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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.
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Antimaláricos , Citocromo P-450 CYP2D6 , Malária Vivax , Monoaminoxidase , Primaquina , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Antimaláricos/uso terapêutico , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Genótipo , Malária Vivax/tratamento farmacológico , Malária Vivax/genética , Monoaminoxidase/genética , Plasmodium vivax/genética , Polimorfismo Genético , Primaquina/uso terapêutico , Recidiva , Estudos RetrospectivosRESUMO
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.
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Malária , Plasmodium , Animais , Brasil/epidemiologia , Formação de Anticorpos , Proteínas de Protozoários , Imunoglobulina M , Estudos Soroepidemiológicos , Antígenos de Protozoários , Malária/veterinária , Primatas , Florestas , Anticorpos Antiprotozoários , Peptídeos , Plasmodium vivaxRESUMO
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.
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Individuals with asymptomatic infection due to Plasmodium vivax are posited to be important reservoirs of malaria transmission in endemic regions. Here we studied a cohort of P. vivax malaria patients in a suburban area in the Brazilian Amazon. Overall 1,120 individuals were screened for P. vivax infection and 108 (9.6%) had parasitemia detected by qPCR but not by microscopy. Asymptomatic individuals had higher levels of antibodies against P. vivax and similar hematological and biochemical parameters compared to uninfected controls. Blood from asymptomatic individuals with very low parasitemia transmitted P. vivax to the main local vector, Nyssorhynchus darlingi. Lower mosquito infectivity rates were observed when blood from asymptomatic individuals was used in the membrane feeding assay. While blood from symptomatic patients infected 43.4% (199/458) of the mosquitoes, blood from asymptomatic infected 2.5% (43/1,719). However, several asymptomatic individuals maintained parasitemia for several weeks indicating their potential role as an infectious reservoir. These results suggest that asymptomatic individuals are an important source of malaria parasites and Science and Technology for Vaccines granted by Conselho Nacional de may contribute to the transmission of P. vivax in low-endemicity areas of malaria.
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Anopheles/parasitologia , Malária Vivax/transmissão , Plasmodium vivax/fisiologia , Animais , Anopheles/fisiologia , Infecções Assintomáticas/epidemiologia , Sangue/parasitologia , Brasil/epidemiologia , Estudos de Coortes , Estudos Transversais , Feminino , Humanos , Malária Vivax/epidemiologia , Malária Vivax/parasitologia , Masculino , Pessoa de Meia-Idade , Plasmodium vivax/genética , Estações do AnoRESUMO
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.
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Malária Falciparum , Plasmodium falciparum , Antígenos de Protozoários/genética , Brasil , Testes Diagnósticos de Rotina , Deleção de Genes , Histidina , Humanos , Malária Falciparum/diagnóstico , Plasmodium falciparum/genética , Proteínas de Protozoários/genéticaRESUMO
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.
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Malária , Parasitos , Plasmodium , Animais , Anticorpos Antiprotozoários , Antígenos de Protozoários , Brasil , Eritrócitos , Florestas , Imunidade Humoral , Malária/veterinária , Plasmodium vivax , Primatas , Proteínas de ProtozoáriosRESUMO
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.
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Antimaláricos/uso terapêutico , Citocromo P-450 CYP2D6/metabolismo , Malária Vivax/tratamento farmacológico , Plasmodium vivax/efeitos dos fármacos , Primaquina/uso terapêutico , Adolescente , Adulto , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Brasil , Criança , Citocromo P-450 CYP2D6/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Plasmodium vivax/genética , Plasmodium vivax/imunologia , Recidiva , Falha de Tratamento , Adulto JovemRESUMO
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.
