RESUMEN
Malaria is increasingly diagnosed in urban centers across the Amazon Basin. In this study, we combined repeated prevalence surveys over a 4-year period of a household-based random sample of 2,774 persons with parasite genotyping to investigate the epidemiology of malaria in Mâncio Lima, the main urban transmission hotspot in Amazonian Brazil. We found that most malarial infections were asymptomatic and undetected by point-of-care microscopy. Our findings indicate that as malaria transmission decreases, the detection threshold of microscopy rises, resulting in more missed infections despite similar parasite densities estimated by molecular methods. We identified genetically highly diverse populations of Plasmodium vivax and P. falciparum in the region; occasional shared lineages between urban and rural residents suggest cross-boundary propagation. The prevalence of low-density and asymptomatic infections poses a significant challenge for routine surveillance and the effectiveness of malaria control and elimination strategies in urbanized areas with readily accessible laboratory facilities.
Asunto(s)
Microscopía , Brasil/epidemiología , Humanos , Prevalencia , Microscopía/métodos , Femenino , Masculino , Adulto , Adolescente , Malaria Vivax/epidemiología , Malaria Vivax/parasitología , Niño , Malaria Falciparum/epidemiología , Malaria Falciparum/parasitología , Malaria Falciparum/prevención & control , Malaria/epidemiología , Malaria/transmisión , Malaria/prevención & control , Malaria/parasitología , Plasmodium vivax/genética , Población Urbana , Preescolar , Plasmodium falciparum/genética , Persona de Mediana Edad , Adulto Joven , Lactante , Historia del Siglo XXIRESUMEN
BACKGROUND: Plasmodium vivax is the main causative agent of malaria in Panama. However, the prevalence of asymptomatic infections in the different endemic regions remains unknown. Understanding the epidemiological behavior of asymptomatic infections is essential for the elimination of malaria. This study aimed to determine the prevalence of asymptomatic malarial infections in one of the main endemic regions of Panama using multiplex real-time reverse transcription RT-MqPCR. METHODS: A cross-sectional study was conducted in three communities in the Guna Yala Comarca. A total of 551 thick blood smears and their respective samples on filter paper were collected from volunteers of different ages and sexes from June 20 to 25, 2016. Infections by the Plasmodium spp. were diagnosed using microscopy and RT-MqPCR. All statistical analyses were performed using the R software. RESULTS: The average prevalence of asymptomatic infections by P. vivax in the three communities detected by RT-MqPCR was 9.3%, with Ukupa having the highest prevalence (13.4%), followed by Aidirgandi (11.1%) and Irgandi (3.3%). A total of 74 samples were diagnosed as asymptomatic infections using RT-MqPCR. Light microscopy (LM) detected that 17.6% (13/74) of the asymptomatic samples and 82.4% (61/74) were diagnosed as false negatives. A 100% correlation was observed between samples diagnosed using LM and RT-MqPCR. A total of 52.7% (39/74) of the asymptomatic patients were female and 85.1% (63/74) were registered between the ages of 1 and 21 years. Factors associated with asymptomatic infection were community (aOR = 0.38 (95% CI 0.17-0.83), p < 0.001) and age aOR = 0.98 (95% CI 0.97-1.00), p < 0.05); F = 5.38; p < 0.05). CONCLUSIONS: This study provides novel evidence of the considerable prevalence of asymptomatic P. vivax infections in the endemic region of Kuna Yala, representing a new challenge that requires immediate attention from the National Malaria Program. The results of this study provide essential information for the health authorities responsible for developing new policies. Furthermore, it will allow program administrators to reorient and design effective malaria control strategies that consider asymptomatic infections as a fundamental part of malaria control and move towards fulfilling their commitment to eliminate it.
Asunto(s)
Malaria Vivax , Plasmodium vivax , Humanos , Panamá/epidemiología , Femenino , Masculino , Adulto , Estudios Transversales , Adolescente , Malaria Vivax/epidemiología , Malaria Vivax/diagnóstico , Malaria Vivax/parasitología , Plasmodium vivax/genética , Plasmodium vivax/aislamiento & purificación , Adulto Joven , Niño , Persona de Mediana Edad , Prevalencia , Infecciones Asintomáticas/epidemiología , Preescolar , Pueblos Indígenas/genética , Lactante , Reacción en Cadena en Tiempo Real de la Polimerasa/métodosRESUMEN
In the Americas, P. vivax is the predominant causative species of malaria, a debilitating and economically significant disease. Due to the complexity of the malaria parasite life cycle, a vaccine formulation with multiple antigens expressed in various parasite stages may represent an effective approach. Based on this, we previously designed and constructed a chimeric recombinant protein, PvRMC-1, composed by PvCyRPA, PvCelTOS, and Pvs25 epitopes. This chimeric protein was strongly recognized by naturally acquired antibodies from exposed population in the Brazilian Amazon. However, there was no investigation about the induced immune response of PvRMC-1. Therefore, in this work, we evaluated the immunogenicity of this chimeric antigen formulated in three distinct adjuvants: Stimune, AddaVax or Aluminum hydroxide (Al(OH)3) in BALB/c mice. Our results suggested that the chimeric protein PvRMC-1 were capable to generate humoral and cellular responses across all three formulations. Antibodies recognized full-length PvRMC-1 and linear B-cell epitopes from PvCyRPA, PvCelTOS, and Pvs25 individually. Moreover, mice's splenocytes were activated, producing IFN-γ in response to PvCelTOS and PvCyRPA peptide epitopes, affirming T-cell epitopes in the antigen. While aluminum hydroxide showed notable cellular response, Stimune and Addavax induced a more comprehensive immune response, encompassing both cellular and humoral components. Thus, our findings indicate that PvRMC-1 would be a promising multistage vaccine candidate that could advance to further preclinical studies.
