En ökad utbredning av vankomycinresistenta enterokocker (VRE) har setts i Sverige sedan 2007. Bakteriemi orsakad av VRE är mycket svårbehandlad, varför snabbare tillförlitlig resistensdiagnostik är betydelsefullt för att minska dödlighet, vårdtider, vårdkostnader och belastning på sjukvårdssystemet. På mikrobiologilaboratoriet, Region Jönköpings län (RJL), tar idag identifiering av fenotypisk vankomycinresistens vid optimala förhållanden 6 timmar, räknat från att enterokocker konstaterats växa i blodet. Resistensgenerna vanA och vanB, som bland andra orsakar vankomycinresistens hos enterokocker, kan genetiskt verifieras med loop-mediated isothermal amplification men tar idag upp till ett dygn då bakteriekolonier används som analysmaterial i arbetsrutinen på molekylärbiologilaboratoriet, RJL. Syftet med studien var att utvärdera bakteriepellet som analysmaterial för genetisk identifiering av vanA och vanB, på Genie® II Mk2 med eazyplex® VRE basic, hos enterokocker från positiva blododlingar. För att utvärdera bakteriepellet som analysmaterial analyserades isolat av Enterococcus faecium (n=17) och Enterococcus faecalis (n=5) från bakteriepellets tillverkade från simulerade positiva blododlingar med eazyplex® VRE basic på Genie® II Mk2, varpå resultaten jämfördes mot isolatens faktiska närvaro/frånvaro av vanA/vanB. Samstämmigheten av de uppmätta- och de förväntade resultaten var fullständig, vilket indikerar att bakteriepellet med hög tillförlitlighet kan användas som analysmaterial till eazyplex® VRE basic för att påvisa vanA och vanB hos enterokocker i blododlingar. / An increased prevalence of vancomycin-resistant enterococci (VRE) has been observed in Sweden since 2007. Treating bacteremia caused by VRE is difficult, which is why faster, and reliable resistance diagnostics are important. At the Microbiology laboratory, Region Jönköping County, the identification of phenotypic vancomycin resistance under optimal conditions takes 6 hours from when growth of enterococci in blood is determined. The genes vanA and vanB, which among others cause vancomycin resistance, can be genetically verified by loop-mediated isothermal amplification, but takes up to one day since bacterial colonies are used as analysis material. The aim of the study was to evaluate bacterial pellet as an analytical material for genetic identification of vanA and vanB, on Genie® II Mk2 with eazyplex® VRE basic, in enterococci from positive blood cultures. To evaluate the bacterial pellet, isolates of Enterococcus faecium (n=17) and Enterococcus faecalis (n=5) from bacterial pellets made from simulated positive blood cultures were analyzed with eazyplex® VRE basic on the Genie® II Mk2, and the results were compared to the actual presence/absence of vanA/vanB in the isolates. The complete coherence between the expected and measured results indicates that the bacterial pellet can be used as an analytical material for eazyplex® VRE basic.

