Natural genetic diversity of the DBL domain of a novel member of the Plasmodium vivax erythrocyte binding-like proteins (EBP2) in the Amazon rainforest.

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.

Monoamine oxidase-A (MAO-A) low-expression variants and increased risk of Plasmodium vivax malaria relapses.

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.

Delayed gametocyte clearance in Plasmodium vivax malaria is associated with polymorphisms in the cytochrome P450 reductase (CPR).

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.

IgM antibody responses against Plasmodium antigens in neotropical primates in the Brazilian Atlantic Forest.

Introduction: Zoonotic transmission is a challenge for the control and elimination of malaria. It has been recorded in the Atlantic Forest, outside the Amazon which is the endemic region in Brazil. However, only very few studies have assessed the antibody response, especially of IgM antibodies, in Neotropical primates (NP). Therefore, in order to contribute to a better understanding of the immune response in different hosts and facilitate the identification of potential reservoirs, in this study, naturally acquired IgM antibody responses against Plasmodium antigens were evaluated, for the first time, in NP from the Atlantic Forest. Methods: The study was carried out using 154 NP samples from three different areas of the Atlantic Forest. IgM antibodies against peptides of the circumsporozoite protein (CSP) from different Plasmodium species and different erythrocytic stage antigens were detected by ELISA. Results: Fifty-nine percent of NP had IgM antibodies against at least one CSP peptide and 87% against at least one Plasmodium vivax erythrocytic stage antigen. Levels of antibodies against PvAMA-1 were the highest compared to the other antigens. All families of NP showed IgM antibodies against CSP peptides, and, most strikingly, against erythrocytic stage antigens. Generalized linear models demonstrated that IgM positivity against PvCSP and PvAMA-1 was associated with PCR-detectable blood-stage malaria infection and the host being free-living. Interestingly, animals with IgM against both PvCSP and PvAMA-1 were 4.7 times more likely to be PCR positive than animals that did not have IgM for these two antigens simultaneously. Discussion: IgM antibodies against different Plasmodium spp. antigens are present in NP from the Atlantic Forest. High seroprevalence and antibody levels against blood-stage antigens were observed, which had a significant association with molecular evidence of infection. IgM antibodies against CSP and AMA-1 may be used as a potential marker for the identification of NP infected with Plasmodium, which are reservoirs of malaria in the Brazilian Atlantic Forest.

Detection of Plasmodium simium gametocytes in non-human primates from the Brazilian Atlantic Forest.

BACKGROUND: Plasmodium species of non-human primates (NHP) are of great interest because they can naturally infect humans. Plasmodium simium, a parasite restricted to the Brazilian Atlantic Forest, was recently shown to cause a zoonotic outbreak in the state of Rio de Janeiro. The potential of NHP to act as reservoirs of Plasmodium infection presents a challenge for malaria elimination, as NHP will contribute to the persistence of the parasite. The aim of the current study was to identify and quantify gametocytes in NHP naturally-infected by P. simium. METHODS: Whole blood samples from 35 NHP were used in quantitative reverse transcription PCR (RT-qPCR) assays targeting 18S rRNA, Pss25 and Pss48/45 malaria parasite transcripts. Absolute quantification was performed in positive samples for 18S rRNA and Pss25 targets. Linear regression was used to compare the quantification cycle (Cq) and the Spearman's rank correlation coefficient was used to assess the correlation between the copy numbers of 18S rRNA and Pss25 transcripts. The number of gametocytes/µL was calculated by applying a conversion factor of 4.17 Pss25 transcript copies per gametocyte. RESULTS: Overall, 87.5% of the 26 samples, previously diagnosed as P. simium, were positive for 18S rRNA transcript amplification, of which 13 samples (62%) were positive for Pss25 transcript amplification and 7 samples (54%) were also positive for Pss48/45 transcript. A strong positive correlation was identified between the Cq of the 18S rRNA and Pss25 and between the Pss25 and Pss48/45 transcripts. The 18S rRNA and Pss25 transcripts had an average of 1665.88 and 3.07 copies/µL, respectively. A positive correlation was observed between the copy number of Pss25 and 18S rRNA transcripts. Almost all gametocyte carriers exhibited low numbers of gametocytes (< 1/µL), with only one howler monkey having 5.8 gametocytes/µL. CONCLUSIONS: For the first time, a molecular detection of P. simium gametocytes in the blood of naturally-infected brown howler monkeys (Alouatta guariba clamitans) was reported here, providing evidence that they are likely to be infectious and transmit P. simium infection, and, therefore, may act as a reservoir of malaria infection for humans in the Brazilian Atlantic Forest.