Asunto(s)
Anticuerpos Antiprotozoarios , Antígenos de Protozoos , Vacunas contra la Malaria , Malaria Vivax , Ratones Endogámicos BALB C , Plasmodium vivax , Proteínas Protozoarias , Animales , Plasmodium vivax/inmunología , Plasmodium vivax/genética , Ratones , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/genética , Malaria Vivax/inmunología , Malaria Vivax/prevención & control , Anticuerpos Antiprotozoarios/inmunología , Vacunas contra la Malaria/inmunología , Femenino , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/genética , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Modelos Animales de Enfermedad , Adyuvantes Inmunológicos , Inmunogenicidad Vacunal , Antígenos de SuperficieRESUMEN
BACKGROUND: Plasmodium vivax is the most predominant malaria species in Latin America, constituting 71.5% of malaria cases in 2021. With several countries aiming for malaria elimination, it is crucial to prioritize effectiveness of national control programs by optimizing the utilization of available resources and strategically implementing necessary changes. To support this, there is a need for innovative approaches such as genomic surveillance tools that can investigate changes in transmission intensity, imported cases and sources of reintroduction, and can detect molecular markers associated with drug resistance. METHODOLOGY/PRINCIPAL FINDINGS: Here, we apply a modified highly-multiplexed deep sequencing assay: Pv AmpliSeq v2 Peru. The tool targets a newly developed 41-SNP Peru barcode for parasite population analysis within Peru, the 33-SNP vivaxGEN-geo panel for country-level classification, and 11 putative drug resistance genes. It was applied to 230 samples from the Peruvian Amazon (2007-2020), generating baseline surveillance data. We observed a heterogenous P. vivax population with high diversity and gene flow in peri-urban areas of Maynas province (Loreto region) with a temporal drift using all SNPs detected by the assay (nSNP = 2909). In comparison, in an indigenous isolated area, the parasite population was genetically differentiated (FST = 0.07-0.09) with moderate diversity and high relatedness between isolates in the community. In a remote border community, a clonal P. vivax cluster was identified, with distinct haplotypes in drug resistant genes and ama1, more similar to Brazilian isolates, likely representing an introduction of P. vivax from Brazil at that time. To test its applicability for Latin America, we evaluated the SNP Peru barcode in P. vivax genomes from the region and demonstrated the capacity to capture local population clustering at within-country level. CONCLUSIONS/SIGNIFICANCE: Together this data shows that P. vivax transmission is heterogeneous in different settings within the Peruvian Amazon. Genetic analysis is a key component for regional malaria control, offering valuable insights that should be incorporated into routine surveillance.
Asunto(s)
Malaria Vivax , Plasmodium vivax , Polimorfismo de Nucleótido Simple , Plasmodium vivax/genética , Plasmodium vivax/aislamiento & purificación , Plasmodium vivax/clasificación , Perú/epidemiología , Malaria Vivax/epidemiología , Malaria Vivax/parasitología , Humanos , Resistencia a Medicamentos/genética , Genoma de Protozoos , Secuenciación de Nucleótidos de Alto Rendimiento , Monitoreo Epidemiológico , GenómicaRESUMEN
Hard-to-reach communities represent Peru's main challenge for malaria elimination, but information about transmission in these areas is scarce. Here, we assessed Plasmodium vivax (Pv) and P. falciparum (Pf) transmission dynamics, resistance markers, and Pf hrp2/3 deletions in Nueva Jerusalén (NJ), a remote, indigenous community in the Peruvian Amazon with high population mobility. We collected samples from November 2019 to May 2020 by active (ACD) and passive case detection (PCD) in NJ. Parasites were identified with microscopy and PCR. Then, we analyzed a representative set of positive-PCR samples (Pv = 68, Pf = 58) using highly-multiplexed deep sequencing assays (AmpliSeq) and compared NJ parasites with ones from other remote Peruvian areas using population genetics indexes. The ACD intervention did not reduce malaria cases in the short term, and persistent malaria transmission was observed (at least one Pv infection was detected in 96% of the study days). In Nueva Jerusalen, the Pv population had modest genetic diversity (He = 0.27). Pf population had lower diversity (He = 0.08) and presented temporal clustering, one of these clusters linked to an outbreak in February 2020. Moreover, Pv and Pf parasites from NJ exhibited variable levels of differentiation (Pv Fst = 0.07-0.52 and Pf Fst = 0.11-0.58) with parasites from other remote areas. No artemisin resistance mutations but chloroquine (57%) and sulfadoxine-pyrimethamine (35-67%) were detected in NJ's Pf parasites. Moreover, pfhrp2/3 gene deletions were common (32-50% of parasites with one or both genes deleted). The persistent Pv transmission and the detection of a Pf outbreak with parasites genetically distinct from the local ones highlight the need for tailored interventions focusing on mobility patterns and imported infections in remote areas to eliminate malaria in the Peruvian Amazon.
