptase-polymerase chain reaction (RT-PCR), showed that both genes are transcribed and that introns are spliced out at predicted positions. Gene expression profiles obtained from microarray analysis indicate that both genes are expressed in the mid-stages of the asexual cycle. In-frame stop codons were detected which interrupted the reading frame of LysTrpA. Whereas the number of the Trp-rich proteins is rather low in P. falciparum, P. chabaudi, P. berghei and P. yoelii, this family seems to have 15 or more members in P. knowlesi and P. vivax.