A fully integrated paperfluidic molecular diagnostic chip for the extraction, amplification, and detection of nucleic acids from clinical samples

Paper diagnostics have successfully been employed to detect the presence of antigens or small molecules in clinical samples through immunoassays; however, the detection of many disease targets relies on the much higher sensitivity and specificity achieved via nucleic acid amplification tests (NAAT). The steps involved in NAAT have recently begun to be explored in paper matrices, and our group, among others, has reported on paper-based extraction, amplification, and detection of DNA and RNA targets. Here, we integrate these paper-based NAAT steps into a single paperfluidic chip in a modular, foldable system that allows for fully integrated fluidic handling from sample to result. We showcase the functionality of the chip by combining nucleic acid isolation, isothermal amplification, and lateral flow detection of human papillomavirus (HPV) 16 DNA directly from crude cervical specimens in less than 1 hour for rapid, early detection of cervical cancer. The chip is made entirely of paper and adhesive sheets, making it low-cost, portable, and disposable, and offering the potential for a point-of-care molecular diagnostic platform even in remote and resource-limited settings.U54 EB015403 - NIBIB NIH HHS; U54 EB015408 - NIBIB NIH HHS; U54-EB015403-S1 - NIBIB NIH HH

Multimodal Optical Imaging for Detection of Cervical Neoplasia

Despite being the most preventable cancer, cervical cancer remains the third leading cause of cancer death worldwide. Over 85% of cervical cancer incidence and mortality occurs in low-resource countries where screening programs for early detection are either inadequate or unavailable. In the developed world, where screening programs are well organized, incidence and mortality rates are greatly reduced. Recent advances in optical imaging have the potential to enable cervical cancer screening at the point-of-care, even in the hands of less experienced providers. High performance optical imaging systems can be constructed at relatively low cost, and image analysis can be automated; thus, these technologies may provide a way to bridge the gap to cervical cancer screening for developing countries. This work focuses on the design, construction, and clinical testing of a novel multimodal optical imaging (combination of wide-field imaging and high-resolution) for early detection of cervical neoplasia. The Multimodal Digital Imager (MDI) acquires in vivo images of cervical tissue in fluorescence, narrow band reflectance, and orthogonal polarized reflectance modes using multiple illumination wavelengths. The High Resolution Microendoscope (HRME) was used to interrogate clinically suspicious areas with subcellular spatial resolution, revealing changes in nuclear to cytoplasmic area ratio. In vivo image data from the wide-field system was combined with image data from a high- resolution microendoscope (HRME) in order to test the effectiveness of the multimodal optical imaging in discriminating between cervical neoplasia and non-neoplastic. Multimodal optical imaging coupled with computer aided diagnostic achieved a sensitivity of 82% and specificity of 85% for discriminating cervical neoplastic from non-neoplastic This work has demonstrated that multimodal optical imaging; combination of wide-field and high-resolution optical imaging of the cervix can assist in the detection of cervical neoplasia and can be implemented effectively in a low-resource setting

Computer Image Analysis Based Quantification of Comparative Ihc Levels of P53 And Signaling Associated With the Dna Damage Repair Pathway Discriminates Between Inflammatory And Dysplastic Cellular Atypia

Epithelial oncogenesis is believed to be generally associated with the accumulation over time of an increasing number of mitotic errors until a threshold number of mutations required for the initiation of cancer is achieved. Preemption of cancer through the morphologic detection of dysplastic cells, i.e. cells with a number of mitotic errors that are still below the threshold for cancer, followed by their surgical removal or eradication, has had an enormous impact on reducing the incidence of cancer of the uterine cervix, skin and colon worldwide, but this strategy has been much less successful with cancers in most other body sites. Inflammation is a relatively common occurrence in the epithelium and is far more common than cancer. A major current obstacle to the preemption of carcinoma is distinguishing morphologically atypical epithelial cells in the presence of inflammation (inflammatory atypia) that mimic dysplasia from morphologically atypical epithelial cells that are truly dysplastic. Formation of double stranded breaks in DNA (DSBs) is an accepted etiology for carcinoma and is, therefore, expected to be associated with dysplasia. Utilizing both algorithmic and artificial intelligence-based computer image analysis of IHC levels, we document the unexpected finding that phosphorylation of molecular markers associated with DSBs is consistently correlated with non-dysplastic iv inflammatory atypia in both squamous (oral cavity) and glandular (Barrett’s metaplasia) epithelia. Using these same image analysis methods, we further show that quantitative immunohistochemistry of the ratio of p-Chk2, a marker of DSB’s, and for mutational failure of the DNA damage repair pathway (p53) required for the proper response to DSBs can distinguish between inflammatory and dysplastic cellular atypia. The ability to use quantitative means to reliably distinguish between inflammatory and dysplastic atypia may facilitate the use of cytological screening for dysplasia to prevent cancer in numerous body sites