Asunto(s)
Malaria Falciparum , Malaria Vivax , Plasmodium falciparum , Plasmodium vivax , Proteínas Protozoarias , Perú/epidemiología , Humanos , Plasmodium falciparum/genética , Plasmodium falciparum/aislamiento & purificación , Plasmodium vivax/genética , Plasmodium vivax/aislamiento & purificación , Malaria Falciparum/epidemiología , Malaria Falciparum/parasitología , Malaria Falciparum/transmisión , Malaria Vivax/epidemiología , Malaria Vivax/parasitología , Malaria Vivax/transmisión , Proteínas Protozoarias/genética , Femenino , Masculino , Niño , Adulto , Antimaláricos/uso terapéutico , Antimaláricos/farmacología , Adolescente , Resistencia a Medicamentos/genética , Persona de Mediana Edad , Pueblos Indígenas/genética , Adulto Joven , Preescolar , Genómica/métodos , Variación Genética , Antígenos de Protozoos/genéticaRESUMEN
In malaria parasites, the erythrocyte binding-like proteins (EBL) are a family of invasion proteins that are attractive vaccine targets. In the case of Plasmodium vivax, the widespread malaria parasite, blood-stage vaccines have been largely focused on a single EBL candidate, the Duffy binding-like domain (DBL) of the Duffy binding protein (DBPII), due to its well-characterized role in the reticulocyte invasion. A novel P. vivax EBL family member, the Erythrocyte binding protein (EBP2, also named EBP or DBP2), binds preferentially to reticulocytes and may mediate an alternative P. vivax invasion pathway. To gain insight into the natural genetic diversity of the DBL domain of EBP2 (region II; EBP2-II), we analyzed ebp2-II gene sequences of 71 P. vivax isolates collected in different endemic settings of the Brazilian Amazon rainforest, where P. vivax is the predominant malaria-associated species. Although most of the substitutions in the ebp2-II gene were non-synonymous and suggested positive selection, the results showed that the DBL domain of the EBP2 was much less polymorphic than that of DBPII. The predominant EBP2 haplotype in the Amazon region corresponded to the C127 reference sequence first described in Cambodia (25% C127-like haplotype). An overview of ebp2-II gene sequences available at GenBank (n = 352) from seven countries (Cambodia, Madagascar, Myanmar, PNG, South Korea, Thailand, Vietnam) confirmed the C127-like haplotype as highly prevalent worldwide. Two out of 43 haplotypes (5 to 20 inferred per country) showed a global frequency of 60%. The results presented here open new avenues of research pursuit while suggesting that a vaccine based on the DBL domain of EBP2 should target a few haplotypes for broad coverage.
Asunto(s)
Variación Genética , Malaria Vivax , Plasmodium vivax , Proteínas Protozoarias , Plasmodium vivax/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/química , Malaria Vivax/parasitología , Humanos , Bosque Lluvioso , Filogenia , Haplotipos , Antígenos de Protozoos/genética , Dominios Proteicos , Receptores de Superficie CelularRESUMEN
OBJECTIVES: Primaquine is essential for the radical cure of Plasmodium vivax malaria and must be metabolized into its bioactive metabolites. Accordingly, polymorphisms in primaquine-metabolizing enzymes can impact the treatment efficacy. This pioneering study explores the influence of monoamine oxidase-A (MAO-A) on primaquine metabolism and its impact on malaria relapses. METHODS: Samples from 205 patients with P. vivax malaria were retrospectively analysed by genotyping polymorphisms in MAO-A and cytochrome P450 2D6 (CYP2D6) genes. We measured the primaquine and carboxyprimaquine blood levels in 100 subjects for whom blood samples were available on the third day of treatment. We also examined the relationship between the enzyme variants and P. vivax malaria relapses in a group of subjects with well-documented relapses. RESULTS: The median carboxyprimaquine level was significantly reduced in individuals carrying low-expression MAO-A alleles plus impaired CYP2D6. In addition, this group experienced significantly more P. vivax relapses. The low-expression MAO-A status was not associated with malaria relapses when CYP2D6 had normal activity. This suggests that the putative carboxyprimaquine contribution is irrelevant when the CYP2D6 pathway is fully active. CONCLUSIONS: We found evidence that the low-expression MAO-A variants can potentiate the negative impact of impaired CYP2D6 activity, resulting in lower levels of carboxyprimaquine metabolite and multiple relapses. The findings support the hypothesis that carboxyprimaquine may be further metabolized through CYP-mediated pathways generating bioactive metabolites that act against the parasite.
