Etablierung, Evaluierung und klinisch-epidemiologische Anwendung virologisch-diagnostischer Methoden während der SARS-CoV-2-Pandemie

Die Ausbreitung des Ende 2019 in China neu aufgetretenen Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) entwickelte sich in kurzer Zeit zu der wissenschaftlich bislang am intensivsten erforschten Pandemie eines viralen respiratorischen Erregers. Basis dieser weltweiten Forschungsbestrebungen war die Verfügbarkeit geeigneter virologisch-diagnostischer Methoden. Nur so war es möglich die klinischen und epidemiologischen Aspekte dieser Infektion rasch und umfassend zu verstehen. Die im Folgenden beschriebene kumulative Habilitationsschrift befasst sich mit wissenschaftlichen Erkenntnissen aus insgesamt 17 Originalarbeiten, die Zielsetzungen aus vier Hauptthemenbereichen zur Etablierung, Evaluierung und klinisch-epidemiologische Anwendung virologisch-diagnostischer Methoden während der SARS-CoV-2-Pandemie untersucht haben. Mittels Nukleinsäure-Amplifikations-Technik-basierter Direktnachweisverfahren wurden Proben von COVID-19-Patienten aus verschiedenen Organsystemen untersucht. In longitudinalen respiratorischen Proben, wie Nasopharynx-Abstrichen und endotrachealer Absaugungen von mild/moderat und schwer verlaufenden SARS-CoV-2-Infektionen, konnte gezeigt werden, dass schwer Erkrankte und immunsupprimierte Lymphompatienten signifikant mehr und länger Virus-RNA ausscheiden. Die Analyse verschiedener Entzündungsparameter dieser Patientengruppen gab einen Hinweis auf die Bedeutung hoher Zytokin-Konzentrationen in der Pathogenese schwer COVID-19-Verläufe. Untersuchungen von Proben der ableitenden Harnwege und des Auges bestätigten einen möglichen Tropismus von SARS-COV-2 in diesen Organsystemen, welche Grundlage für Studien auftretender COVID-19-Symptome wie Pollakisurie und Konjunktivitis waren, aber auch Hygieneempfehlungen für Untersucher und Behandler der entsprechenden medizinischen Fachbereiche begründeten. Zur Evaluierung Antigen-basierter Direktnachweisverfahren wurden insgesamt 11 Antigenschnelltests und vier automatisierte kommerzielle Tests mittels Abstrichproben und angezüchtetem SARS-CoV-2 verglichen. Die Analysen von point-of-care Testen konnten zeigen, dass sich die ermittelte klinische Sensitivität deutlich von der durch die Hersteller angegebenen Werte unterscheidet und eine signifikant schlechtere Erkennung der Omikron Variant of concern nach Bestimmung der analytischen Sensitivitäten bei verschiedenen Antigenschnelltests zu verzeichnen war. Automatisierte Antigentests zeigten diesbezüglich teilweise eine höhere Leistungsfähigkeit basierend auf der Möglichkeit einer semi-/quantitativen Untersuchung und individuellen cut-off Anpassung. In zwei Sero-epidemiologischen Studien an über 7000 Beschäftigten des LMU Klinikums konnte die Leistungsfähigkeit neuer Labormethoden zum indirekten SARS-CoV-2 Nachweis mittels Antikörperdetektion untersucht werden. Die auf diese Weise zuverlässig detektierten Infektionen konnten in Verbindung mit detaillierten Fragebögen in den untersuchten Kohorten ein Risikoprofil für Beschäftigte im Gesundheitswesen kartieren. Das statistisch bestimmte, höchste Risiko für eine SARS-CoV-2 Infektion während der ersten pandemischen Welle hatten demnach Personen, die entweder männlich, Pflegekräfte von COVID-19 Schwerpunktstationen oder Nichtraucher waren oder bekannte Risiko-Kontakte hatten. Durch weitergehende serologische Analysen wurde gezeigt, dass sowohl eine vorbestehende Immunität gegen saisonale Coronaviren aber auch die Höhe der ge-messenen SARS-CoV-2-spezifischen Antikörper mit Empfänglichkeit, Schwere des Verlaufs oder Langzeiteffekten einer SARS-CoV-2-Infektion in Verbindung stehen. Der vierte Hauptthemenbereich dieser Habilitationsarbeit baut auf der Anwendung bereits präpandemisch entwickelter Auswertemethoden zur verbesserten Liquor/Serum-Diagnostik auf. Nach Verfügbarwerden entsprechend geeigneter serologischer Testsysteme zu Beginn der SARS-CoV-2-Pandemie erlaubten diese Rechenalgorithmen eine rasche Etablierung der Bestimmung eines SARS-CoV-2-Antikörperspezifitäts-Index bei COVID-19-Patienten mit ZNS-Symptomatik. Der Inhalt dieser Habilitationsschrift gliedert sich in eine kurze Einleitung und Übersicht relevanter Aspekte der SARS-CoV-2-Pandemie (Kapitel 2), die Formulierung der Forschungsziele und anschließende Zusammenfassung der einzelnen Forschungsprojekte (Kapitel 3 und 4 mit Unterpunkten) sowie Diskussion der Bedeutung der erhobenen Daten. Abschließend sind Sonderdrucke der in Fachzeitschriften veröffentlichten Originalarbeiten angefügt (Kapitel 9)

