Associations between COVID-19 and Glucose-6-Phosphate Dehydrogenase Activity in Brazil.

Glucose-6 phosphate dehydrogenase deficiency (G6PDd) was suggested as a risk factor for severe disease in patients with COVID-19. We evaluated clinical outcomes and glucose-6 phosphate dehydrogenase (G6PD) activity during and after illness in patients with COVID-19. This prospective cohort study included adult participants (≥ 18 years old) who had clinical and/or radiological COVID-19 findings or positive reverse transcription-polymerase chain reaction results. Epidemiological and clinical data were extracted from electronic medical records. Glucose-6 phosphate dehydrogenase activity was measured using SD Biosensor STANDARD G6PD® equipment on admission and 1 year after discharge. Samples were genotyped for the three most common single nucleotide polymorphisms for G6PDd in the Brazilian Amazon. Seven hundred fifty-three patients were included, of whom 123 (16.3%) were G6PD deficient. There was no difference between groups regarding the risks of hospitalization (P = 0.740) or invasive mechanical ventilation (P = 0.31), but the risk of death was greater in patients with normal G6PD levels (P = 0.022). Only 29 of 116 participants (25%) carried the African G6PDd genotype. Of 30 participants tested as G6PD deficient during disease, only 11 (36.7%) results agreed 1 year after discharge. In conclusion, this study does not demonstrate an association of G6PDd with severity of COVID-19. Limitations of the test for detecting enzyme levels during COVID-19 illness were demonstrated by genotyping and retesting after the disease period. Care must be taken when screening for G6PDd in patients with acute COVID-19.

Low handgrip strength is associated with worse functional outcomes in long COVID.

The diagnosis of long COVID is troublesome, even when functional limitations are present. Dynapenia is the loss of muscle strength and power production that is not caused by neurologic or muscular diseases, being mostly associated with changes in neurologic function and/or the intrinsic force-generating properties of skeletal muscle, which altogether, may partially explain the limitations seen in long COVID. This study aimed to identify the distribution and possible associations of dynapenia with functional assessments in patients with long COVID. A total of 113 patients with COVID-19 were evaluated by functional assessment 120 days post-acute severe disease. Body composition, respiratory muscle strength, spirometry, six-minute walk test (6MWT, meters), and hand-grip strength (HGS, Kilogram-force) were assessed. Dynapenia was defined as HGS < 30 Kgf (men), and < 20 Kgf (women). Twenty-five (22%) participants were dynapenic, presenting lower muscle mass (p < 0.001), worse forced expiratory volume in the first second (FEV1) (p = 0.0001), lower forced vital capacity (p < 0.001), and inspiratory (p = 0.007) and expiratory (p = 0.002) peek pressures, as well as worse 6MWT performance (p < 0.001). Dynapenia, independently of age, was associated with worse FEV1, maximal expiratory pressure (MEP), and 6MWT, (p < 0.001) outcomes. Patients with dynapenia had higher intensive care unit (ICU) admission rates (p = 0.01) and need for invasive mechanical ventilation (p = 0.007) during hospitalization. The HGS is a simple, reliable, and low-cost measurement that can be performed in outpatient clinics in low- and middle-income countries. Thus, HGS may be used as a proxy indicator of functional impairment in this population.

Standardization of DNA extraction from paraffinized spleen samples: molecular diagnosis of human malaria.

