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.

Inflammatory Profile Associated with Secondary Infection from Bothrops atrox Snakebites in the Brazilian Amazon.

Bothrops snakebite envenomation (SBE) is consider an important health problem in Brazil, where Bothrops atrox is mainly responsible in the Brazilian Amazon. Local effects represent a relevant clinical issue, in which inflammatory signs and symptoms in the bite site represent a potential risk for short and long-term disabilities. Among local complications, secondary infections (SIs) are a common clinical finding during Bothrops atrox SBE and are described by the appearance of signs such as abscess, cellulitis or necrotizing fasciitis in the affected site. However, the influence of SI in the local events is still poorly understood. Therefore, the present study describes for the first time the impact of SBE wound infection on local manifestations and inflammatory response from patients of Bothrops atrox SBE in the Brazilian Amazon. This was an observational study carried out at the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus (Brazil), involving victims of Bothrops SBE. Clinical and laboratorial data were collected along with blood samples for the quantification of circulating cytokines and chemokines before antivenom administrations (T0) and 24 h (T1), 48 h (T2), 72 h (T3) and 7 days after (T4). From the 94 patients included in this study, 42 presented SI (44.7%) and 52 were without SI (NSI, 55.3%). Patients classified as moderate envenoming presented an increased risk of developing SI (OR = 2.69; CI 95% = 1.08-6.66, p = 0.033), while patients with bites in hands showed a lower risk (OR = 0.20; CI 95% = 0.04-0.96, p = 0.045). During follow-up, SI patients presented a worsening of local temperature along with a sustained profile of edema and pain, while NSI patients showed a tendency to restore and were highlighted in patients where SI was diagnosed at T2. As for laboratorial parameters, leukocytes, erythrocyte sedimentation ratio, fibrinogen and C-reactive protein were found increased in patients with SI and more frequently in patients diagnosed with SI at T3. Higher levels of circulating IL-2, IL-10, IL-6, TNF, INF-γ and CXCL-10 were observed in SI patients along with marked correlations between these mediators and IL-4 and IL-17, showing a plurality in the profile with a mix of Th1/Th2/Th17 response. The present study reports for the first time the synergistic effects of local infection and envenoming on the inflammatory response represented by local manifestations, which reflected on laboratorial parameters and inflammatory mediators and thus help improve the clinical management of SI associated to Bothrops SBE.

Exploring the Profile of Cell Populations and Soluble Immunological Mediators in Bothrops atrox Envenomations.

Bothrops atrox envenomations are common in the Brazilian Amazon. The venom of B. atrox is highly inflammatory, which results in severe local complications, including the formation of blisters. Moreover, there is little information on the immune mechanisms associated with this condition. Thus, a longitudinal study was carried out to characterize the profile of the cell populations and soluble immunological mediators in the peripheral blood and blisters in B. atrox patients s according to their clinical manifestations (mild and severe). A similar response in both B. atrox patient groups (MILD and SEV) was observed, with an increase in inflammatory monocytes, NKT, and T and B cells, as well as CCL2, CCL5, CXCL9, CXCL10, IL-1ß and IL-10, when compared with the group of healthy blood donors. After the administration of antivenom, the participation of patrolling monocytes and IL-10 in the MILD group was observed. In the SEV group, the participation of B cells was observed, with high levels of CCL2 and IL-6. In the blister exudate, a hyperinflammatory profile was observed. In conclusion, we revealed the involvement of cell populations and soluble mediators in the immune response to B. atrox envenomation at the local and peripheral level, which is related to the onset and extent of the inflammation/clinical manifestation.

Pediatric central nervous system infections in the Amazon: clinical and laboratory profiles.

