Morphological and Transcriptional Changes in Human Bone Marrow During Natural Plasmodium vivax Malaria Infections.

BACKGROUND: The presence of Plasmodium vivax malaria parasites in the human bone marrow (BM) is still controversial. However, recent data from a clinical case and experimental infections in splenectomized nonhuman primates unequivocally demonstrated the presence of parasites in this tissue. METHODS: In the current study, we analyzed BM aspirates of 7 patients during the acute attack and 42 days after drug treatment. RNA extracted from CD71+ cell suspensions was used for sequencing and transcriptomic analysis. RESULTS: We demonstrated the presence of parasites in all patients during acute infections. To provide further insights, we purified CD71+ BM cells and demonstrated dyserythropoiesis and inefficient erythropoiesis in all patients. In addition, RNA sequencing from 3 patients showed that genes related to erythroid maturation were down-regulated during acute infections, whereas immune response genes were up-regulated. CONCLUSIONS: This study thus shows that during P. vivax infections, parasites are always present in the BM and that such infections induced dyserythropoiesis and ineffective erythropoiesis. Moreover, infections induce transcriptional changes associated with such altered erythropoietic response, thus highlighting the importance of this hidden niche during natural infections.

Duffy blood system and G6PD genetic variants in vivax malaria patients from Manaus, Amazonas, Brazil.

BACKGROUND: Over a third of the world's population is at risk of Plasmodium vivax-induced malaria. The unique aspect of the parasite's biology and interactions with the human host make it harder to control and eliminate the disease. Glucose-6-phosphate dehydrogenase (G6PD) deficiency and Duffy-negative blood groups are two red blood cell (RBC) variations that can confer protection against malaria. METHODS: Molecular genotyping of G6PD and Duffy variants was performed in 225 unrelated patients (97 with uncomplicated and 128 with severe vivax malaria) recruited at a Reference Centre for Infectious Diseases in Manaus. G6PD and Duffy variants characterizations were performed using Real Time PCR (qPCR) and PCR-RFLP, respectively. RESULTS: The Duffy blood group system showed a phenotypic distribution Fy(a + b-) of 70 (31.1%), Fy(a + b +) 96 (42.7%), Fy(a-b +) 56 (24.9%) and Fy(a-b-) 1 (0.44%.) The genotype FY*A/FY*B was predominant in both uncomplicated (45.3%) and severe malaria (39.2%). Only one Duffy phenotype Fy(a-b) was found and this involved uncomplicated vivax malaria. The G6PD c.202G > A variant was found in 11 (4.88%) females and 18 (8.0%) males, while c.376A > G was found in 20 females (8.88%) and 23 (10.22%) male patients. When combined GATA mutated and c.202G > A and c.376A > G mutated, was observed at a lower frequency in uncomplicated (3.7%) in comparison to severe malaria (37.9%). The phenotype Fy(a-b +) (p = 0.022) with FY*B/FY*B (p = 0.015) genotype correlated with higher parasitaemia. CONCLUSIONS: A high prevalence of G6PD c202G > A and c.376A > G and Duffy variants is observed in Manaus, an endemic area for vivax malaria. In addition, this study reports for the first time the Duffy null phenotype Fy(a-b-) in the population of the Amazonas state. Moreover, it is understood that the relationship between G6PD and Duffy variants can modify clinical symptoms in malaria caused by P. vivax and this deserves to be further investigated and explored among this population.

Prevalence and force of Plasmodium vivax blood-stage infection and associated clinical malaria burden in the Brazilian Amazon.

