Browsing by Subject "Diagnostics"

Laboratory enabled disease diagnosis is one of the cornerstones in patient care and greatly relies in immunological techniques to detect pathogens and quantify biomarkers related to a myriad of clinical conditions. The performance of immunoassays is directly dependent on the binding affinity of the molecules that enable capture and detection of analytical targets of interest and especially on the surfaces that interact with these sensing molecules and the complex elements present in biological samples. The lack of high-quality antibodies and nonspecific protein absorption (NSPA) on test substrates have historically hindered assay sensitivity and overall performance. While antibody development has witnessed a significant evolution over time, mostly driven by the interest in antibody-based drugs, major challenges still exist in the IVD reagent generation process. Regarding NSPA, only recently protein resistant surfaces found their way into immunoassay development applications with extremely promising results.

In this dissertation, we aimed to better understand the surface properties required to deliver a new generation of immunodiagnostics with high sensitivity and broad dynamic range. Our studies demonstrated that there are very specific physicochemical requirements a surface must present to enable inkjet based, simple fabrication of antigen detection tests. Interestingly, we determined that POEGMA based brushes, our prototypical surface, naturally presents a fine balance between protein resistance and hydrophilicity that enables their non-covalent biofunctionalization and use for IVD applications. Once we confirmed POEGMA as the ideal coating for the fabrication of antigen detection tests, our work evolved to address several of the challenges related to antibody generation for diagnostic test development. Towards this end, our work entailed the development of a new antibody pair that targets non-overlapping epitopes of ebolavirus secreted glycoprotein, a truncated version of the structural glycoprotein that is actively secreted from infected cells in early stages of the infection. The generation of these antibodies was achieved by associating scFv phage-display technology with the transient expression of promising scFv candidates as Fc fusions in mammalian cells followed by their seamless purification with an IsoTag based chromatography-free system. These elements when combined with a novel antibody pairing strategy, that leverages the D4 Assay’s multiplexing capabilities and ease of fabrication, warranted the rapid identification of optimal capture and detection reagents. Finally, employing these reagents, we developed and validated an ultra-sensitive ebolavirus detection test based on the D4 Assay test format, which was able to not only match but outperform the sensitivity of qRT-PCR. This exceptional sensitivity which can enable the deployment of life-saving treatment and containment efforts was demonstrated in two independent nonhuman primate models of the disease and attests to the success of this new IVD test development work-flow.

Serological testing—detection of antibodies—plays a key role in the diagnosis, management, and surveillance of infectious diseases. Serological assays can detect both active and past infections which is essential in understanding epidemiological variables such as incidence and fatality rates. Another important metric, antibody avidity, provides additional insight into recency of infection, can be used to discriminate between closely related infectious species, assess vaccine efficacy and provides estimates of who is and who is not immune to certain infections. However, conventional methods of measuring antibody avidity are costly, time consuming, and utilize harsh denaturing reagents that negatively impact automated immuno-ELISA equipment. These challenges have deterred the development of point of care tests for antibody avidity. In this thesis, we investigated the performance of four assay formats for antibody detection developed by inkjet-printing assay reagents on glass surfaces coated with a non-fouling polymer brush. We then adopted the antibody detection formats to determine antibody avidity by measuring resistance of the antibody-antigen bonds to chaotropic agents. We further developed a new technique of measuring antibody avidity by reducing the concentration of capture antigen (cAg) on the immunoassay platform. In this new technique, avidity index was determined as the ratio of fluorescence intensity measured at a lower cAg concentration to intensity measured at a higher cAg concentration. This technique showed strong correlation (R > 0.8) with the conventional method of antibody avidity measurement (resistance to chaotropic agent) in three antibody-antigen systems. Additionally, we showed that the proposed platform can detect key biomarkers for identifying recent HIV1 infections. The targeted biomarkers were based on measuring titers and avidity of antibodies secreted against specific clades of HIV envelope proteins. They included clade C GP140 IgG3, transmitted/founder clade C GP140 IgG4 avidity, clade B GP140 IgG4 avidity, and GP41 immunodominant region (GP41-ID) IgG avidity. The proposed assay detected all four biomarkers with wide dynamic ranges (>103.6) and high sensitivity in diluted pooled human serum. The proposed platform for antibody avidity testing is rapid, easy to use and has high correlation with chaotropic resistance. It therefore has potential to enable measurement of antibody avidity at the point of care for clinical applications.