The goal of this project was to incorporate bacteriophage with Bioluminescent Assay in Real-Time Loop-mediated isothermal amplification (BART-LAMP) for the rapid detection of Mycobacterium avium subspecies paratuberculosis (MAP). As the causative agent of Johne’s Disease, there are no rapid detection methods that are suitable in specificity and sensitivity. A screening assay for phage isolation was developed, and over 400 samples were screened for the isolation of a bacteriophage against MAP. One novel Mycobacterium phage was isolated and characterized using transmission electron miscroscopy, host range studies, restriction enzyme digestion, and pH and temperature stability. It was sequenced, annotated, and underwent an in silico protein analysis. No pathogenic or lysogenic genes were detected, and it was found to be related to Gordonia phage GTE2. BART-LAMP was applied to the detection of the isolated phage using purely extracted DNA and crude phage lysate, showing that phages could be detected successfully. / Beef Cattle Research Council; Agriculture and AgriFood Canada through Growing Forward initiative

abstract: HIV/AIDS is the sixth leading cause of death worldwide and the leading cause of death among women of reproductive age living in low-income countries. Clinicians in industrialized nations monitor the efficacy of antiretroviral drugs and HIV disease progression with the HIV-1 viral load assay, which measures the copy number of HIV-1 RNA in blood. However, viral load assays are not widely available in sub-Saharan Africa and cost between 50-$139 USD per test on average where available. To address this problem, a mixed-methods approach was undertaken to design a novel and inexpensive viral load diagnostic for HIV-1 and to evaluate barriers to its adoption in a developing country. The assay was produced based on loop-mediated isothermal amplification (LAMP). Blood samples from twenty-one individuals were spiked with varying concentrations of HIV-1 RNA to evaluate the sensitivity and specificity of LAMP. Under isothermal conditions, LAMP was performed with an initial reverse-transcription step (RT-LAMP) and primers designed for HIV-1 subtype C. Each reaction generated up to a few billion copies of target DNA within an hour. Presence of target was detected through naked-eye observation of a fluorescent indicator and verified by DNA gel electrophoresis and real-time fluorescence. The assay successfully detected the presence of HIV in samples with a broad range of HIV RNA concentration, from over 120,000 copies/reaction to 120 copies/reaction. In order to better understand barriers to adoption of LAMP in developing countries, a feasibility study was undertaken in Tanzania, a low-income country facing significant problems in healthcare. Medical professionals in Northern Tanzania were surveyed for feedback regarding perspectives of current HIV assays, patient treatment strategies, availability of treatment, treatment priorities, HIV transmission, and barriers to adoption of the HIV-1 LAMP assay. The majority of medical providers surveyed indicated that the proposed LAMP assay is too expensive for their patient populations. Significant gender differences were observed in response to some survey questions. Female medical providers were more likely to cite stigma as a source problem of the HIV epidemic than male medical providers while males were more likely to cite lack of education as a source problem than female medical providers. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2011

<div>Pathogen identification at the point of use is critical in preventing disease transmission and enabling prompt treatment. Current rapid diagnostic tests suffer from high rates of false negatives because they are not capable of detecting the inherently low concentrations of pathogens found in early stages of infection or in environmental reservoirs. The gold standard method for timely pathogen identification is a nucleic acid amplification assay called polymerase chain reaction. Although polymerase chain reaction is extremely sensitive and specific, it requires expensive laboratory equipment and trained personnel to perform the sample preparation, cyclical heating, and amplicon analysis. Isothermal nucleic acid amplification assays are better suited for field use because they operate at a single temperature and are robust to common sample matrix inhibitors. Thus, there is a need to translate isothermal amplification assays to the point of use for rapid and sensitive detection of pathogens in complex samples.</div><div><br></div><div>Here, I outline an approach to bring laboratory-based sample preparation, assays, and analyses to the point of use via portable platforms. First, I characterize a loop-mediated isothermal amplification assay and combine it with lateral flow immunoassay for simple, colorimetric interpretation of results. Next, I optimize an ambient-temperature reagent storage method to eliminate cold-chain requirements and precision pipetting steps. I then incorporate loop-mediated isothermal amplification, lateral flow immunoassay, and reagent drying into two different integrated paperfluidic platforms and demonstrate their ability to separately detect bacteria and viruses in complex sample matrices. Finally, I couple loop-mediated isothermal amplification with particle diffusometry to optically determine pathogen presence by tracking the Brownian motion of particles added to an amplified sample. The combined loop-mediated isothermal amplification and particle diffusometry method is first characterized on a microscope and then translated to a smartphone-based platform. Each of these portable platforms are broadly applicable because they can be easily modified for identification of other pathogens at the point of use.</div>

Quantification of pathogen load, although challenging, is of paramount importance for accurate diagnosis and clinical management of a range of infectious diseases in a point-of-need testing (PONT) scenario such as in resource-limited settings. We formulated a quantification approach to test the standard-curve based absolute quantification ability of isothermal recombinase polymerase amplification (RPA) assay. As a test of principle, a 10-fold dilution series of Leishmania donovani (LD) genomic DNA prepared in nuclease-free-water (NFW), and from culture-spiked-blood (CSB) were tested, and a 15 min assay was performed. A modified algorithm was formulated to derive the detection outcome. The threshold-record times (Tr) in seconds thus obtained were plotted against the initial load of parasite genomes for log-linear regression analysis. The quantitative RPA (Q-RPA) assay was further evaluated against a LD quantitative (q)-PCR assay with DNA extracted from visceral and post-Kala-azar dermal leishmaniasis case specimens and stratified into different ranges of threshold cycle (Ct). The best-fitted regression models were found linear with mean r2/root mean square error (RMSE) values of residual points (in seconds) estimated as 0.996/8.063 and 0.992/7.46 for replicated series of NFW and CSB, respectively. In both series, the lower limit of detection reached less than 0.1 parasite genome equivalent DNA. Absolute agreement between Q-RPA and LD-qPCR was found for test positivity, and strong positive correlations were observed between the Tr and Ct values (r = 0.89; p < 0.0001) as well as between the absolute parasite loads (r = 0.87; p < 0.0001) quantified by respective assays. The findings in this very first Q-RPA assay for leishmaniasis are suggestive of its potential in monitoring LD load in clinical specimens, and the development of rapid Q-RPA assays for other infectious diseases.