Evidencia serológica de la circulación del virus de la hepatitis E y prevalencia de anticuerpos contra la hepatitis A en una población indígena en el norte argentino

In 2005 a universal vaccination program against hepatitis A was introduced in Argentina. Nevertheless, there are still some unvaccinated marginal population groups. There are no data about the seroprevalence of hepatitis E in the northern region of Argentina mainly because of lack of awareness of this emergent pathogen. We aimed to determine the seroprevalence of hepatitis A, and hepatitis E in an indigenous population in northern Argentina. One hundred and twenty six (126) donor serum samples collected near San Salvador de Jujuy were analyzed for anti-HAV IgG and HEV IgG and IgM, alkaline phosphatase and transaminase values. Volunteers were interviewed about their living conditions, animal farming, consumption of tap water or river water, and level of education. Seroprevalence of specific anti-HAV antibodies was high (80.2%, 95% confidence interval, 72.1–86.7%) in children under 5 years of age, indicating early infection in life. Seroprevalence of anti-HEV antibodies was 5.6% (95% CI: 2.3–11.2%), being slightly higher than the values found in healthy patients from other regions of the country. Although we could not characterize the genotype of the circulating HEV strain, the clear epidemiological difference between seroprevalence of HAV and HEV in a community with poor sanitary conditions suggest that the circulating HEV strains spread through a different transmission route than HAV. Furthermore a significant correlation between anti-HEV IgG and swine farming was found (p < 0.05), which supports a zoonotic transmission path. We reassessed the epidemiological pattern of HAV infection and reported evidence of HEV infection for the first-time in a community belonging to the Guarani ethnic group, highlighting the need to include hepatitis E testing in routine diagnostics in the region.En 2005 se inició un programa de vacunación universal contra la hepatitis A en Argentina, pero todavía existen algunas poblaciones marginales no vacunadas. Además, los datos sobre la circulación de hepatitis E en el noroeste argentino son escasos. El objetivo de este trabajo fue determinar la seroprevalencia de la hepatitis A y la hepatitis E en una población autóctona del norte de Argentina. Se colectaron y analizaron 126 muestras de suero en habitantes de las yungas jujeñas; se determinaron transaminasas, fosfatasa alcalina y anticuerpos contra los virus de hepatitis A (HAV) (IgG) y hepatitis E (HEV) (IgG e IgM). Se obtuvieron los consentimientos informados y los voluntarios fueron entrevistados para identificar posibles factores de riesgo, como las condiciones de vida y la cría de animales, entre otros. La seroprevalencia de anticuerpos específicos anti-HAV fue alta (80,2%; intervalo de confianza [IC] 95%: 72,1-86,7%) en niños menores de 5 años, lo que indica infección temprana. La seroprevalencia de anticuerpos anti-HEV fue del 5,6% (IC 95%: 2,3-11,2%), ligeramente más alta que en otras regiones del país en pacientes sanos. Aunque no se caracterizó el genotipo circulante del HEV, la clara diferencia epidemiológica entre la seroprevalencia de ambos virus en una comunidad con malas condiciones sanitarias sugiere que la cepa circulante de HEV se transmite por una vía diferente que la del HAV. Además, encontramos una significativa correlación entre la cría de cerdo y la presencia de anticuerpos IgG anti-HEV (p < 0,05), lo que sugiere una vía de transmisión zoonótica. Reevaluamos el patrón epidemiológico de infección por el HAV y aportamos por primera vez una evidencia de infección por el HEV en una comunidad que pertenece a la etnia guaraní, por lo que destacamos la necesidad de incluir la detección de hepatitis E en la región.Fil: Remondegui, Carlos. Hospital Zonal General Agudos San Roque; ArgentinaFil: Ceballos, Susana. Hospital Zonal General Agudos San Roque; ArgentinaFil: Arce, Lorena Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina; ArgentinaFil: Pintado, Eduardo. Hospital Zonal General Agudos San Roque; ArgentinaFil: Vidaurre, Rene. Hospital Paterson de San Pedro de Jujuy; ArgentinaFil: Nitschko, Hans. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; Alemania. German Center for Infection Research; AlemaniaFil: Osterman, Andreas. German Center for Infection Research; Alemania. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; AlemaniaFil: Vizoso Pinto, María Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina; Argentin