Is zoonotic Plasmodium vivax malaria an obstacle for disease elimination?

BACKGROUND: The groundwork for malaria elimination does not currently consider the potential of Plasmodium zoonotic cycles that involve non-human primates (NHPs) in sylvatic environments. Since vivax malaria is less responsive to control measures, finding Plasmodium vivax infected NHPs adds even more concern. METHODS: Both Free-living monkeys in forest fragments inside the urban area and captive monkeys from a local zoo had blood samples tested for Plasmodium species. RESULTS: In this study, among the Neotropical monkeys tested, three (4.4%), one captive and two free-living, were found to be naturally infected by P. vivax. CONCLUSION: This important finding indicates that it is necessary to estimate the extent to which P. vivax NHP infection contributes to the maintenance of malaria transmission to humans. Therefore, the discussion on wildlife conservation and management must be incorporated into the malaria elimination agenda.

MaHPIC malaria systems biology data from Plasmodium cynomolgi sporozoite longitudinal infections in macaques.

Plasmodium cynomolgi causes zoonotic malarial infections in Southeast Asia and this parasite species is important as a model for Plasmodium vivax and Plasmodium ovale. Each of these species produces hypnozoites in the liver, which can cause relapsing infections in the blood. Here we present methods and data generated from iterative longitudinal systems biology infection experiments designed and performed by the Malaria Host-Pathogen Interaction Center (MaHPIC) to delve deeper into the biology, pathogenesis, and immune responses of P. cynomolgi in the Macaca mulatta host. Infections were initiated by sporozoite inoculation. Blood and bone marrow samples were collected at defined timepoints for biological and computational experiments and integrative analyses revolving around primary illness, relapse illness, and subsequent disease and immune response patterns. Parasitological, clinical, haematological, immune response, and -omic datasets (transcriptomics, proteomics, metabolomics, and lipidomics) including metadata and computational results have been deposited in public repositories. The scope and depth of these datasets are unprecedented in studies of malaria, and they are projected to be a F.A.I.R., reliable data resource for decades.

Improving the Molecular Diagnosis of Malaria: Droplet Digital PCR-Based Method Using Saliva as a DNA Source.

Malaria is an acute febrile disease caused by a protozoan of the genus Plasmodium. Light microscopy (LM) is the gold standard for the diagnosis of malaria. Despite this method being rapid and inexpensive, it has a low limit of detection, which hampers the identification of low parasitemia infections. By using multicopy targets and highly sensitive molecular techniques, it is possible to change this scenario. In this study, we evaluated the performance of droplet digital PCR (ddPCR) to detect Plasmodium DNA obtained from saliva samples (whole saliva and buccal swab) of 157 individuals exposed to malaria transmission from the Brazilian Amazon region. We used the highly sensitive ddPCR method with non-ribosomal multicopy targets for Plasmodium vivax (Pvr47) and Plasmodium falciparum (Pfr364). There was good concordance between the quantitative real-time PCR (qPCR) results from the saliva and blood, except for mixed-species infections. The sensitivity of qPCR was 93% for blood, 77% for saliva, and 47% for swabs. Parasite DNA was not detected in saliva samples in low-density infections compared with the detection in blood samples. ddPCR showed increased sensitivity for detecting Plasmodium in the blood and swabs (99% in blood, 73% in saliva, and 59% in swabs). Notably, ddPCR detected more mixed infections in the blood (15%), saliva (9%), and swabs (18%) than qPCR. Our data showed that the differences between ddPCR and qPCR were the result of a higher number of P. falciparum infections detected by ddPCR. Overall, there was a moderate correlation between parasite densities estimated by the different methods in the blood. Our findings highlight the possibility of using non-invasive sample collection methods for malaria diagnosis by targeting multicopy sequences combined with highly sensitive molecular methods.