Bioanalytical techniques such as immunoassays are ubiquitous in clinical and basic research laboratories and have transformed how we diagnose patients, monitor health, and study disease. Immunoassays typically use capture reagents, such as antibodies or antigens, to detect and quantify a biomarker of interest from a clinical specimen based on highly sensitive and specific binding interactions. While laboratory-based assays, such as enzyme-linked immunosorbent assay (ELISA), are the workhorses of clinical laboratories, they have several shortcomings that limit their overall utility, especially in low resource settings. Of note, ELISA requires multiple timed incubation steps, trained personnel, expensive equipment, and suffers from long times to result. To democratize access to clinical-grade tests, researchers have sought out different methods for point-of-care (POC) testing that are easy to perform and maintain high sensitivity and specificity. This dissertation describes the use of a “zero-background” polymer coating—poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA)—as a substrate for highly sensitive and specific POC diagnostic tests. The POEGMA coating eliminates nearly all non-specific protein adsorption and cellular adhesion, thus leading to high signal-to-noise ratios, even from complex biological samples, such as whole blood. In addition, the POEGMA brush contains all biomolecules necessary to complete an assay after addition of a liquid sample, thus allowing assays to be conducted in a single step. Further, the POEGMA coating stabilizes biomolecules on the surface, which allows tests to be stored at ambient conditions without refrigeration. Assays are read out using a fluorescence detector which quantifies the concentration for a given biomarker of interest. By inkjet printing capture biomolecules at discrete spatial addresses on the POEGMA-slides, multiplexing can be accomplished using a single fluorophore which greatly reduces the complexity and costs for assay readout. This dissertation focuses on adapting and applying this platform to several clinically relevant applications. First, we developed a test for molecular and cellular credentialing of breast cancer tissue at the POC (Chapter 2). With the onset of the coronavirus 2019 (COVID-19) pandemic, we adapted the platform to detect several different relevant biomarkers for COVID-19, including total antibody concentration against several viral proteins (Chapter 3), neutralizing antibodies (Chapter 4), and viral proteins (Chapter 5). All the tests developed for COVID-19 use multiplexed sensing strategies and can be conducted with minimal/no user intervention or clinical infrastructure. Taken together, these studies highlight the great potential for bioanalytical assays built upon POEGMA-coated substrates to be used for clinical applications in disease diagnosis, surveillance, and management.

Background

Melioidosis is a neglected tropical disease that is highly prevalent in Southeast Asia. Misdiagnoses are common as the presenting symptoms are similar to other diseases including upper respiratory infections. When not treated with antibiotics, the disease can lead to severe morbidity or death. Current diagnostics in low- and middle-income countries are often not sensitive nor rapid. Point-of-care rapid diagnostic tests (POC-RDTs) are a potential solution. Few studies have compared the accuracy of POC-RDTs and molecular assays against blood culture. The goal of this study was to conduct such comparisons in detecting Burkholderia pseudomallei infections among infection-suspected patients in Kapit, Sarawak, Malaysia.

Methods

We used an informed consent process as approved by two institutional review boards. In this cross-sectional study, we engaged patients meeting a melioidosis-like case definition that included classical symptoms such as prolonged fever with joint pain and/or abscess. We studied the patients routinely collected clinical specimens with a POC-RDT (Active Melioidosis DetectTM) and a molecular assay compared with the B. pseudomallei bacterial culture for isolation of the bacterial organism.

Results

One hundred patients aged 6 months - 79 years from Kapit Hospital were enrolled in the study from June 12, 2018 to January 8, 2019. Of the 100 sera, 97 urine, and 16 bodily fluid samples (total n= 213) tested with the RDT, 23 samples gave positive results (7 sera, 15 urine, and 1 bodily fluids). Compared to the molecular assay, the POC-RDT had a sensitivity of 40% (95% CI, 5%- 85%), specificity of 94% (95% CI, 87% - 98%), and an accuracy of 90% (95 CI, 82% - 95%) for sera; and a sensitivity of 80% (95% CI, 28%- 99%),a specificity of 65% (95% CI, 55% - 75%), and an accuracy of 87% (95 CI, 77% - 94%) for urine; and a sensitivity of 80% (95% CI, 28%- 99%), a specificity of 65% (95% CI, 55% - 75%), and an accuracy of 81% (95 CI, 54% - 96%) for other bodily fluids. Additionally, when compared to the bacterial culture results, the POC-RDT showed a sensitivity of 38% (95% CI, 9%- 76%), specificity of 95% (95% CI, 88% - 99%), and an accuracy of 90% (95 CI, 82% - 95%) for sera; a sensitivity of 88% (95% CI, 47%- 100%), a specificity of 88% (95% CI, 77% - 95%), and an accuracy of 94% (95 CI, 84% - 98%) for urine; and a sensitivity of 25% (95% CI, 1%- 81%), a specificity of 100% (95% CI, 74% - 100%), and an accuracy of 81% (95 CI, 54% - 96%) for other bodily fluids.

