Novel biorecognition elements against pathogens in the design of state-of-the-art diagnostics

Infectious agents, especially bacteria and viruses, account for a vast number of hospitalisations and mortality worldwide. Providing effective and timely diagnostics for the multiplicity of infectious diseases is challenging. Conventional diagnostic solutions, although technologically advanced, are highly complex and often inaccessible in resource-limited settings. An alternative strategy involves convenient rapid diagnostics which can be easily administered at the point-of-care (POC) and at low cost without sacrificing reliability. Biosensors and other rapid POC diagnostic tools which require biorecognition elements to precisely identify the causative pathogen are being developed. The effectiveness of these devices is highly dependent on their biorecognition capabilities. Naturally occurring biorecognition elements include antibodies, bacteriophages and enzymes. Recently, modified molecules such as DNAzymes, peptide nucleic acids and molecules which suffer a selective screening like aptamers and peptides are gaining interest for their biorecognition capabilities and other advantages over purely natural ones, such as robustness and lower production costs. Antimicrobials with a broad-spectrum activity against pathogens, such as antibiotics, are also used in dual diagnostic and therapeutic strategies. Other successful pathogen identification strategies use chemical ligands, molecularly imprinted polymers and Clustered Regularly Interspaced Short Palindromic Repeats-associated nuclease. Herein, the latest developments regarding biorecognition elements and strategies to use them in the design of new biosensors for pathogens detection are reviewed.This research is affiliated with the VibrANT project that received funding from the EU Horizon 2020 Research and Innovation Programme under the Marie Sklowdowska-Curie Grant, agreement no 765042. In addition, the authors acknowledge the financial support from Fundação para a Ciência e Tecnologia (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit. Débora Ferreira (DF) is the recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020.info:eu-repo/semantics/publishedVersio

Trends in Infectious Diseases

This book gives a comprehensive overview of recent trends in infectious diseases, as well as general concepts of infections, immunopathology, diagnosis, treatment, epidemiology and etiology to current clinical recommendations in management of infectious diseases, highlighting the ongoing issues, recent advances, with future directions in diagnostic approaches and therapeutic strategies. The book focuses on various aspects and properties of infectious diseases whose deep understanding is very important for safeguarding human race from more loss of resources and economies due to pathogens

Surveillance of Emerging Livestock Viruses

Accurate and rapid diagnostic tests are essential for the management and control of emerging infectious diseases. Therefore, the overall goals of studies presented in this dissertation were to provide improved diagnostic and surveillance capabilities for several emerging viral diseases impacting the livestock industry and to provide insight into appropriate control strategies. Recent, severe outbreaks of porcine epidemic diarrhea virus (PEDV) in Asia and North America highlighted the need for well-validated diagnostic tests for identification of PEDV infected animals and evaluation of their immune status. PEDV was first detected in the U.S. in May 2013 and another swine coronavirus, porcine deltacoronavirus (PDCoV) was identified in the U.S. in February 2014. Both pathogens spread rapidly across the U.S., severely impacting the swine industry. As part of this project, well-validated serological assays for the detection of antibodies against PEDV and PDCoV were developed. Monoclonal antibodies (mAbs) against selected PEDV and PDCoV structural proteins were also developed and applied to serological and antigen detection assays. Newly developed fluorescence-based virus neutralization assays also provide valuable tools for assessment of vaccine candidates or protective immunity. The PEDV spike (S) glycoprotein plays a key role in virus entry into target cells and mediates the functions of receptor binding and fusion during entry. Therefore, immunodominant neutralizing epitopes of PEDV were identified using a panel of S-specific mAbs. Epitope mapping by peptide ELISA revealed that seven mAbs recognized linear neutralizing epitopes located in the N-terminus of the S2 glycoprotein subunit. Two mAbs recognized a neutralizing epitope located in the C-terminus of S2, while only one neutralizing mAb reacted against a region of the S1 glycoprotein subunit. These results indicate that the S2 glycoprotein subunit contains major antigenic determinants and, perhaps, the immunodominant neutralizing epitopes of PEDV. Influenza D virus is a recently described virus that affects cattle and limited serological testing capability is currently available. Therefore, new ELISA-based serological assays based on expressed protein antigens representing conserved regions of the nucleoprotein and hemagglutinin-esterase proteins were developed. Results of these assays correlate well with hemagglutination inhibition (HI) and virus neutralization assays, providing new tools for ongoing sero-surveillance and control efforts

Humoral and cellular immune responses after pandemic and seasonal influenza vaccination in humans

Vaccination is the most effective prophylaxis against epidemic and pandemic influenza. Annual seasonal influenza vaccination is recommended for high-risk groups such as younger children <5 years old and occupational workers such as frontline healthcare workers (HCW). In 2009, a novel A/H1N1 influenza virus emerged causing the first pandemic of the 21st century. The AS03-adjuvanted inactivated monovalent A/H1N1 pandemic vaccine was rapidly deployed prior to the peak of pandemic in Bergen. HCW were prioritized for the first round of the vaccination, during the autum of 2009 to maintain the integrity of the healthcare system. The A(H1N1)pdm09 strain was subsequently included in seasonal trivalent inactivated influenza vaccines (TIVs) from 2010/2011 until 2016/2017. The trivalent Live Attenuated Influenza Vaccine (LAIV) was licensed for seasonal use in Europe in 2012 and it is recommended for children 2-17 years old. We conducted two vaccine clinical trials using licensed influenza vaccines. In the first clinical trial we evaluated both the immediate and durable humoral and cellular immune responses in HCW vaccinated with the AS03-adjuvanted pandemic vaccine and subsequent annual seasonal TIVs. In the second clinical trial we investigated in depth the follicular helper T (TFH) cell and antibody responses elicited by LAIV in children and adults. We reported that the pandemic vaccine induced rapid homologous and cross-reactive T cell, B cell and antibody responses against the A(H1N1)pdm09 strain and pre-2009 seasonal influenza A/H1N1 strains. We observed that the baseline A(H1N1)pdm09-specific immune responses significantly increased from 2009 to 2013 and were maintained at high levels after 3–4 repeated vaccinations. Collectively our data from the HCW study provide the immunological evidence for continuing annual influenza vaccination policy in adults. Furthermore, we demonstrated that LAIV induced significant increase in influenza specific systemic and local antibody responses against the three vaccine strains tested as early as day 14 post-vaccination. We also showed that LAIV elicited potent and rapid influenza specific TFH cell responses in children. Our LAIV results provide valuable insights into the immunogenicity of LAIV in different age groups with variable levels of pre-existing immunity.Doktorgradsavhandlin