Background: Central nervous system (CNS) infections are important causes of mortality and morbidity in children, and they are related to severe problems such as hearing loss, neurological sequelae, and death. The objective was to describe clinical and laboratory exam profiles of children who were diagnosed with CNS infections. Methods: We conducted a cross-sectional study based on medical records, which included pediatric patients aged from 3 months to 15 years, with a clinical suspicion of CNS infection between January 2014 to December 2019. The pathogens were confirmed in cerebrospinal fluid (CSF) samples using Gram staining, cell culture, molecular diagnostics (PCR and qPCR), and serology. Results: Out of the 689 enrolled patients, 108 (15.6%) had laboratory-confirmed infections in CSF. The most common bacterial pathogens isolated from the culture were Neisseria meningitidis serogroup C in 19, Streptococcus pneumoniae in 11, and Haemophilus influenzae in seven samples. The viruses identified were Enterovirus, Cytomegalovirus, Varicella-zoster virus, Epstein-Barr virus, and arbovirus. No patient was found to be positive for Herpes simplex virus 1 and 2. Patients with viral infections showed altered levels of consciousness (p = 0.001) when compared to bacterial infections. Conclusion: This study shows the presence of important vaccine-preventable pathogens, and different families of viruses causing CNS infections in the pediatric patients of Manaus.

Molecular diagnosis of opportunistic infections in the central nervous system of HIV-infected adults in Manaus, Amazonas.

Background: Opportunistic infections in the central nervous system (CNS) of people with HIV/AIDS (PLWHA) remain significant contributors to morbidity and mortality, especially in resource-limited scenarios. Diagnosing these infections can be challenging, as brain imaging is non-specific and expensive. Therefore, molecular analysis of cerebrospinal fluid (CSF) may offer a more accurate and affordable method for diagnosing pathogens. Methods: We conducted extensive real-time PCR testing (qPCR) on CSF to evaluate etiological agents in PLWHA with neurological manifestations. Primers targeting DNA from specific pathogens, including cytomegalovirus (CMV), herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), John Cunningham virus (JCV), Toxoplasma gondii, and human T-lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2), were used. Results: Cerebrospinal fluid samples revealed 90 pathogens (36.7%). Toxoplasma gondii was the most frequently detected pathogen, found in 22 samples (30.5%). Other pathogens included Cryptococcus sp. (7.7%), EBV (5.3%), CMV, VZV, and JCV (4.0% each). Conclusion: Despite antiretroviral therapy and medical follow-up, opportunistic central nervous system infections remain frequent in PLWHA. Herpesviruses are commonly detected, but T. gondii is the most prevalent opportunistic pathogen in our study population. Therefore, molecular diagnosis is a crucial tool for identifying opportunistic infections, even in patients undergoing treatment.

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.

Early Antiretroviral Therapy in AIDS Patients Presenting With Toxoplasma gondii Encephalitis Is Associated With More Sequelae but Not Increased Mortality.

Background: Evidence on the optimal time to initiate antiretroviral therapy (ART) in the presence of toxoplasmic encephalitis (TE) is scarce. We compared the impact of early vs. delayed ART initiation on mortality and neurologic complications at discharge in a Brazilian population co-infected with HIV and TE. Methods: We retrospectively evaluated data from 9 years of hospitalizations at a referral center in Manaus, Amazonas. All ART-naïve hospitalized patients were divided into early initiation treatment (EIT) (0-4 weeks) and delayed initiation treatment (DIT) (>4 weeks). The groups were compared using chi-square test and mortality at 16 weeks. Results: Four hundred sixty nine patients were included, of whom 357 (76.1%) belonged to the EIT group. The median CD4+ lymphocyte count and CD4+/CD8+ ratio were 53 cells/mm3 and 0.09, respectively. Mortality rate and presence of sequelae were 4.9% (n = 23) and 41.6% (n = 195), respectively. Mortality was similar between groups (p = 0.18), although the EIT group had the highest prevalence of sequelae at discharge (p = 0.04). The hazard ratio for death at 16 weeks with DIT was 2.3 (p = 0.18). The necessity for intensive care unit admission, mechanical ventilation, and cardiopulmonary resuscitation were similar between groups. Conclusion: In patients with AIDS and TE, early ART initiation might have a detrimental influence on the occurrence of sequelae.