A Comprehensive Analysis of the Genetic Diversity of Plasmodium falciparum Histidine-Rich Protein 2 (PfHRP2) in the Brazilian Amazon.

Early diagnosis and treatment are fundamental to the control and elimination of malaria. In many endemic areas, routine diagnosis is primarily performed microscopically, although rapid diagnostic tests (RDTs) provide a useful point-of-care tool. Most of the commercially available RDTs detect histidine-rich protein 2 (HRP2) of Plasmodium falciparum in the blood of infected individuals. Nonetheless, parasite isolates lacking the pfhrp2 gene are relatively frequent in some endemic regions, thereby hampering the diagnosis of malaria using HRP2-based RDTs. To track the efficacy of RDTs in areas of the Brazilian Amazon, we assessed pfhrp2 deletions in 132 P. falciparum samples collected from four malaria-endemic states in Brazil. Our findings show low to moderate levels of pfhrp2 deletion in different regions of the Brazilian Amazon. Overall, during the period covered by this study (2002-2020), we found that 10% of the P. falciparum isolates were characterized by a pfhrp2 deletion. Notably, however, the presence of pfhrp2-negative isolates has not been translated into a reduction in RDT efficacy, which in part may be explained by the presence of polyclonal infections. A further important finding was the discrepancy in the proportion of pfhrp2 deletions detected using two assessed protocols (conventional PCR versus nested PCR), which reinforces the need to perform a carefully planned laboratory workflow to assess gene deletion. This is the first study to perform a comprehensive analysis of PfHRP2 sequence diversity in Brazilian isolates of P. falciparum. We identified 10 PfHRP2 sequence patterns, which were found to be exclusive of each of the assessed regions. Despite the small number of PfHRP2 sequences available from South America, we found that the PfHRP2 sequences identified in Brazil and neighboring French Guiana show similar sequence patterns. Our findings highlight the importance of continuously monitoring the occurrence and spread of parasites with pfrhp2 deletions, while also taking into account the limitations of PCR-based testing methods associated with accuracy and the complexity of infections.

The genome of the zoonotic malaria parasite Plasmodium simium reveals adaptations to host switching.

BACKGROUND: Plasmodium simium, a malaria parasite of non-human primates (NHP), was recently shown to cause zoonotic infections in humans in Brazil. We sequenced the P. simium genome to investigate its evolutionary history and to identify any genetic adaptions that may underlie the ability of this parasite to switch between host species. RESULTS: Phylogenetic analyses based on whole genome sequences of P. simium from humans and NHPs reveals that P. simium is monophyletic within the broader diversity of South American Plasmodium vivax, suggesting P. simium first infected NHPs as a result of a host switch of P. vivax from humans. The P. simium isolates show the closest relationship to Mexican P. vivax isolates. Analysis of erythrocyte invasion genes reveals differences between P. vivax and P. simium, including large deletions in the Duffy-binding protein 1 (DBP1) and reticulocyte-binding protein 2a genes of P. simium. Analysis of P. simium isolated from NHPs and humans revealed a deletion of 38 amino acids in DBP1 present in all human-derived isolates, whereas NHP isolates were multi-allelic. CONCLUSIONS: Analysis of the P. simium genome confirmed a close phylogenetic relationship between P. simium and P. vivax, and suggests a very recent American origin for P. simium. The presence of the DBP1 deletion in all human-derived isolates tested suggests that this deletion, in combination with other genetic changes in P. simium, may facilitate the invasion of human red blood cells and may explain, at least in part, the basis of the recent zoonotic infections.

Profiling Humoral Immune Response Against Pre-Erythrocytic and Erythrocytic Antigens of Malaria Parasites Among Neotropical Primates in the Brazilian Atlantic Forest.