BACKGROUND: Understanding the epidemiology of malaria through the molecular force of the blood-stage infection of Plasmodium vivax (molFOB) may provide a detailed assessment of malaria transmission. OBJECTIVES: In this study, we investigated risk factors and spatial-temporal patterns of incidence of Plasmodium infection and clinical malaria episodes in three peri-urban communities of Manaus, Western Brazilian Amazon. METHODS: Monthly samples were collected in a cohort of 1,274 individuals between April 2013 and March 2014. DNA samples were subject to Plasmodium species. molFOB was calculated by counting the number of genotypes observed on each visit, which had not been present in the preceding two visits and adjusting these counts by the respective times-at-risk. FINDINGS: Respectively, 77.8% and 97.2% of the population remained free of P. vivax and P. falciparum infection. Expected heterozygosity for P. vivax was 0.69 for MSP1_F3 and 0.86 for MS2. Multiplicity of infection in P. vivax was close to the value of 1. The season was associated with P. vivax positivity [adjusted hazard ratio (aHR) 2.6 (1.9-5.7)] and clinical disease [aHR 10.6 (2.4-47.2)]. P. falciparum infection was associated with previous malarial episodes [HR 9.7 (4.5-20.9)]. Subjects who reported possession of a bed net [incidence rate ratio (IRR) 1.6 (1.2-2.2)] or previous malaria episodes [IRR 3.0 (2.0-4.5)] were found to have significantly higher P. vivax molFOB. MAIN CONCLUSIONS: Overall, P. vivax infection prevailed in the area and infections were mostly observed as monoclonal. Previous malaria episodes were associated with significantly higher P. vivax molFOB.

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.

Influence of CYP2C8, CYP3A4, and CYP3A5 Host Genotypes on Early Recurrence of Plasmodium vivax.

Cytochrome P450 (CYP) enzymes are involved in the biotransformation of chloroquine (CQ), but the role of the different profiles of metabolism of this drug in relation to Plasmodium vivax recurrences has not been properly investigated. To investigate the influence of the CYP genotypes associated with CQ metabolism on the rates of P. vivax early recurrences, a case-control study was carried out. The cases included patients presenting with an early recurrence (CQ-recurrent individuals), defined as a recurrence during the first 28 days after initial infection and plasma concentrations of CQ plus desethylchloroquine (DCQ; the major CQ metabolite) higher than 100 ng/ml. A control group with no parasite recurrence over the follow-up (the CQ-responsive group) was also included. CQ and DCQ plasma levels were measured on day 28. CQ-metabolizing CYP (CYP2C8, CYP3A4, and CYP3A5) genotypes were determined by real-time PCR. An ex vivo study was conducted to verify the efficacy of CQ and DCQ against P. vivax isolates. The frequency of alleles associated with normal and slow metabolism was similar between the cases and the controls for the CYP2C8 (odds ratio [OR] = 1.45, 95% confidence interval [CI] = 0.51 to 4.14, P = 0.570), CYP3A4 (OR = 2.38, 95% CI = 0.92 to 6.19, P = 0.105), and CYP3A5 (OR = 4.17, 95% CI = 0.79 to 22.04, P = 1.038) genes. DCQ levels were higher than CQ levels, regardless of the genotype. Regarding the DCQ/CQ ratio, there was no difference between groups or between those patients who had a normal genotype and those patients who had a mutant genotype. DCQ and CQ showed similar efficacy ex vivo CYP genotypes had no influence on early recurrence rates. The similar efficacy of CQ and DCQ ex vivo could explain the absence of therapeutic failure, despite the presence of alleles associated with slow metabolism.

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.

Malaria impact on cognitive function of children in a peri-urban community in the Brazilian Amazon.

BACKGROUND: In Latin America, where Plasmodium vivax malaria is more prevalent, it is known that this species plays an important role in the sustainability of transmission, and can have an impact on morbidity in terms of anaemia, nutritional status, and cognitive development in children. METHODS: The present study aimed to assess the impact of malaria infection on cognition of children in a peri-urban community in the Brazilian Amazon with moderate endemicity by applying Home Inventory and WPPSI-IV. A non-concurrent cohort study was designed and the cognitive, haematological, and nutritional profiles of the children were assessed. Children with documented malaria history were identified from official reported data. RESULTS: A total of 219 children aged between 2 and 7 years were enrolled. Although 205 (95%) children had normal birth weight, 177 (81%) were malnourished, and 35 (16%) had anaemia. Among the 100 (46%) children who experienced at least one episode of malaria, 89 (89%) children demonstrated low level of cognitive development. The findings showed that Plasmodium vivax malaria was an independent risk factor for low cognitive development. CONCLUSIONS: In addition to the known economic impact of malaria in the Amazon region, the study highlights the deleterious effects P. vivax malaria has on the socio-cultural development of the population.

