Population-wide persistent hemostatic changes after vaccination with ChAdOx1-S

Various vaccines were developed to reduce the spread of the Severe Acute Respiratory Syndrome Cov-2 (SARS-CoV-2) virus. Quickly after the start of vaccination, reports emerged that anti-SARS-CoV-2 vaccines, including ChAdOx1-S, could be associated with an increased risk of thrombosis. We investigated the hemostatic changes after ChAdOx1-S vaccination in 631 health care workers. Blood samples were collected 32 days on average after the second ChAdOx1-S vaccination, to evaluate hemostatic markers such as D-dimer, fibrinogen, α2-macroglobulin, FVIII and thrombin generation. Endothelial function was assessed by measuring Von Willebrand Factor (VWF) and active VWF. IL-6 and IL-10 were measured to study the activation of the immune system. Additionally, SARS-CoV-2 anti-nucleoside and anti-spike protein antibody titers were determined. Prothrombin and fibrinogen levels were significantly reduced after vaccination (−7.5% and −16.9%, p < 0.0001). Significantly more vaccinated subjects were outside the normal range compared to controls for prothrombin (42.1% vs. 26.4%, p = 0.026) and antithrombin (23.9% vs. 3.6%, p = 0.0010). Thrombin generation indicated a more procoagulant profile, characterized by a significantly shortened lag time (−11.3%, p < 0.0001) and time-to-peak (−13.0% and p < 0.0001) and an increased peak height (32.6%, p = 0.0015) in vaccinated subjects compared to unvaccinated controls. Increased VWF (+39.5%, p < 0.0001) and active VWF levels (+24.1 %, p < 0.0001) pointed toward endothelial activation, and IL-10 levels were significantly increased (9.29 pg/mL vs. 2.43 pg/mL, p = 0.032). The persistent increase of IL-10 indicates that the immune system remains active after ChAdOx1-S vaccination. This could trigger a pathophysiological mechanism causing an increased thrombin generation profile and vascular endothelial activation, which could subsequently result in and increased risk of thrombotic events

MOLECULAR DETECTION OF RESPIRATORY PATHOGENS – A SYNDROMIC APPROACH. Focus on immunocompromised adult patients.