Human malaria due to zoonotic transmission has been recorded in the Atlantic Forest, an extra-Amazonian area in Brazil, which are a challenge for malaria control. Naturally acquired humoral immune response against pre-erythrocytic and erythrocytic antigens of Neotropical primates (NP) was evaluated here to improve the knowledge about the exposure of those animals to the malaria transmission and support the identification of the potential reservoirs of the disease in the Atlantic Forest. Blood samples of 154 monkeys from three areas of the Atlantic Forest were used to identify IgG antibodies against peptides of the repeat region of the major pre-erythrocytic antigen, the circumsporozoite protein (CSP), of Plasmodium vivax (PvCSP), Plasmodium brasilianum/Plasmodium malariae (Pb/PmCSP), and Plasmodium falciparum (PfCSP) by ELISA. Antibodies against erythrocytic recombinant antigens of P. vivax, Apical membrane antigen 1 (PvAMA-1), Erythrocyte binding protein 2 (PvEBP-2) and domain II of Duffy binding protein (PvDBPII) were also evaluated. Parameters, such as age, sex, PCR positivity, and captivity, potentially associated with humoral immune response were analyzed. Eighty-five percent of NP had antibodies against at least one CSP peptide, and 76% against at least one P. vivax erythrocytic antigen. A high percentage of adults compared to non-adults were seropositive and showed increased antibody levels. Neotropical primates with PCR positive for P. simium had a significantly higher frequency of positivity rate for immune response against PvEBP-2, PvDBPII and also higher antibody levels against PvDBPII, compared to PCR negative NPs for this species. Monkeys with PCR positive for P. brasilianum/P. malariae showed higher frequency of seropositivity and antibody levels against Pb/PmCSP. Levels of antibodies against Pb/PmCSP, PvEBP-2 and PvDBPII were higher in free-living than in captive monkeys from the same area. All Platyrrhine families showed antibodies against CSP peptides, however not all showed IgG against erythrocytic antigens. These findings showed a high prevalence of naturally acquired antibodies against CSP repeats in all studied areas, suggesting an intense exposure to infected-mosquitoes bites of NP from all families. However, mainly monkeys of Atelidae family showed antibodies against P. vivax erythrocytic antigens, suggesting blood infection, which might serve as potential reservoirs of malaria in the Atlantic Forest.

Dynamics of IgM and IgG responses to the next generation of engineered Duffy binding protein II immunogen: Strain-specific and strain-transcending immune responses over a nine-year period.

BACKGROUND: A low proportion of P. vivax-exposed individuals acquire protective strain-transcending neutralizing IgG antibodies that are able to block the interaction between the Duffy binding protein II (DBPII) and its erythrocyte-specific invasion receptor. In a recent study, a novel surface-engineered DBPII-based vaccine termed DEKnull-2, whose antibody response target conserved DBPII epitopes, was able to induce broadly binding-inhibitory IgG antibodies (BIAbs) that inhibit P. vivax reticulocyte invasion. Toward the development of DEKnull-2 as an effective P. vivax blood-stage vaccine, we investigate the relationship between naturally acquired DBPII-specific IgM response and the profile of IgG antibodies/BIAbs activity over time. METHODOLOGY/PRINCIPAL FINDINGS: A nine-year follow-up study was carried-out among long-term P. vivax-exposed Amazonian individuals and included six cross-sectional surveys at periods of high and low malaria transmission. DBPII immune responses associated with either strain-specific (Sal1, natural DBPII variant circulating in the study area) or conserved epitopes (DEKnull-2) were monitored by conventional serology (ELISA-detected IgM and IgG antibodies), with IgG BIAbs activity evaluated by functional assays (in vitro inhibition of DBPII-erythrocyte binding). The results showed a tendency of IgM antibodies toward Sal1-specific response; the profile of Sal1 over DEKnull-2 was not associated with acute malaria and sustained throughout the observation period. The low malaria incidence in two consecutive years allowed us to demonstrate that variant-specific IgG (but not IgM) antibodies waned over time, which resulted in IgG skewed to the DEKnull-2 response. A persistent DBPII-specific IgM response was not associated with the presence (or absence) of broadly neutralizing IgG antibody response. CONCLUSIONS/SIGNIFICANCE: The current study demonstrates that long-term exposure to low and unstable levels of P. vivax transmission led to a sustained DBPII-specific IgM response against variant-specific epitopes, while sustained IgG responses are skewed to conserved epitopes. Further studies should investigate on the role of a stable and persistent IgM antibody response in the immune response mediated by DBPII.

Prospective assessment of malaria infection in a semi-isolated Amazonian indigenous Yanomami community: Transmission heterogeneity and predominance of submicroscopic infection.

