Clinical microbiology in the time of COVID-19 : Evaluation and application of SARS-CoV-2 laboratory diagnostics

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a large, enveloped, positive-sense, single-stranded RNA virus that is readily transmissible. Symptom severity varies, and the infection can be fatal, especially in vulnerable populations. During the COVID-19 pandemic, clinical diagnostic microbiology has played a key role in the WHO-recommended “test, trace and isolate” strategy to contain the pandemic. Diagnostic laboratories have provided epidemiological insight through serosurveys and sequencing, and laboratories have helped clinicians and the public understand and make interpretations of complex test methods, procedures and results. As the pandemic has progressed, the laboratory methods used for viral RNA, antigen and antibody detection have developed quickly. This thesis evaluates the clinical and analytical performance of RT-PCR and antigen tests for the detection of SARS-CoV-2 and discusses their utility and limitations. It also describes the frequency of severe bacterial infections complicating COVID-19 and reports on sero-surveillance regarding the emergence of the Omicron variant. We assessed the clinical sensitivity of SARS-CoV-2 RT-PCR in comparison with clinical suspicion of SARS-CoV-2 infection that was graded retrospectively. We found that despite the high analytical sensitivity of RT-PCR, its performance against a reference standard based on clinical criteria was only moderate. The evaluation of three commercially available rapid antigen tests was performed by comparing the results to those of RT-PCR and virus culture. As expected, the antigen tests were less sensitive than RT-PCR. We also evaluated a novel diagnostic method for nucleocapsid antigen detection in serum samples. The test sensitivity was high if the sample was taken less than two weeks after symptom onset but declined rapidly after that timepoint. These results suggest that N antigen detection from serum samples can be useful in diagnosing acute COVID-19. We described the frequency, severity, etiology and timing of bacterial infections in hospitalized COVID-19 patients. Bloodstream infections and culture-verified pneumonias were identified in 40/585 (6.5%) cases, and the impact on patient outcomes in our cohort was not statistically significant. This result may inform recommendations on empirical antibiotic treatment and encourage the withholding of routine antibiotic initiation. Lastly, the impact of a novel variant of concern, namely, Omicron, in the Greater Helsinki area was estimated through a serosurvey of IgG-class antibodies conducted on samples sent for routine diagnostic purposes. Vaccination coverage of at least one dose across all age groups was 79% at the time of the study. During the sixteen-week study period, which coincided with Omicron becoming the dominant variant in Finland, the seroprevalence of antibodies against nucleocapsid antigen rose from under 10% to over 30% in age groups under 45 years old. The results presented in this thesis underline the need for continuous microbiological surveillance of emerging infectious diseases as well as the importance of studies on diagnostic accuracy.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) aiheuttaa Coronavirus disease 2019 eli COVID-19 -tautia. SARS-CoV-2 on kookas, vaipallinen, positiivissäikeinen yksijuosteinen RNA-virus, joka tarttuu verrattain helposti. Oirekuvien kirjo on laaja ja COVID-19 voi eritoten iäkkäiden ja rokottamattomien keskuudessa olla kohtalokas. Pandemian aikana kliinisillä, diagnostisilla laboratorioilla on ollut ennennäkemättömän tärkeä rooli osana WHO:n suosittamaa testaamisen, jäljittämisen ja tautitapausten eristämisen -strategiaa. Lisäksi laboratoriot ovat tuottaneet tietoa epidemian kulusta vasta-aineseurannalla ja virusmuunnosten tunnistamiseksi tehdyillä testeillä. Laboratorioiden työ on myös auttanut niin kliinikkoja kuin maallikkojakin tulkitsemaan diagnostisten testien toimintaperiaatteita ja tuloksia. Pandemian edetessä menetelmät, joilla havaitaan SARS-CoV-2 viruksen perimäainesta eli RNA:ta, viruksen antigeeneja tai virusta vastaan muodostuneita vasta-aineita, ovat kehittyneet nopeasti. Tämä on edellyttänyt suuria ponnistuksia menetelmien kehittämiseksi, koestamiseksi ja käyttöönottamiseksi. Tässä väitöskirjassa esitellään tutkimustuloksia RT-PCR- ja antigeenitestien kliinisestä ja analyyttisestä suorituskyvystä sekä arvioidaan testien käyttökelpoisuutta ja rajoitteita. Lisäksi tarkastellaan vakavien bakteeri-infektioiden yleisyyttä sairaalahoitoisilla COVID-19-potilailla sekä Omicron-variantin ilmaantumisen vaikutusta vasta-aine-esiintyvyyteen. Arvioimme SARS-CoV-2 nukleiinihappo-osoitusmenetelmien kliinistä herkkyyttä verraten testiä kliinisin kriteerein määriteltyyn epäilyyn infektiosta. Havaitsimme, että pandemian alkumetreillä, huolimatta nukleiinihappo osoituksen korkeasta analyyttisestä herkkyydestä, testin suorituskyky verrattuna kliiniseen epäilyyn on parhaimmillaankin vain kohtalainen. Kolmen kaupallisen CE-IVD-merkityn pika-antigeenitestin vertailussa totesimme antigeenitestien herkkyyden RT-PCR-testejä matalammaksi. Uusi nukleokapsidiantigeenin seerumista osoittamiseen tarkoitettu menetelmä oli hyvin herkkä analysoitaessa näytteitä, jotka oli otettu kahden viikon sisällä oireiden alkamisesta. Pian tämän aikapisteen jälkeen herkkyys laski. Kuvasimme COVID-19-potilaiden bakteeri-infektioiden yleisyyden, vakavuuden ja aiheuttajakirjon. Veriviljelypositiivisia ja mikrobiologisesti varmennettuja keuhkokuumeita todettiin vain 40/585 (6.8%) potilaalla. Havainnollamme on merkitystä empiirisen antibioottihoidon aloituksen kannalta ja se voi rohkaista antibioottihoidosta pidättäytymiseen. Arvioimme Omicron-muunnoksen ilmaantumisen vaikutusta määrittämällä vasta-aineita satunnaisotannalla valituista seeruminäytteistä. Tutkimusjakso käsitti 16 viikkoa vuodenvaihteessa 2021–2022. Rokotuskattavuus vähintään yhden rokoteannoksen saaneiden henkilöiden osalta oli tutkimusjakson aikana 79 %. Nukleokapsidia vastaan muodostuneiden vasta-aineiden esiintyvyys nousi nopeasti etenkin alle 45-vuotiaiden ikäryhmissä: alle 10 %:sta yli 30 %:iin. Tuloksemme korostavat mikrobiologisen seurannan ja diagnostisten testien suorituskyvyn arvioinnin tärkeytt

