Effect of primaquine dose on the risk of recurrence in patients with uncomplicated Plasmodium vivax: a systematic review and individual patient data meta-analysis.

BACKGROUND: Primaquine is used to eliminate Plasmodium vivax hypnozoites, but its optimal dosing regimen remains unclear. We undertook a systematic review and individual patient data meta-analysis to investigate the efficacy and tolerability of different primaquine dosing regimens to prevent P vivax recurrence. METHODS: For this systematic review and individual patient data meta-analysis, we searched MEDLINE, Web of Science, Embase, and Cochrane Central for prospective clinical studies of uncomplicated P vivax from endemic countries published between Jan 1, 2000, and June 8, 2023. We included studies if they had active follow-up of at least 28 days, and if they included a treatment group with daily primaquine given over multiple days, where primaquine was commenced within 7 days of schizontocidal treatment and was given alone or coadministered with chloroquine or one of four artemisinin-based combination therapies (ie, artemether-lumefantrine, artesunate-mefloquine, artesunate-amodiaquine, or dihydroartemisinin-piperaquine). We excluded studies if they were on prevention, prophylaxis, or patients with severe malaria, or if data were extracted retrospectively from medical records outside of a planned trial. For the meta-analysis, we contacted the investigators of eligible trials to request individual patient data and we then pooled data that were made available by Aug 23, 2021. We assessed the effects of total dose and duration of primaquine regimens on the rate of first P vivax recurrence between day 7 and day 180 by Cox's proportional hazards regression (efficacy analysis). The effect of primaquine daily dose on gastrointestinal symptoms on days 5-7 was assessed by modified Poisson regression (tolerability analysis). The study was registered with PROSPERO, CRD42019154470. FINDINGS: Of 226 identified studies, 23 studies with patient-level data from 6879 patients from 16 countries were included in the efficacy analysis. At day 180, the risk of recurrence was 51·0% (95% CI 48·2-53·9) in 1470 patients treated without primaquine, 19·3% (16·9-21·9) in 2569 patients treated with a low total dose of primaquine (approximately 3·5 mg/kg), and 8·1% (7·0-9·4) in 2811 patients treated with a high total dose of primaquine (approximately 7 mg/kg), regardless of primaquine treatment duration. Compared with treatment without primaquine, the rate of P vivax recurrence was lower after treatment with low-dose primaquine (adjusted hazard ratio 0·21, 95% CI 0·17-0·27; p<0·0001) and high-dose primaquine (0·10, 0·08-0·12; p<0·0001). High-dose primaquine had greater efficacy than low-dose primaquine in regions with high and low relapse periodicity (ie, the time from initial infection to vivax relapse). 16 studies with patient-level data from 5609 patients from ten countries were included in the tolerability analysis. Gastrointestinal symptoms on days 5-7 were reported by 4·0% (95% CI 0·0-8·7) of 893 patients treated without primaquine, 6·2% (0·5-12·0) of 737 patients treated with a low daily dose of primaquine (approximately 0·25 mg/kg per day), 5·9% (1·8-10·1) of 1123 patients treated with an intermediate daily dose (approximately 0·5 mg/kg per day) and 10·9% (5·7-16·1) of 1178 patients treated with a high daily dose (approximately 1 mg/kg per day). 20 of 23 studies included in the efficacy analysis and 15 of 16 in the tolerability analysis had a low or unclear risk of bias. INTERPRETATION: Increasing the total dose of primaquine from 3·5 mg/kg to 7 mg/kg can reduce P vivax recurrences by more than 50% in most endemic regions, with a small associated increase in gastrointestinal symptoms. FUNDING: Australian National Health and Medical Research Council, Bill & Melinda Gates Foundation, and Medicines for Malaria Venture.

Plasmodium vivax apical membrane antigen 1I-II from Nicaragua (2012-2013): genetic and antigenic polymorphism.

