Ultrastructural characterization of alveolar microvascular damage in severe COVID-19 respiratory failure.

Endothelial dysfunction is a key phenomenon in COVID-19, induced by direct viral endothelial infection and secondary inflammation, mainly affecting the microvascular circulation. However, few studies described the subcellular aspects of the lung microvasculature and the associated thrombotic phenomena, which are widely present in severe COVID-19 cases. To that end, in this transversal observational study we performed transmission and scanning electron microscopy in nine lung samples of patients who died due to COVID-19, obtained via minimally invasive autopsies in Sao Paulo, Brazil, in 2020. All patients died due to acute respiratory failure and had microvascular thrombosis at histology. Electron microscopy revealed areas of endothelial damage with basal lamina disruption and virus infection in endothelial cells. In the capillary lumens, the ultrastructure of the thrombi is depicted, with red blood cells stacking, dysmorphism and hemolysis, fibrin meshworks, and extracellular traps. Our description illustrates the complex pathophysiology of microvascular thrombosis at the cellular level, which leads to some of the peculiar characteristics of severe COVID-19.NEW & NOTEWORTHY In this study, electron microscopy was used to explain the pathophysiology of respiratory failure in severe COVID-19. Before the advent of vaccination, as the virus entered the respiratory system, it rapidly progressed to the alveolar capillary network and, before causing exudative alveolar edema, it caused mainly thrombosis of the pulmonary microcirculation with preserved lung compliance explaining "happy hypoxia." Timing of anticoagulation is of pivotal importance in this disease.

Strongyloides infection screening in transplant candidates: What is the best strategy?

BACKGROUND: The potential that Strongyloides stercoralis infection has to cause major morbidity and high mortality when the disseminated form occurs in transplant patients is of particular concern. METHODS: In this study, the objective was to observe S. stercoralis infection in patients who are candidates for transplantation by using parasitological, serological, and molecular techniques and to propose an algorithm for the detection of that infection in transplant candidates. RESULTS: By parasitological techniques, 10% of fecal samples were positive. Anti-Strongyloides antibodies immunoglobulin G were detected in 19.3% and 20.7% of patients by immunofluorescence assay and enzyme-linked immunosorbent assay, respectively. S. stercoralis DNA was observed in 17.3% of samples by conventional polymerase chain reaction and 32.7% of samples by quantitative polymerase chain reaction (qPCR). CONCLUSION: The set of results allows us to reinforce that a positive result by parasitological techniques and/or qPCR indicates that the specific treatment should be applied. However, the improvement of diagnostic techniques may suggest changes in the screening for strongyloidiasis in these patients.

Screening of Strongyloides infection using an ELISA test in transplant candidates.

OBJECTIVES: Hyperinfection or disseminated strongyloidiasis has been frequently reported after transplants and is related to high mortality. This study aimed to screen for strongyloidiasis using serological diagnoses in transplant candidates. METHODS: An ELISA test was performed with filariform larvae of Strongyloides venezuelensis as a source of antigen. RESULTS: In the serum from transplant candidates, anti-Strongyloides IgG antibodies were detected in 35/150 (23.3%) samples by soluble fractions in phosphate buffered saline (PBS), 31/150 (20.7%) samples by soluble fractions in Tris-HCl, 27/150 (18.0%) samples by membrane fractions in PBS and 22/150 (14.7%) samples by membrane fractions in Tris-HCl. CONCLUSIONS: The present results suggest the ELISA test, ideally using soluble fractions of filariform larvae S. venezuelensis in PBS, as an additional strategy for the diagnosis of strongyloidiasis in transplant candidates.

Screening of Strongyloides infection using an ELISA test in transplant candidates

OBJECTIVES: Hyperinfection or disseminated strongyloidiasis has been frequently reported after transplants and is related to high mortality. This study aimed to screen for strongyloidiasis using serological diagnoses in transplant candidates. METHODS: An ELISA test was performed with filariform larvae of Strongyloides venezuelensis as a source of antigen. RESULTS: In the serum from transplant candidates, anti-Strongyloides IgG antibodies were detected in 35/150 (23.3%) samples by soluble fractions in phosphate buffered saline (PBS), 31/150 (20.7%) samples by soluble fractions in Tris-HCl, 27/150 (18.0%) samples by membrane fractions in PBS and 22/150 (14.7%) samples by membrane fractions in Tris-HCl. CONCLUSIONS: The present results suggest the ELISA test, ideally using soluble fractions of filariform larvae S. venezuelensis in PBS, as an additional strategy for the diagnosis of strongyloidiasis in transplant candidates.

