Time-Resolved Förster Resonance Energy Transfer for Rapid Infectious Disease Serodiagnosis‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬

Novel molecular tools for infectious disease diagnostics are constantly under development to reduce the time between onset of symptoms and diagnosis. Not only is it important to receive appropriate treatment, but also to avoid unnecessary use of antibiotics. The availability of rapid diagnostics is also important when epidemics or pandemics emerge. The purpose of this project was to examine the applicability of Förster resonance energy transfer (FRET) in homogeneous immunoassays, and to develop new diagnostic approaches. FRET has widely been applied in proximity-based assays, such as those measuring antigen-antibody binding. In FRET, energy is transferred between two chromophores, the donor and the acceptor, when in close proximity. Utilizing FRET as detection method for immunoassays enables the development of wash-free (homogeneous) simple workflow assays. In this thesis, of the three FRET-based rapid immunoassays that were set up, two served in clinical diagnosis. Study I (I) examined the possibility of detecting antibodies by FRET-pair forming fluorophore-labeled antigens, which upon binding to an antibody would induce a FRET signal. This homogeneous immunoassay, designated FRET-bridge, was successfully optimized for streptavidin (SA). By combining donor-labeled and acceptor-labeled SAs with anti-SA antibodies, FRET signals were recorded with high signal-to-noise ratios. When molecular determinants behind the FRET signals were examined, most of the FRET activity originated from fairly large immunocomplexes rather than from one IgG and two antigens. At the moment, SA represents the only antigen fully functioning in the FRET-bridge assay. This is most likely due to the multivalent nature of the antigen, which seems beneficial in FRET signal formation. Next, we introduced another homogeneous immunoassay (II), the LFRET assay. Here, a FRET pair forming fluorophore-labeled antigen and fluorophore-labeled protein L induce a signal if bound to the Fab (fragment antigen-binding) region of an immunoglobulin. To demonstrate the usefulness of the assay, SA served as test antigen. The assay was next optimized for virus diagnostics by use of Puumala virus (PUUV) nucleocapsid protein as antigen (III). In all, 211 serum samples underwent examination by the LFRET assay, representing acute (n=61) or past PUUV infection (n=27), and seronegative (n=123) individuals. With a simple workflow and an assay time of 30 minutes, the LFRET assay, compared to the reference tests, identified acute PUUV infection at 100% specificity and 95% sensitivity. The fourth study (IV) involved a competitive homogeneous immunoassay for the detection of PUUV antibodies from clinical samples. This assay, CFRET, is based on competition between fluorophore-labeled monoclonal antibodies (MAbs) and serum antibodies. Here, a donor-labeled antigen and an acceptor-labeled MAb form the FRET pair. If the clinical sample contains antibodies against the labeled antigen, they compete with the MAb for antigen binding, resulting in FRET signal decrease. Analysis of assay performance included a panel of 329 samples representing acute (n=101) or past (n=42) infection, and negative samples (n=186). The one-step CFRET assay performed at 99% specificity and 100% sensitivity in diagnosis of hantavirus disease compared to the reference tests, and with a rapid assay time of 30 minutes. The three rapid diagnostic approaches introduced herein represent simplicity, and show that diagnostics need not be time-consuming. Although the assays were optimized for accurate diagnosis of acute infection, both assays also recognized life-long immunity, albeit with lower sensitivity. By optimization, the assays could be developed towards more sensitive detection of past infection as well. The LFRET and CFRET assays thus represent innovative tools for rapid antibody detection, and their potential in serodiagnosis of diverse microbial infections and possibly even in detection of auto- and anti-allergen antibodies calls for further exploration.  