OBJECTIVE: Genetic and antigenic polymorphism of P. vivax apical membrane antigen-1 (pvama1I-II) from Nicaragua was examined. MATERIALS AND METHODS: Infected blood samples from patients were obtained during 2012-2013. A gene fragment comprising domains I-II was amplified and sequenced, and the genetic parameters, haplotype relationships, genetic structure, and amino acid variation in predicted B cell epitopes were analyzed. RESULTS: 65 sequences of pvama1III had 19 nonsynonymous and five synonymous nucleotide changes. Nicaraguan parasites had low diversity, high linkage disequilibrium, and few recombination events. Neutrality tests indicate a positive and divergent selection, and three genetic clusters with loss of haplotypes were demonstrated. Amino acid variation predominated in predicted B cell epitopes and was closely related to that in Latin American parasites. CONCLUSIONS: Nicaraguan P. vivax is a moderately differentiated population under contraction and focalization processes, and the antigenic diversity resembles that of Latin American parasites. This information is relevant for vaccine development and epidemiological surveillance.

Effect of adherence to primaquine on the risk of Plasmodium vivax recurrence: a WorldWide Antimalarial Resistance Network systematic review and individual patient data meta-analysis.

BACKGROUND: Imperfect adherence is a major barrier to effective primaquine radical cure of Plasmodium vivax. This study investigated the effect of reduced adherence on the risk of P. vivax recurrence. METHODS: Efficacy studies of patients with uncomplicated P. vivax malaria, including a treatment arm with daily primaquine, published between January 1999 and March 2020 were identified. Individual patient data from eligible studies were pooled using standardized methodology. Adherence to primaquine was inferred from i) the percentage of supervised doses and ii) the total mg/kg dose received compared to the target total mg/kg dose per protocol. The effect of adherence to primaquine on the incidence of P. vivax recurrence between days 7 and 90 was investigated by Cox regression analysis. RESULTS: Of 82 eligible studies, 32 were available including 6917 patients from 18 countries. For adherence assessed by percentage of supervised primaquine, 2790 patients (40.3%) had poor adherence (≤ 50%) and 4127 (59.7%) had complete adherence. The risk of recurrence by day 90 was 14.0% [95% confidence interval: 12.1-16.1] in patients with poor adherence compared to 5.8% [5.0-6.7] following full adherence; p = 0.014. After controlling for age, sex, baseline parasitaemia, and total primaquine dose per protocol, the rate of the first recurrence was higher following poor adherence compared to patients with full adherence (adjusted hazard ratio (AHR) = 2.3 [1.8-2.9]). When adherence was quantified by total mg/kg dose received among 3706 patients, 347 (9.4%) had poor adherence, 88 (2.4%) had moderate adherence, and 3271 (88.2%) had complete adherence to treatment. The risks of recurrence by day 90 were 8.2% [4.3-15.2] in patients with poor adherence and 4.9% [4.1-5.8] in patients with full adherence; p < 0.001. CONCLUSION: Reduced adherence, including less supervision, increases the risk of vivax recurrence.

Analysis of Fifty Years of Severe Malaria Worldwide Research.

This study analyzed fifty years of severe malaria research worldwide. Malaria is a parasitic disease that continues to have a significant impact on global health, particularly in sub-Saharan Africa. Severe malaria, a severe and often fatal form of the disease, is a major public health concern. The study used different bibliometric indicators such as the number of publications, citations, authorship, and keywords to analyze the research trends, patterns, and progress made in the field of severe malaria. The study covers the period from 1974 to 2021 and includes articles from Scopus. The results of the study indicated that there has been a steady increase in the number of publications on severe malaria over the past fifty years, with a particular increase in the last decade. The study also showed that most of the publications are from USA and Europe, while the disease occurs in Africa, South-East Asia, and the Americas. The study also identified the most frequent keywords used in the publications, and the most influential journals and authors in the field. In conclusion, this bibliometric study provides a comprehensive overview of the research trends and patterns in the field of severe malaria over the past fifty years and highlights the areas that need more attention and research efforts.

Plasmodium vivax MSP1-42 kD Variant Proteins Detected Naturally Induced IgG Antibodies in Patients Regardless of the Infecting Parasite Phenotype in Mesoamerica.