Molecular diagnosis of Strongyloides stercoralis among transplant candidates.

Strongyloidiasis can occur without any symptoms or as a potentially fatal hyperinfection or disseminated infection, principally in immunosuppressed patients. Our study aimed to evaluate the application of conventional polymerase chain reaction (cPCR) and real-time PCR (qPCR). Polymerase chain reaction (PCR) and real-time PCR (qPCR) targeting the 18S rRNA gene for detection of Strongyloides stercoralis infection among transplant candidates were applied in stool samples obtained from 150 transplant candidates, preliminarily analyzed by parasitological methods. S. stercoralis larvae were visualized in 15/150 (10.0%) transplant candidates by parasitological methods. DNA from S. stercoralis was amplified in 26/150 (17.3%) and 49/150 (32.7%) stool samples of transplant candidates, using cPCR and qPCR, respectively. The results suggest that molecular methods, especially qPCR, should be used as an additional tool for diagnostic of S. stercoralis infection among transplant candidates.

Bacteria arise at the border of mycoplasma-infected HeLa cells, containing cytoplasm with either malformed cytosol, mitochondria and endoplasmic reticulum or tightly adjoined smooth vacuoles.

A study with transmission electron microscopy of mycoplasma-contaminated HeLa cells using five cell donors referred to as donors A, B, C, D and E, observations are herein presented. Experiments performed with cells from donors B, C and D, revealed the presence of Mycoplasma hyorhinis after PCR and sequencing experiments. Bacteria probably originated from a cytoplasm with compacted tiny granular particles replacing the normal cytosol territories, or from the contact with the cytoplasm through a clear semi-solid material. The compact granularity (CG) of the cytoplasm was crossed by stripes of smooth and rough endoplasmic reticulum cisternae. Among apparently normal mitochondria, it was noted, in variable proportions, mitochondria with crista-delimited lucent central regions that expand to and occupied the interior of a crista-less organelle, which can undergo fission. Other components of the scenarios of mycoplasma-induced cell demolition are villus-like structures with associated 80-200 nm vesicles and a clear, flexible semi-solid, process-sensitive substance that we named jam-like material. This material coated the cytoplasmic surface, its recesses, irregular protrusions and detached cytoplasmic fragments. It also cushioned forming bacteria. Cyst-like structures were often present in the cytoplasm. Cells, mainly apoptotic, exhibiting ample cytoplasmic sectors with characteristic net-like profile due to adjoined vacuoles, as well as ovoid or elongated profiles, consistently appeared in all cells from the last four cell donors. These cells were named "modified host cells" because bacteria arose in the vacuoles. The possibility that, in some samples, there was infection and/or coinfection of the host cell by another organism(s) cannot be ruled out.

Bacteria arise at the border of mycoplasma-infected HeLa cells, containing cytoplasm with either malformed cytosol, mitochondria and endoplasmic reticulum or tightly adjoined smooth vacuoles

A study with transmission electron microscopy of mycoplasma-contaminated HeLa cells using five cell donors referred to as donors A, B, C, D and E, observations are herein presented. Experiments performed with cells from donors B, C and D, revealed the presence of Mycoplasma hyorhinis after PCR and sequencing experiments. Bacteria probably originated from a cytoplasm with compacted tiny granular particles replacing the normal cytosol territories, or from the contact with the cytoplasm through a clear semi-solid material. The compact granularity (CG) of the cytoplasm was crossed by stripes of smooth and rough endoplasmic reticulum cisternae. Among apparently normal mitochondria, it was noted, in variable proportions, mitochondria with crista-delimited lucent central regions that expand to and occupied the interior of a crista-less organelle, which can undergo fission. Other components of the scenarios of mycoplasma-induced cell demolition are villus-like structures with associated 80-200 nm vesicles and a clear, flexible semi-solid, process-sensitive substance that we named jam-like material. This material coated the cytoplasmic surface, its recesses, irregular protrusions and detached cytoplasmic fragments. It also cushioned forming bacteria. Cyst-like structures were often present in the cytoplasm. Cells, mainly apoptotic, exhibiting ample cytoplasmic sectors with characteristic net-like profile due to adjoined vacuoles, as well as ovoid or elongated profiles, consistently appeared in all cells from the last four cell donors. These cells were named "modified host cells" because bacteria arose in the vacuoles. The possibility that, in some samples, there was infection and/or coinfection of the host cell by another organism(s) cannot be ruled out.