Asunto(s)
Antimaláricos , Citocromo P-450 CYP2D6 , Malaria Vivax , Monoaminooxidasa , Primaquina , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Antimaláricos/uso terapéutico , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Genotipo , Malaria Vivax/tratamiento farmacológico , Malaria Vivax/genética , Monoaminooxidasa/genética , Plasmodium vivax/genética , Polimorfismo Genético , Primaquina/uso terapéutico , Recurrencia , Estudios RetrospectivosRESUMEN
BACKGROUND: Metacaspases comprise a family of cysteine proteases implicated in both cell death and cell differentiation of protists that has been considered a potential drug target for protozoan parasites. However, the biology of metacaspases in Plasmodium vivax - the second most prevalent and most widespread human malaria parasite worldwide, whose occurrence of chemoresistance has been reported in many endemic countries, remains largely unexplored. Therefore, the present study aimed to address, for the first time, the expression pattern of metacaspases in P. vivax parasites. METHODS AND RESULTS: P. vivax blood-stage parasites were obtained from malaria patients in the Brazilian Amazon and the expression of the three putative P. vivax metacaspases (PvMCA1-3) was detected in all isolates by quantitative PCR assay. Of note, the expression levels of each PvMCA varied noticeably across isolates, which presented different frequencies of parasite forms, supporting that PvMCAs may be expressed in a stage-specific manner as previously shown in P. falciparum. CONCLUSION: The detection of metacaspases in P. vivax blood-stage parasites reported herein, allows the inclusion of these proteases as a potential candidate drug target for vivax malaria, while further investigations are still required to evaluate the activity, role and essentiality of metacaspases in P. vivax biology.
Asunto(s)
Malaria Vivax , Plasmodium vivax , Proteínas Protozoarias , Plasmodium vivax/genética , Plasmodium vivax/aislamiento & purificación , Brasil , Humanos , Malaria Vivax/parasitología , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Caspasas/genética , Caspasas/metabolismo , Expresión Génica/genéticaRESUMEN
Malaria remains a public health challenge. Since many control strategies have proven ineffective in eradicating this disease, new strategies are required, among which the design of a multivalent vaccine stands out. However, the effectiveness of this strategy has been hindered, among other reasons, by the genetic diversity observed in parasite antigens. In Plasmodium vivax, the Erythrocyte Binding Protein (PvEBP, also known as DBP2) is an alternate ligand to Duffy Binding Protein (DBP); given its structural resemblance to DBP, EBP/DBP2 is proposed as a promising antigen for inclusion in vaccine design. However, the extent of genetic diversity within the locus encoding this protein has not been comprehensively assessed. Thus, this study aimed to characterize the genetic diversity of the locus encoding the P. vivax EBP/DBP2 protein and to determine the evolutionary mechanisms modulating this diversity. Several intrapopulation genetic variation parameters were estimated using 36 gene sequences of PvEBP/DBP2 from Colombian P. vivax clinical isolates and 186 sequences available in databases. The study then evaluated the worldwide genetic structure and the evolutionary forces that may influence the observed patterns of genetic variation. It was found that the PvEBP/DBP2 gene exhibits one of the lowest levels of genetic diversity compared to other vaccine-candidate antigens. Four major haplotypes were shared worldwide. Analysis of the protein's 3D structure and epitope prediction identified five regions with potential antigenic properties. The results suggest that the PvEBP/DBP2 protein possesses ideal characteristics to be considered when designing a multivalent effective antimalarial vaccine against P. vivax.
Asunto(s)
Antígenos de Protozoos , Variación Genética , Vacunas contra la Malaria , Malaria Vivax , Plasmodium vivax , Proteínas Protozoarias , Plasmodium vivax/genética , Plasmodium vivax/inmunología , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Humanos , Vacunas contra la Malaria/inmunología , Vacunas contra la Malaria/genética , Malaria Vivax/prevención & control , Malaria Vivax/parasitología , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Colombia , Filogenia , Receptores de Superficie CelularRESUMEN
Primaquine (PQ) is the main drug used to eliminate dormant liver stages and prevent relapses in Plasmodium vivax malaria. It also has an effect on the gametocytes of Plasmodium falciparum; however, it is unclear to what extent PQ affects P. vivax gametocytes. PQ metabolism involves multiple enzymes, including the highly polymorphic CYP2D6 and the cytochrome P450 reductase (CPR). Since genetic variability can impact drug metabolism, we conducted an evaluation of the effect of CYP2D6 and CPR variants on PQ gametocytocidal activity in 100 subjects with P. vivax malaria. To determine gametocyte density, we measured the levels of pvs25 transcripts in samples taken before treatment (D0) and 72 hours after treatment (D3). Generalized estimating equations (GEEs) were used to examine the effects of enzyme variants on gametocyte densities, adjusting for potential confounding factors. Linear regression models were adjusted to explore the predictors of PQ blood levels measured on D3. Individuals with the CPR mutation showed a smaller decrease in gametocyte transcript levels on D3 compared to those without the mutation (P = 0.02, by GEE). Consistent with this, higher PQ blood levels on D3 were associated with a lower reduction in pvs25 transcripts. Based on our findings, the CPR variant plays a role in the persistence of gametocyte density in P. vivax malaria. Conceptually, our work points to pharmacogenetics as a non-negligible factor to define potential host reservoirs with the propensity to contribute to transmission in the first days of CQ-PQ treatment, particularly in settings and seasons of high Anopheles human-biting rates.