BACKGROUND: The serological tests using blood stage antigens might be helpful for detecting recent exposure to Plasmodium parasites, and seroepidemiological studies would aid in the elimination of malaria. This work produced recombinant proteins of PvMSP142 variants and evaluated their capacity to detect IgG antibodies in symptomatic patients from Mesoamerica. METHODS: Three variant Pvmsp142 genes were cloned in the pHL-sec plasmid, expressed in the Expi293F™ eukaryotic system, and the recombinant proteins were purified by affinity chromatography. Using an ELISA, 174 plasma or eluted samples from patients infected with different P. vivax haplotypes were evaluated against PvMSP142 proteins and to a native blood stage antigen (NBSA). RESULTS: The antibody IgG OD values toward PvMSP142 variants (v88, v21, and v274) were heterogeneous (n = 178; median = 0.84 IQR 0.28-1.64). The correlation of IgG levels among all proteins was very high (spearman's rho = 0.96-0.98; p < 0.0001), but was lower between them and the NBSA (rho = 0.771; p < 0.0001). In only a few samples, higher reactivity to the homologous protein was evident. Patients with a past infection who were seropositive had higher IgG levels and lower parasitemia levels than those who did not (p < 0.0001). CONCLUSIONS: The PvMSP142 variants were similarly efficient in detecting specific IgG antibodies in P. vivax patients from Mesoamerica, regardless of the infecting parasite's haplotype, and might be good candidates for malaria surveillance and epidemiological studies in the region.

Resistance to Pyrethroids in the Malaria Vector Anopheles albimanus in Two Important Villages in the Soconusco Region of Chiapas, Mexico, 2022.

Chiapas State comprises the largest malaria foci from Mexico, and 57% of the autochthonous cases in 2021, all with Plasmodium vivax infections, were reported in this State. Southern Chiapas is at constant risk of cases imported due to migratory human flow. Since chemical control of vector mosquitoes is the main entomological action implemented for the prevention and control of vector-borne diseases, this work aimed to investigate the susceptibility of Anopheles albimanus to insecticides. To this end, mosquitoes were collected in cattle in two villages in southern Chiapas in July-August 2022. Two methods were used to evaluate the susceptibility: the WHO tube bioassay and the CDC bottle bioassay. For the latter, diagnostic concentrations were calculated. The enzymatic resistance mechanisms were also analyzed. CDC diagnostic concentrations were obtained; 0.7 µg/mL deltamethrin, 12 µg/mL permethrin, 14.4 µg/mL malathion, and 2 µg/mL chlorpyrifos. Mosquitoes from Cosalapa and La Victoria were susceptible to organophosphates and to bendiocarb, but resistant to pyrethroids, with mortalities between 89% and 70% (WHO), and 88% and 78% (CDC), for deltamethrin and permethrin, respectively. High esterase levels are suggested as the resistance mechanism involved in the metabolism of pyrethroids in mosquitoes from both villages. Mosquitoes from La Victoria might also involve cytochrome P450. Therefore, organophosphates and carbamates are suggested to currently control An. albimanus. Its use might reduce the frequency of resistance genes to pyrethroids and vector abundance and may impede the transmission of malaria parasites.

A genetically modified Plasmodium berghei parasite as a surrogate for whole-sporozoite vaccination against P. vivax malaria.

Two malaria parasite species, Plasmodium falciparum (Pf) and P. vivax (Pv) are responsible for most of the disease burden caused by malaria. Vaccine development against this disease has focused mainly on Pf. Whole-sporozoite (WSp) vaccination, targeting pre-erythrocytic (PE) parasite stages, is a promising strategy for immunization against malaria and several PfWSp-based vaccine candidates are currently undergoing clinical evaluation. In contrast, no WSp candidates have been developed for Pv, mainly due to constraints in the production of Pv sporozoites in the laboratory. Recently, we developed a novel approach for WSp vaccination against Pf based on the use of transgenic rodent P. berghei (Pb) sporozoites expressing immunogens of this human-infective parasite. We showed that this platform can be used to deliver PE Pf antigens, eliciting both targeted humoral responses and cross-species cellular immune responses against Pf. Here we explored this WSp platform for the delivery of Pv antigens. As the Pv circumsporozoite protein (CSP) is a leading vaccine candidate antigen, we generated a transgenic Pb parasite, PbviVac, that, in addition to its endogenous PbCSP, expresses PvCSP under the control of a strictly PE promoter. Immunofluorescence microscopy analyses confirmed that both the PbCSP and the PvCSP antigens are expressed in PbviVac sporozoites and liver stages and that PbviVac sporozoite infectivity of hepatic cells is similar to that of its wild-type Pb counterpart. Immunization of mice with PbviVac sporozoites elicits the production of anti-PvCSP antibodies that efficiently recognize and bind to Pv sporozoites. Our results warrant further development and evaluation of PbviVac as a surrogate for WSp vaccination against Pv malaria.