Les infections respiratoires sont les complications infectieuses les plus fréquentes chez les patients immunodéprimés, et sont parmi les causes principales de morbidité et mortalité. Dans le but d’améliorer et d’étendre le diagnostic des pathogènes respiratoires, nous avons introduit au laboratoire une méthode de biologie moléculaire qui permet une approche syndromique, c.-à-d. qu’elle permet de détecter plusieurs micro-organismes responsables d’un même syndrome clinique en une seule étape. Nous avons choisi la technologie des « TaqMan® Array Cards » (TAC), qui permettent de détecter jusqu’à 48 cibles (virales, bactériennes et fongiques) à la fois. Cette technique permet de créer des panels complètement sur-mesure et elle est particulièrement intéressante pour les patients immunodéprimés, une population pour laquelle un diagnostic rapide et correct est crucial pour une prise en charge appropriée du patient. Dans un premier temps, nous avons démontré une valeur ajoutée importante des TACs respiratoires aux méthodes conventionnelles pour le diagnostic d’infections virales respiratoires chez les patients immunodéprimés. Grâce à une meilleure sensibilité et un spectre de détection plus large, le taux de positivité des TACs étaient de 54%, comparé à 19% pour les méthodes conventionnelles. Une deuxième étude nous a permis d’étudier la place des TAC au sein de notre laboratoire clinique. La méthode des TAC a été comparé avec une autre méthode en biologie moléculaire (Simplexa™ Direct assay) pour la détection d’influenza virus et RSV, en termes de performance analytique. Le taux de positivité, les coûts, et le délai de réponse ont également été comparés. La sensibilité et spécificité pour l’influenza et RSV de la méthode Simplexa™ étaient comparables à celles du TAC. Par contre, grâce au large spectre de détection du TAC, le taux de positivité était de 62%, contre 16% pour Simplexa™. Le coût des TAC s’est avéré raisonnable en tenant compte du grand nombre de cibles. Le délai de réponse des TAC dépend largement du nombre d’échantillons respiratoires et de l’organisation du laboratoire. Dans un laboratoire dont le departement de biologie moléculaire n’est pas déployé en dehors des heures d’ouverture normales et non plus pendant le weekend, la combinaison d’une technique moléculaire rapide et disponible 24h/24 et 7j/7 pour la détection de ces pathogènes viraux les plus importants, avec les TAC en deuxième ligne pour les patients à haut risque pourrait offrir une solution. Dans la troisième partie de notre travail, nous avons effectué une étude bi-centrique pour continuer à cartographier l’épidémiologie chez un grand nombre de patients immunodéprimés, et nous avons exploré l’impact clinique des pathogènes respiratoires recherchés. Cette étude a démontré un lien entre la présence d’un pathogène respiratoire et la nécessité d’oxygène supplémentaire. Cette association était toujours valable après l’exclusion des résultats positifs pour influenza, étayant que les virus non-influenza contribuent à une maladie respiratoire sévère dans les patients immunodéprimés. Finalement, nous avons développé notre propre TAC panel respiratoire, en collaboration avec la firme Thermo Fisher Scientific. La première phase, celle du développement de la technique, a compris le choix des pathogènes à inclure, le choix de(s) gène(s) cible(s) pour chaque pathogène, la conception et la validation de chaque ensemble d’amorces et de sondes séparément. Durant cette première phase, nous avons développé et testé 90 PCR différentes. Puis, nous avons sélectionné les 48 PCR à inclure dans notre panel, et ces séquences ont été utilisées pour la fabrication des cartes sur mesure. Ensuite, les cartes ont été validées analytiquement et cliniquement. Finalement, nous avons implémenté cette technique « en routine ». Les patients de l’hôpital Erasme peuvent désormais bénéficier de cette nouvelle technologie de pointe. Avec nos recherches, nous espérons avoir contribué à une amélioration du diagnostic et de la qualité de la prise en charge des patients immunodéprimés atteints d’une infection respiratoire.Respiratory tract infection is the most frequent infectious complication in immunocompromised hosts, and is among the leading causes of morbidity and mortality. In this population, rapid and accurate diagnosis of respiratory pathogens is crucial for appropriate clinical management. The main objective of this work was to improve the detection of respiratory pathogens in this patient group, by means of a syndromic molecular approach. The Taqman® Array Card technology was chosen and viruses, «atypical » bacteria and fungi were selected as targets. We demonstrated that a customized TAC panel had a higher sensitivity in comparison to conventional methods for the detection of respiratory viruses (41% versus 18%; P 0.1). Simplexa™ testing found 16% samples positive with one pathogen each, the TAC assay identified 62% of samples with one or more respiratory pathogens. More than half(54%) of Simplexa™ negative samples were positive by TAC for other pathogens than RSV, Flu A and B. The estimated costs and TAT were 31.8€ and 1.5 hours for Simplexa™ and 56€ and 6 hours for TAC testing. Our two-center study described the epidemiology of respiratory pathogens in a large group of symptomatic immunocompromised patients and provided evidence of a relationship between pathogen detection and the need for supplemental oxygen. This association was still found after the exclusion of the results positive for influenza viruses, substantiating that non-influenza viruses contribute to severe respiratory illness in patients with compromised immunity. Hospital and ICU admission rates were 84% and 11% respectively. The co-infection rates were comparable in our studies, ranging from 12 to 17%. It should be noted however that not all respiratory pathogens were included as targets, so the true co-infection rate is likely higher. The pre-existing TAC used in the above mentioned studies is very complete. Nevertheless, we developed and validated a respiratory TAC, using sequences designed and supported by the manufacturer and included additional pathogens specific for immunocompromised patients. In total, 90 assays were tested in 96-well plates, of which 43 were retained for further validation in TAC format. All tested assays showed excellent analytical sensitivity, with LoD ranging from 1 to 100 copies/µL. An overall specificity of 99.96% was found. Reproducibility ranged from 76%- 100%, with a mean of 91%. For the clinical validation, a total number of 428 samples were tested, with an overall positivity rate of 56.3% and a co-infection rate of 15.9%. 15 results (4% of total number of positives) were not confirmed and considered false positives. One result (0.2%) was considered false negative. This syndromic panel was implemented for routine respiratory testing in the Erasme University Hospital in high risk patient populations. This PhD adds to the growing body of literature regarding the epidemiology of viral, atypical bacterial and fungal respiratory pathogens in adult immunocompromised patients. Our studies have confirmed the added value of a syndromic panel in respiratory diagnostics, and we outlined the development, validation and use of TACs in the clinical diagnostic laboratory in this high risk patient population.Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)info:eu-repo/semantics/nonPublishe