Focal Spot, Summer 1990

https://digitalcommons.wustl.edu/focal_spot_archives/1055/thumbnail.jp

Colposcopic and Cytological changes in intrauterine contraceptive device users: A Prospective study

INTRODUCTION: Intrauterine Contraceptive Device (IUD) is a safe, convenient and inexpensive method of contraception involving neither repetition nor interference with sexual activity. In India, IUD is the second most commonly used family planning method after voluntary female sterilization. IUD has its own merits and demerits. The various complications related to IUD use are, Immediate: Difficulty in insertion, Vasovagal attack, Uterine Cramps, Early: Expulsion, Perforation, Spotting, Menorrhagia, Dysmenorrhea, Vaginal Infection, Actinomyces. Late: Pelvic Inflammatory disease, Pregnancy, Ectopic Pregnancy. Various studies have been conducted since 1980 to study the long-term effect of IUD and its role in predisposition to cervical or endometrial pathology. The studies involve clinical examination, Cytology, Colposcopy and histopathology for detection of cervical pathology. Colposcopic examination involves the systematic evaluation of the lower genital tract with special emphasis on the superficial epithelium and blood vessels of the underlying connective tissue stroma. Cytological study is by means of papsmear. Presently exfoliative cytology and colposcopy are considered to be complementary to each other in the detection of early neoplastic changes. These procedures have contributed to the lowering of incidence of invasive cancer of the cervix in the developed world. AIM OF THE STUDY: To compare the colposcopic changes and cytological changes found in intrauterine contraceptive device users. To create awareness among the IUD users for regular follow up. SUBJECTS AND METHODS: This was a prospective study conducted at Government Kasturbha Gandhi Hospital, Chennai at the department of obstetrics and gynecology from November 2003 to March 2006. This study comprised of study subject [n=298] using intrauterine contraceptive device for more than 6 months and attended family planning clinic for follow up. INCLUSION CRITERIA: 1.Subjects using intrauterine contraceptive device for more than 6 months. 2.The subjects were chosen irrespective of their socio-demographic profile. 3.Never on any other contraceptive method. 4.In active sexual life. EXCLUSION CRITERIA: 1.Subjects with IUD using less than 6 months. 2.Subject with missing IUD 3.Subjects with pregnancy with IUD in situ. METHODS: The subjects were explained about the study and consent was obtained. Detailed history including any complaints, menstrual history, obstetric history and duration of IUD use was obtained. Each subject underwent general physical, systemic, abdominal examination. Then the acceptors was subjected to colposcopic examination. SUMMARY: 1. In this study the mean duration of IUD use is 17.5 months 2. 76 % of the study subjects had inflammatory changes in cytology 3. The incidence of inflammatory change is associated with the duration of the intra uterine contraceptive device use. 4. There is no significant increase in incidence of low grade intraepithelial neoplasia and high grade intraepithelial neoplasia in IUD users. CONCLUSION: The present study indicates that there is a definite change in the cervical architecture in IUD users and the change is also dependent on the duration of use. A simple technique like colposcope is highly useful in diagnosing these changes and combating them by early interference wherever necessary. However the present study and some of the earlier studies does not reveal any increased risk of dysplasia in IUD users over their non using sisters. However the need for regular and periodical follow up cannot be over emphasized particularly those who wish to use the IUD as a long term contraception