Clinical studies of epidemic influenza and pandemic COVID-19 to improve the chain of patient care: from bedside diagnostics to long-term complications :

Tidlig diagnostikk og rask og målrettet behandling kan forbedre utfallet av influensa og Covid-19 sykdom. Gjennom prospektive, kontrollerte observasjonsstudier har vi vist betydningen av rask diagnostikk for å redusere liggetid og starte tidlig antiviral behandling mot influensa, samt definert langtidsplager etter mild Covid-19 infeksjon. Under influensasesongen i 2018/2019 undersøkte vi effekten av å bruke en pasientnær nukleinsyrebasert influensa hurtigtest i akuttmottaket på Haukeland Sykehus sammenlignet med rask, standard laboratoriediagnostikk på Haraldsplass Diakonale Sykehus. Vi fant at den pasientnære hurtigtesten var raskere, og reduserte tidsbruk fra triage til influensatesting. Bruk av hurtigtest var forbundet med mer målrettet isolasjonsbruk og kortere sykehusopphold. Begge sykehus initierte antiviral behandling i >80% av bekreftede influensatilfeller. Pasientnær hurtigtest var ikke forbundet med lavere forbruk av antibiotika (gitt til >70%) eller kortere antibiotikakurer, som antyder at anibiotikastyrings-verktøy i forløpet etter akuttmottak kan være vel så viktig for å forbedre forskrivningspraksis. Bekymring om overforbruk av antibiotika økte under Covid-19 pandemien. Derfor undersøkte vi antibiotikabruk blant innlagte koronapasienter i Bergen under den første bølgen av pandemien. Vi sammenlignet med 2018/2019 influensapasienter fra samme sykehus, samt nasjonale tall over alle Covid-19 relaterte sykehus-innleggelser i Norge i 2020. Vi så at Covid-19 pasienter fikk færrest antibiotikakurer, men det var et høyere forbruk av resistensdrivende antibiotika. Videre så vi en positiv utvikling med redusert antibiotikabruk hos Covid-19 pasienter som ble innlagt i andre bølge av pandemien, sammenlignet med den første. Til slutt undersøkte vi forekomst av restplager, også kalt «long Covid» blant hjemmeisolerte Covid-19 pasienter opp til 18 måneder etter akutt Covid-19 sykdom. Nesten halvparten av pasientene hadde restplager. Vanligst var utmattelse, hukommelse- og konsentrasjonsvansker og tungpust, og ved 12 måneder var forekomsten av disse plagene mye høyere enn blant en aldersjustert seronegativ kontrollgruppe. SARS-CoV-2 spesifikke immunsvar korrelerte også med restplager. Totalt sett så vi at Covid-19 pasienter hadde økt risiko for en rekke symptomer, og at for de fleste tar det lang tid å bli kvitt plagene.Current knowledge indicates that early diagnosis along with timely and targeted management have the potential to improve the outcomes after Influenza and SARS-CoV-2. During the influenza season 2018/2019, we investigated the effect of implementing an ultra-rapid molecular influenza point-of-care test (POCT) in the emergency department (ED) (intervention hospital), compared to the use of rapid laboratory-based diagnostics (control hospital). We showed that influenza POCT was more rapid, reducing the time from triage to testing, allowing correct isolation of patients, and reduced the length of stay. Both hospitals similarly prescribed antivirals to >80% of influenza patients. The influenza POCT was not associated with reduced rate (>70% overall) or duration of antibiotic treatment, suggesting that antibiotic stewardship measures beyond the ED are important to improve targeted antibiotic use. The concern of overuse of antibiotics in respiratory viral infections increased in the SARS-CoV-2 pandemic. Hence, we investigated antibiotic treatment in patients hospitalized during the first COVID-19-wave in Bergen, and compared to antibiotic treatment of our influenza patients and all nationally registered COVID-19 hospitalised patients. COVID-19 patients were prescribed fewer antibiotics than influenza patients, although more resistance-driving antibiotics were used. There was a positive development from the first to second COVID-19 pandemic wave, with reduced antibiotic use. We then investigated the long-term complications of non-severe COVID-19 in home isolated patients up to 18 months after acute infection, named long COVID. We found that up to 18 months, almost half of the patients had one or more residual symptoms, with fatigue, memory problems, concentration problems and dyspnea being most common. The symptom burden at 12 months was significantly higher after infection compared to age- and time-period matched seronegative controls, and we found humoral and cellular SARS-CoV-2 specific immune correlates of symptom sequelae. Overall, our studies demonstrated an excess risk of multiple symptoms, associated with COVID-19, and that recovery from symptoms is slow in most individuals. In conclusion, this work has shown the importance of timely diagnostics for reducing patient length of stay and timely antiviral treatment and defined the long-term complications after mild COVID-19.Doktorgradsavhandlin

Infectious Etiologies of Febrile Illnesses in Cameron.

Ph.D. Thesis. University of Hawaiʻi at Mānoa 2017