Towards Multitarget Testing in Molecular Microbiology

Advantages of PCR assays over more conventional culture-based diagnostics include significantly higher sensitivities and shorter turnaround times. They are particularly useful when patient treatment has already been initiated or for specimens that may contain microorganisms that are slow-growing, difficult to culture, or for which culture methods do not exist. However, due to genome variability, single target testing might lead to false-negative results. This paper focuses on examples from our own experiences and the literature to provide insight into the limitations of single target testing in molecular biology. Lessons learned from these experiences include the careful design of diagnostic assays, preferably multitargeted, the importance of investigating the incidence and epidemiology of infection in detail, the frequent participation in appropriate quality assurance schemes, and the importance of continuous attentiveness by investigators when confronted with inconsistent results. In conclusion, multitargeted testing in microbiological molecular assays should be a rule

MALDI-TOF MS typing of a nosocomial methicillin-resistant Staphylococcus aureus outbreak in a neonatal intensive care unit.

The early detection of a methicillin-resistant Staphylococcus aureus (MRSA) outbreak is decisive to control its spread and rapidly initiate adequate infection control measures. Therefore, prompt determination of epidemiologic relatedness of clinical MRSA isolates is essential. Genetic typing methods have a high discriminatory power but their availability remains restricted. In this study, we aimed to challenge matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a typing tool of a nosocomial MRSA outbreak in a neonatal intensive care unit.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

HCV false positive immunoassays in patients with LVAD: A potential trap!

Background: Left ventricular assist devices (LVAD) are a therapeutic choice for patients with advanced heart failure prior cardiac transplantation. Patients with a LVAD implant are frequently monitored for hepatitis C virus (HCV) as a positive result may be an exclusion criterion for transplantation. Objectives: To determine the rate of false positive results with immunoassays for HCV antibodies in a LVAD population. Study design: Between June 2011 and January 2015, HCV antibody testing using a chemiluminescent immunoassay (CLIA) (Liaison, Diasorin) was performed for 32 patients prior and post LVAD implantation. A HCV reactive result by CLIA was repeated and further tested by an enzyme linked fluorescent assay (ELFA) (VIDAS, bioMérieux). For patients with a positive HCV CLIA and ELFA test, immunoblot and HCV RNA detection were performed. Results: Prior to LVAD implantation, all patients showed a negative HCV serology. After LVAD implantation, 19 patients (59%) had positive results for HCV antibody using CLIA and ELFA technologies. The HCV immunoblot was negative for 17 patients and indeterminate for two patients. For 15 patients, HCV RNA detection was performed and was undetectable. Actually, no HCV infections were observed among those who were tested for HCV RNA. Conclusions: HCV serological tests routinely used in our laboratories are not reliable in patients with cardiac devices. A positive CLIA and/or ELFA reaction in patients with a LVAD should be confirmed by HCV immunoblot and by HCV RNA PCR detection in order to rule out a HCV infection.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Hospital-Wide SARS-CoV-2 Antibody Screening in 3056 Staff in a Tertiary Center in Belgium

status: publishe

Massive Adult Adenoviral Adenoiditis Mimicking Lymphoma

Hypertrophy of the adenoid is a rare condition in adults, often suspicious of malignancy. We present a case of a 31-year-old female with a clinical presentation of a giant nasopharyngeal mass, clinically suspicious for malignancy, given the size and greyish discoloration. She presented with left-side otalgia, hearing loss, and nasal obstruction. After broad investigations on adenoid tissue following adenectomy, a reassuring diagnosis of adenovirus-related adenoiditis could be made. This case demonstrates the importance of broad microbiological testing in ruling out malignancies. The patient recovered completely.&nbsp

Epidemiology and clinical impact of viral, atypical, and fungal respiratory pathogens in symptomatic immunocompromised patients: a two-center study using a multi-parameter customized respiratory Taqman® array card