DIAGNOSIS OF Strongyloides stercoralis INFECTION IN IMMUNOCOMPROMISED PATIENTS BY SEROLOGICAL AND MOLECULAR METHODS.

Strongyloidiasis is a potentially serious infection in immunocompromised patients. Thus, the availability of sensitive and specific diagnostic methods is desirable, especially in the context of immunosuppressed patients in whom the diagnosis and treatment of strongyloidiasis is of utmost importance. In this study, serological and molecular tools were used to diagnose Strongyloides stercoralis infections in immunosuppressed patients. Serum and stool samples were obtained from 52 patients. Stool samples were first analyzed by Lutz, Rugai, and Agar plate culture methods, and then by a quantitative real time polymerase chain reaction (qPCR). Serum samples were evaluated by an enzyme-linked immunosorbent assay (ELISA) using a soluble (AS) or a membrane fractions antigen (AM) obtained from alkaline solutions of the filariform larvae of Strongyloides venezuelensis. Of the 52 immunosuppressed patients, three (5.8%) were positive for S. stercoralis by parasitological methods, compared to two patients (3.8%) and one patient (1.9%) who were detected by ELISA using the AS and the AM antigens, respectively. S. stercoralis DNA was amplified in seven (13.5%) stool samples by qPCR. These results suggest the utility of qPCR as an alternative diagnostic tool for the diagnosis of S. stercoralis infection in immunocompromised patients, considering the possible severity of this helminthiasis in this group of patients.

IMMUNODIAGNOSIS OF HUMAN STRONGYLOIDIASIS: USE OF SIX DIFFERENT ANTIGENIC FRACTIONS FROM Strongyloides venezuelensis PARASITIC FEMALES.

The aim of this study was to evaluate six different antigenic fractions from Strongyloides venezuelensis parasitic females for the immunodiagnosis of human strongyloidiasis. Soluble and membrane fractions from S. venezuelensis parasitic females were prepared in phosphate-buffered saline (SSF and SMF, respectively), Tris-HCl (TSF and TMF, respectively), and an alkaline buffer (ASF and AMF, respectively). Serum samples obtained from patients with strongyloidiasis or, other parasitic diseases, and healthy individuals were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions SSF, TSF, and ASF showed 85.0%, 75.0%, and 80.0% sensitivity and 93.1%, 93.1%, and 87.5% specificity, respectively. Membrane fractions SMF, TMF, and AMF showed 80.0%, 75.0%, and 85.0% sensitivity, and 95.8%, 90.3%, and 91.7% specificity, respectively. In conclusion, the present results suggest that the fractions obtained from parasitic females, especially the SSF and SMF, could be used as alternative antigen sources in the serodiagnosis of human strongyloidiasis.

Immunofluorescence assay for diagnosis of strongyloidiasis in immunocompromised patients.

BACKGROUND: Strongyloides stercoralis is a parasite that causes human strongyloidiasis. The disease ranges from asymptomatic to severe forms, which are often fatal in immunocompromised individuals. Laboratory diagnosis is challenging owing to limitations in the use of conventional parasitological techniques. The present study aimed to evaluate the indirect immunofluorescence assay (IFA) using infective larvae of S. venezuelensis as an antigen for the immunodiagnosis of human strongyloidiasis in immunocompromised patients. METHODS: Serum and stool samples from 200 immunocompromised patients (HIV-positive, HTLV-1-positive, and renal, liver, and/or bone marrow transplantation candidates) were used. Stool samples were examined using three parasitological methods: Lutz, Rugai, and culture agar plate. IFA was performed using sections of infective larvae of S. venezuelensis as antigens, and showed 95.4% sensitivity and 95.8% and specificity. RESULTS: Among the 200 patients, 17 (8.5%) were positive for S. stercoralis by at least one parasitological method, and 43 (21.5%) were positive by IFA. CONCLUSIONS: IFA can be used as a screening method for the detection of S. stercoralis in immunocompromised patients.

