Effect of fluralaner on the biology, survival, and reproductive fitness of the neotropical malaria vector Anopheles aquasalis.

BACKGROUND: Reducing mosquito abundance or interfering with its ability to support the parasite cycle can help to interrupt malaria in areas of significant risk of malaria transmission. Fluralaner is a safe and effective drug for veterinary use indicated for the treatment against fleas and ticks which acts as an antagonist of chloride ion channels mediated by γ-aminobutyric acid (GABA), preventing the entry of these ions into the postsynaptic neuron, leading to hyperexcitability of the postsynaptic neuron of the central nervous system of arthropods. Fluralaner demonstrated insecticidal activity against different insect species. METHODS: The study aimed to evaluate the effects of fluralaner on the biology, survival, and reproductive fitness of Anopheles aquasalis. The following lethal concentrations (LC) were determined for An. aquasalis: LC5 = 0.511 µM; LC25 = 1.625 µM; LC50 = 3.237 µM. RESULTS: A significant decrease (P < 0.001) was evident in the number of eggs, larvae, and pupae in the group exposed to a sublethal dose of fluralaner when compared to a control group (without the drug). Using blood from dogs after administration of fluralaner, it was observed that the drug causes 100% mortality in An. aquasalis in less than 24 h after feeding; this effect remains even after 90 days in all samples. DISCUSSION: Fluralaner showed the same result for up to 60 days, and after that, there was a slight reduction in its effect, evidenced by a decrease in the percentage of dead females; however, still significant when compared to the control group. CONCLUSION: Fluralaner affects the biology and reduction of survival in An. aquasalis in a lasting and prolonged period, and its fecundity with lower dosages, is a strong candidate for controlling disease vectors.

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.

The challenge in detecting risk areas of snakebite when case rates are low: the case of Amazonian coral snakes.

Identifying risk areas for envenomation by animals is relevant for public health, such as strategic distribution of antivenoms. Coral snakes are highly diverse in the Amazon, inhabit natural and human-modified environments, and the outcome of the cases tends to be serious and potentially lethal due to their neurotoxic venom. By integrating species' geographical records and environmental variables, we used species distribution modeling to predict the distribution of coral snake species in the Brazilian Amazonia. We analyzed the relationship between the predicted distribution of coral snake species, along with envenomation data in the region, to propose actions to reduce the number of cases and to provide tools for a better policy of public health. We conclude that the entire Amazon shows high environmental suitability for coral snakes, and such suitability explains little about the incidence of cases. This is probably due to the low human density in the Amazon and to coral snake traits such as secretive habits and non-agressive behavior. Differently from other venomous snakes, the scenario regarding coral snakebites precludes the detection of prominent geographical areas of concern and demands a broad and equitable availability of health centers throughout Amazonia and along other areas of occurrence of the genus Micrurus.

Crosstalk of Inflammation and Coagulation in Bothrops Snakebite Envenoming: Endogenous Signaling Pathways and Pathophysiology.

Snakebite envenoming represents a major health problem in tropical and subtropical countries. Considering the elevated number of accidents and high morbidity and mortality rates, the World Health Organization reclassified this disease to category A of neglected diseases. In Latin America, Bothrops genus snakes are mainly responsible for snakebites in humans, whose pathophysiology is characterized by local and systemic inflammatory and degradative processes, triggering prothrombotic and hemorrhagic events, which lead to various complications, organ damage, tissue loss, amputations, and death. The activation of the multicellular blood system, hemostatic alterations, and activation of the inflammatory response are all well-documented in Bothrops envenomings. However, the interface between inflammation and coagulation is still a neglected issue in the toxinology field. Thromboinflammatory pathways can play a significant role in some of the major complications of snakebite envenoming, such as stroke, venous thromboembolism, and acute kidney injury. In addition to exacerbating inflammation and cell interactions that trigger vaso-occlusion, ischemia-reperfusion processes, and, eventually, organic damage and necrosis. In this review, we discuss the role of inflammatory pathways in modulating coagulation and inducing platelet and leukocyte activation, as well as the inflammatory production mediators and induction of innate immune responses, among other mechanisms that are altered by Bothrops venoms.

Treatment of a Snakebite Injury With Secondary Bacterial Infection: A Case Study.