BACKGROUND: Plasmodium vivax is the main species responsible for human malaria in Brazil, and one of its manifestations is splenic malaria, though there are still challenges in its diagnosis. The present study aimed to standardize Plasmodium sp. DNA extraction from histological slices of spleen and diagnosis using real-time qPCR. METHODS: This study performed a microtomy of a paraffin-embedded spleen as a positive control for P. vivax from a patient who had been previously diagnosed with the parasite. The sample was deparaffinized with xylol and ethanol, then DNA extraction was performed with two commercial kits. qPCR was carried out with the Taqman system for detection of Plasmodium sp. and was made species-specific using PvmtCOX1 gene. From 2015 to 2019, 200 spleen samples were obtained from trauma patients subjected to splenectomy in Manaus, Amazonas. All the samples were tested for cell-free human DNA (cfDNA). RESULTS: The deparaffinization and the Plasmodium vivax DNA extraction method was successfully standardized, and the control sample was positive for P. vivax. Of the 200 samples, all qPCRs were negative, but they were positive for human PCR. CONCLUSION: Paraffinization is practical and efficient for the preservation of samples, but the formation of bonds between proteins and DNA makes extraction difficult. Despite this, in this study, it was possible to standardize a method of DNA extraction for detecting P. vivax.

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.

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.

Antigen-Specific Antibody Signature Is Associated with COVID-19 Outcome.

Numerous studies have focused on inflammation-related markers to understand COVID-19. In this study, we performed a comparative analysis of spike (S) and nucleocapsid (N) protein-specific IgA, total IgG and IgG subclass response in COVID-19 patients and compared this to their disease outcome. We observed that the SARS-CoV-2 infection elicits a robust IgA and IgG response against the N-terminal (N1) and C-terminal (N3) region of the N protein, whereas we failed to detect IgA antibodies and observed a weak IgG response against the disordered linker region (N2) in COVID-19 patients. N and S protein-specific IgG1, IgG2 and IgG3 response was significantly elevated in hospitalized patients with severe disease compared to outpatients with non-severe disease. IgA and total IgG antibody reactivity gradually increased after the first week of symptoms. Magnitude of RBD-ACE2 blocking antibodies identified in a competitive assay and neutralizing antibodies detected by PRNT assay correlated with disease severity. Generally, the IgA and total IgG response between the discharged and deceased COVID-19 patients was similar. However, significant differences in the ratio of IgG subclass antibodies were observed between discharged and deceased patients, especially towards the disordered linker region of the N protein. Overall, SARS-CoV-2 infection is linked to an elevated blood antibody response in severe patients compared to non-severe patients. Monitoring of antigen-specific serological response could be an important tool to accompany disease progression and improve outcomes.

Metabolomic Profiling of Plasma Reveals Differential Disease Severity Markers in COVID-19 Patients.

The severity, disabilities, and lethality caused by the coronavirus 2019 (COVID-19) disease have dumbfounded the entire world on an unprecedented scale. The multifactorial aspect of the infection has generated interest in understanding the clinical history of COVID-19, particularly the classification of severity and early prediction on prognosis. Metabolomics is a powerful tool for identifying metabolite signatures when profiling parasitic, metabolic, and microbial diseases. This study undertook a metabolomic approach to identify potential metabolic signatures to discriminate severe COVID-19 from non-severe COVID-19. The secondary aim was to determine whether the clinical and laboratory data from the severe and non-severe COVID-19 patients were compatible with the metabolomic findings. Metabolomic analysis of samples revealed that 43 metabolites from 9 classes indicated COVID-19 severity: 29 metabolites for non-severe and 14 metabolites for severe disease. The metabolites from porphyrin and purine pathways were significantly elevated in the severe disease group, suggesting that they could be potential prognostic biomarkers. Elevated levels of the cholesteryl ester CE (18:3) in non-severe patients matched the significantly different blood cholesterol components (total cholesterol and HDL, both p < 0.001) that were detected. Pathway analysis identified 8 metabolomic pathways associated with the 43 discriminating metabolites. Metabolomic pathway analysis revealed that COVID-19 affected glycerophospholipid and porphyrin metabolism but significantly affected the glycerophospholipid and linoleic acid metabolism pathways (p = 0.025 and p = 0.035, respectively). Our results indicate that these metabolomics-based markers could have prognostic and diagnostic potential when managing and understanding the evolution of COVID-19.