The prevalence of respiratory viruses in immunocompromised adult patients and the association with clinical outcomes is still underexplored. Our goal was to assess the epidemiology and the potential clinical impact of respiratory viral infections in a high-risk patient population. Two large hospitals performed a respiratory Taqman array card (TAC), targeting 24 viruses, 8 bacteria, and 2 fungi simultaneously, on 435 samples from 397 symptomatic immunocompromised patients. Clinical details were collected retrospectively using a structured case report form. An overall positivity rate of 68% was found (51% mono- and 17% co-infections). Pathogen distribution was as follows: influenza A (20.7%), rhinoviruses (15.2%), coronaviruses (7.8%), Pneumocystis jirovecii (7.4%), RSV (7.1%), and CMV (6.0%) were the most frequently encountered, followed by HSV (5.5%), hMPV (4.4%), parainfluenza viruses (3.9%), influenza B (3.7%), and Aspergillus species (3.7%). Other pathogens were not detected or detected only in ≤ 1% of samples. Hospital and ICU admission rates were 84% and 11%, respectively. The presence of a pathogen was strongly associated with higher need for supplemental oxygen (p = 0.001), but it had no impact on ICU admission, mechanical ventilation requirement, antibacterial therapy, or mortality. In conclusion, our study described the epidemiology of respiratory pathogens in a large group of symptomatic immunocompromised patients and provides evidence of a relationship between pathogen detection and the need for supplemental oxygen. This association was still found after the exclusion of the results positive for influenza viruses, suggesting that non-influenza viruses contribute to severe respiratory illness in patients with compromised immunity.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Clinical evaluation of a multi-parameter customized respiratory TaqMan® array card compared to conventional methods in immunocompromised patients

Background: Respiratory viral infections can cause significant morbidity and mortality in immunocompromised patients. Conventional tests routinely available at most institutions are limited by the number of detectable pathogens, by a poor sensitivity and/or a long turnaround time. Objectives: To compare the performance of routine conventional testing with direct fluorescent antibody assays and viral culture to a customized TaqMan® array card (TAC) real-time PCR method, targeting 24 viruses, 8 bacteria and 2 fungi simultaneously. Study design: We collected 143 respiratory samples from 120 symptomatic immunocompromised patients. Samples for which conventional and TAC results were discordant underwent further verification testing. Results: The TAC assay identified viral pathogens in more samples than did conventional testing (77/143 versus 27/143; McNemar P<. 0.0001), even when TAC results for viruses that could not be detected by conventional testing were excluded from analysis (59/143 versus 26/143; P<. 0.0001). In addition, the TAC assay identified 18 samples with non-viral pathogens. Verification testing confirmed positive TAC results for 50 out of 55 samples for which conventional testing was negative. Two out of three samples with a positive conventional test but negative TAC result were confirmed positive. A viral and a total pathogen co-infection rate of 5.6% and 11.8% were found, respectively. Conclusions: The customized TAC assay resulted in a significantly increased identification of respiratory viruses. This study provides a practical real-life assessment of the performance of the TAC assay in a population for whom rapid and accurate diagnosis of viral and atypical pathogens is crucial for appropriate clinical management.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Performance evaluation of direct fluorescent antibody, Focus Diagnostics Simplexa™ Flu A/B & RSV and multi-parameter customized respiratory Taqman® array card in immunocompromised patients

Background Molecular assays for diagnosis of Flu A, Flu B, and RSV with short turn-around-time (TAT) are of considerable clinical importance. In addition, rapid and accurate diagnosis of a large panel of viral and atypical pathogens can be crucial in immunocompromised patients. Objectives First, to evaluate the performance of the Simplexa™ Direct assay system in comparison with direct fluorescent antibody (DFA) and customized Taqman® Array Card (TAC) testing for RSV, Flu A, and Flu B in immunocompromised patients. Second, to evaluate different algorithms for the detection of respiratory pathogens in terms of cost, turn-around-time (TAT) and diagnostic yield. Study design We collected 125 nasopharyngeal swabs (NTS) and 25 BAL samples from symptomatic immunocompromised patients. Samples for which Simplexa™ and TAC results were discordant underwent verification testing. The TAC assay is based on singleplex RT-PCR, targeting 24 viruses, 8 bacteria and 2 fungi simultaneously. Results The overall sensitivity was significantly lower for DFA testing than for the two molecular methods (p  0.1). For BAL samples only, the sensitivity and specificity of the Simplexa™ assay was 100%. In total, 6.7, 16 and 18% of samples were positive for Flu A, Flu B or RSV by DFA, Simplexa™ and TAC testing, respectively. When considering not only these pathogens but also all results for TAC, the method identified 93 samples with one or more respiratory pathogens (62%). A co-infection rate of 15.3% was found by TAC. The estimated costs and TAT were 8.2€ and 2 h for DFA, 31.8€ and 1.5 h for Simplexa™ and 55€ and 3 h for TAC testing. Conclusions Performing the Simplexa™ test 24 h a day/7 days a week instead of DFA would considerably improve the overall sensitivity and time-to-result, albeit at a higher cost generated in the laboratory. Performing the TAC would increase the diagnostic yield and detection of co-infections significantly.SCOPUS: ar.jinfo:eu-repo/semantics/publishe