BACKGROUND: Pit viper snakebites are challenging as they often cause tissue injury and secondary bacterial infection that may impair full recovery of the affected limb. We describe the evolution of a snakebite injury with secondary infection and the use of specialized dressings to achieve tissue repair and complete closure of the wound. CASE: Ms E., a 45-year-old woman, was bitten by a pit viper that began as a small lesion and progressed to necrosis, cellulitis, edema, and hyperemia of the perilesional skin, local inflammation, and infection. We implemented a combination of topical hydrogel therapy with calcium alginate and hydrofiber with 1.2% silver to promote autolytic debridement, combat local infection, and provide a moist wound environment. The wound required daily local treatment for 2 months due to extensive tissue damage, combined with the proteolytic action of the bothropic venom. CONCLUSION: The care of wounds caused by snakebites is a challenge for the health care team due to tissue loss resulting from the action of the venom and secondary bacterial infection. Close follow-up with the use of systemic antibiotics and topical therapies proved effective in minimizing tissue loss in this case.

Strengthening therapeutic adherence and pharmacovigilance to antimalarial treatment in Manaus, Brazil: a multicomponent strategy using mHealth.

BACKGROUND: Public health initiatives for improving adherence to primaquine based regimens and enhancing effective pharmacovigilance are needed to support the efforts for malaria elimination in real world conditions. METHODS: A multicomponent patient-oriented strategy using a Smart Safety Surveillance (3S) approach including: (1) educational materials for treatment counselling and identification of warning symptoms of haemolytic anaemia; (2) an mHealth component using Short Message Service (SMS) treatment reminders and (3) development and implementation of follow-up phone surveys three days after treatment completion, using a web-based platform linked to the local information system of malaria. Adherence was measured using the Morisky Medication Adherence Scale. Self-reported events were registered using a structured questionnaire and communicated to the Brazilian Health Regulatory Agency. RESULTS: Educational materials were disseminated to 5594 patients, of whom 1512 voluntarily entered the mHealth component through the local information system; 7323 SMS were sent, and 1062 participants completed a follow-up survey after treatment. The mean age of patients was 37.36 years (SD 13.65), 61.24% were male, 98.54% were infected with. Plasmodium vivax and 95.90% received a short regimen of chloroquine plus primaquine (CQ + PQ 7 days), as per malaria case management guidelines in Brazil. From the 1062 surveyed participants 93.31% were considered adherent to the treatment. Most of the patients (95.20%) reported at least one adverse event. Headache, lack of appetite and nausea/vomiting were the most frequently reported adverse events by 77.31%, 70.90% and 56.78% of the patients respectively. A quarter of the patients reported anxiety or depression symptoms; 57 (5.37%) patients reported 5 to 6 warning symptoms of haemolytic anaemia including jaundice and dark urine in 44 (4.14%). Overall, three patients presenting symptoms of haemolytic anaemia attended a hospital and were diagnosed with G6PD deficiency, and one had haemolysis. All of them recovered. CONCLUSIONS: Under real world conditions, a multicomponent patient-oriented strategy using information and communication technologies allowed health care providers to reinforce treatment adherence and enhance safety surveillance of adverse events associated with regimens using primaquine. Active monitoring through phone surveys also reduced under-reporting of ADRs. This approach is low-cost, scalable and able to support prioritized activities of the national malaria programme.

CCL-2 and CXCL-8: Potential Prognostic Biomarkers of Acute Kidney Injury after a Bothrops atrox Snakebite.

In the Brazilian Amazon, the snake Bothrops atrox is the primary cause of snakebites. B. atrox (BaV) venom can cause systemic pathophysiological changes such as acute kidney injury (AKI), which leads to the production of chemokines and cytokines in response to the envenomation. These soluble immunological molecules act by modulating the inflammatory response; however, the mechanisms associated with the development of AKI are still poorly understood. Here, we characterize the profile of these soluble immunological molecules as possible predictive biomarkers of the development of AKI. The study involved 34 patients who had been victims of snakebites by Bothrops sp. These were categorized into two groups according to the development of AKI (AKI(-)/AKI(+)), using healthy donors as the control (HD). Peripheral blood samples were collected at three-time points: before antivenom administration (T0) and at 24 and 48 hours after antivenom (T1 and T2, respectively). The soluble immunological molecules (CXCL-8, CCL-5, CXCL-9, CCL-2, CXCL-10, IL-6, TNF, IL-2, IL-10, IFN-γ, IL-4, and IL-17A) were quantified using cytometric bead array. Our results demonstrated an increase in CXCL-9, CXCL-10, IL-6, IL-2, IL-10, and IL-17A molecules in the groups of patients who suffered Bothrops snakebites (AKI(-) and AKI(+)) before antivenom administration, when compared to HD. In the AKI(+) group, levels of CXCL-8 and CCL-2 molecules were elevated on admission and progressively decreased during the clinical evolution of patients after antivenom administration. In addition, in the signature analysis, these were produced exclusively by the group AKI(+) at T0. Thus, these chemokines may be related to the initiation and extension of AKI after envenomation by Bothrops and present themselves as two potential biomarkers of AKI at T0.