CYP2D6 activity and the risk of recurrence of Plasmodium vivax malaria in the Brazilian Amazon: a prospective cohort study.

BACKGROUND: CYP2D6 pathway mediates the activation of primaquine into active metabolite(s) in hepatocytes. CYP2D6 is highly polymorphic, encoding CYP2D6 isoforms with normal, reduced, null or increased activity. It is hypothesized that Plasmodium vivax malaria patients with defective CYP2D6 function would be at increased risk for primaquine failure to prevent recurrence. The aim of this study was to investigate the association of CYP2D6 polymorphisms and inferred CYP2D6 phenotypes with malaria recurrence in patients from the Western Brazilian Amazon, following chloroquine/primaquine combined therapy. METHODS: The prospective cohort consisted of P. vivax malaria patients who were followed for 6 months after completion of the chloroquine/primaquine therapy. Recurrence was defined as one or more malaria episodes, 28-180 days after the initial episode. Genotyping for nine CYP2D6 SNPs and copy number variation was performed using TaqMan assays in a Fast 7500 Real-Time System. CYP2D6 star alleles (haplotypes), diplotypes and CYP2D6 phenotypes were inferred, and the activity score system was used to define the functionality of the CYP2D6 diplotypes. CYP2D6 activity scores (AS) were dichotomized at ≤ 1 (gPM, gIM and gNM-S phenotypes) and ≥ 1.5 (gNM-F and gUM phenotypes). RESULTS: Genotyping was successfully performed in 190 patients (44 with recurrence and 146 without recurrences). Recurrence incidence was higher in individuals presenting reduced activity CYP2D6 phenotypes (adjusted relative risk = 1.89, 95% CI 1.01-3.70; p = 0.049). Attributable risk and population attributable fraction were 11.5 and 9.9%, respectively. The time elapsed from the first P. vivax malaria episode until the recurrence did not differ between patients with AS of ≤ 1 versus ≥ 1.5 (p = 0.917). CONCLUSIONS: The results suggest that CYP2D6 polymorphisms are associated with increased risk of recurrence of vivax malaria, following chloroquine-primaquine combined therapy. This association is interpreted as the result of reduced conversion of primaquine into its active metabolites in patients with reduced CYP2D6 enzymatic activity.

Chloroquine resistance is associated to multi-copy pvcrt-o gene in Plasmodium vivax malaria in the Brazilian Amazon.

BACKGROUND: The resistance of Plasmodium vivax to chloroquine has become an obstacle to control strategies based on the use of anti-malarials. The current study investigated the association between P. vivax CQ-resistance in vivo with copy number variation and mutations in the promoter region in pvcrt-o and pvmdr1 genes. METHODS: The study included patients with P. vivax that received supervised treatment with chloroquine and primaquine. Recurrences were actively recorded during this period. RESULTS: Among the 60 patients with P. vivax, 25 were CQ-resistant and 35 CQ-susceptible. A frequency of 7.1% of multi-copy pvcrt-o was observed in CQ-susceptible samples and 7.7% in CQ-resistant at D0 (P > 0.05) and 33.3% in CQ-resistant at DR (P < 0.05). For pvmdr1, 10.7% of the CQ-susceptible samples presented multiple copies compared to 11.1% in CQ-resistant at D0 and 0.0% in CQ-resistant at DR (P > 0.05). A deletion of 19 bp was found in 11/23 (47.6%) of the patients with CQ-susceptible P. vivax and 3/10 (23.1%) of the samples with in CQRPv at D0. At day DR, 55.5% of the samples with CQRPv had the 19 bp deletion. For the pvmdr-1 gene, was no variation in the analysed gene compared to the P. vivax reference Sal-1. CONCLUSIONS: This was the first study with 42-day clinical follow-up to evaluate the variation of the number of copies and polymorphisms in the promoter region of the pvcrt-o and pvmdr1 genes in relation to treatment outcomes. Significantly higher frequency of multi-copy pvcrt-o was found in CQRPv samples at DR compared to CQ-susceptible, indicating parasite selection of this genotype after CQ treatment and its association with CQ-resistance in vivo.

