Single centre, cross-sectional assessment of airway hemorrhage in a cohort of patients with COVID19-associated ARDS under Venovenous extracorporeal membrane oxygenation

Objectives: Airway hemorrhage (AH) frequently complicates extracorporeal membrane oxygenation (ECMO) treatment. Inflammation, coagulopathy and antithrombotic therapy are contributing factors. Patients with COVID-19- associated ARDS (CARDS) supported with ECMO present all these features. We aim to characterize the incidence and the clinical and prognostic impact of AH. Method(s): Review of a cohort of patients with CARDS treated with ECMO support at a single ECMO centre between March 2020-February 2022 (n=92). AH was defined as a clinically significant hemorrhage fit demanded interruption of anticoagulation, transfusional support or bronchoscopy. Univariate analysis was performed using GraphPadPrism. Result(s): One third (n= 31) of patients with CARDS treated with ECMO had clinically significant AH. Patients who developed AH had significantly longer ICU length-of-stay (LoS), ECMO run and invasive mechanical ventilation (IMV) duration. Significant differences in coagulation and inflammatory markers were detected between patients with early (<72h) versus late (>9 days) onset of AH (Table 1). Mortality at day 90, demographics, comorbidities, CT scan pattern and clinical severity indexes were similar between patients with and without AH (NAH). Conclusion(s): In patients with severe CARDS treated with ECMO support, the occurrence of airway hemorrhage leads to clinically important morbidity but does not increase mortality. Distinct pathways may be involved in the development of early v. late AH. (Table Presented).

Not yet to be born: A clinical case of pseudothrombocytopenia in a pregnant woman

Background : Pseudothrombocytopenia is a platelet count (PC) erroneously below the reference value, due to platelet aggregation or satellitism, when autoantibodies bind to glycoprotein IIb/IIIa in vitro , in the presence of EDTA. Aims : The authors present a clinical case of pseudothrombocytopenia. Methods : A 32-year-old pregnant woman, gesta 5, para 4, at 36 weeks and 2 days was admitted to the Obstetrics Emergency Room with complains of low back pain, cramps, chills, vomiting and nausea with 1 day evolution. Obstetrics past history of 2 preterm labours. At the inicial observation, she presented no significant clinical changes. Analytically, only revealed thrombocytopenia (126x109/L) and C-RP: 3,93mg/dL. Urine dipstick test confirmed leukocyturia. SARS-CoV-2 RT-PCR test was positive. Cardiotocography (CTG) showed a normal pattern. The patient was hospitalized with CTG monitoring and started cefuroxime for acute pyelonephritis. Prophylactic enoxaparin was started on the 2 nd day. The patient remained clinically stable, analytically highlighting a progressive decrease in PC from the 2 nd day on. On the 4 th day of hospitalization with thrombocytopenia of 11x109/L and sFlt-1/PlGF ratio ongoing, a variety of different diagnosis were raised: pre-eclampsia, HELLP syndrome, heparin induced thrombocytopenia (HIT) or COVID-19-induced thrombocytopenia. Results : HIT test was negative and the blood smear revealed many platelet aggregations. New blood collections in trisodium citrate solution and in Mg 2+ compound collection tubes revealed a PC of 31x10 9 /L and 123x10 9 /L, respectively. A significant difference in the PC remained after COVID-19 cure when measured in an EDTA and in Mg 2+ compound collection tubes. On the 5 th day she was discharged. The delivery happened later, at 39 weeks of gestation. Conclusions : The identification of a pseudothrombocytopenia avoided an iatrogenic preterm labour. When confronted with a patient with no signs or symptoms of bleeding or haemorrhagic dyscrasia and no past history of thrombocytopenia, pseudothrombocytopenia should be considered. Its early detection may avoid extensive investigations and unnecessary therapeutic procedures.

Not yet to be born - A clinical case of pseudothrombocytopenia in a pregnant woman