Methylprednisolone as Adjunctive Therapy for Patients Hospitalized With Coronavirus Disease 2019 (COVID-19; Metcovid): A Randomized, Double-blind, Phase IIb, Placebo-controlled Trial.

BACKGROUND: Steroid use for coronavirus disease 2019 (COVID-19) is based on the possible role of these drugs in mitigating the inflammatory response, mainly in the lungs, triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to evaluate the efficacy of methylprednisolone (MP) among hospitalized patients with suspected COVID-19. METHODS: A parallel, double-blind, placebo-controlled, randomized, Phase IIb clinical trial was performed with hospitalized patients aged ≥18 years with clinical, epidemiological, and/or radiological suspected COVID-19 at a tertiary care facility in Manaus, Brazil. Patients were randomly allocated (1:1 ratio) to receive either intravenous MP (0.5 mg/kg) or placebo (saline solution) twice daily for 5 days. A modified intention-to-treat (mITT) analysis was conducted. The primary outcome was 28-day mortality. RESULTS: From 18 April to 16 June 2020, 647 patients were screened, 416 were randomized, and 393 were analyzed as mITT, with 194 individuals assigned to MP and 199 to placebo. SARS-CoV-2 infection was confirmed by reverse transcriptase polymerase chain reaction in 81.3%. The mortality rates at Day 28 were not different between groups. A subgroup analysis showed that patients over 60 years old in the MP group had a lower mortality rate at Day 28. Patients in the MP arm tended to need more insulin therapy, and no difference was seen in virus clearance in respiratory secretion until Day 7. CONCLUSIONS: The findings of this study suggest that a short course of MP in hospitalized patients with COVID-19 did not reduce mortality in the overall population. CLINICAL TRIALS REGISTRATION: NCT04343729.

Covid-19 Automated Diagnosis and Risk Assessment through Metabolomics and Machine Learning.

COVID-19 is still placing a heavy health and financial burden worldwide. Impairment in patient screening and risk management plays a fundamental role on how governments and authorities are directing resources, planning reopening, as well as sanitary countermeasures, especially in regions where poverty is a major component in the equation. An efficient diagnostic method must be highly accurate, while having a cost-effective profile. We combined a machine learning-based algorithm with mass spectrometry to create an expeditious platform that discriminate COVID-19 in plasma samples within minutes, while also providing tools for risk assessment, to assist healthcare professionals in patient management and decision-making. A cross-sectional study enrolled 815 patients (442 COVID-19, 350 controls and 23 COVID-19 suspicious) from three Brazilian epicenters from April to July 2020. We were able to elect and identify 19 molecules related to the disease's pathophysiology and several discriminating features to patient's health-related outcomes. The method applied for COVID-19 diagnosis showed specificity >96% and sensitivity >83%, and specificity >80% and sensitivity >85% during risk assessment, both from blinded data. Our method introduced a new approach for COVID-19 screening, providing the indirect detection of infection through metabolites and contextualizing the findings with the disease's pathophysiology. The pairwise analysis of biomarkers brought robustness to the model developed using machine learning algorithms, transforming this screening approach in a tool with great potential for real-world application.

Kelch13 mutations in Plasmodium falciparum and risk of spreading in Amazon basin countries.