Tartuntatautien diagnostiikkaan kehitetään jatkuvasti uusia pikatestejä, jotta diagnoosi saataisiin mahdollisimman nopeasti oireiden ilmaannuttua. Nopea diagnoosi varmistaa sen, että oikea hoito voidaan aloittaa mahdollisimman pian, ja samalla vältytään turhilta antibiooteilta. Diagnostiset pikatestit ovat myös tärkeitä epidemioiden tai pandemioiden hallinnassa. Tämän projektin tarkoituksena oli hyödyntää Förster resonance energy transfer (FRET) ilmiötä homogeenisten vasta-ainetestien kehittämisessä. Tutkimuksen tuloksena pystytettiin kolme erityyppistä vasta-ainetestiä, joista kahta sovellettiin kliiniseen diagnostiikkaan. Ensimmäisessä osajulkaisussa (I) tutkittiin, voisiko vasta-aineita tunnistaa sellaisten fluorofori-leimattujen antigeenien avulla, jotka yhteen liittyessään muodostaisivat FRET-parin. Tutkimusasetelma perustui oletukseen, jonka mukaan näiden fluorofori-leimattujen antigeenien sitoutuessa samaan vasta-aineeseen, muodostuu FRET-signaali. Tämä testi, FRET-bridge, kehitettiin alun perin streptavidiini (SA) -malliantigeenille. FRET-signaali muodostui, kun luovuttaja- ja vastaanottajafluoroforileimatut SA:t yhdistyivät SA-vasta-aineiden toimesta. Työssä tutkittiin muodostuvia FRET-signaaleja molekyylitasolla ja huomattiin, että suurin osa signaaleista oli peräisin isoista immuunikomplekseista, eikä niinkään kahdesta antigeenista ja yhdestä vasta-aineesta. Toistaiseksi SA on ainoa antigeeni, joka tässä testissä toimii kunnolla. Tämä johtuu todennäköisesti SA:n multivalentista luonteesta, mikä vaikuttaisi edistävän FRET-signaalin muodostusta. Toisessa ja kolmannessa osajulkaisussa (II, III) tutkittiin homogeenisen vasta-ainetestin soveltuvuutta tartuntatautien diagnostiikkaan. Kyseinen testi, nimeltään LFRET, perustuu fluoroforileimatun antigeenin sekä fluoroforileimatun proteiini L:n interaktioon niiden sitoutuessa saman vasta-aineen Fab (fragment antigen-binding) -osaan. Myös tämä testi optimoitiin käyttäen SA:ia, jonka jälkeen ensimmäisenä virusantigeenina käytettiin Puumala-viruksen (PUUV) nukleokapsidiproteiinia. Työssä testattiin kaikkiaan 211 seeruminäytettä, jotka edustivat akuuttia vaihetta (n=61), vanhaa immuniteettia (n=27) ja PUUV:lle negatiivisia näytteitä (n=123). LFRET-testi tunnisti akuuttia myyräkuumetta 100% tarkkuudella ja 95% herkkyydellä verrattuna referenssitestien tuloksiin. Neljännessä osajulkaisussa (IV) pystytettiin kilpaileva homogeeninen vasta-ainetesti nimeltään CFRET. Tämä testi perustuu kilpailuun antigeeniin sitoutumisesta fluoroforileimatun monoklonaalisen vasta-aineen (MAb) ja seerumin vasta-aineiden välillä. Tässä testissä MAb sekä antigeeni muodostavat FRET-parin. Jos seeruminäyte sisältää vasta-aineita, jotka sitovat kyseistä antigeenia, syntyy kilpailutilanne vasta-aineiden välille, mikä puolestaan johtaa FRET-signaalin laskuun. Työssä testattiin yhteensä 329 näytettä, jotka edustivat akuuttia vaihetta (n=101), vanhaa immuniteettia (n=42) ja PUUV:lle negatiivisia näytteitä (n=186). CFRET tunnisti akuuttia myyräkuumetta 99% tarkkuudella ja 100% herkkyydellä verrattuna referenssitestien tuloksiin. Edellä kuvatut kolme pikatestiä ovat yksinkertaisia ja ne todistavat, että diagnostisen testin ei tarvitse olla aikaavievä. Vaikka sekä LFRET- että CFRET-testi kehitettiin tunnistamaan akuuttia infektiota, molemmat testit tunnistivat myös vanhaa immuniteettia, joskaan ei yhtä herkästi kuin akuuttia infektiota. Hienosäädön myötä on mahdollista, että kyseistä ominaisuutta voitaisiin parantaa entistä herkemmäksi. LFRET- ja CFRET-testit tarjoavat uraauurtavia mahdollisuuksia vasta-aineiden pikadiagnostiikkaan, ja niiden soveltuvuutta useiden eri taudinaiheuttajien diagnostiikkaan sekä auto- että anti-allergeeni vasta-aineiden tunnistamiseen tulisi tutkia lisää