HIV infection increases the risk of acquiring Plasmodium vivax malaria: a 4-year cohort study in the Brazilian Amazon HIV and risk of vivax malaria.

Globally, malaria and human immunodeficiency virus (HIV) are both independently associated with a massive burden of disease and death. While their co-infection has been well studied for Plasmodium falciparum, scarce data exist regarding the association of P. vivax and HIV. In this cohort study, we assessed the effect of HIV on the risk of vivax malaria infection and recurrence during a 4-year follow-up period in an endemic area of the Brazilian Amazon. For the purpose of this study, we obtained clinical information from January 2012 to December 2016 from two databases. HIV screening data were acquired from the clinical information system at the tropical hospital Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD). The National Malaria Surveillance database (SIVEP malaria) was utilized to identify malaria infections during a 4-year follow-up period after diagnosis of HIV. Both datasets were combined via data linkage. Between 2012 and 2016, a total of 42,121 people were screened for HIV, with 1569 testing positive (3.7%). Out of all the patients diagnosed with HIV, 198 had at least one episode of P. vivax malaria in the follow-up. In the HIV-negative group, 711 participants had at least one P. vivax malaria episode. When comparing both groups, HIV patients had a 6.48 [(5.37-7.83); P < 0.0001] (adjusted relative risk) greater chance of acquiring P. vivax malaria. Moreover, being of the male gender [ARR = 1.41 (1.17-1.71); P < 0.0001], Amerindian ethnicity [ARR = 2.77 (1.46-5.28); P < 0.0001], and a resident in a municipality of the Metropolitan region of Manaus [ARR = 1.48 (1.02-2.15); P = 0.038] were independent risk factors associated with an increased risk of clinical malaria. Education ≥ 8 years [ARR = 0.41 (0.26-0.64); P < 0.0001] and living in the urban area [ARR = 0.44 (0.24-0.80); P = 0.007] were associated to a lower risk of P. vivax malaria. A total of 28 (14.1%) and 180 (25.3%) recurrences (at least a second clinical malaria episode) were reported in the HIV-positive and HIV-negative groups, respectively. After adjusting for sex and education, HIV-positive status was associated with a tendency towards protection from P. vivax malaria recurrences [ARR = 0.55 (0.27-1.10); P = 0.090]. HIV status was not associated with hospitalizations due to P. vivax malaria. CD4 + counts and viral load were not associated with recurrences of P. vivax malaria. No significant differences were found in the distribution of parasitemia between HIV-negative and HIV-positive P. vivax malaria patients. Our results suggest that HIV-positive status is a risk factor for vivax malaria infection, which represents an additional challenge that should be addressed during elimination efforts.

Effect of weekly versus daily primaquine on Plasmodium vivax malaria recurrences: A real-life cohort study.

BACKGROUND: Although primaquine (PQ) is indicated for G6PD-deficient patients, data on weekly PQ use in Brazil are limited. METHODS: We aimed to investigate malaria recurrences among participants receiving daily and weekly PQ treatments in a real-life setting of two municipalities in the Amazon between 2019 and 2020. RESULTS: Patients receiving weekly PQ treatment had a lower risk of recurrence than those receiving daily PQ treatment (risk ratio: 0.62, 95% confidence interval: 0.41-0.94), using a model adjusted for study site. CONCLUSIONS: Weekly PQ use did not increase the risk of malaria recurrence. Further studies with larger populations are warranted.

Spread of Gamma (P.1) Sub-Lineages Carrying Spike Mutations Close to the Furin Cleavage Site and Deletions in the N-Terminal Domain Drives Ongoing Transmission of SARS-CoV-2 in Amazonas, Brazil.