BACKGROUND: The first potential focus for artemisinin resistance in South America was recently confirmed with the presence of the C580Y mutation in the Plasmodium falciparum kelch 13 gene (pfk13) in Guyana. OBJECTIVES: This study aimed to strengthen pfk13 monitoring in the Amazon basin countries, to compile the available data and to evaluate the risk of spreading of mutations. METHODS: Sanger sequencing was done on 862 samples collected between 1998 and 2019, and a global map of pfk13 genotypes available for this region was constructed. Then, the risk of spreading of mutations based on P. falciparum case importation between 2015 and 2018 within countries of the Amazon basin was evaluated. RESULTS: No additional pfk13 C580Y foci were identified. Few mutations (0.5%, 95% CI = 0.3%-0.8%) in the propeller domain were observed in the general parasite population of this region despite a high proportion of K189T mutations (49.1%, 95% CI = 46.2%-52.0%) in the non-propeller domain. Case information revealed two patterns of intense human migration: Venezuela, Guyana and the Roraima State in Brazil; and French Guiana, Suriname and the Amapá State in Brazil. CONCLUSIONS: There are few pfk13 mutant foci, but a high risk of dispersion in the Amazon basin, mainly from the Guiana Shield, proportionate to mining activities. Therefore, access to prompt diagnosis and treatment, and continuous molecular monitoring is essential in these geographical areas.

Malaria Trigram: improving the visualization of recurrence data for malaria elimination.

BACKGROUND: Although considerable success in reducing the incidence of malaria has been achieved in Brazil in recent years, an increase in the proportion of cases caused by the harder-to-eliminate Plasmodium vivax parasite can be noted. Recurrences in P. vivax malaria cases are due to new mosquito-bite infections, drug resistance or especially from relapses arising from hypnozoites. As such, new innovative surveillance strategies are needed. The aim of this study was to develop an infographic visualization tool to improve individual-level malaria surveillance focused on malaria elimination in the Brazilian Amazon. METHODS: Action Research methodology was employed to deal with the complex malaria surveillance problem in the Amazon region. Iterative cycles were used, totalling four cycles with a formal validation of an operational version of the Malaria Trigram tool at the end of the process. Further probabilistic data linkage was carried out so that information on the same patients could be linked, allowing for follow-up analysis since the official system was not planned in such way that includes this purpose. RESULTS: An infographic user interface was developed for the Malaria Trigram that incorporates all the visual and descriptive power of the Trigram concept. It is a multidimensional and interactive historical representation of malaria cases per patient over time and provides visual input to decision-makers on recurrences of malaria. CONCLUSIONS: The Malaria Trigram is aimed to help public health professionals and policy makers to recognise and analyse different types of patterns in malaria events, including recurrences and reinfections, based on the current Brazilian health surveillance system, the SIVEP-Malária system, with no additional primary data collection or change in the current process. By using the Malaria Trigram, it is possible to plan and coordinate interventions for malaria elimination that are integrated with other parallel actions in the Brazilian Amazon region, such as vector control management, effective drug and vaccine deployment strategies.

Performance of a sensitive haemozoin-based malaria diagnostic test validated for vivax malaria diagnosis in Brazilian Amazon.

BACKGROUND: Vivax malaria diagnosis remains a challenge in malaria elimination, with current point of care rapid diagnostic tests (RDT) missing many clinically significant infections because of usually lower peripheral parasitaemia. Haemozoin-detecting assays have been suggested as an alternative to immunoassay platforms but to date have not reached successful field deployment. Haemozoin is a paramagnetic crystal by-product of haemoglobin digestion by malaria parasites and is present in the food vacuole of malaria parasite-infected erythrocytes. This study aimed to compare the diagnostic capability of a new haemozoin-detecting platform, the Gazelle™ device with optical microscopy, RDT and PCR in a vivax malaria-endemic region. METHODS: A comparative, double-blind study evaluating symptomatic malaria patients seeking medical care was conducted at an infectious diseases reference hospital in the western Brazilian Amazon. Optical microscopy, PCR, RDT, and Gazelle™ were used to analyse blood samples. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and Kappa values were calculated. RESULTS: Out of 300 patients, 24 test results were excluded from the final analysis due to protocol violation (6) and inconclusive and/or irretrievable results (18). Gazelle™ sensitivity was 96.1 % (91.3-98.3) and 72.1 % (65.0-78.3) when compared to optical microscopy and PCR, respectively whereas it was 83.9 % and 62.8 % for RDTs. The platform presented specificity of 100 % (97.4-100), and 99.0 % (94.8-99.9) when compared to optical microscopy, and PCR, respectively, which  was the same for RDTs. Its correct classification rate was 98.2 % when compared to optical microscopy and 82.3 % for PCR; the test's accuracy when compared to optical microscopy was 98.1 % (96.4-99.7), when compared to RDT was 95.2 % (93.0-97.5), and when compared to PCR was 85.6 % (82.1-89.1). Kappa (95 % CI) values for Gazelle™ were 96.4 (93.2-99.5), 88.2 (82.6-93.8) and 65.3 (57.0-73.6) for optical microscopy, RDT and PCR, respectively. CONCLUSIONS: The Gazelle™ device was shown to have faster, easier, good sensitivity, specificity, and accuracy when compared to microscopy and was superior to RDT, demonstrating to be an alternative for vivax malaria screening particularly in areas where malaria is concomitant with other febrile infections (including dengue fever, zika, chikungunya, Chagas, yellow fever, babesiosis).