Background: Pseudothrombocytopenia (PTCP) is a platelet count (PC) erroneously below the reference value, usually under150x109/L, due to platelet aggregation or platelet satellitism that happens in vitro triggered by the presence of an anticoagulant, mostly seen with EDTA. This phenomenon happens due to the binding of autoantibodies to glycoprotein IIb/IIIa in the presence of EDTA. Aims: The authors present a clinical case of pseudothrombocytopenia. Methods: 32-year-old pregnant woman, gesta 5, para 4, at 36 weeks and 2 days was admitted to the Obstetrics Emergency Room (OER) with complains of low back pain, cramps, chills, vomiting and nausea with1 day evolution. The patient denied fever, cough, respiratory difficulty or urinary symptoms. There was no known epidemiological context for SARS-CoV-2 infection. Obstetric past history of 4 previous eutocic deliveries, 2 of them were preterm labours at 27th and 34th week of gestation. She had been medicated in outpatient care with vaginal progesterone. At clinical observation in the OER the patient was hemodynamically stable with no significant clinical changes, only showing a dubious right Murphy sign. Analytically, revealed thrombocytopenia of126x109/L, lymphopenia of 0,34 x109/L and C-RP: 3,93mg/dL;the remaining inflammatory parameters were within reference values as well as renal function and liver damage analytes. Urine dipstick test revealed Ketone bodies:150mg/dL;Proteins: 25mg/dL;Leucocytes:100 cell/μL;Erythrocytes:10 cell/μL;Nitrites: negative. SARS-CoV-2 RT-PCR test was positive. Cardiotocography (CTG) showed a normal pattern, with fetal heart rate of150-160 bpm and irregular and sparse uterine contractility. The patient was proposed for hospitalization with CTG monitoring and started empirical antibiotherapy with cefuroxime for a presumed acute pyelonephritis. Prophylactic enoxaparin was started on the 2nd day. The pregnant woman remained clinically stable, analytically highlighting a progressive decrease in PC from the 2nd day on, with PC of 85x109/L. On the 4th day of hospitalization with thrombocytopenia of11x109/L, a blood smear and the sFlt-1/PlGF ratio ongoing, a variety of different diagnosis were raised, such as pre-eclampsia, HELLP syndrome, autoimmune disorders, idiopathic thrombocytopenic purpura, heparin induced thrombocytopenia (HIT) or COVID-19-induced thrombocytopenia. At this point, our laboratory was contacted in order to confirm the low PC, since a labour induction was a possibility. Results: HIT test proved to be negative and the blood smear revealed many and huge platelet aggregations. To confirm the PC our laboratory advised a new blood collection in a trisodium citrate solution (S-Monovette. Citrate) and in Mg2+ compound (S-Monovette. ThromboExact) collection tubes, which revealed a PC of 31x109/L and123x109/L, respectively. On the 5th day she was discharged. After COVID-19 infection cure, the PC values remained significantly different when measured in an EDTA and in Mg2+ compound collection tubes. The delivery happened, later, at 39 weeks of gestation. Summary/Conclusion: The identification of a PTCP allowed us to avoid an iatrogenic preterm labour. When confronted with a patient with no signs or symptoms of bleeding or haemorrhagic dyscrasia and no past history of thrombocytopenia, PTCP should be considered as one of the differential diagnosis. It is estimated that approximately 0.1% of the whole population has antiplatelet autoantibodies triggered by EDTA. Early detection of this phenomenon may avoid an extensive investigation and unnecessary therapeutic procedures.

DESFECHOS TROMBOEMBÓLICOS NA INFECÇÃO POR SARS-COV-2: O QUE ESPERAR?