The Amazonas was one of the most heavily affected Brazilian states by the COVID-19 epidemic. Despite a large number of infected people, particularly during the second wave associated with the spread of the Variant of Concern (VOC) Gamma (lineage P.1), SARS-CoV-2 continues to circulate in the Amazonas. To understand how SARS-CoV-2 persisted in a human population with a high immunity barrier, we generated 1,188 SARS-CoV-2 whole-genome sequences from individuals diagnosed in the Amazonas state from 1st January to 6th July 2021, of which 38 were vaccine breakthrough infections. Our study reveals a sharp increase in the relative prevalence of Gamma plus (P.1+) variants, designated Pango Lineages P.1.3 to P.1.6, harboring two types of additional Spike changes: deletions in the N-terminal (NTD) domain (particularly Δ144 or Δ141-144) associated with resistance to anti-NTD neutralizing antibodies or mutations at the S1/S2 junction (N679K or P681H) that probably enhance the binding affinity to the furin cleavage site, as suggested by our molecular dynamics simulations. As lineages P.1.4 (S:N679K) and P.1.6 (S:P681H) expanded (Re > 1) from March to July 2021, the lineage P.1 declined (Re < 1) and the median Ct value of SARS-CoV-2 positive cases in Amazonas significantly decreases. Still, we did not find an increased incidence of P.1+ variants among breakthrough cases of fully vaccinated patients (71%) in comparison to unvaccinated individuals (93%). This evidence supports that the ongoing endemic transmission of SARS-CoV-2 in the Amazonas is driven by the spread of new local Gamma/P.1 sublineages that are more transmissible, although not more efficient to evade vaccine-elicited immunity than the parental VOC. Finally, as SARS-CoV-2 continues to spread in human populations with a declining density of susceptible hosts, the risk of selecting more infectious variants or antibody evasion mutations is expected to increase. IMPORTANCE The continuous evolution of SARS-CoV-2 is an expected phenomenon that will continue to happen due to the high number of cases worldwide. The present study analyzed how a Variant of Concern (VOC) could still circulate in a population hardly affected by two COVID-19 waves and with vaccination in progress. Our results showed that the answer behind that was a new generation of Gamma-like viruses, which emerged locally carrying mutations that made it more transmissible and more capable of spreading, partially evading prior immunity triggered by natural infections or vaccines. With thousands of new cases daily, the current pandemics scenario suggests that SARS-CoV-2 will continue to evolve and efforts to reduce the number of infected subjects, including global equitable access to COVID-19 vaccines, are mandatory. Thus, until the end of pandemics, the SARS-CoV-2 genomic surveillance will be an essential tool to better understand the drivers of the viral evolutionary process.

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).

Effect of weekly versus daily primaquine on Plasmodium vivax malaria recurrences: A real-life cohort study

ABSTRACT Background: Although primaquine (PQ) is indicated for G6PD-deficient patients, data on weekly PQ use in Brazil are limited. Methods: We aimed to investigate malaria recurrences among participants receiving daily and weekly PQ treatments in a real-life setting of two municipalities in the Amazon between 2019 and 2020. Results: Patients receiving weekly PQ treatment had a lower risk of recurrence than those receiving daily PQ treatment (risk ratio: 0.62, 95% confidence interval: 0.41-0.94), using a model adjusted for study site. Conclusions: Weekly PQ use did not increase the risk of malaria recurrence. Further studies with larger populations are warranted.

Short-Course of Methylprednisolone Improves Respiratory Functional Parameters After 120 Days in Hospitalized COVID-19 Patients (Metcovid Trial): A Randomized Clinical Trial.