TOLLIP gene variant is associated with Plasmodium vivax malaria in the Brazilian Amazon.

BACKGROUND: Toll-interacting protein is a negative regulator in the TLR signaling cascade, particularly by impeding the TLR2 and, TLR4 pathway. Recently, TOLLIP was shown to regulate human TLR signaling pathways. Two common TOLLIP polymorphisms (rs5743899 and rs3750920) were reported to be influencing IL-6, TNF and IL-10 expression. In this study, TOLLIP variants were investigated to their relation to Plasmodium vivax malaria in the Brazilian Amazon. METHODS: This cohort study was performed in the municipalities of Careiro and, Manaus, in Western Brazilian Amazon. A total of 319 patients with P. vivax malaria and, 263 healthy controls with no previous history of malaria were included in the study. Genomic DNA was extracted from blood collected on filter paper, using the QIAamp® DNA Mini Kit, according to the manufacturer's suggested protocol. The rs5743899 and rs3750920 polymorphisms of the TOLLIP gene were typed by PCR-RFLP. RESULTS: Homozygous individuals for the rs3750920 T allele gene had twice the risk of developing malaria when compared to individuals homozygous for the C allele (OR 2.0 [95% CI 1.23-3.07]; p = 0.004). In the dominant model, carriers the C allele indicates protection to malaria, carriers of the C allele were compared to individuals with the T allele, and the difference is highly significant (OR 0.52 [95% CI 0.37-0.76]; p = 0.0006). The linkage disequilibrium between the two polymorphisms was weak (r2 = 0.037; D' = 0.27). CONCLUSIONS: These findings suggest that genes involved in the TLRs-pathway may be involved in malaria susceptibility. The association of the TOLLIP rs3750920 T allele with susceptibility to malaria further provides evidence that genetic variations in immune response genes may predispose individuals to malaria.

G6PD deficiency in Latin America: systematic review on prevalence and variants.

Plasmodium vivax radical cure requires the use of primaquine (PQ), a drug that induces haemolysis in glucose-6-phosphate dehydrogenase deficient (G6PDd) individuals, which further hampers malaria control efforts. The aim of this work was to study the G6PDd prevalence and variants in Latin America (LA) and the Caribbean region. A systematic search of the published literature was undertaken in August 2013. Bibliographies of manuscripts were also searched and additional references were identified. Low prevalence rates of G6PDd were documented in Argentina, Bolivia, Mexico, Peru and Uruguay, but studies from Curaçao, Ecuador, Jamaica, Saint Lucia, Suriname and Trinidad, as well as some surveys carried out in areas of Brazil, Colombia and Cuba, have shown a high prevalence (> 10%) of G6PDd. The G6PD A-202A mutation was the variant most broadly distributed across LA and was identified in 81.1% of the deficient individuals surveyed. G6PDd is a frequent phenomenon in LA, although certain Amerindian populations may not be affected, suggesting that PQ could be safely used in these specific populations. Population-wide use of PQ as part of malaria elimination strategies in LA cannot be supported unless a rapid, accurate and field-deployable G6PDd diagnostic test is made available.

Host metabolomic responses in recurrent P. vivax malaria.

Malaria is the leading parasitic disease worldwide, with P. vivax being a major challenge for its control. Several studies have indicated metabolomics as a promising tool for combating the disease. The study evaluated plasma metabolomic profiles of patients with recurrent and non-recurrent P. vivax malaria in the Brazilian Amazon. Metabolites extracted from the plasma of P. vivax-infected patients were subjected to LC-MS analysis. Untargeted metabolomics was applied to investigate the metabolic profile of the plasma in the two groups. Overall, 51 recurrent and 59 non-recurrent patients were included in the study. Longitudinal metabolomic analysis revealed 52 and 37 significant metabolite features from the recurrent and non-recurrent participants, respectively. Recurrence was associated with disturbances in eicosanoid metabolism. Comparison between groups suggest alterations in vitamin B6 (pyridoxine) metabolism, tyrosine metabolism, 3-oxo-10-octadecatrienoate ß-oxidation, and alkaloid biosynthesis II. Integrative network analysis revealed enrichment of other metabolic pathways for the recurrent phenotype, including the butanoate metabolism, aspartate and asparagine metabolism, and N-glycan biosynthesis. The metabolites and metabolic pathways predicted in our study suggest potential biomarkers of recurrence and provide insights into targets for antimalarial development against P. vivax.