Asunto(s)
Antimaláricos , Artemisininas , Malaria Falciparum , Malaria Vivax , Malaria , Humanos , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Malaria Vivax/tratamiento farmacológico , Malaria Falciparum/tratamiento farmacológico , NADPH-Ferrihemoproteína Reductasa , Cloroquina/farmacología , Citocromo P-450 CYP2D6/genética , Artemisininas/farmacología , Primaquina/farmacología , Primaquina/uso terapéutico , Malaria/tratamiento farmacológico , Plasmodium falciparum , Plasmodium vivax/genéticaRESUMEN
Malaria is the deadliest parasitic disease in the world. Traditional control measures have become less effective; hence, there is a need to explore alternative strategies, such as antimalarial vaccines. However, designing an anti-Plasmodium vivax vaccine is considered a challenge due to the complex parasite biology and the antigens' high genetic diversity. Recently, the sporozoite invasion-associated protein 2 (SIAP2) has been suggested as a potential antigen to be considered in vaccine design due to its significance during hepatocyte invasion. However, its use may be limited by the incomplete understanding of gene/protein diversity. Here, the genetic diversity of pvsiap2 using P. vivax DNA samples from Colombia was assessed. Through PCR amplification and sequencing, we compared the Colombian sequences with available worldwide sequences, revealing that pvsiap2 displays low genetic diversity. Molecular evolutionary analyses showed that pvsiap2 appears to be influenced by directional selection. Moreover, the haplotypes found differ by a few mutational steps and several of them were shared between different geographical areas. On the other hand, several conserved regions within PvSIAP2 were predicted as potential B-cell or T-cell epitopes. Considering these characteristics and its role in hepatocyte invasion, the PvSIAP2 protein emerges as a promising antigen to be considered in a multi-antigen-multi-stage (multivalent) fully effective vaccine against P. vivax malaria.
Asunto(s)
Vacunas contra la Malaria , Malaria Vivax , Humanos , Plasmodium vivax/genética , Vacunas contra la Malaria/genética , Antígenos de Protozoos/genética , Proteínas Protozoarias/genética , Variación Genética , Malaria Vivax/prevención & control , Malaria Vivax/parasitología , Selección GenéticaRESUMEN
Colombia aims to eliminate malaria by 2030 but remains one of the highest burden countries in the Americas. Plasmodium vivax contributes half of all malaria cases, with its control challenged by relapsing parasitaemia, drug resistance and cross-border spread. Using 64 Colombian P. vivax genomes collected between 2013 and 2017, we explored diversity and selection in two major foci of transmission: Chocó and Córdoba. Open-access data from other countries were used for comparative assessment of drug resistance candidates and to assess cross-border spread. Across Colombia, polyclonal infections were infrequent (12%), and infection connectivity was relatively high (median IBD = 5%), consistent with low endemicity. Chocó exhibited a higher frequency of polyclonal infections (23%) than Córdoba (7%), although the difference was not significant (P = 0.300). Most Colombian infections carried double pvdhfr (95%) and single pvdhps (71%) mutants, but other drug resistance mutations were less prevalent (< 10%). There was no evidence of selection at the pvaat1 gene, whose P. falciparum orthologue has recently been implicated in chloroquine resistance. Global population comparisons identified other putative adaptations. Within the Americas, low-level connectivity was observed between Colombia and Peru, highlighting potential for cross-border spread. Our findings demonstrate the potential of molecular data to inform on infection spread and adaptation.