Recurrent Plasmodium vivax Cases of Both Short and Long Latency Increased with Transmission Intensity and Were Distributed Year-Round in the Most Affected Municipalities of the RACCN, Nicaragua, 2013-2018.

The characteristics of P. vivax recurrent episodes were examined using a centralized secondary source of malaria records in Nicaragua and in the two most affected municipalities in the RACCN. The study of 36,787 malaria cases due to P. vivax or P. falciparum revealed that, nationwide, 3624 patients had at least one recurrent infection. This was achieved by matching names, gender, age, community/municipality, ethnicity, etc. P. vivax was responsible for 88% of recurrent infections of 25-450 days of latency (51.9% were women and 48.1% were men), and these were assumed to be relapse episodes. Of them, 88.2% and 4.4% occurred in the municipalities of Puerto Cabezas and Rosita, respectively. The proportion of P. vivax patients having presumed relapse episodes rose with elevated transmission rates in both municipalities, reaching 7% in Rosita (2017) and 14.5% in Puerto Cabezas (2018). In both areas, relapse episodes were evident over time and were characterized by the production of a continuous stippling pattern with a slope evolving from one transmission peak to the next. During the dry season, short-latency relapse episodes were more robust, while long-latency ones increased just before the P. vivax transmission season began, with a high proportion of long-latency relapses during this period. The abundance of recurrent P. vivax infections, the wide range of relapse latency lengths, and temporal distribution tended to favor year-round transmission. It is necessary to evaluate compliance with and the effectiveness of primaquine treatment and contemplate the use of an alternative drug, among other actions.

Spatiotemporal Changes in Plasmodium vivax msp142 Haplotypes in Southern Mexico: From the Control to the Pre-Elimination Phase.

For 20 years, Plasmodium vivax has been the only prevalent malaria species in Mexico, and cases have declined significantly and continuously. Spatiotemporal genetic studies can be helpful for understanding parasite dynamics and developing strategies to weaken malaria transmission, thus facilitating the elimination of the parasite. The aim of the current contribution was to analyze P. vivax-infected blood samples from patients in southern Mexico during the control (1993-2007) and pre-elimination phases (2008-2011). Nucleotide and haplotype changes in the pvmsp142 fragment were evaluated over time. The majority of multiple genotype infections occurred in the 1990s, when the 198 single nucleotide sequences exhibited 57 segregating sites, 64 mutations, and 17 haplotypes. Nucleotide and genetic diversity parameters showed subtle fluctuations from across time, in contrast to the reduced haplotype diversity and the increase in the R2 index and Tajima's D value from 2008 to 2011. The haplotype network consisted of four haplogroups, the geographical distribution of which varied slightly over time. Haplogroup-specific B-cell epitopes were predicted. Since only high-frequency and divergent haplotypes persisted, there was a contraction of the parasite population. Given that 84% of haplotypes were exclusive to Mesoamerica, P. vivax flow is likely circumscribed to this region, representing important information for parasite surveillance.

Plasmodium vivax Cysteine-Rich Protective Antigen Polymorphism at Exon-1 Shows Recombination and Signatures of Balancing Selection.

Plasmodium vivax Cysteine-Rich Protective Antigen (CyRPA) is a merozoite protein participating in the parasite invasion of human reticulocytes. During natural P. vivax infection, antibody responses against PvCyRPA have been detected. In children, low anti-CyRPA antibody titers correlated with clinical protection, which suggests this protein as a potential vaccine candidate. This work analyzed the genetic and amino acid diversity of pvcyrpa in Mexican and global parasites. Consensus coding sequences of pvcyrpa were obtained from seven isolates. Other sequences were extracted from a repository. Maximum likelihood phylogenetic trees, genetic diversity parameters, linkage disequilibrium (LD), and neutrality tests were analyzed, and the potential amino acid polymorphism participation in B-cell epitopes was investigated. In 22 sequences from Southern Mexico, two synonymous and 21 nonsynonymous mutations defined nine private haplotypes. These parasites had the highest LD-R2 index and the lowest nucleotide diversity compared to isolates from South America or Asia. The nucleotide diversity and Tajima's D values varied across the coding gene. The exon-1 sequence had greater diversity and Rm values than those of exon-2. Exon-1 had significant positive values for Tajima's D, ß-α values, and for the Z (HA: dN > dS) and MK tests. These patterns were similar for parasites of different origin. The polymorphic amino acid residues at PvCyRPA resembled the conformational B-cell peptides reported in PfCyRPA. Diversity at pvcyrpa exon-1 is caused by mutation and recombination. This seems to be maintained by balancing selection, likely due to selective immune pressure, all of which merit further study.