A Brucella canis é a bactéria responsável pela brucelose nos cães, pode ser transmitida para os seres humanos, ocasionalmente resultando em doença grave, com impacto na saúde pública. A brucelose canina desencadeia inúmeras perdas econômicas em canis comerciais, com a ocorrência de abortamentos, morte embrionária, natimortos e nascimento de filhotes debilitados. O diagnóstico sorológico é rotineiramente realizado, contudo a hemocultura é o teste \"padrão-ouro\". A técnica de reação em cadeia pela polimerase (PCR) pode ser aplicada no diagnóstico direto como alternativa à hemocultura, pela rapidez, alta especificidade e sensibilidade do teste, mas apresenta alto custo com infraestrutura e equipamentos. A amplificação isotérmica mediada por loop (LAMP) constitui outra alternativa para amplificação do DNA em um curto período de tempo, com simplicidade e menor custo. O projeto avaliou comparativamente o desempenho dos testes moleculares de PCR e LAMP com primers direcionados à sequência de inserção IS711 de Brucella spp. em 98 amostras de sangue total, obtidas de 57 cães. Os 57 cães foram divididos em três grupos: infectados por B. canis (cães positivos na hemocultura) não infectados por B. canis (negativos na hemocultura e sem evidências clínicas e epidemiológicas de brucelose) e suspeitos de brucelose (cães negativos na hemocultura, mas com suspeita clínica e/ou epidemiológica da infecção). A sensibilidade e especificidade diagnóstica das reações de LAMP e PCR foram calculadas, utilizando-se os grupos de cães infectados e não infectados, respectivamente. O desempenho dos testes foi analisado, utilizando-se as 98 amostras, comparadas duas a duas, pelos testes estatísticos de Coeficiente Kappa e McNemar. A proporção de amostras positivas foi de 43,87% (43/98) na hemocultura, 46,93% (46/98) na PCR e 16,33% (16/98) na LAMP. A concordância entre a hemocultura e a PCR foi ótima, enquanto que a concordância entre a LAMP e a hemocultura e entre a LAMP e a PCR foi sofrível. A sensibilidade diagnóstica foi de 100% (18/18) na PCR e 44,44% (8/18) na LAMP, enquanto que a especificidade diagnóstica foi de 96% (20/21) na PCR e 100% (21/21) na LAMP. O desempenho da reação de LAMP foi insatisfatório para o diagnóstico da brucelose nos cães, em razão dos baixos valores de sensibilidade do teste. A PCR, por outro lado, apresentou desempenho similar à hemocultura, o que a torna uma alternativa para uso no diagnóstico da brucelose canina. / Brucella canis is the etiological agent responsible for brucellosis in dogs and can be transmitted to human beings, occasionally resulting in severe disease, and leading to impacts on public health. Canine brucellosis triggers numerous economic losses in commercial kennels, causing abortions, embryonic death, stillbirths and birth of debilitated puppies. Serological diagnosis is routinely performed, but blood culture is the gold standard test. Polymerase chain reaction (PCR) can be used to the direct diagnosis of infection in view of its speed and high specificity and sensitivity values, however it has high cost because of the laboratory infrastructure and equipments needed. The loop-mediated isothermal amplification (LAMP) may be an alternative to DNA amplification in a shorter period of time, with simplicity and low cost. This project evaluated the potential of the molecular tests of PCR and LAMP using primers targeting the insertion sequence IS711 of Brucella, using 98 whole blood samples of 57 dogs. The 57 dogs were divided into three groups: infected by B. canis (dogs with positive results in blood culture), non-infected by B. canis (dogs with negative results by blood culture and showing no clinical or epidemiological evidences of brucellosis) and dogs suspected of brucellosis (those with negative blood culture but with clinical and/or epidemiological evidences of infection). The diagnostic sensitivity and specificity of PCR and LAMP were calculated using the infected and non-infected groups, respectively. The performance of the three diagnostic tests was pair compared using the 98 samples using McNemar test and Kappa coefficient. The proportion of positive samples detected by blood culture, PCR and LAMP was respectively 43.87% (43/98), 46.93% (46/98), and 16.33% (16/98). The concordance between blood culture and PCR was almost perfect, while the concordance between LAMP and blood culture and between LAMP and PCR was fair. The diagnostic sensitivity of PCR and LAMP was, respectively, 100% (18/18) and 44.44% (8/18), while the diagnostic specificity of the tests was 96% (20/21) and 100% (21/21), respectively. LAMP performance was not satisfactory for canine brucellosis diagnosis because of the low sensitivity of the test. PCR showed similar performance when compared to blood culture, which makes it a good alternative for use for the diagnosis of canine brucellosis.

