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.

<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.

<p dir="ltr">Since the beginning of the HIV/AIDS epidemic, 85.6 million people worldwide have become infected with HIV; more than half of whom have died from AIDS-related complications.[1] Sustained viral suppression below the clinically relevant threshold (1000 copies per mL) with highly active antiretroviral therapy (HAART) has proven effective at managing and prolonging the life expectancy of people living with HIV (PLHIV). However, in 2022, 11.3 million PLHIV had still not achieved viral suppression and may become susceptible to both HIV transmission and a variety of opportunistic infections. Of particular importance is the complex issue of patient non-compliance in global HIV management due to social, economic, behavioral, and healthcare access barriers, potentially disconnecting many PLHIV from the HIV care continuum. Therefore, to boost patient engagement in clinical care and to improve overall patient outcomes, new approaches to viral load monitoring practices need to be developed to increase access, particularly in regions of high HIV prevalence.</p><p dir="ltr">Nucleic acid amplification tests (NAATs) have emerged as potent tools for monitoring viral load, with reverse transcription quantitative polymerase chain reaction (RT-qPCR) being recognized as the benchmark due to its sensitivity and ability for real-time quantification enabled by fluorescence signal emission. Nevertheless, RT-qPCR is burdened by drawbacks including extended processing times, high operational costs, and the requirement for specialized laboratory facilities. In this study, we propose a novel method for HIV-1 viral load monitoring by integrating reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) with real-time particle diffusometry (PD). This approach allows for the continuous monitoring of changes in the diffusion of 400 nm fluorescent particles during RT-LAMP amplification, targeting the <i>p24</i> gene region of HIV-1 RNA. This enables the real-time detection of amplification curves, achieving a detection sensitivity in water samples as low as 25 virus particles per μL within a short duration of 30 minutes. Additionally, to address challenges related to amplification inhibition in complex human specimens, we developed a power-free sample processing system specifically designed for extracting HIV-1 RNA from both whole blood and plasma.Top of FormBottom of FormThis system modifies a commercially available spin-column protocol by integrating a syringe device and handheld bulb dryer, thus eliminating the requirement for a centrifuge. The adaptation allows for the completion of the entire extraction procedure, encompassing viral lysis, RNA capture, washing, and elution of purified HIV-1 RNA, within a timeframe of less than 16 minutes. Subsequent analyses, including RT-LAMP and RT-qPCR, demonstrate a limit of detection of 100 copies per μL and an average RNA recovery of 32% (for blood) and 70% (for plasma) in the elution fraction. Further investigations emphasize the significant presence of purified RNA in the spin column volume (termed as dead volume), and the cumulative recovered RNA copies align with those obtained using the gold standard centrifugation extraction method. Ultimately, we incorporated the real-time quantitative PD-RT-LAMP assay onto a field-compatible handheld portable platform suitable for field use, featuring built-in quality control measures. This platform enables sample-to-answer viral load testing near the point of care (POC). Subsequently, we undertook essential preparatory steps, such as reagent drying to obviate the need for cold storage, initial device calibration, and hands-on training of laboratory personnel regarding device operation, to validate device performance within a cohort of individuals living with HIV (PLHIV). These innovations facilitate quick and comprehensive viral load determination, offering promise for enhanced HIV management and patient care</p>

<p dir="ltr">The gastrointestinal tract harbors a diverse range of microorganisms, collectively constituting the gut microbiome. <a href="" target="_blank">The maintenance of a symbiotic relationship between the host and these microorganisms is essential to gastrointestinal health. Disruption of the ecological balance within the gut microbiome can result in discomfort or pathological conditions.</a> <a href="" target="_blank">This dissertation explores these alterations within the gastrointestinal tract as potential indicators for specific gastrointestinal diseases.</a> <a href="" target="_blank">In pursuit of this, I collaborated with others to develop a smart ingestible capsule that offers a non-invasive method for enhancing the effectiveness of differential diagnosis and treatment strategies for Inflammatory Bowel Disease (IBD). </a>My contributions encompassed conducting <i>in vitro</i> protein sampling and extraction experiments, as well as enteric coating dissolution tests. Following thorough characterization of the capsule, I advanced to <i>ex vivo</i> sampling experiments. As a proof of concept, the capsule's sampling capabilities have been rigorously validated both <i>in vitro</i> and <i>ex vivo</i> using calprotectin, a key biomarker for monitoring and managing IBD. Future research may explore integrating this technology with other sensors for diverse chemical and gas sensing capabilities, aiming to refine the differential diagnostics of Irritable Bowel Syndrome (IBS) and IBD.</p><p dir="ltr">Simultaneously, the potential transmission of pathogenic microorganisms from the gastrointestinal tract to the environment through fecal matter can lead to substantial public health implications if adequate surveillance is not in place. These pathogens can contaminate water and food sources from various origins, exacerbating the problem. Furthermore, conventional laboratory-based assays, while effective, have extensive turnaround times and require skilled scientists to operate them. In response to this challenge, I have undertaken the development of point-of-care assays, aiming to streamline the detection of fecal contamination. This innovation is designed to mitigate the limitations associated with traditional methods by offering a more rapid and user-friendly approach. The primary objective is to enhance the accessibility of these assays, enabling on-site personnel with varying levels of expertise to utilize them effectively. Through the widespread adoption of these point-of-care assays, the overarching goal is to ensure the consistent provision of safe and reliable water and food supplies to the public.</p>

<p dir="ltr">The necessity for on-farm, point-of-need (PON) nucleic acid amplification tests (NAATs) arises from the prolonged turnaround times and high costs associated with traditional laboratory equipment. This thesis aims to address these challenges by developing devices and a user-interface application designed for the efficient, accurate, and rapid detection of <i>Bacteroidales</i> as an indicator of fecal contamination on fresh produce farms.</p><p dir="ltr">In pursuit of this, I collaborated with lab members to engineer a Field-Applicable Rapid Microbial Loop-mediated isothermal Amplification Platform, FARM-LAMP. This device is portable (164 x 135 x 193 mm), energy-efficient (operating under 20 W), achieves the target 65°C with ± 0.2°C fluctuations, and is compatible with paper-based biosensors for loop-mediated isothermal amplification (LAMP). Subsequently, I led the fabrication of the microfluidic Field-Applicable Sampling Tool, FAST, designed to deliver high-throughput (10 samples per device), equal flow-splitting of fluids to paper-based biosensors, eliminating the need for a laboratory or extensive training. FARM-LAMP achieved 100% concordance with standard lab-based tests when deployed on a commercial lettuce farm and FAST achieved an average accuracy of 89% in equal flow-splitting and 70% in volume hydration.</p><p dir="ltr">A crucial aspect of device development is ensuring that results are easily interpretable by users. To this end, I developed a Python-based image analysis codebase to quantify sample positivity for fecal contamination, ranging from 0% (no contamination) to nearly 100% (definite contamination) and the concentration of field samples. It utilizes calculus-based mathematics, such as first and second derivative analysis, and incorporates image analysis techniques, including hue, saturation, and value (HSV) binning to a sigmoid function, along with contrast limited adaptive histogram equalization (CLAHE). Additionally, I developed a preliminary graphical user interface in Python that defines a prediction model for the concentration of <i>Bacteroidales</i> based on local weather patterns.</p><p dir="ltr">This thesis encompasses hardware development for on-field quantification and the creation of a preliminary user-interface application to assess fecal contamination risk on fresh produce farms. Integrating these devices with a user-interface application allows for rapid interpretation of results on-farm, aiding in the effective development of strategies to ensure safety in fresh produce operations.</p>