Correlation Between Clinical and Pathological Findings of Liver Injury in 27 Patients With Lethal COVID-19 Infections in Brazil.

Liver test abnormalities are frequently observed in patients with coronavirus disease 2019 (COVID-19) and are associated with worse prognosis. However, information is limited about pathological changes in the liver in this infection, so the mechanism of liver injury is unclear. Here we describe liver histopathology and clinical correlates of 27 patients who died of COVID-19 in Manaus, Brazil. There was a high prevalence of liver injury (elevated alanine aminotransferase and aspartate aminotransferase in 44% and 48% of patients, respectively) in these patients. Histological analysis showed sinusoidal congestion and ischemic necrosis in more than 85% of the cases, but these appeared to be secondary to systemic rather than intrahepatic thrombotic events, as only 14% and 22% of samples were positive for CD61 (marker of platelet activation) and C4d (activated complement factor), respectively. Furthermore, the extent of these vascular findings did not correlate with the extent of transaminase elevations. Steatosis was present in 63% of patients, and portal inflammation was present in 52%. In most cases, hepatocytes expressed angiotensin-converting enzyme 2 (ACE2), which is responsible for binding and entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), even though this ectoenzyme was minimally expressed on hepatocytes in normal controls. However, SARS-CoV-2 staining was not observed. Most hepatocytes also expressed inositol 1,4,5-triphosphate receptor 3 (ITPR3), a calcium channel that becomes expressed in acute liver injury. Conclusion: The hepatocellular injury that commonly occurs in patients with severe COVID-19 is not due to the vascular events that contribute to pulmonary or cardiac damage. However, new expression of ACE2 and ITPR3 with concomitant inflammation and steatosis suggests that liver injury may result from inflammation, metabolic abnormalities, and perhaps direct viral injury.

A prenylated dsRNA sensor protects against severe COVID-19.

Inherited genetic factors can influence the severity of COVID-19, but the molecular explanation underpinning a genetic association is often unclear. Intracellular antiviral defenses can inhibit the replication of viruses and reduce disease severity. To better understand the antiviral defenses relevant to COVID-19, we used interferon-stimulated gene (ISG) expression screening to reveal that 2'-5'-oligoadenylate synthetase 1 (OAS1), through ribonuclease L, potently inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We show that a common splice-acceptor single-nucleotide polymorphism (Rs10774671) governs whether patients express prenylated OAS1 isoforms that are membrane-associated and sense-specific regions of SARS-CoV-2 RNAs or if they only express cytosolic, nonprenylated OAS1 that does not efficiently detect SARS-CoV-2. In hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting that this antiviral defense is a major component of a protective antiviral response.

Hemorrhagic and thrombotic manifestations in the central nervous system in COVID-19: A large observational study in the Brazilian Amazon with a complete autopsy series.