In the Amazon basin, indigenous forest-dwelling communities typically suffer from a high burden of infectious diseases, including malaria. Difficulties in accessing these isolated ethnic groups, such as the semi-nomadic Yanomami, make official malaria data largely underestimated. In the current study, we longitudinally surveyed microscopic and submicroscopic malaria infection in four Yanomami villages of the Marari community in the northern-most region of the Brazilian Amazon. Malaria parasite species-specific PCR-based detection of ribosomal and non-ribosomal targets showed that approximately 75% to 80% of all malaria infections were submicroscopic, with the ratio of submicroscopic to microscopic infection remaining stable over the 4-month follow-up period. Although the prevalence of malaria infection fluctuated over time, microscopically-detectable parasitemia was only found in children and adolescents, presumably reflecting their higher susceptibility to malaria infection. As well as temporal variation, the prevalence of malaria infection differed significantly between villages (from 1% to 19%), demonstrating a marked heterogeneity at micro-scales. Over the study period, Plasmodium vivax was the most commonly detected malaria parasite species, followed by P. malariae, and much less frequently P. falciparum. Consecutive blood samples from 859 out of the 981 studied Yanomami showed that malaria parasites were detected in only 8% of the previously malaria-positive individuals, with most of them young children (median age 3 yrs). Overall, our results show that molecular tools are more sensitive for the identification of malaria infection among the Yanomami, which is characterized by heterogeneous transmission, a predominance of low-density infections, circulation of multiple malaria parasite species, and a higher susceptibility in young children. Our findings are important for the design and implementation of the new strategic interventions that will be required for the elimination of malaria from isolated indigenous populations in Latin America.

Novel Insights into Plasmodium vivax Therapeutic Failure: CYP2D6 Activity and Time of Exposure to Malaria Modulate the Risk of Recurrence.

Plasmodium vivax relapse is one of the major causes of sustained global malaria transmission. Primaquine (PQ) is the only commercial drug available to prevent relapses, and its efficacy is dependent on metabolic activation by cytochrome P450 2D6 (CYP2D6). Impaired CYP2D6 function, caused by allelic polymorphisms, leads to the therapeutic failure of PQ as a radical cure for P. vivax malaria. Here, we hypothesized that the host immune response to malaria parasites modulates susceptibility to P. vivax recurrences in association with CYP2D6 activity. We performed a 10-year retrospective study by genotyping CYP2D6 polymorphisms in 261 malaria-exposed individuals from the Brazilian Amazon. The immune responses against a panel of P. vivax blood-stage antigens were evaluated by serological assays. We confirmed our previous findings, which indicated an association between impaired CYP2D6 activity and a higher risk of multiple episodes of P. vivax recurrence (risk ratio, 1.75; 95% confidence interval [CI], 1.2 to 2.6; P = 0.0035). An important finding was a reduction of 3% in the risk of recurrence (risk ratio, 0.97; 95% CI, 0.96 to 0.98; P < 0.0001) per year of malaria exposure, which was observed for individuals with both reduced and normal CYP2D6 activity. Accordingly, subjects with long-term malaria exposure and persistent antibody responses to various antigens showed fewer episodes of malaria recurrence. Our findings have direct implications for malaria control, since it was shown that nonimmune individuals who do not respond adequately to treatment due to reduced CYP2D6 activity may present a significant challenge for sustainable progress toward P. vivax malaria elimination.

Plasmodium infection and its association with biochemical and haematological parameters in free-living Alouatta guariba clamitans (Cabrera, 1940) (Primates: Atelidae) in Southern Brazil.

BACKGROUND: The influence of Plasmodium spp. infection in the health of Southern brown howler monkey, Alouatta guariba clamitans, the main reservoir of malaria in the Atlantic Forest, is still unknown. OBJECTIVES: The aim of this study was to investigate the positivity rate of Plasmodium infection in free-living howler monkeys in an Atlantic Forest fragment in Joinville/SC and to associate the infection with clinical, morphometrical, haematological and biochemical alterations. METHODS: Molecular diagnosis of Plasmodium infection in the captured monkeys was performed by Nested-polymerase chain reaction (PCR) (18S rRNA and coxI). Haematological and biochemical parameters were compared among infected and uninfected monkeys; clinical and morphometrical parameters were also compared. FINDINGS: The positivity rate of Plasmodium infection was 70% among forty captured animals, the highest reported for neotropical primates. None statistical differences were detected in the clinical parameters, and morphometric measures comparing infected and uninfected groups. The main significant alteration was the higher alanine aminotransferase (ALT) levels in infected compared to uninfected monkeys. MAIN CONCLUSIONS: Therefore, Plasmodium infection in howler monkeys may causes haematological/biochemical alterations which might suggest hepatic impairment. Moreover, infection must be monitored for the eco-epidemiological surveillance of malaria in the Atlantic Forest and during primate conservation program that involves the animal movement, such as translocations.