Systematic screening for novel, serologically reactive Hepatitis E Virus epitopes

<p>Abstract</p> <p>Background</p> <p>The National Institutes of Health classified Hepatitis E as an emerging disease since Hepatitis E Virus (HEV) is the major cause of acute hepatitis in developing countries. Interestingly, an increasing number of sporadic cases of HEV infections are described in industrialized countries as zoonosis from domestic livestock. Despite the increasing relevance of this pathogen in clinical virology, commercial antibody assays are mainly based on fragments of HEV open reading frame (ORF) 2 and ORF3. The largest ORF1 (poly-)protein, however, is not part of current testing formats.</p> <p>Methods</p> <p>From a synthesized full length HEV genotype 1 cDNA-bank we constructed a complete HEV gene library consisting of 15 respective HEV ORF domains. After bacterial expression and purification of nine recombinant HEV proteins under denaturating conditions serum profiling experiments using 55 sera from patients with known infection status were performed in microarray format. SPSS software assessed the antigenic potential of these nine ORF domains in comparison to seven commercial HEV antigens (genotype 1 and 3) by performing receiver operator characteristics, logistic regression and correlation analysis.</p> <p>Results</p> <p>HEV antigens produced with our method for serum profiling experiments exhibit the same quality and characteristics as commercial antigens. Serum profiling experiments detected Y, V and X domains as ORF1-antigens with potentially comparable diagnostic significance as the well established epitopes of ORF2 and ORF3. However no obvious additional increase in sensitivity or specificity was achieved in diagnostic testing as revealed by bioinformatic analysis. Additionally we found that the C-terminal domain of the potential transmembrane protein ORF3 is responsible for IgG and IgM seroreactivity. Data suggest that there might be a genotype specific seroreactivity of homologous ORF2-antigens.</p> <p>Conclusions</p> <p>The diagnostic value of identified ORF1 epitopes might not necessarily improve sensitivity and specificity, but broaden the overall quality of existing test systems. ORF2 and ORF3-antigens are still commonly used in diagnostic assays and possibly hold the potential to serologically differentiate between genotype 1 and 3 infections. Our systematic approach is a suitable method to investigate HEV domains for their serologic antigenicity. Epitope screening of native viral domains could be a preferable tool in developing new serologic test components.</p

Case Report: Unilateral Sixth Cranial Nerve Palsy Associated With COVID-19 in a 2-year-old Child