SARS-CoV-2 affects mainly the lungs, however, other manifestations, including neurological manifestations, have also been described during the disease. Some of the neurological findings have involved intracerebral or subarachnoid hemorrhage, strokes, and other thrombotic/hemorrhagic conditions. Nevertheless, the gross pathology of hemorrhagic lesions in the central nervous system has not been previously described in Brazilian autopsy cases. This study aimed to describe gross and microscopic central nervous system (CNS) pathology findings from the autopsies and correlate them with the clinical and laboratory characteristics of forty-five patients with COVID-19 from Manaus, Amazonas, Brazil. Forty-four patients were autopsied of which thirty-eight of these (86.36%) were positive by RT-PCR for COVID-19, and six (13.3%) were positive by the serological rapid test. Clinical and radiological findings were compatible with the infection. The patients were classified in two groups: presence (those who had hemorrhagic and/or thrombotic manifestations in the CNS) and absence (those who did not present hemorrhagic and/or thrombotic manifestations in the CNS). For risk assessment, relative risk and respective confidence intervals were estimated. Macroscopic or microscopic hemorrhages were found in twenty-three cases (52,27%). The postmortem gross examination of the brain revealed a broad spectrum of hemorrhages, from spots to large and confluent areas and, under microscopy, we observed mainly perivascular discharge. The association analyses showed that the use of corticosteroid, anticoagulant and antibiotic had no statistical significance with a risk of nervous system hemorrhagic manifestations. However, it is possible to infer a statistical tendency that indicates that individuals with diabetes had a higher risk for the same outcome (RR = 1.320, 95% CI = 0.7375 to 2.416, p = 0.3743), which was not observed in relation to other comorbidities. It is unknown whether the new variants of the virus can cause different clinical manifestations, such as those observed or indeed others. As a result, more studies are necessary to define clinical and radiologic monitoring protocols and strategic interventions for patients at risk of adverse and fatal events, such as the extensive hemorrhaging described here. It is imperative that clinicians must be aware of comorbidities and the drugs used to treat patients with COVID-19 to prevent CNS hemorrhagic and thrombotic events.

Variants *1, *2, *3 and *17 of CYP2C19 Cytochrome among Helicobacter Pylori Carriers from Manaus, Amazonas State, Brazil.

OBJECTIVE: The objective of the present study was to estimate the frequency of CYP2C19 cytochrome variants *1, *2, *3 and *17 among Helicobacter pylori carriers from Manaus, Amazonas state, who were treated at Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD). METHODS: From the 78 recruited individuals who underwent upper gastrointestinal endoscopy with biopsy and histopathological test, 50 tested positive for H. pylori. Peripheral blood was collected from this group and CYP2C19 *2, *3 and *17 alleles were genotyped using qPCR. RESULTS: Of the 50 H. pylori + individuals, 22 were male and 28 were female. Their age varied from 18 to 67 years old, with the mean age being 40.24. Racial groups were classified by self-declaration, based on the official Instituto Brasileiro de Geografia e Estatística (IBGE) categories: 16% (8) were white, 78% (39) were brown, 4% (2) were black, and 2% (1) were indigenous. H. pylori infection was classified using the system of crosses, with 30% (15) of infections being classified as mild (+), 30% (15) as moderate (++), and 40% (20) as severe (+++). The CYP2C19 genotype results showed an allelic frequency of 11% for *2 (20% of the subjects), 6% for *3 (10% of the subjects) and 17% for *17 (30% of the subjects). Based on the phenotypic profiles, the individuals were classified as poor metabolizers (PM, 10%), intermediate metabolizers (IM, 2%), extensive metabolizers (EM, 58%) and ultra-rapid metabolizers (UM, 20%). CONCLUSIONS: Even though the percentage of allele *3 was higher than expected, the percentage of allele *17, a possible contributor to H. pylori eradication failure, was also significant. The population that self-declared as brown showed a unique genotypic pattern, unlike any other population described in previous studies. Our results show that small populations may have genetic particularities that are relevant to therapeutic outcomes.
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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.

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

Abstract 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.

Das salas de autopsias e microscopias para a vida: mentoria informal em um programa de residência médica de patologia / From autopsy rooms and microscopes to life: informal mentoring in a pathology medical residency program

Resumo: Introdução: Além de demorada, a mentoria em Medicina ocorre em vários níveis e é complexa, pois pode incluir várias combinações de ensino, prática clínica, atendimento ao paciente e pesquisa. Relato de experiência: Trata-se de um relato de experiência individual de uma mentorada em um programa de residência médica em patologia do Hospital Universitário Getúlio Vargas, de Manaus, no Amazonas. Descrevem-se os benefícios individuais e coletivos de uma mentoria fora de um programa formal, orientada pelo discernimento e pela maturidade de relacionamento entre mentor e mentorada. Discussão: Discentes de programa de residência médica podem se beneficiar sobremaneira, pessoal e profissionalmente, de mentorias bem conduzidas. Conclusão: No relacionamento de mentoria, são imprescindíveis qualidades como respeito mútuo, discernimento e abnegação, para que o mentorado cresça e desenvolva uma identidade profissional independente, ainda que atrelada à relação de mentoria, e amadurecida, em seu pleno desenvolvimento, a fim de que ele seja um novo mentor e novos alunos sejam beneficiados.

