Browsing by Author "Richards-Kortum, Rebecca R"

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    A low-cost platform for cervical cancer detection
    (2016-04-11) Grant, Benjamin David; Richards-Kortum, Rebecca R
    Cervical cancer causes an estimated 265,000 deaths annually, 90% of which occur in developing countries. To reduce the global burden of cervical cancer, screening programs and HPV vaccination are needed worldwide. Screening techniques that have had significant success in high resource settings have failed in low-to middle-income countries due to a variety of factors, including lack of highly trained personnel and lack of laboratory infrastructure. Two alternative methods have been employed for cervical cancer screening in low resource settings: visual inspection with acetic acid and human papillomavirus (HPV) DNA testing. While both methods are promising, they both suffer from low specificity. In this thesis, I explored the ability of the high-resolution microendoscope (HRME) to discriminate between neoplastic cervical precancerous lesions and benign conditions in vivo in a pilot study of 59 patients in Barretos, Brazil. By calculating mean nuclear eccentricity and area, neoplastic lesions were discriminated from benign conditions with 92% and specificity of 77%. Further, I designed a new mobile-phone based HRME which reduced the size of the HRME by a factor of four and the prototype cost from $5,000 to $1,700. In addition to imaging techniques, I explored low-cost immunoassays to aid both in cervical cancer screening and HPV vaccination efforts. Detection of the HPV oncoprotein E7 in cervical swabs may offer more specificity than HPV DNA testing, but current testing is both time and equipment intensive. I have developed a paper-based platform for running highly sensitive immunoassays without the need for sophisticated laboratory equipment. In a proof-of-principle malaria antigen assay, this device achieved equivalent limit-of-detection to a standard ELISA. Additionally, I present a modified version of this device for a serological HPV antibody test. In a pilot study of 24 volunteers, the test correctly identified those who had received two or more HPV vaccines with 100% accuracy. Such a test is needed to reduce unnecessary revaccination and to promote efficient vaccination in settings with poor medical records. This work provides an improvement upon the HRME imaging technique and a highly sensitive point-of-care molecular diagnostic platform to aid in cervical cancer screening, detection and prevention in low-resource settings. The preliminary results from clinical pilot studies provide the foundation for further work to help reduce the global cervical cancer burden.
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    Isothermal Nucleic Acid Assays for the Detection of HIV Drug Resistance and Sickle Cell Disease in Low-Resource Settings
    (2020-04-24) Natoli, Mary Elizabeth; Richards-Kortum, Rebecca R
    HIV treatment has become more widely available and effective in the past several decades, with major global health initiatives targeted at increasing diagnosis, treatment, and viral suppression. However, as treatment for HIV has become more widely available and effective, HIV drug resistance has emerged as a major challenge, with a disproportionate effect in low- and middle-income countries (LMICs). A lack of resources in these settings has made it difficult to implement effective strategies to differentiate ARV therapy from true instances of drug resistance. 90% of HIV drug resistance is caused by four specific single-nucleotide polymorphisms (SNPs). Sickle cell disease (SCD) is another disease of global importance that results from a point mutation in the β-globin gene which causes red blood cells to sickle. This in turn leads to painful vaso-occlusion and a host of other clinical consequences. The goal of this thesis work was to develop low-cost nucleic acid tests that can improve early detection of these two conditions in low-resource settings. The first part of this thesis work focused on the development of a method to discriminate M184V, the most common HIV-1 drug resistance mutation, from wild type DNA, and to detect the products in a paper-based enzyme-linked immunosorbent assay (ELISA) format. First, a section of HIV-1 reverse transcriptase is isothermally amplified using a recombinase polymerase amplification (RPA) assay. Next, an oligonucleotide ligation assay (OLA) is used to selectively label the mutant and wild type amplified sequences. Finally, a lateral flow ELISA differentiates between OLA-labeled products with or without M184V. Our method shows 100% sensitivity when tested with samples that contained 200 copies of mutant DNA and 800 copies of wild type DNA prior to amplification. When integrated with sample preparation, this method may detect HIV-1 drug resistance at a low cost and at a rural hospital laboratory. The second part of this thesis work focused on point-of-care detection of sickle cell disease (SCD). SCD is a common, life-threatening disorder caused by a point mutation in the β-globin gene. Early diagnosis through newborn screening is known to reduce mortality; however, the high cost and complexity of conventional diagnostic methods limit the scope and sustainability of newborn screening for SCD in resource-limited areas worldwide. Although several point-of-care tests are currently in development, antibody-based tests cannot be used in patients who have recently received a blood transfusion. Here we describe the development of a rapid, low-cost nucleic acid test that detects the point mutation that causes the formation of sickle hemoglobin (HbS) in one round of isothermal amplification and in an enclosed tube. When tested with a set of clinical samples, our assay demonstrated 100% sensitivity for both the βA globin and βS globin alleles, and 94.7% and 97.1% specificity for the βA globin allele and βS globin allele, respectively (n=91). Finally, sample-to-answer genotyping of genomic DNA is demonstrated from capillary blood in <30 minutes at a cost of <5 USD. This work demonstrates the potential utility of a point-of-care, sample-to-answer nucleic acid test for SCD that will be easily adapted to other disease-causing point mutations in genomic DNA. Overall, this thesis covers the progress made toward several technologies to detect point mutations using isothermal amplification, and contributes to the growing field of scientific knowledge on point mutation detection in resource-limited settings.
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    Multimodal Optical Imaging for Improved Evaluation of Oral Premalignant Lesions
    (2020-03-23) Yang, Eric C; Richards-Kortum, Rebecca R
    Improved evaluation of oral premalignant lesions (OPLs) at risk of malignant transformation is critical to improving oral cancer outcomes. Towards this goal, a multimodal optical imaging system (MMIS) that integrates white-light, autofluorescence (AF), and high-resolution microendoscopy (HRME) imaging to non-invasively evaluate OPLs was developed. The MMIS uses an automated, real-time image analysis algorithm to classify imaged sites as low- or high-risk for high-grade dysplasia or cancer. To analyze HRME images containing regions without quality information, a novel algorithm utilizing deep learning and probabilistic methods was also developed. Finally, two clinical studies were conducted to evaluate the MMIS. In the first study, high-risk OPL patients under surveillance for malignant progression received biopsies if clinically indicated, or if indicated by the MMIS evaluation, at the clinician’s discretion. The results demonstrated that the addition of the MMIS improved identification of high-grade dysplasia, compared to clinical evaluation alone. The second study explored the potential of the MMIS in a community dental setting, where most lesions are benign confounders. The study found that AF+HRME had a higher diagnostic accuracy than the clinical impression of a group of dental residents, and that the addition of AF and HRME to the residents’ clinical impressions improved their diagnostic performance. Together, these studies indicate the potential of the MMIS to improve the clinical evaluation of OPLs in multiple settings.