Molecular variation of Plasmodium vivax dehydrofolate reductase in Mexico and Nicaragua contrasts with that occurring in South America / Variación molecular de la dehidrofolato reductasa de Plasmodium vivax en México y Nicaragua contrasta con la que ocurre en Sudamérica

Abstract: Objective: To research mutations associated to pyrimethamine resistance in dihydrofolate reductase (pvdhfr) of Plasmodium vivax from Mexico and Nicaragua and compare it to that reported in the rest of America. Materials and methods: Genomic DNA was obtained from P. vivax-infected blood samples. A pvdhfr gene fragment was amplified and sequenced. The identified gene variations were compared to those observed in other affected sites of America. Results: No mutations in pvdhfr were detected in P. vivax from Mexico and Nicaragua. One synonymous change and variation in the repeat domain was detected in Nicaraguan parasites. In South America, a high frequency of variant residues 58R and 117N associated to pyrimethamine resistance was reported. Conclusions: The lack of polymorphisms associated with pyrimethamine resistance suggests that drug-resistant P. vivax has not penetrated Mesoamerica, nor have local parasites been under selective pressure. These data contribute to establish the basis for the epidemiological surveillance of drug resistance.

Resumen: Objetivo: Determinar mutaciones en la dihydrofolato reductasa deP. vivax (Pvdhfr) en parásitos de México y Nicaragua, y comparar con lo reportado en América. Material y métodos: Del ADN de sangres infectadas con P. vivax de pacientes, el gen pvdhfr se amplifico y secuenció, y se contrastócon lo observado en América. Resultados: No se detectaron mutaciones asociadas con la resistencia debida a pirimetamina. Los parásitos de Nicaragua tuvieron una mutación sinónima y variación en la región repetida. Se reportaron frecuentes mutaciones asociadas con la resistencia a la pirimetamina en Sudamérica. Conclusiones: La ausencia de polimorfismos en Pvdhfr sugiere que no se han seleccionado ni introducido parásitos resistentes en la zona de estudio, lo que resulta muy útil para la vigilancia epidemiológica.

Molecular variation of Plasmodium vivax dehydrofolate reductase in Mexico and Nicaragua contrasts with that occurring in South America.

OBJECTIVE: To research mutations associated to pyrimethamine resistance in dihydrofolate reductase (pvdhfr) of Plasmodium vivax from Mexico and Nicaragua and compare it to that reported in the rest of America. MATERIALS AND METHODS: Genomic DNA was obtained from P. vivax-infected blood samples. A pvdhfr gene fragment was amplified and sequenced. The identified gene variations were compared to those observed in other affected sites of America. RESULTS: No mutations in pvdhfr were detected in P. vivax from Mexico and Nicaragua. One synonymous change and variation in the repeat domain was detected in Nicaraguan parasites. In South America, a high frequency of variant residues 58R and 117N associated to pyrimethamine resistance was reported. CONCLUSIONS: The lack of polymorphisms associated with pyrimethamine resistance suggests that drug-resistant P. vivax has not penetrated Mesoamerica, nor have local parasites been under selective pressure. These data contribute to establish the basis for the epidemiological surveillance of drug resistance.

OBJETIVO: Determinar mutaciones en la dihydrofolato reductasa de P. vivax (Pvdhfr) en parásitos de México y Nicaragua, y comparar con lo reportado en América. MATERIAL Y MÉTODOS: Del ADN de sangres infectadas con P. vivax de pacientes, el gen pvdhfr se amplifico y secuenció, y se contrastócon lo observado en América. RESULTADOS: No se detectaron mutaciones asociadas con la resistencia debida a pirimetamina. Los parásitos de Nicaragua tuvieron una mutación sinónima y variación en la región repetida. Se reportaron frecuentes mutaciones asociadas con la resistencia a la pirimetamina en Sudamérica. CONCLUSIONES: La ausencia de polimorfismos en Pvdhfr sugiere que no se han seleccionado ni introducido parásitos resistentes en la zona de estudio, lo que resulta muy útil para la vigilancia epidemiológica.

Human Plasmodium vivax diversity, population structure and evolutionary origin.