Children have been described to show neurological symptoms in acute coronavirus disease 2019 (COVID-19) and multisystemic inflammatory syndrome in children (MIS-C). We present a 2-year-old boy's clinical course of unilateral acute sixth nerve palsy in the context of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Onset of the palsy in the otherwise healthy boy occurred seven days after symptoms attributed to acute infection had subsided respectively 3 weeks after onset of respiratory symptoms. SARS-CoV-2 specific IgG was detected in serum as well as in cerebrospinal fluid. The patient showed a prolonged but self-limiting course with a full recovery after three and a half months. This case illustrates in a detailed chronological sequence that sixth cranial nerve involvement may occur as post-infectious, self-limiting complication of pediatric SARS-CoV-2-infection thus expanding the neurological spectrum of symptoms for children with COVID-19. Clinicians should be aware of the possibility of post-infectious sixth nerve palsy related to SARS-CoV-2-infection particularly in view of recent respiratory tract infection or confirmed cases of SARS-CoV-2-infection amongst the patient's close contacts

Travel-associated neurological disease terminated in a postmortem diagnosed atypical HSV-1 encephalitis after high-dose steroid therapy - a case report

Background: Human encephalitis can originate from a variety of different aetiologies, of which infection is the most common one. The diagnostic work-up is specifically challenging in patients with travel history since a broader spectrum of unfamiliar additional infectious agents, e. g. tropical disease pathogens, needs to be considered. Here we present a case of encephalitis of unclear aetiology in a female traveller returning from Africa, who in addition developed an atypical herpes simplex virus (HSV) encephalitis in close temporal relation with high-dose steroid treatment. Case presentation: A previously healthy 48-year-old female presented with confusion syndrome and impaired vigilance which had developed during a six-day trip to The Gambia. The condition rapidly worsened to a comatose state. Extensive search for infectious agents including a variety of tropical disease pathogens was unsuccessful. As encephalitic signs persisted despite of calculated antimicrobial and antiviral therapy, high-dose corticosteroids were applied intravenously based on the working diagnosis of an autoimmune encephalitis. The treatment did, however, not improve the patient's condition. Four days later, bihemispheric signal amplification in the insular and frontobasal cortex was observed on magnetic resonance imaging (MRI). The intracranial pressure rapidly increased and could not be controlled by conservative treatment. The patient died due to tonsillar herniation 21 days after onset of symptoms. Histological examination of postmortem brain tissue demonstrated a generalized lymphocytic meningoencephalitis. Immunohistochemical reactions against HSV-1/2 indicated an atypical manifestation of herpesviral encephalitis in brain tissue. Moreover, HSV-1 DNA was detected by a next-generation sequencing (NGS) metagenomics approach. Retrospective analysis of cerebrospinal fluid (CSF) and serum samples revealed HSV-1 DNA only in specimens one day ante mortem. Conclusions: This case shows that standard high-dose steroid therapy can contribute to or possibly even trigger fulminant cerebral HSV reactivation in a critically ill patient. Thus, even if extensive laboratory diagnostics including wide-ranging search for infectious pathogens has been performed before and remained without results, continuous re-evaluation of potential differential diagnoses especially regarding opportunistic infections or reactivation of latent infections is of utmost importance, particularly if new symptoms occur

Pulmonary function impairment of asymptomatic and persistently symptomatic patients 4 months after COVID-19 according to disease severity

OBJECTIVE Evaluation of pulmonary function impairment after COVID-19 in persistently symptomatic and asymptomatic patients of all disease severities and characterisation of risk factors. METHODS Patients with confirmed SARS-CoV-2 infection underwent prospective follow-up with pulmonary function testing and blood gas analysis during steady-state cycle exercise 4~months after acute illness. Pulmonary function impairment (PFI) was defined as reduction below 80% predicted of DLCOcSB, TLC, FVC, or FEV1. Clinical data were analyzed to identify risk factors for impaired pulmonary function. RESULTS 76 patients were included, hereof 35 outpatients with mild disease and 41 patients hospitalized due to COVID-19. Sixteen patients had critical disease requiring mechanical ventilation, 25 patients had moderate-severe disease. After 4~months, 44 patients reported persisting respiratory symptoms. Significant PFI was prevalent in 40 patients (52.6%) occurring among all disease severities. The most common cause for PFI was reduced DLCOcSB (n = 39, 51.3%), followed by reduced TLC and FVC. The severity of PFI was significantly associated with mechanical ventilation (p 3~mmHg during cycle exercise occurred in 1/5 of patients after mild disease course. CONCLUSION We characterized pulmonary function impairment in asymptomatic and persistently symptomatic patients of different severity groups of COVID-19 and identified further risk factors associated with persistently decreased pulmonary function. Remarkably, gas exchange abnormalities were revealed upon cycle exercise in some patients with mild disease courses and no preexisting pulmonary condition

Dietary habits, traveling and the living situation potentially influence the susceptibility to SARS-CoV-2 infection: results from healthcare workers participating in the RisCoin Study.