Abstract: Introduction: Mentoring in medicine is time-consuming on several levels and complex, as it can include various combinations of teaching, clinical practice, patient care, and research. Experience report: This report is on the individual experience of a mentee in a medical residency program in Pathology at the Getúlio Vargas University Hospital (Manaus-AM). The individual and collective benefits of mentoring outside a formal program are described, guided by the discernment and maturity of the relationship between mentor and mentee. Discussion: Medical residency program students can benefit greatly, both personally and professionally, from well-conducted mentoring. Conclusion: In the mentoring relationship, qualities such as mutual respect, discernment, and selflessness are essential so that the mentee grows and develops a fully developed and independent professional identity, albeit bound to the mentoring relationship, in order for him become a new mentor and to benefit new students.

Correlating Fibrinogen Consumption and Profiles of Inflammatory Molecules in Human Envenomation's by Bothrops atrox in the Brazilian Amazon.

Snakebites are considered a major public health problem worldwide. In the Amazon region of Brazil, the snake Bothrops atrox (B. atrox) is responsible for 90% of the bites. These bites may cause local and systemic signs from acute inflammatory reaction and hemostatic changes, and present common hemorrhagic disorders. These alterations occur due the action of hemostatically active and immunogenic toxins which are capable of triggering a wide range of hemostatic and inflammatory events. However, the crosstalk between coagulation disorders and inflammatory reaction still has gaps in snakebites. Thus, the goal of this study was to describe the relationship between the consumption of fibrinogen and the profile of inflammatory molecules (chemokines and cytokines) in evenomations by B. atrox snakebites. A prospective study was carried out with individuals who had suffered B. atrox snakebites and presented different levels of fibrinogen consumption (normal fibrinogen [NF] and hypofibrinogenemia [HF]). Seventeen patients with NF and 55 patients with HF were eligible for the study, in addition to 50 healthy controls (CG). The 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 in plasma using the CBA technique at three different times (pre-antivenom therapy [T0], 24 h [T1], and 48 h [T2] after antivenom therapy). The profile of the circulating inflammatory response is different between the groups studied, with HF patients having higher concentrations of CCL-5 and lower IFN-γ. In addition, antivenom therapy seems to have a positive effect, leading to a profile of circulating inflammatory response similar in quantification of T1 and T2 on both groups. Furthermore, these results suggest that a number of interactions of CXCL-8, CXCL-9, CCL-2, IL-6, and IFN-γ in HF patients are directly affected by fibrinogen levels, which may be related to the inflammatory response and coagulation mutual relationship induced by B. atrox venom. The present study is the first report on inflammation-coagulation crosstalk involving snakebite patients and supports the better understanding of envenomation's pathophysiology mechanisms and guides in the search for novel biomarkers and prospective therapies.

Case Report: Adrenal Pathology Findings in Severe COVID-19: An Autopsy Study.

Although high mortality has been reported in many COVID-19 studies, very limited postmortem information from complete autopsies is available. We report the findings in the adrenal glands in 28 autopsies with confirmed SARS-CoV-2 infection. Microscopic lesions were identified in the adrenal glands in 12/28 patients (46%). Seven cases showed necrosis, generally ischemic; four showed cortical lipid degeneration; two showed hemorrhage; and one unspecific focal adrenalitis. Vascular thrombosis in one patient and focal inflammation in association with other findings in three patients were observed. No case presented adrenal insufficiency. In conclusion, adrenal lesions are frequent in patients with severe COVID-19. The lesions are mild but could contribute to the lethal outcome.