Conclusion

While study enrollment will continue, data from the first 100 participants, suggests the POC-RDT had poor sensitivity, good accuracy, and high specificity in detecting B. pseudomallei infection. Thus far, the POC-RDT assay seems to work better on urine specimens. Due to low sensitivity, the study data do not support recommending POC-RDT strips as a single diagnostic method. However, as the POC-RDT had high specificity when the test is positive it seems appropriate for clinicians to assume the patient is infected and to prescribe specific antimicrobial therapy. While more participant data are needed, it seems likely that the POC-RDT could be useful in helping physicians to begin treatment early with the high specificity that the POC-RDT has exhibited. If paired with an RDT with high sensitivity, this POC-RDT would add a great value to infection management.

As the cost of medical care increases, people are relying increasingly on internet diagnosis and community care rather than the expertise of medical professionals. Technological and medical advances have facilitated a partial answer through the increase in handheld sensing apparatuses. Yet even with these developments, significant further advancements are required to further drive down fabrication requirements (both in terms of cost and environmental impact) and facilitate fully-integrated and easy to use sensors. Printing electronics could be a powerful tool to accomplish this as printing allows for low-cost fabrication of high-area electronics. The vast majority of printed electronics reports focus on utilization of already developed commercial inks to create devices with new functionalities. This significantly limits development because current inks both necessitate damaging post processing—which precludes the use of delicate substrates, making skin-integration impossible—and many inks require bespoke printing processes, which increases fabrication complexity and thus cost. Further, with the proliferation of single-use medical testing, consideration must be made towards environmental compatibility. Therefore, innovations in electronic ink formulation and printing geared towards addressing the post-processing and environmental impact concerns are needed to enable continued progress towards printed POC sensors. The work contained in this dissertation centers around the development of inks intended to advance electronic biosensing applications. Focus is on using aerosol jet printing to enable the printing of nanomaterials and utilizes the unique properties of these nanomaterials—such as functionality immediately after printing, recyclability, and compatibility with deposition directly on biological surfaces (i.e., human skin)—to develop technologies intended to democratize healthcare. Notably, low temperature printable silver nanowire (AgNW) inks for the creation of biologically integrated electronics are demonstrated. Electrically conductive inks are created that are capable of achieving high conductivities when directly deposited onto living tissue at temperatures compatible with life (20 °C). The conductive lines yielded high resistance to degradation from bending strain, with a mere 8% decrease in conductivity when the plastic film on which they were printed was folded in half. As a demonstration, the AgNW ink was printed onto a human finger and used to illuminate a small light that remained illuminated even when the finger was bent. These results pave the way towards patient-specific medical diagnostics that are comfortable to wear, easy to use, and designed towards the needs of each individual patient. Next, a printing method to deposit biological sensing proteins for biomedical assays is investigated. Traditional techniques require extended time and the use of large quantities of immensely expensive proteins to make biosensors. Herein, a decade-old belief that aerosol jet printing is incompatible with the deposition of proteins is overturned, and, in doing so, highly sensitive biosensors for carcinoembryonic antigen (CEA) that compare favorably the mainstay fabrication technique that is known to impart no damage to the printed biological inks is demonstrated. Finally, the co-printing of a bio-recognition element with the previously mentioned electrically conductive AgNW ink demonstrate the potential for the future investigation of a fully aerosol-jet printed electronic biosensor. To address the environmental waste accumulation concern that plagues the advancement of ubiquitous patient-guided sensing, inks that facilitate the creation of fully-printed, all-carbon recyclable electronics (ACRE) are investigated. The combination of nanocrystalline cellulose, graphene and semiconducting carbon nanotubes enable the first fully recyclable transistor device. The ACRE transistors maintain high stability for over 10 months, display among the best performance of any printed transistor (Ion/Ioff: 104 and Ion 65 µA µm-1) and can be entirely deconstructed for recapture and reuse of the constituent CNT and graphene inks with near 100% nanomaterial retention and the biodegradation of the cellulose-based components. ACRE-based lactate sensors are used as an illustration of utility to show the versatility of the platform. Finally, as a culminating demonstration, a fully-printed chip for the handheld measurement of blood clot time (prothrombin time) was developed. Printing the entirety of the device allows for the creation of a low-cost chip for the simple, fast, and robust measurement of human blood clot times. In addition, a custom-designed, handheld control system with a 3D-printed case was developed to create a fully integrated point-of-care measurement platform towards simplifying medicine dosing strategies. The work described herein marks a significant leap in the development of printed inks to enable custom biological sensing applications. Once fully realized, these applications will mark a watershed, ushering in an era of individualized medicine with ubiquitous sensing to actively track disease progression in real-time. We are at the dawn of a new era in medicine that focuses more on prevention and control as opposed to reaction. One future direction for this work is promoting directly printed and reusable on-skin theragnostics for bespoke patient care such as the delivery and monitoring of pain medication that allows for better oversite over use and misuse.

