Immunologic biomarkers, morbidity and mortality among HIV patients hospitalised in a Tertiary Care Hospital in the Brazilian Amazon.

BACKGROUND: The irregular use of antiretroviral therapy (ART) and late diagnosis still account for a large part of HIV-associated mortality in people living with HIV (PLHIV). Herein, we describe HIV-associated morbidity among hospitalised HIV/AIDS patients with advanced immunosuppression and assess the comorbidities, laboratory parameters, and immunological markers associated with mortality. METHODS: The cross-sectional study was conducted at the Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD) in Manaus, Brazil. In all, 83 participants aged between 12 and 70 years were enrolled by convenience within 72 h of their hospitalisation. Clinical and laboratory data were obtained from electronic medical records. We prospectively measured the cytokines Th1/Th2/Th17 and inflammatory cytokines IL-8, IL-1ß, and IL-12 using cytometric bead array, and the soluble CD14 using in-house enzyme-linked immunosorbent assay. RESULTS: The HIV/AIDS inpatients presented a scenario of respiratory syndromes as the most prevalent comorbidity. Almost all patients had CD4 T counts below 350 cells/mL and the mortality rate was 20.5%. Pulmonary tuberculosis, neurotoxoplasmosis and oropharyngeal-esophageal candidiasis were the most prevalent opportunistic infections. TB and weight loss were more prevalent in HIV/AIDS inpatients who died. The Mann Whitney analysis showed that those who died had higher platelet distribution width (PDW) on admission, which is suggestive for platelet activation. The Poisson multivariate analysis showed the prevalence of TB, digestive syndrome and increases in IL-8 and lactate dehydrogenase (LDH) associated to death. CONCLUSIONS: The advanced immunosuppression characterized by the opportunistic infections presented in these HIV/AIDS inpatients was the major factor of mortality. The role of platelet activation in worse outcomes of hospitalisation and the IL-8 associated with the context of advanced immunosuppression may be promising markers in the prediction of mortality in HIV/AIDS patients.

Increased levels of reactive oxygen species in platelets and platelet-derived microparticles and the risk of respiratory failure in HIV/AIDS patients.

Respiratory failure (RF) is the main cause of hospital admission in HIV/AIDS patients. This study assessed comorbidities and laboratory parameters in HIV/AIDS inpatients with RF (N = 58) in relation to those without RF (N = 36). Tuberculosis showed a huge relative risk and platelet counts were slightly higher in HIV/AIDS inpatients with RF. A flow cytometry assay for reactive oxygen species (ROS) showed lower levels in platelets of these patients in relation to the healthy subjects. However, when stimulated with adrenaline, ROS levels increased in platelets and platelet-derived microparticles of HIV/AIDS inpatients, which may increase the risk of RF during HIV and tuberculosis (HIV-TB) coinfection.

BACH1 promotes tissue necrosis and Mycobacterium tuberculosis susceptibility.

Oxidative stress triggers ferroptosis, a form of cellular necrosis characterized by iron-dependent lipid peroxidation, and has been implicated in Mycobacterium tuberculosis (Mtb) pathogenesis. We investigated whether Bach1, a transcription factor that represses multiple antioxidant genes, regulates host resistance to Mtb. We found that BACH1 expression is associated clinically with active pulmonary tuberculosis. Bach1 deletion in Mtb-infected mice increased glutathione levels and Gpx4 expression that inhibit lipid peroxidation. Bach1-/- macrophages exhibited increased resistance to Mtb-induced cell death, while Mtb-infected Bach1-deficient mice displayed reduced bacterial loads, pulmonary necrosis and lipid peroxidation concurrent with increased survival. Single-cell RNA-seq analysis of lungs from Mtb-infected Bach1-/- mice revealed an enrichment of genes associated with ferroptosis suppression. Bach1 depletion in Mtb-infected B6.Sst1S mice that display human-like necrotic lung pathology also markedly reduced necrosis and increased host resistance. These findings identify Bach1 as a key regulator of cellular and tissue necrosis and host resistance in Mtb infection.