Evaluation of the effect of supervised anti-malarial treatment on recurrences of Plasmodium vivax malaria.

BACKGROUND: Relapses in vivax malaria have posed great challenges for malaria control, and they also account for a great proportion of reported cases. Knowing the real effectiveness of a 7-day primaquine (PQ) scheme is crucial in order to evaluate not only the cost-effectiveness of implementing new anti-hypnozoite drugs, but also how health education strategies can guarantee better compliance and be reinforced. This study aimed to evaluate the effect of daily treatment with chloroquine and PQ supervised by health workers versus prescription without supervision. METHODS: The outcome was the passive detection of new positive thick blood smears up to 180 days, based on the official data records from the National Malaria Control Programme. The recurrences seen in the real life were, therefore, used as a surrogate for true relapses. RESULTS: Patients under supervised treatment had a lower risk of recurrence up to day 180 when compared to the unsupervised treatment (17.9% vs. 36.1%; p = 0.027). CONCLUSIONS: The lack of supervision in the non-supervised group (which followed standard of care in the real life) enabled proper comparison, as consent itself would have lead to greater compliance in this group. Future studies should scale such an analysis to different settings in the Brazilian Amazon.

Should we care about Plasmodium vivax and HIV co-infection? A systematic review and a cases series from the Brazilian Amazon.

BACKGROUND: Malaria and HIV are two important public health issues. However, evidence on HIV-Plasmodium vivax co-infection (HIV/PvCo) is scarce, with most of the available information related to Plasmodium falciparum on the African continent. It is unclear whether HIV can change the clinical course of vivax malaria and increase the risk of complications. In this study, a systematic review of HIV/PvCo studies was performed, and recent cases from the Brazilian Amazon were included. METHODS: Medical records from a tertiary care centre in the Western Brazilian Amazon (2009-2018) were reviewed to identify HIV/PvCo hospitalized patients. Demographic, clinical and laboratory characteristics and outcomes are reported. Also, a systematic review of published studies on HIV/PvCo was conducted. Metadata, number of HIV/PvCo cases, demographic, clinical, and outcome data were extracted. RESULTS: A total of 1,048 vivax malaria patients were hospitalized in the 10-year period; 21 (2.0%) were HIV/PvCo cases, of which 9 (42.9%) had AIDS-defining illnesses. This was the first malaria episode in 11 (52.4%) patients. Seven (33.3%) patients were unaware of their HIV status and were diagnosed on hospitalization. Severe malaria was diagnosed in 5 (23.8%) patients. One patient died. The systematic review search provided 17 articles (12 cross-sectional or longitudinal studies and 5 case report studies). A higher prevalence of studies involved cases in African and Asian countries (35.3 and 29.4%, respectively), and the prevalence of reported co-infections ranged from 0.1 to 60%. CONCLUSION: Reports of HIV/PvCo are scarce in the literature, with only a few studies describing clinical and laboratory outcomes. Systematic screening for both co-infections is not routinely performed, and therefore the real prevalence of HIV/PvCo is unknown. This study showed a low prevalence of HIV/PvCo despite the high prevalence of malaria and HIV locally. Even though relatively small, this is the largest case series to describe HIV/PvCo.

Impact of Plasmodium vivax malaria and antimalarial treatment on cytochrome P450 activity in Brazilian patients.