Imunonanodiagnóstico da pneumonia por Pneumocystis (PPc) uma abordagem inovadora baseada na associação de biossensores antigénicos e nanopartículas de ouro

Pneumocystis jirovecii é um fungo oportunista, de distribuição mundial, capaz de provocar pneumonia fatal em imunocomprometidos. Atualmente, o diagnóstico da pneumonia por Pneumocystis (PPc) baseia-se na visualização microscópica de P. jirovecii e/ou na deteção do seu ADN em amostras respiratórias obtidas por métodos dispendiosos e invasivos. Assim, o desenvolvimento de um método de diagnóstico simples, rápido, económico e menos invasivo, é uma necessidade premente. Neste contexto, este trabalho teve como objetivo desenvolver abordagens alternativas de serodiagnóstico da PPc, aplicando antigénios recombinantes sintéticos (ARS) multi-epítopo específicos de P. jirovecii na deteção de anticorpos séricos anti-P. jirovecii em doentes com e sem infeção por este microrganismo. Dois ARS foram desenhados com base no estudo in silico da imunogenicidade da glicoproteína major de superfície (Msg, major surface glycoprotein) e da serina protease Kex1 de P. jirovecii, duas proteínas com propriedade antigénicas descritas. Após síntese e purificação, esses ARS foram aplicados como ferramentas antigénicas no desenvolvimento e otimização de ELISA (enzyme-linked immunosorbent assays) indiretos para deteção de anticorpos IgG e IgM anti-P. jirovecii em amostras de soro de doentes previamente classificados em grupos clínicos distintos. Seguidamente, realizou-se uma avaliação do desempenho diagnóstico dos ELISA desenvolvidos. Esses ARS foram também conjugados com AuNP previamente funcionalizadas e a interação desses bioconjugados com anticorpos séricos anti-P. jirovecii foi caracterizada por ensaios de eletroforese em gel de agarose (EGA), na presença e ausência de agentes de bloqueamento. Por fim, os bioconjugados foram aplicados na otimização de testes de tiras projetados para detetar anticorpos séricos da classe IgM reativos contra cada um dos ARS produzidos, tendo sido criada uma zona teste com anticorpos anti-IgM humana e uma zona controlo com anticorpos anti-ARS. ELISA desenvolvidos com ambos os ARS mostraram que somente os níveis serológicos de anticorpos da classe IgM estavam significativamente aumentados em doentes com PPc em comparação com doentes sem infeção por este microrganismo. Adicionalmente, o desempenho dos testes ELISA baseados na deteção do nível desses anticorpos no diagnóstico da PPc apresentou sensibilidades de 68.0% e 70.8% e especificidades de 61.8% e 75.0% com o ARS da Msg e o ARS da Kex1, respetivamente. Os ensaios de EGA mostraram que os bioconjugados, após bloqueamento apropriado com caseína, eram capazes de interagir especificamente com anticorpos anti-P. jirovecii presentes no soro de doentes. Os testes de tira desenvolvidos, testados com soros de doentes com e sem infeção por P. jirovecii, apresentaram resultados concordantes com o desempenho esperado, nomeadamente linhas vermelhas na zona teste e controlo com amostras de doentes e apenas uma linha vermelha na zona controlo com amostras de indivíduos sem a doença. Estes resultados suportam a possibilidade de se diagnosticar PPc utilizando os ARS produzidos como ferramentas para deteção serológica de anticorpos IgM anti-P. jirovecii. As alternativas de diagnóstico aqui apresentadas devem ser otimizadas e validadas para sua posterior implementação na prática