More than 200 million malaria clinical cases are reported each year due to Plasmodium vivax, the most widespread Plasmodium species in the world. This species has been neglected and understudied for a long time, due to its lower mortality in comparison with Plasmodium falciparum. A renewed interest has emerged in the past decade with the discovery of antimalarial drug resistance and of severe and even fatal human cases. Nonetheless, today there are still significant gaps in our understanding of the population genetics and evolutionary history of P. vivax, particularly because of a lack of genetic data from Africa. To address these gaps, we genotyped 14 microsatellite loci in 834 samples obtained from 28 locations in 20 countries from around the world. We discuss the worldwide population genetic structure and diversity and the evolutionary origin of P. vivax in the world and its introduction into the Americas. This study demonstrates the importance of conducting genome-wide analyses of P. vivax in order to unravel its complex evolutionary history.

Analysis of Global Research on Malaria and Plasmodium vivax.

Background: Malaria is one of the infectious diseases of greatest interest to the scientific community and of greatest concern to international health authorities. Traditionally, the focus has been on Plasmodium falciparum, the parasite that causes the most severe form of the disease in Africa. However, in the last twenty years, the Plasmodium vivax parasite, responsible for a large number of cases in Latin America, the Middle East, South and Southeast Asia, the Horn of Africa, and Oceania, has also generated enormous interest due, among other things, to the published evidence that it can cause severe malaria. Methods: In this paper, the international scientific publication on malaria and P. vivax has been analyzed using the Scopus database to try to define global trends in this field of study. Results: It has been shown that events such as the emergence of resistance to certain drugs can break a trend. The important role of non-malaria-endemic countries such as the USA or Switzerland in malaria research is also evident. Conclusions: International cooperation will be essential for the eradication of the disease. Moreover, in this sense, the general vision given by the bibliometric analysis of malaria caused by P. vivax is fundamental to paint the picture regarding the current situation and encourage international cooperation and control efforts.

Plasmodium vivax CSP-Pvs25 variants from southern Mexico produce distinct patterns of infectivity for Anopheles albimanus versus An. pseudopunctipennis, in each case independent of geographical origin.

BACKGROUND: The susceptibility of Anopheles albimanus and An. pseudopunctipennis to local Plasmodium vivax has been associated in southern Mexico with two ookinete surface proteins (Pvs25/28) polymorphism. Perhaps parasite population selection (i.e. adaptation to local vectors) contributes to this phenomenon. It is also possible that certain molecular interactions exist between P. vivax and each mosquito species independently of geographical origin. This study aimed to explore the susceptibility of An. albimanus and An. pseudopunctipennis (collected from different geographical sites) to P. vivax cspVk/Pvs25-130 haplotypes from southern Mexico. RESULTS: Of the 120 P. vivax-infected blood samples used to simultaneously feed An. albimanus and An. pseudopunctipennis mosquitoes originating from various geographical sites, 80 produced at least one infected mosquito species. Three parasite haplotypes were identified in infected blood: Vk210/Pvs25-A (12.5%), Vk210/Pvs25-B (20%) and Vk247/Pvs25-B (67.5%). Two parameters (the proportion of infected mosquitoes and number of oocysts/mosquito) showed a similar pattern for each mosquito species (independently of geographical origin). For An. albimanus mosquitoes (from the Pacific coast, Mexican gulf and Lacandon Forest lowlands), these two parameters were higher in specimens infected with P. vivax Vk210/Pvs25-A versus Vk210/Pvs25-B or Vk247/Pvs25-B (P < 0.001). For An. pseudopunctipennis mosquitoes (from the Pacific coast, northeast Mexico and east Guatemala foothills), the same two parameters were higher in specimens infected with Vk247/Pvs25-B or Vk210/Pvs25-B versus Vk210/Pvs25-A (P < 0.001). Higher infection rates were caused by Vk247/Pvs25-B than Vk210/Pvs25-B parasites in An. pseudopunctipennis (P = 0.011) and An. albimanus (P = 0.001). The greatest parasitaemia, gametocytaemia and microgamete formation was observed in Vk247/Pvs25-B infected blood, and each of these parameters correlated with each other and with the number of oocysts in An. pseudopunctipennis from the sympatric colony. CONCLUSIONS: Plasmodium vivax Vk247/Pvs25-B infections were the most prevalent, likely due to the higher parasitaemia produced in the susceptible vector (especially An. pseudopunctipennis). The analysis of mosquito-parasite interactions indicate that An. pseudopunctipennis and An. albimanus each have a unique pattern of transmitting genetic variants of P. vivax, and this is not dependent on geographical origin. The present findings highlight the importance of parasite genotyping to understand transmission dynamics and vectorial participation.