Howler monkeys are the reservoir of malarial parasites causing zoonotic infections in the Atlantic forest of Rio de Janeiro.

BACKGROUND: Although malaria cases have substantially decreased in Southeast Brazil, a significant increase in the number of Plasmodium vivax-like autochthonous human cases has been reported in remote areas of the Atlantic Forest in the past few decades in Rio de Janeiro (RJ) state, including an outbreak during 2015-2016. The singular clinical and epidemiological aspects in several human cases, and collectively with molecular and genetic data, revealed that they were due to the non-human primate (NHP) parasite Plasmodium simium; however, the understanding of the autochthonous malarial epidemiology in Southeast Brazil can only be acquired by assessing the circulation of NHP Plasmodium in the foci and determining its hosts. METHODOLOGY: A large sampling effort was carried out in the Atlantic forest of RJ and its bordering states (Minas Gerais, São Paulo, Espírito Santo) for collecting and examining free-living NHPs. Blood and/or viscera were analyzed for Plasmodium infections via molecular and microscopic techniques. PRINCIPAL FINDINGS: In total, 146 NHPs of six species, from 30 counties in four states, were tested, of which majority were collected from RJ. Howler monkeys (Alouatta clamitans) were the only species found infected. In RJ, 26% of these monkeys tested positive, of which 17% were found to be infected with P. simium. Importantly, specific single nucleotide polymorphisms-the only available genetic markers that differentiate P. simium from P. vivax-were detected in all P. simium infected A. clamitans despite their geographical origin of malarial foci. Interestingly, 71% of P. simium infected NHPs were from the coastal slope of a mountain chain (Serra do Mar), where majority of the human cases were found. Plasmodium brasilianum/malariae was initially detected in 14% and 25% free-living howler monkeys in RJ and in the Espírito Santo (ES) state, respectively. Moreover, the malarial pigment was detected in the spleen fragments of 50% of a subsample comprising dead howler monkeys in both RJ and ES. All NHPs were negative for Plasmodium falciparum. CONCLUSIONS/SIGNIFICANCE: Our data indicate that howler monkeys act as the main reservoir for the Atlantic forest human malarial parasites in RJ and other sites in Southeast Brazil and reinforce its zoonotic characteristics.

Author Correction: An assay for the identification of Plasmodium simium infection for diagnosis of zoonotic malaria in the Brazilian Atlantic Forest.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Pharmacokinetics/pharmacodynamics of chloroquine and artemisinin-based combination therapy with primaquine.