COVID-19-Associated Pulmonary Aspergillosis in a Series of Complete Autopsies from the Brazilian Amazon.

Between April and July 2020, and, therefore, prior to the broad recommendation of corticosteroids for severe COVID-19, a total of 50 full autopsies were performed in Manaus. We confirmed two invasive cases of aspergillosis through histopathology and gene sequencing (4%) in our autopsy series. The confirmed invasive aspergillosis incidence seems much lower than expected based on the "probable and possible" definitions, and an individualized approach should be considered for each country scenario. Interestingly, a prolonged length of stay in the intensive care unit was not observed in any of the cases. Timely diagnosis and treatment of fungal infection can reduce mortality rates.

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.

GPX4 regulates cellular necrosis and host resistance in Mycobacterium tuberculosis infection.

Cellular necrosis during Mycobacterium tuberculosis (Mtb) infection promotes both immunopathology and bacterial dissemination. Glutathione peroxidase-4 (Gpx4) is an enzyme that plays a critical role in preventing iron-dependent lipid peroxidation-mediated cell death (ferroptosis), a process previously implicated in the necrotic pathology seen in Mtb-infected mice. Here, we document altered GPX4 expression, glutathione levels, and lipid peroxidation in patients with active tuberculosis and assess the role of this pathway in mice genetically deficient in or overexpressing Gpx4. We found that Gpx4-deficient mice infected with Mtb display substantially increased lung necrosis and bacterial burdens, while transgenic mice overexpressing the enzyme show decreased bacterial loads and necrosis. Moreover, Gpx4-deficient macrophages exhibited enhanced necrosis upon Mtb infection in vitro, an outcome suppressed by the lipid peroxidation inhibitor, ferrostatin-1. These findings provide support for the role of ferroptosis in Mtb-induced necrosis and implicate the Gpx4/GSH axis as a target for host-directed therapy of tuberculosis.

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.

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.

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.

Histopathological changes in epithelium of hair follicles and acrosyringium caused by measles in child.

Some epidermal alterations in measles has been described, such as keratinocytes apoptotic, parakeratosis, giant-cell formation, intranuclear and cytoplasmic inclusions, dyskeratosis, spongiosis, and intracellular edema. The authors report for the first time in human a case of measles with the presence of multinucleated giant cells in the hair follicle and dyskeratosis in acrosyringium.

Presence of HPV with overexpression of p16INK4a protein and EBV infection in penile cancer-A series of cases from Brazil Amazon.

BACKGROUND: In Brazil, penile cancer (PC) is not uncommon. The highest incidence of PC is in the North and Northeast of the country. In addition to phimosis, the Human Papillomavirus (HPV) and Epstein-Baar Virus (EBV) infections are also related as risk factors for PC. The overexpression of p16INK4a is a surrogate sensitive marker of HPV infection in PC. OBJECTIVES: To correlate p16INK4a overexpression and HPV infection status with EBV infection in a series of PC patients from the Amazon region. METHODS: Tumor tissues from 47 PC cases were analyzed for the presence of HPV and EBV DNA by PCR. All PC patients were diagnosed between 2013 and 2018 at a public reference cancer center hospital in Manaus, Amazonas-Brazil. HPV was genotyped using E7 HPV16/HPV18 type-specific real-time PCR and the PapilloCheck® HPV-Screening assay. p16INK4a expression was evaluated by immunohistochemistry using the automated Ventana® BenchMark Ultra. RESULTS: The mean age of patients at the time of diagnosis was 57.4 years ±SD 17.8 ranging from 20 to 90 years old. Most of the patients (64%) came from rural areas of the Amazonas State. Thirty patients had phimosis (64%). Among the patients with phimosis, 43% (13/30) underwent circumcision, three during childhood and 10 in adulthood. 60% of the patients were smokers or ex-smokers. HPV infection was observed in 45% (21/47) of cases. HPV16 was detected in 13 patients (61%). Other HPV types detected were HPV 6, 11, 42, 51, 53, 68 and 44/55. EBV infection was observed in 30% (14/47) of the patients with PC. Co-infection with HPV and EBV was observed in 28% (6/21) cases. p16INK4a was only investigated in 26 samples. The p16INK4a overexpression was observed exclusively in HPV 16 positive cases and four HPV negative cases. In the survival analysis, the follow-up time was 35.4 months/patient. The mortality rate during the follow up time was 38%. CONCLUSIONS: p16INK4a positivity presented a high correlation to HPV 16 DNA detection, reinforcing its use as a surrogate marker for HPV-driven cancers. Infection with EBV was quite frequent and its role in epithelial penile oncogenesis needs to be demonstrated.