A Brucella canis é a bactéria responsável pela brucelose nos cães, pode ser transmitida para os seres humanos, ocasionalmente resultando em doença grave, com impacto na saúde pública. A brucelose canina desencadeia inúmeras perdas econômicas em canis comerciais, com a ocorrência de abortamentos, morte embrionária, natimortos e nascimento de filhotes debilitados. O diagnóstico sorológico é rotineiramente realizado, contudo a hemocultura é o teste \"padrão-ouro\". A técnica de reação em cadeia pela polimerase (PCR) pode ser aplicada no diagnóstico direto como alternativa à hemocultura, pela rapidez, alta especificidade e sensibilidade do teste, mas apresenta alto custo com infraestrutura e equipamentos. A amplificação isotérmica mediada por loop (LAMP) constitui outra alternativa para amplificação do DNA em um curto período de tempo, com simplicidade e menor custo. O projeto avaliou comparativamente o desempenho dos testes moleculares de PCR e LAMP com primers direcionados à sequência de inserção IS711 de Brucella spp. em 98 amostras de sangue total, obtidas de 57 cães. Os 57 cães foram divididos em três grupos: infectados por B. canis (cães positivos na hemocultura) não infectados por B. canis (negativos na hemocultura e sem evidências clínicas e epidemiológicas de brucelose) e suspeitos de brucelose (cães negativos na hemocultura, mas com suspeita clínica e/ou epidemiológica da infecção). A sensibilidade e especificidade diagnóstica das reações de LAMP e PCR foram calculadas, utilizando-se os grupos de cães infectados e não infectados, respectivamente. O desempenho dos testes foi analisado, utilizando-se as 98 amostras, comparadas duas a duas, pelos testes estatísticos de Coeficiente Kappa e McNemar. A proporção de amostras positivas foi de 43,87% (43/98) na hemocultura, 46,93% (46/98) na PCR e 16,33% (16/98) na LAMP. A concordância entre a hemocultura e a PCR foi ótima, enquanto que a concordância entre a LAMP e a hemocultura e entre a LAMP e a PCR foi sofrível. A sensibilidade diagnóstica foi de 100% (18/18) na PCR e 44,44% (8/18) na LAMP, enquanto que a especificidade diagnóstica foi de 96% (20/21) na PCR e 100% (21/21) na LAMP. O desempenho da reação de LAMP foi insatisfatório para o diagnóstico da brucelose nos cães, em razão dos baixos valores de sensibilidade do teste. A PCR, por outro lado, apresentou desempenho similar à hemocultura, o que a torna uma alternativa para uso no diagnóstico da brucelose canina. / Brucella canis is the etiological agent responsible for brucellosis in dogs and can be transmitted to human beings, occasionally resulting in severe disease, and leading to impacts on public health. Canine brucellosis triggers numerous economic losses in commercial kennels, causing abortions, embryonic death, stillbirths and birth of debilitated puppies. Serological diagnosis is routinely performed, but blood culture is the gold standard test. Polymerase chain reaction (PCR) can be used to the direct diagnosis of infection in view of its speed and high specificity and sensitivity values, however it has high cost because of the laboratory infrastructure and equipments needed. The loop-mediated isothermal amplification (LAMP) may be an alternative to DNA amplification in a shorter period of time, with simplicity and low cost. This project evaluated the potential of the molecular tests of PCR and LAMP using primers targeting the insertion sequence IS711 of Brucella, using 98 whole blood samples of 57 dogs. The 57 dogs were divided into three groups: infected by B. canis (dogs with positive results in blood culture), non-infected by B. canis (dogs with negative results by blood culture and showing no clinical or epidemiological evidences of brucellosis) and dogs suspected of brucellosis (those with negative