clínica, esperando-se um impacto positivo não apenas nas regiões economicamente desenvolvidas, mas também nas comunidades de baixa renda e com acesso escasso às tecnologias existentes para o diagnóstico desta doença oportunista potencialmente fatal.Pneumocystis jirovecii is a ubiquitous opportunistic fungus able to cause fatal pneumonia in immunocompromised patients. Currently, Pneumocystis pneumonia (PcP) diagnosis relies on microscopic visualization of P. jirovecii organisms or its DNA detection in respiratory specimens obtained by invasive and costly techniques. Thus, the development of a simpler, faster, cost-effective and less-invasive diagnostic approach is mandatory. In this context, this work aimed to develop alternative serodiagnostic approaches for PcP, applying innovative P. jirovecii recombinant synthetic (multi-epitope) antigens (RSA) in the detection of specific anti-P. jirovecii antibodies in human serum specimens. Two RSA were designed based on the immunogenicity of P. jirovecii major surface glycoprotein (Msg) and kexin-like serine protease (Kex1), two highly antigenic proteins of this pathogen. After synthesis and purification, these RSA were applied as antigenic tools in indirect enzyme-linked immunosorbent assays (ELISA) for detection of serum anti-P. jirovecii antibodies in patients previously classified in distinct clinical groups. A diagnostic performance assessment of each RSA-based ELISA was performed. The RSA were also conjugated with previously functionalized AuNP and the interaction of these bioconjugates with specific anti-P. jirovecii antibodies was characterized by agarose gel electrophoresis (AGE), in the presence and absence of blocking agents. Then, the bioconjugates were used in the development of two strip-based lateral flow immunoassays, comprising a test zone with anti-human IgM antibodies and a control zone with anti-RSA antibodies, projected to detect the presence of human IgM antibodies reactive to each P. jirovecii RSA produced. ELISA results with both RSA showed that only IgM anti-P. jirovecii levels were significantly increased in patients with PcP compared with patients without P. jirovecii infection. In addition, these IgM RSA-based ELISA allowed a diagnostic performance of PcP with sensitivities of 68.0% and 70.8% and specificities of 61.8% and 75.0% with Msg RSA and Kex1 RSA, respectively. AGE assays showed that after proper blocking with casein, the bioconjugates were able to react specifically with anti-P. jirovecii antibodies present in patients sera. The strip-based tests developed were tested with pools of sera from patients with PcP (positive sample) and from patients without P. jirovecii infection (negative sample). Both samples showed the expected performance namely test and control red-colored lines with the positive sample and only a control red-colored line with the negative sample. These results support the possibility to diagnose PcP using the designed RSA as recognition tools and IgM anti-P. jirovecii antibodies as targets for immunodiagnosis. Optimization and validation of the alternative diagnostic approaches presented in this study are necessary in order to enable their implementation in clinical practice. A positive impact is expected not only on economically advanced regions, but also on low-income communities with scarce access to the technologies currently available to diagnose this life-threatening opportunistic disease