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.

An Immunological Stairway to Severe Tissue Complication Assembly in Bothrops atrox Snakebites.

Snakebites are a serious public health problem and, in the Amazon, the Bothrops atrox snake is the most frequent cause of envenomation. B. atrox venom (BaV) causes pathophysiological changes with intense, local inflammatory processes, such as severe tissue complication (STC). However, mechanisms associated with the inflammatory process in humans are still poorly understood. Thus, in this study, we sought to describe the profile of local and systemic immunological soluble molecules in Bothrops envenomation patients treated at a specialist tertiary healthcare unit in the Brazilian Amazon. An analytical and prospective study was performed with patients who had snakebites with different clinical outcomes (STC and Mild Tissue Complication-MTC) using venous blood and blister exudate in order to measure immunological soluble molecules present in the response process. Twenty STC patients and 20 MTC patients were eligible for the study. In addition, 20 healthy donors (HD) who had never been bitten by a snake were used as controls. The biomarkers CXCL-8, CCL-5, CXCL-9, CCL-2 and CXCL-10; C3a, C4a, and C5a; IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, TNF, IFN-γ and IL-17A were quantified using flow cytometry and ELISA. The circulating response profile differs between the studied groups, with MTC patients presenting a mixed profile and STC patients presenting a more polarized profile for Th1 response. In addition, individuals who develop STC have a more intense local immune response, because the tissue response differs from the circulating immunological soluble molecules and presents Th1/Th2/Th17 response polarization. Furthermore, these results suggest that CCL-2 and CXCL-10 are biomarkers for STC and the response profile they assume against Bothrops snakebite should reflect in the clinical practice for the patient.

Surgical treatment of kidney tumor on a patient with second case of staghorn calculus and associated renal hydronephrosis: case report.

Staghorn, struvite or coral calculus are the ones that completely or partially occupies the renal pelvis and branches to the renal calices. The presence of renal calculi of any kind in the urinary tracts is related to the appearing of malign tumor in the kidneys, especially in the absence or failed treatment. A male patient, 51 years old, native of Manaus, reports about dysuria and polyuria associated with constant fever episodes and urinary tract infections with improvement by using several antibiotics but returning the symptoms after suspending it. The patient return was in a month and no improvement of the pain in the patient's feeling, with the tomography image tests result showing atrophy in the right kidney, which presents dilatation of the collector system, that it's filled with material with density varying between thick liquid and soft parts (average of 45 UH), in addition to calculus in the pelvis and ureteropelvic junction, suggesting a pattern for xanthogranulomatous pyelonephritis and matching previous history of right staghorn calculus. The association between kidney cancer and staghorn calculus should be consider through the treatment. Patients with renal diseases of severe prognosis in both kidneys must be kept under constant surveillance by the urology and nephrology team, the association of both clinics is essential to a fortunate ending like the case reported.

Diabetes insipidus secondary to tuberculous meningoencephalitis with hypothalamic involvement extending to the hypophysis: a case report.

The involvement of Mycobacterium tuberculosis in the central nervous system (CNS) is an uncommon and devastating manifestation of tuberculosis. We report a case of disseminated tuberculosis presenting as meningoencephalitis, hypothalamic involvement with extension to the hypophysis, and secondary insipidus diabetes diagnosed at autopsy.

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.

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.