Membrane fractions from Strongyloides venezuelensis in the immunodiagnosis of human strongyloidiasis.

Strongyloides venezuelensis is a parasitic nematode of rodents frequently used to obtain heterologous antigens for the immunological diagnosis of human strongyloidiasis. The aim of this study was to evaluate membrane fractions from S. venezuelensis for human strongyloidiasis immunodiagnosis. Soluble and membrane fractions were obtained in phosphate saline (SS and SM) and Tris-HCl (TS and TM) from filariform larvae of S. venezuelensis. Ninety-two serum samples (n = 92) were obtained from 20 strongyloidiasis patients (Group I), 32 from patients with other parasitic diseases (Group II), and 40 from healthy individuals (Group III), and were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions (SS and TS) showed 90.0% sensitivity and 88.9% specificity, whereas the membrane fractions (SM and TM) showed 95.0% sensitivity and 94.4% specificity. The present results suggest the possible use of membrane fractions of S. venezuelensis as an alternative antigen for human strongyloidiasis immunodiagnosis.

Membrane fractions from Strongyloides venezuelensis in the immunodiagnosis of human strongyloidiasis / Frações de membrana de Strongyloides venezuelensis para o imunodiagnóstico da estrongiloidíase humana

Strongyloides venezuelensis is a parasitic nematode of rodents frequently used to obtain heterologous antigens for the immunological diagnosis of human strongyloidiasis. The aim of this study was to evaluate membrane fractions from S. venezuelensis for human strongyloidiasis immunodiagnosis. Soluble and membrane fractions were obtained in phosphate saline (SS and SM) and Tris-HCl (TS and TM) from filariform larvae of S. venezuelensis. Ninety-two serum samples (n = 92) were obtained from 20 strongyloidiasis patients (Group I), 32 from patients with other parasitic diseases (Group II), and 40 from healthy individuals (Group III), and were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions (SS and TS) showed 90.0% sensitivity and 88.9% specificity, whereas the membrane fractions (SM and TM) showed 95.0% sensitivity and 94.4% specificity. The present results suggest the possible use of membrane fractions of S. venezuelensis as an alternative antigen for human strongyloidiasis immunodiagnosis.

Strongyloides venezuelensis é um nematódeo parasita de roedores, frequentemente usado como antígeno heterólogo para o diagnóstico imunológico da estrongiloidíase humana. O objetivo deste estudo foi avaliar frações de membrana de S. venezuelensis para o imunodiagnóstico da estrongiloidíase humana. Para tanto, frações solúveis e de membrana foram obtidas em solução salina fosfato (SS e MS) e Tris-HCl (ST e MT) de larvas filarioides de S. venezuelensis. Amostras de soro de 92 indivíduos, sendo 20 com estrongiloidíase (Grupo I); 32 com outras parasitoses (Grupo II), e 40 indivíduos saudáveis (Grupo III), foram analisadas pelo teste Imunoenzimático (ELISA). As frações solúveis (SS e ST) apresentaram 90,0% e 88,9%, enquanto que as frações de membrana (MS e MT) demonstraram 95,0% e 94,4%, de sensibilidade e especificidade, respectivamente. Os resultados obtidos permitem indicar as frações de membranas como antígeno alternativo para o diagnóstico da estrongiloidíase humana.

Parasitological and molecular diagnosis in experimental Strongyloides venezuelensis infection.

Strongyloides venezuelensis is a parasitic nematode of rats which is frequently used as a model to study human and animal strongyloidiasis. The aim of this study was to evaluate the correlation between parasitological and molecular diagnosis in Strongyloides venezuelensis infection. PCR assays were used to detect S. venezuelensis DNA in fecal samples obtained from experimentally infected Rattus norvegicus. The results showed a higher sensitivity of the PCR assay in detecting the infection compared to parasitological methods.