Prevalence of oral trauma in Para-Pan American Games athletes.

The aim of this cross-sectional epidemiological survey was to assess the prevalence of oral trauma in athletes representing 25 countries competing at the most recent Para-Pan American Games (III PARAPAN) held in Rio de Janeiro, Brazil. The study was approved by the appropriate institutional review board. The examiners participated in standardization and calibration training sessions before the field phase began. Invitations were sent to >1200 participating athletes competing in eight sports and to the Medical Committee of the Para-Pan American Sports Organization before and during the III PARAPAN. A convenience sample of 120 athletes was recruited. After signing an informed consent, all athletes answered a questionnaire. Data were collected at the clinical examination and recorded in a specific trauma form. The mean age of the athletes was 32.5 years. Males comprised 79.2% of the sample; females 20.8%. The prevalence of oral trauma among the athletes was 47.5% (N = 57). However, only 15 athletes reported that these traumatic injuries were sports-related. The sport with the highest prevalence of oral trauma was judo (75%); the least was volleyball with no reported traumatic injuries. The most common traumatic injury was enamel fracture (27.4%). The teeth most affected were the maxillary permanent central incisors (N = 19), followed by the maxillary premolars (N = 8). On the basis of the results of this study of oral trauma among athletes examined at the III PARAPAN, a recommendation for enhanced educational efforts and the use of properly fitted mouthguards to prevent traumatic injuries among high-performance athletes with disabilities seems warranted.

Association of CYP2C19, CYP2D6 and CYP3A4 Genetic Variants on Primaquine Hemolysis in G6PD-Deficient Patients.

In the Amazon, the treatment for Plasmodium vivax is chloroquine plus primaquine. However, this regimen is limited due to the risk of acute hemolytic anemia in glucose-6-phosphate dehydrogenase deficiency. Primaquine is a prodrug that requires conversion by the CYP2D6 enzyme to be effective against malaria. A series of cases were performed at an infectious diseases reference hospital in the Western Brazilian Amazon. The STANDARD G6PD (SD Biosensor®) assay was used to infer G6PD status and real-time PCR to genotype G6PD, CYP2C19, CYP2D6 and CYP3A4. Eighteen patients were included, of which 55.6% had African A- variant (G202A/A376G), 11.1% African A+ variant (A376G), 5.6% Mediterranean variant (C563T) and 27.8% were wild type. CYP2C19, CYP2D6 and CYP3A4 genotyping showed no statistically significant differences in the frequency of star alleles between the groups G6PD deficient and G6PD normal. Elevated levels of liver and kidney markers in the G6PDd patients were observed in gNM, gRM and gUM of CYP2C19 and CYP2D6 (p < 0.05). Furthermore, in this study there was no influence of CYPs on hemolysis. These findings reinforce the importance of studies on the mapping of G6PD deficiency and genetic variations of CYP2C19, CYP2D6 and CYP3A4. This mapping will allow us to validate the prevalence of CYPs and determine their influence on hemolysis in patients with malaria, helping to decide on the treatment regimen.

Chemistry and Pharmacology of Bergenin or Its Derivatives: A Promising Molecule.

Bergenin is a glycosidic derivative of trihydroxybenzoic acid that was discovered in 1880 by Garreau and Machelart from the rhizomes of the medicinal plant Bergenia crassifolia (currently: Saxifraga crassifolia-Saxifragaceae), though was later isolated from several other plant sources. Since its first report, it has aroused interest because it has several pharmacological activities, mainly antioxidant and anti-inflammatory. In addition to this, bergenin has shown potential antimalarial, antileishmanial, trypanocidal, antiviral, antibacterial, antifungal, antinociceptive, antiarthritic, antiulcerogenic, antidiabetic/antiobesity, antiarrhythmic, anticancer, hepatoprotective, neuroprotective and cardioprotective activities. Thus, this review aimed to describe the sources of isolation of bergenin and its in vitro and in vivo biological and pharmacological activities. Bergenin is distributed in many plant species (at least 112 species belonging to 34 families). Both its derivatives (natural and semisynthetic) and extracts with phytochemical proof of its highest concentration are well studied, and none of the studies showed cytotoxicity for healthy cells.