Rapid increase in SARS-CoV-2 seroprevalence during the emergence of Omicron variant, Finland

Non peer reviewe

Male predominance in disease severity and mortality in a low Covid-19 epidemic and low case-fatality area - a population-based registry study

Background Men reportedly suffer from a more severe disease and higher mortality during the global SARS-CoV-2 (Covid-19) pandemic. We analysed sex differences in a low epidemic area with low overall mortality in Covid-19 in a population based setting with patients treated in specialized healthcare. Methods We entered all hospitalized laboratory-confirmed Covid-19 cases of all specialized healthcare hospitals of the Capital Province of Finland, into a population-based quality registry and described demographics, severity and case-fatality by sex of the first Covid-19 wave February-June 2020. Results Altogether 5471 patients (49% male) were identified. Patients hospitalized in the specialist healthcare (N = 585, 54% male, OR 1.25; 95% CI 1.05-1.48) were of the same age. Men had less asthma and thyroid insufficiency and more coronary artery disease compared to women. Mean time from symptom onset to diagnosis was at least one day longer for men (p=.005). Men required intensive care unit (ICU) more often (27% vs. 17%) with longer lengths-of-stays at ICU. Male sex associated with significantly higher case-fatality at 90-days (15% vs. 8%) and all excess male deaths occurring after three weeks from onset. Men with fatal outcomes had delays in both Covid-19 testing and hospital admission after a positive test. The delays in patients with the most severe and fatal outcomes differed markedly by sex. In multivariable analysis, male sex associated independently with case-fatality (OR 2.37; 95% CI 1.22-4.59). Conclusions Male sex associated with higher disease severity and case-fatality. Late presentation of male fatal cases could represent different treatment-seeking behaviour or disease progression by sex.Peer reviewe