Ookinete-Specific Genes and 18S SSU rRNA Evidenced in Plasmodium vivax Selection and Adaptation by Sympatric Vectors.

In the southern Pacific coast of Chiapas, Mexico (SM), the two most abundant vector species, Nyssorhynchus albimanus and Anopheles pseudopunctipennis, were susceptible to different Plasmodium vivax Pvs25/28 haplotypes. To broaden our understanding of the existing P. vivax in the area, genes encoding proteins relevant for ookinete development and the 18S rRNA were studied. P. vivax infectivity (percentage of infected mosquitoes and oocyst numbers) was evaluated by simultaneously feeding infected blood samples from patients to Ny. albimanus and An. pseudopunctipennis female mosquitoes. Three infectivity patterns were identified: one group of parasites were more infective to An. pseudopunctipennis than to Ny. albimanus, another group was more infective to Ny. albimanus, while a third group infected both vectors similarly. In 29 parasite isolates, the molecular variations of ookinete-specific genes and the 18S rRNA-type S were analyzed. Using concatenated sequences, phylogenetic trees, and Structure analysis, parasite clustering within SM isolates and between these and those from other geographical origins were investigated. A ML phylogenetic tree resolved two parasite lineages: PvSM-A and PvSM-B. They were associated to a different 18S rRNA variant. PvSM-A parasites had 18S rRNA variant rV2 and correspond to parasites causing high oocyst infection in Ny. albimanus. A new ML tree and Structure analysis, both comprising global sequences, showed PvSM-A clustered with Latin American parasites. Meanwhile, all isolates of PvSM-B had 18S rRNA variant rV1 and remained as unique genetic cluster comprising two subgroups: PvSM-Ba, producing high infection in An. pseudopunctipennis, and PvSM-Bb, causing similar oocyst infection in both vector species. PvSM-A parasites were genetically similar to parasites from South America. Meanwhile, PvSM-B were exclusive to southern Mexico and share ancestry with Asian parasites. The results suggest that these lineages evolved separately, likely by geographic and vector restriction.

Plasmodium genomics: an approach for learning about and ending human malaria.

Malaria causes high levels of morbidity and mortality in human beings worldwide. According to the World Health Organization (WHO), about half a million people die of this disease each year. Malaria is caused by six species of parasites belonging to the Plasmodium genus: P. falciparum, P. knowlesi, P. vivax, P. malariae, P. ovale curtisi, and P. ovale wallikeri. Currently, malaria is being kept under control with varying levels of elimination success in different countries. The development of new molecular tools as well as the use of next-generation sequencing (NGS) technologies and novel bioinformatic approaches has improved our knowledge of malarial epidemiology, diagnosis, treatment, vaccine development, and surveillance strategies. In this work, the genetics and genomics of human malarias have been analyzed. Since the first P. falciparum genome was sequenced in 2002, various population-level genetic and genomic surveys, together with transcriptomic and proteomic studies, have shown the importance of molecular approaches in supporting malaria elimination.

The Identification of Scientific Communities and Their Approach to Worldwide Malaria Research.

It is essential to establish a pattern to detect the strengths and weaknesses of working groups publishing on malaria, to promote coordination to facilitate the eradication of the disease. Given the complexity of the scientific network of groups and institutions studying malaria, it is necessary to use a mathematical algorithm that allows us to know the real structure of research on the disease in the world. In this work, articles with the word "malaria" in the title or author keywords gathered from Elsevier Scopus database were analyzed. By means of specific software, graphs were created. The analysis of the data allowed established different scientific communities, among which two were very diverse: one formed by those groups concerned about the vector transmission and control, and another one focused on the drug resistance of the parasite. Basic, applied, and operational research to eradicate malaria is an ambitious goal of the international institutions and the scientific community. The combination of effort and the establishment of a worldwide-scientific network that allows an effective interconnection (exchange) of knowledge, infrastructure technology, collaborators, financial resources, and datasets will contribute more effectively to end the disease.

The effect of chloroquine dose and primaquine on Plasmodium vivax recurrence: a WorldWide Antimalarial Resistance Network systematic review and individual patient pooled meta-analysis.