Temporal patterns of cytokine and injury biomarkers in hospitalized COVID-19 patients treated with methylprednisolone.

Background: The novel coronavirus disease 2019 (COVID-19) presents with complex pathophysiological effects in various organ systems. Following the COVID-19, there are shifts in biomarker and cytokine equilibrium associated with altered physiological processes arising from viral damage or aggressive immunological response. We hypothesized that high daily dose methylprednisolone improved the injury biomarkers and serum cytokine profiles in COVID-19 patients. Methods: Injury biomarker and cytokine analysis was performed on 50 SARS-Cov-2 negative controls and 101 hospitalized severe COVID-19 patients: 49 methylprednisolone-treated (MP group) and 52 placebo-treated serum samples. Samples from the treated groups collected on days D1 (pre-treatment) all the groups, D7 (2 days after ending therapy) and D14 were analyzed. Luminex assay quantified the biomarkers HMGB1, FABP3, myoglobin, troponin I and NTproBNP. Immune mediators (CXCL8, CCL2, CXCL9, CXCL10, TNF, IFN-γ, IL-17A, IL-12p70, IL-10, IL-6, IL-4, IL-2, and IL-1ß) were quantified using cytometric bead array. Results: At pretreatment, the two treatment groups were comparable demographically. At pre-treatment (D1), injury biomarkers (HMGB1, TnI, myoglobin and FABP3) were distinctly elevated. At D7, HMGB1 was significantly higher in the MP group (p=0.0448) compared to the placebo group, while HMGB1 in the placebo group diminished significantly by D14 (p=0.0115). Compared to healthy control samples, several immune mediators (IL-17A, IL-6, IL-10, MIG, MCP-1, and IP-10) were considerably elevated at baseline (all p≤0.05). At D7, MIG and IP-10 of the MP-group were significantly lower than in the placebo-group (p=0.0431, p=0.0069, respectively). Longitudinally, IL-2 (MP-group) and IL-17A (placebo-group) had increased significantly by D14. In placebo group, IL-2 and IL-17A continuously increased, as IL-12p70, IL-10 and IP-10 steadily decreased during follow-up. The MP treated group had IL-2, IFN-γ, IL-17A and IL-12p70 progressively increase while IL-1ß and IL-10 gradually decreased towards D14. Moderate to strong positive correlations between chemokines and cytokines were observed on D7 and D14. Conclusion: These findings suggest MP treatment could ameliorate levels of myoglobin and FABP3, but appeared to have no impact on HMGB1, TnI and NTproBNP. In addition, methylprednisolone relieves the COVID-19 induced inflammatory response by diminishing MIG and IP-10 levels. Overall, corticosteroid (methylprednisolone) use in COVID-19 management influences the immunological molecule and injury biomarker profile in COVID-19 patients.

Pharmacokinetics of chloroquine in patients with malaria by P. vivax from the Western Brazilian Amazon basin.

The western Amazon basin is an important endemic area for malaria by P. vivax. In recent years, several reports showed the treatment failure with chloroquine, which can be related to resistance. The assessment of chloroquine resistance requires the evaluation of drug exposure, and when possible, the estimation of the pharmacokinetic parameters. However, there is no data on the pharmacokinetics of chloroquine in this endemic area. Moreover, the influence of the early reappearance of parasites in blood on the exposure to the drug was low exploited in the literature. The present study described the pharmacokinetic parameters of chloroquine in whole blood of adult patients with P. vivax malaria from the western Brazilian Amazon basin and compared the area under the curve (AUC) with the parasitological outcome at day 28. A total of 19 patients with parasite recurrence within 28 days and 20 patients with no recurrence were included in the study. Chloroquine was measured by high-performance liquid chromatography (HPLC). The pharmacokinetic parameters were estimated by non-compartmental modeling. The maximum concentration ranged from 1285 to 2030 ng/mL. The terminal half-life varied from 5.3 to 12.8 days. The volume of distribution from 1090 to 2340 L/kg, and the area under the curve to the last measurable concentration from 247 to 432 ng/mL.h. The pharmacokinetic parameters were similar in both groups, which suggests the lack of influence of early reappearance of parasites on chloroquine pharmacokinetics.