AIMS: To investigate the impact of Plasmodium vivax malaria and chloroquine-primaquine chemotherapy on CYP2D6 and CYP2C19 activity in patients from the Brazilian Amazon. METHODS: Adult patients (n = 30) were given subtherapeutic doses of CYP2D6 and CYP2C19 phenotypic probes metoprolol (10 mg) and omeprazole (2 mg) in three different stages of vivax malaria illness: acute disease (study phase 1), post chemotherapy (phase 2) and convalescence (stage 3). Plasma concentrations of probes and CYP-hydroxylated metabolites (α-OH metoprolol and 5-OH omeprazole) were measured using LC/MS/MS. Two pharmacokinetic metrics were used to estimate CYP activity: (a) ratio of plasma concentrations of probe/metabolite at 240 minutes after administration of the probes and (b) ratio of areas under the time-concentration curves for probe/metabolite (AUC0-12h ). For statistical analysis, the pharmacokinetic metrics were normalized to the respective values in phase 3. Taqman assays were used for CYP2D6 and CYP2C19 genotyping. Cytokines levels were measured using cytometric bead array. RESULTS: Both pharmacokinetic metrics for metoprolol and omeprazole, and plasma concentrations of cytokines IL-6, IL-8 and IL-10 varied significantly across the three study phases (ANOVA P < 0.0001). Post hoc tests showed greater metoprolol:α-OH metoprolol ratios in phases 1 and 2 compared to phase 3, larger omeprazole:5-OH omeprazole ratios in phase 1 than in phases 2 and 3, and higher circulating IL-6, IL-8 and IL-10 in phase 1 than in phases 2 and 3. CONCLUSION: P. vivax malaria and treatment altered CYP2D6 and CYP2C19 metabolic phenotypes. CYP2C19 inhibition is attributed to a higher level of circulating proinflammatory cytokines, while suppression of CYP2D6 is ascribed mainly to chloroquine exposure.

Transmission-blocking compound candidates against Plasmodium vivax using P. berghei as an initial screening.

BACKGROUND: Different strategies for improvement of malaria control and elimination are based on the blockage of malaria parasite transmission to the mosquito vector. These strategies include the drugs that target the plasmodial sexual stages in humans and the early developmental stages inside mosquitoes. OBJECTIVES: Here we tested Malaria Box compounds in order to evaluate their activity against male and female gametocytes in Plasmodium berghei, mosquito infection in P. vivax and ookinete formation in both species. METHODS/FINDINGS: The membrane feeding assay and the development of ookinetes by a 24 h ex vivo culture and the ookinete yield per 1000 erythrocytes were used to test transmission-blocking potential of the Malaria Box compounds in P. vivax. For P. berghei we used flow cytometry to evaluate male and female gametocyte time course and fluorescence microscopy to check the ookinete development. The two species used in this study showed similar results concerning the compounds' activity against gametocytes and ookinetes, which were different from those in P. falciparum. In addition, from the eight Malaria Box compounds tested in both species, compounds MMV665830, MMV665878 and MMV665941 were selected as a hit compounds due the high inhibition observed. CONCLUSION: Our results showed that P. berghei is suitable as an initial screening system to test compounds against P. vivax.

Pharmacogenomics of thiopurines: distribution of TPMT and NUDT15 polymorphisms in the Brazilian Amazon.

Reduced function alleles in the TPMT and NUDT15 genes are risk factors for thiopurine toxicity. This study evaluated the influence of Native ancestry on the distribution of TPMT (rs1142345, rs1800460 and rs1800462) and NUDT15 (rs116855232) polymorphisms and compound metabolic phenotypes in 128 healthy males from the Brazilian Amazon. The average proportion of Native and European ancestry differed greatly and significantly between self-declared Amerindians and non-Amerindians, although extensive admixture in both groups was evident. Native ancestry was not significantly associated with the frequency distribution of the TPMT or NUDT15 polymorphisms investigated. The apparent discrepancy with our previous results for NUDT15 rs116855232 in the Ad Mixed American superpopulation of the 1000 Genomes Project is ascribed to the diversity of the Native populations of the Americas. Based on the inferred TPMT/NUDT15 compound metabolic phenotypes, the Clinical Pharmacogenetics Implementation Consortium recommendations for starting thiopurine therapy with reduced doses or to consider dose reduction applied respectively to 3-5% and to 12-20% of the study cohorts.