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Malária/diagnóstico , Adolescente , Adulto , Idoso , Brasil/epidemiologia , Criança , Pré-Escolar , Estudos Transversais , DNA de Protozoário/isolamento & purificação , DNA de Protozoário/metabolismo , Feminino , Humanos , Lactente , Malária/epidemiologia , Malária/parasitologia , Malária/transmissão , Masculino , Pessoa de Meia-Idade , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Plasmodium vivax/genética , Plasmodium vivax/isolamento & purificação , Prevalência , Estudos Prospectivos , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Adulto JovemRESUMO
BACKGROUND: The unexpected high proportion of submicroscopic malaria infections in areas with low transmission intensity challenges the control and elimination of malaria in the Americas. The current PCR-based assays present limitations as most protocols still rely on amplification of few-copies target gene. Here, the hypothesis was that amplification of different plasmodial targets-ribosomal (18S rRNA) and non-ribosomal multi-copy sequences (Pvr47 for Plasmodium vivax and Pfr364 for Plasmodium falciparum)-could increase the chances of detecting submicroscopic malaria infection. METHODS: A non-ribosomal real-time PCR assay targeting Pvr47/Pfr364 (NR-qPCR) was established and compared with three additional PCR protocols, two of them based on 18S rRNA gene amplification (Nested-PCR and R-qPCR) and one based on Pvr47/Pfr364 targets (NR-cPCR). The limit of detection of each PCR protocol, at single and artificial mixed P. vivax/P. falciparum infections, was determined by end-point titration curves. Field samples from clinical (n = 110) and subclinical (n = 324) malaria infections were used to evaluate the impact of using multiple molecular targets to detect malaria infections. RESULTS: The results demonstrated that an association of ribosomal and non-ribosomal targets did not increase sensitivity to detect submicroscopic malaria infections. Despite of that, artificial mixed-malaria infections demonstrated that the NR-qPCR was the most sensitive protocol to detect low-levels of P. vivax/P. falciparum co-infections. Field studies confirmed that submicroscopic malaria represented a large proportion (up to 77%) of infections among asymptomatic Amazonian residents, with a high proportion of infections (~ 20%) identified only by the NR-qPCR. CONCLUSIONS: This study presents a new species-specific non-ribosomal PCR assay with potential to identify low-density P. vivax and P. falciparum infections. As the majority of subclinical infections was caused by P. vivax, the commonest form of malaria in the Amazon area, future studies should investigate the potential of Pvr47/Pfr364 to detect mixed-malaria infections in the field.
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Coinfecção/diagnóstico , Malária/diagnóstico , RNA Ribossômico 18S/genética , Reação em Cadeia da Polimerase em Tempo Real , Adulto , Infecções Assintomáticas , Brasil , Coinfecção/parasitologia , Feminino , Humanos , Limite de Detecção , Malária/sangue , Malária Falciparum/sangue , Malária Falciparum/diagnóstico , Malária Vivax/sangue , Malária Vivax/diagnóstico , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Plasmodium vivax/genética , Plasmodium vivax/isolamento & purificação , Adulto JovemRESUMO
Malaria has provided a major selective pressure and has modulated the genetic diversity of the human genome. The variants of the Duffy Antigen/Receptor for Chemokines (DARC) gene have probably been selected by malaria parasites, particularly the FY*O allele, which is fixed in sub-Saharan Africa and confers resistance to Plasmodium vivax infection. Here, we showed the influence of genomic ancestry on the distribution of DARC genotypes in a highly admixed Brazilian population and confirmed the decreased susceptibility of the FY*A/FY*O genotype to clinical P. vivax malaria. FY*B/FY*O individuals were associated with a greater risk of developing clinical malaria. A remarkable difference among DARC variants concerning the susceptibility to clinical malaria was more evident for individuals who were less exposed to malaria, as measured by the time of residence in the endemic area. Additionally, we found that DARC-negative and FY*A/FY*O individuals had a greater chance of acquiring high levels of antibodies against the 19-kDa C-terminal region of the P. vivax merozoite surface protein-1. Altogether, our results provide evidence that DARC polymorphisms modulate the susceptibility to clinical P. vivax malaria and influence the naturally-acquired humoral immune response to malaria blood antigens, which may interfere with the efficacy of a future vaccine against malaria.