Digital Image Analysis Of Gram Stained Culture Specimens

Digital image analysis for the interpretation of images in clinical microbiology has many potential advantages over current practices. Compared to traditional image interpretation by a medical technologist, digital image analysis offers standardization between laboratories, round-the-clock interpretation, and quantitative results. In the first study of its kind known to the authors, a digital image analysis program was prototyped to interpret a slide containing Gram stained microorganisms. The sample microorganisms were obtained from culture plates during routine processing and subjected to Gram\u27s stain. An initial study learned from 11 Gram-stained slides and classified their microorganisms into the group: Gram-positive, Gram-negative, rods, coccus, and yeast. The sensitivity of identification ranged from 66% to 99% and the specificity ranged from 78% to 99%. The algorithm was next applied to a larger set of 78 slides. The accuracy rate for slide classification was 60 out of 78 or 77%. After using this larger dataset to train the algorithm, the accuracy rate for individual objects was 94% averaged over 5 trials. This suggests the parameters used by the algorithm can differentiate between groups, and the lack of accuracy in classifying the larger database occurred due to limitations in the original training data. Overall, the project demonstrates a unique application of digital image analysis to clinical microbiology

Applications of single-molecule detection in early disease diagnosis and enzymatic reaction study

Various single-molecule techniques were utilized for ultra-sensitive early diagnosis of viral DNA and antigen and basic mechanism study of enzymatic reactions. DNA of human papilloma virus (HPV) served as the screening target in a flow system. Alexa Fluor 532 (AF532) labeled single-stranded DNA probes were hybridized to the target HPV-16 DNA in solution. The individual hybridized molecules were imaged with an intensified charge-coupled device (ICCD) in two ways. In the single-color mode, target molecules were detected via fluorescence from hybridized probes only. This system could detect HPV-16 DNA in the presence of human genomic DNA down to 0.7 copy/cell and had a linear dynamic range of over 6 orders of magnitude. In the dual-color mode, fluorescence resonance energy transfer (FRET) was employed to achieve zero false-positive count. We also showed that DNA extracts from Pap test specimens did not interfere with the system. A surface-based method was used to improve the throughput of the flow system. HPV-16 DNA was hybridized to probes on a glass surface and detected with a total internal reflection fluorescence (TIRF) microscope. In the single-probe mode, the whole genome and target DNA were fluorescently labeled before hybridization, and the detection limit is similar to the flow system. In the dual-probe mode, a second probe was introduced. The linear dynamic range covers 1.44-7000 copies/cell, which is typical of early infection to near-cancer stages. The dual-probe method was tested with a crudely prepared sample. Even with reduced hybridization efficiency caused by the interference of cellular materials, we were still able to differentiate infected cells from healthy cells. Detection and quantification of viral antigen with a novel single-molecule immunosorbent assay (SMISA) was achieved. Antigen from human immunodeficiency virus type 1(HIV-1) was chosen to be the target in this study. The target was sandwiched between a monoclonal capture antibody and a polyclonal detector antibody. The capture antibody was covalently immobilized on modified glass slides. The detector antibody was conjugated with AF532 labeled secondary antibody prior to being used as probe for the antigen. Imaging was performed with a TIRF system. This technique is demonstrated for detecting HIV-1 p24 antigen down to 0.1 pg/mL with a dynamic range of over 4 orders of magnitude. A Langmuir isotherm fit the molecule count dependence on the target concentration. The results also showed that neither sensitivity nor dynamic range was affected by the biological matrix. SMISA is therefore a promising approach for the early diagnosis of virus-induced diseases. Single-molecule enzymatic kinetics and enantioselectivity were monitored in real time by using TIRF microscopy. AF532 labeled poly-L-lysine (PLL) or poly-D-lysine (PDL) was covalently immobilized on a dithiobis (succinimidyl undecanoate) self-assembled monolayer (DSU SAM). Chain shortening due to enzymatic hydrolysis resulted in the reduction of the individual fluorescence intensities. A broad distribution was obtained when 100 single-molecule half-lives were analyzed. However, the detailed hydrolysis process involved also a long-lived component and an induction period that varied significantly among molecules. Charge and steric heterogeneity at the surface are responsible for these features