Asunto(s)
Antimaláricos , Malaria Falciparum , Malaria Vivax , Humanos , Plasmodium vivax/genética , Antimaláricos/farmacología , Colombia/epidemiología , Malaria Vivax/epidemiología , Malaria Vivax/tratamiento farmacológico , Proteínas Protozoarias/genética , Resistencia a Medicamentos/genética , GenómicaRESUMEN
The road to malaria elimination for low- and middle-income countries is paved with obstacles, including the complexity and high costs of advanced molecular methods for genomic analysis. The usefulness of PCR-RFLP as less complex and affordable molecular surveillance tool in low-endemic malaria regions was assessed in a cross-sectional study conducted in Suriname, currently striving for malaria elimination, but plagued by recent P. vivax outbreaks. Molecular analysis of two highly polymorphic genes Pvmsp-1 F2 and Pvmsp-3α was performed for 49 samples, collected during October 2019 through September 2021 from four different regions with varying malaria transmission risks. RFLP-profiling revealed that outbreak samples from three indigenous villages, almost exclusively, harbored a single clonal type, matching the "Palumeu" lineage previously described in 2019, despite multiple relapses and drug pressure exerted by mass drug administration events, suggesting a limited P. vivax hypnozoite reservoir in Suriname. In contrast, isolates originating from Sophie, a mining area in neighboring French Guiana displayed a highly heterogeneous parasite population consistent with its endemic malaria status, demonstrating the differentiating capacity and thus the usefulness of PCR-RFLP for P. vivax genetic diversity studies. Outbreak reconstruction emphasized the impact of undetected human movement and relapses on reintroduction and resurgence of P. vivax malaria and PCR-RFLP monitoring of circulating parasites guided the roll-out of targeted interventions. PCR-RFLP seems a suitable molecular alternative in low-endemic areas with restricted resources for outbreak analysis, for monitoring the spread or containment of circulating strains and for identification of imported cases or potential foci.
Asunto(s)
Malaria Vivax , Proteínas Protozoarias , Humanos , Polimorfismo de Longitud del Fragmento de Restricción , Proteínas Protozoarias/genética , Antígenos de Protozoos/genética , Plasmodium vivax/genética , Estudios Transversales , Variación Genética , Malaria Vivax/epidemiología , Malaria Vivax/parasitología , Reacción en Cadena de la Polimerasa/métodos , RecurrenciaRESUMEN
BACKGROUND: Plasmodium vivax is the main species responsible for human malaria in Brazil, and one of its manifestations is splenic malaria, though there are still challenges in its diagnosis. The present study aimed to standardize Plasmodium sp. DNA extraction from histological slices of spleen and diagnosis using real-time qPCR. METHODS: This study performed a microtomy of a paraffin-embedded spleen as a positive control for P. vivax from a patient who had been previously diagnosed with the parasite. The sample was deparaffinized with xylol and ethanol, then DNA extraction was performed with two commercial kits. qPCR was carried out with the Taqman system for detection of Plasmodium sp. and was made species-specific using PvmtCOX1 gene. From 2015 to 2019, 200 spleen samples were obtained from trauma patients subjected to splenectomy in Manaus, Amazonas. All the samples were tested for cell-free human DNA (cfDNA). RESULTS: The deparaffinization and the Plasmodium vivax DNA extraction method was successfully standardized, and the control sample was positive for P. vivax. Of the 200 samples, all qPCRs were negative, but they were positive for human PCR. CONCLUSION: Paraffinization is practical and efficient for the preservation of samples, but the formation of bonds between proteins and DNA makes extraction difficult. Despite this, in this study, it was possible to standardize a method of DNA extraction for detecting P. vivax.
Asunto(s)
Malaria Vivax , Malaria , Humanos , Bazo , Malaria/diagnóstico , Malaria Vivax/diagnóstico , Malaria Vivax/parasitología , Plasmodium vivax/genética , ADN , Estándares de Referencia , Plasmodium falciparum/genéticaRESUMEN
BACKGROUND: Plasmodium vivax is the second most important cause of human malaria worldwide, and accounts for the majority of malaria cases in South America. A high-quality reference genome exists for Papua Indonesia (PvP01) and Thailand (PvW1), but is lacking for South America. A reference genome specifically for South America would be beneficial though, as P. vivax is a genetically diverse parasite with geographical clustering. RESULTS: This study presents a new high-quality assembly of a South American P. vivax isolate, referred to as PvPAM (P. vivax Peruvian AMazon). The genome was obtained from a low input patient sample from the Peruvian Amazon and sequenced using PacBio technology, resulting in a highly complete assembly with 6497 functional genes. Telomeric ends were present in 17 out of 28 chromosomal ends, and additional (sub)telomeric regions are present in 12 unassigned contigs. A comparison of multigene families between PvPAM and the PvP01 genome revealed remarkable variation in vir genes, and the presence of merozoite surface proteins (MSP) 3.6 and 3.7. Three dhfr and dhps drug resistance associated mutations are present in PvPAM, similar to those found in other Peruvian isolates. Mapping of publicly available South American whole genome sequencing (WGS) data to PvPAM resulted in significantly fewer variants and truncated reads compared to the use of PvP01 or PvW1 as reference genomes. To minimize the number of core genome variants in non-South American samples, PvW1 is most suited for Southeast Asian isolates, both PvPAM and PvW1 are suited for South Asian isolates, and PvPAM is recommended for African isolates. Interestingly, non-South American samples still contained the least subtelomeric variants when mapped to PvPAM, indicating high quality of the PvPAM subtelomeric regions. CONCLUSIONS: Our findings show that the PvPAM reference genome more accurately represents South American P. vivax isolates in comparison to PvP01 and PvW1. In addition, PvPAM has a high level of completeness, and contains a similar number of annotated genes as PvP01 or PvW1. The PvPAM genome therefore will be a valuable resource to improve future genomic analyses on P. vivax isolates from the South American continent.