Bleeding Disorders in Bothrops atrox Envenomations in the Brazilian Amazon: Participation of Hemostatic Factors and the Impact of Tissue Factor.

Bleeding is a common hemostatic disorder that occurs in Bothrops envenomations. We evaluated the changes in coagulation, fibrinolysis components, and platelets in Bothrops atrox envenomations with bleeding. This is an observational study with B. atrox snakebite patients (n = 100) treated in Manaus, Brazilian Amazon. Bleeding was recorded on admission and during hospitalization. We found that the platelet count in our patients presented a weak correlation to tissue factor, factor II, and plasminogen. Tissue factor presented weak correlation to factor V, II, D-dimer, plasminogen, alpha 2-antiplasmin, and moderate correlation to fibrinogen and fibrin/fibrinogen degradation product (FDP). Patients with systemic bleeding (n = 20) presented low levels of factor V, II, fibrinogen, plasminogen, and alpha 2-antiplasmin, and high levels of tissue factor and FDP compared to those without bleeding. Patients with only local bleeding (n = 41) and without bleeding showed similar levels of hemostatic factors. Thrombocytopenia was observed mainly in patients with systemic bleeding and increased levels of serum venom. No association was found between venom levels and systemic bleeding, or between venom levels and clinical severity of envenomation. This is the first report that shows the participation of the extrinsic coagulation pathway in the consumption coagulopathy of B. atrox envenomations with systemic bleeding due to tissue factor release.

Confirmed Invasive Pulmonary Aspergillosis and COVID-19: the value of postmortem findings to support antemortem management.

We present postmortem evidence of invasive pulmonary aspergillosis (IPA) in a patient with severe COVID-19. Autopsies of COVID-19 confirmed cases were performed. The patient died despite antimicrobials, mechanical ventilation, and vasopressor support. Histopathology and peripheral blood galactomannan antigen testing confirmed IPA. Aspergillus penicillioides infection was confirmed by nucleotide sequencing and BLAST analysis. Further reports are needed to assess the occurrence and frequency of IPA in SARS-CoV-2 infections, and how they interact clinically.

The relationship between clinics and the venom of the causative Amazon pit viper (Bothrops atrox).

Snake venoms are complex mixtures of proteins with toxic activities, with many distinct isoforms, affecting different physiological targets, comprised in a few protein families. It is currently accepted that this diversity in venom composition is an adaptive advantage for venom efficacy on a wide range of prey. However, on the other side, variability on isoforms expression has implications in the clinics of human victims of snakebites and in the efficacy of antivenoms. B. atrox snakes are responsible for most of the human accidents in Brazilian Amazon and the type and abundance of protein families on their venoms present individual variability. Thus, in this study we attempted to correlate the individual venom proteome of the snake brought to the hospital by the patient seeking for medical assistance with the clinical signs observed in the same patient. Individual variability was confirmed in venoms of the 14 snakes selected for the study. The abundance of each protein family was quite similar among the venom samples, while the isoforms composition was highly variable. Considering the protein families, the SVMP group presented the best correlation with bleeding disorders and edema. Considering individual isoforms, some isoforms of venom metalloproteinase (SVMP), C-type lectin-like toxins (CTL) and snake venom serine proteinases (SVSP) presented expression levels that with statistically significant positive correlation to signs and symptoms presented by the patients as bleeding disorders, edema, ecchymosis and blister formation. However, some unexpected data were also observed as the correlation between a CTL, CRISP or LAAO isoforms with blister formation, still to be confirmed with a larger number of samples. Although this is still a small number of patient samples, we were able to indicate that venom composition modulates clinical manifestations of snakebites, to confirm at the bedside the prominent role of SVMPs and to include new possible toxin candidates for the development of toxin inhibitors or to improve antivenom selectiveness, important actions for the next generation treatments of snakebites.