Prevalence of glucose 6-phosphate dehydrogenase deficiency in highly malaria-endemic municipalities in the Brazilian Amazon: A region-wide screening study.

Background: Difficulties associated with the assessment of glucose-6-phosphate dehydrogenase deficiency (G6PDd), particularly in remote areas, hinders the safe use of 8-aminoquinolines such as primaquine (PQ) and tafenoquine against Plasmodium vivax malaria due to the risk of haemolysis. Methods: This cross-sectional study was conducted in 41 malaria-endemic municipalities of six states in the Brazilian Amazon, between 2014 and 2018. Male individuals were screened for G6PDd using the qualitative Fluorescent Spot Test using fingerpick-collected whole blood samples. Point and interval estimates of the G6PDd prevalence were calculated for each state. Deficient samples were genotyped for the most prevalent variants in the Amazon. Frequencies of P. vivax malaria recurrences were estimated for G6PDd and non-G6PDd patients. Interpretation: This is one of the largest surveys ever conducted in Latin America, covering the entire malaria endemic area in the Brazilian Amazon. These results indicate that an important proportion of the population is at risk of hemolysis if exposed to PQ and its congener drug tafenoquine. The adoption of G6PDd screening protocols is essential to ensure the safety of individuals treated with those drugs and should also be considered when implementing malaria elimination strategies. Findings: A total of 14,847 individuals were included, of which 5.6% presented G6PDd. The state of Acre had the highest G6PDd prevalence (8.3%), followed by Amapá (5.8%), Pará (5.7%), Rondônia (5.4%), Roraima (4.2%) and Amazonas (4.0%). From 828 genotyped samples, African A+ (6.2%), African A- (39.3%) and wild-type (non-African non-Mediterranean; 54.2%) variants were found. A greater proportion of malaria recurrences was found among G6PD deficient individuals [16.7% vs 4.1%, Risk ratio 3.52 (2.16-5.74) p < 0.01]. Funding: Brazilian Ministry of Health; Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM).

Identification of the asymptomatic Plasmodium falciparum and Plasmodium vivax gametocyte reservoir under different transmission intensities.

BACKGROUND: Understanding epidemiological variables affecting gametocyte carriage and density is essential to design interventions that most effectively reduce malaria human-to-mosquito transmission. METHODOLOGY/PRINCIPAL FINDINGS: Plasmodium falciparum and P. vivax parasites and gametocytes were quantified by qPCR and RT-qPCR assays using the same methodologies in 5 cross-sectional surveys involving 16,493 individuals in Brazil, Thailand, Papua New Guinea, and Solomon Islands. The proportion of infections with detectable gametocytes per survey ranged from 44-94% for P. falciparum and from 23-72% for P. vivax. Blood-stage parasite density was the most important predictor of the probability to detect gametocytes. In moderate transmission settings (prevalence by qPCR>5%), parasite density decreased with age and the majority of gametocyte carriers were children. In low transmission settings (prevalence<5%), >65% of gametocyte carriers were adults. Per survey, 37-100% of all individuals positive for gametocytes by RT-qPCR were positive by light microscopy for asexual stages or gametocytes (overall: P. falciparum 178/348, P. vivax 235/398). CONCLUSIONS/SIGNIFICANCE: Interventions to reduce human-to-mosquito malaria transmission in moderate-high endemicity settings will have the greatest impact when children are targeted. In contrast, all age groups need to be included in control activities in low endemicity settings to achieve elimination. Detection of infections by light microscopy is a valuable tool to identify asymptomatic blood stage infections that likely contribute most to ongoing transmission at the time of sampling.