Asunto(s)
Malaria Vivax , Malaria , Humanos , Plasmodium vivax/genética , Malaria/parasitología , América del Sur , Secuenciación Completa del Genoma , Mutación , Malaria Vivax/parasitología , Proteínas Protozoarias/genéticaRESUMEN
BACKGROUND: In malaria infection, apoptosis acts as an important immunomodulatory mechanism that leads to the elimination of parasitized cells, thus reducing the parasite density and controlling immune cell populations. Here, it was investigated the association of INDEL variants in apoptotic genes-rs10562972 (FAS), rs4197 (FADD), rs3834129 and rs59308963 (CASP8), rs61079693 (CASP9), rs4647655 (CASP3), rs11269260 (BCL-2), and rs17880560 (TP53)-and the influence of genetic ancestry with susceptibility to malaria and parasite density in an admixed population from the Brazilian Amazon. METHODS: Total DNA was extracted from 126 malaria patients and 101 uninfected individuals for investigation of genetic ancestries and genotypic distribution of apoptosis-related variants by Multiplex PCR. Association analyses consisted of multivariate logistic regressions, considering the following comparisons: (i) DEL/DEL genotype vs. INS/DEL + INS/INS; and (ii) INS/INS vs. INS/DEL + DEL/DEL. RESULTS: Individuals infected by Plasmodium falciparum had significantly higher African ancestry proportions in comparison to uninfected controls, Plasmodium vivax, and mixed infections. The INS/INS genotype of rs3834129 (CASP8) seemed to increase the risk for P. falciparum infection (P = 0.038; OR = 1.867; 95% CI 0.736-3.725), while the DEL/DEL genotype presented a significant protective effect against infection by P. falciparum (P = 0.049; OR = 0.446; 95% CI 0.185-0.944) and mixed infection (P = 0.026; OR = 0.545; 95% CI 0.281-0.996), and was associated with lower parasite density in P. falciparum malaria (P = 0.009; OR = 0.383; 95% CI 0.113-1.295). Additionally, the INS/INS genotype of rs10562972 (FAS) was more frequent among individuals infected with P. vivax compared to P. falciparum (P = 0.036; OR = 2.493; 95% CI 1.104-4.551), and the DEL/DEL genotype of rs17880560 (TP53) was significantly more present in patients with mono-infection by P. vivax than in individuals with mixed infection (P = 0.029; OR = 0.667; 95% CI 0.211-1.669). CONCLUSIONS: In conclusion, variants in apoptosis genes are associated with malaria susceptibility and parasite density, indicating the role of apoptosis-related genetic profiles in immune responses against malaria infection.
Asunto(s)
Coinfección , Malaria Falciparum , Malaria Vivax , Parásitos , Humanos , Animales , Predisposición Genética a la Enfermedad , Brasil , Estudios de Casos y Controles , Apoptosis/genética , Malaria Vivax/genética , Malaria Falciparum/genética , Plasmodium vivax/genética , Plasmodium falciparum/genéticaRESUMEN
Malaria is the leading human parasitosis and is transmitted through the bite of anopheline mosquitoes infected with parasites of the genus Plasmodium spp. Among the seven species that cause malaria in humans, Plasmodium vivax is the most prevalent species in Latin America. In recent years, there have been an increasing number of reports of clinical complications caused by P. vivax infections, which were previously neglected and underestimated. P. vivax biology remains with large gaps. The emergence of next-generation sequencing technology has ensured a breakthrough in species knowledge. Coupled with this, the deposition of the P. vivax Sal-1 reference genome allowed an increase in transcriptomics projects by accessing messenger RNA. Thus, the regulation of differential gene expression according to the parasite life stage was verified, and several expressed genes were linked to different biological functions. Today, with the progress associated with RNA sequencing technologies, it is possible to detect nuances and obtain robust results. Discoveries provided by transcriptomic studies allow us to understand topics such as RNA expression and regulation and proteins and metabolic pathways involved during different stages of the parasite life cycle. The information obtained enables a better comprehension of immune system evasion mechanisms; invasion and adhesion strategies used by the parasite; as well as new vaccine targets, potential molecular markers, and others therapeutic targets. In this review, we provide new insights into P. vivax biology by summarizing recent findings in transcriptomic studies.