Fundamentals of SARS-CoV-2 Biosensors

COVID-19 diagnostic strategies based on advanced techniques are currently essential topics of interest, with crucial roles in scientific research. This book integrates fundamental concepts and critical analyses that explore the progress of modern methods for the detection of SARS-CoV-2

Theoretical and Experimental Comparison of Different Formats of Immunochromatographic Serodiagnostics

In this study, a comparative theoretical and experimental analysis of two immuno-chromatographic serodiagnostics schemes, which differ in the immobilization of immunoreagents and the order of the formation of immune complexes, is performed. Based on the theoretical models, the assays are characterized to determine which scheme has a higher quantity of the detected complex and thus ensures the sensitivity of the analysis. The results show that for the effective detection of low-affinity antibodies, the scheme involving the immobilization of the antigen on gold nanoparticles and the antibody-binding protein on the test strip was more sensitive than the predominantly used scheme, which inverts the immunoreagents’ locations. The theoretical predictions were confirmed by the experimental testing of sera collected from tuberculosis patients

Companion animal tuberculosis: clinical presentations, outbreak investigations, improved diagnostics and the early macrophage response

Tuberculosis caused by the Mycobacterium (M.) tuberculosis-complex (MTBC) of organisms remains one of the most prevalent and deadly infectious diseases of man and other animals. The mycobacteria responsible are a highly conserved group of pleomorphic acid-fast bacilli which cause chronic granulomatous infections. Tuberculous infections in humans and cattle often remain latent for prolonged periods of time before progressing to disease that has severe, negative consequences for the health and welfare of the infected host. Some of the organisms within the MTBC are highly specialised and limited to just a single or small number of host species whereas others, such as M. bovis, can infect a broad range of mammals including humans. Companion animals are susceptible to MTBC infections and understanding of the significance and frequency of these infections has grown in recent years. Cats and dogs share unrivalled proximity to their owners and therefore pose a small but real risk for the zoonotic transmission of tuberculous infections. Despite the high frequency of mycobacterial infections observed in companion animals, diagnostic tests to identify the commonly encountered mycobacterial species are lacking. The first aim of this work was therefore to improve on the currently available diagnostic test methodologies for companion animals. A diagnostic PCR assay was developed and applied to 380 histologically confirmed feline and eight canine mycobacteriosis samples. This novel assay specifically targeted the mycobacterial species most frequently identified by mycobacterial culture (M. bovis and M. microti) and was optimised for use with formalin-fixed tissue; a prerequisite for the safe handling of tuberculous tissue from companion animals by UK laboratories. The assay was suitable for both feline and canine tissue with a significantly quicker turnaround time, higher rate of test positive results and a significant increase in the proportion of M. microti diagnoses compared to culture results. Since evaluation of cytokines has shown diagnostic potential in other species, this project explored the potential of cytokine profiling in cats for the rapid and sensitive detection of mycobacteriosis. By evaluating serum/plasma from 116 naturally infected cats, this study demonstrated a consistent elevation in the cytokines associated with macrophage activation and antigenic stimulation compared to control cats. Sub-group analysis showed that elevations in PDGF-BB were specifically associated with M. microti infections whereas elevated TNF-α sensitively identified cats infected with M. bovis. Investigation of an unprecedented outbreak of M. bovis associated with the ingestion of a putatively contaminated raw food product led to the use of the interferon-γ release assay (IGRA) as a screening test for clinically healthy cats, a purpose for which it had not previously been evaluated. Nearly a third of clinically normal IGRA test-positive cats were subsequently found to have structural disease detected by diagnostic imaging. Though this raises questions regarding the specificity of the IGRA in clinically normal cats, it was an invaluable diagnostic tool to evaluate individual cats involved in the outbreak. The work on feline TB was complemented by similar investigations of canine TB. When an outbreak of M. bovis tuberculosis occurred in a kennel of 164 working Foxhounds, a testing strategy to successfully bring the outbreak under control and investigate the cause was developed. Collaborative work undertaken to screen at risk humans exposed to the hounds identified a latently infected person, highlighting the zoonotic risk posed by M. bovis infections in companion animals. Eight novel and existing diagnostic testing methodologies were evaluated for use in dogs of which a cell-based IGRA, and three serological tests comprising a novel comparative peptide ELISA, the Chembio DPP VetTB assay and the Idexx M. bovis Ab ELISA showed diagnostic potential for canine TB. Additional analysis employing a Bayesian latent class modelling approach revealed that the IGRA developed herein was as sensitive and specific as comparable tests in other species. The serological assays were shown to have markedly lower sensitivity than the IGRA but had higher specificities. All four tests had positive and negative predictive value estimates which indicate that these tests can be informative to clinicians who suspect cases of canine TB. A review of 1012 cases of canine TB highlighted an apparently lower incidence of infections in dogs compared to cats, but an increased severity of clinical signs when disease occurred. To investigate this discrepancy a protocol to derive macrophages from canine and feline bone marrow was developed. These cells acquired cell surface molecules indicative of a macrophage phenotype during ten days of culture with recombinant CSF-1. The response of primary macrophages and the DH82 canine histiocytic cell line was assessed following stimulation with LPS, infection with M. bovis Bacille Calmette Guerin, M. bovis AF2122/97 (the reference strain) or a clinical isolate of M. bovis. These investigations consistently revealed that DH82 cells do not accurately represent primary canine macrophage biology which was associated with altered morphology, lack of nitrite production and significantly reduced secretion of the pro-inflammatory cytokines IL-6 and TNF-α. Overall, the work presented in this thesis demonstrates novel advancement of the diagnostic methodologies for identifying cases of companion animal mycobacteriosis and in particular cases of TB. It further begins to explore the immunological basis for the clinical differences seen between species that may further contribute to novel testing and treatment strategies in the future