BACKGROUND: Activation of hypnozoites of vivax malaria causes multiple clinical relapses, which contribute to the Plasmodium vivax burden and continuing transmission. Artemisinin-based combination therapy (ACT) is effective against blood-stage P. vivax but requires co-administration with primaquine to achieve radical cure. The therapeutic efficacy of primaquine depends on the generation of a therapeutically active metabolite via cytochrome P450 2D6 (CYP2D6). Impaired CYP2D6 metabolism has been associated with primaquine treatment failure. This study investigated the association between impaired CYP2D6 genotypes, drug-exposure to the long-acting ACT component (schizonticidal drugs) and tolerance and efficacy. METHODS: Adult patients with acute vivax malaria were enrolled in a recently completed trial and treated with artesunate-mefloquine, chloroquine or artemether-lumefantrine. All received concomitant primaquine (0.5 mg/kg/day for 7-9 days). The association between efficacy and safety and drug exposure was explored using area-under-the-curve (AUC) and half-life (t1/2) estimates obtained by non-compartmental analysis of the long half-life drugs. Parasite recurrences by day 63 were categorized as related relapses or re-infections/unrelated hypnozoite activation by genotyping three microsatellite loci and two polymorphic loci of merozoite surface antigen-1. The CYP2D6 genotype was identified with Taqman assays by real-time PCR to 9 polymorphisms (8 SNPs and one deletion). Impaired CYP2D6 activity was inferred using the Activity Score System. RESULTS: Most recurrences in the ASMQ (67%), CQ (80%) and AL (85%) groups were considered related relapses. Eight of nine (88.9%) of the patients with impaired CYP2D6 activity relapsed with related parasite compared to 18/25 (72%) with normal activity (RR = 1.23, 0.88; 1.72, p = 0.40). There were no associations between the measured PK parameters and recurrence. Patients with longer chloroquine half-lives had more pruritus (RR = 1.09, 1.03; 1.14, p = 0.001). Higher CQ AUCs were associated with reduced falls in haemoglobin by day 14 (Coef - 0.02, - 0.005; - 0.03, p = 0.01). All regimens were well tolerated. CONCLUSION: Genotyping of P. vivax showed that activation of related (homologous) hypnozoites was the most frequent cause of recurrence. The high proportion of the impaired CYP2D6 activity among patients with recurrent infections suggests that slow primaquine metabolism might influence related relapse rates in Brazil among patients receiving primaquine for radical cure, although confirmatory studies are needed. There was no association between drug exposure of the long-acting ACT component (schizonticidal drugs) and risk of related relapse. ACT was well tolerated. These results provide further re-assurance about the safety and efficacy of ACT when combined with short course primaquine to treat uncomplicated malaria vivax in Brazil. Trial registration RBR-79s56s ( http://www.ensaiosclinicos.gov.br/rg/RBR-79s56s/ ).

Humoral immunity prevents clinical malaria during Plasmodium relapses without eliminating gametocytes.

Plasmodium relapses are attributed to the activation of dormant liver-stage parasites and are responsible for a significant number of recurring malaria blood-stage infections. While characteristic of human infections caused by P. vivax and P. ovale, their relative contribution to malaria disease burden and transmission remains poorly understood. This is largely because it is difficult to identify 'bona fide' relapse infections due to ongoing transmission in most endemic areas. Here, we use the P. cynomolgi-rhesus macaque model of relapsing malaria to demonstrate that clinical immunity can form after a single sporozoite-initiated blood-stage infection and prevent illness during relapses and homologous reinfections. By integrating data from whole blood RNA-sequencing, flow cytometry, P. cynomolgi-specific ELISAs, and opsonic phagocytosis assays, we demonstrate that this immunity is associated with a rapid recall response by memory B cells that expand and produce anti-parasite IgG1 that can mediate parasite clearance of relapsing parasites. The reduction in parasitemia during relapses was mirrored by a reduction in the total number of circulating gametocytes, but importantly, the cumulative proportion of gametocytes increased during relapses. Overall, this study reveals that P. cynomolgi relapse infections can be clinically silent in macaques due to rapid memory B cell responses that help to clear asexual-stage parasites but still carry gametocytes.

Improvement of antimalarial activity of a 3-alkylpiridine alkaloid analog by replacing the pyridine ring to a thiazole-containing heterocycle: Mode of action, mutagenicity profile, and Caco-2 cell-based permeability.

The development of new antimalarial drugs is urgent to overcome the spread of resistance to the current treatment. Herein we synthesized the compound 3, a hit-to­lead optimization of a thiazole based on the most promising 3-alkylpyridine marine alkaloid analog. Compound 3 was tested against Plasmodium falciparum and has shown to be more potent than its precursor (IC50 values of 1.55 and 14.7 µM, respectively), with higher selectivity index (74.7) for noncancerous human cell line. This compound was not mutagenic and showed genotoxicity only at concentrations four-fold higher than its IC50. Compound 3 was tested in vivo against Plasmodium berghei NK65 strain and inhibited the development of parasite at 50 mg/kg. In silico and UV-vis approaches determined that compound 3 acts impairing hemozoin crystallization and confocal microscopy experiments corroborate these findings as the compound was capable of diminishing food vacuole acidity. The assay of uptake using human intestinal Caco-2 cell line showed that compound 3 is absorbed similarly to chloroquine, a standard antimalarial agent. Therefore, we present here compound 3 as a potent new lead antimalarial compound.