Identification of novel antigen candidates for a tuberculosis vaccine in the adult zebrafish (Danio rerio)

Public Library of Science open acces

A Generic, Scalable, and Rapid Time-Resolved Förster Resonance Energy Transfer-Based Assay for Antigen Detection—SARS-CoV-2 as a Proof of Concept

Peer reviewe

Real-life clinical sensitivity of SARS-CoV-2 RT-PCR test in symptomatic patients

Background Understanding the false negative rates of SARS-CoV-2 RT-PCR testing is pivotal for the management of the COVID-19 pandemic and it has implications for patient management. Our aim was to determine the real-life clinical sensitivity of SARS-CoV-2 RT-PCR. Methods This population-based retrospective study was conducted in March-April 2020 in the Helsinki Capital Region, Finland. Adults who were clinically suspected of SARS-CoV-2 infection and underwent SARS-CoV-2 RT-PCR testing, with sufficient data in their medical records for grading of clinical suspicion were eligible. In addition to examining the first RT-PCR test of repeat-tested individuals, we also used high clinical suspicion for COVID-19 as the reference standard for calculating the sensitivity of SARS-CoV-2 RT-PCR. Results All 1,194 inpatients (mean [SD] age, 63.2 [18.3] years; 45.2% women) admitted to COVID-19 cohort wards during the study period were included. The outpatient cohort of 1,814 individuals (mean [SD] age, 45.4 [17.2] years; 69.1% women) was sampled from epidemiological line lists by systematic quasi-random sampling. The sensitivity (95% CI) for laboratory confirmed cases (repeat-tested patients) was 85.7% (81.5-89.1%) inpatients; 95.5% (92.2-97.5%) outpatients, 89.9% (88.2-92.1%) all. When also patients that were graded as high suspicion but never tested positive were included in the denominator, the sensitivity (95% CI) was: 67.5% (62.9-71.9%) inpatients; 34.9% (31.4-38.5%) outpatients; 47.3% (44.4-50.3%) all. Conclusions The clinical sensitivity of SARS-CoV-2 RT-PCR testing was only moderate at best. The relatively high false negative rates of SARS-CoV-2 RT-PCR testing need to be accounted for in clinical decision making, epidemiological interpretations, and when using RT-PCR as a reference for other tests.Peer reviewe

The phylodynamics of SARS-CoV-2 during 2020 in Finland

Finland has had a low incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infections as compared to most European countries. Here we report the origins and turnover of SARS-CoV-2 lineages circulating in Finland in 2020. SARS-CoV-2 introduced to Finland in January 2020 and spread rapidly across southern Finland during spring. We observed rapid turnover among Finnish lineages during this period. Clade 20C became the most prevalent among sequenced cases and was replaced by other strains in fall 2020. Bayesian phylogeographic reconstructions suggested 42 independent introductions into Finland during spring 2020, mainly from Italy, Austria, and Spain, which might have been the source for a third of cases. The investigations of the original introductions of SARS-CoV-2 to Finland during the early stages of the pandemic and of the subsequent lineage dynamics could be utilized to assess the role of transboundary movements and effects of early intervention and public health measures.Peer reviewe