Asunto(s)
Malaria Vivax , Malaria , Parásitos , Animales , Humanos , Plasmodium vivax/genética , Malaria Vivax/genética , Malaria Vivax/parasitología , Perfilación de la Expresión GénicaRESUMEN
The PvCelTOS, PvCyRPA, and Pvs25 proteins play important roles during the three stages of the P. vivax lifecycle. In this study, we designed and expressed a P. vivax recombinant modular chimeric protein (PvRMC-1) composed of the main antigenic regions of these vaccine candidates. After structure modelling by prediction, the chimeric protein was expressed, and the antigenicity was assessed by IgM and IgG (total and subclass) ELISA in 301 naturally exposed individuals from the Brazilian Amazon. The recombinant protein was recognized by IgG (54%) and IgM (40%) antibodies in the studied individuals, confirming the natural immunogenicity of the epitopes that composed PvRMC-1 as its maintenance in the chimeric structure. Among responders, a predominant cytophilic response mediated by IgG1 (70%) and IgG3 (69%) was observed. IgM levels were inversely correlated with age and time of residence in endemic areas (p < 0.01). By contrast, the IgG and IgM reactivity indexes were positively correlated with each other, and both were inversely correlated with the time of the last malaria episode. Conclusions: The study demonstrates that PvRMC-1 was successfully expressed and targeted by natural antibodies, providing important insights into the construction of a multistage chimeric recombinant protein and the use of naturally acquired antibodies to validate the construction.
Asunto(s)
Malaria Vivax , Plasmodium vivax , Humanos , Plasmodium vivax/genética , Inmunidad Humoral , Proteínas Protozoarias/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes de Fusión/genética , Inmunoglobulina G , Inmunoglobulina M/genética , Antígenos de Protozoos/genéticaRESUMEN
BACKGROUND: The immunopathology during malaria depends on the level of inflammatory response generated. In this scenario, the TREM-1 has been associated with the severity of infectious diseases and could play an important role in the inflammatory course of malaria. We aimed to describe the allelic and genotypic frequency of four polymorphisms in the trem-1 gene in Plasmodium vivax-infected patients and to verify the association of these polymorphisms with clinical and immunological factors in a frontier area of the Brazilian Amazon. METHODS: We included 76 individuals infected with P. vivax and 144 healthy controls living in the municipality of Oiapoque, Amapá, Brazil. The levels of TNF-α, IL-10, IL-2, IL-4, IL-5, and IFN-γ were measured by flow cytometry, while IL-6, sTREM-1, and antibodies against PvMSP-119 were evaluated by ELISA. The SNPs were genotyped by qPCR technique. Polymorphisms analysis, allelic and genotype, frequencies, and HWE calculation were determined by x2 test in R Software. The association between the parasitemia, gametocytes, antibodies, cytokines, and sTREM-1 with the genotypes of malaria and control groups was performed using the Kruskal-Wallis test, these analyzes were conducted in SPSS Software, at 5% significance level. RESULTS: All SNPs were successfully genotyped. Allelic and genotypic distribution was in Hardy-Weinberg Equilibrium. Furthermore, several associations were identified between malaria and control groups, with increased levels of IL-5, IL-6, IL-10, TNF-α, and IFN-γ in the infected individuals with rs6910730A, rs2234237T, rs2234246T, rs4711668C alleles compared to the homozygous wild-type and heterozygous genotypes of the controls (p-value < 0.05). No association was found for these SNPs and the levels of IL-2, and sTREM-1. CONCLUSIONS: The SNPs on the trem-1 gene are associated with the effector molecules of the innate immunity and may contribute to the identification and effective participation of trem-1 in the modulation of the immune response. This association may be essential for the establishment of immunization strategies against malaria.
Asunto(s)
Malaria Vivax , Malaria , Humanos , Citocinas/genética , Plasmodium vivax/genética , Interleucina-10/genética , Brasil , Receptor Activador Expresado en Células Mieloides 1/genética , Factor de Necrosis Tumoral alfa/genética , Interleucina-6/genética , Interleucina-2/genética , Interleucina-5/genética , Malaria Vivax/genética , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
BACKGROUND: The innate immune response plays an important role during malaria. Toll-like receptors (TLR) are capable of recognizing pathogen molecules. We aimed to evaluate five polymorphisms in TLR-4, TLR-6, and TLR-9 genes and their association with cytokine levels and clinical parameters in malaria from the Brazil-French Guiana border. METHODS: A case-control study was conducted in Amapá, Brazil. P. vivax patients and individuals not infected were evaluated. Genotyping of five SNPs was carried out by qPCR. Circulating cytokines were measured by CBA. The MSP-119 IgG antibodies were performed by ELISA. RESULTS: An association between TLR4 A299G with parasitemia was observed. There was an increase for IFN-ɤ, TNF-É, IL-6, and IL-10 in the TLR-4 A299G and T3911, TLR-6 S249P, and TLR-9 1486C/T, SNPs for the studied malarial groups. There were significant findings for the TLR-4 variants A299G and T3911, TLR-9 1237C/T, and 1486C/T. For the reactivity of MSP-119 antibodies levels, no significant results were found in malaria, and control groups. CONCLUSIONS: The profile of the immune response observed by polymorphisms in TLRs genes does not seem to be standard for all types of malaria infection around the world. This can depend on the human population and the species of Plasmodium.