BACKGROUND: Chloroquine remains the mainstay of treatment for Plasmodium vivax malaria despite increasing reports of treatment failure. We did a systematic review and meta-analysis to investigate the effect of chloroquine dose and the addition of primaquine on the risk of recurrent vivax malaria across different settings. METHODS: A systematic review done in MEDLINE, Web of Science, Embase, and Cochrane Database of Systematic Reviews identified P vivax clinical trials published between Jan 1, 2000, and March 22, 2017. Principal investigators were invited to share individual patient data, which were pooled using standardised methods. Cox regression analyses with random effects for study site were used to investigate the roles of chloroquine dose and primaquine use on rate of recurrence between day 7 and day 42 (primary outcome). The review protocol is registered in PROSPERO, number CRD42016053310. FINDINGS: Of 134 identified chloroquine studies, 37 studies (from 17 countries) and 5240 patients were included. 2990 patients were treated with chloroquine alone, of whom 1041 (34·8%) received a dose below the target 25 mg/kg. The risk of recurrence was 32·4% (95% CI 29·8-35·1) by day 42. After controlling for confounders, a 5 mg/kg higher chloroquine dose reduced the rate of recurrence overall (adjusted hazard ratio [AHR] 0·82, 95% CI 0·69-0·97; p=0·021) and in children younger than 5 years (0·59, 0·41-0·86; p=0·0058). Adding primaquine reduced the risk of recurrence to 4·9% (95% CI 3·1-7·7) by day 42, which is lower than with chloroquine alone (AHR 0·10, 0·05-0·17; p<0·0001). INTERPRETATION: Chloroquine is commonly under-dosed in the treatment of vivax malaria. Increasing the recommended dose to 30 mg/kg in children younger than 5 years could reduce substantially the risk of early recurrence when primaquine is not given. Radical cure with primaquine was highly effective in preventing early recurrence and may also improve blood schizontocidal efficacy against chloroquine-resistant P vivax. FUNDING: Wellcome Trust, Australian National Health and Medical Research Council, and Bill & Melinda Gates Foundation.

Genetic diversity and natural selection of Plasmodium vivax multi-drug resistant gene (pvmdr1) in Mesoamerica.

BACKGROUND: The Plasmodium vivax multidrug resistant 1 gene (pvmdr1) codes for a transmembrane protein of the parasite's digestive vacuole. It is likely that the pvmdr1 gene mutations occur at different sites by convergent evolution. In here, the genetic variation of pvmdr1 at three sites of the Mesoamerican region was studied. Since 1950s, malarious patients of those areas have been treated only with chloroquine and primaquine. METHODS: Blood samples from patients infected with P. vivax were obtained in southern Mexico (SMX), in the Northwest (NIC-NW) and in the northeast (NIC-NE) of Nicaragua. Genomic DNA was obtained and fragments of pvmdr1 were amplified and sequenced. The nucleotide and amino acid changes as well as the haplotype frequency in pvmdr1 were determined per strain and per geographic site. The sequences of pvmdr1 obtained from the studied regions were compared with homologous sequences from the GenBank database to explore the P. vivax genetic structure. RESULTS: In 141 parasites, eight nucleotide changes (two changes were synonymous and other six were nonsynonymous) were detected in 1536 bp. The PvMDR1 amino acid changes Y976F, F1076FL were predominant in endemic parasites from NIC-NE and outbreak parasites in NIC-NW but absent in SMX. Thirteen haplotypes were resolved, and found to be closely related, but their frequency at each geographic site was different (P = 0.0001). The pvmdr1 codons 925-1083 gene fragment showed higher genetic and haplotype diversity in parasites from NIC-NE than the other areas outside Latin America. The haplotype networks suggested local diversification of pvmdr1 and no significant departure from neutrality. The F ST values were low to moderate regionally, but high between NIC-NE or NIC-NW and other regions inside and outside Latin America. CONCLUSIONS: The pvmdr1 gene might have diversified recently at regional level. In the absence of significant natural, genetic drift might have caused differential pvmdr1 haplotype frequencies at different geographic sites in Mesoamerica. A very recent expansion of divergent pvmdr1 haplotypes in NIC-NE/NIC-NW produced high differentiation between these and parasites from other sites including SMX. These data are useful to set a baseline for epidemiological surveillance.