Introduction: According to the World Health Organization (WHO), 500 million arbovirus cases are diagnosed around the world annually, with 2.7 million associated deaths [1]. The burden of disease caused by dengue, chikungunya, and Zika viruses is likely to be underestimated due to a lack of accurate diagnostic tools and knowledge gaps regarding their epidemiology [2, 3]. This thesis uses a subset of data from an on-going 24-month study to evaluate the potential etiology of dengue-like symptoms of patients recruited from medical facilities in Sarawak, Malaysia. A secondary aim is to assess the diagnostic clinical effectiveness of a new detection method, the novel T-Cor 8 Multiplexing Platform (Tetracore, Inc., USA), using qRT-PCR assays as the gold standard method for comparison. The prevalence of arboviral infections as determined by gold-standard qRT-PCR assays and potential risk factors in the study population were also analysed.

Methods: In this cross-sectional study, patients more than seven years of age with dengue-like symptoms were enrolled at medical facilities in the towns of Sibu and Kapit in Sarawak, Malaysia. Blood, urine, and gingival crevicular fluid samples, as well as risk factor data, were collected from participants at the time of enrolment. These samples were studied by qRT-PCR assays and the novel T-Cor 8 Multiplexing Platform.

Results: Seven (14%) of 51 participants’ serum RNA samples tested positive for arbovirus infection by gold-standard qRT-PCR assays. Two participants (4%) were positive for dengue subtype-1, four participants (8%) were positive for dengue subtype-2, and one participant (2%) was positive for dengue subtype-4. No patient samples had molecular evidence of chikungunya or Zika viruses. The T-Cor 8 multiplexing platform demonstrated a 71% sensitivity (95% confidence interval 29-96%), 93% specificity (95% confidence interval 81-99%), and 90% accuracy (95% confidence interval 78-97%) compared to the gold-standard assays on serum RNA samples. From this subset of data, we failed to identify important risk factors for arboviral infection.

Conclusion: From this limited subset of data, we conclude that the T-Cor 8 platform’s simplicity and accuracy in detecting at least dengue virus infections has considerable potential for clinical usefulness in low-resource settings.

Despite its ubiquitous abundance, Hsp90 inhibitors have shown promise in anti-cancer clinical trials, suggesting that Hsp90 inhibitors selectively target tumor cells while exhibiting minimal effects in normal cells. Extracellular expression of heat shock protein 90 (eHsp90) by tumor cells is strongly correlated with malignancy. Development of small molecule probes that can specifically detect eHsp90 in vivo may therefore have utility in the early detection of malignancy. We synthesized a fluorescent cell impermeable Hsp90 inhibitor, HS-131, to target eHsp90 in vivo. HS-131 was characterized biochemically to ensure specificity for eHsp90, and an inactive analog was also synthesized to be used as an in vivo control.

Through confocal microscopy, eHsp90 can be visualized with cell impermeable, fluorophore-tagged Hsp90 inhibitors. High resolution confocal and real time lattice light sheet microscopy showed that probe-bound eHsp90 accumulates in punctate structures on the plasma membrane of breast tumor cells and is subsequently actively internalized. This internalization occurs in the presence and absence of inhibitors. The extent of internalization correlates with tumor cell aggressiveness, and this process can be induced in benign cells by over-expressing p110HER2, leading to malignant transformation of these cells. Internalization of eHsp90 is also increased after inhibition of Hsp70, suggesting that overcompensation of the heat shock response can also upregulate the eHsp90 trafficking mechanism. Whole body 3D cryo fluorescence imaging and histology of flank and spontaneous tumor-bearing mice strongly suggests that eHsp90 expression is a unique phenomenon in vivo.