blood culture but with clinical and/or epidemiological evidences of infection). The diagnostic sensitivity and specificity of PCR and LAMP were calculated using the infected and non-infected groups, respectively. The performance of the three diagnostic tests was pair compared using the 98 samples using McNemar test and Kappa coefficient. The proportion of positive samples detected by blood culture, PCR and LAMP was respectively 43.87% (43/98), 46.93% (46/98), and 16.33% (16/98). The concordance between blood culture and PCR was almost perfect, while the concordance between LAMP and blood culture and between LAMP and PCR was fair. The diagnostic sensitivity of PCR and LAMP was, respectively, 100% (18/18) and 44.44% (8/18), while the diagnostic specificity of the tests was 96% (20/21) and 100% (21/21), respectively. LAMP performance was not satisfactory for canine brucellosis diagnosis because of the low sensitivity of the test. PCR showed similar performance when compared to blood culture, which makes it a good alternative for use for the diagnosis of canine brucellosis.

The work described in this dissertation focuses on developing simple yet effective assays integrating nucleic acid (NA) aptamers with rolling circle amplification (RCA) for the detection of non-NA biomarkers. The first project, a comprehensive literature review, highlights the current state of the art in functional NA-based RCA applications, and identifies shortcomings in the detection of non-NA targets with RCA. Biosensor design is critically evaluated from four key perspectives: regulation, efficiency, and detection of RCA, and the integration of all three components for point of care (POC) applications. The second project investigates how target-binding to a linear aptamer can be utilized to regulate RCA in a simple and inexpensive format. Phi29 DNA polymerase (DP) exhibits difficulty processing DNA strands that are bound to non-NA materials such as proteins. The work uses this restriction of phi29 DP as a feature by utilizing protein-binding aptamers as primer strands (aptaprimers) for RCA. The simplicity is showcased by adapting the method to a cellulose paper-based device for the real-time detection and quantification of PDGF or thrombin within minutes. As the second project is a turn-off sensor, the third project exploits the inherent 3’-exonuclease activity of phi29 DP to generate a simple turn-on assay instead. As target-bound aptamers were shown to be resistant to exonuclease activity, the phi29 DP preferentially digests target-free aptaprimers instead of target-bound aptaprimers. The target-bound aptaprimer could be liberated by a circular template (CT) by incorporating toehold-mediated strand displacement (TMSD), and used for RCA. Sensitivity was improved relative to project two, though the dynamic range was narrow owing to difficulty liberating target-bound aptaprimer at high target concentrations. Project four instead used RecJ, which has 5’-exonuclease activity, to modulate aptaprimer availability. Similarly to project three, target-binding conferred protection on the aptaprimer from 5’-exonuclease digestion by RecJ. By including a free 3’ terminus on the aptaprimer, inhibition of RCA due to target binding was avoided and CT-mediated TMSD was not needed, simplifying the assay. As well, this approach was generalizable as it was demonstrated using both a protein (thrombin) and a small molecule (ochratoxin A) target. This turn-on method further improved the assay compared to project three with a 100-fold enhancement in sensitivity and a restoration of the dynamic range. In sum, this work contributed multiple simple and sensitive approaches for the real-time fluorescent detection of proteins and small molecules with the RCA of linear aptamers. / Thesis / Doctor of Science (PhD)