Background: The use of corticosteroids may help control the cytokine storm occurring in acute respiratory failure due to the severe form of COVID-19. We evaluated the postacute effect of corticosteroids used during the acute phase, such as impairment in pulmonary function parameters, on day 120 (D120)-follow-up, in participants who survived over 28 days. Methods: This is a parallel, double-blind, randomized, placebo-controlled phase IIb clinical trial carried out between April 18 and October 9, 2020, conducted in hospitalized patients with clinical-radiological suspicion of COVID-19, aged 18 years or older, with SpO2 ≤ 94% on room air or requiring supplementary oxygen, or under invasive mechanical ventilation (IMV) in a referral center in Manaus, Western Brazilian Amazon. Intravenous methylprednisolone (MP) (0.5 mg/kg) was given two times daily for 5 days to these patients. The primary outcome used for this study was pulmonary function testing at day 120 follow-up visit. Results: Out of the total of surviving patients at day 28 (n = 246) from the Metcovid study, a total of 118 underwent satisfactory pulmonary function testing (62 in the placebo arm and 56 in the MP arm). The supportive treatment was similar between the placebo and MP groups (seven [11%] vs. four [7%]; P = 0.45). At hospital admission, IL-6 levels were higher in the MP group (P < 0.01). Also, the need for ICU (P = 0.06), need for IMV (P = 0.07), and creatine kinase (P = 0.05) on admission also tended to be higher in this group. In the univariate analysis, forced expiratory volume on 1st second of exhalation (FEV1) and forced vital capacity (FVC) at D120 follow-up were significantly higher in patients in the MP arm, being this last parameter also significantly higher in the multivariate analysis independently of IMV and IL-6 levels on admission. Conclusion: The use of steroids for at least 5 days in severe COVID-19 was associated with a higher FVC, which suggests that hospitalized COVID-19 patients might benefit from the use of MP in its use in the long-term, with less pulmonary restrictive functions, attributed to fibrosis. Trial Registration: ClinicalTrials.gov, Identifier: NCT04343729.

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.

Plasmodium vivax metacaspase 1 (PvMCA1) catalytic domain is conserved in field isolates from Brazilian Amazon.

In the present study, we investigated the genetic diversity of Plasmodium vivax metacaspase 1 (PvMCA1) catalytic domain in two municipalities of the main malaria hotspot in Brazil, i.e., the Juruá Valley, and observed complete sequence identity among all P. vivax field isolates and the Sal-1 reference strain. Analysis of PvMCA1 catalytic domain in different P. vivax genomic sequences publicly available also revealed a high degree of conservation worldwide, with very few amino acid substitutions that were not related to putative histidine and cysteine catalytic residues, whose involvement with the active site of protease was herein predicted by molecular modeling. The genetic conservation presented by PvMCA1 may contribute to its eligibility as a druggable target candidate in vivax malaria.

Pathological findings and morphologic correlation of the lungs of autopsied patients with SARS-CoV-2 infection in the Brazilian Amazon using transmission electron microscopy.

INTRODUCTION: Electron microscopy (EM) is a rapid and effective tool that can be used to create images of a whole spectrum of virus-host interactions and, as such, has long been used in the discovery and description of viral mechanisms. METHODS: Electron microscopy was used to evaluate the pulmonary pathologies of postmortem lung sections from three patients who died from infection with SARS-associated coronavirus 2 (SARS-CoV-2), a new member of the Coronaviridae family. RESULTS: Diffuse alveolar damage (DAD) was predominant in all three patients. The early exudative stage was characterized principally by edema and extravasation of red blood cells into the alveolar space with injury to the alveolar epithelial cells; this was followed by detachment, apoptosis, and necrosis of type I and II pneumocytes. The capillaries exhibited congestion, exposure of the basement membrane from denuded endothelial cells, platelet adhesion, fibrin thrombi, and rupture of the capillary walls. The proliferative stage was characterized by pronounced proliferation of type II alveolar pneumocytes and multinucleated giant cells. The cytopathic effect of SARS-CoV-2 was observed both in degenerated type II pneumocytes and freely circulating in the alveoli, with components from virions, macrophages, lymphocytes, and cellular debris. CONCLUSIONS: Viral particles consistent with the characteristics of SARS-CoV-2 were observed mainly in degenerated pneumocytes, in the endothelium, or freely circulating in the alveoli. In the final stage of illness, the alveolar spaces were replaced by fibrosis.

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).

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.

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.