Taken together, these results suggest that active and differential internalization of eHsp90 in aggressive cancer cells contributes to the selectivity observed upon Hsp90 inhibitor treatment and may provide a novel metastatic biomarker for solid tumors and may lead to the development of a tumor-specific drug delivery system.

Background: Antimicrobial overprescription has been associated with antimicrobial resistance, and is common for lower respiratory tract infections (LRTI) as viral and bacterial infections generally present with similar clinical features. The aim of this study was to identify the prevalence and predictors of antibiotic prescription among children and adults hospitalized with viral LRTI. Methods: A prospective cohort study was conducted among patients hospitalized with viral LRTI in a tertiary care hospital in Southern Province, Sri Lanka from April 2018 to October 2021. Consecutive patients ≥1 year old who met a case definition for LRTI were enrolled. Patients’ demographic, clinical, and laboratory data were recorded. A nasopharyngeal sample and blood sample were collected from all patients for multiplex polymerase chain reaction (PCR) testing for respiratory pathogens and procalcitonin (PCT) detection, respectively. Among patients with viral RTI, demographic and clinical features associated with antibiotic prescription were identified using Chi Square and t-tests, as appropriate. Significant variables (p<0.05) on bivariable analyses were included in a multivariable logistic regression model to identify features associated with antibiotic prescription. The potential impact of procalcitonin testing on antibiotic prescription was simulated using standard PCT cut-offs. Analyses were conducted separately for children (<18 years) and adults using R Statistical Software (R Core Team 2022). Results: A total of 1217 patients were enrolled during the study period; of these, 438 (36.0%) had one or more respiratory viruses detected. Of 438 patients, 48.4% were male and 30.8% were children; the median age was 4 years (IQR 2-7) for children and 61 years (IQR 48-70) for adults. The most commonly detected viruses were influenza A (39.3%), human rhinovirus/ enterovirus (HRV/HEV; 28.3%), and respiratory syncytial virus A (RSV A; 10.3%). Overall, 114 (84.4%) children and 266 (87.8%) adults with respiratory viruses were treated with antibiotics during hospitalization. On multivariable modeling for children, neutrophil percentage (median 63.8% vs 47.3%, p=0.03) was positively associated with antibiotic prescription. Among adults, headache (60.6% vs 35.1%, p=0.003), presence of crepitations/crackles on exam (55.3% vs 21.6%, p<0.001), rhonchi/wheezing on exam (42.9% vs 18.9%, p=0.005), and presence of opacities on chest x-ray (27.4% vs 8.1%, p=0.01) were associated with antibiotic prescription. Access to PCT test results could have potentially decreased inappropriate antibiotic prescription in this study by 83.3%. Conclusions: A high percentage of viral detection and high prevalence of antibiotic prescription were observed among a large inpatient cohort with LRTI. Our results suggest that improved access to point-of-care biomarker testing may improve antimicrobial stewardship for LRTI in this low-resource setting.

Background: It is unclear if strategies such as using different interferon-gamma release assay (IGRA) cutoffs or performing serial testing with a tuberculin skin test (TST) and an IGRA, or two different IGRAs could improve test accuracy in those from countries with high TB prevalence. Methods: We used Bayesian latent class analysis to construct receiving operating characteristic (ROC) curves and calculate their area under the curve (AUC) based on our cohort of HIV-negative, non-US-born individuals 5 years and older residing in the USA with valid results for all three LTBI tests (i.e TST, Quantiferon Gold-In-Tube (QFT) and T-SPOT.TB (TSPOT)). We compared the sensitivity, specificity and predictive values of 1) standard U.S. cutoffs for test positivity and higher or lower cutoffs, and 2) a single LTBI test and two tests: TST-QFT, TST-TSPOT, QFT-TSPOT. Results: A total of 14,167 participants were included in the analysis. The ROC curves had an AUC of 0.81 (95% Credible Interval (CrI) 0.78 - 0.86), 0.89 (95% CrI 0.86-0.93), and 0.92 (95% CrI 0.88-0.96) for TST, QFT and TSPOT respectively. QFT at the 0.35 IU/ml cutoff and TSPOT at a cutoff of 5 spots made the least number of diagnostic errors. The TST-QFT combination had similar sensitivity, specificity, PPV and NPV to a TST at a cutoff of 8 spots and the TST-TSPOT performed similarly to a QFT at a cutoff of 0.7 IU/mL. Conclusions: Using the lowest IGRA cutoffs performed better in our study population by increasing test sensitivity and NPV, therefore minimizing the total number of diagnostic errors. Two tests performed similarly to single testing with an IGRA at higher cutoffs.