<p dir="ltr">Nucleic acid testing has become a prominent method for rapid microbial detection. Unlike polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP) is a simple method of nucleic acid amplification where the reaction can be performed at a constant temperature and the output provided in a colorimetric format. A transparent water bath heater is a desirable instrument to perform the heating and observe the visual results of nucleic acid amplification. However, existing methods of heating the water are not convenient for loading and unloading the nucleic acid samples. Here, we developed a field-deployable water bath heating device—an isothermal heater called IsoHeat for short–which is solely dedicated to performing LAMP reactions and can heat the water up to 85 °C (if needed). Using 3D-printing and LASER-cutting technology, we fabricated different parts of the device and mechanically assembled the parts to develop the entire device. Users can commence the heating by pressing the start button on the screen after entering the target temperature. Subsequently, the device heats up the water bath and maintains the target temperature through a PID algorithm-based control system. We demonstrate that IsoHeat can operate in environmental temperatures ranging from 5-33 °C and it can conduct LAMP reactions in a liquid format as well as in paper-based devices. IsoHeat is more efficient and user-friendly compared to a commercially available immersion-heating device, which is often used to perform LAMP reactions. This newly developed device would be helpful to detect pathogens conveniently in the field (e.g., at the point-of-care for human applications, on farms for plant and animal applications, and in production facilities for food safety applications).</p>

Many environmental compartments are acknowledged transmission routes for infectious diseases, antibiotic resistance, and anthropogenic pollution. The need for environmental sensing has consistently been stressed as a means to minimize public health threats caused by such contaminants. Many analytical detection techniques have been developed and applied for environmental sensing. However, these techniques are often reliant upon centralized facilities and require intensive resources. For these reasons their use can be challenging under resource-constrained conditions characterized by poor water, sanitation, and hygiene (WASH) services. In this dissertation, we developed biotechnology- and/or nanotechnology-advanced analytical tools for environmental sensing that have potential for future application in regions with poor WASH services. First, loop-mediated isothermal amplification (LAMP) and nanopore sequencing were applied to develop assays for the detection of SARS-CoV-2, the causative agent of COVID-19, in wastewater samples. Second, surface-enhanced Raman spectroscopy (SERS) was applied for environmental detection of a range of analytes. Gold nanoparticle (AuNP)-based SERS substrates were fabricated by droplet evaporation-induced aggregation on a hydrophobic substrate. These SERS substrates were then applied for the detection of antibiotic resistance genes (ARGs) and other environmental contaminants (e.g., dye or hydrophobic organic contaminants). In a separate study, Au nanostructured SERS substrates were fabricated and applied for pH sensing in a range of environmental media. Finally, the environmental impact of an AuNP-based colorimetric detection assay was assessed via life cycle assessment. / Doctor of Philosophy / Environmental sensing is an important means to intervene against public health threats of infectious diseases and environmental contaminants. However, currently available analytical tools for environmental samples often require intensive resources that are not available in low- and middle-income countries. In this dissertation, we developed biotechnology and/or nanotechnology advanced analytical tools for environmental sensing that have potential future application applied under resource-constrained conditions. First, we applied loop-mediated isothermal amplification (LAMP) and nanopore sequencing to develop detection assays for SARS-CoV-2, the causative agent of COVID-19, in wastewater samples. Second, we applied surface-enhanced Raman spectroscopy (SERS) to develop assays for environmental analytes. We fabricated SERS substrates by evaporation-induced aggregation of gold nanoparticles (AuNPs) on a hydrophobic substrate and applied these for the detection of antibiotic resistance genes (ARGs) and other environmental contaminants. In addition, Au nanostructured SERS substrates were fabricated and applied for pH sensing in a range of environmental media. Finally, we used life cycle assessment to quantitatively evaluate the environmental impacts of an AuNP-based sensing applications.