Utility of ultra-sensitive qPCR to detect Plasmodium falciparum and Plasmodium vivax infections under different transmission intensities.

BACKGROUND: The use of molecular diagnostics has revealed an unexpectedly large number of asymptomatic low-density malaria infections in many malaria endemic areas. This study compared the gains in parasite prevalence obtained by the use of ultra-sensitive (us)-qPCR as compared to standard qPCR in cross-sectional surveys conducted in Thailand, Brazil and Papua New Guinea (PNG). The compared assays differed in the copy number of qPCR targets in the parasite genome. METHODS: Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) parasites were quantified by qPCR amplifying the low-copy Pf_ and Pv_18S rRNA genes or the multi-copy targets Pf_varATS and Pv_mtCOX1. Cross-sectional surveys at the three study sites included 2252 participants of all ages and represented different transmission intensities. RESULTS: In the two low-transmission areas, P. falciparum positivity was 1.3% (10/773) (Thailand) and 0.8% (5/651) (Brazil) using standard Pf_18S rRNA qPCR. In these two countries, P. falciparum positivity by Pf_varATS us-qPCR increased to 1.9% (15/773) and 1.7% (11/651). In PNG, an area with moderate transmission intensity, P. falciparum positivity significantly increased from 8.6% (71/828) by standard qPCR to 12.2% (101/828) by us-qPCR. The proportions of P. falciparum infections not detected by standard qPCR were 33%, 55% and 30% in Thailand, Brazil and PNG. Plasmodium vivax was the predominating species in Thailand and Brazil, with 3.9% (30/773) and 4.9% (32/651) positivity by Pv_18S rRNA qPCR. In PNG, P. vivax positivity was similar to P. falciparum, at 8.0% (66/828). Use of Pv_mtCOX1 us-qPCR led to a significant increase in positivity to 5.1% (39/773), 6.4% (42/651) and 11.5% (95/828) in Thailand, Brazil, and PNG. The proportions of P. vivax infections missed by standard qPCR were similar at all three sites, with 23%, 24% and 31% in Thailand, Brazil and PNG. CONCLUSION: The proportional gains in the detection of P. falciparum and P. vivax infections by ultra-sensitive diagnostic assays were substantial at all three study sites. Thus, us-qPCR yields more precise prevalence estimates for both P. falciparum and P. vivax at all studied levels of endemicity and represents a significant diagnostic improvement. Improving sensitivity in P. vivax surveillance by us-qPCR is of particular benefit, because the additionally detected P. vivax infections signal the potential presence of hypnozoites and subsequent risk of relapse and further transmission.

Use of a NAT-based assay to improve the surveillance system and prevent transfusion-transmitted malaria in blood banks.

BACKGROUND: Malaria can be transmitted by blood transfusion through donations collected from asymptomatic donors. Transfusion-transmitted malaria (TTM) poses a great risk to blood services worldwide. A good screening tool for Plasmodium spp. detection in blood banks must have a high sensitivity for prevention of TTM. However, in Brazilian blood banks, screening for malaria still relies on microscopy. METHODS: In Brazil, screening for human immunodeficiency virus type 1 (HIV), RNA/DNA for hepatitis C (HCV) and hepatitis B (HBV) viruses is mandatory for every blood donation and uses nucleic acid amplification testing (NAT). The aim of this study was to evaluate the inclusion of an assay for malaria to identify Plasmodium sp. from total nucleic acid (TNA; DNA/RNA) by targeting the 18S rRNA gene of the parasite. RESULTS: Considering the limitations of microscopy and the wide availability of the Brazilian NAT platform in the screening of blood units for HIV, HCV, and HBV, a molecular diagnostic tool was validated for detection of Plasmodium sp. in blood banks; a pilot study showed that using this novel NAT assay could reduce the risk of TTM. CONCLUSION: The prototype HIV/HCV/HBV/malaria NAT assay was effective in detecting infected candidate donors and has good prospects to be applied in routine screening for preventing TTM.

Correction to: Use of a NAT-based assay to improve the surveillance system and prevent transfusion-transmitted malaria in blood banks.

An amendment to this paper has been published and can be accessed via the original article.