Objetivos: Identificar e resumir evidências científicas sobre a incidência de alterações hematológicas e complicações tromboembólicas em pacientes com COVID-19. Material e métodos: Revisão sistemática da literatura utilizando base de dados on-line PubMed. Os termos de pesquisa utilizados foram: Coronavirus OR COVID-19 OR SARS-CoV-2 AND thromboembolism. Foi realizado levantamento bibliográfico do período de janeiro de 2020 a agosto de 2020. Foram considerados critérios de inclusão artigos completos, realizados em humanos, estudos publicados em inglês e espanhol. Foram excluídos artigos que não apresentaram como temática central eventos tromboembólicos na COVID-19. Foram selecionados 273 resultados, e de acordo com os critérios de inclusão e exclusão, restaram 59 artigos. Resultados: Eventos tromboembólicos associados a Covid-19 são descritos em casos graves da doença. Desta forma, é comum o aparecimento de complicação hematológica decorrente de distúrbio de coagulação, o qual predispõe o desenvolvimento de um estado pró-trombótico. As principais alterações hematológicas relatadas nos artigos foram: aumento dos níveis de dímero-D em 86,4% dos artigos, aumento fibrinogênio em 30,5% e interleucina-6 em 25,42%. Foi observado também aumento do tempo de protrombina e proteína C-reativa em 23,72%, aumento de ferritina em 16,94%, linfopenia em 15,25%, aumento de fator VIII em 11,86% e fator de von Willebrand em 8,47% dos artigos selecionados. Discussão: De acordo com os estudos publicados até o momento, dentre as alterações encontradas nos paciente com COVID-19, destacam-se as alterações hematológicas tais como aumento dos níveis de dímero-D, prolongamento do tempo de protrombina e aumento de fibrinogênio. Outras anormalidades laboratoriais encontradas incluíram elevação de marcadores pró-inflamatórios e substâncias que permitem acompanhar a gravidade da doença. Entre eles encontram-se a interleucina-6 e proteína C-reativa. Por desencadear uma síndrome do desconforto respiratório agudo (SDRA), a infecção por SARS-CoV-2 apresenta um quadro de hipoxemia associado, podendo assim agravar o estado de hipercoagulabilidade, isquemia e lesões pulmonares. Esse estado pode ser devido a “tempestade de citocinas” relatada em uma fase mais tardia da doença. Ainda não se sabe ao certo qual o exato mecanismo fisiopatológico da coagulopatia associada a infecção por SARS-CoV-2, mas acredita-se haver presença de lesão endotelial e hipoxemia nos capilares pulmonares, levando a um processo de isquemia e obstrução. Desta forma, ocorre um comprometimento da ação fibrinolítica, desencadeando um estado pró-trombótico, que reduz a capacidade de remoção de depósitos de fibrina no espaço alveolar bloqueando as trocas gasosas. Desta forma, alguns estudos relataram melhora do prognóstico em pacientes graves que fizeram uso profilático de terapia anticoagulante, relatando uma menor taxa de mortalidade. Conclusão: Os estudos já publicados mostram a presença de eventos tromboembólicos nos casos mais graves, devido à prováveis mecanismos relacionados ao aumento da atividade de fatores de coagulação específicos. A melhora do prognóstico em pacientes que fizeram o uso de terapia anticoagulante fortalece a associação de eventos tromboembólicos na patogênese da COVID-19. Novos estudos são necessários para um melhor entendimento dos desfechos tromboembólicos na infecção por SARS-CoV-2.

Bottom-up microwave-assisted seed-mediated synthesis of gold nanoparticles onto nanocellulose to boost stability and high performance for SERS applications

The development of accurate, reliable, inexpensive and fully recyclable analytical platforms is of utmost relevance to several fields from medical diagnosis to environmental screening. Surface-enhanced Raman spectroscopy (SERS) is a compelling detection method with high specificity and sensitivity. In this work, a microwave-assisted synthesis method was used for fast and uniform in situ growth of gold nanoparticles (AuNPs) onto nanocellulose (NC) membranes, through a seed-mediated process. The as-prepared membranes were fully optimized and its application as SERS platforms was demonstrated. A direct comparison with other cellulose-based substrates showed the superior characteristics of NC such as high mechanical strength, high surface area and lower porous content. An Enhancement Factor (EF) up to ~106 was obtained using rhodamine 6G (R6G) 10−6 M as probe molecule and a remarkable shelf life of at least 7 months was achieved, with no special storage requirements. Preliminary results on the label-free detection of spike protein from SARS-CoV-2 virus are shown, through direct measurements on the optimized SERS membrane. We believe that this work evidences the effectiveness of in situ seed-mediated microwave-assisted synthesis as a fabrication method, the high stability of AuNPs and the superior characteristics of NC substrates to be used as SERS platforms. © 2021 Elsevier B.V.

Recommendations for accurate genotyping of SARS-CoV-2 using amplicon-based sequencing of clinical samples

OBJECTIVES: Genotyping of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in monitoring viral evolution and transmission during the pandemic. The quality of the sequence data obtained from these genotyping efforts depends on several factors, including the quantity/integrity of the input material, the technology, and laboratory-specific implementation. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in genomic epidemiology studies. We aimed to establish clear and broadly applicable recommendations for reliable virus genotyping. METHODS: We established and used a sequencing data analysis workflow that reliably identifies and removes technical artefacts;such artefacts can result in miscalls when using alternative pipelines to process clinical samples and synthetic viral genomes with an amplicon-based genotyping approach. We evaluated the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination. RESULTS: We found that at least 1000 viral genomes are necessary to confidently detect variants in the SARS-CoV-2 genome at frequencies of >=10%. The broad applicability of our recommendations was validated in over 200 clinical samples from six independent laboratories. The genotypes we determined for clinical isolates with sufficient quality cluster by sampling location and period. Our analysis also supports the rise in frequencies of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was facilitated by travel during the summer of 2020. CONCLUSIONS: We present much-needed recommendations for the reliable determination of SARS-CoV-2 genome sequences and demonstrate their broad applicability in a large cohort of clinical samples.