PURPOSE To explore occupational and non-occupational risk and protective factors for the coronavirus disease 2019 (COVID-19) in healthcare workers (HCWs). METHODS Serum specimens and questionnaire data were obtained between October 7 and December 16, 2021 from COVID-19-vaccinated HCWs at a quaternary care hospital in Munich, Germany, and were analyzed in the RisCoin Study. RESULTS Of 3,696 participants evaluated, 6.6% have had COVID-19 at least once. Multivariate logistic regression analysis identified working in patient care occupations (7.3% had COVID-19, 95% CI 6.4-8.3, Pr = 0.0002), especially as nurses, to be a potential occupation-related COVID-19 risk factor. Non-occupational factors significantly associated with high rates of the disease were contacts to COVID-19 cases in the community (12.8% had COVID-19, 95% CI 10.3-15.8, Pr < 0.0001), being obese (9.9% had COVID-19, 95% CI 7.1-13.5, Pr = 0.0014), and frequent traveling abroad (9.4% had COVID-19, 95% CI 7.1-12.3, Pr = 0.0088). On the contrary, receiving the basic COVID-19 immunization early during the pandemic (5.9% had COVID-19, 95% CI 5.1-6.8, Pr < 0.0001), regular smoking (3.6% had COVID-19, 95% CI 2.1-6.0, Pr = 0.0088), living with the elderly (3.0% had COVID-19, 95% CI 1.0-8.0, Pr = 0.0475), and frequent consumption of ready-to-eat meals (2.6% had COVID-19, 95% CI 1.1-5.4, Pr = 0.0045) were non-occupational factors potentially protecting study participants against COVID-19. CONCLUSION The newly discovered associations between the living situation, traveling as well as dietary habits and altered COVID-19 risk can potentially help refine containment measures and, furthermore, contribute to new mechanistic insights that may aid the protection of risk groups and vulnerable individuals

In-depth profiling of COVID-19 risk factors and preventive measures in healthcare workers

PURPOSE To determine risk factors for coronavirus disease 2019 (COVID-19) in healthcare workers (HCWs), characterize symptoms, and evaluate preventive measures against SARS-CoV-2 spread in hospitals. METHODS In a cross-sectional study conducted between May 27 and August 12, 2020, after the first wave of the COVID-19 pandemic, we obtained serological, epidemiological, occupational as well as COVID-19-related data at a~quaternary care, multicenter hospital~in Munich, Germany. RESULTS 7554 HCWs participated, 2.2% of whom tested positive for anti-SARS-CoV-2 antibodies. Multivariate analysis revealed increased COVID-19 risk for nurses (3.1% seropositivity, 95% CI 2.5-3.9%, p = 0.012), staff working on COVID-19 units (4.6% seropositivity, 95% CI 3.2-6.5%, p = 0.032), males (2.4% seropositivity, 95% CI 1.8-3.2%, p = 0.019), and HCWs reporting high-risk exposures to infected patients (5.5% seropositivity, 95% CI 4.0-7.5%, p = 0.0022) or outside of work (12.0% seropositivity, 95% CI 8.0-17.4%, p < 0.0001). Smoking was a protective factor (1.1% seropositivity, 95% CI 0.7-1.8% p = 0.00018) and the symptom taste disorder was strongly associated with COVID-19 (29.8% seropositivity, 95% CI 24.3-35.8%, p < 0.0001). An unbiased decision tree identified subgroups with different risk profiles. Working from home as a preventive measure did not protect against SARS-CoV-2 infection. A PCR-testing strategy focused on symptoms and high-risk exposures detected all larger COVID-19 outbreaks. CONCLUSION Awareness of the identified COVID-19 risk factors and successful surveillance strategies are key to protecting HCWs against SARS-CoV-2, especially in settings with limited vaccination capacities or reduced vaccine efficacy